Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Feasibility of a shortened pathway for nitrogen removal from highly nitrogenous wastes Turk, Oussama 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A1 T87.pdf [ 17.54MB ]
Metadata
JSON: 831-1.0062908.json
JSON-LD: 831-1.0062908-ld.json
RDF/XML (Pretty): 831-1.0062908-rdf.xml
RDF/JSON: 831-1.0062908-rdf.json
Turtle: 831-1.0062908-turtle.txt
N-Triples: 831-1.0062908-rdf-ntriples.txt
Original Record: 831-1.0062908-source.json
Full Text
831-1.0062908-fulltext.txt
Citation
831-1.0062908.ris

Full Text

THE FEASIBILITY OF A SHORTENED PATHWAY FOR NITROGEN REMOVAL FROM HIGHLY NITROGENOUS WASTES by OUSSAMA TURK B.E., American University of Beirut, 1973 M.E., American University of Beirut, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Civi l Engineering) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JUNE 1986 ®Oussama Turk, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Q\\i'\^ Ejr\$\t\e£Xin<^ The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 3 U T \ < L 50 \M(o DE-6(3/81) ABSTRACT The o b j e c t i v e of the r e s e a r c h program was to demonstrate the f e a s i b i l i t y of removing n i t r o g e n from h i g h l y n i t r o g e n o u s wastes by a shortened n i t r i f i c a t i o n / d e n i t r i f i c a t i o n pathway. T h i s p r o -cess bypasses the n i t r i t e o x i d a t i o n s tep to n i t r a t e and the c o n -comitant n i t r a t e r e d u c t i o n to n i t r i t e . The success of the s h o r t -cut i s dependent on s e l e c t i v e l y i n h i b i t i n g the n i t r i t e o x i d a t i o n s t ep , a l l o w i n g n i t r i t e to accumulate i n the a e r o b i c env ironment , and be s u b s e q u e n t l y reduced a n a e r o b i c a l l y to a gaseous p r o d u c t . Seven runs l a s t i n g between 13 and 359 days were undertaken us ing b e n c h - s c a l e , a c t i v a t e d s ludge c e l l s operated i n s e r i e s . Parameters i n v e s t i g a t e d as p o t e n t i a l s e l e c t i v e i n h i b i t o r s were: d i s s o l v e d oxygen, n i t r o u s a c i d , a n a e r o b i o s i s , and f r e e ammonia. Of these , o n l y f r e e ammonia (at 5 to 10 mg NH3 - N / L ) was found to be e f f e c t i v e as a d i f f e r e n t i a l i n h i b i t o r of u n a c c l i m a t e d p o p u l a t i o n s . N i t r i t e b u i l d - u p was a c h i e v e d by i n t e r m i t t e n t con-t a c t of the n i t r i t e o x i d i z e r s with the h i g h , f r e e ammonia l e v e l i n the c e l l at the f r o n t - e n d of the system. Comparison of t h i s shortened n i t r o g e n removal pathway with the t r a d i t i o n a l mechanism r e v e a l e d : 1) a 40% r e d u c t i o n of COD demand d u r i n g d e n i t r i f i c a t i o n ; 2) 63% h i g h e r r a t e of d e n i t r i f i c a -t i o n ; 3) Two t h i r d s r e d u c t i o n in biomass y i e l d d u r i n g a n a e r o b i c growth; 4) no apparent n i t r i t e t o x i c i t y e f f e c t s . The degree of n i t r i t e b u i l d - u p was found to be i n v e r s e l y r e l a t e d to the a e r o b i c r e s i d e n c e t ime. i i High n i t r i t e c o n c e n t r a t i o n s c o u l d not be s u s t a i n e d i n d e f i -n i t e l y , due to a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammmonia. Measures i n v e s t i g a t e d to overcome the e f f e c t s of a c c l i -mation were: 1) r e d u c t i o n of the s l u d g e age; 2) e x t e n s i o n of c o n t a c t time to h i g h , f ree ammonia l e v e l s ; 3) r a i s i n g f r e e ammo-n i a l e v e l s ; 4) use of a p o t e n t i a l l y more i n h i b i t o r y waste; 5) doub le s u b s t r a t e i n h i b i t i o n ; 6) p r o v i s i o n of i n t e r m e d i a r y r e c y -c l e , or i n t e r n a l d e n i t r i f i c a t i o n ; 7) temporary r e d u c t i o n of f r e e ammonia l e v e l s ; 8) temporary stoppage of f eed . Of these , the most e f f e c t i v e was i n t e r n a l d e n i t r i f i c a t i o n , due to the c o n t i n u e d a b i l i t y of f r e e ammonia to m a i n t a i n some degree of i n h i b i t i o n to an a c c l i m a t e d p o p u l a t i o n of n i t r i t e o x i d i z e r s . As a r e s u l t , t h e i r n i t r i f y i n g a c t i v i t y lagged that of the ammonia o x i d i z e r s by s e v e r a l h o u r s , a l l o w i n g n i t r i t e b u i l d - u p to s u s t a i n i t s e l f . i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES - ix LIST OF FIGURES .. x ACKNOWLEDGEMENTS . . . . x i i LIST OF ABBREVIATIONS x i i i 1. INTRODUCTION 1 Need For the Regulation of Nitrogen Discharges 1 Eutrophication of Water Bodies 2 Rising Levels of Nitrate in Surface and Ground-water . 2 Health Hazards to Humans, Animals and Aquatic Organisms . . . . . . . . . . . . . . 4 2. RESEARCH OBJECTIVES AND APPROACH 6 3. LITERATURE REVIEW . . 10 Bi o l o g i c a l N i t r i f i c a t i o n . . . . . . . 11 Bi o l o g i c a l Denitr i f i c a t i o n . . 13 Sources of Highly Nitrogenous Wastewater . . . . 14 Trends in Industrial Nitrogen Production . . . . 14 Chemistry of Ammonia . . 16 Chemistry of N i t r i t e 19 N i t r i t e Accumulation . . . 20 Oceanic Subsurface Waters 23 Recirculated Water in Commercial Fish Hatcheries . . . . 24 Subsurface S o i l Strata 24 Bi o l o g i c a l Treatment Systems for Highly Nitrogenous Wastewater 24 i v Page P o s t u l a t e d Causes of N i t r i t e Accumulation During N i t r i f i c a t i o n 25 Free Ammonia 26 N i t r o u s A c i d . 28 Temperature 32 D i s s o l v e d Oxygen 34 Other Causes 34 P o s t u l a t e d Causes of N i t r i t e Accumulation D u r i n g D e n i t r i f i c a t i o n . . . . 36 Predominace of N i t r a t e R e s p i r e r s 36 incomplete D e n i t r i f i c a t i o n 37 E f f e c t s of n i t r i t e . . 38 T o x i c i t y to Microorganisms 38 Acute and Subacute T o x i c i t y to F i s h . . . . . 43 E f f l u e n t C h l o r i n a t i o n . . 46 A b i l i t y to Grow on N i t r i t e As S o l e E l e c t r o n Acceptor 48 4. EXPERIMENTAL APPARATUS AND ANALYTICAL TECHNIQUES 50 Experimental Set-Up 50 Equipment . . . . . . . . . . . . . . . . . 50 Operation 54 S o l i d s Retention Time . . . . . . . . . . . . 55 Feed C h a r a c t e r i s t i c s 55 S y n t h e t i c Feed 55 Leachate . ' 58 A n a l y t i c a l and Sampling Techniques 59 A l k a l i n i t y 59 Ammonia Nitrogen 60 N i t r a t e Nitrogen 60 N i t r i t e Nitrogen 60 T o t a l K j e l d a h l Nitrogen 60 Chemical Oxygen Demand . . . . 60 D i s s o l v e d Oxygen 61 Oxi d a t i o n Reduction P o t e n t i a l . . . . . . . . . 61 Suspended S o l i d s 61 pH . 61 Nitrogen Content of Biomass . 62 Sampling Procedures 62 P r e s e n t a t i o n of Data 62 v P a g e 5 . R E S U L T S 6 4 R u n 1 6 9 O b j e c t i v e s 69 R e s u l t s •• • 6 9 D i s c u s s i o n 73 C o n c l u s i o n s . •• 7 4 R u n 2 7 5 O b j e c t i v e s . 7 5 R e s u l t s . . . 7 5 D i s c u s s i o n . . . 82 C o n c l u s i o n s • 85 R u n 3 86 O b j e c t i v e s 86 R e s u l t s 86 D i s c u s s i o n . . . . . . . . . . 88 C o n c l u s i o n s . . . . . . . . . 88 R u n 4 89 O b j e c t i v e s . . . . . . . . . . . . . . . . . 89 R e s u l t s . . . . . . . . . . . . 89 D i s c u s s i o n . . . . . . . . . . . . . . . . . . . . . 9 6 C o n c l u s i o n s . . . . . . . . . . . . . . • • •• 9 7 R u n 5 . . . . 9 8 O b j e c t i v e s 98 R e s u l t s 9 8 D i s c u s s i o n . . . . . . . . . . . . 1 0 4 C o n c l u s i o n s . . . . . . 104 R u n 6 1 0 5 O b j e c t i v e s . . 1 0 5 R e s u l t s 1 0 6 D i s c u s s i o n . . 1 0 9 C o n c l u s i o n s . . . . 110 R u n 7 • • 1 1 0 O b j e c t i v e s 1 1 0 R e s u l t s • 1 1 0 D i s c u s s i o n • C o n c l u s i o n s 118 v i Page Batch Tests 119 Batch Test No.l 119 Batch Test No.2 120 6. DISCUSSION 127 Causes of N i t r i t e Build-Up 127 Low D i s s o l v e d Oxygen L e v e l s . . . . . . . . . 127 High N i t r o u s A c i d L e v e l s 129 Ana e r o b i o s i s 132 High Free Ammonia L e v e l s . 133 E f f e c t s of N i t r i t e Build-Up 134 Comparison of COD Demand f o r N i t r i t e and N i t r a t e Reduction 135 Comparison of Growth Y i e l d During Anaerobic R e s p i r a t i o n . . . . . . . . . . . . . 137 Comparison of N i t r a t e and N i t r i t e Reduction Rates 142 Comparison of A l k a l i n i t y Consumption and Production . . . . . . . . . 146 E f f e c t s of N i t r i t e on Process Performance . . 149 A b i l i t y to Produce an E f f l u e n t Devoid of N i t r i t e . . . . . . . . . . . . . . . . . . . . . . 150 Aerobic Reduction of N i t r i t e ... . . 151 S t a b i l i t y of N i t r i t e Build-Up . 153 Aerobi c Residence Time . . . . . 154 Continued E f f e c t i v e n e s s of Free Ammonia . . . 157 Reversing the D e c l i n e i n N i t r i t e Build-up . . . 166 Reduction of the Sludge Age 166 Extension of the Contact Time With Free Ammonia 167 R a i s i n g of the Free Ammonia L e v e l 167 Use of a Genuine Waste 168 Double Substrate i n h i b i t i o n 169 P r o v i s i o n of I n t e r n a l Recycle or Intermediary D e n i t r i f i c a t i o n 171 Temporary Reduction of Free Ammonia L e v e l s . . 174 Temporary Stoppage of Feed 174 Others R e s u l t s and Observations . . . . 175 N i t r i f i c a t i o n Rates . 175 "Unaccounted For" Nitrogen Losses . . . . . . . 186 R e l a t i o n s h i p Between ORP and N i t r i t e + N i t r a t e L e v e l s 189 v i i Pag_e I n c o m p l e t e D e n i t r i f i c a t i o n 193 D u r a t i o n o f A n E x p e r i m e n t a l R u n 1 9 5 I m p l i c a t i o n s o f R e s u l t s i n P r a c t i c e . . . . .. 196 7 . C O N C L U S I O N S AND RECOMMENDATIONS 2 0 0 C o n c l u s i o n s 2 0 0 R e c o m m e n d a t i o n s f o r F u t u r e R e s e a r c h N e e d s . . 2 0 2 R E F E R E N C E S . . 2 0 4 A P P E N D I X A - RAW DATA 224 A P P E N D I X B - NITROGEN B A L A N C E S . . . . . . . . . . . . . . . . 2 4 4 A P P E N D I X C - AMMONIA V O L A T I L I Z A T I O N TEST 2 4 9 v i i i LIST OF TABLES Table Page 1 Sources and Le v e l s of Some High Strength Ammonia Wastes . 15 2 Ammonia I n h i b i t i o n of N i t r i f i c a t i o n i n Wastewater Treatment 29 3 N i t r i t e I n h i b i t i o n of N i t r i f i c a t i o n i n Wastewater Treatment 30 4 N i t r i t e I n h i b i t i o n of N i t r i f i c a t i o n i n Pure C u l t u r e s . . . . . . . . . . . . . . . . . . . . . 31 5 Pure C u l t u r e S t u d i e s of I n h i b i t o r y E f f e c t s of N i t r i t e . . . 40 6 S y n t h e t i c Feed Composition 56 7 Strength and C h a r a c t e r i s t i c s of Feed . . . . . . 57 8 Sample Handling 63 9 Summary of Parameters I n v e s t i g a t e d . . . . . . . . . . 65 10 O p e r a t i o n a l C h a r a c t e r i s t i c s 66 11 Sludge Age C h a r a c t e r i s t i c s . 67 12 Ammonia Le v e l s i n F i r s t C e l l 68 13 D i s s o l v e d Oxygen L e v e l s i n Runs 1 and 2 . . . . . . 70 .14 Results of Batch Test No. 2 126 15 Comparison of L i t e r a t u r e Values f o r COD Consumption and C e l l y i e l d During N i t r a t e R e s p i r a t i o n with R e s u l t s of t h i s Study . . . . . 140 16 Comparison Between N i t r i t e and N i t r a t e D i s s i m i l a t o r y Reduction Rates Reported i n the L i t e r a t u r e . 143 17 N i t r i f i c a t i o n Rates Reported i n the L i t e r a t u r e 177 18 N i t r i f i c a t i o n Rates 179 19 Nitrogen Balance Across The System For Runs 2, 4 and 5 18 8 20 Periods of Incomplete D e n i t r i f i c a t i o n During Run 7 194 ix LIST OF FIGURES F i g u r e P a g e 1 F r e e A m m o n i a C o n c e n t r a t i o n s a t V a r i o u s T e m p e r a t u r e s a n d pH V a l u e s 18 2 N i t r o u s A c i d C o n c e n t r a t i o n s a t V a r i o u s T e m p e r a t u r e s a n d pH V a l u e s . 21 3 P r o c e s s T r e a t m e n t S c h e m a t i c C o n f i g u r a t i o n . . . . 5 1 4 P h o t o g r a p h s o f B e n c h - S c a l e S e t - U p . 52 5 R u n 2 / S y s t e m 1 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 76 6 R u n 2 / S y s t e m 2 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 77 7 R u n 4 / S y s t e m 1 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 90 8 R u n 4 / S y s t e m 2 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 91 9 P r o c e s s T r e a t m e n t S c h e m a t i c F o r R u n 5 / S y s t e m 2 99 10 R u n 5 / S y s t e m 1 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 1 0 1 11 R u n 5 / S y s t e m 2 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l 1 0 2 12 R u n 6 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l . . . . 108 13 R u n 7 - E x t e n t o f N i t r i t e B u i l d - U p i n A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l i n F i r s t C e l l . . . . 113 14 B a t c h T e s t N o . l - R u n 4 / S y s t e m 2 N i t r o g e n T r a n s f o r m a t i o n s . . . . . . . . . . . . . 1 2 1 1 5 B a t c h T e s t N o . 2 R e a c t o r . . 1 2 3 16 B a t c h T e s t N o . 2 / R u n 5 - D e n i t r i f i c a t i o n R a t e s F o r N i t r a t e a n d N i t r i t e 125 17 R u n 2 / S y s t e m 2 - E f f e c t o f F r e e A m m o n i a a n d N i t r o u s A c i d L e v e l s o n N i t r i t e B u i l d - U p 130 18 R e l a t i o n s h i p B e t w e e n : F r e e A m m o n i a L e v e l i n A n a e r o b i c C e l l , H o u r s o f S u b s e q u e n t A e r a t i o n a n d N i t r i t e B u i l d - U p . . 1 5 5 x Page 19 Run 7 - E f f e c t of Free Ammonia on N i t r i t e Build-Up A f t e r A c c l i m a t i o n 158 20 Run 4/System 2 - I n h i b i t i o n of N i t r i f i c a t i o n A c t i v i t y due to Shock Load of Free Ammonia . . . . 164 21 E f f e c t of SRT on N i t r i f i c a t i o n Rate . . . . . . . . . 178 22 Run 2/System .1 - E f f e c t of D i s s o l v e d Oxygen on N i t r i f i c a t i o n Rate i n F i r s t Aerobic C e l l . . . . 182 23 Run 2/System 2 - E f f e c t of pH on N i t r i f i c a t i o n Rate 185 24 Ammonia S t r i p p i n g Test R e s u l t s . ,. . . . . . . . . . 190 25 Run 2 - E f f e c t of N i t r a t e + N i t r i t e L e v e l s on ORP . . . . . . . 192 26 Run 7 — Extent of N i t r i t e Build-Up i n Aerobic C e l l s and Free Ammonia L e v e l i n F i r s t C e l l to Day 200 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 x i ACKNOWLEDGEMENTS I wish to express my g r a t i t u d e f o r the c o n s i d e r a b l e a s s i s t a n c e , encouragement and moral support p r o v i d e d by my a d v i s o r Dr. D. S. M a v i n i c , Environmental Engineering D i s c i p l i n e Head at the U n i v e r s i t y of B r i t i s h Columbia. I would a l s o l i k e to thank the f o l l o w i n g f o r t h e i r , much a p p r e c i a t e d , support: - Susan L i p t a k , Environmental Engineering Laboratory Techni-c i a n , f o r her u n f a i l i n g a s s i s t a n c e and understanding during the two and a h a l f years of l a b o r a t o r y work. - Paula Parkinson, Environmental Engineering Laboratory T e c h n i c i a n , f o r a l l her support, e s p e c i a l l y during the two batch t e s t s . - Guy K i r s c h , C i v i l Engineering Workshop T e c h n i c i a n , f o r h i s suggestions and a s s i s t a n c e i n manufacturing the v a r i o u s compo-nents of the bench-scale u n i t s . - C h r i s Wike, M u n i c i p a l i t y of Surrey P u b l i c Works Depart-ment, f o r h i s cooperation and a s s i s t a n c e with l e a c h a t e c o l l e c t i o n d uring the bi-weekly t r i p s to the Port Mann l a n d f i l l . I would a l s o l i k e to thank a l l my f e l l o w environmental engineering graduate students, f o r t h e i r support throughout the r e s e a r c h program. x i i LIST OF ABBREVIATIONS ATP adenosine t r i p h o s p h a t e COD chemical oxygen demand d day DO d i s s o l v e d oxygen g gram hr hour HRT h y d r a u l i c r e t e n t i o n time Ko Michaelis-Menten h a l f s a t u r a t i o n c o e f f i c i e n t f o r oxygen L l i t r e s M molar mM m i l l i m o l a r MLSS mixed l i q u o r suspended s o l i d s MLVSS mixed l i q u o r v o l a t i l e suspended s o l i d s n number of samples (or measurements) N.R. n i t r i f i c a t i o n r a t e ORP o x i d a t i o n r e d u c t i o n p o t e n t i a l s standard d e v i a t i o n SRT s o l i d s r e t e n t i o n time (sludge age) TKN t o t a l K j e l d a h l n i t r o g e n ug micrograms x i i i CHAPTER ONE INTRODUCTION NEED FOR THE R E G U L A T I O N OF NITROGEN D I S C H A R G E S Human a c t i v i t y h a s c a u s e d p e r t u r b a t i o n t o t h e g l o b a l n i t r o -g e n c y c l e . T h e p a s t f e w d e c a d e s h a v e s e e n , a p r o g r e s s i v e i n c r e a s e i n t h e a m o u n t o f f i x e d n i t r o g e n i n t h e e n v i r o n m e n t . T h e e x t e n t a n d l o n g - t e r m i m p a c t o f s u c h i n t e r v e n t i o n r e m a i n s a m a t t e r o f c o n j e c t u r e a n d t h e f o c u s o f o n - g o i n g s t u d i e s ( D e l w i c h e , 1 9 7 7 ; B o l i n a n d A r r h e n i u s , 1 9 7 7 ; R o l s t o n , 1 9 8 1 ; C r u t z e n , 1 9 8 1 ; W i l k i n -s o n a n d G r e e n e , 1 9 8 2 ) . T h e m a j o r p o i n t o f human i n t e r v e n t i o n i n t h e n i t r o g e n c y c l e i s i n t h e p r o c e s s o f n i t r o g e n f i x a t i o n . T h e a c c e l e r a t i o n o f h u m a n a c t i v i t y i n t h e p a s t c e n t u r y h a s l e d t o a d o u b l i n g o f t h e r a t e o f a t m o s p h e r i c n i t r o g e n f i x a t i o n . T h i s h a s come a b o u t p r i m a r i l y f r o m t h e d e v e l o p m e n t o f i n d u s t r i a l n i t r o g e n f i x a t i o n p r o c e s s e s , w h i c h a r e w i d e l y u s e d f o r f e r t i l i z e r p r o d u c -t i o n a n d f r o m t h e e x p a n d i n g a g r i c u l t u r a l p r a c t i c e o f p l a n t i n g n i t r o g e n - f i x i n g l e g u m e c r o p s ( D e l w i c h e , 1 9 8 1 ; C r u t z e n , 1 9 8 1 ) . I t i s b e l i e v e d t h a t , p r i o r t o h u m a n i n t e r v e n t i o n i n t h e n i t r o g e n c y c l e , a b a l a n c e e x i s t e d b e t w e e n t h e r a t e s o f n i t r o g e n f i x a t i o n a n d t h e r e t u r n f l o w o f n i t r o g e n g a s e s t o t h e a t m o s p h e r e v i a n i t r i f i c a t i o n a n d d e n i t r i f i c a t i o n ( C r u t z e n , 1 9 8 1 ) . T h e i m b a -l a n c e t h a t a p p e a r s t o e x i s t i n t h e c y c l e a t p r e s e n t s t e m s f r o m t h e i n a b i l i t y o f t h e d e n i t r i f i c a t i o n s t e p t o k e e p p a c e w i t h t h e e v e r - i n c r e a s i n g r a t e o f n i t r o g e n f i x a t i o n , l e a d i n g t o a n a c c u m u -1 l a t i o n o f f i x e d n i t r o g e n i n t h e t e r r e s t r i a l a n d a q u a t i c e n v i r o n -m e n t s ( D e l w i c h e , 1 9 8 1 ) . Some o f t h e d e t r i m e n t a l i m p a c t s a s s o c i a t e d w i t h r i s i n g l e v e l s o f n i t r o g e n s p e c i e s i n t h e a q u a t i c e n v i r o n m e n t i n c l u d e : 1) E u t r o p h i c a t i o n o f w a t e r b o d i e s ; 2) R i s i n g l e v e l s o f n i t r a t e i n s u r f a c e a n d g r o u n d w a t e r ; 3) H e a l t h h a z a r d s t o h u m a n s , a n i m a l s a n d a q u a t i c o r g a n i s m s . E u t r o p h i c a t i o n o f W a t e r B o d i e s T h e e u t r o p h i c a t i o n o f w a t e r b o d i e s i s c a u s e d b y a n e x c e s s o f n u t r i e n t s ( m o s t l y p h o s p h o r u s a n d n i t r o g e n ) a n d h a s b e e n t h e s u b j e c t o f e x t e n s i v e r e s e a r c h i n t h e p a s t t w e n t y y e a r s . A s a r e s u l t , t h e p r o b l e m s a s s o c i a t e d w i t h i t a r e w e l l u n d e r s t o o d t o d a y . T h e u n d e s i r a b l e c h a n g e s c a u s e d b y e u t r o p h i c a t i o n i n c l u d e : a l g a l b l o o m s , d e p l e t i o n o f d i s s o l v e d o x y g e n (DO) i n b o t t o m w a t e r s , r e d u c t i o n i n w a t e r c l a r i t y , s h o r t e n e d f o o d c h a i n s , l o s s o f a e s t h e t i c a p p e a l , a n d p r o b l e m s w i t h w a t e r t r e a t m e n t ( N . R . C , 1 9 7 8 ; B o l i n a n d A r r h e n i u s , 1 9 7 7 ) . R i s i n g L e v e l s o f N i t r a t e i n S u r f a c e a n d G r o u n d - W a t e r N i t r a t e l e v e l s i n s u r f a c e w a t e r s a n d s h a l l o w a q u i f e r s a p p e a r t o b e r i s i n g s t e a d i l y i n m a n y a r e a s ( N . R . C , 1 9 7 8 ; W . H . O . , 1 9 7 8 ; B r o o k s a n d C e c h , 1 9 7 9 ; F o s t e r e t a l . , 1 9 8 2 ; S o n n e b o r n e t a 1 . , 1 9 8 3 ; W h i t e , 1 9 8 3 ) . T h e a v e r a g e n i t r a t e l e v e l i n t h e T h a m e s R i v e r , w h i c h i s t h e m a j o r w a t e r s u p p l y s o u r c e f o r t h e c i t y o f L o n d o n , E n g l a n d h a s i n c r e a s e d f r o m a b o u t 2 . 5 mg NO3 - N / L i n 1 9 2 8 t o a r o u n d 8 . 0 mg NO3 - N / L i n 1 9 7 7 , b r i n g i n g i t c l o s e t o t h e U n i t e d K i n g d o m a c c e p t a b l e l i m i t o f 1 1 . 3 mg NO3 - N / L ( W i l k i n s o n a n d G r e e n e , 1 9 8 2 ) . I n t h e S t a t e o f I o w a , t h e a v e r a g e n i t r a t e c o n t e n t 2 of the Iowa R i v e r has increased from 1.4 mg NO3-N/L i n 1973 to 5.2 mg NO3-N/L i n 1980 (McDonald and S p l i n t e r , 1982). As e a r l y as 1969, about 3% of a l l p u b l i c water s u p p l i e s i n the U.S. contained n i t r a t e l e v e l s i n excess of the U.S. l i m i t of 10 mg NO3-N/L (Hartman, 1982). I t i s estimated that 5 to 10% of the West German p o p u l a t i o n i s c u r r e n t l y s u p p l i e d with water c o n t a i n i n g i n excess of 10 mg NO3-N/L (Dairmont 1984). I t should, a l s o be remembered that average n i t r a t e l e v e l s do not r e f l e c t c o m p l i c a t i n g f a c t o r s such as year-to-year and seasonal v a r i a t i o n s i n n i t r a t e concen-t r a t i o n s (Wilkinson and Greene, 1982).. A r e c e n t s u r v e y i n the U n i t e d Kingdom found t h a t the con-t r i b u t i o n of domestic water to the n i t r a t e i n take was below 30% i n water s u p p l i e s c o n t a i n i n g l e s s than 50 mg NO3/L (11.3 mg NO3-N/L).. This v a l u e rose to over 80% when the n i t r a t e content of the water was .100 mg NO3/L (22.6 mg NO3-N/L) and over. The annual cost of complying with the 50 mg NO3/L (11.3 mg NO3-N/L) l i m i t f o r d r i n k i n g water s u p p l i e s i n the United Kingdom was estimated at 15 m i l l i o n pounds ($30 m i l l i o n CDN) which would r i s e to about 34 m i l l i o n pounds ($68 m i l l i o n CDN) should the l i m i t be reduced to 40 mg NOJ/L (9.0 mg NO3-N/L) (White, 1983). The r i s i n g l e v e l s of n i t r a t e i n d r i n k i n g water s u p p l i e s i s l i n k e d to a number of human a c t i v i t i e s . They i n c l u d e : l e a c h i n g and s u r f a c e r u n - o f f from f e r t i l i z e d c r o p l a n d s (Commoner, 1977; N.R.C., 1977; Fos t e r et a l . , 1982; McDonald and S p l i n t e r , 1982; Egboka, 1984), l e a c h i n g from s e p t i c tank and t i l e f i e l d systems (N.R.C., 1978, Brooks and Cech, 1979); e f f l u e n t discharge from m u n i c i p a l and i n d u s t r i a l wastewater treatment p l a n t s (N.R.C., 3 1 9 7 8 ; W i l k i n s o n a n d G r e e n e , 1 9 8 2 ) , a n d l e a c h i n g f r o m l a n d f i l l s , b a r n y a r d s a n d m a n u r e l a g o o n s ( E g b o k a , 1 9 8 4 ) . H e a l t h H a z a r d s t o H u m a n s , A n i m a l s a n d A q u a t i c O r g a n i s m s I n h u m a n s , t h e h e a l t h e f f e c t s l i n k e d t o t h e i n g e s t i o n o f n i t r a t e h a v e , u n t i l r e c e n t l y , b e e n m o s t l y a s s o c i a t e d w i t h t h e d e v e l o p m e n t o f m e t h a e m o g l o b i n e m i a ( b l u e b a b y s y n d r o m e ) i n i n f a n t s u n d e r s i x m o n t h s o f a g e . A s a r e s u l t , d r i n k i n g w a t e r q u a l i t y g u i d e l i n e s i n m o s t c o u n t r i e s l i m i t t h e n i t r a t e c o n t e n t t o b e t w e e n 10 a n d 1 1 . 3 mg NO3 - N / L . A n o t h e r h e a l t h r i s k r e c e n t l y i d e n t i f i e d w i t h t h e i n g e s t i o n o f d r i n k i n g w a t e r c o n t a i n i n g h i g h l e v e l s o f n i t r a t e i n v o l v e s t h e f o r m a t i o n o f N - n i t r o s o c o m p o u n d s i n t h e h u m a n b o d y . A s a g r o u p , N - n i t r o s o c o m p o u n d s a r e c o n s i d e r e d t o b e t h e m o s t p o t e n t c a r c i n o -g e n s k n o w n t o d a t e ( N . R . C , 1 9 8 1 ; H a r t m a n , 1 9 8 2 ) . E v i d e n c e i s a l s o a c c u m u l a t i n g t h a t n i t r a t e i n g e s t i o n may b e r e s p o n s i b l e f o r d e l e t e r i o u s i m p a c t s o n t h e t h y r o i d f u n c t i o n ( H a r t m a n , 1 9 8 2 ) . I n h i s r e v i e w o f t h e human h e a l t h e f f e c t s a s s o c i a t e d w i t h t h e i n g e s -t i o n o f n i t r a t e s a n d n i t r i t e s , H a r t m a n ( 1 9 8 2 ) c o n c l u d e d t h a t t h e i r i n g e s t i o n p o s e d a m a j o r h e a l t h h a z a r d . O t h e r p o s s i b l e h e a l t h r i s k s a s s o c i a t e d w i t h t h e p r e s e n c e o f n i t r a t e i n w a t e r i n c l u d e s l o w d o w n o f r e f l e x e s i n c h i l d r e n a n d h i g h e r h y p e r t e n s i o n r i s k among t h e g e n e r a l p o p u l a t i o n ( F r a s e r a n d C h a l m e r s , 1 9 8 1 ) . A m m o n i a a n d n i t r i t e t o x i c i t y t o a q u a t i c o r g a n i s m s a r e w e l l d o c u m e n t e d ( R u s s o e t a _ l . , 1 9 7 4 ; N . R . C , 1 9 7 8 ; A . P . T . , 1 9 8 1 ; D a o u s t a n d F e r g u s o n , 1 9 8 4 ; E r i c k s o n , 1 9 8 5 ) . S e n s i t i v i t y v a r i e s w i t h t h e s p e c i e s a n d w i t h p a r a m e t e r s s u c h a s t e m p e r a t u r e , s a l i n i -4 ty, pH, d i s s o l v e d oxygen (DO) l e v e l s , carbon d i o x i d e l e v e l s , and l i f e stage of the organism (A.P.I., 1981). For example, sub-l e t h a l e f f e c t s have been observed on rainbow t r o u t at f r e e ammo-ni a l e v e l s as low as 0.02 mg NH3-N/L (Thurston et a_l., 1984). In c o n c l u s i o n , our i n c r e a s i n g awareness of the problems a s s o c i a t e d with n i t r o g e n accumulation i n the t e r r e s t r i a l and a q u a t i c r e s e r v o i r s i s g r a d u a l l y l e a d i n g to the development of c o n t r o l s t r a t e g i e s that attempt to r e v e r s e the trend i n n i t r o g e n accumulation and r e s t o r e the n a t u r a l n i t r o g e n balance. One aspect of such s t r a t e g i e s would i n v o l v e the development of c o s t - e f f e c -t i v e , wastewater treatment processes that emphasize d e n i t r i f i c a -t i o n (N.R.C., 1978). 5 CHAPTER TWO RESEARCH OBJECTIVES AND APPROACH T h e g o a l o f t h e r e s e a r c h p r o g r a m w a s t o i n v e s t i g a t e t h e v i a b i l i t y o f i n d u c i n g s e l e c t i v e i n h i b i t i o n , t o s h o r t c i r c u i t t h e t r a d i t i o n a l n i t r i f i c a t i o n / d e n i t r i f i c a t i o n p a t h w a y s f o r n i t r o g e n r e m o v a l f r o m h i g h l y n i t r o g e n o u s w a s t e s : N H j NO2 *- NO3 - NO2 N 2 o x i d a t i o n r e d u c t i o n T h i s i n v o l v e d s h o r t e n i n g t h e t r a d i t i o n a l n i t r i f i c a t i o n / d e n i t r i -f i c a t i o n p a t h w a y b y b l o c k i n g t h e n i t r i f i c a t i o n s e q u e n c e a t t h e n i t r i t e i n t e r m e d i a r y , t h u s b y p a s s i n g t h e n i t r i t e o x i d a t i o n s t e p t o n i t r a t e a n d t h e s u b s e q u e n t n i t r a t e r e d u c t i o n s t e p t o n i t r i t e . N i t r i t e p r o d u c e d u n d e r a e r o b i c c o n d i t i o n s w o u l d b e r e d u c e d a n a e -r o b i c a l l y t o a g a s e o u s p r o d u c t . T h e r e s u l t a n t p a t h w a y NH.J ». NOj *• N 2 o x i d a t i o n r e d u c t i o n c o u l d , r e p o r t e d l y , l e a d t o t h e f o l l o w i n g p r a c t i c a l b e n e f i t s : 1) Up t o 25% r e d u c t i o n i n o x y g e n r e q u i r e m e n t f o r n i t r i f i c a t i o n ; 2) Up t o 40% r e d u c t i o n i n o r g a n i c c a r b o n r e q u i r e m e n t f o r d e n i t r i f i c a t i o n ; 6 3) Up to 50% r e d u c t i o n i n the s i z e of the anaerobic (anoxic)* r e a c t o r r e q u i r e d f o r d e n i t r i f i c a t i o n ; Other o b j e c t i v e s of the research program were to: 1) Confirm the lower t h e o r e t i c a l COD consumption r a t e a s s o c i a t e d with the r e d u c t i o n of n i t r i t e ; 2) Confirm the higher d e n i t r i f i c a t i o n r a t e s reported f o r the r e d u c t i o n of n i t r i t e ; 3) Confirm the reported r e l a t i o n s h i p between n i t r i f i c a t i o n r a t e and low d i s s o l v e d oxygen l e v e l s ; 4) Confirm the r e l a t i o n s h i p between n i t r i f i c a t i o n r a t e and pH; 5) Confirm the r e l a t i o n s h i p between n i t r i f i c a t i o n r a t e and sludge age; 6) Compare a l k a l i n i t y consumption and production r a t e s between n i t r i t e and n i t r a t e p r o d u c t i o n and r e d u c t i o n ; 7) Compare the m i c r o b i a l growth y i e l d between n i t r a t e and n i t r i t e r e d u c t i o n ; 8) E v a l u a t e the t o x i c i t y of n i t r i t e to the treatment process; 9) Conduct n i t r o g e n balances across the system to confirm "unac-countable" n i t r o g e n l o s s e s f r e q u e n t l y reported i n the l i t e r a -t u r e ; 10) Confirm the e x i s t e n c e of a r e l a t i o n s h i p between n i t r i t e •+ n i t r a t e l e v e l s i n the anaerobic c e l l and o x i d a t i o n r e d u c t i o n p o t e n t i a l (ORP); 11) Study the long-term s t a b i l i t y of the proposed s h o r t c u t f o r n i t r o g e n removal. *: The term "anoxic" w i l l not be used i n t h i s t e x t . An anaerobic environment w i l l r e f e r s t r i c t l y to the absence of d i s s o l v e d oxygen from the medium, and w i l l not p r e c l u d e the presence of other e l e c t r o n acceptors. 7 Of the v a r i o u s parameters reported i n the l i t e r a t u r e (see Chapter 3) as capable of inducing n i t r i t e b u i l d - u p , the most promising ones appeared to be: 1) Free ammonia 2) D i s s o l v e d oxygen 3) N i t r o u s a c i d The research program was d i r e c t e d toward studying, among others, the e f f e c t s of these parameters. The s e l e c t e d treatment process had to o f f e r s u f f i c i e n t f l e x i b i l i t y to a l l o w the i n v e s t i g a t i o n of these parameters. To t h i s end, the a c t i v a t e d sludge process was s e l e c t e d . Of the v a r i o u s a c t i v a t e d sludge process c o n f i g u r a t i o n s i n use (i.e. c o m p l e t e l y mixed, p l u g flow, p l u g flow step-feed), the p l u g flow c o n f i g u r a t i o n was adopted. I t was recognized from the outset that a l a b o r a t o r y s c a l e u n i t cannot c o m p l e t e l y d u p l i c a t e t h i s c o n f i g u r a t i o n . However, the system adopted (see Chapter 4) s a t i s f i e d the needs of the study. I t a l l o w e d f o r : 1) The c r e a t i o n of a c o n c e n t r a t i o n g r a d i e n t across the system f o r ammonium and n i t r i t e ; 2) The c o n t r o l of the DO l e v e l s w i t h i n a l l c e l l s ; 3) The complete o x i d a t i o n of a l l ammonia found i n the wastewater; 4) I n t e r m i t t e n t contact of the n i t r i t e o x i d i z e r s to the s p e c i f i c environment w i t h i n each c e l l ; 5) O x i d a t i o n of the r e s i d u a l n i t r i t e to n i t r a t e p r i o r to i t s discharge i n t o the e f f l u e n t . To reduce v a r i a b i l i t y from the feed source, a s y n t h e t i c wastewater was used during the f i r s t f i v e runs. The experimental 8 design for these runs was based on the use of two i d e n t i c a l systems (referred to as Systems 1 and 2); a change was imposed on one system at a time and the effect studied and compared with the performance of the other. A high ammonia municipal l a n d f i l l leachate was used during the l a s t two runs (Runs 6 and 7); these were limited to the operation of a singl e system (as explained in Chapter 5).. 9 CHAPTER THREE LITERATURE REVIEW Although a number of researchers (Prakasam and Loehr, 1972; Murray et a l . , 1975; Voets et a_l., 1975; Laudelout et a_l., 1976; Sauter and Alleman 1980; B l a s z c z y k , 1981) have i d e n t i f i e d the p o t e n t i a l advantages a s s o c i a t e d with the implementation of a sh o r t c u t i n n i t r o g e n removal, v i a the pro d u c t i o n and r e d u c t i o n of n i t r i t e , few s t u d i e s appear to have been undertaken to t e s t the hypothesis and d e v e l o p a process c o n f i g u r a t i o n that would achieve the s h o r t c u t . At the time t h i s research program was e n v i s i o n e d i n e a r l y 1983, the l i t e r a t u r e on the v i a b i l i t y of the shortened pathway fo r n i t r o g e n removal was minimal. Since then, the r e s u l t s of some s t u d i e s have been p u b l i s h e d . T h i s probably r e f l e c t s recent i n t e r -e s t with the t o p i c and i n d i c a t e s that concurrent research i s probably underway at a number of research i n s t i t u t i o n s . L i t e r a -ture on the su b j e c t which has appeared s i n c e the i n t i t i a t i o n of t h i s research program i s presented below. Senanayake (1982) undertook a study to d e v e l o p o p e r a t i o n a l s t r a t e g i e s f o r n i t r o g e n removal v i a n i t r i t e p r o d u c t i o n and reduc-t i o n , using a bench-scale sequencing batch r e a c t o r . He determined that a 3 hour a e r a t i o n c y c l e time produced the highest n i t r i t e l e v e l s i n the system but was unable to s u s t a i n high n i t r i t e c o n c e n t r a t i o n s i n any system beyond a p e r i o d of two months. Batch and bench-scale s t u d i e s , using a co m p l e t e l y mixed 10 a c t i v a t e d sludge system, have been ongoing at the U n i v e r s i t y of Toronto f o r s e v e r a l years. The r e s u l t s , which w i l l be p u b l i s h e d i n the near f u t u r e , seem to i n d i c a t e that the s h o r t c u t i s f e a s i -b l e (Sutherson, 1984 and 1986). Based upon short-term batch t e s t s , B e c c a r i (1983) maintained that implementation of the s h o r t c u t d i d not appear p o s s i b l e , due to the i r r e v e r s i b l e i n h i b i t i o n of n i t r i t e r e d u c t i o n at n i t r i t e l e v e l s above 20 mg NO^-N/L. From the aforementioned, i t i s e v i d e n t that s u b s t a n t i a l research remains to be undertaken i n t h i s area. The l i t e r a t u r e review w i l l , t h e r e f o r e , focus on other aspects that have some bearing on the t o p i c . These i n c l u d e : types of h i g h l y nitrogenous wastes that c o u l d b e n e f i t from the proposed s h o r t c u t i n n i t r o g e n removal, trends i n i n d u s t r i a l n i t r o g e n production, observed instances of n i t r i t e accumulation i n d i f f e r e n t environments, p o s t u l a t e d causes f o r t h i s accumulation, e f f e c t s of n i t r i t e , and a b i l i t y of organisms to reduce n i t r i t e . T h i s w i l l be preceded by a b r i e f overview of n i t r i f i c a t i o n and d e n i t r i f i c a t i o n . BIOLOGICAL NITRIFICATION N i t r i f i c a t i o n i s commonly d e f i n e d as the s e q u e n t i a l b i o l o g i -c a l o x i d a t i o n of ammonia to n i t r i t e and of n i t r i t e to n i t r a t e . T h i s i s c a r r i e d out by two d i f f e r e n t groups of chemoautotrophs, Nitrosomonas N i t r o b a c t e r NHj *- NO2 *• NO3 n i t r i t e being the end product of ammonia o x i d a t i o n by the ammonia o x i d i z e r s (the predominant genus being Nitrosomonas) and a sub-11 s t r a t e f o r the n i t r i t e o x i d i z e r s (the predominant genus being N i t r o b a c t e r ) . Our understanding of the process has progressed s i g n i f i c a n t -l y s i n c e 1877, when S c h l o e s i n g and Muntz f i r s t demonstrated un-e q u i v o c a l l y t h at n i t r i f i c a t i o n was c a r r i e d out by a e r o b i c l i v i n g organisms (S c h l o e s i n g and Muntz, 1877). Due to t h e i r e c o l o g i c a l importance and u b i q u i t o u s nature, n i t r i f y i n g microorganisms have been the focus of study by a d i v e r s e group of d i s c i p l i n e s i n c l u d -ing s o i l s c i e n t i s t s , 1 i m n o l o g i s t s , oceanographers, m i c r o b i o l o -g i s t s , and environmental engineers. T h i s has r e s u l t e d i n an e x t e n s i v e l i t e r a t u r e on the s u b j e c t . A comprehensive review of n i t r i f i c a t i o n i s beyond the scope of t h i s t h e s i s , which w i l l be r e s t r i c t e d to the review of aspects of immediate bearing on the research t o p i c . Reviews on n i t r i f i c a -t i o n appear r e g u l a r l y i n the l i t e r a t u r e and the reader i s r e f e r -red to them f o r a d d i t i o n a l i n f o r m a t i o n . These i n c l u d e : M e i k e l j o h n (1954) and Delwiche (1956a) f o r a g e n e r a l review of the e a r l i e r works, P a i n t e r (1970 and 1977) f o r e x t e n s i v e reviews on the s u b j e c t , Matthias (1980) and Richardson (1985) f o r a review of n i t r i f i c a t i o n i n h i b i t o r s , Focht and Chang (1975) and Sharma and A h l e r t (1977) f o r aspects r e l a t e d to wastewater treatment, Focht and V e r s t r a e t e (1977) and B e l s e r (1979) f o r b i o c h e m i c a l ecology aspects, Schmidt (1982) and Reddy and P a t r i c k (1984) f o r n i t r i f i -c a t i o n i n s o i l s , Bremner and Blackmer (1981) f o r n i t r o u s oxide production, and Drozd (1980) f o r n i t r i f i e r r e s p i r a t i o n mecha-nisms. 12 BIOLOGICAL DENITRIFICATION D e n i t r i f i c a t i o n r e f e r s to a s e r i e s of anaerobic r e s p i r a t i o n processes that reduce n i t r a t e or n i t r i t e to a gaseous product. N i t r i t e i s the f i r s t intermediary i n the d e n i t r i f i c a t i o n process. To-date, about .24 genera of b a c t e r i a have been i d e n t i f i e d as being capable of d e n i t r i f i c a t i o n (Jeter and Ingraham, 1981). N i t r a t e r e s p i r a t i o n r e f e r s to the a b i l i t y of a microorganism to reduce n i t r a t e to n i t r i t e but not f u r t h e r to a gaseous end-product. Such organisms are not deemed to be d e n i t r i f i e r s , s i n c e d e n i t r i f i c a t i o n i s d e f i n e d as the a b i l i t y to produce a gaseous end-product. Around 50 genera of b a c t e r i a are known to c o n t a i n s t r a i n s capable of n i t r a t e r e s p i r a t i o n (Jeter and Ingraham, 1981). Fragmentary evidence of the e x i s t e n c e of d e n i t r i f i c a t i o n s t a r t e d to emerge i n the nineteenth century. In 1856, R e i s e t found that manure possessed the c a p a b i l i t y of producing n i t r o g e n gas when immersed i n water (Reiset, 1856). I n t e r e s t i n d e n i t r i f i -c a t i o n s p r e a d r a p i d l y and l e d i n 1886 to the i s o l a t i o n of the f i r s t known d e n i t r i f y i n g organisms and the o b s e r v a t i o n t h a t the phenomenon occurred i n the absence of oxygen (Gayon and D u p e t i t , 1886). D e n i t r i f i c a t i o n remains, to t h i s day, the focus of intense research, mostly by the same group of d i s c i p l i n e s i n v o l v e d i n n i t r i f i c a t i o n research. As with n i t r i f i c a t i o n , the l i t e r a t u r e on the s u b j e c t i s e x t e n s i v e and beyond the scope of t h i s t h e s i s . The review presented here w i l l be l i m i t e d to aspects with d i r e c t b earing on t h i s research t o p i c . Reviews on the su b j e c t appear r e g u l a r l y . They i n c l u d e : D e l -13 w i c h e ( 1 9 5 6 b ) f o r a r e v i e w o f e a r l i e r w o r k s , P a i n t e r ( 1 9 7 0 ) , F o c h t a n d V e r s t r a e t e (1977) a n d K n o w l e s ( 1 9 8 2 ) f o r g e n e r a l r e -v i e w s / C h r i s t e n s e n a n d H a r r e m o e s ( 1 9 7 7 ) , S h a r m a a n d A h l e r t (1977) a n d S c h r o e d e r ( 1 9 8 1 ) f o r d e n i t r i f i c a t i o n i n w a s t e w a t e r t r e a t m e n t , F i r e s t o n e ( 1 9 8 2 ) , F i l l e r y ( 1 9 8 3 ) a n d R e d d y a n d P a t r i c k ( 1 9 8 4 ) f o r d e n i t r i f i c a t i o n i n s o i l s , B r e z o n i k (1977) f o r n a t u r a l w a t e r s , T h a u e r e t a _ l . (1977) a n d S t o u t h a m e r et al. ( 1 9 8 0 ) f o r e n e r g e t i c s a n d r e s p i r a t i o n m e c h a n i s m s , I n g r a h a m (1981) a n d B r y a n ( 1 9 8 1 ) f o r m i c r o b i o l o g y a n d b i o c h e m i s t r y , R o l s t o n ( 1 9 8 1 ) f o r g a s e o u s p r o -d u c t s , a n d P a y n e ( 1 9 8 1 ) f o r a c o m p r e h e n s i v e r e v i e w w i t h e m p h a s i s o n m i c r o b i o l o g y a n d b i o c h e m i s t r y . SOURCES OF H I G H L Y NITROGENOUS WASTEWATER A n u m b e r o f i n d u s t r i e s p r o d u c e h i g h l y n i t r o g e n o u s w a s t e -w a t e r . T h e y i n c l u d e : t h e n i t r o g e n f i x a t i o n i n d u s t r y w h i c h p r o -d u c e s a m m o n i a , a m m o n i u m s a l t s a n d u r e a ; some c h e m i c a l p r o c e s s i n g i n d u s t r i e s s u c h a s o r g a n i c s a n d i o d i n e m a n u f a c t u r e ; s l a u g h t e r -h o u s e s ; t h e s t e e l i n d u s t r y , e s p e c i a l l y c o k e p l a n t o p e r a t i o n s ; some f o o d p r o c e s s i n g i n d u s t r i e s s u c h a s s u g a r r e f i n e r i e s ; t h e a g r i c u l t u r a l i n d u s t r y , i n c l u d i n g l i v e s t o c k b r e e d i n g . O t h e r s o u r -c e s i n c l u d e l e a c h a t e f r o m m o s t m u n i c i p a l l a n d f i l l s . A s u m m a r y o f n i t r o g e n l e v e l s r e p o r t e d f o r some h i g h l y n i t r o g e n o u s w a s t e s i s p r e s e n t e d i n T a b l e 1 . TRENDS I N I N D U S T R I A L NITROGEN PRODUCTION I n d u s t r i a l p r o d u c t i o n o f f i x e d n i t r o g e n , m o s t l y i n t h e f o r m o f a m m o n i a , b e g a n e a r l y t h i s c e n t u r y a n d i n c r e a s e d s t e a d i l y o v e r t h e y e a r s . B y 1 9 3 0 , w o r l d w i d e p r o d u c t i o n h a d r e a c h e d 1 . 8 m i l l i o n 14 Table 1 - Sources and Levels of Some High Strength Ammonia Wastes Waste Source TKN mg/L NH4-N mg/L BOD mg/L COD mg/L PH Reference ( F i r s t Author) Ammonia p l a n t 16-82 50-150 N.R.C, 1978 Ammonium n i t r a t e p l a n t i64-1640 20 N*R.C, 1978 Ammonium n i t r a t e and urea p l a n t 600 7.5-8.7 N.R.C, 1978 Ammonium sulphate p i a n t 8-820 20 N.R.C, 1978 Chemical processing p l a n t 3 0 - i i o 200-800 7*5-8.5 Ford, 1980 Coke p l a n t 155 7-8 B r i d l e , i981 Coke p l a n t 46-353 413-611 7*4-10.9 Gauthier, 1981 Dynamite f a c t o r y 1171 ne g l . 7.3 AltOna, 1983 F e r t i l i z e r p l a n t 900-1000 300-450 M y c i e l s k i , 1978 Iodine manufacture 60-200 10-20 50-150 7-8 Chou, 1981 L a n d f i l l leachate 1167 10356 16618 6.8 Keenan, 1979 L a n d f i l l leachate 200-600 200-600 80-250 850-1350 8.0-8*5 Knox* 1983 L a n d f i l l leachate 64-284 25-244 20-1650 243-2920 6.7-7.9 Jasper, 1984 Organic chemicals 157-190 96-130 1230 1115-3000 3.4-4.5 Sutton, 1981 Refinery/petrochemical 700-900 1200-1700 Pascik, 1984 Sugar r e f i n e r y 330 1650 3000 P i c a r d , 1980 Urea p i a n t 190-3750 30-300 N.R.C., 1978 tons of n i t r o g e n . In 1962, annual worldwide p r o d u c t i o n was e s t i -mated a t 14 m i l l i o n t ons and i t r o s e t o about 50 m i l l i o n tons by 1975 (N.R.C., 1979) and exceeded 62 m i l l i o n tons by 1981 (F.A.O., 1981). Ammonia i s used t o produce a v a r i e t y of compounds such as f e r t i l i z e r s (mostly i n the form of ammonium n i t r a t e , ammonium phosphate and ammonium s u l p h a t e ) , n i t r i c a c i d and urea (N.R.C., 1978). About three f o u r t h s of the ammonia produced i s used as f e r t i l i z e r (Hauck, 1983). The f r a c t i o n of ammonium compounds discharged as e f f l u e n t from the manufacturing process i s e s t i -mated a t l e s s t han 0.1% of t o t a l p r o d u c t i o n (N.R.C., 1978). The 1,300 ton/day Modderfontein p l a n t , f o r example, reported an ammo-nium l o s s to the e f f l u e n t of about 0.12% of production (Altona et a l . , 19 8 3). The impact of such discharges may be i n s i g n i f i c a n t on a n a t i o n a l l e v e l , but severe on a l o c a l s c a l e . The impact from a 1,000 ton/day ammonia p l a n t d i s c h a r g i n g 0.1% of i t s t o t a l ammonia pr o d u c t i o n i n the e f f l u e n t would be e q u i v a l e n t to the wastewater n i t r o g e n content from a town with a p o p u l a t i o n of 67,000 persons (assuming a per c a p i t a c o n t r i b u t i o n of 15 gm N/day). CHEMISTRY OF AMMONIA Ammonia i s a c o l o u r l e s s gas, which i s very s o l u b l e i n water and i s c h a r a c t e r i z e d with a pungent odour e a s i l y d i s c e r n a b l e at co n c e n t r a t i o n s above 50 mg/L (N.R.C., 1979). Ammonia i s a weak base, NH 3 + H 20 >- NHj + 0H~ (1) with a d i s s o c i a t i o n constant (Kb) of 1.774 x 10" 5 at 25°C (Bates .16 and P i n c h i n g , 1950). I t s conjugate a c i d , ammonium ion (NH4) NHj >• NH3 + H + (2) has a d i s s o c i a t i o n constant (Ka) of 5.6764 x 10"^-^ at 25°C (Bates and P i n c h i n g , 1950). The c o n c e n t r a t i o n of ammonia i n aqueous s o l u t i o n s cannot be measured s e p a r a t e l y from the ammonium i o n , but can be c a l c u l a t e d as shown below, when the l i q u i d tempera-ture, pH and t o t a l ammonia (free ammonia p l u s ammonium ion) are known: [NH 3] % NH3 = x TOO ......................................... (3) [NH 3] + [NHj] from Equation 2 [NH 3] [H +] [NH4] and [NH 3] [H +] [•NH"J] = .(5) Ka S u b s t i t u t i n g the v a l u e of [NH4] i n t o Equation 3 and re a r r a n g i n g 100 Ka % NH 3 = ..................................... (6) Ka + [H +] I t should be noted that the Ka v a l u e is/temperature depend-ent. Equation 6 can be used to estimate the percent f r e e ammonia when the pH and temperature are known. A p l o t of Equation 6 f o r the pH and temperature range n o r m a l l y encountered i n wastewater treatment p l a n t s i s presented i n Fig u r e 1. 17 f 1 1 I 1 1 1 1 1 1 1 1 1 Ko 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 p H F i g . l : F r e e A m m o n i a C o n c e n t r a t i o n a t V a r i o u s T e m p e r a t u r e s a n d p H V a l u e s 18 CHEMISTRY OF NITRITE N i t r i t e i s a h i g h l y r e a c t i v e compound, and as such i t s s a l t s are not commonly found i n nature (Masterton and S l o w i n s k i , 1978). I t s conjugate i s the weak and u n s t a b l e n i t r o u s a c i d HN0 2 *- H + + NOJ (7) A review of p u b l i s h e d i o n i z a t i o n measurements (see P e r r i n , 1982) r e v e a l s v a r i a t i o n between s t u d i e s . As a r e s u l t , a r e l a t i v e -l y wide range of d i s s o c i a t i o n constants are used i n the l i t e r a -t u re. Some examples: Van Cleemput and Baert (1984) used a pKa v a l u e of 3.14; G a r r e t t (1982) adopted a v a l u e of 3.29 at 30°C; A n t h o n i s e n e t a l . (1976) used 3.35 a t 25° C; N.R.C. (1978) r e p o r -ted i t as 3.36; Russo e t a l . (1981) chose 3.39 at 10°C; C a s t e l l a -n i and Niven (1955), N.R.C. (1981) and Boon and Laudelout (1962) used 3.4; F o c h t (1981) adopted 4.2. Such d i f f e r e n c e s l e a d to s u b s t a n t i a l v a r i a t i o n i n c a l c u l a t ° c | v a l u e s of n i t r o u s a c i d c o n c e n t r a t i o n . For example, the n i t r o u s a c i d c o n c e n t r a t i o n c a l c u l a t e d on the b a s i s of a pKa v a l u e of 3.14 would e q u a l about h a l f the v a l u e o b t a i n e d u s i n g a pKa of 3.40. For the purposes of t h i s t h e s i s , i t i s proposed to adopt the r e s u l t s measured by Klemenc and Hayek (1929), as they f a l l between the extremes of measured v a l u e s reported i n the l i t e r a -t u r e . On t h i s b a s i s , a pKa v a l u e of 3.25 a t 25°C i s used. When-ever p o s s i b l e , r e s u l t s reported i n the l i t e r a t u r e , based upon d i f f e r e n t pKa v a l u e s , w i l l be r e c a l c u l a t e d and reported to con-form to the standard adopted i n t h i s t e x t . In such i n s t a n c e s , the o r i g i n a l v a l u e s reported by the authors w i l l be presented i n brackets. T h i s a l l o w s f o r meaningful comparison of reported 19 r e s u l t s . The n i t r o u s a c i d c o n c e n t r a t i o n cannot be measured s e p a r a t e l y from the n i t r i t e i o n but can be c a l c u l a t e d as f o l l o w s : [HN0 2] % HN0 2 = • x 100 .....,....(8) [HN0 2] + [N0 2] from Equation 7 [H +] [N0 2] Ka = (9) [HN0 2] and Ka [HN0 2] [NOj] = (10) [H +] S u b s t i t u t i n g the v a l u e of [N0 2] i n t o Equation 8 and rea r r a n g i n g 100 [H +] % HN0 2 = (11) Ka + [H +] Equation 11 can be used to c a l c u l a t e the percent n i t r o u s a c i d once the pH and temperature are known. A p l o t of Equation 11 fo r the pH and temperature range n o r m a l l y encountered i n waste-water treatment p l a n t s i s presented i n Fi g u r e 2. NITRITE ACCUMULATION N i t r i t e i s an intermediary i n both the n i t r i f i c a t i o n and d e n i t r i f i c a t i o n processes and an end product of n i t r a t e r e s p i r a -t i o n (i.e. d i s s i m i l a t o r y n i t r a t e r e d u c t i o n to n i t r i t e ) . I t does not g e n e r a l l y accumulate i n the environment. This i n d i c a t e s that: 1) N i t r i t e o x i d a t i o n to n i t r a t e i s g e n e r a l l y as f a s t as, i f 20 0.17 I f 1 1 1 1 1 1 1 1 1 1 T-0.00 6.2 6 .3 6.4 6.5 6.6 6.7 6.8 6 .9 7 .0 7.1 7 .2 7 .3 7 .4 7 .5 pH Fig.2 : Nitrous Acid Concentration at Various Temperatures and pH Values 21 n o t f a s t e r t h a n , a m m o n i a o x i d a t i o n t o n i t r i t e 2) N i t r i t e r e d u c t i o n t o a g a s e o u s p r o d u c t i s a s f a s t a s , i f n o t f a s t e r t h a n , n i t r a t e r e d u c t i o n t o n i t r i t e . W h e r e n i t r i t e a c c u m u l a t i o n d o e s o c c u r , t h e p h e n o m e n o n i s o f a t r a n s i e n t n a t u r e t h a t r e s u l t s f r o m a t e m p o r a r y i m b a l a n c e i n t h e n i t r i f i c a t i o n o r d e n i t r i f i c a t i o n s e q u e n c e s . T h e a c c u m u l a t i o n c a n l a s t f r o m s e v e r a l h o u r s t o s e v e r a l y e a r s , d e p e n d i n g u p o n t h e c h a r a c t e r i s t i c s a n d s t a b i l i t y o f t h e e n v i r o n m e n t w h e r e t h e a c c u -m u l a t i o n o c c u r s . F o r t h e p u r p o s e s o f t h i s w o r k , i t i s d e s i r a b l e t o d i s t i n g u i s h b e t w e e n s h o r t - t e r m a c c u m u l a t i o n , t h a t may l a s t u p t o s e v e r a l d a y s , a n d l o n g - t e r m a c c u m u l a t i o n , t h a t c a n b e s u s -t a i n e d f o r s e v e r a l m o n t h s o r m o r e . S h o r t - t e r m n i t r i t e a c c u m u l a t i o n c a n g e n e r a l l y b e e x p l a i n e d b y r e a c t i o n a n d g r o w t h r a t e k i n e t i c s . I n t h e c a s e o f n i t r i f i c a -t i o n , t h e s u d d e n p r e s e n c e o f a m m o n i a i n i t i a l l y f a v o r s t h e g r o w t h o f t h e a m m o n i a o x i d i z e r s . , a t t h e e x p e n s e o f t h e n i t r i t e o x i d i -z e r s . T h i s r e s u l t s i n a t e m p o r a r y l a g b e t w e e n t h e g r o w t h o f t h e t w o g r o u p s o f n i t r i f i e r s , l e a d i n g t o t r a n s i e n t n i t r i t e a c c u m u l a -t i o n . T h e b u i l d - u p r a p i d l y d i s a p p e a r s o n c e t h e n i t r i t e o x i d i z e r s r e - e s t a b l i s h e q u i l i b r i u m c o n d i t i o n s . T h i s p h e n o m e n o n h a s b e e n f r e q u e n t l y r e p o r t e d d u r i n g e x p e r i m e n t s ( K n o w l e s e t a l . , 1 9 6 5 ; S t r a t t o n a n d M c C a r t y , 1 9 6 7 ; A n t h o n i s e n , 1 9 7 4 ) . I t h a s a l s o b e e n o b s e r v e d u p o n s t a r t - u p o f f u l l - s c a l e , n i t r i f y i n g t r e a t m e n t p l a n t s ( A l l e m a n , 1 9 8 4 ) . S h o r t - t e r m n i t r i t e a c c u m u l a t i o n , a s a r e s u l t o f d e n i t r i f i c a -t i o n , h a s a l s o b e e n o b s e r v e d . I t s c a u s e s i n c l u d e : 1) N i t r i t e r e d u c t i o n r a t e s t h a t a r e s l o w e r t h a n n i t r a t e r e d u c t i o n r a t e s . T h i s h a s b e e n f o u n d t o b e t h e c a s e w i t h 22 some Pseudomonas s p e c i e s ( B o l l a g et a_l., 1970; W i l l i a m s et a l . , 1978; B e t l a c h and T i e d j e , 1981). 2) Longer l a g time a s s o c i a t e d with the d e r e p r e s s i o n of the n i t r i t e reductase enzyme, as opposed to the n i t r a t e r e -ductase enzyme, f o l l o w i n g d e p l e t i o n of oxygen i n the medium (Knowles, 1982). Long-term n i t r i t e accumulation i n some micro environments i s w e l l documented. These i n c l u d e : 1) Oceanic subsurface waters; 2) R e c i r c u l a t e d water i n commercial f i s h h a t c h e r i e s ; 3) Subsurface s o i l s t r a t a ; 4) B i o l o g i c a l treatment systems f o r h i g h l y nitrogenous wastewater. Oceanic Subsurface Waters N i t r i t e accumulation i n the oceans seems to be c h a r a c t e r i z e d by two separate n i t r i t e accumulation zones. The f i r s t zone occurs w i t h i n one hundred meter depth and i s g e n e r a l l y l i n k e d with the i n h i b i t i o n of n i t r i t e o x i d a t i o n during n i t r i f i c a t i o n . Other sug-gested causes of n i t r i t e accumulation i n t h i s zone i n c l u d e l e a k -age of n i t r i t e d uring n i t r a t e a s s i m i l a t i o n by phytoplankton and ammonia o x i d a t i o n by m e t h y l o t r o p h i c b a c t e r i a . The second n i t r i t e accumulation zone occurs at g r e a t e r depths, g e n e r a l l y between 150 and 600 m, where the DO l e v e l i s low. N i t r i t e i s b e l i e v e d t o accumulate i n t h i s zone as a r e s u l t of n i t r a t e r e s p i r a t i o n . I t i s presumed that the n i t r a t e r e s p i r e r s predominate over the d e n i t r i -f i e r s i n such h a b i t a t s f o r reasons that have not yet been d i s c o v -ered. (Focht and V e r s t r a t e , 1977; Hahn, 1981; Knowles, 1982). 23 R e c i r c u l a t e d W a t e r I n C o m m e r c i a l F i s h H a t c h e r i e s N i t r i t e a c c u m u l a t i o n i n f i s h c u l t u r e f a c i l i t i e s , p r a c t i c i n g w a t e r r e u s e w i t h b i o l o g i c a l n i t r i f i c a t i o n c a n l a s t s e v e r a l w e e k s o r m o n t h s . I t s c a u s e h a s b e e n a t t r i b u t e d t o t h e i n h i b i t i o n o f t h e n i t r i t e o x i d i z e r s ( C o l l i n s et a _ l . , 1 9 7 5 ; K o n i k o f f , 1 9 7 5 ) . S u b s u r f a c e S o i l S t r a t a N i t r i t e a c c u m u l a t i o n i n s o i l s h a s b e e n a t t r i b u t e d t o a n u m b e r o f c a u s e s , t h e m o s t common b e i n g a n u p s e t i n t h e n i t r i f i -c a t i o n p r o c e s s ( M o r r i l l a n d D a w s o n , 1 9 6 7 ; H a r a d a a n d K a i , 1 9 6 8 ; B e z d i c e k e t a _ l . , 1 9 7 1 ; W e t s e l a a r e t a l . , 1 9 7 2 ; B e r i a n d B r a r , 1 9 7 8 ; C h a l k a n d S m i t h , 1 9 8 3 ) . O t h e r c a u s e s i n c l u d e t h e p r e d o m i -n a n c e o f n i t r a t e r e s p i r e r s o v e r d e n i t r i f i e r s ( F o c h t a n d V e r -s t r a e t e , 1 9 7 7 ; V o l z e t a l . , 19 7 5) a n d i n c o m p l e t e d e n i t r i f i c a t i o n c a u s e d b y u n f a v o r a b l e e n v i r o n m e n t a l f a c t o r s ( B o l l a g e t a l . . , 1 9 7 0 ) . . N i t r i t e a c c u m u l a t i o n i s g e n e r a l l y a s s o c i a t e d w i t h a l k a l i n e s o i l c o n d i t i o n s ( C h a l k a n d S m i t h , 1 9 8 3 ; V a n C l e e m p u t a n d B a e r t , 1 9 8 4 ) . B i o l o g i c a l T r e a t m e n t S y s t e m s F o r H i g h l y N i t r o g e n o u s W a s t e w a t e r N i t r i t e a c c u m u l a t i o n i n b i o l o g i c a l w a s t e w a t e r t r e a t m e n t h a s b e e n r e p o r t e d i n s y s t e m s t r e a t i n g h i g h s t r e n g t h a m m o n i a w a s t e -w a t e r . T h e p h e n o m e n o n i s n o r m a l l y a t t r i b u t e d t o a n u n c o u p l i n g o f t h e n i t r i f i c a t i o n p r o c e s s . A n t h o n i s e n (1974) r e p o r t e d o n a n o x i d a t i o n d i t c h t r e a t i n g p o u l t r y w a s t e , w h e r e n i t r i t e a c c u m u l a t e d t o c o n c e n t r a t i o n s r e a c h i n g 8 0 0 mg N O ^ - N / L f o r p e r i o d s e x c e e d i n g 2 0 0 d a y s . H u t t o n a n d L a R o c c a ( 1 9 7 5 ) o b s e r v e d s i g n i f i c a n t n i t r i t e a c c u -24 m u l a t i o n t h r o u g h o u t t h e i r 70 d a y p i l o t - s c a l e t r e a t m e n t s t u d y o f a n a m m o n i a f e r t i l i z e r w a s t e c o n t a i n i n g u p t o 8 0 0 mg N H ^ - N / L . M y c i e l s k i e t a _ l . ( 1 9 7 8 ) n o t e d s i g n i f i c a n t n i t r i t e a c c u m u l a -t i o n d u r i n g b e n c h - s c a l e t r e a t a b i l i t y s t u d i e s o f f e r t i l i z e r w a s t e , c o n t a i n i n g u p t o 1 0 0 0 mg T K N / L . A l l e m a n a n d I r v i n e ( 1 9 8 0 ) r e p o r t e d o n a 60 d a y b e n c h - s c a l e s t u d y , u s i n g a s e q u e n c i n g b a t c h r e a c t o r ; t h e i r s y s t e m p r o d u c e d a f u l l y n i t r i f i e d e f f l u e n t c o n s i s t i n g s o l e l y o f n i t r i t e . I n b e n c h - s c a l e t r e a t a b i l i t y s t u d i e s o f c o k e - p l a n t w a s t e , B r i d l e e t a _ l . ( 1 9 8 1 ) n o t e d t h e p r e s e n c e o f n i t r i t e i n t h e t r e a t e d e f f l u e n t , w i t h l i t t l e , i f a n y , n i t r a t e p r e s e n t . G a r r e t t ( 1 9 8 2 ) r e p o r t e d e x t e n s i v e l y o n n i t r i t e a c c u m u l a t i o n i n a b e n c h - s c a l e , a t t a c h e d g r o w t h s y s t e m , t r e a t i n g a h i g h s t r e n g t h a m m o n i a w a s t e ; c o n c e n t r a t i o n s r a n g e d b e t w e e n ?.00 a n d 4 0 0 mg N H ^ - N / L . S i l v e r s t e i n a n d S c h r o e d e r ( 1 9 8 3 ) o b s e r v e d t h a t h i g h c o n c e n -t r a t i o n s o f a m m o n i u m i o n s , i n a b e n c h - s c a l e s e q u e n c i n g b a t c h r e a c t o r , may h a v e i n h i b i t e d N i t r o b a c t e r a c t i v i t y . POSTULATED C A U S E S OF N I T R I T E ACCUMULATION DURING N I T R I F I C A T I O N S e v e r a l p a r a m e t e r s h a v e b e e n p o s t u l a t e d t o c a u s e n i t r i t e a c c u m u l a t i o n d u r i n g n i t r i f i c a t i o n . T h e y i n c l u d e : 1) F r e e a m m o n i a 2) N i t r o u s a c i d 3) T e m p e r a t u r e 4) D i s s o l v e d o x y g e n 5) A n u m b e r o f p a r a m e t e r s d i s c u s s e d u n d e r " O t h e r C a u s e s " 25 F r e e A m m o n i a W a r i n g t o n ( 1 8 9 1 ) w a s f i r s t t o d e m o n s t r a t e t h a t a m m o n i a i n h i -b i t e d t h e n i t r i t e o x i d i z e r s . I n 1 9 1 6 , M e y e r h o f ( c i t e d i n L e e s , 1 9 6 3 ) o b s e r v e d t h a t t h e t o x i c i t y o f a m m o n i a w a s pH d e p e n d e n t ; i t i n c r e a s e d w i t h i n c r e a s i n g p H . He s u g g e s t e d t h a t t o x i c i t y w a s c a u s e d b y t h e 1 i p i d - s o l u b l e f r e e a m m o n i a a n d n o t t h e l i p i d -i n s o l u b l e ammonium i o n . I t h a s s i n c e b e e n c o n f i r m e d b y a n u m b e r o f i n v e s t i g a t o r s ( A l e e m a n d A l e x a n d e r , 1 9 6 0 ; B o o n a n d L a u d e l o u t , 1 9 6 2 ; P r a k a s a m e t a _ l . , 1 9 7 8 ) t h a t f r e e a m m o n i a i s m o r e i n h i b i t o r y t o N i t r o b a c t e r t h a n i s i t s i o n i z e d f o r m . T h i s t i e s i n w i t h t h e r e s u l t s o f S u z u k i e t a _ l . ( 1 9 7 4 ) , who c o n f i r m e d t h a t t h e a c t u a l s u b s t r a t e o x i d i z e d b y N i t r o s o m o n a s i s f r e e a m m o n i a a n d n o t ammo-n i u m i o n . M e y e r h o f h a d a l s o o b s e r v e d t h a t a m m o n i a w a s m o r e i n h i -b i t o r y t o N i t r o b a c t e r t h a n t o N i t r o s o m o n a s . T h i s h a s a l s o s i n c e b e e n c o n f i r m e d b y o t h e r r e s e a r c h e r s ( M e i k e l j o h n , 1 9 5 4 ) . T h e r e s u l t s o f a m a j o r i n v e s t i g a t i o n i n t o t h e c a u s e s o f n i t r i t e a c c u m u l a t i o n i n w a s t e w a t e r t r e a t m e n t w a s r e p o r t e d b y A n t h o n i s e n e t a l . ( 1 9 7 6 ) . I t l i n k e d n i t r i t e b u i l d - u p t o t h e p r e s e n c e o f f r e e a m m o n i a , w h i c h w a s f o u n d t o a c t a s a s p e c i f i c i n h i b i t o r o f n i t r i t e o x i d a t i o n i n t h e c o n c e n t r a t i o n r a n g e o f 0 . 