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The effects of temperature, pH and retention time on volatile fatty acids production from primary… Gupta, Ashok Kumar 1986

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THE EFFECTS OF TEMPERATURE, pH AND RETENTION TIME ON VOLATILE FATTY ACIDS PRODUCTION FROM PRIMARY SLUDGE by ASHOK KUMAR GUPTA B . S c . ( C i v i l Engg.) 1980, M . S c . ( C i v i l Engg.) 1982, U n i v e r s i t y of D e l h i , D e l h i , I n d i a A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF v MASTER OF APPLIED SCIENCE i n FACULTY OF GRADUATE STUDIES DEPARTMENT OF CIVIL ENGINEERING We accept t h i s t h e s i s as conforming t o the S q u i r e d standard UNIVERSITY OF BRITISH COLUMBIA January,1986 © ASHOK KUMAR GUPTA, January,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 C i V M U gM5fr. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date JaMx^a/t-A-j ' DE-6(3/81) A B S T R A C T A t y p i c a l b i o l o g i c a l phospho rus remova l p r o c e s s c o n s i s t s o f a l ternate anaerob ic and ae rob i c z o n e s . Recent research at the U n i v e r s i t y o f B r i t i sh C o l u m b i a (UBC) has ind ica ted that the add i t i on of vo la t i l e fa t ty ac ids ( V F A ' s ) to the anaerob ic zone of B i o - P p r o c e s s e s i m p r o v e s the ove ra l l phosphorus r e m o v a l e f f i c i e n c y , at least when t rea t ing weak s e w a g e . A t the f u l l - s c a l e b i o l o g i c a l phospho rus r e m o v a l plant at K e l o w n a , B.C., and at a p i l o t - s c a l e f a c i l i t y at U B C , anaerob i c f e rmen ta t i on of p r imary s ludge has been used to p roduce V F A ' s to add to the anaerob i c zone of these p r o c e s s e s . The p r imary ob jec t i ve of th is research w a s to i m p r o v e the k n o w l e d g e o f the ac id phase of anaerob ic d i ges t i on (the p roduc t i on o f V F A ' s ) to help ach ieve o p t i m u m des ign and ope ra t i on o f the fe rmen te r . The e f f e c t s o f t empera tu re , re ten t ion t ime and pH on the p roduc t i on o f s h o r t - c h a i n V F A ' s f r o m p r imary s e w a g e s ludge we re s tud ied at l a b - s c a l e . Ox ida t i on reduc t ion po ten t ia l (ORP) w a s a l s o m o n i t o r e d throughout the s tudy to exp lo re the re la t i onsh ip be tween ORP and the p roduc t i on of V F A ' s . A c o m p l e t e l y random 3 X 3 X 2 fac to r i a l exper imen ta l des ign w a s used to de te rm ine the expe r imen ta l s e q u e n c e . Three l i t re anaerob ic reac to rs w e r e run on a f i l l and d raw b a s i s in a t e m p e r a t u r e - c o n t r o l l e d r o o m . Reac to rs w e r e f e d once a day w i t h p r imary s ludge brought f r o m the U B C p i lo t p lant . ii iii Results showed that the net VFA production consistently improved with increase in temperature in the range of 10°C to 30°C. At low temperatures (10°C and 20°C) the net VFA production improved with the increase in retention t ime; however extension of the retention time to 9 days at 30°C appeared detrimental to VFA production. The effect of pH control , at a value of 7.0, was not consistent with both retention time and temperature and had a confounding effect. At 10°C and 20°C temperatures, a relationship was observed between ORP and VFA production. No definite relationship between the net VFA production and ORP was found at 30° C. Table of Contents ABSTRACT ii LIST OF TABLES vi LIST OF FIGURES vii ACKNOWLEDGEMENT .„' ix 1.0 INTRODUCTION 1 1.1. E X C E S S BIOLOGICAL PHOSPHORUS REMOVAL 1 1.2. ANAEROBIC DIGESTION 4 1.3. OPTIMIZATION OF ACID FORMERS 5 1.4. METHANOGEN INHIBITION .7 2.0 RESEARCH OBJECTIVES 9 3.0 DESIGN OF EXPERIMENTS 12 3.1. EXPERIMENTAL DESIGN 12 3.2. SEQUENCE OF EXPERIMENTS 13 4.0 EXPERIMENTAL FACILITIES 16 4.1. EXPERIMENTAL S E T - U P 16 4.2. FEED USED IN THE STUDY 16 4.2.1 COLLECTION AND STORAGE OF THE FEED 16 4.2.2 MAINTENANCE OF THE COD OF THE FEED 18 4.3. EXPERIMENTAL PROCEDURE 18 4.3.1 FILL AND DRAW REACTORS: 18 4.4. SAMPLING AND A N A L Y S I S 20 4.4.1 SAMPLING ! 20 4.4.2 ANALYT ICAL TECHNIQUES USED 20 4.4.2.1 VOLATILE FATTY ACIDS 21 iv V 4.4.2.2 T O T A L ORGANIC CARBON 21 5.0 RESULTS AND DISCUSSION 22 5.1. RESULTS 22 5.2. S T A T I S T I C A L A N A L Y S I S 23 5.2.1 STANDARDIZED ERRORS 23 5.2.2 A N A L Y S I S OF VARIANCE (ANOVA) 27 5.2.3 DUNCAN'S MULTIPLE RANGE T E S T 33 5.3. DISCUSSION 33 5.3.1 T O T A L VFA PRODUCTION .„ 37 5.3.2 A C E T I C ACID PRODUCTION 40 5.3.3 PROPIONIC ACID PRODUCTION 43 5.3.4 EFFECT OF pH CONTROL 46 5.3.5 EFFECT OF TEMPERATURE 49 5.3.6 EFFECT OF RETENTION TIME 50 5.3.7 BEST COMBINATION OF T R E A T M E N T VARIABLES 50 5.3.8 MICROBIOLOGICAL E X P L A N A T I O N FOR THE BEHAVIOUR OF REACTOR A T 30 °C 51 5.3.9 RELATIONSHIP BETWEEN SOLUBLE TOC AND V F A PRODUCTION 55 5.3.10 DAILY PRODUCTION OF V F A 58 5.3.11 OXIDATION REDUCTION POTENTIAL(ORP) 61 6.0 CONCLUSIONS AND RECOMMENDATIONS 73 6.1. CONCLUSIONS 73 6.2. RECOMMENDATIONS .....74 REFERENCES 76 APPENDIX I 80 List of Tables Table Page Table 3.1 Layout of Sequence of Experiments 14 Table 4.1 Typical Characteristics of the influent primary sludge 19 Table 5.1 Summary of Results for 10°C Temperature Run 24 Table 5.2 Summary of Results for 20°C Temperature Run 25 Table 5.3 Summary of Results for 30°C Temperature Run 26 Table 5.4 Standardized Errors 28 Table 5.5 Analys is of Variance for Net Acet ic Ac id Production 29 Table 5.6 Analys is of Variance for Net Propionic Ac id Production 30 Table 5.7 Analys is of Variance for Total Net Volati le Fatty Ac id Production .31 Table 5.8 Ranking of Means of Net Acet ic Ac id Production Under Different Operating Condit ions using Duncan's Multiple Range Test 34 Table 5.9 Ranking of Means of Net Propionic Ac id Production Under Different Operating Condit ions using Duncan's Mult iple Range Test 35 Table 5.10 Ranking of Means of Total Net VFA Production Under Different Operating Condit ions using Duncan's Multiple Range Test 36 vi List of Figures Figure Page Fig. 2.1 Causes and Effects 11 Fig. 4.1 Sketch of Anaerobic Reactor (Adapted from Comeau, 1984) 17 Fig. 5.1 Total Net VFA Concentration vs. Retention Time at Control led pH 38 Fig. 5.2 Total Net VFA Concentration vs. Retention Time at Uncontrol led pH .39 Fig. 5.3 Net Acet ic Ac id Concentration vs . Retention Time at Control led pH ..41 Fig. 5.4 Net Acet ic Ac id Concentration vs. Retention Time at Uncontrol led pH 42 Fig. 5.5 Net Propionic Ac id Concentration vs . Retention Time at Control led pH 44 Fig. 5.6 Net Propionic Ac id Concetration vs . Retention Time at Uncontrol led pH 45 Fig. 5.7 Total Net VFA concentration vs . Soluble Total Organic Carbon 56 Fig. 5.8 Net Daily Production of Total VFA vs. Retention Time at Control led pH 59 Fig. 5.9 Net Daily Production of Total VFA vs. Retention Time at Uncontrol led pH 60 Fig. 5.10 Relationship Between Mean ORP vs. Net Acet ic Ac id Production 62 Fig. 5.11 Relationship Between Mean ORP vs. Net Propionic Ac i d Production ..63 vii vi i i Fig. 5.12 Relationship Between Mean ORP vs. Total Net VFA Production 64 Fig. 5.13 Relationship Between ORP and Net VFA production for 10°C and Control led pH Operation 66 Fig. 5.14 Relationship Between ORP and Net VFA production for 10°C and Uncontrolled pH Operation 67 Fig. 5.15 Relationship Between ORP and Net VFA production for 20°C and Control led pH Operation 68 Fig. 5.16 Relationship Between ORP and Net VFA production for 20°C and Uncontrolled pH Operation 69 Fig. 5.17 Relationship Between ORP and Net VFA production for 30°C and Control led pH Operation 70 Fig. 5.18 Relationship Between ORP and Net VFA production for 30°C and Uncontrolled pH Operation 71 ACKNOWLEDGEMENT I am s i n c e r e l y thankfu l to my s u p e r v i s o r , Dr. W . K. O l d h a m , fo r his gu idance and encouragement dur ing th is s tudy . I a l so a c k n o w l e d g e deep l y the mora l suppor t and a s s i s t a n c e r e c e i v e d f r o m the f e l l o w graduate s tudent , P. F. C o l e m a n , at the va r i ous s tages of th is r esea rch . I a l so thank Sue J a s p e r , S u s a n L ip tak , Pau la P a r k i n s o n and T i m o t h y M a for their help in the lab wo rk and Barry Rab inow i t z fo r br ing ing p r imary s ludge f r o m the p i lo t plant e v e r y w e e k . Thanks are a l so due to Fred K o c h , Y v e s C o m e a u and T. V a s s o s fo r their pa r t i c i pa t i on in the d i s c u s s i o n s and to Dr. D. S . M a v i n i c fo r r e v i e w i n g the thes is in a ve ry shor t t i m e . ix CHAPTER 1 INTRODUCTION Phospho rus r e m o v a l has r e c e i v e d cons ide rab le a t ten t ion in the recent pas t . Th is is ma in l y because phosphorus r e m o v a l f r o m w a s t e w a t e r is an essen t i a l c o m p o n e n t o f t reatment where the rece i v i ng wa te r b o d i e s are in danger o f eu t roph ica t ion due to phospho rus enr i chment . M a n y ex i s t i ng w a s t e w a t e r t rea tment f a c i l i t i e s have been upgraded in recent y e a r s to inc lude phosphorus r e m o v a l . A number o f c h e m i c a l p rec ip i t a t i on techn iques are p resen t l y in use in the va r i ous t reatment p lants around the w o r l d . O f la te , h o w e v e r , cons ide rab le in terest ' is be ing s h o w n in the e x c e s s b i o l o g i c a l phospho rus r e m o v a l p r o c e s s . Th is b i o l o g i c a l p r o c e s s is a ve ry p r o m i s i n g t e c h n o l o g y and has , in a ve ry short t i m e , c o m e to be f a v o u r a b l y c o m p a r e d w i th the c h e m i c a l p rec i p i t a t i on , o w i n g to the lat ter 's e x p e n s i v e nature and the p r o b l e m of inorgan ic res idues (which o f t e n p o s e d i f f i cu l t d i s p o s a l p r o b l e m s ) . 1.1. EXCESS BIOLOGICAL PHOSPHORUS REMOVAL A c c o r d i n g to Lev in and S h a p i r o (1965), it w a s S a s t r y et a l . (1959 ) in India, w h o f i rs t repor ted phospho rus r e m o v a l in e x c e s s of m e t a b o l i c requ i rements in the w a s t e ac t i va ted s ludge p lant . T h e s e i nves t i ga to r s c o n c l u d e d that, in the plant where they o b s e r v e d e x c e s s phospho rus r e m o v a l , the magn i tude o f the r e m o v a l appeared to be l inked to the in tens i t y o f ae ra t ion . Lev in and S h a p i r o (1965) we re the f i rs t to h y p o t h e s i z e a b i o l o g i c a l m e c h a n i s m fo r P uptake. H o w e v e r , it w a s Barnard 1 2 (1974,1976) who first noted that the common feature of all processes exhibiting excess P removal was that at some point in the process, the mixed liquor had been subjected to an anaerobic condit ion of such an intensity that P release from the sludge back to the supernatant occured. He stated that anaerobic P release is a prerequisite for the excess biological P removal. A typical B i o - P removal process consists of alternate anaerobic and aerobic zones. In the absence of free or inorganic forms of oxygen, simple carbon substrates such as volati le fatty acids (VFA's) are stored in certain bacterial cel ls as poly-j3-hydroxybutyrate (PHB) and simultaneously, phosphorus is released. PHB has been reported as a stored carbon compound by Nicholls and Osborn (1979), Deinema et al . (1980), Fukase et a l . (1982) and Comeau (1984). Fukase et al . (1982) and Arv in (1985) observed that there is some form of a relationship between the amount of volati le fatty acids added and the amount of phosphorus released as phosphate into solution under anaerobic condit ions. When the anaerobic zone is fo l lowed by an aerobic zone, some bacteria take phosphate from solution and store it in polyphosphate pools. The amount of phosphorus uptake in the aerobic zone can be correlated with the amount of phosphorus released under anaerobic condit ions, which in turn can be correlated with the amount of VFA available in the anaerobic zone (Wentzell , 1984). Hence, the biological phosphorus removal capacity of a plant can be improved by the addition of VFA in the anaerobic zone. In North Amer ica , COD values of the wastewaters are generally quite low (in the 200-300mg/l range). Siebritz et al . (1982) found that the readily biodegradable COD comprises about 20% of the total COD of the 3 South Afr ican wastewaters. If same is true for North American wastewaters, then the readily biodegradable GOD concentration in North American wastewaters would be in the range of 40-60mg/ l . A l lowing for the dilution effect of a recycle rate of 1:1 with respect to the influent f low, the readily biodegradable COD concentration available to the organisms in the anaerobic zone is reduced to 20-30mg/ l , the minimum required to ensure anaerobic P release, assuming that no nitrate enters the zone (Rabinowitz and Oldham, 1985). Therefore, to ensure that the speci f ic substrates required for the excess biological P removal mechanism is available to the bacteria in the anaerobic zone, the addition of VFA 's is almost essent ia l . VFA concentration in the influent can be increased either by the addition of synthetic VFA or by primary sludge fermentation. Rensink et a l . (1984) used acetic acid addition to improve phosphorus removal from 45% to 97%. Oldham (1984) and Rabinowitz (1985) report that the fermentation of primary sludge can be used succesful ly to produce volati le fatty acids. At the fu l l -sca le biological phosphorus removal plant in Kelowna, B.C., and at a p i lo t -sca le faci l i ty at the University of British Columbia, primary sludge was purposefully fermented and the acid rich fermentor liquor was used as a VFA source in the anaerobic zone of the bioreactor. To optimize the use of anaerobic digestion for VFA 's rather than methane production, improved knowledge of the acid phase of digestion and the impact of digestion products on the process is required. Unfortunately, much of the research on anaerobic digestion is focussed on maintaining a viable population of methane formers. However, little is known about optimizing acid production, while suppresing methane 4 p r o d u c t i o n , to max im ize the ava i l ab i l i t y o f V F A ' s . 