Appl Biochem Biotechnol (2015) 175:2000–2011 DOI 10.1007/s12010-014-1406-0
Simultaneous Heterotrophic Nitrification and Aerobic Denitrification by the Marine Origin Bacterium Pseudomonas sp. ADN-42 Ruofei Jin & Tianqi Liu & Guangfei Liu & Jiti Zhou & Jianyu Huang & Aijie Wang
Received: 2 September 2014 / Accepted: 13 November 2014 / Published online: 29 November 2014 # Springer Science+Business Media New York 2014
Abstract Recent research has highlighted the existence of some bacteria that are capable of performing heterotrophic nitrification and have a phenomenal ability to denitrify their nitrification products under aerobic conditions. A high-salinity-tolerant strain ADN-42 was isolated from Hymeniacidon perleve and found to display high heterotrophic ammonium removal capability. This strain was identified as Pseudomonas sp. via 16S rRNA gene sequence analysis. Gene cloning and sequencing analysis indicated that the bacterial genome contains N2O reductase function (nosZ) gene. NH3-N removal rate of ADN-42 was very high. And the highest removal rate was 6.52 mg/L· h in the presence of 40 g/L NaCl. Under the condition of pure oxygen (DO >8 mg/L), NH3-N removal efficiency was 56.9 %. Moreover, 38.4 % of oxygen remained in the upper gas space during 72 h without greenhouse gas N2O production. Keeping continuous and low level of dissolved oxygen (DO 8 mg/L), NH3-N concentration decreased from 174.5 to 75.1 mg/L within 72 h with a NH3-N removal efficiency of 56.9 %. The concentration of NH3-N decreased significantly after 12 h and the removal rate of
Appl Biochem Biotechnol (2015) 175:2000–2011
2007 20
200
3.0
NO2--N concentration(mg/L)
16
160 140
2.5
2.0
12
120 100
8
80 60
4
40
OD600
NH3-N concentration (mg/L)
180
1.5
1.0
0.5
20 0
0
20
40
60
80
100
120
0
0.0
Time (h)
(a)
(b) Fig. 4 The SND performance and gas production by Pseudomonas sp. ADN-42. a Closed squares, NH3-N; closed triangles, NO2−-N; open squares, OD600. b Closed squares, O2; open circles, N2; open squares, CO2; open triangles, N2O
Pseudomonas sp. ADN-42 was 2.6 mg/L·h in the first 36 h. NO2−-N dose reached maximum concentration 6.3 mg/L at 24 h and then decreased to 2.4 mg/L at the end of 72 h. This may be due to the high nitrite reductase activity, and the nitrite produced by nitrate reductase was quickly reduced by nitrite reductase. Furthermore, no NH2OH and NO3−-N was detected, which may be due to the instability of NH2OH. The strain grew very well in the medium and OD600 reached 1.9 at 48 h, which indicated that the strain could adapt to the environment and enter into the logarithmic phase rapidly. In the meantime, high NH3-N removal rate suggested that the logarithmic growth phase of the strain was consistent with the rapid NH3-N removal period. This was different from the conclusion of Zhang et al. [25], who reported that nitrification occurred at aging cells, but consistent with the results of Su et al.[17]. Wehrfritz et al. suggested that during the oxidation of ammonia to nitrite by ammonia monooxygenase, electrons were not transferred to coenzyme Q by heterotrophic denitrifying bacteria. Instead,
2008
Appl Biochem Biotechnol (2015) 175:2000–2011
coenzyme Q obtained electrons via the oxidation of externally added organic carbon source. Therefore, the removal of ammonia nitrogen by heterotrophic nitrification bacteria was coupled with the consumption of organic carbon source [22]. Then the strain grew into the stationary phase and OD600 no longer increased any more after 54 h, which also explained the phenomenon that NH3-N concentration slightly increased after 48 h. It may be due to the decomposition of strain so that part of the nitrogen containing in the cell could return into the water again. As shown in Fig. 4b, the content change of N2, N2O, and O2 in upper gas space was investigated through GC/TCD. The concentration of N2 was 52.1 mg/L and 38.4 % of oxygen remained in the upper gas space, which indicated that the reaction has always been under aerobic conditions. Nitrogen production of strain YZN-001 was 35.2 mg/L after 24 h and oxygen remained in upper gas space was 411.9 mg/L, which meant that the consumption of oxygen was 59.9 % [26]. Similar conclusion was