reducing production of excess biomass during wastewater treatment

PII: S0043-1354(98)00325-X Wat Res Vol 33, No 5, pp 1119±1132, 1999 # 1999 Elsevier Science Ltd All rights reserved Printed in Great Britain 0043-1354/99/$ - see front matter REVIEW PAPER REDUCING PRODUCTION OF EXCESS BIOMASS DURING WASTEWATER TREATMENT EUAN W LOW* and HOWARD A CHASE Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge, CB2 3RA, U.K (First received May 1998; accepted in revised form July 1998) AbstractÐExcess biomass produced during the biological treatment of wastewaters requires costly disposal As environmental and legislative constraints increase, thus limiting disposal options, there is considerable impetus for reducing the amount of biomass produced This paper reviews biomass production during wastewater treatment and identi®es methods for reducing the quantity of biomass produced E€orts to reduce biomass production during aerobic metabolism must promote the conversion of organic pollutants to respiration products with a concomitant increase in the aeration requirements Promoting further metabolism of assimilated organic carbon will release additional respiration products and reduce the overall biomass production e.g by inducing cell lysis to form autochtonous substrate on which cryptic growth occurs or by encouraging microbial predation by bacteriovores Uncoupling metabolism such that catabolism of substrate can continue unhindered while anabolism of biomass is restricted would achieve a reduction in the biomass yield Metabolite overproduction in substrate excess conditions has been demonstrated in several bacterial species and can result in an increase in the substrate uptake while resulting in a decreased yield and increased carbon dioxide evolution rate Addition of protonphores to uncouple the energy generating mechanisms of oxidative phosphorylation will stimulate the speci®c substrate uptake rate while reducing the rate of biomass production Increasing the biomass concentration such that the overall maintenance energy requirements of the biomass within the process are increased can signi®cantly reduce the production of biomass Suitable engineering of the physical conditions and strategic process operation may result in environments in which biomass production may be reduced It is noted that as biomass settling characteristics are a composite product of the microbial population, any changes which result in a shift in the microbial population may a€ect the settling properties Reduced biomass production may result in an increased nitrogen concentration in the e‚uent Anaerobic operation alleviates the need for costly aeration and, in addition, the low eciency of anaerobic metabolism results in a low yield of biomass, its suitability for wastewater treatment is discussed A quantitative comparison of these strategies is presented # 1999 Elsevier Science Ltd All rights reserved Key wordsÐactivated sludge, biomass production, biomass reduction, wastewater, uncoupled metabolism NOMENCLATURE D kd K QW qm dilution rate (hÀ1) decay coecient (hÀ1) equilibrium constant volumetric rate of biomass removal (L hÀ1) speci®c substrate uptake related to maintenance energy requirements (g gÀ1 hÀ1) *Author to whom all correspondence should be addressed [Tel.: +44 1223 330132; Fax: +44 1223 334796; Email: ewl21@cam.ac.uk] AbbreviationsÐBOD, biological oxygen demand, ATP, adenosine triphosphate, NADH, nicotineamide adenine dinucleotide, COD, chemical oxygen demand, MLSS, Mixed Liquor Suspended solids ÀrS rate of substrate uptake (g LÀ1 hÀ1) ÀrSG rate of substrate uptake for incorporation in new biomass (g LÀ1 hÀ1) rX rate of biomass production (g LÀ1 hÀ1) ÀrXd rate of biomass loss due to cell death and lysis (g LÀ1 hÀ1) S substrate concentration (g LÀ1) initial substrate concentration (g LÀ1) S0 t time (h) V reactor volume (L) X biomass concentration (g LÀ1) Xv concentration of viable biomass (g LÀ1) YATP biomass yielded per gram of ATP consumed (g biomass g ATPÀ1) YG true biomass yield (g biomass g substrateÀ1) 1119 1120 Euan W Low and Howard A Chase Table Nutritional classi®cation of microorganisms employed in wastewater treatment according to the origin of their cellular carbon, energy source and reducing equivalents Nutritional classi®cation Origin of cell carbon Chemolithotrophs (autotrophs) YS m Terminal electron acceptor organic carbon organic carbon inorganic carbon Chemoorganotrophs (heterotrophs) Energy source inorganic compounds, e.g NH3, H2S and Fe2+ aerobic: oxygen anaerobic: organic compounds anoxic: nitrate, sulphate oxygen observed biomass yield (g biomass g substrateÀ1) speci®c growth rate (hÀ1) cing biomass production on these aspects are also discussed BIOLOGICAL PROCESSES WITHIN WASTEWATER TREATMENT INTRODUCTION Biological wastewater treatment involves the transformation of dissolved and suspended organic contaminants to biomass and evolved gases (CO2, CH4, N2 and SO2) which are separable from the treated waters Excess biomass produced within processes must be disposed of and may account for 60% of total plant operating costs (Horan, 1990) In addition, recent European legislation requires that more wastewaters receive biological treatment prior to discharge (91/2711EEC, 1991), resulting in a considerable increase in the production of biomass, but this is also restricting options for its disposal (Boon and Thomas, 1996) There is therefore considerable impetus to develop strategies for reducing the amount of biomass produced The purpose of this review is to develop a comprehensive account of how pollutant metabolism leads to biomass production during the treatment of wastewaters in order to reveal strategies which can reduce both biomass production and as a result, the disposal requirements Wastewaters typically contain a complex mixture of components which are degraded by a diverse range of microbial cells in biochemical reactions The availability of oxygen will in¯uence the metabolic pathways utilised by the cells As biological, biochemical and physical phenomena all in¯uence nutrient removal, these shall be considered in conjunction with strategies for process operation, with the objectives of identifying mechanisms which may reduce disposal requirements Essential aspects of aerobic biological wastewater treatment include aeration requirements, population dynamics and sludge settling properties, the consequences of redu- Secondary sludges contain inert solids and biological solids, collectively called biomass, the latter being derived through metabolism of pollutants The purpose of sludge wastage is to purge the inert solids and remove excess biological solids in order to prevent accumulation of these solids within the system Reducing the production of excess biomass will reduce the required wastage rate Substances contaminating waters, such as organic carbon and certain nitrogen compounds, are assimilated by microorganisms to provide energy or to be utilised in biosynthesis, thus removing the contaminants from the waters The microorganisms employed can be classi®ed according to how they meet their nutritional requirements and this classi®cation is based on their sources of energy, carbon and terminal electron acceptor (Table 1) Most wastewater treatment involves aerobic metabolism of organic pollutants by chemoorganotrophic bacteria The chemolithotrophs which ``nitrify'' ammonia via nitrite to nitrate also require an aerobic environment In the absence of dissolved oxygen, nitrate can be used as the terminal electron acceptor releasing nitrogen gas and this process is termed denitri®cation Biomass contains a diverse and interactive microbial population consisting of cells, either in an isolated manner or in an agglomerate of cells forming a ¯oc or bio®lm These heterogeneous microbial cells are undergoing life cycles and reproducing, with relationships between the di€erent types of cells being characterised by symbiotic, cooperative, aggressive and competitive behaviour As a result of these relationships, the microbial population is dynamic and evolutionary Aerobic, anaerobic and Table Allocation of substrate by a cell in each stage of its life cycle Cell state Substrate utilization in energy generation in assimilation for maintenance Viable, growing and respiring Non-viable, respiring Dead for anabolism for biosynthesis [ [ x [ x x [ x x Reducing production of excess biomass anoxic environments will determine the availability of reducing equivalents thus in¯uencing