1.2. ANAEROBIC DIGESTION A n a e r o b i c d i g e s t i o n is a b i o l o g i c a l p r o c e s s in wh i ch o rgan ic mat ter is conve r t ed to methane and ca rbon d iox ide in the absence o f mo lecu la r oxygen. l t can be c o n s i d e r e d as a three s tage p r o c e s s requi r ing at least three m e t a b o l i c g roups o f b a c t e r i a : 1. Fe rmen ta t i ve bac te r ia w h i c h h y d r o l y z e c o m p l e x o rgan ic p o l y m e r s to p roduce s imp le r shor t cha in fa t ty a c i d s ; 2. H y d r o g e n p roduc ing ace togen i c bac te r ia wh i ch conver t these vo la t i l e ac ids to ace ta te , ca rbon d iox ide and h y d r o g e n ; 3. M e t h a n o g e n i c bac te r ia w h i c h ca tabo l i ze the ace ta te , ca rbon d iox ide and hyd rogen to p roduce methane and ca rbon d iox ide ( W o o d s et a l . , 1980). The n o n - m e t h a n o g e n i c phase of anaerob ic d i g e s t i o n is c o m m o n l y c a l l e d the a c i d - f o r m i n g phase . In i t ia ly , the i nso lub le po r t i on of the c o m p l e x o rgan ic subs t ra te is h y d r o l y z e d by ex t r a - ce l l u l a r e n z y m e s to s i m p l e so l ub le c o m p o u n d s . C e l l u l o s e and s tarch are h y d r o l y z e d to s i m p l e sugars wh i l e p ro te ins are h y d r o l y z e d to am ino a c i d s . Fa t ty ac i ds are the on l y c o m p o u n d s that are not a t tacked by these e x t r a - c e l l u l a r e n z y m e s . The f e rmen ta t i on o f c a r b o h y d r a t e s , a m i n o ac ids and long cha in fa t t y ac i ds leads to the f o r m a t i o n o f short cha in vo la t i l e fa t t y ac i ds ( V F A ' s ) , other neutral c o m p o u n d s , h y d r o g e n and ca rbon d iox ide (Chynowe th and M a h , 1971). The m o s t c o m m o n s h o r t - c h a i n , v o l a t i l e fa t t y ac i ds p roduced are ace t i c ac id and p r o p i o n i c a c i d . Other impor tant ac i ds p roduced 5 are formic acid, butyric ac id, voleric acid and isovaleric acid. Hydrogen, although produced in this phase, is barely detectable because it is rapidly oxidized by all known methanogenic bacteria during the reduction of carbon dioxide to methane (Mah et al., 1977). Short chain volat i le fatty acids produced in the acid forming phase becomes substrate for a group of strictly anaerobic methanogenic bacteria. These bacteria ferment VFA's to methane and carbon dioxide. Acet ic acid is a very important intermediary. Approximately 70% of the methane in nature is produced via the methyl group of acetic acid (McCarty, 1964). Propionic acid is also a major intermediary. Although many organisms are required in anaerobic digest ion, the groups of bacteria catabolizing acetic acid and propionic acid are the most important in methane fermentation. 1.3. OPTIMIZATION OF ACID FORMERS Little is known about the optimization of the acid producing bacteria in conjunction with suppression of the methane formers. Most of the important information on the acid phase has been obtained from studies using soluble substrates such as glucose (Andrews and Pearson ,1965; Ghosh and Pohland, 1974; Cohen et al., 1984; Zoetemeyer et al., 1979,1982). Hence, the usefulness of the results from these studies, to optimize the design and operation of the fermenter using primary sludge as a substrate, is questionable. There are few studies available where primary sludge has been used as a substrate to study the the acid phase. Important among them are the studies done by Eastman and Ferguson (1981), O'Rouke (1968), Chynoweth and Mah (1971) and Borchardt (1971). Important 6 o b s e r v a t i o n s made in the s tud ies us ing so lub le subs t ra te , as w e l l as in the s tud ies us ing p r imary s ludge as the subs t ra te , are d i s c u s s e d b e l o w . A n d r e w s and P e a r s o n (1965) and G h o s h and Poh land (1974), us ing so lub le subs t ra te , have f o u n d that the f e r m e n t a t i o n s tep is e x c e e d i n g l y rap id w i t h a m inumum ce l l r es i dence t ime of o n l y a f e w hours be ing requ i red . A n d r e w s and P e a r s o n (1965) a l so c o n c l u d e d that the t ype of vo la t i l e ac id p roduced f r o m a g i ven subs t ra te may be m a r k e d l y i n f l uenced by va r ia t i on of the o r g a n i s m res idence t i m e . Z o e t e m e y e r (1982) found that the re la t i ve p roduc t i on of ind iv idua l fa t ty ac i ds depends on the d i lu t ion rate and more s t r o n g l y on the cul ture pH va lue . E a s t m a n and Fe rguson (1981) repor t that the d i s t r i bu t ion o f the vo la t i l e ac i ds is a f f e c t e d by opera t ing c o n d i t i o n s , e s p e c i a l l y p H . C a r b o h y d r a t e s and n i t rogenous mate r ia l s are deg raded in the ac id phase (Eas tman and F e r g u s o n , 1981). L i p i ds require a longer de ten t ion t ime and hence inh ib i t ion o f l ip id deg rada t i on occu rs at shor t de ten t ion t i m e s (Chynowe th and M a h , 1971). B i s s e l e et a l . (1975), quo ted by Ur ibe la r rea arid Pare i l l eux (1981), repor ted the ex i s tence of 22 n o n - m e t h a n o g e n i c o r g a n i s m s able to cove r t d i f f e ren t ca rbon s o u r c e s ( ce l l u l ose , p r o t e i n s , ca rbohyd ra tes etc.) into vo la t i l e fa t t y a c i d s . A c c o r d i n g to Borchard t (1971), the o p t i m u m pH fo r ac id p roduc t i on f r o m raw s ludge d i g e s t i o n is around 7. In the s a m e s t u d y , the o p t i m u m o x i d a t i o n - r e d u c t i o n po ten t ia l fo r a c i d p roduc t i on w a s repo r ted as - 5 1 0 mV (ca lome l ref.). 7 McCarty (1963) and Andrews and Pearson (1965) found that the degradation of butyric acid by the methanogenic bacteria occurs at a faster rate than acetic acid and much faster than propionic ac id ; this results in a very low butyric concentration and often no detection is possib le. 1.4. METHANOGEN INHIBITION Methanogenic bacteria are extremely sensit ive to environmental condit ions. The optimal pH range for methanogens is between 7.0 and 7.2. McCarty (1964) reports that the gas production is quite sat isfactory as long as the pH is maintained between between 6.6 and 7.6. When pH drops below 6.6, there is signifiant inhibition of the methanogenic bacteria. At a pH of about 6.2, the acid condit ion exhibits acute toxici ty to these bacteria. It is interesting to note that this pH does not stop acid production. The fermentative bacteria wi l l continue to produce acids until the pH drops to 4.5 or 5.0 (Pfeffer, 1974). As methanogens are strict anaerobes, even a small amount of molecular oxygen can be inhibitory to these bacteria. Hence they require a highly reduced environment for their growth. Dirisian et a l . (1963) found that optimum redox potential for methane formers is between -520 mV and -530 mV (calomel ref. electrode, (E^-.)). Converse et al . (1971) using minimum aeration, studied the effect of ORP on the treatment of swine wastes. When operating with an of -435 mV, some methane production occured, but when the E^ reached -360 mV, methane production was completely inhibited. One system was completely anaerobic,(no aeration), with an E^ of -500 mV; the o f f -gas from this system was 51.1% methane by volume. 8 A m m o n i a , par t i cu la r l y when in N H 3 f o r m , is i nh ib i to ry at high concen t ra t i on s . A t concen t ra t i on s be tween 1500 and 3000 mg/ l , of to ta l a m m o n i a n i t rogen and a pH greater than 7.4, N H 3 can b e c o m e inh ib i to ry . A t concen t ra t i on s above 3000 mg/l the a m m o n i u m ion i t se l f b e c o m e s tox i c regard les s o f pH (McCar ty , 1961). Methanogens are ve ry s l o w g row ing o r gan i sms . A l t hough the m i n i m u m ce l l re s idence t ime repor ted f o r methane p roduc ing bacter ia is as l o w as 2.5 to 4 days (And rews and Pea r son , 1965), a c t i ve methane f e rmen ta t i o n requires much longer ce l l ~ re s idence t ime . Ea s tman and Ferguson (1981) report that, in a batch test us ing p r imary s ludge at 3 5 ° C and pH=6 , ve ry l i t t le gas p roduct i on took p lace by the eighth day of ope ra t i on and act i ve methanogenes i s s ta r ted on l y on the twent i e th day of ope ra t i on . CHAPTER 2 RESEARCH OBJECTIVES A s d i s cu s sed in the p rev ious chapter, imp roved k n o w l e d g e of the ac id phase of anaerob ic d i ge s t i on is requi red to ach ieve o p t i m u m des ign and ope ra t i on of the fe rmenter . Un fo r tuna te l y , much of the i n f o rma t i on ava i l ab le on anaerob ic d i ge s t i on is re lated to the o p t i m i z a t i o n o f the methane f o r m i n g bac te r i a . L i t t le is k nown about the o p t i m i z a t i o n of the ' ac id f o r m e r s in con junc t i on w i t h suppre s i on of the methane f o r m e r s . The main ob jec t i ve o f this s tudy w a s to exp lo re the re la t ionsh ip be tween net v o l a t i l e f a t t y ac id p roduc t i on f r o m p r imary s ludge and fe rmente r temperature , re tent ion t ime and pH. The e f f e c t o f pH w a s s tud ied on a con t r o l (pH=7) vs . no con t r o l s i t ua t i on . The e f f e c t o f the t reatment va r i ab le s (i.e. pH, temperature and retent ion t i m e ) w a s s tud ied not on ly w i t h re spect to net to ta l v o l a t i l e f a t t y ac id p r oduc t i on , but a l so the net p roduc t i on of the ind iv idua l ac id s (e.g. acet i c a c i d , p r op i on i c a c i d , buty r i c ac id). It w a s a l so dec i ded to mon i t o r the ox ida t i on reduct ion po tent i a l (ORP) throughout the s tudy, in order to exp lo re the p o s s i b i l i t y of a re la t i onsh ip be tween vo l a t i l e f a t t y ac id (VFA ) p roduc t i on and ORP. If there ex i s t s a re l a t i on sh ip , it w o u l d be use fu l to dete rmine whether ORP can be used as a con t ro l parameter to mon i t o r V F A p roduc t i on . 9 10 The bas ic purpose w a s to ach ieve an understand ing of the ac id phase of anaerob ic d i ge s t i on to max im i ze the V F A p roduc t i on in the fe rmenter . Th i s w o u l d be he lpfu l in imp rov i n g the b i o l o g i c a l e f f i c i e n c y o f a s ewage t reatment plant be ing des i gned to r emove phosphorus f r o m a w a s t e w a t e r , w i t h a l o w concen t ra t i on of s o l ub l e , s imp le subst rate. S ludge retent ion t ime (SRT) and hydrau l ic re tent ion t ime (HRT) are t w o d i f f e ren t parameter s . The f o rme r governs the t ype o f o rgan i sms wh i l e the HRT governs amount of f o o d used by the o r gan i sms . Because of the exp lo ra to r y nature of the s tudy and to s i m p l i f y i t , it w a s dec ided to have a c o m p l e t e l y m ixed s y s t e m hav ing the same SRT and HRT. To a vo i d c o n f u s i o n , the te rm re tent ion t ime is used ins tead of SRT and HRT. Be f o r e des i gn ing these expe r iment s , it w a s ve ry important to k n o w the causes (or t reatment va r i ab le s ) and the e f f e c t s (or pe r f o rmance var iab les ) . F igure 2.1 s h o w s the causes and e f f e c t s in the present s tudy. C A U S E S E F F E C T S RETENTION TIME NET ACETIC ACID PRODUCTION TEMPERATURE SOME MECHANISM > NET PROPIONIC ACID PRODUCTION WITH/WITHOUT pH CONTROL NET TOTAL VFA PRODUCTION Figure 2.1 : CAUSES AND E F F E C T S . CHAPTER 3 DESIGN OF EXPERIMENTS To k n o w whether there is any in te rac t ion be tween the t reatment va r i ab le s , it w a s dec ided to des ign the exper iment s on a s t a t i s t i c a l bas i s . 