reported in Pseudomonas alcaligenes AS-1 and Pseudomonas stutzeri SU2 which could convert nitrate nitrogen to nitrogen gas under the condition of high concentration of oxygen with a nitrogen production of 175.6 mg/L after 44 h and the consumption of oxygen was 50.5 %. N2 was considered to be the primary product and reached 52.1 mg/L in the end [18]. Aerobic denitrifying bacteria producing greenhouse gas N2O during the process of denitrification was reported in many literatures. Strain L7 could produce 15N2O when using 15 NH4Cl as nitrogen source [27]. Thiosphaera pantotropha and A. faecalis No. 4 could convert ammonia nitrogen into N2O by SND process [1, 5]. But N2O could not be detected in this experiment, which might be ascribed to the high N2O reductase activity transforming N2O to N2 in a short time. The analysis of nosZ also explained this phenomenon. The detection of intermediate and end products proved that Pseudomonas sp. ADN-42 was a bacterium with high heterotrophic nitrification and aerobic denitrification capacity. Influence of DO on Simultaneous Heterotrophic Nitrification and Aerobic Denitrification It was generally acknowledged that aerobic and anoxic conditions were DO >2.0 mg/L and DO 8
3.2
0
3.2
Final DO (mg/L)
>8
0
0
3.2
–: not measured
Appl Biochem Biotechnol (2015) 175:2000–2011
2009
Table 2 Nitrogen balance during the heterotrophic nitrification aerobic denitrification process Initial N (mg/L)
Final amount of N (mg/L)
NH3-N
NH3-N
NO2−-N
NO3−-N NH2OH N2
174.5 (±3.3) 80.2 (±4.2) 2.3 (±1.1) 0
0
N2O Biological nitrogen N lost
52.1 (±1.1) 0
35.4 (±3.2)
4.4 (±2.9)
(Nap) nitrate reductases [12]. Nar was more sensitive to oxygen inhibition. Most aerobic denitrifying bacteria mainly reduced NO3 – into NO 2− by Nap [9, 20]. According to the results of this experiment, it could be inferred that certain increase of DO may promote the growth of the strains and denitrification, and the continuous low DO may be better for denitrification. Thus, it was better to keep DO at 3 mg/L. This may be due to the effect of Nap. Munch et al. observed that an efficient SND process occurred at a DO concentration lower than 0.5 mg/L [11]. The importance of maintaining a low DO concentration was confirmed by Pochana and Keller, who showed that NH3-N, NO2−-N, NO3−-N, and soluble CODCr were removed in 4 h when DO concentration was between 0.3 and 0.8 mg/L [14]. Another study suggested that the aerobic fermentation denitrifying bacteria had strong denitrification ability under micro-aerobic conditions [28]. Feng et al. observed the possibility that high DO may inhibit the removal of total nitrogen [4]. Based on the detection of end products, it was better to keep continuous low DO during the reaction process. Nitrogen Balance Analysis Batch experiments were carried out to evaluate the intermediate products and calculate the nitrogen balance in the heterotrophic nitrification-aerobic denitrification process by Pseudomonas sp. ADN-42. As shown in Table 2, initially, NH3-N was the main form of Nsource and the TN concentration was 174.5 mg/L. Finally, NH3-N decreased to 80.2 mg/L accompanied with the trace amount accumulation of NH2OH, NO2−-N, NO3−-N, and gas products. By comparing the initial and final TN concentration, the nitrogen content of nitrogen product was 170.0 mg/L (final TN concentration = NH3-N + NO2−-N + NO3−-N + NH2OH + N2 + N2O + biological nitrogen = 80.2 + 2.3 + 52.1 + 35.4=170 mg/L). The reaction pathway of Pseudomonas sp. ADN-42 is deduced to be NH3-N → NH2OH → NO2−-N(⇔NO3−-N) → NO → NO2 → N2. And 2.5 % of the initial nitrogen was lost, which was probably removed in the form of other gases such as NO and NO2. On the other hand, 37.5 % of the NH3-N was converted into biological nitrogen and N2 accounted for the proportion of nitrogen product content as high as 30.6 %. This was in accordance with the results that A. faecalis No. 4 converted about 50 % of the NH3-N into biological nitrogen [5]. Kim et al. pointed out that the proportion was 24.8 % in Bacillus strains at a C/N ratio of 8 [7]. These observations indicated that the assimilation made a significant contribution to the heterotrophic nitrogen removal, especially at high C/N ratios.