the metabolic eciency Microenvironments in cell agglomerates may cause zoni®cation (Scuras et al., 1997), encouraging or inhibiting growth of di€erent classes of microbes A cell's ability to assimilate substrate in biosynthesis will be a€ected by the stage in its life cycle (Table 2) As a portion of biomass is composed of non-viable bacteria, the maintenance requirements of living, non-viable bacteria may make a signi®cant contribution to substrate metabolism Microbial metabolism liberates a portion of the carbon from organic substrates in respiration and assimilates a portion into biomass To reduce the production of biomass, wastewater processes must be engineered such that substrate is diverted from assimilation for biosynthesis to fuel exothermic, non-growth activities Dead cells that are still intact are unavailable to other bacteria as a food source and in this context contribute to inert biomass However, both living and dead bacteria can be utilised in trophic reactions (as a food source) by higher bacteriovoric organisms such as protozoa, metazoa and nematodes Cell lysis will release cell contents into the medium, thus providing an autochtonous substrate which contributes to the organic loading This organic autochthonous substrate is reused in microbial metabolism and a portion of the carbon is liberated as products of respiration and so results in a reduced overall biomass production The growth which subsequently occurs on this autochthonous substrate can not be distinguished from growth on the original organic substrate and is therefore termed cryptic growth (Mason et al., 1986) Since metabolism of organic carbon yields both biomass and carbon dioxide and when that carbon assimilated into biomass can be made available as a substrate, then the repeated metabolism of the same carbon will reduce the overall biomass production Carbon utilisation during cryptic growth on the autochthonous substrate formed from cell lysis products as the only carbon source has been studied in Klebsiella pneumoniae maintained in a chemostat culture (Mason and Hamer, 1987) Dilution rates of 0.69 and 1.46 hÀ1 resulted in 0.42 and 0.52 mg of carbon being assimilated into the synthesis of new cells per mg of lysed cellular carbon respectively Several processes have been developed to bene®t from the reduced overall biomass produced that can be achieved by promoting further metabolism of the organic carbon Biodegradation of biomass By exploiting the e€ects of cell death, autolysis and subsequent cryptic growth to reduce overall biomass yields, Sakai et al (1992) sought to balance cell growth and decay Activated sludge from a municipal sewage treatment plant was acclimated for 1121 more than weeks in a 1.8 L aeration tank using a ®ll and draw cultivation method A high mixed liquor suspended solids (MLSS) concentration (11 g LÀ1) was maintained on a continuously-fed, synthetic waste (0.81 g COD LÀ1 dÀ1) In addition to gravitational settling, biomass retention was enhanced by supplementing the biomass with ferromagnetic powder (11 g LÀ1 of average particle diameter 0.4 0.1 mm) and using magnetically forced sedimentation During the subsequent period of 30 d, biomass concentration remained constant and no excess sludge was produced In a full-scale process, the high solids concentration would necessitate e€ective biomass retention within the aeration basin to prevent overloading of the secondary clari®er Scale-up would diminish the ecacy of magnetically forced sedimentation Further metabolism of organic carbon by digestion of wasted excess biomass has been introduced for reducing overall biomass production (Mason et al., 1992) Ganczarczyk et al (1980) found that for the semi-continuous aerobic digestion of a waste biomass at 208C, a 50% reduction of the biomass was obtained for digestor retention times in excess of 16 d The rate of naturally occurring cell death is assumed to be proportional to the viable cell concentration ÀrXd=kdXv Typically the kd values in wastewater treatment are in the order of 0.03± 0.06 dÀ1 (Horan, 1990) and therefore the ability to promote cell death and lysis could be advantageous and can be achieved by engineering hostile environments Aerobic, thermophilic digestion of wasted biomass is exothermic and can therefore be autothermal with appropriate heat retention and heat exchange Thermophilic temperatures induce lysis of those cells less tolerant to heat and promote the biodegradation of certain compounds that are recalcitrant in less extreme environments Also the thermophilic temperatures may pasteurise the biomass reducing the content of pathogenic organisms Mason and Hamer (1987) sought to identify optimal conditions for the digestion of cell lysis products by a mixed thermophilic bacterial population Baker's yeast as the sole organic carbon source was suspended in a mineral medium This medium was continuously fed to a reactor in which the temperature and oxygen supply could be varied Cell lysis and biodegradation was optimal under oxygen-limited conditions at 608C with a residence time of d, with a 52% reduction in biomass with respect to the amount of biomass entering the system However, as the physiology of baker's yeast is di€erent to the predominantly bacterial biomass used in biological wastewater treatment, the use of baker's yeast in this context is inappropriate Canales et al (1994), employing a membrane bioreactor demonstrated that shorter sludge ages increased the biomass viability However when the 1122 Euan W Low and Howard A Chase Fig Process ¯owsheet for the aerobic process with thermally induced cell lysis of excess biomass to form autochthonous substrate (Canales et al., 1994) biomass passed through a thermal treatment loop (residence time h, 908C) nearly 100% of cells were killed and partial cell lysis was induced Thus a portion of biomass, recycled via the thermal treatment loop and back to the reactor (Fig 1), formed autochthonous substrate (lysis products) on which cryptic growth occurred and this further metabolism contributed to a 60% reduction in the overall biomass production Using a di€erent mechanism to achieve cell lysis, but with similar results, Yasui and Shibata (1994) enhanced cell lysis by contacting a portion of the recycled biomass with ozone (Fig 2) The aeration basin with a biomass concentration of 4200 mg LÀ1 was fed with 1000 mg BOD LÀ1 dÀ1, culture was removed from the aeration basin and recirculated via the ozonation stage at a dilution rate of 0.3 dÀ1 in which a dose of 0.05 mg O3 mg biomassÀ1 was applied During weeks of operation, no biomass was wasted from the process and yet the reactor biomass concentration remained constant Application of this concept on a full-scale process receiving 550 kg BOD dÀ1 found the requirement of biomass to be treated was 3.3 times more than the quantity of biomass to be eliminated (Yasui et al., 1996) No excess biomass needed to be wasted over 10 months of operation, a marginal increase of refractory total organic carbon was measured in the ®nal e‚uent Bacteriovoric metabolism Additional metabolism can also be achieved by bacteriovory by higher organisms such as protozoa Fig Process ¯owsheet for the aerobic process with enhanced cell lysis by contacting excess biomass with ozone to form autochthonous substrate (Yasui and Shibata, 1994) and metazoa Ciliated protozoa have been demonstrated to be an indicator of good e‚uent quality (Salvado et al., 1995) and the presence of protozoa or metazoa are accepted as indicators of a healthy population in waste water treatment systems (Horan, 1990) Protozoa are considered to be the most common predators of bacteria, making up around 5% of the total dry weight of a wastewater biomass, 70% of these are ciliates (Ratsak et al., 1996) Ratsak et al (1994) demonstrated predatory grazing on biomass by employing the ciliated Tetrahymena pyriformis to graze on Pseudomonas ¯uorescens and reported a 12±43% reduction in the overall biomass production Similarly Lee and Welander (1996) employed protozoa and metazoa to achieve a 60±80% decrease in the overall biomass production in a mixed microbial culture In both of these experiments, bacterial cells were cultured in a primary reactor vessel and the e‚uent was fed to a second reactor vessel in which the bacteriovores metabolised the bacterial cells To achieve a similar reduction in the overall biomass production in wastewater processes requires increased bacterial grazing by the bacteriovores Curds (1973) developed a model which predicted oscillating prey and predator population sizes for bacteria and protozoa, where these were caused by diurnal variations of sewage ¯ow and composition This is supported by experimental studies on population dynamics of prey±predator relationships which indicated oscillating population sizes (Lynch and Poole, 1979) The use of predatory activity to reduce the overall biomass production requires some caution Cech et al (1994) report that for a mixed population in a one stage laboratory scale reactor a concomitant decrease in phosphorous removal occurred while there was a marked increase in predator numbers BIOCHEMISTRY WITHIN WASTEWATER TREATMENT While no single species is capable of utilising all the assorted organic and inorganic compounds found in wastewaters, the heterogeneous microbial population in a wastewater process can utilise a wide range of substrates Despite such diversity, all microorganisms have the common purpose of using catabolism to conserve free energy by distributing it among compounds which can store and carry the energy to where it is required in the cell Three alternate pathways have been identi®ed in chemoorganotrophs for reducing organic compounds, the most commonly used pathway being glycolysis, typically to pyruvate with the concomitant formation of energy carrying compounds such as adenosine 5'-triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH) and reduced ¯avin adenine dinucleotide (FADH2) While this mechanism yields a small amount of energy, it does not require oxygen and can therefore occur in anaerobic Reducing production of excess biomass environments The bacterial genus Pseudomonas, which Horan (1990) describes as a signi®cant oxidiser of carbon in wastewater treatment, utilise the Entner±Doudoro€ pathway which is similar to glycolysis in producing pyruvate, but is less ecient in ATP generation Pyruvate is utilised in the citric acid cycle to produce molecules of NADH and FADH2 which carry pairs of electrons with a high transfer potential The donation of these electrons to molecular oxygen in a controlled regime allows a large amount of free energy to be transferred In addition to providing useful energy for meeting the cell's needs, intermediaries of the citric acid cycle can be withdrawn to form materials required in biosynthesis Thus catabolised carbon is removed from metabolic pathways during respiration as CO2 and as metabolites for synthesis of biomass The concentrations of certain compounds regulate the rate of reactions of the citric acid cycle within eukaryotes and may also so within prokaryotes The process employed to conserve the free energy transferred to NADH or FADH2 is the chemosmotic process of oxidative phosphorylation (Mitchell, 1972) This involves respiratory assemblies containing a series of electron carriers located across the cell's cytoplasmic membrane and while these transfer electrons from NADH or FADH2 to O2, protons are simultaneously pumped out of the cell cytoplasm Thus a proton-motive gradient is generated across the membrane providing the driving force for the ¯ow of protons back into the cytoplasm The enzyme complex ATPase provides a pathway for these protons catalysing the transfer of the potential energy to provide the activation energy in the phosphorylation of ADP to create a high free energy covalent bond in ATP ATP within the cell provides energy for a variety of cell func- tions The energy liberated during the conversion of ATP back to ADP + Pi fuels endergonic functions such as cell anabolism, reproduction, motility and maintenance functions such as active transport of substrates and regulation of intracellular concentrations (Fig 3) Oxidation of the electron carriers NADH or FADH2 results in these carriers being again available to transport a subsequent pair of electrons (Stryer, 1988) For anaerobic catabolism to continue in the absence of oxygen as the terminal electron acceptor, NAD+ must be reduced through fermentative processes which utilise organic compounds as reducing agents Few of these processes are coupled to ATP formation, so overall ATP generation is much lower than in aerobic processes Consequently, anaerobic metabolism is considerably less ecient than aerobic metabolism, resulting in much lower biomass yields Uncoupled metabolism Intracellular regulation of catabolic and anabolic processes by bacteria is necessary to ensure an ecient ¯ow of energy Within the mitochondria of higher organisms the concentration of ATP is known to inhibit activity in the citric acid cycle, in e€ect producing a feed-back control loop (Stryer, 1988) However, there is less certainty about the presence of respiratory controls in bacteria Senez (1962) suggested that bacterial anabolism is coupled to catabolism of substrate through rate limiting respiration However uncoupled metabolism would occur if respiratory control did not exist and instead the biosynthetic processes were rate limiting Therefore excess free energy would be directed away from the production of biomass To consume this available energy, several possibilities were considered, including, the dissipation of energy as heat by adenosine triphosphatase systems, the activation of alternative metabolic pathways by-passing free energy conserving reactions and the accumulation of polymerised products in storage form or as secreted waste Stouthamer (1979) reports that uncoupled metabolism has been observed: Fig The role of the ATP±ADP cycle in cell metabolism The high-energy phosphate bonds of ATP are used in coupled reactions for carrying out energy-requiring functions; ultimately, inorganic phosphate (Pi) is released ADP is rephosphorylated to ATP during energy yielding reactions of catabolism (adapted from Atkinson and Mavituna, 1991) 1123 in the presence of inhibitory compounds in the presence of excess energy source at unfavourable temperatures in minimal media during transition periods, in which cells are adjusting to changes in their environment Russel and Cook (1995) de®ne ``uncoupling'' as being the inability of chemosmotic oxidative phosphorylation to generate the maximum theoretical amount of metabolic energy in the form of ATP For clarity in this work this is rede®ned as ``uncoupled oxidative phosphorylation'' to di€erentiate it from other mechanisms of ``uncoupling'' metabolism Russel and Cook also suggested that 1124 Euan W Low and Howard A Chase ATP lost to non-growth reactions be termed ATP spilling Decreasing the ATP available for biosynthesis would in turn reduce biomass production and ability to replicate these uncoupling processes in wastewater treatment would therefore be advantageous Further, if microorganisms exhibit similar behaviour to mitochondria in the regulation of the activity in the citric acid cycle, then a reduction of cellular ATP concentration would provide a stimulus to the feed-back control loop to promote catabolism of the pollutants Anderson and Meyenburg (1980) demonstrated that in aerobic batch cultures of E coli although biosynthesis was very tightly coupled to respiration, respiration was not tightly coupled with anabolism They concluded that growth was limited by the rate of respiration Cook and Russel (1994) found that in cultures of Streptoccus bovis containing an excess of glucose, glucose was consumed faster than could be explained by growth or maintenance The energy spilling e€ect appeared to be due to a futile cycle of protons through the cell membrane Marr's (Marr, 1991) analysis of the literature on E coli supports the conjecture that its growth rate is set by the supply of a precursor metabolite and of the cellular structures synthesised from it rather than by the supply of ATP Metabolism in excess carbon conditions In reviewing the physiological and energetic aspects of bacterial metabolite overproduction, Tempest and Neijssel (1992) noted that metabolite overproduction occurred in many bacterial species when grown in chemostat cultures under conditions of nutrient limitation and carbon substrate excess This e€ect occurred for Klebsiella pneumoniae, Escherichia coli, Pseudomonas ¯uorescens, Pseudomonas putida, Paracoccis denitri®cans, Bacillus subtilis and Bacillus stearothermophilus Carbon substrate uptake and carbon dioxide evolution rates were greatly elevated under nutrient limited conditions For K pneumoniae growing under magnesium limited conditions, the speci®c substrate uptake rates were 3.5 times greater when compared with carbon limited