The 11th SEATUC Symposium

STUDY ON NITROGEN REMOVAL CAPACITY OF ANAMMOX

EXISTED IN SLUDGE OF SOY SAUCE WASTEWATER
Dang Van Dien (1), Le Cong Nhat Phuong (2), Nguyen Phuoc Dan (3)
(1)Ho Chi Minh City University of Food Industry, Vietnam
(2) Institute of Tropical Biology, Vietnam Academy of Science and Technology, Vietnam
(3) Ho Chi Minh City University of Technology, Vietnam

Email: diendv@cntp.edu.vn

ABSTRACT The main process of aerotank is conversion from

This study investigates nitrogen (ammonium)
removal efficiency of Anammox bacteria existed in
sludge of soy sauce wastewater treatment plant. A UASB
reactor with using volume of 10 liters was run in two
stages. The sludge was fed with synthetic wastewater in
the first stage while swine wastewater was used in the
second stage. In both stages, the reactor was operated
with a flowrate of 10 liters/day to investigate the nitrogen
removal efficiency (NRE) at various nitrogen
concentration of 100, 200, 300, 400 mg/l in four
experiments (the scale of NH4+-N:NO2--N = 1:1),
respectively. In 210 days of operation in the first stage,
with maximum nitrogen concentration was 400 mg/l
(200 mg/l of NH4+-N and 200 mg/l of NO2--N), the
ammonium conversion efficiency (ACE) was 85.9%
while NRE reached 85%. Influent pH in the first stage
was controlled at 80.2. Similarly, after 260 days of
operation in the second stage, the highest performance
was achieved at the nitrogen concentration of 400 mg/l,
ACE was 88.77%, total NRE was over than 80%. Inlet
pH in the second stage was kept at 7.40.2. The
produced gas sample was collected and sent to laboratory
for analysis. The gas analysis result showed that
percentage of nitrogen gas was over than 99% when
NRE obtained over than 75% in both stages.
KEYWORDS: Anammox, ammonium removal, swine
wastewater.
1. INTRODUCTION
The wastewater treatment processes are focusing on
removal of COD, BOD but ammonium removal. In Ho
Chi Minh city, ammonium concentration in effluent of
most wastewater treatment plants is over than the limit
(10mg/l QCVN40:2011, B column). The popular method
applied to treat wastewater recently is using aerotank.
ammonium to nitrate. This results in high nitrate
concentration from ammonium pollution. While nitrogen
pollution needs to be treated thoroughly, the aerotank
seems to be costly because it requests additional
denitrification process to follow the aerotank. This leads
to chemical and energy demand for the operation. This is
also the main reason to prevent Vietnamese enterprises to
invest the standard wastewater treatment plant.
Discovery of a new kind of microorganism to find
out a modern technology to remove ammonium was
studied many years ago. Most of the autotrophic
biological processes for nitrogen removal are based on
Anammox, in which, partial nitritation plays a role as a
pre-treatment phase to facilitate the Anammox process.
In 1995, the first experiment to confirm Anammox
process was described by researchers of Delft university
of technology (Mulder et. al. 1995). Van de Graaf et al.
have studied growth of Anammox in a fluidized bed
reactor. That study stated that the main product of the
Anammox reaction is N2, but about 10% of the N-feed is
converted to NO3-. The overall nitrogen balance gave a
ratio of NH4+ conversion to NO2-conversion and NO3-
production of 1:1.31. Acetylene, phosphate and oxygen
were shown to be strong inhibitors of the Anammox
activity. In Germany, there was a species named
Candidatus Kuenenia stuttgartiensis (Schmid et al, 2003).
Nutchanat and Suwanchai experimented on
Anammox enrichment from different conventional
sludges with three sets of sequencing batch reactor
(SBR) and UASB. The cultivated biomass in all SBRs
was classified in the group of Planctomycetales bacteria
with respect to the anaerobic ammonium-oxidizing
bacteria, Candidatus Brocadia anammoxidans and
Candidatus Kuenenia stuttgartiensis.
2. MATERIALS AND METHODS

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2.1. Sludge UASB (Upflow Anaerobic Sludge Blanket). Each model

