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Attached growth nitrification using Ringlace media Setter, Kevin J. 1995

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ATTACHED GROWTH NITRIFICATION USING RINGLACE® MEDIA B y KEVIN J. SETTER B.A.Sc. (Bio-Resource Engineering) The University O f British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Bio-Resource Engineering We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A Apri l 1995 © Kevin J. Setter , 1995 In p resen t ing this thesis in partial fu l f i lment o f t h e r e q u i r e m e n t s f o r an advanced d e g r e e at the Univers i ty o f Brit ish C o l u m b i a , I agree that t h e Library shall make it f reely available f o r re ference and s tudy. I fu r ther agree that pe rmiss ion f o r ex tens ive c o p y i n g o f this thesis f o r scholar ly pu rposes may be g r a n t e d by t h e head o f m y d e p a r t m e n t o r by his o r her representat ives. It is u n d e r s t o o d that c o p y i n g o r pub l i ca t i on o f this thesis fo r f inancial gain shall n o t be a l l o w e d w i t h o u t m y w r i t t e n permiss ion . D e p a r t m e n t The Univers i ty o f Brit ish C o l u m b i a Vancouver , Canada DE-6 (2/88) ABSTRACT This thesis reports on an attempt to grow nitrifying bacteria attached on the submerged growth media Ringlace® in the aerobic zone of a pilot scale Biological Enhanced Phosphorus Removal (BEPR) process treating municipal sewage. The Ringlace® media was a fibrous rope modified PVC material stretched over frames that were immersed into the process. Nitrification on the support media was meant to enhance overall process nitrifying capacity. The overall process operating parameters HRT, SRT, and DO were manipulated to investigate their effects on the nitrification rate of the bacteria attached to the support media. Growth on the media consisted of an array of higher life forms and bacteria. Worm infestation on the media appeared to increase with increased DO concentration. Cyclic anaerobic periods of 12 to 24 hours controlled worm infestations at operating DO levels of 3.5 mg/L and lower but proved to be unsuccessful at tested DO concentrations above that. Sludge settling in the test process improved over the control process during a run with 6 hour HRT, 4 day SRT, 5.5 mg/L DO level, and 18 °C temperature. During this run the control process average specific nitrification rate was 2 mg-N/gTSS-hr and the test process average rate was 0.5 mg-N/gTSS-hr. This run had the highest DO level of the experiment and the Ringlace® support media had the highest amount of worms observed on it for the entire experiment. It is believed that the significant reduction in nitrification rate and the improved sludge settling were a result of interference from the worms. The results of batch testing Ringlace® frames removed from the flow through system throughout the experiment showed the biomass attached to the Ringlace® support media ii never exhibited specific ammonia uptake rate significantly greater than zero at the 5 % significance level. The suspended growth portion did display specific ammonia uptake rates which were approximately the same as rates reported in the literature for similar suspended growth studies. In selected batch tests the biomass on the media was shown to take up soluble carbon indicated by B O D 5 measurements. iii TABLE OF CONTENTS Page ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENT ix CHAPTER 1 INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 3 2.1 A T T A C H E D G R O W T H S Y S T E M S 4 2.2 E N V I R O N M E N T A L P A R A M E T E R E F F E C T S O N A T T A C H E D G R O W T H N I T R I F I C A T I O N 6 2.3 O P E R A T I N G P A R A M E T E R E F F E C T S O N A T T A C H E D G R O W T H N I T R I F I C A T I O N 8 2.4 P R E V I O U S S T U D I E S O N A T T A C H E D G R O W T H S Y S T E M S 10 2.5 O B J E C T I V E S 13 CHAPTER 3 MATERIALS AND METHODS 14 3.1 E X P E R I M E N T A L S E T - U P 14 3.1.1 Ringlace® 16 3.1.2 Ringlace® experimental configuration 17 3.1.2.1 Volume ratio and specific density 17 3.1.2.2 Modular design 18 3.1.3 Process H R T and SRT operation and control 19 3.1.4 D O control 20 3.1.5 Temperature logging 21 3.2 A N A L Y T I C A L P R O C E D U R E S 21 3.2.1 Batch test description and rationale 21 3.2.1.1 Batch test apparatus 22 3.2.1.2 Batch test procedure 24 3.2.2 Pilot scale process sampling schedule 25 3.2.3 Sample analysis methods 26 3.2.3.1 N O : ; and N H 3 26 iv Table of Contents Page 3.2.3.2 Suspended solids 26 3.2.3.3 Volatile suspended solids 26 3.2.3.4 Soluble B O D s 26 3.3 S T A T I S T I C S 27 3.3.1 N O x and N H 3 sampling precision 27 3.3.2 M L V S S 27 3.3.3 Solids retention time of runs 29 CHAPTER 4 RESULTS AND DISCUSSION 30 4.1 B A T C H T E S T R A T E S U M M A R Y 30 4.2 D E T A I L E D B A T C H T E S T I N G A N A L Y S I S 38 4.2.1 R u n l 38 4.2.2 Run 2 40 4.2.3 Run 3 41 4.2.4 Run 4 42 4.2.5 Run 5 44 4.2.6 Run 6 45 4.3 S U R R O U N D I N G C A R B O N E F F E C T 46 4.4 S U R F A C E A R E A E F F E C T 49 4.5 S L U D G E S E T T L I N G C H A R A C T E R I S T I C S 50 4.6 R1NGLACE® B I O M A S S 53 4.6.1 Ringlace® biomass characterization 53 4.6.2 Worm effect and growth 56 4.6.3 Worm population control 57 4.6.4 Ringlace® solids mass estimation 58 CHAPTER 5 CONCLUSIONS 62 5.1 C O N C L U S I O N S 62 5.1.1 P R I M A R Y C O N C L U S I O N 62 5.1.2 S E C O N D A R Y C O N C L U S I O N S 63 5.2 R E C O M M E N D A T I O N S 64 REFERENCES 65 APPENDIX A - BATCH TEST RESULTS 69 APPENDIX B - PROCESS OPERATING AMMONIA DATA 76 v LIST OF TABLES TABLE Page Table 3.1. Volume Fractions o f Process Zones 15 Table 3.2. H R T and SRT Factor Levels 19 Table 3.3. Batch Reactor Equipment 23 Table 3.4. Pilot Scale Process Sampling Schedule 25 Table 3.5. N O x - N and N H 3 - N Sampling Precision 27 Table 3.6. Process M L V S S as a Percentage of M L S S 28 Table 3.7. Volatile Fraction of Media Solids 28 Table 3.8. Average and Standard Deviation of SRT 29 Table 4.1. A N O V A of Total Ammonia Uptake Rate Data 32 Table 4.2. Ammonia Uptake Rates from Literature 34 Table 4.3. A N O V A of Run 1 Ammonia Uptake Rate Data 40 Table 4.4. Soluble B O D 5 (mg/L)Values for Six Batch Tests at Assorted D O , SRT, and H R T Conditions 48 Table 4.5. A N O V A of SVI , Run 4, 5, and 6 51 Table 4.6. A N O V A of M L S S , and Effluent Suspended Solids, Run 6 51 Table 4.7. Mass Estimation of Solids on Media „ 59 vi LIST OF FIGURES FIGURE PAGE Figure 3.1. Simplified U C T Process 15 Figure 3.2. Cubical Frame For Mounting Ringlace® 18 Figure 3.3. Typical D O Curve Obtained With Min /Max Control 21 Figure 3.4. Batch Reactor 23 Figure 3.5. Batch Reactor Monitoring and Control 24 Figure 4.1. Batch Test Rate Data Summary, Run 1 31 Figure 4.2. Batch Test Rate Data Summary, Run 3,4, 5 31 Figure 4.3. Batch Test Rate Data Summary, Run 6 32 Figure 4.4. Process Sludge Volume Index, Run 4, 5, and 6 52 Figure 4.5. Process M L S S of Run 4, 5, and 6 52 Figure 4.6. Process Effluent SS Run 4, 5, and 6 53 Figure 4.7. Stalk Ciliates 54 Figure 4.8. Rotifers 55 Figure 4.9. Worms On Media 55 Figure 4.10. Cubical Frame With Attached Biomass, 1 60 Figure 4.11. Cubical Frame With Attached Biomass, 2 61 vii LIST OF ABBREVIATIONS A A-side settled Anoxic zone supernatant A - A A-side attached solids batch test A-S A-side suspended solids batch test A-S/A A-side suspended and attached solids batch test BEPR biological enhanced phosphorus removal BNR biological nutrient removal BOD 5 five day biochemical oxygen demand B-S B-side suspended solids batch test df degrees of freedom DO dissolved oxygen eff effluent eff SS effluent suspended solids F F statistic Fcrit critical F statistic H 2 S 0 4 sulphuric acid HRT hydraulic retention time M C R T mean cell residence time MLSS mixed liquor suspended solids MLVSS mixed liquor volatile suspended solids N H 3 - N ammonia nitrogen NOx-N nitrate and nitrite nitrogen ORP oxidation reduction potential PO4-P ortho phosphate phosphorus RBC rotating biological contactor SRT solids retention time SVI sludge volume index V F A volatile fatty acid WWTP wastewater treatment plant viii ACKNOWLEDGEMENT I wish to express my sincere appreciation to the following people for their support and assistance throughout the entire study. This work was made a smooth process by them. Dr. William K . Oldham, Professor of the Civi l Engineering Department. Dr. Oldham provided guidance, advice and the opportunity for me to attempt a Master's degree. Dr. K . Victor L o Professor from the Bio-Resource Engineering department, and Dr. William D . Ramey of the Microbiology Department for serving on my committee and offering advice and assistance. Ping Liao and Adeline Chen, staff o f the Bio-Resource Engineering lab and Jufang Zhou, Paula Parkinson, Susan Harper, staff of the Civi l Engineering lab, all for their excellent assistance and advice in the analysis of data. Bud Bennett of Ringlace® products for advice and the supply of Ringlace® to the study. Frederic A . Koch, research associate in the Environmental Engineering group for constructive criticism and assistance in experimental planning. Ron Dolling for comic relief and assistance in electronics related maintenance at the pilot plant. Guy Kirsch, and Dick Postgate of the civil shop for pilot plant maintenance, and help with constructing the experiment. Fellow graduate students Allen Gibb, Heather Atherton, and Angus Chu for their friendship, advice and support during my study. Special thanks to my girlfriend Margarita for her delicious lunches, her perseverance through bad days when batch tests went wrong, and for listening to my thoughts. This research was carried out under a grant from the Science Council o f British Columbia. Their financial support is gratefully acknowledged. ix CHAPTER ONE INTRODUCTION Stringent government regulations and increasing populations have forced municipalities to achieve higher effluent quality from wastewater treatment plants. Increased standards mean that nitrogen as well as carbon wil l have to be removed. The removal o f nitrogen requires the conventional activated sludge process be upgraded to a biological nutrient removal ( B N R ) process. To meet these new upgrades existing activated sludge treatment plants wil l require retrofitting to B N R . The B N R process biologically removes nitrogen and phosphorus from wastewater. Nitrogen removal is achieved by sequential oxidation and reduction of nitrogen compounds in the mixed liquor. Ammonia nitrogen in raw wastewater enters the process and is first oxidized to nitrite and then nitrate by autotrophic bacteria. This oxidation procedure carried out in the aerobic zone o f the process is called nitrification. The nitrite and nitrate compounds are then recycled with the mixed liquor to the anoxic zone where heterotrophic bacteria reduce them to nitrogen gas that can be released to the atmosphere. This second step is called denitrification. The space consuming step in the B N R process is nitrification. Nitrifying autotrophic bacteria have a long regeneration time. A long regeneration time means the solids retention time (SRT) must be long enough to allow for sufficient regeneration o f the autotrophic bacteria. A long SRT requires large process tank volumes i f the M L S S concentration is to be kept reasonably low. Large tank volume presents a problem when existing processes with limited space and funds Chapter 1. Introduction cannot afford to expand to the tank sizes required for nitrification in a B N R plant retrofit. One solution to the problems caused by large tank size is to increase the SRT without increasing the tank volume. SRT will be increased by the attachment of nitrifying bacteria within the aerobic zone to a solid stationary support media. Attachment wil l eliminate the unintentional loss of the nitrifying bacteria through sludge wasting or washout over the effluent weir. The attached nitrifiers wi l l then have time to fully regenerate and the total nitrifier population in the system wil l increase which wil l increase overall process nitrification capacity. A s a result o f increased population the conventional SRT necessary for nitrification based on the concentration of suspended solids nitrifiers will be sufficiently reduced thus lowering the process design tank volume. Nitrification in attached biomass processes can be sensitive to operational parameters such as H R T and SRT. A better understanding of the effect that these parameters have on attached growth processes would help in process operation and control. In the application of attached media to a full scale plant, information about the effects o f these operating parameters wi l l be very useful to operators faced with process upsets and failures. 2 CHAPTER TWO LITERATURE REVIEW The discovery of the nitrification process began in the late 1800's when the first experiments lead to isolation of the autotrophic bacteria Nitrosomonas and Nitrobacter. Painter (1970) reported these two bacteria were not the only bacteria capable of nitrification however; they were the most common species isolated from the nitrifying process. The unit cell yield chemical equations for converting ammonium to nitrite and nitrite to nitrate by Nitrosomonas and Nitrobacter respectively are shown below (Barnes et al., 1983). Nitrosomonas cell yield. 55 N H 4 + + 76 0 2 + 109 H C C V - C 5 H 7 N 0 2 + 54 N 0 2 ' + 57 H 2 0 + 104 H 2 C 0 3 Nitrobacter cell yield. 