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Development and application of a denitrification test

Water Science and Technology Vol 41 No 12 pp 113–120 © IWA Publishing 2000

based on gas production
B.R. Buys,* A. Mosquera-Corral,** M. Sánchez** and R. Méndez**
*Wageningen Agricultural University, Department of Environmental Technology, P.O. Box 8129, 6700 EV,
Wageningen, The Netherlands
**Institute of Technology, Department of Chemical Engineering, University of Santiago de Compostela,
Campus Sur s/n. 15706 Santiago de Compostela, Spain

Abstract In order to assess the toxicity of compounds in wastewater and activities in reactors a precise,
reproducible, fast and simple batch test is used. This research was focused on the development of such a
test to determine denitrifying activity. The test is based on measuring the rate of gas production during
denitrification, by tracking both the pressure and the gas composition in the headspace of a sealed bottle.
The optimum pH for batch activity was found to equal the pH in the lab-scale reactor (9.5) from which the
sludge originated. The minimum COD/N ratio required for linear gas production curves with acetate/nitrate
was 3.5 g COD/g N. A significant increase in specific activity at low solids concentrations (<2 g VSS/l) was
observed. No clear relation between activity, DNA and RNA content of the sludge after the test and initial
substrate to biomass (S0/X0) ratio could be established, indicating that other factors play a role in
determining the activity, possibly adsorption and substrate mass transfer. Standardization of batch tests
should therefore include the solids concentration.
Keywords Activity test; alkaline denitrification; biomass concentration; DNA; RNA; S0/X0 ratio

Introduction
Batch activity tests are frequently used to assess biomass activities in treatment systems,
the influence of toxic compounds and environmental parameters and to measure kinetic
model parameters. Anaerobic batch tests are often performed by adding a large amount of
substrate to the sludge and monitoring the resulting gas production with a Mariotte flask
system (Soto et al., 1992). Denitrification activities may be measured in a similar way, even
though lag phases of several days may occur, often coinciding with N2O accumulation
(Sánchez, 1999; Sánchez et al., 2000). Therefore an alternative system that yields results on
a time scale of hours instead of days is proposed here. The Mariotte flask system is not sen-
sitive enough to measure small amounts of gas produced accurately and therefore demands
the addition of large amounts of substrate. By using a pressure sensor, small amounts of gas
may be measured accurately in pressurized vials, so that experiments may also be per-
formed at relatively low substrate concentrations. An additional advantage is that the sys-
tem is suitable for automation, and in this manner on-line results of gas production versus
time are easily obtained (Beaubien et al., 1995) and of gas composition if on-line equipment
is available for this purpose. A clear disadvantage is the lack of information on possible
nitrite accumulation (Glass and Silverstein, 1998; Almeida et al., 1995). This problem can
not be circumvented by measuring the nitrite reduction rate separately, because nitrite
accumulation may be caused by the competition of nitrate and nitrite for electrons if the
oxidative cell metabolism is saturated and thus may depend on the presence of nitrate.
Batch tests may induce changes in the biomass depending on the inital substrate to bio-
mass ratio (S0/X0) applied, X0 being the active biomass in the sludge. When this ratio is suf-
ficiently low (S0/X0 <2–4 gCOD/gVSS) no or negligible cell multiplication will take place
(Liu, 2000; Chudoba et al., 1992). Moreno et al. (1999) reported on the effect of the S0/X0
ratio (between 0.054–10.8 gCOD/gVSS) on the results of anaerobic batch tests. The highest 113
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specific activities (i.e. volumetric activity divided by biomass concentration) were connect-
ed to the highest S0/X0 ratios applied. The yield observed was found to decrease as the
S0/X0 ratio increased, in accordance with the substrate spilling theory of Chudoba et al.
(1992): at (very) high S0/X0 ratios substrate may be consumed without energy being gener-
ated. However, the yield was also found to depend on the biomass concentration: a lower
yield was found at low biomass concentration even when the S0/X0 ratio was kept constant.
B.R. Buys et al.