0 8 t o 0 . 8 3 mg NH3 - N / L ( 0 . 1 t o 1 mg NH3/ L ) , a n d a s a g e n e r a l i n h i b i -t o r o f n i t r i f i c a t i o n a t c o n c e n t r a t i o n s e x c e e d i n g 8 . 3 mg NH3-N/L (10 mg NH3/ L ) . B a s e d u p o n t h e s e r e s u l t s , A n t h o n i s e n a n d c o l l e a -g u e s p r o d u c e d a s u b s t r a t e t o l e r a n c e c h a r t f o r u s e i n p r e d i c t i n g t h e l i k e l i h o o d o f n i t r i t e a c c u m u l a t i o n d u r i n g w a s t e w a t e r t r e a t -m e n t . T h e c h a r t a c c o u n t e d f o r s u c h v a r i a b l e s a s t e m p e r a t u r e , pH a n d t o t a l a m m o n i a ( f r e e a m m o n i a a n d a m m o n i u m i o n ) c o n c e n t r a t i o n s . 26 They a l s o noted that such f a c t o r s as temperature, number of a c t i v e organisms and a c c l i m a t i o n caused v a r i a t i o n i n the f r e e ammonia l e v e l i n h i b i t o r y to the n i t r i t e o x i d i z e r s . The adequacy of the s u b s t r a t e t o l e r a n c e c h a r t , i n p r e d i c t i n g n i t r i t e accumulation, was i n v e s t i g a t e d by V e r s t r a e t e e_t a l . (1977), i n the course of a 4-month long study using bench-scale fi11-and-draw systems. They reported c l o s e agreement with the chart. The r e s u l t s of other s t u d i e s a l s o produced i n d i r e c t sup-p o r t f o r the cha r t as a p r e d i c t i v e t o o l of n i t r i t e accumulation. For example, i n a study using sequencing batch b i o l o g i c a l reac-t o r s , Alleman and I r v i n e (1980) reported that n i t r i t e was the dominant o x i d i z e d n i t r o g e n s p e c i e s observed through the d u r a t i o n of the study. T h i s was a t t r i b u t e d to the mode of ope r a t i o n , which r e s u l t e d i n a f r e e ammonia peak of 0.6 mg NH^-N/L that occurred once during every 8.5 hour c y c l e and i n h i b i t e d the a c t i v i t y of the n i t r i t e o x i d i z e r s . As a r e s u l t of these and other s t u d i e s , the p r e d i c t i v e chart produced by Anthonisen and c o l l e a g u e s has s i n c e gained wide acceptance and i s now reproduced i n books on the s u b j e c t (Barnes and B l i s s , 1983). A number of s t u d i e s have confirmed the r e s u l t s of Anthonisen and c o l l e a g u e s , regarding the a c t i o n of f r e e ammonia as a ge n e r a l i n h i b i t o r of n i t r i f i c a t i o n a c t i v i t y at co n c e n t r a t i o n s above 8.3 mg NH3-N/L (10 mgNH-j/L). Pure c u l t u r e s t u d i e s of Nitrosomonas, by Neufeld et a_l. (1980), reported optimum n i t r i f i c a t i o n r a t e s at about 8 mg NH3-N/L, with s u b s t r a t e i n h i b i t i o n at higher concen-t r a t i o n s . Reporting on n i t r i f i c a t i o n s t u d i e s , using bench-scale a c t i v a t e d sludge systems fed chemical wastewater with high ammo-nium l e v e l s , Ford et a l . (1980) found a good c o r r e l a t i o n between the f r e e ammonia l e v e l and degree of n i t r i f i c a t i o n up to a con-c e n t r a t i o n of 30 mg NH3-N/L; above t h i s l e v e l , i n h i b i t i o n of ammonia o x i d a t i o n was noted. T a b l e 2 summarizes the r e s u l t s of these and other s t u d i e s d e a l i n g with f r e e ammonia i n h i b i t i o n of n i t r i f i c a t i o n . F u rther evidence l i n k i n g n i t r i t e accumulation to the s e l e c -t i v e i n h i b i t i o n of f r e e ammonia can be found i n the research conducted on n i t r i f i c a t i o n i n s o i l s . High ammonia l e v e l s i n s o i l s n o r m a l l y r e s u l t from n i t r o g e n f e r t i l i z e r a p p l i c a t i o n . Numerous s t u d i e s , spanning a p e r i o d of about twenty years, have found that ammonia a p p l i c a t i o n over a l k a l i n e s o i l s , or i n c o n j u n c t i o n with l i m i n g a c t i v i t y , r e s u l t e d i n i n h i b i t i o n of n i t r i t e o x i d a t i o n a c t i v i t y l e a d i n g to n i t r i t e accumulation (Nommik and N i l s s o n , 1963; Hauck and Stephenson, 1965; M o r r i l l and Dawson, 1967; Harada and K a i , 1968; Bezdicek e t a_l., 1971; Wetselaar et al.., 197.2; Chalk et a l . , 1975; B e r i and Brar, 1978; Smith and Chalk, 1980) . Nit r o u s A c i d The i n h i b i t o r y e f f e c t s of n i t r i t e , or ra t h e r n i t r o u s a c i d , on microorganisms other than the n i t r i f i e r s , i s di s c u s s e d l a t e r (see E f f e c t s of N i t r i t e ) . Reported i n h i b i t o r y l e v e l s on the n i t r i f i e r s i n wastewater treatment s t u d i e s are presented i n Table 3. Ta b l e 4 summarizes the r e s u l t s of s t u d i e s with pure c u l t u r e s of n i t r i f i e r s . Some researchers maintain that n i t r i t e (i.e. n i t r o u s acid) can a c t as a d i f f e r e n t i a l i n h i b i t o r of n i t r i f i c a t i o n by s e l e c t i -v e l y i n h i b i t i n g the n i t r i t e o x i d i z e r s . Prakasam and Loehr (1972) 28 Table 2 - Ammonia I n h i b i t i o n of N i t r i f i c a t i o n i n Wastewater Treatment T o t a l Free pH % I n h i b i t i o n Remarks Reference Ammonia Ammonia ( F i r s t Author) mg N/L mg N/L 0.02 over 90% f o r n i t r i t e o x i d i z e r s bench-scale A.S. Prakasam 1972 650 24 8 .0 t r a n s i e n t NO^ accumulation batch A i S . Wong-Chong 800 3.2 7 .0 no NC>2 accumulation 1975 500 20 8 .2 over 90% f o r n i t r i t e o x i d i z e r s o x i d a t i o n d i t c h Murray 1975 0 ,08-8.0 i n h i b i t o r y to n i t r i t e batch, bench & Anthoni sen o x i d i z e r s f u l l - s c a l e 1976 s t u d i e s 8-80 i n h i b i t o r y to both groups of • n i t r i f i e r s 490 13 7 ,8 100% f o r both groups of n i t r i - f i11-and-draw V e r s t r a e t e f i e r s 1977 284 0,28 6 .3 over 90% for n i t r i t e o x i d i z e r s up to 1060 0.7-40 6.5 -8.0 no e f f e c t e n r i c h e d Wong-Chong up to 840 14-32 8 .0 n i t r i t e accumulation observed c u l t r u e s 1978 13 0.6 7,8 -8.1 over 95% n i t r i t e accumulation S . B. R. Alleman 1980 40 i5.5 9 .2 t r a n s i e n t n i t r i t e accumulation SiB.R. Sauter 1980 10 .2 i n h i b i t i o n of n i t r i f i c a t i o n 24 i n h i b i t o r y to ammonia o x i d i z e r s bench-scale A.S. Ford 1980 7 8 .0 reduction i n ammonia o x i d a t i o n n i t r i f y i n g Neufeld 1980 c u l t u r e s T a b l e 3 - N i t r i t e I n h i b i t i o n o f N i t r i f i c a t i o n i n W a s t e w a t e r T r e a t m e n t N i t r i t e mg N/L N i t r o u s A c i d u g N/L pH % I n h i b i t i o n R e m a r k s R e f e r e n c e ( F i r s t A u t h o r ) 3 0 0 a p p a r e n t i n h i b i t i o n o f n i t r i t e o x i d a t i o n b e n c h s c a l e A . S . P r a k a s a m 1 9 7 2 5 0 0 7 5 0 6 . 1 a p p a r e n t i h i b i t i o n o f a m m o n i a o x i d a t i o n o x i d a t i o n d i t c h M u r r a y 1 9 7 5 5 0 - 6 3 3 i n h i b i t o r y t o b o t h g r o u p s o f n i t r i f i e r s b a t c h , b e n c h & A n t h o n i s e n 1 9 7 6 f u i l - s c a l e s t u d i e s u p t o 3 0 0 2 9 0 6 . 3 m i n i m a l e f f e c t o h a m m o n i a o x i d i z e r s f i l l - a n d - d r a w V e r s t r a e t e 1977 2 6 5 0 100% i n h i b i t i o n o f n i t r i f i c a t i o n e n r i c h e d c u l t u r e W o n g - C h o h g 1978 6 3 - 2 2 6 i n h i b i t o r y t o n i t r i t e o x i d i z e r s b e n c h - s c a l e A . S * F o r d 1 9 8 0 5 6 - 1 4 0 o v e r 80% i n h i b i t i o n t o n i t r i t e o x i d i z e r s b e n c h - s c a l e a t t a c h e d g r o w t h G a r r e t 1 9 8 2 2 0 - 2 5 1 . 4 7 . 5 i n h i b i t o r y t o n i t r i t e o x i d i z e r s b a t c h t e s t s B e c c a r i 1 9 8 3 N o t e : N i t r O u s a c i d c o n c e n t r a t i o n c a l c u l a t e d a s p e r c o n s t a n t u s e d i n t e x t . V a l u e s may d i f f e r f r o m t h o s e , r e p o r t e d b y t h e a u t h o r s * T a b l e 4 - N i t r i t e I n h i b i t i o n o f N i t r i f i c a t i o n i n P u r e C u l t u r e s S p e c i e s Mode o f I n h i b i t i o n % I n h i b . PH NC-2-N mg N/L H N 0 2 u g N/L R e m a r k s R e f e r e n c e ( F i r s t A u t h o r ) N i t r o b a c t e r n i t r i t e o x i d a t i o n r a t e 0 30 7 . 6 7 . 6 2 8 0 1 4 0 0 12 59 B o o n 1 9 6 0 N i t r o b a c t e r o x y g e n u p t a k e 0 25 7 . 5 7.5 224 3 5 0 12 B u t t 1 9 6 0 N i t r o b a c t e r g r o w t h i n h i b . 0 130 5 0 0 l a g p h a s e e x p o n e n t i a l g r o w t h A l e e m 1 9 6 0 N i t r o b a c t e r m i n i m u m i n h i b . 3 0 0 B o c k 1 9 7 8 N i t r o s o m o n a s g r o w t h 100 7 . 8 2 5 0 0 68 l o g g r o w t h L o v e l e s s 1 9 6 8 N i t r o s o m o n a s m i n i m u m i n h i b . 1 0 0 0 - 5 0 0 0 B o c k 1 9 7 8 N i t r o s o m o n a s g r o w t h 100 7 0 0 0 q u o t i n g D r o z d 1 9 8 0 e a r l i e r w o r k N o t e : N i t r o u s a c i d c o n c e n t r a t i o n c a l c u l a t e d a s p e r c o n s t a n t u s e d i n t e x t . V a l u e s may d i f f e r f r o m t h o s e r e p o r t e d b y t h e a u t h o r s . noted a r e l a t i o n s h i p between the degree of n i t r i t e b u i l d - u p and the n i t r o u s a c i d c o n c e n t r a t i o n i n a batch system t r e a t i n g p o u l t r y manure. The r e l a t i o n s h i p does not appear to have been s i g n i f i c a n t s i n c e the authors, s t a t e d i n a subsequent p u b l i c a t i o n , that no c l e a r i n d i c a t i o n c o u l d be found l i n k i n g n i t r o u s a c i d to n i t r i t e b u i l d - u p (Anthonisen et a_l., 1976). Murray et a_l. (1975) l i n k e d high n i t r i t e l e v e l s i n an oxida-t i o n d i t c h , t r e a t i n g p i g waste, to an i n i t i a l high f r e e ammonia l e v e l which caused n i t r i t e b u i l d - u p , and a subsequent r i s e i n n i t r o u s a c i d l e v e l . G a r r e t t (1982) reported on the r e s u l t s of a 40-month long bench-scale study of attached growth n i t r i f i c a t i o n , i n which he l i n k e d n i t r i t e accumulation to the presence of n i t r o u s a c i d at l e v e l s of 7.9 + 3.1 ug HN0 2-N/L (9.3 + 3.7 ug HN0 2-N/L, a u t h o r s ' v a l u e s ) . These l e v e l s i n h i b i t e d the n i t r i t e o x i d i z e r s and l e d to n i t r i t e accumulation exceeding 80% of the t o t a l o x i d i z e d n i t r o g e n s p e c i e s formed.. Temperature I t i s w e l l e s t a b l i s h e d that i n the temperature range of 5 to 30°C, the growth rate constant f o r both groups of n i t r i f i e r s v a r i e s e x p o n e n t i a l l y with temperature, i n gene r a l agreement with the Arrhenius law (Wild e t a l . , 1971; P a i n t e r , 1977). The ra t e of change with temperature, however, appears to vary between the two groups. Knowles et a_l. (1965) estimated that the growth r a t e of N i t r o b a c t e r increased from 0.52 to 1.88 d - ^ , as the temperature rose from 8 to 30°C. The r e s p e c t i v e i n c r e a s e f o r Nitrosomonas was 32 from 0.24 to 1.97 d - 1 . Thus at 8°C, the growth rate for Nitrobacter was more than double that of Nitrosomonas, while at 30°C, the rate was slightly lower than that of Nitrosomonas. These results suggest that an increase in temperature may enhance nitrite build-up. This was confirmed by the analysis carried out by Quinlan (1980) of several published studies dealing with temperature, ammonia and nitrification rates for both groups of nitrifiers. Based upon the analyses, relationships were developed for the temperature range of 0° to 50°C and free ammonia concen-tration range of 0.1 to 10 mg NH3-N/L. The results confirmed that thermal enrichment leads to nitrite accumulation during n i t r i f i -cation. In contrast to the aforementioned, some investigators have linked nitrite accumulation to low temperatures. Alleman (1984) reported, without providing supporting documentation, that .sever-al full -scale treatment plants exhibited instances of nitrite accumulation at low temperatures. Randall and Buth (1984a) found that l i t t l e nitrification occurred in a bench-scale activated sludge system in the temperature range of 6 to 10°C. Nitrite accumulation occurred at temperatures between 12 and 14°C and complete nitrification was achieved at temperatures above 17°C. The authors did not report the duration of their experiment. In an extensive study of temperature effects on nitr i f ica-tion rates in fi11-and-draw activated sludge reactors, Painter and Loveless (1983) concluded that the growth rate of Nitrosomo- nas exceeded that of Ni trobacter in about half the cases where a comparison was possible, while the reverse was true for the remainder of the cases. 33 D i s s o l v e d Oxygen The reported Michaelis-Menten h a l f s a t u r a t i o n c o e f f i c i e n t f o r oxygen (Ko) f o r N i t r o b a c t e r ranges between 0.5 and 1.84 w h i l e that f o r Nitrosomonas v a r i e s between 0.25 and 0.5 mg/L (Boon and Laudelout, 1962; L o v e l e s s and P a i n t e r , 1968; Peeters, 19.69; Focht and Chang, 1975). T h i s i m p l i e s that N i t r o b a c t e r i s more s e n s i t i v e to low DO c o n c e n t r a t i o n s than i s Nitrosomonas. The i n h i b i t i o n of n i t r i t e o x i d a t i o n , that may a r i s e at low DO l e v e l s , was s t u d i e d by Laudelout et a l . (1976). Experimental r e s u l t s , i n c o n j u n c t i o n with s i m u l a t i o n s t u d i e s , showed that inadequate a e r a t i o n induced a temporal s h i f t of ammonium and n i t r i t e o x i d a t i o n , l e a d i n g to t r a n s i e n t n i t r i t e accumulation l a s t i n g s e v e r a l days. In batch n i t r i f i c a t i o n s t u d i e s using f l u i d i z e d sand f i l t e r s , Tanaka et a_l. (1981) a t t r i b u t e d temporary n i t r i t e accumulation to the e f f e c t of oxygen l i m i t a t i o n . The use of pure oxygen, i n l i e u of a i r , reduced the extent of the problem. They confirmed t h e i r f i n d i n g s by a d d i t i o n a l s t u d i e s (Tanaka and Dunn, 1982). The cause of n i t r i t e accumulation was a t t r i b u t e d e i t h e r to a higher Ko v a l u e f o r the n i t r i t e o x i d i z e r s than f o r the ammonia o x i d i z e r s , or to the p r e s e n c e of the n i t r i t e o x i d i z e r s i n the i n n e r b i o f i l m regions, where l i m i t e d oxygen d i f f u s i o n occurred. Other Causes A number of other parameters have been l i n k e d to n i t r i t e b u i l d - u p . They i n c l u d e : 34 1) M e t a l s R a n d a l l a n d B u t h ( 1 9 8 4 b ) r e p o r t e d o n t h e r e s u l t s o f b e n c h -s c a l e , a c t i v a t e d s l u d g e n i t r i f i c a t i o n s t u d i e s o f u n k n o w n d u r a -t i o n , c a r r i e d o u t a t t h r e e t e m p e r a t u r e s ( 1 4 , 17 a n d 3 0 ° C ) a n d a t v a r i a b l e n i c k e l c o n c e n t r a t i o n s (0 t o 15 m g / L ) . . T h e y c o n c l u d e d t h a t a t 1 4 ° C , n i c k e l w a s m o r e t o x i c t o t h e n i t r i t e o x i d i z e r s t h a n t o t h e a m m o n i a o x i d i z e r s , c a u s i n g n i t r i t e b u i l d - u p i n t h e s y s t e m . A t t h e o t h e r t w o t e m p e r a t u r e s , n i c k e l w a s n o t s h o w n t o a c t a s a d i f f e r e n t i a l i n h i b i t o r . T h e a u t h o r s s u g g e s t e d t h a t t h e s y n e r g i s -t i c e f f e c t o f t e m p e r a t u r e a n d n i c k e l may h a v e c a u s e d n i t r i t e b u i l d - u p . 2) I n c o m p l e t e D e n i t r i f i c a t i o n T h i s p h e n o m e n o n h a s a p p a r e n t l y b e e n o b s e r v e d a t a n i n d u s -t r i a l w a s t e w a t e r t r e a t m e n t p l a n t a n d w a s r e p o r t e d b y A l l e m a n ( 1 9 8 4 ) , b a s e d u p o n a p e r s o n a l c o m m u n i c a t i o n . I t i n v o l v e s t h e a c c u m u l a t i o n o f n i t r i t e d u r i n g d e n i t r i f i c a t i o n a n d t h e s u b s e q u e n t i n a b i l i t y o f t h e n i t r i t e o x i d i z e r s t o c o p e w i t h i t i n t h e a e r a t e d z o n e , l e a d i n g t o n i t r i t e a c c u m u l a t i o n . A l l e m a n r e f e r r e d t o t h e p h e n o m e n o n a s " c r y p t i c n i t r a t e r e d u c t i o n " . 3) S l u d g e A g e B a s e d u p o n b a t c h s t u d i e s , B e c c a r i e t a l ( 1 9 7 9 ) r e p o r t e d t h a t l o w s l u d g e a g e c a u s e d n i t r i t e a c c u m u l a t i o n . T h e y n o t e d 100% n i t r i t e b u i l d - u p f o r s y s t e m s o p e r a t i n g a t s l u d g e a g e s r a n g i n g b e t w e e n 2 . 5 a n d 4 d a y s , w h i l e n o n i t r i t e b u i l d - u p o c c u r r e d a t s l u d g e a g e s a b o v e 4 d a y s . A l l e m a n ( 1 9 8 4 ) r e p o r t e d o n a f u l l - s c a l e n i t r i f y i n g f a c i l i t y , w h e r e a r e d u c t i o n i n M L S S c o n c e n t r a t i o n f r o m a b o u t 5 6 0 0 t o 2 8 0 0 m g / L o v e r a t w o w e e k p e r i o d c a u s e d a t e m p o r a r y 35 r i s e i n e f f l u e n t n i t r i t e l e v e l s from under 1 t o o v e r 9 mg NO^-N/L, i n c o n j u n c t i o n with a r i s e i n e f f l u e n t ammonia l e v e l s to about 6 mg NH4-N/L. 4) COD Loading Prakasam and Loehr (1972) b e l i e v e d that the COD l o a d i n g r a t e may determine the end product of n i t r i f i c a t i o n . At l o a d i n g r a t e s ranging between 0.15 to 0.8 kg COD/day.kg VSS, n i t r i t e appeared to predominate, w h i l e n i t r a t e predominated at lower l o a d i n g r a t e s . They concluded that l o a d i n g r a t e s c o u l d be used to s e l e c t the end-product of n i t r i f i c a t i o n . 5) Phosphorus Studies on n i t r i f i c a t i o n i n s o i l s appear to i n d i c a t e that the n i t r i t e o x i d i z e r s are more s e n s i t i v e to phosphorus d e f i c i e n c y than are the ammonia o x i d i z e r s . In - s o i l s c o n t a i n i n g l e s s than 4 mg P/kg, t h i s c o u l d l e a d to n i t r i t e accumulation ( V e r s t r a e t e , 1981) . POSTULATED CAUSES OF NITRITE ACCUMULATION DURING DENITRIFICATION N i t r i t e accumulation has a l s o been observed to occur as a r e s u l t of d e n i t r i f i c a t i o n . P o s s i b l e causes i n c l u d e : 1) Predominance of n i t r a t e r e s p i r e r s ; 2) Incomplete d e n i t r i f i c a t i o n . Predominance of N i t r a t e R e s p i r e r s N i t r i t e i s the end-product of n i t r a t e r e s p i r a t i o n . N i t r i t e accumulation, due to the predominance of the n i t r a t e r e s p i r e r s over the d e n i t r i f i e r s , i s commonly observed i n marine waters (as 36 d i s c u s s e d e a r l i e r ) . I t i s n o t c l e a r why t h e n i t r a t e r e s p i r e r s w o u l d p r e d o m i n a t e o v e r t h e d e n i t r i f i e r s i n s u c h h a b i t a t s . One t h e o r y s u g g e s t s t h a t t h e M i c h a e l i s - M e n t e n h a l f s a t u r a t i o n c o e f f i -c i e n t f o r t h e o x i d a t i o n o f c a r b o n a c e o u s s u b s t r a t e s i s l o w e r f o r t h e n i t r a t e r e s p i r e r s , a l l o w i n g t h e m t o c o m p e t e m o r e e f f e c t i v e l y f o r t h e s c a r c e c a r b o n a c e o u s s u s b s t r a t e s a v a i l a b l e ( F o c h t a n d V e r s t r a e t e , 1 9 7 7 ) . I n c o m p l e t e D e n i t r i f i c a t i o n N i t r i t e , a s a n i n t e r m e d i a r y i n t h e d e n i t r i f i c a t i o n p r o c e s s , c a n a c c u m u l a t e d u r i n g d i s s i m i l a t o r y n i t r a t e r e d u c t i o n . T h i s h a s b e e n a t t r i b u t e d t o a n u m b e r o f c a u s e s . P a y n e ( 1 9 7 3 ) a n d D e l w i c h e a n d B r y a n ( 1 9 7 6 ) c i t e d p u r e c u l t u r e s t u d i e s w h e r e n i t r i t e a c c u m u -l a t i o n h a d b e e n o b s e r v e d i n c u l t u r e s o f M i c r o c o c c u s d e n i t r i f i c a n s a n d P s e u d o m o n a s p e r f e c t o m a r i n u s ( b o t h a r e d e n i t r i f i e r s ) i n t h e p r e s e n c e o f n i t r a t e . T h i s s u g g e s t e d t h a t t h e s y n t h e s i s o f n i t r i t e r e d u c t a s e w a s t r i g g e r e d b y n i t r i t e a c c u m u l a t i o n o r t h a t n i t r i t e r e d u c t a s e w a s s u p p r e s s e d b y t h e p r e s e n c e o f n i t r a t e . N i t r a t e s u p p r e s s i o n w a s c o n s i d e r e d t h e m o r e l i k e l y c a u s e , s i n c e n o r m a l e n z y m e a c t i v i t y w a s o b s e r v e d i n i t s a b s e n c e . B e t l a c h a n d T i e d j e (1981) a n d T i m m e r m a n n a n d V a n H a u t e ( 1 9 8 3 ) , o n t h e o t h e r h a n d , r e j e c t e d t h e s e f i n d i n g s , b a s e d u p o n t h e i r r e s e a r c h w i t h o t h e r o r g a n i s m s . T h e y f o u n d n o e v i d e n c e o f n i t r i t e a c c u m u l a t i o n i n t h e p r e s e n c e o f n i t r a t e . B o l l a g e t a l . ( 1 9 7 0 ) a t t r i b u t e d n i t r i t e b u i l d - u p i n s o i l t o e n v i r o n m e n t a l f a c -t o r s t h a t r e s u l t e d i n u n f a v o r a b l e g r o w t h c o n d i t i o n s t o t h e d e n i -t r i f i e r s . S u c h f a c t o r s i n c l u d e d c h a n g e s i n p H , t e m p e r a t u r e a n d n i t r a t e l e v e l s . 37 EFFECTS OF NITRITE From an e c o l o g i c a l and p u b l i c h e a l t h standpoint, the produc-t i o n and discharge of n i t r i t e i n t o the environment, even i n s m a l l q u a n t i t i e s , may be viewed as more hazardous than the discharge of n i t r a t e . Canadian D r i n k i n g Water G u i d e l i n e s recommend that n i -t r i t e l e v e l s not exceed 1 mg NO^-N/L, whereas the n i t r a t e l i m i t i s s e t at 10 mg NO3-N/L. N i t r i t e t o x i c i t y to a l l forms of l i f e i s w e l l e s t a b l i s h e d and p e r t i n e n t aspects w i l l be reviewed i n t h i s S e c t i o n . Where ap p r o p r i a t e , comparisons to n i t r a t e hazards w i l l be made. T o x i c i t y to Microorganisms I n t e r e s t i n n i t r i t e as an i n h i b i t o r of b a c t e r i a l growth stems l a r g e l y from i t s use i n cured meat f o r c o l o u r enhancement. Lewis and Moran f i r s t recognized i t s a n t i m i c r o b i a l c a p a c i t y i n 1928, w h i l e conducting s t u d i e s on meat c u r i n g (Tarr, 1941).. In 1929, G r i n d l e y observed that n i t r i t e was more e f f e c t i v e i n con-t r o l l i n g the growth of C l o s t r i d i u m botulinum, the botulism-caus-ing microbe, at low pH and suggested that the a n t i m i c r o b i a l a c t i v i t y of n i t r i t e c o u l d be l i n k e d to n i t r o u s a c i d rather than to the n i t r i t e ion (Sofos et a_l., 1979). This i n s i g h t was subse-q u e n t l y confirmed by other i n v e s t i g a t o r s , who found n i t r o u s a c i d to be more t o x i c to a number of organisms than was n i t r i t e ion (Meikeljohn, 1940; T a r r , 1941; C a s t e l l a n i and Niven, 1955; Perigo and Roberts, 1968; Roberts and Ingram, 1973; Wodzinski et a l . , 1978; Page and S o l b e r g , 1979; M e i j e r et a_l., 1979). I t i s now b e l i e v e d that n i t r i t e (i.e. n i t r o u s acid) p l a y s a major r o l e i n safeguarding cured meat from b o t u l i s m , as i t r e t a r d s the growth 38 of C l o s t r i d i u m botulinum and d e l a y s the p r o d u c t i o n of i t s deadly neurotoxin (Sofos et a_l.f 1979). Shank et a l . (1962) recorded l o s s of n i t r i t e i n h i b i t i o n with some C l o s t r i d i u m species as the pH dropped below 5. They a t t r i -buted i t to the decomposition of n i t r o u s a c i d to non-toxic n i -t r a t e and n i t r i c oxide under a c i d i c c o n d i t i o n s . They p o s t u l a t e d that the maximum l e v e l of b a c t e r i c i d a l a c t i v i t y occurred i n the pH range of 4.5 to 5.5 and dropped r a p i d l y a t l e v e l s above.and below t h i s range. Some i n v e s t i g a t o r s have a l s o found n i t r i t e to be more i n h i b i t o r y to some organisms under anaerobic c o n d i t i o n s (Caste H a n i and Niven, 1955; Bo H a g and Henninger, 1978). The p h y s i o l o g i c a l b a s i s f o r n i t r i t e i n h i b i t i o n i s not c l e a r -l y understood. I t i s probable that the a c t i o n i s complex and i n v o l v e s s e v e r a l pathways and mechanisms (Rowe et .al.., 1979; Sofos et al..-, 1979; Rake and Eagon, 1980; Meiberg et a l ., 1980; Benedict, 1980). Tab l e 5 summarizes i n h i b i t o r y n i t r i t e l e v e l s reported i n the l i t e r a t u r e . Wherever p o s s i b l e , n i t r o u s a c i d l e v e l s were c a l c u -l a t e d . Where n i t r o u s a c i d l e v e l s reported by the authors were based on a d i s s o c i a t i o n constant d i f f e r e n t from the one adopted here, the v a l u e was r e - c a l c u l a t e d . An i n s p e c t i o n of the t a b l e shows a thousand-fold v a r i a t i o n i n i n h i b i t o r y l e v e l s between some organisms. T h i s d i f f e r e n c e i s reduced to a c e r t a i n extent, when reported as n i t r o u s a c i d . G e n e r a l l y , n i t r o u s a c i d appears to be i n h i b i t o r y to most microorganisms at c o n c e n t r a t i o n s above 0.1 mg HNO2-N/L and to a number of them at c o n c e n t r a t i o n s as low as O.Olmg HNO2-N/L. The n i t r i t e c o n c e n t r a t i o n needed to achieve a n i t r o u s a c i d l e v e l of 0.01 mg HN0 2-N/L i s around 175 mg NO^-N/L 39 Table 5 - Pure Culture Studies of Inhibitory Effects of N i t r i t e Species Mode of % PH N i t r i t e Nitrous Reference Inhibition Inhib, Acid ( F i r s t Author) mg N/L ug N/L Pseudomonas growth ioo 6.6-6.9 140 29-58 MeikelJohn, 30 8.0 140 2.3 1940 C. Botulinum growth 100 5.6 41 182 Tarr, 1942 S. aureas growth 100 5.1 41 571 Flavobacterium growth 100 6,1-6.7 41 14-53 Achromobacter growth 100 5.6-5,9 41 92-182 Pseudomonas growth ioo 5.4-5.8 41 12-288 T. de n i t r i f i c a h s anaerobic 40 7 4.9 0.8 Baalsrud, growth 90 7 8.4 1.4 1954 94 7 13.0 5.3 S. aureas growth 100 5.1-6.9 24-1217 260-350 Cas t e l l a n i , Streptococcus spp. growth minimal 6.6-6.7 609-3043 200-1200 1955 Other species growth minimal 6.6-6.7 12-7609 10-3000 Chlorella pyrenoidosa CO? fixation 67.5 3.9 2.4 400 H i l l e r , 1965 13.3 4.5 2.4 100 Aerobacter aerogenes growth 100 6.8 140 32 Hadj ipetrou, 40 6.8 70 16 1965 M. de n i t r i f i c a n s growth 100 6.8-7.0 140 20-40 Bovell, 1967 74 6.8-7.0 0.7-70 up to 20 100 6.8-7.0 700 120-180 C. sporogenes growth no growth 7.0 i280 179 Perigo, 1968 no growth 6.0 200 302 T a b l e 5 - P u r e C u l t u r e S t u d i e s o f i n h i b i t o r y E f f e c t s o f N i t r i t e ( C o n t ' d ) S p e c i e s Mode o f I n h i b i t i o n % I n h i b . PH N i t r i t e mg N/L N i t r o u s A c i d u g N/L R e f e r e n c e ( F i r s t A u t h o r ) S o i l i s o l a t e s a n a e r o b i c ioo 7 . 2 6 0 9 6 1 B o l l a g , i 9 7 0 g r o w t h m i n i m a l 7 . 2 152 15 S . a u r e a s g r o w t h m i n i m a i 7 , 3 2 0 0 0 1 5 2 B u c h a n a n * s i g n i f i c a n t 6 . 3 2 0 0 0 1 5 1 6 1 9 7 2 C . b o t u l i n u m g r o w t h m i n i m a i 6 . 2 3 0 0 2 9 3 R o b e r t s , ioo 6 . 0 3 0 0 4 6 4 1 9 7 3 100 5 . 8 2 5 0 6 1 2 100 5 . 6 1 5 0 5 1 8 1 0 0 5 . 4 50 3 0 6 P s e u d o m o n a s d i s s i m i l a t o r y 0 7 . 2 2 0 0 30 V a n g n a i , n i t r i t e 2 0 - 6 0 7 ; 2 6 0 0 80 1974 r e d u c t i o n 100 7 . 2 7 0 0 - 8 0 0 1 0 0 C ; p e r f r i h g e n s g r o w t h 100 7 . 2 i 6 2 - 3 0 4 1 4 - 2 7 R i h a , 1 9 7 5 ioo 6 . 3 i 6 - 8 i 1 1 - 5 7 P r o p i o n i b a c t e r i u m p . a n a e r o b i c 100 6 , 8 i 2 6 30 V a n G e n t - R . , g r o w t h 1 9 7 5 C , p e r f r i r i g e n s a d o l a s e 67 7 . 6 2 0 0 8 O 1 L e a r y , a c t i v i t y 1 9 7 6 P . a e r u g i n o s a a n a e r o b i c s l o w d o w n 7 . 0 210 36 W i l l i a m s * g r o w t h i 9 7 8 S o i l i s o l a t e s a n a e r o b i c 6 7 . 0 8 2 5 1 1 5 G a r c i a , 1977 g r o w t h ido 7 , 0 8 2 5 1 1 5 Table 5 - Pure C u l t u r e Studies of I n h i b i t o r y E f f e c t s of N i t r i t e (Cont'd) Species Mode of % pH N i t r i t e N i t r o u s Reference I n h i b i t i o n i n h i b * A c i d ( F i r s t Author) mg N/L ug N/L P. d e n i t r i f i c a r i s growth 100 6.9 140 30 VanVerseveld 1977 Cyanobacteria C0 2 uptake 95-100 6.0 14 25 Wodzinski, 82-93 7.7 420 16 1978 PseudOmonas anaerobic growth 0 7.0 70 12 B o l l a g , 1978 100 7.0 140 24 P. aeruginosa oxygen uptake 98 7.0 1400 240 Rowe, 1979 aerobic glucose transport 100 7.0 350 60 anaerobic glucose t r a n s p o r t 0 7.0 1400 240 S* typhimurium growth 10 7.2 800 64 Page* 1979 93 6.0 800 1200 P. d e n i t r i f i c a n s oxygen uptake 0 7*0 28 4 M e i j e r , 1979 some i n h i b * 6.0 28 40 Hyphomicrobium X enzyme a c t i v i t y 100 7.0 42 6 Meiberg, 1980 P. aeruginosa anaerobic growth i n h i b i t i o n 7.5 210 10 C a r l s o n , P. d e n i t r i f i c a n s anaerobic growth 0 7.5 504 25 1983 S. aureus (starved anaerobic glucose 100 6*0 406 550 Simone* 1983 c e l l ) t r ansport some i n h i b . 7.0 41 6 Note: N i t r o u s a c i d concentration c a l c u l a t e d as per constant used i n t e x t . Values may d i f f e r from those reported by the authors* a t p H 7 ( 2 0 ° C ) . I t i n c r e a s e s t o 5 5 5 mg N O ^ - N / L a t p H 7 . 5 a n d t o 1 7 6 0 mg N O ^ - N / L a t p H 8 . A c u t e a n d S u b a c u t e T o x i c i t y t o F i s h I n t e r e s t i n t h e t o x i c e f f e c t s o f n i t r i t e t o f i s h d e v e l o p e d a s a r e s u l t o f t h e i n c r e a s i n g u s e o f w a t e r r e c i r c u l a t i o n s y s t e m s i n f i s h c u l t u r e o p e r a t i o n s . A c o n s t i t u e n t t h a t n o r m a l l y r e q u i r e s r e m o v a l f r o m t h e r e c i r c u l a t e d w a t e r i s a m m o n i a , w h i c h a c c u m u l a t e s a s a r e s u l t o f i t s e x c r e t i o n b y f i s h . T r a d i t i o n a l l y , a m m o n i a h a s b e e n r e m o v e d f r o m s u c h s y s t e m s b y b i o l o g i c a l n i t r i f i c a t i o n . T h i s c a n o c c a s i o n a l l y r e s u l t i n t r a n s i e n t n i t r i t e a c c u m u l a t i o n c a u s i n g f i s h m o r t a l i t y ( K o n i k o f f , 1 9 7 5 ; C o l l i n s e t a l . , 1 9 7 5 ; B r o w n a n d M c L e a y , 1 9 7 5 ; C o l t a n d T c h o b a n o g l o u s , 1 9 7 6 ; P e r r o n e a n d M e a d e , 1 9 7 7 ; W e d e m e y e r a n d Y a s u t a k e , 1 9 7 8 ; T o m a s s o e t a l . , 1 9 7 9 ; T u c k e r a n d S c h w e d l e r , 19 8 3 ; E d d y e t a l . , 1 9 8 3 ) . T h e t o x i c i t y o f n i t r i t e t o f i s h i s c a u s e d p r i m a r i l y b y i t s a b i l i t y t o c o n v e r t h a e m o g l o b i n t o m e t h a e m o g l o b i n , a f o r m o f b l o o d p l a s m a i n c a p a b l e o f t r a n s p o r t i n g o x y g e n . T h i s l e a d s t o c y a n o s i s a n d h y p o x i a . N i t r i t e t o x i c i t y h a s b e e n e x t e n s i v e l y s t u d i e d a n d a w i d e r a n g e o f a c u t e t o x i c i t y v a l u e s h a v e b e e n r e p o r t e d . T h e v a l u e s r a n g e f r o m a 9 6 h r L C 5 0 o f 0 . 2 3 mg N O ^ - N / L f o r p o s t - a l e v i n r a i n b o w t r o u t ( B r o w n a n d M c L e a y , 1 9 7 5 ) t o a 96 h r L C 5 0 o f 10 mg N O ^ - N / L f o r j u v e n i l e s t e e l h e a d t r o u t ( W e d e m e y e r a n d Y a s u t a k e , 1 9 7 8 ) , . O t h e r s t u d i e s f o u n d n o t o x i c e f f e c t s a t 5 mg N O ^ - N / L f o r c a t f i s h ( T o m a s s o e t a l . , 1 9 7 9 ) a n d a t 120 mg N O ^ - N / L f o r c h i n o o k s a l m o n f i n g e r l i n g s i n s e a w a t e r ( C r a w f o r d a n d A l l e n , 1 9 7 7 ) . T h e w i d e d i s c r e p a n c y i n t o x i c l e v e l s r e p o r t e d i n t h e l i t e r a -t u r e ( s e e a l s o R u s s o e t a l . , 1974 a n d N . R . C , 1 9 7 8 ) w a s l a r g e l y 4 3 c l a r i f i e d i n 1 9 7 7 b y P e r r o n e a n d M e a d e , who d i s c o v e r e d t h a t c h l o -r i d e i o n s p r o t e c t e d f i s h f r o m t h e t o x i c e f f e c t s o f n i t r i t e . T h e y r e p o r t e d n o m o r t a l i t y o f y e a r l i n g c o h o s a l m o n s u b j e c t e d t o 2 9 . 8 mg N O ^ - N / L i n w a t e r c o n t a i n i n g 2 6 1 mg C l ~ / L f o r 4 8 h o u r s , a s o p p o s e d t o 5 8 . 3 % m o r t a l i t y f o r t h o s e e x p o s e d t o 3 . 8 mg N O ^ - N / L i n w a t e r c o n t a i n i n g 2 . 5 mg C l ~ / L ( P e r r o n e a n d M e a d e , 1 9 7 7 ) . T h e i r f i n d i n g s h a v e s i n c e b e e n c o n f i r m e d b y s e v e r a l o t h e r s r e s e a r c h e r s ( W e d e m e y e r a n d Y a s u t a k e , 1 9 7 8 ; T o m a s s o e t a _ l . , 1 9 7 9 ; B a t h a n d E d d y , 1 9 8 0 ; R u s s o e t a l . , 1 9 8 1 ; E d d y e t a l . , 1 9 8 3 ; S c h w e d l e r a n d T u c k e r , 1 9 8 3 ; G a i n o e t a l . , 1 9 8 4 ) . T h e p r e s e n c e o f c h l o r i d e i o n s h a s b e e n s h o w n t o r e d u c e n i t r i t e t r a n s p o r t f r o m t h e w a t e r i n t o t h e b l o o d . I t i s p o s t u l a t e d t h a t t h e c h l o r i d e u p t a k e m e c h a n i s m p r e s e n t i n f i s h g i l l s h a s some a f f i n i t y f o r n i t r i t e , t h u s a l l o w i n g i t s t r a n s p o r t i n t o t h e b l o o d w h e n e x t e r n a l c h l o r i d e l e v e l s a r e l o w ( B a t h a n d E d d y , 1 9 8 0 ) , . T h i s may e x p l a i n t h e p r o t e c t i v e i n f l u e n c e a l s o a f f o r d e d b y HCO3, w h i c h i s k n o w n t o c o m p e t e f o r t h e c h l o r i d e u p t a k e m e c h a n i s m ( B a t h a n d E d d y , 1 9 8 0 ) . R e c e n t l y , E d d y ^et a l . ( 1 9 8 3 ) r e p o r t e d t h a t b r o m i d e o f f e r e d a s m u c h p r o t e c t i o n a g a i n s t n i t r i t e t o x i c i t y a s d o e s c h l o r i d e . T h e c h l o r i d e l e v e l s n e e d e d t o o f f e r p r o t e c t i o n a g a i n s t n i -t r i t e t o x i c i t y h a v e b e e n q u a n t i f i e d b y s e v e r a l a u t h o r s . T o m a s s o e t a l . ( 1 9 7 9 ) r e p o r t e d t o t a l s u p p r e s s i o n o f n i t r i t e - i n d u c e d m e t h a e m o g l o b i n f o r m a t i o n i n c a t f i s h a t a C 1 ~ : N 0 2 - N r a t i o o f a r o u n d 4 0 : 1 , w h i l e B a t h a n d E d d y ( 1 9 8 0 ) f o u n d a r a t i o o f 2 0 : 1 t o b e s u f f i c i e n t f o r r a i n b o w t r o u t u p t o 7 mg N O ^ - N / L . E d d y et a l . ( 1 9 8 3 ) r e p o r t e d c o m p l e t e a l l e v i a t i o n o f n i t r i t e t o x i c i t y i n j u -v e n i l e r a i n b o w t r o u t a t a r a t i o o f 1 5 : 1 f o r n i t r i t e l e v e l s u p t o 44 10 mg N O ^ - N / L . T u c k e r a n d S c h w e d l e r ( 1 9 8 3 ) r e c o m m e n d e d a p r a c t i -c a l r a t i o o f 1 0 : 1 , w h i c h t h e y f o u n d s u f f i c i e n t t o m a i n t a i n m e t h a e m o g l o b i n l e v e l s b e l o w 30% f o r n i t r i t e l e v e l s u p t o 3 mg N 0 2 - N / L . T h e y a l s o r e p o r t e d o n a c o m m e r c i a l p o n d o p e r a t i o n t h a t h a d i n t r o d u c e d t h e p r a c t i c e o f c h l o r i d e s a l t a d d i t i o n t o a l l e v i a t e n i t r i t e t o x i c i t y t o c a t f i s h . N i t r i t e t o x i c i t y t o f i s h a p p e a r s t o a l s o b e pH d e p e n d e n t . I t i n c r e a s e s w i t h d r o p p i n g pH l e v e l s ( C o l t a n d T c h o b a n o g l o u s , 1 9 7 6 ; W e d e m e y e r a n d Y a s u t a k e , 1 9 7 8 ; R u s s o e t a l . , 1 9 8 1 ) . C o l t a n d T c h o b a n o g l o u s ( 1 9 7 6 ) a t t r i b u t e t h e t o x i c i t y t o n i t r o u s a c i d r a t h e r t h a n t h e n i t r i t e i o n . T h e y a l s o n o t e d t h a t n i t r o u s a c i d t o x i c i t y w a s m o r e p r o n o u n c e d a t h i g h e r t e m p e r a t u r e s . R u s s o e t a l . ( 1 9 8 1 ) a g r e e d t h a t n i t r o u s a c i d w a s m o r e t o x i c t h a n n i t r i t e i o n , b u t c o n c l u d e d t h a t b o t h s p e c i e s c o n t r i b u t e t o t h e t o x i c i t y . S u b - l e t h a l m e t h a e m o g l o b i n e m i a i n f i s h i s r e p o r t e d t o o c c u r a t v e r y l o w n i t r i t e l e v e l s . B r o w n a n d M c L e a y ( 1 9 7 5 ) a n d W e d e m e y e r a n d Y a s u t a k e ( 1 9 7 8 ) o b s e r v e d t h i s e f f e c t a t n i t r i t e c o n c e n t r a -t i o n s a s l o w a s 0 . 0 1 5 mg N O ^ - N / L , w h i l e S m i t h a n d R u s s o ( 1 9 7 5 ) d e t e c t e d i t a t a l e v e l o f 0 . 0 3 mg N O ^ - N / L . T h e c a u s e o f t o x i c i t y a t s u c h l o w l e v e l s i s l i n k e d t o i t s a c c u m u l a t i o n i n t h e b l o o d p l a s m a . N i t r i t e l e v e l s i n f r e s h w a t e r f i s h b l o o d w a s f o u n d t o e x c e e d s u r r o u n d i n g e n v i r o n m e n t a l l e v e l s b y f a c t o r s o f 10 ( E d d y e t a l . , 1 9 8 3 ) t o 6 0 ( M a r g i o c c o e t a _ l . , 1 9 8 3 ) . Two t h e o r i e s h a v e b e e n a d v a n c e d t o e x p l a i n t h i s p h e n o m e n o n . T h e f i r s t i s b a s e d o n t h e p r e m i s e t h a t n i t r o u s a c i d , a n d n o t n i t r i t e i o n , d i f f u s e s a c r o s s t h e g i l l o f f i s h . D i f f u s i o n o f n i -t r o u s a c i d p r o c e e d s u n t i l a n e q u i l i b r i u m i s r e a c h e d b e t w e e n t h e 45 l e v e l i n t h e b l o o d a n d t h a t i n t h e w a t e r . T h e b r a n c h i a l b l o o d p H i s a r o u n d 7 . 9 t o 8 . 0 a n d t h a t o f f r e s h w a t e r i s g e n e r a l l y l o w e r . I n o r d e r t o a c h i e v e a n i t r o u s a c i d b a l a n c e , t h e n i t r i t e i o n c o n c e n t r a t i o n i n t h e b l o o d w o u l d h a v e t o b e s u b s t a n t i a l l y h i g h e r t h a n i n t h e w a t e r , s i n c e t h e HNO2/NO2 e q u i l i b r i u m s h i f t s t o w a r d t h e n i t r i t e i o n w i t h i n c r e a s i n g pH v a l u e s . T h u s , a d i f f e r e n c e o f o n e pH u n i t c o u l d l e a d t o a t e n - f o l d a c c u m u l a t i o n o f n i t r i t e i n t h e b l o o d . T h e s e c o n d t h e o r y a c c o u n t s f o r t h e p r o t e c t i v e e f f e c t o f c h l o r i d e d i s c u s s e d e a r l i e r . I t s u g g e s t s t h a t C l " e i t h e r s u p -p r e s s e s b r a n c h i a l d i f f u s i o n o f n i t r o u s a c i d o r t h a t t h e b r a n c h i a l c h l o r i d e i o n u p t a k e s y s t e m o f f r e s h w a t e r f i s h h a s a n a f f i n i t y f o r n i t r i t e , a l l o w i n g i t t o d i f f u s e i n t h e a b s e n c e o f c h l o r i d e ( E d d y e t a l . , 1 9 8 3 ) . B o t h o f t h e a f o r e m e n t i o n e d t h e o r i e s e x p l a i n t h e o b s e r v a t i o n t h a t s e a w a t e r f i s h a r e n o t a s s u s c e p t i b l e t o n i t r i t e t o x i c i t y a s a r e f r e s h w a t e r f i s h ( C r a w f o r d a n d A l l e n , 1 9 7 7 ; E d d y e t a l . . , 1 9 8 3 ) ; s e a w a t e r c o n t a i n s a h i g h c h l o r i d e c o n c e n t r a t i o n a n d h a s a r e l a t i v e l y h i g h p H . E f f l u e n t C h l o r i n a t i o n T h e p r e s e n c e o f n i t r i t e i n a s e w a g e e f f l u e n t i m p a c t s u p o n t h e d i s i n f e c t i o n p r o c e s s i n t w o w a y s : 1. T h e c h l o r i n e d e m a n d 2. T h e e f f e c t i v e n e s s o f d i s i n f e c t i o n 1. C h l o r i n e Demand F r e e c h l o r i n e o x i d i z e s n i t r i t e t o n i t r a t e C l 2 + H 20 + NO2 »- NO3 + 2HC1 ....(12) c r e a t i n g a n i n c r e a s e d c h l o r i n e d e m a n d ( W h i t e , 1 9 8 1 ) . D h a l i w a l a n d 46 B a k e r ( 1 9 8 3 ) r e p o r t e d a n i n c r e a s e i n c h l o r i n e d e m a n d f r o m a b o u t 12 t o o v e r 3 0 m g / L a t a t r e a t m e n t p l a n t w h e n t h e e f f l u e n t n i t r i t e l e v e l r o s e f r o m z e r o t o a r o u n d 1 2 mg N O^-N/ L . T h e a m m o n i a e f -f l u e n t l e v e l r e m a i n e d b e l o w 2 mg NH^-N/L d u r i n g t h a t p e r i o d . C o m b i n e d c h l o r i n e d o e s n o t o x i d i z e n i t r i t e a s r e a d i l y , a n d a s s u c h , n i t r i t e d o e s n o t e x e r t a c h l o r i n e d e m a n d o n t h e c o m b i n e d c h l o r i n e s p e c i e s ( W h i t e , 1 9 8 1 ) . D h a l i w a l a n d B a k e r ( 1 9 8 3 ) r e p o r -t e d t h a t , i n t h e p r e s e n c e o f 4 mg NH^-N/L o r m o r e i n t h e e f -f l u e n t , n i t r i t e d i d n o t e x e r t a n y s i g n i f i c a n t c h l o r i n e d e m a n d . T h i s w a s a t t r i b u t e d t o t h e r e a c t i o n o f a m m o n i a w i t h c h l o r i n e t o f o r m c h l o r a m i n e s , w h i c h d o n o t r e a d i l y o x i d i z e n i t r i t e . 2 . E f f e c t i v e n e s s o f D i s i n f e c t i o n G a s s e r ( 1 9 8 4 ) r e p o r t e d o n a n e x p e r i m e n t c a r r i e d o u t a t t h e R e d d i n g w a s t e w a t e r t r e a t m e n t p l a n t i n E n g l a n d , t o t e s t t h e e f f e c -t i v e n e s s o f c h l o r i n e d i s i n f e c t i o n . T h e y f o u n d t h a t t h e a b s e n c e o f a m m o n i a i n t h e e f f l u e n t a n d t h e p r e s e n c e o f 3 . 4 mg N O ^ - N / L ( h i g h e s t n i t r i t e l e v e l r e p o r t e d ) r e s u l t e d i n t h e h i g h e s t c o l i f o r m s u r v i v a l r a t e s a n d l o w e s t r e s i d u a l c h l o r i n e l e v e l s . T h e a b s e n c e o f b o t h a m m o n i a a n d n i t r i t e f r o m t h e e f f l u e n t r e d u c e d t h e e f f l u e n t c o l i f o r m c o u n t b y a f a c t o r o f a b o u t 1 0 . T h e l o w e s t e f f l u e n t c o l i f o r m c o u n t s a n d h i g h e s t c h l o r i n e r e s i d u a l l e v e l s w e r e , h o w e v e r , a s s o c i a t e d w i t h t h e p r e s e n c e o f 3 . 0 mg N H ^ - N / L a n d 3 . 4 mg N O ^ - N / L i n t h e e f f l u e n t . I t a p p e a r s f r o m t h e a f o r e m e n t i o n e d t h a t t h e p r e s e n c e o f n i t r i t e i n t h e e f f l u e n t c a n e n h a n c e t h e d i s i n f e c t i o n p r o c e s s u n d e r c e r t a i n c o n d i t i o n s , b u t b e d e t r i m e n t a l t o i t u n d e r o t h e r c o n d i t i o n s , d e p e n d i n g u p o n t h e p r e s e n c e o r a b s e n c e o f a m m o n i a 47 f r o m t h e e f f l u e n t . A B I L I T Y TO GROW ON N I T R I T E AS SOLE ELECTRON ACCEPTOR N e a r l y a l l d e n i t r i f i e r s c a n g r o w w i t h n i t r i t e a s s o l e e l e c -t r o n a c c e p t o r , a l t h o u g h some d e g r e e o f a c c l i m a t i o n may b e r e -q u i r e d ( P a y n e a n d G r a n t , 1 9 8 1 ) . N u m e r o u s s t u d i e s h a v e c o n f i r m e d t h e a b i l i t y o f b i o l o g i c a l t r e a t m e n t s y s t e m s t o r e d u c e n i t r i t e t o n i t r o g e n g a s , e v e n a t c o n c e n t r a t i o n s a b o v e 5 0 0 mg N O ^ - N / L ( P r a k a -sam a n d L o e h r , 1 9 7 2 ; A n t h o n i s e n , 1 9 7 4 ; V o e t s e t al., 1 9 7 5 ; M u r r a y e t a l . , 1 9 7 5 ; B l a s z c z y k e t a l . , 1 9 8 1 ; N a g a s h i m a e_t a l . , 1 9 8 1 a a n d b ; B l a s z c z y k , 1 9 8 3 ) . . T h e e x i s t e n c e o f m i c r o o r g a n i s m s c a p a b l e o f r e d u c i n g n i t r i t e , b u t n o t n i t r a t e , t o n i t r o g e n g a s w a s f i r s t r e p o r t e d b y Y o u a t t ( 1 9 5 4 ) w i t h a s o i l i s o l a t e . T h i s w a s l a t e r c o n f i r m e d b y S k e r m a n e t a l . ( 1 9 5 8 ) a n d o t h e r s . V a n g n a i a n d K l e i n ( 1 9 7 4 ) r e p o r t e d t h e i s o l a t i o n f r o m ; s o i l o f t h r e e m i c r o o r g a n i s m s c a p a b l e o f d i s s i m i l a -t o r y n i t r i t e r e d u c t i o n b u t n o t n i t r a t e r e d u c t i o n . I n t h e i r r e v i e w o n d e n i t r i f i e r s , J e t e r a n d I n g r a h a m ( 1 9 8 1 ) l i s t e d t h r e e g e n e r a c o n t a i n i n g s t r a i n s c a p a b l e o f d i s s i m i l a t o r y n i t r i t e b u t n o t n i -t r a t e r e d u c t i o n . I n s p i t e o f t h e a c c u m u l a t e d e v i d e n c e , some r e s e a r c h e r s c o n -t e n d t h a t n i t r i t e r e d u c t i o n i s n o t p o s s i b l e i n a c t i v a t e d s l u d g e s y s t e m s . B e c c a r i e_t a _ l . ( 1 9 8 3 ) r e p o r t e d o n t h e r e s u l t s o f 4 - h o u r l o n g b a t c h d e n i t r i f i c a t i o n t e s t s c a r r i e d o u t w i t h a b i o m a s s n o t a c c l i m a t e d t o t h e r e d u c t i o n o f n i t r i t e . T h e y o b s e r v e d t o t a l i r r e v e r s i b l e i n h i b i t i o n o f n i t r i t e r e d u c t i o n a t n i t r i t e l e v e l s o f 20 mg N O ^ - N / L . T h e y n o t e d , h o w e v e r , t h a t g r a d u a l l y i n c r e a s i n g t h e n i t r i t e l e v e l f r o m 5 t o 4 5 mg N O ^ - N / L o v e r a 4 h o u r p e r i o d 48 r a i s e d the i n h i b i t o r y t h r e s h o l d v a l u e . They concluded that t h e i r r e s u l t s seemed to r u l e out the p o s s i b i l i t y of a c h i e v i n g n i t r o g e n removal v i a the n i t r i t e s h o r t c u t . 49 CHAPTER FOUR EXPERIMENTAL SET-DP AND ANALYTICAL TECHNIQUES E X P E R I M E N T A L S E T - U P E q u i p m e n t T h e e x p e r i m e n t a l a p p a r a t u s c o n s i s t e d o f b e n c h - s c a l e , a c t i -v a t e d s l u d g e m o d u l e s o p e r a t e d i n s e r i e s , t o e m u l a t e a p l u g f l o w c o n f i g u r a t i o n . A l l m o d u l e s ( o r c e l l s ) w e r e o f e q u a l d i m e n s i o n . A m i n i m u m o f f o u r a n d m a x i m u m o f e i g h t m o d u l e s w e r e u s e d p e r s y s t e m p e r r u n . A s c h e m a t i c o f a t y p i c a l f o u r - c e l l c o n f i g u r a t i o n i s s h o w n i n F i g u r e 3 , a n d p h o t o g r a p h s o f t h e s e t - u p a r e p r e s e n t e d i n F i g u r e 4 . E a c h c e l l c o n s i s t e d o f a 3 . 1 8 mm t h i c k , c a s t a c r y l i c t u b i n g w i t h a n i n t e r n a l d i a m e t e r o f 8 2 . 5 5 mm a n d a h e i g h t o f 5 3 0 mm. T h e s e d i m e n s i o n s w e r e s e l e c t e d f o l l o w i n g a p r e l i m i n a r y i n v e s t i g a -t i o n , t o d e t e r m i n e t h e o p t i m u m h e i g h t t o d i a m e t e r r a t i o n e e d e d t o a c h i e v e n e a r - u n i f o r m DO l e v e l s w i t h i n t h e c e l l . E a c h c e l l a p p r o -x i m a t e d a c o m p l e t e l y - m i x e d , a c t i v a t e d s l u d g e mode o f o p e r a t i o n , w h i l e t h e s y s t e m a s a w h o l e a p p r o x i m a t e d a p l u g f l o w c o n f i g u r a -t i o n . M i x i n g w i t h i n e a c h c e l l w a s a c h i e v e d b y t h e u s e o f a v a r i a -b l e s p e e d c o n e d r i v e n m i x e r ( S a r g e n t - W e l c h S c i e n t i f i c C o . ) . A e r a -t i o n w a s p r o v i d e d b y a p o r o u s g l a s s c o a r s e b u b b l e d i f f u s e r ( K i m b l e P r o d u c t s ) a n d w a s c o n t r o l l e d b y a n A a r b o r g a i r f l o w m e t e r . H o l e s w e r e d r i l l e d t h r o u g h t h e s i d e s o f t h e t u b e s t o a c c o m m o d a t e EFFLUENT CONTAINER (SOL) CONTAINER (of 4<»c) ( 2 0 L ) Figure 3= Process Treatment Schematic Configuration F i g . 4 : Photographs of Ben ch - S ca l e Set Up 52 N o . 3 s i l i c o n s t o p p e r s w h e r e v e r n e e d e d . T h e s t o p p e r s w e r e p e r f o -r a t e d t o a l l o w t h e i n s e r t i o n o f 4 . 7 6 mm d i a m e t e r N a l g e o r 4 . 8 9 mm d i a m e t e r T y g o n t u b i n g . T h e t u b i n g w a s u s e d t o c o n n e c t t h e c e l l s t o e a c h o t h e r a n d a l l o w f l o w o f t h e 1 i q u i d t h r o u g h t h e s y s t e m . T h e i n l e t a n d o u t l e t t o e a c h c e l l w e r e a t o p p o s i t e h e i g h t s ( i . e . , a n i n l e t a t t h e b o t t o m w i t h a n o u t l e t a t t h e t o p a n d v i c e v e r s a ) , i n a d d i t i o n , a n o v e r f l o w p i p i n g a r r a n g e m e n t w a s i n c l u d e d i n a l l c e l l s . T h e o v e r f l o w t u b i n g w a s l o c a t e d a b o v e t h e n o r m a l l i q u i d l e v e l a n d i t s f u n c t i o n w a s t o p r e v e n t l i q u i d o v e r f l o w f r o m a c e l l d u e t o b l o c k a g e o f t h e c o n n e c t i n g t u b i n g . T h e n o r m a l l i q u i d v o l u m e p e r c e l l w a s a r o u n d 2 . 5 L . A p r o g r e s s i v e r e d u c t i o n i n l i q u i d h e i g h t ( a b o u t 3 t o 5 mm r e d u c t i o n p e r c e l l ) r e s u l t e d f r o m h y d r a u l i c l o s s e s t h r o u g h t h e s y s t e m . T h e f i n a l c l a r i f i e r c o n s i s t e d o f a o n e - l i t r e c a p a c i t y J m h o f f s e t t l i n g c o n e . S l u d g e w a s w i t h d r a w n f r o m t h e b o t t o m , w h i l e e f -f l u e n t w a s a l l o w e d t o d i s c h a r g e f r o m t h e t o p v i a a 3 . 1 8 mm d i a m e -t e r t e e . A l 1 s l u d g e r e c y c l e 1 i n e s w e r e o f 3 . 1 8 mm d i a m e t e r T y g o n t u b i n g . A r a k e m e c h a n i s m c o n n e c t e d t o a 1 r p m D a y t o n m o t o r w a s u s e d f o r c l e a n i n g t h e s i d e s o f t h e c l a r i f i e r . A t i m e r c o n t r o l l e d t h e o p e r a t i o n o f t h e r a k i n g m e c h a n i s m . A l l p u m p s , e x c e p t o n e , w e r e M a s t e r f l e x . T h e e x c e p t i o n w a s a D e s a g a m u l t i - c h a n n e l p e r i s t a l t i c p u m p , w h i c h , w a s u s e d f o r a b o u t s i x m o n t h s , a n d t h e n e x c h a n g e d i n f a v o u r o f M a s t e r f l e x p u m p s . T h r e e - w a y v a l v e s w e r e i n s t a l l e d o n a l l f e e d a n d r e c y c l e l i n e s t o a l l o w f o r f l o w m e a s u r e m e n t . T h e y w e r e a l s o i n s t a l l e d b e t w e e n c e l l s t o a l l o w f o r s a m p l e c o l l e c t i o n f r o m i n d i v i d u a l c e l l s . T h e e n t i r e t r e a t m e n t s y s t e m w a s r a c k m o u n t e d o n t o p o f a l a b o r a t o r y b e n c h . T h e s p a c e b e t w e e n t h e u n d e r s i d e o f c e l l s a n d t h e t o p o f t h e b e n c h w a s u s e d f o r l a y i n g o f t u b i n g , c l e a n i n g p u r p o s e s , a n d s a m p l e c o l l e c t i o n . O p e r a t i o n T h e f e e d w a s k e p t r e f r i g e r a t e d a t 4 ° C i n a 30 L c a p a c i t y b u c k e t l o c a t e d b e s i d e t h e b e n c h . No m i x i n g w a s p r o v i d e d w i t h i n t h e b u c k e t a s t h e f e e d c o n s t i t u e n t s w e r e s o l u b l e . F e e d w a s a d d e d d a i l y t o t h e b u c k e t t o s a t i s f y a b o u t o n e d a y s s u p p l y , a n d t h e r e m a i n d e r w a s k e p t r e f r i g e r a t e d i n t h e c o l d r o o m . F e e d f r o m t h e b u c k e t w a s p u m p e d t o t h e f i r s t c e l l v i a 3 . 1 8 mm d i a m e t e r t u b i n g . E f f l u e n t w a s c o l l e c t e d i n a 20 L c o n t a i n e r w h i c h w a s e m p t i e d d a i l y . A t o t a l o f s e v e n r u n s w e r e c o n d u c t e d i n t h e E n v i r o n m e n t a l E n g i n e e r i n g L a b o r a t o r y o v e r a p e r i o d o f t w e n t y e i g h t m o n t h s , s t a r t i n g S e p t e m b e r 1 3 , 1 9 8 3 . T e m p e r a t u r e i n s i d e t h e l a b o r a t o r y w a s n o r m a l l y m a i n t a i n e d a t a b o u t .20 +_ ; 2 ° C . F r e s h s l u d g e w a s u s e d a t t h e b e g i n n i n g o f e a c h r u n . T h e s l u d g e f o r R u n s 1 t o 6 w a s o b t a i n e d f r o m t h e p i l o t - s c a l e a c t i v a t e d s l u d g e p l a n t o p e r a t e d b y t h e D e p a r t m e n t o f C i v i l E n g i n e e r i n g . T h e p i l o t p l a n t , l o c a t e d b e s i d e B . C . R e s e a r c h a t t h e S o u t h e n d o f t h e C a m p u s , w a s b e i n g o p e r a t e d t o a c h i e v e b i o l o g i c a l p h o s p h o r u s a n d n i t r o g e n r e m o v a l . S l u d g e f o r R u n 7 w a s o b t a i n e d f r o m t h e S q u a m i s h a c t i v a t e d s l u d g e w a s t e w a t e r t r e a t m e n t p l a n t ; t h i s p l a n t i s l o c a t e d a b o u t 70 km n o r t h o f V a n c o u v e r a n d t h e s l u d g e c o n s i s t e d o f a n o n - n i t r i f y i n g b i o m a s s . T h e i n s i d e w a l l s o f a l l c e l l s w e r e c l e a n e d r e g u l a r l y t o m i n i m i z e b i o m a s s a d h e r e n c e t o t h e s u r f a c e . W a s t a g e w a s d o n e f r o m t h e l a s t c e l l o f t h e s y s t e m . 54 S o l i d s R e t e n t i o n T i m e S o l i d s r e t e n t i o n t i m e (SRT) w a s c a l c u l a t e d b y a c c o u n t i n g f o r t h e s o l i d s l o s t i n t h e e f f l u e n t a n d M L S S w a s t a g e . A s a r e s u l t , v a r i a t i o n e x i s t e d i n SRT v a l u e s , e v e n w h e n w a s t a g e v o l u m e s r e m a i n e d c o n s t a n t . A e r o b i c SRT w a s c a l c u l a t e d a s a f u n c t i o n o f a c t u a l a e r o b i c h y d r a u l i c r e t e n t i o n t i m e d i v i d e d b y t h e t o t a l a c t u a l h y d r a u l i c r e t e n t i o n t i m e . F E E D C H A R A C T E R I S T I C S S y n t h e t i c F e e d A s y n t h e t i c f e e d w a s u s e d d u r i n g t h e f i r s t f i v e r u n s . I t c o n s i s t e d o f a r e c i p e d e v i s e d t o r e f l e c t a h i g h - s t r e n g t h , a m m o n i a w a s t e , c o n t a i n i n g a m u l t i - c a r b o n f e e d s o u r c e f o r u s e b y t h e h e t e -r o t r o p h i c m i c r o o r g a n i s m s . T h e f e e d c o m p o s i t i o n w a s b a s e d p a r t l y o n s o m e e l e m e n t s o f t h e r e c i p e s r e p o r t e d i n t h e l i t e r a t u r e (OCDE, 1 9 7 1 ; P o d u s k a a n d A n d r e w s , 1 9 7 4 ; F a u p e t a _ l . , 1 9 7 8 ) . T h e r e c i p e w a s r e v i s e d s e v e r a l t i m e s d u r i n g t h e f i r s t m o n t h s ( i . e . , R u n 1 a n d t h e e a r l y p a r t o f R u n 2 ) , m a i n l y t o i n c r e a s e i t s C O D : T K N r a t i o . T a b l e s 6 a n d 7 a r e a s u m m a r y o f i t s c o m p o s i t i o n a n d d i l u t e d s t r e n g t h t h r o u g h o u t t h e s t u d y . T h e s y n t h e t i c f e e d w a s p r e p a r e d a s a s t o c k c o n c e n t r a t e i n 7 . 5 L b a t c h e s , o f w h i c h 2 5 0 t o 5 0 0 m l ( d e p e n d i n g o n t h e s t r e n g t h d e s i r e d ) w a s a d d e d d a i l y t o t h e f e e d b u c k e t a n d d i l u t e d t o 2 0 L w i t h t a p w a t e r ( d a i l y f e e d f l o w r a n g e d b e t w e e n 10 a n d 2 0 L / d f o r e a c h s y s t e m ) . B u f f e r a n d a l k a l i n i t y , i n t h e f o r m o f s o d i u m b i c a r b o n a t e o r s o d i u m h y d r o x i d e , w a s a d d e d d i r e c t l y t o t h e f e e d b u c k e t t o a l l o w d a i l y a d j u s t m e n t s t o t h e p H o f t h e s y s t e m ( t o 55 T a b l e 6 - S y n t h e t i c F e e d C o m p o s i t i o n F o r R u n s 1 t o 5 Compound To 2 / 1 2 / 8 3 TO 3 0 / 1 2 / 8 3 TO 1 8 / 4 / 8 4 TO 6 / 7 / 8 4 TO 1 3 / 1 0 / 8 4 TO 5 / 1 2 / 8 4 Beef extract 4 . 4 6 . 0 9 . 3 9 . 3 9 . 3 9 . 3 Yeast extract 0 . 8 2 . 0 4 . 7 4 . 7 4 . 7 4 . 7 Bactopeptone 2 . 7 3 . 3 4 . 7 4 . 7 4 . 7 5 . 3 D-glucose anh. 1 . 3 1 . 6 2 . 0 2 . 0 2 . 0 4 . 0 Starch 1 . 3 1 . 3 0 0 0 0 Lactose 0 0 1 . 6 1 . 6 1 . 6 2 . 7 Urea 1 . 3 4 . 0 0 0 0 0 Sodium acetate 8 . 0 ^ ) 8 . 0 8 . 0 8 . 0 8 . 0 8 . 0 N H 4 C 1 6 . 7 2 6 , . 7 3 0 . 7 1 3 . 3 2 1 . 3 - 2 5 . 3 K H 2 P 0 4 3 . 1 3 . 1 3 . 1 2 . 0 2 . 4 2. .4 Calcium sulphate 0 . 5 0 . 5 0 . 5 ( 2 > 0 . 4 0 . 4 0 . 4 Magnesium sulphate 0 . 3 0 . . 4 0 . 4 0 . . 4 ( 3 ) 0 . 4 0 . 4 Trace mineral soln. 3 . 3 4 . 0 4 . 0 3 . 7 3 . 7 3 . 7 A l l v a l u e s a r e i n g r a m s , e x c e p t t r a c e m i n e r a l s o l u t i o n w h i c h i s i n m i l l i l i t r e s T h e f e e d w a s d i l u t e d i n 1 L o f t a p w a t e r (1) S t a r t e d o n 2 1 / 1 0 / 8 3 (2) R e p l a c e d w i t h C a C l 2 . H 2 0 f r o m . 2 6 / 2 / 8 4 o n w a r d s (3) R e p l a c e d w i t h M g C l 2 . H 2 0 f r o m 1 8 / 4 / 8 4 o n w a r d s T r a c e m i n e r a l s o l u t i o n c o m p o s i t i o n : To 1 L o f w a t e r , a d d , i n g r a m s : 1 9 . 5 F e C l 3 , 4 . 1 M n S 0 4 . H 2 0 , 3 . 3 Z n C l 2 , 2 . 1 C u C l 2 . 2 H 2 0 , 2 . 9 C o C l 2 . H 2 0 , 2 . 9 N a 2 M o 0 4 . 2 H 2 0 , 177 N a 3 c i t r a t e , 1 . 2 N a 2 B 4 O 7 . 1 0 H 2 O 56 T a b l e 7 - S t r e n g t h a n d C h a r a c t e r i s t i c s o f D i l u t e d S y n t h e t i c F e e d a n d L a n d f i l l L e a c h a t e D a y s COD TKN NH4-N p H ( C u m u l a t i v e ) ( m g / L ) ( m g / L ) ( m g / L ) D i l u t e d S y n t h e t i c F e e d R u n 1 0 - 15 16 - 4 1 R u n 2 0 - 17 18 - 23 24 - 5 1 5 1 - 8 0 8 1 - 147 R u n 3 0 - 1 3 R u n 4 0 - 38 39 - 44 4 5 - 98 R u n 5 0 - 29 34 - 76 L a n d f i l l L e a c h a t e R u n 6 0 - 44 R u n 7 0 - 3 5 9 150 - 180 300 - 350 3 5 0 - 4 0 0 3 5 0 2 2 0 - 2 4 0 3 1 0 6 0 0 6 0 0 6 0 0 6 0 0 7 0 0 120 250 120 120 120 120 250 1 3 5 1 3 0 130 - 1 8 5 1 8 5 1 8 5 2 1 0 100 200 100 100 100 100 200 85 85 8 5 - 140 1 4 0 1 4 0 1 6 5 n e g l i g i b l e n e g l i g i b l e 2 6 0 2 5 5 2 5 5 - 2 8 5 7.6 - 7.7 6.8 - 7.2 7.7 - 7.8 7.8 v a r i a b l e v a r i a b l e v a r i a b l e 8 . 