The sludge used in this study was taken from an include the following components:
anaerobic sludge digester of soy sauce wastewater - An feeding water tank with a capacity of 45 liters.
treatment plant of Chinsu Foods - Masan Group (Tan - A dosing pump to feed wastewater from the tank into
Binh Industrial park). Conduct testing SS (suspended the UASB column.
solids), MLSS (mixed liquid suspended solids) followed - An UASB reactor made of gray PVC, cylindrical,
by filtering to remove sands, litters. The sludge was then with a height of 1 meter, diameter of 0.116 meter,
incubated in a dark room and supplied with substrates in useful volume of 10 liters. Along the height of the
an adapted medium, just after 4 days of incubation, the column 6 sampling valves with a distance of 0.015
top water was poured out to remove mold, scum, grease meter were attached. At the center of the reactor
and other organic compounds. After 20 days of bottom, inlet valve was securely attached to the
incubation, a lot of gas escaped from the sludge as hopper spraying inside to increase the disturbance
observed, such sludge can be used for UASB model to and mix wastewater and sludge. At the top the the
accelerate the adaptation process. The sludge at that reactor, a serrated hopper was installed and connected
moment was black color with very fine granules, SS was to the gas collector by plastic pipes.
11.6 g/l, which would be used for Anammox enrichment. - A gas collector made of plastic PE, 5 liter capacity air
2.2. Anammox enrichment medium receiver.
The first medium was synthetic with the components - A tank for receiving effluent with capacity of 45
showed in Table 1. liters.
Table 1. Characteristics of synthetic mediums used for - Speed of water surges: 0.042 m / h
Anammox enrichment - Flowrate: 10 liters / day
Ingredient Concentration
N-NH4 (NH4Cl) 50; 100;150;200 mg/l
N-NO2 (NaNO2) 50; 100; 150; 200 mg/l
KHCO3 50 250 mg/l
KH2PO4 27 100 mg/l
CaCl2.2H2O 180 mg/l Eff.
MgSO4.7H2O 120 mg/l
FeSO4.7H2O 9 mg/l
Gas collector

EDTA2Na 5mg/l
Na2S. 9H2O < 125 mg/l Feeding

Ct
(Van de Graaf et al.,1996) water
The second medium was swine waste water after
biogas reactor of Dong A pig breeding enterprise (Di An Dosing
district, Binh Duong province). Properties of swine is Pump
indicated in Table 2. Figure 1. Experimental model
Table 2. Characteristics of swine waste water after
biogas reactor 2.4. Operation of reactor
The models were operated at the Institute of
Parameter Value
Tropical Biology - Linh Trung Ward - Thu Duc District -
pH 6.8 8.5 mg/l Ho Chi Minh city.
DO 0 0.2 mg/l The volume of sludge used to insert into the reactor
COD 1,800 3,200 mg/l type was calculated relating to the mixed volume in the
BOD5 1,000 1,800 mg/l model as the following formula:
N-NH4 200 800 mg/l Vsludge * SSsludge = Vreactor * MLSS
N-NO2 0 5 mg/l Where:
N-NO3 3 15 mg/l Vsludge: sludge volume needed for the reactor (liter)
Total P 30 80 mg/l SSsludge: VSS of sludge (mg / liter)
SS 1,500 4,200 mg/l Vreactor: UASB reactor volume (liter)
The initial volume of sludge to be inserted into each
2.3. Experimental set-up reactor was: 2.9 liters, equivalent to 33% volume of the
The experiment to enrich Anammox bacteria were reactor. The sludge was poured into the reactors after 20
operated with two parallel models were designed as days of incubation

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The first reactor was operated with synthetic Table 6. Efficiency of ammonium conversion and nitrite
medium, within 210 days. The model was run in 4 phases removal
with the data illustrated in Table 3. Days Efficiency of N-NH4 Efficiency of N-NO2
Table 3. Operation of reactor with synthetic medium conversion (%) removal (%)
Days N-NH4 N-NO2 pH 80 11.7 14.0
(mg/l) (mg/l)
120 21.4 23.5
80 50 50 80.2 180 52.1 54.2
120 100 100 80.2 210 78.1 79.8
180 150 150 80.2 In the first 80 days, Anammox bacteria had not
210 200 200 80.2 adapted yet, so the efficiency was very low. The
The second model was operated with swine adaptation of Anammox bacteria started from the next 30
wastewater, within 160 days. The model was also run in days of the operation time though efficiency was still
4 phases with the data illustrated in Table 4. low. The efficiency reached higher value from the 170th
Table 4. Operation of reactor with swine waste water day of operation. The change of the efficiency was
Days N-NH4 N-NO2 pH presented in Figure 2.
(mg/l) (mg/l)

