reducing production of excess biomass during wastewater treatment

Promoting further metabolism of assimilated organic carbon will release additional respiration products and reduce the overall biomass production e.g.. Increas-ing the biomass concentrat

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 dis-posal As environmental and legislative constraints increase, thus limiting disposal options, there is con-siderable 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 conver-sion of organic pollutants to respiration products with a concomitant increase in the aeration require-ments 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 sub-strate excess conditions has been demonsub-strated 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 Increas-ing 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 dilution rate (hÿ1)

kd decay coecient (hÿ1)

K equilibrium constant

QW volumetric rate of biomass removal (L hÿ1)

qm speci®c substrate uptake related to

mainten-ance energy requirements (g gÿ1hÿ1)

ÿrS rate of substrate uptake (g Lÿ1hÿ1)

ÿrS G rate of substrate uptake for incorporation in new biomass (g Lÿ1hÿ1)

rX rate of biomass production (g Lÿ1hÿ1)

ÿrXd rate of biomass loss due to cell death and lysis (g Lÿ1hÿ1)

S substrate concentration (g Lÿ1)

S0 initial substrate concentration (g Lÿ1)

t time (h)

V reactor volume (L)

X biomass concentration (g Lÿ1)

Xv concentration of viable biomass (g Lÿ1)

YATPbiomass yielded per gram of ATP consumed (g biomass g ATPÿ1)

YG true biomass yield (g biomass g substrateÿ1)

Printed in Great Britain 0043-1354/99/$ - see front matter

PII: S0043-1354(98)00325-X

*Author to whom all correspondence should be addressed.

[Tel.: +44 1223 330132; Fax: +44 1223 334796;

E-mail: ewl21@cam.ac.uk].

AbbreviationsÐBOD, biological oxygen demand, ATP,

adenosine triphosphate, NADH, nicotineamide adenine

dinucleotide, COD, chemical oxygen demand, MLSS,

Mixed Liquor Suspended solids

1119

YS observed biomass yield (g biomass g

sub-strateÿ1)

m speci®c growth rate (hÿ1)

INTRODUCTION

Biological wastewater treatment involves the

trans-formation of dissolved and suspended organic

con-taminants 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

ad-dition, recent European legislation requires that

more wastewaters receive biological treatment prior

to discharge (91/2711EEC, 1991), resulting in a

con-siderable 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

com-prehensive 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

meta-bolic pathways utilised by the cells As biological,

biochemical and physical phenomena all in¯uence

nutrient removal, these shall be considered in

con-junction 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-cing biomass production on these aspects are also discussed

BIOLOGICAL PROCESSES WITHIN WASTEWATER

TREATMENT

Secondary sludges contain inert solids and bio-logical 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 assimi-lated by microorganisms to provide energy or to be utilised in biosynthesis, thus removing the contami-nants 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 bac-teria The chemolithotrophs which ``nitrify'' ammo-nia 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 mi-crobial population consisting of cells, either in an isolated manner or in an agglomerate of cells form-ing 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 1 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 Energy source Terminal electron acceptor Chemoorganotrophs (heterotrophs) organic carbon organic carbon aerobic: oxygen

anaerobic: organic compounds anoxic: nitrate, sulphate Chemolithotrophs (autotrophs) inorganic carbon inorganic compounds, e.g NH 3 , H 2 S and Fe 2+ oxygen

Table 2 Allocation of substrate by a cell in each stage of its life cycle

in energy generation in assimilation for maintenance for anabolism for biosynthesis

anoxic environments will determine the availability

of reducing equivalents thus in¯uencing the

meta-bolic eciency Microenvironments in cell

agglom-erates 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

com-posed of non-viable bacteria, the maintenance

requirements of living, non-viable bacteria may

make a signi®cant contribution to substrate

metab-olism

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

reac-tions (as a food source) by higher bacteriovoric

organisms such as protozoa, metazoa and

nema-todes Cell lysis will release cell contents into the

medium, thus providing an autochtonous substrate

which contributes to the organic loading This

or-ganic autochthonous substrate is reused in

mi-crobial 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

pro-ducts 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 hÿ1 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

mu-nicipal sewage treatment plant was acclimated for

more than 4 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 Lÿ1) was maintained on a continuously-fed, synthetic waste (0.81 g COD Lÿ1dÿ1) In addition

to gravitational settling, biomass retention was enhanced by supplementing the biomass with ferro-magnetic powder (11 g Lÿ1of average particle diam-eter 0.420.1 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 pro-cess, 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 diges-tion 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 con-centration ÿrXd=kdXv Typically the kd values in wastewater treatment are in the order of 0.03± 0.06 dÿ1 (Horan, 1990) and therefore the ability to promote cell death and lysis could be advantageous and can be achieved by engineering hostile environ-ments

