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Characterization of an aerobic denitrifier Enterobacter cloacae strain HNR and its nitrate reductase gene

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Abstract

Enterobacter cloacae strain HNR was found to grow well and denitrify aerobically at high NO3−—N concentrations. When the concentrations of NO3-—N were 200, 300 and 500 mg/L, the removal efficiencies of NO3-—N were 83%, 74.5% and 75%, respectively. More importantly, the intermediates accumulation of NO2-—N and NH4+—N was not obvious during the aerobic denitrification processes, resulting in a high TN removal of 82%, 74% and 70%, respectively. Meanwhile, strain HNR also presented the ability of heterotrophic nitrification. With initial NH4+—N concentrations of 20 and 80 mg/L, the NH4+—N removal efficiency reached 78% and 76%, respectively. The key nitrate reductase enzyme gene relating to denitrification was successfully amplified by polymerase chain reaction (PCR) from strain HNR, and identified it as napA, which encodings the large catalytic subunit A of periplasmic nitrate reductase (NAPA). The sequence analysis of napA indicates that NAPA is a hydrophilic, non-transmembrane protein. The existence of napA might be crucial for strain HNR to denitrify nitrate under aerobic conditions. This study showed prospect to develop novel technology for nitrogen removal by application of E. cloacae strain HNR.

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References

  • APHA (1998) Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, Washington, DC

    Google Scholar 

  • Bai H, Liao S, Wang A, Huang J, Shu W, Ye J (2019) High-efficiency inorganic nitrogen removal by newly isolated Pannonibacter phragmitetus B1. Bioresour Technol 271:91–99

    Article  CAS  Google Scholar 

  • Bell LC, Richardson DJ, Ferguson SJ (1990) Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha : The periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS Lett 265:85–87

    Article  CAS  Google Scholar 

  • Burgin AJ, Hamilton SK (2007) Have we overemphasized the role of denitrification in aquatic ecosystems? a review of nitrate removal pathways. Front Ecol Environ 5:89–96

    Article  Google Scholar 

  • Burow KR, Nolan BT, Rupert MG, Dubrovsky NM (2010) Nitrate in groundwater of the United States, 1991–2003. Environ Sci Technol 44:4988–4997

    Article  CAS  Google Scholar 

  • Chen J, Strous M (2013) Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution. Biochim Biophys Acta 1827:136–144. https://doi.org/10.1016/j.bbabio.2012.10.002

    Article  CAS  PubMed  Google Scholar 

  • Chen S, He S, Wu C, Du D (2018) Characteristics of heterotrophic nitrification and aerobic denitrification bacterium Acinetobacter sp. T1 and its application for pig farm wastewater treatment. J Biosci Bioeng 127:201–205

    Article  Google Scholar 

  • Duan J, Fang H, Bing S, Chen J, Lin J (2015) Characterization of a halophilic heterotrophic nitrification–aerobic denitrification bacterium and its application on treatment of saline wastewater. Bioresour Technol 179:421–428

    Article  CAS  Google Scholar 

  • Franz J, Bereau T, Pannwitt S, Anbazhagan V, Lehr A, Nubbemeyer U, Dietz U, Bonn M, Weidner T, Schneider D (2017) Nitrated fatty acids modulate the physical properties of model membranes and the structure of transmembrane proteins. Chem - Eur J 23:9690–9697

    Article  CAS  Google Scholar 

  • Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, NJ, USA, pp 571–607

    Chapter  Google Scholar 

  • Giannopoulos G, Sullivan MJ, Hartop K, Rowley G, Gates A, Watmough NJ, Richardson DJ (2017) Tuning the modular Paracoccus denitrificans respirome to adapt from aerobic respiration to anaerobic denitrification. Environ Microbiol 19:4953–4964

    Article  CAS  Google Scholar 

  • Guo LJ, Zhao B, An Q, Tian M (2016) Characteristics of a novel aerobic denitrifying bacterium, Enterobacter cloacae strain HNR. Appl Biochem Biotechnol 178:947–959. https://doi.org/10.1007/s12010-015-1920-8

    Article  CAS  PubMed  Google Scholar 

  • He D, Zheng M, Ma T, Li C, Ni J (2015) Interaction of Cr(VI) reduction and denitrification by strain Pseudomonas aeruginosa PCN-2 under aerobic conditions. Bioresour Technol 185:346–352. https://doi.org/10.1016/j.biortech.2015.02.109

    Article  CAS  PubMed  Google Scholar 

  • He T, Li Z, Sun Q, Xu Y, Ye Q (2016) Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion. Bioresour Technol 200:493–499. https://doi.org/10.1016/j.biortech.2015.10.064