3.1. EXPERIMENTAL DESIGN A c o m p l e t e l y r andomi zed 3 X 3 X 2 f a c t o r i a l exper imenta l de s i gn w a s used. S i nce the des ign w a s c o m p l e t e l y r andom, t reatment c o m b i n a t i o n s we re a l l o ca ted r andomly throughout the exper imenta l de s i gn , pr ior to the execut ion of the exper iment s . Howeve r , the c o m p l e t e r andom i za t i on w a s re s t r i c ted b y , equ ipment l im i t a t i on s ( ava i l ab i l i t y o f pH con t ro l l e r and a temperature c o n t r o l l e d room) . In a l l , e ighteen c o m b i n a t i o n s o f pH, temperature and retent ion t i m e were s tud ied . Three cond i t i on s o f each of temperature and retent ion t ime and 2 cond i t i on s o f pH we re s tud ied . The deta i l s are g i ven b e l o w : RETENTION TIME: The e f f e c t o f re tent ion t ime on the p roduc t i on of vo l a t i l e f a t t y ac ids w a s s tud ied f o r 3, 6 and 9 days re tent i on t i m e s . Higher re tent ion pe r i od s we re not s tud ied because of the p o s s i b i l i t y o f s i gn i f i can t methane f o r m a t i o n . TEMPERATURE: V o l a t i l e f a t t y ac id s p roduc t i on at three d i f f e ren t temperatures w a s s tud ied . Keep ing in mind the c l i m a t i c c ond i t i o n s in B r i t i sh C o l u m b i a , it 12 13 w a s dec i ded to cove r the temperature range f r o m 10°C to 3 0 ° C . The three temperatures that we re s tud ied are 10°C , 2 0 ° C and 3 0 ° C . pH: It is w e l l - k n o w n that w i t h the p roduc t i on of v o l a t i l e f a t t y ac id s , the pH of the reactor f a l l s . Hence, it w a s dec ided to s tudy the pH e f f e c t for ' the uncon t ro l l ed pH cond i t i o n and the con t r o l l ed pH c o n d i t i o n . The con t ro l pH s tud ied w a s 7. A c o m p l e t e layout of the exper iment is g i ven in Tab le 3.1. T o ob ta i ned the layout de s c r i bed in Tab le 3.1, the ideal r andom des ign w a s c o m p r o m i s e d because r andomi za t i on of temperature w a s l i m i t e d by the a va i l a b i l i t y o f on l y a s ing le temperature c on t r o l l ed r o o m . Hence, c o m p l e t e r andom i za t i on w a s l im i t ed by nes t ing each run's par t icu lar re tent ion t ime X pH c o m b i n a t i o n w i t h i n a s ing le temperature va lue. The e f f e c t of this c o m p r o m i s e w o u l d be to con f ound run to run va r i a t i on s such as culture c ond i t i on i n g w i t h pH X re tent ion t i m e X temperature i n te rac t i ons . Howeve r , to reduce the p o s s i b i l i t y of culture c o n d i t i o n i n g , it w a s dec ided to in termix the content s of the three reacto r s be tween each exper imenta l run. The layout of exper iment s w a s checked out by the Department o f S t a t i s t i c s at the Un i ve r s i t y of Br i t i sh C o l u m b i a and c o n f i r m e d that the a f o r e m e n t i o n e d layout w a s the best one, g i ven the seve re equ ipment and t i m e l i m i t a t i o n s . 3.2. SEQUENCE OF EXPERIMENTS A s is ev ident f r o m Tab le 3.1, s ix set s o f exper iment s were run. In each set , three anaerob ic reac to r s were opera t ing . Deta i l s o f each 14 Table 3.1 Layout of Sequence of Experiments RUN # REACTOR# 1 REACTOR# 2 REACTOR# 3 1 9/10/pHu 3/10/pHu 9/10/pHc 2 9/30/phu 6/30/pHu 3/30/pHc 3 9/30/pHc 6/3 0/pHc 3/30/pHu 4 6/20/pHc 9/20/pHc 9/20/pHu 5 3/20/pHc 3/20/pHu 6/20/pHu 6 6/10/pHc 6/10/pHu 3/10/phc KEY: R e t e n t i o n Time ( d a y s ) / Temperature °C/ pH C o n t r o l l e d o r U n c o n t r o l l e d 15 set of exper iment s are g i ven be l ow . FIRST SET; The f i r s t set of exper iment s w a s run at 10°C. The f i r s t reactor had 9 day s retent ion t ime and uncon t ro l l ed pH. The s e cond reactor a l s o had uncon t ro l l ed pH but on l y 3 days re tent i on t ime . The th i rd reactor had 9 days re tent ion t ime and con t r o l l ed pH. SECOND SET: The s e cond set of expe r iment s w a s run at 3 0 ° C . The f i r s t reactor had a retent ion t ime of 9 day s and uncon t ro l l ed pH. The s e cond reactor a l s o had uncont ro l l ed pH but s ix day s re tent i on t ime . The third reactor had three days re tent i on t ime and con t r o l l ed pH. THIRD SET: The third set o f exper iment s w a s p e r f o r m e d ot 3 0 ° C . The f i r s t reactor had nine days re ten t i on t ime and con t r o l l ed pH. The second reactor a l s o had con t r o l l ed pH but s i x days re tent ion t ime . The third reactor had uncon t ro l l ed pH and three day s re tent ion t i m e . FOURTH SET: This set w a s run at 2 0 ° C . The f i r s t reactor had s ix day s retent ion t ime and c o n t r o l l e d pH. The s e cond reactor a l so had c o n t r o l l e d pH, but the retent ion t ime w a s nine days . The third reactor had uncon t ro l l ed pH and nine days re tent ion t ime. FIFTH SET; The f i f t h set o f exper iment s w a s a l so run at 2 0 ° C . The f i r s t reactor had three days re tent i on t ime and c o n t r o l l e d pH. The second reactor a l so had three day s re tent ion t ime but uncon t ro l l ed pH. The third reactor had s ix day s re tent ion t ime and uncont ro l l ed pH. SIXTH SET: The s ixth and last set of exper iment s we re conduc ted at 10°C. The f i r s t reactor wa s run at s ix days re tent ion t ime and con t r o l l ed pH. The s e cond reactor wa s a l so run at s ix day s re tent ion t i m e but uncont ro l l ed pH. The th ird reactor w a s run at three day s re tent i on t ime and con t r o l l ed pH. 16 CHAPTER 4 EXPERIMENTAL FACILITIES 4.1. EXPERIMENTAL SET-UP A s d i s cu s sed in Chapter 3, each exper imenta l run c o n s i s t e d of three ident i ca l anaerob ic reacto r s set up in a temperature c o n t r o l l e d r o o m . Three Pyrex E r l enmeyer f l a s k s of 2.8- l i t re c a p a c i t y each were used as anaerob ic reac to r s . A n a e r o b i c c ond i t i on s were ensured by a rubber bung w i t h sea led open ings f o r a pH p robe , ORP probe, t he rmomete r and a f eed i ng/was t i n g tube. A d d i t i o n a l y , an open ing cove red w i t h a septum w a s used to insert a needle that w a s a t tached to a n i t r o g e n - f i l l e d b a l l o o n , in order to rep lace the vo l ume o f s amp le w i t hd rawn by an inert gas. Reactor s we re ma in ta ined c o m p l e t e l y m ixed at al l t imes by means o f magnet ic s t i r re r s . The pH w a s c o n t r o l l e d by a F i sher A u t o m a t i c T i t ra to r M o d e l 380 us ing d i lute NaOH. A sketch o f the anaerob ic reactor s is s h o w n in Figure 4.1. ' . 4.2 . FEED USED IN THE STUDY 4.2.1 C O L L E C T I O N A N D S T O R A G E OF THE FEED P r imary s ludge f r o m the B i o - P p i l o t plant at the Un i ve r s i t y of B r i t i sh C o l u m b i a w a s used as f eed throughout the s tudy . P r imary s ludge w a s c o l l e c t e d in sea led p l a s t i c conta ine r s f r o m the p i lo t plant eve ry week 16 ,n F ig. 4.1 Sketch of Anae rob i c Reactor (Adapted f r o m C o m e a u , 1984) 18 and s to red in the l abora to ry at 4 ° C unti l needed fo r da i l y f eed i ng . 4.2.2 M A I N T E N A N C E OF THE COD OF THE FEED P re l im ina r y ana l y s i s of the p r imary s ludge f r o m the p i lo t plant s h o w e d that the COD of the s ludge va r i ed in the 1800 mg/l to 2400 mg/l range. To have a cons tant qua l i t y of s ludge throughout the s tudy, it w a s dec i ded to mainta in the COD of the s ludge at 2000 mg/ l . To th is end, COD ana l y s i s w a s p e r f o r m e d be f o r e feed ing new batch of p r imary s ludge every week . Dur ing s teady state ope ra t i on , COD ana l y s i s w a s done every day be fo re f eed i ng . If requ i red, the s ludge w a s then d i lu ted by water or w a s concent ra ted by r emov i n g supernatant to ach ieve a COD value c l o s e to 2000 mg/ l . T y p i c a l cha rac te r i s t i c s of the inf luent p r imary s ludge are g iven in Tab le 4.1. 4.3 . EXPERIMENTAL PROCEDURE A t the beg inn ing of the s tudy, a m ixed m i c r o b i a l culture w a s obta ined f r o m the f l o w - t h r o u g h fe rmenter at the Un i ve r s i t y of B r i t i sh C o l u m b i a B i o - P p i l o t plant. Th i s culture w a s used to start the anaerob ic reac to r s . 4.3.1 FILL A N D D R A W R E A C T O R S : Reactor s were run on a f i l l and d raw bas i s . Wa s t i n g and f eed i n g w a s done once per day. S ludge wa s t i n g v o l u m e w a s ca l cu la ted on the bas i s o f the des i red retent ion t ime . A s the reacto r s we re c o m p l e t e l y m i xed at a l l t i m e s , the w a s t e d s ludge w a s the s ame as the reactor con ten t s and hence w a s used to c a r r y - o u t the nece s s a r y t e s t s . 19 T a b l e 4.1 T y p i c a l C h a r a c t e r i s t i c s o f t h e I n f l u e n t P r i m a r y Sludge PARAMETER MEAN VALUE RANGE U n f i l t e r e d COD (mg/L) 2000.0 1950 .0 t o 2030. 0 T o t a l Suspended S o l i d s (mg/L) 1000.0 950 .0 t o 1100. 0PH 6.8 6 .5 t o 6. 9 A l k a l i n i t y (mg/L as CaC0 3) 260.0 240 .0 t o 270. 0 A c e t i c A c i d (mg/L) 20.0 14 .0 t o 25. 0 P r o p i o n i c A c i d (mg/L) n i l n i l B u t y r i c A c i d (mg/L) n i l n i l 20 A t the end o f each exper imenta l run, the con ten t s of the three reacto r s we re i n t e r - m i x e d and red i s t r i bu ted . This w a s done to reduce the p o s s i b i l i t y o f c ond i t i on i ng the m i c r ob i a l culture. 4.4. SAMPLING AND ANALYSIS 4,4.1 S A M P L I N G To ensure that the m i c r o o r g a n i s m s we re a c c l i m a t e d to a new set of c ond i t i o n s , each exper imenta l s e t - u p w a s ope ra ted fo r at least t w o retent ion t ime s be fo re de ta i l ed data gather ing w a s c o m m e n c e d . In each exper imenta l run, s teady s tate s amp l i n g w a s done fo r a pe r i od o f 7 days . Because the reactor s were c o m p l e t e l y m i x e d , wa s t e s a m p l e s w e r e used to represent the content s o f the reac to r s . S a m p l e s f o r v o l a t i l e f a t t y ac id s , T O C , and a l ka l i n i t y were c o l l e c t e d once per day dur ing the s teady state ana l y s i s . Rep l i ca te s amp l i n g w a s done f o r V F A ' s to a sce r ta in the s amp l i n g error. A l t hough ORP and pH read ings we re mon i t o red quite f r equen t l y during the day, no s i gn i f i can t va r i a t i on w a s ob se r ved ove r the 24 -hour pe r i od . Hence fo r the purpose of data ana l y s i s , one set of ORP and pH read ings w e r e r eco rded just be fo re the s chedu led w a s t i n g and f e e d i n g eve ry day. S a m p l e s fo r v o l a t i l e f a t t y ac id s were f i l t e r e d through 0.45/nm membrane f i l t e r s and were i m m e d i a t e l y f r o zen to ensure that no b i o l o g i c a l a c t i v i t y o c cu red during the s to rage p e r i o d . A l i q o u t s o f the s ame s amp le s 21 w e r e used f o r T O C ana l y s i s . 4,4.2 A N A L Y T I C A L TECHN IQUES USED A n a l y s e s fo r a l ka l i n i t y , s o l i d s and COD were car r ied out in acco rdance w i t h "S tandard M e t h o d s " (1980). The pH w a s mon i t o r ed us ing pH meter w i t h c o m b i n e d g la s s and re fe rence e l e c t r ode s in one probe. O x i d a t i o n - R e d u c t i o n potent ia l (ORP) w a s measured w i t h a B r oad l ey J a m e s Co rp . c o m b i n e d ORP probe w i t h per iphera l j unc t i on , us ing Ag/AgC I as a re fe rence coup le . F isher A c c u m e t M o d e l 320 expanded s ca le pH meters were used to measure pH and ORP. 4.4.2.1 V O L A T I L E F A T T Y A C I D S V o l a t i l e Fatty A c i d s ( VFA ' s ) were de te rm ined by gas ch romatog raphy a cco rd i ng to the procedure de sc r i bed in the S u p e l c o Bu l le t in 751E (1982), us ing a 60/80 c a r b o - p a c k c/0.3% c a rbowax 20M/0.1%H 3 PO 4 pack ing f o r the co l umn . A gas ch romatog raph (Hewlett Packard M o d e l 5750) w a s used f o r a l l ana l y se s . The v o l a t i l e f a t t y ac ids ana l y s ed inc lude ace t i c , p r op i on i c and buty r i c a c i d s . S tandards were prepared f r o m the reagent -g rade ace t i c ac id (99.9% pure), p r op i on i c ac id (96% pure) and buty r i c ac id (98% pure). M i c r o s y r i n g e s (Hami l ton M o d e l 75N # 87900, 5MI) we re used to inject 1.0jul o f p r e - f i l t e r e d s amp l e s . Jus t be fo re i n jec t i on , the pH w a s brought b e l o w 3, .by adding phosphor i c a c i d , to obta in a 1% ac i d s o l u t i o n . 4.4.2.2 T O T A L O R G A N I C C A R B O N A s ment i oned ear l ier , a l i quot s of the V F A s a m p l e s were used f o r T O C ana l y s i s . To ta l Organ ic Ca rbon w a s de te rm ined by us ing Beckman M o d e l 915A TOC ana lyzer . 