60 50 50 7.40.2 %
100 100 100 7.40.2 90
80
130 150 150 7.40.2 70
160 200 200 7.40.2 60
N-NO2 concentrations in Table 4 were added to the 50
N-NO2
reactor by using NaNO2 as used in Table 1 to form N- 40
N-NH4
NH4 : N-NO2 ratio at 1:1. 30
Growth of microorganisms, volume of generated gas 20
were observed and outlet samples were tested in the 10
laboratory with parameters including N-NH4, N-NO2, N- 0
NO3, COD, SS, pH. 80 days 120 days 180 days 210 days Time
2.5. Analytical methods and calculations 50 mg/l 100 mg/l 150 mg/l 200 mg/l
The analytical methods for parameter N-NH4, N-
NO2, N-NO3, COD, pH were presented in the Table 5. Figure 2. Efficiency of N-NH4 conversion and N-NO2
Table 5. Analytical methods removal with synthetic medium
Parameter Method During operation time, besides N-NH4 conversion
and N-NO2 removal, a small amout of N-NO3 was
pH Electrode produced, COD was reduced. Gas production and change
N-NH4 US EPA Nessler method, maximum of sludge color were also obviously seen.
absorbance wavelength = 430 nm The change of Anammox sludge color was described
N-NO2 Colormetric method with Griss agent, in Figure 3. (microscope Olympus CH30, objective lens
maximum absorbance wavelength = 520 x 10)
nm
N-NO3 Colormetric method with phenoldisulfonic
acid, maximum absorbance wavelength =
430 nm
COD Potassium permanganate acidic method
SS Gravimetric analysis method
Initial sludge After 80 days
3. RESULTS AND DISCUSSIONS
3.1. Results of the first reactor run with synthetic
medium

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%
90
80
70
60
50
N-NO2
40
N-NH4
After 120 days After 210 days 30
Figure 3. Change of color of sludge operated with 20
synthetic medium 10
Table 7. COD reduction, N-NO3 generation and change 0
of sludge operated with synthetic medium 60 days 100 days 130 days 160 days Time
50 mg/l 100 mg/l 150 mg/l 200 mg/l
Time SS N-NO3 % COD Color of Gas
(day) (mg/l) (mg/l) reduction sludge volume
Figure 5. Efficiency of N-NH4 conversion and N-NO2
(mg/l) (l/d) removal with swine wastewater
80 6,200 0.5 1.8 Black 0.05 During operation time, besides of N-NH4 conversion
120 5,500 1.3 17.9 Black 0.50 and N-NO2 removal, a small amout of N-NO3 was
180 3,600 4.5 9.8 Brown 0.65 produced, COD was reduced. Gas production and change
of sludge color were also obviously seen.
210 1,260 5.3 18.7 Maroon 1.25
Table 10. COD reduction, N-NO3 generation and change
of sludge operated with swine wastewater
When N-NH4 conversion efficiency was over than Time SS N-NO3 % COD Color of Gas
75%, meaning Anammox reaction took place, gas (day) (mg/l) (mg/l) reduction sludge volume
samples were collected and sent to analysis laboratory. (mg/l) (l/d)
Table 8. Result of gas sample analysis
60 6,200 0.7 2.9 Black 0.10
Gas Unit Result Method
100 5,400 1.1 14.3 Black 0.50
N2 % 99.3 GC-Supe;co chromatography
product, p. 767, 1995-Alltech, 130 3,200 3.6 13.7 Brown 0.75
chromatography catalog 350, p.24. 160 1,350 5.8 25.5 Maroon 1.30
(Center for Analysis Service of Experiment in Ho Chi The change of Anammox sludge color was described
Minh City) in Figure 6. (microscope Olympus CH30, objective lens
3.2. Results of the second reactor run with swine x 10)
wastewater
Table 9. Efficiency of ammonium conversion and nitrite
removal
Days Efficiency of N-NH4 Efficiency of N-NO2
conversion (%) removal (%)
60 17.5 21.6
100 33.5 43.9 Initial sludge After 80 days
130 52.6 65.7
160 80.8 83.1

In the first 60 days, Anammox bacteria had not

adapted yet, so the efficiency was very low. The
adaptation of Anammox bacteria started from the next 30
days of the operation time though efficiency was still After 120 days After 210 days
low. The efficiency reached higher value from the 170th Figure 6. Change of color of sludge operated with swine
day of operation. The change of the efficiency was wastewater
presented in Figure 5. When N-NH4 conversion efficiency was over than
75%, meaning Anammox reaction took place, gas
samples were collected and sent to analysis laboratory.