conditions, yet the biomass yield decreased to 41% of that obtained with the carbon limited conditions whilst the carbon dioxide evolution rate doubled (Table 3) Two explanations were o€ered; the ®rst being that energy dissipation by leakage of ions, such as protons or K+, through the cytoplasm membrane weakens the potential across it and thus subsequently uncouples oxidative phosphorylation The second mechanism is that the organisms induce a metabolic reaction pathway (the methylglyoxal bypass) that circumvents the energy conserving steps of glycolysis These observations suggest that production rates of intermediary metabolites and ATP by catabolism can be in excess of their consumption rate during anabolism (due to limitations arising from other sources) Energy is consequently dissipated and uncoupled metabolism may result in a reduction in the yield of biomass In general, organic carbon availability limits cell growth in wastewater processes, but excess carbon conditions can be engineered by increasing the food to microorganism ratio A de®ciency of other growth factors could also contribute to uncoupled metabolism This is especially applicable during the treatment of those industrial e‚uents which require nutrient addition to sustain biological treatment However, while achieving the low biomass yields by engineering conditions of excess carbon, additional treatment of the wastewaters would be necessary to reduce the concentration of organic carbon to acceptable levels Uncoupling of oxidative phosphorylation Dissipation of the proton-motive driving force required for oxidative phosphorylation can be induced by increasing the proton-conducting capacity of the membrane Zakharov and Kuz'mina (1992) found oxidative phosphorylation to be thermolabile in Thermus thermophilus and suggested that elevated temperatures increased the proton permeability of the membrane Maintaining a population at higher temperatures is likely to cause a shift toward a thermophilic population Unacclimatised biomass introduced to the substrate at higher temperatures may achieve reduced biomass production with thermally induced uncoupled oxidative phosphorylation Oxidative phosphorylation can also be uncoupled by futile cycles which transfer protons across the membrane Ammonia is typically present in wastewaters and also produced by decomposition of nitrogenous organic matter Further, Stouthamer (1979) proposed that the uncoupling e€ect of ammonium on oxidative phosphorylation in mitochondria could be explained by a futile ion cycle The movement of ammonium ions into the cytoplasm is driven by the same proton-motive driving force as utilised by oxidative phosphorylation, this driving Table Glucose and oxygen consumption rates and corresponding yield values of chemostat cultures of Klebsiella pneumoniae growing aerobically on glucose in a simple salts medium at a ®xed rate (D = 0.17 hÀ1; 358C; pH 6.8) (Tempest and Neijssel, 1992) Limitation Speci®c consumption rate (mmol hÀ1 g dry weight cellsÀ1) glucose Glucose Magnesium Potassium oxygen carbon dioxide 2.1 7.4 10.3 4.2 11.2 17.4 5.5 11.1 16.9 Index of carbon recovery in biomass 1.00 0.41 0.39 Reducing production of excess biomass force is dissipated by the dissociation of protons from the ammonium ions which forms ammonia and then di€use back through the cytoplasm membrane Nitri®cation of ammonia in wastewater treatment produces nitrite; Almeida et al (1995) studying nitrite inhibition of denitri®cation with a pure culture of P ¯uorescens as a model system, found that nitrite accumulation caused growth to be uncoupled from denitri®cation and it was suggested that the nitrite ion acts as a protonphore Yarbrough et al (1980) also reported that sodium nitrite inhibited oxidative phosphorylation in E coli Uncoupling oxidative phosphorylation has been more thoroughly studied with organic protonphores which are similarly capable of shuttling protons across the membrane and have been reported to have high uncoupling potential (Neijssel, 1977; Stockdale and Sewyn, 1971) Research on a variety of respiring cells (bacteria, rat brain and angiosperm) in the presence of the organic protonphore, dinitrophenol, showed that respiration could be increased by between 1.5 and times that of the controls (Simon, 1953) Stockdale and Sewyn (1971) gave a comprehensive and quantitative report on the uncoupling of oxidative phosphorylation in rat liver mitochondria At low protonphore concentrations, some degree of respiratory control is retained and the rate of respiration is limited by the energy coupling system At higher protonphore concentrations, respiratory control is lost and the rate of respiration becomes limited by the rate of oxidation Further increases in concentration inhibit respiration, Stockdale suggested that this was by direct action on a protein in the respiratory chain Loomis and Lipmann (1948) and Simon (1953) have similarly noted that these higher concentrations have inhibited the respiratory process Low and Chase (1998a) supplemented a chemostat monoculture of P putida with the protonphoric uncoupler of oxidative phosphorylation, para-nitrophenol The e€ect of this addition was to dissipate energy within the cells and thus reduce the energy available for endothermic processes Under these conditions cells continued to satisfy their maintenance energy requirements prior to making energy available for anabolism, thus reducing the observed biomass yield The optimum pH range for activated sludge treating domestic sewage is pH 7.0±7.5, with an e€ective process range of pH 6.0±9.0 (Eckenfelder and Connor, 1961) Simon (1953) observes that acidic conditions improve the uncoupling activity of organic protonphores and there is a greater association of protons with protonphoric compounds at lower pH Low and Chase (1998a) found that decreases in pH alone had no e€ect on biomass production, but caused additional protonphore induced reduction of biomass production At pH 6.2 the eciency of biomass production was 1125 reduced by 77% when the feed was supplemented with 100 mg para-nitrophenol LÀ1 Research with organic protonphores has usefully shown that the dissipation of energy, through uncoupling biochemical processes such as oxidative phosphorylation, can directly reduce biomass production However, the actual use of organic protonphores to achieve this is impractical for several reasons, which include the inherent toxicity of protonphores Also, the protonphore would need to be removed from the waters prior to discharge However, further experimentation to establish alternative methods of uncoupling metabolism is desirable ATP consumption Uncoupling oxidative phosphorylation reduces the production of ATP With reduced ATP availability cells continue to satisfy their maintenance energy requirements prior to making energy available for anabolism The yield of biomass per gram of ATP (YATP) for an organism is determined by the cell composition, the speci®c growth rate and the maintenance coecient (Stouthammer and Bettenhaussen, 1973) As these vary between species, the YATP can not be expected to be constant for di€erent microorganisms Uncoupling oxidative phosphorylation within a mixed culture may favour species which are more ecient in generating and using ATP i.e have a high YATP While these species may displace less ecient species, it is generally accepted that di€erent microorganisms have di€erent anities for substrates Thus metabolically less ecient species, which have higher anities for growth-limiting substrates, may survive However, with a reduced ATP availability, a shift in the population dynamics is a likely event Measurement of ATP generation and consumption would be a valuable method for comparing the eciencies of di€erent systems ATP is an intermediate in metabolism with a high turnover rate (typically within a minute of formation Stryer, 1988) so measurement of the rate of ATP production is dicult For complete metabolism of a given substrate, the theoretical yield of ATP by a given microorganism can be predicted However, the composition of wastewaters are variable and microbial populations are unde®ned In addition, the removal of metabolic intermediates during aerobic respiration for biosynthesis complicates the determination of the actual ATP yield Presently accurate measurements of YATP are limited to anaerobic systems where the net ATP gain per mole of substrate is accurately known from the metabolic balances and in vitro studies of the enzymic