22 CHAPTER 5 RESULTS AND DISCUSSION 5.1. RESULTS Fermenter behav iour during s teady s tate ope ra t i on w a s quite s tab le . No s i gn i f i can t trends were o b s e r v e d in any parameter over the f ina l s e ven days pe r i od o f ana l y s i s . On l y acet ic ac id and p rop i on i c ac id we re found to be p roduced f r o m the f e rmenta t i on of the p r imary s ludge. Buty r i c a c i d w a s a l w a y s f ound b e l o w the de tec t i on l im i t (which w a s 3 mg/l). A s per McCar ty (1963 ) and A n d r e w s and Pearson(1965), the degradat ion o f the bu ty r i c ac id by the methanogen i c bacte r i a occu r s at a f a s te r rate than acet i c ac id and much fa s te r than p rop i on i c a c i d ; wh i ch resu l t s in a ve ry l o w buty r i c ac id concen t r a t i on and o f ten no de tec t i on is p o s s i b l e . The max imum net ace t i c ac id p roduc t i on noted in this exper iment w a s 103 mg/ l , c o r r e spond i ng to 9 day s re tent ion t i m e , 3 0 ° C temperature and c on t r o l l ed pH c o m b i n a t i o n . M a x i m u m net p rop ion i c ac id p roduc t i on w a s 84 mg/l f o r the uncon t ro l l ed pH, 9 days re tent ion t ime and 3 0 ° C temperature c o m b i n a t i o n , wh i ch a l so accoun ted f o r the l owe s t net ace t i c a c i d p roduc t i on (35 mg/l ) in th i s exper iment . L o w e s t p rop i on i c ac id p r oduc t i on w a s 5 mg/l f o r the uncon t ro l l ed pH, 3 days re tent ion t i m e and 10° C temperature c o m b i n a t i o n . 22 23 The pH in the d i f f e ren t reacto r s wa s quite s tab le during the f ina l 7 days of each s amp l i n g pe r i od . The l owe s t average pH reco rded in the exper iment w a s 5.63 wh i ch co r r e sponded to the highest to ta l net V F A product ion in the study (152 mg/l as ace t i c ac id at 6 day s re tent ion t ime and 3 0 ° C temperature) . Ox ida t i on - reduct ion po tent i a l in al l the reac to r s w a s a l so very s table during the s teady state pe r i od . S i n ce the reac to r s were s t r i c t l y anaerob ic , ORP va lues in a l l the reac to r s w e r e very l o w . The highest ORP reco rded in the exper iment w a s - 2 65mv (Ag/AgC I r e fe rence ) wh i l e the l owe s t ORP reco rded w a s - 395mv (Ag/AgC I re fe rence) . Resu l t s of the exper iment are s ummar i z ed in Tab le s 5.1, 5.2 and 5.3 f o r 10°C , 2 0 ° C and 3 0 ° C temperature run r e s p e c t i v e l y . C o m p l e t e data reco rd of the net V F A p roduc t i on is g i ven in A p p e n d i x 1. 5.2. STATISTICAL ANALYSIS 5.2.1 S T A N D A R D I Z E D ERRORS The s tandard i zed e r ro r s , as ca l cu l a ted f o r the net V F A p roduc t i on , are l i s ted in Tab le 5.4. The s amp l i n g error is the va r i a t i on be tween s amp l e s , w i t h i n day s , wh i l e exper imenta l error is the va r i a t i on that occu r s be tween days w i t h i n reac to r s . S amp l i n g error is ca l cu la ted as the square root of the rat io of the mean square (MS ) error and number o f s amp le s taken per day, wh i l e exper imenta l error is ca l cu la ted as the square root of the rat io o f the mean square error and number of day s s amp le s c o l l e c t e d . Mean square T a b l e 5.1 Summary o f R e s u l t s f o r 10 C Temp. Run Ret. Time (days) Average pH Net A c e t i c A c i d (mg/L) Net P r o p i o n i c A c i d (mg/L) T o t a l Net VFA (mg/L as A c e t i c A c i d ) Mean S o l . TOC (mg/L) * Mean ORP (mV) Mean Range Mean Range Mean Range 3 6.43 44 34 - 50 5 3 - 7 48 37 - 56 93 -263 3 7.00 48 38 - 56 19 13 - 28 63 54 - 79 98 -274 6 5.97 50 42 - 55 20 15 - 25 66 58 - 73 108 -303 6 7.00 60 53 - 70 31 25 - 15 85 77 - 93 118 -326 9 5.93 53 46 " 60 19 15 - 23 68 61 - 78 110 -295 9 7.00 71 63 - 80 31 25 - 40 97 85 - 104 123 -385 *: Ag/AgCl r e f . T a b l e 5.2 Summary o f R e s u l t s f o r 20° C Temp. Run Ret. Time (days) Average PH Net A c e t i c A c i d (mg/L) Net P r o p i o n i c A c i d (mg/L) T o t a l Net VFA (mg/L as A c e t i c A c i d ) Mean S o l . TOC (mg/L) * Mean ORP (mV) Mean Range Mean Range Mean Range 3 6.25 48 41 - 54 18 11 - 22 63 53 - 70 104 -342 3 7.00 63 57 - 68 31 25 - 36 88 78 - 95 113 -358 6 5.88 79 70 - 84 29 22 - 33 103 95 - 110 118 -366 6 7.00 74 67 - 82 43 37 - 48 109 98 - 118 135 -391 9 6.01 66 60 - 70 60 55 - 65 115 105 - 122 140 -378 9 7.00 82 75 - 86 52 J 48 - 56 124 114 - 129 143 -359 *: Ag/AgCl r e f . T a b l e 5.3 Summary of R e s u l t s f o r 30° C Temp. Run Ret. Time (days) 3 3 6 6 9 9 Average PH 5.89 7.00 5.63 7.00 6.13 7.00 Net A c e t i c A c i d (mg/L) Mean 75 77 88 94 35 103 Range Net P r o p i o n i c A c i d (mg/L) Mean 68 70 78 84 30 95 82 84 95 100 40 110 47 32 80 73 84 42 Range 42 28 75 70 80 35 50 36 88 77 95 46 T o t a l Net VFA (mg/L as A c e t i c Acid) Mean 113 103 152 153 103 137 Range 104 94 140 142 95 123 121 111 160 163 116 147 Mean S o l . TOC (mg/L) 130 123 163 162 123 153 Mean ORP (mV) -396 -352 -354 -376 -391 -334 *: Ag/AgCl r e f . 27 er rors used in the ca l cu l a t i on o f s tandard i zed er rors f o r net ace t i c , p r op i on i c and tota l V F A p roduc t i on s are g i ven in Tab le s 5.5, 5.6 and 5.7 r e s p e c t i v e l y . In a l l c a se s , the s amp l i n g error wa s les s than the exper imenta l error. S i nce the exper imenta l error is the larger error, it has been used in all the s t a t i s t i c a l te s t s . 5.2.2 A N A L Y S I S OF V A R I A N C E (ANOVA") To k n o w whether there is any i n te rac t i on be tween the t reatment var iab les (pH, Retent ion T i m e and Temperature ) , ana l y s i s o f va r iance ( A N O V A ) w a s p e r f o r m e d on the net V F A p r oduc t i on data. Tab les 5.5, 5.6 and 5.7 out l ine the resu l t s o f the ana l y s i s of va r i ance and the type of c o n c l u s i o n s drawn f r o m its i n te rpretat ions fo r net a c e t i c , p rop i on i c and to ta l V F A p roduc t i on r e s p e c t i v e l y . F - S t a t i s t i c s ( 95% c o n f i d e n c e interva l ) va lues used in the ana l y s i s of va r iance ( A N O V A ) has been obta ined f r o m the Handbook of Tab les f o r P r obab i l i t y and S t a t i s t i c s , CRC P res s (1966) For al l three ca se s (Tables 5.5, 5.6 and 5.7), the rat io of mean squa re s (MS ) be tween days to the mean sqaures w i t h i n day s is greater than the F - S t a t i s t i c ( 95% c o n f i d e n c e interva l ) va lue. Hence the exper imenta l error (or va r i a t i on that occur s when the Retent ion T i m e , pH and Temperatures are he ld con s tan t ) is greater than errors caused by the s a m p l i n g and subsequent l abo ra to ry ana l y s i s ; e.g. f o r net to ta l V F A p roduct i on (Table 5.7). M S B e t w e e n Days = ^ > F _ S t a t i s t j c ( 9 5 % C | } = 1 4  M S W i t h i n Days (108,126) 28 T a b l e 5.4 S t a n d a r d i z e d E r r o r s PARAMETER SAMPLING ERROR (mg/L) EXPERIMENT ERROR (mg/L) Net Acetic Acid Production (mg/L) ±1.72 ±2.24 Net Propionic Acid Production (mg/L) ±1.47 ±1.59 Net Total V o l a t i l e Fatty Acids Production (mg/L as Ac. Acid) ±2.11 ±2.76 29 T a b l e 5.5 A n a l y s i s o f V a r i a n c e f o r n e t A c e t i c A c i d P r o d u c t i o n 3x3x2 FACTORIAL WITH NESTED NUMBER OF SAMPLES CLASSIFICATION WITH TAKEN EACH DAY EQUAL Sources o f V a r i a t i o n s Deg. o f Freedom (df) Sum o f Squares (SS) Mean Squares (MS) FSTAT FTEST Between R e a c t o r s 17 81881.86 4816.58 137.02 S i g n . R e t e n t i o n Time 2 9369.31 4684.65 133.26 S i g n . Temperature 2 24782.64 12391.32 352.49 S i g n . pH 1 13906.29 13906.29 395.59 S i g n . Ret. Time x Temp. 4 8414.48 2103.62 59.84 S i g n . Ret. Time x pH 2 11571.50 5785.75 164.59 S i g n . Temp, x pH 2 3517.93 1758.96 50.04 S i g n . Ret. Time x pH x Temp. 4 10319.71 2579.93 73 .39 S i g n . Between Days w i t h i n R e a c t o r s 108 3796.57 35.15 5.95 S i g n . Between Samples w i t h i n Days 126 744.00 5.90 30 T a b l e 5.6 A n a l y s i s o f V a r i a n c e f o r n e t P r o p i o n i c A c i d P r o d u c t i o n 3x3x2 FACTORIAL WITH NESTED CLASSIFICATION WITH EQUAL NUMBER OF SAMPLES TAKEN EACH DAY Sources o f V a r i a t i o n s Deg. o f Freedom (df) Sum o f Squares • (ss) Mean Squares (MS) FSTAT FTEST Between R e a c t o r s 17 1.22E+05 7182.84 466.92 S i g n . R e t e n t i o n Time 2 2.67E+04 13358.65 868.37 S i g n . Temperature 2 6.38E+04 31920.80 2074.99 S i g n . pH 1 4.63E+03 4630.29 301.05 S i g n . Ret. Time x Temp. 4 1.08E+04 2712.18 176.30 S i g n . Ret. Time x pH 2 4.32E+03 2159.65 140.39 S i g n . Temp, x pH 2 1.36E+04 6820.54 443.36 S i g n . Ret. Time x pH x Temp. 4 2.69E+03 673.49 43 .78 S i g n . Between Days w i t h i n R e a c t o r s 108 1.66E+03 15.38 3.54 S i g n . Between Samples w i t h i n Days i 1 126 5.47E+02 4.34 31 T a b l e 5.7 A n a l y s i s o f V a r i a n c e f o r T o t a l N e t V o l a t i l e F a t t y A c i d s P r o d u c t i o n 3x3x2 FACTORIAL WITH NESTED CLASSIFICATION WITH EQUAL NUMBER OF SAMPLES TAKEN EACH DAY Sources of V a r i a t i o n s Deg. of Freedom (df) Sum o f Squares (SS) Mean Squares (MS) FSTAT FTEST Between R e a c t o r s 17 224008 13177 250.07 S i g n . R e t e n t i o n Time 2 49756 24878 472.13 S i g n . Temperature 2 131248 65624 1245.39 S i g n . PH 1 12718 12718 241.35 S i g n . R e t. Time x Temp. 4 18933 4733 89.83 S i g n . Ret. Time x pH 2 3066 1533 29.09 S i g n . Temp, x pH 2 1767 883 16.76 S i g n . Ret. Time x pH x Temp. 4 6520 1630 30.93 S i g n . Between Days w i t h i n R e a c t o r s 108 5691 53 5.92 S i g n . Between Samples w i t h i n Days 126 1121 9 32 A n o t h e r important th ing to examine is whether the change in the net V F A p roduc t i on caused by va ry ing the pH, temperature or retent ion t ime is greater than wh i ch w o u l d occur by chance. If the rat io of the mean squares be tween reactor s to the mean squares be tween days w i th i n reacto r s is greater than F - S t a t i s t i c ( 9 5% C.l.) va lue , then the V F A p roduc t i on i n one reactor is d i f f e ren t than that in another reactor because o f a change in s o m e comb i na t i on of pH, re tent ion t ime and temperature . In al l the three ca se s (Tables 5.5, 5.6 and 5.7) the rat io o f the mean squares be tween reac to r s to the mean squares be tween days w i th in reacto r s is greater than F - S t a t i s t i c ( 95% C.l.) va lue. For examp le , in case of t o ta l V F A p roduc t i on (Table 5.7), ^ ^ B e t w e e n Reacto r s „,.„ „_ _ _ _ . . _ , . , = 250.07 > F - S t a t i s t i c ( 9 5 % C.l.) = 1.69 ' ^ B e t w e e n Days Hence, the va r i a t i on in the net V F A p roduct ion s be tween reactor s is not because of chance, but is because o f t reatment va r i ab le s . To c l a r i f y h o w the changes in pH, re tent ion t ime and temperatu re caused the va r i a t i on be tween r eac to r s , the va r ia t ion is pa r t i t i oned into eight s ou r ce s (Tables 5.5, 5.6 and 5.7). The f i r s t s tep is to examine if the e f f e c t o f a change in one parameter is dependent on the va lues of the other t w o . If the rat io of the mean squares due t o (pH)x(Ret.Time)x(Temp.) i n te rac t ion to the mean squares be tween reactor s is greater than F - S t a t i s t i c ( 95% C.l.) va lue, then the e f f e c t o f a change in one parameter is dependent on the va lues o f the other t w o va r i ab le s . In a l l the three ca se s (Tables 5.5, 5.6 and 5.7), (pH)x(Ret.Time)x(Temp.) in te ract ion is s i g n i f i c a n t ; e.g. f o r to ta l V F A p roduc t i on (Table 5.7), 33 (pH)x(Ret.Time)x(Temp,) = ^ > F _ S t a t j s t i c ( 9 5 % c , } = 2 5 ' ^ B e t w e e n Days Because the (pH)x(Ret.Time)x(Temp.) i n te rac t ion is s i g n i f i c an t , then the other s e ven sources w i l l au tomat i ca l l y be s i gn i f i c an t , as s hown in the Tab le 5.7. 5.2.3 D U N C A N ' S MULT IPLE R A N G E T E S T Net V F A p roduc t i on data w a s examined us ing Duncan ' s Mu l t i p l e Range tes t , to rank the mean ac i d p roduc t i on . This w a s done to c l a r i f y the nature o f (pH)x(Ret.Time)x(Temp.) i n te rac t ion . Results o f th i s test are s hown in Tab le 5.8, 5.9 and 5.10 f o r net ace t i c , p r o p i o n i c , and to ta l V F A p r oduc t i on , r e s pec t i v e l y . C o m b i n a t i o n s o f pH, re tent ion t ime and tempera tu re s , account ing fo r s t a t i s t i c a l l y i nd i f fe rent V F A p roduc t i on s , have been grouped together. For e xamp le , the f o l l o w i n g c o m b i n a t i o n s are l i s t ed in one co l umn of Table 5.8 because they were f ound to be s t a t i s t i c a l l y i nd i f fe ren t as d i s c u s s ed above . 