8 - 9 . 0 8 . 9 - ' 9 . 0 8 . 5 - 9 . 1 7 . 0 - 7 . 2 7.0 - 7.2 * F r o m D a y 38 o n w a r d s * * S u p p l e m e n t a l c a r b o n a n d p h o s p h o r u s a d d e d d i r e c t l y t o s y s t e m NOTE: R u n 7 f e e d w a s s u p p l e m e n t e d w i t h 7 . 5 g a n h y d r o u s ammonium c h l o r i d e / 1 9 . 5 L l e a c h a t e 57 r a i s e o r d r o p t h e pH a s d e s i r e d ) . T h e q u a n t i t y o f s o d i u m b i c a r - ' b o n a t e a d d e d r a n g e d b e t w e e n 1 . 2 a n d 3 . 5 g / L o f f e e d , w h i l e t h e q u a n t i t y Of s o d i u m h y d r o x i d e r a n g e d b e t w e e n 0 . 1 5 a n d 1.7 g / L o f f e e d . L e a c h a t e F e e d f o r R u n s 6 a n d 7 c o n s i s t e d o f m u n i c i p a l l e a c h a t e c o l -l e c t e d e v e r y t w o w e e k s f r o m t h e 15 h e c t a r e P o r t M a n n l a n d f i l l , l o c a t e d i n S u r r e y , B . C . o n a n o l d f l o o d p l a i n o f t h e F r a s e r R i v e r . F o u r w e l l s , o n t h e n o r t h e r n p e r i p h e r y o f t h e s i t e , a r e u s e d f o r i n t e r c e p t i n g l e a c h a t e , w h i c h i s t h e n p u m p e d t o t h e G r e a t e r V a n c o u v e r S e w e r a g e a n d D r a i n a g e D i s t r i c t s e w e r a g e s y s t e m . B a s i c c h a r a c t e r i s t i c s o f t h i s l e a c h a t e a r e p r e s e n t e d i n T a b l e 7 . M o r e c o m p r e h e n s i v e c h a r a c t e r i z a t i o n o f t h i s w a s t e c a n b e f o u n d e l s e -w h e r e ( J a s p e r e t a l . , 1 9 8 6 ; D e d h a r a n d M a v i n i c , 1 9 8 5 ) . . L e a c h a t e w a s i n i t i a l l y c o l l e c t e d f r o m w e l l #3 a t t h e l a n d f i l l s i t e ; t h e d i s c h a r g e l i n e f r o m t h e w e l l w a s p r o v i d e d w i t h a t e e f o r s a m p l e c o l l e c t i o n a n d w a s u s e d i n p r e v i o u s r e s e a r c h a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . I n t h e e a r l y s t a g e s o f R u n 7 , t h e w a l I s o f t h e w e l l s e e m t o h a v e c o l l a p s e d a s l e a c h a t e d i s c h a r g e s t o p p e d , a n d e f f o r t s b y P e r s o n n e l f r o m t h e S u r r e y W o r k s D e p a r t m e n t t o r a i s e t h e s u b m e r s i b l e pump f r o m t h e w e l l p r o v e d u n s u c c e s s f u l . A s a r e s u l t , w e l l #3 w a s a b a n d o n e d a n d w e l l # 2 , l o c a t e d a b o u t 2 0 0 m f r o m w e l l # 3 , w a s u s e d f o r l e a c h a t e c o l l e c t i o n f o r t h e r e m a i n d e r o f t h e r u n . T h e a m m o n i u m c o n t e n t o f w e l l #2 w a s a b o u t 1 0 0 mg N/L l o w e r t h a n t h a t o f w e l l #3 a n d i t b e c a m e n e c e s s a r y t o s u p p l e m e n t t h e f e e d w i t h 7 . 5 g o f ammonium c h l o r i d e / 1 9 . 5 L o f l e a c h a t e t o m a k e u p f o r i t s l o w e r a m m o n i u m c o n t e n t . Ammonium a c c o u n t e d f o r 58 about 95 to 98% of the TKN content of the feed from both w e l l s . The l e a c h a t e from both w e l l s was d e f i c i e n t i n organic carbon and phosphorus. Supplemental carbon was added during Runs 6 and 7. The c a r b o n was added d i r e c t l y t o one of the c e l l s , u s u a l l y an anaerobic one. The carbon source c o n s i s t e d of methanol (7 ml/day) and sodium a c e t a t e (8 g/day) d i l u t e d i n water and pumped at the r a t e of 170 ml/day (COD of about 14,000 mg/d). A minor q u a n t i t y of anhydrous g l u c o s e was a l s o i n c o r p o r a t e d i n the feed during the e a r l y stages of Run 6. Supplemental phosphorus, at the r a t e of about 170 mg P/day, was added d i r e c t l y to the carbon feed during Runs 6 and 7. The pH of the system d u r i n g Runs 6 and 7 was c o n t r o l l e d by the a d d i t i o n of 0.5 to 0.75 N sodium hydroxide s o l u t i o n d i r e c t l y to C e l l 1. The a c t u a l amount of sodium hydroxide a d d i t i o n ranged between 0.4 and 1.5 g/L of feed. I t was pumped f o r f i f t e e n minutes per hour, using a timer c o n t r o l mechanism. As a r e s u l t , the pH l e v e l i n C e l l 1 v a r i e d by about h a l f a u n i t per c y c l e ; i t was h i g h e s t a t the end of the pump c y c l e and l o w e s t p r i o r t o resumption of pumping. The v a r i a t i o n i n the pH l e v e l of the other c e l l s was much l e s s pronounced. ANALYTICAL AND SAMPLING TECHNIQUES The m a j o r i t y of t e s t s were c a r r i e d out i n g e n e r a l accordance with Standard Methods (15th E d i t i o n , 1980). Exceptions and c l a r i -f i c a t i o n s are noted hereunder. A l k a l i n i t y The a l k a l i n i t y p H - t i t r a t i o n end-point was set at 3.7 ( i n -stead of 4.5), s i n c e i t corresponded to the i n f l e c t i o n p o i n t on 59 t h e t i t r a t i o n c u r v e f o r b o t h t h e s y n t h t e t i c f e e d a n d M L S S . A m m o n i a N i t r o g e n A m m o n i a n i t r o g e n w a s a n a l y z e d b y t i t r a t i o n , f o l l o w i n g d i s -t i l l a t i o n . T e n d r o p s o f a s i l i c o n a n t i f o a m a g e n t (Dow C o r n i n g a n t i f o a m B e m u l s i o n ) w a s a d d e d t o a l l s a m p l e s p r i o r t o d i s t i l l a -t i o n . N i t r a t e N i t r o g e n N i t r a t e - n i t r o g e n was a n a l y z e d b y t h e c a d m i u m r e d u c t i o n m e t h -o d , u s i n g a T e c h n i c o n A u t o A n a l y z e r I I . C o l u m n r e d u c t i o n e f f i -c i e n c y w a s c o n t i n u o u s l y m o n i t o r e d d u r i n g e a c h t e s t b y c o m p a r i n g t h e e f f i c i e n c y o f n i t r a t e s t a n d a r d s w i t h n i t r i t e s t a n d a r d s . A c o r r e c t i o n f a c t o r w a s t h e n a p p l i e d t o t h e n i t r a t e v a l u e s o b t a i n e d , t o a c c o u n t f o r t h e n i t r i t e p r e s e n t i n t h e s a m p l e s . C o r r e c t i o n s w e r e n o t n e e d e d w h e n t h e s a m p l e c o n t a i n e d n o n i t r i t e . N i t r i t e N i t r o g e n N i t r i t e - n i t r o g e n w a s a n a l y z e d u s i n g a T e c h n i c o n A u t o A n a l y -z e r I I w i t h o u t a c a d m i u m r e d u c t i o n c o l u m n . T o t a l K j e l d a h l N i t r o g e n T o t a l K j e l d a h l n i t r o g e n (TKN) w a s a n a l y z e d o n t h e T e c h n i c o n A u t o A n a l y z e r I I u s i n g t h e c o l o r i m e t r i c , s e m i - a u t o m a t e d b l o c k d i g e s t e r m e t h o d ( E . P . A . , 1 9 7 9 ) . M e r c u r i c s u l p h a t e w a s n o t a d d e d d u r i n g t h e d i g e s t i o n s t e p . D u p l i c a t e s a m p l e s w e r e d i g e s t e d . E a c h s a m p l e w a s a n a l y z e d i n t r i p l i c a t e . C h e m i c a l O x y g e n d e m a n d C h e m i c a l O x y g e n D e m a n d (COD) w a s a n a l y z e d b y t h e d i c h r o m a t e 60 r e f l u x method. Sample s i z e was 20.0 ml and mercuric sulphate was added to reduce c h l o r i d e i n t e r f e r e n c e . N i t r i t e was accounted f o r by s u b t r a c t i n g 1.1 mg COD/mg NO^-N present i n the sample. Samples were analyzed i n d u p l i c a t e . D i s s o l v e d Oxygen D i s s o l v e d oxygen (DO) was measured using a YSI 5739 submer-s i b l e probe, with a high s e n s i t i v i t y membrane, connected to a YSI model 54 DO meter. Oxi d a t i o n Reduction P o t e n t i a l O x i d a t i o n Reduction P o t e n t i a l (ORP) was measured using a s i l v e r - s i l v e r c h l o r i d e , g e l - f i l l e d combination r e f e r e n c e e l e c -trode (Broadley-James C o r p o r a t i o n ) , connected to a C o l e Palmer high-impedance d i g i t a l v o l t m e t e r with a s e n s i t i v i t y i n the m i l l i -v o l t range. The ORP p o t e n t i a l measured i s : ORP = E h - E Q (13) where En = hydrogen e l e c t r o d e p o t e n t i a l E Q = standard p o t e n t i a l (reference) Suspended S o l i d s Suspended s o l i d s were measured using 934-AH Whatman g l a s s m i c r o f i b r e f i l t e r s . A l l samples were analyzed i n d u p l i c a t e . EH pH measurements were made using a permanently g e l - f i l l e d d o u b l e - j u n c t i o n , s i l v e r c h l o r i d e r e f e r e n c e e l e c t r o d e (Orion or F i s h e r Dura probe) and a F i s h e r Model 210 pH meter. 61 Nitrogen Content of Biomass The n i t r o g e n content of the MLVSS was determined by c o l l e c t -ing a s u f f i c i e n t volume of MLVSS and undertaking a suspended s o l i d s a n a l y s i s with a p o r t i o n of the sample. The remainder was f i l t e r e d through a m i c r o f i b r e f i l t e r and the contents separated from the f i l t e r and d r i e d at 104°C f o r one hour. The dry sample was then ground and homogenized and p a r t of the sample was a c i d d i g e s t e d i n d u p l i c a t e f o r TKN a n a l y s i s . Sampling Procedures With the exception of TKN and e f f l u e n t suspended s o l i d s , a l l samples were analyzed immediately a f t e r c o l l e c t i o n . The procedure f o r sample p r e p a r a t i o n and sample p r e s e r v a t i o n techniques are presented i n Tab l e 8. E f f l u e n t samples were c o l l e c t e d d a i l y from the contents of the e f f l u e n t c o n t a i n e r ( f o l l o w i n g thorough shaking) f o r purposes of TSS a n a l y s i s . A composite of these d a i l y samples was analyzed two to three times weekly. Samples f o r MLSS/MLVSS a n a l y s i s were c o l l e c t e d on the day of a n a l y s i s , i n equal volumes, from a l l c e l l s i n the system. The d i s c r e t e samples were combined i n t o a s i n g l e composite sample f o r a n a l y s i s . A comparison between the s o l i d s l e v e l s i n each r e a c t o r , undertaken during the shakedown p e r i o d p r i o r to s t a r t - u p of the study, showed a + 10% v a r i a t i o n from the o v e r a l l average. P r e s e n t a t i o n of Data Data from a l l phases of t h i s p r o j e c t are summarized and presented i n Ta b l e s 10, 11 and 12, and i n Appendices A and B. 62 Table 8 — Sample Handling Test F i l t r a t i o n ^ Storage P r e s e r v a t i v e N i t r a t e yes no phenyl mercuric acetate N i t r i t e yes no phenyl mercuric acetate Ammoni a no no none E f f l u e n t TKN yes up to 1 month phenyl mercuric acetate at 4°C Feed TKN yes up to 1 month cone. H 2S0 4 @ 4°C COD yes no none A l k a l i n i t y no no none E f f l u e n t S.S. yes ( 2) up to 1 week 4°C MLSS/MLVSS yes<2) no none (1) Whatman No. 4 f i l t e r (2) Whatman No. 934-AH g l a s s m i c r o f i b r e f i l t e r 63 CHAPTER FIVE RESULTS T h e r e s u l t s a r e p r e s e n t e d f o r e a c h r u n s e p a r a t e l y , i n o r d e r t o p r o v i d e a n i n s i g h t a s t o t h e p r o g r e s s i o n o f t h e r e s e a r c h p r o g r a m . To t h i s e n d , t h e o b j e c t i v e s o f e a c h r u n , a s i d e n t i f i e d a t t h e t i m e , w i l l b e o u t l i n e d p r i o r t o t h e p r e s e n t a t i o n o f t h e r e s u l t s . T h i s w i l l b e f o l l o w e d b y a b r i e f d i s c u s s i o n a n d c o n c l u -s i o n s e c t i o n , a s i t p e r t a i n s t o t h e p r o g r e s s i o n o f t h e r e s e a r c h p r o g r a m . T h e r e s u l t s o f t w o b a t c h t e s t s , u n d e r t a k e n d u r i n g R u n s 4 a n d 5 , a r e p r e s e n t e d a t t h e e n d o f t h i s C h a p t e r . A c o m p r e h e n s i v e d i s c u s s i o n o f t h e r e s u l t s i s p r e s e n t e d i n C h a p t e r 6 . T h e p a r a m e -t e r s i n v e s t i g a t e d d u r i n g e a c h r u n a r e s u m m a r i z e d i n T a b l e 9 a n d t h e r e a d e r i s a d v i s e d - t o c o n s u l t i t w h i l e r e a d i n g t h i s c h a p t e r , i n o r d e r t o m a i n t a i n a f o c u s o n t h e v a r i o u s p a r a m e t e r s d i s c u s s e d . F o r c l a r i t y , a n d t o m a i n t a i n c o n t i n u i t y , r e s u l t s w h i c h a r e i n c i d e n t a l t o t h e m a i n t h r u s t o f t h e r e s e a r c h p r o g r a m ( e . g . , COD c o n s u m p t i o n r a t e s , d e n i t r i f i c a t i o n r a t e s , o c c u r r e n c e o f a e r o b i c d e n i t r i f i c a t i o n , a l k a l i n i t y c o n s u m p t i o n a n d p r o d u c t i o n v a l u e s , n i t r i f i c a t i o n r a t e s a n d O R P / n i t r a t e r e l a t i o n s h i p s ) h a v e n o t b e e n i n c l u d e d i n t h i s c h a p t e r . T h e y a r e p r e s e n t e d i n C h a p t e r 6 u n d e r t h e a p p r o p r i a t e h e a d i n g s a n d a r e a l s o s u m m a r i z e d i n T a b l e 9 . T h e o p e r a t i o n a l c h a r a c t e r i s t i c s a n d d a t a f o r e a c h r u n ( w i t h t h e e x c e p t i o n o f some p a r a m e t e r s o f R u n 1 , a s e x p l a i n e d l a t e r ) a r e p r e s e n t e d i n T a b l e s 1 0 , 11 a n d 1 2 , a n d i n A p p e n d i x A . 64 T a b l e 9 - S u m m a r y o f P a r a m e t e r s I n v e s t i g a t e d R u n D u r a t i o n M a j o r P a r a m e t e r s O t h e r P a r a m e t e r s N o . ( d a y s ) I n v e s t i g a t e d I n v e s t i g a t e d * 5 5 l o w DO e f f e c t o f N H 3 i n t e r m i t t e n t c o n t a c t t o NH: 147 3 4 13 98 76 l o w DO a n a e r o b i o s i s N H 3 l e v e l s a n a e r o b i o s i s + N H 3 h i g h H N O , i n t e r m i t t e n t c o n t a c t t o N H 3 d u r a t i o n o f a e r a t i o n t i m e h i g h H N 0 2 l o n g SRT s h o r t SRT h i g h N H 3 e x t e n s i o n o f c o n t a c t t i m e t o N H 3 r e d u c t i o n o f a e r a t i o n t i m e i n t e r n a l r e c y c l e i n t e r m e d i a r y d e n i t r i f i c a t i o n a b s e n c e o f n i t r i t e i n e f f l u e n t a l k a l i n i t y a e r o b i c d e n i t r i f i c a t i o n COD c o n s u m p t i o n t o x i c i t y o f n i t r i t e N.R. v s . DO N.R. v s . pH ORP v s . NO; COD c o n s u m p t i o n a e r o b i c d e n i t r i f i c a t i o n ORP v s . NO; N - n i t r o s a m i n e s a e r o b i c d e n i t r i f i c a t i o n 44 c o m p l e x w a s t e ( l a n d f i l l l e a c h a t e ) d o u b l e s u b s t r a t e i n h i b i t i o n ( N H 3 + N a C l 0 3 ) i n c o m p l e t e d e n i t r i f i c a t i o n 7 3 5 9 i n t e r m e d i a r y d e n i t r i f i c a t i o n h i g h N H 3 l o w NHo c o m p l e x w a s t e ( l a n d f i l l l e a c h a t e ) t e m p o r a r y s t o p p a g e o f f e e d a b s e n c e o f n i t r i t e i n e f f l u e n t l o n g t e r m s t a b i l i t y o f a c c l i m a t e d b i o m a s s B a t c h T e s t N o . l d u r a t i o n o f a e r a t i o n t i m e e x t e n s i o n o f c o n t a c t t i m e t o N H 3 v a r i a b l e N H 3 l e v e l s n i t r i t e d i s a p p e a r a n c e r a t e s * a d h e r e n c e o f n i t r i f i e r s t o c e l l w a l l s * i n c o m p l e t e d e n i t r i f i c a t i o n B a t c h T e s t N o . 2 COD c o n s u m p t i o n r a t e s * a n a e r o b i c g r o w t h y i e l d * d e n i t r i f i c a t i o n r a t e s * t o x i c i t y o f n i t r i t e * N . R . : n i t r i f i c a t i o n r a t e s p a r a m e t e r s d i s c u s s e d i n C h a p t e r 6 65 T a b l e 10 - O p e r a t i o n a l C h a r a c t e r i s t i c s o f E a c h R u n R u n S y s t e m P e r i o d n F e e d R e c y c l e A c t u a l A n a e r o b i c N o . N o . F l o w F l o w H R T / C e l l C e l l ( s ) ( d a y s ) ( L / d ) ( L / d ) ( h o u r s ) 1 1 1 _ 36 10 20 2 . 0 1 1 1 37 - 4 3 - 10 30 1 . 5 1 1 1 44 - 5 5 — 10 20 2 . 0 1 1 2 1 _ 4 5 _ 10 5 4 . 0 n o n e 1 2 46 - 55 - 10 10 3 . 0 1 2 1 1 75 18 9 . 7 + 0 . 7 1 9 . 4 + 0 . 7 2 . 0 v a r . 2 1 76 — .147 26 9 . 9 + 0 . 4 1 0 . 8 + 0 . 5 3 . 0 1 2 2 1 _ 7 5 18 9 . 7 + 0 . 7 1 9 . 5 + 1 . 0 2 . 0 n o n e 2 2 76 - 147 28 9 . 3 + 0 . 6 10. . 6 + 0 . 9 3 . 0 1 3 N/A .1 - 13 3 20 1 4 1 . 7 n o n e 4 1 1 _ 32 15 1 0 . 0 + 0 . 7 1 0 . 5 + 0 . 7 3 . 0 1 4 1 33 - 67 15 1 0 . 0 + 0 . 7 2 0 . 1 + 1 . 3 2 . 0 1 + 2 4 1 67 8 1 6 1 0 . 0 + 0 . 7 1 0 . 0 + 0 . 8 3 . 0 1 + 2 4 1 82 - 98 5 1 0 . 0 + 0 . 7 2 2 . 0 + 0 . 3 2 . 0 .1 4 .2 1 - 98 43 9 . 9 + 0 . 6 '10-. 5 + 0 . 8 3 . 0 1 •+ 2 ( 1 ) 5 1 1 — 6 2 16 1 0 . 5 + 0 . 7 2 6 . 8 + 3 . 0 1 . 6 1 5 2 1 20 3 10 . 3 + 0 . 5 2 3 . 6 1 . 8 1 5 2 20 - 76 16 1 0 . 3 + 0 . 5 (2) 1 . 6 ( 3 ) 1 ( 4 ) 6 N/A 1 - 4 1 14 "19.. 9 + 0 . 2 19 . 0 + 0 . 3 1 . 5 1 + 7 7 N/A 1 _ 3 3 8 9 1 1 6 . 3 + 0 . 6 1 3 . 7 + 1 . 6 2 . 0 1 + 3 ( 5 ) n : n u m b e r o f m e a s u r e m e n t s (1) C e l l .2 c o n v e r t e d t o a n a e r o b i c o n D a y 67 (2) i n t e r n a l r e c y c l e f 1 ow b e t w e e n c e 1 1 s 2 a n d 1 = 2 7 . 8 + 2 . 9 L / d a n d c l a r i f i e r r e c y c l e f l o w = 2 . 2 + 0 . 1 L / d (3) A c t u a l HRT f o r c e l l s 3 a n d 4 = 4 . 9 h o u r s (4) C e l l 3 c o n v e r t e d t o a n a e r o b i c o n D a y 53 (5) C e l l 1 c o n v e r t e d t o a n a e r o b i c o n D a y 28 a n d C e l l 3 o n D a y 76 66 Table 11 - Sludge Age C h a r a c t e r i s t i c s Run System Pe r i o d n Aer o b i c s T o t a l s NO. No. SRT SRT (days) (days) (days) 2 1 4 75 12 10.5 3.5 15.8 6.7 2 1 76 - 147 10 9.9 1.4 13.1 1.9 2 2 4 - 75 10 11.3 4.8 11.3 4 .8 2 2 76 — 147 10 11.9 1.5 15.8 2.0 3 N/A 1 - 13 3 9.1 — 9.1 -4 1 19 - 48 13 22.2 7.1 36.4 8.5 4 1 49 - 52 2 10.5 - 20.9 -4 1 53 — 98 15 3.8 1.9 6.4 2.4 4 2 17 - 48 14 28.2 6.3 3 7.6 8.4 •4 2 49 - 52 2 14.6 — 19.4 -4 2 53 — 98 15 5.4 4.3 9.2 5.4 5 1 1 - 62 17 7.6 1.0 10.1 1.3 5 2 1 - 76 21 7.0 1.2 10.5 1.3 6 N/A 3 — 19 5 9.4 0.8 12.5 1.1 6 N/A 20 - 39 7 14 .9 2.3 19.9 3.1 7 N/A 24 - 286 74 12.5 2.6 18.7 3.9 7 N/A 287 - 321 9 7.1 1.8 10 .7 2.7 7 N/A 322 338 6 11.3 1.8 16 .9 .2 .7 s: standard d e v i a t i o n n: number of measurements * From Day 29 onwards (n=9) 67 Table 12 - Ammonia Level in F i r s t C e l l Run System Period n Average s Average s NO. No. (days) mg NH3-N/L mg NH^-N/L 2 1 1 - 28 7 0.26 0.09 25 6 2 1 29 - 37 2 0.12 - 11 — 2 1 38 - 75 8 0.92 0.14 43 7 2 1 76 - 147 27 2.00 0.92 100 7 2 2 1 — 75 17 0.20 0.10 19 7 2 2 76 - 136 23 5.74 1.34 102 18 2 2 137 - 147 4 2.59 0.63 100 7 3 N/A 3 1 13..9 86 _ 3 N/A 4 — 13 4 0,38 - 45 -4 1 3 _ 5 3 3.5 65 4 1 7 - 8 2 13.1 - 98 -4 1 9 — 12 4 3.3 - 99 -4 1 13 — 67 25 4.5 1.6 51 .12 4 1 68 - 81 6 7.3 1.5 88 9 4 1 82 - 98 5 7.8 1.3 48 3 4 2 3 - 8 7 4.8 2.3 64 9 4 2 9 - 12 4 3.2 — 99 — 4 2 13 — 39 13 4.9 1.4 50 7 •4 2 40 - 98 22 11.4 4.8 84 14 5 1 10 20 4 8.5 3 .2 113 29 5 1 .21 — 62 13 5.2 1.1 44 5 5 2 10 20 4 7 .9 1.1 93 24 5 .2 !21 — 76 20 6.6 1.6 49 9 6 N/A 1 _ 8 6 4.8 2.7 88 14 6 N/A 9 - 22 8 18.8 7.3 191 33 6 N/A 23 - 38 7 8.6 3. 8 145 41 7 N/A 21 30 5 1.1 0.6 119 21 7 N/A 31 - 70 10 19.1 3.3 127 12 7 N/A 7.1 - 91 .5 10.6 1.4 156 4 7 N/A 92 - 122 9 12.7 8.3 * 16 2 9 7 N/A 123 - 153 9 17 .4 7.2 * 147 8 7 N/A 158 - 227 21 7 .7 3.9 .139 8 7 N/A 228 - 248 6 25.5 8.4 149 4 7 N/A 249 - 282 10 0.9 0.1 149 7 7 N/A 283 - 286 1 7.4 - 155 -7 N/A 287 — 317 9 1.0 0.4 78 25 7 N/A 318 - 338 6 12.9 5.7 * 134 7 7 N/A 339 - 350 ** - 0.0 — 0 — 7 N/A 351 - 356 2 0.5 — 118 — 7 N/A 3 57 - 359 2 8.8 - 138 — n: number of samples s: standard deviation * Wide fluctuation in pH levels ** Feed stopped during this period 68 RUN 1 O b j e c t i v e s T h e o b j e c t i v e s o f t h i s r u n w e r e t o : 1) C o n f i r m t h e f i n d i n g s o f p r e v i o u s i n v e s t i g a t o r s , l i n k i n g n i t r i t e b u i l d - u p t o t h e a c t i o n o f f r e e a m m o n i a a s a d i f f e r e n t i a l i n h i b i t o r o f n i t r i f i c a t i o n a c t i v i t y i n t h e c o n c e n t r a t i o n r a n g e o f 0 . 1 t o 1 mg NH3 - N / L . 2) D e t e r m i n e i f t h e i n h i b i t o r y f r e e a m m o n i a l e v e l n e e d b e m a i n -t a i n e d a c r o s s t h e e n t i r e s y s t e m i n o r d e r t o s u s t a i n n i t r i t e b u i l d - u p . . 3) B a s e d u p o n t h e r e l a t i v e l y h i g h o x y g e n h a l f - s a t u r a t i o n c o e f f i -c i e n t r e p o r t e d i n t h e l i t e r a t u r e f o r t h e n i t r i t e o x i d i z e r s , d e t e r m i n e i f l o w DO l e v e l s ( b e 1 ow 1 . 0 m g / L ) c a n b e u s e d a s a m e c h a n i s m t o s e l e c t i v e l y r e d u c e t h e g r o w t h o f t h e n i t r i t e o x i d i z e r s , t h u s l e a d i n g t o n i t r i t e a c c u m u l a t i o n . R e s u l t s T h i s r u n c o m m e n c e d o n S e p t e m b e r 1 3 , 1 9 8 3 a n d l a s t e d f o r 5 5 d a y s . I t f o l l o w e d a 4 0 - d a y o p e r a t i o n a l s h a k e d o w n o f t h e e x p e r i -m e n t a l s e t - u p . To g a i n m a x i m u m i n s i g h t a s t o t h e p a r a m e t e r s t h a t c o u l d i n d u c e a n d s u s t a i n n i t r i t e a c c u m u l a t i o n , t h e t w o s y s t e m s w e r e o p e r a t e d u n d e r d i f f e r e n t m o d e s . S y s t e m 1 w a s o p e r a t e d i n a p r e - d e n i t r i f i c a t i o n mode b y m a i n t a i n i n g t h e f i r s t c e l l a n a e r o b i c . T h e DO l e v e l i n t h e t h r e e r e m a i n i n g a e r o b i c c e l l s w a s k e p t h i g h , g e n e r a l l y a b o v e 3 m g / L . T h e r e c y c l e r a t e w a s s e t a t t w i c e f e e d f l o w i n o r d e r t o d i l u t e t h e a m m o n i u m l e v e l s w i t h i n t h e a n a e r o b i c c e l l . DO l e v e l s m a i n -t a i n e d d u r i n g t h e r u n a r e p r e s e n t e d i n T a b l e 1 3 . 69 Table 13 - D i s s o l v e d Oxygen L e v e l s i n Runs 1 and 2 System Period (days) Aero b i c C e l l No. n Mean DI Ql Median Q3 D9 Run 1 1 20 - 55 1 26 3.68 2.2 3.0 3.6 4.4 5.1 1 20 - 55 2 26 3.43 1.1 2.1 3.1 4.6 6 .2 1 20 - 55 3 26 4.88 2.4 4.0 4.6 6.0 6.6 2 20 - 55 1 65 0.75 0.35 0 .45 0.60 0.80 1.3 2 20 - 55 2 65 0.80 0.40 0.50 0.60 1.00 1.4 2 20 - 55 3 65 0.69 0.40 0.45 0.50 0.70 1.3 2 20 - 55 4 53 1.27 0.25 0.30 0.45 1.15 4.6 Run 2 1 1 - 36 1 36 1.15 0.55 0.70 1.0 1.5 1.9 1 37 - 119 1 35 1.89 0.80 0.95 1..7 2 .2 3.3 2 1 - 73 1 52 1.54 0.95 1.10 1.4 1.7 2.7 2 74 - 119 .1 20 1.54 0.80 0.90 1.4 2.0 2.3 n: number of readings DI: low d e c i l e QL: low q u a r t i l e Q3: high q u a r t i l e D9: high d e c i l e \ 70 S y s t e m 2 w a s o p e r a t e d i n a f u l l y a e r o b i c m o d e a n d t h e DO l e v e l w a s g e n e r a l l y m a i n t a i n e d b e l o w 0 . 6 m g / L i n a l l c e l l s . T h e r e c y c l e r a t e w a s s e t a t h a l f t h e f e e d f l o w i n o r d e r t o m a i n t a i n h i g h a m m o n i u m l e v e l s i n t h e f i r s t c e l l . T o t a l i n h i b i t i o n o f n i t r i f i c a t i o n a c t i v i t y m a n i f e s t e d i t s e l f i n b o t h s y s t e m s u p o n s t a r t - u p . T h i s w a s a t t r i b u t e d t o i n s u f f i -c i e n t a c c l i m a t i o n t i m e t o t h e h i g h ammonium c o n t e n t o f t h e f e e d ( a b o u t 2 1 5 mg N H ^ - N / L a n d 2 5 0 mg T K N / L ) . U p o n f a i l u r e o f n i t r i f i -c a t i o n a c t i v i t y t o e s t a b l i s h i t s e l f w i t h i n t w o w e e k s , h a l f t h e b i o m a s s o f e a c h s y s t e m w a s r e p l a c e d w i t h f r e s h s l u d g e a n d t h e f e e d c o n c e n t r a t i o n w a s h a l v e d . T h i s l e d t o a r a p i d r i s e i n n i t r i -f i c a t i o n a c t i v i t y a n d t h e f e e d c o n c e n t r a t i o n w a s g r a d u a l l y i n -c r e a s e d t o i t s o r i g i n a l l e v e l o v e r a t w o w e e k p e r i o d . T h e a e r o b i c SRT o f b o t h s y s t e m s w a s m a i n t a i n e d a t a r o u n d 20 d a y s . N i t r i t e b u i l d - u p o c c u r r e d i n S y s t e m 1 i m m e d i a t e l y u p o n e s -t a b l i s h m e n t o f n i t r i f i c a t i o n a c t i v i t y . T h e a c c u m u l a t i o n l a s t e d f o r t h e r e m a i n d e r o f t h e r u n . E f f l u e n t n i t r i t e l e v e l s r a n g e d b e t w e e n 80 a n d 1 5 0 mg N O ^ - N / L a n d r e p r e s e n t e d b e t w e e n 5 5 a n d 80% o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t . I n S y s t e m . 2 , o n t h e o t h e r h a n d , n i t r i t e w a s v i r t u a l l y a b s e n t f r o m t h e e f f l u e n t a n d p r e s e n t i n c o n c e n t r a t i o n s b e l o w 5 mg N O ^ - N / L i n t h e f i r s t t w o a e r o b i c c e l l s . T h r o u g h o u t t h e r u n , t h e f r e e a m m o n i a l e v e l w a s h i g h e s t i n t h e f i r s t c e l l o f e a c h s y s t e m . I t r a n g e d b e t w e e n 2 a n d 5 mg NH3-N / L i n S y s t e m 1 , a n d 0 . 5 t o 3 mg NH3 - N / L i n S y s t e m 2 . D u r i n g t h e f i r s t t e n d a y s , f o l l o w i n g t h e e s t a b l i s h m e n t o f n i t r i f i c a t i o n a c t i v i t y , t h e f r e e a m m o n i a l e v e l i n t h e f i r s t c e l l o f S y s t e m 1 c o n s i s t e n t l y e x c e e d e d 4 mg NH3 - N / L , w h i l e i n S y s t e m 2 i t r e m a i n e d 71 b e l o w 1 mg NH3 - N / L . I t s h o u l d b e p o i n t e d o u t t h a t t h e d a t a c o l -l e c t e d d u r i n g t h i s r u n w a s i n c o m p l e t e , e s p e c i a l l y i n t e r m s o f pH m e a s u r e m e n t s . B a s e d u p o n t h e r e s u l t s o f s u b s e q u e n t r u n s , i t i s s u s p e c t e d t h a t f r e e a m m o n i a l e v e l s i n t h e f i r s t c e l l o f S y s t e m 1 may h a v e e x c e e d e d 5 mg NH3-N/L a t some t i m e s . T h e f r e e a m m o n i a l e v e l i n t h e r e m a i n i n g c e l l s o f e a c h s y s t e m w a s a l w a y s l o w e r t h a n i n t h e f i r s t c e l l ; i t r a n g e d b e t w e e n 0 . 2 a n d 0 . 9 mg NH3-N/L f o r t h e s e c o n d c e l l o f S y s t e m 1 a n d 0 . 3 t o 3 . 4 mg NH3-N/L i n t h e s e c o n d c e l l o f S y s t e m 2 . T h e f r e e a m m o n i a l e v e l i n t h e r e m a i n i n g a e r o b i c c e l l r a n g e d b e t w e e n 0 . 0 a n d 0 . 1 mg NH3 - N / L . B y D a y 3 5 f u l l n i t r i f i c a t i o n w a s a c h i e v e d i n b o t h s y s t e m s , a s d e t e r m i n e d b y t h e a b s e n c e o f a n y r e s i d u a l a m m o n i u m i n t h e " l a s t t w o c e l l s o f e a c h s y s t e m . I n o r d e r t o i n d u c e n i t r i t e b u i l d - u p i n S y s t e m 2 , t h e a e r o b i c SRT w a s r e d u c e d o n D a y 35 f r o m 20 t o 1 0 d a y s . A s i m i l a r r e d u c t i o n i n SRT w a s i m p l e m e n t e d o n S y s t e m 1 . T h i s d i d n o t l e a d t o a r i s e i n n i t r i t e l e v e l s i n S y s t e m 2 . On D a y 4 1 , t h e a e r o b i c SRT i n b o t h s y s t e m s w a s f u r t h e r r e d u c e d t o a r o u n d 5 d a y s . T h i s l e d t o a r i s e i n n i t r i t e l e v e l s i n S y s t e m 2 . B y D a y 4 6 , t h e e f f l u e n t o f S y s t e m 2 c o n t a i n e d a b o u t 6% n i t r i t e - N . On t h i s d a y t h e f i r s t c e l l o f S y s t e m .2 w a s c o n v e r t e d f r o m a e r o b i c t o a n a e r o b i c i n a f u r t h e r e f f o r t t o i n d u c e n i t r i t e b u i l d - u p . T h e s e c h a n g e s l e d t o a r e d u c -t i o n o f n i t r i f i c a t i o n a c t i v i t y i n S y s t e m . 2 . R e c o v e r y h a d n o t b e e n a c h i e v e d b y t h e e n d o f t h e r u n , a l t h o u g h s o m e d e g r e e o f n i t r i t e a c c u m u l a t i o n o c c u r r e d . B y D a y 5 5 , n i t r i t e a c c o u n t e d f o r a b o u t o n e t h i r d o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e e f f l u e n t o f S y s t e m 2 , w h i l e i t e x c e e d e d 70% i n S y s t e m 1 ( t h e a c t u a l p e r c e n t 7 2 could not be confirmed due to overestimation of the nitrate fraction, as explained in the Discussion). Discussion Insufficient data was collected during this run to permit a proper evaluation of the conditions that induced and maintained nitrite build-up in System 1. In addition, it was later estab-lished (during Run 2) that the cadmium reduction column used for nitrate analysis was not 100% efficient in reducing nitrate to nitrite; and as a result, the nitrate concentration was overesti-mated when nitrite was present. A correction factor had to be applied to account for the inefficiency in the cadmium reduction column. Therefore, the data collected during this run, with the exception of DO, can only be interpreted qualitatively and not quantitatively. In spite of these difficulties, a major objective was at-tained; nitrite accumulation was induced and sustained for a period of time. It remained to identify the factor(s) responsible for its occurrence. The occurrence of nitrite build-up in System 1, and not in System 2, appeared to be linked to the presence of an anaerobic c e l l at the front-end of System 1. The cause of nitr i te build-up may, therefore, have been due to: 1) Anaerobiosis alone; 2) Anaerobiosis preventing ammonia oxidation from taking place in that c e l l , leading to the establishment of higher free ammonia levels in an anaerobic c e l l (as opposed to an aerobic one). Regardless of the actual cause of inhibition, the realiza-73 t i o n t h a t a n a e r o b i o s i s m i g h t p l a y a r o l e a s a s e l e c t i v e i n h i b i t o r o f t h e n i t r i t e o x i d i z e r s f o c u s e d a t t e n t i o n o n t h e n e e d t o i n v e s -t i g a t e t h e e f f e c t s o f t h r e e o p e r a t i o n a l v a r i a b l e s l i n k e d t o t h e e s t a b l i s h m e n t o f a n a n a e r o b i c e n v i r o n m e n t . T h e s e w e r e : 1) E x p o s u r e t i m e t o a n a e r o b i o s i s ; 2) I n t e r v a l t i m e b e t w e e n s u c c e s s i v e e x p o s u r e s t o a n a e r o b i o s i s ; 3) R o l e o f f r e e a m m o n i a i n t h e a n a e r o b i c e n v i r o n m e n t . C o n c l u s i o n s Some o b s e r v a t i o n s m a d e d u r i n g t h i s p h a s e o f t h e r e s e a r c h p r o g r a m i n c l u d e d : 1) N i t r i t e a c c u m u l a t i o n c o u l d a p p a r e n t l y b e i n d u c e d b y r e l a t i v e l y s i m p l e m e a n s . 2) N i t r i t e b u i l d - u p c o u l d b e s u s t a i n e d . 3) N i t r i t e b u i l d - u p c o u l d b e a c h i e v e d w h i l e m a i n t a i n i n g a f u l l y n i t r i f i e d e f f l u e n t . 4) T h e p r e s e n c e o f a n a n a e r o b i c c e l l a p p e a r e d t o p l a y a r o l e i n t h e p h e n o m e n o n o f n i t r i t e a c c u m u l a t i o n . M o r e o v e r , n i t r i t e b u i l d - u p c o u l d b e s u s t a i n e d , i n s p i t e o f t h e v i r t u a l a b s e n c e o f f r e e a m m o n i a i n t h e l a s t t w o a e r o b i c c e l l s a n d i t s p r e s e n c e a t c o n c e n t r a t i o n s b e l o w 1 mg NH3-N/L i n t h e f i r s t a e r o b i c c e l l . 5) DO c o n c e n t r a t i o n s a s l o w a s 0 . 6 m g / L d i d n o t a p p e a r t o p l a y a n i d e n t i f i a b l e r o l e i n i n h i b i t i n g n i t r i t e o x i d a t i o n . H i g h DO l e v e l s d i d n o t p r e v e n t n i t r i t e b u i l d - u p . 6) T h e f r e e a m m o n i a l e v e l n e e d e d t o c a u s e i n h i b i t i o n o f n i t r i t e o x i d a t i o n a p p e a r e d t o b e h i g h e r t h a n r e p o r t e d i n t h e l i t e r a -t u r e ( 0 . 1 t o 1 . 0 mg NH3 - N / L ) . 74 7) T h e r e c y c l e r a t e a n d S R T m a y h a v e p l a y e d a r o l e i n n i t r i t e b u i l d - u p . RUN 2 O b j e c t i v e s T h e o b j e c t i v e s o f t h i s r u n w e r e t o c o n f i r m s o m e o f t h e r e s u l t s o b s e r v e d i n R u n 1 a n d t o i d e n t i f y t h e p a r a m e t e r s r e s p o n -s i b l e f o r n i t r i t e a c c u m u l a t i o n i n a c t i v a t e d s l u d g e : s y s t e m s . T h e m a j o r o b j e c t i v e s w e r e a s f o l l o w s : 1) D e t e r m i n e t h e f e a s i b i l i t y o f i n d u c i n g n i t r i t e b u i l d - u p i n a f u l l y a e r o b i c s y s t e m . 2) C o n f i r m t h e f i n d i n g s o f R u n 1 , r e g a r d i n g t h e v i a b i l i t y o f i n d u c i n g n i t r i t e b u i l d - u p b y m a i n t a i n i n g l o w DO l e v e l s . 3) A s s e s s t h e a b i l i t y o f a n a e r o b i o s i s , i n t h e a b s e n c e o f f r e e a m m o n i a , i n i n d u c i n g n i t r i t e b u i l d - u p . 4) C o n f i r m t h a t a n a n a e r o b i c c e l l a t t h e f r o n t - e n d o f a n a c t i -v a t e d s l u d g e s y s t e m w o u l d i n d u c e a n d s u s t a i n n i t r i t e b u i l d - u p i n t h e a b s e n c e o f i n h i b i t o r y f r e e a m m o n i a l e v e l s i n t h e r e -m a i n i n g a e r o b i c c e l l s . 5) D e t e r m i n e t h e m i n i m u m f r e e a m m o n i a l e v e l n e e d e d t o m a i n t a i n n i t r i t e b u i l d - u p . R e s u l t s T h i s r u n , c o n s i s t i n g o f t w o f o u r - c e l l s y s t e m s , s t a r t e d o n N o v e m b e r 1 0 , 1 9 8 3 a n d l a s t e d f o r 147 d a y s . To m e e t t h e o b j e c t i v e s s e t o u t f o r t h i s r u n , t h e e x p e r i m e n t a l p r o g r a m was d i v i d e d i n t o f o u r d i s t i n c t s t a g e s . F i g u r e s 5 a n d 6 s h o w t h e c a l c u l a t e d f r e e a m m o n i a l e v e l s i n t h e f i r s t c e l l o f e a c h s y s t e m a n d t h e r e s u l t a n t 75 10 1-100 A E R O B I C T I M E ( d a y s ) F i g . 5 : Run 2 / S y s t e m 1 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 76 T I M E ( d a y s ) F i g . 6 : Run 2 / S y s t e m 2 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 77 d e g r e e o f n i t r i t e b u i l d - u p i n e a c h o f t h e t h r e e r e m a i n i n g c e l l s , a s a f u n c t i o n o f t h e t o t a l o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e c e l l ( i . e . , n i t r i t e - N p l u s n i t r a t e - N ) . T h e n i t r o g e n c o n t e n t o f t h e f e e d a v e r a g e d 120 mg T K N / L ( 1 0 0 mg N H ^ - N / L ) d u r i n g t h e f i r s t t h r e e s t a g e s , a n d a b o u t d o u b l e t h a t c o n c e n t r a t i o n d u r i n g S t a g e 4 . S t a g e 1 : D a y 1 - 2 8 T h e o b j e c t i v e o f t h i s f o u r - w e e k l o n g s t a g e w a s t o a c c l i m a t e t h e b i o m a s s a n d c o n f i r m t h e p o t e n t i a l f o r n i t r i t e a c c u m u l a t i o n u n d e r f u l l y a e r o b i c c o n d i t i o n s . B o t h s y s t e m s w e r e o p e r a t e d i n a n i d e n t i c a l mode u n d e r f u l l y a e r o b i c c o n d i t i o n s . C o m p l e t e n i t r i f i -c a t i o n w a s a c h i e v e d w i t h i n a f e w d a y s o f s t a r t - u p . N i t r i t e w a s a b s e n t f r o m a l l c e l l s , i n b o t h s y s t e m s , d u r i n g t h i s s t a g e . Ammo-n i a w a s a b s e n t f r o m t h e l a s t t w o c e l l s o f e a c h s y s t e m . F r e e a m m o n i a l e v e l s w e r e h i g h e s t i n t h e f i r s t c e l l a n d d e c l i n e d r a p i d -l y i n t h e r e m a i n i n g c e l l s . I t a v e r a g e d 0 . 2 6 mg N H ^ - N / L i n t h e f i r s t e e l 1 o f S y s t e m 1 a n d 0 . 2 1 mg NH3 - N / L i n t h e f i r s t c e l l o f S y s t e m 2 . No n i t r i t e w a s d e t e c t e d i n a n y o f t h e c e l l s d u r i n g t h i s s t a g e . I n v i e w o f t h e I n a b i l i t y o f a c h i e v i n g n i t r i t e a c c u m u l a t i o n u n d e r t h e s e o p e r a t i n g c o n d i t i o n s , t h e e x p e r i m e n t a l p r o g r a m p r o -c e e d e d t o t h e s e c o n d s t a g e . S t a g e 2 : D a y 2 9 - 3 7 T h e o b j e c t i v e o f t h i s s t a g e w a s t o d e t e r m i n e i f a n a e r o b i o s i s a l o n e , i n t h e a b s e n c e o f f r e e a m m o n i a , c o u l d i n d u c e n i t r i t e b u i l d - u p . T e s t i n g t h i s h y p o t h e s i s c a l l e d f o r c o n v e r t i n g a n a e r o -b i c c e l l , c o n t a i n i n g l i t t l e f r e e a m m o n i a , i n t o a n a n a e r o b i c o n e . T h e s e c o n d c e l l o f S y s t e m 1 w a s c h o s e n , s i n c e i t c o n t a i n e d l i t t l e 7 8 r e s i d u a l f r e e a m m o n i a . T h e f r e e a m m o n i a l e v e l i n t h i s c e l l a t n o t i m e e x c e e d e d 0 . 1 5 mg NH3-N/L d u r i n g t h i s s t a g e . S y s t e m 2 w a s m a i n t a i n e d f u l l y a e r o b i c a n d a c t e d a s a c o n t r o l . No d e t e c t a b l e i n c r e a s e i n n i t r i t e l e v e l s w a s m e a s u r e d i n a n y o f t h e c e l l s o f S y s t e m 1 a s a r e s u l t o f t h e c o n v e r s i o n . B y D a y 3 7 , i t b e c a m e e v i d e n t t h a t a n a e r o b i o s i s a l o n e w o u l d n o t i n d u c e n i t r i t e b u i l d - u p a n d t h e e x p e r i m e n t a l p r o g r a m p r o c e e d e d t o t h e n e x t s t a g e . S t a g e 3 : D a y 3 8 - 7 5 T h e o b j e c t i v e o f t h i s s t a g e w a s t o c o n f i r m t h a t t h e p r e s e n c e o f a f r o n t - e n d , a n a e r o b i c c e l l w o u l d i n d u c e n i t r i t e b u i l d - u p . T h e f r e e a m m o n i a l e v e l i n S y s t e m 1 w a s r a i s e d t o a r o u n d 1 mg NH3 - N / L , b y c o n v e r t i n g C e l l 1 f r o m a e r o b i c t o a n a e r o b i c . C e l l 2 w a s c o n -v e r t e d b a c k t o a n a e r o b i c m o d e . S y s t e m 2 w a s m a i n t a i n e d f u l l y a e r o b i c a n d a c t e d a s a c o n t r o l . T h e c h a n g e i n t r o d u c e d i n S y s t e m I d i d n o t l e a d t o a n y n i t r i t e b u i l d - u p . On D a y 4 4 , C e l l 2 o f S y s t e m 1 w a s a l s o c o n v e r t e d t o a n a n a e r o b i c m o d e . T h i s e f f e c t i v e l y d o u b l e d t h e a c t u a l a n a e r o b i c r e t e n t i o n t i m e p e r c y c l e f r o m .2 t o 4 h o u r s ( o u t o f a t o t a l H R T / c y c l e o f 8 h o u r s ) . A t r a n s i e n t n i t r i t e b u i l d - u p w a s o b s e r v e d , l a s t i n g o n l y a f e w d a y s a n d a m o u n t i n g t o a b o u t 10% o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e e f f l u e n t . T h e p e r i o d f a l l i n g b e t w e e n D a y s 50 a n d 55 c o i n c i d e d w i t h t h e l o w e s t M L S S c o n c e n t r a t i o n a t t a i n e d d u r i n g t h i s r u n ( s e e A p p e n d i x A ) . I t w a s c a u s e d b y a c o m b i n a t i o n o f l o w c a r b o n c o n t e n t i n t h e f e e d a n d p o o r e f f l u e n t q u a l i t y . A s a r e s u l t , t h e a e r o b i c SRT i n S y s t e m 1 d r o p p e d t e m p o r a r i l y t o a b o u t 3 . 3 d a y s . T h i s , i n c o m b i n a -79 t i o n w i t h t h e c h a n g e s i m p o s e d o n S y s t e m 1 e a r l i e r , i n d u c e d a n o t h -e r t r a n s i e n t n i t r i t e b u i l d - u p , w h i c h a m o u n t e d t o a b o u t 50% o f t h e o x i d i z e d n i t r o g e n p r e s e n t i n t h e e f f l u e n t . T h e b u i l d - u p d e c l i n e d s t e a d i l y a n d d i s a p p e a r e d w i t h i n t e n d a y s . B y D a y 7 5 i t w a s e v i d e n t t h a t t h e e f f o r t s , t o - d a t e , h a d f a i l e d t o i n d u c e a n d m a i n t a i n n i t r i t e b u i l d - u p a n d t h e e x p e r i m e n -t a l p r o g r a m p r o c e e d e d t o S t a g e 4 . S t a g e 4 : D a y 7 6 - 1 4 7 T h e o b j e c t i v e o f t h i s s t a g e w a s t o i n d u c e a n d m a i n t a i n n i t r i t e b u i l d - u p b y i n c r e a s i n g t h e f r e e a m m o n i a c o n c e n t r a t i o n a t t h e f r o n t - e n d o f t h e s y s t e m . B o t h s y s t e m s w e r e c o n v e r t e d t o a n i d e n t i c a l mode o f o p e r a t i o n . C e l l 1 w a s m a i n t a i n e d a n a e r o b i c , w h i l e t h e r e m a i n i n g t h r e e c e l l s w e r e a e r o b i c . T h e n i t r o g e n f e e d c o n c e n t r a t i o n w a s d o u b l e d d u r i n g t h i s s t a g e t o a r o u n d 2 5 0 mg T K N / L a n d t h e r e c y c l e r a t e w a s d r o p p e d b y 5 0 % . T h e e f f e c t o f t h e s e m e a s u r e s w a s t o s u b s t a n t i a l l y r a i s e t h e f r e e a m m o n i a l e v e l i n t h e f i r s t c e l l o f e a c h s y s t e m . To f u r t h e r i n d u c e n i t r i t e b u i l d - u p , t h e f e e d r a t e w a s r a i s e d b y 20% f o r a f e w d a y s . T h e s e s u d d e n c h a n g e s l e d t o a r i s e i n n i t r i t e l e v e l s i n b o t h s y s t e m s . T h e e x t e n t o f a c c u m u l a t i o n d i f f e r e d a p p r e c i a b l y a s s e e n f r o m F i g u r e s 5 a n d 6 . I n t h e e f f l u e n t o f S y s t e m 1 , n i t r i t e r o s e t o a b o u t 40% o f t h e o x i d i z e d n i t r o g e n s p e c i e s a n d t h e n d e c l i n e d s t e a d i l y . I n S y s t e m 2 , n i t r i t e r o s e s t e a d i l y f o r a p e r i o d o f t w o w e e k s , u n t i l i t a c c o u n t e d f o r o v e r 90% o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e e f f l u e n t . T h i s l e v e l w a s m a i n t a i n e d f o r a b o u t 3 0 d a y s i n t h e l a s t c e l l a n d f o r o v e r 5 0 d a y s i n t h e f i r s t a e r o b i c c e l l . 80 One n o t i c e a b l e d i f f e r e n c e b e t w e e n t h e t w o s y s t e m s was t h a t t h e f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c c e l l d i f f e r e d a p p r e c i a b -l y . A s s h o w n i n T a b l e 1 2 , a n d F i g u r e s 5 a n d 6 , i t a v e r a g e d 2 . 0 mg NH3-N/L i n S y s t e m 1 a n d 5 . 7 mg NH3-N/L i n S y s t e m 2 , u p t o D a y 1 3 6 , a f t e r w h i c h i t d e c l i n e d t o a n a v e r a g e 2 . 6 mg NH3 - N / L . T h e d e c l i n e i n f r e e a m m o n i a l e v e l s i n S y s t e m 2 w a s c a u s e d b y a g r a d u -a l r e d u c t i o n i n t h e a l k a l i n i t y c o n t e n t o f t h e f e e d , i m p l e m e n t e d f r o m D a y 80 o n w a r d s . T h e f e e d a l k a l i n i t y w a s r e d u c e d t o e s t a b -l i s h : 1) t h e m i n i m u m f r e e a m m o n i a l e v e l n e e d e d t o s u s t a i n n i t r i t e b u i l d - u p ; a n d 2) t h e a b i l i t y o f n i t r o u s a c i d t o s u s t a i n n i t r i t e b u i l d - u p . A s c a n b e s e e n f r o m F i g u r e 6 , t h e e f f e c t o f t h i s r e d u c t i o n i n a l k a l i n i t y m a n i f e s t e d i t s e l f m o s t c l e a r l y f r o m D a y 1 3 5 o n w a r d s , w h e n t h e f r e e a m m o n i a l e v e l i n t h e f i r s t c e l l s t a r t -e d t o d r o p . N i t r o u s a c i d l e v e l s i n t h e f i r s t a e r o b i c c e l l s t a r t e d r i s i n g e a r l i e r , f r o m D a y 1 2 0 o n w a r d s , f r o m u n d e r 5 t o o v e r 30 u g HNO2-N/L. T h e a v e r a g e a e r o b i c s l u d g e a g e d u r i n g t h i s f o u r t h s t a g e w a s 9 . 9 d a y s f o r S y s t e m 1 a n d 1 1 . 9 d a y s f o r S y s t e m 2 , a s s h o w n i n T a b l e 1 1 . B y D a y 1 2 0 , t h e n i t r i t e l e v e l i n t h e l a s t t w o e e l I s o f S y s t e m 2 s t a r t e d a g r a d u a l , b u t s t e a d y , d e c l i n e t h a t p e r s i s t e d t o t h e e n d o f t h e r u n . T h e d e c l i n e i n n i t r i t e b u i l d - u p i n t h e f i r s t a e r o b i c c e l l s t a r t e d a r o u n d D a y 1 4 0 . B y t h e e n d o f t h e r u n , S y s t e m 2 w a s f a i l i n g t o m a i n t a i n n i t r i t e b u i l d - u p . A s s h o w n i n F i g u r e 5 , n i t r i t e b u i l d - u p i n S y s t e m 1 a l s o d e c l i n e d p r o g r e s s i v e -l y w i t h t i m e . 8 1 D i s c u s s i o n 1) N i t r i t e A c c u m u l a t i o n N i t r i t e a c c u m u l a t i o n w a s i n d u c e d i n S y s t e m 2 a t t h e b e g i n i n g o f s t a g e 4 a n d w a s s u s t a i n e d f o r a p e r i o d o f a b o u t 50 d a y s . I n S y s t e m 1 , o n t h e o t h e r h a n d , n i t r i t e a c c u m u l a t i o n w a s i n d u c e d a t m o d e r a t e l e v e l s b u t c o u l d n o t b e s u s t a i n e d . I n . b o t h s y s t e m s , n i t r i t e a c c u m u l a t i o n w a s i n d u c e d b y a r i s e i n t h e f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c c e l l . O n c e i n d u c e d , a n u m b e r o f f a c t o r s may h a v e b e e n r e s p o n s i b l e f o r s u s t a i n i n g t h e n i t r i t e b u i l d - u p . T h e s e a r e d i s c u s s e d b r i e f l y h e r e , b u t a r e c o v e r e d i n m o r e d e t a i l i n C h a p t e r 6 . T h e s e f a c t o r s i n c l u d e d : a) Low s l u d g e a g e b) L o w DO l e v e l c) H i g h n i t r o u s a c i d l e v e l i n t h e a e r o b i c c e l l s d) H i g h f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c c e l l a) Low S l u d g e A g e T h i s f a c t o r d i d n o t a p p e a r t o h a v e b e e n r e s p o n s i b l e f o r s u s t a i n i n g n i t r i t e b u i l d - u p , s i n c e t h e a e r o b i c s l u d g e a g e f o r S y s t e m 2 , w h i c h s u s t a i n e d n i t r i t e b u i l d - u p , w a s 20 % h i g h e r t h a n f o r S y s t e m 1 , w h i c h d i d n o t s u s t a i n i t . b) L o w DO L e v e l F r o m t h e r e s u l t s o f R u n 1 , t h e r e w a s s t r o n g i n d i c a t i o n t h a t DO l e v e l s b e l o w 1 m g / L i n h i b i t e d a m m o n i a o x i d a t i o n . D u r i n g R u n 1 , c o n s i d e r a b l e e n e r g y was d i r e c t e d t o w a r d s m o n i t o r i n g a n d m a n u a l l y c o n t r o l l i n g DO l e v e l s i n t h e f o u r c e l l s o f S y s t e m 2 . I n v i e w o f t h e d i s c o u r a g i n g r e s u l t s o b t a i n e d i n R u n 1 , w h e r e n o n i t r i t e a c c u m u l a t i o n c o u l d b e i n d u c e d , i n s p i t e o f r e l a t i v e l y l o w DO 82 l e v e l s , the resumption of t h i s t ime consuming a c t i v i t y c o u l d not be j u s t i f i e d . As a r e s u l t , i t was d e c i d e d to l i m i t r i g i d DO c o n t r o l to the f i r s t a e r o b i c c e l l of each system. T h i s d e c i s i o n was based on the o b s e r v a t i o n t h a t over 80% of the n i t r i f i c a t i o n process was n o r m a l l y completed w i t h i n the f i r s t a e r o b i c c e l l . Thus , i f low DO l e v e l s were to s e l e c t i v e l y i n h i b i t the n i t r i t e o x i d i z e r s , the e f f e c t would be apparent i n the f i r s t a e r o b i c c e l l . F u r t h e r m o r e , i t was d e c i d e d t o m a i n t a i n a DO l e v e l i n the f i r s t a e r o b i c c e l l o f a b o u t 1 mg/L i n System 1 and 1.5 m g / L i n S y s t e m 2, i n o r d e r to compare the e f f e c t of DO on the n i t r i f i c a t i o n process (d i scussed i n Chapter 6). These l e v e l s , which were h i g h e r than those m a i n -t a i n e d i n System 2 d u r i n g Run 1, were l a r g e l y m a i n t a i n e d t h r o u g h -out the r u n , as shown i n T a b l e 13. The DO l e v e l i n System "2, d u r i n g the f i r s t three s tages , was e q u a l to t h a t ma in ta ined d u r i n g stage 4, ye t no n i t r i t e a c c u m u l a t i o n mani fe s ted i t s e l f u n t i l s t a g e 4, when the f r e e ammonia l e v e l was r a i s e d . In Sys tem 1, t h e DO l e v e l d u r i n g s t a g e s 1 and 2, when no n i t r i t e b u i l d - u p o c c u r r e d , was 40% lower than d u r i n g s tages 3 and 4, when n i t r i t e a c c u m u l a t i o n mani f e s t ed i t s e l f (as a r e s u l t of the r i s i n g f r e e ammonia l e v e l s ) . On t h i s b a s i s , low DO l e v e l s d i d not appear to be a f a c t o r t h a t i n i t i a t e d and s u s t a i n e d n i t r i t e a c c u m u l a t i o n . c) High N i t r o u s A c i d L e v e l i n the A e r o b i c C e l l In an e f f o r t to g a i n an u n d e r s t a n d i n g of the e f f e c t of n i t r o u s a c i d on n i t r i t e a c c u m u l a t i o n , the pH of the feed was reduced d u r i n g s tage 4. T h i s l e d to a r i s e i n the n i t r o u s a c i d l e v e l i n the a e r o b i c c e l l s of both systems, e s p e c i a l l y System 2. 83 I t r o s e i n the f i r s t a e r o b i c c e l l of System 2, from an a v e r a g e 7 ug HN02-N/L (n=16, s=4) between Days 79 and 119, to 20 ug HN0 2-N/L ( n = l l , s=9) between Days 120 and 142. T h i s c o i n c i d e d with the r a p i d d e c l i n e i n n i t r i t e l e v e l s i n that system (see F i g u r e 6) and suggested that n i t r o u s a c i d d i d not s u s t a i n n i t r i t e b u i l d - u p . d) High Free Ammonia L e v e l i n the Anaerobic C e l l Free ammonia appeared to be the parameter r e s p o n s i b l e f o r t r i g g e r i n g n i t r i t e b u i l d - u p . I t a l s o seemed to be the major f a c t o r r e s p o n s i b l e f o r s u s t a i n i n g the b u i l d - u p . The f r e e ammonia l e v e l needed to i n i t i a t e and s u s t a i n n i t r i t e b u i l d - u p appeared to be higher than reported i n the l i t e r a t u r e , by a f a c t o r of 10 (5 to 8 mg NH3-N/L as opposed to 0.1 to 1.0 mg NH3-N/L). 2) R e l a t i o n s h i p Between Duration of A e r a t i o n and N i t r i t e B u i l d - u p F i g u r e 6 showed t h a t , f o r a g i v e n f r e e ammonia l e v e l , n i -t r i t e accumulation was h i g h e s t i n the f i r s t a e r o b i c c e l l and lowest i n the l a s t a e r o b i c c e l l . I t seemed that the a b i l i t y of the n i t r i t e o x i d i z e r s to r e g a i n t h e i r n i t r i f y i n g a c t i v i t y was d i r e c t l y r e l a t e d to the time e l a p s e d from t h e i r e x i t from the high f r e e ammonia environment of the anaerobic c e l l . 3) E f f e c t of A n a e r o b i o s i s A n a e r o b i o s i s , per se, d i d not appear to p l a y a r o l e i n n i t r i t e b u i l d - u p . It's e f f e c t appeared to be i n d i r e c t ; the estab-lishment of anaerobic c o n d i t i o n s at the front-end of the system l e d to a s u b s t a n t i a l r i s e i n pH and ammonium l e v e l s i n that c e l l , r e s u l t i n g i n high f r e e ammonia l e v e l s that s e l e c t i v e l y i n h i b i t e d the n i t r i t e o x i d i z e r s . 84 W h a t r e m a i n e d t o b e d e t e r m i n e d w a s : a) t h e m i n i m u m f r e e a m m o n i a c o n t a c t t i m e n e e d e d t o i n d u c e s e l e c t i v e i n h i b i t i o n a n d , b) t h e e f f e c t o f p r o l o n g i n g t h e c o n t a c t t i m e . 4) S l u d g e A g e T h e i m p o r t a n c e o f s l u d g e a g e i n d e t e r m i n i n g t h e p r e d o m i n a n c e o f a p a r t i c u l a r o x i d i z e d n i t r o g e n s p e c i e s c o u l d n o t b e c o n f i r m e d d u r i n g t h i s r u n . T h e a v e r a g e a e r o b i c s l u d g e a g e i n b o t h s y s t e m s w a s m a i n t a i n e d b e t w e e n 10 a n d 12 d a y s . I t s h o u l d b e n o t e d t h a t a t t h i s s l u d g e a g e , n i t r i t e w a s a b s e n t f r o m S y s t e m 2 d u r i n g t h e f i r s t h a l f o f t h e r u n a n d p r e s e n t d u r i n g t h e s e c o n d h a l f . C o n c l u s i o n s 1) N i t r i t e a c c u m u l a t i o n w a s i n d u c e d a n d s u s t a i n e d b y i n t e r m i t t e n t c o n t a c t o f t h e n i t r i t e o x i d i z e r s t o i n h i b i t o r y l e v e l s o f f r e e a m m o n i a . 2) T h e p r o c e s s c o n f i g u r a t i o n , u s i n g a n a n a e r o b i c c e l l a t t h e f r o n t e n d o f t h e p r o c e s s , m a i n t a i n e d f r e e a m m o n i a l e v e l s t h a t w e r e s e l e c t i v e l y i n h i b i t o r y t o t h e n i t r i t e o x i d i z e r s . 3) A n a e r o b i o s i s , p e r s e , d i d n o t i n d u c e n i t r i t e b u i l d - u p . T h e p r e s e n c e o f a n a n a e r o b i c c e l l a t t h e f r o n t - e n d f a c i l i t a t e d t h e d e v e l o p m e n t o f n i t r i t e b u i l d - u p , b y c r e a t i n g a n e n v i r o n m e n t c o n t a i n i n g r e l a t i v e l y h i g h f r e e a m m o n i a l e v e l s . 4) E l a p s e d t i m e , f o l l o w i n g e x i t f r o m t h e a n a e r o b i c c e l l , a p p e a r e d t o d e t e r m i n e t h e e x t e n t o f n i t r i t e b u i l d - u p . S h o r t a e r a t i o n t i m e l e d t o n i t r i t e b u i l d - u p , w h e r e a s l o n g e r a e r a t i o n p e r i o d s r e s u l t e d i n r e d u c e d n i t r i t e b u i l d - u p . 5) T h e l o s s o f n i t r i t e b u i l d - u p i n S y s t e m 2 , t o w a r d t h e e n d o f 85 the run, may have been caused by the d e c l i n e i n f r e e ammonia l e v e l i n the anaerobic c e l l . 6) D i s s o l v e d oxygen l e v e l s as low as 1 mg/L d i d not cause detec-t a b l e n i t r i t e b u i l d - u p . 7) The degree of i n h i b i t i o n of n i t r i t e o x i d a t i o n appeared to be r e l a t e d to the f r e e ammonia l e v e l i n the anaerobic c e l l . 8) Free ammonia l e v e l s of 6 mg NH^-N/L, or higher, seemed neces-sary to s u s t a i n a high degree of n i t r i t e b u i l d - u p w i t h i n the system. RUN 3 O b j e c t i v e s The o b j e c t i v e of t h i s run was to d e t e r m i n e i f n i t r i t e a c c u -m u l a t i o n c o u l d be su s t a i n e d by the presence of n i t r o u s a c i d and the absence of f r e e ammonia. Res u l t s The run commenced on A p r i l 30, 1984 and l a s t e d f o r o n l y t h i r t e e n days. Two, f o u r - c e l l systems were operated under f u l l y a e r o b i c c o n d i t i o n s . Major problems with the ope r a t i o n of one system p r e c l u d e d the generation of any meaningful data d u r i n g t h i s s h o r t run. As a r e s u l t , r e f e r e n c e w i l l be l i m i t e d t o the one system f o r which meaningful data was obtained. The feed flow was set at 20 L/d and the r e c y c l e r a t e at 14 L/d. I n f l u e n t n i t r o g e n c o n c e n t r a t i o n was around 135 mg TKN/L (85 mg NH^-N/L). F u l l n i t r i f i c a t i o n was e s t a b l i s h e d w i t h i n 24 hours. On Day 2, the pH i n the system was r a i s e d r a p i d l y (from about 6.8 to around 8.0) to induce n i t r i t e b u i l d - u p through the i n h i b i t o r y 86 a c t i o n o f f r e e a m m o n i a ; t h e l a t t e r r e a c h e d a c o n c e n t r a t i o n o f 1 . 2 mg NH3-N/L i n a l l c e l l s . T h i s i n d u c e d r a p i d n i t r i t e b u i l d - u p , a t t a i n i n g a b o u t 2 0 mg N 0 2 - N / L i n a l l c e l l s b y t h e e n d o f D a y 2 ( a m o u n t i n g t o 2 5 % o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t ) . T h e pH l e v e l w a s t h e n r a p i d l y d r o p p e d b y t h e a d d i t i o n o f s u l p h u r i c a c i d i n a l l f o u r c e l l s a n d i n t h e f e e d b u c k e t ( t o pH 6 . 7 - 7 . 0 ) . T h i s l e d t o a r i s e i n t h e n i t r o u s a c i d c o n c e n t r a t i o n f r o m u n d e r 0 . 1 u g HN0 2 - N / L t o a b o u t 5 t o 10 u g HN0 2 - N / L i n a l l f o u r c e l l s ; s i m u l t a n e o u s l y , t h e r e w a s a d r o p i n f r e e a m m o n i a l e v e l t o 0 . 1 mg NH3-N/L i n t h e f i r s t c e l l , a n d b e l o w t h a t i n t h e r e m a i n i n g c e l l s . T h i s r e s u l t e d i n a n i m m e d i a t e d e c l i n e i n n i t r i t e l e v e l s i n a l l c e l l s . T h e pH w a s d r o p p e d f u r t h e r ( t o a r o u n d 6 i n a l l c e l l s ) a n d t h e n i t r o u s a c i d l e v e l r e a c h e d a c o n c e n t r a t i o n o f 168 u g HN0 2 - N / L i n t h e f o u r t h c e l l , w i t h n o a p p a r e n t a r r e s t o f t h e d e c l i n e i n t h e r a t e o f n i t r i t e d i s a p p e a r a n c e , . B y D a y 3 , i t w a s e v i d e n t t h a t n i t r i t e a c c u m u l a t i o n w a s r a p i d l y d i s a p p e a r i n g ( i t h a d d r o p p e d f r o m 24% t o 13% w i t h i n 12 h o u r s i n C e l l 2 , f o r e x a m p l e ) . A t t h a t p o i n t , t h e pH o f t h e s y s t e m w a s q u i c k l y r a i s e d t o a r o u n d 8 . 5 b y t h e a d d i t i o n o f s o d i u m c a r b o n a t e t o a l l c e l l s . T h i s l e d t o a r i s e i n f r e e a m m o n i a l e v e l s t o t h e r a n g e o f 1 0 t o 14 mg NH3 - N / L i n a l l f o u r e e l I s , r e s u l t i n g i n o v e r 95 % i n h i b i t i o n o f n i t r i f i c a t i o n a c t i v i t y . A s t h e pH g r a d u a l l y d r o p p e d a n d n i t r i f i c a t i o n a c t i v i t y r e s u m e d , n i t r i t e a c c u m u l a t i o n w a s o b s e r v e d w i t h i n a f e w d a y s . B y D a y 7 , n i t r i f i c a t i o n a c t i v i t y h a d l a r g e l y r e c o v e r e d a n d n i t r i t e a c c o u n t e d f o r a b o u t 85 % o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e e f f l u e n t . A s e c o n d a t t e m p t w a s t h e n m a d e t o i n d u c e a n d m a i n t a i n h i g h n i t r o u s a c i d l e v e l s . T h e a l k a l i n i t y i n t h e f e e d 87 w a s i m m e d i a t e l y r e d u c e d a n d t h e pH w i t h i n t h e s y s t e m g r a d u a l l y d r o p p e d o v e r t h e f o l l o w i n g d a y s ; t h i s l e a d t o a c o n s i s t e n t r i s e i n n i t r o u s a c i d l e v e l s t h r o u g h o u t t h e s y s t e m , a n d a d e c l i n e i n f r e e a m m o n i a l e v e l s t o u n d e r 0 . 1 mg NH3 - N / L . B y D a y s 10 a n d 1 1 , t h e pH h a d d r o p p e d t o b e t w e e n 6 . 5 a n d 6 . 8 , a n d t h e n i t r o u s a c i d c o n c e n t r a t i o n r e a c h e d 10 u g H N 0 2 - N / L i n t h e f i r s t e e l 1 , 2 1 u g H N 0 2 - N / L i n t h e s e c o n d c e l l , 3 5 u g H N 0 2 - N / L i n t h e t h i r d c e l l a n d o v e r 4 0 u g H N 0 2 - N / L i n t h e f o u r t h c e l l . T h e d e c l i n e i n n i t r i t e b u i l d - u p s t a r t e d m a n i f e s t i n g i t s e l f f r o m D a y 8 o n w a r d s a n d w a s n o t a r r e s t e d b y t h e r i s e i n n i t r o u s a c i d l e v e l s w i t h i n t h e s y s t e m . B y D a y 1 3 , n i t r i t e h a d d i s a p p e a r e d f r o m t h e e f f l u e n t a n d a c c o u n t e d f o r a b o u t 10 % o f t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e f i r s t t w o c e l l s . A t t h i s t i m e , t h e r u n w a s t e r m i n a t e d . D i s c u s s i o n N i t r i t e b u i l d - u p was a c h i e v e d b y r a i s i n g t h e f r e e a m m o n i a l e v e l i n t h e f i r s t c e l l . T h e r a p i d d e c l i n e i n n i t r i t e a c c u m u l a -t i o n w a s a s s o c i a t e d w i t h t h e d i s a p p e a r a n c e o f f r e e a m m o n i a a n d t h e r i s e i n n i t r o u s a c i d l e v e l s . T h e p r e s e n c e . o f r e l a t i v e l y h i g h n i t r o u s a c i d l e v e l s d i d n o t r e v e r s e , o r e v e n a r r e s t , t h e r a t e o f n i t r i t e d e e l i n e . On D a y s 9 a n d 1 0 , n i t r o u s a c i d l e v e l s i n t h e s y s t e m e x c e e d e d , b y f a c t o r s o f 1 t o 4 , t h e c o n c e n t r a t i o n s r e p o r -t e d b y G a r r e t t ( 1 9 8 2 ) t o b e i n h i b i t o r y t o t h e n i t r i t e o x i d i z e r s ( 7 . 9 + 3 . 1 u g H N 0 2 - N / L ) . C o n c l u s i o n s T h e r e s u l t s o f t h i s r u n t e n d e d t o c o n f i r m t h a t n i t r o u s a c i d w a s n o t a p a r a m e t e r r e s p o n s i b l e f o r n i t r i t e a c c u m u l a t i o n . 