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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 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 NH 4 + -N:NO 2 - -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 NH 4 + -N and 200 mg/l of NO 2 - -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.

The main process of aerotank is conversion from 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 N 2 , but about 10% of the N-feed is converted to NO 3 - . The overall nitrogen balance gave a ratio of NH 4 + conversion to NO 2 - conversion and NO 3 - 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

The sludge used in this study was taken from an anaerobic sludge digester of soy sauce wastewater treatment plant of Chinsu Foods - Masan Group (Tan Binh Industrial park). Conduct testing SS (suspended solids), MLSS (mixed liquid suspended solids) followed by filtering to remove sands, litters. The sludge was then incubated in a dark room and supplied with substrates in an adapted medium, just after 4 days of incubation, the top water was poured out to remove mold, scum, grease and other organic compounds. After 20 days of incubation, a lot of gas escaped from the sludge as

observed, such sludge can be used for UASB model to accelerate the adaptation process. The sludge at that

moment was black color with very fine granules, SS was 11.6 g/l, which would be used for Anammox enrichment.

2.2. Anammox enrichment medium

The first medium was synthetic with the components showed in Table 1. Table 1. Characteristics of synthetic mediums used for Anammox enrichment

Ingredient

Concentration

N-NH 4 (NH 4 Cl) N-NO 2 (NaNO 2 ) KHCO 3 KH 2 PO 4 CaCl 2 .2H 2 O MgSO 4 .7H 2 O FeSO 4 .7H 2 O

50; 100;150;200 mg/l

50; 100; 150; 200 mg/l

50

250 mg/l

27

100 mg/l

180

mg/l

120

mg/l

9 mg/l

EDTA2Na

5mg/l

Na 2 S. 9H 2 O

< 125 mg/l

(Van de Graaf et al.,1996) The second medium was swine waste water after biogas reactor of Dong A pig breeding enterprise (Di An district, Binh Duong province). Properties of swine is indicated in Table 2. Table 2. Characteristics of swine waste water after biogas reactor

Parameter

Value

pH

DO

COD

BOD 5

N-NH

N-NO

N-NO

6.8 8.5 mg/l 0 0.2 mg/l 1,800 3,200 mg/l 1,000 1,800 mg/l 200 800 mg/l 0 5 mg/l 3 15 mg/l 30 80 mg/l 1,500 4,200 mg/l

4

2

3

 

Total P

SS

2.3.

Experimental set-up

The experiment to enrich Anammox bacteria were operated with two parallel models were designed as

UASB (Upflow Anaerobic Sludge Blanket). Each model include the following components:

- An feeding water tank with a capacity of 45 liters.

- A dosing pump to feed wastewater from the tank into the UASB column.

- An UASB reactor made of gray PVC, cylindrical, with a height of 1 meter, diameter of 0.116 meter, useful volume of 10 liters. Along the height of the column 6 sampling valves with a distance of 0.015 meter were attached. At the center of the reactor bottom, inlet valve was securely attached to the hopper spraying inside to increase the disturbance and mix wastewater and sludge. At the top the the reactor, a serrated hopper was installed and connected to the gas collector by plastic pipes.

- A gas collector made of plastic PE, 5 liter capacity air receiver.

- A tank for receiving effluent with capacity of 45 liters.

- Speed of water surges: 0.042 m / h

- Flowrate: 10 liters / day

Eff. Feeding Gas collector water Dosing Pump
Eff.
Feeding
Gas collector
water
Dosing
Pump

Figure 1. Experimental model

2.4. Operation of reactor The models were operated at the Institute of Tropical Biology - Linh Trung Ward - Thu Duc District - Ho Chi Minh city. The volume of sludge used to insert into the reactor type was calculated relating to the mixed volume in the model as the following formula:

V sludge * SS sludge = V reactor * MLSS

Where:

V sludge : sludge volume needed for the reactor (liter) SS sludge : VSS of sludge (mg / liter) V reactor : UASB reactor volume (liter) The initial volume of sludge to be inserted into each reactor was: 2.9 liters, equivalent to 33% volume of the reactor. The sludge was poured into the reactors after 20 days of incubation

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The first reactor was operated with synthetic medium, within 210 days. The model was run in 4 phases with the data illustrated in Table 3. Table 3. Operation of reactor with synthetic medium