9 days Ret. T ime/20°C temp./ uncont ro l l ed pH, 3 days Ret. T ime/20°C temp./ con t r o l l ed pH, and 6 days Ret. T ime/10°C temp./ con t r o l l ed pH 5.3. DISCUSSION The layout of th i s s tudy enab les us to c l a r i f y the re la t i onsh ip a m o n g three con t ro l va r iab les and their e f f e c t s on net V F A p roduc t i on . S t a t i s t i c a l ana l y s i s of the data s h o w s that, in al l c a se s , the va r ia t ion due to (pH)x(Ret.Time)x(Temp.) i n te rac t i on is s i g n i f i c an t l y greater than the Table 5.8 Ranking of Means of Net Acetic Acid Production Under Different Operating Conditions Using Duncan's Multiple Range Test 9/30/U 6/30/U 9/20/U 3/30/C 3/20/C 6/10/C 6/30/C > 9/30/C > 9/20/C > 3/30/U > 6/20/U > 6/10/C > 3/10/C > 3/10/U 6/2 0/C 9/10/C 3/20/U KEY: Retention time (days)/Temp. °C/pH Controlled or Uncontrolled. T a b l e 5 . 9 Ranking o f Means o f Net P r o p i o n i c A c i d P r o d u c t i o n Under D i f f e r e n t O p e rating C o n d i t i o n s U s i n g Duncan's M u l t i p l e Range T e s t 3/30/C 6/10/U 9/10/C 3/10/C 9/30/U > 6/30/U > 6/30/C > 9/20/U > 9/20/C > 3/30/U > 6/20/C > 6/10/C > 9/10/U > 3/10/U 9/30/C 3/20/C 3/20/U 6/20/U KEY: R e t e n t i o n time (days)/Temp. °C/pH C o n t r o l l e d o r U n c o n t r o l l e d . Table 5.10 Ranking of Means of Total Net VFA Production Under Different Operating Conditions using Duncan's Multiple Range Test 9/20/C 9/10/U 6/20/U 9/20/U 6/10/U 9/30/C > 6/30/C > 6/30/U > 3/30/C > 3/20/C > 3/20/U > 9/20/U 3/30/U 6/10/C 3/10/C 6/20/C 3/10/U 9/10/C KEY: Retention time (days)/Temp. °C/pH Controlled or Uncontrolled. 37 exper imenta l error or the va r ia t ion be tween days w i th i n the reactor s (the va r i a t i on w e w o u l d get if all r eac to r s had the s ame retent ion t i m e , temperature and pH). This result c o n f i r m s that the e f f e c t of change in one t reatment var iab le on the vo l a t i l e f a t t y ac ids p r oduc t i on , is very much dependent on the va lues of the other t w o t reatment va r i ab le s . The re fo re , the e f f e c t s of the parameters cannot be s tud ied sepa ra te l y . The f o l l y of s ing le parameter s tud ies is that in teract ions are c on f ounded w i t h the ma in e f f e c t i.e. its e f f e c t w o u l d be v i e w e d as if caused by the parameter under s tudy. In truth, it is caused by the i n te rac t i on be tween the parameters under s tudy and the cond i t i on s w i t h i n the reactor . 5.3.1 T O T A L V F A PRODUCT ION In fo rmat ion obta ined f r o m the p lo t s of the mean to ta l net V F A concen t ra t i on for each set of re tent ion t ime , temperature and pH va lues (Figs. 5.1 and 5.2) agrees w e l l w i t h the s t a t i s t i c a l ana l y s i s of the data. For examp le , at 3 0 ° C , 9 days re tent ion t ime and uncon t ro l l ed pH, the to ta l net V F A p roduc t i on w a s 103 mg/l as acet ic ac id (Fig. 5.2). Howeve r , when the pH con t r o l w a s added, the p roduc t i on j umped to 137 mg/l (Fig. 5.1). If the re tent ion t ime is reduced t o 6 days , the d i f f e r e n c e be tween the t w o reac to r s (having d i f f e ren t pH c o n d i t i o n s ) b e c o m e s i n s i gn i f i cant (152 mg/l and 153 mg/l). if the re tent ion t ime is d ropped to 3 days , the uncon t r o l l ed pH reactor has s i g n i f i c a n t l y more tota l net V F A p roduct i on than the c o n t s r o l l e d reactor (103 and 113 mg/l as acet i c ac id) . 38 JO < U 130 cu u < < CD 6 c o O > ,*o CD 160 - i 150-j 140 120 no -3 100 9 0 -8 0 -7 0 -6 0 -50 - i 4 0 -30 20 ^ 10 0 3 4 5 6 7 Retention Time (Days) Legend A A t 10° c X A t 20° c • A t 3 0 ' C 8 9 10 Fig. 5.1 Total Net VFA Concentration vs . Retention Time at Controlled pH 39 TS < o % o < < CD u c o o 160 150 HO 130-120-110 -100 9 0 -8 0 -70 60 50 40 3 0 -2 0 -10 -0 Legend A M 10° c X At 20° c • Af 30' C 3 4 5 6 7 Retention Time (Days) x • 8 10 Fig. 5.2 Total Net V F A Concent rat ion v s . Re ten t i on T i m e at Uncon t ro l l ed pH 40 Repeat ing the s ame procedure fo r 2 0 ° C (Figs. 5.1 and 5.2), 9 days re tent ion t ime s h o w s a s i gn i f i cant d i f f e r ence be tween con t r o l l ed and uncont ro l l ed pH cond i t i on s , but not of the same magni tude as w a s s hown at 3 0 ° C (115 vs . 124 mg/l as acet ic acid). If the re tent ion t ime is d ropped to 6 days , the d i f f e r ence be tween the t w o reactor s is s t i l l s i gn i f i can t but much sma l l e r (103 vs. 109 mg/l as acet i c acid). If w e drop the retent ion t ime to 3 day s , the d i f f e r e n c e be tween the t w o reactor s ' (having d i f f e ren t pH c o n d i t i o n s ) b e c o m e s more s i gn i f i can t (63 vs. 88 mg/l as acet i c ac id) and the c o n t r o l l e d reactor p roduces more V F A ' s than the uncont ro l l ed reactor , unl ike the 3 0 ° C c o m p a r i s o n . A t 10°C (Fig. 5.1 and 5.2), the same pattern occu r s as wa s f ound at 2 0 ° C , but t o a much larger degree. A t al l three re tent ion t ime s t e s ted (3, 6 and 9 days) , the pH con t r o l l ed reactor p roduces much more net V F A ' s than the uncont ro l l ed reactor . 5.3.2 A C E T I C A C I D PRODUCT ION The in te rac t ion be tween pH, re tent ion t ime and temperature w a s f ound to have a c on found i ng e f f e c t on net acet i c ac id p roduc t i on a l s o . For examp le , at 3 0 ° C and 9 day s re tent ion t i m e , the net ace t i c ac id p roduct i on w i t h no pH con t ro l w a s 35 mg/ l , the l owe s t p roduc t i on ach ieved in this s tudy (Fig. 5.4). Howeve r when pH con t ro l w a s added, the p roduct i on j umped to 103 mg/l , the h ighest a ch ieved in th is s tudy (Fig. 5.3). If the re tent ion t ime is reduced to 6 days , d i f f e r e n c e be tween the acet i c ac id p roduc t i on o f the c o n t r o l l e d and uncon t ro l l ed pH reactor s is s t i l l s i gn i f i can t , but much sma l l e r (94 v s . 88 mg/l). If the re tent ion t ime is further reduced to 3 days , the net ace t i c ac id p roduc t i on in the t w o reactor s (having 41 110 ioo H 90 80 H 70-o 60 H .2 50 A X ^ 40H Z 30 H S 20-10-2 -r 4 Legend A At 10° c X At 20° c • At 3 0 ' C 5 6 7 Retention Time (days) 8 10 Fig. 5.3 Net A c e t i c A c i d Concent rat ion v s . Re ten t i on T i m e at Con t ro l l ed pH 42 100 90 rj) 80 E. o c o o "o < o U < 70 60 H 50 40 A d) 30 H c o CD 20 H 10 4 Legend A At 10° c X At 20° c • At 3 0 ' C 2 ^ 6 ) 3 4 5 6 7 8 Retention Time (days) 10 Fig. 5.4 Net A c e t i c A c i d Concent ra t ion v s . Retent ion T i m e at Uncont ro l l ed PH 43 d i f f e ren t pH c ond i t i o n s ) is not s i g n i f i c an t l y d i f f e ren t (71 v s . 75 mg/l). A t 2 0 ° C , 9 days retent ion t ime s h o w s a s i gn i f i c an t d i f f e r e n c e in the net ace t i c ac id p roduc t i on of the con t r o l l ed and uncont ro l l ed pH reactor s but not to the s ame magnitude as w a s s hown at 3 0 ° C (81 vs. 66 mg/l). If the re tent ion t ime is d ropped to 6 days , the uncont ro l l ed pH reactor p roduces more acet i c ac id than c on t r o l l ed reactor (74 vs . 79 mg/l), but the d i f f e r e n c e be tween the t w o is very s m a l l . A t 3 days re tent ion t i m e , the c o n t r o l l e d pH reactor p roduces much more net ace t i c ac id than the uncon t ro l l ed reactor (82 v s . 66 mg/l). A t 10°C , the s ame pattern occur s as w a s f ound at 3 0 ° C , but to a much le s se r degree (Figs. 5.3 and 5.4). A t a l l the three re tent i on t ime s t e s t ed (3, 6 and 9 days ) , the pH c o n t r o l l e d reactor p roduces more net ace t i c a c i d than the uncont ro l l ed reactor . 5.3.3 PROPIONIC AC ID PRODUCT ION A s in the case of net ace t i c ac id and to ta l V F A p roduc t i on , the i n te rac t i on be tween pH, retent ion t ime and temperature w a s found to have c on f ound i n g e f f e c t on net p rop i on i c ac id p roduc t i on as w e l l (Figs. 5.5 and 5.6). A t 10°C , the net p rop ion i c ac id p roduc t i on imp rove s w i t h the add i t i on of the pH con t ro l f o r al l the three retent ion t ime s s tud ied. For e xamp le , at 3 day s re tent ion t ime , the net p roduc t i on o f p r op i on i c a c i d i m p r o v e d f r o m 5 to 19 mg/l w i t h the add i t i on of pH c o n t r o l . S i m i l a r l y f o r 6 day s and 9 day s re tent i on t i m e s , the net p rop i on i c ac id p roduc t i on i m p r o v e d f r o m 20 to 31 mg/l and 19 to 31 mg/ l , r e s p e c t i v e l y , when pH con t r o l w a s added. 44 90 80-\ CO 70 J , 6 60 c o o 50 < d 40 o Q_ 30 % c 20 o 2 10 Legend A At 10° c X At 20° c • At 3 0 ' C 3 4 5 6 7 Retention Time (days) 9 10 Fig. 5.5 Net Prop ion ic A c i d Concent ra t ion vs . Re ten t i on T i m e at Con t ro l l ed PH 45 F ig . 5.6 Net P rop ion i c A c i d Concet ra t i on vs. Re tent i on T i m e at Uncon t ro l l ed PH 46 A t 2 0 ° C , and 3 days re tent ion t i m e , net p r op i on i c ac id p roduc t i on imp roved f r o m 18 to 31 mg/l w i th the add i t i on of the pH c o n t r o l . A t 6 days re tent ion t ime a l so , the pH c o n t r o l l e d reactor w a s found to produce more net p rop ion i c ac id than the uncon t ro l l ed pH reactor . Howeve r , at 9 days re tent ion t ime , the rever se pattern w a s ob se r ved . C o n t r o l l e d pH reactor p roduced les s net p rop ion i c ac id than the uncon t ro l l ed reactor (52 vs . 60 mg/l). A t 3 0 ° C , the net p rop i on i c ac id p roduc t i on d ropped w i t h the add i t i on of the pH con t ro l f o r al l the three retent ion t i m e s s tud ied. For examp le , the net p rop i on i c ac id p roduc t i on d ropped f r o m 47 to 32 mg/l and f r o m 80 to 73 mg/l f o r 3 day s and 6 days re tent i on t i m e s r e s p e c t i v e l y , when pH con t ro l w a s added. A t 9 day s re tent ion t i m e , the drop in the p r op i on i c ac id p roduc t i on w a s much more . The p roduc t i on d ropped by 5 0 % ( f rom 84 to 42 mg/l) w i t h the add i t i on of pH c o n t r o l . 5.3,4 EFFECT OF pH CONTROL Con t r o l of pH to a va lue o f 7 w a s f ound to have a c on found i ng e f f e c t w i t h temperature and retent ion t i m e on the net V F A p roduc t i on . A t 10°C , pH con t ro l imp roved the net t o ta l V F A p roduct i on s i g n i f i c a n t l y . For examp le , pH con t ro l i m p r o v e d the net t o ta l V F A p roduc t i on f r o m 48 to 63 mg/l as ace t i c a c i d , f o r 3 days re tent i on t ime and 10°C temperature . S i m i l a r l y , f o r 6 and 9 day s retent ion t i m e s , w i t h the add i t i on of the pH c o n t r o l , to ta l net V F A p roduc t i on imp roved f r o m 66 t o 85 mg/l as a ce t i c a c i d , and 68 to 97 mg/l as ace t i c a c i d , r e s p e c t i v e l y (Figs. 5.1 and 5.2). 47 A t higher tempera tu re s , the con t ro l of pH is not e f f e c t i v e in imp rov i n g the net tota l V F A p roduc t i on . A t 2 0 ° C , on ly f o r 3 day s re tent ion t ime d id the pH con t ro l l ed reactor produce s i g n i f i c an t l y more net to ta l V F A (63 vs . 88mg/l as acet ic ac id ) ; at higher re tent ion t i m e s , there w a s no s i gn i f i c an t improvement in the net to ta l V F A concen t r a t i on . S i m i l a r l y , at 3 0 ° C a l so the pH con t ro l w a s not e f f e c t i v e in imp rov i n g the net to ta l V F A p roduc t i on . A t 3 day s re ten t i on t i m e , the net V F A p roduc t i on dec reased f r o m 113 to 103 mg/l as a ce t i c ac id w i t h pH c o n t r o l , wh i l e at 9 days re tent ion t i m e , on ly a sma l l increase in the net V F A p roduc t i on was ach ieved w i t h pH c o n t r o l . A t 6 day s re tent ion t i m e , no s i gn i f i c an t change in the to ta l net V F A p roduct i on w a s o b s e r v e d . Con t r o l of pH w a s f ound to have a s i g n i f i c an t e f f e c t on the re la t i ve p roduc t i on of acet i c and p rop i on i c ac id s . For e xamp le , at 3 0 ° C , the e f f e c t o f pH con t ro l on ace t i c ac id p roduct i on is i n s i gn i f i c an t at a re tent ion t ime of 3 days (75 vs. 77 mg/l), but the p r op i on i c ac id p roduc t i on w a s s i g n i f i c an t l y d i f f e ren t .w i th the uncon t ro l l ed reactor p roduc ing much more net p rop ion i c a c i d than the c on t r o l l ed reactor (47 vs . 