Aerobic, thermophilic digestion of wasted bio-mass is exothermic and can therefore be autother-mal 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 recal-citrant in less extreme environments Also the ther-mophilic temperatures may pasteurise the biomass reducing the content of pathogenic organisms Mason and Hamer (1987) sought to identify opti-mal conditions for the digestion of cell lysis pro-ducts 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 tempera-ture and oxygen supply could be varied Cell lysis and biodegradation was optimal under oxygen-lim-ited conditions at 608C with a residence time of 5 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 bio-reactor demonstrated that shorter sludge ages increased the biomass viability However when the

biomass passed through a thermal treatment loop

(residence time 3 h, 908C) nearly 100% of cells were

killed and partial cell lysis was induced Thus a

por-tion of biomass, recycled via the thermal treatment

loop and back to the reactor (Fig 1), formed

auto-chthonous substrate (lysis products) on which

cryp-tic growth occurred and this further metabolism

contributed to a 60% reduction in the overall

bio-mass 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 Lÿ1

was fed with 1000 mg BOD Lÿ1dÿ1, culture was

removed from the aeration basin and recirculated

via the ozonation stage at a dilution rate of 0.3 dÿ1

in which a dose of 0.05 mg O3mg biomassÿ1 was

applied During 6 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 dÿ1found 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

and metazoa Ciliated protozoa have been demon-strated 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 bio-mass production in a mixed microbial culture In both of these experiments, bacterial cells were cul-tured in a primary reactor vessel and the e‚uent was fed to a second reactor vessel in which the bac-teriovores metabolised the bacterial cells

To achieve a similar reduction in the overall bio-mass 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 popu-lation dynamics of prey±predator repopu-lationships which indicated oscillating population sizes (Lynch and Poole, 1979)

The use of predatory activity to reduce the over-all 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 chemoor-ganotrophs for reducing organic compounds, the most commonly used pathway being glycolysis, typically to pyruvate with the concomitant for-mation of energy carrying compounds such as ade-nosine 5'-triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH) and reduced ¯avin adenine dinucleotide (FADH2) While this mechan-ism yields a small amount of energy, it does not require oxygen and can therefore occur in anaerobic

Fig 1 Process ¯owsheet for the aerobic process with

ther-mally induced cell lysis of excess biomass to form

auto-chthonous substrate (Canales et al., 1994).

Fig 2 Process ¯owsheet for the aerobic process with

enhanced cell lysis by contacting excess biomass with

ozone to form autochthonous substrate (Yasui and

Shibata, 1994).

environments The bacterial genus Pseudomonas,

which Horan (1990) describes as a signi®cant

oxidi-ser of carbon in wastewater treatment, utilise the

Entner±Doudoro€ pathway which is similar to

gly-colysis in producing pyruvate, but is less ecient in

ATP generation

Pyruvate is utilised in the citric acid cycle to

pro-duce molecules of NADH and FADH2which carry

pairs of electrons with a high transfer potential

The donation of these electrons to molecular

oxy-gen in a controlled regime allows a large amount of

free energy to be transferred In addition to

provid-ing useful energy for meetprovid-ing the cell's needs,

inter-mediaries of the citric acid cycle can be withdrawn

to form materials required in biosynthesis Thus

catabolised carbon is removed from metabolic

path-ways during respiration as CO2 and as metabolites

for synthesis of biomass The concentrations of

cer-tain compounds regulate the rate of reactions of the

citric acid cycle within eukaryotes and may also do

so within prokaryotes

The process employed to conserve the free energy

transferred to NADH or FADH2is the

chemosmo-tic process of oxidative phosphorylation (Mitchell,

1972) This involves respiratory assemblies

contain-ing a series of electron carriers located across the

cell's cytoplasmic membrane and while these

trans-fer electrons from NADH or FADH2 to O2,

pro-tons are simultaneously pumped out of the cell

cytoplasm Thus a proton-motive gradient is

gener-ated across the membrane providing the driving

force for the ¯ow of protons back into the

cyto-plasm 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 concen-trations (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 pro-cesses 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, an-aerobic 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 res-piration 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 con-sidered, 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 se-creted waste

Stouthamer (1979) reports that uncoupled metab-olism has been observed:

1 in the presence of inhibitory compounds

2 in the presence of excess energy source

3 at unfavourable temperatures

4 in minimal media

5 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 phos-phorylation 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€eren-tiate it from other mechanisms of ``uncoupling'' metabolism Russel and Cook also suggested that

Fig 3 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

func-tions; ultimately, inorganic phosphate (P i ) is released.

ADP is rephosphorylated to ATP during energy yielding

reactions of catabolism (adapted from Atkinson and

Mavituna, 1991).

