Journal of Ocean University of China

, Volume 19, Issue 1, pp 241–248 | Cite as

Screening and Characterization of Nitrite-Degrading Bacterial Isolates Using a Novel Culture Medium

  • Qingshan Ma
  • Zengguo HeEmail author


In this study, a novel culture medium that simulates shrimp pond conditions was established to screen nitrite-degrading isolates. The medium was supplemented with nitrite as a nitrogen source and shrimp feed as the major carbon source, to achieve the high nitrogen and low carbon nutritional status found in shrimp farming ponds. Screening using this medium identified potent denitrifying Bacillus isolates, among which Bacillus subtilis M7-1 was considered best. M7-1 was able to completely degrade nitrite-N in 24 h without much consumption of dissolved oxygen. Efficient denitrification activity took place in liquid cultures within a set of non-stringent ranges of pH (5.0-9.0), salinity (0-30) and temperature (25-35℃). The denitrifying enzyme gene was amplified, se-quenced and further identified as nirS type. In biosecurity assessments, M7-1 had no negative effects on shrimps at a dose of 106cfu mL−1. M7-1 could therefore be used in aquaculture to reduce and control the nitrogen concentration, and to promote the development of sustainable and healthy culture systems.

Key words

aerobic denitrification nitrite screening culture medium Bacillus subtilis 


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This research was supported by the Special Fund for Qingdao Marine Biomedical science and Technology Innovation Center, China (No. 2017-CXZX01-3-13).


