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Studies on the metabolism of polyhydroxyalkanoic acid and phosphate in the biological excess phosphate… Wu, Manhong 1993

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STUDIES ON THE METABOLISM OF POLYHYDROXYALKANOIC ACIDAND PHOSPHATE IN THE BIOLOGICAL EXCESS PHOSPHATEREMOVAL PROCESSbyMANHONG WUB.Sc., Fudan University, Shanghai, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Microbiology and Immunology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 1993© Manhong Wu, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of  H <, c,ro bi. Mod j ctAcii  17 rt\ rmkrvA0MThe University of British ColumbiaVancouver, CanadaDate  Oct. '5 Aq 93DE-6 (2/88)ABSTRACTThe basic objectives of my thesis were to examine the role of PHA in phosphatesequestering sludge metabolism and to test the effect of nutrient combinations on theintracellular level of PHA. The role of PHA in the process was studied by measuringlevels of PHA in both functional and non-functional sewage treatment plant conditions.The role of nutrients was studied by testing the effects on PHA of nutrient combinationswhich would be expected to affect the level of NADH. Despite previous correlationbetween PHA storage and phosphate sequestering, it was found that even with good PHAstorage in the anaerobic condition some sludges showed poor phosphate uptake in thecorresponding aerobic condition. PHA accumulation requires NADH and is dependent ona proper NADH and NAD balance in the cells. However, most biochemical studies havebeen limited to pure components such as acetate which have limited capacity to generateNADH under anaerobic conditions. Thus this study focused on nutrient combinationswhich would provide a suitable carbon source as well as proper reducing equivalentbalance. Several nutrient combinations were tested including the fermentation productspyruvate, butyrate and ethanol. Fluoroacetate was used as an inhibitor of the glyoxylatecycle. It seemed that the glyoxylate cycle was not the only source of NADH for PHAsynthesis.TABLE OF CONTENTS^ pageABSTRACT^ iiTABLE OF CONTENTS^ iiiLIST OF TABLES vLIST OF FIGURES^ viLIST OF ABBREVIATIONS^ ixACKNOWLEDGMENTS x1. Introduction ^  11.1 Biological phosphate removal^  11.2 Biological phosphate removal process^  41.3 Thesis objective^  82. Material and Methods^  92.1 Sludge sources for bench scale reactors^  92.2. Bench scale system^ 92.3 Oxidation reduction potential (ORP)^  102.4 PHA analysis^  102.5 Volatile fatty acid (VFA)^ 122.6 P-32 uptake ^  132.7 Fluorescein isothiocyanate (FITC) microscopy^  133. Results and Discussion ^  143.1 PHA storage and phosphate uptake^  143.1.1 Normal PHA metabolism in the plants  141113.1.2 PHA metabolism under different working conditions^ 163.1.3 Factors that may limit phosphate uptake or PHA consumption^ 223.1.4 Morphological distinct microorganism^ 233.2 Biochemical model test ^ 323.2.1 The similarity of the three reactors ^ 343.2.2 Continuous and pulse feeding of nutrients 373.2.3 The inhibitory effect of different concentrations of fluoroacetate^ 393.2.4 PHB and PHV synthesis^  453.2.5 Combination of acetate and pyruvate in the presence of inhibitinglevels of fluoroacetate^ 453.2.6 Combination of acetate and butyrate in the presence of fluoroacetate ^ 503.2.7 Other combinations of nutrients and acetate^ 504. Conclusions^  53References^ 54ivLIST OF TABLESpageTable I: PHA of Three Zones in UBC Pilot Plant.^ 15Table II: PHA in Anaerobic Zone, A Side, Modified UCT Plant.^20Table III: PHA in Anaerobic Zone, B side, Modified UCT Plant. 20Table IV: MLSS of Modified UCT Pilot Plant.^ 21Table V: PHA in Anaerobic Zone, A side, FGR/SGR Plant.^ 47Table VI: Nutrient Effects on PHA Synthesis in the Presence of Fluoroacetate.^51Table VII: Reducing Equivalents Associated with Different Organic Molecules^51LIST OF FIGURESpageFigure 1A: Flow schematic of the conventional activated sludge process.^5Figure 1B: Flow schematic of the five-stage Bardenpho process.^5Figure 1C: Flow schematic of the FGR/SGR pilot plant in UBC.^7Figure 1D: Flow schematic of the modified UCT pilot plant in UBC.^7Figure 2: A. typical chromatograph of a sludge sample.^ 11Figure 3: Phosphate uptake of anaerobic sludge (Sample date:February 23, 1993).^ 17Figure 4: Phosphate uptake of anaerobic sludge (Sample date:November 9, 1992).^ 18Figure 5: The effect of metal ions on phosphate uptake of anaerobic sludge.^24Figure 6A - B: FITC stain of distinct microorganism observed in theanaerobic B side when the modified UCT plant failed (1000x, magnification).^26Figure 6C - D: FITC stain of distinct microorganism observed in theanaerobic B side when the modified UCT plant failed (1000x, magnification).^27viFigure 6E - F: FITC stain of distinct microorganism observed in theanaerobic B side when the modified UCT plant failed(400x, magnification).^ 28Figure 6G - H: FITC stain of distinct microorganism observed in theanaerobic A side when the modified UCT plant failed(400x, magnification).^ 29Figure 61 - J: FITC stain of distinct microorganism observed in theanaerobic A side when the modified UCT plant failed (1000x, magnification).^30Figure 6K - L: FITC stain of distinct microorganism observed in theanaerobic A side when the modified UCT plant failed (1000x, magnification).^31Figure 7: Proposed biochemical model for PHA storage underanaerobic conditions.^ 33Figure 8: Redox profile of the parallel reactors during batch experiments.^35Figure 9: PHA synthesis in three parallel reactors.^ 36Figure 10: Comparison of the effects of nutrient feeding style.^38Figure 11: Effect of fluoroacetate concentration on PHA synthesis.^40Figure 12: Effect of an acetate and pyruvate combination on PHAsynthesis showing good fluoroacetate inhibition.^ 41vi iFigure 13: Effect of an acetate and pyruvate combination on PHAsynthesis in a system showing poor fluoroacetate inhibition.^42Figure 14: PHB net synthesis in the presence of acetate with or withoutfluoroacetate.^ 43Figure 15: PHV net synthesis in the presence of acetate with or withoutfluoroacetate.^ 44Figure 16: PHB and PHV synthesis with pure acetate feeding.^46Figure 17: PHB and PHV synthesis with combined acetate, fluoroacetateand propionate feeding.^ 48viiiLIST OF ABBREVIATIONATP^-adenosine triphosphateADP^-adenosine diphosphateAPI^-Analytical Profile IndexBEPR^-biological excess phosphate removalEMP^-Embden -Meyerhof-ParnasFGR/SGR^-fixed growth reactor/suspended growth reactorFITC^-Fluorescein isothiocyanateGLC^-gas-liquid chromatographyMLSS^-Mixed-liquor suspended solidNAD^-nicotinamide adenine dinucleotideNADH^-reduced NADORP^-oxidation-reduction potentialPHA^-polyhydroxyalkanoic acidPHB^-polyhydroxybutyric acidPHV^-polyhydroxyvaleric acidUBC^-University of British ColumbiaUCT^-University of Cape TownVFA^-volatile fatty acidixACKNOWLEDGMENTSI would like to thank the following persons for their support and guidance throughout myresearch and study.Dr. William Ramey for his supervision, patience, encouragement and support throughoutmy studies.Dr. William K. Oldham, professor of the UBC Department of Civil Engineering, andDr. Barbara Dill of the Department of Microbiology, for providing valuable advice andserving as my committee members.Frederick A. Koch, research associate in the Environmental Engineering Group, foroverseeing the operation of the University of British Columbia modified UCT pilot plantand providing valuable information.Al Gibb, for overseeing the operation of the University of British Columbia FGR-SGRpilot plant and providing valuable information.Tai Man Louie, fellow graduate student for numerous discussions and explanation and forassistance for taking samples from the plants.Terry Jock Mah, for providing valuable information and assistance for analysis of VFA.Yeow Chern Chou, for kindly lending his computer to me.x1. IntroductionEutrophication of water by excess phosphate is a world wide pollution problem.In order to prevent phosphate enrichment, phosphates must be removed from municipaland industrial waste water by physico-chemical and biological methods before the water isdischarged into