400 N 0 2 ' + N H 4 + + 4 H 2 C 0 3 - +HCCV + 195 0 2 - C 5 H 7 N 0 2 + 3 H 2 0 + 400 N 0 3 " In the mid seventies Barnard (1975) reported using these naturally occurring reactions for treating municipal wastewater in a single sludge system. Nitrification reactions shown above along with naturally occurring denitrification reactions could remove nitrogen from wastewater without chemical addition. Total nitrogen removals were reported to be 94 %. A major problem in applying B N R to municipal wastewater treatment is the fact that Heterotrophic bacteria have rapid growth rates relative to autotrophic bacteria and tend to outgrow the suspended population of autotrophic bacteria at short SRT. Therefore the SRT Chapter 2. Literature Review must be lengthened to prevent washout or unintentional wasting of the nitrifiers before they have had time to regenerate. Potential solutions to this problem led to the idea of growing nitrifiers on fixed surfaces within the suspended sludge reactors. This concept is commonly referred to as a suspended/attached growth system. 2.1 ATTACHED GROWTH SYSTEMS The attached growth system involves a solid support medium on which biomass develops in thin layers. The support media is situated in the process such that mixed liquor contacts the outermost biomass layer surface. Nutrients required for life in the layers are obtained by diffusion into the surface layer from the mixed liquor flowing past (Barnes et al., 1983). A profile of the nutrients exists across the layers highly concentrated at the solid water interface and decreasing inwards. The outer layers consist of bacteria able to thrive on materials easily obtained from the solid-liquid interface where nutrients are most abundant. The bacteria in the inner layers are more capable of making use of by-products from the outer layer bacterial metabolism. The entire biomass in all the layers is a diverse array of species and not easily characterized. Three methods of attached growth are commonly used; the trickling filter, the rotating biological contactor (RBC) , and the submerged media carrier. The trickling filter media is stationary and waste liquid is trickled over the surface by gravity flow. Nitrifying bacteria growing on the trickling filter media surface obtain nutrients and dissolved oxygen from the liquid flowing past. The R B C consists of a disc plate partially submerged and rotated in the process 4 Chapter 2. Literature Review mixed liquor. Nitrifying bacteria growing on the surface obtain oxygen from the atmosphere when their section of the disc rotates out of the mixed liquor. When the section rotates back into the mixed liquor, the bacteria absorb nutrients from the liquor. The submerged media carrier is a stationary process where the media is completely submerged in the process mixed liquor. Nutrients and dissolved oxygen are obtained from the surrounding mixed liquor. A biomass made up of heterotrophic and autotrophic bacteria plus higher life forms grows on the surface of all three support media systems. Heterotrophic bacteria have a faster growth rate than the autotrophic bacteria and can successfully out compete autotrophs for oxygen under conditions where soluble carbon is available for heterotrophic growth (EPA Nitrogen Control manual, 1993). Soluble carbon in the aerobic zone of a sewage treatment process will be highest in the feed end. Stimulated by the soluble carbon, the heterotrophic bacteria will grow the fastest and thus be most active nearest the feed end. In contrast Autotrophic bacteria will then be most active further down stream after the heterotrophic bacteria have completed soluble carbon consumption and the competition for oxygen decreases. The relative positioning of support media in the aerobic zone is thought to be important. Support media should be placed in the region where the soluble carbon consumption is complete and where suspended autotrophic bacteria growth allows a higher possibility for attachment. Schlegel (1988) observed the greatest degree of nitrification along a submerged attached growth system at the point where soluble carbon removal was complete. 5 Chapter 2. Literature Review 2.2 ENVIRONMENTAL PARAMETER EFFECTS ON ATTACHED GROWTH NITRIFICATION Many environmental parameters affect the activity of nitrifiers on media in attached processes applied to sewage treatment. These parameters include organic carbon loading, specific surface area, the nature of higher life forms, and the process temperature. Organic carbon loading, soluble or particulate, is an important parameter for attached nitrification. Recently, Figueroa et al. (1991) observed that even very low levels o f soluble and particulate B O D 5 decreased attached growth nitrification in pilot scale trickling filters. Nitrification rates decreasing linearly decreased in the form of a finite discontinuity from 1.5 mg-N/m 2 -d to 0.7 mg-N/m 2-d when total B O D 5 levels in the influent reached 10 mg/L and continued decreasing linearly above 10 mg/L B O D 5 . The inhibitory effect of organic carbon loading was also observed in an earlier study where submerged carriers were used for tertiary nitrification (Rusten, 1984; Haug and McCarty, 1972; Bonhomme et al., 1990). In these studies nitrification was observed after soluble carbon was lowered in the influent to the reactors and there was reduced competition for oxygen by heterotrophic bacteria. This observation was consistent with nitrification rates decreasing when total B O D 5 increased in the reactor feed. Boiler et. al. (1990) investigated a pilot scale tertiary R B C addition to an existing activated sludge treatment plant. After achieving nitrification in the R B C at a stable level of 1.8 mg of N H 4 - N / m 2 - d, introducing a cloth pre-filtration step to remove particulate material 6 Chapter 2. Literature Review contained in the secondary effluent feed resulted in nitrification rates increasing to 2.9 mg of N H 4 -N / m 2 - d. To explain the particulate material effect, Boiler et. al. (1990) speculated that particles preferentially adhered to the surface crowded the nitrifiers and lowered the media's nitrification capacity but they did not define the particles as bacteria. However, in a pilot scale R B C study Figueroa (1992) tested synthetic influent levels of total B O D 5 > 20 mg/L and found that inhibition of nitrification by particulate B O D 5 was identical to that by soluble B O D 5 . Figueroa suggested that the inhibition by the particulate B O D 5 was due to competitive inhibition by heterotrophs on and around the support media, the mechanism previously identified for soluble B O D 5 inhibition. Specific surface area in the treatment reactors affects nitrification because larger specific surface area increases the real surface available for bacterial attachment. This advantage is countered by the increased clogging and bridging which occurs when the support media is too plentiful and there is insufficient void space for the sludge to flow through the media. The ideal specific surface areas are at least 100 m 2 /m 3 o f media cage volume and typically 150 m 2 /m 3 (Schlegel, 1988; Rusten, 1984; Bonhomme, 1990; Andersson, 1990). In a pilot scale plant of 63 m 3 volume the support media Ringlace® was shown to shed excessive biomass and minimize clogging (Lessel, 1991). Ringlace® sheds biomass because of the swaying motion of the long vertical strands submerged in the process mixed liquor. Infestations of higher life forms on support media have been a reported problem. Protozoa dominated the downstream end of the support media submerged within an activated sludge process (Schlegel, 1988) and limited autotrophic growth altogether. Andersson (1990) tested attached growth media submerged in the last four aerobic zones of an eight zone process 7 Chapter 2. Literature Review treating municipal wastewater. After a three week period microscopic studies showed bacteria, protozoa, and ciliates growing on the media. After a longer period of time bacteria dominated the media in the first aerobic zone while worms and large protozoa dominated the media in the last aerobic zone. Nitrifying bacteria attachment never occurred on the media. Temperature also affects nitrification because metabolism of nitrifiers increases as the temperature is increased over the range 5 ° C t o 3 5 ° C (Shammas, 1986). The most optimum temperature for nitrification lies in the range 30 - 36 °C (Barnes, 1983). Since nitrification rates follow an Arrhenius type relation as a function pf temperature (EPA Nitrogen Control manual, 1993) these temperature effects can be mathematically compensated. However, MLVSS interacts with temperature and proper compensation should use a modification to the temperature coefficient of the Arrhenius equation (Shammas, 1986). Shammas (1986) modified the equation to include an adjustment to the temperature coefficient based on MLVSS concentration. 2.3 OPERATING PARAMETER EFFECTS ON ATTACHED GROWTH NITRIFICATION Particular parameters which can be manipulated and have an effect on nitrification in a process provide a means of controlling nitrification in a process. These parameters are called operating parameters. SRT, HRT, and DO concentration are some of these common operating parameters. The operating parameter SRT affects nitrification as it determines the time available for 8 Chapter 2. Literature Review suspended nitrifiers to grow and thus influences their population in the process. A t constant influent substrate loading the SRT directly affects the M L V S S concentration because reduced wasting wil l lead to higher concentrations of microorganisms. Randall et al. (1992) ran a B E P R process at 1.5 to 15 days SRT and 10 to 20 °C temperatures. Effluent filtered ammonia increased when SRT was decreased to 5 days at a temperature of 15 °C. The operating parameter H R T affects nitrification by controlling the ammonia loading to the aerobic zone. Decreasing H R T wil l increase the flow through the aerobic tanks and increase ammonia concentration. The rate limiting step for nitrification in municipal wastewater treatment processes is the oxidation of ammonia nitrogen to nitrite by Nitrosomonas (Barnes, 1983). The effect of increased aerobic zone ammonia concentration on the growth rate of Nitrosomonas can be described by Monod kinetics (Barnes, 1983). The Monod kinetic equation describing the effects of both N H 4 + - N and D O on the growth rate of Nitrosomonas follows: (equation 1) where = specific growth rate, (mg/mg-d) •max = maximum value of u when substrate concentration is not limiting, (mg/mg-d) K M = N H 4 + - N half-saturation constant, (mg/L) K D = D O half-saturation constant, (mg/L) 9 Chapter 2. Literature RevieM' Equation 1 shows that an increase in the concentration of ammonia will increase the growth rate of Nitrosomonas. Increased growth rate results from increased efficiency of ammonia transport across the cell walls of the nitrifying population. Decreasing H R T increases the ammonia loading to the process resulting in a higher ammonia concentration available to the nitrifying population. Therefore decreased H R T wil l improve the nitrification rate with respect to the mass of nitrifiers in the suspended sludge. The effect of the operating parameter D O on the growth rate of Nitrosomonas is shown by the Monod kinetic relationship of equation 1. B y replacing N H 4 + - N with N 0 2 " ' - N in equation 1 the effect of oxygen concentration on the growth rate of Nitrobacter can also be shown. As D O concentration increases for either of these two nitrifying bacterial species the respective growth rate will increase. D O concentration influences growth because oxygen is the final electron acceptor in the oxidation of ammonia and nitrite. However, the effective concentration range must be quite broad since high levels of oxygen concentration have not been reported to inhibit nitrification and nitrification still proceeds at concentrations as low as 0.2 mg/L (Barnes, 1983). The E P A Manual for Nitrogen Control (1993) suggests operating the aerobic zones o f the nitrification process at 2.0 mg/L of dissolved oxygen and maintaining more than 1.0 mg/L to avoid oxygen limited nitrification. 2.4 P R E V I O U S S T U D I E S O N A T T A C H E D G R O W T H S Y S T E M S Various studies have reported promising nitrification results with attached growth media 10 Chapter 2. Literature Review submerged in aerobic sludge. Valve et al. (1991) studied submerged free floating foam carriers in a nitrogen removal process treating municipal sewage. Carrier samples were periodically removed from the process and batch tested to measure ammonia oxidation rates. Suspended sludge ammonia oxidation rates were measured the same way. Average rate value comparisons showed that the rates from attached growth on isolated carriers were 20-30 % lower than the rates using isolated suspended sludge. Based on four batch tests the average ammonia oxidation rate with the attached growth on the carrier was 1.13 mg N H 3 - N / g V S S - h r and the average rate with suspended sludge was 1.44 mg NH 3 -N /gVSS-h r . Lessel (1991) reported a study at Geiselbullach, Germany. In that study three different materials including Ringlace® were tested for increasing the M L S S in the aeration tank of a 63 m 3 volume test process. Each material showed an increase in the overall process nitrification reaction. The ammonia oxidation rate calculated from process parameter values was roughly 8.5 mg NH 3 -N/gTSS-hr for the test with Ringlace®. The Ringlace® showed less clogging and sinking problems than the other two materials and was chosen for installation into a full scale process for further testing. In the full scale process, Ringlace® improved sludge settling characteristics and thus allowed process operation at a higher suspended solids concentration. Due to the introduction o f phosphorus precipitating chemicals to the full scale process during the experiment the biofilm was tested for only a short period of time when the process was still unstable. Because of the short study time and unstable operation Lessel concluded that the effect of Ringlace® could not be seen clearly by the test results. 