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© IWA Publishing 2014 Water Science & Technology
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Nitrate removal from groundwater by hydrogen-fed autotrophic denitrification in a bio-ceramsite reactor Dan Chen, Kai Yang, Hongyu Wang and Bin Lv
ABSTRACT In this work, the denitrification performance of a bio-ceramsite reactor based on autohydrogenotrophic denitrification was investigated. The effects of various experimental parameters such as nitrate loading, carbon to nitrogen ratio (C/N), water temperature and pH were evaluated during the operation. The unique aspect of this research is that the bio-reactor uses ceramsite as a carrier, which can provide a habitat for autohydrogenotrophic biocoenoses to accrete and grow. The results indicated that the denitrification rate increased as nitrate loading (below 130 mg
NO 3 -N/L)
increased. However, the activity of autohydrogenotrophic denitrifying bacteria was
inhibited when nitrate loading was further increased to higher than 130 mg NO 3 -N/L. Denitrification
Dan Chen Kai Yang Hongyu Wang (corresponding author) School of Civil Engineering, Wuhan University, Wuhan 430072, China E-mail: [email protected] Bin Lv School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China
efficiency changed slightly with C/N, this system performed well if C/N was more than 0.9. The W
optimum temperature for the reactor was 25–35 C. This denitrification system was positively related to pH, as a neutral or alkaline environment was more preferable for the reactor. During the operation, effluent nitrite levels were always maintained below 1.75 mg NO 2 -N/L. Key words
| autohydrogenotrophic denitrifying bacteria, bio-ceramsite reactor, denitrification efficiency, nitrate
INTRODUCTION With the rapid development of industrialization and the extensive use of synthetic fertilizers in intensive agriculture, nitrate contamination in groundwater has increased in many countries over years (Jiang et al. ). For instance, the United States and some European countries’ groundwater nitrate pollution level has increased to 200 mg/L. Nitrate contamination in groundwater is threatening the health of humans by causing methemoglobinemia and stomach cancer (Wan et al. ). Currently, conventional nitrate removal methods include biological, chemical and physical-chemical methods. Although physical-chemical methods like electrodialysis and ion exchange are simple and easy to control, these methods are associated with drawbacks, such as high capital and operating costs (Ghafari et al. ; Xia et al. ), and the toxic by-products require further disposal. Because of its high efficiency and low pollution, biological denitrification is considered to be one of the most economical and promising approaches (Mousavi et al. ) to treat nitrate-polluted water. Biological denitrification can be involved in autotrophic and heterotrophic methods. Hydrogen-fed autotrophic denitrification based on an inorganic carbon doi: 10.2166/wst.2014.167
source, involves hydrogen as the electron donor, and nitrate as the electron accepter for the bacterial metabolic chain (Tang et al. ). Since hydrogen is naturally clean and harmless, and does not require subsequent treatment processes (Hwang et al. ), hydrogenotrophic denitrifiers become a promising approach for nitrate removal. In this work, an H2-fed bio-ceramsite reactor has been developed to treat nitrate-polluted groundwater. The purpose of this work is to investigate the performance of autohydrogenotrophic denitrifying bacteria in this system under different conditions containing nitrate loading, pH, temperature and carbon to nitrogen ratio (C/N). The results of this research will offer a theory and technical support to autohydrogenotrophic denitrification for groundwater polluted by nitrate.
MATERIAL AND METHODS Experimental set-up A schematic of the reactor used in this work is shown in Figure 1. The total volume (total height 100 cm, diameter
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Domestication of autohydrogenotrophic denitrifying microflora
Figure 1
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Schematic of hydrogen-fed bio-ceramsite reactor.
Start-up of the bio-ceramsite reactor was initiated by seeding with anaerobic activated sludge from an anaerobic tank of Erlangmiao Municipal Wastewater Treatment Plant in Wuhan, China. At the beginning of start-up, all the ceramsite must be covered with anaerobic sludge. The synthetic sludge as bacterial seed was cultured for 2 days, then the system was changed to continuous flow. Meanwhile, sufficient hydrogen must be introduced into the reactor. The domestication of bacteria was accomplished when the denitrification rate reached 80% and a compacted biofilm formed on the surface of the ceramsite after 3 weeks. The mixed liquor suspended solids (MLSS) concentration and total organic carbon (TOC) content of the inoculum were 8.60 and 3.14 mg/L, respectively. The MLSS content of the effluent was 13.26 mg/L, and the TOC content of the effluent was 6.87 mg/L. In the process of the operation, sodium bicarbonate (inorganic carbon source) sufficient for the growth of microorganisms was added to the reactor. Analytical methods
11 cm) of the reactor, which is made of organic glass, is 6.2 L. The working volume of the reactor is 2.3 L. The height of the packing filled with 3–5 mm ceramsite is 50 cm. During the experiment, synthetic wastewater was pumped to the bottom of the continuous flow reactor. After making a good contact with the biofilm, it would flow out of the top of the reactor. An intermittent hydrogen supply system had been installed at the bottom of the ceramsite in order to maintain constant hydrogen pressure in the bio-reactor. H2 was introduced to this reactor, and the efficiency of H2 was 15.62%. This system was sealed so that the reactor was under anoxic conditions. The dissolved oxygen (DO) concentration in the reactor was less than 0.3 mg/L during the operation.