the environment. The biological excess phosphate removal (BEPR)method is recognized as a preferable option in the sense of cost and efficiency (Canviro etat, 1986; Morrison, 1988), because the continuous addition of chemicals is expensive andthe inorganic precipitated residues from chemical treatment are still a burden to theenvironment. By biological treatment methods the phosphate is incorporated into biomasswhich is simply removed from the water by settling without generating toxic inorganicresidues.1.1 Biological phosphate removalThe BEPR method for wastewater is accomplished by introducing an anaerobiczone to the conventional treatment plant and recycling the sludge between anaerobic zoneand aerobic zone. It was first reported by Srinath and coworkers in 1959 as theobservations of enhanced phosphate removal in an activated sludge plant (Srinath et al,1959). Later Shapiro and Levin conducted intensive investigations on phosphate uptakeand release. They reported that in an anaerobic zone, phosphate was released while in asubsequent aerobic zone phosphate including part or all of the phosphate in the influentwas taken up (Shapiro, 1967; Levin et at, 1972). They proposed that this phenomenonwas due to biological activities. At that time there were some nonbiological explanationsof the process, today there is little doubt that this phenomenon is the result of microbialaction. In 1975, Fuh and Chen implicated Acitietobacter spp. in enhanced phosphateremoval systems. Their observation has been supported by others, (Osborn et at, 1986,Deinema et al., 1985, Gersberg and Allen, 1985, Lotter and Murphy, 1985, and Buchan,1983). However, it has been argued that Acinetobacter is not the only bacterium which isresponsible for BEPR. In 1990 Chu studied the potential populations of phosphatesequestering microorganism from several BEPR plants in B. C., including the Kelownaplant, Squamish plant and the UBC modified UCT (University of Cape Town, SouthAfrica) pilot plant. He reported that the sludge contained many genera of bacteria besidesAcinetobacter and many of these bacteria could be involved in BEPR activity. Hisobservations include some species previously implicated by others such as Pseudomonasvesicularis (Suresh et al, 1985), Klebsiella pneumoniae (Gersberg, 1985), Micrococcus(Ye et al, 1988), Aeromoncts hydrophila (Brodisch and Joyner, 1983), Arthrobacterglobiformis (Shoda et al, 1980), Moraxella spp. and Enterobacter spp. (Lotter andMurphy, 1985). These general identified groups of microorganisms are considered to becapable of accumulating polyphosphate (poly-P), polyhydroxyalkanoate (PHA) and/orglycogen. This belief is consistent with published models of BEPR activities and reportsthat PHA in sludge is a mixture of C4, CS, C6 and C7 components (Comeau, 1988a and1988b; Wallen et al., 1972 and 1974).Several models have been proposed to explain the biochemical basis of the BEPRprocess and the role of poly-P and PHA metabolism (Nicholls, 1978; Osborn eta!, 1979;Deinema, 1980; Marais et al, 1983). Two significant models were developed byComeau/Wentzel and Mino (Comeau et al, 1986; Mino, 1987; Wentzel et al., 1991).Both models indicate that the stimulation of BEPR requires sequential anaerobic/aerobiccycling of the sludge. Although it is well known that the phosphate release and PHAstorage are the two major events in the anaerobic zone, the function of the anaerobicphase is not fully understood. It is generally believed that the abundant readilybiodegradable organic carbon in this anaerobic zone, e.g., acetate, glucose, amino acid,will be converted to acetyl-CoA and stored as polyhydroxyalkanoate (PHA) (Comeau,1986) and/or glycogen (Mino, 1987). Stored poly-P is believed to be hydrolyzed tophosphate to supply the energy for the activation of acetate to acetyl-CoA and the2maintenance of the membrane potential (Comeau, 1988b). Therefore, the models tie theobserved anaerobic P-release to the conversion of soluble organic carbon to PHA.Comeau et al. (1988b) studied their model system by feeding pure acetate and volatilefatty acids as sole carbon source and concluded that these nutrients are major substratesfor BEPR bacteria in anaerobic zone. Mino (1987) used artificial sludge fed with glucoseas the sole carbon source and suggested glucose was a major substrate which would bestored as PHA or as glycogen. In both models the magnitude of P-release in the anaerobicphase was directly linked to the magnitude of phosphate uptake later in the aerobic phase.Since better PHA storage should allow better anaerobic P-release it has been generallyassumed that better PHA storage should subsequently lead to better phosphate removal inthe following aerobic phase.In both models, the BEPR bacteria with internal carbon reserves should haveadvantages over those bacteria without carbon reserves during the nutrient limitingaerobic phase, because these stored nutrients could be hydrolyzed to produce energy andcarbon for growth and the uptake and the storage of phosphate as poly-P. However, themodels differ with regard to the generation of the reducing equivalents of nicotinamideadenine dinucleotide (NADH) which are required to convert acetate topolyhydroxybutyrate (PHB). Comeau/Wentzel proposed that acetate could be partiallyoxidized through the TCA cycle to provide the major source of NADH for the reductionof acetyl-CoA to PHB. Mino suggested that the metabolism of glycogen via the EMPpathway would provide the principle NADH to store fatty acid as PHB. The actual supplyprobably depends on the source of nutrient (Mah, 1991) and might use NADH from bothtypes of pathways (Mah, 1991). Comeau/Wentzel also suggested that the ratio ofNADH/NAD and of ATP/ADP were the factors which regulated the metabolism of PHA.Gerber (1986) tested a variety of nutrients and found that phosphate released inthe anaerobic zone was primarily dependent on the nature of substrates. Acetate andpropionate were concluded to be the best substrates for BEPR bacteria, although many3other substrates gave comparable activity. The problem associated with these studies wasthat the tested substrates used in these experiments were pure compounds fed into thesystem as single pulses of nutrient. The natural nutrient is probably a mixture ofcontinuously supplied compounds. These mixed nutrients in the real situation wouldprobably provide a better balance of reducing power for PHA synthesis than any givensingle nutrient. In addition, the tricarboxylic acid cycle (TCA) or Embden-Meyerhoffpathway (EMP) is probably not the only source of NADH because the breakdown ofnutrients like butyrate may provide extra reducing power through the unique degradationpathways (Schubert, 1988). Consequently, the important potential of interactions betweennutrients used for synthesizing PHA remains unclear. This type of nutrient interaction mayaccount for the observation made by Comeau (Comeau, 1988b) and Mah (Mah, 1991)that the amount of stored PHA is greater than the quantity of specific supplied nutrient,even though PHA synthesis in each of their systems showed absolute dependence on thesupplied nutrients.1.2 Biological phosphate removal processThere are several types of enhanced biological phosphate removal processes, allbased on the conventional activated sludge systems (Fig. 1A). In the PhoStrip process(Levin and Shapiro, 1965; Fig. 1B) only a small fraction of sludge from the anaerobic zonewhich is called phosphate-stripper is recycled to the aerobic basin, the rest is treated withlime to precipitate the phosphate. In the original Bardenpho process, the anoxic zone wasseparated from the aerobic zone (Barnard, 1973). Barnard (Barnard, 1974) postulatedthat the key requirement for enhanced biological phosphate removal was the exposure ofsludge microorganisms to anaerobic conditions, under which phosphate release occurred.A five-stage modified Bardenpho process was developed with an additional anaerobicstage placed at the head of the process (Mclaren and Wood, 1976; Davelaar4low -Peffluentprimaryanoxicinfluent^1.