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Stoichiometry and Kinetics 217

substances, such as oxygen or light energy, or the formation of products,
such as methane or carbon dioxide.
The expression of yield can be combined with the constant growth rate
equation (dX/dt = X) to give the rate of substrate utilisation:
ds
dt
=

Y
X
When the amount of substrate utilisation for cell maintenance is expected
to be signicant, i.e. at high temperatures, long residence times and high
cell concentrations, the above equation should be modied:
ds
dt
=

Y
X k
m
X
where k
m
is the specic maintenance coecient (Beneeld and Randall
1980).
Kinetic constants should be determined experimentally using bench or
pilot scale methods (Giona et al. 1979). However, because of the biologi-
cal and chemical nature of such experiments, the wastewater engineer and
designer often prefer to rely on data presented in the literature. The varia-
tion in the kinetic constants quoted in the literature, due to the variation
in wastewater quality and environmental conditions, is large. For example,
Y = 0.350.45 mg VSS mg COD
1
, k
d
= 0.050.10 d
1
, k
s
= 25100 mg
l
1
, and
m
= 3.510.0 d (Mandt and Bell 1982). Thus, clearly, kinetic ana-
lysis can only be performed with any degree of condence on experimentally
collected data.
3.1.3. The BOD test
The biochemical oxygen demand (BOD
5
) test was introduced in Sec. 1.4.2
and is a quantitative measure of the oxygen uptake by aerobic micro-
organisms, mainly bacteria, as they oxidise organic matter. The test
initiates the heterotrophic microbial activity that occurs both during
the self-purication process in rivers and in the breakdown of organic
wastewaters in biological treatment processes. The test provides a useful
laboratory model describing what happens during the biodegradation of
organic matter.
Microbial growth
Unlike the wastewater treatment plant or river, the BOD bottle is a
closed microbial ecosystem which is devoid of external inuences such as
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218 The Role of Organisms
light, turbulence, nutrient imbalance, and factors that aect the oxy-
gen balance. The test is done under controlled conditions, in darkness
at 20