On a UV-Visible spectrophotometer (nanbeijt, China), nitrate (NO 3 -N) and nitrite (NO2 -N), were measured using ultraviolet spectrophotometry, spectrophotometry based on N-(1-naphthyl) ethylenediamine dihydrochloride, respectively. The pH in influent and effluent was measured by pH meter (PC-320), and DO and water temperature in the reactor were measured by YSI550A DO meter. TOC concentration was determined by TOC analyzer (TOCVCPH). The microbial biomass was monitored by OD600. The cell mass was counted by Acridine orange direct count.
RESULTS AND DISCUSSION Denitrification effect under different nitrate loadings
Synthetic influent water The composition of synthetic wastewater was (g/L): KH2PO4 0.975, NaHCO3 7.5, and piped water, pH 7. Nitrate loading ranged from 30 to 130 mg NO 3 -N/L. The composition of the microelement was (mg/L): ZnCl2 0.52, CoCl2 · 6H2O 1.90, MnSO4 · 7H2O 1.00, MgCl2 · 6H2O 0.25, NiCl2 · 6H2O 0.24, CuCl2 · 2H2O 0.29, FeSO4 · 7H2O 0.25, CaCl2 · 6H2O 0.50, Na2MoO4 · 2H2O 0.36, and H3BO3 0.30.
The effect of nitrate loading on nitrate reduction is apparent in Figure 2. The applied nitrate loading varied from 30 to 150 mg NO 3 -N/L, with hydraulic retention time (HRT), H2 pressure, C/N, water temperature, and pH at steadystate values of 24 h, 0.01 MPa, 1.0, 25 C, and 7, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. W
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Figure 2
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Nitrate removal by hydrogen-fed autotrophic denitrification
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different nitrate
loading conditions.
It was observed that the denitrification rate increased as initial nitrate loading (below 130 mg NO 3 -N/L) increased. When initial nitrate loading was further increased to 150 mg NO 3 -N/L, the denitrification rate was relatively low at the initial phase. Then, the highest denitrification rate (7.41 mg NO 3 -N/L/h) was observed in the reactor when nitrate loading was reduced to 130 mg NO 3 -N/L. This showed that denitrification was inhibited when nitrate loading was higher than 130 mg/L in the reactor. During the operation, there was no nitrite accumulated in the treated water and the nitrite level ranged from 0.06 to 1.40 mg NO 2 -N/L. Low nitrate reductase activity conflicted with the rapid decrease of nitrate concentration (Deiglmayr et al. ).
Figure 3
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The denitrification was found to be inhibited at high nitrate concentration. In this work, the denitrification rate was slower when nitrate loading was higher than 130 mg NO 3 -N/L. The reason for this might be that the activity of autohydrogenotrophic denitrifying bacteria was suppressed under this environment. Nevertheless, nitrite was not accumulated and the level was below 1.40 mg NO 2 -N/L. As a consequence, there might be two kinds of microflora in this hydrogenotrophic denitrification system. One was nitrate-reducing bacteria (these bacteria convert NO 3 -N to NO 2 -N, using NO3 -N as electron acceptor, and H2 as electron donor (Equation (1)), which could only reduce nitrate to nitrite. The other was nitrite-reducing bacteria (these bac teria convert NO 2 -N to N2, using NO2 -N as electron acceptor, and H2 as electron donor (Equation (2)), which reduce nitrite to nitrogen (Ghafari et al. ). For this reason, nitrite has been observed to accumulate, most probably because the nitrite reduction rate was too low to catch up with the production rate of nitrite (Wilderer et al. ). In this work, nitrite was not accumulated, due to the two rates having obvious difference between them so that the intermediate NO 2 -N could be timeously converted into N2 NO 3 þ H2 ! NO2 þ H2 O
(1)
þ 2NO 2 þ 3H2 þ 2H ! N2 þ 4H2 O
(2)
Denitrification effect under different C/Ns The effect of C/N on nitrate reduction is shown in Figure 3. The applied C/N varied from 0.3 to 2.2, with HRT, H2 pressure, pH, water temperature, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 7, 25 C, W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different C/N conditions (SSA (Sum of Squares for factor A(C/N)) ¼ 170.87, SSE (Sum of Squares for Error) ¼ 22.83,
SST (Sum of Squares for Total) ¼ 193.70, F (level of significance) ¼ 59.87 > Fcrit).
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and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiency changed slightly with C/N in the reactor. Denitrification efficiencies were 89.32% ± 0.04%, 95.88% ± 0.01%, 93.85% ± 0.03% and 94.59% ± 0.02% when C/N were 0.3, 0.9, 1.5 and 2.2, respectively. Denitrification efficiency fell by 6.56% with C/N reduced from 0.9 to 0.3 due to the fact that C/N of 0.3 was not sufficient for bacteria to grow (OD600 reduced from 0.032 to 0.020 when C/N reduced from 0.9 to 0.3). Ghafari et al. () observed that bicarbonate was the effective carbon source for a faster growth and adaption of autohydrogenotrophic denitrifying bacteria. In addition, Ghafari et al. () reported that the optimum bicarbonate concentration was 1,100 mg NaHCO3/L for an initial nitrate concentration of 20 mg NO 3 -N/L. Chen & Lin () defined the theoretical stoichiometric equations for denitrification and the theoretical C/N ratios were established as 0.71. Similar research showed C/N ¼ 1.1 (Gómez et al. ) and 2.2 (Fan et al. ) for optimum denitrification. In the present research, the bacteria could grow well if C/N was more than 0.9. If C/N was greater than the theoretical value, the extra consumption of an inorganic carbon source could participate in microbe assimilation, which cause biomass growth (cell mass increased from 31 × 106 to 57 × 106 cfu/mL when C/N increased from 0.9 to 2.2) in the bio-ceramsite reactor. Moreover, the effluent nitrite level ranged from 0.08 to 0.83 mg NO 2 -N/L.