Conclusions The strain ADN-42 isolated from a marine sponge H. perleve was a Gram-negative Pseudomonas strain. The cells were observed to appear as short rod bacteria. Srain ADN-42 contained N2O reductase function gene. Around 75.8 % ammonium was removed when the
2010
Appl Biochem Biotechnol (2015) 175:2000–2011
initial NH3-N was 489.9 mg/L and NaCl was 40 g/L. It could degrade ammonia nitrogen by SND in the presence of pure oxygen with the production of N2 and CO2. And no greenhouse gas N2O was determined during the whole operation. This study suggested that continuous low DO may be better for denitrification, it was better to keep DO at 3 mg/L. Acknowledgments This work was supported by the Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (QA201313) and the Fundamental Research Funds for the Central Universities (DUT14LAB09).
References 1. Beline, F., & Martinez, J. (2002). Nitrogen transformations during biological aerobic treatment of pig slurry: effect of intermittent aeration on nitrous oxide emissions. Bioresource Technology, 83, 225–228. 2. Chiu, Y.-C., Lee, L.-L., Chang, C.-N., & Chao, A.-C. (2007). Control of carbon and ammonium ratio for simultaneous nitrification and denitrification in a sequencing batch bioreactor. International Biodeterioration and Biodegradation, 59, 1–7. 3. Feng, L.-J., Xu, J., Xu, X.-Y., Zhu, L., Xu, J., Ding, W., et al. (2012). Enhanced biological nitrogen removal via dissolved oxygen partitioning and step feeding in a simulated river bioreactor for contaminated source water remediation. International Biodeterioration and Biodegradation, 71, 72–79. 4. Joo, H.-S., Hira, M., & Shoda, M. (2005). Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4. Journal of Bioscience and Bioengineering, 100, 184–191. 5. Khardenavis, A.-A., Kapley, A., & Purohit, H.-J. (2007). Simultaneous nitrification and denitrification by diverse Diaphorobacter sp. Applied Microbiology and Biotechnology, 77, 403–409. 6. Kim, J.-K., Park, K.-J., Cho, K.-S., Nam, S.-W., Park, T.-J., & Bajpai, R. (2005). Aerobic nitrificationdenitrification by heterotrophic Bacillus strains. Bioresource Technology, 96, 1897–1906. 7. Kim, M., Jeong, S.-Y., Yoon, S.-J., Cho, S.-J., Kim, Y.-H., Kim, M.-J., et al. (2008). Aerobic denitrification of Pseudomonas putida AD-21 at different C/N ratios. Journal of Bioscience and Bioengineering, 106, 498–502. 8. Lalucat, J., Bennasar, A., Bosch, R., Garcia-Valdes, E., & Palleroni, N.-J. (2006). Biology of Pseudomonas stutzeri. Microbiology and Molecular Biology Reviews, 70, 510–547. 9. Mevel, G., & Prieur, D. (2000). Heterotrophic nitrification by a thermophilic Bacillus species as influenced by different culture conditions. Canadian Journal of Microbiology, 46, 465–473. 10. Munch, E.-V., Lant, P., & Keller, J. (1996). Simultaneous nitrification and denitrification in bench-scale sequencing batch reactors. Water Research, 30, 277–284. 11. Philippot, L. (2002). Denitrifying genes in bacterial and archaeal genomes. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1577, 355–376. 12. Plosz, B.-G., Jobbagy, A., & Grady, C.-P.-L. (2003). Factors influencing deterioration of denitrification by oxygen entering an anoxic reactor through the surface. Water Research, 37, 853–863. 13. Pochana, K., & Keller, J. (1999). Study of factors affecting simultaneous nitrification and denitrification (SND). Water Science and Technology, 39, 61–68. 14. Robertson, L.-A., Vanniel, E.-W.-J., Torremans, R.-A.-M., & Kuenen, J.-G. (1988). Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha. Applied and Environment Microbiology, 54, 2812–2818. 15. SEPA. (2002). Water and Wastewater Monitoring Methods (pp. 210–280). Beijing: Chinese Environmental Science Publishing House. 16. Su, J.-J., Liu, B.-Y., & Liu, C.-Y. (2001). Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. Journal of Applied Microbiology, 90, 457–462. 17. Su, J.-J., Yeh, K.-S., & Tseng, P.-W. (2006). A strain of Pseudomonas sp. isolated from piggery wastewater treatment systems with heterotrophic nitrification capability in Taiwan. Current Microbiology, 53, 77–81. 18. Taylor, S.-M., He, Y.-L., Zhao, B., & Huang, J. (2009). Heterotrophic ammonium removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium, Providencia rettgeri YL. Journal of Environment Science (China), 21, 1336–1341.
