Journal of Water and Environment Technology, Vol.12, No.

4, 2014

Denitrification and Dissimilatory Nitrate Reduction to

Ammonium (DNRA) Activities in Freshwater Sludge and
Biofloc from Nile Tilapia Aquaculture Systems

Pokchat CHUTIVISUT*, Wiboonluk PUNGRASMI*, Sorawit POWTONGSOOK**, ***

*Department of Environmental Engineering, Faculty of Engineering, Chulalongkorn

University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand
**Center of Excellence for Marine Biotechnology, Department of Marine Science, Faculty of
Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330,
Thailand
***National Center of Genetic Engineering and Biotechnology, 113 Thailand Science Park,
Phahonyothin Road, Khlong Luang, Pathum Thani 12120, Thailand

ABSTRACT
Suspended organic sludge from freshwater and biofloc Nile tilapia systems were examined for
the presence of denitrifying and dissimilatory nitrate reduction to ammonium (DNRA) activities
under nitrate and sulfide stimulation. Initial nitrate concentrations at 25 and 100 mg NO3–-N/L
were added to the freshwater sludge and biofloc samples to simulate low and high nitrate levels
that are normally found in aquaculture systems. The results showed that freshwater sludge and
biofloc both had denitrifying activity immediately after nitrate addition. However, ammonium
accumulated in the biofloc reactors but not in the freshwater reactors, indicating the activity of
DNRA in the high C/N biofloc particles. The influence of sulfide on nitrate reduction was also
studied by adding different concentrations of sulfide along with 100 mg NO3–-N/L. The results
showed that elevated sulfide concentrations partially inhibited denitrification in the freshwater
sludge and caused nitrite and ammonium accumulation, in which ammonium formation was
probably responsible by DNRA activity. In sulfide-added biofloc reactors, ammonium
accumulated at the same level as found in the biofloc reactors without sulfide. Therefore, DNRA
bacteria residing in the biofloc aquaculture system were more likely to be heterotrophs that did
not use sulfide as their electron donor.

Keywords: biofloc, denitrification, dissimilatory nitrate reduction to ammonium (DNRA),

freshwater aquaculture sludge, Nile tilapia, sulfide

INTRODUCTION
Aquaculture systems generally produce inorganic nitrogen and organic suspended solid
wastes that are capable of causing adverse effects if directly discharged to the
environment. Recirculating aquaculture system (RAS) has been developed to overcome
this problem, in which, with proper wastewater treatment process, water can be reused
in aquaculture systems and the water discharge can be minimized to almost zero. Most
of the treatment systems in RAS target the removal of ammonium, nitrite, and nitrate, as
well as the separation of particulate organic matters (POMs) derived from fish feed, fish
feces, and suspended solids (Cripps and Bergheim, 2000). However, instead of
discarding the solids as organic wastes, POMs can further be used as the source of
carbon for anoxic denitrification process (van Rijn et al., 1995). Normally, external
carbon has to be added to a denitrifying reactor due to the low dissolved organic carbon
in the aquaculture system. However, with the use of POMs, the growth of denitrifying
bacteria can be promoted in a sustainable way and the organic sludge can also be
stabilized along the process. These POMs are removed in a separate compartment and

Address correspondence to Sorawit Powtongsook, Center of Excellence for Marine Biotechnology,

National Center of Genetic Engineering and Biotechnology, Email: sorawit@biotec.or.th
Received May 11, 2013, Accepted January 17, 2014.
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 Journal of Water and Environment Technology, Vol.12, No.4, 2014

degraded through anaerobic decomposition. Denitrifying reactors supplied with digested

sludge have been found to operate successfully without the need for external carbon
addition (Aboutboul et al., 1995; Klas et al., 2006; Tal et al., 2009). Hence, this is the
method of choice when it comes to nitrate removal in aquaculture systems.