11 Chapter 2. Literature Review Lessel (1992) later published a second report on the use o f Ringlace® at Geiselbullach Germany concluding the study of Ringlace® at the full scale. Worms and other aerobic higher life forms grew prolifically on the support media and sometimes needed to be controlled by maintaining prolonged anaerobic periods. Stable nitrification was observed only twice over a three year test operation. However, the final conclusion was that Ringlace® improved sludge characteristics and allowed higher operating biomass concentrations such that nitrification was assured. However, the Ringlace® was not tested independent o f the sludge to determine i f the attached biomass itself contained nitrifiers. Sen et al. (1993) ran full-scale tests on Ringlace® at the Annapolis W W T P in Maryland. This study looked at effects of media positioning within the aerobic section o f a plug flow nitrogen removal process. A parallel control process was run along side the test process. Comparing differences in the ammonia levels between test process and control process at succeeding points along the aeration section before and after the Ringlace® was used to assess the effect o f the support media on nitrification in the process. These tests showed that under optimum concentrations of ammonium, nitrification in the process with Ringlace® increased 60 % over the control process. Maximum ammonia oxidation rate was reported as 1.7 kg/d-1000m of media. Assuming a media solids value of 10 g/m, which was observed by Sen (1993) in bench scale studies, the ammonia oxidation rate equated to 7 mg N H 3 - N / g attached solids-hr. The Ringlace® was not tested independent of the sludge to determine i f the attached biomass contained nitrifiers or whether the enhancement of nitrification was due to other differences between the systems. Worm infestations were observed on the media and may have affected the 12 Chapter 2. Literature Review comparative processes. Anaerobic periods greater than 12 hours reportedly sloughed off the worms. 2.5 OBJECTIVES The results of the previous studies on Ringlace® (Lessel, 1991; Lessel, 1992; Sen et al., 1993) showed promise for its use as an attached growth media to improve nitrification. However, none o f the tests of Ringlace® measured the ammonia oxidation rate of the attached biomass separate from the ammonia oxidation rate of the process suspended sludge. Valve et al. (1990) had shown improved nitrification with a different support media tested independent o f the suspended sludge. Improved process nitrification in the Ringlace® tests may have been a result of some other effect of the media. Therefore the occurrence of nitrifying bacteria growth on the Ringlace® needed to be clarified and measured. The general research objective was to grow nitrifying bacteria on Ringlace® submerged in the first aerobic zone of a B N R process. The specific research objective was to determine the effect of H R T and SRT on the specific ammonia uptake rates exhibited by isolated attached growth on the Ringlace® and determine the degree to which attached nitrifiers enhanced the overall nitrification capacity o f the process. 13 CHAPTER THREE MATERIALS AND METHODS 3.1 EXPERIMENTAL SET-UP The experiment was performed at a B E P R pilot scale wastewater treatment plant located on the U B C campus. The process treated domestic sewage produced from student residences and a small neighbourhood of houses. The process consisted of two parallel 2200 L trains called the A and B sides. The A-side had Ringlace® installed in the first aerobic cell (Figure 3.1) and the B -side was operated without Ringlace® to serve as control. The configuration of the process was shown in Figure 3.1. The volume fractions of each zone are listed in Table 3.1. Twice a day, at times when nutrients were richest, the feed sewage was pumped from the sewer into two holding tanks totalling 9000 L in volume. Sodium bicarbonate was added to these holding tanks to provide buffering for nitrification. The average alkalinity level in the feed to the process was 200 mg/L as C a C 0 3 . The p H in the first aerobic cell fluctuated between 6.7 and 7.2. The influent passed from the storage tanks through a fermenter process consisting o f one complete mix fermenter and settling tank in series, connected by an underflow recycle. The fermenter volume was 400 L and the settling tank 360 L . The fermenter process was varied as part of another study examining effects of solids and hydraulic retention time on V F A production from August 1993 until June 1994 end (Atherton, 1995). 14 Chapter 3. Materials And Methods Settler Anae Aerobic Cells 1 2 3 ~m Anoxic Location of Ringlace in Process FIGURE 3.1. SIMPLIFIED UCT PROCESS TABLE 3.1. VOLUME FRACTIONS OF PROCESS ZONES Zone Volume, (L) Fraction of Total Reactor Percent o f Total Reactor. (%) Anaerobic 250 2/18 11 Anoxic 610 5/18 28 Aerobic 1 610 5/18 28 Aerobic 2 365 3/18 16.5 Aerobic 3 365 3/18 16.5 Secondary Clarifier 520 15 Chapter 3. Materials And Methods The plant was equipped with a data logging and control system known as a S C A D A (Supervisory Control And Data Acquisition) system ( T H E F I X , by Intellution). Process temperature, O R P , and D O were logged continuously. The D O data was used for controlling the D O level in the aeration tanks. 3.1.1 Ringlace® Ringlace® is a synthetic fibrous rope type product with loops approximately 1.5 cm long protruding from the centre all along the length. The material is a modified P V C called polyvinylidene chloride. Ringlace® Products of Oregon, the supplier to this study, reported that the material was water resistant and chemically very stable. The manufacturer estimated a useful life of more than 10 years at temperatures up to 50 °C and exposure to concentrations up to 65 % for chemicals such as Nitric acid, Caustic Soda, Acetic Acid , Benzene, Acetone, and Cyclohexane The flexible fibrous loops are supposed to provide a larger surface area for the attached growth and in combination with the swaying motion o f the flexible rope are supposed to improve shedding action to prevent clogging in the process. The Ringlace® ropes were stretched tightly over frames which were immersed into the process mixed liquor. The rope tension pushed the fibrous loops along the length outwards from the centre. After a few hours to a day a slight slack developed in the rope that allowed a swaying motion which flexed the surface continuously and broke older dead biomass loose into clumps that would float away. This sloughing effect was meant to keep existing biomass fresh and minimize the large thick anaerobic biomass layers that characteristically develop on solid inflexible structures. Ringlace® Products of Oregon, 16 Chapter 3. Materials And Methods recommended following the Lessel (1991) study as a guide to designing the Ringlace® configuration. 3.1.2 Ringlace® experimental configuration 3.1.2.1 Volume ratio and specific density Lessel (1991) used the parameter volume ratio to determine the cage size holding the Ringlace® and the parameter specific density to determine Ringlace® quantity within that cage. Volume ratio is the percentage o f the aeration zone volume that the cage containing the Ringlace® occupies. The supplier to Lessel recommended a volume ratio in the range of 18 to 28 %. Lessel (1991) used 25 % to allow adequate water circulation around the cage and contact with the attached biomass. In this study a mixer fitted beside the cage limited the cage volume to 96 L to give a 16 % volume ratio for the 610 L first aerobic cell. This 16 % volume ratio was 2 % lower than the range proposed for Lessel (1991) but the small difference was considered insignificant. Specific density is a measurement o f media length per unit cage volume in units of m/m 3. The supplier for Lessel (1991) recommended using 250 to 500 m/m 3 . The maximum specific density o f 500 m/m 3 equates to a distance o f 4.5 cm between the ropes in the cage. This space is the minimum distance necessary to minimize potential clogging and bridging. A 530 m/m 3 specific density was used in this study to give roughly a 4.5 cm distance between the strands. 17 Chapter 3. Materials And Methods 3.1.2.2 Modular design The Ringlace® was installed in the process in a modular fashion. There were 16 cubical frames laid in 4 layers o f 4 cubical frames each. The individual cubical frames were threaded with 3.2 m of Ringlace® (Figure 3.2) and mounted onto a large frame in the process by sliding the cubical frame's tubular corners onto rods extending upwards from the large frame base. This design allowed removal of selected sections containing 1/16 of the total cage volume for analysis in batch testing outside the process (section 3.2.1). Cubical Frame FIGURE 3.2. CUBICAL FRAME FOR MOUNTING RINGLACE® The bottom cubical frames were 41 cm from the process floor and the top cubical frames were 12 cm from the liquid surface. Two fine bubble diffusers supplied aeration to the cell and were located directly below the large frame base. 18 Chapter 3. Materials And Methods 3.1.3 Process HRT and SRT operation and control The process was operated at each possible combination o f the H R T and SRT factor levels listed in Table 3.2. When the process was stable at a combination, batch tests were performed and operation was changed to the next H R T , SRT combination and the cycle repeated. Process stability was indicated by the stable response of weekly and or daily measured process parameters, M L S S , ammonia, nitrite and nitrate, and ortho-phosphorus. Zaloum (1992) studied the M L V S S response time in a wastewater treatment process with fixed influent flow due to changes in H R T and SRT using the Danckwert's equation (Danckwert, 1953). Zaloum (1992) recommended as a general rule o f thumb to wait 3 times the difference in SRT change as an adjustment period. This waiting period rule was followed in this study. However, the same rule did not apply to changes in H R T and in this study an H R T change adjustment period lasted until measurements of the above mentioned process parameters were stable. TABLE 3.2. HRT AND SRT FACTOR LEVELS U ^ a c t o r ^ ^ e v e l s ^ ^ l H R T (hours) 6 ^ J V ^ ^ M e d i m n j ^ ^ 9 ^ J ^ K g h ^ l 12 SRT (days) 8 14 20 The flow rates were monitored weekly and the process H R T was controlled by adjusting the feed pump rates weekly i f required. The process SRT was controlled by daily wasting of calculated amounts of mixed liquor from the last aerobic cell based on equation 2 (Metcalf & 19 Chapter 3. Materials And Methods Eddy, 1993). The effluent SS (Xe) and M L S S were measured as outlined in section 3.2.2. Each day a sample was taken the M L S S value resulted from one sample and the eff SS value was an average o f two samples. Calculated wasting values carried over from previous days were used to cover days when no measurements for the variables were taken. For example, the weekend wasting values were the Friday values calculated from Friday's measurements. 8 . ™ -Q X + Q X (equation 2) Where, 6C = Mean cell residence time, (days) X = Mixed liquor suspended solids, (mg/L) X e = Effluent suspended solids, (mg/L) Q w = Sludge wastage flow rate, (m3/day) Q e = Effluent flow rate, (m3/day) V = Process volume, (m 3) 3.1.4 DO control Aeration was supplied by two fine bubble diffusers in the first cell and one diffuser in each successive cell. D O levels from each cell were measured and data logged to a computer by a system similar to that shown in Figure 3.5. The D O levels were separately controlled in each cell by using minimum and maximum set points to switch valves on and off in the air lines feeding each cell. The desired D O level was then the average value between these alternating set points. 20 Chapter 3. Materials And Methods A n example of a typical D O data on-line plot is shown in Figure 3 3 Hi 2.0 L o w 1.0 time FIGURE 3.3. T Y P I C A L DO C U R V E OBTAINED W I T H M I N / M A X C O N T R O L 3.1.5 Temperature logging The S C A D A system logged the temperature monitored by two probes inserted into each process. One probe was located at the influent and the other at the effluent. The temperature probes were designed and assembled by the electronics staff of the U B C Civil Engineering department. 