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19. Van, A.-N.-E., Sherrill, L.-A., Iglewski, B.-H., & Haidaris, C.-G. (2009). Compensatory peripasmic nitrate reductase activity supports anaerobic growth of Pseudomonas aeruginosa. Canadian Journal of Microbiology, 55, 1133–1144. 20. Wan, C.-L., Yang, X., Lee, D.-J., Du, M.-A., Wan, F., & Chen, C. (2011). Aerobic denitrification by novel isolated strain using NO2−-N as nitrogen source. Bioresource Technology, 102, 7244–7248. 21. Wehrfritz, J.-M., Carter, J.-P., Spiro, S., & Richardson, D.-J. (1997). Hydroxylamine oxidation in heterotrophic nitrate-reducing soil bacteria and purification of a hydroxylamine-cytochrome coxidoreductase from a Pseudomonas species. Archives of Microbiology, 166, 421–424. 22. Wehrfritz, J.-M., Reilly, A., Spiro, S., & Richardson, D.-J. (1993). Purification of hydroxylamine oxidase from Thiosphaera pantotropha, identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification. FEBS Letters, 335, 246–250. 23. Yun, H.-J., & Kim, D.-J. (2003). Nitrite accumulation characteristics of high strength ammonia wastewater in an autotrophic nitrifying biofilm reactor. Journal of Chemical Technology and Biotechnology, 78, 377–383. 24. Zhang, G.-Y., Chen, M.-C., Han, R.-Y., & Ming, H. (2003). Isolation, identification and phylogenetic analysis of a heterotrophic nitrifier. ACTA Microbiologica Sinica of China, 43, 156–161. 25. Zhang, J.-B., Wu, P.-X., Hao, B., & Yu, Z.-N. (2011). Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001. Bioresource Technology, 102, 9866–9869. 26. Zhang, Q.-L., Liu, Y., Ai, G.-M., Miao, L.-L., Zheng, H.-Y., & Liu, Z.-P. (2012). The characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium, Bacillus methylotrophicus strain L7. Bioresource Technology, 108, 35–44. 27. Zhu, X.-Y., & Chen, Y.-G. (2011). Reduction of N2O and NO generation in anaerobic-aerobic (low dissolved oxygen) biological wastewater treatment process by using sludge alkaine fermentation liquid. Environmental Science and Technology, 45, 2137–2143.