--onoerobic '-4- primaryaerobicinfkient settling^effluentbasinaerobicbasinreturn sludgewaste sludgeFigure IA: Flow schematic of the conventional activated sludge process (Toerienet al., 1990).P- enrichedmixed liquor return^ waste sludgereturn sludgeFigure 1B: Flow schematic of the five-stage Bardenpho process. (Toerien et al.,1990)5et cd., 1978). Barnard 1976 also found that nitrate had a detrimental effect on phosphateremoval. This effect later was confirmed by the others (Simpkins and Mclaren, 1978;Nicholls and Osborn, 1979; Hascoet and Florentz,1985). Subsequently the UCT processwas developed by Ekama et al. (1984) to counteract this nitrate detrimental effect. Theprocess is similar to the three-stage Bardenpho process except that the return sludge isdischarged to the anoxic reactor rather than the anaerobic reactor, and the mixed liquor isrecycled from the anoxic reactor to the anaerobic reactor. The principal advantage of thischange is the opportunity for NO3- in the return sludge to be denitrified in the anoxicbasin before entering the anaerobic basin. The modified UCT process contains an anoxicreactor divided into two separate compartments so that denitrification of the return sludgeand mixed liquor recycle streams can be controlled separately (Toerien eta!., 1990).Two BEPR pilot plants are currently operated in UBC, a modified UCT plant anda Fixed Growth Reactor-Suspended Growth Reactor (FGR-SGR) plant (Fig. 1C and Fig.1D). They both include the essential three compartments of biological treatment plant, ananaerobic zone, an anoxic zone for denitrification and a following aerobic zone. Afermenter is put in front of the anaerobic zone in each plant so that the organic compoundswill be fermented and degraded to the volatile fatty acid substrates which can be used byBEPR bacteria in the anaerobic zone. The major differences between these two plantscome from the aeration mechanism and the fermenters. The modified UCT plant providesthe aeration to the aerobic basin by air bubbling and stirring. The FGR-SGR plantprovides air by a trickling filter. The latter one is believed to have greater aerationcapacity and better sludge settlement. The UCT plant uses a intermittently mixed upflowclarifier (IMUC) fermenter while the FGR-SGR uses a standard continuously mixedfermenter. Both plants are operated in a dual mode to allow comparative analysis oftreatment in an experimental A side and a corresponding untreated B side.6Return SludgeWaste---)IMUC EffluentStorage Anaerobic AnoxicReturn SludgeAerobic ClarifierFigure IC: Flow schematic of the modified UCT pilot plant in UBC.Return Sludge1 ••••■•■••••■--) V--) EffluentFermenter —› --> —).11■■■•••■Storage Anaerobic Anoxic Trickling Aerobic^ClarifierFilterFigure ID: Flow schematic of the FGR/SGR pilot plant in UBC.71.3 Thesis objectiveBEPR process has been widely used to remove phosphate from wastewater.Intensive research on its biological mechanism has been conducted. Yet because of thecomplicated nature of the system, the ecology and physiology of the microbial processeshave not been thoroughly understood (Toerien et al., 1990). The presence of PHA isthought to be important and the effects of several nutrients such as acetate and propionateon the BEPR system have been well studied by Comeau (1988b), Gerber (1986) andothers. However, as mentioned earlier, most of these studies only involved purecompounds or a limited combination of VFA. The basic objective of this thesis was tostudy the role of PHA in the BEAR response and test the effect of nutrient combinationson the accumulation of intracellular level of PHA.82. Material and Methods2.1 Sludge sources for bench scale reactorsDepending on the experiment, about 30 liters of sludge were collected from eitherthe FGR-SGR pilot plant or the UCT plant and carried in plastic tanks to the laboratory.This sludge was allowed to settle and decanted to make approximately 12 liters ofconcentrated sludge with a ratio of supernatant to settled solid of about two to one(volume to volume). This concentrated sludge would either be used immediately for theexperiments if it originated from the anaerobic zones or allowed to sit overnight beforeuse if it was taken from the aerobic zone.2.2 Bench scale systemIn each experiment the concentrated sludge was distributed into three 4-litrecylindrical plexiglass reactors and incubated with gentle stirring until the redox was -100mV. One reactor was fed with various nutrient combinations to determine the effect ofthe nutrient combination on PHA level in the sludge. The other two reactors were used ascontrols. One control was used as a reference point for the potential ability to accumulatePHA. It was fed with pure acetate. The other control was either not fed or fed with amixture of acetate and inhibitor to determine the basal level of PHA synthesis in thesystem. In each case the nutrient was pumped at a constant rate through a thick-walltubing (diameter 1 mm) to the appropriate reactor. The pump (Dayton Electric MFG.Co., Chicago, USA) was equipped with three identical heads (Master flex, Model 701321) so that the nutrient was fed into three reactors in the same way. The toxin wasdissolved in distilled water prior to addition and added through a port located at the top ofthe reactors. In each reactor the sludge was slowly stirred with a multibladed motordriven propeller. Every half hour approximately 7.5 ml of sludge was withdrawn from a9sampling port located near the bottom of each reactor and immediately treated by theaddition of 2 ml of commercial 6% hypochlorite bleach solution (Chemtech) to stopfurther biological activities Mah, 1991; Poindexter and Eley, 1983).2.3 Oxidation reduction potential (ORP)An oxidation reduction potential (ORP) probe (Broadley-James Corp., Santa Ana,CA.) connected to a Fisher Accumet pH meter (Model 600) was used to continuouslymonitor ORP in each reactor. The data were recorded automatically using the Fermacsoftware package (BioChem technology Ltd., Malvern, PA). Each probe was calibratedby buffered quinhydrone standards to give the readings of 86 mV in pH 7 buffer and 263mV in pH 4 buffer. The ORP data were used to assess the relative anaerobic and aerobicstates of the sludge.2.4 PHA analysisPHA was isolated according to the method of Poindexter and Eley (1983) by firstadding hypochlorite to dissolve the membranes of the cells. The PHA granules in thehypochlorite-treated mixture were then collected by centrifuging the sample at 10,000 rpmfor 20 minutes, the supernatant was decanted and the remaining pellet was freeze-driedovernight. After the dried pellet samples containing the PHA granules were weighed todetermine the solid content, the solid was heated in acidified methanol (3% H2SO4) andchloroform for 3.5 hours. The PHA was methylated and extracted to chloroform. Thischloroform solution was then analyzed by injecting 2 pi to the gas liquid chromatography(GLC) using the method published by Comeau (1988a) and modified by Mah (1991). APerkin-Elmer, Sigma 3B dual FID chromatograph equipped with a DB-wax column andHewlett Packard 3396A integrator was used for the analysis. A typical chromatogram ofa sludge sample is shown as Fig. 2. The PHA peaks were specifically identified by gas10Retention Time (Minute)Figure 2: A typical chromatograph of a sludge sample. A: PHB, 8.063 minutes; B:PHV, 10.635 minutes; C: oxo-valeric acid, 10.952 minutes; D: benzoic acid asinternal standard, 12.220 minutes.11chromatography/mass spectrometry and routinely identified by the relative retention time.The corresponding amount of 13-hydroxybutyrate (HB) was estimated from a standardcurve. This standard curve was made by plotting the ratio of the areas of the BIB peak andthe internal standard of benzoic acid (100 mg/L) against different known concentrations ofHB sodium salt. Since P-hydroxyvalerate (HV) standard was not available, the responseof HV was first directly read from the same standard curve as BIB and then calibrated bythe formula ofcalculated final [HV] = (-0.00087 x [HV] read from standard+1.296) x [HV] readfrom standard(Mah, personal communication). The final calculated concentration of PHA wasnormalized by dividing by the