The distinction between “low” and “high” S0/X0 is inherently vague when it is expressed as
gCOD/gVSS because not all of the VSS consist of active biomass. The fraction of active
biomass of the solids depends particularly on the sludge loading rate in the reactor
(Jorgensen et al., 1992; Janssen, 1979): the higher the loading rate, the higher the active bio-
mass fraction. A low COD/VSS ratio may therefore be a high S0/X0 ratio if the active
biomass fraction of VSS is small.
Biomass may also be estimated by quantifying the DNA content of the sludge, because
DNA content is maintained at relatively constant proportions within microorganisms
(Ingraham et al., 1983; Liebeskind and Dohmann, 1994). The RNA of microorganisms is
mainly involved in protein synthesis and therefore the RNA content varies considerably
depending on the growth conditions (Ingraham et al., 1983). The ribose of RNA may serve
as carbon source upon carbon exhaustion under aerobic conditions (Mason and Egli, 1993).
Changes in RNA content during a batch experiment may be the result of a change in cell
numbers or a change in activity without an increase in cell numbers, and this makes results
hard to interpret. Both RNA and DNA are quickly degraded upon cell lysis, which makes
them specific for non-lysed cells. A distinction between actively respiring and non-active
cells can not however be made. In the present work the results on the changes in DNA and
RNA content that occur during batch experiments, related to the maximum specific activity
are presented.
In the experiments carried out in this work with denitrifying biomass, a negative correla-
tion between biomass concentration and specific activity in batch tests was also observed.
In literature there is a large range of biomass concentrations at which batch tests are per-
formed. In some cases low biomass concentrations are chosen (10–100 mg VSS/l), on the
one hand because the small dimensions of lab-scale reactors may limit the amount of solids
that is available for batch assays, on the other hand because biomass may need to be diluted
in order to decrease the activity into the range of the activity measuring device. The specific
activity is supposed to be independent of the biomass concentration.
The purpose of the present investigation is to develop a simple batch test for the determi-
nation of denitrifying activities and to apply this technique to the investigation of the effects
of several test parameters (pH, COD/N ratio, S0/X0 ratio and solids concentration).

Materials and method

Inoculum
Sludge samples for batch assays were taken from a lab-scale reactor that was fed with a syn-
thetic influent consisting of 1000 mg NO3––N/l, 1300 mg acetate-C/l (3.5 gCOD/gN) and
other compounds as shown in Table 1. The selected COD/N ratio to carry out the tests is
higher than the value determined by the denitrification catabolic reaction for 100% of
nitrate reduction to nitrogen gas without biomass growth which is 2.85 gCOD/gN accord-
ing to the stoichiometric expression (Eq. (1)). In reality more carbon is needed to sustain
biomass growth.
5 CH3COOH + 8 NO3– + 8 H+ → 10 CO2 + 14 H2O + 4 N2 (1)
The reactor was initially seeded with activated sludge from an aerobic treatment plant for
114 domestic wastewater. The upflow reactor was operated at a volumetric loading rate of
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Table 1 Composition of the influent for the denitrification reactor

Compound Concentration (mg/l) Compound Concentration (mg/l)

NH3–N 50 P 12
BO33– 0.17 Mg2+ 10.1
Ca2+ 60–200 Mn2+ 0.28
Co2+ MoO42–

B.R. Buys et al.

0.30 1.38
Cu2+ 0.93 Ni2+ 0.15
Fe2+ 0.92 Zn2+ 0.32

0.6–3.0 kg N/m3 · d and functioned at close to 100% nitrate and acetate removal efficiency.
pH in the reactor was not controlled and was 9.5±0.2.

Batch experiments procedure

The batch tests were prepared (first feed) as follows: the sludge was washed with 50 mM
phosphate (KH2PO4) solution 2–3 times to remove possible presence of substrates; a mix-
ture of biomass and phosphate buffer was added to 60 or 110 ml vials; a concentrated solu-
tion containing ammonium and trace elements (Table 1) was added; pH was adjusted to 9.5;
N2 gas was flushed into the vials for about 10–15 minutes. The vials were then put in a
shaker at 37ºC for 15 minutes in order to reach gas/liquid equilibrium. Pressure was
equalised to the atmospheric one. The headspace volume (“ 10 ml) was calculated using the
ideal Gas Law from the pressure difference that resulted from the injection of 1.00±0.01 ml
of phosphate solution (in triplicate), keeping the vials in a waterbath controlled at 37ºC. The
tests were started by injecting 1 ml of either concentrated NaAc/NaNO3 or NaAc/NaNO2,
in order to reach the desired initial concentrations (100–1000 mg N/l) and COD/N ratios in
the vials. The vials were then put in a shaker (150–225 rpm) at 37ºC. Batch experiments
were performed in duplicate. The production of N2 gas was tracked by measuring the pres-
sure in the headspace and the gas composition with a frequency depending on the activity of
every test. N2 production was calculated from the headspace volume, gas composition and
pressure data by applying the ideal gas law. Second feed was injected without changing the
liquid phase.