ATP lost to non-growth reactions be termed ATP

spilling Decreasing the ATP available for

biosyn-thesis would in turn reduce biomass production and

ability to replicate these uncoupling processes in

wastewater treatment would therefore be

advan-tageous 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

stimu-lus to the feed-back control loop to promote

cata-bolism 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

review-ing the physiological and energetic aspects of

bac-terial metabolite overproduction, Tempest and

Neijssel (1992) noted that metabolite

overproduc-tion 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,

Para-coccis denitri®cans, Bacillus subtilis and Bacillus

stearothermophilus Carbon substrate uptake and

carbon dioxide evolution rates were greatly elevated

under nutrient limited conditions For K

pneumo-niae growing under magnesium limited conditions,

the speci®c substrate uptake rates were 3.5 times

greater when compared with carbon limited

con-ditions, 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 pro-cesses, but excess carbon conditions can be engin-eered by increasing the food to microorganism ratio A de®ciency of other growth factors could also contribute to uncoupled metabolism This is es-pecially 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 Dissipa-tion 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 ther-mophilic population Unacclimatised biomass intro-duced 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 waste-waters and also produced by decomposition of ni-trogenous organic matter Further, Stouthamer (1979) proposed that the uncoupling e€ect of am-monium on oxidative phosphorylation in mitochon-dria 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 3 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 h ÿ1 ; 358C; pH 6.8) (Tempest and Neijssel, 1992) Limitation Speci®c consumption rate (mmol h ÿ1 g dry weight cells ÿ1 ) Index of carbon recovery in biomass

glucose oxygen carbon dioxide

force is dissipated by the dissociation of protons

from the ammonium ions which forms ammonia

and then di€use back through the cytoplasm

mem-brane 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

proto-nphores 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

or-ganic protonphore, dinitrophenol, showed that

res-piration could be increased by between 1.5 and 3

times that of the controls (Simon, 1953) Stockdale

and Sewyn (1971) gave a comprehensive and

quan-titative report on the uncoupling of oxidative

phos-phorylation in rat liver mitochondria At low

protonphore concentrations, some degree of

respir-atory 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

concen-tration inhibit respiration, Stockdale suggested that

this was by direct action on a protein in the

respir-atory chain Loomis and Lipmann (1948) and

Simon (1953) have similarly noted that these higher

concentrations have inhibited the respiratory

pro-cess

Low and Chase (1998a) supplemented a

chemo-stat monoculture of P putida with the protonphoric

uncoupler of oxidative phosphorylation,

para-nitro-phenol 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

mainten-ance 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

associ-ation 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

reduced by 77% when the feed was supplemented with 100 mg para-nitrophenol Lÿ1

Research with organic protonphores has usefully shown that the dissipation of energy, through uncoupling biochemical processes such as oxidative phosphorylation, can directly reduce biomass pro-duction However, the actual use of organic proto-nphores to achieve this is impractical for several reasons, which include the inherent toxicity of pro-tonphores 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 phos-phorylation 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 YATPcan not be expected

to be constant for di€erent microorganisms Uncou-pling oxidative phosphorylation within a mixed cul-ture 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 micro-organisms 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 consump-tion would be a valuable method for comparing the eciencies of di€erent systems ATP is an inter-mediate in metabolism with a high turnover rate (typically within a minute of formation Stryer, 1988) so measurement of the rate of ATP pro-duction 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 mi-crobial populations are unde®ned In addition, the removal of metabolic intermediates during aerobic respiration for biosynthesis complicates the determi-nation of the actual ATP yield Presently accurate measurements of YATPare limited to anaerobic sys-tems 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 biologi-cally useful energy for fuelling their endothermic reactions (Fig 3) An increase of the energy require-ments for non-growth activities, in particular main-tenance functions, would decrease the amount of

energy available for biosynthesis of new biomass.

Exothermic maintenance functions include the

turn-over of cell materials and osmotic work to maintain

concentration gradients In addition, energy

require-ments 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 1 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

monocul-tures 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 Lÿ1) to acclimatised biomass, the biomass

pro-duction 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

deter-mined (amongst other things) by the supply of

sub-strate In substrate-limited wastewater processes, it

is reasonable to expect that microorganisms'

allo-cation of the available carbon source will

preferen-tially 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

re-cycle, 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

sub-strate 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;

there-fore the observed biomass yield (YS) from the

sub-strate consumed is;

1

YSˆqmm‡Y1

However, this relationship can not adequately

describe most wastewater treatment processes,

which seek to enhance the concentration of the

cat-alytic 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…S0ÿ S† ˆYmX

and it was stated that in the case of total biomass recycle, with zero growth rate, the maintenance coecient can be evaluated from

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 bio-mass removal (QW) divided by the reactor volume i.e.,