dried weight and expressed as jig PHA/mg dried weight ofsludge. With pure 1-113 standards, the detection limit was estimated to be 10 lig/L(Comeau, 1988a).2.5 Volatile fatty acid (VFA)VFA was analyzed by GLC. Sludge samples were taken, then separated bymicrocentrifuge for 2 minutes at full speed before one ml of each supernatant wasremoved and acidified by adding 10 jti of 10% H3PO4. One microliter of each sample ofthe acidified supernatant was then directly injected into a column packed with 60/80Carbopack C/0.3% Carbowax (Supelco Canada Ltd., Oakville, Ontario) for analysis of Clto CS VFA compounds. The gas chromatograph was operated isothermally with an oventemperature of 1200C, injection and detector part temperatures of 2000C. The lowestconcentration of acetate that can be detected by this method was about 0.1 mg/L (Mah,personal communication).122.6 P-32 uptakeA radioactive phosphate assay was used to measure phosphate uptake (Chu, 1988;Mah, 1991). Three ml of sludge were put into a 16 x 150 mm test tube containing 0.5 KiP-32 (Sigma Chemical Co.) and 10 mg/L cold phosphate ( KH2PO4 ). The test tube wasthen aerated in a platform shaker. Every 15 minutes a 150 j_t1 sample was taken andcentrifuged in the microcentrifuge for 1 minute, a 100 1,t1 aliquot of supernatant was thenremoved and placed onto a filter paper disc. The filter paper disc was dried in the ovenand put into a vial with scintillation liquid. The radioactivity in the supernatant would thenbe counted using a Beckman LS6000IC scintillation counter to determine total insolubleradioactive phosphate. The ratio of radioactivity to cold phosphate as determined by thestandard methods for Wastewater (Standard Methods for Wasterwater, 18th Ed., 1992)was then used to calculate total levels of insoluble phosphate that was taken up by thebiomass.2.7 Fluorescein isothiocyanate (FITC) microscopyA loopful of sludge was placed onto the glass slide, air dried and heat fixed. Themount was then stained by fluorescein isothiocyanate (FITC) according to Pital's method(Pital et al, 1966). The slide was examined by an incident-light fluorescent microscopy(Zeiss, West Germany) with through-the-objective irradiation from a mercury vapor shortwavelength arc lamp light source, equipped with a blue excitation filter (Filter set, 48 7910; Exciter filter, BP 450-490; Barrier filter, 515-565). A 40x Neofluar objective and anoil-immersion 100x planapo objective were used with standard 10x occulars. Thephotographs were taken by a camera connected to the microscope. The exposure timewas 30 seconds. The film used was Fujicolor 200.133. Results and Discussion3.1 PHA storage and phosphate uptakeA direct relationship between phosphate release/phosphate uptake and PHAstorage/consumption has been previously observed. The enhanced phosphate uptakeunder aerobic condition by BEPR bacteria was proposed to be limited by the availability ofstored PHA since the rate of phosphate uptake was negligible when the sludge PHAconcentration was about 5 mg HB/L (Comeau, 1988b). Thus it has been widely believedthat PHA storage and phosphate uptake are tightly coupled and it is generally assumedthat better PHA storage should lead to better phosphate uptake. According to this belief,for optimum BEPR, PHA storage should be maximized in the anaerobic zone of the BEPRprocess. Efforts have been made to test this belief, and greater phosphate release in theanaerobic zone does correlate to greater consumption of PHA. However, the reciprocalinteractions had not been studied. Therefore, the effect of a diminished phosphate uptakeresponse on stored level of sludge PHA was studied when the A side of the modified UCTpilot plant quit taking up phosphate during the course of this thesis.3.1.1 Normal PHA metabolism in the plantsWithin the models, Acittetobacter is accepted as typical of the BEPR bacterium.This genus was initially reported as a strict aerobe (Juni, 1978), but some species withinthis genus can use nitrate as an electron acceptor (Chu, 1988 and Lotter, 1985). In eithercase, in the anaerobic phase with no oxygen or nitrate, nutrients will be stored as PHAand/or glycogen (Marais et al., 1983). In the aerobic phase, anaerobically stored PHA willbe hydrolyzed to supply energy and carbon to uptake phosphate which will be used forgrowth and stored as poly-P. Therefore, the PHA level will be higher in the anaerobic14basin than in the aerobic basin. This relative decrease in PHA concentration betweenreactor zones was observed in the operated UBC pilot plants.Table I indicates the observed PHA level of the three zones in the two plants. Inboth plants, the PHA storage was maximum in the anaerobic zones and lowest in theaerobic zones, which was consistent with the model that PHA is synthesized and brokendown as BEPR activated sludge goes through the anaerobic and aerobic cycle. Bothpolyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) were consumed aerobically,and PHV use was slightly preferable. The FGR-SGR plant consumed about 9.8 ligPHA/mg dried weight of sludge, the modified UCT plant consumed 5.5 i..tg PHA/mg driedweight of sludge. The observed PHB and PHV levels in the anaerobic zone of A sidewere similar to the average values of 5.5 1.1g PHB/mg MLSS and 6.1 i_tg PHV/mg MLSSreported by Comeau (1988b). However, the PHA level of the aerobic zone studied byComeau (1988b) was about 50% lower than the present level. These values were notabsolutely consistent, they varied from day to day. The major cause of the fluctuation waspresumably daily variation of sludge composition. However, the general relative patternof PHA storage and consumption was consistent as long as the plants worked normally.Table I PHA of Three Zones in UBC Pilot PlantsPlant SidePHA in each zone(ug/mg dried weight of sludge)Anaerobic Anoxic AerobicPHB PHV PHA PHB PHV PHA PHB PHV PHAFGR/SGR A 7.1 8.0 15.1 5.0 4.5 9.4 3.1 2.2 5.3B 6.2 5.8 12.0 5.1 3.2 8.4 1.8 1.3 3.1Modified UCT A 5.3 5.5 10.8 5.2 4.5 9.7 3.1 2.2 5.3B 4.0 4.9 8.9 4.2 4.0 8.2 3.5 2.6 6.1(Sample date: April 20, 1993)15During the times of these PHA observations the BEPR sludge from the pilot plantwas consistently capable of taking up phosphate under aerobic condition. Fig. 3 showsthe sludge of the B side from the modified UCT plant took up about 24.5 mg/L phosphatefrom the supernatant in 20 minutes, and the sludge from the A side took up 20 mg/Lphosphate. When the phosphate in the supernatant was depleted, the curve flattened out.These observations were consistent with on line monitoring data which showed that thesoluble phosphate concentration in the effluent was 0.02 mg/L to 0.05 mg/L. The total Pconcentration was around 4.5 mg/L in the influent and was reduced to 0.25 mg/L to 0.6mg/L in the effluent.3.1.2 PHA metabolism under different working conditionsIn November 1992, the A side of the modified UCT plant failed due to unknowncauses. The observed on-line phosphate level in the effluent rose to 1.6 mg/L.Subsequent radioactive P-32 uptake experiments confirmed a slow rate and a low extentof phosphate uptake on the A side (Fig. 4), where only about 8 mg/L per hour phosphatewas removed compared to the 18 mg/L per hour removed by the still effective B side.This failure of phosphate uptake was probably associated with the incidental introductionof oxygen to the system during operation of a high rate ultrafflter used to produce pureeffluent for a continuous on-line phosphate and nitrate analysis (Koch, personalcommunication), since the entrapment of oxygen from air into the anaerobic zone wasdetrimental to the system (Paepcke, 1983; Pitman, 1984). Before determining thisprobable mechanical cause of the failure, microbial factors were investigated to determinepossible biochemical causes and consequences of the failure on the BEPR process. SincePHA and phosphate release in the anaerobic zone were essential to the normal BEPRresponse, samples were taken to check the PHA level in the anaerobic zone and to1625.00-o20.00-(I)0.00 1^1^I^i0 0^10.0 20.0 30.0 40.0Time (Minute) 50.015.00-60.0Figure 3: Phosphate uptake of anaerobic sludge (Sample date: February 23, 1993).