3.2 A N A L Y T I C A L P R O C E D U R E S 3.2.1 Batch test description and rationale Batch tests are a way of indirectly measuring the nitrification rate of nitrifying bacteria through measuring the disappearance of ammonia nitrogen in an aerated batch reactor containing 21 Chapter 3. Materials And Methods these organisms. The nitrification rate was measured in batch tests for the suspended sludge from both the test process and control process. These batch tests were called A-side suspended (A-S) and Pi-side suspended (B-S) respectively. The nitrification rate measured for the biomass attached to Ringlace® media separated from the suspended sludge was measured in batch tests called A-side attached (A-A). The A - A batch tests used settled anoxic zone supernatant, which contained a small amount of suspended solids, plus one cubical frame of Ringlace® media removed from the process first aerobic zone. To eliminate the nitrification rate in the A - A batch tests caused by the anoxic zone supernatant suspended solids, the anoxic supernatant was batch tested in tests called A-side (A). The nitrification rate was measured for a combination of the test process suspended sludge and the attached biomass in a batch test called A-side suspended/attached (A-S/A). The A-S/A test showed the nitrification rates from suspended sludge and attached biomass working together. All batch test nitrification rates were reported normalized to the amount of total suspended solids in the respective batch reactor. Batch testing was performed in this manner could specifically distinguish the nitrification rate of the attached biomass from the rate of the suspended sludge. Comparison of the batch test rates would directly indicate the specific nitrification capacity of the attached biomass. 3.2.1.1 Batch test apparatus Batch test reactors were 24 L bench scale tanks sized to contain one Ringlace® cubical frame at the process volume ratio and specific density. The batch reactor is shown in Figure 3.4. The equipment used in this batch reactor are listed in Table 3.3. Samples were drawn with a ' 22 Chapter 3. Materials And Methods syringe and tube. Mechanical stirring blades were mounted above the reactor to provide continuous mixing and an aquarium aeration stone provided air diffusion. D O 470 mm ORP Air dia. = 255 mm Volume = 24 L FIGURE 3.4. BATCH REACTOR TABLE 3.3. BATCH REACTOR EQUIPMENT Item Make Model D O Meter and Probe Y S I 54A (meter), 5739 (probe) O R P Probe Broadley James No . 9176 p H and Temperature Horiba D-13 U S Fine Bubble Aerator Aquarium Ai r Stone 6" Computer I B M X T , 8086 23 Chapter 3. Materials And Methods A i r V a l v e s o* o C o n t r o l D O O R P v y © FIGURE 3.5. BATCH REACTOR MONITORING AND CONTROL At time of testing batch reactors were arranged to measure up to three batch tests at a time (Figure 3.5). The batch reactor D O control was similar to the D O control in the pilot scale process using min/max switching of air valves. O R P and D O data were logged on the I B M X T computer throughout the batch test by a program written for this experiment. 3.2.1.2 Batch test procedure The Batch tests were run in two stages. Stage one tested A - S and A - S / A . Stage two tested A - A , A, and B-S . The p H and temperature data were collected at the beginning and end of the batch test. Two samples were taken at the beginning of each test to measure suspended solids. For selected batch tests, samples were collected at the beginning and end for B O D 5 24 Chapter 3. Materials And Methods measurements (Table 4.4). The Ringlace® cubical frames, (Figure 3.2) were extracted at random from the process for batch testing. Drawing numbered paper slips from an envelope was used for random selection. After selection, each paper slip was removed from the envelope to avoid re-picking a frame that might not have had enough time to re-establish growth. Mixed liquor for each batch test was taken from the respective process train anoxic zone. Each reactor filled with sludge and/or Ringlace® was immediately taken to the bench, connected to the computer logging and control system, and aerated to best represent conditions in the process first aerobic cell. 3.2.2 Pilot scale process sampling schedule The process sampling schedule is outlined in Table 3.4 where an 'x' represents a sample was taken. Sampling was not done on the weekends. Samples were all taken as grab samples. TABLE 3.4. PILOT SCALE PROCESS SAMPLING SCHEDULE 1 Parameter Mondav Tuesdav Wednesday Thursday Fridav NO x , P 0 4 , N H 3 all zones eff. only all zones all zones eff. only Process MLSS, X X X X Effluent SS, Xe X X X X X PH X 25 Chapter 3. Materials And Methods 3.2.3 Sample analysis methods 3.2.3.1 NCL-N and NH,-N Samples were filtered with Whatman # 4 filter paper, pore size range of 20 - 25 um. N O x - N samples were stored in 10 ml tubes and preserved by adding phenyl mercuric acetate to lower the p H to 3-4. N H 3 - N samples were preserved in 10 ml tubes by adding 3 % H 2 S 0 4 to lower the p H to 3-4. Stored tubes were refrigerated at 4 ° C and assayed within one week. Lab measurements were performed on an Auto Analyzer using standard colorimetric methods (American Public Health Association, 1992). The test for N O x - N used method No . 353.2 and the test for N H 3 - N used method No. 350.1. 3.2.3.2 Suspended solids, effluent and total Suspended solids were measured at the experimental site. Samples were filtered through Whatman 934 -AH paper, pore size 1.5 um, and oven dried at 105°C before differences in mass measurement were used to calculate the suspended solids value (method N o . 2540 D , American Public Health Association, 1992). 3.2.3.3 Volatile suspended solids After measuring suspended solids, samples were fired at 550°C for 30 minutes to vaporize the volatile fraction. Differences in mass before and after vaporization were used to calculate the volatile fraction (method N o . 2540 E , American Public Health Association, 1992). 3.2.3.4 Soluble BOD, Whatman # 4 filter paper with pore size 20 - 25 um was used to filter samples. Samples were then diluted with seed water and incubated at 20°C for 5 days (method N o . 5210 A , 26 Chapter 3. Materials And Methods American Public Health Association, 1992). A Y S I probe (model N o . 5730) and meter (model N o . 54) were used to measure dissolved oxygen concentration in test bottles. 3.3 STATISTICS 3.3.1 N O x and N H 3 sampling precision The precision of sampling, preservation, and lab measurements of N O x and N H 3 was tested. The results are reported in Table 3.5. Ten samples of N O x and N H 3 were taken simultaneously during one batch test. The same preservation and handling technique was used for each sample and all were sent to the lab for analysis. The standard deviation of the N O x and N H 3 data was low (Table 3.5). T A B L E 3.5. NOx-N A N D NH 3 -N S A M P L I N G PRECISION 1 Statistic N O x - N ( m s - N / D N H , - N ( m s - N / U Sample Mean 1.05 4.20 Sample Standard Deviation 0.0570 0.0827 95 % Confidence Interval • +/- 0.0400 +/- 0.0570 Sample Size, N 10 10 3.3.2 M L V S S Mixed Liquor Volatile Suspended Solids ( M L V S S ) values were measured for the entire 27 Chapter 3. Materials And Methods first run covering a period from the beginning of July to the beginning of October 1994. These measurements were reported in Table 3.6 as a percentage o f the M L S S of the same sample. TABLE 3.6. PROCESS MLVSS AS A PERCENTAGE OF MLSS Sample Statistic A-side (%) B-side (%) Mean 81 84 Standard Deviation 3.5 2.3 95 % Confidence Interval +/- 1.3 +/- 1.2 Sample Size, N 28 14 Table 3.7 shows the volatile fraction of solids scraped from the media. This data was obtained from the first six batch tests of the first run during the summer of 1993 when the process had a 20 day SRT, 12 hour H R T , 3.5 mg/L D O level, and temperatures were near 2 0 ° C for this period. TABLE 3.7. VOLATILE FRACTION OF MEDIA SOLIDS Sample Statistic V O L A T I L E S O L I D S (%) Mean 87 Standard Deviation 6.0 95 % Confidence Interval +/- 3.6 Sample Size, N 11 28 Chapter 3. Materials And Methods 3.3.3 Solids retention time of runs Table 3.8 shows the average and the standard deviation for the SRTs used in this study. The SRTs were calculated by applying the sludge age formula (section 3.1.3) to the wasting and suspended solids data collected throughout the study. Samples for effluent SS and M L S S were taken and analyzed as outlined in section 3.2.2. The eff SS and M L S S data were used to calculate the wasting values as outlined in section 3.1.3. The high standard deviation within the 20 day SRT, 12 hour H R T on B-side occurs because errors made in wasting solids were compensated by adding or subtracting from the required wasting on subsequent days. The pilot plant maintenance and operations staff were undergoing training at this time. TABLE 3.8. AVERAGE AND STANDARD DEVIATION OF SRT Nominal Nominal Observed SRT Sample SRT H R T A-side (days) B-side (days) Size fdavs) (hours') Averaae Std. Dev. Averaae Std. Dev. N 20 12 20.1 1.70 21.3 5.80 45 14 12 13.6 1.30 13.6 1.50 37 8 12 7.20 2.50 7.90 1.10 37 8 9 8.40 1.80 9.20 3.50 35 4 9 4.20 1.10 4.10 1.00 89 4 6 4.50 0.60 4.10 1.00 35 29 CHAPTER FOUR RESULTS AND DISCUSSION 4.1 BATCH TEST RATE SUMMARY The ammonia uptake rate was measured from 2 to 4 times for each combination of H R T and SRT (Table 3.2) during each pilot scale run by performing the batch tests described in section 3.2.1. The resulting rate data were plotted in summary graphs (Figure 4.1, 4.2, 4.3). The following rules apply to all these rate summary plots. Where no data point appears for A-side Attached (A-A) , the value was less than zero. The A - A batch test rates were normalized specific to the equivalent attached plus suspended solids and have units o f mg-N/(g TSS + g attached solids) - hr. The A-side suspended attached (A-S /A) batch test also contained attached solids but the rates were normalized to suspended solids only. These calculations allowed comparison of suspended solids nitrification rates between A - S / A and A and B side suspended (A-S, B-S) batch tests. There are no recorded rates for run 2 due to operating problems during this run (section 4.2.2). N o nitrification rate was indicated for the attached biomass in the first run (Figure 4.1). Worms discovered on the media during this run were thought to possibly be inhibiting attached nitrifier growth, therefore the operating parameter D O was manipulated in an attempt to suffocate the worms. From this point on in the study, D O was varied along with H R T and SRT to test effects on attached bacteria nitrification rates. 30 CO CO ^3 CD <D a: o o Q- 1 CO HRT =12 hrs, DO = 3.5 mg/L SRT = 20 d *^ SBS i SI l T = 1 4 d SRT = 8 d © — - ® — A - —- » A * s -B-E3 A 1 H ft r I 2 — *-t «-r 1 j X 2 0 4 0 6 0 8 0 1 0 0 Days From Beginning of 12 hr HRT 120 25 20 -4—' (0 15 « E cu 10 ID O O A-S B-S is A-S/A T A-A Temp FIGURE 4.1. BATCH TEST RATE DATA SUMMARY, RUN 1 Run 3 20 HRT = 9 hrs Run 4 Run 5 40 60 80 100 Days From Beginning of 9 hr HRT 120 A-S A B-S H A-S/A x A-A Temp FIGURE 4.2. BATCH TEST RATE DATA SUMMARY, RUN 3, 4, 5 31 HRT = 6 hrs SRT = 4 d • -DO = 5.5 mg/L ® A A i B * , n X 5 B , L 1 0 2 0 3 0 Days From Beginning of 6 hr HRT 4 0 A-S A B-S eg A-S/A x A-A -<*-Temp FIGURE 4.3. BATCH TEST RATE DATA SUMMARY, RUN 6 TABLE 4.1. ANOVA OF TOTAL AMMONIA UPTAKE RATE DATA T e s t D a t a S e t s F s ta t is t ic Fcr i t , @ 5 % level A - S & B-S 3.5 4.1 A - S & A - S / A 0.25 4 .1 A - S & A - A 7 2 . 9 4.1 A - A & A / S / A 58 .7 4.1 A - A & Z e r o 0.6 4.1 no te : df = 39 f o r all tes t d a t a se ts . 32 Chapter 4. Results and Discussion Mixed liquor nitrification rates (Figure 4.1, 4.2, 4.3). indicate nitrifying bacteria were present in the process but did not preferentially attach to the media under the experimentally adjusted operating parameters SRT, H R T , and D O . Throughout the entire experiment the attached growth on the media Ringlace® did not exhibit an ammonia uptake rate significantly greater than zero at the 5 % significance level (Table 4.1) and may have even released ammonia since some A - A rates were less than zero. Since A - S / A rate data were normalized to suspended solids, A - S / A rates should be at least equal to or greater than the A - S values since the sludge was taken from the same zone at the same time. A - S and A - S / A rates were not significantly different at the 5 % significance level (Table 4.1). The minor differences in the graphs were within the normal range of measurement error. Since each experimentally adjusted operating parameter, H R T , SRT, and D O affects nitrification, it is necessary to discuss what the effect of each parameter was and to discuss how each might have affected attached growth nitrification. SRT is the operating parameter that describes the average time a particle of SS remains in the process. The length of time nitrifying bacteria spend in the process determines their ability to effectively reproduce and maintain functional numbers. Changes in SRT in this study affected the suspended solids ammonia uptake rates in the predictable manner as the rates lowered with the lowering SRT throughout the study (Figure 4.1, 4.2, 4.3). Suspended nitrification began to fail on A-side and B-side as soon as the SRT was reduced to 4 days (Appendix B) . These shorter SRT were expected to improve conditions for attached nitrifiers by decreasing the suspended nitrifier population and increasing available ammonia. Although the nitrifiers in the suspended 33 Chapter 4. Results and Discussion solids were stressed and nitrification began to fail, the lower S R T did not increase attached growth nitrification. The ammonia uptake rates of the suspended biomass in this study were less than the rates reported in the literature for similar processes operated under similar SRTs and temperature (Table 4.2). However, since the pilot scale process in this study was achieving close to total ammonia conversion (Appendix B) , a smaller than normal percentage of the M L V S S was autotrophs because there wasn't enough food to support a higher population. Therefore the relatively lower rates could indicate that the nitrification in the process was not strong enough or there were too few nitrifiers to establish conditions favourable for growth on the Ringlace® media. TABLE 4.2. AMMONIA UPTAKE RATES FROM LITERATURE Reference Sludee Aee fdavs) Temperature (°C) Rate fme-N/eVSS-hri 1 Kos, (1992) 8 .7-9 .5 1 3 - 1 5 3.6 II 2.4 II 4.0 II H 3.4 II 2.2 4.2 Randall, (1992) 5 15 . 