32 mg/l). H o w e v e r , at 9 days retent ion t i m e , the increase in ace t i c ac id p roduc t i on at the c o n t r o l l e d pH was greater than the l o s s of p r op i on i c ac id p roduc t i on . A t c o n t r o l l e d pH, the ace t i c ac id p roduc t i on i m p r o v e d f r o m 35 mg/l (measured in the uncont ro l l ed reactor ) to 103 mg/l (Figs. 5.3 and 5.4), wh i l e the p r op i on i c ac id p roduc t i on d ropped f r o m 84 mg/l (measured in uncon t r o l l ed reactor ) to 42 mg/l w i t h the add i t i on of c on t r o l (Figs. 5.5 and 5.6). A t 6 day s retent ion t i m e , the acet i c and p r op i on i c ac id s changes be tween pH con t ro l and no c on t r o l are of the s ame magn i tude but o p p o s i t e in s i gn . The re fo re there w a s i n s i gn i f i can t change be tween the to ta l V F A 48 p roduc t i on in the t w o reac to r s . E f f e c t s of pH and temperature in teract ion on the net V F A p roduc t i on can be exp la ined by tak ing into account the degree of env i ronmenta l s t re s s the bacter ia l ce l l s might be undergo ing at d i f f e ren t temperatures and the po s s i b l e p resence o f methane p roduc ing bacte r i a in the reactor s operated at 30° C. A t 10° C, the popu la t i on of ac id p roduc ing bacter ia is p s y ch roph i l i c . P s y ch roph i l e s have max imum temperatures fo r g rowth o f app rox imate l y 2 0 ° C and an o p t i m u m temperature o f about 15° C (Gaudy and Gaudy, 1980). Th i s means that the ac id p roduc ing bacter ia l popu la t i on is not under much env i r onmenta l s t re s s at 10° C, and if o p t i m u m pH is p r o v i d e d , it w i l l enhance e n z y m a t i c a c t i v i t i e s in the bacter ia l c e l l , wh i ch w i l l s ubsequent l y increase the V F A p roduc t i on . Resu l t s of the 10° C ope ra t i on agrees w i t h this exp lanat ion . A s de s c r i bed ear l ier , when the pH w a s c o n t r o l l e d to a va lue of 7, V F A p roduc t i on w a s f ound to imp rove s i g n i f i c an t l y . A t 2 0° C, mos t of the ac id p roduc ing bacte r i a again are p s y ch roph i l i c . A s men t i oned earl ier.the p s ych roph i l e s have max imum temperatures f o r g rowth o f 2 0° C and o p t i m u m temperature o f 15° C. It means that a c i d p roduc ing bacter ia at th is high temperature are a l ready under con s i de rab l e env i r onmenta l s t re s s and may not be in a p o s i t i o n t o r e spond p o s i t i v e l y to the p resence o f env i r onmenta l s t re s s caused by the o p t i m u m pH cond i t i o n s . A s de s c r i bed b e f o r e , the resu l t s of 2 0° C ope ra t i on does not s h o w any s i gn i f i can t increase in the V F A p roduc t i on at the c o n t r o l l e d pH. 49 A t 30° C the ac id produc ing bacter ia l popu l a t i on is me soph i l i c . M e s o p h i l e s have a max imum temperature fo r g rowth of app rox ima te l y 4 5 ° C and an op t imum temperature of about 3 7° C (Gaudy and Gaudy, 1980). Th i s means that the popu la t i on of ac id p roduc ing bacte r i a at 3 0° C, wh i ch is ma in l y me soph i l i c , w i l l not be under much env i r onmenta l s t re s s . If o p t i m u m pH is p rov i ded under such a temperature reg ime, it shou ld increase the V F A p roduc t i on due to inc reased enzymat i c a c t i v i t i e s in the bacter ia l c e l l . Howeve r , the resu l t s of 3 0 ° C temperature run does not agree w i t h th is hypo thes i s . A s men t i oned ear l ier , no s i gn i f i can t imp rovemen t w a s no t i c ed in the net V F A p roduc t i on w i t h pH con t ro l to the value of 7. This can be exp la ined by tak ing into account the p o s s i b l e p re sence of the methanogen i c bacter ia in the reactor s operated at 3 0 ° C. Th i s methanogen ic bacter ia l popu la t i on can use V F A ' s as subs t ra te , wh i ch w o u l d neutra l ize the inc reased V F A p roduc t i on at c on t r o l l ed pH due to i nc rea sed enzymat i c a c t i v i t i e s in the ac id p roduc ing bacter ia l c e l l . A de ta i l ed exp lanat ion suppor t ing the presence o f the methanogen ic bacte r i a in the 3 0 ° C operated reac to r s , is g i ven later in this chapter. 5.3.5 EFFECT OF T E M P E R A T U R E Temperature w a s found to have a ve ry p ronounced e f f e c t on the net a ce t i c , p rop i on i c and tota l V F A p roduc t i on s . A s e xpec ted , the net to ta l V F A p roduc t i on i m p r o v e d c o n s i s t e n t l y w i t h the increase in temperature , be tween 10°C to 3 0 ° C . The on ly excep t i on w a s the behav iour o f the reac to r hav ing 9 days re tent ion t ime , uncont ro l l ed pH and 3 0 ° C temperature. In th i s part icu lar c a se , the net to ta l V F A p roduc t i on d ropped f r o m 115 mg/l as acet i c ac id at 2 0 ° C to 103 mg/l as ace t i c a c i d at 3 0 ° C (Fig. 5.2). The ma in reasons fo r the drop in the net to ta l V F A p roduc t i on w a s 50 the unexpected drop in the net acet ic ac id p roduc t i on . The net acet i c ac id p roduc t i on d ropped f r o m 60 mg/l to 35 mg/l w i t h the increase in temperature f r o m 2 0 ° C to 3 0 ° C (Fig. 5.4). A po s s i b l e exp lanat ion fo r this abnorma l behav iour is g i ven later in this chapter. O the rw i s e , the net p roduc t i on of ace t i c , p rop i on i c and to ta l V F A c o n s i s t e n t l y imp roved w i t h temperature . 5.3.6 EFFECT OF RETENTION T IME Retent ion t ime w a s f ound to have a c o n f o u n d i n g e f f e c t w i t h temperature on the net t o ta l V F A p roduc t i on . A t 10°C and 2 0 ° C temperatu re , the net to ta l V F A p roduc t i on imp roved w i t h an increase in re tent ion t i m e (for 3, 6 and 9 days re tent ion t ime s te s ted ) . Howeve r , at 3 0 ° C , the net to ta l V F A p roduc t i on i nc reased when the re tent i on t ime w a s ra i sed f r o m 3 to 6 days.but it sub sequent l y dec reased when the retent ion t ime w a s further increased to 9 days . The net p roduc t i on d ropped f r o m 152 to 103 mg/l as acet i c ac id f o r the uncon t ro l l ed pH reac to r and f r o m 153 to 137 mg/l as acet i c ac id f o r the pH c o n t r o l l e d reactor (Fig. 5.1 and 5.2). A p o s s i b l e m i c r o b i o l o g i c a l exp lanat ion is g i ven later in th is chapter. 5.3.7 BEST C O M B I N A T I O N OF T R E A T M E N T V A R I A B L E S Of the 18 c o m b i n a t i o n s o f pH, temperature and re tent ion t ime t e s t e d , the max imum net to ta l V F A concen t ra t i on w a s a ch i e ved at 3 0 ° C and 6 day s re tent ion t ime (153 mg/l as a ce t i c ac id). The con t r o l of pH to a va lue o f 7 d id not make any s i gn i f i c an t change in the net to ta l V F A p roduc t i on . 51 Max imum net acet i c ac id p roduc t i on (103 mg/l), w a s ach ieved at 3 0 ° C , 9 day s re tent ion t ime and c o n t r o l l e d pH. Uncon t r o l l ed pH, at 3 0 ° C and 9 day s retent ion t i m e , accounted for the m a x i m u m p rop i on i c ac id p roduc t i on (84 mg/l). A s d i s c u s s ed ear l ier , the best c o m b i n a t i o n s fo r the m a x i m u m net t o ta l V F A , acet ic and p rop ion i c ac id s p roduc t i on s c l ea r l y s hows that the con t ro l o f pH to a va lue of 7 does not have a s i g n i f i c an t e f f e c t on the net t o ta l V F A p roduc t i on . Howeve r , it ce r ta in l y a f f e c t s the re la t i ve d i s t r i bu t i on o f the ace t i c ac id and p rop i on i c ac id concen t r a t i on s . Z o e t e m e y e r et al (1982) and Eas tman and Ferguson (1981) have a r r i ved at the s im i l a r c onc l u s i o n s about the e f f e c t o f pH. In both of the above s tud ie s , it w a s found that pH a f f e c t e d the re la t i ve d i s t r i bu t i on of the ind iv idua l v o l a t i l e f a t t y ac id s . 5.3.8 M I C R O B I O L O G I C A L E X P L A N A T I O N FOR THE BEHAV IOUR OF R E A C T O R  A T 30^C A s men t i oned ear l ier , it w a s f ound that, at 9 days re tent ion t ime , 3 0 ° C reactor temperature and uncon t ro l l ed pH, the net acet i c ac id p roduc t i on unexpec ted l y d ropped to 35 mg/ l , the l owe s t p roduc t i on r eco rded in the s tudy (Fig. 5.4). A m i c r o b i o l o g i c a l exp lanat ion fo r the f e rmente r behav iour at these opera t ing c ond i t i o n s can be g i ven , tak ing into account the po ten t i a l a c t i v i t y o f methane p roduc ing bac te r i a . Methane p roduc ing bacter ia are ve ry s en s i t i v e to env i r onmenta l c ond i t i o n s . The o p t i m u m pH of methane f o r m e r s l ies be tween 7 and 7.2. Methane bacte r i a g r ow quite s l o w l y c o m p a r e d to a c i d - p r o d u c i n g bacte r i a 52 and so a longer retent ion t ime is required fo r them to mainta in an adequate popu la t i on for the substrate present. M i n i m u m ce l l re s idence t ime fo r methane produc ing bacter ia has been repor ted be tween 2.5 to 4 days ( A n d r e w s and Pearson,1965). A c c o r d i n g to Lawrence and McCarty (1969) , the m i n imum ce l l re s idence t ime requi red fo r the g rowth o f methane bacter ia depends on the type of subst rate and temperature. A t 3 5 ° C, the p red i c ted average va lues of the m i n imum so l i d s re tent ion t ime f o r the methanogen ic bacter ia r e spon s i b l e fo r the a s s i m i l a t i o n o f ace t i c , p r op i on i c and buty r i c ac id s are 3.1, 3.2, 2.7 days r e s pec t i v e l y . The m i n i m u m ce l l r e s idence t ime fo r acet i c a c i d a s s i m i l a t i o n w a s f ound to increase to 4.2 day s w i t h the reduct ion in temperature to 3 0 ° C (Lawrence and McCarty,1969) . The rate o f degradat ion o f the acet i c ac id and p rop i on i c a c i d are a l so d i f f e ren t . A c c o r d i n g to A n d r e w s and Pearson(1965), the rate o f deg radat ion of a ce t i c ac id is much higher than that of p rop i on i c ac id . H o w e v e r , Lawrence and McCar ty (1969 ) reported that when favou rab le env i r onmenta l c ond i t i o n s are p rov i ded fo r the p rop ion i c a c i d , a s s im i l a t i n g methanogen ic bac te r i a , the rate of degradat ion of p rop ion i c ac id is comparab le to that o f ace t i c ac id . Redox potent ia l in the uncon t ro l l ed reactor w a s ve ry l o w (about - 3 9 1 m V vs. Ag/AgC I ref.), the m i n imum reco rded in the w h o l e s tudy. Methane p roduc t i on has been repor ted to occur at an E^. o f - 4 3 5 m V ( -390mV vs . Ag/AgC I ref.) ( P f e f f e r , 1979). Hence, there is a high p robab i l i t y of methane f o r m a t i o n at 9 day s re tent ion t ime and 3 0 ° C temperature. L owe r net to ta l V F A p roduc t i on in the uncon t ro l l ed reactor may be interpreted as the result of the use o f the acet i c ac id as subst rate by methane f o r m i n g bacter ia . A c c o r d i n g to A n d r e w s and Pea r son (1965) and M c C a r t y (1963), acet i c ac id is much more read i l y degradab le by methane f o r m e r s than is p rop i on i c a c i d . A ve r a ge pH in the reactor w a s 6.13, w h i c h 53 is not a f avou rab le pH fo r methane f o r m e r s . A c c o r d i n g to Lawrence and McCar ty (1969 ) , the unfavourab le env i r onmenta l c ond i t i on s a f f e c t s the p r op i on i c a c i d degradat ion more than the ace t i c ac id deg radat ion . The re fo re , there w a s p robab l y no los s in p rop i on i c ac id p roduc t i on . Resu l t s of this s tudy are quite compa rab l e to the f i nd ings of A n d r e w s and Pearson(1965). The main d i f f e r e n c e be tween the work of A n d r e w s and Pear son ' s and this s tudy is that they used on ly one temperature , and hence no s t a t i s t i c a l de s i gn of exper iment s w a s nece s sa r y . M o r e o v e r , they used syn thet i c f eed wh i l e in the present s tudy p r imary s ludge has been used. They a l so o b s e r v e d that the start of methane p roduc t i on a f f e c t e d the re la t i ve concen t r a t i on s of a ce t i c ac id and p rop i on i c ac i d d i f f e r e n t l y . Methane p roduc t i on w a s a c c o m p a n i e d by a" sudden decrease in the net ace t i c ac id concen t r a t i on . Howeve r , there w a s a net increase in p r op i on i c a c i d concent ra t i on o f 5.7 meq/ l . when the SRT w a s inc reased f r o m 2.4 to 4.3 days . The on l y known vo l a t i l e a c i d , f r o m w h i c h p r op i on i c ac id can be p roduced by methane f o r m e r s , is va le r i c a c i d , and the net measured dec rease fo r this ac id w a s on l y 2.2 meq/ l . S i nce one meq of p r op i on i c a c i d is p roduced per meq of va l e r i c ac id m e t a b o l i z e d , A n d r e w s and Pearson(1965) conc luded that there w a s a change in the nature of ac id p roduc ing f e rmen ta t i o n w i th re spect to re s idence t ime . Howeve r , they cou ld not e s t ab l i s h the exact reason f o r the change in f e r m e n t a t i o n . The change cou l d be due to a sh i f t in the t ype of m i c r o o r g a n i s m s present , the u t i l i z a t i on o f endogenous re se rve s by ex i s t i n g m i c r o o r g a n i s m s w i t h the p roduc t i on o f d i f f e ren t t y p e s of ac i d s , or changes in the me tabo l i c pa thway s o f ex i s t i ng m i c r o o r g a n i s m s . 