Proceedings of the 19th IAHR-APD Congress 2014, Hanoi, Vietnam

ISBN 978604821338-1

Treatment of Nitrogen Polution in Aquaculture Wastewater by Addition of Carbon Source

PHAM THI HOA

School of Biotechnology, International University, Hochiminh City, Vietnam, Email address: pthoa@hcmiu.edu.vn

ABSTRACT
In this research, nitrogen in aquaculture wastewater change as respond to the addition of carbon source to the system
were studied in microcosm experiments. Nitrogen transformation was studied by looking at the change in different
forms of nitrogen including the total nitrogen (N), total ammonium nitrogen (TAN), nitrite and nitrate. These
parameters, in high concentrations, are very likely to negatively affect pond water quality. Nitrogen from aquaculture
wastewater also badly affects the receiving waterbodies and ecosystem health. Results from this study show that the
addition of carbon source and aeration in pond water reduced TAN, nitrite and total N concentrations. It proved that
this simple technique can help to solve nitrogen problem in ponds.

Keywords: Nitrogen transformation; total nitrogen, total ammonium nitrogen (TAN), nitrite, nitrate

1. INTRODUCTION A main factor regulating nitrification is oxygen

penetration into sediment (Reddy and Patrick, 1984;
Nitrogen (N) exists in many different forms. It can be
Rysgaard et al., 1994). Nitrate may in turn be assimilated
nutrients and can also be toxicants. Therefore, nitrogen
by phytoplankton. The phytoplankton is deposited on the
plays an important role in the dynamics of aquaculture
pond floor. The resulting detritus may be remineralized
system. Ammonia is toxic if it is accumulated as the end
by bacteria to form TAN or buried in the sludge (NSED)
product of protein catabolism. The unionized gaseous
(Fig. 1).
form of nitrogen, ammonia is more toxic to aquatic
organisms at high pH and temperature. The importance
of the ammonium adsorption process is the combination
of nitrifying bacteria with particles (Seitzinger, 1990).
Another potentially toxic nitrogenous compound is
nitrite which may accumulate in aquaculture pond.
During nitrification and denitrification, nitrite is released
as an intermediate product. Methemoglobin presents
toxicity of nitrite which does not have the capacity to
carry oxygen, which is formed by the competitive
binding of nitrite to hemoglobin (Hargreaves, 1998).
The reduction of atmospheric dinitrogen by
Figure 1.Nitrogen cycle in aquaculture pond (Burford, Lorenzen,
heterocystous cyanobacteria may release nitrogen to 2004). Arrows represent pathways and boxes indicate the key N
aquaculture ponds. The phytoplankton community and components: TAN=total ammonia N; NOX=nitrate plus nitrite;
ammonia concentration is two deciding factor for the DON=dissolved organic N; and NSED=N buried in the sludge.
quantity of N added to aquaculture ponds. The N can
Nitrogen (N) biogeochemistry of aquaculture ponds is
enter the water column as TAN from aquatic organism
dominated by biogeochemical transformations of N
excretion and as dissolved organic N (DON) from
added to ponds in the form of inorganic or organic
formulated feed. TAN may be assimilated by
fertilizers and formulated feeds. Nitrogen application in
phytoplankton, volatilized as gaseous ammonia and
excess of pond assimilatory capacity can lead to the
converted to nitrite/nitrate (NO X) via nitrification
deterioration of water quality through the accumulation
processes (Burford, Lorenzen, 2004). Nitrification is two-
of nitrogenous compounds (e.g., ammonia and nitrite)
step oxidation of ammonia to nitrate. Nitrosomonas
with toxicity to fish or shrimp (J.A. Hargreaves, 1998).
converts ammonium form of nitrogen into nitrite form.
Similarly, Nitrobacter converts nitrite form into nitrate If carbon and nitrogen are well balanced in the solution,
form. Both of them are chemoautotrophic, gram- ammonium in addition to organic nitrogenous waste will
negative, motile rods with long generation times (20 to 40 be converted into bacterial biomass (Schneider et al.,
h). The reactions proceed as follows: 2005). By adding carbohydrates to the pond,
heterotrophic bacterial growth is stimulated and nitrogen
2NH 4+ + 3O2 2NO 2- + 4H+ + 2H2O
uptake through the production of microbial proteins
and takes place (Avnimelech, 1999). Immobilization of
ammonium by heterotrophic bacteria occurs much more
2NO2- + 1O2 2NO 3-
rapidly because the growth rate and microbial biomass