Figure 4
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Denitrification effect under different water temperatures Figure 4 shows the results for the effect of water temperature on nitrate reduction. The applied water temperature varied from 15 to 30 C, with HRT, H2 pressure, C/N, pH, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 1.0, 7, and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiency decreased as water temperature increased. Denitrification efficiencies were 94.17% ± 0.02% and 96.21% ± 0.01% at the temperature of 25 and 30 C. As water temperature reduced to 20 and 15 C, the reduction rate of nitrate decreased to 75.04% ± 0.03% and 73.43% ± 0.01%, respectively. This showed that the higher temperatures used allowed better performance of the hydrogenotrophic cultures (Vasiliadou et al. ). During the operation, the effluent nitrite level ranged from 0.13 to 0.90 mg NO 2 -N/L. Due to denitrifying bacteria’s capacity to survive in extreme environmental conditions, denitrification processes can occur in the range of 2–50 C (Brady & Weil ). Some of the temperature values applied in studies on hydrogenotrophic denitrification varied between 10 and 30 C (Karanasios et al. ). Another study reported by Rezania et al. (a, b) showed that the denitrification rate increased as temperature increased from 12 to 25 C. In the research of Kurt et al. (), experimental evidence suggested that temperature affected the denitrification process by affecting bacteria behavior. In the present work, denitrification efficiency always maintained a high level W
W
W
W
W
W
W
W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different water temperature conditions (SSB (Sum of Squares for factor B (temperature)) ¼ 3097.55, SSE (Sum of
Squares for Error) ¼ 33.66, SST (Sum of Squares for Total) ¼ 3131.22, F (level of significance) ¼ 736.15 > Fcrit).
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W
above 70% with a water temperature range of 15–30 C. The reason might be that these bacteria were not severely sensitive to water temperature in the bio-reactor. No matter how the water temperature changed, the autohydrogenotrophic denitrifying bacteria grew well (OD600 were 0.038, 0.036, 0.041, 0.042 when temperatures were 15 , 20 , 25 , 30 C, respectively). Therefore, this denitrification system has an excellent ability to resist temperature change. W
W
W
W
Denitrification effect under different pH The effect of pH on this system is obvious, as can be seen in Figure 5. The applied pH values varied from 6 to 9, with HRT, H2 pressure, C/N, water temperature, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 1.0, 25 C, and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiencies were 94.86% ± 0.03% and 95.93% ± 0.02% at pH 7 and 8, respectively. This showed that the system worked well under this environment. However, the reduction rate of nitrate decreased to 75.06% ± 0.01% as pH increased to 9. Furthermore, the efficiency decreased to 81.89% ± 0.03% with a pH of 6. The results illustrated that denitrification was excellent under neutral or alkalescent environments. The effluent nitrite levels were always maintained below 1.75 mg NO 2 -N/L during the operation. The hydrogenotrophic denitrification process is positively related to pH, with an optimum value range of 7.6–8.6 (Lee & Rittmann ). Another study by Xia et al. () showed that the optimum pH for autotrophic denitrification was 7.2–8.2,
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with the maximum efficiency at pH 7.7. Ho et al. () demonstrated that nitrate could be reduced effectively with no nitrite accumulation when carbon dioxide was applied, while the pH of the bio-reactor remained at 7. In this work, a low pH value (6) inhibited denitrification because the decomposition of carbonate ion stripping could strongly affect the hydrogenotrophic denitrification process. Moreover, a high pH value (9) could cause a significant decrease in the nitrate removal rate due to the fact that the activity of nitrate reductase and nitrite reductase might be inhibited (Rezania et al. a, b). For this reason, it was necessary to keep the pH at 7–8 to obtain steady effluent quality.
W
Figure 5
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CONCLUSIONS In this work, an H2-fed bio-ceramsite reactor has been developed, in which autohydrogenotrophic denitrifying bacteria use ceramsite as a carrier and forms a biofilm on the surface of the ceramsite in order to treat nitrate contaminated groundwater. The performance of this system was investigated using laboratory-scale apparatus. The highest denitrification rate (7.41 mg NO 3 -N/L/h) was observed at nitrate loading of 130 mg NO 3 -N/L. However, that this system was inhibited when nitrate loading was higher than 130 mg NO 3 -N/L. C/N was not a significant factor for the reactor. The reactor could perform well if the carbon source was sufficient for the growth of the denitrifying bacteria. This denitrification system has an excellent ability to resist temperature change, and the optimum temperature was 25–35 C. Nitrate reduction was significantly affected by pH in the reactor, and the optimum pH was 7–8. It was W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different pH conditions (SSC (Sum of Squares for factor C(pH)) ¼ 2171.36, SSE (Sum of Squares for Error) ¼ 14.64,
SST (Sum of Squares for Total) ¼ 2186.00, F (level of significance) ¼ 1186.41 > Fcrit).