Denitrification, however, is not the only process for nitrate reduction. Other nitrogen
respiration pathways such as anaerobic ammonium oxidation (anammox), which is the
transformation of nitrite and ammonium into dinitrogen gas, and dissimilatory nitrate
reduction to ammonium (DNRA), which is the reduction of nitrate to nitrite followed by
the reduction of nitrite to ammonium, have also been proven to contribute a significant
proportion to nitrate removal in various environments (Kartal et al., 2007; Smith et al.,
2007; Dong et al., 2011). Favorable conditions for these pathways are hypothesized to
depend mainly on the concentration and type of electron donor, the amount of inorganic
nitrogen compounds present, along with the redox potential found in those systems (van
Rijn et al., 2006; Kraft et al., 2011). To date, there had been several reports on the
presence of anammox bacteria in aquaculture treatment systems that use degraded
sludge as the substrates for denitrification (Tal et al., 2006; Lahav et al., 2009).
However, the involvement of the DNRA process in aquaculture systems is still
uncertain. So far, DNRA has been found in the sediment below cages of an open water
fish farm with activity up to 7-fold greater than denitrification (Christensen et al., 2000).
The activity of DNRA was also detected along with autotrophic denitrification in
mariculture sludge under high sulfide concentration (Sher et al., 2008). The
understanding of the factors influencing the DNRA process in RAS, however, is still
limited. The occurrence of DNRA is undesirable in wastewater treatment systems
because it competes with denitrification and reduces the process efficiency by
producing ammonium, hence nitrogen waste is still retained in the water. To control the
occurrence of DNRA, conditions affecting its presence and activity need to be studied in
more detail. The nitrogen pathways that can occur in aquaculture systems under oxic
and anoxic conditions are shown in Fig. 1.

Organic + - -
NH4 NO2 NO3 Nitrification Oxic
Nitrogen

Anoxic - - +
NO3 NO2 NH4 DNRA

- + + Organic
Anammox N2 NO2 NH4
Nitrogen

NO

N2O

N2

Denitrification
Fig. 1 - Nitrogen pathways that can occur in aquaculture systems under
oxic and anoxic conditions.

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During the organic sludge digestion, sulfide was also found to be released and
stimulates the community of autotrophic denitrifying bacteria under anoxic conditions
(Cytryn et al., 2003; Schwermer et al., 2010; Shao et al., 2010). Since a sulfide-rich
environment has been hypothesized to favor DNRA (Brunet and Garcia-Gil, 1996; Sher
et al., 2008), the presence of the DNRA process is to be expected in this system and
should also be an important pathway for nitrate reduction. Apart from sulfide, the high
C/N ratio is another factor that promotes the growth of DNRA bacteria (van Rijn et al.,
2006; Schreier et al., 2010). The high C/N condition can be found in the biofloc system,
which is a type of aquaculture that uses external carbon substrates to enhance the
growth of heterotrophic bacteria in the fish tank (Crab et al., 2012). Ammonium uptake
by microbial biomass in the biofloc, thus, eliminates nitrogen wastes without the need
for slow-growing autotrophic nitrifiers. This high C/N ratio should also support the
growth of DNRA bacteria, hence, under oxygen depleted conditions, ammonium
regeneration is also likely to occur in the biofloc system.

The objective of this study was to investigate the occurrence of denitrification and
DNRA processes in sludge taken from freshwater and biofloc aquaculture systems.
Activities of denitrifying and DNRA bacteria in both sludge types were compared by
monitoring the change in nitrate, nitrite, and ammonium concentrations in batch reactors.
The effects of sulfide on nitrate reduction in both freshwater sludge and biofloc were
also studied by adding different concentrations of sulfide to the sludge samples. Results
of the experiment were discussed in terms of the presence of different nitrogen
transformation pathways in the systems as well as the effects of environmental
conditions on the occurrence of these microbial processes.

MATERIALS AND METHODS

Sources of aquaculture sludge
Freshwater aquaculture sludge was taken from 4-m3 indoor Nile tilapia culture tank
with internal nitrification media for ammonium removal. Fish density in the tank was
equal to 9.5 kg/m3 with suspended solid concentration of 126.0 ± 17.4 mg/L. A biofloc
sample was collected from 800-L Nile tilapia tank with fish density of 9 kg/m3. Starch
was added daily to this biofloc aquaculture system as the external carbon source to
provide a C:N ratio of 20:1. Suspended solid concentration in the biofloc aquaculture
system was equal to 498.3 ± 30.1 mg/L.

