Study on Biological Characteristics of Ammonia Oxidizing Bacteria Isolated from Wastewater Sludge at Some Wood Processing Facilities in Vietnam

   Aim. This study investigates the biological characteristics of ammonia-oxidizing bacteria (AOB) isolated from wastewater sludge collected at several wood processing facilities in Vietnam.

   Methodology. To achieve the objectives of the study, composite sludge samples were collected from wastewater treatment units and processed within 6 hours to preserve microbial integrity. Ammonium-oxidizing bacteria were isolated using serial dilution and selective AMS medium, followed by screening for ammonium removal capability. Physiological characteristics of the isolates were evaluated under varying pH, temperature, and ammonium concentrations. Molecular identification was performed by PCR amplification and sequencing of the 16S rRNA gene. The combined use of microbiological and molecular techniques provided a comprehensive understanding of the diversity and functional traits of ammonium-oxidizing bacteria in sludge.

   Results. From six sludge samples collected at wood processing facilities in Yen Bai, Thanh Hoa, and Dong Nai provinces, a total of eleven bacterial strains with ammonium-oxidizing capabilities were isolated. Dong Nai samples yielded the highest number of isolates, suggesting favorable conditions for ammonia-oxidizing bacteria in this region. Among the isolates, three strains – Bacillus subtilis BT183.1.B1, Bacillus velezensis BT751.1.B2, and Bacillus amyloliquefaciens BT751.2.B1 – exhibited superior ammonium removal efficiency and were selected for detailed characterization. These strains showed optimal growth at neutral pH (7.0–7.5), temperatures between 35 °C and 37 °C, and ammonium concentrations up to 700 mg/L. Growth and activity decreased significantly at more extreme pH values, temperatures, and ammonium concentrations. The ability of these Bacillus strains to tolerate high ammonium levels and environmental variability highlights their potential for application in biological treatment of nitrogen-rich industrial wastewater, particularly in the wood processing sector.

   Research implications. Vietnam ranks fifth globally, second in Asia, and first in Southeast Asia in wood and forest product exports. The rapid growth of the wood industry has brought substantial economic benefits but has also caused environmental challenges, particularly pollution from wood processing wastewater containing high levels of ammonium – a pollutant difficult to treat biologically. These findings contribute to expanding the diversity of ammonium-oxidizing bacteria and highlight their potential application in treating ammonium-rich wastewater from wood processing industries.

1. General Statistics Office of Vietnam. Vietnam statistical yearbook 2022. Hanoi: Statistical Publishing House, 2023.

2. Tran M. H., Nguyen T. T., Nguyen T. D., Nguyen C. T., Nguyen T. Q., Nguyen H. C. The effects of ammonium loading rates and salinity on ammonium treatment of waste-water from super-intensive shrimp farming. In: Vietnam Journal of Science and Technology, 2023, vol. 61, no. 5, pp. 854–864. DOI: 10.15625/2525-2518/16427

3. Sun Y., Ma Y., Cao Y., Liu S. Nitrogen removal performance and changes in microbial community structure of a single-stage partial nitrification-anammox (PNA) process for treating municipal wastewater with an extremely low carbon- to-nitrogen ratio. In: Research Square, 2024. DOI: 10.21203/rs.3.rs-5369986/v1

4. Mai W., Chen J., Liu H., Liang J., Tang J., Wei Y. Advances in Studies on Microbiota Involved in Nitrogen Removal Processes and Their Applications in Wastewater Treatment. In: Frontiers in Microbiology, 2021, vol. 12. DOI: 10.3389/fmicb.2021.746293

5. Duan S., Zhang Y., Zheng S. Heterotrophic nitrifying bacteria in wastewater biological nitrogen removal systems : A review. In: Environmental Science and Technology, 2021, vol. 52, no. 3, pp. 1–37. DOI: 10.1080/10643389.2021.1877976

6. Kowalchuk G. A., Stephen J. R. Ammonia-oxidizing bacteria: A model for molecular microbial ecology. In: Annual Review of Microbiology, 2001, vol. 55, pp. 485–529. DOI: 10.1146/annurev.micro.55.1.485

7. Gao Z., Zhu T., Liu C., Zhang J. Ammonium removal characteristics of heterotrophic nitrifying bacterium Pseudomonas stutzeri GEP-01 with potential for treatment of ammonium-rich wastewater. In: Bioprocess and Biosystems Engineering, 2020, vol. 43, no. 1, pp. 959–969. DOI: 10.1007/s00449-020-02292-x

8. Ding C., He T. Bacillus thuringiensis EM-A1: A novel bacterium for high concentration of ammonium elimination with low nitrite accumulation. In: Chemosphere, 2023, vol. 338, DOI: 10.1016/j.chemosphere.2023.139465

9. American Public Health Association (APHA). Standard Methods for the Examination of Water and Wastewater. Washington: American Public Health Association, American Water Works Association, Water Environment Federation, 2017.