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demonstrated that this H2-fed bio-ceramsite reactor was effective for the treatment of nitrate-polluted groundwater.
ACKNOWLEDGEMENTS This work was financially supported by the open fund of the State Key Lab of Urban Water Resources and Environment (HIT) (No. QA200810, QAK201014); National Natural Science Foundation of China (NSFC) (No. 51008239, 51378400); Natural Science Foundation of Hubei Province, China (No. 2013CFB289, 2013CFB308); and Major Science and Technology Program for Water Pollution Control and Treatment (No. 2009ZX07317-008-003).
REFERENCES Brady, N. C. & Weil, R. R. The Nature and Properties of Soils. 13th edn, Prentice Hall, New Jersey. Chen, K. C. & Lin, Y. F. Relationship between denitrifying bacteria and methanogenic bacteria in a mixed culture system of acclimated sludges. Water Res. 27, 1749–1759. Deiglmayr, K., Philippot, L. & Kandeler, E. Functional stability of the nitrate-reducing community in grassland soils towards high nitrate supply. Soil Biol. Biochem. 38, 2980–2984. Fan, B., Qu, J. H., Lei, P. J. & Li, D. P. Studies on nitrate removal by a combined heterotrophy electrode biofilm reactor. Acta Scientiae Circumstantiae 21, 257–262. Ghafari, S., Hasan, M. & Aroua, M. K. Bio-electrochemical removal of nitrate from water and wastewater: A review. Bioresour. Technol. 99, 3965–3974. Ghafari, S., Hasan, M. & Aroua, M. K. Effect of carbon dioxide and bicarbonate as inorganic carbon sources on growth and adaptation of autohydrogenotrophic denitrifying bacteria. J. Hazard. Mater. 162, 1507–1513. Ghafari, S., Hasan, M. & Aroua, M. K. A kinetic study of autohydrogenotrophic denitrification at the optimum pH and sodium bicarbonate dose. Bioresour. Technol. 101, 2236–2242. Gómez, M. A., González-López, J. & Hontoria-García, E. Influence of carbon source on nitrate removal of contaminated groundwater in a denitrifying submerged filter. J. Hazard. Mater. 80, 69–80.
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Ho, C. M., Tseng, S. K. & Chang, Y. J. Autotrophic denitrification via a novel membrane-attached biofilm reactor. Lett. Appl. Microbiol. 33, 201–205. Hwang, J. H., Cicek, N. & Oleszkiewicz, J. A. Achieving biofilm control in a membrane biofilm reactor removing total nitrogen. Water Res. 44, 2283–2291. Jiang, W., Qu, J. H., Lei, P. J., Liu, S. X. & Meng, G. H. Nitrate nitrogen removal from ground water by autotrophic denitrification in a packed bed reactor. China Environ. Sci. 21, 133–136. Karanasios, K. A., Vasiliadou, I. A., Pavlou, S. & Vayenas, D. V. Hydrogenotrophic denitrification of potable water: a review. J. Hazard. Mater. 180, 20–37. Kurt, M., Dunn, I. J. & Bourne, J. R. Biological denitrification of drinking water using autotrophic organisms with H2 in a fluidized-bed biofilm reactor. Biotechnol. Bioeng. 29, 493–501. Lee, K. C. & Rittmann, B. E. Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollowfiber membrane-biofilm reactor. Water Res. 37, 1551–1556. Mousavi, S., Ibrahim, S., Aroua, M. K. & Ghafari, S. Development of nitrate elimination by autohydrogenotrophic bacteria in bio-electrochemical reactors – A review. Biochem. Eng. J. 67, 251–264. Rezania, B., Cicek, N. & Oleszkiewicz, J. A. a Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions. Biotechnol. Bioeng. 92, 900–906. Rezania, B., Oleszkiewicz, J. A., Cicek, N. & Mo, H. b Hydrogen-dependent denitrification in an alternating anoxicaerobic SBR membrane bio-reactor. Water Sci. Technol. 51, 403–409. Tang, Y. N., Zhou, C., Ziv-El, M. & Rittmann, B. E. A pH-control model for heterotrophic and hydrogen-based autotrophic denitrification. Water Res. 45, 232–240. Vasiliadou, I. A., Karanasios, K. A., Pavlou, S. & Vayenas, D. V. Experimental and modelling study of drinking water hydrogenotrophic denitrification in packed-bed reactors. J. Hazard. Mater. 165, 812–824. Wan, D. J., Liu, H. J., Liu, R. P. & Qu, J. H. Study of a combined sulfur autotrophic with proton-exchange membrane electrodialytic denitrification technology: Sulfate control and pH balance. Bioresource Technol. 102, 10803–10809. Wilderer, P., Jones, W. & Dau, U. Competition in denitrification systems affecting reduction rate and accumulation of nitrite. Water Res. 21, 239–245. Xia, S. Q., Zhong, F. H., Zhang, Y. H., Li, H. & Yang, X. Bio-reduction of nitrate from groundwater using a hydrogenbased membrane biofilm reactor. J. Environ. Sci. 22, 257–262.