—*— A side: working side,— + —B side: working side.1720.0018.00-16.00-14.00-12.00-10.00-8.00-6.00-4.00-2.00--a=Z/5ExNE0.00 ^fI0 0 10.0^20. ^30. ^40.0^50.0Time  60.0Figure 4: Phosphate uptake of anaerobic sludge (Sample date: November 9, 1992).NE A side: non-working side;^+ B side: working side;^XCombination of sludge from both side.18determine whether low or depleted PHA levels of the sludge caused the sludge to stoptaking up phosphate.Tables II and III show the PHA content of anaerobic zones in the modified UCTBEPR pilot plant in U13C over a period of time including working and nonworkingconditions. Surprisingly the PHA level in the non-working A side was consistentlycomparable or higher than the levels in the preceding working periods of the A side. Thisincrease in PHA level might have been due to the reduced use of PHA storage forphosphate uptake in the failed system but the sludge biomass remained approximatelyconstant (Table IV) despite wasting. Therefore, the cells in the sludge must have grownand there must have been some alternative mechanism for actively replenishing the PHApool despite the limited capacity of polyphosphate storage. In addition, the still functionalworking B side showed the same increasing trend of PHA at that time, as if the increase inPHA were due to some common factors such as the season rather than the poor operationof the A side. The results were unexpected since it seemed that PHA and polyphosphatewere somehow uncoupled, so that sustained high PHA storage did not require highphosphate uptake and storage.One possibility to account for this unexpected uncoupling could be the aerobicstorage and consumption of glycogen since the anaerobic consumption of glycogen mightprovide substrate level phosphorylation and energy for PHA accumulation withoututilizing the poly-P pool.(Satoh et al., 1992). This mechanism would not needpolyphosphate to replenish energy supplies in the anaerobic condition and would beconsistent with the poor sequestering of phosphate in the aerobic condition. However,there are complications associated with this statement. The model systems which havebeen reported to have important glycogen accumulations have been fed with wastewaterwhich either contained only glucose (Fukase et aL, 1982; Tsuno et al., 1987; Mino et al.,1987; Arun et al., 1988) or a significant proportion of glucose, 25% to 45% of the COD(Ramanathan, 1988) and are probably unrealistic nutrient conditions. In addition, Comeau19Table II PHA in Anaerobic Zone, A Side, Modified UCT PlantOperationalConditionDate PHA of Anaerobic Zone(ug/mg dried weight of sludge)PHB PHV PHAWorking March 31/92 5.0 5.5 10.5June 29/92 8.0 6.8 14.8July 3/92 6.3 4.4 10.7July 11/92 5.7 5.4 11.1July 18/92 8.2 13.9 22.1September 14/92 8.4 7.5 15.9Non-Working November 18/92 9.2 7.6 16.8November 26/92 18.1 12.0 30.1December1/92 17.1 9.7 26.8Working February 23/93 12.1 , 8.8 20.9April 20/93 5.3 5.5 10.8Table Ill^PHA of Anaerobic Zone, B Side in Modified UCT PlantOperational conditionof A sideDate PHA (ug/mg dried weight of sludge)PHB PHV PHAWorking May 8/92 5.2 10.9 16.1May 13/92 7.0 4.3 11.3Non-working November 18/92 14.5 15.5 29.9November 26/92 24.3 19.0 43.3December 1/92 19.9 14.6 34.5Partially Working January 19/93 9.7 9.6 19.3January 26/93 11.1 9.6 20.6February 3/93 14.7 13.2 27.9February 4/93 12.9 13.1 26.0Mixture of working& non-working sludgeFebruary 11/93 11.0 8.7 19.7February 16/93 15.2 10.7 25.9Working February 23/93 13.9 8.7 22.6March 10/93 18.7 .^17.6 36.3April 20/93 4.0 4.9 8.920Table IV MLSS of Modified UCT Pilot Plant*Date MLSS(mg/L)A Side B SideMay/1992 3811 2895November/1992 2579 2338December/1992 2980 3570January/1993 3400 3460February/1993 3851 4070March/1993 4250 4971April/1993 5113 3979(* Data provided by Koch, F.(personal communication))21(1988b) reported that only a small amount of glycogen could be detected in the UBCmodified pilot plant, and that amount remained constant in the three zones, so it wasprobably not the basis of the energy for anaerobic phosphate uptake. Unfortunately theglycogen content was not followed in this study. Therefore, potential changes in storedcarbohydrate content are unknown.An alternative possibility is that an essential limited factor was missing or depletedfrom the wastewater on the "A" side because of the mechanical perturbations of thesystem.3.1.3 Factors that may limit phosphate release/uptake or PHA storage/consumptionIn order to test whether the poor behavior of the plant was caused by someunknown soluble biochemical factors which were missing from the sludge, samples weretaken from both non-working A side and working B side. One and a half ml of sludgefrom the non-working A side were then combined with 1.5 ml of sludge from the workingB side before the potential to take up phosphate was measured in the P-32 uptakeexperiment. Pure samples of sludge from the A side alone and the B side alone wereincluded as controls. Fig. 4 showed that the ability of the combined sludge to take upphosphate was between the level of two controls. These results indicated that the solublepart of sludge from B side did not immediately improve the ability of sludge from A sideto uptake phosphate and the "A" side sludge simply diluted the "B" side sludge withoutother adverse effect. It seemed that the plant failure was not caused by some solubleinhibitory factors in the "A" side sludge, and the limited uptake on the "A" side was notdue to the absence of some readily available soluble factors which could be supplied to the"A" sludge by the added "B" sludge either. This conclusion was consistent with somerelated studies testing the effect of metal ions on the non-working A side sludge.22It has been reported that potassium, magnesium and calcium cations need to becotransported together with phosphate for both the import and the export of phosphatefrom the cells (Groenestijn et al. 1988). These cations are major poly-P counterionswhich will neutralize the negative charge of poly-P. Limitation of these ions can interferein the poly-P response (Groenestijn et al., 1988). The addition of Mg2+, Ca2+ and themixed trace elements to sludge from the poorly operating A side did not help thephosphate uptake either (Fig. 5). Therefore, the acute limitation of cations that werenecessary for phosphate uptake was not the cause of the problem.3.1.4 A morphologically distinct microorganismThe BEPR activated sludge is a mixture of microorganisms (Doria-Serrano et al.,1992). Acinetobacter has been recognized as a principal phosphate removing agent, butother organisms have also been implicated in the process (Cloete, et al., 1988) and thedifferent organisms are expected to interact with each other. For example, Aerornonaspunctata was reported to enhance the phosphate uptake capability of Acinetobactercalcoaceticus by converting carbohydrates present in the sewage to a substrate requiredfor phosphate removal (Karin, 1985). Similarly, Nocardia has been reported to enhanceAcinetobacter yields in pure culture (Lemmer, 1988). Therefore if a special group ofmicroorganism becomes limited, the system may quit working. In addition, thepopulations of the microorganism in the sludge may change as the sludge nutrientconditions change. For example Wentzel and coworkers enriched Acinetobacter bysimply switching the system from domestic sewage to an acetate/yeast extract/mineralsalts/micronutrients feed. After approximately a month of stable operation almost 100%of the organisms cultured were aerobically identified by the Analytical Profile Index (API)method as Acinetobacter (Wentzel et al, 1987). Chu (1990) found that differentpopulations of pure microorganisms were enriched by simply switching aliquots of the236.000.15.00-Z-5_J0'1 4.00-E3.00-oa)cc 2.00-a)1.00-010.0^20.0^30.0^40.0. Time (Minute)50.0^60.0Figure 5: The effect of metal ions on aerobic phosphate uptake of by sludge takenfrom the anaerobic zone.