1
yield per unit substrate of heterotrophs are a factor 10
higher than that of nitrifying bacteria (Hargreaves, 2006).
The assimilation of inorganic nitrogen into microbial
protein is a common process (Avnimelech et al., 1992).
However, it is still not clear the detail transformation of
different forms of nitrogen with the addition of carbon
source. Therefore, nitrogen transformation in the
aquaculture wastewater added C source will be studied
in this subject through the microcosm in laboratory.
Results from this study can develop better understanding
about nitrogen tranformation in aquaculture pond. In
addition, it can be used to develop nitrogen
transformation model in aquaculture farms in Vietnam.
The data obtained can be used as reference for further
studies on management nitrogen in aquaculture pond.
2. MATERIALS AND METHODS
2.1 Experimental design
Aquatic microcosms were assembled in 20-L plastic pot.
Input water collected from a freshwater catfish pond in
aquaculture farm of Agriculture and Forestry University.
Input water was measured for total nitrogen, total
ammonia nitrogen (TAN), nitrate and nitrite before
adding into microcosm. Each microcosm was received 16
liter of input water. There were two kinds of microcosm
experiments used in this study. One type of microcosm
was the control reactor which had only input water. The
second type of microcosm had input water added with
carbon source called sample. There were two replicates
for each experiment. All microcosms were maintained in
outside condition for ten days. All microcosmswere
aerated continuously during the experiment. The carbon
source added to promote bioflocswas glucose. Glucose
concentration was based on C: N ratio (~ 20:1).
Nitrogen transformation was followed by the change in
the nitrogen concentration. Nitrite, nitrate concentration
were measured routinely at three to seven days intervals
throughout the course of the experiment. Total ammonia
nitrogen, total nitrogen, chlorophyll, biomass and the
change of algae population followed by algae species
count measured at two times: initial and final. Following
these measurements, the remained water sample was
returned to the microcosm from which it was taken.
Small volumes of distilled water were added to the
microcosms as needed to compensate for evaporation.
2.2 Total nitrogen determination
For total N determination using the modified Kjeldahl
method (TCVN 5987-1995), accurately add 50 mL sample
into a digestion flask. In each batch, include two blanks
(whole procedure with 50 mL distilled water), two
replicates of sample (control and sample). Add 10 mL of
concentrated H2SO4 and 5 0.5 g of catalyst mixture
(CuSO 4 and K2SO4) respectively and mix by swirling.
Place the digestion flasks stand beside the block digester
and fit the exhaust manifold on top of it. Place the flasks
with rack and exhaust manifold on the preheated
digestion block in the fume-hood. Digestion for 3 hours
and then allow to cool in 30 minutes. Accurately 50 mL of
boric acid solution add into an Erlenmeyer flask
corresponding to the number of samples and blanks and
then add two drops of indicator solution (Tashiro
indicator). Erlenmeyer flask places under the condenser
of the steam-distillation unit. Take care that the end of the
condenser is immersed in the boric acid solution to
prevent any loss of ammonia. Start the distillation
process by setting the distillation time about 8 minutes.
When the distillation is complete, i.e. when about 200 mL
of distillate has been collected, lower the Erlenmeyer
flask. Continue with the next sample. Titrate the receiver
flask solution from green over colorless to a pink end
point with 0.02 N H 2SO4. Record the amount of H2SO4
used. Always standardize the acid to obtain the extract
normality of the titrant. Calculate Kjeldahl nitrogen
concentration (CN) by the equation [1]:
mg N/L = x C x 14,01 x 1000
[1]
where:
V0 = total mL of sample
V1 = mL of H 2SO4 required for titration of sample
V2 = mL of H 2SO4 required for titration of blank
C = normality of H 2SO4 (0.02 N)
14,01 = atomic weight of nitrogen
2.3 Determination of total ammonia nitrogen (TAN)
For TAN determination using the modified direct
distillation and titration method (TCVN 5988-1995),
accurately add 50 mL sample into a flask. In each batch,
include blank (whole procedure with 50 mL distilled
water), two samples (control and sample). Add three
drops indicator solution (phenolphthalein indicator) and
0.03 g MgO respectively into flask. Accurately 50 mL of
boric acid solution add into an Erlenmeyer flask
corresponding to the number of samples and blanks and
then add two drops of indicator solution (Tashiro
indicator). Erlenmeyer flask places under the condenser
of the steam-distillation unit. Take care that the end of the
condenser is immersed in the boric acid solution to
prevent any loss of ammonia. Start the distillation
process by setting the distillation time about 4 minutes.
When the distillation is complete, i.e. when about 200 mL
of distillate has been collected, lower the Erlenmeyer
flask. Continue with the next sample. Titrate the receiver
flask solution from green over colorless to a pink end
point with 0.02 N H 2SO4. Record the amount of H2SO4
used. Always standardize the acid to obtain the exact
normality of the titrant. Calculate TAN concentration
(CN) by the equation [2]:
mg N/L = x C x 14,01 x 1000 [2]
where:
V0 = total mL of sample
V1 = mL of H 2SO4 required for titration of sample
V2 = mL of H 2SO4 required for titration of blank