Analytical methods
The pressure in the headspace was measured using a differential pressure transducer
0–5 Psi, linearity 0.5% of full scale, Centrepoint Electronics. Gas composition in CH4, N2,
N2O and CO2 was determined by gas chromatography (Hewlett-Packard 5890II) provided
with a thermal conductivity detector. Nitrates and nitrites were measured at the end of the
experiment by capillary electrophoresis equipped with a UV detection system (Water
Capillary Ion Analyzer). Dissolved organic and inorganic carbon were determined in a
Shimadzu analyzer (TOC-5000 model), acetate with a gas chromatograph (Hewlett-
Packard, 5890A). Total and volatile suspended solids (TSS and VSS) were measured
according to Standard Methods (APHA, 1985). DNA was extracted with perchloric acid
and quantified according to Liebeskind and Dohmann (1994), using herring sperm DNA
(Sigma) for the calibration curve. RNA was quantified by the orcinol method after alkaline
extraction and using RNA from baker’s yeast (Sigma) for the calibration curve (Herbert et
al., 1971). Duplicate extractions and duplicate colour formation yielded the same results.

Results and discussion

The differences in gas production curves between initial concentrations of 100 and 1000 mg
NO3––N/l (COD/N=3.5 gCOD/gN) are shown in Figure 1. The activity was calculated from 115
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B.R. Buys et al.

Figure 1 Influence of initial substrate concentration on shape of gas production curves. Left: 100 mg N/l;
right: 1000 mg N/l. COD/N=3.5 gCOD/gN and [VSS]” 1 g/l. The dashed lines represent the amount of
nitrogen added as nitrate at start

Figure 2 Influence of COD/N ratio between 0-6.7 gCOD/gN on gas production.The horizontal dashed line
indicates the amount of N added as nitrate at start

the slope of the gas production curve. It has been observed that upon dosing 100 mg N/l, a
straight line was obtained. Dosing 1000 mg N/l resulted in a significant increase in activity
during the test, shown in Figure 1 from 0.48–1.21 gN/gVSS · d. This was probably due to
biomass growth during the test. When the COD/N ratio was decreased to values lower than
3.5 gCOD/gN, discontinuities were observed in the gas production rate (Figure 2). This was
likely to be caused by depletion of the external carbon source, so that the biomass switched
to using internal carbon, which slowed down the denitrification rate. For COD/N ratios
higher than 3.5, no differences in gas production rate were detected.
A minimum of 2.85 gCOD/gN is required for denitrification of all the nitrate added,
according to equation [1]. It being assumed that all excess carbon can be used for
anabolism, the maximum possible yield in batch assays at COD/N=3.5, can be estimated at
(3.5–2.85)/3.5 ∗ 100=18% of the acetate-carbon added. In the reactors from which the
sludge originated (also operated at COD/N=3.5 gCOD/gN), the carbon balance showed an
accumulation of 10–20% of the influent acetate-carbon in the reactor. The microbial yield
was only part of this 10–20%, due to precipitation of carbonates (mainly CaCO3) in the
sludge. Since the minimum COD/N ratio to obtain linear curves is 3.5, the yield in batch
assays seemed to be higher than the yield in the reactor. Another explanation is that the bio-
mass initially could take up more acetate than could be used for denitrification and that this
acetate was stored intracellularly. These storage products were not used until the extracellu-
lar acetate pool was exhausted. The rate at which carbon was released from the storage
product, apparently is slower than the transport of dissolved acetate into the cell, which
116 caused the decrease in denitrification rate at COD/N= 2.9 gCOD/gN.
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Table 2 Solids concentration, maximum specific activity and % of DNA at the end of the test. The
superscripts indicate 1st and 2nd feed

[VSS]_start (g/l) Specif. activity (g N/g VSSo · d) DNA (%) (% of VSS_end)

0.44 1.7201–1.9392 0.88

0.43 1.860 1.71
0.85 0.982 1.25

B.R. Buys et al.