  1. Arig, N., Suzer, C., Gokvardar, A., Basaran, F., Coban, D., Yildirim, S., Kamaci, H. O., Firat, K., and Saka, S., 2013. Effects of probiotic (Bacillus sp.) supplementation during larval development of Gilthead Sea Bream (Sparus aurata, L.). Turkish Journal of Fisheries & Aquatic Sciences, 13 (3): 407–414.CrossRefGoogle Scholar
  2. Bernheimer, A. W., 1988. Assay of Hemolytic Toxins, Methods in Enzymology. Academic Press, Amsterdam, 213–217.Google Scholar
  3. Braker, G., Fesefeldt, A., and Witzel, K. P., 1998. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Applied and Environmental Microbiology, 64 (10): 3769–3775.CrossRefGoogle Scholar
  4. Camargo, J. A., and Alonso, Á., 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: A global assessment. Environment International, 32 (6): 831–849.CrossRefGoogle Scholar
  5. Cheng, S. Y., and Chen, J. C., 1999. Hemocyanin oxygen affinity, and the fractionation of oxyhemocyanin and deoxy-hemocyanin for Penaeus monodon exposed to elevated nitrite. Aquatic Toxicology, 45 (1): 35–46.CrossRefGoogle Scholar
  6. Crab, R., Avnimelech, Y., Defoirdt, T., Bossier, P., and Verstraete, W., 2007. Nitrogen removal techniques in aquacul-ture for a sustainable production. Aquaculture, 270 (1): 1–14.CrossRefGoogle Scholar
  7. Duan, J., Fang, H., Su, B., Chen, J., and Lin, J., 2015. Characterization of a halophilic heterotrophic nitrification-aerobic denitrification bacterium and its application on treatment of saline wastewater. Bioresource Technology, 179: 421–428.CrossRefGoogle Scholar
  8. GB 7493-87, 1987. Water quality-determination of nitrogen (nitrite)-Spectrophotometric method. The State Environmental Protection Administration of China, Beijing, 144–148.Google Scholar
  9. Gui, M., Chen, Q., and Ni, J., 2017. Effect of sulfamethoxazole on aerobic denitrification by strain Pseudomonas stutzeri PCN-1. Bioresource Technology, 235: 325–331.CrossRefGoogle Scholar
  10. Hargreaves, J. A., 1998. Nitrogen biogeochemistry of aquacul-ture ponds. Aquaculture, 166 (3-4): 181–212.CrossRefGoogle Scholar
  11. Holt, J., 1994. Bergey’s Manual of Determinative Bacteriology, 9th edition. Lippincott Williams & Wilkins, Philadelphia, 1–22.Google Scholar
  12. Hotchkiss, J. H., Helser, M. A., Maragos, C. M., and Weng, Y. M., 1992. Nitrate, nitrite, and N-nitroso compounds: Food safety and biological implications. Acs Symposium Series-american Chemical Society, 484: 400–418.CrossRefGoogle Scholar
  13. Huang, F., Pan, L., Lv, N., and Tang, X., 2017. Characterization of novel Bacillus strain N31 from mariculture water capable of halophilic heterotrophic nitrification-aerobic denitrification. Journal of Bioscience and Bioengineering, 124 (5): 564–571.CrossRefGoogle Scholar
  14. Hui, C., Guo, X., Sun, P., Khan, R. A., Zhang, Q., Liang, Y., and Zhao, Y. H., 2018. Removal of nitrite from aqueous solution by Bacillus amyloliquefaciens biofilm adsorption. Bioresour-ce Technology, 248: 146–152.CrossRefGoogle Scholar
  15. Jensen, F. B., 2003. Nitrite disrupts multiple physiological functions in aquatic animals. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 135 (1): 9–24.CrossRefGoogle Scholar
  16. Ji, B., Yang, K., Zhu, L., Jiang, Y., Wang, H., Zhou, J., and Zhang, H., 2015. Aerobic denitrification: A review of important advances of the last 30 years. Biotechnology and Biopro-cess Engineering, 20 (4): 643–651.CrossRefGoogle Scholar
  17. Khin, T., and Annachhatre, A. P., 2004. Novel microbial nitrogen removal processes. Biotechnology Advances, 22 (7): 519–532.CrossRefGoogle Scholar
  18. Lalloo, R., Ramchuran, S., Ramduth, D., Görgens, J., and Gardiner, N., 2007. Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. Journal of Applied Microbiology, 103 (5): 1471–1479.CrossRefGoogle Scholar
  19. Li, P., Zhang, S., and Liu, D., 2005. Study progress of bacterial aerobic denitrification. Journal of Microbiology, 25: 60–64.Google Scholar
  20. Liu, X., Steele, J. C., and Meng, X. Z., 2017. Usage, residue, and human health risk of antibiotics in Chinese aquaculture: A review. Environmental Pollution, 223: 161–169.CrossRefGoogle Scholar
  21. Ma, B., Wang, S., Cao, S., Miao, Y., Jia, F., Du, R., and Peng, Y., 2016. Biological nitrogen removal from sewage via anammox: Recent advances. Bioresource Technology, 200: 981–990.CrossRefGoogle Scholar
  22. Ma, F., Wang, H. Y., Zhou, D. D., and Zuo, W., 2005. Denitrification characteristics of an aerobic denitrifying bacterium Pseudomonas chloritidismutans strain X31. Journal of South China University of Technology, 33 (7): 42–46.Google Scholar
  23. Maag, M., and Vinther, F. P., 1996. Nitrous oxide emission by nitrification and denitrification in different soil types and at different soil moisture contents and temperatures. Applied Soil Ecology, 4 (1): 5–14.CrossRefGoogle Scholar