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Table 11. Result of gas sample analysis discovered in a denitrifying fluidized reactor, FEMS
Gas Unit Result Method Microbiol. Ecol., no. 16, pp. 177-184, 1995.
N2 % GC-Supe;co chromatography Nutchanat Chamchoi, Suwanchai Nitisoravut:
99.84 product, p. 767,1995-Alltech, Anammox enrichment from different conventional
chromatography catalog 350, p.24. sludges, ScienceDirect, no. 66, pp. 2225-2232, 2007.
Schmid M., Twachtmann U., Klein M., Strous M.,
(Center for Analysis Service of Experiment in Ho Chi
Juretschko S., Jetten M. S. M., Metzger J., Schleifer K.,
Minh City)
Wagner M.: Molecular evidence for genus level diversity
of bacteria capable of catalyzing anaerobic ammonium
3.2. Discussions
oxidation, Systematic and Applied Microbiology, no. 23,
After 210 days of operation of the first reactor with
pp. 93106, 2000.
synthetic medium, the N-NH4 conversion and N-NO2
Schmid, M., Walsh, K., Webb, R., Rijpstra, W.I.C.,
removal efficiency gradually increased from 0 to 74.3%
van de Pas-Schoonen, K., Verbruggen, M.J. et al.:
and from 0 to 79.8%, respectively.
Candidatus "Scalindua brodae", sp nov., Candidatus
After 160 days of operation of the second reactor
"Scalindua wagneri", sp nov., two new species of
with swine wastewater, the N-NH4 conversion and N-
anaerobic ammonium oxidizing bacteria. Systematic and
NO2 removal efficiency also gradually increased from 0
Applied Microbiology, no. 26, pp. 529-538, 2003.
to 80.1% and from 0 to 83.1%, respectively.
van de Graaf, A.A., de Druijn, P., Robertson, L.A
SS in both reactors decreased, from 6,200 to 1,260
Jetten, M.S.M., & Kuenen, J.G.: Autotrophic growth of
mg/l in the first reactor and from 6,200 to 1,350 mg/l in
anaerobic, ammonium-oxidizing microorganisms in a
the second reactor. The sludge color changed from black
fluidized bed reactor. Microbiology no. 142, pp. 2187-
to sepia. This is a distinguish signal of Anammox
2196, 1996.
bacteria, as studied in previous researches (Schmidt et
Van Dongen L.G.J.M., Jetten M.S.M. & Van
al., 2000, Van Dongen et al., 2001).
Loosdrecht M.C.M.,: The Combined Sharon /
Anammox process for treatment of ammonium rich
4. CONCLUSIONS
wastewater, Water Science and Technology, no. 44,
Sludge taken from the septic tank of soy sauce
pp.153-160, 2001.
wastewater treatment plant, after enriched with synthetic
medium and swine wastewater, has accumulated
Anammox bacteria with specific sepia color.
Enrichment time in reactor run with swine
wastewater shorter than that in reactor operated with
synthetic medium.
Anammox bacteria which existed in soy sauce
wastewater treatment plant had a good adaptation at
ammonium concentrations in a range of 50 200 mg/l.
Nitrogen removal efficiency reached 79.8% and 83.1%
in synthetic medium and swine wastewater, respectively.
Nitrogen presented in sample gas was 99.3% and 99.8%,
in synthetic medium and swine wastewater, respectively
respectively.
With the result achieved, Anammox process can be
surely applied to a swine wastewater treatment plant as
well as other ammonium rich wastewater treatment
plants in practice.
The schematic proposed for ammonium rich
wastewater treatment plants is described as below:
Inf. primary treatment UASB partial
nitritation Anammox reactor aerotank clarifier
filtering disinfection water bodies.

REFERENCES
Mulder, A., van de Graaf, A.A., Robertson, L.A.,
Kuenen, J.G.: Anaerobic ammonium oxidation