0.87 1.7021–1.1182 1.34
2.32 1.1061–1.0102 1.60
2.97 0.936 2.04
2.97 1.0151–1.0922 2.44

Figure 3 Influence of pH on denitrification with nitrate and nitrite

The optimum pH was found to be 9.5, which was equal to the pH in the reactor from which
the sludge was collected: at pH=7 accumulation of N2O occurred (at maximum ~5% of the
nitrate-N added) and at pH=11 denitrification activity was very low, which is shown in
Figure 3. In all cases all the nitrate added was fully converted to nitrogen gas, taking more
than 40 hours at pH=11 (final pH=9.8) and more than 10 hours at pH=7 (final pH=7.7).
The decrease in gas production rate with nitrate at pH=9.5 after about 1.5 hours (Figure
3), was caused by a COD/N ratio lower than 3.5 gCOD/gN. At pH=9.5 there was hardly any
difference between the maximum nitrate and nitrite reduction rate. In several other experi-
ments the nitrite reduction rate was found to be significantly higher than the nitrate reduc-
tion rate. A possible explanation for this observation is that electron transfer is rate limiting:
for nitrite reduction 40% fewer electrons need to be transferred.
In many experiments a clear effect of solids concentrations on specific activity was
observed, often masking effects of other variables that were being studied. In Figure 4 and
Table 2 the results of an experiment in which the solids concentration was varied between
0.5–3.5 g VSS/l and the initial substrate concentration fixed at 100 mg N/l and 346 mg
COD/l are shown.
In some cases a second feed of the same concentration was added. The activity upon the
second feed was in some cases greater (about 10%) and in other cases substantially less than
the activity obtained during the first feed. A higher activity value may be attributed to
growth during the assay. At the end of this experiment, DNA was extracted from the bio-
mass (Table 2). The DNA content of the biomass of the original sludge was in this case not
determined. The low DNA content of 0.88% in the case of two feeds (Table 2) was not like-
ly to be caused by an experimental error, since the method in general yielded excellent 117
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Table 3 Solids concentrations, initial substrate concentrations, specific activity and %DNA/%RNA at the
end of the test. %RNA at start was 5.1% of VSS. The superscripts indicate 1st and 2nd feed

[VSS]_start [NO3––N]-start Specif. activity 1st feed-2nd feed RNA % DNA %

(g/l) (mg N/l) (gN/gVSS_start · d) (% of VSS_end) (% of VSS_end)

0.74 1001+1002 0.441–1.831–0.882–2.952 4.68

0.77 100 0.41–1.68 3.91
B.R. Buys et al.

3.15 100 0.71 4.64 1.69

3.24 100 0.68 5.00 1.87
0.52 1000 0.22–2.34 10.0 2.52
0.53 750 0.19–0.09–1.73 8.14 2.20
3.13 1000 0.27–0.04–0.07–0.19 5.66 1.52
3.14 1000 0.36–1.46–2.68 2.76
3.20 1001+5002 0.041–0.852–2.472 6.37 2.76
3.15 100 0.73
3.21 100 0.69
3.35 100 0.52–0.10–0.05

Figure 4 Influence of S0/X0 ratio on denitrification, at constant initial substrate concentration

duplicates. In order of magnitude the DNA contents are comparable to those obtained by
Ingraham et al. (1983) who reported 2.8% of VSS for an average E.Coli B/r cell. In any case
it is clear that the DNA content of the sludge was not related to the specific activity and that
the DNA content can change during the test. This was confirmed in another experiment
where both initial substrate concentration and solids concentration were varied (Figure 5).
In this experiment gas production curves were not always linear: changes in the specific
activity during the test are given in Table 3. In the cases when two feeds were added, the
total amount of substrate was used for the S0/X0 calculation.
At high S0/X0 ratios, a high specific activity was expected to be accompanied by a high
RNA content: biomass is likely to adapt to the conditions of high substrate concentration by
increasing the protein synthesis system, in which RNA plays an essential role. Indeed from
Table 3 it can be seen that the highest S0/X0 ratio coincided with the highest RNA content at
the end of the assay and also with a high specific activity, however this was not the case: the
highest activity measured (1st row Table 3) was measured at relatively low S0/X0 ratio and
almost unchanged RNA content compared to the sludge at start. In one case a high activity
was accompanied by a decrease in RNA content (8th row in Table 3). The DNA content of
118 the sludge tended to be high when the initial substrate concentration was high (rows 5, 6 and
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B.R. Buys et al.