pathways Maintenance energy requirements Through catabolism, cells make available biologically useful energy for fuelling their endothermic reactions (Fig 3) An increase of the energy requirements for non-growth activities, in particular maintenance functions, would decrease the amount of 1126 Euan W Low and Howard A Chase energy available for biosynthesis of new biomass Exothermic maintenance functions include the turnover of cell materials and osmotic work to maintain concentration gradients In addition, energy requirements for cell motility can not be di€erentiated from maintenance energy requirements Increasing the quantity of substrate utilised by maintenance functions in order to decrease the observed yield has been considered previously Watson (1970) observed that the presence of M NaCl in a culture of Saccharomyces cerevisiae, increased the maintenance energy requirements with consequent decreases in the observed yield Strachan et al (1996), seeking to reduce excessive bio®lm growth in a membrane bioreactor, similarly found that addition of NaCl to chemostat monocultures increased maintenance energy requirements and therefore reduced the yield of biomass However, Hamoda and Al-attar (1995) found that while the organic removal eciency and the e‚uent quality of an activated sludge did not deteriorate as a result of constant addition of NaCl (up to 30 g LÀ1) to acclimatised biomass, the biomass production was not found to be reduced It was suggested that during acclimation, the biomass had adapted to the saline environment The energy available to microorganisms is determined (amongst other things) by the supply of substrate In substrate-limited wastewater processes, it is reasonable to expect that microorganisms' allocation of the available carbon source will preferentially be orientated toward satisfying their maintenance energy requirements Several models have been proposed to account for the e€ects of satisfying maintenance energy requirements in cell cultures under conditions of substrate-limited growth and their relevance in biological wastewater treatment is now reviewed In considering a mass balance on the carbon source in a chemostat system without biomass recycle, Pirt (1975) proposed that a portion of the total carbon source is consumed for maintenance and a portion is utilised in anabolism If all the substrate was employed for anabolism, then this would theoretically give the maximum growth yield, YG; this is termed the true growth yield For a given steady-state with a given amount of biomass, the rate at which the carbon source is consumed for maintenance, qm, is assumed to be constant; therefore the observed biomass yield (YS) from the substrate consumed is; qm ˆ ‡ m YS YG …1† However, this relationship can not adequately describe most wastewater treatment processes, which seek to enhance the concentration of the catalytic biomass Bouillot et al (1990) in seeking to evaluate the maintenance coecient for a model system of P ¯uorescens metabolising a synthetic waste in a system with biomass recycle developed Pirt's relationship equation 1, D…S0 À S † ˆ mX ‡ qm X YG …2† and it was stated that in the case of total biomass recycle, with zero growth rate, the maintenance coecient can be evaluated from qm ˆ D…S0 À S † X …3† It was also proposed that for the system with partial biomass recycle, the maintenance coecient be evaluated from Pirt's relationship (equation 1) by assuming that, at steady-state, the speci®c growth rate is equal to the volumetric rate of biomass removal (QW) divided by the reactor volume i.e., qm V ˆ ‡ YS QW YG …4† However, the maintenance coecient obtained from this method with partial biomass recycle (qm=0.035 g gÀ1 hÀ1) was signi®cantly di€erent from those obtained with complete biomass recycle (qm=0.042 g gÀ1 hÀ1) and that obtained in a chemostat with no recycle (qm=0.028 g gÀ1 hÀ1) In the situation with complete biomass recycle, described by equation 3, the physiology of the cells will be similar to that in the resting stage of batch growth This approach is similar to that employed by Muller and Babel (1996) to study the energy requirements for survival Operation with partial recycle results in an increase in biomass concentration in the reactor and as substrate utilisation for satisfying maintenance functions depends on the amount of biomass present, the ration of substrate utilised in satisfying these functions will increase However, equation does not correctly describe this situation Low and Chase (1998a) observed that as wastewater processes typically seek to enhance biomass concentration within the reactor, biomass concentration is divorced from biomass production, so meaningful determination of the empirical term m is complicated A model was sought which excluded the speci®c growth rate, but incorporated the biomass concentration in order to provide a more suitable description of a system with partial biomass recycle It was proposed that for a continuously fed, perfectly mixed biological reactor, the mass balance on the utilisation of the energy source is presented as the sum of the substrate utilised by anabolism and the substrate utilised by the biomass for satisfying maintenance requirements; ÀrS ˆ À1 rX À qm X YG …5† Reducing production of excess biomass 1127 EFFECTS OF PHYSICAL ENVIRONMENT ON METABOLISM Fig Concentrations of oxygen and organic substrates across a cell agglomerate resulting in zoni®cation of metabolic activity and thus the biomass production per unit volume may be represented by rX ˆ YG …rS À qm X † …6† Low and Chase (1998b) found that dissipating energy with protonphores, cells preferentially satisfy the energy requirements associated with maintenance functions and that cell synthesis will occur using the remaining substrate available With a constant supply of substrate and a situation where growth is substrate limited at a constant level, then rS can also be assumed constant It follows from equation that if YG and qm are constant and biomass growth is substrate limited, then biomass production decreases proportionally with biomass concentration YG and qm can be determined by measurement of biomass production at di€erent biomass concentrations In the aeration basin of the activated sludge process, the biomass concentration is a function of the sludge return rate and therefore is an accessible control parameter Increasing the reactor biomass concentration from to g LÀ1 reduced biomass production by 12% and analysis of a similar system observed that increasing biomass concentration from 1.7 g LÀ1 to 10.3 g LÀ1 reduced biomass production by 44% Metabolic eciency is dependent on the terminal electron acceptor used Therefore, aerobic, anaerobic and anoxic environments, either engineered in the bulk conditions or naturally occurring in di€erent zones within cell aggregates (Fig 4), will result in di€erent yields of ATP and subsequently di€erent extents of biomass production Mixing regimes and relative velocities between cell agglomerates and the liquid in¯uence the size of cell agglomerates, thus permit the possible management of an optimum size Anaerobic wastewater treatment produces considerably less biomass than aerobic treatment (Table 4) and with the bene®t of methane gas as a by-product But organic compounds act as the reducing agents during fermentation producing malodorous volatile fatty acids which may overwhelm the bu€ering capacity of the process, resulting in a drop in pH Further, the fastidious bacteria capable of reducing these volatile fatty acids can not survive below pH 6.2 and their inability to remove the volatile fatty acids further exacerbates the drop in pH (Noaves, 1987) At typical ambient wastewater temperatures of around 5±208C low rates of reaction occur, while at thermophilic conditions the microbiological population is unstable Therefore it is desirable to operate at mesophilic conditions The temperature of the water can be raised by transfer of heat from the combustion of produced methane, however this process is only autothermic for wastewaters with high concentrations of organic pollutants Therefore, despite the bene®ts of low biomass production and methane generation, anaerobic processes require careful control and have been developed for wastewaters with high concentrations of organic pollutants In addition anaerobically treated waters normally require an aerobic polishing stage prior to discharge and as a consequence the process has not been widely adopted in the