First received 4 December 2013; accepted in revised form 17 March 2014. Available online 29 March 2014
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© IWA Publishing 2014 Water Science & Technology
|
69.12
|
2014
Nitrate removal from groundwater by hydrogen-fed autotrophic denitrification in a bio-ceramsite reactor Dan Chen, Kai Yang, Hongyu Wang and Bin Lv
ABSTRACT In this work, the denitrification performance of a bio-ceramsite reactor based on autohydrogenotrophic denitrification was investigated. The effects of various experimental parameters such as nitrate loading, carbon to nitrogen ratio (C/N), water temperature and pH were evaluated during the operation. The unique aspect of this research is that the bio-reactor uses ceramsite as a carrier, which can provide a habitat for autohydrogenotrophic biocoenoses to accrete and grow. The results indicated that the denitrification rate increased as nitrate loading (below 130 mg
NO 3 -N/L)
increased. However, the activity of autohydrogenotrophic denitrifying bacteria was
inhibited when nitrate loading was further increased to higher than 130 mg NO 3 -N/L. Denitrification
Dan Chen Kai Yang Hongyu Wang (corresponding author) School of Civil Engineering, Wuhan University, Wuhan 430072, China E-mail: [email protected] Bin Lv School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China
efficiency changed slightly with C/N, this system performed well if C/N was more than 0.9. The W
optimum temperature for the reactor was 25–35 C. This denitrification system was positively related to pH, as a neutral or alkaline environment was more preferable for the reactor. During the operation, effluent nitrite levels were always maintained below 1.75 mg NO 2 -N/L. Key words
| autohydrogenotrophic denitrifying bacteria, bio-ceramsite reactor, denitrification efficiency, nitrate
INTRODUCTION With the rapid development of industrialization and the extensive use of synthetic fertilizers in intensive agriculture, nitrate contamination in groundwater has increased in many countries over years (Jiang et al. ). For instance, the United States and some European countries’ groundwater nitrate pollution level has increased to 200 mg/L. Nitrate contamination in groundwater is threatening the health of humans by causing methemoglobinemia and stomach cancer (Wan et al. ). Currently, conventional nitrate removal methods include biological, chemical and physical-chemical methods. Although physical-chemical methods like electrodialysis and ion exchange are simple and easy to control, these methods are associated with drawbacks, such as high capital and operating costs (Ghafari et al. ; Xia et al. ), and the toxic by-products require further disposal. Because of its high efficiency and low pollution, biological denitrification is considered to be one of the most economical and promising approaches (Mousavi et al. ) to treat nitrate-polluted water. Biological denitrification can be involved in autotrophic and heterotrophic methods. Hydrogen-fed autotrophic denitrification based on an inorganic carbon doi: 10.2166/wst.2014.167
source, involves hydrogen as the electron donor, and nitrate as the electron accepter for the bacterial metabolic chain (Tang et al. ). Since hydrogen is naturally clean and harmless, and does not require subsequent treatment processes (Hwang et al. ), hydrogenotrophic denitrifiers become a promising approach for nitrate removal. In this work, an H2-fed bio-ceramsite reactor has been developed to treat nitrate-polluted groundwater. The purpose of this work is to investigate the performance of autohydrogenotrophic denitrifying bacteria in this system under different conditions containing nitrate loading, pH, temperature and carbon to nitrogen ratio (C/N). The results of this research will offer a theory and technical support to autohydrogenotrophic denitrification for groundwater polluted by nitrate.
MATERIAL AND METHODS Experimental set-up A schematic of the reactor used in this work is shown in Figure 1. The total volume (total height 100 cm, diameter
D. Chen et al.
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Nitrate removal by hydrogen-fed autotrophic denitrification
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Domestication of autohydrogenotrophic denitrifying microflora
Figure 1
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Schematic of hydrogen-fed bio-ceramsite reactor.
Start-up of the bio-ceramsite reactor was initiated by seeding with anaerobic activated sludge from an anaerobic tank of Erlangmiao Municipal Wastewater Treatment Plant in Wuhan, China. At the beginning of start-up, all the ceramsite must be covered with anaerobic sludge. The synthetic sludge as bacterial seed was cultured for 2 days, then the system was changed to continuous flow. Meanwhile, sufficient hydrogen must be introduced into the reactor. The domestication of bacteria was accomplished when the denitrification rate reached 80% and a compacted biofilm formed on the surface of the ceramsite after 3 weeks. The mixed liquor suspended solids (MLSS) concentration and total organic carbon (TOC) content of the inoculum were 8.60 and 3.14 mg/L, respectively. The MLSS content of the effluent was 13.26 mg/L, and the TOC content of the effluent was 6.87 mg/L. In the process of the operation, sodium bicarbonate (inorganic carbon source) sufficient for the growth of microorganisms was added to the reactor. Analytical methods
11 cm) of the reactor, which is made of organic glass, is 6.2 L. The working volume of the reactor is 2.3 L. The height of the packing filled with 3–5 mm ceramsite is 50 cm. During the experiment, synthetic wastewater was pumped to the bottom of the continuous flow reactor. After making a good contact with the biofilm, it would flow out of the top of the reactor. An intermittent hydrogen supply system had been installed at the bottom of the ceramsite in order to maintain constant hydrogen pressure in the bio-reactor. H2 was introduced to this reactor, and the efficiency of H2 was 15.62%. This system was sealed so that the reactor was under anoxic conditions. The dissolved oxygen (DO) concentration in the reactor was less than 0.3 mg/L during the operation.