^Control;^+ Mg2+(0.1g/L);^)i(Ca2±(0.1g/L);^1=3 Pfennig's medium trace elements solution(lx) (Lapage eta/,1970);^Mg2+ (0.1g/L)+Ca2+ (0.1g/L);^I Mg2+ (0.1g/L) +Pfennig's medium trace elements solution (1x). The Pfennig's medium traceelements solution contains MnC12.4H20 (3 mg/L), H3B03 (30 mg/L),CoC12.6H20 (20 mg/L), CuC12.2H20 (1 mg/L), NiC12.6H20 (2 mg/L),Na2Mo04.2H20 (3 mg/L).24same samples to different media. When predominant populations of bacteria change, thePHA might be stored in a group of non BEPR bacteria which do not use the storage touptake phosphate and accumulate poly-P.To test whether the population of microorganism potentially changed during thefailed operation of the plant, a series of samples from both the A side and the B side of theplant were microscopically compared (Fig. 6). Several differences were observed betweenthese two sides. The B side (Fig. 6A & B) had a greater variety of morphologicallydistinct types of microorganism. For example, it showed more single rod-shaped cellsthan the A side (Fig. 6A-D, 6I-L). The B side also showed more red fluorescing bacteriathan the A side (Fig. 6A-L). The floc in A side looked more solid than in B side (Fig. 6Eto F; 6G to H). Most interestingly, a morphological distinct type of bacteria was onlyobserved in the failed A side (Fig. 6G to L). In FITC stained samples, this distinctbacterium seemed to be large, spherical cells approximately 2 1AM in diameter. Theyformed tight microcolonies, reminiscent of Geodermatophilus spp. (Levy et al., 1973;Ishiguro et al., 1970). Geodermatophilus belongs to the family of Actinomycetales, andpossesses a life cycle which consists of two forms, C form and R form. In the presence ofa wide variety of monovalent and divalent inorganic cations or organic amines, thecoccoid C form predominates (Ishiguro, 1974). In the absence of these factors the C formwill differentiate into motile R form. The tetra-coccoid microcolonies observed in theplant resembled the C form. The only similar type of microcolony in B side sample hadsmaller cells which attached loosely to each other (Fig. 6B), instead of forming the tightmicrocolonies observed on the A side. Attempts to isolate the Geodermatophilus-likecells on glucose plates failed. The plant recovered before further samples were plated.Therefore, the identity of the bacteria and the capacity of these bacteria to store PHA inthe absence of polyphosphate response could not be directly tested. However,Geodermatophihis is capable of storing PHA (Ishiguro, 1970) and usually forms the Cform morphology in the presence of excess levels of ammonium. The appearance of the25Figure 6K - L: FITC stain of distinct microorganism observed in the anaerobic Aside when the UCT plant failed (1000x, magnification). The GeodermatophiThs-like microcolonies were indicated by the letters "G" The red fluorescing cellswere indicated by the arrows.31Figure 61 - J: FITC stain of distinct microorganism observed in the anaerobic Aside when the UCT plant failed (1000x, magnification) The Geodermatophihis-like microcolonies were indicated by the letters "G". The red fluorescing cellswere indicated by the arrows30Figure GG - H FITC stain of distinct microorganism observed in the anaerobic Aside when the modified UCT plant failed (400x, magnification). The microcolonieswere indicated by the letters "e"s29Figure 6E - F: FITC stain of distinct microorganism observed in the anaerobic Bside when the A side of the modified UCT plant failed (400x, magnification) Themicrocolonies were indicated by the letters "e"s. The red fluorescing cells wereindicated by arrows28Figure 6C - D. FITC stain of distinct microorganism observed in the anaerobic Bside when the A side of the modified UCT plant failed (1000x, magnification)The red fluorescing cells (which appear in the picture as yellow) were indicatedby the arrows.27AFigure 6A - B: FITC stain of distinct microorganism observed in the anaerobic Bside when the A side of the modified UCT plant failed (1000x, magnification).The red fluorescing cells were indicated by the arrows. Different types ofmicroorganism were indicated by the letters "a, b, c".26Geodermatophihts-like microorganism in A side may indicate that the problem in A-sidephosphate metabolism was due to a problem in the metabolism of ammonium or excessivelevels of cations. However, the ammonium levels at that time were normal (F. Koch,personal communication). Therefore the problems in ammonium metabolism was unlikely.In addition, the limited available observations did not distinguish whether these uniquecolonies were the cause or the consequence of the plant failure. Geodermatophilus wasnot been previously reported as one of the BEPR bacteria. Further investigations mightattempt to isolate the R form of Geodermatophilus on CB medium according to Ishiguro's(1970) method.3.2 Biochemical model testSeveral biochemical models have been proposed to describe the BEPR process(Comeau, 1988; Mino, 1987; Mah, 1991). An original objective of this thesis was to testthe models, with particular regard to potential sources of NADH in the biochemicalpathway of PHA synthesis.It had been proposed that the glyoxylate cycle plays a central role during anaerobicPHA storage by providing balanced levels of NADH reducing power and inadvertentlyproducing propionyl-CoA as a waste product (Fig. 7, Mah, 1991). Proper nutrientcombinations might be capable of substituting for the glyoxylate cycle to provide NADHeven in the presence of fluoroacetate. Fluoroacetate can block the glyoxylate cycle byinhibition of the enzyme aconitase (Morrison and Peters, 1954) and was observed toimpair the PHA synthesis in pure acetate fed sludge (Mah, 1991). In pure enzyme andpure culture systems, monofluoroacetate has been shown to be inhibitory to cell growth atconcentrations of 0.2 g_tM and 10 JAM respectively (Beatty, 1980; Morrison and Peters,1954). However, appropriate nutrient combinations could overcome the inhibitory effectof fluoroacetate on PHA synthesis by presumably providing both adequate carbon source32IsocitrateitratAcety CoA ANADH\".NADOxaloacetateNADHNADMalate,Acetate \CAD -NADHcx-Ketoglutarate^)NADCNADHSuccinyl CoA Glyoxylate Acctoacetyl„E^CoA NADHNADi3-Hydroxy-butyrl CoA Acetyl CoA (?NAD ^FumarateNADHFADHFADInhibitors:a = cx-ketoglutarateb = fluorocitratec = anaerobiosis.d = malonatePHBPHVPyruvate fiNADHNADLactate NADHNADSuccinate Hydroxy-yalcriy1 CoA NAD^NADHOxo-valcryl CoA Propionyl CoAFigure 7: Proposed biochemical model for PHA storage under anaerobic conditions.Points of inhibition indicated by double lines. Inhibitors indicated by lower case letters.(Mah, 1991).and the necessary NADH balance for PHA synthesis. The following tests were intendedto provide some information about possible biochemical pathways involved in PHAsynthesis.3.2.1 The similarity of the three reactorsA major difficulty in dealing with samples taken from the pilot plants is the dailyand monthly variation of influent and operational conditions. Some of these problemswere minimized by including parallel controls in the experiments, so that change could beattributed to the treatment rather than the sludge variation.To test whether these parallel controls were comparable, the sludge wasdistributed evenly into three parallel reactors. Once these reactors reached the low stableredox levels which are characteristic of an anaerobic environment, they were continuouslyfed at a constant rate with a solution of pure acetate. Fig. 8 shows the general trend of theredox behavior the same in all three reactors. The redox dropped abruptly at thebeginning of the run, and then leveled off or decreased slowly in all three reactors. Therewere some minor differences in absolute redox values due to differences of the individualprobe but the overall patterns were similar and comparable readings were observed whenthe probes were interchanged. The "nitrate knee" which is the characteristic indicator tooperationally define the transition between anoxic and anaerobic conditions in the sludge(Koch and Oldham, 1985), was not observed. The absence of this indicator might be dueto the expectation that only minor levels of nitrate will be present since samples weretaken from the anaerobic zone and any incidental nitrate forming aeration during transportand setting up the reactor was slight.Fig. 9 shows that each of the three parallel control reactors synthesized PHA to thesame extent and pattern. The level of PHA started to increase as soon as the nutrient wasadded and the storage was saturated by one and half hour. Approximately the same34200.0087.50-r.