Days

N-NH4

N-NO2

pH

(mg/l)

(mg/l)

80

50

50

80.2

120

100

100

80.2

180

150

150

80.2

210

200

200

80.2

The second model was operated with swine wastewater, within 160 days. The model was also run in 4 phases with the data illustrated in Table 4. Table 4. Operation of reactor with swine waste water

Days

N-NH4

N-NO2

pH

(mg/l)

(mg/l)

60

50

50

7.40.2

100

100

100

7.40.2

130

150

150

7.40.2

160

200

200

7.40.2

N-NO 2 concentrations in Table 4 were added to the reactor by using NaNO 2 as used in Table 1 to form N- NH 4 : N-NO 2 ratio at 1:1. Growth of microorganisms, volume of generated gas were observed and outlet samples were tested in the laboratory with parameters including N-NH 4 , N-NO 2 , N- NO 3 , COD, SS, pH. 2.5. Analytical methods and calculations The analytical methods for parameter N-NH 4 , N- NO 2 , N-NO 3 , COD, pH were presented in the Table 5. Table 5. Analytical methods

Parameter

Method

pH

Electrode US EPA Nessler method, maximum absorbance wavelength = 430 nm Colormetric method with Griss agent, maximum absorbance wavelength = 520 nm Colormetric method with phenoldisulfonic acid, maximum absorbance wavelength = 430 nm Potassium permanganate acidic method Gravimetric analysis method

N-NH

4

N-NO

2

N-NO

3

COD

SS

3. RESULTS AND DISCUSSIONS 3.1. Results of the first reactor run with synthetic medium

Table 6. Efficiency of ammonium conversion and nitrite removal

Days

Efficiency of N-NH4 conversion (%)

Efficiency of N-NO2 removal (%)

80

11.7

14.0

120

21.4

23.5

180

52.1

54.2

210

78.1

79.8

In the first 80 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 low. The efficiency reached higher value from the 170th day of operation. The change of the efficiency was presented in Figure 2.

% 90 80 70 60 50 N-NO2 40 N-NH4 30 20 10 0 80 days
%
90
80
70
60
50
N-NO2
40
N-NH4
30
20
10
0
80
days
120
days
180
days
210
days
Time
50
mg/l
100
mg/l
150
mg/l
200
mg/l

Figure 2. Efficiency of N-NH 4 conversion and N-NO 2 removal with synthetic medium During operation time, besides N-NH 4 conversion and N-NO 2 removal, a small amout of N-NO 3 was produced, COD was reduced. Gas production and change of sludge color were also obviously seen. The change of Anammox sludge color was described in Figure 3. (microscope Olympus CH30, objective lens x 10)

sludge color was described in Figure 3. (microscope Olympus CH30, objective lens x 10) Initial sludge

Initial sludge

sludge color was described in Figure 3. (microscope Olympus CH30, objective lens x 10) Initial sludge

After 80 days

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The 11th SEATUC Symposium After 120 days After 210 days Figure 3. Change of color of

After 120 days

The 11th SEATUC Symposium After 120 days After 210 days Figure 3. Change of color of

After 210 days

Figure 3. Change of color of sludge operated with synthetic medium Table 7. COD reduction, N-NO 3 generation and change of sludge operated with synthetic medium

Time

SS

N-NO3

% COD

Color of

Gas

(day)

(mg/l)

(mg/l)

reduction

sludge

volume

 

(mg/l)

(l/d)

80

6,200

0.5

1.8

Black

0.05

120

5,500

1.3

17.9

Black

0.50

180

3,600

4.5

9.8

Brown

0.65

210

1,260

5.3

18.7

Maroon

1.25

When N-NH 4 conversion efficiency was over than 75%, meaning Anammox reaction took place, gas samples were collected and sent to analysis laboratory. Table 8. Result of gas sample analysis

Gas

Unit

Result

Method

N 2

%

99.3

GC-Supe;co chromatography product, p. 767, 1995-Alltech, chromatography catalog 350, p.24.

(Center for Analysis Service of Experiment in Ho Chi Minh City) 3.2. Results of the second reactor run with swine wastewater Table 9. Efficiency of ammonium conversion and nitrite removal

Days

Efficiency of N-NH4 conversion (%)

Efficiency of N-NO2 removal (%)

60

17.5

21.6

100

33.5

43.9

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 low. The efficiency reached higher value from the 170th day of operation. The change of the efficiency was presented in Figure 5.