88 RUN 4 O b j e c t i v e s T h e o b j e c t i v e o f t h i s r u n w a s t o d e t e r m i n e t h e e f f e c t o f l o n g a n d s h o r t SRT o n t h e a b i l i t y t o m a i n t a i n n i t r i t e b u i l d - u p . R e s u l t s T h i s r u n s t a r t e d o n M a y 1 7 , 1 9 8 4 a n d l a s t e d f o r 98 d a y s . To m e e t t h e o b j e c t i v e s o f t h e r u n , t h e e x p e r i m e n t a l p r o g r a m w a s d i v i d e d i n t o t w o d i s t i n c t s t a g e s . T h e n i t r o g e n f e e d c o n t e n t w a s m a i n t a i n e d a t 1 3 0 mg T K N / L ( a b o u t 8 5 mg N H ^ - N / L ) d u r i n g t h e f i r s t 38 d a y s o f S t a g e 1 , a n d a t 1 8 5 mg T K N / L ( a r o u n d 140 mg N H ^ - N / L ) f r o m D a y 4 5 o n w a r d s ( s e e T a b l e 1 2 ) . S t a g e 1 : D a y 1 - 5 2 T h e o b j e c t i v e o f t h i s s t a g e w a s t o a s s e s s t h e e f f e c t o f r e l a t i v e l y l o n g S R T ' s o n t h e a b i l i t y t o s u s t a i n n i t r i t e b u i l d - u p . B o t h s y s t e m s w e r e o p e r a t e d u n d e r f u l l y a e r o b i c c o n d i t i o n s u n t i l n i t r i f i c a t i o n a c t i v i t y e s t a b l i s h e d i t s e l f , a f t e r w h i c h t h e y w e r e c o n v e r t e d t o a p r e - d e n i t r i f y i n g m o d e o f o p e r a t i o n . T h e s l u d g e a g e w a s m a i n t a i n e d a s h i g h a s p o s s i b l e b y k e e p i n g s l u d g e w a s t a g e t o a m i n i m u m ( b y l i m i t i n g w a s t a g e t o s a m p l e c o l l e c t i o n a n d s o l i d s e s c a p i n g o v e r t h e w e i r ) . . A s a r e s u l t , t h e s l u d g e a g e r e m a i n e d a b o v e 36 d a y s i n b o t h s y s t e m s d u r i n g t h i s s t a g e . F i g u r e s 7 a n d 8 s h o w t h e d e g r e e o f n i t r i t e b u i l d - u p a t t a i n e d i n e a c h a e r o b i c c e l l a n d t h e c a l c u l a t e d f r e e a m m o n i a l e v e l i n t h e f i r s t c e l l . N i t r i f i c a t i o n a c t i v i t y e s t a b l i s h e d i t s e l f r a p i d l y u p o n s t a r t - u p . T h e e f f l u e n t a m m o n i a l e v e l d r o p p e d t o b e l o w 1 mg N/L w i t h i n t w o d a y s , a t w h i c h t i m e a n a t t e m p t w a s m a d e t o i n i t i a t e 89 100 O CO < I CN o T I M E ( d a y s ) F i g . 7 : Run 4 / . S y s t e m 1 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 90 T I M E (days) F i g . 8 : Run 4 / S y s t e m 2 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 91 n i t r i t e b u i l d - u p . I t i n v o l v e d t h e a d d i t i o n o f a b o u t 1 0 0 mg N H ^ -N/L t o e a c h c e l l i n c o n j u n c t i o n w i t h s u f f i c i e n t s o d i u m b i c a r b o -n a t e , t o m a i n t a i n a pH l e v e l i n t h e r a n g e o f 7 . 7 t o 8 . 2 a c r o s s t h e s y s t e m . A s a r e s u l t , t h e f r e e a m m o n i a c o n c e n t r a t i o n r o s e t o a b o u t 4 mg NH3-N/L i n b o t h s y s t e m s . T h i s p r o v e d t o b e i n s u f f i -c i e n t , a s n i t r i t e b u i l d - u p w a s m i n i m a l , a n d r e c o v e r y w a s o c c u r -r i n g b y D a y 3. T h e pH w a s f u r t h e r r a i s e d t o a r o u n d 8 . 5 i n a l l c e l l s , r e s u l t i n g i n a r i s e i n f r e e a m m o n i a l e v e l t o a b o u t 7 mg NH3-N/L i n t h e f i r s t c e l l o f e a c h s y s t e m . T h i s , i n c o n j u n c t i o n w i t h a c o n v e r s i o n o f t h e f i r s t c e l l o f S y s t e m 1 f r o m a e r o b i c t o a n a e r o b i c , l e d t o a s h a r p d r o p i n n i t r i f i c a t i o n a c t i v i t y i n b o t h s y s t e m s . B y D a y 7 , n i t r i f i c a t i o n a c t i v i t y i n S y s t e m 1 h a d v i r t u a l l y c e a s e d a n d , a s a r e s u l t , t h e f r e e a m m o n i a l e v e l r o s e t o t h e r a n g e o f 1 0 t o 14 mg NH3 - N / L . I n S y s t e m 2 , o n t h e o t h e r h a n d , t h e f r e e a m m o n i a l e v e l r e m a i n e d b e l o w 3 n-.g IJH3-N/L a n d n i t r i f i c a t i o n a c t i v i t y p r o g r e s s e d s l o w l y , w i t h m i n i m a l n i t r i t e b u i l d - u p . I n a n e f f o r t t o i n d u c e g r e a t e r n i t r i t e b u i l d - u p i n S y s t e m 2 , t h e f i r s t c e l l w a s c o n v e r t e d t o a n a e r o b i c o n D a y 7 . T h i s l e d t o a v i r t u a l c e s s a t i o n o f n i t r i f i c a t i o n a c t i v i t y b y D a y 9 , d u e t o t h e r i s e i n f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c e e l 1 t o a b o u t 8 mg NH3 - N / L . T h e pH i n b o t h s y s t e m s w a s g r a d u a l l y l o w e r e d b y r e d u c i n g t h e b i c a r b o n a t e c o n t e n t o f t h e f e e d . T h i s l e d t o a d e c l i n e i n f r e e a m m o n i a l e v e l s t o a r o u n d 2 mg NH3-N/L a n d a r e s u m p t i o n o f n i t r i -f i c a t i o n a c t i v i t y . B y D a y 1 6 , n i t r i f i c a t i o n a c t i v i t y h a d r e c o v e r e d i n b o t h s y s t e m s . I t s r e s u m p t i o n w a s a c c o m p a n i e d b y a h i g h d e g r e e o f n i t r i t e b u i l d - u p , a m o u n t i n g t o o v e r 90 % o f t h e o x i d i z e d n i t r o g e n 92 s p e c i e s . T h e o c c u r r e n c e o f m a s s i v e n i t r i t e b u i l d - u p , f o l l o w i n g c o m p l e t e i n h i b i t i o n o f n i t r i f i c a t i o n a c t i v i t y , w a s a l s o o b s e r v e d i n p r e v i o u s r u n s . F o r t h e n e x t 1 5 d a y s , t h e f r e e a m m o n i a l e v e l i n t h e a n a e r o -b i c c e l l o f e a c h s y s t e m w a s m a i n t a i n e d b e t w e e n 5 a n d 6 mg NH3-N / L . T h i s w a s t h e l e v e l f o u n d a p p r o p r i a t e i n m a i n t a i n i n g n i t r i t e b u i l d - u p d u r i n g R u n 2 . A s c a n b e s e e n f r o m F i g u r e 7 , i t d i d n o t p r e v e n t a r a p i d d e c l i n e i n n i t r i t e l e v e l s i n S y s t e m 1 f r o m m a n i -f e s t i n g i t s e l f ( s t a r t i n g a b o u t D a y 2 3 ) . I n a n e f f o r t t o a r r e s t i t , t h e r e c y c l e f l o w r a t e w a s d o u b l e d o n D a y 3 3 , t o r e d u c e t h e a c t u a l H R T / c e l l d u r i n g e a c h c y c l e ; t h i s r e d u c e d t h e i n t e r v a l b e t w e e n s u c c e s s i v e b i o m a s s c o n t a c t t o t h e f r e e a m m o n i a i n t h e a n a e r o b i c c e l l . I n a d d i t i o n , C e l l 2 w a s c o n v e r t e d f r o m a e r o b i c t o a n a e r o b i c . T h e s e m e a s u r e s , w h i c h e f f e c t i v e l y : r e d u c e d t h e a e r o b i c s y s t e m HRT p e r c y c l e f r o m 9 t o 4 h o u r s a n d i n c r e a s e d t h e a n a e r o -b i c s y s t e m HRT p e r c y c l e f r o m 3 t o 4 h o u r s , a r r e s t e d t h e d e c l i n e b u t d i d n o t r e v e r s e i t . T h e d e c l i n e i n n i t r i t e b u i l d - u p i n S y s t e m 2 w a s a p p a r e n t b y D a y 3 5 , a n d i t s d e c l i n e w a s a s r a p i d a s i n S y s t e m 1 . I n a n e f f o r t t o a r r e s t t h e d e c l i n e i n b o t h s y s t e m s , t h e a m m o n i a f e e d c o n c e n -t r a t i o n w a s i n c r e a s e d f r o m 130 t o 1 8 5 mg T K N / L b e t w e e n D a y s 40 a n d 4 5 . T h i s l e d t o a s h a r p r i s e i n f r e e a m m o n i a l e v e l s i n S y s t e m 2 , t o a b o u t 20 mg NH3 - N / L , a n d t e m p o r a r i l y r e v e r s e d t h e d e c l i n e i n t h a t s y s t e m . S u r p r i s i n g l y , n i t r i f i c a t i o n a c t i v i t y w a s n o t s u b s t a n t i a l l y i n h i b i t e d b y t h e h i g h f r e e a m m o n i a l e v e l , a n d t h e p r o c e s s r e c o v e r e d w i t h i n a f e w d a y s . T h e r i s e i n f r e e a m m o n i a l e v e l i n S y s t e m 1 w a s n o t a s d r a m a t i c ; i t b a r e l y r o s e a b o v e 4 mg 93 NH3 - N / L . T h i s may h a v e b e e n d u e t o t h e h i g h e r d i l u t i o n r e s u l t i n g f r o m t h e d o u b l i n g o f t h e r e c y c l e r a t e . T h e d e c l i n e i n n i t r i t e b u i l d - u p i n b o t h s y s t e m s r e s u m e d o n D a y 4 5 . B y D a y 5 2 i t w a s a p p a r e n t t h a t t h e d e c l i n e i n b o t h s y s t e m s c o u l d n o l o n g e r b e a r r e s t e d w i t h o u t m o r e d r a s t i c m e a s u r e s . A s a r e s u l t , t h e e x p e r i m e n t a l p r o g r a m m o v e d t o t h e n e x t s t a g e , w h i c h i n v o l v e d a d r a s t i c r e d u c t i o n i n t h e s l u d g e a g e . S t a g e 2 : D a y 5 3 - 9 8 F r o m D a y 53 t o 5 6 , s l u d g e w a s t a g e w a s i n c r e a s e d f r o m b o t h s y s t e m s . T h i s e f f e c t i v e l y d r o p p e d t h e a e r o b i c s l u d g e a g e t o 2 . 6 d a y s i n S y s t e m 1 a n d t o 3 . 8 D a y s i n S y s t e m 2 . T h e d e c l i n e i n n i t r i t e a c c u m u l a t i o n w a s a r r e s t e d , b u t n o t r e v e r s e d . E f f l u e n t a m m o n i a l e v e l s r e m a i n e d b e l o w 1 mg N/L d u r i n g t h i s p e r i o d . F r o m D a y 5 7 t o 6 0 , t h e w a s t a g e r a t e w a s f u r t h e r i n c r e a s e d i n b o t h s y s t e m s , r e s u l t i n g i n a t e m p o r a r y a e r o b i c SRT o f 1 . 0 d a y i n S y s t e m 1 a n d 1 . 9 d a y i n S y s t e m 2 . I n s p i t e o f t h e s e d r a s t i c m e a s u r e s , t h e e f f l u e n t a m m o n i a l e v e l i n S y s t e m 1 r e m a i n e d b e l o w 1 mg N / L . I n S y s t e m 2 , o n t h e o t h e r h a n d , i t i n c r e a s e d r a p i d l y t o ' o v e r 90 mg N / L , r e f l e c t i n g a m a j o r l o s s o f n i t r i f i c a t i o n a c t i v i -t y . A s a r e s u l t o f t h e s e m e a s u r e s , t h e d e c l i n e i n n i t r i t e b u i l d -u p w a s r e v e r s e d i n b o t h s y s t e m s . I n S y s t e m 1 , t h e r e v e r s a l w a s n o t s i g n i f i c a n t ; h o w e v e r , t h e r a t e o f s l u d g e w a s t a g e w a s r e d u c e d , t h u s a l l o w i n g t h e a e r o b i c s l u d g e a g e t o a g a i n r i s e t o a r o u n d 4 . 5 d a y s . B y D a y 67 t h e d e c l i n e r e s u m e d a n d t h e a e r o b i c s l u d g e a g e w a s a g a i n d r o p p e d t o 0 . 8 d a y s a n d m a i n t a i n e d a t u n d e r 2 d a y s u p t o D a y 7 2 ( i . e . , f o r f i v e d a y s ) . I n a d d i t i o n , t h e r e c y c l e f l o w w a s r e d u c e d f r o m 20 t o 94 10 L / d , i n o r d e r t o r a i s e t h e f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c c e l l s . T h i s l e d t o a t r a n s i e n t r i s e i n e f f l u e n t a m m o n i a c o n c e n -t r a t i o n a n d a t e m p o r a r y r e v e r s a l i n n i t r i t e d e c l i n e . N i t r i t e d e c l i n e r e s u m e d o n D a y 77 ( s e e F i g u r e 7 ) , a n d n o f u r t h e r a t t e m p t w a s m a d e t o a r r e s t i t , b u t t h e a e r o b i c s l u d g e a g e w a s k e p t b e l o w 6 d a y s f o r t h e r e m a i n d e r o f t h e r u n . T h e s e c o n d c e l l o f S y s t e m 1 w a s c o n v e r t e d b a c k t o a n a e r o b i c m o d e , t h e r e c y c l e r a t e w a s o n c e a g a i n d o u b l e d ( t h u s r e d u c i n g t h e f r e e a m m o n i a l e v e l i n t h e a n a e -r o b i c c e l l ) a n d t h e a e r o b i c s l u d g e a g e w a s m a i n t a i n e d a t a b o u t 5 t o 6 d a y s . A s a r e s u l t , t h e a c t u a l a e r o b i c HRT f o r t h e s y s t e m r e m a i n e d a t 6 h o u r s p e r c y c l e , w h i l e t h e a n a e r o b i c HRT d r o p p e d f r o m 6 t o 2 h o u r s . T h e f r e e a m m o n i a l e v e l r e m a i n e d b e t w e e n 6 a n d 8 mg NH3-N/L t o t h e e n d o f t h e r u n , a t w h i c h t i m e n i t r i t e h a d d i s a p p e a r e d f r o m t h e l a s t t w o c e l l s o f t h e s y s t e m . T h e r e v e r s a l i n n i t r i t e d e c l i n e w a s m o r e p r o n o u n c e d i n S y s t e m 2 t h a n i n S y s t e m 1 , b u t t h e b u i l d - u p d i d n o t r e a c h t h e l e v e l s a c h i e v e d a t t h e b e g i n i n g o f t h e r u n ( s e e F i g u r e 8) , i n s p i t e o f a g r a d u a l r i s e i n f r e e a m m o n i a l e v e l s f r o m 6 t o 17 mg NH3-N/L ( f r o m D a y 49 t o 5 8 ) . T h e h i g h f r e e a m m o n i a l e v e l l e d t o a d r o p i n n i t r i f i c a t i o n a c t i v i t y w h i c h m a n i f e s t e d i t s e l f o n D a y 5 8 . To a l l o w n i t r i f i c a t i o n a c t i v i t y t o r e c o v e r , M L S S w a s t a g e w a s t e m p o r a r i l y r e d u c e d b e t w e e n D a y s 60 a n d 67,. T h e i n h i b i t i o n w a s g r a d u a l l y o v e r c o m e a n d , b y D a y 6 7 , n i t r i f i c a t i o n a c t i v i t y h a d l a r g e l y r e c o v e r e d . T h i s c o i n c i d e d w i t h a r e s u m p t i o n i n t h e d e -c l i n e i n n i t r i t e a c c u m u l a t i o n . T h e a e r o b i c s l u d g e a g e w a s m a i n -t a i n e d a t a b o u t 4 . 3 d a y s f r o m D a y 5 3 t o t h e e n d o f t h e r u n . I n o r d e r t o r e v e r s e t h e d e c l i n e i n n i t r i t e b u i l d - u p i n S y s t e m 2 , a s e c o n d c e l l w a s c o n v e r t e d t o a n a e r o b i c o n D a y 6 7 , 95 d r o p p i n g t h e a c t u a l a e r o b i c s y s t e m HRT f r o m 9 t o 6 h o u r s / c y c l e , w h i l e r a i s i n g t h e a n a e r o b i c HRT f r o m 3 t o 6 h o u r s . F u r t h e r m o r e , t h e a l k a l i n i t y o f t h e f e e d w a s r a i s e d , r e s u l t i n g i n h i g h e r pH l e v e l s i n t h e s y s t e m s . T h i s l e d t o a s h a r p r i s e i n f r e e a m m o n i a l e v e l s , r e a c h i n g 22 mg NH3-N/L b y D a y 74; t h i s , i n t u r n , c a u s e d i n h i b i t i o n o f n i t r i f i c a t i o n a c t i v i t y , a t w h i c h t i m e t h e a l k a l i n i -t y o f t h e f e e d was r e d u c e d . T h e i n h i b i t i o n , h o w e v e r , w a s l e s s s e v e r e t h a n t h a t e x p e r i e n c e d o n D a y 6 0 , a n d r e c o v e r y w a s a c h i e v e d b y D a y 77. T h e s e m e a s u r e s r e v e r s e d t h e d e c l i n e . On D a y 8 8 , t h e d e c l i n e m a n i f e s t e d i t s e l f a g a i n i n t h e l a s t c e l l , a l t h o u g h n o d e c l i n e w a s e v i d e n t b y t h e e n d o f t h e r u n i n t h e f i r s t a e r o b i c c e l l . T h e r u n w a s t e r m i n a t e d o n D a y 9 8 , a t w h i c h t i m e a b a t c h t e s t ( B a t c h T e s t N o . l ) w a s c o n d u c t e d o n S y s t e m 2. T h e - r e s u l t s o f t h e b a t c h t e s t a r e p r e s e n t e d a t t h e e n d o f t h e C h a p t e r . D i s c u s s i o n T h e i n d u c e m e n t o f n i t r i t e b u i l d - u p a t t h e b e g i n i n g o f t h e r u n w a s a c h i e v e d b y t h e p r o c e d u r e s e s t a b l i s h e d i n t h e p r e v i o u s r u n s a n d p r e s e n t e d n o d i f f i c u l t i e s . T h e i n a b i l i t y o f e i t h e r s y s t e m t o s u s t a i n h i g h n i t r i t e c o n c e n t r a t i o n s f o r a n y a p p r e c i a b l e e x t e n t o f t i m e (7 d a y s i n S y s t e m 1 a n d 19 d a y s i n S y s t e m 2), i n s p i t e o f t h e p r e s e n c e o f f r e e a m m o n i a l e v e l s r a n g i n g b e t w e e n 6 a n d 7 mg NH3-N/L i n t h e a n a e r o b i c c e l l , w a s u n e x p e c t e d . U n l i k e t h e s i t u a t i o n o b s e r v e d i n S y s t e m 2 d u r i n g R u n 2, t h e n i t r i t e o x i d i z e r s o v e r c a m e t h e i n h i b i t o r y e f f e c t s o f f r e e a m m o n i a w i t h r e l a t i v e s p e e d . T h i s s u g g e s t e d t h a t t h e n i t r i t e o x i d i z e r s w e r e a b l e t o g r o w a t a r a t e f a s t e r t h a n t h e w a s h o u t r a t e a n d s e e m e d t o 96 i n d i c a t e t h a t t h e p r e s e n c e o f f r e e a m m o n i a , e v e n a t r e l a t i v e l y h i g h c o n c e n t r a t i o n s , d i d n o t t o t a l l y i n h i b i t t h e i r a c t i v i t y . T h e pH i n t h e a n a e r o b i c c e l l o f b o t h s y s t e m s r a n g e d b e t w e e n 8 . 1 a n d 8 . 7 f r o m D a y 14 o n w a r d s . T h i s w a s h i g h e r ( b y a t l e a s t 0 . 5 u n i t ) t h a n t h e pH l e v e l i n a l l o t h e r r u n s t o - d a t e . T h i s s u g g e s t e d t h a t p H , p e r s e , d i d n o t p l a y a n y d i s c e r n i b l e r o l e i n p r e v e n t i n g t h e d e c l i n e i n n i t r i t e l e v e l s . E f f o r t s t o r e v e r s e t h e d e c l i n e i n n i t r i t e l e v e l s i n c l u d e d : 1) E x t e n d i n g t h e a n a e r o b i c r e s i d e n c e t i m e ( a n d h e n c e t h e c o n t a c t t i m e t o h i g h f r e e a m m o n i a l e v e l s ) f r o m 2 5 t o 5 0 % o f t o t a l d e t e n -t i o n t i m e ; 2) R e d u c i n g t h e a e r o b i c SRT f r o m o v e r 20 d a y s t o u n d e r 5 d a y s . T h e s e e f f o r t s l e d o n l y t o a t e m p o r a r y a r r e s t i n t h e d e c l i n e o f n i t r i t e l e v e l s . T h e m e a s u r e s u n d e r t a k e n t o r e v e r s e t h e d e c l i n e i n n i t r i t e l e v e l s w e r e s e v e r e a n d p r o b a b l y b e y o n d t h e c a p a b i l i t y o f f u l l - s c a l e t r e a t m e n t f a c i l i t i e s . E v e n t h e n , t h e d e c l i n i n g t r e n d c o u l d n o t b e r e v e r s e d i n S y s t e m 1 a n d t h e m e a -s u r e s t a k e n w e r e b a r e l y e f f e c t i v e i n S y s t e m 2 . T h e e f f o r t s l e d , a t b e s t , t o t e m p o r a r y a r r e s t s o r r e v e r s a l s . T h e r e s u l t s a p p e a r e d t o r e f l e c t t h e d i f f i c u l t y i n r e v e r s i n g o r a r r e s t i n g t h e d e c l i n e i n n i t r i t e b u i l d - u p , o n c e i n p r o g r e s s . C o n c l u s i o n s 1) R e d u c i n g t h e a e r o b i c s l u d g e a g e f r o m 2 8 t o 4 d a y s w a s n o t s u f f i c i e n t t o s u s t a i n n i t r i t e b u i l d - u p . Low s l u d g e a g e t e m p o -r a r i l y a r r e s t e d t h e d e c l i n e i n n i t r i t e b u i l d - u p , b u t c o u l d n o t r e v e r s e i t i n d e f i n i t e l y . 2) T h e n i t r i t e o x i d i z e r s a p p e a r e d c a p a b l e o f t o l e r a t i n g e v e r -i n c r e a s i n g l e v e l s o f f r e e a m m o n i a , t h u s c a u s i n g a n i r r e v e r s i b -97 l e d e c l i n e i n n i t r i t e a c c u m u l a t i o n f o r m o s t o p e r a t i o n a l s y s -t e m s t e s t e d . 3) T h e c o m b i n a t i o n o f : l o w s l u d g e a g e , s h o r t a e r a t i o n t i m e , l o n g a n a e r o b i c r e s i d e n c e t i m e , e x t e n d e d c o n t a c t t o h i g h f r e e ammo-n i a l e v e l s , a n d h i g h e r f r e e a m m o n i a l e v e l s a l l o w e d S y s t e m 2 t o s u s t a i n n i t r i t e a c c u m u l a t i o n t o t h e e n d o f t h e r u n , i n s p i t e o f t h e a p p a r e n t " a c c l i m a t i o n " o f t h e n i t r i t e o x i d i z e r s t o f r e e a m m o n i a . RUN 5 O b j e c t i v e s F r o m t h e p r e v i o u s r u n s , i t w a s a p p a r e n t t h a t t h e d u r a t i o n o f t h e a e r a t i o n p e r i o d ( i . e . , a c t u a l a e r o b i c H R T / c y c l e ) p l a y e d a m a j o r r o l e i n s u s t a i n i n g n i t r i t e a c c u m u l a t i o n ( s e e a l s o B a t c h T e s t N o . l ) . T h e m a j o r o b j e c t i v e s o f t h i s r u n w e r e t o d e t e r m i n e w h e t h e r r e d u c i n g t h e a v i a t i o n t i m e p r i o r t o d e n i t r i f i c a t i o n , b y i n c o r p o r a t i n g a n i n t e r n a l r e c y c l e s t e p , c o u l d : 1) s u s t a i n n i t r i t e b u i l d - u p b y p r e v e n t i n g t h e n i t r i t e o x i d a t i o n s t e p f r o m p r o g r e s -s i n g s u f f i c i e n t l y p r i o r t o d e n i t r i f i c a t i o n , w h i l e , .2) p e r m i t t i n g t h e d i s c h a r g e o f a f u l l y n i t r i f i e d e f f l u e n t , c o n t a i n i n g n o n i -t r i t e , a s a r e s u l t o f l o n g a e r o b i c d e t e n t i o n t i m e i n t h e r e m a i n -i n g a e r o b i c c e l l s ( s e e F i g u r e 9 f o r t h e s c h e m a t i c s e t - u p f o r t h i s r u n ) . R e s u l t s T h e r u n c o m m e n c e d o n S e p t e m b e r 2 1 , 1 9 8 4 a n d l a s t e d 76 d a y s . S y s t e m 1 , u s e d a s a c o n t r o l , was t e r m i n a t e d o n D a y 62 b e c a u s e o n e o f t h e p u m p s c o u l d n o l o n g e r be u s e d . B y t h a t t i m e , t h e c o n t r o l 98 Volume • 2.5 L 10 L / d Per Cycle Figure 9 : Process Treatment Schematic For Run 5 / System 2 had largely served its purpose (as evidenced from Figures 10 and 11), and no loss was suffered as a result. The two systems were operated in an identical manner up to Day 20. An average aerobic SRT of 7 to 8 days was maintained in both systems throughout the run. The nitrogen content of the feed averaged 185 mg TKN/L (140 mg NH -^N/L) from Day 2 to Day 29, and 210 mg TKN/L (165 mg NH4-N/L) from Day 34 onwards. The free ammonia level ranged between 5.5 to 6.5 mg NH3-N/L in the first cel l of each system. Nitrite accumulation levels and associated free ammonia concentrations are presented in Figures 10 and 11. By Day 2, complete nitrification was achieved in both sys-tems, at a feed strength of 180 mg TKN/L, and the first cel l of each system was then converted to an anaerobic one. In addition, the pH was raised in an effort to achieve inhibition of nitr i te oxidation. This led to a gradual rise in free ammonia levels.and, by Day 10, major inhibition of ammonia oxidation was evident in both systems. By Day 13, the degree of inhibition exceeded 85% in both systems, and the alkalinity content of the feed was tempora-r i ly reduced. Recovery of ammonia oxidation in both systems was evident by Day 17, at which time the alkal inity content of the feed was raised. The resumption of nitrification activity was accompanied by nitrite accumulation. By Day 20, nitrite accounted for 96% of the oxidized nitro-gen species present in the effluent of System 1 and 87% of that in System 2. On that day, internal recycle was implemented in System 2 and the overall recycle rate for both systems was t r i -pled from 10 to 30 L/d. In System 2, this was achieved by spl i t -ting the recycle into two streams; a clarifier recycle of 2 L/d, 100 100 TIME ( d a y s ) F i g . 1 0 : Run 5 / S y s t e m 1 - E x t e n t of N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l Z i H o o < CM o 101 15 C E L L 1 ( a n a e r o b i c ) T I M E ( d a y s ) F i g . l l : Run 5 / S y s t e m 2 - E x t e n t of N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 102 and an internal recycle from Cel l 2 of 28 L/d. Thus, the total recycle rate to Cel l 1 was similar in both systems. The use of a split recycle in System 2 resulted in a reduction of actual HRT in cells 1 and 2 from 3 to 1.5 hours each and an increase in actual HRT in ce l ls 3 and 4 from 3 to 5 hours each. The actual HRT in each cel l of System 1, on the other hand, was reduced from 3 to 1.5 hours. As noted from Figures 10 and 11, nitrite build-up in the first aerobic cel l of System 2 was sustained for a much longer period of time than in System 1, in spite of its lower i n i t i a l degree of nitrite accumulation. However, the accumulation could not be sustained indefinitely and a gradual decline in nitrite build-up was evident from Day 35 onwards. In an effort to reverse the decline in nitrite build-up in System 2, Cell 3 was converted to an anaerobic mode on Day 55, and carbon feed in the form of 100 ml/d of 5% acetic acid, was supplied directly to that c e l l . It was hoped that, by reducing a larger fraction of the nitrite anaerobical ly (i.e., in Cells 1 and 3), the decline could be re-versed. This resulted in a temporary arrest, after which the decline resumed. No further effort was made to reverse the dec-line in nitrite accumulation. By Day 62, the degree of nitr i te build-up in the f i rs t c e l l of each system was similar, at around 35%. This level of accumu-lation was sustained in System 2 to the end of the run. Nitrite was virtually absent from the effluent of both systems from Day 40 onwards. A batch test (Batch Test No.2) was conducted on Day 48 and is discussed later in this Chapter. 103 Discussion By incorporating internal recycle, System 2 was capable of sustaining nitrite accumulation for a longer period than the control. Although nitrite build-up in System 2 eventually de-clined, it seemed able to sustain itself, for an extended period of time in the f i rs t aerobic c e l l , at a level of around 35%. Furthermore, the virtual disappearnce of nitrite from the last aerobic cel l of System 2 by Day 41 suggested the possibility of operating the system in a mode which produced a high level of nitrite in the first aerobic c e l l , while producing a fully n i t r i -fied effluent, virtually devoid of any nitrite. The absence of nitr i te in the effluent of System 2 by Day 41 was largely due to a combination of: 1) removal of a large portion of nitr i te within the system by denitrification and, 2) relatively long aeration time in Cells 3 and 4, effectively allowing for oxidation of the remaining nitrite to nitrate. The operation of a four cel l system did not provide suffi-cient f lexibi l i ty to optimize the variables involved, namely: hydraulic retention time per ce l l , pre- and post- denitrifica-tion, and the complete oxidation of nitrite to nitrate prior to its discharge. This may have been a contributing factor in the inability to arrest the decline in nitrite levels once they occurred, and suggested that a more flexible, multi-celled, plug-flow arrangement could produce the desired results. Conclusions 1) The implementation of internal recycle and intermediary deni-trification extended the duration of nitrite build-up. 104 2) T h e a b i l i t y t o p r o d u c e a f u l l y n i t r i f i e d e f f l u e n t , d e v o i d o f n i t r i t e , w h i l e m a i n t a i n i n g n i t r i t e b u i l d - u p w i t h i n t h e f r o n t -e n d o f t h e s y s t e m , w a s c o n f i r m e d . 3) F o u r c e l l s p e r s y s t e m w e r e i n s u f f i c i e n t t o s t u d y t h e e f f e c t s o f t h e s e l e c t e d v a r i a b l e s , w h i l e e n s u r i n g a n e f f l u e n t d e v o i d o f n i t r i t e . RUN 6 T h e u n f o l d i n g e v i d e n c e o f a c c l i m a t i o n o f t h e n i t r i t e o x i d i -z e r s t o f r e e a m m o n i a , l e d t o a s u s p i c i o n t h a t t h e c o m p o s i t i o n o f t h e s y n t h e t i c f e e d , w h i c h d i d n o t c o n t a i n i n h i b i t o r s o t h e r t h a n a m m o n i u m , may h a v e c o n t r i b u t e d t o t h e a c c l i m a t i o n p r o c e s s , d u e t o t h e a b s e n c e o f p o t e n t i a l l y i n h i b i t o r y c o m p o u n d s t h a t may b e p r e s e n t i n a g e n u i n e w a s t e . I n o r d e r t o i n v e s t i g a t e t h i s t h e o r y , l e a c h a t e f r o m t h e P o r t M a n n l a n d f i l l i n S u r r e y , B . C . , w a s u s e d . I t w a s c h a r a c t e r i z e d b y a r e l a t i v e l y h i g h a m m o n i u m c o n t e n t a n d l o w COD ( s e e T a b l e 7 ) . I t w a s h o p e d t h a t t h i s m u n i c i p a l l e a c h a t e m i g h t c o n t a i n s u b s t a n c e s t h a t c o u l d e i t h e r e n h a n c e t h e s e l e c t i v e i n h i b i t i o n o f f r e e a m m o n i a , o r " p e r t u r b " t h e n i t r i t e o x i d i z e r s , a n d p r e v e n t t h e m f r o m a c c l i m a t i n g , a s r e a d i l y , t o f r e e a m m o n i a . O b j e c t i v e s T h e o b j e c t i v e s o f t h i s r u n w e r e t o : 1) D e t e r m i n e i f n i t r i t e a c c u m u l a t i o n c o u l d b e s u s t a i n e d w i t h t h e c o m b i n a t i o n o f i n t e r m e d i a r y d e n i t r i f i c a t i o n a n d a g e n u i n e w a s t e , s u c h a s m u n i c i p a l l a n d f i l l l e a c h a t e . 2) I n t h e e v e n t t h a t n i t r i t e a c c u m u l a t i o n c o u l d n o t b e s u s t a i n e d , e s t a b l i s h t h e v i a b i l i t y o f s u b j e c t i n g t h e n i t r i t e o x i d i z e r s t o 10 5 a n o t h e r s e l e c t i v e i n h i b i t o r , a n d u t i l i z e t h e c o n c e p t o f d o u b l e s u b s t r a t e i n h i b i t i o n t o s e l e c t i v e l y i n h i b i t t h e n i t r i t e o x i d i -z e r s . R e s u l t s T h e r u n c o m m e n c e d o n D e c e m b e r 7 , 1 9 8 4 a n d l a s t e d 44. d a y s . A s i n g l e e i g h t - c e l l s y s t e m w a s o p e r a t e d . T h i s c o n f i g u r a t i o n w a s a d o p t e d i n o r d e r t o p r o d u c e m a x i m u m f l e x i b i l i t y i n o p e r a t i o n . F u r t h e r m o r e , h a v i n g i n v e s t i g a t e d , i n t h e p a s t f i v e r u n s , m o s t o f t h e p a r a m e t e r s r e s p o n s i b l e f o r i n i t i a t i n g a n d s u s t a i n i n g n i t r i t e b u i l d - u p , a s w e l l a s t h o s e t h a t m i g h t r e v e r s e i t , t h e n e e d f o r o p e r a t i n g a c o n t r o l s y s t e m w a s n o l o n g e r d e e m e d i m p e r a t i v e . I t w a s f e l t t h a t , a t t h i s s t a g e , t h e f l e x i b i l i t y o f o p e r a t i n g a n e i g h t c e l l s y s t e m o u t w e i g h e d t h e b e n e f i t s o f m a i n t a i n i n g a c o n -t r o l . T h e n i t r o g e n c o n t e n t o f t h e w a s t e w a s c o m p o s e d a l m o s t e n t i r e l y o f a m m o n i u m ( o v e r 9 5 % ) , a n d a v e r a g e d 2 6 0 mg N H ^ - N / L . C e l l 1 w a s m a i n t a i n e d a n a e r o b i c , t o a c h i e v e h i g h f r e e ammo-n i a l e v e l s . C a r b o n f e e d f o r d e n i t r i f i c a t i o n p u r p o s e s w a s a d d e d t o C e l l 6 , w h i c h w a s a l s o m a i n t a i n e d a n a e r o b i c . T h e r e m a i n i n g s i x c e l l s w e r e a e r o b i c . T h e f e e d a n d r e c y c l e f l o w w e r e s e t a t 20 L / d e a c h . S o d i u m h y d r o x i d e ( 0 . 5 N) s o l u t i o n w a s a d d e d d i r e c t l y t o C e l l 1 t o m a i n t a i n h i g h pH l e v e l s w i t h i n t h e c e l l . U n l i k e t h e p r e v i o u s f i v e r u n s , w h e r e d e n i t r i f i c a t i o n o c c u r r e d i n t h e f i r s t c e l l , i n R u n s 6 a n d 7, t h e d e n i t r i f i c a t i o n s t e p w a s c a r r i e d o u t i n a s e p a r a t e c e l l w i t h i n t h e s y s t e m ( i n o r d e r t o a c h i e v e i n t e r -m e d i a r y d e n i t r i f i c a t i o n ) . T h u s , t h e s o l e p u r p o s e o f t h e a n a e r o b i c c e l l a t t h e f r o n t - e n d o f t h e s y s t e m w a s t o c r e a t e a h i g h f r e e a m m o n i a e n v i r o n m e n t , w i t h v i r t u a l l y n o d e n i t r i f i c a t i o n t a k i n g 106 place. As a result, the nitrite + nitrate content of the recycle remained almost unchanged (on a mass balance basis) following its residence in the first anaerobic ce l l . In order to obtain a baseline from which to estimate the degree of build-up caused by free ammonia, it was necessary to subtract the nitrite + nitrate content of the first anaerobic cel l from that of the aerobic ones. Results of the run, presented in Figure 12 (as well as Figure 13 for Run 7), refer to the "net" nitrite build-up, after subtracting the contribution from the anaerobic ce l l -Full nitrification was achieved by Day 3. Nitrite accumula-tion was minimal and transient in nature, in spite of a free ammonia level in Cel l 1 ranging between 1.7 and 9.4 mg NH3-N/L (caused by variation in pH levels between 7.4 and 8.3). On Day 10,.the pH in Cel l 1 was raised above 9 resulting in a free ammonia level exceeding 20 mg NH3-N/L. This caused a major reduc-tion in nitrification activity, characterized by a rather slow recovery lasting 24 days. During this period, the free ammonia level in Cel l 1 ranged between 1 and 32 mg NH3-N/L. Nitrite build-up manifested itself as a result of raising the free ammo-nia level and, by Day 22, exceeded 85% of the oxidized nitrogen present in the first aerobic ce l l . The build-up was transient, however, and the decline became evident from Day 36 onwards. In an effort to reverse the decline in nitr i te build-up, a 1 molar solution of sodium chlorate was added, on Day 39, at a rate of 0.17 L/d to the first aerobic cel l (Sodium chlorate was added to selectively inhibit the nitrite oxidizers). The duration of sodium chlorate addition lasted about 17 hours. The calculated 107 1 , . I I [-0 10 20 30 -40 TIME (days) F ig .12 : Run 6 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 108. h i g h e s t sodium c h l o r a t e c o n c e n t r a t i o n reached, w i t h i n p a r t s of the system, was 6.2 mM. The r e s u l t was a t o t a l i n h i b i t i o n of n i t r i f i c a t i o n a c t i v i t y , with no s i g n of r e c o v e r y by Day 44, when the run was f i n a l l y terminated. Thus, d e s p i t e the evidence c i t e d i n the l i t e r a t u r e (see Chapter 6), sodium c h l o r a t e d i d not act as a s p e c i f i c i n h i b i t o r of the n i t r i t e o x i d i z e r s , but as a g e n e r a l i n h i b i t o r of n i t r i f i c a t i o n . D i s c u s s i o n The r e s u l t s of t h i s run i n d i c a t e d that the n i t r i t e o x i d i z e r s were a b l e to r e g a i n t h e i r n i t r i f y i n g a b i l i t y r a p i d l y , i n s p i t e of the r e l a t i v e l y high l e v e l s of f r e e ammonia and the presence of a m u n i c i p a l l a n d f i l l l e a c h a t e . A c c l i m a t i o n seemed to occur once again, d e s p i t e the p o t e n t i a l f o r i n h i b i t i o n from v a r i o u s impuri-t i e s i n the l e a c h a t e . E f f o r t s to maintain n i t r i t e b u i l d - u p , by the use of double s u b s t r a t e i n h i b i t i o n (free ammonia and sodium c h l o r a t e ) , r e s u l t e d i n t o t a l i n h i b i t i o n of n i t r i f i c a t i o n a c t i v i t y . T h i s was s u r p r i -s i n g , s i n c e sodium c h l o r a t e had been known (since 1945) to act as a s p e c i f i c i n h i b i t o r of n i t r i t e o x i d a t i o n with l i t t l e , i f any, e f f e c t on the ammonia o x i d i z e r s (Lees, 1963). The i n i t i a t i o n of n i t r i t e b u i l d - u p , from Day 16 onwards, d i d not l e a d to an immediate a b i l i t y to reduce n i t r i t e a n a e r o b i c a l l y . No n i t r i t e reducing a b i l i t y was observed u n t i l Day 27. From then on, n i t r i t e r e d u c t i o n remained minimal, and n i t r a t e was reduced p r e f e r e n t i a l l y to n i t r i t e . In view of the r e l a t i v e s h o r t n e s s of the run, a l o n g w i t h the major v a r i a t i o n s i n f r e e ammonia l e v e l s , the i n h i b i t i o n of n i t r i -109 f i c a t i o n a c t i v i t y t h r o u g h o u t t h e r u n , a n d t h e n o n - s t e a d y s t a t e c o n d i t i o n s t h a t p r e v a i l e d t h r o u g h o u t , m i n i m a l q u a n t i t a t i v e i n t e r -p r e t a t i o n o f r e s u l t s w a s p o s s i b l e . T h e s h o r t n e s s o f t h e r u n a n d t h e a b r u p t c e s s a t i o n o f n i t r i f i c a t i o n a c t i v i t y , f o l l o w i n g s o d i u m c h l o r a t e a d d i t i o n , p r e c l u d e d a n e x t e n s i v e e v a l u a t i o n o f i n t e r n a l d e n i t r i f i c a t i o n a s a m e c h a n i s m t o s u s t a i n n i t r i t e b u i l d - u p . C o n c l u s i o n s 1) N i t r i t e a c c u m u l a t i o n c o u l d n o t b e s u s t a i n e d w i t h a g e n u i n e w a s t e w a t e r , s u c h a s m u n i c i p a l l a n d f i l l l e a c h a t e . 2) T h e u s e o f d o u b l e s u b s t r a t e i n h i b i t i o n ( f r e e a m m o n i a a n d s o d i u m c h l o r a t e ) t o s u s t a i n n i t r i t e b u i l d - u p p r o v e d u n s u c c e s s -f u l , a s i t l e d t o t h e t o t a l i n h i b i t i o n o f n i t r i f i c a t i o n a c t i v i t y . . RUN 7 O b j e c t i v e s T h e o b j e c t i v e s o f t h i s r u n w e r e t o : 1) D e t e r m i n e t h e d e g r e e o f n i t r i t e b u i l d - u p t h a t c o u l d b e s u s -t a i n e d i n d e f i n i t e l y , b y t h e u s e o f i n t e r m e d i a r y d e n i t r i f i c a -t i o n ( i n t h e p r e s e n c e o f a p o p u l a t i o n o f n i t r i t e o x i d i z e r s a c c l i m a t e d t o f r e e a m m o n i a ) . 2) I n v e s t i g a t e o t h e r m e c h a n i s m s t h a t c o u l d r e v e r s e t h e a c c l i m a -t i o n p r o c e s s t o f r e e a m m o n i a . R e s u l t s T h e r u n c o m m e n c e d o n J a n u a r y 2 1 , 1 9 8 5 a n d l a s t e d 3 5 9 d a y s . A s i n g l e , s i x - c e l l s y s t e m w a s o p e r a t e d a n d f e d w i t h m u n i c i p a l l e a c h a t e f r o m t h e P o r t M a n n l a n d f i l l . R e s u l t s o f t h e p r e v i o u s r u n 110 indicated that a six-cel l system would provide the desired f lex i -b i l i ty and reduce the operating difficulties experienced with eight cells; these difficulties were more frequent plugging of lines (i.e., spills) and more potential for liquid overflow, due to the smaller freeboard resulting from the higher hydraulic headloss across the system. The nominal feed and recycle rates were set at 15 L/d. The system was operated in a fu l ly aerobic mode unti l Day 28, at which time Cell 1 was converted to anaerobic. Exogenous carbon feed, in the form of sodium acetate and methanol, was added from Day 1, in order to acclimate the biomass to this carbon source. Carbon feed was added to Cel l 1 unti l Day 56, when i t was trans-ferred to Cel l 3 (which was converted at that time to an anaero-bic mode of operation). The amount of carbon feed was gradually increased until Day 70, after which slight adjustments were made until Day 140, in an effort to meet the denitrification requirements within Cell 3. After that date, the carbon feed rate remained constant. Sodium hydroxide (0.5 N) was fed to Cell 1 from Day 31 onwards, to maintain the desired pH level within that ce l l . Ammonium accounted for over 95 % of the nitrogen content of the waste. Its concentration varied between 255 and 280 mg NH4-N/L during the run. The biomass used for this run contained a very low popula-tion of nitrifiers. As a result, one month was needed to gradual-ly build-up the nitrifier population. On Day 31, Cell 1 was con-verted to an anaerobic mode and sodium hydroxide addition was 111 i n i t i a t e d . T h i s l e d to a d r a m a t i c r i s e i n f r e e ammonia l e v e l s , as shown i n F i g u r e 13, and r e s u l t e d i n some i n h i b i t i o n of n i t r i f i c a -t i o n a c t i v i t y and a t r a n s i e n t r i s e i n n i t r i t e l e v e l s . Although the f r e e ammonia l e v e l exceeded 20 mg NH3-N/L, n e i t h e r the reduc-t i o n i n n i t r i f i c a t i o n a c t i v i t y nor r i s e i n n i t r i t e b u i l d - u p were a p p r e c i a b l e . N i t r i f i c a t i o n a c t i v i t y was reduced by about a t h i r d , w h i l e n i t r i t e b u i l d - u p reached a maximum of 79% i n the f i r s t a e r o b i c c e l l on Day 45, a f t e r which i t s t a r t e d an immediate and r a p i d d e c l i n e . T h i s was i n c o n t r a s t to a l l p r e v i o u s runs, where n i t r i t e b u i l d - u p reached higher l e v e l s and was sustained f o r longer p e r i o d s , p r i o r to i t ' s d e c l i n e . On Day 56, C e l l 3 was converted to anaerobic. By then, the e x t e n t of n i t r i t e b u i l d - u p had dropped to about 35% i n the f i r s t a e r o b i c c e l l and v i r t u a l l y disappeared from the remaining c e l l s . The a b i l i t y to reduce n i t r i t e a n a e r o b i c a 1 l y was not present i n the biomass u n t i l Day 70, when some r e d u c t i o n i n n i t r i t e was observed i n C e l l 3. From the day n i t r i t e r e d u c t i o n e s t a b l i s h e d i t s e l f , the degree of n i t r i t e accumulation i n the f i r s t a e r o b i c c e l l r o s e s t e a d i l y , u n t i l i t r e a c h e d 60% by Day 91, a f t e r which i t d e c l i n e d s h a r p l y to under 30% by Day 94. The d e c l i n e was temporary, and n i t r i t e b u i l d - u p resumed and reached over 50 % by Day 116 and over 60% by Day 126. I t exceeded 80% by Day 150 and remained above t h a t l e v e l f o r o v e r 45 days. By Day 199, a d e c l i n e i n n i t r i t e l e v e l s manifested i t s e l f . However, no attempt was made to r e v e r s e i t by r a i s i n g the f r e e ammonia l e v e l or reducing the sludge age. By Day 227, n i t r i t e b u i l d - u p had d e c l i n e d to j u s t over 45% of the o x i d i z e d n i t r o g e n species present i n C e l l 2. An attempt 112 100 C E L L 1 T I M E ( d a y s ) F ig .13 : Run 7 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l 113 was then made, o v e r the next 20 days, to r e v e r s e t h i s d e c l i n e by r a i s i n g the pH and hence the f r e e ammonia l e v e l , from an average 7.7 mg NH3-N/L to about 20 mg NH3-N/L. The d e c l i n e was not ar-rested . On Day 247, the f r e e ammonia l e v e l reached 40 mg NH3-N/L, r e s u l t i n g i n minimal r e v e r s a l of n i t r i t e b u i l d - u p . An attempt was then made to determine whether evidence of "washout" of the ac c l i m a t e d p o p u l a t i o n or l o s s of a c c l i m a t i o n c o u l d be detected f o l l o w i n g a temporary r e d u c t i o n i n f r e e ammonia l e v e l s . Therefore, on Day 249, sodium hydroxide a d d i t i o n was stopped and no f u r t h e r e f f o r t was made, during the next 35 days, to r e v e r s e the d e c l i n e . As a r e s u l t , the f r e e ammonia l e v e l i n the f i r s t c e l l dropped to an a v e r a g e 0.9 mg NH3-N/L i n the f i r s t a e r o b i c c e l l . A temporary r i s e i n n i t r i t e l e v e l s (to around 60%) was observed between Days 251 and 275, a f t e r which the d e c l i n e resumed. This temporary r i s e was unexpected, and i t s occurrence was viewed as encouraging, s i n c e i t may have s i g n a l l e d a s h i f t i n the n i t r i t e o x i d i z e r p o p u l a t i o n towards l e s s t o l e r a n c e to f r e e ammonia. This would have r e f l e c t e d the changing environmental c o n d i t i o n s , namely, a low f r e e ammonia environment. On Day 282, sodium hydroxide a d d i t i o n was resumed. I t caused no d i s c e r n a b l e change i n the ra t e of d e c l i n e of n i t r i t e b u i l d - u p . Sodium hydroxide feed was, again, t e m p o r a r i l y stopped on Day .286; at which time, C e l l 1 was c o n v e r t e d t o an a e r o b i c mode to f u r t h e r reduce f r e e ammonia l e v e l s . T h i s mode of oper a t i o n was maintained f o r 31 days (from Day 287 to 318). The f r e e ammonia l e v e l i n the f i r s t c e l l remained around 1.0 mg NH3-N/L. By Day 318, n i t r i t e 114 h a d v i r t u a l l y d i s a p p e a r e d f r o m t h e s y s t e m . T h e s e r e s u l t s r e f u t e d t h e p o s t u l a t e o f a " w a s h o u t " o f a c c l i m a t e d p o p u l a t i o n s a n d a s h i f t t o w a r d s a l e s s t o l e r a n t n i t r i t e o x i d i z e r p o p u l a t i o n . On D a y 3 1 8 , C e l l 1 w a s r e c o n v e r t e d t o a n a n a e r o b i c m o d e a n d s o d i u m h y d r o x i d e a d d i t i o n w a s r e s u m e d . T h e f r e e a m m o n i a l e v e l i n t h e a n a e r o b i c c e l l r o s e t o a n a v e r a g e o f 1 3 mg NH3 - N / L f o r t h e n e x t 2 0 d a y s . T h i s l e d t o a s l i g h t a n d g r a d u a l r i s e i n n i t r i t e l e v e l s , r e a c h i n g o v e r 25% b y D a y 3 3 8 . A s e c o n d a t t e m p t w a s t h e n m a d e t o r e v e r s e t h e a p p a r e n t a c c l i m a t i o n , b y t e m p o r a r i l y s t o p p i n g a l l f e e d a n d o p e r a t i n g t h e s y s t e m i n a n e n d o g e n o u s r e s p i r a t i o n m o d e , w i t h a l l c e l l s a e r o b i c f o r a p e r i o d o f t h i r t e e n d a y s ( u n t i l D a y 3 5 1 ) . R e s u m p t i o n o f f e e d o n D a y 3 5 1 l e d t o a d r a m a t i c r i s e i n n i t r i t e l e v e l s , i n s p i t e o f f u l l y a e r o b i c c o n d i t i o n s a n d l o w f r e e a m m o n i a l e v e l s . T h i s o c c u r r e d i n c o n j u n c t i o n w i t h a m a j o r r e d u c -t i o n i n n i t r i f i c a t i o n a c t i v i t y ( a s c o m p a r e d t o n i t r i f i c a t i o n a c t i v i t y p r i o r t o D a y 3 3 8 ) . I n o r d e r t o d e n i t r i f y t h e n i t r i t e , C e l l 4 w a s r e v e r t e d t o a n a n a e r o b i c m o d e o n D a y 3 5 3 , a n d c a r b o n f e e d t o t h a t c e l l w a s r e s u m e d . R e c o v e r y o f n i t r i f i c a t i o n a c t i v i -t y o v e r t h e n e x t f e w d a y s o c c u r r e d s i m u l t a n e o u s l y w i t h a r a p i d d e c l i n e i n n i t r i t e a c c u m u l a t i o n . I n o r d e r t o a r r e s t t h e d e c l i n e , C e l l 1 w a s r e c o n v e r t e d t o a n a n a e r o b i c m o d e o n D a y 3 5 6 a n d s o d i u m h y d r o x i d e a d d i t i o n t o t h a t c e l l w a s r e s u m e d . R a i s i n g o f f r e e a m m o n i a l e v e l s f r o m 0 . 5 mg NH3 - N / L t o 9 mg NH3 - N / L b e t w e e n D a y s 3 5 6 a n d 3 5 9 , d i d n o t h a v e a n y v i s i b l e e f f e c t i n r e v e r s i n g t h e d e c l i n e . T h e r u n w a s t e r m i n a t e d o n D a y 3 5 9 . 1 1 5 D i s c u s s i o n T h e n i t r i t e o x i d i z e r s a c c l i m a t e d r a p i d l y t o f r e e a m m o n i a l e v e l s o f o v e r 2 0 mg NH3 - N / L . T h i s r e s u l t e d i n a r a p i d r e v e r s a l i n n i t r i t e b u i l d - u p d u r i n g t h e i n i t i a l p h a s e o f t h e r u n . T h e f r e e a m m o n i a l e v e l a v e r a g e d 19 mg NH3 - N / L f r o m D a y 35 t o 7 0 . I n s p i t e o f t h i s , n i t r i t e b u i l d - u p c o u l d n o t b e s u s t a i n e d a n d i t d r o p p e d i n t h e f i r s t a e r o b i c c e l l f r o m 80% t o 35%. O n c e n i t r i t e r e d u c t i o n a b i l i t y m a n i f e s t e d i t s e l f i n t h e a n a e r o b i c c e l l ( C e l l 3), o n D a y 7 0 , n i t r i t e l e v e l s i n t h e f i r s t a e r o b i c c e l l c o m m e n c e d a s l o w b u t s t e a d y r i s e . T h e i m p l e m e n t a t i o n o f i n t e r m e d i a r y d e n i t i r i f i c a t i o n a r r e s t e d a n d r e v e r s e d t h e d e c l i n e i n n i t r i t e b u i l d - u p o n t w o o c c a s i o n s ( D a y s 7 6 , a n d 9 4 ) . T h i s w a s p r o b a b l y a c h i e v e d d u e t o t h e r e m o v a l o f n i t r i t e f r o m t h e s y s t e m b e f o r e i t s ' o x i d a t i o n t o n i t r a t e , . T h e r e s u l t s c o n f i r m e d t h e f i n d i n g s o f p r e v i o u s r u n s ; t h a t f r e e a m m o n i a m a i n t a i n e d some d e g r e e o f i n h i b i t i o n t o a n a c c l i -m a t e d p o p u l a t i o n o f n i t r i t e o x i d i z e r s , s i n c e a t i m e - l a g e x i s t e d i n t h e i r a b i l i t y t o o x i d i z e n i t r i t e , w h e n c o m p a r e d t o t h e a m m o n i a o x i d i z e r ' s a b i l i t y t o o x i d i z e a m m o n i a t o n i t r i t e . T h e d u r a t i o n o f t h i s l a g , w h i c h l a s t e d a t l e a s t t w o h o u r s , w a s s u f f i c i e n t f o r t h e o x i d a t i o n o f m o s t o f t h e a m m o n i a p r e s e n t t o n i t r i t e , b u t o n l y a f r a c t i o n o f t h e n i t r i t e t o n i t r a t e . T h e i n c o r p o r a t i o n o f a d e n i -t r i f i c a t i o n s t e p a t t h i s p o i n t a l l o w e d f o r t h e r e m o v a l o f m o s t o f t h e n i t r o g e n f r o m t h e s y s t e m , w i t h a l a r g e p o r t i o n o f i t h a v i n g b e i n g o x i d i z e d o n l y t o t h e n i t r i t e i n t e r m e d i a r y ( a b o u t 90% o f t h e a m m o n i a p r e s e n t i n t h e w a s t e w a s o x i d i z e d t o n i t r i t e + n i t r a t e i n t h a t f i r s t a e r o b i c c e l l ) . T h e r e v e r s a l i n n i t r i t e d i s a p p e a r a n c e o c c u r r e d i n s p i t e o f 116 f a l l i n g f r e e a m m o n i a l e v e l s . T h e a v e r a g e f r e e a m m o n i a l e v e l d r o p p e d f r o m 1 9 . 1 mg NH3-N/L b e t w e e n -Days 35 t o 7 0 , t o 1 0 . 6 mg NH3-N/L b e t w e e n D a y s 70 t o 9 1 . T h e e x t e n t o f n i t r i t e b u i l d - u p a t t h i s p o i n t a p p e a r e d t o b e r a t h e r i n s e n s i t i v e t o m a j o r v a r i a t i o n s i n f r e e a m m o n i a l e v e l s , a s c a n b e s e e n f r o m F i g u r e 1 3 . T h e r e s u l t s c o n f i r m e d t h a t n i t r i t e b u i l d - u p c o u l d b e s u s -t a i n e d , f o r a p e r i o d o f t i m e , i n t h e p r e s e n c e o f a n i t r i f i e r b i o m a s s a c c l i m a t e d t o h i g h l e v e l s o f f r e e a m m o n i a , p r o v i d e d i n t e r m e d i a r y d e n i t r i f i c a t i o n w a s m a i n t a i n e d . F u r t h e r m o r e , t h e e f f l u e n t c o n s i s t e d o f a f u l l y n i t r i f i e d d i s c h a r g e , c o n t a i n i n g n o n i t r i t e . T h e d e c l i n e w h i c h m a n i f e s t e d i t s e l f o n D a y 1 9 9 c o u l d n o t b e r e v e r s e d , i n s p i t e o f r a i s i n g f r e e a m m o n i a l e v e l s t o 40 mg NH3-N / L . U n t i l D a y 2 4 9 , w h e n s o d i u m h y d r o x i d e a d d i t i o n w a s f i r s t s t o p p e d , t h e n i t r i t e l e v e l i n t h e f i r s t a e r o b i c c e l l d i d n o t d r o p b e l o w 3 5 % , c o n f i r m i n g t h e r e s u l t s o f e a r l i e r r u n s . T h i s d a t a b a s e s e e m e d t o s u g g e s t t h a t a m i n i m u m d e g r e e o f n i t r i t e a c c u m u l a t i o n c a n b e s u s t a i n e d " i n d e f i n i t e l y " , e v e n i n t h e p r e s e n c e o f a b i o -m a s s f u l l y a c c l i m a t e d t o f r e e a m m o n i a . E f f o r t s t o r e v e r s e t h e a c c l i m a t i o n p r o c e s s , b y t e m p o r a r i l y a l l e v i a t i n g t h e i n h i b i t o r y e f f e c t s o f f r e e a m m o n i a ( f r o m D a y s 2 4 9 t o 3 1 8 ) , p r o v e d f r u i t l e s s . T h e b i o m a s s d i d n o t l o s e i t s a c c l i m a -t i o n t o f r e e a m m o n i a , i n s p i t e o f a s u b s t a n t i a l r e d u c t i o n i n f r e e a m m o n i a l e v e l s i n t h e f i r s t c e l l ( f r o m o v e r 2 5 . 5 t o u n d e r 1 mg NH3-N/L ) f o r a n e x t e n d e d p e r i o d o f t i m e ( a l m o s t 70 d a y s ) . T h e t e m p o r a r y r i s e i n n i t r i t e l e v e l s , f o l l o w i n g r e s u m p t i o n o f f e e d o n D a y 3 5 1 , w a s p r o b a b l y c a u s e d b y t h e l o s s o f a c t i v i t y 117 of a l a r g e f r a c t i o n of the n i t r i f i e r p o p u l a t i o n during the endo-genous r e s p i r a t i o n p e r i o d , and not due to the l o s s of a c c l i m a t i n g a b i l i t y . T h i s i s p l a u s i b l e s i n c e n i t r i t e b u i l d - u p occurred i n the absence of i n h i b i t o r y l e v e l s of f r e e ammonia. S i m i l a r t o Run 6, the a b i l i t y to reduce n i t r i t e d i d not manifest i t s e l f immediately. Over one month was needed f o r n i -t r i t e r e d u c t i o n to occur. In a d d i t i o n , incomplete d e n i t r i f i c a t i o n ( i . e . , r e d u c t i o n of n i t r a t e to n i t r i t e ) was a l s o e v i d e n t during two p e r i o d s . T h i s w i l l be d i s c u s s e d i n more d e t a i l i n Chapter 6. Conclusions 1) N i t r i t e b u i l d - u p c o u l d be maintained f o r an extended p e r i o d of time i n the presence of a n i t r i f i e r biomass a c c l i m a t e d to high l e v e l s of f r e e ammonia; however, n i t r i t e b u i l d - u p c o u l d not be su s t a i n e d i n d e f i n i t e l y . 2) The e f f l u e n t c o n s i s t e d of a f u l l y n i t r i f i e d e f f l u e n t , c o n t a i n -ing no n i t r i t e . 3) The d e c l i n e i n n i t r i t e b u i l d - u p was r e v e r s e d , twice, by the implementation of intermediary d e n i t r i f i c a t i o n . 4) R a i s i n g the f r e e ammonia l e v e l d i d not r e v e r s e the d e c l i n e i n t h i s run. 5) A c c l i m a t i o n c o u l d not be rev e r s e d by a l l e v i a t i n g the i n h i b i t o -ry e f f e c t s of f r e e ammonia f o r a p e r i o d as long as 70 days. 6) A c c l i m a t i o n c o u l d not be rev e r s e d by operating the system i n an endogenous r e s p i r a t i o n mode f o r a p e r i o d of 13 days. 118 BATCH TESTS Two b a t c h t e s t s were c o n d u c t e d i n the c o u r s e o f the s e v e n r u n s . Batch T e s t N o . l T h i s ba tch t e s t was conducted at the end of Run 4. I t s o b j e c t i v e s were t o : 1) Determine the e f f e c t of a e r a t i o n time on l o s s of n i t r i t e b u i l d - u p , and q u a n t i f y the r a t e of n i t r i t e o x i d a t i o n to n i t r a t e ; 2) Assess the e f f e c t of p r o l o n g i n g the c o n t a c t t ime w i t h h i g h f r e e ammonia l e v e l s on the degree of n i t r i t e b u i l d - u p ; 3) Determine the occurrence of any s u b s t a n t i a l adherence of n i t r i f i e r s to c e l l w a l l s i n the a e r o b i c c e l l s (such p o p u l a -t i o n s would not be s u b j e c t to f r e e ammonia i n h i b i t i o n ) ; 4) Compare the n i t r i t e o x i d a t i o n r a t e between c e l l s s u b j e c t e d to d i f f e r e n t f r e e ammonia l e v e l s at s t a r t - u p . The t e s t i n v o l v e d the i s o l a t i o n of a l l four c e l l s of System 2 (which was s u s t a i n i n g n i t r i t e b u i l d - u p ) , thus c o n v e r t i n g each one to a b a t c h r e a c t o r . Feed and r e c y c l e f l ow was stopped and a i r was i n t r o d u c e d i n t o the two c e l l s ( C e l l s 1 and 2) t h a t had been m a i n t a i n e d i n an a n a e r o b i c s t a t e d u r i n g the cont inuous run . Thus , a t s t a r t - u p , C e l l s 1 and 2 were a n a e r o b i c w h i l e the c o n t e n t s of C e l l s 3 and 4 had u n d e r g o n e on a v e r a g e , t h r e e and s i x h o u r s o f a e r a t i o n r e s p e c t i v e l y . F u r t h e r m o r e , the organisms i n C e l l 1 had been s u b j e c t e d to about t h r e e hours of a n a e r o b i o s i s i n the p r e s -ence of f r e e ammonia, w h i l e t h o s e i n C e l l 2 had been s u b j e c t e d to s i x hours of a n a e r o b i o s i s . The degree of n i t r i t e b u i l d - u p was 119 83% and 55% in Cells 3 and 4 respectively. Other characteristics of the system at start-up are presented in Figure 14. Samples were collected hourly from each reactor and were analyzed for ammonia, nitrite, nitrate and pH. The test lasted until the nitrogen species present in a l l four reactors were oxidized to nitrate. The results of the test are presented in Figure 14. As noted, a l l four cells behaved almost identically, once the lag effect of aeration time is taken into account (i.e., Cells 1 and 2 lag Cel l 3 by 3 hours, which in turn lags Cel l 4 by 3 hours). This indicated that none of the parameters investigated exerted any appreciable effect on the rate of decline in nitrite build-up. These findings are discussed in more detail in Chapter 6. Specifically, the first objective of this test is discussed under the heading "Aerobic Residence Time", the second objective in "Extension of the Contact Time With Free Ammonia", the third objective in "Adherence of Nitrifiers to Cell Walls", and the fourth objective in "Raising of the Free Ammonia Levels". Batch Test No.2 This test was conducted on Day 48 of Run 5, with the objec-tive of: 1) Comparing the denitrification rates of nitrite and nitrate; 2) Confirming the lower COD requirements associated with the dissimilatory reduction of nitrite, as compared to nitrate; 3) Comparing biomass production rates between the dissimilatory reduction of nitrite and nitrate. 120 5 . 8 m g N H . - N / L 6 . 3 ( t y p . ) R6 .3 <t C o n d i t i o n s o l S t a r t u p i C e l l 1 c o n t e n t s a n a e r o b i c f o r 3 h o u r s C e l l 2 c o n t e n t s a n a e r o b i c f o r 6 h o u r s C e l l 3 c o n t e n t s a e r o b i c f o r 3 h o u r s C e l l A c o n t e n t s a e r o b i c f o r 6 h o u r s 100 - 8 0 CO 6 0 " E 40 Z 2 0 A 5 6 T I M E (hou rs ) F ig .14 : B a t c h Test N o . l - Run 4 / S y s t e m 2 N i t r o g e n T r a n s f o r m a t i o n s 121 T h e t i m i n g o f B a t c h T e s t N o . 2 w a s n o t i d e a l ( s e e F i g u r e 1 0 ) . I t w o u l d h a v e b e e n d e s i r a b l e t o a l l o w a l o n g e r i n t e r v a l t i m e b e t w e e n t h e d i s a p p e a r a n c e o f n i t r i t e i n t h e r e c y c l e f l o w o f S y s t e m 1 ( i . e . , i n C e l l 4) a n d t h e i n i t i a t i o n o f t h e b a t c h t e s t , t o e n s u r e t h e c o m p l e t i o n o f t h e s h i f t i n m i c r o b i a l p o p u l a t i o n ( t h i s c a n n o r m a l l y b e e x p e c t e d a s a r e s u l t o f c h a n g e s i n o p e r a -t i n g c h a r a c t e r i s t i c s ) . T h e d e c i s i o n t o p r o c e e d w i t h t h e b a t c h t e s t a t t h a t d a t e w a s p r o m p t e d b y t w o c o n s i d e r a t i o n s ; t h e e v e r -d e c l i n i n g l e v e l s o f n i t r i t e i n S y s t e m 2 , a n d t h e c o n s t r a i n t s i m p o s e d f r o m t h e s c h e d u l i n g o f o t h e r a c t i v i t i e s i n t h e l a b o -r a t o r y . F o u r , o n e - l i t r e b a t c h r e a c t o r s w e r e s e t - u p a s s h o w n i n F i g u r e 15 ( C o m e a u , 1 9 8 4 ) . S l u d g e w a s w i t h d r a w n s e p a r a t e l y f r o m e a c h c o n t i n u o u s l y r u n s y s t e m a n d a d d e d t o a d e s i g n a t e d b a t c h r e a c t o r . T h e l i q u i d a n d a i r w i t h i n e a c h r e a c t o r w a s f l u s h e d w i t h h e l i u m g a s t o r e m o v e a l l t r a c e s o f o x y g e n p r i o r t o c o m m e n c e m e n t o f t h e t e s t . E a c h r e a c t o r w a s s p i k e d a t s t a r t - u p w i t h a p r e d e t e r -m i n e d a m o u n t o f c o n c e n t r a t e d COD a n d n i t r a t e , o r n i t r i t e , s o l u -t i o n . P o s i t i v e h e l i u m p r e s s u r e w a s m a i n t a i n e d i n a l l r e a c t o r s t h r o u g h o u t t h e t e s t b y c o n n e c t i o n t o a b a l l o o n f i l l e d w i t h h e l i u m g a s . T h e s o l u t i o n c a r b o n c o n t e n t w a s i d e n t i c a l t o t h a t u s e d d u r i n g t h e c o n t i n u o u s r u n . T h e c h a r a c t e r i s t i c s o f t h e t w o c o n t i -n u o u s l y - r u n s y s t e m s a t t h e t i m e w e r e a s f o l l o w s : S y s t e m 1 : T h e r e c y c l e f l o w c o n t a i n e d e x c l u s i v e l y n i t r a t e f r o m D a y 4 1 o n w a r d s . S y s t e m 2 : T h e r e c y c l e f l o w f r o m C e l l 2 c o n t a i n e d i n e x c e s s o f 80% n i t r i t e , w h i l e t h e r e c y c l e f r o m t h e f i n a l c l a r i f i e r 1 2 2 Figure 15 - Batch Test No. 2 Reactor ( w h i c h r e f l e c t e d t h e n i t r o g e n c o n t e n t o f t h e l a s t c e l l ) c o n t a i n e d o x i d i z e d n i t r o g e n i n o n l y t h e n i t r a t e f o r m . I n v i e w o f t h e f l o w r a t e s a s s o c i a t e d w i t h e a c h r e c y c l e l i n e ( s e e F i g u r e 9 ) , t h e o v e r a l l r e c y c l e f l o w c o n t a i n e d a b o u t 75% n i t r i t e - N a n d 25% n i t r a t e - N . C o n d i t i o n s a t s t a r t - u p o f t h e t e s t w e r e a s f o l l o w s : R e a c t o r 1 : B i o m a s s f r o m S y s t e m 1 , a c c l i m a t e d t o n i t r a t e r e d u c t i o n , s p i k e d w i t h n i t r i t e s o l u t i o n . R e a c t o r 2 : B i o m a s s f r o m S y s t e m 1 , a c c l i m a t e d t o n i t r a t e r e d u c t i o n , s p i k e d w i t h n i t r a t e s o l u t i o n . R e a c t o r 3 : B i o m a s s f r o m S y s t e m 2 , a c c l i m a t e d t o n i t r i t e a n d n i t r a t e r e d u c t i o n , s p i k e d w i t h n i t r i t e s o l u t i o n . R e a c t o r 4 : B i o m a s s f r o m S y s t e m 2 , a c c l i m a t e d t o n i t r i t e a n d n i t r a t e r e d u c t i o n , s p i k e d w i t h n i t r a t e s o l u t i o n . T h e r e s u l t s a r e p r e s e n t e d i n F i g u r e 16 a n d T a b l e 1 4 . T h e y c o n f i r m e d t h e l o w e r COD r e q u i r e m e n t s a n d f a s t e r d i s s i m i l a t o r y r e d u c t i o n r a t e s a s s o c i a t e d w i t h t h e u s e o f n i t r i t e a s e l e c t r o n a c c e p t o r , a s o p p o s e d t o n i t r a t e . I t a l s o r e v e a l e d t h e l o w e r b i o m a s s y i e l d r e s u l t i n g f r o m t h e d i s s i m i l a t o r y r e d u c t i o n o f n i -t r i t e . T h e s e f i n d i n g s a r e d i s c u s s e d i n m o r e d e t a i l i n C h a p t e r 6 u n d e r " E f f e c t s o f N i t r i t e B u i l d - U p " . 