U.K Downstream digestion of wasted excess biomass by further metabolism can be operated anaerobically and bene®t from lower yields to reduce the volume of biomass to be dewatered and disposed Table Indication of yields for various substrates under aerobic and anaerobic operation (adapted from Tchobanoglous and Burton, 1991) Substrate Process Yield (mg volatile suspended solids/mg BOD5) range Domestic sludge Protein Fatty acids Carbohydrate Domestic wastewater anaerobica anaerobica anaerobica anaerobica aerobicb typical 0.04±0.1 0.05±0.09 0.04±0.07 0.02±0.04 0.4±0.8 0.06 0.024 0.05 0.075 0.6 a Values are for anaerobic processes operating at 208C bValues are for a conventional activated sludge process operating at 208C 1128 Euan W Low and Howard A Chase DRAWBACKS TO REDUCED BIOMASS PRODUCTION Fig Process ¯owsheet for the oxic settling anaerobic system employed by Chudoba et al (1992) to reduce biomass production of Modern engineering and control strategies should be able to overcome ostensible issues of unreliability and malodorous emissions Chudoba et al (1992) included an anaerobic zone in the biomass recycle stream of a laboratory-scale activated sludge process A reduction in excess biomass production was observed in this so-called oxic settling anaerobic system (Fig 5) This was explained by endogenous metabolism to meet the cells' energy requirements and microorganisms consuming intracellular stocks of ATP in the anaerobic zone thus limiting biosynthesis Comparison of the oxic settling anaerobic process with a conventional activated sludge process, each with a sludge age of d, found the yields of biomass obtained were in the ranges from 0.13 to 0.29 g suspended solids/g COD and from 0.28 to 0.47 g suspended solids/g COD, respectively PROCESS CONTROL Two major parameters can be regulated in activated sludge processes to achieve the desired e‚uent quality These are the return biomass ¯owrate to the aeration basin and the biomass wastage ¯owrate Return of biomass in¯uences biomass concentration in the aeration basin Manipulation of the wastage rate is employed in control strategies providing either a constant Food to Microorganisms (F/M) ratio or to regulate the mean residence time of cells within the process, often referred to as the sludge age The F/M ratio describes the amount of substrate that a given amount of biomass is utilising It follows from equation that a low F/M ratio would result in lower biomass production Sludge age is de®ned as the ratio of the total amount of biomass in the process to the rate of biomass wastage However, since determination of the amount of biomass in the clari®er stage is dicult, this is more commonly measured as the ratio of biomass in the reactor to the rate of biomass wastage Biomass disposal requirements are typically lower at higher sludge ages (Horan, 1990) This may be due to maintenance e€ects, endogenous respiration during cell starvation and through further metabolism of biomass releasing more organic carbon as carbon dioxide Bene®ts can be realised from strategies which reduce the production of biomass These include; economic savings from the reduced costs of treatment and disposal of excess biomass, improved operational eciencies and a reduced environmental burden with lower disposal requirements However, other economic, operational and environmental costs may be incurred and these must be considered Settling properties in activated sludge processes Conventional mixed aeration processes such as activated sludge processes require that ¯oc agglomerates have good settling characteristics and are desirable for achieving a high quality e‚uent and to provide a concentrated biomass for recycling to the reactor to enhance the concentration therein These characteristics are thought to be strongly in¯uenced by reactor conditions through biomass population dynamics and surface chemistry (Foster, 1985) Variations in settleability has been correlated to the balance between ¯oc-forming and ®lamentous classes of microorganisms (Jenkins et al., 1993) Exocellular polymer production and cation concentration have also been correlated with settleability (Urbain et al., 1993, and Higgins and Novak, 1997, respectively) It is probable that employing any strategy which reduces biomass production may also a€ect the growth rates of individual species di€erently and so alter the population dynamics Altering the stresses on the population dynamics may in turn adversely alter the biomass settling characteristics, e.g changing surface chemistry and so causing poor ¯occulation or encouraging a proliferation of ®lamentous bacteria leading to bulking of the biomass Introducing stresses to a mixed microbial population requires care to ensure that the quality of the ®nal e‚uent or the ecacy of the process operation are not compromised Oxygen requirements In conventional activated sludge processes the oxygen transfer yields range from 0.6 to 4.2 kg O2/ kWh according to the method of aeration (Horan, 1990) Aeration typically accounting for more than 50% of total plant energy requirements (Groves et al., 1992) Reducing biomass disposal requirements by removing pollutants from wastewaters as respiration products will increase the oxygen demand and so the increased energy costs need to be considered Oxygen is utilised in respiration to provide the terminal electron acceptor during catabolism Examination of a simple balance on oxygen requirements illustrates how the various oxygen requirements sum to create the total oxygen demand, Reducing production of excess biomass b ˆ a…6 À 7X96Yobs † Total oxygen demand ˆ Oxygen required for energy extraction to satisfy maintenance functions ‡ Oxygen required for energy extraction to fuel biosynthesis ‡ Oxygen incorporated into new biomass ‡ Oxygen incorporated into metabolic by À products ‡ Oxygen required for nitrification Similarly the total oxygen supply can be determined by considering the various inputs of oxygen into the system, Total oxygen supplied ˆ Gaseous oxygen dissolved ‡ Molecular oxygen released from substrates ‡ Oxygen released during denitrification If oxygen is in excess, CO2 and H2O will be released as respiration products, whereas insucient oxygen availability may result in fermentative pathways forming organic by-products Nitrifying processes impose an additional oxygen requirement; however, a portion of this oxygen can subsequently be made available by microbial denitri®cation The total oxygen demand may also be o€set by gaseous oxygen dissolved in the in¯uent waters and by catabolism of contaminants liberating bound molecular oxygen However, additional oxygenation is typically required to supplement these sources to meet the total oxygen demand Low and Chase (1998c) assessed the e€ects of reducing biomass production on oxygen requirements Biomass was assumed to have an empirical formula of C5H7NO2 (Horan, 1990) and for a non-nitrifying process, with metabolic by-products assumed to have the empirical formula (±CH2O±)n, the complete metabolism of glucose was presented as, aC6 H12 O6 ‡ bO2 Mwt : 180 32 ‡ cNH3 À À4wCO2 ‡ xH2 O À 17 44 18 ‡ yC5 H7 NO2 ‡ z À CH2 O 113 30 1129 …8† Experimentation in a chemostat monoculture of P putida system with and without uncoupled metabolism was used to verify equation A carbon balance was conducted to measure the extent of carbon utilisation and to determine reaction stoichiometry, oxygen requirements were evaluated from the reaction stoichiometry Oxygen uptake rates were also evaluated by the dynamic gassing out method Both experimental methods compared well with both theoretical values predicted (by equation 8) for the measured observed yield Consequently equation indicates a rise in the oxygen demand to permit respiration of organic carbon, diverted from assimilation into biomass, to oxidise it to carbon dioxide Similarly, if lysed are utilised for cryptic growth and it is assumed that nitrogen is released as ammonium, a simpli®ed stoichiometry may be presented as, aC5 H7 NO2…aq† Mwt : 113 ‡ bO2 À À4wC5 H7 NO2…s† ‡ xCO2 À 32 113 44 ‡ yH2 O ‡ z À NH3 18 17 …9† This reaction shows that cryptic growth is associated with an increased oxygen requirement The biomass yielded by cryptic growth is Ycryptic=w/a and so a relationship between the gaseous oxygen requirements and the biomass yielded by cryptic growth can be obtained by solving simultaneous equations based on the stoichiometry as, b ˆ 5a…1 À Ycryptic † …10† Ycryptic characterises the eciency of lysis