Wat. Res. Vol. 33, No. 5, pp.

1119±1132, 1999
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(98)00325-X 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 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 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. 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 ÿrS rate of substrate uptake (g Lÿ1 hÿ1)

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 gÿ1 hÿ1)
*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
ÿrSG rate of substrate uptake for incorporation in
new biomass (g Lÿ1 hÿ1)
rX rate of biomass production (g Lÿ1 hÿ1)
ÿrXd rate of biomass loss due to cell death and
lysis (g Lÿ1 hÿ1)
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)
YATP biomass yielded per gram of ATP consumed
(g biomass g ATPÿ1)
YG true biomass yield (g biomass g substrateÿ1)
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
Chemoorganotrophs (heterotrophs) organic carbon
Chemolithotrophs (autotrophs) inorganic carbon
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-
Table 2. Allocation of substrate by a cell in each stage of its life cycle
Cell state
for maintenance
Viable, growing and respiring
Non-viable, respiring
Dead
Energy source Terminal electron acceptor
organic carbon aerobic: oxygen
anaerobic: organic compounds
anoxic: nitrate, sulphate
inorganic compounds, e.g. NH3, H2S and Fe2+ oxygen
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
Substrate utilization
in energy generation in assimilation
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 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
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 Lÿ1) was maintained on a continuously-fed,
synthetic waste (0.81 g COD Lÿ1 dÿ1). In addition
to gravitational settling, biomass retention was
enhanced by supplementing the biomass with ferro-
magnetic powder (11 g Lÿ1 of average particle diam-
eter 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 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
1122 Euan W. Low and Howard A. Chase
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).
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 Lÿ1 dÿ1, 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 O3 mg 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 dÿ1 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 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 relationships
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
 Reducing production of excess biomass
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 FADH2 which 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 meeting 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 FADH2 is 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-
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 (Pi) is released.
ADP is rephosphorylated to ATP during energy yielding
reactions of catabolism (adapted from Atkinson and
Mavituna, 1991).
1123
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
1124 Euan W. Low and Howard A. Chase
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
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 hÿ1; 358C; pH 6.8) (Tempest and Neijssel, 1992)
Limitation Speci®c consumption rate (mmol hÿ1 g dry weight cellsÿ1)
glucose oxygen
Glucose 2.1 4.2
Magnesium 7.4 11.2
Potassium 10.3 17.4
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
Index of carbon recovery in biomass
carbon dioxide
5.5 1.00
11.1 0.41
16.9 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 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
1125
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 YATP can 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 YATP are 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
1126 Euan W. Low and Howard A. Chase
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 qm 1 fed, perfectly mixed biological reactor, the mass bal-
ˆ ‡ …1†
YS m YG
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,
mX
D…S0 ÿ S † ˆ ‡ qm X …2†
YG
and it was stated that in the case of total biomass
recycle, with zero growth rate, the maintenance
coecient can be evaluated from
D…S0 ÿ S †
qm ˆ …3†
X
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 qm V 1
ˆ ‡ …4†
YS QW YG
However, the maintenance coecient obtained
from this method with partial biomass recycle
(qm=0.035 g gÿ1 hÿ1) was signi®cantly di€erent
from those obtained with complete biomass recycle
(qm=0.042 g gÿ1 hÿ1) and that obtained in a chemo-
stat with no recycle (qm=0.028 g gÿ1 hÿ1). 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
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;
ÿ1
ÿrS ˆ rX ÿ qm X …5†
YG
 Reducing production of excess biomass
Fig. 4. Concentrations of oxygen and organic substrates
across a cell agglomerate resulting in zoni®cation of meta-
bolic activity.
and thus the biomass production per unit volume
may be represented by
rX ˆ YG …rS ÿ qm X † …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
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 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 Lÿ1 reduced biomass production by 44%.
Table 4. Indication of yields for various substrates under aerobic and anaerobic
operation (adapted from Tchobanoglous and Burton, 1991)
Substrate Process
Domestic sludge anaerobica
Protein anaerobica
Fatty acids anaerobica
Carbohydrate anaerobica
Domestic wastewater aerobicb
a
Values are for anaerobic processes operating at 208C. bValues are for a conven-
tional activated sludge process operating at 208C.