124 0 60 120 180 240 300 360 T I M E (minutes) F I G . 1 6 : B a t c h Test N o . 2 / R u n 5 D e n i t r i f i c a t i o n R a t e s f o r N i t r i t e a n d N i t r a t e 125 Table 14 - Resu l t s of Batch Test No.2 Reactor Biomass N0£ Max. mg COD mg VSS Produced No. Acclimated Present D e n i t . Consumed per To Reduction As Rate per of mg NO^-N mg NO^-N mg COD Reduced Reduced Consumed 1 n i t r a t e n i t r i t e 36.3 3.35 0.45 0.136 2 n i t r a t e n i t r a t e 30.0 5.31 1.54 0.360 3 n i t r i t e n i t r i t e 48.8 3.34 0.54 0.162 4 n i t r i t e n i t r a t e 29.7 6.86 1.64 0.238 D e n i t r i f i c a t i o n r a t e expressed as mg NOm—N reduced/g VSS.hr 126 CHAPTER SIX DISCUSSION This research program d e a l t with numerous aspects p e r t a i n i n g to the proposed n i t r o g e n removal s h o r t c u t . These can be grouped under the f o l l o w i n g headings, each of which w i l l be d i s c u s s e d s e p a r a t e l y i n t h i s chapter: 1) Causes of n i t r i t e b u i l d - u p 2) E f f e c t s of n i t r i t e b u i l d - u p 3) S t a b i l i t y of n i t r i t e b u i l d - u p 4) Reversing the d e c l i n e i n n i t r i t e b u i l d - u p 5) Other r e s u l t s and o b s e r v a t i o n s CAUSES OF NITRITE BUILD-UP Four p o s s i b l e causes f o r n i t r i t e accumulation were i n v e s t i -gated: 1) Low d i s s o l v e d oxygen l e v e l s 2) High n i t r o u s a c i d l e v e l s 3) A n a e r o b i o s i s 4) High f r e e ammonia l e v e l s Low D i s s o l v e d Oxygen Leve l s T h i s was i n v e s t i g a t e d during Runs 1 and 2. In Run 1, a frequency p l o t of DO c o n c e n t r a t i o n s i n System 2 (where DO c o n t r o l was maintained) d i s p l a y e d a p o s i t i v e skewness. This d i s t r i b u t i o n was t y p i c a l f o r a l l c e l l s of System 2 and r e f l e c t e d the opera-127 t i o n a l c o n s t r a i n t s i m p o s e d u p o n DO l e v e l s ; t h e y w e r e c o n t r o l l e d a n d m a i n t a i n e d , a s m u c h a s p o s s i b l e , b e l o w 1 m g / L . S i n c e DO l e v e l s c a n n o t f a l l b e l o w 0 m g / L , b u t c o u l d r i s e t o a r o u n d 8 m g / L , i t f o l l o w s t h a t a n a s y m m e t r i c d i s t r i b u t i o n , w o u l d r e s u l t . A s y m m e t r i c d i s t r i b u t i o n s d o n o t l e n d t h e m s e l v e s t o s t a t i s t i -c a l a n a l y s i s w i t h o u t t r a n s f o r m a t i o n t o a n o r m a l d i s t r i b u t i o n . T h e y c a n , h o w e v e r , b e a d e q u a t e l y d e s c r i b e d b y o t h e r m e a s u r e s s u c h a s f r a c t i l e s . T a b l e 13 s u m m a r i z e s t h e r e s u l t s o f a f r a c t i l e a n a l y s i s . T h e m e a n DO l e v e l s a r e t y p i c a l l y h i g h e r t h a n t h e m e d i a n v a l u e s , c o n f i r m i n g t h e p o s i t i v e s k e w n e s s o f t h e d a t a . A l t h o u g h t h e v a l u e s f o r S y s t e m 1 c o r r e s p o n d e d t o a n o r m a l d i s t r i b u t i o n , t h e y w e r e a n a l y z e d a s f r a c t i l e s f o r p u r p o s e s o f u n i f o r m i t y a n d c o m p a r i s o n . A s c a n b e n o t e d f r o m T a b l e .1 .3, DO l e v e l s i n C e l l s .2 a n d 4 o f S y s t e m 1 ( w h i c h a c h i e v e d n i t r i t e a c c u m u l a t i o n ) e x c e e d e d 2 m g / L a t l e a s t 9 0 % o f t h e t i m e , w h i l e i n C e l l 3 i t e x c e e d e d i t 75% o f t h e t i m e . I n c o n t r a s t , t h e DO l e v e l i n a l l c e l l s o f S y s t e m 2 ( w h e r e n o n i t r i t e a c c u m u l a t i o n o c c u r r e d ) w e r e b e l o w 1 m g / L a b o u t 75% o f t h e t i m e a n d b e l o w 0 . 6 m g / L o v e r 50% o f t h e t i m e . E v i d e n t -l y , t h e s e l o w DO v a l u e s d i d n o t i n d u c e a n y d e t e c t a b l e n i t r i t e b u i l d - u p i n S y s t e m 2 . I t d i d , h o w e v e r , s e e m t o i n h i b i t a m m o n i u m o x i d a t i o n a c t i v i t y , s i n c e a m m o n i u m l e v e l s i n a l l c e l l s o f S y s t e m 2 f a r e x c e e d e d t h e l e v e l i n t h e c o r r e s p o n d i n g a e r o b i c c e l l s o f S y s t e m 1 . I n R u n 2 , t h e DO l e v e l i n t h e f i r s t a e r o b i c c e l l o f S y s t e m 2 a v e r a g e d 1 . 4 m g / L t h r o u g h o u t t h e r u n , w h e r e a s i t a v e r a g e d 1 . 0 m g / L i n t h e c o r r e s p o n d i n g c e l l o f S y s t e m 1 d u r i n g t h e f i r s t t w o 128 stages of that run (Days 1 to 37). Nitrite build-up was not sustained in System 1, and was only sustained in System 2 during the latter part of the run (as a result of raising NH3). In summary, nitrite build-up could not be induced nor sus-tained at DO levels as low as 0.6 mg/L. The results of Runs 1 and 2 appear to rule out this condition as being able to cause accu-mulation of nitrite. Findings of other investigators (Laudelout et a l . , 1976; Tanaka e_t a_l., 1981; Tanaka and Dunn, 1982) linking nitrite build-up to low DO levels could not be confirmed, even at the relatively low DO levels maintained during Runs 1 and 2. High Nitrous Acid Levels The ability of nitrous acid to induce and sustain nitrite build-up was investigated in Runs 2 and 3. In the latter part of Run 2, the pH was lowered within the system, which thus raised the nitrous acid concentration. .This led to a gradual rise in nitrous acid levels in the f irst aerobic cel l of System .2 (which was sustaining nitrite accumulation), from an average 7 ug HN02-N/L, between Days 79 to 119, to over 28 ug HN02-N/L, between Days 136 and 142. A negative correlation* (Figure 17) seemed to exist between the nitrous acid level in that cel l and the total concen-tration of nitrite species (N02 + HN02). This indicated that' nitrite build-up increased as the nitrous acid level decreased. No correlation could be established between the nitrous acid levels in the remaining aerobic cells and the degree of nitrite build-up (see Figure 17). *: The curves presented in this chapter are best-fit nonlinear monotone curves, and the equations from which they are derived are not intended for use for modelling purposes since the assumptions of regression analysis were not rigorously met. 129 Day 8 6 t o 1 4 4 , • Y - 9 7 . 3 - 1 6 4 X " r * 1 . 9 8 X r. R - 0 . 8 6 n - 2 5 THIRD AEROBIC CELL • Y - 1 0 5 . 4 - 1 0 9 . 5 X " ' * 0 . 3 4 X • R - 0 . 8 8 n - 2 5 D a y 8 6 t o 1 4 4 SECOND AEROBIC CELL Y - 1 1 3 . 6 - 6 0 . 8 X " ' - 1 . 0 7 X R - 0 . 8 5 n - 2 5 D a y 8 6 t o 1 4 4 FIRST AEROBIC CELL (mgNHj-N/L) I I I 1 - 1 0 0 0 1 2 3 4 5 6 7 8 9 0 A M M O N I A IN A N A E R O B I C C E L L D a y 8 6 t o 1 4 7 n - 2 4 THIRD AEROBIC CELL - 8 0 - 6 0 - 4 0 . 2 0 o z o CO < D a y 8 6 t o 1 4 7 n - 2 5 SECOND AEROBIC CELL 1 0 0 8 0 6 0 - 4 0 - 2 0 o z u_ o CO < Z i o z Y - I 0 0 . 5 - 0 . 5 8 X + 0 . 0 0 4 8 X R - 0 . 4 8 n - 2 6 D a y 8 6 t o 1 4 7 FIRST AEROBIC CELL 1 0 0 - 6 0 - 4 0 - 2 0 O >? co < Z I CN o z 1-0 10 2 0 3 0 4 0 5 0 H N 0 2 - N C u g / L ) Fig. 17: Effect of Free Ammonia and Nitrous Acid on Nitrite Build-Up - Run 2/System 2 130 In Run 3, the absence of free ammonia and presence of r e l a -t i v e l y large concentrations of nitrous acid r e s u l t i n g from pH reduction, ranging from 10 to 40 ug HN02-N/L in a l l c e l l s (with a maximum of 168 ug HN02-N/L), f a i l e d to sustain n i t r i t e build-up. In summary, n i t r o u s a c i d d i d not appear to p l a y a r o l e in i n i t i a t i n g or sustaining n i t r i t e build-up in any of the runs. The r e s u l t s appear to be in agreement with the findings of O'Kelley et a_l. (1970) who determined that the substrate for Nitrobacter was nitrous acid and not n i t r i t e ion. In this l i g h t , increasing the concentration of nitrous acid in the medium may have resulted in a higher rate of metabolism and a c t i v i t y for the n i t r i t e oxidizers, leading to a reduction in n i t r i t e build-up. The i n a b i l i t y to l i n k n i t r i t e build-up to the presence of nitrous acid is in contradiction to the recent findings of Gar-rett (1982). In view of this apparent contradiction i n r e s u l t s , a brief review of Garrett's work i s in order. His r e s u l t s were based on a 40-month laboratory-scale n i t r i f i c a t i o n study, using attached growth medium (on thin a c r y l i c plates). Six, 10 L capa-c i t y flow-through reactors, with no suspended s o l i d s recycle, were fed synthetic waste at a rate of 10 L/d. The ammonia feed concentration ranged between 200 and 400 mg N/L. One of the major conclusions of h i s work was that n i t r o u s acid concentrations above 7.9 _+ 3.1 ug HN02-vN/L (authors values based on a d i f f e r e n t dissociation constant: 9.3 + 3.7 ug HN02-N/L) at 30°C, s e l e c t i v e l y inhibited Nitrobacter metabolism and led to n i t r i t e accumulation in excess of 80 % of the t o t a l o x i -dized nitrogenous compounds. The amount of data found in his published thesis is limited. 131 For example, out of 1231 operating days f o r Reactor 1, G a r r e t t s e l e c t e d a t o t a l of 246 days, c o v e r i n g e i g h t p e r i o d s , f o r a n a l y -s i s . The d u r a t i o n of each p e r i o d ranged between 12 and 44 days. For each of these p e r i o d s , a s i n g l e average v a l u e was reported per parameter s t u d i e d (e.g., pH, NOj, NH^, and so on). S t a n d a r d d e v i a t i o n and sample number were not reported. The author j u s t i -f i e d u s i n g o n l y p a r t of the d a t a on the grounds t h a t i t r e p r e s e n -ted p e r i o d s where "the r e a c t o r s operated without any upsets and showed the same amount of ammonia co n v e r s i o n to n i t r i t e and n i t r a t e without any change i n ammonia or organic carbon concen-t r a t i o n s " . Having l i n k e d n i t r i t e b u i l d - u p to the presence of n i t r o u s a c i d , G a r r e t t f a i l e d to r e c o n c i l e h i s c o n c l u s i o n with an obvious que s t i o n , namely, the mechanism r e s p o n s i b l e f o r causing n i t r i t e accumulation to occur i n the f i r s t p l a c e . He r e j e c t e d f r e e ammo-nia as the cause of n i t r i t e accumulation, based on h i s f i n d i n g s t h a t f r e e ammonia l e v e l s as h i g h as 2.2 mg NH^-N/L were not i n h i b i t o r y to the n i t r i t e o x i d i z e r s . Because of these d i s c r e p a n -c i e s , i t i s reasonable to s t a t e that i n s u f f i c i e n t s u b s t a n t i a t i o n was presented to support h i s hypothesis. A n a e r o b i o s i s The r e s u l t s from Run 2 (stage 2) c l e a r l y i n d i c a t e d that a n a e r o b i o s i s , per se, was not a f a c t o r d i r e c t l y r e s p o n s i b l e f o r causing n i t r i t e b u i l d - u p . The presence of an anaerobic e n v i r o n -ment at the front-end of the system was i n d i r e c t l y r e s p o n s i b l e for n i t r i t e b u i l d - u p , s i n c e i t a l l o w e d ammonium to accumulate i n that c e l l . T h i s , i n c o n j u n c t i o n with m a n i p u l a t i o n of pH l e v e l s , 132 permitted the f r e e ammonia c o n c e n t r a t i o n i n the anaerobic c e l l to r i s e to l e v e l s that s e l e c t i v e l y i n h i b i t e d the n i t r i t e o x i d i z e r s . Extension of the p e r i o d of a n a e r o b i o s i s from 3 to 6 hours d i d not appear to enhance n i t r i t e b u i l d - u p , as confirmed during Batch Test No.l, conducted at the end of Run 4; r e s u l t s f o r C e l l 1, subjected to 3 hours of a n a e r o b i o s i s , and C e l l 2, subjected to 6 hours a n a e r o b i o s i s , were i d e n t i c a l (see F i g u r e 14).. High Free Ammonia Le v e l s The f r e e ammonia l e v e l i n the f i r s t c e l l was c o n t r o l l e d by means o f : 1) pH adjustment 2) changes i n r e c y c l e flow r a t e 3) changes i n TKN (and ammonium) feed c o n c e n t r a t i o n pH adjustment was most commonly used (see Tables 10 and 12). U n l i k e pH adjusment, the other two modes of f r e e ammonia c o n t r o l e n t a i l e d a concomitant change i n the ammonium ion c o n c e n t r a t i o n . T h i s r a i s e d the p o s s i b i l i t y t h a t the ammonium ion may a l s o have pl a y e d a r o l e i n i n h i b i t i n g the n i t r i t e o x i d i z e r s . The r e s u l t s of Run 2, however, tend to c o n t r a d i c t t h i s p o s t u l a t e . The t o t a l ammonia (ammonium ion p l u s f r e e ammonia) c o n c e n t r a t i o n i n the f i r s t c e l l of each system was s i m i l a r during that run (see Tabl e 12), yet System 2 sustained n i t r i t e accumulation (during stage 4) w h i l e System 1 d i d not. The r e s u l t s o f Run 7 a l s o c o n f i r m s t h i s f i n d i n g ; the t o t a l ammonia l e v e l i n the f i r s t c e l l was r e l a t i v e l y constant during the run, yet n i t r i t e b u i l d - u p was onl y observed f o l l o w i n g a r i s e i n the pH (and hence the f r e e ammonia) l e v e l . The r e s u l t s from the seven runs i n d i c a t e that f r e e ammonia 133 w a s r e s p o n s i b l e f o r i n i t i a t i n g a n d s u s t a i n i n g n i t r i t e a c c u m u l a -t i o n i n t h e s y s t e m . F r e e a m m o n i a l e v e l s w e r e g e n e r a l l y c o n t r o l l e d b y pH m a n i p u l a t i o n . T h e e x c e p t i o n s w e r e R u n s 2 , 4 a n d 5 w h e r e t o t a l ammonium l e v e l s i n t h e f e e d w e r e r a i s e d o n c e d u r i n g e a c h r u n ( s e e T a b l e 7 ) . F r o m t h e r e s u l t s o b t a i n e d i t w a s n o t p o s s i b l e t o d i s c e r n a n y d i f f e r e n c e b e t w e e n t h e u s e o f pH a d j u s t m e n t o r s p i k i n g o f t h e f e e d , a s m e c h a n i s m s t o c h a n g e t h e a m m o n i a f o r m w i t h i n t h e s y s t e m . I n i t i a t i o n o f some d e g r e e o f n i t r i t e b u i l d -u p ( a r o u n d 5 0 % ) , w i t h a n o n - a c c l i m a t e d b i o m a s s , a n d i t s s h o r t -t e r m a c c u m u l a t i o n w a s a c h i e v e d b y r a i s i n g f r e e a m m o n i a l e v e l s t o a r o u n d 1 t o 3 mg NH3 - N / L , a s e v i d e n c e d d u r i n g R u n s 2 ( S y s t e m 1 ) , 3 a n d 5 ( S y s t e m 1 ) . I n o r d e r t o i n d u c e a n d s u s t a i n h i g h e r l e v e l s o f n i t r i t e b u i l d - u p w i t h a n o n - a c c l i m a t e d b i o m a s s , i t w a s g e n e r -a l l y n e c e s s a r y t o r a i s e t h e f r e e a m m o n i a l e v e l t o a r o u n d 8 mg NH3 - N / L , w h i c h n o r m a l l y r e s u l t e d i n a m a j o r r e d u c t i o n o f f i r s t s t a g e n i t r i f i c a t i o n a c t i v i t y . U p o n r e s u m p t i o n o f n i t r i f i c a t i o n a c t i v i t y ( u s u a l l y w i t h i n 2 t o 7 d a y s ) , n i t r i t e b u i l d - u p w a s o b s e r v e d . T h e f r e e a m m o n i a c o n c e n t r a t i o n n e e d e d t o s u c c e s s f u l l y i n i t i a t e a n d s u s t a i n n i t r i t e b u i l d - u p w a s s u b s t a n t i a l l y h i g h e r t h a n t h a t r e p o r t e d b y A n t h o n i s e n e t a _ l . ( 1 9 7 6 ) ( w h i c h r a n g e d f r o m 0 . 1 t o 1 . 0 mg NH3 - N / L ) , a n d c o n f i r m e d b y o t h e r s ( V e r s t r a e t e e t a l . , 1 9 7 7 ; A l l e m a n a n d I r v i n e , 1 9 8 0 ) . No s a t i s f a c t o r y e x p l a n a t i o n f o r t h i s d i s c r e p a n c y c a n b e a d v a n c e d . E F F E C T S OF N I T R I T E B U I L D UP A n u m b e r o f p o t e n t i a l a d v a n t a g e s a n d d i s a d v a n t a g e s , a s s o c i a t e d w i t h n i t r i t e b u i l d - u p , w e r e i n v e s t i g a t e d a n d w i l l b e d i s c u s s e d h e r e . T h e y i n c l u d e : 134 1) C o m p a r i s o n o f COD d e m a n d f o r n i t r i t e a n d n i t r a t e r e d u c t i o n 2) C o m p a r i s o n o f g r o w t h y i e l d i n t h e p r e s e n c e o f n i t r i t e a n d n i t r a t e d u r i n g a n a e r o b i c r e s p i r a t i o n 3) C o m p a r i s o n o f n i t r a t e a n d n i t r i t e r e d u c t i o n r a t e s 4) C o m p a r i s o n o f a l k a l i n i t y c o n s u m p t i o n a n d p r o d u c t i o n r a t e s d u r i n g t h e p r o d u c t i o n a n d r e d u c t i o n o f n i t r i t e a n d n i t r a t e 5) E f f e c t s o f n i t r i t e o n t h e t r e a t m e n t p r o c e s s p e r f o r m a n c e 6) A b i l i t y t o p r o d u c e a n e f f l u e n t d e v o i d o f n i t r i t e 7) A e r o b i c r e d u c t i o n o f n i t r i t e C o m p a r i s o n o f COD Demand F o r N i t r i t e a n d N i t r a t e R e d u c t i o n T h e COD d e m a n d f o r n i t r a t e a n d n i t r i t e r e d u c t i o n w e r e c o m -p a r e d i n R u n s 2 a n d 4 a n d i n B a t c h T e s t N o . 2 ( R u n 5 ) . I n R u n 2 , f r o m D a y 82 o n w a r d s , t h e o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t i n t h e r e c y c l e f l o w t o t h e a n a e r o b i c c e l l o f S y s t e m 1 c o n s i s t e d e n t i r e l y o f n i t r a t e , w h e r e a s i n S y s t e m 2 , i t w a s a b o u t 90 % n i t r i t e . T h i s p r o v i d e d a n o p p o r t u n i t y t o c a r r y o u t a l o n g -t e r m c o m p a r i s o n b e t w e e n t h e c a r b o n r e q u i r e m e n t s f o r t h e r e d u c t i o n o f n i t r a t e a n d t h a t o f n i t r i t e . T h e a v e r a g e COD d e m a n d f o r t h e r e d u c t i o n o f n i t r a t e w a s 4 . 9 5 mg COD c o n s u m e d / m g NO3-N r e d u c e d (s - 0 . 6 , n = 1 0 ) , w h e r e a s t h e r a t i o f o r t h e r e d u c t i o n o f n i t r i t e w a s 2 . 8 0 mg COD c o n s u m e d / m g NO^-N r e d u c e d ( s = 0 . 4 , n = 1 0 ) . T h e d i f f e r e n c e w a s s t a t i s t i c a l l y s i g n i f i c a n t a n d i n d i c a t e d t h a t n i t r i t e - N r e d u c t i o n r e q u i r e d a b o u t 43 % l e s s o r g a n i c c a r b o n t h a n d i d n i t r a t e - N r e d u c t i o n . T h i s w a s c o n f i r m e d d u r i n g R u n 4 . A s i m i l a r t e s t c a r r i e d o u t o n D a y 90 e x h i b i t e d a CODrNO^-N r a t i o o f 4 . 0 : 1 f o r S y s t e m 1 ( w h i c h w a s r e d u c i n g n i t r a t e e x c l u s i v e l y ) a n d a r a t i o o f 2 . 3 : 1 f o r S y s t e m 2 1 3 5 (which was reducing mostly nitrite). The COD consumption for nitrite-N reduction was 42% lower than for nitrate-N reduction. A comparison between the COD consumption rates for the reduction of nitrite and that of nitrate undertaken in Batch Test No.2 is presented in Table 14. The consumption rates for nitrite were similar for both reactors undergoing nitrite reduction (Re-actors 1 and 3), but were significantly different between the two reactors undergoing nitrate reduction (Reactors 2 and 4). The COD consumption rate for nitrate in Reactor 4, containing nitrite acclimated sludge, was about 30% higher per mg of NO^ -N reduced than was the COD consumption rate for nitrate in Reactor 2, which contained nitrate acclimated sludge. This suggested the absence of efficient nitrate reducers in the nitrite acclimated sludge. As a result., the COD consumption data for Reactor 4 was not used for comparison purposes, since it was not deemed to be represen-tative of nitrate reduction rates. Comparison of results from the three other reactors showed that the reduction of nitrite-N required about 37% less COD than did the reduction of nitrate-N. The above results for Runs 2 and 4 and Batch Test No.2 are close to the theoretical difference of 40 %, based upon the difference in number of electrons needed to reduce each species to nitrogen gas: NO3 3» N0 2 s> N2 + 5 +3 0 The reduction of nitrate to nitrogen gas requires the trans-fer of five electrons from a reduced compound, whereas the reduction of nitrite requires the transfer of only three elec-136 trons; t h i s represents a 40 % r e d u c t i o n i n number of e l e c t r o n s t r a n s f e r r e d . Since organic carbon i s no r m a l l y used as e l e c t r o n donor, i t f o l l o w s that the r e s u l t a n t r e d u c t i o n i n carbon demand i s a l s o i n the order of 40%. The r e s u l t s are i n gen e r a l agreement with those reported by Bl a s z c z y k et a l . (1981), who found that the d e n i t r i f i c a t i o n of n i t r i t e r e q u i r e d 37% l e s s a c e t i c a c i d than d i d the d e n i t r i f i c a -t i o n of n i t r a t e . However, i t c o n t r a d i c t s the f i n d i n g s of Halmo and E i m h j e l l e n (1981), who reported higher methanol requirements fo r n i t r i t e r e d u c t i o n than f o r n i t r a t e r e d u c t i o n i n a bench-scale system. These authors a l s o reported that the n i t r o g e n content of the e f f l u e n t was higher than i n the i n f l u e n t , during the p e r i o d of n i t r i t e b u i l d - u p , but o f f e r e d no s a t i s f a c t o r y e x p l a n a t i o n f o r t h i s discrepancy. Although the COD consumption to NO^-N r e d u c t i o n r a t i o was always h i g h e r than the t h e o r e t i c a l v a l u e o f 3.7 : 1 ( i t a v e r a g e d 4.95 : 1 d u r i n g Run 2 and 5.3 : 1 d u r i n g B a t c h T e s t No.2) , i t was in agreement with some of those reported i n the l i t e r a t u r e (see T a b l e 15). The h i g h e r r a t i o may have been caused by the r e m o v a l of p a r t of the a v a i l a b l e COD by means other than d i s s i m i l a t o r y n i t r a t e r e d u c t i o n . These i n c l u d e , carbon storage w i t h i n the c e l l , s u l p h a t e r e d u c t i o n , and a e r o b i c r e s p i r a t i o n (at the s u r f a c e of the r e a c t o r ) . Comparison of Growth Y i e l d During Anaerobic R e s p i r a t i o n The amount of b a c t e r i a l growth (i.e., VSS production) i s d i r e c t l y p r o p o r t i o n a l to the amount of ATP which can be obtained from the c a t a b o l i s m of the energy y i e l d i n g s u b s t r a t e (Stouthamer, 137 1 9 7 6 ) . When t h e t y p e o f e l e c t r o n a c c e p t o r s ( i . e . , 0 2, NO3 o r N0 2) i s t h e f a c t o r r e g u l a t i n g g r o w t h , g r o w t h y i e l d s t u d i e s p r o v i d e i n f o r m a t i o n c o n c e r n i n g t h e r e l a t i v e e f f i c i e n c i e s o f ATP p r o d u c -t i o n d u r i n g r e s p i r a t i o n w i t h t h a t p a r t i c u l a r e l e c t r o n a c c e p t o r . C o m p a r i s o n o f g r o s s b a c t e r i a l y i e l d b e t w e e n n i t r i t e a n d n i t r a t e r e s p i r a t i o n w a s u n d e r t a k e n d u r i n g B a t c h T e s t N o . 2 ( R u n 5 ) . T h e b a c t e r i a l y i e l d w a s m e a s u r e d i n d i r e c t l y a s t h e V S S o f t h e b i o m a s s , r e c o g n i z i n g t h a t t h e V S S i s n o t c o m p o s e d e n t i r e l y o f v i a b l e b a c t e r i a l g r o w t h , b u t c o n t a i n s o t h e r c o m p o n e n t s s u c h a s d e a d c e l l s a n d e x t r a c e l l u l a r m a t e r i a l . V S S p r o d u c t i o n r a t e s , d e f i n e d a s t h e r a t i o o f V S S p r o d u c e d p e r u n i t o f NO^-N r e d u c e d , a r e p r e s e n t e d i n T a b l e 1 4 . A s c a n b e s e e n f r o m c o m p a r i n g t h e r e s u l t s o f R e a c t o r s 1 a n d 2 w i t h t h o s e o f R e a c t o r s 3 a n d 4 , t h e r e d u c t i o n o f n i t r i t e - N p r o d u c e d a b o u t t h r e e t i m e s l e s s b i o m a s s t h a n d i d t h e r e d u c t i o n o f n i t r a t e - N . A 40% d i f f e r e n c e i n b i o m a s s p r o d u c t i o n w a s a n t i c i p a t e d , s i n c e t h e r e -d u c t i o n o f n i t r i t e r e q u i r e s 40% l e s s COD t h a n d o e s t h e r e d u c t i o n o f n i t r a t e ( r e s u l t i n g i n a t h e o r e t i c a l 40% r e d u c t i o n i n b i o m a s s , i f g r o w t h y i e l d s a r e e q u a l ) . T h e t h r e e - f o l d d i f f e r e n c e i n V S S p r o d u c t i o n w a s c o n s i s t e n t i n a l l t e s t s , r e g a r d l e s s o f t h e s t a t e o f a c c l i m a t i o n o f t h e b i o m a s s ; n i t r i t e a n d n i t r a t e a c c l i m a t e d b i o m a s s s h o w e d v e r y s i m i l a r s o l i d s p r o d u c t i o n r a t e s f o r b o t h n i t r i t e a n d n i t r a t e . T h e s e r e s u l t s s t r o n g l y s u g g e s t t h a t t h e b a c t e r i a l g r o w t h y i e l d , d u r i n g n i t r i t e r e s p i r a t i o n , w a s s u b s t a n t i a l l y l o w e r t h a n d u r i n g n i t r a t e r e s p i r a t i o n . T h e a m o u n t o f b a c t e r i a l g r o w t h i s d i r e c t l y p r o p o r t i o n a l t o t h e a m o u n t o f ATP w h i c h c a n b e o b t a i n e d f r o m t h e c a t a b o l i s m o f t h e e n e r g y y i e l d i n g s u b s t r a t e . S i n c e 138 i d e n t i c a l s u b s t r a t e was used i n a l l four r e a c t o r s , the r e s u l t s obtained are a d i r e c t r e f l e c t i o n of the r e l a t i v e e f f i c i e n c i e s of ATP production, a s s o c i a t e d with the e l e c t r o n acceptor present ( i . e . , n i t r i t e or n i t r a t e ) . A comparison of these r e s u l t s with l i t e r a t u r e v a l u e s , f o r other wastewater treatment systems, was not p o s s i b l e , s i n c e no s i m i l a r s t u d i e s appear to have been reported ( r e s u l t s from pure c u l t u r e work are presented l a t e r ) . S e v e r a l s t u d i e s have, however, reported growth y i e l d v a l u e s from the use of n i t r a t e as s o l e e l e c t r o n acceptor i n b i o l o g i c a l wastewater treatment systems. Most of them appear to have used methanol as e l e c t r o n donor, and Tab l e 15 summarizes some of the reported v a l u e s . As noted, the VSS production r a t e s reported i n the l i t e r a t u r e are g e n e r a l l y lower than the ones obtained during Batch Test No.2. T h i s discrepancy i s p a r t i a l l y due to the d i f f e r -ing experimental c o n d i t i o n s . A l l the l i t e r a t u r e v a l u e s represent net y i e l d s obtained from c o n t i n u o u s l y - r u n systems, where b a c t e r i -a l decay i s c o n t i n u o u s l y taking p l a c e . The growth y i e l d obtained from the batch t e s t , on the other hand, represents gross y i e l d , s i n c e i t i s of short d u r a t i o n with a l l s u b s t r a t e s present i n excess. Furthermore, the r e s u l t s of Moore and Schroeder (1970 and 1971) suggests that the growth y i e l d i s dependent, to some ex-t e n t , on o t h e r v a r i a b l e s such as SRT and n i t r a t e c o n t e n t of the feed. Some pure c u l t u r e s t u d i e s have reported on the comparative growth y i e l d between n i t r i t e and n i t r a t e r e s p i r a t i o n . In s t u d i e s on Aerobacter aerogenes, Hadjipetrou and Stouthamer (1965) found that the growth y i e l d (g/mole of glucose) during n i t r i t e r e s p i r a -139 T a b l e 15 - C o m p a r i s o n o f L i t e r a t u r e V a l u e s f o r COD C o n s u m p t i o n a n d C e l l Y i e l d R e s u l t i n g f r o m D i s s i m i l a t o r y N i t r a t e R e d u c t i o n w i t h R e s u l t s o f t h i s S t u d y mg COD mg V S S P r o d u c e d C o n s u m e d p e r p e r mg NO^,-N mg NOi^-N mg COD R e d u c e d R e d u c e d C o n s u m e d E l e c t r o n D o n o r SRT (d) R e m a r k s R e f e r e n c e ( F i r s t A u t h o r ) T h i s S t u d y 5 . 3 1 . 5 0 . 3 6 c o m p l e x 1 0 B a t c h T e s t No . 2 5 . 0 c o m p l e x 15 R u n 2 / S y s t e m 1 4 . 0 c o m p l e x 6 R u n 4 / S y s t e m 1 L i t e r a t u r e 4 . 5 4 . 8 3 . 4 0 . 6 0 . 9 0 . 6 5 0 . 1 2 0 . 1 7 0 . 1 7 m e t h a n o l m e t h a n o l m e t h a n o l 2 8 12 f l o w t h r o u g h n o r e c y c l e M o o r e 1 9 7 0 5 . 5 3 . 6 1 . 4 0 . 5 m e t h a n o l m e t h a n o l 5 . 6 5 . 6 1 0 mg N O ^ - N / L 40 mg NO3-N/L M o o r e 1 9 7 1 0 . 6 m e t h a n o l l o n g p a c k e d - b e d R e q u a 1 9 7 3 0 . 1 7 - 0 . 2 m e t h a n o l 1 - 7 f l o w t h r o u g h n o r e c y c l e S t e n s e l 1 9 7 3 0 . 7 - 1 . 4 m e t h a n o l 3 - 6 p i l o t - s c a l e S u t t o n 1 9 7 5 0 . 5 - 0 . 8 m e t h a n o l 4 - 1 4 f l o w t h r o u g h w i t h r e c y c l e E n g b e r g 1 9 7 5 0 . 7 0 . 3 3 g l u c o s e f 1 o w t h r o u g h n o r e c y c l e S h a h 1 9 7 8 2 . 2 - 1 0 30 i n d u s t r i a l b a t c h t e s t s M o n t e i t h c a r b o n w a s t e s 1 9 8 0 4 . 5 q u o t i n g o t h e r w o r k G o r o n s z y 1 9 8 3 4 . 1 - 5 . 9 B e c c a r i 1 9 8 3 1 4 0 t i o n w a s a b o u t h a l f o f t h a t o b s e r v e d d u r i n g n i t r a t e r e s p i r a t i o n . S i m i l a r r e s u l t s w e r e a l s o r e p o r t e d b y V a n G e n t - R u i j t e r s e t a l . ( 1 9 7 5 ) . K o i k e a n d H a t t o r i ( 1 9 7 5 ) , o h t h e o t h e r h a n d , f o u n d n i -t r i t e r e d u c t i o n t o b e e q u a l t o n i t r a t e r e d u c t i o n , w h e n e x p r e s s e d i n t e r m s o f e l e c t r o n e q u i v a l e n c y . I n a n e f f o r t t o v e r i f y t h e v a l i d i t y o f t h e r e s u l t s o b t a i n e d d u r i n g B a t c h T e s t N o . 2 , c o n c e r n i n g V S S p r o d u c t i o n , d a t a f o r t h e t w o , c o n t i n u o u s l y - r u n s y s t e m s , d u r i n g R u n 5 , w e r e a l s o c o m p a r e d . Due t o s t a r t - u p o f e x o g e n o u s c a r b o n a d d i t i o n t o S y s t e m 2 o n D a y 5 5 , a n d t h e u n s t e a d y - s t a t e c o n d i t i o n s t h a t p r e v a i l e d d u r i n g t h e f i r s t t h i r t y f i v e d a y s o f o p e r a t i o n , i t w a s n o t p o s s i b l e t o o b t a i n s u f f i c i e n t d a t a f o r a s t a t i s t i c a l l y s i g n i f i c a n t c o m p a r i -s o n . T h e c o l l e c t e d d a t a d o e s s h o w , h o w e v e r , t h a t o v e r a l l s o l i d s p r o d u c t i o n i n t h e n i t r i t e p r o d u c i n g s y s t e m w a s a b o u t 22% l o w e r t h a n i n t h e n i t r a t e p r o d u c i n g s y s t e m . T h i s d i f f e r e n c e , w h i l e s i g n i f i c a n t , i s n o t a s h i g h a s t h a t o b t a i n e d d u r i n g B a t c h T e s t N o . 2 . R e a s o n s f o r t h i s s m a l l e r d i f f e r e n c e may i n c l u d e : 1) E x c e s s COD r e m a i n e d i n t h e a n a e r o b i c c e l l , f o l l o w i n g n i t r i t e r e d u c t i o n , d u e t o t h e l o w COD r e q u i r e m e n t s a s s o c i a t e d w i t h i t s r e d u c t i o n ( t h e a n a e r o b i c c e l l w a s v i r t u a l l y d e v o i d o f o x i d i z e d n i t r o g e n , w h e r e a s i n t h e o t h e r s y s t e m , a r e s i d u a l r e m a i n e d ) . A s a r e s u l t , t h e r e m a i n i n g COD w a s o x i d i z e d a e r o b i c a l l y , r e s u l t i n g i n s u b s t a n t i a l l y h i g h e r b i o m a s s p r o d u c t i o n r a t e s p e r mg COD c o n s u m e d . T h i s p r o b a b l y o f f s e t p a r t o f t h e g a i n s a c h i e v e d d u r i n g n i t r i t e r e d u c t i o n . 2) G r o w t h o f t h e n i t r i f i e r p o p u l a t i o n p o r t i o n o f t h e b i o m a s s o c c u r s o n l y u n d e r a e r o b i c c o n d i t i o n s . T h e i r g r o w t h r a t e , 1 4 1 t h e r e f o r e , may h a v e f u r t h e r m a s k e d t h e d i f f e r e n c e i n b i o m a s s p r o d u c t i o n r a t e s e x h i b i t e d u n d e r a n a e r o b i c c o n d i t i o n s . W h i l e i t i s n o t p o s s i b l e t o g e n e r a l i z e f r o m t h e r e s u l t s o f o n e s t u d y , i t w o u l d s e e m t h a t t h e v o l a t i l e m i x e d - l i q u o r s u s p e n d e d s o l i d s f r a c t i o n c o u l d b e r e d u c e d s i g n i f i c a n t l y i n t r e a t m e n t s y s -t e m s p r a c t i s i n g n i t r i t e p r o d u c t i o n a n d r e d u c t i o n . T h i s w o u l d r e s u l t i n a c o n c o m i t a n t r e d u c t i o n i n s l u d g e h a n d l i n g a n d d i s p o s a l c o s t s . C o m p a r i s o n o f N i t r a t e a n d N i t r i t e R e d u c t i o n R a t e s T h e d e n i t r i f i c a t i o n r a t e i s r e p o r t e d a s t h e h i g h e s t r a t e o f d i s s i m i l a t o r y n i t r i t e o r n i t r a t e r e d u c t i o n p e r u n i t m a s s p e r u n i t t i m e A NO^-N o r NO3-N D e n i t r i f i c a t i o n r a t e = , (.14) g V S S .. h r A n u m b e r o f s t u d i e s h a v e c o n f i r m e d t h a t d i s s i m i l a t o r y n i -t r i t e r e d u c t i o n i s m o r e r a p i d t h a n d i s s i m i l a t o r y n i t r a t e r e d u c -t i o n , i n a c c l i m a t e d b i o l o g i c a l s y s t e m s . A s u m m a r y o f r e p o r t e d v a l u e s i s p r e s e n t e d i n T a b l e 1 6 . G e n e r a l l y , i t a p p e a r s t h a t t h e d e n i t r i f i c a t i o n r a t e f o r n i t r i t e i s t w i c e a s f a s t a s f o r n i t r a t e . T h i s c o n t r a d i c t s t h e f i n d i n g s o f some p u r e c u l t u r e s t u d i e s , r e p o r t e d e a r l i e r , w h i c h d e m o n s t r a t e d t h a t t h e d i s s i m i l a t o r y n i -t r i t e r e d u c t i o n r a t e f o r some P s e u d o m o n a s s p e c i e s w a s s l o w e r t h a n t h e d i s s i m i l a t o r y n i t r a t e r e d u c t i o n r a t e ( s e e N i t r i t e A c c u m u l a -t i o n i n C h a p t e r 3). T h e a p p a r e n t c o n t r a d i c t i o n b e t w e e n t h e r e -s u l t s o f some p u r e c u l t u r e s t u d i e s a n d b i o l o g i c a l w a s t e w a t e r t r e a t m e n t s t u d i e s h i g h l i g h t s a m a j o r d i f f e r e n c e b e t w e e n p u r e a n d 1 4 2 T a b l e 16 - Comparison Between N i t r i t e and N i t r a t e D i s s i m i l a t o r y Reduction Rates Reported i n the L i t e r a t u r e D e n i t r i f i c a t i o n Rate* Reference Remarks ( F i r s t N i t r i t e N i t r a t e NOj-NrNOj-N Author) 0.11-0.40 0.06-0.11 1.8 - 3.6 h i g h l y nitrogenous Prakasam p o u l t r y waste 1972 0.02 0.0015 13 batch s t u d i e s on Voets high s t r e n g t h waste 1975 2 animal waste Murray 1975 20 12 1.7 batch s t u d i e s on Nagashima fermentation waste 1981a 21.8-24.4 8.6-13.5 1.8 - 2.5 batch s t u d i e s B e c c a r i 1983 69 32 2.2 batch t e s t s Timmermans 1983 4 batch t e s t s Abnf^yad 19 8 3 * as mg NO^-N/g•VSS (or TSS).hr 143 a p p l i e d research. Pure c u l t u r e research d e a l s with the c h a r a c t e r -i s t i c s of a s p e c i f i c s t r a i n of organism, whereas a p p l i e d research s e l e c t s f o r the s t r a i n ( s ) best a b l e to compete i n a p a r t i c u l a r environment. For example, i n an anaerobic packed bed r e a c t o r fed s y n t h e t i c waste, B l a s z c z y k (1983) found that the p r e v a l e n c e of a p a r t i c u l a r s p e c i e s of microorganism was dependent upon the type of carbon source used (i.e., methanol, ethanol or glucose) and e l e c t r o n acceptor present (i.e., n i t r a t e or n i t r i t e ) . The h i g h e s t r a t e of d e n i t r i f i c a t i o n can o n l y be determined when a l l s u b s t r a t e s are present i n excess. T h i s c o n d i t i o n was never met i n any of the continuous runs, s i n c e e i t h e r s o l u b l e organic carbon or n i t r i t e p l u s n i t r a t e was u s u a l l y exhausted i n the anaerobic c e l l . As a r e s u l t , a batch t e s t had to be under-taken where a l l s u b s t r a t e s would be present i n excess. F u r t h e r -more, i n order to compare the d e n i t r i f i c a t i o n r a t e s of n i t r i t e and n i t r a t e , i t was necessary to compare the d e n i t r i f i c a t i o n r a t e of a n i t r i t e a c c l i m a t e d biomass to that of a n i t r a t e a c c l i m a t e d biomass. Such an occasion arose during Run 5, at which time Batch Test No.2 was conducted. The r e s u l t s are presented i n Tab l e 14 and F i g u r e 16. As noted, the r e d u c t i o n of n i t r i t e approached a zero order r a t e down to around 2 mg NO^-N/L, i n c o n t r a s t to n i t r a t e r e d u c t i o n . The i n a b i l i t y of n i t r a t e r e d u c t i o n to maintain a zero order r a t e below 40-50 mg NO3-N/L was s u r p r i s i n g , s i n c e both n i t r a t e and COD were s t i l l present i n excess (105 minutes w i t h i n the t e s t , they averaged 58 mg NO3-N/L and 424 mg COD/L i n Reactor 2). S e v e r a l s t u d i e s have confirmed that n i t r a t e r e d u c t i o n approaches a z e r o o r d e r r a t e down to l e v e l s of 1-2 mg NOj-N/L and 1-5 mg COD/L (Requa and Schroeder, 1973; EPA, 1975; Sutton et 144 a l . y 1975; Engberg and Schroeder, 1975). This anomaly was proba-bly due to exhaustion of "readily available" carbon sources. Unlike the studies reported in the literature, which used metha-nol as carbon source, the carbon source used for the batch test was similar to that used during the continuous run (see Table 6), and may have contained substrates not as readily degradable by anaerobic respiration. For comparison of denitrification rates between nitrite and nitrate, the zero order part of the curve was used. As noted, nitrite reduction proceeded at a much faster rate than did the reduction of nitrate. The nitrite acclimated biomass (Reactor 3) reduced nitrite at a rate 63% faster than nitrate reduction, by a nitrate acclimated biomass (Reactor 2). These results are in general agreement with the findings of Nagashima et al. (1981a) and are slightly lower than those reported by Murray et al . (1975), Beccari et a l , (1983) and Timmermans et a l . (1983),. It is also interesting to note that the denitrification of nitrite was achieved within the same time-frame for both the nitrite and nitrate acclimated biomass. The denitrification of nitrate, on the other hand, was much slower for the biomass not acclimated to its reduction (Reactor 4). This suggests that the denitrifier population that had established itself in the ni-trite producing system was composed mostly of organisms with l i t t l e ability to reduce nitrate. This contradicts the findings of Nagashima e_t a_l. (1984a and b) , who noted that nitrite accl i -mated biomass, exposed to nitrate, achieved higher nitrate reduc-tion rates than did nitrate acclimated biomass; conversely, ni -145 trate acclimated biomass, exposed to n i t r i t e , achieved lower n i t r i t e reduction rates than did n i t r i t e acclimated biomass. The a b i l i t y to reduce n i t r i t e during Runs 6 and 7 did not esta b l i s h i t s e l f immediately. In Run 6, over ten days were needed, while in Run 7 about 30 days were needed for n i t r i t e reduction to appear, following the establishment of anaerobic conditions in the presence of n i t r i t e ; this was the case in spite of the presence of an adequate carbon source. Nitrate, on the other hand, was reduced shortly after establishing anaerobic conditions. Comparison of A l k a l i n i t y Consumption and Production Theoretical a l k a l i n i t y consumption during the two stages of n i t r i f i c a t i o n can be calculated from the following equations (EPA, 1975): 55NH| + 760 2 + IO9HCO3 ^ C 5H 7N0 2 +54N02 + 57H20 + 104H2CO3 (15) 400NO2 + NH4 + 4H 2C0 3 + HC0 3 + 19502 > C 5H 7N0 2 + 3H20 + 400NO3 (16) It is evident from these equations that most of the a l k a l i -nity consumption occurs during the oxidation of ammonia to n i -t r i t e , with n e g l i g i b l e consumption during the oxidation of n i -t r i t e to nitrate ( i t amounts to about 0.1% of t o t a l consumption). Based on these equations, 7.21 mg of a l k a l i n i t y as CaC0 3 is consumed/mg N03-N produced. A l k a l i n i t y production from the dis s i m i l a t o r y reduction of nitrate and n i t r i t e to nitrogen gas, with methanol as substrate, can be calculated as follows (McCarty e_t a_l., 1969): 146 NO3 + I.O8CH3OH + 0 . 2 4 H 2 C O 3 >• 0 . 0 5 6 C 5 H 7 N O 2 + 0 . 4 7 N 2 + 1 . 6 8 H 2 0 + HCO3 (17) N 0 2 + 0 . 6 7 C H 3 O H + 0 . 5 3 H 2 C O 3 ' » 0 . 0 4 C 5 H 7 N O 2 + 1 . 2 3 H 2 0 + 0 . 4 8 N 2 + HCO3 . . . ( 1 8 ) I t i s e v i d e n t f r o m t h e s e e q u a t i o n s t h a t t h e r e d u c t i o n o f e i t h e r n i t r a t e - N o r n i t r i t e - N t o n i t r o g e n g a s p r o d u c e s a n e q u a l a m o u n t o f a l k a l i n i t y ( 3 . 5 7 mg o f a l k a l i n i t y a s CaCO^ p r o d u c e d / m g NO^-N r e d u c e d t o N 2 ) . I t f o l l o w s t h e n , t h a t t h e f i r s t s t e p i n t h e d e n i t r i f i c a t i o n p r o c e s s ( i . e . , t h e r e d u c t i o n o f n i t r a t e t o n i -t r i t e ) d o e s n o t p r o d u c e a n y a l k a l i n i t y . B a s e d o n t h e a f o r e m e n t i o n e d e q u a t i o n s , a l k a l i n i t y c o n s u m p -t i o n a n d r e d u c t i o n a r e l i n k e d s o l e l y w i t h : a m m o n i a o x i d a t i o n t o n i t r i t e , a n d n i t r i t e r e d u c t i o n t o n i t r o g e n g a s . T h e r e f o r e , a t r e a t m e n t p r o c e s s r e l y i n g u p o n t h e s h o r t e n e d n i t r o g e n r e m o v a l p a t h w a y s h o u l d e x h i b i t s i m i l a r a l k a l i n i t y r e l a t i o n s h i p s t o o n e o p e r a t i n g a l o n g t h e m o r e t r a d i t i o n a l n i t r i f i c a t i o n / d e n i t r i f i c a -t i o n p a t h w a y . T h i s p r e m i s e w a s i n v e s t i g a t e d i n t h e c o u r s e o f R u n 2 . A l k a l i n i t y c o n s u m p t i o n w a s m e a s u r e d d u r i n g t h e e a r l i e r s t a g e s o f t h e r u n , w h e n b o t h s y s t e m s w e r e o p e r a t i n g i n a f u l l y a e r o b i c mode w i t h n o n i t r i t e p r e s e n t ; t h i s w a s u n d e r t a k e n t o e s t a b l i s h a l k a l i n i t y t o n i t r a t e - N c o n s u m p t i o n r a t i o s . T h e s e v a l -u e s a v e r a g e d 6 . 4 5 mg a s C a C C ^ / m g NO3-N p r o d u c e d (s = 0 . 1 5 , n = 6 ) . T h i s w a s l o w e r t h a n t h e t h e o r e t i c a l v a l u e o f 7 . 2 1 mg a s C a C 0 3 / m g NO3-N p r o d u c e d b u t w a s i n g e n e r a l a g r e e m e n t w i t h n u m e r -o u s o t h e r e x p e r i m e n t a l r e s u l t s , w h e r e t h e a m o u n t o f a l k a l i n i t y c o n s u m e d h a s u s u a l l y b e e n f o u n d t o b e b e l o w 7 mg C a C C ^ / m g NO3-N 1 4 7 ( S c e a r c e e t a _ l . , 1 9 8 0 ) . M u l b a r g e r (1971) r e p o r t e d a n a v e r a g e 6 . 1 mg o f a l k a l i n i t y c o n s u m e d p e r mg NO3-N p r o d u c e d . A t t h e 1 3 . 5 m ^ / s B l u e P l a i n s a c t i v a t e d s l u d g e p l a n t , a n a v e r a g e 5 . 3 mg o f a l k a l i -n i t y w a s r e m o v e d p e r mg TKN o x i d i z e d ( B a i l e y e t a _ l . , 1 9 8 3 ) . T h e d i s c r e p e n c y b e t w e e n t h e t h e o r e t i c a l v a l u e a n d e x p e r i m e n t a l r e -s u l t s h a s b e e n a t t r i b u t e d t o a n u m b e r o f f a c t o r s . Among t h e m : 1) I n c i d e n t a l d e n i t r i f i c a t i o n w h i c h l e a d s t o t h e p r o d u c t i o n o f a l k a l i n i t y ; 2) A m m o n i f i c a t i o n ( m i n e r a l i z a t i o n ) o f o r g a n i c n i t r o g e n w h i c h p r o d u c e s 3 . 5 7 mg o f a l k a l i n i t y a s C a C 0 3 / m g o r g a n i c n i t r o g e n m i n e r a l i z e d ( B i s h o p a n d F a r m e r , 1 9 7 9 ) . i n t h e l a t t e r s t a g e s o f t h e r u n , t h e e l e c t r o n a c c e p t o r i n t h e a n a e r o b i c e e l 1 o f S y s t e m 1 w a s n i t r a t e , w h e r e a s i n s y s t e m 2 i t w a s l a r g e l y n i t r i t e . T h i s a l l o w e d f o r a c o m p a r i s o n o f o v e r a l l a l k a l i n i t y r e l a t i o n s h i p s b e t w e e n n i t r i t e a n d n i t r a t e . U s i n g t h e r e s u l t s o b t a i n e d e a r l i e r , d u r i n g f u l l y a e r o b i c c o n d i t i o n s , a s a n e s t i m a t e o f a l k a l i n i t y c o n s u m p t i o n d u r i n g n i t r i f i c a t i o n , a l k a l i -n i t y p r o d u c t i o n i n S y s t e m 1 ( a s a r e s u l t o f n i t r a t e r e d u c t i o n ) w a s c a l c u l a t e d a t a b o u t 3 . 1 mg a s C a C 0 3 p r o d u c e d / m g NO3-N r e d u c e d (s = 0 . 1 5 , n = 5 ) . T h i s w a s l o w e r t h a n t h e t h e o r e t i c a l v a l u e o f 3 . 5 7 mg CaCO^ p r o d u c e d / m g NO3-N r e d u c e d , b u t i n c l o s e a g r e e m e n t w i t h e x p e r i m e n t a l r e s u l t s r e p o r t e d i n t h e l i t e r a t u r e ( E P A , 1 9 7 5 ; G o r o n s z y a n d B a r n e s , 1 9 8 2 ) . T h e a l k a l i n i t y c o n s u m p t i o n a n d p r o -d u c t i o n v a l u e s o b t a i n e d f o r n i t r a t e w e r e u s e d t o e s t i m a t e t h e a l k a l i n i t y r e l a t i o n s h i p s f o r S y s t e m 2 , w h i c h w a s p r o d u c i n g a n d r e d u c i n g n i t r i t e ; t h e s e v a l u e s w e r e t h e n c o m p a r e d t o t h e a c t u a l a l k a l i n i t y m e a s u r e m e n t s m a d e i n t h a t s y s t e m . T h e c a l c u l a t e d v a -148 lues, averaged for five readings, came within 3.2% of the mea-sured ones. This seemed to confirm that the overall alkalinity relationships for nitrite and nitrate were similar. Effects of Nitrite on Process Performance The effect of nitrite on process performance was monitored during Run 2 and Batch Test No.2 (Run 5). Throughout the period of nitrite build-up in System 2 during Run 2, there was no indication of any inhibition to the treatment process due to the presence of nitrite at concentrations as high as 100 mg NO -^N/L. Based upon the Bartlett test, nitrification rates (presented later in Table 18) were not statistically dif-ferent between the system producing mostly nitrate and that producing mostly nitrite, nor were these rates statistically different from those obtained, during the fully aerobic mode of operation, when no nitrite was present in either system. The results of Batch Test No.2 (see Figure 16) indicated that nitrite, per se, did not appear to inhibit heterotrophic denitrifying biological activity at concentrations as high as 115 mg NO -^N/L; the denitrification rates for nitrite, which was 64% higher than for nitrate, remained linear down to very low levels. Had nitrite caused inhibition, a slower reduction rate would have manifested itself in i t ia l ly , until the nitrite levels dropped to a non-inhibitory concentration, after which the rate would have increased. Denitrification activity, which is a reflection of heterotrophic activity, occurred in a l l runs. This was an indi-rect confirmation that some heterotrophs were capable of growing in a medium containing relatively high levels of nitrite. 149 T h e a b o v e r e s u l t s a g r e e w i t h t h e f i n d i n g s o f s e v e r a l s t u d i e s t h a t h a v e c o n f i r m e d t h e a b i l i t y o f a n u m b e r o f m i c r o - o r g a n i s m s t o t o l e r a t e n i t r i t e l e v e l s a b o v e 1 0 0 mg N O ^ - N / L , p r o v i d e d n i t r o u s a c i d l e v e l s r e m a i n e d l o w ( B o l l a g a n d H e n n i n g e r , 1 9 7 8 ; Rowe e t a l . , 1 9 7 9 ) . T h e s e r e s u l t s a l s o a g r e e w i t h o t h e r s t u d i e s w h i c h d e m o n s t r a t e d t h e a b i l i t y o f b i o l o g i c a l s y s t e m s t o w i t h s t a n d n i -t r i t e c o n c e n t r a t i o n s a s h i g h a s 5 0 0 mg N O ^ - N / L ( N a g a s h i m a e t a l . , 1 9 8 1 ; B l a s z c z y k e t a l . , 1 9 8 1 ) . A b i l i t y t o P r o d u c e A n E f f l u e n t D e v o i d o f N i t r i t e T h e d i s c h a r g e o f a n e f f l u e n t c o n t a i n i n g n i t r i t e may n o t b e a c c e p t a b l e , d u e t o i t s p o t e n t i a l t o x i c i t y ( s e e E f f e c t s o f N i t r i t e i n C h a p t e r 3 ) . A s a r e s u l t , t h e e x p e r i m e n t a l p r o g r a m i n v e s t i -g a t e d t h e f e a s i b i l i t y o f r e c o n c i l i n g t h e c o n t r a d i c t o r y o b j e c t i v e s o f p r o d u c i n g a d e n i t r i f i e d e f f l u e n t , d e v o i d o f n i t r i t e , w i t h t h e n e e d t o m a i n t a i n n i t r i t e a c c u m u l a t i o n i n t h e s y s t e m . T h i s w a s a t t e m p t e d d u r i n g R u n s 5 a n d 7 . I n b o t h r u n s , i t i n v o l v e d t h e p r o d u c t i o n a n d r e d u c t i o n o f n i t r i t e a t t h e f r o n t - e n d o f t h e s y s t e m , a n d t h e u s e o f t h e r e m a i n i n g c e l l s f o r o x i d i z i n g a n y r e s i d u a l n i t r i t e t o n i t r a t e . I n b o t h r u n s , t h e o b j e c t i v e w a s a t t a i n e d . T h i s w a s l a r g e l y f a c i l i t a t e d b y t h e f a c t t h a t m o s t o f t h e f e e d n i t r o g e n c o n t e n t w a s r e m o v e d a t t h e f r o n t - e n d o f t h e s y s t e m d u r i n g t h e p e r i o d o f n i t r i t e b u i l d - u p ( i t a m o u n t e d t o a r o u n d 9 5 % i n R u n 7 ) . T h i s a p p e a r s t o b e t h e f i r s t d o c u m e n t e d d e m o n s t r a t i o n o f a s y s t e m t h a t s u c c e s s f u l l y o p e r a t e d a l o n g t h e s h o r t e n e d n i t r o g e n p a t h w a y , w h i l e p r o d u c i n g a f u l l y n i t r i f i e d e f f l u e n t d e v o i d o f n i t r i t e . 1 5 0 Aerobic Reduction of N i t r i t e In terms of p o t e n t i a l energy y i e l d , n i t r a t e i s not q u i t e as f a v o r a b l e a t e r m i n a l e l e c t r o n acceptor as i s m o l e c u l a r oxygen. As a r e s u l t , d i s s i m i l a t o r y n i t r a t e r e d u c t i o n i s u s u a l l y repressed by the presence of oxygen (Pai n t e r , 1970; Delwiche and Bryan, 1976; Pocht and V e r s t r a e t e , 1977). Although d e n i t r i f i c a t i o n i n most b a c t e r i a l s p e c i e s appears to be i n h i b i t e d by the presence of even low DO l e v e l s , some s t r a i n s appear to be r e l a t i v e l y i n s e n s i t i v e to the presence of oxygen. K r u l and Veeningen (1977), i s o l a t e d two s t r a i n s from a c t i v a t e d sludge that were capable of s y n t h e s i -zing n i t r a t e reductase at DO l e v e l s above 4 mg/L. Strand (1982), reported that a s t r a i n of Zoogloea ramigera was capable of s i g n i -f i c a n t a e r o b i c d e n i t r i f i c a t i o n up to a DO l e v e l of 4.4 mg/L. From a s t r i c t l y thermodynamic p e r s p e c t i v e , the f r e e energy y i e l d from the r e d u c t i o n of n i t r i t e i s h i g h e r than t h a t from the r e d u c t i o n of m o l e c u l a r oxygen (McCarty et a_l., 1972). This theo-r e t i c a l l y a l l o w s n i t r i t e to act as an e q u a l l y f a v o u r a b l e e l e c t r o n acceptor as m o l e c u l a r oxygen, and e l i m i n a t e s the need f o r an anaerobic environment f o r i t s r e d u c t i o n . Free energy y i e l d s , however, g i v e o n l y an approximate idea of the energy that organ-isms can o b t a i n from a redox r e a c t i o n ; more meaningful are the ATP y i e l d s obtained with v a r i o u s e l e c t r o n acceptors. There are i n d i c a t i o n s that a e r o b i c r e d u c t i o n of n i t r i t e i s more common than the a e r o b i c r e d u c t i o n of n i t r a t e (bearing i n mind that the l i t e -r a t u r e on n i t r i t e r e d u c t i o n i s not as e x t e n s i v e as on n i t r a t e r e d u c t i o n ) . R e s u l t s from pure c u l t u r e s t u d i e s have reported the i s o l a t i o n of s t r a i n s of b a c t e r i a capable of reducing n i t r i t e i n 151 the presence of oxygen (Skerman e_t a_l., 1958; Vangnai and K l e i n , 1974; J u s t i n and K e l l y , 1978). Mechsner and Wuhrmann (1963) and K r u l (1976) i s o l a t e d d e n i t r i f y i n g s t r a i n s from a c t i v a t e d sludge systems that c o u l d reduce n i t r i t e a e r o b i c a l l y . Anthonisen (1974) a l l u d e d to i t s occurrence i n an o x i d a t i o n d i t c h t r e a t i n g high s t r e n g t h ammonia waste. Voets et a_l. (1975) c a r r i e d out s e v e r a l batch t e s t s to determine n i t r o g e n removal r a t e s f o r a h i g h l y nitrogenous waste. They found that n i t r i t e r e d u c t i o n was p o s s i b l e under a e r o b i c c o n d i t i o n s , u n l i k e n i t r a t e r e d u c t i o n , which o n l y occurred i n the absence of oxygen. The p e r i o d between Days 96 and 137, during Run 2, o f f e r e d an i d e a l s e t t i n g f o r studying the occurrence of a e r o b i c d e n i t r i f i c a -t i o n of n i t r i t e by comparing System 1, which contained almost e x c l u s i v e l y n i t r a t e , with System 2, which contained mostly n i -t r i t e . To e s t a b l i s h the presence or absence of a e r o b i c d e n i t r i -f i c a t i o n i n a n i t r i f y i n g system, i t was necessary to account f o r the f a t e of a l 1 n i t r o g e n s p e c i e s a c r o s s the system, and a de-t a i l e d n i t r o g e n balance (discussed l a t e r i n t h i s chapter) was maintained f o r such purposes. The "unaccounted f o r " n i t r o g e n l o s s amounted to 7.1% (s=6.1, n=16) and 6.1% (s=6.6, n=16) i n Systems 1 and 2, r e s p e c t i v e l y . The l o s s e s were minor, w e l l w i t h i n the l i m i t s of experimental e r r o r , and not s t a t i s t i c a l l y d i f f e r e n t from each other. Thus, no evidence of a e r o b i c d e n i t r i f i c a t i o n , due to the presence of n i t r i t e , c o u l d be e s t a b l i s h e d . T h i s was a l s o confirmed from other, a d d i t i o n a l experimentation d i s c u s s e d l a t e r i n "Unaccountable Nitrogen Losses". 152 STABILITY OF NITRITE BUILD-UP N i t r i t e b u i l d - u p was su s t a i n e d i n a l l runs by i n t e r m i t t e n t contact of the n i t r i t e o x i d i z e r s to the i n h i b i t o r y " f r e e ammonia" l e v e l s maintained i n the f i r s t c e l l of the system. This observa-t i o n was a l l u d e d to e a r l i e r by Alleman and I r v i n e (1980) and Sauter and Alleman (1980) and confirmed r e c e n t l y by Senanayake (1982) i n h i s s t u d i e s using s e q u e n t i a l batch r e a c t o r s . These researchers a t t r i b u t e d the accumulation of n i t r i t e i n t h e i r sys-tem to the pro d u c t i o n of a temporary, i n h i b i t o r y f r e e ammonia c o n c e n t r a t i o n during each ope r a t i n g c y c l e (i.e., i n t e r m i t t e n t contact to f r e e ammonia). Apart from the aforementioned o b s e r v a t i o n s , made on a t r e a t -ment process that i n h e r e n t l y produces r e l a t i v e l y l a r g e v a r i a t i o n s i n f r e e ammonia l e v e l s during each c y c l e , there appears to be no rep o r t s of e f f o r t s undertaken s p e c i f i c a l l y to achieve n i t r i t e b u i l d - u p by i n t e r m i t t e n t contact to i n h i b i t o r y free ammonia l e -v e l s using the c o n v e n t i o n a l , a c t i v a t e d sludge treatment process. As can be seen from F i g u r e s 6, 8 and 13 f o r Runs 2, 4 and 7, n i t r i t e b u i l d - u p was su s t a i n e d i n the f i r s t a e r o b i c c e l l f o r peri o d s of about 50, 90 and 130 days r e s p e c t i v e l y . These were the most s u c c e s s f u l runs i n terms of s u s t a i n i n g n i t r i t e b u i l d - u p . In Runs 4 and 7, the d e c l i n e was re v e r s e d , i n s p i t e of the apparent presence of a biomass that had become a c c l i m a t e d to f r e e ammonia. T h i s was a c h i e v e d d u r i n g Run 4 by a c o m b i n a t i o n of v e r y s h o r t SRT, high f r e e ammonia l e v e l s and reduced a e r o b i c HRT. In Run 7, i t was achieved by intermediary d e n i t r i f i c a t i o n ( i e . shortening the a e r o b i c HRT). U l t i m a t e l y , i n a l l runs, except p o s s i b l y Run 4, the b u i l d - u p c o u l d not be sustained i n d e f i n i t e l y and an i r r e v e r -153 s i b l e decline eventually occurred. The s t a b i l i t y of the desired process was l a r g e l y linked to two parameters: 1) Aerobic residence time 2) Continued effectiveness of free ammonia Aerobic Residence Time Extension of the a e r a t i o n time seemed to have been a major factor responsible in causing the decline in n i t r i t e accumula-tion. The a b i l i t y of the n i t r i t e oxidizers to regain their n i t r i -fying a c t i v i t y was d i r e c t l y related to the time elapsed from their exit from the high, free ammonia environment of the anaero-bic c e l l . This was confirmed in a l l runs. The r e s u l t s of Run 2, for example, can be rearranged, as shown in Figure 18, to ref-l e c t the e f f e c t of aerobic residence time on the degree of n i -t r i t e build-up. It is apparent from the graph that the degree of n i t r i t e build-up was inversely proportional to the aerobic r e s i -dence time. The results of Batch Test No.l (Run 4) confirmed the effect of aerobic residence time on the degree of n i t r i t e build-up. As shown in Figure 14, a l l four reactors showed an inverse l i n e a r relationship between aerobic residence time and degree of n i t r i t e b uild-up, down to n i t r i t e l e v e l s of 10 mg NO^-N/L. The rate of decline with time (as a function of percent of oxidized nitrogen species) was equal in a l l four reactors and averaged 11% per hour of aeration. This was close to the percent drop observed in System 2 at the end of Run 2 ( i t averaged 10 - 14% per hour). An observation of the results for Reactors 1 and 2 revealed that ammonia oxidation was completed within the f i r s t 4 hours of 154 3 h o u r s ( c e l l 2 ) 6 h o u r s ( c o i l 3 ) R u n 2 / S y s t e m 2 D a t a p o i n t s d e l e t e d f o r c l a r i t y a r e s h o w n i n F i g . 17 3 h o u r s o f a e r a t i o n : Y - 1 1 3 . 6 - 6 0 . 8 3 X " ' - 1 . 0 7 X n - 2 5 R - 0 . 8 5 6 h o u r s o f a e r a t i o n : Y - 1 0 5 . 4 - 1 0 9 . 5 X " ' + 0 . 3 4 X n - 2 5 R - 0 . 8 8 9 h o u r s o f a e r a t i o n : Y - 9 7 . 3 - 1 6 4 . 0 X " 1 • 1 . 9 8 X n - 2 5 R - 0 . 8 6 1 2 3 4 5 6 7 8 9 A M M O N I A L E V E L IN A N A E R O B I C C E L L ( m g N / L ) r 1 0 0 - 9 0 - 8 0 - 7 0 1-60 ^ l_L_ o 5 0 CO < - 4 0 - 3 0 - . 