2.8 Tondaj, (1992) - 15 2.0 This study, (1993) 8 15 1.4 34 Chapter 4. Results and Discussion HRT also affects the ammonia loading to the aerobic zone of the process (Metcalf and Eddy, 1991). Decreased HRT increases flow through the process, therefore more ammonia is loaded into the first aerobic zone and decreasing the amount of time suspended nitrifiers have to consume ammonia in the first aerobic zone is decreased. As discussed in the literature review, higher ammonia loading increases the growth rate of Nitrosomonas and thus increases the overall nitrification rate. The changes to HRT in this study were expected to show up as constant changes in ammonia uptake rates between HRT changes (Figure 4.1, 4.2, 4.3) but the changes are complicated by concurrent changes in other variables. The first tested HRT change from 12 hours (Figure 4.1) to 9 hours (Figure 4.2) showed only a slight rate difference. This limited effect might have been due to a concurrent change of the DO from 3.5 mg/L down to 1.5 mg/L. However, for the suspended portion a 1.5 mg/L DO concentration should not have had a rate limiting effect since DO above 1.0 mg/L does not limit the growth rate of Nitrosomonas suspended in sewage treatment process mixed liquor (EPA Manual for Nitrogen Control, 1993). The subsequent change in HRT from 9 hours (Figure 4.2) to 6 hours (Figure 4.3). The B-side suspended rates increased as expected for the change in HRT, but the A-side suspended rates in Figure 4.3 unexpectedly decreased. In this case DO was concurrently increased from 3.5 mg/L to 5.5 mg/L but since both values were significantly above rate limiting concentrations the change in DO should not have decreased the nitrification. Temperature also increased during the 9 hour to 6 hour HRT change from Figure 4.2 to Figure 4.3 and might have affected the B-side suspended nitrification rate increase. However, 3 5 Chapter 4. Results and Discussion both sides were at similar temperatures and so the rate increase effect should have occurred on A-side suspended rates as well. The difference in the reaction of suspended rates from run 5 to run 6 is more likely due to a severe worm infestation which occurred on the solid surfaces of the A-side (section 4.6.2). Worm numbers were insignificant on the B-side at this time. It might also be possible that ammonia measurements suffered interference effects if worms produced a large amount of ammonia through their faecal matter and impaired the ammonia uptake rate measurement. Oxygen is a key component in nitrifier metabolism serving as the final electron acceptor in the electron transport chain. Dissolved oxygen concentration in the process influences Nitrosomonas growth rate according to a study in the EPA Manual for Nitrogen Control (1993) by increasing the growth rate with increased DO. The minimum DO concentration for normal activated sludge process operation with suspended solids alone is 1.0 mg/L. Since the DO concentrations in this study were above this minimum level, oxygen should have been sufficient for suspended growth nitrification. However, diffusional resistance to oxygen transfer into the biofilm might have limited the amount of available oxygen from getting to the attached nitrifying bacteria within the biofilm. Even the high levels of 5.5 mg/L used in this study might not have been adequate to overcome diffusional resistance. This belief is consistent with the observation that increased DO in run 6 did not enhance nitrification or promote attached nitrifier growth in A-side. Throughout the study the temperature was monitored as an uncontrollable environmental parameter. Changes in the suspended solids nitrification rates appear to correlate directly to 3 6 Chapter 4. Results and Discussion changes in temperature within the runs (Figure 4.1, 4.2, 4.3). However, the temperature values in this study were within the acceptable 5 to 25 °C range for nitrification to occur in B N R processes (Shammas, 1986; Painter, 1970; Randall et al., 1993), therefore temperature should not have been a limiting factor to attached growth nitrification. It was originally intended to remove any temperature effects by applying a modified form (Shammas, 1986) of the Arrhenius type equation provided by the E P A Nitrogen Control manual (1993). However, for the attached growth data in this study the Shammas (1986) equation could not be applied because a logarithmic term in the equation does not work with zero and negative numbers. The positioning of Ringlace® within the aerobic zone changes the conditions under which nitrification occurs. In a B E P R process part of the carbon entering the aerobic zone is in a soluble form but most carbon enters as a stored form within heterotrophic bacteria (Randall et. al., 1992). In this study soluble carbon levels entering the aerobic zone were typically 45 to 55 mg/L B O D 5 (Table 4.4). Heterotrophic bacteria respiring in the carbon rich feed end of the aerobic zone create competition for oxygen with the autotrophic bacteria in that zone suggesting that conditions for attached growth nitrifiers would be better further down stream where there is less competition. However, in placing attached growth media in the downstream end of a B N R process Andersson (1990) reported higher life form infestation on the media. Sen et. al. (1993) placed Ringlace® media between the 80 and 90 % downstream points in the aerobic zone of a B E P R process and also observed worm infestations. In this study the support media was placed at the feed end of the aerobic zone (Figure 3.1) consistent with the belief that competition for oxygen in that.zone limited nitrifier development. 37 Chapter 4. Results mid Discussion Nitrification did not occur on the media however, measurable oxygen was consistently present and the poor nitrification was probably due to some other factors such as the worms. Worm infestations were observed on the media when it was in the front end of the aerobic zone. Since concurrent worm infestations were observed on support media placed in both ends of the aerobic zone in this and previous studies, the problem of higher life form presence can not be eliminated by simply switching sections in the aerobic zone. 4.2 DETAILED BATCH TESTING ANALYSIS 4.2.1 Run 1 The first goal in run 1 was to initiate growth on the media. The second goal was to measure specific ammonia uptake rate of developed media growth after stable process operation at 12 hour H R T and 20, 14, and 8 day SRTs. Batch test rate data for this pilot plant run were plotted in Figure 4.1. This run began on June 3, 1993 and ended October 7, 1993. The test duration covered the summer months when the temperature was highest and favoured nitrifier activity. The D O was constant at 3.5 mg/L for the three tested sludge ages. The Ringlace® media was placed into the process for the first time and growth accumulation on the surface was observed. Full accumulation occurred over a four to five day period. The level of growth was determined by a dry measure of biomass as described in section 4.6.4. Further weight determinations throughout the runs were subjective estimates, based on 38 Chapter 4. Results and Discussion comparison to picture records. The Ringlace® that was firmly stretched over the frame before entering the process developed a slight slack. This slack did not develop further over the study duration. Figure 4.1 showed how changes in SRT affected the suspended sludge ammonia uptake rates in a predictable manner with the rates decreasing as the SRT was reduced. The SRT changes did not, however, result in nitrifying bacteria growing on the Ringlace® as nitrification did not occur on the media for this run. The A N O V A test statistic at the 5 % level from Table 4.3 below indicated the A-S, A-S/A, and the B-S uptake rates were similar and all significantly different from A-A. Table 4.3 also indicated A - A was not significantly different from zero at the 5 % level. Therefore SRT changing from 20 to 14 to 8 days at a 12 hour HRT and 3.5 mg/L DO did not promote nitrifying bacterial growth on Ringlace® media in the aerobic zone of a BNR process. As shown in Figure 4.1, temperature appeared to have increased the nitrification rates as the temperature increased. Temperature and SRT could have had an interacting effect on nitrification as solids levels changed with SRT (Appendix A) and MLSS and temperature affected nitrification (Shammas, 1986). Regardless, no nitrification occurred on the media. Worm growth was discovered on the media under microscopic observation. The worms were insufficient to discolour the media as they did in subsequent runs but on average covered roughly 40 % of the field of view in the microscope. 3 9 Chapter 4. Results and Discussion TABLE 4.3. ANOVA OF RUN 1 AMMONIA UPTAKE RATE DATA Test Data Sets F statistic Fcrit. (cb. 5% level 1 A - S & B-S 0.41 4.49 A - S & A - S / A 0.44 4.49 A - A & A - S 249 4.49 A - A & A / S / A 102 4.49 A - A & Zero 1.78 4.49 Note: df = 17 for all Test Data Sets 4.2.2 Run 2 The addition of the experimentally manipulated variable D O required a step by step experimental approach. Batch test results from stable process operation during each run were analyzed and this analysis was used to predict the most favourable parameter values to enhance nitrification on this support media for each subsequent run. The goal of the second run was to investigate low H R T and low D O level effect. A low 6 hour H R T was chosen to improve growth conditions on the support media by increasing available ammonia nitrogen around the Ringlace®. In addition, the dissolved oxygen level was decreased from 3.5 mg/L to 1.5 mg/L in an attempt to suffocate the worms. The process failed under the tested H R T condition because the clarifiers hydraulically overloaded causing solids losses too high to maintain the desired SRT and no data was obtained as a result. The run started in late October, 1993 and was shut down for changes in the first week 40 Chapter 4. Results and Discussion of December, 1993. To achieve the 6 hour H R T and assured clarifier operation, the flow rates would need to be lowered. For lower flow rates the process would have to be shut down and physically shortened. Therefore testing the 6 hour H R T was postponed until a later date. 4.2.3 Run 3 The run 3 goal was to continue from run 2 creating a more difficult environment for suspended growth nitrifiers and a more favourable one for the attached growth. In this run the H R T was lowered from 12 hours to 9 hours, instead of 6 hours as in run 2, to allow proper clarifier operation. The H R T was lowered to provide more ammonia nitrogen to the support media in the first aerobic zone. The D O was kept low at 1.5 mg/L as in run two to continue attempts at suffocating the worms. The SRT remained at 8 days. The batch test specific ammonia uptake rate and temperature data for run 3 were plotted in Figure 4.2. The run began on December 10, 1993 and finished on January 20, 1994. The mid data point for attached growth showed a slight degree o f nitrification (Figure 4.2). However this limited nitrification can be accounted for by the suspended solids that were transferred with the media into the batch reactor plus the suspended solids in the anoxic zone supernatant used to make up the batch reactor volume. The solids growth on the media in run 3 was higher than the other runs (Figure 4.11) as if the richer influent nutrient conditions over the Christmas holidays promoted rapid process biomass growth. A l l three parameters H R T , D O , and temperature changed from the previous run to this run. This simultaneous multiple parameter change made interpretation of the effects of each 41 Chapter 4. Results and Discussion parameter on nitrification rates difficult. Decreasing D O and temperature both should have effectively decreased nitrification rates (Beccari et al., 1992). Decreased H R T should have increased rates by making more ammonia nitrogen available to nitrifying bacteria. Surprisingly the suspended rates were similar to what they were before the parameter changes (Figure 4.1) and the attached rates remained effectively zero (Table 4.1). Therefore under the operating conditions for this run, nitrification was not observed to occur on Ringlace® media. Worm growth on the media was visible under the microscope but the large infestations indicated by red coloration visible with the naked eye were not observed. 4.2.4 Run 4 Up to this point in the study ammonia uptake had still not been observed on the media (Figure 4.1, 4.2). Therefore the goal in this run was to create a more difficult environment for suspended growth nitrifiers in order to decrease competition for N H 4 and improve selection for attached growth nitrifiers. A more difficult environment was created by lowering the SRT to 4 days. The other parameters H R T and D O remained at 9 hours and 1.5 mg/L respectively. The specific ammonia uptake rate data for run 4 was plotted in Figure 4.2. The run started on January 20, 1994 and finished on March 10, 1994. During run 4 on day 60 (Figure 4.2) road salt from de-icing was observed in the sewage feed. On day 62 chemicals smelling like paint thinner, possibly from a construction site upstream were noticed in the sewage. On day 74 the first batch test proceeded. Due to technical lab problems the second batch test scheduled for day 81 was not completed. 