1127
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
Yield (mg volatile
suspended solids/mg
BOD5)
range typical
0.04±0.1 0.06
0.05±0.09 0.024
0.04±0.07 0.05
0.02±0.04 0.075
0.4±0.8 0.6
1128 Euan W. Low and Howard A. Chase
Fig. 5. Process ¯owsheet for the oxic settling anaerobic
system employed by Chudoba et al. (1992) to reduce bio-
mass 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 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 require-
ments illustrates how the various oxygen require-
ments sum to create the total oxygen demand,
 Reducing production of excess biomass
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 ÿ products ‡ Oxygen required for nitrification
Similarly the total oxygen supply can be deter-
mined 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 path-
ways forming organic by-products. Nitrifying pro-
cesses 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 cata-
bolism of contaminants liberating bound molecular
oxygen. However, additional oxygenation is typi-
cally required to supplement these sources to meet
the total oxygen demand.
Low and Chase (1998c) assessed the e€ects of redu-
cing 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 ÿÿ4wCO
ÿ 2 ‡ xH2 O
17 44 18
‡ yC5 H7 NO2 ‡ z ÿ CH2 O
113 30 …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 sol-
ving simultaneous equations based on the stoichi-
ometry as,
1129
b ˆ a…6 ÿ 7:96Yobs † …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 stoichi-
ometry, 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 oxy-
gen demand to permit respiration of organic car-
bon, 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 am-
monium, a simpli®ed stoichiometry may be pre-
sented as,
aC5 H7 NO2…aq†
Mwt : 113
‡ bO2 ÿÿ4wC
ÿ 5 H7 NO2…s† ‡ xCO2
32 113 44
‡ yH2 O ‡ z ÿ NH3
18 17 …9†
This reaction shows that cryptic growth is associ-
ated 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 ÿ 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 oxy-
gen requirement will be incurred.
Nutrient removal
The stoichiometry of equation 7 shows that a re-
duction 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 pro-
duction may result in a lower amount of other ma-
terials (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 treat-
ment may be required. Also, the inclusion a nitri®-
1130 Euan W. Low and Howard A. Chase
cation stage will further increase oxygen require-
ments.
DISCUSSION
The magnitude of additional capital and operat-
ing costs o€set by reduced disposal costs will deter-
mine 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 techni-
cal 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 ap-
plicable methods would involve:
1. in¯uencing the choice of terminal electron accep-
tor
2. manipulation of mixing regimes within the reac-
tor
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 pro-
duction achieved by applying various strategies suit-
able for aerobic wastewater treatment. All these
strategies are similar in encouraging further metab-
olism 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
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
adversely alter the biomass settling characteristics.
Complete biomass retention will result in the ac-
cumulation of inert solids within wastewater pro-
cesses process and reduce the e€ective reactor
volume. There exists a minimum rate at which bio-
mass 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 reac-
tions. 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 metab-
olism 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¯u-
ence biochemical processes within cells. This pro-
vides 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 con-
ditions not only decreases the yield of biomass, but
also greatly elevates the speci®c rate of carbon con-
sumption. 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, ad-
ditional treatment of the wastewaters would be
necessary to reduce the concentration of organic
carbon to acceptable levels. Uncoupling oxidative
Reduction in production of excess biomass (%) Reference
100 Sakai et al. (1992)
60 Canales et al. (1994)
100 Yasui and Shibata (1994)
50 Ganczarczyk et al. (1980)
52 Mason and Hamer (1987)
12±43 Ratsak et al. (1994)
60±80 Lee and Welander (1996)
59±61 Tempest and Neijssel (1992)
45 Low and Chase (1998a)
12 Low and Chase (1998b)
44 Bouillot et al. (1990)
44 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 pro-
ton leakage through the thermolabile cytoplasm
membranes, inducing futile ion cycles or by ad-
dition of protonphores. Raising temperature of
wastewaters is typically uneconomical and an oper-
ating 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 waste-
water 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 catabo-
lism results in a low biomass yield whilst alleviating
the need for costly aeration. While domestic waste-
waters are too dilute to receive anaerobic treatment,
industrial e‚uents which contain high organic sub-
strate concentrations are suitable for such treat-
ment. 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 pro-
cesses can be engineered to maximise the e€ects of
further metabolism by cell lysis, cryptic growth and
bacteriovore predation. The eciency of metab-
olism 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 com-
posite 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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