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: ].
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 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

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 2. 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 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 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 2 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 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 bioreactor demonstrated that shorter sludge ages
increased the biomass viability. However when the


1122

Euan W. Low and Howard A. Chase

Fig. 1. 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 3 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 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 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. 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).

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 do
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:
1.
2.
3.
4.
5.

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 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 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 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 3
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 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 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;
1
qm
1
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.,
1
qm V
1
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 6 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. 4. 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 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 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 4. 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. 5. 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
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 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 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 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 8. 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 8 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 7 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 9
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:
1. in¯uencing the choice of terminal electron acceptor
2. manipulation of mixing regimes within the reactor
3. regulation of process ¯owrates to a€ect relative
concentrations of substrate and biomass

4. appropriate selection of reactor design
5. addition of chemicals such as protonphores or
salts
Table 5 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 5. 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.

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