Simultaneous Heterotrophic Nitrification and Aerobic Denitrification by the Marine Origin Bacterium Pseudomonas sp. ADN-42 Ruofei Jin & Tianqi Liu & Guangfei Liu & Jiti Zhou & Jianyu Huang & Aijie Wang
Received: 2 September 2014 / Accepted: 13 November 2014 / Published online: 29 November 2014 # Springer Science+Business Media New York 2014
Abstract Recent research has highlighted the existence of some bacteria that are capable of performing heterotrophic nitrification and have a phenomenal ability to denitrify their nitrification products under aerobic conditions. A high-salinity-tolerant strain ADN-42 was isolated from Hymeniacidon perleve and found to display high heterotrophic ammonium removal capability. This strain was identified as Pseudomonas sp. via 16S rRNA gene sequence analysis. Gene cloning and sequencing analysis indicated that the bacterial genome contains N2O reductase function (nosZ) gene. NH3-N removal rate of ADN-42 was very high. And the highest removal rate was 6.52 mg/L· h in the presence of 40 g/L NaCl. Under the condition of pure oxygen (DO >8 mg/L), NH3-N removal efficiency was 56.9 %. Moreover, 38.4 % of oxygen remained in the upper gas space during 72 h without greenhouse gas N2O production. Keeping continuous and low level of dissolved oxygen (DO 8 mg/L), NH3-N concentration decreased from 174.5 to 75.1 mg/L within 72 h with a NH3-N removal efficiency of 56.9 %. The concentration of NH3-N decreased significantly after 12 h and the removal rate of
Appl Biochem Biotechnol (2015) 175:2000–2011
2007 20
200
3.0
NO2--N concentration(mg/L)
16
160 140
2.5
2.0
12
120 100
8
80 60
4
40
OD600
NH3-N concentration (mg/L)
180
1.5
1.0
0.5
20 0
0
20
40
60
80
100
120
0
0.0
Time (h)
(a)
(b) Fig. 4 The SND performance and gas production by Pseudomonas sp. ADN-42. a Closed squares, NH3-N; closed triangles, NO2−-N; open squares, OD600. b Closed squares, O2; open circles, N2; open squares, CO2; open triangles, N2O
Pseudomonas sp. ADN-42 was 2.6 mg/L·h in the first 36 h. NO2−-N dose reached maximum concentration 6.3 mg/L at 24 h and then decreased to 2.4 mg/L at the end of 72 h. This may be due to the high nitrite reductase activity, and the nitrite produced by nitrate reductase was quickly reduced by nitrite reductase. Furthermore, no NH2OH and NO3−-N was detected, which may be due to the instability of NH2OH. The strain grew very well in the medium and OD600 reached 1.9 at 48 h, which indicated that the strain could adapt to the environment and enter into the logarithmic phase rapidly. In the meantime, high NH3-N removal rate suggested that the logarithmic growth phase of the strain was consistent with the rapid NH3-N removal period. This was different from the conclusion of Zhang et al. [25], who reported that nitrification occurred at aging cells, but consistent with the results of Su et al.[17]. Wehrfritz et al. suggested that during the oxidation of ammonia to nitrite by ammonia monooxygenase, electrons were not transferred to coenzyme Q by heterotrophic denitrifying bacteria. Instead,
2008
Appl Biochem Biotechnol (2015) 175:2000–2011
coenzyme Q obtained electrons via the oxidation of externally added organic carbon source. Therefore, the removal of ammonia nitrogen by heterotrophic nitrification bacteria was coupled with the consumption of organic carbon source [22]. Then the strain grew into the stationary phase and OD600 no longer increased any more after 54 h, which also explained the phenomenon that NH3-N concentration slightly increased after 48 h. It may be due to the decomposition of strain so that part of the nitrogen containing in the cell could return into the water again. As shown in Fig. 4b, the content change of N2, N2O, and O2 in upper gas space was investigated through GC/TCD. The concentration of N2 was 52.1 mg/L and 38.4 % of oxygen remained in the upper gas space, which indicated that the reaction has always been under aerobic conditions. Nitrogen production of strain YZN-001 was 35.2 mg/L after 24 h and oxygen remained in upper gas space was 411.9 mg/L, which meant that the consumption of oxygen was 59.9 % [26]. Similar conclusion was reported in Pseudomonas alcaligenes AS-1 and Pseudomonas stutzeri SU2 which could convert nitrate nitrogen to nitrogen gas under the condition of high concentration of oxygen with a nitrogen production of 175.6 mg/L after 44 h and the consumption of oxygen was 50.5 %. N2 was considered to be the primary product and reached 52.1 mg/L in the end [18]. Aerobic denitrifying bacteria producing greenhouse gas N2O during the process of denitrification was reported