On a UV-Visible spectrophotometer (nanbeijt, China), nitrate (NO 3 -N) and nitrite (NO2 -N), were measured using ultraviolet spectrophotometry, spectrophotometry based on N-(1-naphthyl) ethylenediamine dihydrochloride, respectively. The pH in influent and effluent was measured by pH meter (PC-320), and DO and water temperature in the reactor were measured by YSI550A DO meter. TOC concentration was determined by TOC analyzer (TOCVCPH). The microbial biomass was monitored by OD600. The cell mass was counted by Acridine orange direct count.
RESULTS AND DISCUSSION Denitrification effect under different nitrate loadings
Synthetic influent water The composition of synthetic wastewater was (g/L): KH2PO4 0.975, NaHCO3 7.5, and piped water, pH 7. Nitrate loading ranged from 30 to 130 mg NO 3 -N/L. The composition of the microelement was (mg/L): ZnCl2 0.52, CoCl2 · 6H2O 1.90, MnSO4 · 7H2O 1.00, MgCl2 · 6H2O 0.25, NiCl2 · 6H2O 0.24, CuCl2 · 2H2O 0.29, FeSO4 · 7H2O 0.25, CaCl2 · 6H2O 0.50, Na2MoO4 · 2H2O 0.36, and H3BO3 0.30.
The effect of nitrate loading on nitrate reduction is apparent in Figure 2. The applied nitrate loading varied from 30 to 150 mg NO 3 -N/L, with hydraulic retention time (HRT), H2 pressure, C/N, water temperature, and pH at steadystate values of 24 h, 0.01 MPa, 1.0, 25 C, and 7, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. W
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Figure 2
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Nitrate removal by hydrogen-fed autotrophic denitrification
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different nitrate
loading conditions.
It was observed that the denitrification rate increased as initial nitrate loading (below 130 mg NO 3 -N/L) increased. When initial nitrate loading was further increased to 150 mg NO 3 -N/L, the denitrification rate was relatively low at the initial phase. Then, the highest denitrification rate (7.41 mg NO 3 -N/L/h) was observed in the reactor when nitrate loading was reduced to 130 mg NO 3 -N/L. This showed that denitrification was inhibited when nitrate loading was higher than 130 mg/L in the reactor. During the operation, there was no nitrite accumulated in the treated water and the nitrite level ranged from 0.06 to 1.40 mg NO 2 -N/L. Low nitrate reductase activity conflicted with the rapid decrease of nitrate concentration (Deiglmayr et al. ).
Figure 3
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The denitrification was found to be inhibited at high nitrate concentration. In this work, the denitrification rate was slower when nitrate loading was higher than 130 mg NO 3 -N/L. The reason for this might be that the activity of autohydrogenotrophic denitrifying bacteria was suppressed under this environment. Nevertheless, nitrite was not accumulated and the level was below 1.40 mg NO 2 -N/L. As a consequence, there might be two kinds of microflora in this hydrogenotrophic denitrification system. One was nitrate-reducing bacteria (these bacteria convert NO 3 -N to NO 2 -N, using NO3 -N as electron acceptor, and H2 as electron donor (Equation (1)), which could only reduce nitrate to nitrite. The other was nitrite-reducing bacteria (these bac teria convert NO 2 -N to N2, using NO2 -N as electron acceptor, and H2 as electron donor (Equation (2)), which reduce nitrite to nitrogen (Ghafari et al. ). For this reason, nitrite has been observed to accumulate, most probably because the nitrite reduction rate was too low to catch up with the production rate of nitrite (Wilderer et al. ). In this work, nitrite was not accumulated, due to the two rates having obvious difference between them so that the intermediate NO 2 -N could be timeously converted into N2 NO 3 þ H2 ! NO2 þ H2 O
(1)
þ 2NO 2 þ 3H2 þ 2H ! N2 þ 4H2 O
(2)
Denitrification effect under different C/Ns The effect of C/N on nitrate reduction is shown in Figure 3. The applied C/N varied from 0.3 to 2.2, with HRT, H2 pressure, pH, water temperature, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 7, 25 C, W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different C/N conditions (SSA (Sum of Squares for factor A(C/N)) ¼ 170.87, SSE (Sum of Squares for Error) ¼ 22.83,
SST (Sum of Squares for Total) ¼ 193.70, F (level of significance) ¼ 59.87 > Fcrit).
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and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiency changed slightly with C/N in the reactor. Denitrification efficiencies were 89.32% ± 0.04%, 95.88% ± 0.01%, 93.85% ± 0.03% and 94.59% ± 0.02% when C/N were 0.3, 0.9, 1.5 and 2.2, respectively. Denitrification efficiency fell by 6.56% with C/N reduced from 0.9 to 0.3 due to the fact that C/N of 0.3 was not sufficient for bacteria to grow (OD600 reduced from 0.032 to 0.020 when C/N reduced from 0.9 to 0.3). Ghafari et al. () observed that bicarbonate was the effective carbon source for a faster growth and adaption of autohydrogenotrophic denitrifying bacteria. In addition, Ghafari et al. () reported that the optimum bicarbonate concentration was 1,100 mg NaHCO3/L for an initial nitrate concentration of 20 mg NO 3 -N/L. Chen & Lin () defined the theoretical stoichiometric equations for denitrification and the theoretical C/N ratios were established as 0.71. Similar research showed C/N ¼ 1.1 (Gómez et al. ) and 2.2 (Fan et al. ) for optimum denitrification. In the present research, the bacteria could grow well if C/N was more than 0.9. If C/N was greater than the theoretical value, the extra consumption of an inorganic carbon source could participate in microbe assimilation, which cause biomass growth (cell mass increased from 31 × 106 to 57 × 106 cfu/mL when C/N increased from 0.9 to 2.2) in the bio-ceramsite reactor. Moreover, the effluent nitrite level ranged from 0.08 to 0.83 mg NO 2 -N/L.