  24. Medeiros, R. S., Lopez, B. A., Sampaio, L. A., Romano, L. A., and Rodrigues, R. V., 2016. Ammonia and nitrite toxicity to false clownfish Amphiprion ocellaris. Aquaculture International, 24 (4): 985–993.CrossRefGoogle Scholar
  25. Miron, D. D. S., Moraes, B., Becker, A. G., Crestani, M., Spane-vello, R., Loro, V. L., and Baldisserotto, B., 2008. Ammonia and pH effects on some metabolic parameters and gill histology of silver catfish, Rhamdia quelen (Heptapteridae). Aquaculture, 277 (3): 192–196.CrossRefGoogle Scholar
  26. Nimrat, S., Suksawat, S., Boonthai, T., and Vuthiphandchai, V., 2012. Potential Bacillus probiotics enhance bacterial numbers, water quality and growth during early development of white shrimp (Litopenaeus vannamei). Veterinary Microbiology, 159 (3-4): 443–450.CrossRefGoogle Scholar
  27. Pai, S. L., Chong, N. M., and Chen, C. H., 1999. Potential applications of aerobic denitrifying bacteria as bioagents in wastewater treatment. Bioresource Technology, 68 (2): 179–185.CrossRefGoogle Scholar
  28. Ren, Y. X., Yang, L., and Liang, X., 2014. The characteristics of a novel heterotrophic nitrifying and aerobic denitrifying bacterium, Acinetobacter junii YB. Bioresource Technology, 171: 1–9.CrossRefGoogle Scholar
  29. Šiljeg, M., Foglar, L., and Kukučka, M., 2010. The ground water ammonium sorption onto Croatian and Serbian clinop-tilolite. Journal of Hazardous Materials, 178 (1): 572–577.CrossRefGoogle Scholar
  30. Song, Z. F., An, J., Fu, G. H., and Yang, X. L., 2011. Isolation and characterization of an aerobic denitrifying Bacillus sp. YX-6 from shrimp culture ponds. Aquaculture, 319 (1): 188–193.CrossRefGoogle Scholar
  31. Sun, Y., Feng, L., Li, A., Zhang, X., Yang, J., and Ma, F., 2017. Ammonium assimilation: An important accessory during aerobic denitrification of Pseudomonas stutzeri T13. Biore-source Technology, 234: 264–272.CrossRefGoogle Scholar
  32. Sun, Z., Lv, Y., Liu, Y., and Ren, R., 2016. Removal of nitrogen by heterotrophic nitrification-aerobic denitrification of a novel metal resistant bacterium Cupriavidus sp. S1. Bioresour-ce Technology, 220: 142–150.CrossRefGoogle Scholar
  33. Third, K. A., Gibbs, B., Newland, M., and Cord-Ruwisch, R., 2005. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor. Water Research, 39 (15): 3523–3530.CrossRefGoogle Scholar
  34. Tomasso, J. R., 1994. Toxicity of nitrogenous wastes to aquacul-ture animals. Reviews in Fisheries Science, 2 (4): 291–314.CrossRefGoogle Scholar
  35. Tseng, I. T., and Chen, J. C., 2004. The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus under nitrite stress. Fish & Shellfish Immunology, 17 (4): 325–333.CrossRefGoogle Scholar
  36. Van Rijn, J., Tal, Y., and Schreier, H. J., 2006. Denitrification in recirculating systems: Theory and applications. Aquacultural Engineering, 34 (3): 364–376.CrossRefGoogle Scholar
  37. Wan, W., He, D., and Xue, Z., 2017. Removal of nitrogen and phosphorus by heterotrophic nitrification-aerobic denitrification of a denitrifying phosphorus-accumulating bacterium En-terobacter cloacae HW-15. Ecological Engineering, 99: 199–208.CrossRefGoogle Scholar
  38. Wongkiew, S., Hu, Z., Chandran, K., Lee, J. W., and Khanal, S. K., 2017. Nitrogen transformations in aquaponic systems: A review. Aquacultural Engineering, 76: 9–19.CrossRefGoogle Scholar
  39. Yang, X. P., Wang, S. M., Zhang, D. W., and Zhou, L. X., 2011. Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium, Bacillus sub-tilis A1. Bioresource Technology, 102 (2): 854–862.CrossRefGoogle Scholar
  40. Zhang, Q. L., Liu, Y., Ai, G. M., Miao, L. L., Zheng, H. Y., and Liu, Z. P., 2012. The characteristics of a novel heterotrophic nitrification-aerobic denitrification bacterium, Bacillus me-thylotrophicus strain L7. Bioresource Technology, 108 (3): 35–44.CrossRefGoogle Scholar
  41. Zhou, Q., Li, K., Jun, X., and Bo, L., 2009. Role and functions of beneficial microorganisms in sustainable aquaculture. Bio-resource Technology, 100 (16): 3780–3786.CrossRefGoogle Scholar
  42. Zhou, W., Sun, Y., Wu, B., Zhang, Y., Huang, M., Miyanaga, T., and Zhang, Z., 2011. Autotrophic denitrification for nitrate and nitrite removal using sulfur-limestone. Journal of Environmental Sciences, 23 (11): 1761–1769.CrossRefGoogle Scholar
  43. Zokaeifar, H., Babaei, N., Che, R. S., Kamarudin, M. S., Sijam, K., and Balcazar, J. L., 2014. Administration of Bacillus sub-tilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Lito-penaeus vannamei. Fish & Shellfish Immunology, 36 (1): 68–74.CrossRefGoogle Scholar

Copyright information

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2020

Authors and Affiliations

  1. 1.School of Medicine and PharmacyOcean University of ChinaQingdaoChina
  2. 2.Marine Biomedical Research Institute of QingdaoQingdaoChina

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