Figure 5 Influence of S0/X0 on maximum specific activity expressed as gN/gVSS · d or gDNA/gVSS*100,
RNA and DNA content at the end of the test. COD/N=4.3 gCOD/gN, pH=9.5, S0 and X0 variable. %RNA at
start was 5.1% of VSS. See also Table 3

9 in Table 3). In one case a high initial substrate concentration and a low activity coincided
with a low DNA content and a small increase in RNA content (7th row in Table 3). The
decrease in RNA content during the test at low S0/X0 (between 0–2) was repeated in anoth-
er experiment with COD/N=3.5 gCOD/gN where 30–40% decrease of RNA content was
observed.
When only nitrate was added without a carbon source, the RNA might be used as a car-
bon source, thus explaining the decrease. In the other cases this is unlikely, since carbon
was added in excess of the stoichiometric needed (2.85 gCOD/gN, Eq. (1)). Only at the
highest S0/X0 ratio applied (S0/X0=2), was the RNA content at the end of the experiment
equal to the content at the start.
Whilst working with enriched cultures of nitrifying organisms Wong-Chong and Loehr
(1975) observed a similar effect: the specific nitrifying activity increased with decreasing
biomass concentration. They worked with initial nitrogen concentrations of 100–1200 mg
N/l and biomass concentrations of 0.2–4.0 g VSS/l. Linear nitrogen depletion curves were
observed and no increase in biomass concentration (as VSS) could be measured after the
test, presumably indicating negligible growth.
The availability of trace elements, which could favour the activity at low biomass con-
centrations, does not seem to play an important role, because no differences in maximum
activity were found in assays with and without trace elements and/or EDTA. A possible
explanation of the effect of biomass concentration on specific activity in batch assays is the
fast adsorption of acetate to biomass, instantaneously after dosing. At low biomass concen-
trations, a smaller part of the acetate will be adsorbed, resulting in a higher dissolved acetate
concentration. Assuming that (a) only dissolved acetate can enter cells, (b) acetate transfer
is diffusion determined, (c) acetate transport is rate limiting, at lower biomass concentra-
tion the biomass is exposed to a higher dissolved acetate concentration during the test. This
then results in a higher mass transfer rate and therefore in a greater specific activity.

Conclusions
Measuring pressure and gas composition is a sensitive method for studying denitrification
in batch assays. Upon dosing sufficiently small amounts of substrate (100 mg N/l) at
COD/N≥3.5 gCOD/gN, linear gas production curves were obtained, whereas higher initial
concentrations result in either lag-phases or significant biomass growth during the assay. 119
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The optimum pH was found to be 9.5, equal to the pH in the reactor, whilst at pH=7 accu-
mulation of N2O occurred. The inverse relationship between specific activity and biomass
concentration that was found, can not be explained by the initial substrate to biomass ratio
S0/X0: both high and low specific activities were measured at the same S0/X0 ratio. The
RNA content of sludge tended to decrease during a batch experiment at low S0/X0. At high
S0/X0 ratio, both RNA and DNA content increased. However, no clear relationship between
B.R. Buys et al.

RNA and DNA content of the sludge and maximum specific activity could be established.
The inverse relationship between biomass concentration and maximum specific activity
may be caused by adsorption of the acetate and mass transfer limitation.

Acknowledgements
This research was financed by the European Commission through the BIOTOBIO TMR
project (Contract FMRX-CT97-0114), which enhances the mobility of researchers in the
European Union. The authors thank Margarita Vilas Cruz, Mar Orge Alvarez, Luisa
Grandal Alves and Rosa Arcos for their valuable contribution in the experimental work.

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