20 - 1 0 10 CM o F i g . 1 8 : R e l a t i o n s h i p B e t w e e n : F r e e A m m o n i a L e v e l in A n a e r o b i c C e l l , H o u r s o f S u b s e q u e n t A e r a t i o n a n d N i t r i t e B u i l d - U p 155 aeration; the additional aeration time merely served to oxidize the accumulated nitrite to nitrate. Furthermore, while the con-centration of nitrite rose steadily during the first 3 hours of aeration, it declined in terms of percent of oxidized nitrogen species present. It appeared that an "optimum" aeration time existed, where most of the ammonia was oxidized to nitrite, while l i t t l e of the nitrite produced had yet been oxidized to nitrate. From the aforementioned, it is clear that extension of the aerobic residence time alleviates the inhibitory effects of free ammonia, leading to a reduction in the degree of nitrite build-up. This observation is of practical significance in optimizing the design and operation of activated sludge systems dedicated to nitrogen removal via nitrite production and reduction. It may also explain some of the discrepancies reported in the literature concerning inhibitory free ammonia levels. In their work on nitrification/denitrification, using bench-scale, sequencing batch reactors, Alleman and Irvine (1980) re-ported that extension of the aeration time reduced the degree of nitrite accumulation in a system that was fed synthetic waste, containing 60 mg TKN/L. They found that an aeration phase of 3 hours produced an effluent containing almost exclusively nitrite, whereas an extended aeration phase alleviated the inhibition to the Nitrobacter species and produced a fully nitrified effluent. The cause of inhibition was believed to be high levels of free ammonia, estimated at 0.6 mg NH3-N/L, which developed during each f i l l period. They concluded that manipulation of the duration of the aeration phase would establish the form of oxidized nitrogen species present in the effluent. These results were later con-156 firmed by Senanayake (1982), who reported n i t r i t e b u i l d - u p i n bench-scale, sequencing batch r e a c t o r s with an a e r a t i o n c y c l e of 3 hours, but no b u i l d - u p f o r longer a e r a t i o n periods. He noted, however, that n i t r i t e b u i l d - u p e v e n t u a l l y disappeared even i n the system operated under the 3 hour a e r a t i o n c y c l e . Expanding upon the f i n d i n g s of p r e v i o u s s t u d i e s conducted by h i s research group, Alleman (1984) reported that f i v e hours of a e r a t i o n time was s u f f i c i e n t to o x i d i z e a l l the n i t r i t e to n i t r a t e . Continued E f f e c t i v e n e s s of Free Ammonia The success of the process was dependent upon the continued e f f e c t i v e n e s s of the s e l e c t i v e i n h i b i t o r " f r e e ammonia". The r e s u l t s of Run 2 seemed to suggest that f r e e ammonia maintained i t s i n h i b i t o r y e f f e c t through most of the run (up to Day 138). This i s f u r t h e r confirmed by o b s e r v a t i o n of F i g u r e 17, which shows a good c o r r e l a t i o n between the f r e e ammonia l e v e l i n the anaerobic c e l l and the degree of n i t r i t e b u i l d - u p i n the three remaining a e r o b i c c e l l s . The r e s u l t s have been rearranged i n Fi g u r e 18, to account f o r both the e f f e c t s of f r e e ammonia and a e r a t i o n time. As noted, the degree of n i t r i t e accumulation was p r o p o r t i o n a l to the f r e e ammonia l e v e l i n the anaerobic c e l l . With an a c c l i m a t e d biomass, no such r e l a t i o n s h i p was e v i -dent. .In Run 7, f o r example, a c c l i m a t i o n manifested i t s e l f as e a r l y as Day 42, and, as can be observed from F i g u r e 19, no c o r r e l a t i o n e x i s t s between v a r i a t i o n s i n f r e e ammonia l e v e l s and degree of n i t r i t e b u i l d - u p . During p e r i o d s of n i t r i t e b u i l d - u p , l a r g e f l u c t u a t i o n s i n f r e e ammonia l e v e l s appeared to have l i t t l e e f f e c t i n s u s t a i n i n g the b u i l d - u p . T h i s was most e v i d e n t between 157 1 0 0 - 9 0 - 8 0 7 0 I 6 0 O z IX. O 5 0 co < 4 0 T O z 3 0 - 2 0 - 1 0 F r o m D a y 4 2 o n w a r d s 10 2 0 3 0 m g N H 3 - N / L IN A N A E R O B I C C E L L 4 0 F ig .19 : Run 7 - E f f e c t o f F r e e A m m o n i a o n N i t r i t e B u i l d - U p A f t e r A c c l i m a t i o n 158 Days 70 and 200, when the f r e e ammonia l e v e l v a r i e d between 0.5 and 20 mg NH3-N/L, with l i t t l e apparent c o r r e l a t i o n to the degree of n i t r i t e b u i l d - u p . The a b i l i t y of the n i t r i t e o x i d i z e r s , as w e l l as the ammonia o x i d i z e r s , to withstand ever i n c r e a s i n g c o n c e n t r a t i o n s of f r e e ammonia was e v i d e n t i n Runs 4, 5, 6 and 7. The ammonia and n i t r i t e o x i d i z e r s were a b l e , over a p e r i o d of s e v e r a l days, weeks or months, to re g a i n t h e i r a c t i v i t y i n the presence of f r e e ammonia l e v e l s that had i n i t i a l l y caused i n h i b i t i o n . T h i s was probably the major cause of the d e c l i n e i n n i t r i t e b u i l d - u p observed during Runs 2, 4, 5, 6 and 7, as w e l l as the probable cause of i n i t i a l l o s s of n i t r i t e b u i l d - u p during Run 7. The a b i l i t y of both n i t r i f y i n g groups to regain t h e i r n i t r i -f y i n g a b i l i t y , with time, has been p r e v i o u s l y reported i n the l i t e r a t u r e . Ford et a l . (1980) reported t o t a l i n h i b i t i o n of n i t r i f i c a t i o n a c t i v i t y at f r e e ammonia l e v e l s o f 24 mg NH3-N/L, but noted that system recovery was p o s s i b l e , even at l e v e l s as high as 56 mg NH3-N/L. The r e c o v e r y i n n i t r i f y i n g a c t i v i t y may have been due to: 1) Adherence to c e l l w a l l s 2) A c c l i m a t i o n 1) Adherence to C e l l W a l l s Although r e g u l a r c l e a n i n g of c e l l w a l l s was c a r r i e d out during a l l runs, the p o t e n t i a l f o r adherence of the n i t r i f i e r s to the c e l l w a l l s was a constant source of concern. Organisms a t -tached to a e r o b i c c e l l w a l l s are not sub j e c t to the f r e e ammonia " s t r e s s " w i t h i n the anaerobic c e l l , and as such, t h e i r growth on 159 the w a l l s c o u l d have been a c o n t r i b u t i n g f a c t o r i n the d e c l i n e i n n i t r i t e l e v e l s . N i t r i f i e r adherence to c e l l w a l l s i s w e l l docu-mented (Meikeljohn, 1954; P a i n t e r , 1970; Watson et a_l., 1981). The r e s u l t s of Batch Test No.l (Run 4), appeared to r u l e out the occurrence of any a p p r e c i a b l e adherence of n i t r i f i e r s to the a e r o b i c c e l l w a l l s . Had they adhered to any a p p r e c i a b l e extent, the r e s u l t s of the batch t e s t would have e x h i b i t e d a slower o x i d a t i o n r a t e f o r Reactors 1 and 2, r e f l e c t i n g the lower n i t r i t e o x i d i z e r p o p u l a t i o n w i t h i n these two anaerobic c e l l s (since i t would have been u n l i k e l y f o r the n i t r i f i e r s to have adhered to the w a l l s i n an environment dev o i d of oxygen). As can be observed from F i g u r e 14, t h i s was not the case. 2) A c c l i m a t i o n The n i t r i t e o x i d i z e r s may have a c c l i m a t e d to the high f r e e ammonia l e v e l s , thus rendering them more t o l e r a n t to ever i n -c r e a s i n g ammonia co n c e n t r a t i o n s . Adaptation (or a c c l i m a t i o n ) can be caused by (Glass, 1982): 1) Mutations: Non l e t h a l mutations may impart on an organism the a b i l i t y to s u r v i v e i n the presence of e x t e r n a l b a c t e r i o s t a t i c or b a c t e r i c i d a l f a c t o r s . I t i s a h e r i t a b l e change that perma-n e n t l y a f f e c t s the chromosome. Mutations can be spontaneous or induced by e x t e r n a l f a c t o r s such as r a d i a t i o n and chemicals. 2) Gene T r a n s f e r : Some c e l l s harbour s m a l l extrachromosomal gene-t i c elements that are s t a b l y i n h e r i t e d (plasmids). Plasmids may mediate the t r a n s f e r of g e n e t i c m a t e r i a l from one organism to another by a process termed conjugation. In g e n e r a l , natu-r a l l y o c c u r r i n g b a c t e r i a l plasmids are d i s p e n s i b l e but may be 160 b e n e f i c i a l to the host c e l l . Plasmids are i m p l i c a t e d i n im-p a r t i n g r e s i s t a n c e to t o x i c metals, m e t a l l o i d compounds and a n t i b i o t i c s . 3) Gene Expression: During the b a c t e r i a l l i f e c y c l e , adjustments i n c e l l u l a r metabolism may be necessary when the bacterium i s confronted with an unusual growth substance or a l t e r e d e x t e r -n a l c o n d i t i o n s . Thus, w h i l e the m a j o r i t y of genes are expres-sed c o n s t i t u t i v e l y , some are a c t i v e l y c o n t r o l l e d and are not n o r m a l l y expreseed during the l i f e c y c l e of the c e l l . These genes express themselves o n l y under a l t e r e d e x t e r n a l c o n d i -t i o n s . The cause of adaptation to f r e e ammonia cannot be determined from the research undertaken. L i t t l e i s known about the a d a p t i v e a b i l i t y of the n i t r i f i e r s , although s t u d i e s i n d i c a t e that a c c l i -mation may p l a y an important r o l e i n t h e i r s u r v i v a l (Sharma and A h l e r t , 1977). Anthonisen (1974) noted the a b i l i t y of the n i t r i f i e r s to adapt to f r e e ammonia and a t t r i b u t e d t h i s a b i l i t y to a c c l i m a t i o n . Senanayake (1982) reported on the a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia over a two-month p e r i o d using a bench-s c a l e sequencing batch r e a c t o r fed 60 mg TKN/L. Wong-Chong and Loehr (1978) observed that f r e e ammonia i n h i -b i t i o n i n batch s t u d i e s of enriched, n i t r i f i e r c u l t u r e s was d i r e c t l y r e l a t e d to a c c l i m a t i o n . Acclimated c u l t u r e s c o u l d t o l e r -ate c o n c e n t r a t i o n s as high as 40 mg NH-j-N/L, w h i l e unacclimated ones were i n h i b i t e d at c o n c e n t r a t i o n s of 3.5 mg NH-j-N/L. Tomlinson et a l . (1966) commented on the a c c l i m a t i o n of the 161 n i t r i f i e r s to hex a v a l e n t chromium, i n a fi l 1 - a n d - d r a w r e a c t o r . A d d i t i o n of 5 mg/L of chromium caused complete l o s s of n i t r i f i c a -t i o n a c t i v i t y . F o l l o w i n g a r e s t p e r i o d of s e v e r a l days, a second a d d i t i o n of 5 mg/L of chromium r e s u l t e d i n minimal r e d u c t i o n of n i t r i f i c a t i o n a c t i v i t y . Knoetze ejb a_l. (1980) reported on the a c c l i m a t i o n of n i t r i -f i e r s to cyanide i n a bench s c a l e r o t a t i n g b i o l o g i c a l c o n t a c t o r , fed with domestic sewage. Cyanide at a c o n c e n t r a t i o n of 0.5 mg/L caused 61% i n h i b i t i o n to a non-acclimated c u l t u r e . F i v e days l a t e r , the same t e s t was r e p e a t e d on the same c u l t u r e and the degree of i n h i b i t i o n had dropped to 26%. A t h i r d t e s t c a r r i e d out 15 days l a t e r , r e s u l t e d i n 57% i n h i b i t i o n w h i l e a f o u r t h t e s t , conducted immediately f o l l o w i n g the t h i r d t e s t and at double the cyanide c o n c e n t r a t i o n , r e s u l t e d i n o n l y 18% i n h i b i t i o n . They concluded that a n i t r i f i e r p o p u l a t i o n i n constant contact with cyanide would adapt and gain r e s i s t a n c e to i n h i b i t i o n , but would l o s e t h i s r e s i s t a n c e i f l e f t to m u l t i p l y i n i t s absence. Dedhar and M a v i n i c (1985) r e c e n t l y reported on the e f f e c t s of z i n c a d d i t i o n to a bench-scale, p r e - d e n i t r i f y i n g a c t i v a t e d sludge system, fed l a n d f i l l l e a c h a t e . The unacclimated n i t r i f i e r biomass was i n h i b i t e d by i n f l u e n t z i n c l e v e l s of 14.9 to 17.6 mg/L. The i n h i b i t i o n was overcome by reducing the z i n c l e v e l i n the i n f l u e n t to 5.5 mg/L; a subsequent gradual r i s e of z i n c l e v e l s t o 19.5 mg/L, o v e r a 30 day p e r i o d , caused no d i s c e r n a b l e i n h i b i t i o n . They a t t r i b u t e d t h i s l o s s of i n h i b i t o r y e f f e c t s to a c c l i m a t i o n . Huey (1982) reported that 1.7 uM of t a n n i c a c i d reduced the 162 growth r a t e of unacclimated c u l t u r e s of Nitrosomonas by 50%, but that the organism was a b l e to adapt and good growth was achieved a f t e r s e v e r a l s u b c u l t u r e s . Haug and McCarty (1972) and Stankewich (1972) reported on a c c l i m a t i o n of the n i t r i f i e r s to pH v a l u e s as low as 5.8 to 6.0. Focht and V e r s t r a e t e (1977) c i t e d s e v e r a l s t u d i e s which i n d i c a t e d that n i t r i f i e r s a c c l i m a t e d to the temperature regime of t h e i r h a b i t a t and adapted to low temperatures. M a v i n i c and Koers (1982) noted the a b i l i t y of the n i t r i f i e r s t o adapt to a c o m b i n a t i o n of low temperatures (5°C) and low pH (about 4). The a b i l i t y of the n i t r i f i e r s to r e g a i n t h e i r a c t i v i t y a p p e a r s to be l i n k e d , a t l e a s t i n p a r t , to t h e i r a b i l i t y t o a c c l i m a t e . F i g u r e 20 g r a p h i c a l l y demonstrates the e f f e c t of a c c l i m a t i o n during Run 4. As can be seen, non-acclimated c u l t u r e s were over 95% i n h i b i t e d by a f r e e ammonia c o n c e n t r a t i o n of 8.1 mg NH-j-N/L at the b e g i n i n g of the run. By Day 42, a shock l o a d i n g of 15.8 mg NH^-N/L produced o n l y a 65% r e d u c t i o n i n n i t r i f i c a t i o n a c t i v i t y . By Day 75, and at an a e r o b i c sludge of o n l y 3 to 5 days, a f r e e ammonia c o n c e n t r a t i o n of 22.1 mg NH3-N/L c o u l d o n l y achieve a 57% r e d u c t i o n i n n i t r i f i c a t i o n a c t i v i t y . The trend towards i n c r e a s i n g t o l e r a n c e to i n c r e a s i n g f r e e ammonia l e v e l s i s e v i d e n t . Another c o n f i r m a t i o n of the occurrence of a c c l i m a t i o n was obtained during Batch Test No.l (Run 4). As shown i n F i g u r e 14, the f r e e ammonia l e v e l s i n R e a c t o r s 1 and 2, a t s t a r t - u p , a v e r -aged 6 mg NH3-N/L. This c o n c e n t r a t i o n d i d not appear to cause a l a g i n n i t r i f i c a t i o n a c t i v i t y . At Hour 3, f o r example, the n i -t r i t e and n i t r a t e l e v e l s i n Reactors 1 and 2 were s i m i l a r to 163 A n a e r o b i c C e l l 22.1 F r e e A m m o n i a ( m g N H ^ - N / L ) 15.8 14.6 8.1 l F i r s t A e r o b i c C e l l 9 5 % 6 5 % 3 8 % 57% I % I n h i b i t i o n o f N i t r i f i c a t i o n A c t i v i t y A e r o b i c S l u d g e A g e ( d a y s ) too D A Y S F i g . 2 0 : Run 4 / S y s t e m 2 - I nh ib i t i on o f N i t r i f i c a t i o n A c t i v i t y D u e t o S h o c k L o a d o f F r e e A m m o n i a 16.4 t h o s e of R e a c t o r 3 a t s t a r t - u p (which had a f r e e ammonia l e v e l of 0.2 mg NH-j-N/L at s t a r t - u p ) . The i n i t i a l , high f r e e ammonia l e v e l i n R e a c t o r s 1 and 2 was e x p e c t e d to cause a l a g i n n i t r i f i c a t i o n a c t i v i t y because the environment w i t h i n those two Reactors was more i n h i b i t o r y than that w i t h i n Reactor 3 (where an ammonia l e v e l of about 0.2 mg NH3-N/L was maintained at a l l times during the continuous run). C l e a r l y t h at was not the case. A d d i t i o n a l evidence of a c c l i m a t i o n came i n Run 5. A f r e e ammonia l e v e l of 8.3 mg NH3-N/L on Day 10, caused o v e r 75% r e d u c -t i o n i n n i t r i f i c a t i o n a c t i v i t y i n System 2, whereas a f r e e ammo-n i a , "shock l o a d " of 13.6 mg NH3-N/L, on Day 73 of t h a t run, r e s u l t e d i n under 20% i n h i b i t i o n of n i t r i f i c a t i o n a c t i v i t y . Fur-thermore, r e c o v e r y time i n the f i r s t i nstance was i n the order of ten days, w h i l e i n the l a t t e r , i t was i n the order of three days. The e f f e c t s of a c c l i m a t i o n were a l s o e v i d e n t during Run 7, .v.hen the n i t r i f y i n g a c t i v i t y of both groups of organisms was not s i g n i f i c a n t l y i n h i b i t e d , i n s p i t e of f r e e ammonia l e v e l s ranging between 20 and 30 mg NH3-N/L; t h i s c o n c e n t r a t i o n range was found to be i n h i b i t o r y to both groups of unacclimated organisms i n a l l runs, and confirmed by s e v e r a l i n v e s t i g a t o r s (Prakasam and Loehr, 1972; Anthonisen et a_l., 1976; V e r s t r a e t e et a U , 1977). A c c l i m a t i o n may be a major f a c t o r r e s p o n s i b l e f o r the wide discrepancy i n f r e e ammonia v a l u e s reported as being i n h i b i t o r y to the n i t r i f i e r s (see Table 2). I t i s probable that a number of e a r l i e r s t u d i e s were not of s u f f i c i e n t d u r a t i o n to a l l o w the e f f e c t s of a c c l i m a t i o n to manifest themselves. I t i s c l e a r from t h i s study that both groups of organisms were capable of a c c l i m a -t i n g to f r e e ammonia l e v e l s as h i g h as 40 mg NH3-N/L. 165 REVERSING THE DECLINE IN NITRITE BUILD-UP The d e c l i n e i n n i t r i t e accumulation was probably caused by the a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia. A number of p o t e n t i a l mechanisms were i n v e s t i g a t e d during Runs 4, 5, 6 and 7 to r e v e r s e or minimize the e f f e c t s of a c c l i m a t i o n . They i n c l u d e d : 1) Reduction of the sludge age 2) Extension of the contact time with f r e e ammonia 3) R a i s i n g of the f r e e ammonia l e v e l 4) Use of a genuine waste 5) Double s u b s t r a t e i n h i b i t i o n 6) P r o v i s i o n of i n t e r n a l r e c y c l e or intermediary d e n i t r i f i c a t i o n 7) Temporary r e d u c t i o n of f r e e ammonia l e v e l s 8) Temporary stoppage of feed Reduction of the Sludge Age In an e f f o r t to r e v e r s e the d e c l i n e i n n i t r i t e b u i l d - u p , the ae r o b i c sludge age i n System 1 during Run 4 was dropped, on Day 53 from over 22 days to an average 2.9 days f o r the next 28 days (to Day 85); s t a r t i n g on Day 86, i t rose to about 6 days, t i l l t h e end of the run on Day 98. As noted i n F i g u r e 7, the d r a s t i c r e d u c t i o n i n a e r o b i c SRT l e d to a temporary r e v e r s a l of the d e c l i n i n g n i t r i t e l e v e l s , but the d e c l i n i n g trend r e - e s t a b l i s h e d i t s e l f , once again, on Day 79. These f i n d i n g s c o n t r a d i c t those of B e c c a r i et a_l. (1979) who reported, on the r e s u l t s of a run of u n s p e c i f i e d d u r a t i o n , a predominance of n i t r i t e at sludge ages 166 between 2.5 and 4.5 days and i t s complete o x i d a t i o n to n i t r a t e at sludge ages i n excess of 5 days. In a l l runs, n i t r i t e accumulation was induced at sludge ages ranging between 10 to 30 days, and was su s t a i n e d f o r extended p e r i o d s of time at a e r o b i c sludge ages of 10 to 13 days, as c o n f i r m e d i n Runs 2 and 7, but c o u l d not be s u s t a i n e d once a c c l i -mation manifested i t s e l f , even at sludge ages of under 3 days. Extension of the Contact Time With Free Ammonia The contact time with the i n h i b i t o r y , f r e e ammonia l e v e l i n the anaerobic c e l l was doubled i n both systems during Run 4, by c o n v e r t i n g the f i r s t two c e l l s of each system to an anaerobic mode. This d i d not r e v e r s e the d e c l i n e i n n i t r i t e b u i l d - u p nor d i d i t r e s u l t i n any measurable i n c r e a s e i n i n h i b i t o r y e f f e c t s . T h i s was confirmed from the r e s u l t s of Batch Test No.l, under-taken at the end of t h a t Run. The v i r t u a l i d e n t i c a l r e s u l t s obtained f o r Reactors 1 and 2 (see F i g u r e 14) i n d i c a t e that the degree of n i t r i t e b u i l d - u p was not a f f e c t e d by d o u b l i n g the contact time to f r e e ammonia from 3 to 6 hours. I t can be c o n c l u d e d t h a t c o n v e r t i n g 50% of the system t o an anaerobic mode, to a l l o w 50% contact time to i n h i b i t o r y f r e e ammonia l e v e l s , was i n s u f f i c i e n t to r e v e r s e the d e c l i n e i n n i -t r i t e l e v e l s . R a i s i n g of the Free Ammonia L e v e l As d i s c u s s e d e a r l i e r and shown i n F i g u r e 19, no c o r r e l a t i o n c o u l d be e s t a b l i s h e d between the f r e e ammonia l e v e l and the degree of n i t r i t e accumulation, once a c c l i m a t i o n manifested i t -s e l f . Attempts to r e v e r s e the d e c l i n e i n n i t r i t e b u i l d - u p , by 167 r a i s i n g the f r e e ammonia l e v e l , were undertaken i n s e v e r a l runs. In Run 4, the f r e e ammonia l e v e l i n the a n a e r o b i c c e l l of System 2 was r a i s e d from an average 4.6 to 11.4 mg NH3-N/L and main-t a i n e d at t h a t l e v e l from Day 40 to 98; i n Run 6, i t was r a i s e d from 4.8 to 18.8 mg NH3-N/L from Day 9 to 22. In both c a s e s , t h e d e c l i n e was not reversed. During Run 7, the f r e e ammonia l e v e l was r a i s e d s u b s t a n t i a l l y during two p e r i o d s : between Days 35 and 70 and between Days 228 and 24.8. The r e s u l t a n t average f r e e ammonia l e v e l s were 19.1 and 25.5 mg NH3-N/L r e s p e c t i v e l y , with peaks as high as 40 mg NH3-N/L. In both i n s t a n c e s , the d e c l i n e was not a r r e s t e d by t h i s technique. I n d i r e c t evidence of i t s l a c k of e f f e c t can be i n t e r p r e t e d from the r e s u l t s of Batch Test No.l (Run 4). D i f f e r i n g f r e e ammonia l e v e l s at the s t a r t of the t e s t , ranging from 0.2 to 6.3 mg NH3-N/L i n . t h e four r e a c t o r s , had no apparent e f f e c t on the r a t e of ammonia and n i t r i t e o x i d a t i o n . I t appears that r a i s i n g f r e e ammonia l e v e l s was i n e f f e c t i v e i n m a i n t a i n i n g or r e v e r s i n g n i t r i t e b u i l d - u p when undertaken by i t s e l f with a c c l i m a t e d c u l t u r e s ; however, t h i s technique may have been p a r t i a l l y e f f e c t i v e when undertaken i n c o n j u n c t i o n with i n t e r n a l d e n i t r i f i c a t i o n and r e d u c t i o n of SRT (Run 4/System 2). Use of a Genuine Waste It was p o s t u l a t e d that the use of a genuine waste c o n t a i n i n g p o t e n t i a l l y i n h i b i t o r y compounds might cause s u f f i c i e n t p e r t u r b a -t i o n i n the biomass to prevent or minimize a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia due to the presence of other " i n h i b i t o r y " substances. The use of a l a n d f i l l l e a c h a t e ' f e e d source, during Runs 6 and 7, d i d not prevent a c c l i m a t i o n of the 168 n i t r i t e o x i d i z e r s to f r e e ammonia. Double Substrate I n h i b i t i o n The simultaneous impact of two or more s u b s t r a t e s , each known to act ,as a s e l e c t i v e i n h i b i t o r of the n i t r i t e o x i d i z e r s , was i n v e s t i g a t e d . The o b j e c t i v e was to determine i f the presence of two i n h i b i t o r y s u b s t r a t e s c o u l d act s y n e r g i s t i c a l l y to p r o v i d e s u f f i c i e n t " s t r e s s " on the organisms and prevent t h e i r a c c l i m a -t i o n to f r e e ammonia. The p o s s i b l e use of n i t r o u s a c i d and low DO l e v e l s , as s e l e c t i v e i n h i b i t o r s , had been r u l e d out, based on the r e s u l t s of e a r l i e r runs. The l i t e r a t u r e suggested that sodium c h l o r a t e was capable of s e l e c t i v e l y i n h i b i t i n g the n i t r i t e o x i d i -zers. As a r e s u l t , the combination i n v e s t i g a t e d was that of f r e e ammonia and sodium c h l o r a t e . Double s u b s t r a t e i n h i b i t i o n was attempted during Run 6, i n an e f f o r t to r e v e r s e the d e c l i n i n g trend i n n i t r i t e b u i l d - u p . The a d d i t i o n of sodium c h l o r a t e (reaching a maximum c a l c u l a t e d con-c e n t r a t i o n of 6.2 mM w i t h i n three c e l l s , and 4.1 mM i n the r e -maining f i v e c e l l s of the system) r e s u l t e d i n the t o t a l suppres-s i o n of n i t r i f i c a t i o n a c t i v i t y . T h i s was unexpected, s i n c e i t had been reported that sodium c h l o r a t e suppressed the growth of N i t r o b a c t e r i n the c o n c e n t r a t i o n range of 0.01 to 0.001 mM, and i n h i b i t e d n i t r i t e o x i d a t i o n , and not ammonia o x i d a t i o n , at a co n c e n t r a t i o n of 5 mM (Lees, 1963). These r e s u l t s were supported more r e c e n t l y by B e l s e r and Mays (1980), who confirmed that, at co n c e n t r a t i o n s of 10 mM, sodium c h l o r a t e acted as a s p e c i f i c i n h i b i t o r of n i t r i t e o x i d a t i o n , with l i t t l e i n h i b i t o r y e f f e c t on ammonia o x i d a t i o n . The use of sodium c h l o r a t e i n a c t i v a t e d sludge 169 systems, to i n h i b i t the n i t r i t e o x i d i z e r s , has a l s o been reported i n the l i t e r a t u r e . Voets et a_l. (1975), r e g u l a r l y added a 1 mM s o l u t i o n of sodium c h l o r a t e to i n h i b i t n i t r i t e o x i d a t i o n i n mixed l i q u o r , w h i l e studying n i t r o g e n removal from a h i g h l y nitrogenous waste. C l e a r l y , the r e s u l t s obtained c o n t r a d i c t e d those f i n d i n g s and i n d i c a t e d that sodium c h l o r a t e acted as a t o t a l i n h i b i t o r of both groups of n i t r i f i e r s . T h i s apparent c o n t r a d i c t i o n with the l i t e r a t u r e was e x p l a i n e d r e c e n t l y by Hynes and Knowles (1983). They found that c h l o r a t e d i d not i n h i b i t pure c u l t u r e s of N i t r o - somonas but d i d i n h i b i t Nitrosomonas c u l t u r e s growing i n mixed c u l t u r e s with N i t r o b a c t e r . T h e i r research r e v e a l e d that c h l o r a t e was reduced to c h l o r i t e , by N i t r o b a c t e r , under both a e r o b i c and anaerobic c o n d i t i o n s and that c h l o r i t e , and not c h l o r a t e , was the i n h i b i t o r y compound to both groups of organisms. E a r l i e r work, by other m i c r o b i o l g i s t s , was c a r r i e d out on pure c u l t u r e s of e i t h e r organism, but not on mixed c u l t u r e s . Thus, pure c u l t u r e s of Nitrosomonas were not exposed to the c h l o r i t e ion produced by N i t r o b a c t e r c u l t u r e s , as a r e s u l t of c h l o r a t e r e d u c t i o n . The work of Hynes and Knowles once again h i g h l i g h t s the major d i f f e r e n c e between pure c u l t u r e s t u d i e s , favoured by micro-b i o l o g i s t s , and m i x e d - c u l t u r e research that i s inherent i n b i o l o -g i c a l wastewater treatment. I t u n d e r l i n e s the extreme c a u t i o n that should be e x e r c i s e d when e x t r a p o l a t i n g r e s u l t s from pure c u l t u r e s t u d i e s f o r use i n m i x e d - c u l t u r e work. The f i n d i n g s of Hynes and Knowles r e s o l v e s the c o n t r a d i c -t i o n s between the r e s u l t s of Run 6 and t h o s e of pure c u l t u r e 170 work, but f a i l s t o r e c o n c i l e i t w i t h t hose of V o e t s and c o l -leagues, who reported no l o s s of ammonia o x i d a t i o n a b i l i t y i n m i x e d - l i q u o r , subjected to sodium c h l o r a t e a d d i t i o n . The major d i f f e r e n c e between t h e i r study and Run 6 l i e s i n the concentra-t i o n of sodium c h l o r a t e added; t h e i r v a l u e s were s i x times lower. T h e i r r e s u l t s , however, seem to c o n t r a d i c t the f i n d i n g s of Hynes and Knowles, who reported that ammonia o x i d a t i o n appeared to be 50 times more s e n s i t i v e to sodium c h l o r i t e than was n i t r i t e o x i d a t i o n . C l e a r l y , the matter has not been f u l l y r e s o l v e d yet, and a d d i t i o n a l s t u d i e s are needed to determine: 1) whether sodium c h l o r a t e does indeed act as a s e l e c t i v e i n h i b i t o r at low concen-t r a t i o n s , and 2) the c o n c e n t r a t i o n range f o r such s e l e c t i v e i n h i b i t i o n . Even i f i t i s found to a c t as a s e l e c t i v e i n h i b i t o r at c o n c e n t r a t i o n s lower than 4 mM, c a u t i o n w i l l have to be exer-c i s e d i n i t s use, s i n c e minor misadjustments of the dosing r a t e c o u l d l e a d to g e n e r a l i n h i b i t i o n of n i t r i f i c a t i o n a c t i v i t y . A review of the l i t e r a t u r e has f a i l e d to i d e n t i f y other com-pounds that can be used as s e l e c t i v e i n h i b i t o r s of the n i t r i t e o x i d i z e r s . P r o v i s i o n of I n t e r n a l Recycle or Intermediary D e n i t r i f i c a t i o n As d i s c u s s e d e a r l i e r , e xtension of the a e r a t i o n time a l l e -v i a t e d the s t r e s s on the n i t r i t e o x i d i z e r s and r e s u l t e d i n l o s s of n i t r i t e b u i l d - u p . T h i s suggested that the implementation of an i n t e r n a l r e c y c l e , or an i n t e r n a l d e n i t r i f i c a t i o n step, might remove the accumulated n i t r i t e from the system p r i o r to i t s o x i d a t i o n to n i t r a t e ; t h i s might prevent the n i t r i t e o x i d i z e r p o p u l a t i o n from growing, i n s p i t e of t h e i r a c c l i m a t i o n to f r e e 171 ammonia. The i n t e r n a l r e c y c l e step was implemented i n System 2, d u r i n g Run 5. T h i s extended the d u r a t i o n of n i t r i t e b u i l d - u p i n comparison to System 1, where no i n t e r n a l r e c y c l e was implemented (see F i g u r e s 10 and 11). By the end of the run, however, n i t r i t e l e v e l s i n the f i r s t a e r o b i c c e l l had dropped to around 35% of the t o t a l o x i d i z e d n i t r o g e n s p e c i e s present. The use of intermediary d e n i t r i f i c a t i o n was i n v e s t i g a t e d during Run 7. I t succeeded i n r e v e r s i n g the i n i t i a l d e c l i n e i n n i t r i t e l e v e l s i n the f i r s t a e r o b i c c e l l from under 35% on Day 70, (see F i g u r e 1.3.) to over 75% by Day 144, and i t permitted the maintenance of r e l a t i v e l y high n i t r i t e l e v e l s i n the system p r i o r to d e n i t r i f i c a t i o n . N i t r i t e b u i l d - u p i n the a e r o b i c c e l l , preced-ing i n t e r n a l d e n i t r i f i c a t i o n , exceeded 80%, with over 90% of the ammonia content of the feed o x i d i z e d i n that c e l l . The r e s u l t s of Run 5 were l e s s f a v o u r a b l e than those of Run 7. T h i s i s d i f f i c u l t to e x p l a i n s i n c e the n i t r i t e o x i d i z e r s were a c c l i m a t e d to f r e e ammonia i n both runs and there was l i t t l e p r a c t i c a l d i f f e r e n c e between i n t e r n a l r e c y c l e and intermediary d e n i t r i f i c a t i o n , as implemented during these two runs. The major d i f f e r e n c e s between the two runs were: 1) Duration of run 2) L e v e l of f r e e ammonia i n the f i r s t c e l l 3) Type of feed used 4) Feed TKN c o n c e n t r a t i o n Run 5 l a s t e d f o r 76 days and was fed a s y n t h e t i c r e c i p e , whereas Run 7 l a s t e d f o r 359 days and was f e d P o r t Mann l e a c h a t e . 17.2 The f r e e ammonia l e v e l i n C e l l 1, of System 2, d u r i n g Run 5, averaged 6.5 mg NH-j-N/L, whereas i t v a r i e d between 5 and 20 mg NHj-N/L during the f i r s t 250 days of Run 7. The feed TKN concen-t r a t i o n averaged 210 mg TKN/L during Run 5 (from Day 34 onwards) and about 265 mg NH^-N/L during Run 7. The i n i t i a l r e c o v e r y of n i t r i t e b u i l d - u p from Day 70 on-wards, during Run 7, was dramatic. I t was achieved probably as a r e s u l t of implementation of i n t e r n a l d e n i t r i f i c a t i o n which was attempted f o l l o w i n g the o b s e r v a t i o n s made i n e a r l i e r runs, con-cer n i n g the l a g time i n n i t r i t e o x i d a t i o n . I t was observed that n i t r i t e o x i d a t i o n r a t e s , by a p o p u l a t i o n a c c l i m a t e d to f r e e ammonia, s t i l l lagged ammonia o x i d a t i o n r a t e s , r e s u l t i n g i n a temporary but s i g n i f i c a n t accumulation of n i t r i t e l a s t i n g s e v e r a l hours f o l l o w i n g the e x i t from the high ammonia environment of the anaerobic c e l l . The concept of intermediary d e n i t r i f i c a t i o n , or i n t e r n a l r e c y c l e , was based upon t a k i n g advantage of t h i s s h o r t l a g , to d e n i t r i f y the n i t r i t e produced w i t h i n the f i r s t two to three hours of a e r a t i o n . N i t r i t e subsequently removed from the system, by d e n i t r i f i c a t i o n , was no longer a v a i l a b l e as s u b s t r a t e to the n i t r i t e o x i d i z e r s . T h i s a l l o w e d n i t r i t e l e v e l s i n the f i r s t a e r o b i c c e l l during Run 7 to r i s e s t e a d i l y during the next 80 days t o o v e r 80% of the o x i d i z e d n i t r o g e n s p e c i e s p r e s e n t and s u s t a i n i t s e l f at that l e v e l f o r a f u r t h e r 45 days, a f t e r which i t d e c l i n e d steadily.. No apparent reason was found f o r the even-t u a l d e c l i n e i n n i t r i t e l e v e l s ; however, t h i s procedure showed promise as a p o s s i b l e s o l u t i o n to the dilemma at hand. 173 Temporary Reduction of Free Ammonia Level s An attempt was made d u r i n g Run 7 to r e v e r s e the a p p a r e n t a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia by tempora-r i l y stopping sodium hydroxide a d d i t i o n to the system and a l l o w -ing the f r e e ammonia l e v e l i n the f i r s t anaerobic c e l l to drop to around 1 mg NH3-N/L. This a c c e l e r a t e d the d e c l i n e i n n i t r i t e b u i l d - u p . A f t e r a p e r i o d of two sludge ages (35 days), sodium hydroxide a d d i t i o n was resumed. I t d i d not l e a d to any apparent r e v e r s a l i n n i t r i t e d e c l i n e , and four days f o l l o w i n g resumption of i t s a d d i t i o n , sodium hydroxide feed was again t e m p o r a r i l y stopped f o r the next 31 days. On Day 318, sodium hydroxide a d d i -t i o n was resumed. I t r e s u l t e d i n minimal n i t r i t e b u i l d - u p . C l e a r l y , the a c c l i m a t e d biomass was not "washed away" over a p e r i o d of a l m o s t 70 days, as a r e s u l t of r e d u c i n g the f r e e ammo-nia l e v e l s w i t h i n the system. Temporary Stoppage of Feed This procedure was attempted on Day 338, during Run 7, i n an e f f o r t to r e v e r s e the apparent a c c l i m a t i o n of the n i t r i t e o x i d i -zers to f r e e ammonia. Feeding to the system was stopped for a p e r i o d of 13 days, and a l l c e l l s were converted to an a e r o b i c mode of op e r a t i o n . The mixed l i q u o r was maintained under endoge-nous (i.e., s t a r v a t i o n ) c o n d i t i o n s during that p e r i o d of time. On Day 351, feed was resumed and r e s u l t e d i n a t r a n s i e n t r i s e i n n i t r i t e l e v e l s , accompanied by a sharp r e d u c t i o n i n ammonia o x i d a t i o n a c t i v i t y , both of which appeared to be l i n k e d to a g e n e r a l l o s s of n i t r i f i c a t i o n c a p a b i l i t y (as a r e s u l t of endoge-nous c o n d i t i o n s ) . The n i t r i t e l e v e l d e c l i n e d r a p i d l y i n the next 174 few days, and on Day 356, sodium hydroxide a d d i t i o n was resumed. I t d i d not a r r e s t the d e c l i n e i n n i t r i t e l e v e l s , i n s p i t e of r a p i d l y r i s i n g f r e e ammonia l e v e l s (to about 9 mg NH^-N/L). OTHER RESULTS AND OBSERVATIONS This s e c t i o n d e a l s with a number of t o p i c s t a n g e n t i a l to the main t h r u s t of the research program, but of r e l e v a n c e to the broader aspects of the t o p i c . N i t r i f i c a t i o n Rates The ammonia o x i d a t i o n r a t e i s reported as the h i g h e s t r a t e of ammonia o x i d a t i o n per u n i t mass per u n i t time A NHJ-N Ammonia o x i d a t i o n r a t e = (20) g VSS.hr This equation i s a l s o r e f e r r e d t o, rather l o o s e l y by many res e a r c h e r s , as the n i t r i f i c a t i o n r a t e . In r e a l i t y , most measured v a l u e s , i n a c t i v a t e d sludge systems, should be d e f i n e d as ammonia o x i d a t i o n r a t e , s i n c e they do not r e f l e c t the r a t e of n i t r i t e o x i d a t i o n . For example, the absence of n i t r i t e from the medium i n d i c a t e s o n l y that the r a t e of n i t r i t e o x i d a t i o n i s at l e a s t as r a p i d as that of ammonia o x i d a t i o n ; i t does not p r e c l u d e the p o s s i b i l i t y that the n i t r i t e o x i d a t i o n step may be f a s t e r than that of ammonia o x i d a t i o n . The presence of n i t r i t e i n the medium, on the other hand, i s an i n d i c a t i o n that the r a t e of n i t r i t e o x i d a t i o n i s slower than that of ammonia o x i d a t i o n . In order to conform with "common p r a c t i c e " , the term n i t r i -f i c a t i o n r a t e w i l l be used when r e f e r r i n g to ammonia o x i d a t i o n 175 r a t e s . The h i g h e s t r a t e of ammonia o x i d a t i o n can o n l y be d e t e r -mined when a l l s u b s t r a t e s are present i n excess. In the course of t h i s work, t h i s c o n d i t i o n was u s u a l l y met o n l y i n the f i r s t a e r o b i c c e l l , as ammonia was n o r m a l l y exhausted w i t h i n the second a e r o b i c c e l l . C a l c u l a t e d n i t r i f i c a t i o n r a t e s (i.e., ammonia o x i -d a t i o n rates) are, t h e r e f o r e , l i m i t e d to those measured i n the f i r s t a e r o b i c c e l l . T a ble 17 summarizes some of the n i t r i f i c a t i o n r a t e s pub-l i s h e d i n the l i t e r a t u r e . They are broken down under three c a t e -g o r i e s to r e f l e c t experimental set-up. G e n e r a l l y , i n - s i t u (con-t i n u o u s l y - r u n systems) t e s t s do not r e f l e c t maximum n i t r i f i c a t i o n r a t e s , but lower r a t e s , due to s u b s t r a t e exhaustion partway through the p e r i o d of a e r a t i o n . Pure c u l t u r e r e s u l t s r e f l e c t maximum r a t e s a t t a i n a b l e when the e n t i r e biomass c o n s i s t s of ammonia o x i d i z e r s . Batch t e s t s on a c c l i m a t e d biomass g e n e r a l l y p r o v i d e the most r e a l i s t i c r e s u l t s , s i n c e they r e f l e c t the maxi-mum n i t r i f y i n g a b i l i t y of a biomass when a l l s u b s t r a t e s are present i n excess. What i s e v i d e n t from Table 17, i s the wide range i n reported v a l u e s . They vary from a low of 0.7 to a high of 600 mg NH^-N o x i d i z e d / g VSS.hr. The n i t r i f i c a t i o n r a t e i s dependent, among others, on the n i t r i f i e r f r a c t i o n w i t h i n the biomass. T h i s , i n turn, i s depen-dent on parameters such as sludge age and the COD:TKN r a t i o of the feed. T h i s i s best i l l u s t r a t e d i n F i g u r e 21 ( t a b u l a t e d i n Table 18), which presents average n i t r i f i c a t i o n r a t e s f o r a l l runs, c a l c u l a t e d f o r p e r i o d s of "steady-state" c o n d i t i o n s (i.e., no apparent f r e e ammonia i n h i b i t i o n , s t a b l e SRT, no changes i n feed COD:TKN r a t i o s , and no process upsets). 176 Table 17 - N i t r i f i c a t i o n Rates Reported i n the L i t e r a t u r e N i t r i f i c a t i o n Type of Remarks Reference Rate Feed ( F i r s t Author) Pure C u l t u r e Studies 220 - 640 s y n t h e t i c S r i n a t h , 1976 130 - 320 s y n t h e t i c H a l l , 1980 24 - 41 s y n t h e t i c Neufeld, 1980 Batch Tests 7.7 sewage Wild, 1971 2.3 - 7.4 p o u l t r y S r i n a t h , 1976 4.5 - 5.5 sewage Beer, 1977 0.8 i n d u s t r i a l B r i d l e , 1979 7 - 8* s y n t h e t i c sequencing batch r e a c t o r Alleman, 1980 5 - 7* s y n t h e t i c Braam, 1981 0.9 coke Sutton, 1981 0.5 - 20 sewage P a i n t e r , 198 3 5 - 10 sewage second stage A.S. B a i l e y , 1983 I n - S i t u Tests 6 sewage 2.5 hrs a e r a t i o n Sutton, 1975 2 sewage 6 hrs a e r a t i o n 1.3 sewage 10 hrs a e r a t i o n Young, 1979 0.7 - 1.5 sewage 5 hrs a e r a t i o n Jones, 1980 1.7 - 3.7 i n d u s t r i a l 7.5 hrs a e r a t i o n Ford, 1980 0.7 - 1.6 sewage range f o r 5 p l a n t s B a r t h , 1981 N i t r i f i c a t i o n r a t e i s expressed as mg NH|-N o x i d i z e d or mg NO3-N produced/g VSS.hr * Expressed as MLSS instead of VSS 177 20 00 Legend • C0D:TKN=3.1-3.4 O COD:TKN=2.2-2.4 A C0D:TKN=4.6 2 / 2 R u n / S y * t » m 3 . 4 C O D , T K N , 4 / 1 4 . 6 4 / 2 4 . 6 C O D . T K N - 3 . 1 - 3 . 4 , Y - 1 4 . 2 9 • 2 8 . I X " ' - 0 . 3 7 8 X R - 0 . 9 1 n - 5 H 5 H o u _c CO CO > Q LU <J O O Oct o. Z i E 5 10 15 20 25 30 35 SRT ( d a y s ) 40 F i g . 2 1 : E f f e c t o f S R T o h N i t r i f i c a t i o n R a t e Table 18 - N i t r i f i c a t i o n Rates Run System P e r i o d N i t r i f i c a t i o n n S COD:TKN SRT No. No. (days) Rate mg:mg (d) 2 1 96 - 147 17.1 20 1.0 2.4 12.6 2 2 18 - 69 15.0 13 4.4 2.2 11.3 2 2 70 - .147 14.3 28 3.6 2.4 15.8 4 1 16 - 39 7.8 6 0.2 4.6 34 .3 4 1 45 - 98 17.2 15 4.5 3.2 6.4 4 2 16 - 39 5.8 13 0.7 4.6 .37.6 4 2 74 — 98 11.4 4 0.5 3.2 8.8 5 1 34 - 62 1.5.2 5 1.9 3.4 10.0 5 2 59 - 69 13.2 4 ~ 3.4 10 .5 7 N/A 105 - 112 10.6 3 - 2.2 20 7 N/A 140 - 230 8.4 27 1.1 3.1 19 .'2 N i t r i f i c a t i o n r a t e i s expressed as mg NO^-N produced/g VSS.hr n: number of measurements s: standard d e v i a t i o n NOTE: 1) No values are given f o r Run 3 because i t was too s h o r t . 2) No V a l u e s are g i v e n f o r Run 6 because n i t r i f i c a t i o n was i n h i b i t e d during most of the run. 179 The e x i s t e n c e of an i n v e r s e r e l a t i o n s h i p between n i t r i f i c a -t i o n r a t e s and SRT i s noteworthy. C l e a r l y , t h i s r e l a t i o n s h i p should r e v e r s e i t s e l f below the minimum SRT needed to maintain n i t r i f i c a t i o n . ' The e f f e c t of CODrTKN r a t i o on the n i t r i f i c a t i o n process was not as c l e a r from the r e s u l t s obtained as that of the SRT. The n i t r i f i c a t i o n r a t e s a s s o c i a t e d with the lowest COD:TKN r a t i o (2.1 to 2.2) appear to be higher than those of other r a t i o s . S e v e r a l authors have confirmed that as the r a t i o i s lowered, the ammonia f r a c t i o n i n the wastewater i n c r e a s e s , l e a d i n g to a p r o p o r t i o n a l r i s e i n the n i t r i f i e r f r a c t i o n , causing the n i t r i f i c a t i o n r a t e to increase (EPA, 1975; Sherrard et al., 1982). The o b s e r v a t i o n that n i t r i f i c a t i o n r a t e s increase with: decreasing SRT, and lowering of the COD:TKN r a t i o , appears to be i n g e n e r a l agreement with the f i n d i n g s of Sherrard et. a l . (1982). According to the graphs they developed, from f i r s t p r i n c i p l e s , the n i t r i f i c a t i o n r a t e i n c r e a s e s by over 260%, as the SRT drops from 20 to 5 days, and the COD:TKN content of the feed i s reduced from 4.6 to 3.2. Th i s i s i n c l o s e agreement with the r e s u l t s of System 1 i n Run 4, which showed that the n i t r i f i c a t i o n r a t e increased by about 215%, as the a e r o b i c sludge age dropped from 22 to 4 days, and the COD:TKN content of the feed was reduced from 4.6 to 3.2. Sherrard and c o l l e a g u e s a t t r i b u t e d the increased n i t r i f i c a t i o n r a t e at lower SRT to a lower decay rate c o e f f i c i e n t f o r the n i t r i f i e r s . The impact of the f o l l o w i n g parameters on n i t r i f i c a t i o n r a t e s were a l s o i n v e s t i g a t e d during Run 2: 180 1) D i s s o l v e d oxygen 2) pH i n the a e r o b i c c e l l 1) D i s s o l v e d Oxygen As seen i n F i g u r e 22, f o r System 1 i n Run 2, a s i g n i f i c a n t c o r r e l a t i o n e x i s t e d between the DO l e v e l and n i t r i f i c a t i o n r a t e s . The r e l a t i o n s h i p approximated a f i r s t order one up to a DO l e v e l of around 2.5 mg/L, above which the n i t r i f i c a t i o n r a t e appeared to be i n d e p e n d e n t of DO l e v e l s . The e s t i m a t e d Ko v a l u e was 0.8 mg/L. T h i s i s higher than the reported v a l u e s f o r Nitrosomonas i n pure c u l t u r e s , which range between 0.25 and 0.5 mg/L ( L o v e l e s s and P a i n t e r , 1968; Peeters et a_l., 1969; Focht and Chang, 1975; P a i n t e r , 1977). P a i n t e r (1977) reported on an a c t i v a t e d sludge system where the Ko v a l u e was found to be 0.3 mg/L, i n c l o s e agreement with pure c u l t u r e r e s u l t s . R e c e ntly however, MacFarlane and Herbert (1984) reported the r e s u l t s of chemostat s t u d i e s , where the n i t r i f i c a t i o n a c t i v i t y increased s t e a d i l y with i n c r e a s -i n g DO l e v e l s up to a c o n c e n t r a t i o n of 6 mg/L. T h i s l e d to the suggestion that the Ko v a l u e f o r Nitrosomonas i s probably higher than commonly accepted. These r e s u l t s are i n c l o s e agreement with those of Terashima and Ishikawa (1984), who reported a Ko v a l u e of 0.8 mg/L f o r a n i t r i f y i n g , o x i d a t i o n d i t c h process. They a l s o found that the f i r s t order r e l a t i o n s h i p approximated a zero-order one up to a DO l e v e l of 2 mg/L. A number of a c t i v a t e d sludge s t u d i e s have contended that n i t r i f i c a t i o n i s i n h i b i t e d at DO l e v e l s below 1.5 to 2.0 mg/L (Wuhrmann, 1968; Sharma and A h l e r t , 1977). However, others have reported maximum n i t r i f i c a t i o n r a t e s at DO l e v e l s as low as 1 181 0.0 0.5 1.0 1.5 2.0 2.5 D I S S O L V E D O X Y G E N ( m g / L ) F i g . 2 2 : Run 2 / S y s t e m 1 - E f f e c t o f D i s s o l v e d O x y g e n o n N i t r i f i c a t i o n R a t e in F i r s t A e r o b i c C e l l 182 mg/L (Wild et a_l., 1971). O b v i o u s l y , no consensus appears to have emerged concerning the e f f e c t of low DO c o n c e n t r a t i o n s on n i t r i -f i c a t i o n r a t e s . Some p o s t u l a t e d f a c t o r s which may be r e s p o n s i b l e f o r t h i s d i vergence i n reported r e s u l t s i n c l u d e : 1) the e f f e c t s of m u l t i p l e - s u b s t r a t e l i m i t i n g k i n e t i c s , where the minimum DO l e v e l needed f o r n i t r i f i c a t i o n decreases with i n c r e a s i n g ammonia l e v e l s (Stenstrom and Poduska, 1980) and, 2) the presence of a DO co n c e n t r a t i o n g r a d i e n t across a b i o f l o c ( P a i n t e r , 1977). Regardless of these c o n t r a d i c t i o n s , i t i s g e n e r a l l y accepted that the n i t r i f i c a t i o n process i s s e n s i t i v e to low DO l e v e l s , and as such, they are n o r m a l l y maintained above 2 mg/L at treatment p l a n t s p r a c t i s i n g n i t r i f i c a t i o n (Downing, 1968; P a i n t e r , 1977). The r e s u l t s obtained during Run .2 tend to confirm t h i s . I t should be remembered that m a i n t a i n i n g maximum n i t r i f i c a t i o n r a t e s i s not always necessary i n n i t r i f y i n g treatment p l a n t s , due to the re s e r v e c a p a c i t y u s u e l l y a v a i l a b l e i n the form of excess a e r a t i o n volume and SRT. I t may be a c c e p t a b l e under such circumstances to o p e r a t e the p r o c e s s a t DO l e v e l s below 2 mg/L. Examples a r e a number of long sludge age (44 to 164 days) o x i d a t i o n d i t c h e s , a c h i e v i n g complete n i t r i f i c a t i o n at DO l e v e l s below 0.5 mg/L (Rittman and Langeland, 1985). 2) pH The p e r i o d , ranging between Days 88 and 144 f o r System 2 i n Run 2, was s e l e c t e d f o r a n a l y s i s because: 1) i t was of su f -f i c i e n t l y long d u r a t i o n to a l l o w the c o l l e c t i o n of data, 2) no operat i n g c h a r a c t e r i s t i c s other than system pH were a l t e r e d , 3) the pH range w i t h i n the system was being g r a d u a l l y reduced (no 183 sudden changes i n pH occurred). As shown i n F i g u r e 23, optimum n i t r i f i c a t i o n appeared to occur at pH 6.8 and the r a t e d e c l i n e d s t e a d i l y at v a l u e s above and below i t . These r e s u l t s c o n t r a d i c t the f i n d i n g s of most i n v e s t i g a t o r s , who re p o r t optimum n i t r i f i c a -t i o n r a t e s i n the pH range of 7.5 to 8.0 (Boon and Laudelout, 1962; P a i n t e r and L o v e l e s s , 1968; S r i n a t h et a_l., 1976; Shieh and LaMotta, 1979; Neufeld et a l . , 1980; Jones and Paskins 1982; Pa i n t e r and L o v e l e s s , 1983). Some authors reported optima o u t s i d e t h i s range. Wong-Chong and Loehr (1975) found i t to range between 7.0 and 7.5, w h i l e W i l d et a_l. (1971) and P a i n t e r and L o v e l e s s (1983) reported optima as high as 8.4 and 8.5, r e s p e c t i -v e l y . This apparent anomaly was r e c e n t l y c l a r i f i e d by Quin l a n (1984), who analyzed the data of three p u b l i s h e d s t u d i e s d e a l i n g with pure c u l t u r e s of Nitrosomonas. The author developed a c o r r e -l a t i o n between maximum n i t r i f i c a t i o n r a t e s , pH and ammonium l e v -e l s . The c o r r e l a t i o n showed that the pH, corresponding to maximum n i t r i f i c a t i o n r a t e s , s h i f t e d from about 9.1 to 8.3 as the ammo-nium l e v e l increased from 3.2 to 238 mg N H | - N / L . Although the optimum pH v a l u e obtained during Run 2 was s t i l l much lower than that i n d i c a t e d by Quinlan, i t seemed to s u b s t a n t i a t e the concept of a downward-moving optimum pH with i n c r e a s i n g ammonium l e v e l s . On t h i s b a s i s , i t can be suggested that use of pH as a parameter, in determining optimum n i t r i f i c a t i o n c o n d i t i o n s , should be tem-pered with an understanding of i t s l i m i t a t i o n s . 184 2 5 D a y 8 8 t o 1 4 4 Y - - 2 1 0 9 3 + 8 8 5 8 . 1 X - 1 2 3 6 . J X 2 * 5 7 . 3 5 2 X 3 R - 0 . 8 5 n - 2 4 F i g . 2 3 : Run 2 / S y s t e m 2 E f f e c t o f p H o n N i t r i f i c a t i o n R a t e 2 0 P r e - d e n i t r i f l c a t l o n m o d e > Q UJ U z> 15 Cx o ex. a. Z i CO E 10 6 . 4 6 . 6 6 . 8 7 7 . 2 pH IN A E R O B I C C E L L 7.A 7 , 6 18 5 "Unaccounted For" Nitrogen Losses "Unaccounted f o r " n i t r o g e n l o s s e s i n n i t r i f y i n g treatment systems are f r e q u e n t l y reported i n the l i t e r a t u r e . Wuhrmann (1963) reported on the o p e r a t i o n of two s i m i l a r a c t i v a t e d sludge p i l o t p l a n t s fed 4,000 mg TKN/L. The n o n - n i t r i f y i n g p l a n t , oper-a t e d a t a low DO l e v e l (0.7 mg/L) a c h i e v e d a n i t r o g e n b a l a n c e whereas the n i t r i f y i n g p l a n t , operated at a high DO l e v e l (6.7 mg/L) had a s i g i n i f i c a n t underbalance of n i t r o g e n (-26%). Numer-ous i n v e s t i g a t o r s have reported such l o s s e s i n the i n t e r v e n i n g years (Wong-Chong and Loehr, 1975; Murray et a_l., 1975; Cooper et. a l . , 1977). Woods et a_l. (1981), f o r example, reported an unac-countable n i t r o g e n l o s s of 43% at the Crossness n i t r i f y i n g sewage treatment p l a n t i n England. This l o s s has been a t t r i b u t e d to a number of causes such as: e r r o r s i n a n a l y s i s and measurements, biomass a s s i m i l a t i o n , l o c a l -i z e d anaerobic d e n i t r i f i c a t i o n , ammonia v o l a t i l i z a t i o n , i n c i d e n -t a l d e n i t r i f i c a t i o n i n the a e r o b i c medium ( d e n i t r i f i c a t i o n w i t h i n the inner p a r t s of the b i o f l o c , or i n non-aerated p a r t s of the tank), simultaneous n i t r i f i c a t i o n / d e n i t r i f i c a t i o n by Nitrosomonas ( t h i s phenomenon was f i r s t observed by Corbet i n 1935), and a c t i v e d e n i t r i f i c a t i o n under a e r o b i c c o n d i t i o n s (discussed ear-l i e r ) . The f i r s t three causes (and, to a c e r t a i n extent, the fourth) do not s t r i c t l y f a l l i n the category of unaccounted f o r n i t r o g e n l o s s e s , s i n c e i t i s p o s s i b l e , by r i g i d experimental c o n t r o l , to account f o r them. To e s t a b l i s h the occurrence of the other forms of unaccounted f o r n i t r o g e n l o s s e s , i t was necessary to keep t r a c k of the f a t e of a l l n i t r o g e n s p e c i e s a c r o s s the system, and a d e t a i l e d n i t r o g e n balance was maintained during 186 Runs 2, 4 and 5. I t i n v o l v e d monitoring f o r : 1) Nitrogen l o s t to d e n i t r i f i c a t i o n i n the anaerobic c e l l ; 2) Nitrogen a s s i m i l a t i o n i n t o biomass; 3) Nitrogen removed from the system through sludge wastage; 4) Nitrogen content of the e f f l u e n t ( d i s s o l v e d and suspended); 5) Changes in n i t r o g e n content of the biomass; 6) Nitrogen content of the feed; 7) Nitrogen v o l a t i l i z a t i o n (as f r e e ammonia). The summary of r e s u l t s i s presented i n T a b l e 19. Raw data i s g i v e n i n Appendix A. As noted, the unaccounted f o r l o s s e s were minor and the occurrence of a e r o b i c d e n i t r i f i c a t i o n of n i t r i t e was deemed u n l i k e l y , as d i s c u s s e d i n an e a r l i e r s e c t i o n . The minor l o s s e s may have been due to any of the o t h e r f a c t o r s d i s -cussed e a r l i e r ; the most l i k e l y one, e s p e c i a l l y during Run 2, was ammonia v o l a t i l i z a t i o n i n the feed bucket and i n the anaerobic c e l l . The occurrence of ammonia v o l a t i l i z a t i o n i n the feed bucket was confirmed d u r i n g Run 4, when i t was observed, during the e a r l y stages of the Run, that the TKN c o n c e n t r a t i o n i n the feed bucket dropped s l i g h t l y , but s t e a d i l y , between s u c c e s s i v e d a i l y a d d i t i o n s of feed. The change was monitored f o r the remainder of the run. The r e s u l t s r e v e a l e d an a v e r a g e 5.2% (s = 2.3, n = 23) r e d u c t i o n i n feed TKN c o n c e n t r a t i o n during the 24 hour i n t e r v a l s e p a r a t i n g feed p e r i o d s , r e p r e s e n t i n g an average l o s s of .9 mg NH4-N/L. The l o s s of n i t r o g e n i n the r e f r i g e r a t e d feed bucket was probably due to ammonia v o l a t i l i z a t i o n , which was enhanced by the r e l a t i v e l y high pH l e v e l of the feed (pH ranged between 8.5 and 187 Table 19 - Nitrogen Balance Across the System f o r Runs 2, 4 and 5 Run No. System No. n Nitrogen Loss (as % of Nitrogen Gain i n f l u e n t N) s 2 1 36 5.2 7.0 2 2 32 4.1 5.6 4 1 27 1.9 6.1 4 2 29 3.3 8.0 5 1 5 ,2.6 6.6 5 2 5 0.7 7.1 n: number of samples s: standard d e v i a t i o n 188 9.0). T h i s l o s s was accounted f o r i n the n i t r o g e n balance c a l c u -l a t i o n s f o r Runs 4 and 5. A p r e l i m i n a r y study of ammonia v o l a t i l i z a t i o n r a t e s was undertaken p r i o r to commencement of the experimental program. The experimental procedure i s g i v e n i n Appendix C. The r e s u l t s , presented i n F i g u r e 24, show increased ammonia s t r i p p i n g with i n c r e a s i n g : pH, ammonium c o n c e n t r a t i o n and a i r flow. The r e s u l t s confirm the p o t e n t i a l f o r minor l o s s e s due to ammonia v o l a t i l i z a -t i o n from the system. In summary, unaccounted f o r n i t r o g e n l o s s e s during Runs 2, 4 and 5, were minimal and w i t h i n the l i m i t s of experimental e r r o r , c o n s i d e r i n g the r a t h e r numerous an a l y s e s and measurements r e -q u i r e d to c a l c u l a t e the o v e r a l l n i t r o g e n balance across the system. The frequent r e p o r t i n g of such l o s s e s i n the wastewater treatment l i t e r a t u r e may, to some extent, r e f l e c t incomplete n i t r o g e n balances and experimental e r r o r s , r a ther than the occur-rence of t r u l y unaccountable l o s s e s due to: a e r o b i c d e n i t r i f ica^-t i o n , simultaneous n i t r i f i c a t i o n / d e n i t r i f i c a t i o n , and i n c i d e n t a l d e n i t r i f i c a t i o n w i t h i n the biomass. R e l a t i o n s h i p Between ORP and N i t r i t e + N i t r a t e L e v e l s The use of ORP probes, to monitor r e s i d u a l n i t r a t e l e v e l s i n anaerobic c e l l s , i s not n o r m a l l y p r a c t i s e d i n wastewater t r e a t -ment. Past and on-going r e s e a r c h at the U n i v e r s i t y of B r i t i s h Columbia has e s t a b l i s h e d the v i a b i l i t y of ORP as a t o o l f o r such monitoring. C o r r e l a t i o n s have been found to e x i s t between ORP v a l u e s and r e s i d u a l n i t r a t e l e v e l s (Koch and Oldham, 1984). In an e f f o r t to confirm the e x i s t e n c e of such a r e l a t i o n s h i p i n the 189 AIR F L O W (ml /m inute ) F i g . 2 4 : A m m o n i a S t r i p p i n g Test R e s u l t s 190 present work, ORP measurements were taken i n the anaerobic c e l l of each system during Run 2 and Run 4, and compared with r e s i d u a l n i t r i t e + n i t r a t e l e v e l s recorded i n those c e l l s . The r e s u l t s f o r Run 2 a r e p r e s e n t e d i n F i g u r e 25. pH v a l u e s i n the anaerobic c e l l averaged 7.68 (s = 0.11, n = 35) f o r System 1 and 8.15, (s = 0.16, n = 26) f o r System 2. As noted from F i g u r e 25, a c o r r e l a t i o n was e s t a b l i s h e d between ORP and r e s i d u a l n i -t r i t e + n i t r a t e l e v e l s i n System 1. No such c o r r e l a t i o n c o u l d be confirmed f o r System 2 s i n c e the n i t r i t e + n i t r a t e l e v e l s i n that system d i d not vary a p p r e c i a b l y . T h i s anomaly may have been caused by the d i f f e r e n c e i n o p e r a t i n g c o n d i t i o n s between the two systems. In System 1, n i t r a t e + n i t r i t e l e v e l s i n the anaerobic c e l l g e n e r a l l y exceeded 5 mg N/L. In the anaerobic c e l l of System 2, however, the r e s i d u a l c o n c e n t r a t i o n was c l o s e to 0 mg N/L from Day 88 onwards. In a d d i t i o n , s u l f a t e r e d u c t i o n to s u l f i d e was p r obably o c c u r r i n g i n System 2, as observed from a b l a c k d e p o s i -t i o n on the c e l l w a l l s and the t y p i c a l r o t t e n egg odours generat-ed from the c e l l . I t i s probable, t h e r e f o r e , that the s u l f a t e / s u l f i d e couple predominated i n System 2, a f f e c t i n g the r e l a t i o n -s h i p between n i t r a t e and ORP. An o b s e r v a t i o n of F i g u r e 25 shows that s u f f i c i e n t s c a t t e r e x i s t e d i n the data to p r e c l u d e the use of ORP measurements, to a c c u r a t e l y p r e d i c t n i t r i t e + n i t r a t e l e v e l s i n the anaerobic c e l l . ORP measurements, taken during Run 4, r e v e a l e d a d e c l i n i n g trend i n ORP l e v e l s i n both systems, i n s p i t e of f a i r l y constant pH and n i t r i t e + n i t r a t e l e v e l s . In System 1, f o r example, ORP l e v e l s dropped s t e a d i l y from -150 mV on Day 7 (at pH 8.44 and 0.1 mg NO^-N/L) to -474 mV on Day 53 (at pH 8.32 and 0.17 mg NOrJ-191 £>• 1 • -S y s t e m 2 A n a e r o b i c C e l l 100 50 0 - 3 5 0 - 4 0 0 100 h50 0 Y - - 3 8 3 + 6 . 