C = normality of H 2SO4 (0.02 N)
14,01 = atomic weight of nitrogen
2.4 Determination of nitrite
2
For nitrite determination using molecular absorption
spectrometric method (TCVN 6178-1996), accurately take
40 mL sample into a 50 mL falcon. If the sample contains
suspended solids, filter through a membrane filter.To
50.0 mL sample, or to a portion diluted to 50.0 mL, add 1
mL color reagent (85% phosphoric acid, sulfanilamide
and N-(1-naphthyl)-ethylenediaminedihydrochloride)
and mix. Between 20 min and 30 min after adding color
reagent to samples and standards, measure absorbance at
540 nm providing a light path of 10 cm and getting OD of
nitrite. Prepare a standard curve by plotting absorbance
of standards against NO 2-N concentration. Sample
concentration is computed directly from curve. Standard
nitrite solution was prepared by dissolving 1.232 0.0002
g NaNO 2 in water and dilution to 1000 mL. Stock nitrite Figure 2.The total nitrogen concentration in control and sample.
solution always standardizes to obtain the exact mg NO2- Error bar is SD.
N/mL in stock NaNO 2 solutionby standard methods.
Table 1. Total N concentration (mg_N/L) of control and sample at
day 0 and 9
2.5 Determination of nitrate
Determination of nitrate is based on their reduction to Day 0 Day 9
nitrite in the presence of Zn/NaCl (Badiadka Narayana
and Kenchaiah Sunil, 2009). To a 50.0-mL sample in a
Control 6.54 2.642 4.48 0.647
250-mL Erlenmeyer flask, add 20 g of Zn/NaCl (5:1
w/w) granular mixture, and was allowed to stand for 30
minutes with occasionally stirring to form nitrite, then Sample 6.54 2.642 2.38 0.324
the solution was filtered using filter paper. The filtered
Values are mean and standard deviation (Mean SD).
sample measures nitrate concentration by the same
method in nitrite determination (TCVN 6178-1996) and
getting OD of nitrate. Prepare a standard curve by 3.2 Total ammonia nitrogen (TAN)
plotting absorbance of standards against NO 3--N In the control, TAN accumulation after 3 days, then
concentration. Sample concentration is computed directly reduced after 7 days. In the sample, TAN concentration
from curve. Standard nitrate solution was prepared by reduced with time, and smaller than TANconcentration
dissolving 0.7218 0.0001 g KNO 3 in water and dilution in the control after 7 days (Fig. 3). TAN may be
to 1000mL. transformed via a number of pathways: assimilated by
phytoplankton (CHL), volatilized as gaseous ammonia
2.6 Data analysis and converted to nitrite/nitrate (NO X) via nitrification
processes (Burford, K. Lorenzen, 2004). The addition of
SPSS software program was used for statistical
carbon source in certain doses allows elimination
assessment. Data were performed using the one-way
potential toxic as ammonia, especially prevent short-term
variance analysis (ANOVA). All significant levels were
TAN accumulation (after 3 days).This promoted nitrogen
set at p < 0.05.
uptake by bacterial growth decreases the ammonium
concentration more rapidly than nitrication
3. RESULTS AND DISCUSSION
(Hargreaves, 2006).
3.1 Total Nitrogen
In general, total nitrogen concentration tended to
decrease with time. As shown in Fig. 2, total N
concentration in added C source sample was reduced
more than total N concentration in control tank. There
was a significant difference (p < 0.05) between control
and sample at day 9. This result agreed with Avnimelech
et al. (1992) who reported that when bacteria were
provided additional C source, they would get nitrogen in
the water to form proteins. Therefore, bacteria would
reduce inorganic nitrogen concentration in the water.
Pond water normally contains some amount of carbon. Figure 3. The TAN concentration in control and sample
However, the ratio of C and N usually is low (C:N < 20:1)
(Avnimelech, 2012). Thus, total N concentration in added 3.3 Nitrite
C source sample decreased considerable because
The results presented in figure 4 showed that nitrite
microorganism activities happen stronger. The previous
concentration for 3 days decreased in sample more than
research, Crab et al. (2009) reported that the control of
control. However, if the reaction time extended to 5, 7
organic N form in pond by adjustment the ratio of C / N
days, nitrite concentration also reduced in control. It was
is feasible and reliable.
clearly that the transformation of nitrite happened slowly
in C unsupplied water.The addition of glucose and

3
aeration continuously in pond water which creates
Control 3.72 3.11 1.09 0.43
bioflocs allows elimination the potential toxic as nitrite.
0.143 0.565 0.038 0.000

Sample 3.72 1.47 1.36 0.97

0.143 0.546 0.019 0.019

Values are mean and standard deviation (Mean SD).

4. CONCLUSION
The microcosm developed herein simulated fairly well
the water quality in freshwater pond, constituting a basic
for understanding the nitrogen transformation in
response to the addition of carbon source in aquaculture
pond. It became clear that, in general, concentrations of
Figure 4.The nitrite concentration in control and sample. Error bar potentially toxic TAN, NO 2-N decreased in the water
is SD. pond added carbon source which had aeration every day.
Table 2. Nitrite concentration (mg_N/L) in day 0, 3, 5 Nevertheless, this study is recommended that further
and 7 for control and sample research should make detail kinetics of nitrogen
tranformation in order to prepare mathematical model to
Day 0 Day 3 Day 5 Day 7 control nitrogen in ponds.

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