42 Chapter 4. Results and Discussion The only operational parameter change from the previous run 3 was the SRT. Decreasing S R T brought down the M L S S level (Appendix A ) . The environmental parameter change from run 3 was the temperature which also decreased by roughly 3 °C. The decrease in SRT and temperature caused the suspended sludge nitrification rates to decrease (Figure 4.2). After two weeks both processes had begun to fail at near complete removal of ammonia nitrogen by suspended sludge alone (Appendix B) . The attached batch test rates were effectively zero except for the first batch test which appeared to be close to half of the A - S / A rate which would be statistically significant (Figure 4.2). This limited A - A rate could be explained by suspended solids entered into the reactor from the anoxic supernatant used as fill and from suspended solids transferred on the media. However, specific to suspended solids alone the ammonia uptake rate in the A - A batch test was 0.75 mg-N/gTSS-hr which was lower than the A - S batch test rate of 1.24 mg-N/gTSS-hr. The lower A - A rate might have been caused by a lower amount of autotrophs in the suspended solids portion of the A - A batch test because of a dilution effect of attached solids sloughing off into the reactor. If the attached solids did not contain autotrophs then the effect would have been to dilute the numbers of autotrophs in the reactor suspended solids and thus lowering the specific rate. This view is consistent with the observation that the A - A batch test suspended solids level was higher than other A - A tests throughout the study (Appendix A ) The A - S / A in this run were close to half the A - S rates. This difference in rate could have been caused by competition for oxygen from the support media growth. The extra mass of heterotrophs and worms that grew on the support media added to this batch test made up 43 Chapter 4. Results and Discussion approximately one third of the total mass of solids in the reactor. With the S R T down to 4 days the suspended portion of autotrophs would find this extra mass a significant competitor for the low level supply of oxygen in this run. Worm growth was not visible as red coloration on the media surface but small numbers were observed microscopically (Figure 4.9). 4.2.5 Run 5 Ammonia uptake by attached growth on the media had still not been observed at this point in the study. Consideration was now given to the possibility'that oxygen might limit attached nitrifier populations because of diffusion resistance through the bio-film i f the free oxygen level surrounding the media was not high enough to penetrate the attached biomass. Therefore the goal of this run was to increase D O to remove possible oxygen diffusion limitation through the attached growth film surface. The D O level was increased to 3.5 mg/L, the SRT was left at 4 days to discourage suspended nitrifier growth and the H R T remained at 9 hours. The specific ammonia uptake rates were plotted in Figure 4.2. The run started on March 10, 1994 and finished on Apri l 28, 1994. Part of the ammonia data for the first batch test of A - S for run 5 was lost due to a lab equipment problem so the remaining four data points were used to determine that uptake rate. From run 4 to run 5 the only parameter changes were the operating parameter D O and the environmental parameter temperature. Both parameters increased. The ammonia uptake rates for the B-side control process increased slightly from the previous run (Figure 4.2). A - S / A rates 44 Chapter 4. Results and Discussion did not appear to change from run 4 to run 5 but the A - S rates dropped to near those of the A -S/A. This drop could have been caused by interference from the worms which grew more abundant in this run than the previous run. B-side rates appeared to increase in response to temperature. However, the slight increase in B-side rates could also have been a response to the increased D O . The ammonia uptake rates for A - A batch test showed zero and negative rates. Therefore nitrification on attached growth media Ringlace® did not occur under the conditions of this study for this run. This run had a worm outbreak which made the media frame cubes completely red in colour. The amount of worms were higher than any other run to this date. The infestation of worms increased in this run as did the operating D O level and the A-side process nitrification continued to fail (Appendix B) . 4.2.6 Run 6 Attached growth ammonia uptake had still not been observed in any previous run. One of the postulated causes for the problem was still believed to be potential oxygen diffusion resistance as in run 5. Another postulated cause of the problem was the limited ammonia nitrogen supply to the media in the first zone. Therefore one goal in this run was to increase the D O level from 3.5 mg/L to 5.5 mg/L to be sure oxygen diffusion limitation through the attached growth film surface was not occurring. The other goal was to decrease the H R T from 9 hours to 6 hours to ensure enough ammonia nitrogen was available to the attached growth. The SRT was left at 4 days to discourage suspended nitrifier growth. 45 Chapter 4. Results and Discussion The specific ammonia uptake rate data for run 6 was plotted in Figure 4.3. The run was started on May 17, 1994 and finished on June 25, 1994. Rates on the B-side were high enough to completely remove the 15 mg/L average ammonia level entering the process during run 6 and appear to be a response to the increasing trend in the temperature across the run and the increased D O from run 5 to run 6. The A - S and A - S / A nitrification rates were less than half the B-S nitrification rates (Figure 4.3) and were consistent with the A-side process ammonia removal almost completely failing during this run (Appendix B) . The poor nitrification performance o f the A-side was believed due to the outbreak of worms growing on the media. The rates of ammonia uptake for the A - A batch test Showed zero uptake, some tests showed rates as i f ammonia was produced. Therefore nitrification on attached growth media Ringlace® did not occur under the conditions of this study for this run. The worms in the A-side process during this run were at extremely high levels. The worm density was so high that the media strands were completely bridged across to neighbouring strands. 4.3 SURROUNDING CARBON EFFECT The surrounding organic carbon in the process mixed liquor was divided into two fractions, particulate and soluble. Particulate organic carbon represented microorganisms and soluble organic carbon represented nutrients. Recent work in attached growth processes has indicated particulate and soluble carbon levels as low as 20 mg/L B O D 5 were inhibitory to 46 Chapter 4. Results and Discussion attached growth nitrification (Figueroa et al., 1992; Andersson et al., 1994). The soluble organic carbon levels from assorted batch tests were reported in Table 4.4. Levels at the beginning of the batch test were roughly 50 mg/L soluble B O D 5 . This data suggests that although the suspended nitrifier portion nitrified well, successfully removing almost all ammonia, the possibility nitrification was inhibited on the attached growth by the high level of soluble carbon exists. Despite the null results from this study some similar previous studies on attached growth media submerged in aerobic mixed liquor did report nitrification! The B O D 5 : T K N ratio in these reports indicate soluble organic loading was not a deterring factor. O f special interest to this study was the report by Sen et al. (1993) using Ringlace® as an attached growth media for nitrification. In that report the B O D 5 : T K N ratio was 5. The B O D 5 : T K N ratio for this study was 2 and consequently the B O D 5 loading effect on nitrification should not have been an inhibiting factor. However in that study the nitrification on the media was not separately tested. The reported results might have simply been due to enhanced suspended nitrification rates. The soluble B O D 5 measurements from Table 4.4 were taken before and after each batch test with the percentage of soluble B O D 5 removed between these measurements recorded in the table. O f particular interest were the percent removal recordings for batch tests A (A-side anoxic zone supernatant) and A - A (A-side attached growth). On average the A - A batch test removed twice the percentage of soluble B O D 5 as did A suggesting soluble carbon consumption by the biomass on the support media signifying possible heterotrophic growth on the Ringlace® media. 47 Chapter 4. Results and Discussion TABLE 4.4. SOLUBLE BOD5 (mg/L) VALUES FOR SIX BATCH TESTS AT ASSORTED DO, SRT, AND HRT CONDITIONS Run 1 1 1 4 5 5 Avg DO (mg/L) 3.5 3.5 3.5 1.5 3.5 3.5 SRT (days) 8 8 8 4 4 4 HRT (hours) 12 12 12 9 9 9 A-S initial 50.10 51.45 35.23 65.30 67.00 31.00 50.02 A-S final 24.90 23.96 18.43 35.35 28.83 10.50 23.66 % rpmnvpri 50 SO 53 4n 47 7n 45 on 57 00 fifi 10 53 an A-S/A initial 50.70 47.44 46.27 60.13 67.55 31.70 50.63 A-S/A final 32.55 25.84 18.47 32.70 37.50 11.90 26.49 % rpmm/pri 35 an 45 50 fin m 45 fif) 44 5n fi? 50 49 on A-A initial 50.25 44.96 38.87 56.00 52.93 30.00 45.50 A-A final 34.46 24.71 18.10 19.33 31.18 18.40 24.36 % rpmm/pri 31 40 45 53 4 fi5 5n 41 10 38 30 45 Rn A initial 54.05 51.30 32.70 46.02 A final 44.95 37.88 24.20 35.68 % rpmm/pri 1fiR0 9fi 9n 9fi nn ?3 nn B-S initial 47.9 52.91 53.77 67.08 58.43 52.50 55.43 B-S final 31.0 35.00 21.37 29.95 24.18 33.60 29.19 % removed 35.3 33.80 60.30 55.40 58.60 36.00 46.60 Soluble carbon enhanced heterotrophic growth might have limited autotrophic attachment in this study but other factors might also have come into play. Autotrophic bacteria in suspension around the Ringlace® surface might have been impeded from attaching due to surface specificity. Studies summarized in text books (Beachey, 1980; Denyer, 1993) have indicated some chemical 48 Chapter 4. Results and Discussion characteristics between cell walls and surfaces cause species specific adhesion. Covalent bonding, ionic bonding or hydrophobicity mismatches between a cell wall and a surface may prevent a bond (Beachey, 1980). Extra-cellular material produced by enzymatic activity could change chemical characteristics at the outer cell wall surface making it an incompatible surface for bonding (Denyer, 1993). The biomass that attached to the media in this study was a slime type layer as indicated by visual inspection only. In attempting to offer a plausible answer to the negative results of this study it is possible that the nitrifying bacteria may have lacked chemical characteristics at the cell wall surface to allow attachment to the slime layer on the media. 4.4 SURFACE AREA EFFECT Large specific surface area wil l increase the amounts of attached biomass in the process. More biomass equates to a greater capacity to absorb nutrients including ammonia i f nitrifiers make up part of the attached biomass. The amount of specific surface area for Ringlace® was smaller than other submerged type media studies. Specific surface area o f other submerged media studies were typically 100 to 150m 2 /m 3 (Andersson, 1990; Schlegel, 1988;Rusten, 1984; Bonhomme, 1990; Andersson et al., 1990). The Ringlace® surface does not allow easy area calculation due to an irregular shape. The surface of the lace strands were covered with layers o f organisms that extend out from the rope centre. The section view of the media cutting horizontally across looked like a rough jagged star shape with many points and the surface area could be calculated after assuming a cylinder of 49 Chapter 4. Results and Discussion this cross section. After working with this approximation a maximum value of 60 m 2 /m 3 was reached. This number was still one third to half the other studies reported above and could mean that there was insufficient surface area for significant nitrifying bacteria attachment. However, since the value was at least one third to one half the other studies, an ammonia uptake rate of the same ratio would be expected. For this reason specific surface area was not considered a limiting factor to nitrifying bacterial attachment. 4.5 SLUDGE SETTLING CHARACTERISTICS A reported advantage with using attached growth media was to raise process suspended solids concentration because settling conditions were improved (Lessel et al., 1993). Higher process suspended solids concentration could equate to larger nitrifying organism populations in the suspended sludge resulting in improved ammonia uptake rates. In this study settling conditions were observed to improve for the test process A-side over the control process B-side. During run 6 the decreased S V I value, indicating a more compressible sludge, was significantly lower at the 5 % level in A-side than B-side (Figure 4.4, Table 4.5). B -side effluent SS were higher than A-side during this run (Table 4.6, Figure 4.5) and at times sludge age control was not possible in B-side due to uncontrolled effluent solids losses resulting in SRTs lower than 4 days. However during run 6 the M L S S were not significantly different from A to B sides (Table 4.6) and ammonia uptake rates were higher for B-side than A-side (Figure 4.3). The large worm infestation which occurred on the media in A-side may have limited the 50 Chapter 4. Results and Discussion ammonia uptake rates despite the improved settling. The worms could also be the reason for better settling since samples with more worms settled better. This effect could be due to the direct consumption of floes by the worms. The worms could also have detached from the media to become a part of the suspended sludge and help it to settle better. TABLE 4.5. ANOVA OF SVI, RUN 4, 5, AND 6 Statistic (A-side vs B-side) Run #4 SVI Run # 5 SVI Run #6 SVI F 0.10 0.50 19 F critical 4.0 4.0 4.0 a level 5% 5% 5 % degrees of freedom 69 69 66 TABLE 4.6. ANOVA OF MLSS AND EFFLUENT SUSPENDED SOLIDS, RUN 6 Statistic (A-side vs B-side) Run #6 MLSS Run #6 Eff SS F 0.0 6.5 F critical 4.0 4.0 a level 5% 5 % degrees of freedom 69 51 51 Chapter 4. Results and Discussion S V I •5 150 > Run #4 Run #5 o Run #6 o o ® ® A X a X X as # o x % X " *L ° en i » —WW O _ i X ^ - f ^ X x * xX . X X 1 1 1 1 — i — i — i — i — i — i -X —1—1—1—1— 0 20 40 60 80 100 120 140 160 Days at A-side * B-side (control) F I G U R E 4 . 