product utilisation to form biomass, equation 10 indicates that if this eciency is low, then an increased oxygen requirement will be incurred …7† The stoichiometric coecients are dependent on the overall eciency of metabolism Inecient metabolism may be caused by a low eciency of free energy conservation, dissipation of free energy or increased maintenance energy requirements To compensate, a greater portion of substrate must be utilised to provide energy resulting in an increased formation of respiration products or by-products and a reduced biomass production A relationship between the gaseous oxygen requirements and the yield of biomass from glucose was obtained by solving simultaneous equations based on the stoichiometry as, Nutrient removal The stoichiometry of equation shows that a reduction of biomass production will result in less nitrogen being removed from waters by assimilation into biomass Also, the stoichiometry of equation shows that further metabolism of cellular material will result in nitrogen being released to the waters So, strategies which seek to reduce biomass production may result in a lower amount of other materials (e.g nitrogenous compounds and phosphorous) being removed from the waters by assimilation into biomass The consequent discharge of these materials can cause eutrophication and deoxygenation in the receiving waters Thus for compliance with discharge consents, tertiary treatment may be required Also, the inclusion a nitri®- 1130 Euan W Low and Howard A Chase cation stage will further increase oxygen requirements DISCUSSION The magnitude of additional capital and operating costs o€set by reduced disposal costs will determine the feasibility of each strategy The optimal solution will be speci®c to any individual process Proposed methods for reducing biomass production must be acceptable to plant operators Also technical and economic constraints reduce the number of parameters available for control, eliminating options of extensive changes to reactor conditions such as temperature or pH Therefore the most applicable methods would involve: in¯uencing the choice of terminal electron acceptor manipulation of mixing regimes within the reactor regulation of process ¯owrates to a€ect relative concentrations of substrate and biomass appropriate selection of reactor design addition of chemicals such as protonphores or salts Table summarises the reduction in biomass production achieved by applying various strategies suitable for aerobic wastewater treatment All these strategies are similar in encouraging further metabolism of the organic carbon such that it is allocated to respiration products rather than assimilated into biomass Reducing biomass production in aerobic wastewater treatment by increasing the oxidation of the organic contaminants to respiration products will increase the total oxygen demand and is likely to result in an increase in the aeration costs These strategies may also cause a decrease in nitrogen assimilation into biomass and release nitrogen into the waters, increasing downstream nitri®cation and denitri®cation requirements There must be an awareness that introducing changes which stress the existing microbial ecosystem may cause adaptation in either a population shift in the biomass or microbial species acclimatisation through competitive selection Such changes may adversely alter the biomass settling characteristics Complete biomass retention will result in the accumulation of inert solids within wastewater processes process and reduce the e€ective reactor volume There exists a minimum rate at which biomass must be wasted, to purge inert solids from the system Enhancing biomass retention within a process such that further metabolism of organic carbon reduces the overall biomass production requires an increased amount of biomass to catalyse these reactions This may be achieved in situ with an increased biomass concentration or by employing a larger reactor volume to accommodate the extra biomass Cell lysis is promoted with the input of ozone or thermal energy but at additional cost The digestion of wasted biomass in a separate unit will incur both capital and operating costs Protozoan and metazoan predation has a valuable role in maintaining a healthy biomass and can reduce the overall biomass production with the further metabolism of organic carbon Methods to enhance and regulate populations of bacteriovores need to be developed to remove instabilities in population sizes Manipulation of the microbial environment and the presence of certain inhibitory substances in¯uence biochemical processes within cells This provides an opportunity to reduce biosynthesis within the cells The review of biochemistry, common to all organisms, highlighted the coupled processes of anabolism and catabolism with energy conservation being the vital link between the two Therefore, uncoupling anabolism from catabolism would be a powerful method to reducing biosynthesis Inducing metabolite overproduction in carbon excess conditions not only decreases the yield of biomass, but also greatly elevates the speci®c rate of carbon consumption However, this requires that the feed be de®cient in essential nutrients and so the strategy is more appropriate to treatment of industrial e‚uents where nutrient addition is practised Also, additional treatment of the wastewaters would be necessary to reduce the concentration of organic carbon to acceptable levels Uncoupling oxidative Table Summary and quantitative comparison of strategies for reducing the production of excess biomass Strategy Enhanced solids retention Thermally induced lysis and cryptic growth Ozone induced lysis and cryptic growth Aerobic, mesophilic digestion (208C) Aerobic, thermophilic digestion, (608C) Protozoan grazing Protozoan and metazoan grazing Bacterial metabolite overproduction Uncoupled oxidative phosphorylation Increased energy requirements for maintenance functions Oxic settling anaerobic Reduction in production of excess biomass (%) Reference 100 60 100 50 52 12±43 60±80 59±61 45 12 44 44 Sakai et al (1992) Canales et al (1994) Yasui and Shibata (1994) Ganczarczyk et al (1980) Mason and Hamer (1987) Ratsak et al (1994) Lee and Welander (1996) Tempest and Neijssel (1992) Low and Chase (1998a) Low and Chase (1998b) Bouillot et al (1990) Chudoba et al (1992) Reducing production of excess biomass phosphorylation reduces ATP production and so reduces biomass production and may also result in stimulated substrate catabolism This may be achieved by elevating the temperature to cause proton leakage through the thermolabile cytoplasm membranes, inducing futile ion cycles or by addition of protonphores Raising temperature of wastewaters is typically uneconomical and an operating cost is also associated with the addition of protonphores Microorganisms satisfy their maintenance energy requirements in preference to producing additional biomass Therefore, increasing the reactor biomass concentration can achieve an increase in the amount of organic carbon consumed in satisfying maintenance energy requirements and results in a lower generation of excess biomass during wastewater treatment No alterations are required to the process with only a marginal increase in operating costs originating from increased biomass recycle pumping requirements The low metabolic eciency of anaerobic catabolism results in a low biomass yield whilst alleviating the need for costly aeration While domestic wastewaters are too dilute to receive anaerobic treatment, industrial e‚uents which contain high organic substrate concentrations are suitable for such treatment Similarly, anaerobic digestion of biomass wasted from wastewater treatment processes will reduce the ®nal disposal requirements CONCLUSIONS There is a complex combination of processes which contribute to biomass production These processes can be engineered to maximise the e€ects of further metabolism by cell lysis, cryptic growth and bacteriovore predation The eciency of metabolism may be reduced and the ration of substrate utilised for satisfying maintenance functions increased In addition, stresses can be introduced to populations by engineering anaerobic or anoxic zones or by causing starvation An optimal solution may lie in a combination of these strategies A composite comprehension of these processes and their interactions reveals that there is considerable scope for reducing biomass production and therefore the disposal requirements of excess biomass