Evaluation of sludge digestion and nitrate reduction

Sludge digestion and nitrate reduction experiments were conducted using 4 sets of 1-L
DURAN® bottles (Schott, Germany), 2 reactors for freshwater sludge and 2 reactors for
biofloc sample, at room temperature (30°C), with approximately 10 g (dry weight) of
sludge per liter. The reactors were operated in batch mode by providing nitrate at the
initial concentrations of 25 and 100 mg NO3–-N/L to each sludge sample using NaNO3
as the source of NO3–-N. Each reactor was supplemented with nutrients containing (per
liter): 3.8 g of Na2HPO4, 1.5 g of K2HPO4, 0.1 g of MgSO4·7H2O, and 2 mL of trace
element solution (van Rijn et al., 1996). Trace element solution added consisted of (per
liter): 50.0 g of EDTA, 22.0 g of ZnSO4·7H2O, 7.34 g of CaCl2·2H2O, 5.06 g of
MnCl2·4H2O, 4.99 g of FeSO4·7H2O, 0.195 g of NaMoO4·2H2O, 1.57 g of CuSO4·5H2O,
and 1.61 g of CoCl2·6H2O (Vishniac and Santer, 1957). The medium was stirred with a

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magnetic stirrer to maintain the homogenous state of the medium. Water samples were
taken every 30 or 60 minutes depending on the initial concentration of nitrate.

Effects of sulfide on nitrate reduction

Effects of sulfide on nitrate reduction was studied by adding sulfide (Na2S·H2O) at the
initial concentrations of 50 and 100 mg S2–/L in freshwater sludge, and 25 and 50 mg
S2–/L in biofloc samples. The experiments were conducted in batch mode using 4 sets of
1-L Duran® bottles at room temperature (30°C). Nitrate was added as NaNO3 at 100 mg
NO3–-N/L for all sulfide concentrations. The culture medium supplemented and water
samplings were similar to those of the previous experiment.

Analytical methods
Nitrate was analyzed using an ultraviolet spectrophotometric method (PowerWave XS2
Microplate Spectrophotometer, BioTek Instruments, USA) according to Standard
Methods for Water and Wastewater Analysis (APHA, 1992). Nitrite and ammonium
were both analyzed colorimetrically using the sulfanilamide method (APHA, 1992) and
the salicylate-hypochlorite method (Bower and Holm-Hansen, 1980), respectively.
Sulfide fixation was performed by adding 2.5-mL of sample to 1-mL of 5% zinc acetate
solution (Cytryn et al., 2003), then the concentration of sulfide was measured
colorimetrically using the methylene blue method (Cline, 1969).

RESULTS AND DISCUSSION

Nitrate reduction in freshwater sludge and biofloc reactors
Nitrate reduction in freshwater sludge and biofloc reactors was monitored by measuring
the changes in nitrate, nitrite, and ammonium concentrations in the reactors. The results
are shown in Fig. 2. For freshwater sludge reactors, nitrate decreased rapidly at the rates
of 205.7 ± 52.8 and 165.9 ± 82.7 µg-N/L-min for the initial concentrations of 25 and
100 mg NO3–-N/L (as indicated in Table 1), respectively. A slight increase in nitrite was
found during nitrate reduction for both of the freshwater sludge reactors, indicating the
transformation of nitrate to nitrite. Ammonium in both of the freshwater sludge reactors
was low and comparable to the concentration found in the fish tank from where the
sludge was originally taken (0.04 ± 0.04 mg-N/L, as shown in Table 2). Changes in
nitrate, nitrite, and ammonium concentrations indicated that denitrifying bacteria were
already present in the freshwater aquaculture system and could function readily under
anoxic conditions, which can be seen from the concomitant reduction of nitrate and
nitrite. The activity of DNRA could not be detected under either nitrate concentration,
which otherwise should result in the accumulation of ammonium in the system. For the
biofloc reactors, nitrate reduction rates were equal to 51.7 ± 28.5 and 94.6 ± 94.9
µg-N/L-min for the nitrate concentrations of 25 and 100 mg NO3–-N/L, respectively.
These reduction rates were slower than those found in the freshwater sludge reactors,
while nitrite and ammonium were found to accumulate in the biofloc reactors. Peaks of
nitrite and ammonium both exceeded the toxicity threshold for aquatic animals, which is
not desirable for water recycling. The absence of ammonium accumulation in
freshwater sludge showed that ammonium release from sludge decomposition was
unlikely to occur at this stage, and that ammonium increase in the biofloc reactors was
likely caused by DNRA bacteria that originally resided in the biofloc sample. Hence,
the differences in nitrogen transformation patterns in freshwater sludge and biofloc