C and by plotting the oxygen uptake against time the character-

istic BOD curve is obtained (Fig. 1.18). Similar to the microbial growth
curve (Fig. 3.5), it can be divided into the various growth phases: lag, log
(acceleration), stationary, and endogenous respiration phases. There is a
fth phase in the BOD curve, that of nitrication (Fig. 1.19). Of course,
the exact nature and extent of each growth phase is dependent on the com-
position of the sample being tested, thus in certain cases one or more phases
may not be in evidence.
The lag phase is dependent upon the nature of the micro-organisms
present in the sample. Its duration is a function of the number of
bacteria present and whether or not they are acclimatised to the substrate.
Normally, only small numbers are present and these have to acclimatise to
their new environment in the BOD bottle, resulting in a delay before log
growth and maximum oxidation occurs. Initially, the soluble substrate is
rapidly assimilated and while cell numbers remain constant, cell size and
mass increase. There is relatively little oxygen utilised during this phase as
synthesis reactions require much less oxygen per unit of substrate utilised
than does oxidation to carbon dioxide. Therefore, in order to avoid under-
estimating the BOD, the lag phase should be as short as possible, which is
achieved by using laboratory grown micro-organisms to seed samples that
are acclimatised both to the test temperature and to the substrate. Organ-
isms transferred from a log growth phase will have a shorter lag phase than
if transferred from any other growth stage (Young and Clark 1965). The
eect on the BOD of prolonged lag phases because of insucient bacteria
when a sample has not been seeded, using unacclimatised seed or in the
presence of inhibitory or toxic substances, is shown in Fig. 3.14 (HMSO
1983a).
During the log-growth phase the organic matter present in the sam-
ple is utilised by the heterotrophs for energy and growth. Once the mass
of micro-organisms is large enough and is acclimatised to the substrate,
the synthesis reaction rate declines and the oxidation reaction rate in-
creases. During the log growth phase the available food supply (substrate)
is in excess, resulting in a high food to micro-organism ratio (f/m), so
that microbial growth is unrestricted. However, the micro-organisms are
at their most vulnerable during this period because their generation time
is reduced to a minimum, cell size is decreasing, and their resistance
to toxic and inhibitory substances is low (Young and Clark 1965). This
phase generally lasts between 2436 hours during which time about 50% of
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Stoichiometry and Kinetics 219
Fig. 3.14. Lag growth phases in the BOD test (adapted HMSO 1983a).
the organic matter will be oxidised. Eventually, with the micro-organisms
having reached maximum concentration, the substrate becomes limiting (a
low f/m ratio) and more dicult to assimilate. With a consequent decrease
in growth rate, there is a decrease in the rate of oxygen uptake. The rate
continues to decrease and levels out at near zero uptake rate over a period
of up to 30 hours, resulting in a plateau called the stationary growth phase.
In the stationary phase all the available soluble substrate has been
utilised. Continued oxygen uptake rate after this phase is due to endogenous
respiration and the presence of predators, such as protozoans feeding on
the bacterial biomass, produced during the log-growth phase (Busch 1958;
Schroeder 1968). Bhatla and Gaudy (1965) found that the occurrence and
duration of the plateau is usually linked to the population of predators,
especially the protozoans, present at the end of the log-growth phase. For
example, if protozoan growth lagged signicantly behind bacterial growth,
then a long plateau could be expected.
With a low f/m ratio, the declining bacterial population is forced
into endogenous respiration. This normally occurs 35 days after incu-
bation commences. Endogenous respiration is the oxidation of the por-
tion of organic matter that was converted into cellular tissue during the
log-growth phase. The rst compounds to be oxidised are the expend-
able storage products such as glycogen and PHB (poly--hydroxybutyrate),
leaving the more essential cellular constituents such as amino acids, pro-
teins, and nucleic acids to degrade only when these have been utilised
(Painter 1983). Oxidation occurs at a much slower rate than during the
log-growth phase. By the fth or sixth day, the reduced bacterial popula-
tion begins to limit the growth of protozoans, which are also forced into
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220 The Role of Organisms
endogenous respiration. All the micro-organisms continue their endogenous
respiration for about a further 15 days after which time biochemical oxida-
tion should be over 90% complete.
The nal stage in the BOD curve is only observed in samples containing
a high concentration of ammonia and a signicant population of nitrify-
ing bacteria (Sec. 3.5). With the exception of partially nitried euents,
nitrication proceeds very slowly during the initial stages of the BOD test,
with no appreciable demand for oxygen. Thus, in practice, the eect of
nitrication on the 5-day test (BOD
5
) is usually negligible. This is due to
the very slow reproduction rate of the nitrifying bacteria, which is between
26 days for most genera and results in a noticeable nitrication oxygen
demand only after 10 days incubation. Nitrication can exert an apprecia-
ble oxygen demand in the BOD
5
when partially nitried sewage euents
are tested. Such samples contain a high concentration of ammonia as well
as an established population of nitrifying bacteria.
If the oxidation of organic matter is allowed to proceed in the BOD
bottle, complete oxidation is achieved between 20100 days depending on
substrate. The total oxidation of all organic carbon, nitrogen, and hydrogen
is referred to as the ultimate BOD (L). In practice, L is approximated
mathematically from the rate of oxygen uptake during the initial incubation
period, rather than actually incubating samples for such a lengthy period.
(Fig. 1.22).
BOD kinetics
The kinetics of the BOD test have been fully explained in Sec. 1.4.2.1 and
are based on rst-order reaction principles. However, the concept of the
oxygen uptake rate in the BOD test conforming to a rst-order reaction has
been widely disputed (Rivera et al. 1965; Young and Clark 1965; Landine
1971; Stones 1981, 1982). Stones (1982) applied both rst- and second-order
equations to the results of an experiment in which a 0.01 dilution of settled
domestic sewage was incubated at 20