REFERENCES 91/2711EEC (1991EEC) Council directive concerning urban waste water treatment O.J Euro Commun L355, 40±45 Almeida J S., Julio S M., Reis M A M and Carrondo M J T (1995) Nitrite inhibition of denitri®cation by Pseudomonas ¯uorescens Biotechnol Bioeng 46, 194± 201 1131 Anderson K B and Meyenburg K (1980) Are growth rates of Escherichia coli in batch cultures limited by respiration? J Bacteriol 144, 114±123 Atkinson B and Mavituna F (1991) Biochemical Engineering and Biotechnology Handbook, 2nd edn., pp 130±131 Stockton, New York Boon A and Thomas V (1996) Resource or rubbish The Chemical Engineer 612, 25±32 Bouillot P., Canales A., Pareilleux A., Huyard A and Goma G (1990) Membrane bioreactors for the evaluation of maintenance phenomena in wastewater treatment J Ferm Bioeng 69(3), 178±183 Canales A., Pareilleux A., Rols J L., Goma C and Huyard A (1994) Decreased sludge production strategy for domestic wastewater treatment Wat Sci Tech 30(8), 96±106 Cech J S., Hartman P and Macek M (1994) Bacteria and protozoa population dynamics in biological phosphate removal systems Wat Sci Tech 29(7) 109±117 Chudoba P., Morel A and Capedeville B (1992) The case of both energetic uncoupling and metabolic selection of microorganisms in the OSA activated sludge system Environ Tech 13, 761±770 Cook G M and Russel J B (1994) Energy-spilling reactions of Streptococcus bovis and resistance of its membrane to proton conductance Appl Environ Microbiol 60(6), 1942±1948 Curds C R (1973) A theoretical study of factors in¯uencing the microbial population dynamics of the activated-sludge processÐI Wat Res 7,1269±1284 Eckenfelder W W and Connor D J (1961) Biological Waste Treatment Pergamon Press, Oxford Foster C F (1985) Factors involved in the settlement of activated sludge, I Nutrients and surface polymers Wat Res 19(10), 1259±1264 Ganczarczyk J., Hamada M F and Hong-Lit W (1980) Performance of aerobic digestion at di€erent sludge solid levels and operation patterns Wat Res 14, 627± 633 Groves K P., Daigger G T., Simpkin T J., Redmon D T and Ewing L (1992) Evaluation of oxygen-transfer eciency and alpha-factor on variety of di€used aeration systems Wat Sci Tech 644(5), 691±698 Hamoda M F and Al-attar I M S (1995) E€ects of high sodium chloride concentrations on activated sludge treatment Wat Sci Tech 31(9), 61±72 Higgins M J and Novak J T (1997) The e€ect of cations on the settling and dewatering of activated sludges: laboratory results Wat Env Res 69(2), 215±224 Horan N J (1990) Biological Wastewater Treatment Systems Wiley, Chichester Jenkins D., Richard M G and Daigger G T (1993) Manual on the Causes and Control of Activated Sludge Bulking and Foaming, 2nd edn Lewis, Chelsea, MI Lee N M and Welander T (1996) Use of protozoa and metazoa for decreasing sludge production in aerobic wastewater treatment Biotech Lett 18(4), 429±434 Loomis W F and Lipmann F (1948) Reversible inhibition of the coupling between phosphorylation and oxidation J Biol Chem 173, 807±808 Low E W and Chase H A (1998a) Reduced biomass production during wastewater treatment, I Promotion of energy dissipation during metabolism, Wat Res., to be published Low E W and Chase H A (1998b) The e€ect of maintenance energy requirements on biomass production during wastewater treatment, Wat Res., accepted for publication Low E W and Chase H A (1998c) Reduced biomass production during wastewater treatment, II E€ects on oxygen requirements, Wat Res., to be published Lynch J M and Poole N J (1979) Microbial Ecology: a Conceptual Approach Blackwell, New York 1132 Euan W Low and Howard A Chase Marr A G (1991) Growth rate of Escherichia coli Microbiol Rev 55(2), 316±333 Mason C A., Hamer G and Bryers J D (1986) The death and lysis of microorganisms in environmental processes FEMS Microbiol Rev 39, 373±401 Mason C A., Haner A and Hamer G (1992) Aerobic thermophilic waste sludge treatment Wat Sci Tech 25(1), 113±118 Mason C A and Hamer G (1987) Cryptic growth in Klebsiella pneumoniae Appl Microbiol Biotech 25, 577±584 Mitchell P (1972) Chemiosmotic coupling and energy transduction: a logical development of biochemical knowledge Bioenergetics 3, 5±24 Muller R H and Babel W (1996) Measurement of growth at very low rates (mr0), an approach to study the energy requirements for the survival of Alcaligenes eutrophus JMP 134 Appl Environ Microbiol 62(1), 147±151 Neijssel O M (1977) The e€ect of 2,4-dinitrophenol on the growth of Klebsiella aerogenes NCTC 418 in aerobic chemostat cultures FEMS Lett 1, 47±50 Noaves R F V (1987) Microbiology of anaerobic digestion Wat Sci Tech 18(12), 1±4 Pirt S J (1975) Principles of Microbe and Cell Cultivation, 1st edn., pp 66±74 Blackwell, Oxford Ratsak C H., Kooi B W and van Verseveld H W (1994) Biomass reduction and mineralization increase due to the ciliate Tetrahymena pyriformis grazing on the bacterium Pseudomonas ¯uorescens Wat Sci Tech 29(7), 119±128 Russel J B and Cook G M (1995) Energetics of bacterial growth: balance of anabolic and catabolic reactions Microbiol Rev 59(1), 48±62 Sakai Y., Tani K and Tkahashi F (1992) Sewage treatment under conditions odf balancing microbial growth and cell decay with a high concentration of activated sludge supplemeted with ferromagnetic powder J Ferm Bioeng 76(6), 413±415 Salvado H., Gracia M P and Amigo J M (1995) Capability of ciliated protozoa as indicators of e‚uent quality in activated sludge plants Wat Res 29(4), 1041±1050 Scuras S., Daigger G T and Leslie Grady Jr C P (1997) Modelling Competition in the Activated Sludge Floc Microenvironment, Proceedings of the 2nd International Conference on Microorganisms in Activated Sludge and Bio®lm Processes 1997, pp 151± 152 Senez J C (1962) Some considerations on the energetics of bacterial growth Bacteriol Rev 26, 95±107 Simon E W (1953) Mechanisms of dinitrophenol toxicity Biol Rev 28, 453±479 Stentiford E and Taylor M (1995) Sludge drying In Advances in Water and E‚uent Treatment, Vol 2, pp 55±63 Biwater, U.K Stockdale M and Sewyn J (1971) E€ects of ring substituents on the activity of phenols as inhibitors and uncouplers of mitochondrial respiration Eur J Biochem 21, 565±574 Stouthamer A H., (1979) Correlation of growth yields In Microbial Biochemistry, International Review of Biochemistry, ed J R Quayle, Vol 21, pp 1±47 University Park, Baltimore Stouthammer A and Bettenhaussen C (1973) Utilisation of energy for growth and maintenance in continuous and batch cultures Biochim Biophys Acta 30, 53±70 Strachan L F., Freitas dos Santos L M., Leak D J and Livingston A G (1996) Minimization of biomass in an extractive membrane bioreactor Wat Sci Tech 34(5± 6), 273±280 Stryer L (1988) Biochemistry, 3rd edn Freeman, New York Tchobanoglous G and Burton F L (1991) Wastwater Engineering: Treatment, Disposal and Reuse Metcalf and Eddy, McGraw-Hill Tempest D W and Niejssel O M (1992) Physiological and energetic aspects of bacterial metabolite overproduction FEMS Microbiol Lett 100, 169±176 Urbain V., Block J C and Manem J (1993) Bio¯occulation in activated sludge: an analytical approach Wat Res 27(5), 829±838 Watson T G (1970) E€ects of sodium chloride on steadystate growth and metabolism of Saccharoymes cerevisiae J Genet Microbiol 64, 91±99 Yarbrough J M., Rake J B and Eagon R G (1980) Bacterial inhibitary e€ects of nitrite: inhibition of active transport, but not of group translocation, and of intracellur enzymes Appl Environ Microbiol 39(4), 831± 834 Yasui H and Shibata M (1994) An innovative approach to reduce excess sludge production in the activated sludge process Wat Sci Tech 30(9), 11±20 Yasui H., Nakamura K., Sakuma S., Iwasaki M and Sakai Y (1996) A full-scale operation of a novel activated sludge process without excess sludge production Wat Sci Tech 34(3), 395±404 Zakharov S D and Kuz'mina V P (1992) ATP-synthase activity of the thermophilic bacterium Thermus Thermophilus HB-8 membranes Biokhimiya 57(4), 539± 545  ... for reducing the amount of biomass produced The purpose of this review is to develop a comprehensive account of how pollutant metabolism leads to biomass production during the treatment of wastewaters... in lower biomass production Sludge age is de®ned as the ratio of the total amount of biomass in the process to the rate of biomass wastage However, since determination of the amount of biomass. .. 4.2 11.2 17.4 5.5 11.1 16.9 Index of carbon recovery in biomass 1.00 0.41 0.39 Reducing production of excess biomass force is dissipated by the dissociation of protons from the ammonium ions