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reactors were possibly caused by differences in the microbial communities that were
responsible for nitrate reduction. The high C/N ratio in the biofloc aquaculture system
was one of the major parameters that could influence these differences, as it had been
proposed as the favorable condition for DNRA process (van Rijn et al., 2006; Schreier
et al., 2010).

Nitrate Nitrite Ammonium Nitrate Nitrite Ammonium

a b

Nitrite and ammonium concentrations

30 1 100 1
Nitrate concentration (mg-N/L)

25
0.8 80 0.8

20
0.6 60 0.6

(mg-N/L)
15
0.4 40 0.4
10

0.2 20 0.2
5

0 0 0 0
0 60 120 180 240 300 360 420 0 60 120 180 240 300 360 420 480 540 600
Time (min) Time (min)

Nitrate Nitrite Ammonium Nitrate Nitrite Ammonium

c d

Nitrite and ammonium concentrations

25 10 100 10
Nitrate concentration (mg-N/L)

20 8 80 8

15 6 60 6

(mg-N/L)
10 4 40 4

5 2 20 2

0 0 0 0
0 60 120 180 240 300 360 420 480 540 600 0 60 120 180 240 300 360 420 480 540 600
Time (min) Time (min)

Fig. 2 - Nitrate, nitrite, and ammonium concentrations during nitrate reduction by (a)
freshwater sludge with 25 mg-NO3–-N/L; (b) freshwater sludge with 100
mg-NO3–-N/L; (c) biofloc with 25 mg-NO3–-N/L; and (d) biofloc with 100
mg-NO3–-N/L. All experiments were conducted without sulfide addition.

Table 1 - Percentage of nitrate removal and rate of nitrate reduction.

Initial NO3-/ S2 Final NO3- concentration Percentage of NO3- Rate of NO3- reduction
-
concentration (mg- NO3 -N/L) removal (µg-N/L-min)
Freshwate Biofloc Freshwate Biofloc Freshwater Biofloc
r sludge r sludge sludge
25 mg NO3--N/L 2.0 6.7 92.4 68.5 205.7±52.8 51.7±28.5
100 mg NO3--N/L 3.1 46.6 96.7 32.7 165.9±82.7 94.6±94.9
25 mg-S2-/L*,** - 14.8 - 26.4 - -
50 mg-S2-/L* 4.7 70.8 63.0 17.6 - -
100 mg-S2-/L*,*** 48.1 - 55.1 - - -
*With 100 mg NO3--N/L
**At 25 mg-S2-/L, only biofloc sample was tested
***At 100 mg-S2-/L, only freshwater sludge was tested

Table 2 - Characteristics of water in freshwater and biofloc aquaculture systems.