10. Sheela B., Khasim B. S., Yellaji R. O. Biore-mediation of ammonia using ammonia oxidizing bacteria isolated from sewage. In: International Journal of Environmental Bioremediation and Biodegradation, 2014, vol. 4, pp. 146–150.

11. Frank J. A., Reich C. I., Sharma S., Weisbaum J. S., Wilson B. A., Olsen G. J. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. In: Applied and Environmental Microbiology, 2008, vol. 74, no. 8, pp. 2461–2470. DOI: 10.1128/AEM.02272-07

12. Srinivasan R., Karaoz U., Volegova M., MacKichan J., Kato-Maeda M., Miller S., Lynch S. V. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. In: PLOS ONE, 2015, vol. 10, no. 2, e0117617. DOI: 10.1371/journal.pone.0117617

13. Nguyen T. T., Tran V. A., Do T. T. H. Study on heterotrophic nitrifying bacteria in aquaculture wastewater in Vietnam. In: Journal of Environmental Biotechnology, 2024, vol. 12, no. 2, pp. 45–52.

14. Nguyen D. H., Bui V. T., Vo C. D. Nitrate-oxidizing bacteria in aquaculture systems: A case study in Thanh Hoa and Soc Trang provinces. In: Journal of Water Science and Technology, 2022, vol. 85, no. 4, pp. 867–875.

15. Pham T. H., Le M. T., Tran Q. H. Ammonium removal efficiency of indigenous Bacillus strains in high-load wastewater. In: Environmental Technology, 2023.

16. Zhang L., Liu Y., Yang M., Ni B.-J. High ammonium removal efficiency by Bacillus strains isolated from municipal wastewater treatment plants. In: Bioresource Technology, 2018, vol. 256, pp. 70–76.

17. Yang J., Chen X., Guo W., Ngo H. H., Zhang J. Simultaneous nitrification and denitrification by Bacillus megaterium in aerobic granular sludge system. In: Journal of Environmental Management, 2019, vol. 240, pp. 136–143.

18. Harshvardhan K., Jha B. Biodegradation of low-density polyethylene by marine bacteria from pelagic waters, Arabian Sea, India. In: Marine Pollution Bulletin, 2013, vol. 77, no. 1–2, pp. 100–106.

19. Kim D. J., Kim S. H., Lee D. I. Effect of pH and temperature on nitrogen removal efficiency and microbial structure of biofilms in a nitrifying trickling filter. In: Journal of Biotechnology, 2005, vol. 115, no. 3, pp. 317–328.

20. Bellucci M., Curtis T. P. The effect of heterotrophic and autotrophic conditions on nitrifying bacterial populations in activated sludge. In: Water Research, 2011, vol. 45, no. 13, pp. 4399–4406.

21. Nguyen H. D., Nguyen T. V., Dinh T. T. H., Phan D. H., Tran H. D. Ammonia oxidation capacity of bacillus bacteria in swine waste-water after biogas treatment. In: Hue University Journal of Science: Natural Science, 2022, vol. 131, no. 1D, pp. 77–87.

22. Xu M., Chen L, Xin Y., Wang X., Wang Z., Meng X., Zhang W., Sun W., Geng B., Li L. Characteristics and Mechanism of Ammonia Nitrogen Removal by Heterotrophic Nitrification Bacterium Klebsiella pneumoniae LCU1 and Its Application in Wastewater Treatment. In: Microorganisms, 2025, vol. 13, no. 2, pp. 297. DOI: 10.3390/microorganisms13020297

23. Wang L., Si Z., Yu H., Yu L., Lin Q., Liu W., Jiang L., Jiang S., Peng S. Unveiling the dual role of heterotrophic ammonia-oxidizing bacteria: enhancing plant regrowth through modulating cytokinin delivery. In: Frontiers in Microbiology, 2023, vol. 14. DOI: 10.3389/fmicb.2023.1268442

24. Qiang L., Hao R., Wang X., Li J., Lin Q., Peng S. Dual function of heterotrophic ammonia-oxidizing bacteria in facilitating maize compensatory growth under limited rewatering after drought. In: BMC Biotechnology 25, 2025, vol. 71. DOI: 10.1186/s12896-025-01006-z

25. Martikainen P. J. Heterotrophic nitrification – An eternal mystery in the nitrogen cycle. In: Soil Biology and Biochemistry, 2022, vol. 168. DOI: 10.1016/j.soilbio.2022.108611