in many literatures. Strain L7 could produce 15N2O when using 15 NH4Cl as nitrogen source [27]. Thiosphaera pantotropha and A. faecalis No. 4 could convert ammonia nitrogen into N2O by SND process [1, 5]. But N2O could not be detected in this experiment, which might be ascribed to the high N2O reductase activity transforming N2O to N2 in a short time. The analysis of nosZ also explained this phenomenon. The detection of intermediate and end products proved that Pseudomonas sp. ADN-42 was a bacterium with high heterotrophic nitrification and aerobic denitrification capacity. Influence of DO on Simultaneous Heterotrophic Nitrification and Aerobic Denitrification It was generally acknowledged that aerobic and anoxic conditions were DO >2.0 mg/L and DO 8
3.2
0
3.2
Final DO (mg/L)
>8
0
0
3.2
–: not measured
Appl Biochem Biotechnol (2015) 175:2000–2011
2009
Table 2 Nitrogen balance during the heterotrophic nitrification aerobic denitrification process Initial N (mg/L)
Final amount of N (mg/L)
NH3-N
NH3-N
NO2−-N
NO3−-N NH2OH N2
174.5 (±3.3) 80.2 (±4.2) 2.3 (±1.1) 0
0
N2O Biological nitrogen N lost
52.1 (±1.1) 0
35.4 (±3.2)
4.4 (±2.9)
(Nap) nitrate reductases [12]. Nar was more sensitive to oxygen inhibition. Most aerobic denitrifying bacteria mainly reduced NO3 – into NO 2− by Nap [9, 20]. According to the results of this experiment, it could be inferred that certain increase of DO may promote the growth of the strains and denitrification, and the continuous low DO may be better for denitrification. Thus, it was better to keep DO at 3 mg/L. This may be due to the effect of Nap. Munch et al. observed that an efficient SND process occurred at a DO concentration lower than 0.5 mg/L [11]. The importance of maintaining a low DO concentration was confirmed by Pochana and Keller, who showed that NH3-N, NO2−-N, NO3−-N, and soluble CODCr were removed in 4 h when DO concentration was between 0.3 and 0.8 mg/L [14]. Another study suggested that the aerobic fermentation denitrifying bacteria had strong denitrification ability under micro-aerobic conditions [28]. Feng et al. observed the possibility that high DO may inhibit the removal of total nitrogen [4]. Based on the detection of end products, it was better to keep continuous low DO during the reaction process. Nitrogen Balance Analysis Batch experiments were carried out to evaluate the intermediate products and calculate the nitrogen balance in the heterotrophic nitrification-aerobic denitrification process by Pseudomonas sp. ADN-42. As shown in Table 2, initially, NH3-N was the main form of Nsource and the TN concentration was 174.5 mg/L. Finally, NH3-N decreased to 80.2 mg/L accompanied with the trace amount accumulation of NH2OH, NO2−-N, NO3−-N, and gas products. By comparing the initial and final TN concentration, the nitrogen content of nitrogen product was 170.0 mg/L (final TN concentration = NH3-N + NO2−-N + NO3−-N + NH2OH + N2 + N2O + biological nitrogen = 80.2 + 2.3 + 52.1 + 35.4=170 mg/L). The reaction pathway of Pseudomonas sp. ADN-42 is deduced to be NH3-N → NH2OH → NO2−-N(⇔NO3−-N) → NO → NO2 → N2. And 2.5 % of the initial nitrogen was lost, which was probably removed in the form of other gases such as NO and NO2. On the other hand, 37.5 % of the NH3-N was converted into biological nitrogen and N2 accounted for the proportion of nitrogen product content as high as 30.6 %. This was in accordance with the results that A. faecalis No. 4 converted about 50 % of the NH3-N into biological nitrogen [5]. Kim et al. pointed out that the proportion was 24.8 % in Bacillus strains at a C/N ratio of 8 [7]. These observations indicated that the assimilation made a significant contribution to the heterotrophic nitrogen removal, especially at high C/N ratios.
Conclusions The strain ADN-42 isolated from a marine sponge H. perleve was a Gram-negative Pseudomonas strain. The cells were observed to appear as short rod bacteria. Srain ADN-42 contained N2O reductase function gene. Around 75.8 % ammonium was removed when the
2010
Appl Biochem Biotechnol (2015) 175:2000–2011
initial NH3-N was 489.9 mg/L and NaCl was 40 g/L. It could degrade ammonia nitrogen by SND in the presence of pure oxygen with the production of N2 and CO2. And no greenhouse gas N2O was determined during the whole operation. This study suggested that continuous low DO may be better for denitrification, it was better to keep DO at 3 mg/L. Acknowledgments This work was supported by the Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (QA201313) and the Fundamental Research Funds for the Central Universities (DUT14LAB09).