1 3 X - 0 . 0 2 6 6 X ' S y s t e m 1 A n a e r o b i c C e l l i i r -20 40 60 80 N O T - N ( m g / L ) F i g . 2 5 : Run 2 - E f f e c t o f N i t r a t e + N i t r i t e L e v e l s o n O R P I—.50 -100 h - 1 5 0 - 2 0 0 - 2 5 0 - - 3 0 0 — 350 - 400 100 192 N/L). During that p e r i o d , n i t r a t e p l u s n i t r i t e l e v e l s at no time exceeded 0.9 mg NO^-N/L, w h i l e pH l e v e l s remained i n the range of 8.1 to 8.5. From the aforementioned, i t seems that ORP measurements can be used, under c e r t a i n c o n d i t i o n s , as an approximate i n d i c a t i o n of n i t r a t e + n i t r i t e l e v e l s i n the anaerobic c e l l , as long as other reducing couples do not predominate. Incomplete D e n i t r i f i c a t i o n During Run 7, incomplete d e n i t r i f i c a t i o n was observed during two p e r i o d s ; from Day 31 to Day 70, and from Day 303 to 317. On both occasions, n i t r i t e l e v e l s rose a p p r e c i a b l y i n the anaerobic c e l l , w h i l e the n i t r a t e l e v e l s dropped (see T a b l e 20). T h i s phenomenon can be a t t r i b u t e d e i t h e r to: 1) the predominance of n i t r a t e r e s p i r e r s (who are o n l y capable of reducing n i t r a t e to n i t r i t e but not to n i t r o g e n gas) over d e n i t r i f i e r s , or 2) incom-p l e t e r e d u c t i o n of n i t r a t e . The s c a r c i t y of carbonaceous sub-s t r a t e s has been p o s t u l a t e d as a cause of the predominance of n i t r a t e r e s p i r e r s , over d e n i t r i f i e r s , i n some marine waters (Focht and V e r s t r a e t e , 1977). The p e r i o d from Day 31 to 70 occur-red w h i l e the biomass was being a c c l i m a t e d to n i t r o g e n r e d u c t i o n . No d e n i t r i f y i n g or n i t r a t e reducing a b i l i t y had manifested i t s e l f up to that time. The second n i t r i t e accumulation p e r i o d c o i n c i d e d with the disappearance of n i t r i t e i n the a e r o b i c c e l l , preceding the anaerobic c e l l (by i t s v i r t u a l t o t a l o x i d a t i o n to n i t r a t e ) . This f i r s t l e d to a r i s e i n r e s i d u a l , o x i d i z e d n i t r o g e n l e v e l s i n the anaerobic c e l l due, probably, to the exhaustion of the carbon s u b s t r a t e i n that c e l l . Within a few days, n i t r i t e accumulation 19 3 Table 20 - Periods of Incomplete D e n i t r i f i c a t i o n During Run 7 Day I n f l u e n t N i t r i t e L e v e l E f f l u e n t N i t r i t e L e v e l To Anaerobic C e l l From Anaerobic C e l l (mg NO2-N/L) (mg NO^-N/.L) 31* 0.0 30.5 35 24.0 40.0 38 59.0 75.0 42 44.0 64.0 45 54.0 73.0 49 2.0 58.0 52 1.0 3.0 56 0.0 3.0 300** 4.0 0.6 303 5.5 9.0 307 6.5 25.5 310 14.0 25.5 314 15.0 39.5 317 15.0 .52.5 321 7.0 2 .7 * D e n i t r i f y i n g a b i l i t y had not manifested i t s e l f u n t i l Day 31 ** Major d e c l i n e i n n i t r i t e b u i l d - u p had s t a r t e d on Day 287 194 was observed. N i t r i t e formation during d e n i t r i f i c a t i o n was r e -ported to occur i n h a l f of 30 i n d u s t r i a l wastes t e s t e d as poten-t i a l carbon sources f o r b i o l o g i c a l d e n i t r i f i c a t i o n (Monteith e_t a l . , 1980). It's occurrence i n a wastewater treatment f a c i l i t y , has a l s o been r e c e n t l y reported by Alleman (1984). Duration of an Experimental Run A question a r i s i n g from t h i s experimental program d e a l s with the d u r a t i o n of a run, when working with mixed c u l t u r e s of bacte-r i a . I t i s t r a d i t i o n a l l y accepted that a p e r i o d of two to three s l u d g e ages a r e s u f f i c i e n t t o m o n i t o r the e f f e c t of a change on a b i o l o g i c a l system. Many s t u d i e s found i n the l i t e r a t u r e r e p o r t the r e s u l t s of r e l a t i v e l y s h o r t - d u r a t i o n runs, g e n e r a l l y l a s t i n g up to two to three months. Some are a l s o based on extremely short d u r a t i o n t e s t s , such as one day batch t e s t s , using unacclimated biomass (Beccari et a l . , 1983). A major o b s e r v a t i o n made during t h i s research program was that s t e a d y - s t a t e , as f a r as n i t r i t e b u i l d - u p was concerned, was never achieved, even during Run 7 (which l a s t e d one year). A p e r i o d of apparent s t e a d y - s t a t e c o n d i t i o n s d i d seem to occur when the a c c l i m a t e d system appeared to be moving g r a d u a l l y , but s t e a d i l y , towards s t e a d y - s t a t e from Day 70 to Day 147 (see F i g u r e 13). I t was deemed to have reached s t e a d y - s t a t e by then, s i n c e the l e v e l of n i t r i t e b u i l d - u p remained constant f o r a c o n s i d e r -a b l e p e r i o d of time a f t e r t h i s date (up to Day 199). T h i s steady-s t a t e c o n d i t i o n then maintained i t s e l f f o r a p e r i o d of about three sludge ages (from Day 147 to Day 199). The run c o u l d have been terminated at t h i s stage (and considered as completely 195 s u c c e s s f u l ) s i n c e i t had met a l l the accepted " c r i t e r i a " ; i t had l a s t e d almost seven months, a biomass a c c l i m a t e d to f r e e ammonia had e s t a b l i s h e d i t s e l f as e a r l y as Day 49, the d e c l i n e i n n i t r i t e accumulation was s u c c e s s f u l l y r e v e r s e d on two occasions i n the presence of an a c c l i m a t e d biomass, and s t e a d y - s t a t e had been deemed to have been a c h i e v e d . A p l o t of the r e s u l t s to Day 199, when the run c o u l d have been terminated, i s presented i n F i g u r e 26. I t c l e a r l y d e p i c t s a t o t a l l y d i f f e r e n t outcome from that of F i g u r e 13, which presents the r e s u l t s of the e n t i r e run. I t seems, t h e r e f o r e , that no s i n g l e c r i t e r i o n can be used i n e s t i m a t i n g the d u r a t i o n of a run, but t h a t a u s e f u l r u l e of thumb would be to extend i t s d u r a t i o n f o r as l o n g as p o s s i b l e ( w i t h i n reason, of course), e s p e c i a l l y when a c c l i m a t i o n can be expected to o c c u r . I m p l i c a t i o n s of R e s u l t s i n P r a c t i c e T h i s research program has i d e n t i f i e d a process c o n f i g u r a t i o n and mechanism that c o u l d induce and maintain n i t r i t e b u i l d - u p i n a system, t r e a t i n g h i g h l y nitrogenous wastes. I t i n v o l v e s the presence of a p H - c o n t r o l l e d , anaerobic c e l l at the front-end of the system and the implementation of intermediary d e n i t r i f i c a t i o n or i n t e r n a l r e c y c l e at the p o i n t , w i t h i n the system, where the n i t r i t e l e v e l s are highest. The proposed process c o n f i g u r a t i o n would n o r m a l l y produce an e f f l u e n t d e v o i d of n i t r i t e , s i n c e the r e s i d u a l n i t r i t e i n the system would be o x i d i z e d to n i t r a t e i n the remaining a e r o b i c c e l l s . The advantages of t h i s " s h o r t c u t " have been confirmed to i n c l u d e : 1) lower COD consumption during d e n i t r i f i c a t i o n , 196 1 0 0 C E L L 1 T I M E ( d a y s ) F i g . 2 6 : Run 7 - E x t e n t o f N i t r i t e B u i l d - U p in A e r o b i c C e l l s a n d F r e e A m m o n i a L e v e l in F i r s t C e l l t o D a y 2 0 0 197 2) f a s t e r d e n i t r i f i c a t i o n r a t e s , and 3) lower biomass production during d e n i t r i f i c a t i o n . In a d d i t i o n , the presence of n i t r i t e i n the system d i d not seem to be d e t r i m e n t a l to the t r e a t m e n t p r o -cess. However, the long term s t a b i l i t y of the process c o u l d not be maintained and the numerous measures undertaken were unable to prevent the e v e n t u a l d e c l i n e of n i t r i t e b u i l d - u p . T h i s d e c l i n e was probably caused by a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia. The outcome of t h i s research program suggests that, i f a way can be found to permanently overcome the apparent a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia, a c o s t - e f f e c t i v e t e c h n o l o -gy may e v o l v e f o r the removal of n i t r o g e n from h i g h l y nitrogenous wastewater; t h i s would be based upon n i t r i t e p r o d u c t i o n and r e d u c t i o n . Implementation of t h i s technology w i l l depend, i n l a r g e p a r t , upon the need to undertake n i t r o g e n removal from wastewater. At present, the p r a c t i c e of n i t r o g e n removal i s l i m i t e d to a few l o c a l i t i e s or s p e c i a l i z e d treatment circum-stances. I t should be borne i n mind that, u n l i k e l a b o r a t o r y - s c a l e u n i t s , f u l l - s c a l e treatment f a c i l i t i e s r a r e l y operate under "st e a d y - s t a t e " c o n d i t i o n s . Continuous changes occur as a r e s u l t of seasonal temperature f l u c t u a t i o n s , v a r i a t i o n i n feed flow and s t r e n g t h , the o c c a s i o n a l " s l u g " of t o x i c waste, operator i n a t t e n -t i o n , and so on. These c o n d i t i o n s lead to upsets of the treatment process, and sometimes, to the "washout" of the biomass. These upsets may p r e c l u d e the e v e n t u a l f a i l u r e of the s h o r t c u t , and thus a l l o w the i n d e f i n i t e maintenance of some degree of n i t r i t e accumulation. T h i s s u p p o s i t i o n can o n l y be confirmed by a p i l o t 198 or f u l l - s c a l e process e v a l u a t i o n . Based upon the r e s u l t s of t h i s experimental program, the i d e a l p i l o t or f u l l - s c a l e treatment process c o n f i g u r a t i o n would be a p l u g - f l o w system, c o n s i s t i n g of a number of short HRT c e l l s (3 to 5); t h i s would o f f e r s u f f i c i e n t f l e x i b i l i t y f o r o p t i m i z i n g n i t r i t e p r o d u c t i o n and r e d u c t i o n , f o l l o w e d by one, long HRT c e l l f o r o x i d a t i o n of any r e s i d u a l n i t r i t e to n i t r a t e . I n t e r n a l recy-c l e , w i t h i n the short HRT c e l l s , should be adopted when the e l e c t r o n donor needed f o r d e n i t r i f i c a t i o n i s present i n the waste, and intermediary d e n i t r i f i c a t i o n when an exogenous e l e c -tron donor (carbon source) i s used. 19 9 CHAPTER SEVEN CONCLUSIONS AND RECOMMENDATIONS Conclusions A process c o n f i g u r a t i o n f o r n i t r o g e n removal was developed i n a system t r e a t i n g h i g h l y nitrogenous wastes. T h i s process was based on s h o r t - c i r c u i t i n g the t r a d i t i o n a l n i t r i f i c a t i o n / d e n i t r i -f i c a t i o n pathway by means of inducing and s u s t a i n i n g n i t r i t e b u i l d - u p . Based upon the research program c a r r i e d out i n t h i s study, the f o l l o w i n g c o n c l u s i o n s can be put^ f o r t h : 1) I n h i b i t i o n of the n i t r i t e o x i d a t i o n step was i n i t i a t e d and sustained by i n t e r m i t t e n t contact to r e l a t i v e l y high f r e e ammonia l e v e l s i n an anaerobic c e l l , l o c a t e d at the front-end of the system. 2) I n h i b i t i o n of the n i t r i t e o x i d i z e r s c o u l d not be achieved e i t h e r with low d i s s o l v e d oxygen l e v e l s or high n i t r o u s a c i d l e v e l s . 3) The degree of n i t r i t e accumulation was i n v e r s e l y p r o p o r t i o n a l to the reside n c e time i n the a e r o b i c zone. 4) Intermediary d e n i t r i f i c a t i o n , or i n t e r n a l r e c y c l e , was needed to minimize residence time i n the aerated zone. 5) The process c o n f i g u r a t i o n was capable of removing most of the ni t r o g e n content of the waste by the shortened pathway, w h i l e o x i d i z i n g the remaining r e s i d u a l n i t r o g e n to n i t r a t e . 6) COD requirements f o r n i t r i t e r e d u c t i o n were about 40% lower 200 than f o r n i t r a t e r e d u c t i o n . 7) D i s s i m i l a t o r y n i t r i t e r e d u c t i o n r a t e s were 63% higher than d i s s i m i l a t o r y n i t r a t e r e d u c t i o n r a t e s . 8) M i c r o b i a l growth y i e l d , during anaerobic r e s p i r a t i o n , was three times lower with n i t r i t e as the e l e c t r o n acceptor, than with n i t r a t e as the e l e c t r o n acceptor. 9) A l k a l i n i t y consumption and production r e l a t i o n s h i p s f o r n i -t r i t e p roduction and r e d u c t i o n were s i m i l a r to those f o r n i t r a t e p r o d u c t i o n and red u c t i o n . 10) N i t r i t e l e v e l s as high as 115 mg NO^-N/L caused no d i s c e r n i -b l e i n h i b i t i o n to the treatment process. 11) The degree of n i t r i t e b u i l d - u p , with unacclimated biomass, appeared to be p r o p o r t i o n a l to the f r e e ammonia l e v e l i n the anaerobic c e l l . 12) The f r e e ammonia l e v e l needed to i n i t i t i a t e n i t r i t e b u i l d - u p was more than t e n f o l d higher than reported i n the l i t e r a t u r e . 13) Long-term s t a b i l i t y ( i n d e f i n i t e steady-state) of the process c o u l d not be maintained, due to e v e n t u a l apparent a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia. 14) With an a c c l i m a t e d biomass, f r e e ammonia l e v e l s as high as 40 mg NH3-N/L d i d not appear to i n h i b i t e i t h e r the ammonia or the n i t r i t e o x i d a t i o n steps. 15) A f i r s t order r e l a t i o n s h i p e x i s t e d between n i t r i f i c a t i o n r a t e and d i s s o l v e d oxygen up to about 2.5 mg/L. 16) The optimum pH f o r n i t r i f i c a t i o n need not always be i n the range of 7.5 to 8.0, but can be s u b s t a n t i a l l y lower, depend-ing on the c o n c e n t r a t i o n of ammonia i n the system. 17) E f f o r t s to r e v e r s e the d e c l i n e i n n i t r i t e b u i l d - u p , due to 201 e v e n t u a l a c c l i m a t i o n , i n c l u d e d : r e d u c t i o n of sludge age, extension of contact time to f r e e ammonia, r a i s i n g of f r e e ammonia l e v e l , use of a p o t e n t i a l l y more i n h i b i t o r y waste (i.e., m u n i c i p a l l a n d f i l l l e a c h a t e ) , double s u b s t r a t e i n h i b i -t i o n (free ammonia + sodium c h l o r a t e ) , i n t e r n a l r e c y c l e (or intermediary d e n i t r i f i c a t i o n ) , temporary r e d u c t i o n of f r e e ammonia l e v e l , and temporary stoppage of feed. None of the mechanisms were capable of "permanently" r e v e r s i n g the de-c l i n e i n n i t r i t e b u i l d - u p . 18) Changes caused by a c c l i m a t i o n may take s e v e r a l months to e x h i b i t themselves and r a i s e s the very important q u e s t i o n of a r e a l i s t i c " l e n g t h of run", when working with mixed bacte-r i a l c u l t u r e s i n a b i o l o g i c a l treatment system. Recommendation f o r Future Research Needs The two primary research needs are to determine the nature of the apparent a c c l i m a t i o n of the n i t r i t e o x i d i z e r s to f r e e ammonia and e x p l o r e means of p r e v e n t i n g or overcoming i t . The f i r s t task c a l l s f o r b a s i c s c i e n t i f i c research, w h i l e the second task can, to a c e r t a i n extent, be pursued along the t r a d i t i o n a l l i n e of a p p l i e d research. The concept of double s u b s t r a t e i n h i b i -t i o n , i n c o n j u n c t i o n with intermediary d e n i t r i f i c a t i o n (or i n t e r -n a l r e c y c l e ) , probably o f f e r s the most promise i n that regard. A number of other aspects a s s o c i a t e d with t h i s t o p i c w i l l a l s o have to be c l a r i f i e d , determined and/or confirmed. They i n c l u d e : 1) E f f e c t of temperature: I t i s necessary to study the e f f e c t of temperatures other than 20°C, on the s t a b i l i t y of the process, 20 2 e s p e c i a l l y c o l d e r operating temperatures. 2) Confirmation of the p r e l i m i n a r y f i n d i n g s regarding biomass y i e l d s : The low biomass y i e l d s , f o r d i s s i m i l a t o r y n i t r i t e r e d u c t i o n recorded during Batch Test No.2, w i l l have to be confirmed. T h i s can best be achieved by operating s e v e r a l systems i n p a r a l l e l , with i d e n t i c a l carbon feed, but d i f f e r e n t e l e c t r o n acceptors (i.e., n i t r a t e , n i t r i t e and oxygen). 3) P i l o t - s c a l e c o n f i r m a t i o n of the bench-scale r e s u l t s : To be undertaken once a s u c c e s s f u l p r o c e s s - t r a i n has been estab-l i s h e d and proven over a much-longer oper a t i n g p e r i o d (say 8 to 12 months). ) 4) Use of n i t r a t e , n i t r i t e and ammonia probes: The s u c c e s s f u l implementation of t h i s technology w i l l c a l l f o r the use of probes and automated c o n t r o l l e r s . S t u d i e s are a l s o needed to determine the r e l i a b i l i t y of such probes. 203 REFERENCES Abufayad, A. A. 1983. U t i l i z a t i o n of primary sewage sludge as a source of carbon f o r d e n i t r i f i c a t i o n i n sequencing batch r e a c t o r s . D o c t o r a l d i s s e r t a t i o n , Univ. of C a l i f o r n i a , Davis. Aleem, M.I.H. and M. Alexander 1960. N u t r i t i o n and p h y s i o l o g y of N i t r o b a c t e r agi 1 i s . Appl. M i c r o b i o l . , 8_: 80-84. Alleman, J. E. 1984. E l e v a t e d n i t r i t e occurence i n b i o l o g i c a l wastewater treatment systems. Wat. S c i . Tech., 17_: 409-419. Alleman, J. E. and R. L. I r v i n e 1980. N i t r i f i c a t i o n i n the sequenching batch b i o l o g i c a l r e a c t o r . J. Wat. P o l l u t . C o n t r o l Fed., 52: 2747-2754. A l t o n a , R. E., J . Bosman, C. J . Breyer-Menke and N. A. L e v e r 1983. D i s p o s a l of wastewater from Modderfontein f a c t o r y : review of the b i o l o g i c a l n i t r o g e n removal systems. Water SA, 9: 125-130. American Petroleum I n s t i t u t e (A.P.I.) 1981. The sources, chemis-t r y , f a t e and e f f e c t s of ammonia i n a q u a t i c environments. Anthonisen, A. C. 1974. The e f f e c t s of f r e e ammonia and f r e e n i t r o u s a c i d on the n i t r i f i c a t i o n process. D o c t o r a l d i s s e r t a t i o n , C o r n e l l Univ., Ithaca, New York. A n t h o n i s e n , A. C , R. C. L o e h r , T. B. S. Prakasam and E. G. S r i n a t h 1976. I n h i b i t i o n of n i t r i f i c a t i o n by ammonia and n i t r o u s a c i d . J. Wat. P o l l u t . C o n t r o l Fed., 4_8_: 835-852. B a a l s r u d , K. and K. S. B a a l s r u d 1954. Studies on T h i o b a c i l l u s d e n i t r i f i c a n s . A r c h i v . fur M i k r o b i o l o g i e , 2J): 34-62. B a i l y , W. F., J.. D. Bonomo and E. R. Jones 1983. N i t r i f i c a t i o n f a c i l i t i e s s t a r t - u p and i n i t i a l o p eration. J. Wat. P o l l u t . C o n t r o l Fed., 55: 221-228. Barnes, D. and P. J. B l i s s 1983. B i o l o g i c a l c o n t r o l of n i t r o g e n i n wastewater treatment. E. & F. N. Spon. Barth, E. F. and H. D. S t e n s e l 1981. I n t e r n a t i o n a l n u t r i e n t c o n t r o l technology f o r m u n i c i p a l e f f l u e n t s . J. Wat. P o l l u t . C o n t r o l Fed., 53_: 1691-1701. B a t e s , R. G. and G. D. P i n c h i n g 1950. D i s s o c i a t i o n c o n s t a n t of aqueous ammonia at 0° to 50° from E.m.f. s t u d i e s of the ammonium s a l t of a weak a c i d . J . Am. Chem. S o c , 7_2: 1393-1396. Bath, R. N. and F. B. Eddy 1980. T r a n s p o r t of n i t r i t e a c r o s s 204 f i s h g i l l s . J . Exp. Z o o l . , 214: 110-121. B e c c a r i , M., D. Marani and R. Ramadori 1979. A c r i t i c a l a n a l y s i s of n i t r i f i c a t i o n a l t e r n a t i v e s . Wat. Research, 1_3: 185-192. B e c c a r i , M., R. P a s s i n o , R. Ramadori and V. Ta n d o i 1983. K i n e t i c s of d i s s i m i l a t o r y n i t r a t e and n i t r i t e r e d u c t i o n i n suspended growth c u l t u r e . J . Wat. P o l l u t . C o n t r o l Fed., 55: 58-64. Beer, C. 1977. Tests f o r n i t r i f y i n g and d e n i t r i f y i n g a b i l i t y of a c t i v a t e d sludge. B u l l e t . E n v i r o n . Contamination & Tox i c o l o g y , 18_: 558-564 . Beg, S. A., R. H. S i d d i q i and S. I l i a s 1982. I n h i b i t i o n of n i t r i f i c a t i o n by a r s e n i c , chromium and f l u o r i d e . J. Wat. P o l l u t . C o n t r o l Fed., 54: 482-488. B e l s e r , L. W. 1979. P o p u l a t i o n ecology of n i t r i f y i n g b a c t e r i a . Annual Rev. of M i c r o b i o l . , ^ 3.: 309-333. B e l s e r , L. W. and E. L. Mays 1980. S p e c i f i c i n h i b i t i o n of n i t r i t e o x i d a t i o n by c h l o r a t e and i t s use i n as s e s s i n g n i t r i f i c a t i o n i n s o i l s and sediments. Appl. E n v i r o n . Micro-b i o l . , 3_9: 505-510. B e n e d i c t , R. C. 1980. B i o c h e m i c a l b a s i s f o r n i t r i t e - i n h i b i t i o n of C l o s t r i d i u m botulinum i n cured meat. J. Food P r o t e c t i o n , _43: 877-891. Benninger., R. W. and J. H. Sherrard 1978. N i t r i f i c a t i o n and a l k a l i n i t y r e l a t i o n s h i p s i n a c t i v a t e d sludge. J . Wat. Pollut.. C o n t r o l Fed., 50: 2132-2142. B e r i , V. and S. S. Brar 1978. urease a c t i v i t y i n s u b t r o p i c a l , a l k a l i n e s o i l s of I n d i a . S o i l S cience, 126: 330-335. B e t l a c h , M. R. and J. M. T i e d j e 1981. K i n e t i c e x p l a n a t i o n f o r accumulation of n i t r i t e , n i t r i c oxide and n i t r o u s oxide during b a c t e r i a l d e n i t r i f i c a t i o n . Appl. E n v i r o n . Microbiol.., 42: 1074-1084. B e z d i c e k , D. F., J . M. MacGregor and W. P. M a r t i n 1971. The i n f l u e n c e of s o i l f e r t i l i z e r geometry on n i t r i f i c a t i o n and n i t r i t e accumulation. S o i l S c i . Soc. Am. P r o c , 35: 997-1002. Bishop, P. L. and M. Farmer 1979. Fate of n u t r i e n t s during a e r o b i c d i g e s t i o n . J.. E n v i r o n . Eng. D i v., P r o c . Am. Soc. C i v i l Eng., 104: 967-979. B l a s z c z y k , M. 1983. E f f e c t of organic carbon and high n i t r i t e and n i t r a t e c o n c e n t r a t i o n s on the s e l e c t i o n of d e n i t r i f y i n g b a c t e r i a . II Continuous c u l t u r e s i n packed bed r e a c t o r s . Acta M i c r o b i o l . P o l o n i c a , 32: 65-71. 205 B l a s z c z y k , M., M. Przytocka-Jusaik, U. Kruszewska and R. M y c i e l s k i 1981. D e n i t r i f i c a t i o n of high c o n c e n t r a t i o n s of n i t r i t e s and n i t r a t e s i n s y n t h e t i c medium with d i f f e r e n t sources of organic carbon. Acta M i c r o b i o l . P o l o n i c a , 3_0_: 49-58.. Bock, E. 1978. L i t h o a u t o t r o p h i c and chemoorganotrophic growth of n i t r i f y i n g b a c t e r i a , p. 310-314. J_n D. • S c h l e s s i n g e r (ed.). M i c r o b i o l o g y - 1978. Am. Soc. f o r M i c r o b i o l . , Washington, D.C. B o l i n , B. and E. Arrhenius 1977. Nitrogen - an e s s e n t i a l l i f e f a c t o r and a growing environmental hazard. Ambio, 6_: 96-105. B o l l a g , J.-M. and N. Henninger 1978. E f f e c t s of n i t r i t e t o x i c i t y on s o i l b a c t e r i a under a e r o b i c and anaerobic c o n d i t i o n s . S o i l B i o l . Biochem., 1J0: 377-381. B o l l a g , J.-M., M. L. Orcutt and B. B o l l a g 1970. D e n i t r i f i c a t i o n by i s o l a t e d s o i l b a c t e r i a under v a r i o u s environmental c o n d i -t i o n s . S o i l S c i . Soc. Am. P r o c , 3_4: 875-879. Boon, B. and H. Laudelout 1962. K i n e t i c s of n i t r i t e o x i d a t i o n by N i t r o b a c t e r winogradskyi. Biochemical J., 85_: 440-447. B o v e l l , C. 1967. The e f f e c t of sodium n i t r i t e on the growth of Micrococcus d e n i t r i f i c a n s . A r c h i v . f u r M i k r o b i o l o g i e , 59: 13-19 . Braam, F. and A. Klapwijk 1981. Effect- of copper on n i t r i f i c a -t i o n i n a c t i v a t e d sludge. Wat. Research, 15; 1093-1098. Bremner, J. M. and A. M. Blackmer 1981. T e r r e s t r i a l n i t r i f i c a t i o n as a source of atmospheric n i t r o u s oxide, p. 151-170. I_n C. C. Delwiche (ed.). N i t r i f i c a t i o n , d e n i t r i f i c a t i o n and atmospheric n i t r o u s oxide. John Wiley & Sons. Brezonik, P. L. 1977. D e n i t r i f i c a t i o n i n n a t u r a l waters. Prog. Wat. Tech., 8: 373-392. B r i d l e , T. R., W. K. B e d f o r d and B. E. Jank 1981. B i o l o g i c a l n i t r o g e n c o n t r o l of coke p l a n t wastewaters. Prog. Wat. Tech., 12: 667-680. Brimblecombe, P. and D. H. Stedman 1982. H i s t o r i c a l evidence f o r a dramatic i n c r e a s e i n the n i t r a t e component of a c i d r a i n . Nature, 298: 460-462. Brooks, D. and I. Cech 1979. N i t r a t e s and b a c t e r i a l d i s t r i b u t i o n i n r u r a l domestic water supplies.. Wat. Research 13_: 33-41.. Brown, D. A. and D. J . McLeay 1975. E f f e c t of n i t r i t e on methemoglobin and t o t a l hemoglobin of j u v e n i l e rainbow t r o u t . The P r o g r e s s i v e F i s h - C u l t u r i s t , 37: 36-38. 206 Bryan, B. A. 1981. P h y s i o l o g y and biochemistry of d e n i t r i f i c a -t i o n , p. 67-84. I_n C. C. D e l w i c h e (ed.). Deni t r i f i c a t i o n , n i t r i f i c a t i o n and atmospheric n i t r o u s oxide. John Wiley & Sons. Buchanan, R. L. and M. S o l b e r g 1972. I n t e r a c t i o n of sodium n i t r a t e , oxygen and pH on growth of Staphylococcus aureus. J . Food S c i . , 31_: 81-85. Butt, W. D. and H. Lees 1960. The biochemistry of n i t r i f y i n g organisms. Biochem. J . , 7j6: 425-427. C a r l s o n , C. A. and J. Ingraham 1983. Comparison of d e n i t r i f i c a -t i o n by Pseudomonas s t u t z e r i , Pseudomonas aeruginosa, and Paracoccus d e n i t r i f i c a n s . Appl. E n v i r o n . M i c r o b i o l . , 45: 1247-1253. C a s t e l l a n i , A. G. and C. F. Niven J r . 1955. F a c t o r s a f f e c t i n g the b a c t e r i o s t a t i c a c t i o n of sodium n i t r i t e . Appl. M i c r o b i o l . , 3: 154-159. C h a l k , P. M. and C. J . Smith 1983. Chemoden i t r i f i ca t i on. I_n J . R. F r e n e y and J . R. Simpson (eds.). Gaseous l o s s of n i t r o g e n from p l a n t - s o i l system ( V o l . 9). Martinus N i j h o f f / D r . W. Junk. C h i i v e r s , C , H. Inskip, C. C a y g i l l , B. Bartholomew, P. F r a s e r and M. H i l l 1984. A survey of d i e t a r y n i t r a t e i n w e l l - w a t e r u s e r s . I n t . J . Epidemiol.., JL3_: 324-331. Chou, C. C.-S. and T. Daicho 1981. B i o - s u r f n i t r i f i c a t i o n of concentrated ammonia-brine waste from an i o d i n e manufac-t u r i n g p l a n t . Proc. 36th Ind. Waste Conf., Purdue Univ., 48-55. C h r i s t e n s e n , M. H. and P. Harremoes 1977. B i o l o g i c a l d e n i t r i f i -c a t i o n of sewage: a l i t e r a t u r e review. Prog. Wat. Tech., £: 509-555. C o l l i n s , M. T., J . B. G r a t z e k , E. B. S h o t t s , D. L. Dawe, L. M. Campbell and D. R. Senn 1975. N i t r i f i c a t i o n i n an a q u a t i c r e c i r c u l a t i n g system. J. F i s h . Res. Bd. Canada, 32: 2025-2031. C o l t , J. and G. Tchobanoglous 1976. E v a l u a t i o n of the short-term t o x i c i t y of nitrogenous compounds to channel c a t f i s h , I c t a l u r u s Punctatus. Aquaculture, IB: 209-224. Comeau, Y. 1984. Biochemical models f o r b i o l o g i c a l phosphorus removal from wastewater. M.A.Sc. T h e s i s , Univ. of B r i t i s h Columbia, Canada. Commoner, B. 1977. C o s t - r i s k - b e n e f i t a n a l y s i s of n i t r o g e n f e r t i l i z a t i o n : a case h i s t o r y . Ambio, j>: 159-161. 207 Cooper, P. F., E. A. Drew, D. A. B a i l e y , and E. V. Thomas 1977. Recent advances i n sewage e f f l u e n t d e n i t r i f i c a t i o n : Part I. Wat. P o l l u t . C o n t r o l , 7_6: 287-300. Corbet, A. S. 1935. The formation of hyponitrous a c i d as an intermediate compound i n the b i o l o g i c a l or photochemical o x i d a t i o n of ammonia to n i t r o u s a c i d . Biochem. J.,29: 1086-1096. Crawford, R. E. and G. H. A l l e n 1977. Seawater i n h i b i t i o n of n i t r i t e t o x i c i t y to chinook salmon. Trans. Am. F i s h S o c , 106: 105-109. Crutzen, P. J. 1981. Atmospheric chemical processes of the oxides of n i t r o g e n , i n c l u d i n g n i t r o u s oxide, p. 17-44.In C. C. Delwiche (ed.), D e n i t r i f i c a t i o n , n i t r i f i c a t i o n and atmos-p h e r i c n i t r o u s oxide. John Wi l e y & Sons. Dairmont, T. 1984. N i t r a t e l e v e l s i n d r i n k i n g water i n a wine producing area of Germany (F.R.G.). The Science of the T o t a l Environment 3_7: 253-257. Daoust, P-Y. and H. W. F e r g u s o n 1984. The p a t h o l g y of c h r o n i c ammonia t o x i c i t y i n rainbow t r o u t , Salmo g a i r d n e r i Richard-son. J . of F i s h D i s e a s e s 199-205. Dedhar, S. 1985, Ammonia removal from a l a n d f i l l l e a c h a t e by b i o l o g i c a l n i t r i f i c a t i o n and d e n i t r i f i c a t i o n . M.A.Sc. Th e s i s , Univ. of B r i t i s h Columbia, Canada. Dedhar, S. and D. S. M a v i n i c 1985. Ammonia removal from a l a n d f i l l l e a c h a t e by n i t r i f i c a t i o n and d e n i t r i f i c a t i o n . Wat. P o l l u t . Research J . Canada, 2J3 ( V o l . 3)-: 126-137. D e l w i c h e , C. C. 1956a. N i t r i f i c a t i o n , p.218-232. I_n W. D. McElroy and B. G l a s s (eds.). Symposium on i n o r g a n i c n i t r o g e n metabolism; f u n c t i o n of m e t a l l o - f l a v o p r o t e i n s . John Hopkins Press.. D e l w i c h e , C. C. 1956b. D e n i t r i f i c a t i on, p. 233-258. I_n W. D. McElroy and B. G l a s s (eds.). Symposium on i n o r g a n i c n i t r o g e n metabolism; f u n c t i o n of m e t a l l o - f l a v o p r o t e i n s . John Hopkins Press. Delwiche, C. C. 1977. Energy r e l a t i o n s i n the g l o b a l n i t r o g e n c y c l e . Ambio, £: 106-111. Delwiche, C. C. 1981. The n i t r o g e n c y c l e and n i t r o u s oxide, p. 1-15. Tn C. C. Delwiche (ed.) , D e n i t r i f i c a t i o n , n i t r i f i c a -t i o n and atmospheric n i t r o u s oxide. John W i l e y & Sons. D e l w i c h e , C. C. and B. A. Bryan 1976. Den i t r i f i ca t i on. Ann. Rev. M i c r o b i o l . , 30: 241-262. 208 D h a l i w a l , B. S. and R. A. Baker 1983. R o l e of ammonia-N i n secondary e f f l u e n t c h l o r i n a t i o n . J. Wat. P o l l u t . C o n t r o l Fed., 55: 454-456. Downing, A. L. 1968. F a c t o r s to be considered i n the design of a c t i v a t e d sludge p l a n t s , p. 190-202. I_n E. F. Gloyna and W. W. E c k e n f e l d e r (eds.). Advances i n water q u a l i t y improvement. Univ. of Texas Press. Drozd, J. W. 1980. R e s p i r a t i o n i n the ammonia-oxidizing chemo-a u t o t r o p h i c b a c t e r i a , p. 87-111. I_n C. J . Knowles (ed.). D i v e r s i t y of b a c t e r i a l r e s p i r a t o r y systems. CRC Press. Eddy, F. B., P. A. K u n z l i k and R. N. Bath 1983. Uptake and l o s s of n i t r i t e from the blood of rainbow t r o u t , Salmo g a i r d n e r i Richardson, and the a t l a n t i c salmon, Salmo s a l a r L. i n f r e s h water and i n d i l u t e sea water. J . F i s h B i o l . , 2_3: 105-116. Egboka, B. C. E. 1984. N i t r a t e contamination of s h a l l o w groundwaters i n Ontario, Canada. The Science of the T o t a l Environment, 35_: 53-70. Engberg, D. J. and E. D. Schroeder 1975. K i n e t i c s and s t o i c h i o -metry of b a c t e r i a l d e n i t r i f i c a t i o n as a f u n c t i o n of c e l l r e sidence time. Water Research, 9_: 1051-1054. Environmental P r o t e c t i o n Agency (E.P.A.) 1975. Process manual fo r n i t r o g e n c o n t r o l . Technology T r a n s f e r , U.S.A.. Environmental P r o t e c t i o n Agency (E.P.A.) .1979. Methods f o r chemical a n a l y s i s of water and wastes. EPA 600/4-79-020, U.S.A. Er i c k s o n , R. J. 1985. An e v a l u a t i o n of mathematical models f o r the e f f e c t s of pH and temperature on ammonia t o x i c i t y to a q u a t i c organisms. Wat. Research, 19.' 1047-1058. Faup, G.-M., M. A. P i c a r d and C. Del Zappo 1978. N i t r i f i c a t i o n -d e n i t r i f i c a t i o n of wastewaters with a high organic and ammonia n i t r o g e n content (waste e f f l u e n t s from sugar r e f i n e r i e s ) . Prog. Wat. Technol., ,10: 493-501. F i l l e r y , I. R. P. 1983. B i o l o g i c a l d e n i t r i f i c a t i o n , p. 33-64.In J . R. F r e n e y and J . R. Simpson (eds.). Gaseous l o s s of n i t r o g e n from p l a n t s o i l systems (Vol.. 9). Martinus N i j h o f f / D r . W. Junk. F i r e s t o n e , M. K. 1982. B i o l o g i c a l d e n i t r i f i c a t i o n , p. 289-326. In F. J. Stevenson (ed.). Nitrogen i n a g r i c u l t u r a l s o i l s , V o l . 2 2 . Am. Soc. of Agronomy. F o c h t , D. D. 1981. S o i l den i t r i f i ca t i on, p. 499-516. I_n J . M. Lyons et a_l. (eds.). Genetic engineering of symbiotic n i t r o g e n f i x a t i o n and c o n s e r v a t i o n of f i x e d n i t r o g e n , V o l . 17. Plenum Press. 209 Focht, D. D. and A. C. Chang 1975. N i t r i f i c a t i o n and d e n i t r i f i -c a t i o n processes r e l a t e d to wastewater treatment. Adv. Appl. Microb., 19: 153-186. Focht, D. D. and W. V e r s t r a e t e 1977. Biochemical ecology of n i t r i f i c a t i o n and d e n i t r i f i c a t i o n . Adv. Microb. E c o l . , 1_: 135-214. Food and A g r i c u l t u r a l O r g a n i z a t i o n (F.A.O.) 1981. F e r t i l i z e r handbook, V o l . 31. United Nations. F o r d , D. L., R. L. C h u r c h w e l l and J . W. K a c h t i c k 1980. Compre-hensive a n a l y s i s of n i t r i f i c a t i o n of chemical p r o c e s s i n g wastewaters. J. Wat. P o l l u t . C o n t r o l Fed., 52: 2726-2746. F o s t e r , S. S. D., A. C. C r i p p s and A. S m i t h - C a r i n g t o n 1982. N i t r a t e l e a c h i n g to groundwater. P h i l . Trans. R. Soc. London 296: 477-489. F r a s e r , P. and C. C h i i v e r s 1981. H e a l t h aspects of n i t r a t e i n d r i n k i n g water. The Science of the T o t a l Environ., 18: 103-116. Gaino, E., A. A r i l l o and P. Mensi 1984. Involvement of the g i l l c h l o r i d e c e l l s of t r o u t under acute n i t r i t e i n t o x i c a t i o n . Comp. Biochem. P h y s i o l . , 77A:: 611-617. G a r c i a , J.-L. 1977. Analyse de d i f f e r e n t s groupes composant l a m i c r o f l o r e d e n i t r i f i a n t e des s o l s de r i z i e r e du Senegal. Ann. M i c r o b i o l . ( I n s t . Pasteur),, 128 A: 433-446. G a r r e t t , J. T. J r . 1982. Attached growth n i t r i f i c a t i o n . D o c t o r a l d i s s e r t a t i o n , U n i v e r s i t y of Texas, A u s t i n , Texas. Gasser, J. A. 1984. D i s i n f e c t i o n of n i t r i f i e d e f f l u e n t s . J. Wat. P o l l u t . C o n t r o l Fed., 5_6: 386-387. G a u t h i e r , J . J., D. D. J o n e s , L. W. W i l s o n and C. R. M a j o r s 1981. Combined b i o l o g i c a l treatment of coke-plant waste-water and b l a s t - f u r n a c e r e c y c l e - w a t e r system blowdown. Proc. 36th Ind. Waste Conf. Purdue, 77-9,1. Gayon, M. U. and G. Dupetit 1886. Reduction des n i t r a t e s par l e s i n f i n i m e n t p e t i t e s . Memoires S o c i e t e des Sciences Physiques et N a t u r e l l e s de Bordeaux, 3: 201-307. G l a s s , R. E. 1982. Gene Function - E^ c o l i and i t s H e r i t a b l e T r a i t s . Univ. of C a l i f o r n i a Press,. Goronszy, M. C. and D. Barnes 1982. Nitrogen removal i n continuous-flow s e q u e n t i a l l y aerated a c t i v a t e d sludge systems,. Process Biochemistry, 17_: 13-19. Grennfelt,,P. and J. S c h j o l d a g e r 1984. Photochemical oxidants i n 210 the atmosphere: a mounting menace. Ambio, 13_: 61-67. Had j i p e t r o u , L. P. and A. H. Stouthamer 1965. Energy production during n i t r a t e r e s p i r a t i o n by Aerobacter aerogenes. J . Gen. M i c r o b i o l . , 3_£: 29-34. Hahn, J . 1981. N i t r o u s o x i d e i n the oceans. I_n C. C. D e l w i c h e (ed.). D e n i t r i f i c a t i o n , n i t r i f i c a t i o n and atmospheric n i t r o u s oxide. John W i l e y & Sons. H a l l , E. R. and K. L. Murphy 1980. E s t i m a t i o n of n i t r i f y i n g biomass and k i n e t i c s i n wastewater. Wat. Research, 14: 297-304. Halmo, G. and K. E i m h j e l l e n 1981. Low temperature removal of n i t r a t e by b a c t e r i a l d e n i t r i f i c a t i o n . Wat. Research, 15: 989-998. Harada, T. and H. Kai 1968. Studie s on the environmental c o n d i t i o n s c o n t r o l l i n g n i t r i f i c a t i o n i n s o i l . S o i l Sci.. and P l a n t N u t r i t i o n , 14: 20-26. Hartman, P. E. 1982. N i t r a t e s , n i t r i t e s : i n g e s t i o n , pharmacody-namics, and t o x i c i t y , p. 211-294. In F. de S e r r e s and A. Ho l l a e n d e r (eds.). Chemical Mutagens. Plenum Press. Hauck, R. D. 1983. Agronomic and t e c h n o l o g i c a l approaches to minimizing gaseous n i t r o g e n l o s s e s from c r o p l a n d s , p. 285-312. I n J . R. F r e n e y and J . R. Simpson (eds.) Gaseous l o s s of n i t r o g e n from p l a n t s o i l systems ( V o l . 9).. Martinus N i j h o f f / D r . W. Junk. Hauck, R. D. and H. F. Stephenson 1965. N i t r i f i c a t i o n of ni t r o g e n f e r t i l i z e r s . E f f e c t of n i t r o g e n sources, s i z e and pH of the gr a n u l e , and c o n c e n t r a t i o n . J . A g r i c . Food Chem., 13: 486-492. Haug, R. T. and P. L. McCarty 1972. N i t r i f i c a t i o n with submerged f i l t e r s . J . Wat. P o l l u t . C o n t r o l Fed., 44: 2086-2102. H i l l e r , R. G. and J. A. Bassham 1965. I n h i b i t i o n of CC>2 f i x a t i o n by n i t r o u s a c i d . Biochim. et B i o p h y s i c a Acta. 109: 607-610. Huey, J. 1982. Growth responses of Nitrosomonas Europaea. D o c t o r a l d i s s e r t a t i o n , Univ. of Iowa, Iowa C i t y , Iowa. Hutton, W. C. and S. A. LaRocca 1975. B i o l o g i c a l treatment of concentrated ammonia wastewaters. J . Wat. P o l l u t . C o n t r o l Fed., 47:: 9 8 9-997. Hynes, R. K. and R. Knowles 1983. I n h i b i t i o n of chemoautotrphic n i t r i f i c a t i o n by sodium c h l o r a t e and sodium c h l o r i t e : a reexamination. Appl. E n v i r o n . M i c r o b i o l . , 4_5: 1178-1182. Ingraham, J . L. 1981. M i c r o b i o l o g y and g e n e t i c s of d e n i t r i f i e r s , 211 p. 45-67. I_n C. C. D e l w i c h e (ed.). Deni t r i f i c a t i o n , n i t r i f i -c a t i o n and atmospheric n i t r o u s oxide. John Wi l e y & Sons. Jasper,S., J. W. Atwater and D. S. M a v i n i c 1986. C h a r a c t e r i z a t i o n and treatment of l e a c h a t e from a West Coast l a n d f i l l . Dept. of C i v i l Eng., Univ. of B r i t i s h Columbia, Canada. J a s p e r , S., J . W. Atwater and D. S. M a v i n i c 1985. L e a c h a t e pr o d u c t i o n and c h a r a c t e r i s t i c s as a f u n c t i o n of water input and l a n d f i l l c o n f i g u r a t i o n , p.64-83. Int. Conf. on New D i r e c t i o n s and Research i n Waste Treatment and R e s i d u a l s Management, Univ. of B r i t i s h Columbia, Canada. J e t e r , R. M. and J . L. Ingraham 1981. The d e n i t r i f y i n g p r o -k a r y o t e s , p. 913-925. I_n M. P. S t a r r e t a l . (eds.). The prokaryotes ( V o l . 1). S p r i n g e r - V e r l a g . Jones, G. L. and A. R. Paskins 1982. I n f l u e n c e of high p a r t i a l pressure of carbon d i o x i d e and/or oxygen on n i t r i f i c a t i o n . J . Chem. Technol. B i o t e c h n o l . , 3^: 213-223. Jone s , P.. H. and H. M. Sabra 1980. E f f e c t of systems s o l i d s r e t e n t i o n time (SSRT or sludge age) on n i t r o g e n removal from a c t i v a t e d - s l u d g e systems. Wat. P o l l u t . C o n t r o l , 79: 106-116. J u s t i n , P. and D. P. K e l l y 1978. M e t a b o l i c changes i n T h i o b a c i 1 —  l u s d e n i t r i f i c a n s accompanying the t r a n s i t i o n from a e r o b i c to anaerobic growth i n continuous chemostat c u l t u r e . J. General Microbiol.., 107: 131-137. Keenan, J . D., R. L. S t e i n e r and A. A. F u n g a r o l i 1979. S u b s t r a t e i n h i b i t i o n of n i t r i f i c a t i o n . J. E n v i r o n . S c i . H e a l t h , A14: 377-397. Klemenc, A. and Hayek 1929. Zur kenntnis der d i s s o z i a t i o n s konstante der s a l p e t r i g e n saure. Monatshefte fuer chemie, 53-54: 407-412. Knoetze, C , T. R. D a v i e s and S. G. W i e c h e r s 1980. C h e m i c a l i n h i b i t i o n of b i o l o g i c a l n u t r i e n t removal processes. Water SA, 6: 171-180. Knowles, G., A. L. Downing and M. J . B a r r e t t 1965. D e t e r m i n a t i o n of k i n e t i c constants f o r n i t r i f y i n g b a c t e r i a i n mixed c u l t u r e with the a i d of an e l e c t r o n i c computer. J. Gen. M i c r o b i o l . , 3J3: 263-278. Knowles, R. 1982. D e n i t r i f i c a t i o n . M i c r b i o l . Rev., 4_6: 43-70. Knox, K. 1983. T r e a t a b i l i t y s t u d i e s on l e a c h a t e from a co-d i s p o s a l l a n d f i l l . E n v i r o n . P o l l u t . (Series B), 5_: 157-174. Koch, F. A. and W. K. Oldham 1984. ORP - a t o o l f o r m o n i t o r i n g , c o n t r o l and o p t i m i z a t i o n of b i o l o g i c a l n u t r i e n t removal 212 systems. Wat. S c i . Technol., 17_, P a r i s Conference. Koike, I. and A. H a t t o r i 1975. Energy y i e l d of d e n i t r i f i c a t i o n : an estimate from growth y i e l d i n continuous c u l t u r e s of Pseudomonas d e n i t r i f i c a n s under n i t r a t e - , n i t r i t e - and n i t r o u s o x i d e - l i m i t e d c o n d i t i o n s . J . Gen. M i c r o b i o l . , 88: 11-19. K o n i k o f f , M. 1975. T o x i c i t y of n i t r i t e to channel c a t f i s h . The Pr o g r e s s i v e F i s h - C u l t u r i s t , 32_z 96-98. Krul., J. M. 1976. D i s s i m i l a t o r y n i t r a t e and n i t r i t e r e d u c t i o n under a e r o b i c c o n d i t i o n s by an a e r o b i c a l l y and a n a e r o b i c a 1 l y grown A l c a l i g e n e s sp. and by a c t i v a t e d sludge. J . Appl. Bact., 40: 245-260 . K r u l , J . M. and R. Veeningen 1977. The s y n t h e s i s of the d i s s i m i -l a t o r y n i t r a t e reductase under a e r o b i c c o n d i t i o n s i n a num-ber of d e n i t r i f y i n g b a c t e r i a , i s o l a t e d from a c t i v a t e d sludge and d r i n k i n g water. Wat. Research, 11: 39-43. L a u d e l o u t , H., R. Lambert and M. L. Pham 1976. I n f l u e n c e du pH et de l a p r e s s i o n p a r t i e l l e d'oxygene sur l a n i t r i f i c a t i o n . Ann. M i c r o b i o l . (Inst. Pasteur), 127 A: 367-382. Lees, H. 1963. i n h i b i t o r s of n i t r i f i c a t i o n , p. 615-629. l_n R. M. Hochster and J. H. Quastel (eds.). M e t a b o l i c I n h i b i t o r s , V o l I I . Academic Press.. L o v e l e s s , J.. E. and H. A. P a i n t e r 1968. The i n f l u e n c e of metal ion c o n c e n t r a t i o n s and pH v a l u e on the growth of a N i t r o s o -monas s t r a i n i s o l a t e d from a c t i v a t e d sludge. J . Gen. M i c r o b i o l . , 52: 1-14. Macfarlane, G. T. and R. A. Herbert 1984. E f f e c t of oxygen t e n s i o n , s a l i n i t y , temperature and organic matter concentra-t i o n on the growth and n i t r i f y i n g a c t i v i t y of an e s t u a r i n e s t r a i n of Nitrosomonas. FEMS M i c r o b i o l . L e t t e r s , 23: 107-111. Margiocco, C , A. A r i l l o , P. Mensi and G. Schenone 1983. N i t r i t e bioaccumulation i n Salmo G a i r d n e r i Rich, and hematological consequences. Aquatic T o x i c o l o g y , 3_: 261-270. Masterton, W. L. and E. J. S l o w i n s k i 1978. Chemical p r i n c i p l e s with q u a l i t a t i v e a n a l y s i s . W. B. Saunders Co. Matthias, S. 1980. N i t r i f i c a t i o n i n h i b i t o r s — p o t e n t i a l and l i m i t a t i o n s . Am. Soc. of Agronomy S p e c i a l P u b l i c a t i o n No. 38. M a v i n i c , D. S. and D. A. Koers 1982. F a t e of n i t r o g e n i n a e r o b i c sludge d i g e s t i o n . J. Wat. P o l l u t . C o n t r o l Fed., 5_4: 352-360. McCarty, P. L. 1972. E n e r g e t i c s of organic matter degradation, 213 p. 91-118. In R. M i t c h e l l (ed.). Water P o l l u t i o n M i c r o b i o l o -gy. J . W i l e y . McCarty, P. L. f L. Beck and P. St.Amant 1969. B i o l o g i c a l d e n i -t r i f i c a t i o n of wastewaters by a d d i t i o n of organic m a t e r i a l s . Proc. 24th i n d u s t r i a l Waste Conf., Purdue Univ., 1271-1285. McDonald, D. B. and S p l i n t e r , R. C. 1982. Long-term t r e n d s i n n i t r a t e c o n c e n t r a t i o n i n Iowa water s u p p l i e s . J. Am. Wat. Works Ass. 7_4: 437-440. Mechsner, Von K. and K. Wuhrmann 1963. B e i t r a g zur kenntnis der m i k r o b i e l l e n d e n i t r i f i k a t i o n . Path. M i c r o b i o l . 26^: 579-591. M e i b e r g , J . B. M., P. M. B r u i n e n b e r g and W. Harder 1980. E f f e c t of d i s s o l v e d oxygen t e n s i o n on the metabolism of methylated amines i n Hyphomicrbbium X i n the absence and presence of n i t r a t e : Evidence f o r 'aerobic' d e n i t r i f i c a t i o n . J . Gen. M i c r o b i o l . , 120: 453-463. M e i j e r , E. M., J . W. Van Der Zwaan, R. Wever and A. H. Stouthamer 1979. Anaerobic r e s p i r a t i o n and energy c o n s e r v a t i o n i n Paracoccus d e n i t r i f i c a n s . Eur. J. Biochem., 9jS: 69-76. Me i k e l j o h n , J. 1940. Aerobic d e n i t r i f i c a t i o n . Ann. Appl. B i o l . , 27: 558-573. Me i k e l j o h n , J. 1954. Some aspects of the p h y s i o l o g y of the n i t r i f y i n g b a c t e r i a , p. 68-83. J_n A u t o t r o p h i c microorgan-isms. Fourth Symp. Soc. Gen. M i c r o b i o l . , Cambridge Univ. Press. Minoru, N., S. Noguchi and T. Suzuki 1981a. A c c l i m a t i o n of sludge f o r e f f i c i e n t removal of n i t r o g e n from fermentation wastewater. J . Ferment. Technol., 59: 49-53. Minoru, N., S. Noguchi and T. Suzuki 1981b. O p e r a t i o n a l c o n d i t i o n s e l i m i n a t i n g the e v o l u t i o n of n i t r o u s oxide i n a d e n i t r i f i c a t i o n p rocess. J . Ferment. Technol., 5_9: 55-58. M o n t e i t h , H. D., T. R. B r i d l e and P. M. S u t t o n 1980. I n d u s t r i a l waste carbon sources f o r b i o l o g i c a l d e n i t r i f i c a t i o n . Prog. Wat. T e c h n o l . , 12: 127-141. Moore, S. F. and E. D. Shroeder 1970. An i n v e s t i g a t i o n of the e f f e c t s of residence time on anaerobic b a c t e r i a l d e n i t r i f i -c a t i o n . Wat. Research, 4_: 685-694. Moore, S. F. and E. D. S c h r o e d e r 1971. The e f f e c t of n i t r a t e feed r a t e on d e n i t r i f i c a t i o n . Wat. Research, 5: 445-452. M o r r i l l , L. G. and J. E. Dawson 1967. P a t t e r n s observed f o r the o x i d a t i o n of ammonium to n i t r a t e by s o i l organisms. S o i l S c i . Soc. Am. P r o c , 31: 757-760. .214 Mulbarger, M. C. 1971. N i t r i f i c a t i o n and d e n i t r i f i c a t i o n i n a c t i v a t e d sludge systems. J. Wat. P o l l u t . C o n t r o l Fed., 43; 2059-2070 . Murray, I., J . W. P arsons and K. Robinson 1975. I n t e r -r e l a t i o n s h i p s between n i t r o g e n balance, pH and d i s s o l v e d oxygen i n an o x i d a t i o n d i t c h t r e a t i n g farm animal waste. Wat. R e s e a r c h , 9: 25-30. M y c i e l s k i , R., B. Krogulska and M. B l a s z c z y k 1978. N i t r i f i c a t i o n of i n d u s t r i a l wastewaters with high n i t r o g e n c o n c e n t r a t i o n . Acta M i c r o b i o l . P o l o n i c a , 2_7: 393-402. Nagashima, M., S. Noguchi and T. Suzuki 1981a. A c c l i m a t i o n of sludge f o r e f f i c i e n t removal of n i t r o g e n from fermentation wastewater. J. Ferment. Technol., 59: 49-53. Nagashima, M., S. Nogushi and T. Suzuki 1981b. O p e r a t i o n a l c o n d i t i o n s e l i m i n a t i n g the e v o l u t i o n of n i t r o u s oxide i n a d e n i t r i f i c a t i o n process. J. Ferment. Technol., 5_9: 55-58. N a t i o n a l Research C o u n c i l (N.R.C.) 1978. N i t r a t e s : an e n v i r o n -mental assessment. Environmental Stud i e s Board, Commission on N a t u r a l Resources, Washington, D.C. N a t i o n a l Academy of S c i e n c e s . N a t i o n a l R e s e a r c h C o u n c i l (N.R.C.) 1979. Ammonia. Assembly of L i f e Sciences, N a t i o n a l Research C o u n c i l . U n i v e r s i t y Park P r e s s (Baltimore). N a t i o n a 1 . R e s e a r c h C o u n c i l (N.R.C.) 1981. The h e a l t h e f f e c t s of n i t r a t e , n i t r i t e and N - n i t r o s o compounds (Part 1). Assembly of L i f e Sciences. Washington, D.C. N a t i o n a l Academy of S c i e n c e s . N e u f e l d , R. D., A. J . H i l l and D. 0. Adekoya 1980. P h e n o l and f r e e ammonia i n h i b i t i o n to Nitrosomonas a c t i v i t y . Wat. Research, lj4: 1695-1703. Nommik, H. and K. 0. N i l s s o n 1963. N i t r i f i c a t i o n and movement of anhydrous ammonia i n s o i l . Acta A g r i c u l t u r a e S c a n d i n a v i c a , 13: 205-219. O ' K e l l e y , J . C , G. E. Becker and A. Nason 1970. C h a r a c t e r i z a -t i o n of the p a r t i c u l a t e n i t r i t e oxidase and i t s component a c t i v i t i e s from the chemoautotroph N i t r o b a c t e r agi1 i s . Biochim. Biophys. Acta, 205: 409-425. O'Leary, V.. and M. S o l b e r g 1976. E f f e c t of sodium n i t r i t e i n h i b i t i o n on i n t r a c e l l u l a r t h i o l groups and on the a c t i v i t y . Appl. E n v i r o n . M i c r o b i o l . , 3_1: 208-212. O r g a n i z a t i o n f o r Economic Co-operation and Development (OCDE) 1971. P o l l u t i o n by d e t e r g e n t s . 2 rue Andre-Pascal 75 P a r i s . 215 Page, G. V. and M. S o l b e r g 1979. Redox potential-dependent n i t r i t e metabolism by S a l m o n e l l a typhimurium. Appl. E n v i r o n . M i c r o b i o l . , 37: 1152-1156. P a i n t e r , H. A. 1970. A review of l i t e r a t u r e on i n o r g a n i c n i t r o -gen metabolism i n microorganisms. Wat. Research, 4_: 393-450. P a i n t e r , H. A. 1977. M i c r o b i a l t r ansformations of i n o r g a n i c n i t r o g e n . Prog. Wat. Tech., 8_: 3-29. P a i n t e r , H. A. and J. E. L o v e l e s s 1983. E f f e c t of temperature and pH v a l u e on the growth-rate constants of n i t r i f y i n g b a c t e r i a i n the a c t i v a t e d - s l u d g e process. Wat. Research, 17: 237-248. Pascik, I. and T. Mann 1984. The two-step n i t r i f i c a t i o n of ammonia-rich waste water. Wat. S c i . Tech., Ij5: 215-223. Payne, W. J. 1973. Reduction of nitrogenous oxides by microorganisms. B a c t e r i d . Rev., 3J_: 409-452. Payne, W. J . 1981. D e n i t r i f i c a t i o n . John Wiley & Sons. Payne, W. J. and M. A. Grant 1981. Overview of d e n i t r i f i c a t i o n , p. 411-427. I_n J . M. Lyons e_t al. (eds.). G e n e t i c E n g i n e e r -ing of Symbiotic n i t r o g e n f i x a t i o n and c o n s e r v a t i o n of f i x e d n i t r o g e n . Vol.. 17, Plenum Press. P e e t e r s . T. L., A.. P. Van G o o l and H. L a u d e l o u t 1969. K i n e t i c study of oxygen-limited r e s p i r a t i o n i n n i t r i f y i n g b a c t e r i a . B a c t e r i o l . P r o c , Am. S o c f o r M i c r o b i o l . , p. 141. Perigo, J. A. and T. A. Roberts 1968. I n h i b i t i o n of C l o s t r i d i a by n i t r i t e . J . Food Technol., 3;.: 91-94. P e r r i n , D. 1982. I o n i s a t i o n constants of i n o r g a n i c a c i d s and bases i n aqueous s o l u t i o n . IUPAC Chemical Data S e r i e s No. 29. Pergamon Press. Perrone, S. J. and T. Meade 1977. P r o t e c t i v e e f f e c t of c h l o r i d e on n i t r i t e t o x i c i t y to coho salmon (Oncorhynchus k i s u t c h ) . J . F i s h . Res. Board, Can., J 3 4 : 486-492. P i c a r d , M. A. and G. M. Faup 1980. Removal of n i t r o g e n from i n d u s t r i a l waste waters by b i o l o g i c a l n i t r i f i c a t i o n - d e n i t r i -f i c a t i o n . Wat. P o l l u t . C o n t r o l , ISz 213-224. Poduska, R. A. and J. F. Andrews 1974. Dynamics of n i t r i f i c a t i o n i n the a c t i v a t e d sludge process. Proc. 29th Ind. Waste Conf., Purdue Univ., p. 1005-1025. Prakasam, T. B. and R. C. Loehr 1972. M i c r o b i a l n i t r i f i c a t i o n and d e n i t r i f i c a t i o n i n concentrated wastes. Wat. Research,-6: 859-869. 216 Quinlan, A. V. 1980. The thermal s e n s i t i v i t y of n i t r i f i c a t i o n as a f u n c t i o n of the c o n c e n t r a t i o n of n i t r o g e n s u b s t r a t e . Wat. Research, L4: 1501-1507. Quinlan, A. V. 1984. P r e d i c t i o n of the optimum pH f o r ammonia-N o x i d a t i o n by Nitrosomonas europaea i n w e l l - a e r a t e d n a t u r a l and domestic-waste waters. Wat. Research, 1J5: 561-566. Rake, J . B. and R. G. Eagon 1980. I n h i b i t i o n , but not uncou-p l i n g , of r e s p i r a t o r y energy c o u p l i n g of three b a c t e r i a l s p e c i e s by n i t r i t e . J . B a c t e r i d . , 144; 975-982. R a n d a l l , C. W. and D. Buth 1984a. N i t r i t e b u i l d - u p i n a c t i v a t e d sludge r e s u l t i n g from temperature e f f e c t s . J. Wat. P o l l u t . • C o n t r o l Fed., 5_6: 1039-1044. R a n d a l l , C. W. and D. Buth 1984b. N i t r i t e b u i l d - u p i n a c t i v a t e d sludge r e s u l t i n g from combined temperature and t o x i c i t y e f f e c t s . J . Wat. P o l l u t . C o n t r o l Fed., 56: 1045-1049. Reddy, K. R. and W. H. P a t r i c k 1984. N i t r o g e n t r a n s f o r m a t i o n s and l o s s i n f l o o d e d s o i l s and sediments. CRC C r i t i c a l Reviews i n Environmental C o n t r o l ,1_3_: 273-309. R e i s e t , M. J. 1856. Experiences sur l a p u t r e f a c t i o n et sur l a formation des fumiers. Comptes Rendus ( P a r i s ) , 42: 53-59. Requa, D. A. and E. D. S c h r o e d e r 1973. K i n e t i c s of packed-bed d e n i t r i f i c a t i o n . J . Wat. P o l l u t . C o n t r o l Fed., 45: 1696-1707. Richardson, M. 1985. N i t r i f i c a t i o n i n h i b i t i o n i n the treatment of sewage. Royal Soc. of Chemistry (U.K.). Riha, W. E. and M. S o l b e r g 1975. C l o s t r i d i u m p e r f r i n g e n s i n h i b i t i o n by sodium n i t r i t e as a f u n c t i o n of pH, inoculum s i z e and heat. J . Food S c i . , 4JD: 439-442. Rittmann, B. E. and W. E. Langeland 1985. Simultaneous d e n i t r i f i c a t i o n with n i t r i f i c a t i o n i n s i n g l e - c h a n n e l o x i d a t i o n d i t c h e s . J . Wat. P o l l u t . C o n t r o l Fed., 57: 300-308 . R o b e r t s , T. A. and M. Ingram 1973. I n h i b i t i o n of growth of C l . botulinum at d i f f e r e n t pH v a l u e s by sodium c h l o r i d e and sodium n i t r i t e . J. Food Technol., 8: 467-475. R o l s t o n , D. E. 1981. N i t r o u s oxide and n i t r o g e n gas production i n f e r t i l i z e r l o s s , p. 127-149. T_n C. C. D e l w i c h e (ed.) , D e n i t r i f i c a t i o n , n i t r i f i c a t i o n and atmospheric n i t r o u s oxide. John Wil e y & Sons. Rowe, J . J., J . M. Yarbrough, J . B. Rake and R. G. Eagon 1979. N i t r i t e i n h i b i t i o n of a e r o b i c b a c t e r i a . Current M i c r o b i o l . , 2: 51-54. 217 Russo, R. C , C. E. Smith and R. v . T h u r s t o n 1974. Acute t o x i c i t y of n i t r i t e to rainbow t r o u t (Salmo g a i r d n e r i ) . J . F i s h . Res. Board Can., 31: 1653-1655. Russo, R. C , R. V. T h u r s t o n and K. Emerson 1981. Acute t o x i c i t y of n i t r i t e to rainbow t r o u t (Salmo g a i r d n e r i ) : e f f e c t s of pH, n i t r i t e s p e c i e s , and anion s p e c i e s . Can. J . F i s h Aquat. S c i . , 3_8: 387-393. Sauter, L. J. and J. E. Alleman 1980. A s t r e a m l i n e d approach to b i o l o g i c a l n i t r o g e n removal. Proc. Am. Soc. C i v i l Eng., E n v i r o n . Eng. Div., New York, pp. 296-306. S c e a r c e , S. N., R. W. B e n n i n g e r , A. S. Weber and J . H. S h e r r a r d 1980. P r e d i c t i o n of a l k a l i n i t y changes i n the a c t i v a t e d sludge p r o c e s s . J . Wat. P o l l u t . C o n t r o l Fed., 52: 399-405. S c h l o e s i n g , T. and A. Muntz 1877. Sur l a n i t r i f i c a t i o n par l e s ferments organises. Comptes Rendus ( P a r i s ) , j[4: 301-303. Schmidt, E. L. 1982.. N i t r i f i c a t i o n i n s o i l , p. 253-288. I_n F. J . Stevenson (ed.). Nitrogen i n a g r i c u l t u r a l s o i l s ( V o l . 22). Am. Soc. of Agronomy. Schroeder, E. D. 1981. D e n i t r i f i c a t i o n i n wastewater management, p. 105-125. I n C. C. D e l w i c h e (ed.) . D e n i t r i f i c a t i o n , n i t r i -f i c a t i o n and atmospheric n i t r o u s oxide. John W i l e y & Sons. Schwedler, T. E. and C. S. Tucker 1983. E m p i r i c a l r e l a t i o n s h i p s between percent methemoglobin i n channel c a t f i s h and d i s -s o l v e d n i t r i t e and c h l o r i d e i n ponds. Trans. Am. F i s h . S o c , 112: 117-119. Senanayake, S. 1982. Substrate i n h i b i t i o n f o r s t r e a m l i n e d n i t r o -gen removal i n the sequencing batch r e a c t o r . M.Sc. t h e s i s , Univ. of Maryland., C o l l e g e Park, Maryland. Shah, D. B. and G. A. Coulman 1978. K i n e t i c s of n i t r i f i c a t i o n and d e n i t r i f i c a t i o n r e a c t i o n s . B i o t e c h n o l . B i o e n g i n e e r i n g , 20: 4 3-7 2. Shank, J . L., H. S i l l i k e r and R. H. Harper 1962. The e f f e c t of n i t r i c oxide on b a c t e r i a . Appl. M i c r o b i o l . , 1£: 185-189. Sharma, B. and R. C. A h l e r t 1977. N i t r i f i c a t i o n and n i t r o g e n removal. Wat. Research, 11: 897-925. S h e r r a r d , J . H., D. K. Manning and D. A. Smith 1982. N i t r i f i c a -t i o n r a t e s i n a c t i v a t e d sludge. J. E n v i r o n . Eng. Div., Pro c . Am. Soc. C i v i l Eng., 10 8: 1074-1078. S h i e h , W. K. and E. J . La Motta 1979.. E f f e c t of i n i t i a l s u b s t r a t e c o n c e n t r a t i o n on the r a t e of n i t r i f i c a t i o n i n a batch experiment. B i o t e c h n o l . Bioengineer i n g , 2_1: 201-211. 