NH4+ NO2- NO3- DO pH Temp SS Tank Carbon
(mg-N/L (mg-N/L (mg-N/L (mg/L) (ºC) (mg/L) volume added
(L)
Fresh- 0.04 0.09 10.0 4.2 6.8 29.7 126.0 4,000 -
water ±0.04 ±0.03 ±0.7 ±0.1 ±0.0 ±0.1 ±17.4
Bioflo 0.13 0.35 32.8 7.1 7.0 29.6 498.3 700 Starch
±0.02 ±0.01 ±1.0 ±0.2 ±0.0 ±0.1 ±30.1

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When comparing the results from these two sludge types, denitrifying bacteria in the
freshwater sludge seem to be more active than those found in the biofloc reactors. This
might be due, in part, to the differences in the fish tank volumes from where the two
sludge samples were originally taken. With the larger freshwater aquaculture system (as
indicated in Table 2), water mixing by aeration was not effective enough to suspend
most of the sludge in the water column. Accumulation of the bottom sludge layer was
hence usually found, which has microaerobic or anoxic conditions that favor the growth
of denitrifying bacteria. The low nitrate concentration (10.0 ± 0.7 mg-N/L, as shown in
Table 2) in the freshwater compared to the biofloc aquaculture system also supports this
hypothesis that denitrifiers were present and functionally active in the freshwater sludge
sample. On the contrary, the biofloc aquaculture system had a much smaller fish tank
volume which meant that the water mixing was sustained well by the strong aeration.
An accumulation of nitrate was found in the biofloc aquaculture system (32.8 ± 1.0
mg-N/L, Table 2) as a result of the aerobic nitrification process in the biofloc particles
(Nootong et al., 2011). The bottom sludge layer accumulation in the fish tank thus plays
a significant role in controlling the fate of nitrate in aquaculture systems.

Effects of sulfide on nitrate reduction

Percentages of nitrate removal after sulfide addition were found to be lower than those
without sulfide for both freshwater sludge and biofloc reactors (Table 1). Despite the
fact that sulfide can contribute as an electron donor for autotrophic denitrifying bacteria
(Cytryn et al., 2005a; Cytryn et al., 2005b; Shao et al., 2010), certain concentrations of
sulfide also inhibit the enzymes responsible for denitrification (Brunet and Garcia-Gil,
1996). Slower rates of nitrate reduction under high sulfide (as shown in Fig. 3) could be
the result of the partial inhibition of the denitrifying bacterial community. Nevertheless,
the fluctuation of nitrate under elevated sulfide conditions made the calculation of exact
nitrate reduction rates somewhat unreliable, especially for the biofloc reactors in which
nitrate only decreased slightly. However, the decrease in sulfide concentration
simultaneously with nitrate was found in the freshwater sludge reactors, which was
possibly caused by sulfide-oxidizing denitrifiers. Furthermore, sulfide at 50 and 100
mg-S2–/L in the freshwater sludge reactors could induce significant accumulation of
nitrite up to 24.2 and 32.9 mg-N/L (Fig. 3), respectively, which were substantially
higher than those found in the freshwater sludge reactors without sulfide addition (Fig.
2). The increase in nitrite suggested that nitrite reduction was the rate-limiting step in
the denitrification process under this condition, where nitrate seems to be continuously
transformed to nitrite. Ammonium in the sulfide-added freshwater sludge reactors also
accumulated to 9.5 mg-N/L for 50 mg-S2–/L and 12.1 mg-N/L for 100 mg-S2–/L. This
implies that another microbial pathway, which was likely to be DNRA, competed with
denitrifying bacteria under the high sulfide conditions in the freshwater sludge reactors.
This result was in accordance with the previous findings that sulfide could drive DNRA
activity, as suggested by the works of Brunet and Garcia-Gil (1996) and Sher et al.
(2008).

As for the biofloc reactors, sulfide also had certain inhibitory effect on nitrate reduction
by lowering the removal of nitrate to 26.4 and 17.6 percent for 25 and 50 mg-S2–/L,
respectively (as shown in Table 1). In addition, for 25 mg-S2–/L, nitrite accumulated to
14.2 mg-N/L but hardly increased in the biofloc reactor with 50 mg-S2–/L, suggesting
that nitrate reduction step was hindered by the higher sulfide concentration. Furthermore,

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ammonium in both of the reactors elevated to the same level as found in the biofloc
reactors without sulfide (Fig. 3). This indicated that sulfide had little effect on the
activity of DNRA in the biofloc reactors and these DNRA bacteria were unlikely to use
sulfide as their electron donor. Compared with the freshwater sludge reactors which had
DNRA activity following sulfide addition (as indicated in Fig. 3), DNRA bacteria in the
biofloc reactors were thus likely to be the group of heterotrophs, while DNRA
community in the freshwater sludge reactors were more likely to be sulfide oxidizers.