C and the residual dissolved oxy-

gen concentrations determined at daily intervals over 15 days, from which
k
l
values were determined (Table 3.4). His results clearly show that the
values of k
1.1
and k
1.2
, computed using rst-order reaction kinetics, de-
crease rapidly as oxidation proceeds. This is in contrast to the k
1.3
values,
computed using second-order reaction kinetics, which are virtually con-
stant from the second to the tenth day of incubation, with the observed
decline after 10 days attributable to the onset of nitrication. There is little
doubt that the second-order reaction equation gives a better mathematical
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Stoichiometry and Kinetics 221
Table 3.4. Values of the velocity coecients
k
1.1
, k
1.2
, and k
1.3
computed using rst- and
second-order reaction kinetics (Stones 1981,
1982, 1985).
Days k
1.1
k
1.2
k
1.3
1 0.350 0.200 0.0397
2 0.318 0.170 0.0390
3 0.295 0.149 0.0386
4 0.277 0.132 0.0383
5 0.263 0.119 0.0381
6 0.252 0.109 0.0381
7 0.244 0.100 0.0381
8 0.236 0.092 0.0381
9 0.230 0.085 0.0382
10 0.224 0.080 0.0382
12 0.218 0.070 0.0391
15 0.212 0.059 0.0404
k
1.1
, velocity coecient calculated using a
rst-order reaction equation in which it is as-
sumed that the rate of biochemical oxidation
varies with the unsatised BOD.
k
1.2
, velocity coecient calculated using a
rst-order reaction equation in which it is as-
sumed that the rate of biochemical oxidation
varies with the residual DO concentration.
k
1.3
, velocity coecient calculated using a
second-order reaction equation in which it is
assumed that the rate of biochemical oxida-
tion varies with both the unsatised BOD and
the residual DO concentration.
description of the log-growth phase. However, the overall BOD curve is
made up of a minimum of three growth phases each having an entirely
dierent growth rate. Thus, BOD decay in natural environments rep-
resents a complex interaction between a diverse assemblage of bacteria
and a heterogeneous organic substrate (Swamee and Ojha 1991). This is
supported by other studies that show that no single xed value, whether
rst-, second-, or half-order, can appropriately describe all wastes (Young
and Clark 1965; Adrian and Saunders 1992, 1998; Adrian et al. 1999).
Therefore, the use of a rst-order reaction equation is to be preferred as it
more accurately describes the summation of all the individual growth rates
that make up the BOD curve. For this reason the rst-order decay model
is widely used in water quality modelling to describe the deoxygenation of
organic wastes.
January 19, 2004 14:33 World Scientic Biology of Wastewater Treatment (New Edition) bwt
222 The Role of Organisms
Borsuk and Stow (2000) have proposed an alternative to the simple xed
reaction order approach by allowing L to remain a free parameter. Thus,
rather than assuming a rst-order decay process a priori, they acknowl-
edged that the BOD exertion is a mixture of decay processes and allow the
data itself to determine the reaction order. BOD exertion is modelled as a
rst-order decay process in which the oxygen consumption is proportional
to the concentration of BOD remaining (L
t
):
dL
t
dt
= k
1
L
t
with L
t
a free parameter as proposed by Borsuk and Stow, this is rewritten
as:
dL
t
dt
= k
n
L
n
t
This integrates to:
L
t
= {L
1n
0
k
t
n
(1 n)}
1
1 n
where L
0
is the ultimate BOD (mg l
1
), L
t
the BOD remaining after time
t (mg l
1
), k
1
the rst-order reaction rate constant (d
1
), t the time (d
1
),
n a pseudo-order parameter, and k
n
the mixed order reaction rate constant
((mg l
1
)
(1n)
d
1
).
Substituting (L
0
Y ) for L
t
:
Y = L
0
{L
1n
0
k
t
n
(1 n)}
1
1 n
where Y is the BOD exerted at time t (L
0
L
t
) mg l
1
.
The authors used a Bayesian approach to estimate the parameters and
found that the mixed order model described above resulted in a better t
to observed data and produced more realistic predictions of the ultimate
BOD than rst-order expressions.
Further reading
General : Beneeld and Randall 1980; Andrews 1983; Jank and Bridle 1983;
Atkinson and Mavituna 1983; Schugerl 1987; Bulock and Kristiansen
1987.
Stoichometry: Lawrence and McCarty 1970; McCarty 1972, 1975; Chris-
tensen and McCarty 1975; Schugerl 1987.
Kinetics: Giona et al. 1979; Schugerl 1987; Sinclair 1987.