    Article  CAS  PubMed  Google Scholar 

  • He T, Xie D, Li Z, Ni J, Sun Q (2017) Ammonium stimulates nitrate reduction during simultaneous nitrification and denitrification process by Arthrobacter arilaitensis Y-10. Bioresour Technol 239:66–73

    Article  CAS  Google Scholar 

  • He X, Sun Q, Xu T, Dai M, Wei D (2019) Removal of nitrogen by heterotrophic nitrification–aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04. Bioproc Biosyst Eng 42:853–866

    Article  CAS  Google Scholar 

  • Huang T, Guo L, Zhang H, Su J, Wen G, Zhang K (2015) Nitrogen-removal efficiency of a novel aerobic denitrifying bacterium, Pseudomonas stutzeri strain ZF31, isolated from a drinking-water reservoir. Bioresour Technol 196:209–216. https://doi.org/10.1016/j.biortech.2015.07.059

    Article  CAS  PubMed  Google Scholar 

  • Ji B, Yang K, Wang H, Zhou J, Zhang H (2015) Aerobic denitrification by Pseudomonas stutzeri C3 incapable of heterotrophic nitrification. Bioproc Biosyst Eng 38:407–409. https://doi.org/10.1007/s00449-014-1271-9

    Article  CAS  Google Scholar 

  • Jin R, Liu T, Liu G, Zhou J, Huang J, Wang A (2015) Simultaneous heterotrophic nitrification and aerobic denitrification by the marine origin bacterium Pseudomonas sp. ADN-42. Appl Biochem Biotechnol 175:2000–2011. https://doi.org/10.1007/s12010-014-1406-0

    Article  CAS  PubMed  Google Scholar 

  • Kourtev PS, Nakatsu CH, Allan K (2009) Inhibition of nitrate reduction by chromium (VI) in anaerobic soil microcosms. Appl Environ Microbiol 75:6249–6257

    Article  CAS  Google Scholar 

  • Kraft B, Strous M, Tegetmeyer HE (2011) Microbial nitrate respiration-genes, enzymes and environmental distribution. J Biotechnol 155:104–117. https://doi.org/10.1016/j.jbiotec.2010.12.025

    Article  CAS  PubMed  Google Scholar 

  • Leaver JT, Richardson DJ, Butler CS (2008) Enterobacter cloacae SLD1a-1 gains a selective advantage from selenate reduction when growing in nitrate-depleted anaerobic environments. J Ind Microbiol Biot 35:867–873

    Article  CAS  Google Scholar 

  • Li C, Yang J, Wang X, Wang E, Li B, He R, Yuan H (2015) Removal of nitrogen by heterotrophic nitrification-aerobic denitrification of a phosphate accumulating bacterium Pseudomonas stutzeri YG-24. Bioresour Technol 182:18–25. https://doi.org/10.1016/j.biortech.2015.01.100

    Article  CAS  PubMed  Google Scholar 

  • Li W, Shan XY, Wang ZY, Lin XY, Li CX, Cai CY, Abbas G, Zhang M, Shen LD, Hu ZQ (2016) Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities. Water Res 88:758–765

    Article  CAS  Google Scholar 

  • Möller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653

    Article  Google Scholar 

  • Moir JW, Wood NJ (2001) Nitrate and nitrite transport in bacteria. Cell Mol Life Sci 58:215–224

    Article  CAS  Google Scholar 

  • Patureau D, Bernet N, Delgenès JP, Moletta R (2000) Effect of dissolved oxygen and carbon–nitrogen loads on denitrification by an aerobic consortium. Appl Microbiol Biotechnol 54:535–542

    Article  CAS  Google Scholar 

  • Peel JW, Reddy KJ, Sullivan BP, Bowen JM (2003) Electrocatalytic reduction of nitrate in water. Water Res 37:2512–2519

    Article  CAS  Google Scholar 

  • Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ (2001) Functional, biochemical and genetic diversity of prokaryotic nitrate reductases. Cell Mol Life Sci 58:165–178

    Article  CAS  Google Scholar 

  • Ridley H, Watts CA, Richardson DJ, Butler CS (2006) Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions. Appl Environ Microbiol 72:5173–5180

    Article  CAS  Google Scholar 

  • Ruiz-Bevia F, Fernandez-Torres MJ (2019) Effective catalytic removal of nitrates from drinking water: An unresolved problem? J Clean Prod 217:398–408

    Article  CAS  Google Scholar 

  • Sparacino-Watkins C, Stolz JF, Basu P (2014) Nitrate and periplasmic nitrate reductases. Chem Soc Rev 43:676–706. https://doi.org/10.1039/c3cs60249d

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Su JF, Shi JX, Ma F (2017) Aerobic denitrification and biomineralization by a novel heterotrophic bacterium, Acinetobacter sp. H36. Mar Pollut Bull 116:209–215. https://doi.org/10.1016/j.marpolbul.2017.01.014