Figure 4
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Denitrification effect under different water temperatures Figure 4 shows the results for the effect of water temperature on nitrate reduction. The applied water temperature varied from 15 to 30 C, with HRT, H2 pressure, C/N, pH, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 1.0, 7, and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiency decreased as water temperature increased. Denitrification efficiencies were 94.17% ± 0.02% and 96.21% ± 0.01% at the temperature of 25 and 30 C. As water temperature reduced to 20 and 15 C, the reduction rate of nitrate decreased to 75.04% ± 0.03% and 73.43% ± 0.01%, respectively. This showed that the higher temperatures used allowed better performance of the hydrogenotrophic cultures (Vasiliadou et al. ). During the operation, the effluent nitrite level ranged from 0.13 to 0.90 mg NO 2 -N/L. Due to denitrifying bacteria’s capacity to survive in extreme environmental conditions, denitrification processes can occur in the range of 2–50 C (Brady & Weil ). Some of the temperature values applied in studies on hydrogenotrophic denitrification varied between 10 and 30 C (Karanasios et al. ). Another study reported by Rezania et al. (a, b) showed that the denitrification rate increased as temperature increased from 12 to 25 C. In the research of Kurt et al. (), experimental evidence suggested that temperature affected the denitrification process by affecting bacteria behavior. In the present work, denitrification efficiency always maintained a high level W
W
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W
W
W
W
W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different water temperature conditions (SSB (Sum of Squares for factor B (temperature)) ¼ 3097.55, SSE (Sum of
Squares for Error) ¼ 33.66, SST (Sum of Squares for Total) ¼ 3131.22, F (level of significance) ¼ 736.15 > Fcrit).
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above 70% with a water temperature range of 15–30 C. The reason might be that these bacteria were not severely sensitive to water temperature in the bio-reactor. No matter how the water temperature changed, the autohydrogenotrophic denitrifying bacteria grew well (OD600 were 0.038, 0.036, 0.041, 0.042 when temperatures were 15 , 20 , 25 , 30 C, respectively). Therefore, this denitrification system has an excellent ability to resist temperature change. W
W
W
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Denitrification effect under different pH The effect of pH on this system is obvious, as can be seen in Figure 5. The applied pH values varied from 6 to 9, with HRT, H2 pressure, C/N, water temperature, and nitrate loading at steady-state values of 24 h, 0.01 MPa, 1.0, 25 C, and 30 mg NO 3 -N/L, respectively. The time profiles of the mixed liquor NO 3 -N and NO2 -N were monitored in order to investigate how nitrate loading affected the performance of autohydrogenotrophic denitrifying bacteria. It was observed that denitrification efficiencies were 94.86% ± 0.03% and 95.93% ± 0.02% at pH 7 and 8, respectively. This showed that the system worked well under this environment. However, the reduction rate of nitrate decreased to 75.06% ± 0.01% as pH increased to 9. Furthermore, the efficiency decreased to 81.89% ± 0.03% with a pH of 6. The results illustrated that denitrification was excellent under neutral or alkalescent environments. The effluent nitrite levels were always maintained below 1.75 mg NO 2 -N/L during the operation. The hydrogenotrophic denitrification process is positively related to pH, with an optimum value range of 7.6–8.6 (Lee & Rittmann ). Another study by Xia et al. () showed that the optimum pH for autotrophic denitrification was 7.2–8.2,
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with the maximum efficiency at pH 7.7. Ho et al. () demonstrated that nitrate could be reduced effectively with no nitrite accumulation when carbon dioxide was applied, while the pH of the bio-reactor remained at 7. In this work, a low pH value (6) inhibited denitrification because the decomposition of carbonate ion stripping could strongly affect the hydrogenotrophic denitrification process. Moreover, a high pH value (9) could cause a significant decrease in the nitrate removal rate due to the fact that the activity of nitrate reductase and nitrite reductase might be inhibited (Rezania et al. a, b). For this reason, it was necessary to keep the pH at 7–8 to obtain steady effluent quality.
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Figure 5
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CONCLUSIONS In this work, an H2-fed bio-ceramsite reactor has been developed, in which autohydrogenotrophic denitrifying bacteria use ceramsite as a carrier and forms a biofilm on the surface of the ceramsite in order to treat nitrate contaminated groundwater. The performance of this system was investigated using laboratory-scale apparatus. The highest denitrification rate (7.41 mg NO 3 -N/L/h) was observed at nitrate loading of 130 mg NO 3 -N/L. However, that this system was inhibited when nitrate loading was higher than 130 mg NO 3 -N/L. C/N was not a significant factor for the reactor. The reactor could perform well if the carbon source was sufficient for the growth of the denitrifying bacteria. This denitrification system has an excellent ability to resist temperature change, and the optimum temperature was 25–35 C. Nitrate reduction was significantly affected by pH in the reactor, and the optimum pH was 7–8. It was W
Concentrations of NO 3 -N and NO2 -N in influent and effluent at different pH conditions (SSC (Sum of Squares for factor C(pH)) ¼ 2171.36, SSE (Sum of Squares for Error) ¼ 14.64,
SST (Sum of Squares for Total) ¼ 2186.00, F (level of significance) ¼ 1186.41 > Fcrit).