Nitrate Nitrite Sulfide Ammonium Nitrate Nitrite Sulfide Ammonium

f
Nitrate, nitrite, and sulfide concentrations

100
e 14 120 14

Ammonium concentration (mg-N/L)

12 100 12
80
(mg-N/L and mg-S2-/L)

10 10
80
60 8 8
60
40 6 6
40
4 4
20
2 20 2

0 0 0 0
0 60 120 180 240 300 360 420 0 60 120 180 240 300 360 420
Time (min) Time (min)

Nitrate Nitrite Sulfide Ammonium Nitrate Nitrite Sulfide Ammonium

g h
Nitrate, nitrite, and sulfide concentrations

150 5 200 5

Ammonium concentration (mg-N/L)

125
4 160 4
(mg-N/L and mg-S2-/L)

100
3 120 3
75
2 80 2
50

1 40 1
25

0 0 0 0
0 60 120 180 240 300 360 420 0 60 120 180 240 300 360 420
Time (min) Time (min)

Fig. 3 - Nitrate, nitrite, ammonium, and sulfide concentrations during nitrate reduction
under sulfide-rich environment by (e) freshwater sludge with 50 mg-S2–/L; (f)
freshwater sludge with 100 mg-S2–/L; (g) biofloc with 25 mg-S2–/L; and (h)
biofloc with 50 mg-S2–/L. All experiments were conducted with the initial
nitrate concentration at 100 mg-NO3–-N/L.

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 Journal of Water and Environment Technology, Vol.12, No.4, 2014

CONCLUSIONS
When comparing nitrate reduction rates between freshwater sludge and biofloc without
sulfide addition, it was found that the freshwater sludge had higher nitrate reduction
rates without any undesirable ammonium and nitrite accumulation. However, with
sulfide added to the freshwater sludge reactors, significant increases in both nitrite and
ammonium were observed. This indicates the partial inhibition of denitrification along
with an increase in DNRA activity under elevated sulfide conditions. For the biofloc
reactors, DNRA was detected at the same level for all of the reactors with or without
sulfide addition. Since sulfide had little influence on DNRA activity in the biofloc
reactors, these DNRA bacteria were likely to be heterotrophs that did not use sulfide as
the source of energy. This was in contrast to the DNRA bacteria found in the freshwater
sludge reactors which seem more likely to be sulfide oxidizers. The understanding of
the DNRA process could provide useful information that can be used to solve the
periodic ammonium accumulation in the denitrifying reactor. However, confirmation
with more precise techniques, e.g. molecular methods with genes related to the DNRA
pathway, is recommended to prove the presence and activity of DNRA bacteria in
aquaculture systems. Nevertheless, the results of this study have shown that denitrifying
and DNRA activities exist in both freshwater and biofloc aquaculture systems and can
perform activities that drive the transformation of nitrogen when conditions are
favorable.

ACKNOWLEDGEMENTS
This research was financially supported by the Integrated Innovation Academic Center,
Chulalongkorn University Centenary Academic Development Project (CU56-FW14). It
received partial support from the Higher Education Research Promotion and National
Research University Project of Thailand, Office of the Higher Education Commission
and the Ratchadaphiseksomphot Endowment Fund (FW1017A). Additional support was
obtained from the Chulalongkorn University Graduate Scholarship to commemorate the
72nd Anniversary of His Majesty King Bhumibol Adulyadej. Equipment used was
provided by the Thai Government Stimulus Package 2 (TKK2555) under the Project for
the Establishment of Comprehensive Center for Innovative Food, Health Products and
Agriculture.

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