References 1. Beline, F., & Martinez, J. (2002). Nitrogen transformations during biological aerobic treatment of pig slurry: effect of intermittent aeration on nitrous oxide emissions. Bioresource Technology, 83, 225–228. 2. Chiu, Y.-C., Lee, L.-L., Chang, C.-N., & Chao, A.-C. (2007). Control of carbon and ammonium ratio for simultaneous nitrification and denitrification in a sequencing batch bioreactor. International Biodeterioration and Biodegradation, 59, 1–7. 3. Feng, L.-J., Xu, J., Xu, X.-Y., Zhu, L., Xu, J., Ding, W., et al. (2012). Enhanced biological nitrogen removal via dissolved oxygen partitioning and step feeding in a simulated river bioreactor for contaminated source water remediation. International Biodeterioration and Biodegradation, 71, 72–79. 4. Joo, H.-S., Hira, M., & Shoda, M. (2005). Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4. Journal of Bioscience and Bioengineering, 100, 184–191. 5. Khardenavis, A.-A., Kapley, A., & Purohit, H.-J. (2007). Simultaneous nitrification and denitrification by diverse Diaphorobacter sp. Applied Microbiology and Biotechnology, 77, 403–409. 6. Kim, J.-K., Park, K.-J., Cho, K.-S., Nam, S.-W., Park, T.-J., & Bajpai, R. (2005). Aerobic nitrificationdenitrification by heterotrophic Bacillus strains. Bioresource Technology, 96, 1897–1906. 7. Kim, M., Jeong, S.-Y., Yoon, S.-J., Cho, S.-J., Kim, Y.-H., Kim, M.-J., et al. (2008). Aerobic denitrification of Pseudomonas putida AD-21 at different C/N ratios. Journal of Bioscience and Bioengineering, 106, 498–502. 8. Lalucat, J., Bennasar, A., Bosch, R., Garcia-Valdes, E., & Palleroni, N.-J. (2006). Biology of Pseudomonas stutzeri. Microbiology and Molecular Biology Reviews, 70, 510–547. 9. Mevel, G., & Prieur, D. (2000). Heterotrophic nitrification by a thermophilic Bacillus species as influenced by different culture conditions. Canadian Journal of Microbiology, 46, 465–473. 10. Munch, E.-V., Lant, P., & Keller, J. (1996). Simultaneous nitrification and denitrification in bench-scale sequencing batch reactors. Water Research, 30, 277–284. 11. Philippot, L. (2002). Denitrifying genes in bacterial and archaeal genomes. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1577, 355–376. 12. Plosz, B.-G., Jobbagy, A., & Grady, C.-P.-L. (2003). Factors influencing deterioration of denitrification by oxygen entering an anoxic reactor through the surface. Water Research, 37, 853–863. 13. Pochana, K., & Keller, J. (1999). Study of factors affecting simultaneous nitrification and denitrification (SND). Water Science and Technology, 39, 61–68. 14. Robertson, L.-A., Vanniel, E.-W.-J., Torremans, R.-A.-M., & Kuenen, J.-G. (1988). Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha. Applied and Environment Microbiology, 54, 2812–2818. 15. SEPA. (2002). Water and Wastewater Monitoring Methods (pp. 210–280). Beijing: Chinese Environmental Science Publishing House. 16. Su, J.-J., Liu, B.-Y., & Liu, C.-Y. (2001). Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. Journal of Applied Microbiology, 90, 457–462. 17. Su, J.-J., Yeh, K.-S., & Tseng, P.-W. (2006). A strain of Pseudomonas sp. isolated from piggery wastewater treatment systems with heterotrophic nitrification capability in Taiwan. Current Microbiology, 53, 77–81. 18. Taylor, S.-M., He, Y.-L., Zhao, B., & Huang, J. (2009). Heterotrophic ammonium removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium, Providencia rettgeri YL. Journal of Environment Science (China), 21, 1336–1341.
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