    Article  CAS  PubMed  Google Scholar 

  • Su JJ, Liu BY, Liu CY (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. J Appl Microbiol 90:457–462

    Article  CAS  Google Scholar 

  • Sun Y, Li A, Zhang X, Ma F (2015) Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13. Appl Microbiol Biotechnol 99:3243–3248. https://doi.org/10.1007/s00253-014-6221-6)

    Article  CAS  PubMed  Google Scholar 

  • Wood NJ, Alizadeh T, Richardson DJ, Ferguson SJ, Moir JWB (2002) Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus. Mol Microbiol 44:157–170

    Article  CAS  Google Scholar 

  • Xu XJ, Chen C, Wang AJ, Ni BJ, Guo WQ, Yuan Y, Huang C, Zhou X, Wu DH, Lee DJ, Ren NQ (2017) Mathematical modeling of simultaneous carbon-nitrogen-sulfur removal from industrial wastewater. J Hazard Mater 321:371–381. https://doi.org/10.1016/j.jhazmat.2016.08.074

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Ren YX, Liang X, Zhao SQ, Wang JP, Xia ZH (2015) Nitrogen removal characteristics of a heterotrophic nitrifier Acinetobacter junii YB and its potential application for the treatment of high-strength nitrogenous wastewater. Bioresour Technol 193:227–233. https://doi.org/10.1016/j.biortech.2015.05.075

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Ren YX, Zhao SQ, Liang X, Wang JP (2016) Isolation and characterization of three heterotrophic nitrifying-aerobic denitrifying bacteria from a sequencing batch reactor. Ann Microbiol 66:737–747

    Article  CAS  Google Scholar 

  • Yao S, Ni J, Chen Q, Borthwick AG (2013) Enrichment and characterization of a bacteria consortium capable of heterotrophic nitrification and aerobic denitrification at low temperature. Bioresour Technol 127:151–157. https://doi.org/10.1016/j.biortech.2012.09.098

    Article  CAS  PubMed  Google Scholar 

  • Zayed G, Winter J (1998) Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification. Appl Microbiol Biotechnol 49:469–474

    Article  CAS  Google Scholar 

  • Zhang H, Zhao Z, Chen S, Kang P, Wang Y, Feng J, Jia J, Yan M, Wang Y, Xu L (2018) Paracoccus versutus KS293 adaptation to aerobic and anaerobic denitrification: Insights from nitrogen removal, functional gene abundance, and proteomic profiling analysis. Bioresour Technol 260:321–328

    Article  CAS  Google Scholar 

  • Zhang W, Yan C, Shen J, Wei R, Gao Y, Miao A, Xiao L, Yang L (2019) Characterization of aerobic denitrifying bacterium Pseudomonas mendocina strain GL6 and its potential application in wastewater treatment plant effluent. Int J Environ Res Public Health 16:364

    Article  CAS  Google Scholar 

  • Zhao B, Cheng DY, Tan P, An Q, Guo JS (2018) Characterization of an aerobic denitrifier Pseudomonas stutzeri strain XL-2 to achieve efficient nitrate removal. Bioresour Technol 250:564–573. https://doi.org/10.1016/j.biortech.2017.11.038

    Article  CAS  PubMed  Google Scholar 

  • Zhao B, He YL, Zhang XF (2010) Nitrogen removal capability through simultaneous heterotrophic nitrification and aerobic denitrification by Bacillus sp. LY. Environ Technol 31:409–416

    Article  CAS  Google Scholar 

  • Zhao Y, Feng C, Wang Q, Yang Y, Zhang Z, Sugiura N (2011) Nitrate removal from groundwater by cooperating heterotrophic with autotrophic denitrification in a biofilm-electrode reactor. J Hazard Mater 192:1033–1039

    Article  CAS  Google Scholar 

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Acknowledgements

Enterobacter cloacae strain HNR was isolated from activated sludge from the aeration tank of Jiguanshi municipal wastewater treatment plant (Chongqing, China). We are very grateful to Jiguanshi municipal wastewater treatment plant (Chongqing, China) for allowing us to use the activated sludge. This work was supported by National Key Research and Development Program of China (2019YFD1100504, 2019YFD1100501), and Technical Innovation and Application Demonstration Project of CQ CSTC (Grant No. cstc2018jscx-msybX0308).

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  1. Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing, 400045, People’s Republic of China

    Bin Zhao, Qiao Dan, Long Jie Guo, Qiang An & Jin Song Guo

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  1. Bin Zhao
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Zhao, B., Dan, Q., Guo, L.J. et al. Characterization of an aerobic denitrifier Enterobacter cloacae strain HNR and its nitrate reductase gene. Arch Microbiol 202, 1775–1784 (2020). https://doi.org/10.1007/s00203-020-01887-y

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