54 In the c o n t r o l l e d reactor , the net to ta l v o l a t i l e ac id p roduc t i on i m p r o v e d because of the increase in the net ace t i c ac id p roduc t i on . Redox po ten t i a l in the con t r o l l ed reactor wa s - 3 3 4 m V (Ag/AgCI ref.). The l owe s t redox po tent i a l reported (concurrent ly w i t h methane p roduc t i on ) is - 3 6 0 m V , E c ( - 315mV vs. Ag/AgC I ref . ) (Pfef fer 1979). O p t i m u m pH fo r methane f o r m e r s is be tween 7 and 7.2. A c c o r d i n g to Borchardt(1971), the o p t i m u m pH fo r the ac id f o rme r s is 7. Hence, pH cond i t i on s in the con t r o l l ed pH reactor were c l o s e to o p t i m u m for methane f o r m e r s as w e l l as ac id f o r m e r s . A c c o r d i n g to A n d r e w s and Pearson(1965), methane bacter ia can produce not on l y methane and carbon d i ox i de but a l so other vo l a t i l e ac id s . A c e t i c ac id is p roduced both f r o m the p r imary subs t ra te , by the ac id p roduc ing bac te r i a , and f r o m the longer chain v o l a t i l e ac id s , by the methane bac te r i a . A c e t i c ac id is an eventual p roduct in the m e t a b o l i s m o f a l l the other v o l a t i l e ac id s . The re f o re , the extra ace t i c a c i d s o p roduced , at c o n t r o l l e d pH, might have c o m p e n s a t e d fo r the acet i c ac id lo s t to methane f o r m i n g bac te r i a , thus g i v i ng a higher net to ta l V F A p roduc t i on . The l o s s of p r op i on i c ac id at the c o n t r o l l e d pH is due to the inc reased a c t i v i t y o f the p r op i on i c a c i d a s s im i l a t i n g methane bacte r i a under f avou rab le env i ronmenta l c o n d i t i o n s , as repor ted by Lawrence and McCarty (1969) . Z o e t e m e y e r et al.(1982) a l s o s tud ied the in f luence o f pH and temperature on ac i dogen i c d i s s i m i l a t i o n o f g l u co se in an anaerob ic d i ges to r . In their f i r s t s tudy they s tud ied the e f f e c t o f pH on a c i dogene s i s of g l u co se . Z o e t e m e y e r et al.(1982a) s tud ied seven c o n d i t i o n s o f c on t r o l l ed pH (4.5, 5.0, 5.7, 6.0, 6.4, 6.9, 7.9). Howeve r , in s tead of us ing exper imenta l de s i gn , they s tud ied al l the seven c o n t r o l l e d pH cond i t i o n s at a s ing le temperature(30° C) and conc l uded that o p t i m u m pH fo r the a c i d i f i c a t i o n of g l u co se is be tween 5.7 and 6.0. In their next s tudy, Z o e t e m e y e r et 55 al.(1982b) s tud ied the e f f e c t of temperature on the a c i d o g e n e s i s o f g l uco se , w i t h f ou r teen cond i t i on s of c on t r o l l ed temperatures in a range of 2 1° C to 6 0 ° C be ing used. A l l the exper iment s we re conduc ted at a c o n t r o l l e d pH of 5.8, w h i c h was found to be the o p t i m u m pH for a c i d i f i c a t i o n o f g lucose in their p rev ious s tudy. On the bas i s o f these exper iment s they conc l uded that the op t imum temperature w i th i n the mesoph i l i c range is b e tween 3 6 ° C and 3 8 ° C, and for the the rmoph i l i c range is be tween 51° C and 5 3 ° C. C o m p a r i n g the two s tud ies done by Z o e t e m e y e r et al.(1982) w i t h this s tudy, one can see the impor tance of us ing s t a t i s t i c a l de s i gn in exper iment s dea l i ng w i t h b i o l o g i c a l s y s t e m s . In th i s s tudy , us ing f a c t o r i a l exper imenta l de s i gn , it has been p r o ved s t a t i s t i c a l l y that temperature and pH have an i n te rac t i ve e f f e c t on the V F A p roduc t i on . Hence, the e f f e c t of one parameter on the V F A p roduc t i on can not be s tud ied sepa ra te l y . It means that at 3 0 ° C , a pH of 5.8 may be o p t i m u m fo r V F A p r o d u c t i o n ; but it doe s not nece s sa r i l y mean that at other temperatu res , the o p t i m u m pH fo r the a c i d i f i c a t i o n o f g l uco se is the s ame. In other w o r d s , the acceptance of pH = 5.8 being op t imum fo r al l the fou r teen temperatu res s tud ied by Z o e t e m e y e r et al.(1982) w a s not nece s s a r i l y co r rec t . The re fo re the c o n c l u s i o n s drawn on the bas i s of such exper iment s about the o p t i m u m c o n d i t i o n o f temperature and pH are not de fen s i b l e . 5.3.9 RELAT IONSH IP B E T W E E N SOLUBLE T O C A N D V F A PRODUCT ION Tota l o rgan ic ca rbon w a s measured in al l the exper iment s to de te rm ine if a re la t ionsh ip be tween to ta l o rgan ic ca rbon and to ta l net V F A p r oduc t i on ex i s t s . If a re la t i onsh ip does ex i s t , TOC measu rement s can be used as an ind icator o f the net to ta l V F A p roduc t i on . 56 170 160 150 140 -130 120-110-100 -90-80 70 6 0 -50-4 0 30 20-10-Legend A Soluble T O C X Unaccounted TOC 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 Mean Net Total VFA Prod, (mg/l Acetic Acid) F ig . 5.7 Net To ta l V F A concentrat ion v s . S o l ub l e T o t a l Organic Carbon 57 The re la t i onsh ip be tween to ta l s o l ub le T O C and to ta l net V F A p roduc t i on in the f e rmente r s is s hown in F ig. 5.7. It is quite c lear f r o m F ig . 5.7 that to ta l V F A p roduc t i on has an a lmos t l inear re la t ionsh ip w i t h s o l ub l e TOC . The f o l l o w i n g equat ion f o r the line of best f i t ( cor re la t ion c o e f f i c i e n t , r=0.98) w a s ob ta ined us ing the m e t h o d o f l ea s t - s qua re s . y = 0.659x + 60 where , y = So lub le TOC in mg/l x = To ta l net V F A cone, in mg/l as ace t i c ac id . To determine whether the s o l u b i l i z a t i o n of the organ ic mattter in the ac id phase is pure ly due to the p roduc t i on of the acet i c and p rop i on i c ac id s , it w a s dec i ded to subtract the T O C cont r ibu ted by the to ta l V F A p roduc t i on f r o m the so lub le T O C va lue s ; the remainder o f the T O C w a s then p l o t t ed against the to ta l net V F A p roduc t i on . T O C cont r ibu ted by to ta l V F A p roduc t i on w a s ca l cu la ted by mu l t i p l y i n g t o ta l net V F A cone, in mg/l as ace t i c ac id by 24/60 (as each mo lecu l e o f ace t i c a c i d (mo l . w t . = 60) has t w o carbon atoms(at . w t . - 12)). Hence, Unaccounted TOC = TOC - 24/60(Total net V F A cone.) A re la t ionsh ip be tween unaccounted TOC and net tota l V F A p roduc t i on is a l so p l o t t ed in F ig . 5.7.The f o l l o w i n g equat ion f o r the l ine o f best f i t ( cor re la t ion Coef f . , r=0.88) w a s obta ined us ing the l ea s t - s qua re s m e t h o d , 58 y ' = 0.255x + 60 where , y ' = TOC - 24/60 (VFA ) x = To ta l net V F A cone, in mg/l as a ce t i c a c i d . A very sma l l increase in the value of unaccounted T O C w i th the increase in the tota l net V F A p roduc t i on was o b s e r v e d . It means that s ometh i n g other than ace t i c ac id and p rop i on i c a c i d w a s a l so being s o l ub i l i z ed in a very s m a l l amount. No de f i n i t e c o n c l u s i o n can be drawn about the nature of these un iden t i f i ed p roduc t s , as on l y V F A ana l y s i s f o r a ce t i c , p r op i on i c and buty r i c ac ids w a s done. They may be other vo l a t i t l e f a t t y ac id s (l ike va le r i c , i s o va l e r i c , i s obu ty r i c etc.), w h i c h are k n o w n to be p roduced in the ac id phase, but as they are not s i gn i f i c an t t o the B IO -P p r o c e s s , no ana l y s i s w a s undertaken. The p o s s i b i l i t y o f s o m e other organ ic c o m p o u n d s be ing p roduced cannot be ru led out. 5.3,10 DA I LY PRODUCT ION OF V F A G i v e n a f i x ed v o l ume of f e rmente r , through w h i c h va r y i ng da i l y v o l u m e s o f p r imary s ludge are c y c l e d , it is o f interest to k n o w how the tota l mas s of V F A p roduced per unit t ime va r i e s . Net to ta l p roduc t i on in mg/day fo r the con t r o l l ed and uncon t ro l l ed pH c o n d i t i o n s have been p l o t t ed in F ig s . 5.8 and 5.9 r e s pec t i v e l y . These p l o t s c l ea r l y s h o w that net to ta l V F A p r o d u c t i o n , in mg/day, imp rove s c o n s i s t e n t l y by l owe r i n g the retent ion t ime and ra i s ing the temperature fo r both the c o n t r o l l e d and uncont ro l l ed pH c o n d i t i o n s . In this s tudy , the max imum net da i l y p roduc t i on o f V F A w a s o b s e r v e d at 3 0 ° C and 3 day s re tent ion t i m e . 59 150 - r Acid) 150 -140 -Acetic 1 3 0 -120 -< o /day 100 -9 0 -• (m 9/ 8 0 -• (m 9/ 7 0 -TJ O t 5 0 -VFA Pr 5 0 -4 0 -o 3 0 ^ 2 0 -Net 10 -0 -3 4 5 6 7 Retention Time (Days) Legend A At 10° c X At 2 0 ° c • At 3 0 ° C 8 9 10 F ig. 5.8 Net Da i ly P roduct ion of Tota l V F A v s . Retent ion T i m e at Cont ro l l ed PH 60 160 q Acid) 150 -140-Acetic 130-120-As 110-/day o o O cn Co J , 8 0 -7 0 -po. 6 0 -VFA Pr 5 0 -40-o 3 0 -2 0 -Net "'l""T o o 2 3 4 5 6 7 Retention Time (Days) Legend A At 10° c X At 20° c • At 30° C 10 Fig. 5.9 Net Da i l y P roduc t i on of To ta l V F A v s . Re tent i on T i m e at Uncon t ro l l ed pH 61 In con s i de r i ng the resu l t s of these c o m p l e t e - m i x exper iments on the po tent i a l e f f e c t i v e n e s s of the b i o l o g i c a l phosphorus r e m o v a l p roce s s in a f u l l - s c a l e t reatment p lant, the f o l l o w i n g po in t s must be kept in mind. 1. If an un l im i ted source of p r imary s ludge were ava i l ab le as substrate f o r the anaerob ic o r gan i sms in the fermenter.and if the v o l ume of the f e rmente r were f i xed (eg. an ex i s t i ng tank w a s be ing used fo r this pu rpose ) then the shorter re tent ion s y s t e m w o u l d p r o v i de the greatest mas s o f V F A ' s per unit t ime , fo r f eed ing to the ma in phosphorus r e m o v a l p roce s s (as s hown in F ig s . 5.8 and 5.9). 2. If it is important to max im i ze the p roduc t i on o f V F A ' s f r o m the tota l a va i l ab le mass of p r imary s l udge , then the c oncen t r a t i on o f V F A ' s p roduced in the c o m p l e t e - m i x anaerob ic fe rmenter is the govern ing c r i te r i a f o r max im i z i ng the input of s i m p l e subs t ra te t o the b i o l o g i c a l phosphorus remova l f a c i l i t y . S i n ce p r imary s ludge a v a i l a b i l i t y in most mun i c ipa l w a s t e w a t e r s is l o w , it is the m a x i m i z a t i o n o f concen t ra t i on as s h o w n in F igs. 5.1 and 5.2 that is important to the b i o l o g i c a l P - r e m o v a l p roce s s . Hence, the des i gn s i z i ng and temperatu re of the f e rmente r is of paramount impor tance . 5.3.11 O X I D A T I O N REDUCT ION P O T E N T I A U O R P I ORP measurement s we re quite s tab le dur ing the f i na l 7 days of s teady s tate ana l y s i s in a l l the 18 reac to r s . No t rends o f any var ia t ions in the ORP readings were no t i ced ove r the pe r i od o f 7 day s . ORP in the reac to r s va r i ed f o r m - 265 to - 3 9 5 m V (Ag/AgCI ref.) in th is s tudy. A s ment i oned ear l ie r , o x i d a t i o n - r e d u c t i o n po tent i a l wa s m o n i t o r e d throughout the exper iment s to exp lo re the p o s s i b i l i t y of us ing it 62 F ig . 5.10 Re lat ionsh ip Between Mean ORP vs . Net A c e t i c A c i d P roduct ion 63 F i g . 5.11 Re lat ionsh ip Between Mean ORP vs . Net P r o p i o n i c A c i d P roduct i on 64 180 -i CD Z 20 J — i 1 1 1 1 1 1 1 — - 4 0 0 - 3 8 0 - 3 6 0 - 3 4 0 - 3 2 0 - 3 0 0 - 2 8 0 - 2 6 0 ORP (mV). Ag/AgCI REF. F ig . 5.12 Re lat ionsh ip Between Mean ORP v s . T o t a l Net V F A P roduct ion 65 as an i nd i ca to r of V F A p roduc t i on . Re la t i on sh ip s be tween ORP and net a ce t i c , p r op i on i c and tota l V F A p roduc t i on have been p l o t t ed in F igs. 5.10, 5.11 and 5.12 r e spec t i v e l y . Upon exam ina t i on , it is i m p o s s i b l e to d i s ce rn a de f i n i t e re l a t i on sh ip be tween the net ac id p roduc t i on and ORP. The on ly c o n c l u s i o n one can draw is that there is no de f i n i te re la t ionsh ip be tween the t w o . H o w e v e r , s ince measured ORP is a f f e c t e d by temperature, pH and other va r i ab l e s , it w a s dec i ded to exp lo re the ORP vs. net V F A p roduc t i on re la t i onsh ip f o r the d i f f e ren t set s of temperature and pH comb i na t i o n s . F ig s . 5.13 to 5.18 s h o w the re la t i on sh ip s be tween ORP and V F A p roduc t i on fo r d i f f e ren t c o m b i n a t i o n s o f temperature and pH. F igs . 