-25.00- \.-137.50--250.000.00__ _,.•• -- ---.:--.:—...-- ._---•:_—_-- —^. --:r"kflilitrastIrrsor• ." ---12.40^4.80ATime (Hour)Figure 8: Redox profile of the parallel reactors during batch experiments. A:Reactor 1; B: Reactor 2; C: Reactor 3.3580.0070.00-60.00-50.00-40.00-30.00-20.00^)+E--10.00^00 0.5i^I^i^11.0 1.5 2.0 2.5Time(Hour) 3.0^35Figure 9: PHA synthesis in three parallel reactors.^Reactor 1;Reactor 2;^4- Reactor 3. Acetate feeding was started at time 0,then continuously at 5 mg/minute.36amount of PHA was synthesized in the same period in all three reactors. The PUB andPHV synthesis pattern were also the same (data not shown). In contrast to the naturalsludge samples, the PHB synthesis accounted for most of the PHA synthesis and onlysmall amounts of PHV synthesis was observed. However, it seemed that the threereactors were equivalent and that parallel reactors could give an adequate comparison forassessing the effect of various nutrients in the presence and absence of fluoroacetate.3.2.2 Continuous and pulse feeding of nutrientsAs mentioned in the introduction, pulse feeding of nutrients to the systems hasbeen used by previous studies, but the natural nutrients in the plant situation would likelyinvolve a more continuous nutrient supply. In order to imitate the real situation, this studyplanned to use a continuous feeding style of nutrient. A test was done to see whether adifferent feeding style would create a different PHA synthesis pattern. One reactor wasfed with a single pulse of 1.2 grams of acetate to provide a final concentration of 300mg/L. The other one was fed continuously with low level of acetate so that after 3 hoursthe total addition was 300 mg/L. The results are shown in Fig. 10. It seemed that thedifferent feeding pattern did create a different PHA synthesis pattern. For pulse feedingsystem there was a lag phase before the PHA content started to increase as if the highconcentration of substrate delayed the synthesis and the system needed to acclimate to thesubstrate. For the continuous feeding system, the system adapted to the substrate fasterand the PHA synthesis started at least within half an hour after the feeding started. Noobvious lag phase was observed. This result was not consistent with the previousobservation made by Comeau (1988). Comeau fed the system 75 mg/L of acetate as apulse and after three hours the amount of acetate provided was consumed completely. Nolag period of PHA synthesis was reported in his study. The PHA pattern he observed wasmore like the continuous feeding system in this studies. However, this difference may be3770.00cu60.00—50.00—40.00—30.00—20.00—10.00(1.0.00 I^I^1^100^0.5 1.0 1.5 2.0Time (Hour) 2.5^3.0Figure 10: Comparison of the effects of nutrient feeding style. •^Control (nonutrient); CD 300 mg/L acetate ( as pulse);^4- Acetate (continuously at 5mg/minute.). Acetate feeding was started at time 0.38due to different concentrations of acetate used since the 300 mg/L acetate supplied in thisstudy was 4 times higher than 75 mg/L in Comeau's system. After the lag phase, the rateof PHA synthesis for the pulse feeding system was similar to the initial rate observed forthe continuous feeding system. The continuous feeding system was used in the followingstudies.3.2.3 The inhibitory effect of different concentrations of fluoroacetateDespite previous reports to the contrary (Mah, 1991) Fig. 11 shows thatfluoroacetate did not inhibit the synthesis of PHA completely but simply reduced the levelof the synthesis. This limiting inhibition was not increased by increasing the concentrationof fluoroacetate from 50 p.M to 250 pM. Since inhibition by fluoroacetate is competitiveand can be readily reversed by higher concentrations of substrate (Morrison and Peters,1954) some of this poor inhibition might be due to the high concentration of accumulatedunused acetate in the reactor by the end of each continuously fed run, since the inhibitionwas usually better at the beginning and the accumulated acetate by the end of each runmight be expected to compete with fluoroacetate.Surprisingly the extent of fluoroacetate inhibition varied from day to day,sometimes fluoroacetate inhibited better, sometimes it had no inhibition (Fig. 12 and Fig.13). This inconsistency of inhibition might be due to different daily nutrient conditions.For example, pyruvate eliminates fluoroacetate inhibition (Mah, 1991). In the poorlyinhibited cases there might have been some analogous nutrients present which could allowthe cells to bypass the inhibitory effect. This belief is consistent with the pattern ofresidual PHA accumulated in the fluoroacetate treated samples. For example in 4 out ofthe 5 cases which displayed good residual PHA synthesis, that fluoroacetate eventuallyhad more inhibitory effect on PHV synthesis than on PHB synthesis (Fig. 14 and Fig. 15).39-o 50.00-a)-o40.00-< 30.00a_20.00^00 0.5^1.0 1.5^2.0Time (Hour) 2.5 3.0^3580.0070.00-60.00--015Figure 11: Effect of different concentration of fluoroacetate on PHA synthesis.Es^Acetate;^D Acetate + 501.1M fluoroacetate;^-I- Acetate + 250 ptMfluoroacetate. Acetate feeding was started at time 0, then continuously at 5mg/minute; fluoroacetate was added as a pulse at time 0.4050.0045.00-40.00-35.00-30.00-25.00-20.00-15.0010.00 00^0.5^1.0 351.5^2.0Time(Hour) 2.5Figure 12: Acetate and pyruvate combination which indicated good fluoroacetateinhibition on PHA synthesis. a Acetate; +^Acetate + fluoroacetate;* Acetate + fluoroacetate + pyruvate. Acetate or/and pyruvate feeding wasstarted at time 0, then continuously at 5 mg/minute. The molar ratio of acetate topyruvate was 1:4. 50 ktM flu oroacetate was added as a pulse at time 0.4135.00a)-o 30.00-co4o-.11E-1 25.00--o 20.00-o.r...-E 15.00-I^ f^ I1.5 2.0 2.5Time (Hour)13.0^355.00^00 0.5^1.011Figure 13: Acetate and pyruvate combination on PHA synthesis whichindicated poor fluoroacetate inhibition.^IN^Acetate;^0 Acetate +fluoroacetate;^+ Acetate + fluoroacetate + pyruvate. Acetate or/andpyruvate feeding was started at time 0, then continuously at 5 mg/minute. Themolar ratio of acetate to pyruvate was 1:4. 50 [tM fluoroacetate was added as apulse at time 0.420.002.50 3.50Time(Hour) 4.15016.00 14.00-12.00-10.00-8.00-6.00-4.00-2.00--0(7)-0'r----0Figure 14: PHB net synthesis in the presence of acetate with or withoutfluoroacetate. Acetate feeding was started at 2.75 hours, and continuously at 5mg/minute. Fluoroacetate was added as a pulse at 2.75 hours.Acetate (PHB concentration at 2.75 hours was 8.64 jig/mg dried weight ofsludge); + Acetate + 50 jiM fluoroacetate (PHB concentration at 2.75 hourswas 9.29 jig/mg dried weight of sludge).43Cl)-o7/3 8.004E5I. 7.00-6.00-5.00---- 4.00-=-oCl)2.00-E00 1.00-co0.00 ^ri^2.50-oCl)-o3.50Time(Hour) 4.150Figure 15: PHV net synthesis in the presence of acetate with or withoutfluoroacetate. Acetate feeding was started at 2.75 hours, and continuously at 5mg/minute. Fluoroacetate was added as a pulse at 2.75 hours.Acetate (PHB concentration at 2.75 hours was 8.64 pg/mg dried weight ofsludge); + Acetate + 50 1AM fluoroacetate (PHB concentration at 2.75 hourswas 9.29 pg/mg dried weight of sludge).44This type of PHA pattern would be consistent and expected if the fluoroacetate wasactually inhibiting the glyoxylate cycle and preventing synthesis of the propionyl-CoA,which was believed to be important for the polyhydroxyvalerate synthesis (Mah, 1991).The single exception to this observation might be due to the presence of propionate orsome other hydroxyvalerate precursors in the sludge. In any case the random poorinhibition complicated the study but some analysis was attempted by selecting cases wherethe fluoroacetate control fed with pure acetate showed the expected inhibition.3.2.4 PHB and PHV synthesisWith pure acetate feeding, higher PHB than PHV accumulation was observed (Fig.16). The