1

YSˆqQmV

W ‡Y1

However, the maintenance coecient obtained from this method with partial biomass recycle (qm=0.035 g gÿ1hÿ1) was signi®cantly di€erent from those obtained with complete biomass recycle (qm=0.042 g gÿ1hÿ1) and that obtained in a chemo-stat with no recycle (qm=0.028 g gÿ1hÿ1) 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 re-cycle results in an increase in biomass concentration

in the reactor and as substrate utilisation for satis-fying maintenance functions depends on the amount

of biomass present, the ration of substrate utilised

in satisfying these functions will increase However, equation 6 does not correctly describe this situ-ation

Low and Chase (1998a) observed that as waste-water processes typically seek to enhance biomass concentration within the reactor, biomass concen-tration 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 bio-mass concentration in order to provide a more suit-able description of a system with partial biomass recycle It was proposed that for a continuously fed, perfectly mixed biological reactor, the mass bal-ance on the utilisation of the energy source is pre-sented as the sum of the substrate utilised by anabolism and the substrate utilised by the biomass for satisfying maintenance requirements;

ÿrSˆYÿ1

and thus the biomass production per unit volume

may be represented by

rXˆ YG…rSÿ qmX † …6†

Low and Chase (1998b) found that dissipating

energy with protonphores, cells preferentially satisfy

the energy requirements associated with

mainten-ance functions and that cell synthesis will occur

using the remaining substrate available With a

con-stant supply of substrate and a situation where

growth is substrate limited at a constant level, then

rScan also be assumed constant

It follows from equation 6 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 3 to 6 g Lÿ1 reduced biomass production by

12% and analysis of a similar system observed that

increasing biomass concentration from 1.7 g Lÿ1 to

10.3 g Lÿ1reduced biomass production by 44%

EFFECTS OF PHYSICAL ENVIRONMENT ON

METABOLISM

Metabolic eciency is dependent on the terminal electron acceptor used Therefore, aerobic, anaero-bic and anoxic environments, either engineered in the bulk conditions or naturally occurring in di€er-ent 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 con-siderably 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 mal-odorous 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 vol-atile fatty acids further exacerbates the drop in pH (Noaves, 1987) At typical ambient wastewater tem-peratures of around 5±208C low rates of reaction occur, while at thermophilic conditions the micro-biological 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 waste-waters with high concentrations of organic pollu-tants Therefore, despite the bene®ts of low biomass production and methane generation, anaerobic pro-cesses require careful control and have been devel-oped 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 anaerobi-cally and bene®t from lower yields to reduce the volume of biomass to be dewatered and disposed

Fig 4 Concentrations of oxygen and organic substrates

across a cell agglomerate resulting in zoni®cation of

meta-bolic activity.

Table 4 Indication of yields for various substrates under aerobic and anaerobic

operation (adapted from Tchobanoglous and Burton, 1991)

suspended solids/mg BOD 5 ) range typical Domestic sludge anaerobic a 0.04±0.1 0.06

Carbohydrate anaerobic a 0.02±0.04 0.075 Domestic wastewater aerobic b 0.4±0.8 0.6

a Values are for anaerobic processes operating at 208C b Values are for a conven-tional activated sludge process operating at 208C.

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

bio-mass 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

con-suming 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

5 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

acti-vated sludge processes to achieve the desired

e‚u-ent quality These are the return biomass ¯owrate

to the aeration basin and the biomass wastage

¯ow-rate Return of biomass in¯uences biomass

concen-tration in the aeration basin Manipulation of the

wastage rate is employed in control strategies

pro-viding 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

utilis-ing It follows from equation 6 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

bio-mass 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

typi-cally lower at higher sludge ages (Horan, 1990)

This may be due to maintenance e€ects,

endogen-ous respiration during cell starvation and through

further metabolism of biomass releasing more

or-ganic carbon as carbon dioxide

DRAWBACKS TO REDUCED BIOMASS PRODUCTION

Bene®ts can be realised from strategies which reduce the production of biomass These include; economic savings from the reduced costs of treat-ment and disposal of excess biomass, improved op-erational 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 con-sidered

Settling properties in activated sludge processes Conventional mixed aeration processes such as activated sludge processes require that ¯oc agglom-erates 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 ®lamen-tous classes of microorganisms (Jenkins et al., 1993) Exocellular polymer production and cation concentration have also been correlated with settle-ability (Urbain et al., 1993, and Higgins and Novak, 1997, respectively) It is probable that employing any strategy which reduces biomass pro-duction may also a€ect the growth rates of individ-ual 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 chem-istry and so causing poor ¯occulation or encoura-ging 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 respir-ation 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 ments illustrates how the various oxygen require-ments sum to create the total oxygen demand,

Fig 5 Process ¯owsheet for the oxic settling anaerobic

system employed by Chudoba et al (1992) to reduce

bio-mass production.