% 90 80 70 60 50 N-NO2 40 N-NH4 30 20 10 0 Time 60
%
90
80
70
60
50
N-NO2
40
N-NH4
30
20
10
0
Time
60
days
100
days
130
days
160
days
50
mg/l
100
mg/l
150
mg/l
200
mg/l

Figure 5. Efficiency of N-NH 4 conversion and N-NO 2 removal with swine wastewater During operation time, besides of N-NH 4 conversion and N-NO 2 removal, a small amout of N-NO 3 was produced, COD was reduced. Gas production and change of sludge color were also obviously seen.

Table 10. COD reduction, N-NO 3 generation and change of sludge operated with swine wastewater

Time

SS

N-NO3

% COD

Color of

Gas

(day)

(mg/l)

(mg/l)

reduction

sludge

volume

 

(mg/l)

(l/d)

60

6,200

0.7

2.9

Black

0.10

100

5,400

1.1

14.3

Black

0.50

130

3,200

3.6

13.7

Brown

0.75

160

1,350

5.8

25.5

Maroon

1.30

The change of Anammox sludge color was described in Figure 6. (microscope Olympus CH30, objective lens x 10)

in Figure 6. (microscope Olympus CH30, objective lens x 10) Initial sludge After 120 days After

Initial sludge

Olympus CH30, objective lens x 10) Initial sludge After 120 days After 80 days After 210

After 120 days

CH30, objective lens x 10) Initial sludge After 120 days After 80 days After 210 days

After 80 days

lens x 10) Initial sludge After 120 days After 80 days After 210 days Figure 6.

After 210 days

Figure 6. Change of color of sludge operated with swine wastewater When N-NH 4 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

Gas

Unit

Result

Method

N 2

%

GC-Supe;co chromatography

 

99.84

product, p. 767,1995-Alltech, chromatography catalog 350, p.24.

(Center for Analysis Service of Experiment in Ho Chi Minh City)

3.2. Discussions After 210 days of operation of the first reactor with synthetic medium, the N-NH 4 conversion and N-NO 2 removal efficiency gradually increased from 0 to 74.3% and from 0 to 79.8%, respectively. After 160 days of operation of the second reactor with swine wastewater, the N-NH 4 conversion and N- NO 2 removal efficiency also gradually increased from 0 to 80.1% and from 0 to 83.1%, respectively. SS in both reactors decreased, from 6,200 to 1,260 mg/l in the first reactor and from 6,200 to 1,350 mg/l in the second reactor. The sludge color changed from black to sepia. This is a distinguish signal of Anammox bacteria, as studied in previous researches (Schmidt et al., 2000, Van Dongen et al., 2001).

4. CONCLUSIONS Sludge taken from the septic tank of soy sauce 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

discovered in a denitrifying fluidized reactor, FEMS Microbiol. Ecol., no. 16, pp. 177-184, 1995. Nutchanat Chamchoi, Suwanchai Nitisoravut:

Anammox enrichment from different conventional sludges, ScienceDirect, no. 66, pp. 2225-2232, 2007. Schmid M., Twachtmann U., Klein M., Strous M., Juretschko S., Jetten M. S. M., Metzger J., Schleifer K., Wagner M.: Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation, Systematic and Applied Microbiology, no. 23, pp. 93106, 2000. Schmid, M., Walsh, K., Webb, R., Rijpstra, W.I.C., van de Pas-Schoonen, K., Verbruggen, M.J. et al.:

Candidatus "Scalindua brodae", sp nov., Candidatus "Scalindua wagneri", sp nov., two new species of anaerobic ammonium oxidizing bacteria. Systematic and Applied Microbiology, no. 26, pp. 529-538, 2003. van de Graaf, A.A., de Druijn, P., Robertson, L.A Jetten, M.S.M., & Kuenen, J.G.: Autotrophic growth of anaerobic, ammonium-oxidizing microorganisms in a fluidized bed reactor. Microbiology no. 142, pp. 2187- 2196, 1996. Van Dongen L.G.J.M., Jetten M.S.M. & Van Loosdrecht M.C.M.,: “The Combined Sharon / Anammox process for treatment of ammonium rich wastewater,” Water Science and Technology, no. 44, pp.153-160, 2001.