4 . PROCESS SLUDGE VOLUME INDEX RUN 4 , 5 , AND 6 M L S S X * Run #6 • K Run #4 Run # 5 OB O * - n " r T X OS O <s> X X x f f i % ° i r S2 J E x X X 2E ' _ X SS X X a ? ° ° 1 1 1 1 1 1 1 1 h 1 1 1 1 0 20 40 60 80 100 120 140 160 Days x A-side * B-side (control) F I G U R E 4 . 5 . PROCESS MLSS OF RUN 4 , 5 , AND 6 52 Chapter 4. Results arid Discussion Effluent SS 50 0 Run tt 4 Run tt 5 x Run #6 o x x X X % m X X " x x • _ X X ® m o x * «L X „ X ^ T X o x J ? % 5 1 1 1 1 x X x * % * < » x o 2 — 1 — 1 — 1 — . — 1 - , * x u x * . T " ^ V X X . I S . 1 20 40 60 80 100 120 140 160 * A-side * B-side (control) FIGURE 4 . 6 . PROCESS EFFLUENT SUSPENDED SOLIDS RUN 4 , 5 , AND 6 4.6 R I N G L A C E ® BIOMASS 4.6.1 Ringlace biomass characterization The stalked ciliated protozoa, bacteria, and worms that were commonly found in sewage wastewater treatment processes (Esteban et al., 1991; Andersson, 1990) were found growing on the media (Figure 4.7, 4.8, 4.9). Morphology was not attempted for the bacteria but microscopic photographs were used to observe the higher life forms. Worms were identified as a species of the Oligochaete genus Dero (personal communication with Dr. W . D . Ramey of the U B C Microbiology department). There was some similarity to both Dero obtusa and Dero digitata but 53 Chapter 4. Results and Discussion the specific morphology was slightly different than either of these species, as i f they represented a distinct local variant or species. 1 cm = 100 urn F I G U R E 4.7. S T A L K C I L I A T E S 54 Chapter 4. Results and Discussion 1 cm = 240 um FIGURE 4.8. ROTIFERS Typical rotifers found in the scrapings from the media (Figure 4.8), Here the patches seen around the rotifers were bacterial floes. 1 cm = 240 nm FIGURE 4.9. WORMS ON MEDIA 55 Chapter 4. Results and Discussion 4.6.2 Worm effect and growth Many different species of microscopic worms are known to occur naturally in sewage. Normally they are not a problem since their numbers stay low enough not to interfere with process microbial activity. However, under certain environmental and operational parameter conditions the populations can grow excessively large and interfere with normal process operation. It is possible that the worms and other higher life forms growing on the media limited the nitrifiers growth. The problem could be direct interference in nitrification if the worms secrete inhibitory molecules. These worms could interfere with the establishment of nitrifiers on the media by grazing them off faster than the nitrifiers could attach and regenerate. The heterotrophs however, with their fast growth rate, could regenerate quicker than they were being grazed and were able to maintain a presence. This mechanism could explain why there were bacteria and soluble carbon uptake (Table 4.4) on the media, autotrophs in suspension around the media but no nitrifiers on the media. Thus in addition to occupying space the worms might have consumed the other attached life limiting the nitrifiers. The fluctuation in DO level in this study appeared to affect the worm growth on the media. The worm level decreased in scrapings from the media after run 1 when the DO level decreased. Then from run 4 to run 6 when the DO level was first 1.5 mg/L then 3.5 mg/L and finally 5.5 mg/L the worm growth increased to a point where infestation levels were quantifiable through an unassisted visual inspection. Worms are a red colour and increasing amounts of worms directly correlate to increased intensity of colour in the biofilm. In run 4 some media 56 was Chapter 4. Results and Discussion portions were slightly red and in run 5 the surface was completely red but in run 6 the film intensely red and the worms were thick enough to cause bridging between the strands of the media. Worm population explosions were encountered in other Ringlace® studies (Lessel et al., 1993; Sen et al., 1993) and in studies with other submerged support media (Andersson, 1990). Andersson et al. (1990) observed worm development on the media when the DO level ranged from 5 to 7 mg/L. The worms may have inhibited nitrification on the support media in these studies but since the nitrification on the support media was not directly tested there is no data to decide the effect. An industry theory holds that worm outbreaks occur following the major shifts in temperature that usually take place in a transition season such as Spring and Fall. In this study the fluctuation in worm population also corresponded to the temperature swings of transition seasons. During the Fall towards the end of run 1 worms were noticed forming a red colouring on the Ringlace® when temperature fell roughly 4°C (Figure 4.1). In the Spring increased worms were noticed from run 4 to 6 when the temperature jumped roughly 4°C (Figure 4.2, 4.3). The Fall outbreak was not as large as the Spring outbreak and might have been affected by the lower operating DO level. 4.6.3 Worm population control Lessel (1991) and Sen et. al. (1993) controlled worm outbreaks by briefly limiting the available oxygen . The method briefly puts the entire process into an anaerobic condition by turning the air off in the aerobic stage and suffocating the worms. The fraction of worms actually 57 Chapter 4. Results and Discussion killed by the treatment compared to the fraction which simply moved to the process surface was not determined. When this method was used in run 6 gobs of worms could be seen floating on' the surface and attached to the weirs. Based on visual inspection these attempts at removing the worms from the media worked for 2 to 3 week periods during low DO operation. During high DO (5.5 mg/L, run 6) the method failed. In addition , operating the process to cause an anaerobic worm kill had a negative effect for the BEPR process mechanism and loss of phosphate to the effluent occurred. Ramey and Fong (1994) showed that three salts, sodium chloride, sodium nitrate, and calcium chlorite were effective at killing the worms. These tests showed that 1.5 hours contact time for 1.5 % and 3 hours for 1.2 % concentration were needed to assure a complete worm kill. This salt effect was consistent with a report which used sea water to flush secondary effluent fed trickling filters infested with worms (Andersson, 1994). The Ramey and Fong (1994) study suggested using a saline solution for worm population control. It appeared that the proper sodium chloride concentration would be 1.5 % and a contact time would be at least 3 hours. This chemical treatment was not attempted at the pilot scale in this study. 4.6.4 Ringlace® solids mass estimation To calculate a specific ammonia uptake rate for the attached nitrifiers it was necessary to measure the solids biomass on the media. An attempt was made to obtain a reasonable solids mass estimate under stable process operation. Seven centimetre long Ringlace® samples were attached to a 2 dimensional frame that was attached to the top four frame cubes in the process. 58 Chapter 4. Results and Discussion Samples were taken out at weekly intervals for two months and measured destructively for solids content. The measurement period took place over the months July, August, and September 1993. Further mass determinations throughout the experiment were subjective estimates, based on comparison to picture records (Figure 4.10, 4.11). T A B L E 4.7. MASS ESTIMATION O F SOLIDS O N M E D I A Samole Statistic Solids Content (s/m) Mean 9.5 Standard Deviation 3.7 95 % Confidence Interval +/- 2.0 Sample Size, N 13 As seen from the standard deviation the results were scattered quite widely around the mean. This variation could be due to the shedding action and experimental errors in the sampling and measuring technique. The photograph in Figure 4.10 taken during a batch test in run 1 shows the typical amount of biomass observed throughout the study except for the month January 1994. The solids seen on the media in Figure 4.10 corresponds to the 9.5 g/m mass measurements. The photograph in Figure 4.11 taken in January 1994 shows the solids thickness on the media when the process MLSS was extraordinarily high. This unique event in this study was believed to be due to high organic levels in the sewage stream over the Christmas holidays. The attached 59 V Chapter 4. Results and Discussion biomass quantity shown in Figure 4.11 was estimated by visual inspection to be 1.5 times the normal amount of biomass shown in Figure 4.10. FIGURE 4.10. CUBICAL FRAME WITH ATTACHED BIOMASS, 1 6 0 Chapter 4. Results and Discussion F I G U R E 4.11. C U B I C A L F R A M E W I T H A T T A C H E D B I O M A S S , 2 61 CHAPTER FIVE ' CONCLUSIONS AND RECOMMENDATIONS 5.1 CONCLUSIONS The first and general objective for this thesis was to grow nitrifying bacteria on the attached growth media Ringlace®. Measurement of specific ammonia uptake rate in batch tests would indicate the degree to which attached organisms were functioning. By comparing ammonia uptake rates for Ringlace® alone to other combinations of Ringlace® and suspended sludge from a control and test process, the attached uptake rate significance could be determined. After reviewing the batch test results the following conclusions were made. 5.1.1 PRIMARY CONCLUSION Observed specific ammonia uptake rates for the attached biomass on Ringlace® were never significantly different from zero at the 5 % significance level and significant attached nitrification was never observed under any manipulation of the operating parameters HRT, SRT, and DO tested in this experiment. 62 Chapter 5. Conclusions And Recommendations 5.1.2 SECONDARY CONCLUSIONS a) Suspended nitrification rates were similar to the values reported in the literature and occurred at average soluble B O D 5 levels of 50 mg/L, a B O D 5 : T K N ratio of 2, an SRT of 4 days, an H R T of 6 hours, a D O of 5.5 mg/L, and Temperature of 18°C. b) Nitrifier activity in suspended solids varied directly with temperature. c) The attached biomass consisted primarily of worms, and protozoa plus some bacteria. The worms appeared to increase in population with the increase in dissolved oxygen (DO) concentration. d) Soluble carbon was taken up by attached growth on the Ringlace®. e) Sludge settling for the test process improved over the settling for the control process during periods of high D O and consequent high worm concentration. The Mixed Liquor Suspended Solids ( M L S S ) and effluent suspended solids (eff SS) remained the same for both the test process and the control process even though sludge settling was different. f) Process anaerobic periods of 24 hours were successfully used to control worm growth on the media at D O levels < 3.5 mg/L but were not effective at D O levels of 5.5 mg/L. 63 Chapter 5. Conclusions And Recommendations 5.2 RECOMMENDATIONS Future research should focus on the following areas: 1) Specific measurements of nitrification should be done on the attached growth media reported to be nitrifying in full-scale processes treating municipal sewage. This research is necessary to decide whether Ringlace® can grow attached populations of actively nitrifying bacteria. 2) The level of soluble carbon removal achievable with this media should be investigated. Ringlace® could possibly be used for enhancing carbon removal at activated sludge plants. 3) The attachment specificity of nitrifier cell walls for slime surfaces typically found on attached growth media resulting from heterotrophic growth should be studied in order to understand the requirements for reasonable levels of attachment. These studies would also assist in selecting chemically appropriate media. 4) The front end of the aerobic zone could have presented conditions unfavourable to the attachment of nitrifying bacteria to the Ringlace® media. Conditions further down stream within the aerobic zone nearer the stage where rapid oxidation of soluble carbon decreases might be more conducive to attached nitrifier growth. 64 REFERENCES American Public Health Association (A .P .H .A . ) , (1992) Standard Methods For The Examination Of Water And Wastewater. 18th edition, Washington, D C . Andersson B . (1990) Tentative Nitrogen Removal With Fixed Bed Processes In Malmo Sewage Treatment Plant. Wat. Sci. Tech., 22, No . 1-2, pp. 239-250. Andersson B . , Aspegren H . , Parker D . S., and Lutz M . P. (1994) High Rate Nitrifying Trickling Filters. Wat. Sci. Tech., in submission. Arvin E , and Harremoes P. (1990) Concepts And Models For Biofilm Reactor Performance. "Wat. Sci. Tech., 22, No . 1-2, pp. 171-192. Barnard J. L . (1994) Protocal For Determination Of Nitrification Rates In Activated Sludge Plants. British Columbia Water & Waste Association, 22nd Annual Conference, Victoria, B . C . , May. Barnard J. L . (1975) Biological Nutrient Removal Without The Addition of Chemicals. Water Research, 9, pp. 485-490. Barnes D. , Bliss P. J. (1983) Biological Control Of Nitrogen In Wastewater Treatment, Printed in Great Britain, University Press, Cambridge. Beachey E . H . editor (1980) Bacterial Adherence, pp. 5-7, 348-353, Published by Chapman and Hall , London. Beccari M . , D i Pinto A . C , Ramadori R., and Tomei M . C. (1992) Effects OfDissolived Oxygen And Diffusion Resistances On Nitrification Kinetics, Wat. Sci. Tech., 26, N o . 