5.13 and 5.14 s h o w the re la t i onsh ip be tween ORP and net V F A p roduc t i on at 10°C f o r c on t r o l l ed and uncon t ro l l ed pH cond i t i on s . In both ca se s , a higher net V F A p roduc t i on w a s a s s o c i a t e d w i t h l ower ORP. In the uncon t ro l l ed pH reactor , the net ace t i c ac id and to ta l V F A p roduc t i on s tar ted d ropp ing when the ORP w a s reduced b e l o w - 3 0 0 m V , ind icat ing that the c r i t i ca l ORP l ies s o m e w h e r e c l o s e to - 3 0 0 m V fo r net ac id p roduc t i on at 10°C and uncont ro l l ed pH. But at c o n t r o l l e d pH, the net p roduc t i on o f acet i c ac id and tota l V F A keep on i nc reas ing , even when the ORP w a s as l o w as - 3 8 5 m V (Ag/AgCI ref.). S i m i l a r l y , f o r 2 0 ° C temperature and uncont ro l l ed pH (Fig. 5.16), the net t o ta l V F A p roduc t i on inc reased w i t h dec rea s i ng ORP. But the net ace t i c ac id p roduc t i on d ropped when the ORP w a s further l owe red than - 3 6 6 m V , w h i l e net p r op i on i c ac id p roduc t i on inc reased as the ORP w a s l o w e r e d f r o m - 3 6 6 m V to - 3 7 8 m V (Ag/AgCI ref.). For the c on t r o l l ed pH ope ra t i on at 2 0 ° C (Fig. 5.15), the c r i t i c a l ORP w a s around - 3 6 0 m V (Ag/AgCI ref.) f o r net V F A p roduc t i on . 66 F ig. 5.13 Relat ionship Between ORP and Net V F A p r oduc t i on for 10°C and Con t ro l l ed pH Operat ion 67 70-, u o o 60 H % u o 8 5 0 H 40 30 20 H io H cn J , c q ~o D o Q. > C D -310 Legend A Total VFA X Acetic Acid O Propionic Acid -300 -290 - 2 8 0 Mean ORP (mv) - 2 7 0 •260 Fig. 5.14 Relationship Between ORP and Net VFA production for 10°C and Uncontrolled pH Operation Fig. 5.15 Relat ionsh ip Be tween ORP and Net V F A p r o d u c t i o n for 2 0 ° C and Cont ro l l ed pH Operat ion 69 Fig. 5.16 Re lat ionsh ip Between ORP and Net V F A p r oduc t i on for 20°C and Uncont ro l l ed pH Operat ion 70 F ig. 5.17 Re lat ionsh ip Between ORP and Net V F A p roduc t i on for 3 0 ° C and Con t ro l l ed pH Operat ion 71 F i g . 5.18 Relat ionsh ip Between ORP and Net V F A p roduc t i on for 3 0°C and Uncont ro l l ed pH Operat ion 72 A t 3 0 ° C temperature and, fo r c o n t r o l l e d as w e l l as uncon t ro l l ed pH, no de f i n i te re la t i onsh ip w a s ob se r ved be tween mean ORP and net V F A p roduct i on (Fig. 5.17 and 5.18). A c c o r d i n g to Lawrence and McCar ty (1969 ) , the m i n imum ce l l re s idence t ime requi red fo r ace t i c ac id a s s i m i l a t i n g methane bacter ia is 4.2 days . A c c o r d i n g to P f e f f e r (1979), the ORP must be at least as l o w as - 3 1 5 m V (Ag/AgCI r e f . ) f o r ' methane p roduc t i on t o occur . In al l the exper iment s conduc ted at 3 0 ° C , the ORP lay between - 3 3 4 m V to - 3 9 6 m V (Ag/AgCI ref.). M o r e o v e r , it is a l so known that the methane f o r m i n g bacte r i a are more ac t i ve at higher temperatures . Hence, there is a higher p r obab i l i t y of methanogenes i s occu r ing at 3 0 ° C operated reac to r s , wh ich might exp la in the lack of a de f i n i t e re la t i onsh ip be tween V F A p roduc t i on vs . ORP. Hughes (1979) has exp re s sed doubt about the s i g n i f i c ance of ORP in the methanogen ic phase o f anaerob ic d i ge s t i on . The ma in redox pairs ( C O 2 / H 2 ) are generated by a w i d e range o f o r gan i sms and thus their c oncen t r a t i on externa l l y w i l l d i f f e r w i d e l y at d i f f e ren t t i m e s . They w i l l a l s o d i f f e r f r o m the concent ra t i on w i t h i n the methane bac te r i a , where reduct ion is ca r r ied out. However , if the measu rement s were c o m b i n e d w i t h gas y i e l d , ( C O 2 / H 2 / C H 4 ) , f a t t y ac ids and A T P , measu rement s of ORP might b e c o m e a u se fu l c on t r o l me thod . 73 CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS 6 .1. CONCLUSIONS The f o l l o w i n g ' c o n c l u s i o n s can be made f r o m the d i s cu s s i on of the re su l t s : 1. Of the 18 c o m b i n a t i o n s of re tent ion t ime , temperature and pH t e s t e d , the max imum net to ta l V F A concen t ra t i on was ach ieved at 3 0 ° C and 6 days re tent ion t ime. The con t ro l of pH to a va lue of 7 d id not make any s i gn i f i can t change in the to ta l net V F A p roduc t i on . 2. Temperatu re , pH and retent ion t ime are in teract i ve in their e f f e c t s on V F A p roduc t i on . Hence, the e f f e c t o f change in one var iab le is dependent on the va lues o f the other t w o va r i ab le s . In e s sence then, the e f f e c t of one parameter on the net V F A p roduc t i on cannot be de te rm ined in i s o l a t i o n . 3. A t the l ower temperature (10°C) , the con t ro l o f pH to a va lue o f 7 s eems to improve the net to ta l V F A p roduc t i on . But at higher temperature (30°C), it doe s not a f f e c t the net p roduc t i on s i g n i f i c an t l y . 4. The con t r o l of pH w a s f ound to a f f e c t the re la t i ve net p roduc t i on o f ace t i c and p rop ion i c ac id s . 5. Net to ta l V F A p roduc t i on c o n s i s t e n t l y imp roved w i t h the increase in temperature be tween 10°C to 3 0 ° C . 6. A t l ower temperature ( 10°C and 2 0 ° C ) the increase in re tent ion t ime he lped to improve the net V F A p r o d u c t i o n / 73 74 7. Extend ing the re tent ion t ime to 9 days at 3 0 ° C appears to be det r imenta l to the net V F A p roduct i on because o f its use as substrate by methane p roduc ing bac te r i a . 8. A de f i n i te re la t ionsh ip be tween so lub le TOC and to ta l net V F A p roduc t i on w a s found to ex i s t . The re fo re , s o lub le TOC can be used as a v iab le ind icator of the tota l net V F A p roduc t i on , once the re l a t i on sh ip has been de te rmined fo r the s p e c i f i c s ewage and p roce s s . 9. There s eems to be s o m e f o r m of re la t ionsh ip be tween net V F A p roduc t i on and ORP at 10°C and 2 0 ° C . On average, net V F A p roduc t i on increased w i t h the l ower i ng of ORP. 10. A t 3 0 ° C temperature, no de f i n i te re la t i onsh ip w a s ob se r ved be tween ORP and net V F A p roduc t i on . 11. The use of ORP mesu rement s as ind icator o f V F A p roduc t i on appears to be doubt fu l un less temperature and pH va lues are taken into account . 6.2. RECOMMENDATIONS 1. Because o f the exp l o ra to r y nature of this s tudy, on l y three c ond i t i o n s o f each of temperature and retent ion t ime and two c ond i t i o n s of pH w e r e s tud ied. In future s tud ies , more c ond i t i o n s of temperature , re tent ion t ime and pH shou ld be s tud ied to de te rmine the o p t i m u m c o m b i n a t i o n s of va r i ab le s f o r m a x i m u m net V F A p roduc t i on . 2. There is a potent ia l f o r a s tudy dea l ing w i t h the i n ve s t i g a t i on o f the un iden t i f i ed p roduct s f o r m e d during the ac id phase o f anaerob ic d i g e s t i o n . This requires ana l y s i s f o r V F A ' s other than ace t i c , p r op i on i c and butyr ic ac ids and other c ompound s . 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" , Wate r Research, 16(1982), 313 -321 . 80 A P P E N D I X DA I LY NET V O L A T I L E F A T T Y A C I D S PRODUCT ION D A T A RECORD DAYS IN STEADY STATE TREATMENT VARIABLES ******************* RET. TIME TEMP. C pH C/U NET NET ACETIC PROP. ACID ACID PROD. PROD, (mg/L) (mg/L) NET VFA PROD. (mg/L as a c e t i c a c i d ) 1 1 2 2 3 3 4 4 5 5 6 6 7 9 9 9 9 9 9 9 9 9 9 9 9 9 1 0 10 10 10 10 10 10 10 10 10 10 10 10 U U U U U U u u u u u u u 50 52 59 4 6 54 6 0 48 4 9 54 5 5 5 0 53 5 5 1 5 18 23 2 0 1 7 2 0 18 1 5 1 5 2 2 2 0 1 6 2 2 6 2 . 2 6 6 . 6 7 7 . 6 6 2 . 2 6 7 . 8 7 6 . 2 6 2 . 6 61 . 2 6 6 . 2 7 2 . 8 6 6 . 2 6 6 . 0 7 2 . 8 80 10 u 50 19 6 5 . 4 1 1 2 2 3 3 4 4 5 5 6 6 7 7 3 3 3 3 3 3 3 3 3 3 3 3 3 3 10 1 0 10 1 0 10 10 10 10 10 10 10 10 10 10 u u u u u u u u u u u u u u 48 42 40 50 35 48 50 50 45 38 48 34 4 5 46 5 5 6 6 4 4 5 7 6 3 4 4 5 5 5 2 . 1 4 6 . 1 4 4 . 9 5 4 . 9 3 8 . 2 5 1 . 2 5 4 . 1 5 5 . 7 4 9 . 9 4 0 . 4 51 . 2 3 7 . 2 4 9 . 1 5 0 . 1 1 1 2 2 3 3 4 4 5 5 9 9 9 9 9 9 9 9 9 9 10 10 10 1 0 10 10 10 10 10 10 C C C C C C c c c c 7 5 78 70 65 70 80 63 7 5 7 5 68 30 32 3 5 28 4 0 2 5 2 7 28 33 33 9 9 . 3 1 0 3 . 9 9 8 . 4 8 7 . 7 1 0 2 . 4 1 0 0 . 3 8 4 . 9 9 7 . 7 1 0 1 . 8 9 4 . 8 10 10 10 10 c c c c 68 70 7 5 66 3 5 38 28 27 9 6 . 4 1 0 0 . 8 9 7 . 7 8 7 . 9 30 30 30 3 0 3 0 30 3 0 30 3 0 30 30 30 3 0 3 0 U U u u u u u u u u u u u u 33 30 3 2 3 5 4 0 38 39 30 33 34 3 3 3 6 38 3 7 8 4 8 0 88 8 0 93 8 2 81 8 2 8 0 8 5 84 9 5 8 6 8 2 1 0 1 . 1 9 4 . 9 1 0 3 . 4 9 9 . 9 1 1 5 . 4 1 0 4 . 5 1 0 4 . 7 9 6 . 5 9 7 . 9 1 0 2 . 9 1 0 1 . 1 1 1 3 . 0 1 0 7 . 7 1 0 3 . 5 3 0 30 30 30 3 0 3 0 3 0 U U U U U U U 90 9 5 8 2 8 5 8 6 9 0 9 3 8 3 8 0 8 5 78 76 76 7 9 1 5 7 . 3 1 5 9 . 9 1 5 0 . 9 1 4 8 . 2 1 4 7 . 6 1 5.1 . 6 1 5 7 . 1 30 30 30 30 30 30 30 U U U U U U U 8 5 90 8 8 8 9 78 8 5 8 9 88 8 0 8 0 7 5 7 7 8 0 8 3 1 5 6 . 4 1 5 4 . 9 1 5 2 . 9 1 4 9 . 8 1 4 0 . 4 1 4 9 . 9 1 5 6 . 3 30 30 30 30 30 30 30 30 30 30 30 30 30 30 C C C C C C C C C C C C C C 70 7 5 78 7 5 79 81 8 2 7 2 7 0 7 7 77 7 9 8 0 8 4 3 0 28 3 0 32 3 0 3 3 34 34 3 3 3 0 3 3 2 8 3 6 3 3 9 4 . 3 9 7 . 7 1 0 2 . 3 1 0 0 . 9 1 0 3 . 3 1 0 7 . 8 1 0 9 . 6 9 9 . 6 9 6 . 8 1 0 1 . 3 1 0 3 . 8 101 . 7 1 0 9 . 2 1 1 0 . 8 30 30 30 3 0 C C c c 100 110 9 5 105 40 4 5 3 5 4 0 1 3 2 . 4 1 4 6 . 5 1 2 3 . 4 1 3 7 . 4 30 30 30 3 0 3 0 3 0 30 30 3 0 30 C C C C C C C C C C 1 06 108 1 0 5 9 5 1 0 5 9 9 1 08 1 00 1 0 5 100 40 4 5 42 4 3 4 5 4 0 43 4 5 42 4 6 1 3 8 . 4 1 4 4 . 5 1 3 9 . 1 1 2 9 . 9 1 4 1 . 5 1 3 1 . 4 1 4 2 . 9 1 3 6 . 5 1 3 9 . 1 1 3 7 . 3 30 3 0 3 0 3 0 3 0 3 0 30 30 3 0 3 0 3 0 30 3 0 3 0 C C C C C C C C C C C C C C 90 8 5 92 94 96 98 98 9 0 9 2 8 4 94 98 99 100 70 7 0 7 3 7 5 7 5 7 3 7 5 74 7 2 7 2 7 5 7 3 73 77 1 4 6 . 8 1 4 1 . 8 1 5 1 . 2 1 5 4 . 8 1 5 6 . 8 1 5 7 . 2 1 5 8 . 8 1 5 0 . 0 1 5 0 . 4 1 4 2 . 4 1 5 4 . 8 1 5 7 . 2 1 5 8 . 2 1 6 2 . 4 30 U 70 48 1 0 8 . 9 30 30 30 30 30 30 30 30 30 30 30 3 0 3 0 U U U U U U U U U U U U U 68 78 7 9 8 0 7 5 74 7 5 71 82 8 0 74 7 4 7 2 44 4 3 4 6 4 9 5 0 4 9 5 0 4 5 48 47 42 48 4 7 1 0 3 . 7 1 1 2 . 9 1 1 6 . 3 1 1 9 . 7 1 1 5 . 5 1 1 3 . 7 1 1 5 . 5 1 0 7 . 5 1 2 0 . 9 1 1 8 . 1 1 0 8 . 1 1 1 2 . 9 1 1 0 . 1 20 2 0 2 0 2 0 2 0 20 2 0 2 0 2 0 2 0 2 0 20 2 0 C C C C C C C C C C C C C 7 2 68 6 7 7 5 78 8 2 8 2 7 0 7 0 7 2 7 5 7 4 78 3 9 3 7 4 0 4 2 4 4 4 3 44 41 40 44 4 8 48 4 4 1 0 3 . 6 9 8 . 0 9 9 . 4 1 0 9 . 1 1 1 3 . 7 1 1 6 . 9 1 1 7 . 7 1 0 3 . 2 1 0 2 . 4 1 0 7 . 7 1 1 3 . 9 1 1 2 . 9 1 1 3 . 7 20 7 6 4 5 1 1 2 . 5 20 2 0 20 20 20 20 20 20 2 0 2 0 20 2 0 2 0 20 C C C C C ' C C C C C C C C C 80 78 7 5 8 4 8 5 8 6 8 5 78 8 0 8 0 8 3 8 4 8 6 8 0 5 5 56 48 52 51 5 3 5 0 5 0 5 0 51 54 53 5 3 4 9 1 2 4 . 6 1 2 3 . 4 1 1 3 . 9 1 2 6 . 2 1 2 6 . 4 1 2 9 . 0 1 2 5 . 5 1 1 8 . 5 1 2 0 . 5 1 2 1 . 4 1 2 6 . 8 1 2 7 . 0 1 2 9 . 0 1 1 9 . 7 2 0 20 2 0 2 0 2 0 20 20 20 2 0 2 0 U U U U U U U U U U 63 60 6 5 68 7 0 7 0 6 9 6 0 6 3 6 3 58 58 61 57 6 2 6 2 6 5 5 5 59 61 1 1 0 . 0 1 0 7 . 0 1 1 4 . 5 1 1 4 . 2 1 2 0 . 3 1 2 0 . 3 1 2 1 . 7 1 0 4 . 6 1 1 0 . 8 1 1 2 . 5 20 20 20 2 0 U U u u 65 69 69 70 56 6 2 6 0 6 2 1 1 0 . 4 1 1 9 . 3 1 1 7 . 6 1 2 0 . 3 20 20 2 0 20 20 20 2 0 2 0 2 0 20 20 2 0 2 0 2 0 C C C C C C C C C C C C C C 63 6 5 68 57 6 5 63 60 58 67 63 59 6 5 66 66 28 28 3 0 34 3 2 3 2 2 7 2 5 3 0 3 3 3 2 3 2 3 6 2 9 8 5 . 7 8 7 . 7 9 2 . 7 8 4 . 6 9 0 . 9 8 8 . 9 81 . 9 7 8 . 3 91 . 3 8 9 . 8 8 4 . 9 9 0 . 9 9 5 . 2 8 9 . 5 2 0 2 0 2 0 20 20 2 0 2 0 U U U U U U U 50 46 48 41 52 53 52 2 0 18 2 2 1 5 1 1 18 20 6 6 . 2 6 0 . 6 6 5 . 8 5 3 . 2 6 0 . 9 6 7 . 6 6 8 . 2 20 2 0 2 0 2 0 2 0 2 0 20 U U U U U U U 44 42 4 5 47 52 50 54 18 1 5 20 18 18 20 2 0 5 8 . 6 5 4 . 2 6 1 . 2 61 . 6 6 6 . 6 6 6 . 2 7 0 . 2 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 20 2 0 2 0 20 U U U U U U U U U U U U U U 7 5 2 2 8 0 70 84 8 4 8 2 78 78 8 2 7 2 81 8 4 8 0 2 5 2 2 28 33 30 31 28 2 5 3 0 26 33 2 9 3 2 3 0 9 5 . 3 9 5 . 8 1 0 2 . 7 9 6 . 8 1 0 8 . 3 1 0 9 . 1 1 0 4 . 7 9 8 . 3 1 0 2 . 3 1 0 3 . 1 9 8 . 8 1 0 4 . 5 1 0 9 . 9 1 0 4 . 3 10 10 10 10 C C C C 60 62 59 58 3 0 31 3 5 33 8 4 . 3 8 7 . 1 8 7 . 4 8 4 . 8 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1 0 10 1 0 1 0 10 10 1 0 10 c c c c c c c c c c u u u u u u u u u u u u u u 53 6 5 61 59 64 57 5 9 5 6 7 0 6 0 48 4 3 53 52 54 4 9 50 5 0 42 5 0 5 3 5 5 52 4 5 2 9 2 5 3 0 3 2 3 0 34 31 3 3 2 8 3 0 2 0 2 5 18 2 2 2 0 1 7 19 19 2 0 2 5 2 4 19 15 18 7 6 . 5 8 5 . 3 8 5 . 3 8 4 . 9 8 8 . 3 8 4 . 6 8 4 . 1 8 2 . 8 9 2 . 7 8 4 . 3 6 4 . 2 6 3 . 3 6 7 . 6 6 9 . 8 7 0 . 2 6 2 . 8 6 5 . 4 6 5 . 4 5 8 . 2 7 0 . 3 7 2 . 5 7 0 . 4 6 4 . 2 5 9 . 6 10 10 C C 4 5 38 15 19 5 7 . 2 5 3 . 4 10 1 0 10 10 10 10 1 0 10 1 0 10 10 10 c c c c c c c c c c c c 43 52 48 51 52 48 40 4 5 56 50 50 48 2 2 2 2 1 3 1 3 20 1 6 1 7 20 28 2 0 18 21 6 0 . 8 6 9 . 8 5 8 . 5 61 . 5 6 8 . 2 61 . 0 5 3 . 8 61 . 2 7 8 . 7 6 6 . 2 6 4 . 6 6 5 . 0 

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