218 S i l v e r s t e i n , J. and E. D. Schroeder 1983. Performance of SBR a c t i v a t e d sludge process with n i t r i f i c a t i o n / d e n i t r i f i c a t i o n . J . Wat. P o l l u t . C o n t r o l Fed., 5_5: 377-384. Simone, A. and M. S o l b e r g 1983. Mechanism of g l u c o s e t r a n s p o r t i n h i b i t i o n by n i t r i t e i n C l o s t r i d i u m p e r f r i n g e n s and Staphylococcus aureus. Abstr. Annu. Meeting, Am. Soc. f o r M i c r o b i o l . , p. 265. Skerman, V. B. D., B. J . Carey and I. C. MacRae 1958. The i n f l u e n c e of oxygen on the r e d u c t i o n of n i t r i t e by washed suspensions of adapted c e l l s of achromobacter 1 i q u e f a c i e n s . Can. J . M i c r o b i o l . , 4_: 243-256. Smith, C. E. and R. C. Russo 1975. N i t r i t e - i n d u c e d methemoglobi-nemia i n rainbow t r o u t . The P r o g r e s s i v e F i s h - C u l t u r i s t , 37: 150-152. S o f o s , J . N., F. F. B u s t a and C. E. A l l e n 1979. B o t u l i s m c o n t r o l by n i t r i t e and sorbate i n cured meats: A review. J. Food P r o t e c t i o n , 4j2: 739-770. Sonneborn, M., J . Mandelkow, D. Schon and H. H o f f m e i s t e r 1983. H e a l t h e f f e c t s of i n o r g a n i c d r i n k i n g water c o n s t i t u e n t s , i n c l u d i n g h a r d n e s s , i o d i d e , and f l u o r i d e . CRC C r i t i c a l Reviews i n E n v i r o n . C o n t r o l , _13: 1-22. S r i n a t h , E. G., R. C. Loehr and T. B. Prakasam 1976. N i t r i f y i n g organism c o n c e n t r a t i o n and a c t i v i t y . J. Env. Eng. Div., P r o c . Am. Soc. C i v i l Eng., 10 2: 449-463. Standard Methods f o r the Examination of Water and Wastewater 1980. Am. P u b l i c Health Assoc., 15th e d i t i o n . Stankewich, M. J. 1972. B i o l o g i c a l n i t r i f i c a t i o n with the high p u r i t y oxygenation process. Proc. 27th Ind. Waste Conf., Purdue Univ., pp.. 1-23. S t e n s e l , H. D., R. C. Loehr and A. W. Lawrence 1973. B i o l o g i c a l k i n e t i c s of suspended-growth d e n i t r i f i c a t i o n . J. Wat. P o l l u t . C o n t r o l Fed., 4j>: 249-261. S t e n s t r o m , M. K. and R. A. Poduska 1980. The e f f e c t of d i s -s o l v e d oxygen c o n c e n t r a t i o n on n i t r i f i c a t i o n . Wat. Research, 14_: 643-649. Stouthamer, A. H. 1976. Biochemistry and g e n e t i c s of n i t r a t e r e d u c t a s e i n b a c t e r i a , pp. 315-375. I_n A. H. Rose and D. W. Tempest (eds.). Advances in m i c r o b i a l p h y s i o l o g y , V o l . 14. Academic Press. Stouthamer, A. H., J . v a n ' t R i e t and L. F. Oltmann 1980. R e s p i r a -t i o n w i t h n i t r a t e as a c c e p t o r , p. 19-48. I_n C. J . Knowles (ed.). D i v e r s i t y of b a c t e r i a l r e s p i r a t o r y systems, V o l . I I . 219 CRC Press. Strand, S. E. 1982. Concurrent oxygen uptake and d e n i t r i f i c a t i o n by m i c r o b i a l f i l m s and suspensions. D o c t o r a l d i s s e r t a t i o n , P e n n s y l v a n i a State Univ. S t r a t t o n , F. E. and McCarty, P. L. 1967. P r e d i c t i o n of n i t r i f i -c a t i o n e f f e c t s on the d i s s o l v e d oxygen balance of streams. E n v i r o n . S c i . Te.ch., 1: 405-410. Sutherson, S. 1984 and 1986. Personal communication. D o c t o r a l candidate, Department of C i v i l Eng., Univ. of Toronto. S u t t o n , P. M., K. L. Murphy and R. N. Dawson 1975. Low-tempera-ture b i o l o g i c a l d e n i t r i f i c a t i o n of wastewater. J. Wat. P o l l u t . C o n t r o l Fed., 47: 122-134. S u t t o n , P. M., K. L. Murphy, B. E. Jank and B. A. Monaghan 1975. E f f i c a c y of b i o l o g i c a l n i t r i f i c a t i o n . J. Wat. P o l l u t . C o n t r o l Fed., 47.: .2665-2673.. S u t t o n , P. M., T. R. B r i d l e , W. K. B e d f o r d and J . A r n o l d 1981. N i t r i f i c a t i o n and d e n i t r i f i c a t i o n of an i n d u s t r i a l waste-water. J. Wat. P o l l u t . C o n t r o l Fed., 53: 176-184. S u z u k i , I., U. D u l a r and C. Kwok 1974. Ammonia or ammonium i o n as s u b s t r a t e f o r o x i d a t i o n by Nitrosomonas europaea c e l l s and e x t r a c t s . J . B a c t e r i o l . , 120: 556-558. Tanaka, H. and I.. J . Dunn 1982.. K i n e t i c s of b i o f i l m n i t r i f i c a -t i o n . B i o t e c h n o l . Bioengineer., 24: 669-689. Tanaka, H., S. Uzman and I. J . Dunn 1981. K i n e t i c s of n i t r i f i c a -t i o n using a f l u i d i z e d sand bed r e a c t o r with attached growth. B i o t e c h n o l . Bioengineer., 2_3: 1683-1702. Tarr., H. L. A. 1941. The a c t i o n of n i t r i t e s on b a c t e r i a . J . F i s h . Res. Board Canada, 5: 176-186. T a r r , H. L. A. 1942. The a c t i o n of n i t r i t e s on b a c t e r i a : f u r t h e r experiments. J . F i s h . Res. Board Can., (5 : 74-89 . Terashima, Y. and M. Ishikawa 1984. The k i n e t i c a n a l y s i s of BOD and n i t r o g e n removal i n an o x i d a t i o n d i t c h . Wat. S c i . Technol., 17: 291-302. Thauer, R. K., K. Jungermann and K. Decker 1977. Energy c o n s e r -v a t i o n i n chemotrophic anaerobic b a c t e r i a . B a c t e r i o l . Rev., 41: 100-180. T h u r s t o n , R. V., R. C. Russo, R. J . Luedtke, C. E. Smith, E. L. Meyn, C. Chakoumakos, K. C. Wang and C. J . D. Brown 1984. C h r o n i c t o x i c i t y of ammonia to rainbow t r o u t . T r a n s . Am. F i s h . S o c , 113: 56-73. 220 Timmermans, P. and A. Van Haute 1983. D e n i t r i f i c a t i o n with methanol. Wat. Research, 17_: 1249-1255. Tomasso, J . R., B. A. Simco and K. B. D a v i s 1979. C h l o r i d e i n h i b i t i o n of n i t r i t e - i n d u c e d methemoglobinemia i n channel c a t f i s h ( I c t a l u r u s punctatus). J. F i s h . Board Can., 36; 1141-1144. T o m l i n s o n , T. G., A. G. Boon and C. N. A. Trotman 1966. I n h i b i t i o n of n i t r i f i c a t i o n i n the a c t i v a t e d sludge process of sewage d i s p o s a l . J . Appl. Bacterid..., 2_9: 266-291. Tucker, C. S. and T. E. Schwedler 1983. A c c l i m a t i o n of channel c a t f i s h ( I c t a l u r u s punctatus) to n i t r i t e . B u l l . E n v i r o n . Contam. T o x i c o l . , 3}2: 516-521. Van Cleemput, 0. and L. Baert 1984. N i t r i t e : a key compound i n N l o s s processes under a c i d c o n d i t i o n s . P l a n t and S o i l , 76: 233-241. Van Gent-Ru i j t e r s, M. L., W. de V r i e s and A. H. Stouthamer 1975. I n f l u e n c e of n i t r a t e on fermentation p a t t e r n , molar growth y i e l d s and s y n t h e s i s of cytochrome b i n Prop i o n i b a c t e r i u m  pentosaceum. J. Gen. M i c r o b i o l . , J38: 36-48. Van V e r s e v e l d , H. W., E. M. M e i j e r and A. H. Stouthamer 1977,. Energy c o n s e r v a t i o n during n i t r a t e r e s p i r a t i o n i n Paracoccus  d e n i t r i f icans.. Arch. M i c r o b i o l . , 112: 17-23. Vangnai, S. and D. A. K l e i n 1974. A study of n i t r i t e - d e p e n d e n t d i s s i m i l a t o r y micro-organisms i s o l a t e d from Oregon S o i l s . S o i l B i o l . Biochem., 6: 335-339. V e r s t r a e t e , W. 1981. N i t r i f i c a t i o n , p. 303-313. In F. E. C l a r k and T. Rosswall (eds.). T e r r e s t r i a l Nitrogen C y c l e s , E c o l . B u l l . , V o l . 33, Stockholm. V e r s t r a e t e , W., H. Vanstaen and J. P.. Voets 1977. Adaptation t o n i t r i f i c a t i o n of a c t i v a t e d sludge systems t r e a t i n g h i g h l y nitrogenous waters. J. Wat. P o l l u t . C o n t r o l Fed., 4_9: 1604-1608 . Voets, J . P., H. Vanstaen and W. V e r s t r a e t e 1975. Removal of ni t r o g e n from h i g h l y nitrogenous wastewater. J. Wat. P o l l u t . C o n t r o l Fed., £7: 394-398. V o l z , M. G., L. W. B e l s e r , M. S. A r d a k a n i and A. D. McLaren 1975. N i t r a t e r e d u c t i o n and a s s o c i a t e d m i c r o b i a l p o p u l a t i o n s i n a ponded Hanford sandy loam. J. En v i r o n . Q u a l i t y , 99-102. Wang, W. C , Y. L. Yung, A. A. L a c i s , T. Mo and J . E. Hansen 1976. Greenhouse e f f e c t s due to man-made p e r t u r b a t i o n s of tr a c e gases. Science, 194, 4266: 685-690. 221 W a r i n g t o n , R. 1891. On n i t r i f i c a t i o n - P a r t I V . J . C h e m . S o c , 59: 484-529. W a t s o n , S . W . , F . W. V a l o i s a n d J . B . W a t e r b u r y 1981. T h e f a m i l y N i t r o b a c t e r a c e a e , p p . 1005-1022. In M . P . S t a r r e_t a l . ( e d s . ) . T h e P r o k a r y o t e s , V o l 1, S p r i n g e r - V e r l a g . W e d e m e y e r , G. A . a n d W. T . Y a s u t a k e 1978, P r e v e n t i o n a n d t r e a t m e n t o f n i t r i t e t o x i c i t y i n j u v e n i l e s t e e l h e a d t r o u t ( S a l m o g a i r d n e r i ) . J . F i s h . R e s . B o a r d C a n . , 3J5: 822-827. W e t s e l a a r , R . , J . B . P a s s i o u r a a n d B . R. S i n g h 1972. C o n s e q u e n -c e s o f b a n d i n g n i t r o g e n f e r t i l i z e r s i n s o i l . P l a n t a n d S o i l , 36: 159-175. W h i t e , R . J . 1983. N i t r a t e i n B r i t i s h w a t e r s . A q u a , 2: 51-57. W h i t e , G . C , R. D . B e e b e , V . F . A l f o r d a n d H . A . S a n d e r s 1981. P r o b l e m s o f d i s i n f e c t i n g n i t r i f i e d e f f l u e n t s . N a t i o n a l C o n -f e r e n c e . o n E n v i r o n m e n t a l E n g i n e e r i n g . P r o c Am. S o c C i v i l E n g . , E n v i r o n . E n g . D i v . S p e c i a l t y C o n f e r e n c e , p p . 497-512. W i l d , H . E . J r . , C . N . S a w y e r a n d T . C . M c M a h o n 1971. F a c t o r s a f f e c t i n g n i t r i f i c a t i o n k i n e t i c s . J . W a t . P o l l u t . C o n t r o l F e d . , 43: 1845-1854. W i l k i n s o n , W. B . a n d L . A . G r e e n e 1982. T h e w a t e r i n d u s t r y a n d t h e n i t r o g e n c y c l e . P h i l . T r a n s . R. S o c . L o n d o n , B 296: 459-475. W i l l i a m s , D. R . , J . J . R o w e , P . R o m e r o a n d R. G . E a g o n 1978. D e n i t r i f y i n g P s e u d o m o n a s a e r u g i n o s a : some p a r a m e t e r s o f g r o w t h a n d a c t i v e t r a n s p o r t . A p p l . E n v i r o n . M i c r o b i o l . , 36: 257-263. W o d z i n s k i , R. S . , D. P.. L a b e d a a n d M . A l e x a n d e r 1978. E f f e c t s o f l o w c o n c e n t r a t i o n s o f b i s u l f i t e - s u l f i t e a n d n i t r i t e o n m i c r o o r g a n i s m s . A p p l . E n v i r o n . M i c r o b i o l . , 3_5: 718-723. W o n g - C h o n g , G . M . a n d R. C . L o e h r 1975. T h e k i n e t i c s o f m i c r o b i a l n i t r i f i c a t i o n . W a t . R e s e a r c h , _9: 1099-1106. W o n g - C h o n g , G . M . a n d R. C . L o e h r 1978. K i n e t i c s o f m i c r o b i a l n i t r i f i c a t i o n : n i t r i t e - n i t r o g e n o x i d a t i o n . W a t . R e s e a r c h , 12: 605-609. W o o d , L . B . , B . J . H u r l e y a n d P . J . M a t t h e w s 1981. S o m e o b s e r v a -t i o n s o n t h e b i o c h e m i s t r y a n d i n h i b i t i o n o f n i t r i f i c a t i o n . W a t . R e s e a r c h , 1E>: 543-551. W o r l d H e a l t h O r g a n i z a t i o n 1978. N i t r a t e s , n i t r i t e s a n d N - n i t r o s o c o m p o u n d s . E n v i r o n m e n t a l H e a l t h C r i t e r i a 5, G e n e v a . W u h r m a n n , K. 1963.. E f f e c t o f o x y g e n t e n s i o n o n b i o c h e m i c a l r e a c t i o n s i n s e w a g e p u r i f i c a t i o n p l a n t s , p . 27-40. I n W.W. 222 E c k e n f e l d e r and J . McCabe (eds.) . Advances i n B i o l o g i c a l Treatment . P r o c . 3rd Conference on B i o l o g i c a l Waste Treatment , New York . Wuhrmann, K. 1968. O b j e c t i v e s , t e c h n o l o g y , and r e s u l t s of n i t r o g e n and phosphorus removal p r o c e s s e s , p. 21-48. In E . E . G l o y n a and W. W. Eckenf e l d e r (eds.) . Advances i n Water Q u a l i t y Improvement. U n i v . of Texas P r e s s . Y o u n g , J . C . / L . 0. Thompson and D. R. C u r t i s 1979. C o n t r o l s t r a t e g y f o r b i o l o g i c a l n i t r i f i c a t i o n systems. J . Wat. P o l l u t . C o n t r o l F e d . , 51: 1824-1840. Y o u a t t , J . B. 1954. D e n i t r i f i c a t i o n of n i t r i t e by a s p e c i e s of Achromobacter . N a t u r e , 173: 826-827. 223 APPENDIX A RAW DATA General Comments A l l c o n c e n t r a t i o n s are reported as mg/L.. A l l flows are reported as L/d. Temperature i s reported i n degrees C e l s i u s . pH i s reported i n pH u n i t s . ORP i s reported i n mV. SRT r e f e r s to t o t a l SRT and i s reported i n days. EFF TSS r e f e r s to e f f l u e n t t o t a l suspended s o l i d s . The numbers (1 to 8) presented at the top of each t a b l e r e f e r to c e l l l o c a t i o n i n the system. 224 RUN 1: DISSOLVED OXYGEN DATA SUMMARY SYSTEM 1 SYSTEM 2 (A.M.) SYSTEM 2 (P.M.) DAY 2 3 4 1 2 3 4 1 2 3 4 21 21 22 22 23 24 24 25 25 26 .27 28 29 30 30 31 32 4.2 1.1 4.3 33 4 .0 '6.5 6.0 34 3.7 7.0 6.6 35 3.2 2.4 5.5 36 2.5 3.2 4.5 37 2.6 4.5 6.2 38 3.1 3.2 6.1 39 3.1 2.8 5.4 •40 4.2 2.1 1.6 41 4.4 2.8 4.7 42 4.0 2.8 1.2 43 3.3 2.0 4.3 44 45 3.9 1.9 4.1 46 4.4 1.6 4.4 47 3.6 2.9 3.5 48 5.1 3.1 3.6 49 4.4 3.5 4.0 50 4.5 2.8 3.6 51 5.1 3.2 5.5 52 6.0 6.3 6.5 53 2.8 4.0 6.3 54 3.3 4.9 5.5 55 .2.1 4.6 4.6 1.60 0.60 0 .60 0 .50 0.45 0.70 0 .85 0.45 1.05 0.60 0 .65 1 .15 1.90 0.40 0 .70 1 .55 0.85 1.00 0 .55 0 .45 1.05 2.15 0 .75 0 .50 0.65 0.55 0 .50 0 .50 0.75 0.30 0 .55 0.40 0.90 0.60 0.70 0 .45 0.60 0.35 0.45 1 .90 0.50 0.45 0 .40 0 .30 0 .40 0.50 0.45 0 .35 0.. 55 0.55 0 .60 4 .25 0.50 0.55 0 .70 3 .00 0.35 0.50 1 .35 5 .50 0.75 0.65 3 .30 5 .10 0.15 0.40 0 .40 0 .20 0.30 0.40 0 .40 0 .20 0.65 0.50 0 .45 3 .10 0.45 1.00 0 .45 0 .20 0.80 1.10 2 .20 6 .20 0.60 1.30 0 .45 5 .60 0.80 1.15 0 .35 0 .30 0.60 2.50 0 .35 0 .30 0.45 1.70 0.40 0 .30 1.20 0.50 4.25 0.35 0 .50 0 .45 0 .25 4.50 0 .70 0 .60 1.40 1 .75 1 .00 0.45 1 .40 1 .70 0.90 1 .25 1 .30 2.95 1 .15 1 .00 0.60 0 .55 0 .80 1.30 1 .30 1 .50 1.25 1 .40 1 .85 1.60 0 .95 1.20 0 .70 0 .70 0 .70 2 .00 0 .70 0 .50 0 .75 0 .70 0 .60 0 .55 0 .55 0 .60 1 .10 0 .60 0 .45 1 .15 0 .45 1 .10 0 .65 0.50 0 .65 0 .80 0 .50 0 .55 0 .65 0 .50 0 .50 0 .45 0 .80 0 .55 0 .60 0 .45 0 .60 0 .70 0 .55 0 .55 0 .80 0 .35 0 .45 0 .35 0 .80 0 .40 0 .50 A .35 0 .55 0 .40 0 .55 1.80 0 .30 0 .45 0 .40 0 .30 0 .55 0 .50 0 .50 0 .35 0 .70 0 .70 0.45 0 .35 0 .25 0 .50 0 .50 0 .35 0 .45 0 .80 1 .80 5 .00 0 .35 0 .50 0 .45 .0 .25 0 .40 0 .50 0 .55 4 .60 (1 .15 0 .50 0 .40 1 .40 0 .45 0 .50 0 .35 0. .25 0 .40 0 .50 0 .45 0 .30 0 .40 0 .55 0 .35 0 .30 0 .55 0 .75 0 .40 5 .30 0 .70 0 .85 0 .35 0 .25 1 .30 1 .40 0 .40 0 .25 0 .45 0 .80 0 .40 0 .25 5 .20 0 .60 0 .70 3 .30 0 .65 0 .85 2 .05 1 .45 1 .05 0 .85 1 .45 1. .30 0 .45 1 .00 0 .95 0 .75 o. .70 0 .70 225 RUN 2 - SYSTEM 1: SUMMARY OF DATA DAY TKN IN FLOW IN FLOW ORP RECYC 1 DO 2 1 2 PH 3 4 1 NH4 2 -N 3 4 1 N03-N 2 3 4 1 N02 2 !-N 3 4 MLSS VSS EFF TSS SR' 8 104 10.0 20.5 0.8 7.5 18 4 1 0 85 98 103 106 1 0 0 0 12 121 10.1 20.2 0.8 7.2 18 12 1 0 90 108 108 108 1 0 0 0 2296 1889 81 17 15 124 10.1 20.7 0.8 7.3 23 2 0 0 83 105 109 0 1 0 0 1639 1382 55 9.4 19 10.1 19.3 1.7 7.3 7*3 28 8 0 0 96 117 120 122 0 0 0 0 22 9.9 19.6 1.0 7.5 7.:5 27 9 0 99 114 124 0 0 0 1202 1002 37 9.7 26 10.1 19; 3 0.6 7,4 7.4 35 24 18 73 85 88 91 0 0 0 0 29 10.1 19.0 3.0 7.5 7.7 10 0 0 98 112 112 112 5 0 0 0 1006 804 19 12 34 10.1 19.3 58 1.6 7.4 7.4 7.8 i i 14 0 0 99 99 105 105 1 1 0 0 36 117 10.1 19.3 67 1.9 7.4 7.4 7.6 13 15 2 91 94 107 108 0 0 0 0 1004 755 20 16 40 118 10.5 18.7 -10 3.4 7.6 7.4 7.5 50 36 46 60 74 83 3 1 0 0 43 126 10.4 19.3 20 2.2 7.7 7.8 42 22 5 0 54 72 90 96 2 1 0 0 814 604 15 10 47 .8.9 18.8 -171 2,7 7.7 7.5 56 52 43 35 .26 28 39 44 4 2 3 5 50 9.1 18.6 -116 2.1 7,9 7.8 7 .5 7.5 39 39 19 3 43 40 58 74 1 1 2 1 684 500 19 18 54 119 9.6 18.6 -162 3.0 7.8 7.8 7.6 45 48 32 18 13 11 19 31 9 8 14 21 567 406 55 6.7 58 119 9.4 18.9 -151 4.0 7.8 7.7 7 .3 7.5 38 39 21 5 23 19 34 46 4 2 9 11 69 9.1 20.6 -106 0.8 7,9 7,8 7 .3 7.7 33 35 7 2 35 31 58 63 0 0 1 0 953 725 13 31 71 9.0 9.9 -89 27 48 65 0 4 0 73 -112 1.1 76 117 7.5 9.7 -96 0.6 7.7 7.8 7 .4 7.8 44 48 13 0 22 18 58 64 0 0 0 0 1347 1007 26 24 78 -268 0.9 7.9 7.7 7.9 80 244 11; 5 9.9 -341 1.0 8.0 7.6 7.7 i29 56 21 0 .3 2 40 53 43 82 253 10.0 9.4 -330 1.0 7.9 7.2 7 .0 7.2 105 36 0 0 8 118 1 39 31 2 1584 1242 19 14 84 9.9 10.2 -269 0.8 7.8 7.0 6 .7 6.7 109 40 7 4 117 0 25 16 0 87 253 9.9 11.2 -281 2.1 7.8 6.9 6 .6 6.5 118 35 7 6 122 0 40 19 0 89 253 9.8 10.9 -223 1.4 7.7 6.9 6 .6 6.7 90 23 2 18 36 78 111 4 42 28 4 1746 1442 21 16 92 9.8 10.6 -210 0.9 7.7 7.0 6 .7 6.5 90 26 3 22 55 106 125 8 29 9 0 94 254 9.5 11.0 -204 0.8 7.6 6.9 6 .6 6;3 91 30 5 0 20 61 112 128 9 27 5 0 96 234 9.4 i i . o -187 0.9 7.5 6.9 6 .5 6.2 90 24 4 0 16 62 115 123 10 28 3 0 1772 1498 27 16 101 254 9.8 11.1 -155 1.5 7.5 6.8 6 .4 6.2 70 16 i 24 48 105 125 5 45 12 0 103 25i 1 0 i l 11.3 -138 1.6 7.6 6.9 6 .5 6.1 93 18 2 22 58 103 121 4 34 11 0 1613 1361 32 12 106 263 10.3 10.1 -137 2.0 7.6 6.8 6 .3 6.0 88 12 0 0 i9 74 116 127 4 25 1 0 108 249 10.2 10.1 -i41 1.9 7.7 6.7 6 .2 5.7 l i 7 30 11 1 110 246 10.3 10.1 -122 7.5 6.7 6 .0 5.2 138 43 18 7 12 65 114 122 2 28 2 0 1756 1497 79 11 RUN 2 - SYSTEM 1: SUMMARY OF DATA (CONT'D) DAY TKN FLOW FLOW ORP DO pH NH4-N N03-N N02-N MLSS VSS EFF SRT IN IN RECYC 1 2 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 113 246 10 .1 11 ,3 -133 2 ;2 7 .6 6 .9 6 .5 6 .5 83 18 0 0 10 47 86 109 3 34 16 0 115 244 10 .0 11 .2 -137 7 .6 6 .9 6 .6 6 .7 86 16 0 0 13 53 98 115 4 32 12 0 1640 1412 60 12 117 254 10 .0 11 .1 -110 7 .6 7 .0 6 .7 6 .7 87 15 0 0 13 63 107 118 8 26 6 0 120 9 .9 11 .0 -152 i .9 7 .9 7 .4 7 .2 7 ,4 97 13 0 b 5 65 116 H 9 4 22 1 0 124 252 10 i l 10 .7 -189 7 .8 7 ;2 7 .0 7 .1 100 29 9 l 4 57 86 100 1 20 14 9 1757 1471 71 12 127 243 9 ;9 10 .8 -234 7 .8 7 .2 7 .0 7 .2 98 16 0 4 63 106 111 2 18 1 0 i29 243 9 .7 10 .5 -230 7 .8 7 .2 7 .0 7 .2 100 26 1 4 62 110 113 0 20 0 0 131 260 9 .8 10 .6 -273 7 .9 7 .3 7 .2 7 .4 100 16 0 0 4 54 93 108 2 21 7 0 1711 1458 47 13 134 256 9 .7 11 .2 -271 7 .7 7 ;2 7 .0 7 .2 92 18 0 2 72 115 117 2 13 1 0 136 248 9 .8 io .7 -325 7 .8 7 .2 7 .0 7 .2 111 27 0 1 66 117 120 0 15 1 0 137 262 9 .9 10 .5 -259 7 .8 6 .9 6 .4 6 ;7 i l 5 31 2 1 64 115 120 0 13 0 0 138 9 .8 11 .5 -259 7 .5 6 .9 6 .5 6 .6 104 26 0 1 80 114 116 0 9 0 0 1910 1652 42 14 141 252 9 .6 11 .5 -250 7 .6 6 .9 6 .6 6 .8 98 20 b 0 72 107 114 0 7 0 0 143 256 9 .7 10 .7 -292 7 .6 6 .8 6 .3 6 .5 106 28 l 1 67 i l 3 116 0 11 1 0 145 9 .7 10 .9 7 .4 6 ;7 5 .7 5 .2 i03 30 2 2 77 i l 3 118 0 9 0 0 1823 1571 62 13 RUN 2 - SYSTEM 2: SUMMARY OF DATA DAY TKN FLOW FLOW ORP DO pH NH4-N N03-N N02-N MLSS VSS EFF SRT IN IN RECYC : 1 2 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 8 104 10^0 20.5 1.2 7.4 10 2 0 0 93 104 104 106 1 0 0 0 2439 1991 50 23 12 121 10.1 20.2 i.o 7.5 14 3 0 95 107 110 109 1 0 0 0 15 124 10.1 20.7 1.0 7.3 7;5 24 0 0 0 76 99 103 1 2 1818 1514 46 10 19 10.1 18.9 2.3 7.3 7.5 16 4 0 0 100 114 117 120 4 3 0 0 22 9.9 19.3 2.4 7.3 7.5 20 3 0 100 115 120 3 4 0 1265 1054 42 9 .0 26 10.1 19.3 1.5 7.3 7.5 19 6 0 0 93 110 115 115 1 0 0 0 29 10.1 18.7 2.2 7.5 7.5 26 17 9 85 97 108 112 0 0 0 0 906 723 33 9 .9 34 10.1 18.7 1.1 7.4 7.5 21 10 0 0 88 100 109 109 0 0 0 36 117 10il 18.7 1.7 7.5 7 .2 7.8 18 5 0 0 87 105 113 112 2 2 0 0 888 628 29 12 40 118 10.5 18.1 1.4 7.4 7.8 20 10 90 106 112 112 2 2 0 0 43 126 10.4 20.0 1;3 7.5 8.0 15 3 0 0 95 111 108 112 2 1 0 0 695 505 30 8 .5 47 8.9 19.9 1.4 7.5 15 0 0 98 111 111 114 0 0 0 0 50 170 9.1 20.3 2.5 7.4 7.5 42 32 23 12 106 109 125 133 2 2 1 1 697 521 30 9 .4 54 119 9.6 20*6 1.8 7.3 7 .2 7.8 16 5 0 0 91 104 105 106 0 0 0 0 702 529 80 5 .0 58 119 9.4 20.6 1.7 7.3 7 .2 7 .4 7.6 15 0 0 89 103 109 107 4 1 0 0 69 9.1 17.7 2.9 7.3 7 0 0 95 103 107 i07 6 0 0 0 977 779 35 13 71 8.9 14.0 2.2 110 0 76 117 7.5 13.7 2.0 7.3 17 0 0 84 i07 106 104 8 0 0 0 981 781 29 13 78 -273 7.8 7 .8 7.8 80 244 11.5 9.7 -341 7.8 7 .7 7.8 149 117 81 43 4 2 14 30 41 82 253 10.0 9.4 8.0 7 .4 7 .2 6.9 132 74 27 2 2 26 51 52 1068 880 19 11 84 9.9 9.6 -322 8.0 7 .3 7 .0 6.8 119 63 12 3 2 32 58 46 87 253 9.9 9.9 -356 8;1 7 .1 6 .7 6.6 122 39 9 2 1 66 77 64 89 253 9.8 10.9 -300 1.9 8.1 6 .9 6 .8 6.8 94 23 2 0 0 12 27 1 62 79 73 14i5 1201 25 15 92 9.8 10.6 -333 1.3 8.3 7 .2 6 .9 6.9 93 26 2 0 0 6 13 1 62 88 90 94 254 9.9 11.0 -330 1.7 8.4 7 .1 6 .8 6.8 95 28 4 0 76 100 H i 0 69 87 94 96 234 9.8 11.2 -340 1.2 8.2 7 .2 6 .8 6.6 97 40 9 0 2 5 7 0 56 82 89 1768 1553 20 17 101 254 9.3 11;2 -316 2.6 8.2 7.0 6 .7 6.7 82 23 3 0 1 6 8 3 66 84 88 103 25l 9.3 11.4 -327 1.6 8.2 7 .2 6 .8 6.7 90 25 3 0 1 7 7 0 62 85 88 1955 1713 17 17 106 263 9.5 9.6 -342 2.5 8.3 7 .2 6 .8 6.6 97 35 7 0 0 3 6 8 0 64 86 96 108 249 9.4 9.5 -346 2;3 8.3 7 .3 6 .9 6.7 109 55 21 12 110 246 9.5 9.7 -346 8.2 7 .4 7 .0 6.8 138 64 36 22 0 1 1 9 3 55 83 89 1982 1776 17 17 RUN 2 - SYSTEM 2: SUMMARY OF DATA (CONT'D) DAY TKN FLOW FLOW ORP DO pH NH4-N N03-N N02-N MLSS VSS EFF SRT IN IN RECYC 1 2 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 113 246 9 .3 10.7 -340 2 .0 8 .2 7 .3 6 .9 6 .8 98 46 16 6 0 2 4 7 1 50 79 88 115 244 9 .2 10.7 -397 8 .2 7 .3 7 .0 6 .8 98 46 13 0 2 5 8 0 48 80 89 117 254 9 .1 10.7 -347 8 .3 7 .3 7 .0 7 .0 93 39 9 0 1 5 10 0 4 9 80 89 1945 1740 21 17 120 9 .1 11.0 -354 1 .3 8 .5 7 .6 7 .4 7 .6 95 31 0 0 0 0 3 6 0 56 86 93 124 252 9 .3 ll.O -321 8 .1 7 .2 7 .3 7 .4 89 21 0 0 0 2 7 14 0 70 87 83 2388 2li3 41 16 127 243 9 .1 11.2 -315 8 .2 7 .2 7 .3 7 .4 87 5 0 0 4 9 20 1 84 88 85 129 243 8 ;9 10.9 -304 8 .2 7 .1 7 .3 7 .3 85 17 3 0 0 2 13 24 0 86 92 86 131 260 8 .9 11.0 -320 8 .3 7 .2 7 .4 7 .5 84 4 0 0 1 3 15 25 1 85 87 79 2099 1801 47 15 134 256 8 .9 10.5 -336 8 .2 7 .0 7 .4 7 ,5 88 2 0 0 6 23 38 0 96 87 72 136 248 8 .9 9,7 -291 8 .1 7 .0 7 .3 7 .6 103 3 0 0 5 29 48 0 98 86 67 137 262 9 .2 9.6 -317 8 .2 6 .1 6 .8 7 .1 108 7 1 0 6 29 53 0 92 83 63 138 9 .1 10.9 -3l4 7 .9 6 .7 6 .8 7 .0 95 7 0 0 7 28 53 0 90 82 59 139 8 .6 io.5 -305 8 .0 6 .9 7 a 7 .3 0 3 28 55 0 87 77 54 1904 1670 32 16 14i 252 8 .8 10.7 -306 8 .0 6 .8 6 .9 7 .2 91 5 0 2 9 37 64 0 85 69 43 143 256 8 .9 9.8 -304 7 .9 6 .7 6 .6 6 .8 103 11 2 0 16 46 84 0 81 64 32 145 8 .8 9.8 7 .6 6 .6 6 .2 6 ,0 104 16 4 1 23 62 105 0 66 45 10 2035 1790 12 18 148 6 .8 6 .5 25 78 48 20 RUN 3 SUMMARY OF DATA DAY TKN FLOW FLOW pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 0 .5 137 9 .7 13 ;3 7.0 6 .7 6 .5 6.5 21 12 4 1 39 49 55 54 3 1 0 0 3421 2767 21 1 .0 117 19 .9 13 .5 8.0 8 .0 8 .0 8.0 45 28 13 1 36 46 57 69 14 17 20 21 1 .5 6.7 6 .8 6.5 14 17 21 2 .0 7.0 7.2 17 23 25 2 .5 6.5 6 .2 5.3 9 11 17 3 .0 125 19 .8 13 .7 8.5 8 .6 8 .6 8.7 U6 86 86 86 4 5 6 7 8 9 11 12 3 .5 8.1 8 .1 8.1 8 io 13 5 .0 118 20 .3 13 .7 7.7 7 .7 7 .1 7.7 f>6 61 52 46 5 6 7 9 32 41 48 56 2180 1835 70 11 5 .5 7.7 7 .6 7.6 6 7 11 45 57 78 6 .0 149 20 .4 7.6 7 .5 7 .4 7.5 45 30 19 10 6 io 12 16 54 67 76 86 7 .0 120 20 .5 13 .7 7.7 7 .4 7 .4 7.6 35 14 5 1 14 is 16 18 61 81 90 96 2015 1677 66 9.2 8 .0 7.3 7 .2 7 .4 7.7 18 22 26 30 56 73 77 77 8 .5 7.0 6 .9 7 .0 7.2 21 27 32 36 46 59 64 67 9 .0 6.9 6 .6 6 .5 6.5 24 30 36 43 38 48 50 50 10 .0 20 .6 6.8 6 .5 6 .3 6.1 35 20 13 ib 31 41 48 60 29 36 35 33 1528 13i3 96 7 11 .0 6.0 70 15 12 .0 20 .6 13 .4 6.6 6 .2 5 .8 5.5 44 31 26 26 41 53 65 72 12 11 4 1 RON 4 - SYSTEM 1: SUMMARY OF DATA DAY TKN FLOW FLOW ORP pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 4 i 2 3 4 1 2 3 4 TSS 2 9 .7 9.8 7 .8 7 .9 7.8 7 .8 2*. 4 0 58 75 90 93 6 9 3 0 3 9 .5 8 .1 8 .3 8.2 7 ,9 69 63 51 46 56 64 76 10 12 14 15 3 8 .5 8 .4 8.4 8 .5 42 67 14 20 4 120 9 .4 9.7 8 .2 8 .3 8,1 7 .9 58 50 30 41 50 59 68 15 17 20 23 5 -86 8 .2 8 .3 8.2 8 ,1 68 64 44 1 30 0 12 6 -i26 7 .9 8 .1 8.3 8 .2 80 79 63 11 21 0 0 7 9 .2 9.8 -150 8 .4 8 .6 8.6 8 .6 93 95 96 0 1 2 4 0 0 0 0 8 -121 8 .4 8 .3 8.4 8 .5 104 107 109 9 9 .2 9.6 -128 7 .6 8 .0 8.1 8 .2 104 110 112 0 1 2 2 0 1 2 3 10 9 .2 -76 7 .2 7 .8 7.9 7 .9 108 109 109 0 2 2 3 0 2 4 7 11 9 .2 9.8 -104 7 .1 7 .4 7.5 7 ,4 102 101 95 0 2 3 4 0 6 13 19 12 9 .1 11.2 -181 7 .7 7 .5 7.4 7 .0 81 71 51 0 2 4 4 0 15 33 47 13 -211 7 .9 7 .4 7.4 7 ,2 52 34 8 14 9 .3 10.5 -181 8 .1 7 .6 7.5 7 .4 51 20 6 0 3 4 5 0 31 48 52 16 9 .2 9.6 -244 8 .3 7 .5 7.7 7 .8 49 9 0 2 7 7 1 37 50 50 2177 1807 18 130 9 ,5 10.8 -266 8 .6 7 .8 7.9 7 .9 51 11 0 2 5 7 0 38 47 48 1994 1652 22 7.7 21 127 9 .4 10.9 8 .5 7 .8 8,0 8 .0 49 5 0 3 5 8 0 42 49 46 2169 1802 23 45 23 133 9 .5 10.4 -290 8 .5 7 .6 7.8 7 .8 50 4 3 0 5 8 14 0 45 47 45 2060 1743 22 32 25 130 9 .5 10.8 -324 8 .5 7 .5 8.0 7 .8 50 5 3 0 6 12 17 0 43 43 39 2505 2122 17 33 28 127 9 ,5 11.3 -363 8 .5 7 .8 7,8 8 .0 49 6 2 0 5 14 27 0 43 37 27 2588 2203 27 49 30 129 9 .4 11.4 -176 8 .5 7 .7 8.1 8 .1 44 6 3 0 14 24 35 0 36 28 19 2547 2169 34 15 32 133 9 .6 11.8 -255 8 ,4 8 .1 8.2 8 .1 47 4 3 0 15 30 45 0 35 23 10 2698 2290 33 36 35 136 10 .4 19.5 -269 8 .3 8 .3 8.0 8 .1 32 37 2 2 0 0 10 22 0 1 27 20 2830 2425 29 36 37 137 10 .4 9.6 -252 8 .4 8 .4 7.8 7 ,9 32 33 3 4 0 0 i i 21 i 0 29 20 2934 2520 18 37 39 130 11 .1 23.7 -284 8 .4 8 .4 7.8 8 .0 36 33 2 2 0 0 11 25 0 0 28 19 2966 2544 28 35 42 153 10 ;9 19,1 -297 8 .4 8 .4 7.8 8 .1 4 4 40 2 4 0 0 8 22 0 0 36 27 3191 2786 24 41 44 157 10 ,6 18,5 -317 8 .4 8 .3 7.8 8 .0 38 39 2 1 0 0 10 28 0 0 34 22 3296 2865 23 38 46 187 10 .4 18.3 -326 8 .1 8 .0 7.4 7 .7 51 49 2 i 0 0 16 35 0 0 4o 24 3569 3162 25 32 49 186 10 .3 19.6 -345 8 ,0 7 .9 7.3 7 .7 58 58 2 2 0 0 31 58 0 0 31 6 3829 3323 27 47 51 185 9 .8 20.2 -430 8 .1 8 , i 7.6 7 .9 57 54 5 0 0 0 36 63 0 0 24 i 3672 3187 26 24 53 187 11 .4 20.4 -474 8 .3 8 .3 7.5 7 .9 56 56 3 0 0 0 35 66 0 0 23 1 3776 3346 24 18 RUN 4 - SYSTEM 1: SUMMARY OF DATA (CONT'D) DAY TKN FLOW FLOW ORP pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 56 11*6 20.6 8 .5 8 .4 7.6 7 .9 55 53 0 0 0 38 64 0 0 23 1 2551 2232 27 5 .3 58 185 9.4 20.2 8 .3 8 .3 7.7 8 .0 52 50 3 1 0 0 31 50 0 0 19 3 1571 1389 28 3 .5 60 186 10.9 21.0 8 .2 8 .1 7.7 7 .6 59 53 14 2 0 0 14 32 0 0 22 25 758 661 15 2 .0 63 187 9.7 20.1 8 .1 8 .0 7.6 7 .6 53 53 23 8 1 0 10 25 1 0 18 24 1014 907 16 4 .5 65 189 10.0 20.5 8 .3 8 .3 8.3 8 .4 100 90 79 54 0 0 3 8 0 0 5 6 67 184 10.7 20.8 8 .1 8 .1 7.6 7 .7 54 51 10 1 0 0 21 44 0 0 19 10 1389 1233 17 9 .2 70 10.0 9.3 8 .4 8 .4 7.6 7 .5 88 84 25 1 0 19 52 1 0 31 31 72 9.7 8.8 8 .3 8 .3 7.8 7 .5 91 90 41 16 41 27 37 918 827 16 3 .7 74 11*0 10.8 8 .4 8 *3 7.9 7 .6 98 101 67 4 18 13 36 1076 944 23 3 .3 77 183 11*5 10.3 -398 8 .2 8 .2 7.9 7 .5 97 95 50 17 0 0 5 17 0 0 24 52 1024 928 18 9 .5 79 182 9.8 10.6 -462 8 .2 8 .2 7*5 7 .4 77 8i 26 11 0 0 9 35 0 0 24 32 1211 1079 25 8 .2 81 9.4 10.0 -409 8 .4 8 .3 7.8 7 .8 76 78 14 0 0 15 41 0 0 33 25 1345 1142 20 7 .0 84 186 10.3 22.3 -315 8 .7 8 .2 8.2 8 .3 43 5 1 0 2i 44 50 0 19 6 1 1322 1131 40 7 .9 88 184 9*5 21.6 8 .7 8 .2 8.2 8 *3 48 11 3 0 24 44 5i b 14 6 1 1338 1140 39 7 .9 91 182 10.4 21.8 -349 8 .6 8 .2 8.3 8 .3 49 11 2 0 31 49 53 0 10 2 1 1120 982 27 8 .2 95 189 10.6 22.1 8 .5 8 .1 8.0 8 a 51 17 3 2 31 49 55 1 5.5 2 0 1176 1031 37 7 .8 98 183 i l i l 22.3 8 .6 8 .3 8.1 8 .2 49 10 2 2 34 51 56 0 5 1 0 1275 1094 37 7 .6 RUN 4 - SYSTEM 2: SUMMARY OF DATA DAY TKN FLOW FLOW ORP pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 4 1 2 3 4 i 2 3 4 TSS 2 9 ,8 9 .4 8 .1 8 .0 7.9 7.8 26 4 0 56 69 83 93 7 11 9 0 3 9 .7 8 .2 8 .3 8.2 8,0 66 60 45 43 53 63 73 14 16 18 20 3 8 .5 8 .5 8,6 8.7 56 90 40 66 16 24 4 120 9 .5 9 .4 8 .4 8 .3 8.2 8.0 62 54 37 35 44 51 61 16 20 22 25 5 8 .3 8 .3 8.4 8.2 76 70 57 28 59 8 11 6 8 .1 8 .1 8.0 8.2 63 55 39 37 76 10 15 7 9 .3 9 .4 8 .0 8 .0 7.8 7.6 51 36 27 14 43 52 63 69 15 20 24 24 8 -79 8 .5 8 .5 8.5 8.4 73 74 56 9 9 .4 9 .3 -62 7 .5 8 .0 8.1 8.1 93 101 97 0 3 5 7 0 0 0 0 10 9 .4 -22 7 .1 7 .9 7.9 7.9 107 112 112 0 4 8 12 0 0 0 0 11 9 .4 9 .5 -24 7 .0 7 .6 7.6 7.6 104 106 104 0 6 12 19 0 0 0 0 12 9 .3 10 .9 -82 7 .6 7 .6 7.5 7,4 93 88 72 0 9 17 27 0 2 5 8 13 -85 7 .8 7 .8 7.7 7.5 68 61 35 14 9 .5 10 .1 -104 8 ,0 7 .7 7.5 7.4 57 34 17 0 9 18 28 0 16 28 34 16 9 .4 9 .8 -120 8 .4 7 .8 7.6 7.8 47 19 1 11 22 31 0 23 31 26 2259 1879 18 130 9 .7 11 .0 -111 8 .6 7 .8 7.8 8.0 48 17 3 0 7 16 27 0 28 34 29 2345 1956 20 39 21 127 9 .7 11 .0 8 .5 7 .8 7.8 8.0 44 15 4 0 1 6 12 0 31 43 41 2445 2042 20 45 23 133 9 .7 10 .6 -144 8 .5 7 .7 7.7 7.8 47 12 4 0 4 6 11 0 35 45 45 2370 2018 16 41 25 130 9 .7 10 .9 -140 8 .5 7 .5 7.7 7.9 47 11 4 0 3 6 11 0 39 46 44 2576 2192 17 38 28 127 9 .6 11 .4 -i49 8 ,4 7 .7 7.9 7.9 47 6 3 0 0 3 9 1 46 49 45 2930 2496 26 52 30 129 9 .5 10 .9 -122 8 .4 7 .8 7.9 8.1 50 7 4 0 5 8 11 0 42 47 47 2801 2385 28 27 32 133 9 .7 11 .0 -138 8 ,4 7 ,9 8.0 8.1 52 io 3 0 3 5 8 0 40 49 49 2698 2290 39 34 35 136 9 .8 9 .8 -114 8 .4 7 .9 7.9 8.1 52 16 5 0 4 8 13 0 33 45 46 2929 2512 42 33 37 137 9 .6 10 ,0 -117 8 .4 8 .1 7.9 8,0 44 16 4 0 5 13 17 0 32 43 41 2912 2505 35 34 39 130 10 .3 l l .5 -120 8 .5 8 .1 7.8 7.9 46 25 2 0 6 13 23 0 21 35 36 2962 2522 43 36 42 153 10 ,0 10 .2 -105 8 .8 8 .7 8.5 8.3 72 64 23 0 4 12 22 0 11 19 28 2866 2480 41 34 44 157 9 .7 9 .2 -156 8 .8 8 .7 8.5 8.4 90 80 51 0 4 10 20 0 8 16 23 2700 2333 34 33 46 187 9 .5 10 ,2 -297 8 .4 8 .4 8.2 8.0 104 89 57 0 5 12 21 0 14 26 37 2615 2282 37 28 49 186 10 .1 10 .8 -351 8 .3 7 ,9 7.6 7.6 74 50 5 1 14 25 43 0 22 38 41 2946 2571 34 40 51 185 9 .6 9 .7 -354 8 .4 7 .8 7,7 8.0 73 35 1 0 23 44 69 0 23 29 13 2853 2477 32 22 53 187 11 .1 10 .6 -123 8 ,5 7 .9 7,7 7,9 75 38 1 b 25 52 81 0 18 22 5 2812 2468 34 17 RUN 4 - SYSTEM 2: SUMMARY OF DATA (CONT'D) DAY TKN FLOW FLOW ORP pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 TSS 56 l i . 4 10.7 8 .7 8 .3 7 .8 7 *9 73 44 53 1 1 27 53 78 0 13 17 6 2067 1808 36 5.1 58 i85 9.2 10.1 8 .7 8 .7 8 .6 8 .5 91 83 43 0 4 14 32 0 4 6 6 1353 1185 37 3.4 60 186 10.7 11.0 8 .5 8 .5 8 .6 8 .7 127 127 90 0 1 2 3 0 3 8 14 819 726 29 2.6 63 187 9.6 10.0 8 .5 8 .6 8 .1 8 .0 110 100 72 0 4 12 12 0 13 25 47 1135 1019 25 25 65 189 9.9 10.3 8 *5 8 .1 7 .7 7 .7 77 51 12 0 7 11 17 1 26 49 68 67 184 10.6 11.2 8 .3 7 .7 7 .6 7 .8 73 30 6 0 13 23 37 1 40 55 47 1209 1061 31 15 70 9.9 10.5 8 .7 8 .7 7 .7 7 *7 72 69 i7 1 0 18 42 1 0 41 38 72 9.6 12*5 8 .7 8 .7 8 .6 8 .3 84 88 66 5 16 14 33 998 883 37 4.7 74 10.9 9*9 8 .7 8 .7 8 .4 8 .2 110 105 94 3 9 13 30 1123 992 38 11 77 183 11*4 11.4 -147 8 .3 8 .3 8 .0 7 .1 92 93 65 39 0 0 6 10 0 0 29 59 1182 1045 29 9.0 79 182 9.9 10.8 -188 8 .4 8 .4 7 .8 7 .5 75 73 32 4 0 0 4 11 0 0 21 58 1298 1153 25 8.4 81 9.4 10:2 -277 8 .5 8 .4 8 .0 7 .8 79 73 24 0 0 4 10 0 0 38 58 1302 1150 16 9.1 84 186 10.4 11.5 -204 8 .7 8 .7 8 .4 8 .0 73 71 46 20 0 0 4 7 0 0 21 49 1534 1336 21 7.1 88 184 9.7 11.1 8 .4 8 .4 7 .8 7 .7 69 71 20 4 0 0 6 16 0 0 38 53 1536 1356 15 9.2 91 182 10.7 10.6 -424 8 .4 8 .3 7 .8 7 .7 78 78 19 5 0 0 11 26 0 0 34 42 1506 1337 15 8.9 95 189 10.9 11.1 8 .2 8 .0 7 .6 7 *5 81 76 17 4 0 0 7 28 0 0 40 48 1765 1564 15 9.1 98 183 11*4 11.8 8 .2 8 .2 7 .6 7 .5 75 74 12 5 0 0 7 28 0 0 45 43 1895 1663 17 9.8 RUN 5 - SYSTEM 1: SUMMARY OF DATA DAY TKN FLOW FLOW pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC 1 2 3 4 1 2 3 1 2 3 4 1 2 3 4 TSS 1 90 12 .7 23 .5 7 .5 7.7 7 .5 2 180 10 .7 25.8 6 .8 7.1 6 .8 3 10 .7 25 .8 7 .5 7.5 7 .3 6 10 .7 21 .0 7 .9 8.0 7 .9 10 188 10 .5 9 .8 8 .4 8.4 8 .2 13 183 10 .9 10 .4 8 .0 8.2 8 .2 17 180 9 .6 9 .1 8 .1 7.5 7 .5 20 185 10 .8 10 .4 8 .3 7.7 7 .7 24 185 10 .7 22 .5 8 .4 7.6 7 .7 27 189 10 .8 29 .5 8 .4 7.7 7 .8 29 188 10 .5 29 .7 8 .5 7.8 7 .9 31 196 10 .8 30 .2 8 .6 7.8 7 .9 34 208 10 .9 30 .5 8 .6 7.8 7 .8 38 208 10 .7 29 .1 8 .5 7,8 7 .6 41 2i2 10 .0 28 .4 8 .4 7.7 7 .7 42 9 .9 28 .1 45 214 9 .9 26 .6 8 .5 7,8 7 .9 51 213 9 .7 29 .6 8 .4 7.7 7 .8 55 10 .2 24 .8 8 .5 7,8 7 .8 59 10 .1 24 .5 8 .5 7.8 7 .7 62 9 ,9 24 .8 8 .5 7.8 7 ,7 7.5 6.8 11 0 107 108 7.1 48 34 15 56 74 92 113 7.8 83 67 53 0.2 18 25 37 8.0 124 123 109 0.1 5.5 17 32 8.0 147 149 145 0 1.8 4 7 7.3 101 78 51 0,3 0 3 4.5 7.7 79 21 2 0 1 2 2.5 7.7 45 5 0 0,3 0.3 1 1.8 7.8 38 4 0 0.6 36 40 41 7.9 35 3 0 0.2 0.7 1.5 2.4 7.8 40 6 0 0.2 0.8 2.8 4.5 7.8 47 6 0 0.5 40 45 47 7.6 50 13 0,4 7.5 16 29 7.6 45 4 8.3 9.3 28 44 58 63 7.8 48 3 8 31 53 60 7.8 38 2 17 54 65 67 7.8 51 5 2 43 53 54 7.6 47 9 0.5 26 40 52 7.7 46 6 2,7 29 43 54 13 12 0 3 5 4 2506 2079 0 .1 2 0.4 0.2 2088 1774 83 9. 0 0 .1 0 0 0 1813 1560 90 6. 3 0 4 8 11 1603 1356 92 6. 8 0 .4 23 51 72 1429 1228 52 11 .4 0 .6 58 75 76 1655 1375 38 10 .5 0 .9 42 49 49 1713 1451 16 11 .8 0 .5 35 39 39 1783 1519 42 10 .0 0 .6 35 38 37 1834 1578 27 11 .2 i .0 38 40 39 2012 1753 31 11 .1 0 .4 39 42 40 2058 1787 37 10 .9 0 .7 28 34 24 1940 1675 53 10 .7 0 .6 21 i3 1.5 1844 1594 50 11 .5 0 .8 0 0 .9 22 6 0 1940 1623 50 11 .8 1 16 1.5 0 1777 1539 71 10 .2 1 17 5 0 1744 1527 78 9. 4 0 .3 13 9.5 0 1974 1727 89 9. 2 2 .4 14. 9 0 1666 1460 119 7. 3 RUN 5 - SYSTEM 2: SUMMARY OF DATA DAY TKN FLOW FLOW INTER PH NH4-N NO 3 -N N02-N MLSS MLVSS EFF ' SRT IN IN RECYC RECY 1 2 3 4 i 2 3 1 2 3 4 1 2 3 4 TSS 1 90 12.0 24.2 7 .6 7 .7 7.5 7 .3 2 180 10.2 25.4 7 .0 7 . i 6.9 6 .7 11 0 115 117 13 12 3 10.3 25.4 7 .5 7 .6 7.5 7 .1 47 29 12 53 72 90 108 0 0 0 1 2690 2178 6 10.2 21.2 8 .0 8 .0 8.1 8 .0 52 24 1 0 41 63 69 0 1 0 0 2255 1879 35 11.1 10 188 10.1 10.6 8 .4 8 .3 8.2 7 .9 83 63 31 0.3 19 48 77 0 0 0 0 2069 1767 35 10.6 13 i83 10*5 10.9 8 . i 8 .2 8.3 8 .1 127 121 105 0 12 26 39 0 1 2 1 2033 1737 49 10.9 17 180 9.2 9.2 8 .3 7 .8 7.7 7 .4 90 60 30 ( .6 5.5 13 19 0 .4 32 59 7 i 1575 1350 48 10.3 20 185 10 . 4 11;1 8 .5 7 .8 8.0 8 .0 73 22 2 0.4 3 6 9.5 0 .4 52 69 69 1610 1390 47 10.1 24 185 10.3 5.2 13.7 8 .5 7 .7 8.0 8 .1 50 5 0 0.2 2 5 11 2 49 52 49 1739 1473 21 10.1 27 189 10.3 2.2 26.9 8 .5 7 .8 8.1 8 .2 39 4 0 0.1 2.2 5 19 0 .6 34 37 26 1822 1550 35 10.6 29 188 10.1 2.0 26.1 8 .5 7 .7 8.1 8 . i 35 3 0 0.2 2.3 3 18 0 .5 36 35 26 1893 1623 39 10.1 31 196 10.4 2.1 29.7 8 .5 7 .9 8.3 8 .3 42 7.5 0 0.1 2.9 17 31 0 .6 36 29 17 1959 1692 48 9.6 34 208 10.5 2.3 30.0 8 .6 7 .9 8.3 8 ;3 46 8 0 0.1 2.5 21 38 0 .4 38 30 16 2004 1735 43 9.8 38 208 10.5 2.1 31.4 8 .5 7 .8 8.2 8 .1 46 7 0.3 5.2 35 55 0 .6 34 20 3 2065 1757 27 11.0 41 212 9*9 2.2 28.9 8 .5 7 .7 8.2 8 .0 41 4 0.3 8.3 45 59 0 .6 33 8 0 1942 1678 55 10.1 42 9.8 2.2 29.2 0.1 8.2 62 0 .2 33 0 43 6.5 32 45 214 9.8 2.2 28.7 8 .5 8 .0 8.1 8 .0 68 33 0.4 6.2 40 70 0 .7 26 32 8 1535 1283 75 8.7 49 7 32 50 5 46 20 36 51 213 9.7 2.2 27.4 8 .6 7 .9 8.1 8 .1 38 4 0 8.2 19 36 58 1 .5 26 18 0 1486 1219 61 9.4 52 13 38 53 13 38 54 8 .5 7 .8 56 17 13 23 55 10.4 2.4 22.4 8 .6 7 .9 8.0 8 .3 61 22 2 i 0.7 16 13 53 0 .9 25 19 6 1629 1408 57 10.9 56 12 20 57 8 .6 56 i4 42 56 21 9 59 10.2 2.2 23.0 8 .5 7 .8 7.9 8 .2 62 18 14 0.1 22 15 37 0 .2 19 2 1 2019 1766 21 13.4 60 27 18 61 31 17 62 10.2 2.4 22.5 8 .6 7 .9 7.9 8 .4 54 6 5 2.2 32 13 26 0 .5 16 1 0 2589 2282 21 12;2 63 25 14 RUN 5 - SYSTEM 2: SUMMARY OF DATA (CONT'D) DAY TKN FLOW FLOW INTER pH NH4-N N03-N N02-N MLSS MLVSS EFF SRT IN IN RECYC RECY 1 2 3 4 1 2 3 1 2 3 4 1 2 3 4 TSS 64 25 14 65 28 14 66 10.3 2.5 30.2 8.6 7.9 7.8 8.3 40 1 1 i . l 37 5 25 0.3 12 1 0 2295 1999 40 10.6 67 28 12 68 / 29 9 69 10.1 2.1 29.7 8.7 8.0 8.0 8.4 52 9 6 1.2 27 9 17 0,8 11 1 0 1979 1757 78 8.6 71 8.7 47 28 11 72 8.8 95 29 10 73 10.3 2.2 29.7 8.5 104 2111 1846 43 9.9 74 8.5 55 32 13 75 34 12 76 10.5 2.1 29.7 8.6 7.9 7.9 8,3 53 9 l l 2.3 35 14 34 0,5 13 1 0 1981 1787 26 10.8 RUN 6: SUMMARY OF DATA DAY FLOW FLOW FEED FEED pH NH4-N IN RECYC TKN NH4-N 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 19 .6 8 .0 3 19 ;5 18 .6 224 7 .4 4 8 ,0 6 19 .3 18 .1 178 176 8 .2 7 - 8 .3 8 7 .7 io 19 .6 18 *7 277 7 .9 11 9 .6 12 8 .0 13 19 .5 18 .9 255 265 7 .2 14 8 .1 16 19 .0 18 .7 256 255 8 .2 19 19 .2 18 .7 253 255 8 .5 22 19 .2 18 .7 258 270 8 .4 24 i9 .2 19 ,0 256 295 7 .6 27 19 ,4 19 .2 259 270 8 .1 29 19 .2 19 .0 257 254 8 .1 31 19 .6 19 .2 264 262 8 .3 34 19 .6 19 .3 248 8 .2 36 19 .3 19 .3 272 8 .0 38 19 .5 19 .2 252 8 .1 41 290 8 .3 44 7.5 7 .3 7 .2 7 .2 7 . i 7 ,6 7.8 7 .6 7 .5 7.9 7 .6 7 i5 7 ;5 7 .1 7 .3 7 .6 7.7 7 .5 7 .4 7 .3 7 .3 7 .6 7 .3 8 .2 7.5 7 .5 7 .6 7 • 6 7 .6 7 .6 7 .6 8.1 8 .0 8 .1 8 .1 8 i l 8 .0 8 .0 8.3 8 .2 8 .2 8 .3 8 .2 8 .1 8 .2 8.1 8 .0 8 .0 8 .1 8 i l 8 ;1 8 .2 7.7 8 .0 8 .0 8 .1 8 .0 7 .9 7 i9 8.0 8 ,0 7 .9 7 ,9 7 .8 7 .8 7 .8 7.8 7 .8 7 .8 7 .8 7 .7 7 ,9 7 .8 7.9 7 .9 7 i7 7 .6 7 .5 7 .9 7 .8 8.7 8 .2 7 .9 9 .0 8 .5 8 .3 8 .2 7.8 7 ,5 7 .2 7 .3 7 .1 7 .3 7 .4 7.3 7 i l 7 .1 7 i l 7 ;2 8 .0 7 .8 8.4 8 .4 8 .5 8 .3 8 .7 8 .5 8 i5 77 33 104 63 15 2 83 63 14 0 i40 123 65 23 138 123 192 177 212 212 193 174 206 169 216 i96 179 155 203 187 173 159 222 206 190 209 189 168 151 158 132 97 65 141 108 68 33 155 115 82 44 78 5 0 0 161 102 72 57 114 50 14 2 245 224 177 188 RUN 6: SUMMARY OF DATA (CONT'D) DAY MLSS MLVSS EFF SRT N03-N N02-N TSS 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 4092 3026 3 3225 2512 38 74 90 108 129 147 163 153 157 0 1.5 4 6 9 5.5 5 0 4 86 107 139 151 11 13 18 15 6 3918 2301 137 11 .0 33 47 70 93 114 83 73 73 0.5 6 6 7 2 0.5 0 0 10 4176 2051 125 12 .2 89 107 125 150 174 189 168 190 0 1.5 1.5 5 4 2 0 0 13 4573 1707 188 12 .8 47 58 64 77 79 86 84 94 2.5 1.5 2 3.5 3 2.5 2.5 3 16 5035 1550 142 12 .5 29 36 39 45 53 58 55 60 10 12 13 16 19 20 21 22 19 4293 1441 130 14 .0 10 14 17 20 26 30 20 24 20 23 25 29 33 36 36 39 22 1.5 2 4 5 8.5 10 1 3.5 16 19 20 20 24 28 29 33 24 3532 1214 140 19 .7 1.5 4 6.5 7 11 18 2.5 10 37 41 50 50 59 67 68 76 27 4001 1439 127 23 .4 0.5 1 4.5 6 10 11 2 2 66 72 86 101 113 127 119 134 29 3517 1382 145 17 .2 0.5 2 3 7 9 12 0 2 71 79 97 113 126 141 128 143 31 4859 1496 119 16 .1 1 2 3 6 8 12 0 7 65 73 90 105 112 128 114 128 34 4346 1642 135 22 .9 0 1 9 20 30 36 0 1 93 97 119 130 133 136 107 92 36 6685 1820 115 22 .5 0 5 14 20 19 24 0 0 40 56 72 86 90 100 96 88 38 6053 1725 145 17 .4 47 65 78 87 96 110 79 96 19 38 50 59 68 65 51 38 39 122 141 173 176 16 24 16 3 RUN 7: SUMMARY OF DATA DAY TEMP FLOW FLOW FEED FEED MLSS VSS EFF SRT pH IN RECYC TKN NH4-N TSS 1 2 3 4 5 6 1 2917 1950 7 1095 829 122 17 21.0 15 .4 16 .7 3785 891 96 22 .4 7 .0 7 .1 7 .2 7 .4 7 .5 7 .5 21 15 .4 16 .5 259 267 3341 1048 48 7 .1 7 .2 7 .2 7 .2 7 .3 7 .3 24 21.0 15 .8 16 .9 265 260 2979 1080 55 31 .1 7 .2 7 .3 7 .2 7 .2 7 .2 7 .5 28 21.5 15 .6 .16 .5 265 259 3427 1232 57 21 .3 7 .2 7 .2 7 .2 7 .3 7 .2 7 .1 29 7 .3 7 .6 7 .7 7 .8 7 .8 7 .6 30 7.6 7 .5 7 .3 7 .5 7 .4 7 .2 31 21.0 15 .8 16 .9 284 260 3413 1218 74 20 .7 8 .1 7 .6 7 .4 7 .4 7 .3 7 .1 35 21.0 15 .6 16 .7 282 275 6381 1526 109 23 .2 8 .6 8 .3 8 .0 7 .7 7 .5 7 .3 38 21.0 15 .6 17 .0 253 260 7535 1685 175 15 .5 8 .7 8 .2 7 .9 7 .6 7 .4 7 .4 42 21.0 16 .1 16 .9 274 274 6298 1272 149 11 .2 8 .6 8 .1 7 .7 7 .6 7 .6 7 .6 45 21.0 15 .8 17 .1 272 268 10127 1170 154 15 .5 8 .7 8 .6 8 .2 8 .0 7 .8 7 .5 49 21.0 15 .9 17 .0 276 273 10343 1402 170 17 .3 8 .8 8 .5 7 .8 7 .6 7 .8 7 .8 52 15 .6 17 .2 276 273 8396 1814 147 14 .7 8 .7 7 .6 7 .3 7 .3 7 .8 7 .9 56 21.0 15 .3 17 .2 279 273 9414 1939 72 19 .2 8 .6 7 .5 7 .2 7 .5 8 .1 8 .2 59 21.0 15 .9 17 .5 266 257 11712 2258 83 18 .1 8 .6 7 .5 7 .4 7 .6 7 .8 7 .9 63 15 .7 17 .6 280 272 13581 2400 78 20 .2 8 .5 7 .2 7 .1 7 .2 7 .6 7 .6 70 21.0 15 .8 14 .1 284 270 12983 2234 122 12 .2 8 .7 7 .6 7 .9 7 .9 7 .5 7 .8 73 21.0 16 .1 14 .1 272 276 14928 2478 101 23 .3 8 .3 7 .2 7 .8 7 .8 7 .5 7 .8 76 21.0 16 .1 14 .0 284 269 15580 2845 81 22 .2 8 .2 7 .2 7 .7 7 .7 7 .4 7 .7 80 21.5 16 .6 13 .2 285 17218 3183 62 21 .4 8 .3 7 .3 7 .8 7 .2 7 .6 7 .8 84 21.5 16 .6 14 .0 280 19213 3321 88 20 .9 8 .2 7 .1 7 .9 7 .2 7 .6 7 .7 91 21.5 16 .3 12 .5 280 16521 2560 127 16 .8 8 .3 7 .2 8 .2 7 .6 7 .9 8 .2 94 21.0 16 .2 15 .0 263 16498 2673 106 19 .8 8 .0 7 .7 8 .3 7 .8 8 .0 8 .2 98 21.0 16 .2 13 .1 277 16257 2637 58 21 .4 8 .4 7 .6 7 .5 8 .7 8 .6 8 .5 101 21.0 16 .3 7 .8 278 16557 2604 75 19 .2 7 .9 7 .2 8 .1 7 .7 8 .0 8 .0 105 21.0 16 .3 14 .9 275 17229 2634 77 20 .9 8 .7 7 .4 8 .5 7 .9 8 .2 U 108 21.0 16 .5 10 .4 267 16362 2571 98 19 .1 8 .3 7 .5 8 .5 8 .2 8 .4 8 .4 112 21.0 16 .7 13 .4 274 16133 2590 84 17 .8 8 .6 7 .7 9 .1 8 .7 8 .8 8 .7 116 21.0 16 .9 12 .1 240 14763 2538 87 19 .8 8 .3 7 .6 9 .0 8 .9 8 .3 8 .2 119 21.0 16 .7 12 .5 270 14805 2714 79 20 .1 7 .8 7 .2 8 .3 8 .1 8 .1 8 .1 122 21.5 16 .3 12 .1 256 15365 2798 124 21 .4 8 .2 8 .2 8 .8 8 .3 8 .6 8 .6 126 22.0 16 .4 12 .0 264 14975 2724 123 17 .7 8 .5 7 .6 8 .9 8 .6 8 .7 8 .7 129 21.5 16 .5 12 .2 272 13639 2650 117 18 .9 8 .3 7 .3 9 .1 8.8 8 .6 8 .6 133 22.0 16 .6 12 .2 281 13842 2772 97 17 .8 8 .4 7 .4 8 .7 8 .4 8 .6 8 .7 136 21.5 16 .7 12 .4 256 14453 2917 118 18 .4 8 .8 8 .0 9 .1 9 .0 9 .2 8 .9 140 22.0 16 .8 12 .1 286 13838 2880 148 17 .9 8 .6 7 .7 9 .0 8 .7 8 .7 8 .7 143 21.5 16 .8 12 .0 256 13508 2914 160 17 .7 8 .6 7 .6 9 .0 8 .8 8 .8 8 .7 146 20.0 16 .5 11 .7 257 12703 2736 152 17 .1 8 .5 7 .8 8 .9 8 .6 8 .7 8 .6 150 21.0 16 .9 11.8 257 13846 2759 173 13 .9 7 .6 6 .9 8 .1 7 .7 7 .6 153 21.0 16 .7 11 .4 277 12110 2716 164 18 .0 8 .3 7 .8 8 .7 8 .3 8 .5 8 .5 157 21.0 15 .7 13 .6 270 11500 2597 171 18 .0 7 .2 7 .3 8 .0 7 .5 7 .7 7 .9 160 20.0 15 .6 13 .6 287 11118 2634 119 18 .0 7 .9 7 .2 8 .3 7 .9 8 .0 8 .0 164 22.5 16 .4 14 .0 265 10388 2417 143 12 .0 7 .9 7 .2 8 .4 8 .1 8 .1 8 .1 166 21.5 16 .3 13 .9 276 10616 2475 123 24 .2 7 .9 7 .2 8 .5 8 .1 8 .1 8 .1 168 22.0 16 .4 13 .9 263 10626 2512 121 24 .2 7 .9 7 .2 8 .3 8 .0 8 .1 8 .0 171 22.5 16 .6 14 .2 280 10730 2595 126 20 .6 8 .2 7 .3 8 .5 8 .0 8 .0 174 22.0 16 .3 13 .5 275 11024 2728 145 41 .8 8 .0 7 .3 8 .3 8 .0 8 .0 8 .0 178 23.0 16 .8 14 .2 275 10807 2735 140 17 .4 8 .1 7 .4 8 .6 8 .2 8 .2 8 .2 181 24.0 16 .6 14 .0 280 11283 2860 114 18 .1 8 .3 7 .5 8 .8 8 .4 8 .4 8 .5 240 RUN 7: SUMMARY OF DATA (CONT'D) DAY TEMP FLOW IN FLOW RECYC FEED NH4-! MLSS N MLVSS EFF SRT TSS 1 2 PH 3 4 5 6 185 22 .5 16 .6 14.0 281 10592 3000 82 19 .2 8 .1 7 .6 8 .7 8 .2 8 .4 8 .5 188 22 .5 16 .5 13.6 283 10770 3225 62 20 .2 7 .8 7 .7 8 .8 8 .2 8 .4 8 .1 192 21 .5 16 .3 14.3 260 9608 3105 91 18 .4 8 .0 7 .3 8 .7 8 .3 8 .4 8 .4 195 21 .5 16 .1 14.1 289 8540 2954 181 16 .5 7 .9 7 .5 8 .5 7 .9 8 .1 8 .1 199 20 .5 16 .1 14.2 270 7704 2668 110 17 .1 7 .9 7 .4 8 .1 7 .5 7 .8 7 .8 202 20 .0 16 .3 14.0 266 7743 2632 127 16 .2 8 .3 7 .6 8 .2 7 .5 7 .8 7 .9 206 20 .0 16 .5 12.2 285 7809 2477 133 16 .5 8 .0 7 .5 8 .1 7 .6 7 .7 8 .0 209 22 .0 16 .3 12.1 285 8917 2696 133 16 .2 8 .0 7 .4 8 .1 7 .4 7 .8 7 .8 213 20 .5 16 .5 12.7 258 8879 2659 85 18 .6 8 .2 7 .4 8 .2 7 .5 7 .8 7 .8 216 20 .5 16 .0 12.0 281 9025 2602 53 20 .6 8 .3 7 .4 8 .3 7 .8 8 .0 7 .9 220 21 .0 16 .6 12.3 270 9574 2417 75 19 .3 8 .6 7 .5 8 .5 8 .0 8 .1 8 .1 224 21 .0 16 .3 12.2 288 9735 2238 70 19 .6 8 .3 7 .3 8 .4 7 .8 7 .9 7 .9 227 20 .0 16 .9 12.5 275 9754 2254 62 20 .5 8 .1 7 .5 8 .3 7 .6 7 .9 7 .8 230 20 .5 16 .4 12.1 267 10770 2343 70 20 .1 8 .6 7 .5 8 .4 7 .9 8 .1 8 .0 234 20 .0 16 .8 12.6 270 11741 2443 115 18 .6 8 .7 7 .6 8 .5 7 .9 8 .2 8 .1 237 19 .0 16 .6 12.3 265 12145 2446 125 18 .3 8 .7 7 .6 8 .4 7 .8 8 .1 8 .0 241 20 .0 16 .9 12.7 275 12481 2538 116 18 .4 8 .5 7 .6 8 .3 7 .7 8 .0 8 .0 244 19 .0 16 .7 11.7 260 14357 2943 85 19 .9 8 .9 7 .7 8 .4 7 .8 8 .0 8 .0 248 20 .0 17 .2 12.3 258 15131 3099 123 19 .2 8 .6 8 .0 8 .5 7 .9 8 .1 8 .0 251 19 .0 17 .0 12.5 277 14819 3529 96 19 .6 7 .2 7 .4 7 .8 7 .6 7 .4 7 .4 255 19 .5 13 .2 11.7 262 11991 3318 79 19 .6 7 .2 7 .3 7 .8 7 .4 7 .6 7 .6 258 19 .0 16 .9 11.7 259 11442 3533 78 19 .3 7 .3 7 .3 7 .8 7 .4 7 .6 7 .6 262 20 .0 17 .5 12.6 270 10238 3585 67 18 .9 7 .2 7 .4 7 .9 7 .5 7 .6 7 .6 265 19 .0 17 .0 12.3 265 9109 3492 81 18 .4 7 .2 7 .3 7 .9 7 .4 7 .6 7 .6 268 20 .0 17 .6 12.8 265 7875 3219 164 15 .1 7 .2 7 .2 7 .8 7 .4 7 .5 7 .6 272 18 .5 17 .2 12.6 273 7281 3201 85 17 .9 7 .1 7 .3 7 .9 7 .4 7 .5 7 .5 275 20 .5 17 .5 13.1 245 6449 3077 149 16 .3 7 .1 7 .2 7 .9 7 .4 7 .5 7 .5 279 20 .0 17 .4 13.1 259 5745 2815 211 13 .8 7 .2 7 .3 7 .7 7 .2 7 .4 7 .5 282 17 .8 13.4 269 4723 2522 238 10 .5 7 .2 7 .4 7 .8 7 .3 7 .2 7 .4 286 20 .0 17 .1 13.4 268 6715 2747 129 15 .0 8 .1 7 .5 8 .2 7 .5 7 .6 7 .8 289 20 .0 17 .6 13.6 262 5398 2472 490 8 .2 7 .6 7 .0 7 .7 7 .4 7 .7 7 .8 293 17 .5 17 .0 14.1 251 4887 2489 348 9 .2 7 .5 7 .2 7 .7 7 .3 7 .6 7 .7 297 18 .5 17 .2 13.8 275 4060 2191 327 8 .8 7 .5 7 .2 7.8 7 .4 7 .4 7 .5 300 16 .0 11 .0 14.3 263 4510 2386 95 13 .6 7 .5 7 .2 7 .7 7 .5 7 .7 7 .9 303 20 .5 16 .3 12.8 247 3835 2088 249 9 .5 7 .3 6 .9 7 .6 7 .7 7 .7 7 .7 307 20 .5 16 .1 12.6 272 3860 2112 107 15 .1 7 .4 7 .0 7 .7 7 .7 7 .8 7 .8 310 20 .5 15 .0 14.7 267 3543 1980 206 10 .5 7 .5 7 .1 7 .6 7 .5 7 .7 7 .8 314 21 .0 15 .6 14.6 267 3158 1820 329 7 .8 7 .4 7 .1 7 .5 7 .4 7 .6 7 .7 317 21 .0 16 .5 13.8 249 3095 1837 100 13 .6 7 .4 7 .1 7 .6 7 .6 7 .7 7 .7 321 15 .0 15 .4 12.9 274 4363 2055 173 11 .6 8 .3 7 .5 8 .1 7 .4 7 .6 7 .8 324 21 .0 16 .6 13.6 279 6505 2434 69 17 .8 8 .6 7 .4 8 .2 7 .6 8 .0 8 .1 328 15 .0 15 .6 13.0 253 7408 2445 95 17 .2 8 .6 7 .6 8 .2 7 .6 7 .8 8 .0 331 21 .0 16 .9 13.9 246 8320 2465 112 17 .0 8 .7 7 .6 8 .2 7 .7 8 .1 8 .1 335 18 .5 16 .1 13.3 261 8260 2442 67 18 .9 8 .2 7 .4 8 .1 7 .4 7 .8 8 .0 338 19 .5 16 .3 13.3 269 9364 2888 79 19 .1 8 .4 7 .5 8 .4 8 .4 8 .4 8 .2 353 20 .5 17 .3 14.2 284 7 .0 6 .8 6 .8 6 .7 6 .8 6 .9 356 17 .0 15 .4 12.7 266 7 .2 7 .1 7 .2 7 .5 7 .5 7 .4 359 20 .5 16 .3 13.2 273 8 .2 7 .2 7 .2 7 .7 7 .5 7 .6 241 RUN 7: SUMMARY OF DATA (CONT'D) DAY 1 2 3 NH4-N 4 5 6 1 2 N03-N 3 4 5 6 1 2 N02-N 3 4 5 6 17 61 14 0 95 105 127 158 160 159 6 12 14 3 0 . 0 21 101 66 2 112 120 139 169 168 193 9 13 16 21 28 13 24 104 56 1 120 135 162 200 207 2 3 4 5 4 0 28 105 73 1 135 152 175 205 225 240 1 1 1 1 1 0 29 146 114 63 30 138 90 36 31 135 65 20 72 98 96 155 188 219 31 23 22 6 2 1 33 143 34 140 35 137 74 17 58 67 75 97 123 155 40 48 46 52 51 47 38 111 40 1 24 30 45 45 63 80 75 94 104 115 120 113 42 122 27 1 38 50 58 86 10 3 125 64 78 93 98 99 86 45 115 64 1 21 7 13 37 66 99 73 59 81 104 120 104 49 116 2 0 47 34 85 139 191 220 53 58 91 81 33 4 52 123 41 33 94 148 66 117 178 205 3 33 101 81 28 2 56 125 33 29 92 139 47 134 186 189 3 38 95 49 3 0 59 113 23 22 36 101 99 0 82 103 3 39 38 72 21 1 63 122 31 26 67 121 27 23 121 135 3 33 79 76 12 1 70 144 70 61 21 68 2 0 56 64 2 29 9 2 7 1 73 156 62 48 17 69 3 0 47 51 1 33 4 0 2 0 76 161 72 65 11 53 2 1 31 41 1 38 2 0 2 0 80 155 62 49 10 50 1 31 39 40 1 45 1 6 1 0 84 151 63 63 19 55 1 52 62 64 1 40 2 8 0 0 91 155 67 60 23 57 2 40 59 62 0 51 4 17 1 0 94 167 100 73 17 70 30 45 64 64 0 22 1 11 1 0 98 170 76 2 25 83 164 80 83 85 0 42 22 3 0 0 101 174 62 46 23 93 23 79 84 85 0 46 3 3 0 0 105 168 SO 34 30 99 33 83 88 88 0 53 2 2 0 0 108 166 38 26 22 108 35 71 73 74 0 61 7 1 0 0 112 154 11 2 21 97 20 59 63 66 0 67 2 1 0 0 116 151 11 0 1 65 3 5 15 12 0 77 11 5 0 0 119 157 12 2 4 70 1 3 12 15 0 77 3 3 0 0 122 149 79 56 1 24 0 13 15 12 1 35 0 4 1 0 126 143 20 8 9 54 1 19 23 25 0 87 0 1 0 0 129 141 24 23 1 39 0 0 12 13 0 83 0 0 1 0 133 161 40 19 20 59 0 41 50 56 1 76 1 42 23 7 136 140 15 2 9 53 1 31 33 31 0 83 24 3 0 0 140 148 21 12 4 44 0 14 15 16 0 93 0 1 0 0 143 142 11 2 2 36 0 8 10 11 0 102 1 1 0 0 146 147 15 9 4 35 0 15 21 20 0 102 6 7 1 0 150 144 15 3 0 25 0 1 5 5 0 101 0 3 0 0 153 159 39 29 4 28 0 10 13 14 0 95 0 3 0 0 157 112 61 87 1 15 0 9 15 18 1 53 0 3 5 1 160 141 23 15 8 28 0 21 24 23 0 100 11 6 ' 1 0 164 134 15 2 0 22 0 4 4 5 0 98 0 0 0 0 166 144 16 16 3 22 0 10 11 11 0 101 1 1 0 0 168 134 23 5 2 23 0 4 5 6 0 96 1 1 0 0 171 141 16 8 0 19 0 4 5 6 1 104 0 1 0 0 174 138 20 10 2 25 0 9 10 10 1 99 0 1 0 0 178 140 19 10 0 20 0 4 5 5 1 96 0 0 0 0 242 RUN 7: SUMMARY OF DATA (CONT'D) DAY 1 2 NH4-N 3 4 5 6 1 2 3 N03. 4 -N 5 6 1 2 3 N02-N 4 5 6 181 141 19 11 2 20 0 7 7 8 1 104 0 0 0 0 185 148 20 15 3 21 0 10 10 9 1 103 0 0 0 0 188 143 24 16 3 21 0 10 10 10 1 100 0 0 0 0 192 123 18 11 3 18 0 8 8 9 1 97 0 0 0 0 195 141 31 25 0 18 0 10 11 12 0 95 0 1 0 0 199 120 49 41 5 25 0 10 12 13 1 71 0 1 0 - 0 202 133 58 46 11 • 43 0 21 26 27 0 50 0 3 0 0 206 144 73 62 11 57 0 23 28 28 1 36 0 2 0 0 209 152 51 40 19 71 4 39 42 43 0 44 1 3 0 0 213 138 47 33 18 75 6 39 41 40 0 37 1 3 0 0 216 148 37 27 18 67 26 46 45 1 62 1 1 0 0 220 131 31 25 8 51 0 25 23 24 1 65 0 0 0 0 224 146 33 33 22 69 21 61 63 64 0 64 2 1 0 0 227 146 52 43 26 76 16 62 65 65 0 43 1 3 0 0 230 151 34 29 30 90 38 74 76 76 1 48 2 1 0 0 234 150 41 32 12 77 1 37 37 36 1 42 1 1 0 0 237 142 46 36 19 114 13 54 54 54 0 34 1 1 0 0 241 148 64 60 14 76 1 41 44 44 0 26 0 0 0 0 244 151 53 46 10 78 0 39 39 37 0 27 0 0 0 0 248 151 63 57 0 4 49 0 28 31 31 0 38 0 1 0 0 251 92 141 142 13 29 1 22 58 53 0 2 0 3 0 0 255 155 71 2 0 87 1 40 46 44 0 10 0 0 0 0 258 153 60 2 16 97 1 39 40 41 0 13 0 0 0 0 262 152 38 2 10 102 0 30 31 31 0 22 0 0 0 0 265 159 54 2 16 84 0 35 37 38 0 29 0 0 0 0 268 142 44 1 8 65 0 25 26 25 0 45 0 0 0 0 272 148 55 2 9 52 0 22 24 24 0 52 0 0 0 0 275 137 36 2 5 45 0 12 14 15 1 61 0 0 0 0 279 146 64 4 9 50 0 20 26 27 0 40 0 1 0 0 282 146 85 26 17 58 2 15 41 40 1 19 0 2 0 0 286 155 81 23 15 67 0 27 41 41 0 20 0 1 0 0 289 98 50 35 ' 66 101 0 30 31 31 5 8 0 0 0 0 293 94 61 47 62 100 1 36 39 38 2 4 0 0 0 0 297 129 95 56 84 2 33 61 62 0 2 1 1 0 0 300 78 38 16 54 101 2 30 27 25 1 4 1 0 0 0 303 56 4 0 93 142 10 26 26 28 6 6 9 0 0 0 307 66 12 2 101 141 9 48 50 51 4 7 26 0 0 0 310 64 10 2 85 128 6 40 40 41 8 14 26 0 0 0 314 69 13 3 88 127 1 58 59 59 8 15 39 0 0 0 317 47 2 2 125 144 8 66 68 70 3 15 53 0 0 0 321 143 72 12 28 92 6 55 63 60 1 7 3 0 0 0 324 143 47 2 23 91 0 44 47 50 1 24 0 0 0 0 328 131 55 2 19 71 1 41 49 46 1 12 1 1 0 0 331 129 46 2 10 76 0 35 33 35 1 19 0 0 0 0 335 135 65 2 23 78 1 51 52 50 0 16 0 2 0 0 338 125 19 2 20 85 1 7 18 32 1 24 0 0 0 0 353 118 79 71 11 14 9 58 60 79 71 82 87 108 114 97 356 117 100 67 29 45 73 0 18 33 16 28 36 71 57 45 359 138 36 0 26 75 99 24 30 38 1 16 25 30 14 2 243 APPENDIX B NITROGEN BALANCE General Comments Column 2: TKN IN represents the n i t r o g e n content of the feed ( i t d i d not c o n t a i n any o x i d i z e d n i t r o g e n s p e c i e s ) . Column 3: TN OUT represents a l l n i t r o g e n s p e c i e s measured i n the e f f l u e n t (both d i s s o l v e d and suspended). Column 4: TN DENITR represents the measured o x i d i z e d n i t r o g e n l o s s i n the anaeorobic c e l l ( s ) . Column 5: VSS represents the change i n VSS c o n c e n t r a t i o n of the biomass with time. Column 6: VSS WASTED represents the d a i l y amount of VSS wasted. Column 7: VSS NET represents the net change i n VSS content w i t h i n the system and i s equal t n the d i f f e r e n c e between Column 6 and Column 5. Column 8: % N CONENT OF VSS represents the measured n i t r o g e n content of the VSS. Column 9: TN WASTED represents the amount of n i t r o g e n l o s t with wasted VSS and i s e q u a l t o Column 7 times Column 8. Column 10: NITROGEN BALANCE represents the d i f f e r e n c e between the incoming n i t r o g e n (Column 2) and the i d e n t i f i a b l e n i t r o g e n removed from the system i n i t s d i f f e r e n t forms (Columns 3, 4 and 9). Column 11: % NITROGEN WASTED i s the same as Column 10, but ex-p r e s s e d i n terms of % l o s s or g a i n w i t h r e s p e c t t o the incoming n i t r o g e n . 244 NITROGEN BALANCE CALCULATIONS DAY TKN TN TN - VSS - VSS VSS N TN NITROGEN NITROGEN IN OUT DENITR VSS WASTED NET CONTENT WASTED BALANCE BALANCE mg/d mg/d mg/d mg/d mg/d mg/d % mg/d mg/d % RUN 2/SYSTEM 1 12 1240 1090 0 724 1463 -739 9 .3 69 81 6 .5 19 1255 1214 0 543 1031 -488 9 .8 48 -7 -0 .6 29 1192 1141 0 283 678 -395 10 .0 40 11 1 .0 34 1189 1058 0 70 464 -394 10 .1 40 91 7 .6 36 1179 1089 0 70 464 -394 10 .1 40 50 4 .2 40 1240 1020 140 221 581 -360 10 .0 36 44 3 .6 43 1315 992 169 221 581 -360 10 .0 36 118 9 .0 47 1058 782 203 143 284 -141 10 .0 14 59 5 .6 50 1079 698 260 143 284 -141 10 .0 14 107 9 .9 54 1140 613 468 300 523 -223 10 .8 24 35 3 .0 58 1114 552 501 -435 237 -672 10 .2 69 -8 -0 .8 69 1079 590 399 -435 237 -672 10 .2 69 21 2 .7 76 876 509 328 -403 420 -823 11 .0 91 -54 -6 .2 80 2811 1313 997 -336 893 -1229 10 .9 134 367 13 .0 82 2517 1194 997 -336 893 -1229 10 .9 134 192 7 .7 84 2500 1176 1128 -286 885 -1171 11 .2 131 65 2 .6 87 2500 1255 1299 -286 885 -1171 11 .2 131 185 7 .4 89 2467 1131 807 -286 885 -1171 11 .2 131 398 16 .0 92 2467 1199 711 -20 958 -1038 11 .1 115 442 18 .0 94 2418 1219 810 -80 958 -1038 11 .1 115 274 11 .4 96 2200 1175 824 -80 958 -1038 11 .1 115 86 3 .9 101 2477 1229 782 196 1165 -969 12 .0 116 350 14 .2 103 2530 1260 838 196 1165 -969 12 .0 116 316 12 .5 106 2704 1316 812 -194 1407 -1601 11 .8 189 387 14 .4 110 2529 1295 975 -194 1407 -1601 11 .8 189 70 2 .7 113 2480 1129 952 120 1234 -1114 11 .0 123 276 11 .1 115 2435 1168 936 120 1234 -1114 11 .0 123 208 8 .5 117 2535 1198 878 120 1234 -1114 11 .0 123 336 13 .3 124 2535 1157 1081 -83 1261 -1344 11 .4 153 144 5 .7 127 2413 1102 1075 17 1101 -1084 11 .7 127 109 4 .6 129 2362 1098 1095 17 1101 -1084 11 .7 127 42 1 .8 131 2540 1065 1008 17 1101 -1084 11 .7 127 '340 13 .3 134 2476 1131 1251 -276 1163 -1439 10 .2 147 -53 -2 .1 136 2423 1202 1262 -276 1163 -1439 10 .2 147 -168 -6 .9 137 2586 1189 1236 -276 1163 -1439 10 .2 147 14 0 .5 141 2424 1097 1313 116 1252 -1136 11 .2 127 -113 -4 .7 143 2476 1141 1198 116 1252 -1136 11 .2 127 10 0 .4 145 2461 1148 1258 116 1252 -1136 11 .2 127 -72 -2 .9 245 NITROGEN BALANCE CALCULATIONS (CONT'D) DAY TKN TN TN - VSS - VSS VSS N TN NITROGEN NITROGEN IN OUT DENITR VSS WASTED NET CONTENT WASTED BALANCE BALANCE mg/d mg/d mg/d mg/d mg/d mg/d % mg/d mg/d % RUN 2/SYSTEM 2 12 1240 1090 0 681 1451 770 9.3 72 78 6.3 19 1255 1184 0 656 1167 511 9.5 49 22 1.7 29 1192 1131 0 473 732 259 9.7 25 36 3.0 36 1179 1129 0 137 546 409 10.0 41 9 0.8 40 1240 1188 0 176 593 417 10.0 42 10 1.0 43 1315 1169 0 176 593 417 10.0 42 104 7.9 47 1058 1013 0 -23 554 577 10.0 58 -13 -1.2 54 1140 1054 0 174 762 588 10.0 59 27 2.4 58 1114 1020 0 -347 597 944 10.5 99 -5 -0.4 69 1079 980 0 -347 597 944 10.5 99 0 0.0 76 876 801 0 0 589 589 11.0 65 10 1.0 89 2467 975 1073 -459 800 1259 11.1 1*0 279 11.3 94 2510 1018 1159 -504 939 1443 11.6 167 164 6.5 96 2291 1018 1071 -504 939 1443 11.6 167 35 1.6 101 2352 889 1010 -227 988 1215 11.8 143 310 13.1 115 3564 1979 1094 454 2439 1985 21.1 215 276 14.8 125 3743 2158 987 566 2191 1625 21.8 186 412 17.4 139 3519 2200 938 383 1756 1373 22.0 162 219 10.9 149 3806 2073 994 187 1589 1402 22.0 160 579 22.5 155 3473 2195 1036 226 1636 1410 22.0 161 81 4.2 160 3614 2119 1057 226 1636 1410 22.0 161 277 15.4 171 3392 1976 1082 -554 1897 2451 21.3 270 64 2.1 181 3351 1991 1147 617 2004 1387 21.9 154 59 3.8 187 3265 2002 1190 96 1839 1743 22.4 194 -121 -5.8 200 3393 1923 1130 96 1839 1743 22.4 19 4 146 6.3 210 3142 1792 1183 190 1660 1470 21.2 155 12 1.1 225 4674 1990 2177 -269 1871 2140 21.3 230 277 11.2 231 4907 2098 2268 -314 2010 2324 21.8 257 28 3 11.4 235 4434 1951 2157 -314 2010 2324 21.8 257 69 3.2 141 2215 950 1130 -171 984 1155 11.3 131 4 0.2 143 2266 1009 1102 -171 984 1155 11.3 131 24 1.2 145 2252 1007 1132 -171 984 1155 11.3 131 -18 -0.8 246 NITROGEN BALANCE CALCULATIONS (CONT'D) DAY TKN TN TN - VSS - VSS VSS N TN NITROGEN NITROGEN IN OUT DENITR VSS WASTED NET CONTENT WASTED BALANCE BALANCE mg/d mg/d mg/d mg/d mg/d mg/d % mg/d mg/d % RUN 4/SYSTEM 1 18 1232 521 577 775 2247 1472 9.3 136 -2 -0.2 21 1195 541 599 -500 384 884 9.8 87 -32 -2.7 23 1260 573 583 295 553 258 10.2 26 78 6.2 25 1230 564 599 -1895 580 2475 10.3 255 -186 -15.2 28 1201 525 583 -270 446 716 10 .4 74 19 1.6 30 1214 542 598 170 1492 1322 10.6 140 -66 -5.5 32 1273 563 620 -605 735 1340 10.6 142 -52 -4.1 35 1424 474 810 -450 655 1105 10.7 118 22 1.5 37 1423 482 759 -475 677 1152 10.8 124 58 4.1 39 1447 512 1008 -120 731 851 10.8 92 -165 -11.4 42 1665 582 912 -807 655 1462 10.9 159 12 0.7 44 1669 536 881 -395 744 1139 11.0 125 127 -> ,6 46 1958 631 1077 -1485 963 2448 11.0 269 -19 -1.0 49 1916 674 1256 -805 698 1503 11.3 170 -184 -9.6 51 1820 633 1269 453 1384 931 11.6 108 -190 -10 .5 53 2088 749 1331 -795 1782 2577 11.9 307 -299 -14.3 56 2129 752 1332 3713 5278 1565 12.2 191 -146 -6.9 58 1732 507 1060 4215 5183 968 12.5 121 44 2.5 60 2009 630 1184 3640 5121 1481 12.4 184 11 0.5 63 1808 571 945 -820 1748 2568 12.2 313 -21 -1.2 65 1891 748 322 -965 1162 2127 12.1 257 564 29.9 67 1965 590 1101 -665 1162 1827 12.0 219 55 2.7 77 2097 985 696 53 981 928 11.9 110 306 14.6 79 1789 832 715 -755 1219 1974 11.7 231 11 0.6 84 1930 552 1128 37 1444 1407 12.3 173 77 4.0 88 1744 514 1079 -23 1439 1462 12.6 184 -33 -2.0 91 1911 595 1175 527 1292 765 12.7 97 44 2.3 95 2007 621 1149 -123 1293 1416 12.8 181 56 2.7 98 2029 641 1183 -210 1394 1604 12.9 207 -2 -0.1 247 NITROGEN BALANCE CALCULATIONS (CONT'D) DAY TKN TN TN - VSS - VSS VSS N TN NITROGEN NITROGEN IN OUT DENITR VSS WASTED NET CONTENT WASTED BALANCE BALANCE mg/d mg/d mg/d mg/d mg/d mg/d % mg/d mg/d % RUN 4/SYSTEM 2 18 1257 566 599 -385 585 970 9.3 90 2 0.2 21 1228 561 574 -287 412 699 9.6 67 26 2.1 23 1283 583 579 120 499 379 10.0 38 83 6.4 25 1257 577 592 -870 653 1523 10.0 152 -64 -5.1 28 1224 541 594 -1013 454 1467 10.1 148 -59 -4.8 30 1228 577 609 555 901 346 10.2 35 7 0.5 32 1283 589 621 -680 840 1520 10.3 157 -84 -6.6 35 1339 618 558 30 774 744 10.4 77 86 6.4 37 1312 598 575 35 734 699 10.5 73 66 5.0 39 1336 628 663 -85 811 896 10.6 95 -50 -3.8 42 1517 759 507 140 727 587 10.7 63 188 12.4 44 1526 953 393 735 741 6 10.8 1 179 11.7 46 1777 1112 590 170 827 657 11.0 72 3 0.2 49 1875 883 881 -963 606 1569 11.4 179 -68 -3.7 51 1780 801 777 470 1148 678 11.6 79 123 6.9 53 2046 945 886 45 1476 1431 11.8 169 46 2.3 56 2102 1009 891 2200 4193 1993 11.9 237 -35 -1.7 58 1708 817 374 3115 4349 1234 12.0 148 369 21.5 60 1985 1195 183 2295 3695 1400 11.8 165 438 22.1 63 1788 1257 583 -977 349 1326 11.5 152 -204 -11.4 65 1870 957 873 -105 683 788 11.3 89 -49 -2.6 67 1943 943 906 -105 683 788 11.4 90 4 0.3 77 2083 1235 783 -177 1137 1314 12.0 158 -93 -4.5 79 1798 732 733 -540 1308 1848 12.2 225 108 6.0 84 1952 825 647 -620 1744 2364 12.3 291 189 9.6 88 1779 716 762 -50 1470 1520 12.4 188 113 6.3 91 1953 768 709 63 1506 1443 12.6 182 294 15.0 95 2064 857 823 -568 1589 2157 12.8 276 108 5.2 98 2090 908 875 -330 1641 1971 12.8 252 55 2.6 KJN 5/SYSTEM 1 10 1982 1312 391 741 1990 1249 9.7 121 158 7.9 17 1828 1154 699 474 1134 660 9.9 65 -90 -4.9 24 1980 585 1094 -319 1201 1520 11.8 179 122 6.1 31 2111 500 1252 -431 1497 1928 11.9 229 130 6.2 39 2217 608 1512 -148 1475 1623 10 .9 177 -80 -3.6 IUN 5/SYSTEM 2 10 1889 851 874 587 1723 1136 10.2 116 48 2.5 17 1746 1057 793 596 1494 898 9.8 88 -192 -11 24 1908 651 1003 -176 1224 1400 11.5 161 93 4.9 31 2136 555 1251 -313 1728 2041 12.1 247 83 3.8 39 2186 616 1360 -33 1659 1683 10 .9 183 27 1.1 248 APPENDIX C AMMONIA VOLATILIZATION TEST A p r e l i m i n a r y study of ammonia v o l a t i l i z a t i o n r a t e s , using one of the r e a c t o r s , was undertaken p r i o r to commencement of the experimental program. The reactor was f i l l e d with mixed l i q u o r obtained from the U.B.C. p i l o t wastewater treatment p l a n t which was operating i n a mode to b i o l g i c a l l y remove n i t r o g e n and phosphorus. A predetermined amount of ammonium c h l o r i d e was added to the r e a c t o r immediately p r i o r to s t a r t - u p . The pH was adjusted p r i o r to commencement of the t e s t and measured at the end of the experiment. The d u r a t i o n of each t e s t v a r i e d , from a minimum of 28 minutes to a maximum of 5 hours. The r e a c t o r was s e a l e d at s t a r t - u p and the c o l l e c t e d gas was d i r e c t e d through a l i n e to two b o r i c a c i d baths, connected i n s e r i e s , to t r a p a l l of the v o l a t i -l i z e d ammonia. The contents of both baths were t i t r a t e d at the end of the experiment to determine the amount of ammonia c o l -l e c t e d . Upon s t a r t - u p of each t e s t , a i r was bubbled at a pre-determined r a t e through a flowmeter to the r e a c t o r . A l l t e s t s were conducted at 20°C. R e s u l t s of the t e s t are presented i n Chapter 6. 249 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0062908/manifest

Comment

Related Items