observation agrees with previous results (Comeau, 1988; Mah, 1991). In theactual pilot plant situation, PHV comprises up to 60% of PHA (Table V), as if the systemuses nutrients other than acetate. One of these nutrients could be propionate sincepropionate can be readily used (Comeau, 1988b). Fig. 17 shows that a combination ofacetate and propionate feeding in the presence of fluoroacetate only allowed PHVsynthesis. However, the propionate level in the actual plant is barely detectable, andshould not be adequate to yield the observed proportion of PHV (Koch, personalcommunication). In the actual fermentor the fermentation products were only 30%propionate, the rest were 70% acetate and 1% butyrate, the direct use of this combinationof VFA should not produce the observed composition of 60% PHV in the sludge(Comeau, 1988b). This effect could be explained if there was preferential turnover ofPHB compared to PHV but earlier direct observations on the pilot plant sludge indicatepreferential use of PHV. Therefore it would seem that preferential use of PHB would beunlikely. An alternative explanation is that there were specific nutrients in the sludge otherthan propionate which increase the accumulation of PHV relative to the accumulation ofPHB. If these proposed nutrients were consumed immediately after production by451 iI 118.00im10.00-=< 8.00Ia-6.00^00 0.5^1.01^11.5 2.0Time(Hour) 2.5^3.0^35Figure 16: PHB and PHV synthesis with pure acetate feeding. NI PHB;+^PHV. Acetate feeding was started at time 0, then continuously at5 mg/minute.46Table V PHA in Anaerobic zone, A side, FGR/SGR PlantDate PHA (ug/mg dried weight of sludge)PHB PHV PHAMarch 15/93 8.5 13.9 22.5March 17/93 4.7 8.3 13.0March 31/93 5.8 8.1 13.9April 20/93 7.1 8.0 15.1April 30/93 4.9 5.4 10.3May 5/93 14.6 15.9 30.5May 6/93 10.5 11.4 21.94760.0055.00-50.00-45.00-40.00-35.0030.00-25.00-issIN20.00 ,^I^i^i^I^100^0.5 1.0 1.5 2.0 2.5 3.0^35Time (Hour) Figure 17: PHB and PHV synthesis with combined acetate, fluoroacetate andpropionate feeding.^PHB; L PHV. Acetate and propionate feeding werestarted at time 0, then continuously at 5 mg/minute. 504M fluoroacetate wasadded as a pulse at time 0.48fermentation, they would be hard to detect. The following experiments were designed totest for effects of these nutrients.3.2.5 Combination of acetate and pyruvate in the presence of inhibiting levels offluoroacetatePyruvate may be one of the intermediate sludge substrate. Pyruvate could beoxidized to acetyl-CoA to generate NADH, and oxidized in a separate pathway topropionyl-CoA (Fig. 7) In this way, the generation of PHV in sludge fed pyruvate andacetate could be independent of the glyoxylate cycle needed by acetate fed sludge (Mah,1991), and the presence of fluoroacetate should not affect the PHA synthesis if bothpyruvate and acetate are present. An experiment was done to test this assumption (Fig. 12and Table VI). Fig. 12 showed that it might be true. The PHA synthesis was higher whenacetate was present without fluoroacetate, which indicated that fluoroacetate inhibited thePHA synthesis. However, with both acetate and pyruvate present, the PHA synthesis wasthe highest, which indicates that the fluoroacetate inhibitory effect was overcome. Inaddition, with acetate feeding alone, 18.7 vtg/mg dried weight PHA was synthesized. Inthe presence of fluoroacetate, only 11.6 1.1g/mg dried weight PHA was synthesized. Ifpyruvate was added with acetate, 23.9 ig/mg dried weight PHA was synthesized despitethe presence of fluoroacetate (Table VI). The results showed a PHB/PHV ratio of 3.4 inthe presence of acetate, pyruvate and fluoroacetate; 3.5 in the presence of only acetate;7.7 in the presence of acetate and fluoroacetate. These results indicate that the presenceof pyruvate restored the synthesis that was impaired by the presence of fluoroacetate anddidn't change the pattern of PHB and PHV synthesis. This observation agrees with Mah'sresult. Mah (1991) fed 10 mg/L acetate with 50 mg/L pyruvate in the presence offluoroacetate. He observed that PHB and PHV accumulated to similar extents. These49results suggest that specific nutrient combination can eliminate the apparent acetatedependence on the glyoxylate cycle.3.2.6 Combination of acetate and butyrate in the presence of fluoroacetateSome degradation pathways for butyrate provide NADH (Schubert, et al., 1988).If the fluoroacetate inhibition of PHA synthesis in acetate fed cells is due to an imbalanceof NADH production caused by the absence of glyoxylate cycle, then butyrate should beable to bypass the inhibition of PHA synthesis. However tests from each plant showedunreliable fluoroacetate inhibition, and the synthesis with the combination of fluoroacetate,acetate, and butyrate was always less than or equal to the synthesis in the acetate controlor the samples with fluoroacetate and acetate (Table VI).3.2.7 Other combinations of nutrients and acetateA variety of other nutrients including ethanol, lactate, gluconate and glucuronatewere tested for effects on fluoroacetate inhibition. These nutrients were selected toachieve a net redox balance (Table VII) when each was co-utilized with acetate as feed tothe system. Each of these nutrients also had the potential to be a source of NADH toovercome the inhibition of fluoroacetate and develop a different pattern of relative PHVand PT-TB synthesis. In each test either the control showed limited inhibition, or the totalPHA synthesis was too small to distinguish the effects of the different treatment. Sincethe basal level of PHA in these samples was already high, some of the problem wasprobably due to PHA synthesis occurring with the endogenous sludge nutrients in theinitial settling period before nutrient feeding when the sludge redox was being lowered tothe anaerobic condition. If this belief is real, it implies that significant PHA synthesis50Table VI Nutrient Effects on PHA Synthesis in the Presence of FA%ample Source Run Date Nutrient -Nutrient Consumed (mg/L) Net synthesis of PHA(Ug/mg dried weight of sludge)Dried Weightmg/ml-Ratio(molarmolar)Acet. Butyrate PHB PHV PHA Acet./PHA PHB:PHV-FGR/SGRAnaerobiczoneMarch^/93 Acet. 98 0 14.5 4.2 1 8. 7 2.94 21 3.5^.Acet.+FA. 89 0 10.0 1.5 11.6 3.15 10.2 7.7Acet.+Pyruv.+FA. 83 N/A 17.7 6.3 23.9 3.19 13.4 3.4 -Modified UCTAerobic zoneMarch 18/93 Acet. 128 0 17.8 5.9 23.7 3.02 9.1 3.4Acet.+FA. 110 0 14.9 3.2 18.1 2.57 13.1 4.7Acet.+Pyruv.+FA. 90 N/A 16.3 3.6 19.9 3.98 6.3 5.3Modified UCTAerobic zoneMarch 16/93 Acet. 106 0 21.9 4.5 26.4 3.05 8.8 5.3Acet.+FA. 77 0 16.6 6.0 22.6 2.36 6 3.2Acet.+Butyr.+FA. 61 37 7.5 3.2 10.7 2.42- 10 2FGR/SGRAerobic zoneMarch 23/93 Acet. 90 0 7.4 0.0 7.4 3.40 9.7 0.7:0Acet.+FA. 66 0 7.0 4.9 11.8 326 10.8 1.7Acet.+Butyr.+FA. 61 36 6.8 1.0 7.8 3.14 7.9 66* Acet.=AcetatePyruv.=PyruvateButyr..-butyrateFA=fluoroacetateTable VII Reducing Equivalents Associated withDifferent Organic MoleculesMolecules Formula [ft per 06Gluconate C6H1207 +2Glucuronate C6H1007 0Ethanol CH3CH2OH -8Lactate CH3CHOHCOO -4Acetate CH3COOH 0(Clark, D.P., 1989)51occurred in relatively poor anaerobic conditions. It also implies that less background PHAsynthesis would occur if the studies concentrated on the nutrient depleted aerated sludge.However, since the intent of the study was to look at the synthesis with endogenousnutrients, nutrient depleted sludge would not be an adequate sludge source.The results in this study seemed to indicate that the glyoxylate cycle might operateunder some anaerobic conditions in the BEPR process. They also suggest that this cycle isnot the only source of NADH under pilot plant conditions. Further investigation needs tobe conducted, since most of the experiments were done during a period of the pooroperation of the pilot plant, and the effect of the pilot plant condition on the tests isunknown. Some nutrients, e.g. butyrate, lactate, ethanol, etc. are currently being retestedin a separate study (Tian Shu Yuan, personal communication).524. ConclusionsGood PHA storage in the anaerobic zone was not consistent with good phosphateuptake in the aerobic zone. 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