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demonstrated that this H2-fed bio-ceramsite reactor was effective for the treatment of nitrate-polluted groundwater.
ACKNOWLEDGEMENTS This work was financially supported by the open fund of the State Key Lab of Urban Water Resources and Environment (HIT) (No. QA200810, QAK201014); National Natural Science Foundation of China (NSFC) (No. 51008239, 51378400); Natural Science Foundation of Hubei Province, China (No. 2013CFB289, 2013CFB308); and Major Science and Technology Program for Water Pollution Control and Treatment (No. 2009ZX07317-008-003).
REFERENCES Brady, N. C. & Weil, R. R. The Nature and Properties of Soils. 13th edn, Prentice Hall, New Jersey. Chen, K. C. & Lin, Y. F. Relationship between denitrifying bacteria and methanogenic bacteria in a mixed culture system of acclimated sludges. Water Res. 27, 1749–1759. Deiglmayr, K., Philippot, L. & Kandeler, E. Functional stability of the nitrate-reducing community in grassland soils towards high nitrate supply. Soil Biol. Biochem. 38, 2980–2984. Fan, B., Qu, J. H., Lei, P. J. & Li, D. P. Studies on nitrate removal by a combined heterotrophy electrode biofilm reactor. Acta Scientiae Circumstantiae 21, 257–262. Ghafari, S., Hasan, M. & Aroua, M. K. Bio-electrochemical removal of nitrate from water and wastewater: A review. Bioresour. Technol. 99, 3965–3974. Ghafari, S., Hasan, M. & Aroua, M. K. Effect of carbon dioxide and bicarbonate as inorganic carbon sources on growth and adaptation of autohydrogenotrophic denitrifying bacteria. J. Hazard. Mater. 162, 1507–1513. Ghafari, S., Hasan, M. & Aroua, M. K. A kinetic study of autohydrogenotrophic denitrification at the optimum pH and sodium bicarbonate dose. Bioresour. Technol. 101, 2236–2242. Gómez, M. A., González-López, J. & Hontoria-García, E. Influence of carbon source on nitrate removal of contaminated groundwater in a denitrifying submerged filter. J. Hazard. Mater. 80, 69–80.
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Ho, C. M., Tseng, S. K. & Chang, Y. J. Autotrophic denitrification via a novel membrane-attached biofilm reactor. Lett. Appl. Microbiol. 33, 201–205. Hwang, J. H., Cicek, N. & Oleszkiewicz, J. A. Achieving biofilm control in a membrane biofilm reactor removing total nitrogen. Water Res. 44, 2283–2291. Jiang, W., Qu, J. H., Lei, P. J., Liu, S. X. & Meng, G. H. Nitrate nitrogen removal from ground water by autotrophic denitrification in a packed bed reactor. China Environ. Sci. 21, 133–136. Karanasios, K. A., Vasiliadou, I. A., Pavlou, S. & Vayenas, D. V. Hydrogenotrophic denitrification of potable water: a review. J. Hazard. Mater. 180, 20–37. Kurt, M., Dunn, I. J. & Bourne, J. R. Biological denitrification of drinking water using autotrophic organisms with H2 in a fluidized-bed biofilm reactor. Biotechnol. Bioeng. 29, 493–501. Lee, K. C. & Rittmann, B. E. Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollowfiber membrane-biofilm reactor. Water Res. 37, 1551–1556. Mousavi, S., Ibrahim, S., Aroua, M. K. & Ghafari, S. Development of nitrate elimination by autohydrogenotrophic bacteria in bio-electrochemical reactors – A review. Biochem. Eng. J. 67, 251–264. Rezania, B., Cicek, N. & Oleszkiewicz, J. A. a Kinetics of hydrogen-dependent denitrification under varying pH and temperature conditions. Biotechnol. Bioeng. 92, 900–906. Rezania, B., Oleszkiewicz, J. A., Cicek, N. & Mo, H. b Hydrogen-dependent denitrification in an alternating anoxicaerobic SBR membrane bio-reactor. Water Sci. Technol. 51, 403–409. Tang, Y. N., Zhou, C., Ziv-El, M. & Rittmann, B. E. A pH-control model for heterotrophic and hydrogen-based autotrophic denitrification. Water Res. 45, 232–240. Vasiliadou, I. A., Karanasios, K. A., Pavlou, S. & Vayenas, D. V. Experimental and modelling study of drinking water hydrogenotrophic denitrification in packed-bed reactors. J. Hazard. Mater. 165, 812–824. Wan, D. J., Liu, H. J., Liu, R. P. & Qu, J. H. Study of a combined sulfur autotrophic with proton-exchange membrane electrodialytic denitrification technology: Sulfate control and pH balance. Bioresource Technol. 102, 10803–10809. Wilderer, P., Jones, W. & Dau, U. Competition in denitrification systems affecting reduction rate and accumulation of nitrite. Water Res. 21, 239–245. Xia, S. Q., Zhong, F. H., Zhang, Y. H., Li, H. & Yang, X. Bio-reduction of nitrate from groundwater using a hydrogenbased membrane biofilm reactor. J. Environ. Sci. 22, 257–262.
First received 4 December 2013; accepted in revised form 17 March 2014. Available online 29 March 2014
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