8, pp. 1099-1104. Boiler M . , Gujer W., and Nyhuis G . (1990) Tertiary Rotating Biological Contactors For Nitrification. Wat. Sci. Tech., 22, No . 1-2, pp. 89-100. Bonhomme M . , Rogalla F., Boisseau G. , and Sibony J. (1990) Enhancing Nitrogen Removal With Fixed Growth Biomass. Wat. Sci. Tech., 22, No . 1-2, pp. 127-137. Cooper P. F., and Williams S. C. (1990) High-Rate Nitrification In A Biological Fluidized Bed. Wat. Sci. Tech., 22, No . 1-2, pp. 431-442. Danckwert P. M . (1953) Continuous Flow Systems - Distribution Of Residence Time, J. Chem. Eng. Sci., 2, pp. 1-13. 65 References Denver S. P., Gorman S. P., Sussman M . editors (1993) MicrobialBiofilms: Formation and Control, pp. 51-54, The Society for Applied Bacteriology Technical Series No . 30, Blackwell Scientific Publications, Oxford, London. Environmental Protection Agency U.S . (1993) Manual For Nitrogen Control. Office of Research and Development, Office of Water, Washington D C , 20460, EPA/62 5/R-93/010. Esteban G. , Tellez C , and Bautista L . M . (1991) Dynamics Of Ciliated Protozoa Communities In Activated Sludge Process. Water Research, 25, No . 8, pp. 967-972. Figueroa L . A . , and Silverstein J. (1991) Pilot Scale Trickling Filter Nitrification At The Longmont Wastewater Treatment Plant. Env. Engineering, pp. 302-307. Figueroa L A . , and Silverstein J. (1992) The Effect Of Particulate Organic Matter On Biofilm Nitrification. Water Env. Research, 64, No . 5, pp. 728-733. Finnson A . , Andersson B . (1991) A review Of Some Trickling Filter Applications In Systems For Nitrogen Removal, Nitrogenrensning Med Biofilmprosesser Seminar, January 29-30, Oslo Finland. Gasser J. A . , Chen C. L . , and Miele R. P. (1974) Fixed-Film Nitrification of Secondary Effluent. Presented at the E E D - A S C E Specialty Conference, Perm. State Univ., Pa. Hall E . R., Murphy K . L . (1985) Sludge Age and Substrate Effects On Nitrification Kinetics. Journal W P C F , 57, No . 5, pp. 413-418. Haug R. T., and McCarty P. L . (1972) Nitrification with the Submerged Filter. J W P C F , 44, pp. 2086-2102. Hem L . J., Rusten B . , and Odegaard H . (1994) Nitrification In A Moving Bed Biofilm Reactor. Water Research, 28, N o . 6, pp. 1425-1433. Kos M . , Wanner J., Sorm I., and Grau P. (1992) R-D-N Activated Sludge Process. Wat. Sci. Tech., 25, No . 4-5, pp. 151-160. Lessel T. H . (1991) First Proctical Experiences With Submerged Rope Type Bio-Film Reactors For Upgrading and Nitrification. Wat. Sci. Tech., 23, Kyoto, pp. 825-834. Lessel T. H . , Kopmann T., and Eichenau (1993) Experiences With Submerged Fixed-Bed Reactors For Nitrification Stages. Translation from Korrespondenz Abwasser, pp. 1652. 66 References Metcalf & Eddy, Inc., revised by Tchobanoglous G . , and Burton F. L . (1991) Wastewater Engineering Treatment, Disposal, and Reuse. Third edition, McGraw H i l l , Inc. McClintock S. A . , Randall C. W. , and Pattarkine V . M . (1993) Effects Of Temperature And Mean Cell Residence Time On Biological Nutrient Removal Processes, Water Env. Research, 65, N o . 2, pp. 110-118. McHarness, D . D . , Haug, R.T. , and McCarty P. L . (1975) Field Studies of Nitrification with Submerged Filters. JWPCF, 47, No . 2, pp. 291-309. Painter H . A . (1970) A Review of Literature on Inorganic Nitrogen Metabolism in Microorganisms. Water Research, 4, pp. 393, 410-424, 429-434, 441-450. Parker D . S., Lutz M . P., and Pratt A . M . (1990) New Trickling Filter Applications In The U.S.A. Wat. Sci. Tech., 22, No . 1-2, pp. 215-226. Ramey W . D . , and Fong F. (1994) The Mortality Effects Of Different Concentration of Various Salts On Sludge Worms. Department of Microbiology and Immunology, U B C . Randall C. W. , Barnard J. L . , and Stensel H . D . (1992) Design And Retrofit Of Wastewater Treatment Plants For Biological Nutrient Removal. Water Quality Management Library, 5, Technomic Publishing Co. Inc., Lancaster, P A . U S A . Randall C. W. , Pattarkine V . M . , and McClintock S. A . (1992) Nitrification Kinetics In Single-Sludge Biological Nutrient Removal Activated Sludge Systems. Wat. Sci. Tech., 25, N o . 6, pp. 195-214. RustenB. (1984) Wastewater Treatment With Aerated Submerged Biological Filters. J W P C F , 56, N o . 5, pp. 424-431. Schlegel S. (1988) The Use of SubergedBiological Filters For Nitrification. Wat. Sci. Tech., 20, N o . 4-5, pp. 117-187. Sen D. , Farren G . D . , Rhodes R , Copithorn, and Randall C. W . (1993) Full Scale Evaluation Of Nitrification And Denitrification On Fixed Film Media (Ringlace) For Design Of Single Sludge Nitrogen Removal System. Anahiem Conference, pp. 137-148. Shammas N . K h . (1986) Interactions Of Temperature, pH, And Biomass On The Nitrification Process. Journal W P C F , 58, No . 52, pp. 52-59. Tendaj M . , Reinius L . G . , and Hultgren (1992) Some Observation On Nitrification And 67 References Denitrification Following Full-Scale Trials At Henriksdal AndBromma Sewage Treatment Plants In Stockholm, Wat. Sci. Tech., 25, No . 4-5, pp. 195-202. Tsuno H . , Somiya I., Matsumoto N . , and Sasai S. (1992) Attached Growth Reactor For BOD Removal And Nitrification With Polyurethane Foam Medium. Wat. Sci. Tech., 26, No. 9-11, pp. 2035-2038. Valve M . , and Rantanen P. (1991) Some Observation On Reaction Rates With Free Swimming Carrier Material, Nitrogenrensning med Biofilmprosesser, Oslo, January 29-30. Zaloum R. (1992) Significance And Establishment Of Adequate Transition Periods In Wastewater Experimentation Env. Tech., 13, pp. 605-619. 68 APPENDIX A BATCH TEST RESULTS TABLE PAGE A l . Specific Ammonia Uptake Rates 70 A2 . R A 2 Values for Ammonia Uptake Rates 71 A 3 . Batch Test Parameter Data 72 A4 . Suspended Solids for all Batch Tests 72 FIGURE A l . Typical Batch Test Curves for A - S 73 A2 . Typical Batch Test Curves for B-S 73 A 3 . Typical Batch Test Curves for A - S / A 74 A4. Typical Batch Test Curves for A - A 74 A5 . Typical Batch Test Curves for A 75 69 TABLE A1. SPECIFIC AMMONIA UPTAKE RATES Run Batch Specific NH3 Uptake (mg-N/gSS-hr) # # A-S B-S A-S/A A-A A 1 1.97 2.49 1.70 -0.26 -2 1.93 2.33 1.72 0.01 -3 1.38 1.73 1.27 0.08 -1 4 2.01 2.18 2.02 -0.20 -5 2.23 2.23 2.50 0.06 -6 2.45 2.35 2.45 0.00 -7 1.45 1.50 1.03 -0.12 -8 1.71 1.32 1.24 -0.05 -9 1.73 1.77 1.68 0.01 -1 1.53 1.57 1.58 0.11 -3 2 1.92 1.52 1.71 0.35 -3 1.59 1.54 1.63 0.16 -4 1 1.24 1.05 0.66 0.24 -2 1.01 0.91 0.67 0.09 -1 0.18 1.04 0.66 0.05 -8.75 5 2 0.56 1.52 0.66 -0.44 2.21 3 0.61 1.11 0.69 0.03 -0.21 1 0.50 1.52 0.48 0.09 0.57 6 2 0.48 1.92 0.34 0.10 -0.91 3 0.46 2.37 0.08 0.36 -0.08 Notes: 1. For an explanation of A-S, A-S/A, A-A, A, and B-S see section 3.2.1. 70 TABLE A2. RA2 VALUES FOR AMMONIA UPTAKE RATES Run Batch RA2 Values for ammonia uptake rates # # A-S B-S A-S/A A-A A 1 0.99 0.98 0.99 0.60 2 0.96 0.96 0.96 0.00 3 0.98 0.97 0.98 0.04 4 0.98 0.98 0.99 0.00 1 5 0.99 0.97 0.99 0.04 6 0.99 0.99 0.98 0.00 7 0.99 0.99 0.95 0.27 8 0.98 0.97 0.97 0.03 9 0.99 0.99 0.99 0.02 1 0.99 0.99 0.99 0.10 3 2 0.99 0.97 0.99 0.64 3 0.99 0.93 0.99 0.25 4 1 0.89 0.93 0.75 0.30 0.44 2 0.95 0.95 0.93 0.79 0.02 1 * 0.00 0.96 0.62 0.02 0.23 5 2 0.81 0.97 * 0.03 0.73 0.18 3 ** 0.46 0.93 * 0.06 0.01 0.00 1 0.63 0.98 0.76 0.30 0.13 6 2 0.93 0.97 0.83 0.09 0.68 3 0.87 0.96 * 0.08 0.03 0.00 notes: 1. The RA2 value for a horizontal regression line is zero because RA2 = SSR/SSTO and SSR goes to. zero as all values on the regression line approach the average of the data. 2. A-S * An error at the lab caused data to be faulty. 3. A-S ** Errors in batch test sample timing. 4. A-S/A Rate data approaches horizontal line. 5. A-A All data approached horizontal line. 71 TABLE A3. BATCH TEST PARAMETER DATA Run Batch HRT SRT DO Temp Day Date # # hr days mg/L deg C 1 12 20 3.5 18.9 35 Jul 7 2 12 20 3.5 19.1 42 Jul 14 3 12 14 3.5 19.5 56 Jul 28 1 4 12 14 3.5 20.0 63 Aug 4 5 12 14 3.5 21.4 70 Aug 11 6 12 14 3.5 21.0 77 Aug 18 7 12 8 3.5 18.3 106 Sep 15 8 12 8 3.5 19.3 113 Sep 22 9 12 8 3.5 18.6 120 Sep 29 1 9 8 1.5 14.7 26 Jan 5 3 2 9 8 1.5 14.8 33 Jan 12 3 9 8 1.5 15.6 40 Jan 19 4 1 9 4 1.5 12.8 74 Feb 23 2 9 4 1.5 13.7 88 Mar 9 1 9 4 3.5 13.0 100 Mar 21 5 2 9 4 3.5 16.8 109 Mar 30 3 9 4 3.5 14.5 116 Apr 6 1 6 4 5.5 17.5 15 Jun 1 6 2 6 4 5.5 17.2 29 Jun 15 3 6 4 5;5 19.0 36 Jun 22 TABLE A4. SUSPENDED SOLIDS FOR ALL BATCH TESTS Run Batch Suspended Solids (mg/L) # # A-S B-S A-S/A A-A A 1 1 3180 2350 3180 226 2 3063 2716 3063 213 3 2532 2775 2640 226 4 2372 2415 2239 213 5 2340 2360 2250 100 6 1980 2078 1804 333 7 1655 1470 1450 185 8 1455 1495 1455 410 9 1620 1340 1550 240 3 1 3197 2898 3460 135 2 3020 3415 2977 123 3 3077 2983 2927 140 4 1 1160 1875 1041 609 158 2 1906 1560 1763 166 155 5 1 2334 1943 1890 245 170 2 1730 1528 1743 349 107 3 1953 1789 1950 158 147 6 1 2127 3285 1915 400 287 2 3054 2910 2955 421 315 3 2550 2035 2463 199 206 72 A-Side Suspended Day 26 HRT = 9 hr SRT = 8 days 6 0 8 0 Time (min) 1 4 0 NOx NH3 NH3 Uptake : 4.90 mg-N/L-hr per gram SS : 1.53 mg-N/gSS-hr FIGURE A 1 . TYPICAL BATCH TEST CURVE FOR A-S B-Side Suspended Day 26 HRT = 9 hr SRT = 8 days 6 0 8 0 Time (min) 1 4 0 NOx A NH3 NH3 Uptake : 4.54 mg-N/L-hr per gram SS : 1.57 mg-N/gSS-hr FIGURE A 2 . TYPICAL BATCH TEST CURVE FOR B-S 73 A-Side Suspended/Attached Day 26 HRT = 9 hr S R T = 8 days 60 80 Time (min) NOx A NH3 NH3 Uptake : 5.46 rag-N/L-hr per gram SS : 1.58 mg-N/gSS-hr per gram TS : 1.16 mg-N/gTS-hr FIGURE A3. TYPICAL BATCH TEST CURVE FOR A-S/A A-Side Attached Day 26 HRT = 9 hr S R T = 8 days 10 20 40 NOx A NH3 60 80 Time (min) 100 120 140 NH3 Uptake : 0.16 mg-N/L-hr per gram TS : 0.11 mg-N/gTS-hr FIGURE A4. TYPICAL BATCH TEST CURVE FOR A-A 74 A-Side Supernatant Only Day 20 HRT = 9 hr SRT 4 days 20 40 60 80 Time (min) NOx . NH3 100 120 140 NH3 Uptake : 0.24 mg-N/L/hr per gram SS : 2.21 mg-N/gSS/hr FIGURE A5. TYPICAL BATCH TEST CURVE FOR A 75 APPENDIX B PROCESS OPERATING AMMONIA DATA FIGURE PAGE B I . A-side Process Ammonia Data, Run 1, 20 D A Y SRT 77 B2 . B-side Process Ammonia Data, Run 1, 20 D A Y SRT 77 B3 . A-side Process Ammonia Data, Run 1, 14 D A Y SRT 78 B4. B-side Process Ammonia Data, Run 1, 14 D A Y SRT 78 B5. A-side Process Ammonia Data, Run 1, 8 D A Y SRT 79 B6. B-side Process Ammonia Data, Run 1,8 D A Y SRT 79 B7. A-side Process Ammonia Data, Run 3 80 B8. B-side Process Ammonia Data, Run 3 80 B9 . A-side Process Ammonia Data, Run 4 81 BIO. B-side Process Ammonia Data, Run 4 81 B11. A-side Process Ammonia Data, Run 5 82 B12. B-side Process Ammonia Data, Run 5 82 B13. A-side Process Ammonia Data, Run 6 83 B14. B-side Process Ammonia Data, Run 6 83 76 HRT = 12 hr SRT = 20 d DO = 3.5 mg/L 1 0 + 2 0 3 0 Days since beginning of Run 5 0 ANOX © AERO-1 T AERO-2 • AERO-3 X EFF FIGURE B1. A-SIDE PROCESS AMMONIA DATA, RUN 1, 20 DAY SRT HRT = 12 hr SRT = 20d DO = 3.5 mg/L 5 4-2 0 3 0 Days since beginning of Run ANOX ® AERO-1 • AERO-2 B AERO-3 X EFF FIGURE B2. B-SIDE PROCESS AMMONIA DATA, RUN 1, 20 DAY SRT 77 10 HRT = 12 hr SRT = 14 d DO = 3.5 mg/L Days since beginning of Run E l ANOX © AERO-1 V AERO-2 • AERO-3 X EFF FIGURE B3. A-SIDE PROCESS AMMONIA DATA, RUN 1,14 DAY SRT HRT = 12 hr SRT = 14 d DO = 3.5 mg/L 10 20 30 Days since beginning of Run 40 ANOX © AERO-1 V AERO-2 H AERO-3 X EFF FIGURE B4. B-SIDE PROCESS AMMONIA DATA, RUN 1, 14 DAY SRT 78 HRT = 12 hr SRT = 8 d DO = 3.5 mg/L 10 2.5 0 E l .. K ! E l S I * " E l E l E E l E l © © ® © ® © I . i l l 1 -JK— 0 5 10 15 20 25 30 35 Days since beginning of Run E l ANOX © AERO-1 W AERO-2 B AERO-3 X EFF FIGURE B5. A-SIDE PROCESS AMMONIA DATA, RUN 1, 8 DAY SRT HRT = 12 hr SRT = 8 d DO = 3.5 mg/L 10 15 20 25 Days since beginning of Run ANOX ® AERO-1 V AERO-2 B AERO-3 X EFF FIGURE B6. B-SIDE PROCESS AMMONIA DATA, RUN 1, 8 DAY SRT 79 20 HRT = 9 hr SRT = 8 d DO = 1.5 mg/L 15 E,10 m I 10 — ¥ X — — » * I X H » X -—H E X - S ; -20 30 Days since beginning of Run 40 ANOX © AERO-1 V AERO-2 E3 AERO-3 X effluent FIGURE B7. A-SIDE PROCESS AMMONIA DATA, RUN 3 HRT = 9 hr SRT = 8 d DO = 1.5 mg/L X * iS-X-X'S-X 1 g X § H 1-10 20 30 Days since beginning of Run 40 ANOX @ AERO-1 V AERO-2 ED AERO-3 X effluent FIGURE B8. B-SIDE PROCESS AMMONIA DATA, RUN 3 80 HRT = 9 hr SRT = 4 d DO = 1.5 mg/L - 8 • IS! ' • B * mm ^ o — H a i a a z — — v 10 20 30 Days since beginning of Run 40 50 ia ANOX AERO-1 • AERO-2 • AERO-3 Z Effluent FIGURE B9. A-SIDE PROCESS AMMONIA DATA, RUN 4 HRT = 9 hr SRT = 4 d DO = 1.5 mg/L • _ E l K I X E l X T X * • : • • E E l E E 3 S ....jgj.... T • f i E l I mm Ei X E S E t . _ - x ' * X ixB x l x x * > x • X • I 0 10 20 30 40 Days since beginning of Run E l ANOX • AERO-1 • AERO-2 • AERO-3 X Effluent FIGURE B10. B-SIDE PROCESS AMMONIA DATA, RUN 4 81 35 HRT = 9 hr SRT = 4 d DO = 3.5 mg/L 30 25 z 20 E . £ 15 I ® X 10 +••• X x x x x " I " I i 10 15 20 Days since beginning of Run 25 30 IS ANOX AERO-1 • AERO-2 AERO-3 X Effluent FIGURE B11. A-SIDE PROCESS AMMONIA DATA, RUN 5 HRT = 9 hr SRT = 4 d DO = 3.5 mg/L 30 — 25 — 0 -I 1 1 1 1 1 1 1 1 H 1 1 1 1 h 0 4 8 12 16 . 20 24 28 Days since beginning of Run IS ANOX « AERO-1 T AERO-2 . • AERO-3 X Effluent FIGURE B12. B-SIDE PROCESS AMMONIA DATA, RUN 5 82 20 HRT = 6 hr SRT = 4 d DO = 5.5 mg/L 15 E 10 : E ! X E3 E l * X i f* x f 5 4-10 20 30 Days since beginning of Run 40 E l ANOX • AERO-1 V AERO-2 X Effluent FIGURE B13. A-SIDE PROCESS AMMONIA DATA, RUN 6 HRT = 6 hr SRT = 4 d DO = 5.5 mg/L 10 20 30 Days since beginning of Run E l ANOX . • AERO-1 T AERO-2 X Effluent FIGURE B14. B-SIDE PROCESS AMMONIA DATA, RUN 6 83 

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