Isolation of bacteria involved in ammonium uptake to develop a bioformulation for wastewater treatment.
Author affiliations
DOI:
https://doi.org/10.15625/2615-9023/23405Keywords:
Ammonium, bioformulation, isolation, Pseudomonas protegens WV5.7, wastewater treatmentAbstract
Ammonium contamination in wastewater poses significant threats to aquatic ecosystems and public health. In this study, bacterial strains capable of ammonium uptake were isolated from seafood processing wastewater and evaluated for their potential application in producing bioformulations. From 25 strains isolated, 4 strains demonstrated ammonium removal efficiencies greater than 90% at an initial concentration of 100 ppm. The most efficient strain, WV5.7, achieved ammonium removal rates of 99 and 88.6% at 100 ppm and 200 ppm ammonium, respectively, after 24 hours of cultivation in mineral medium. Based on 16S-rRNA gene sequencing and biochemical characterization, strain WV5.7 was identified as Pseudomonas protegens WV5.7. Suitable conditions for ammonium uptake included aerobic cultivation at pH 7, with the temperature range from 28 oC to 31 oC, and salinity below 1%. Seven carrier materials were applied for storing strain WV5.7, including sugarcane bagasse, sawdust, coconut coir, soybean residue, talc, and mixtures of talc with bagasse or sawdust (1:1, w/w). Among these, talc powder and the sawdust-talc mixture were the most effective carriers, maintaining ammonium uptake activity and viable cell numbers reaching above 106 CFU/g after 180 days of storage. The addition of vitamin B12 to the bacterial suspension did not significantly enhance bacterial viability or activity. Bioformulations retained their ammonium removal capability in both mineral medium and a lab-scale domestic wastewater treatment model. These findings highlighted P. protegens WV5.7 as a promising candidate for the development of microbial products to treat ammonium-rich wastewater.
Ammonium contamination in wastewater poses significant threats to aquatic ecosystems and public health. In this study, bacterial strains capable of ammonium uptake were isolated from seafood processing wastewater and evaluated for their potential application in producing bioformulations. From 25 strains isolated, 4 strains demonstrated ammonium removal efficiencies greater than 90% at an initial concentration of 100 ppm. The most efficient strain, WV5.7, achieved ammonium removal rates of 99 and 88.6% at 100 ppm and 200 ppm ammonium, respectively, after 24 hours of cultivation in mineral medium. Based on 16S-rRNA gene sequencing and biochemical characterization, strain WV5.7 was identified as Pseudomonas protegens WV5.7. Suitable conditions for ammonium uptake included aerobic cultivation at pH 7, with the temperature range from 28 oC to 31 oC, and salinity below 1%. Seven carrier materials were applied for storing strain WV5.7, including sugarcane bagasse, sawdust, coconut coir, soybean residue, talc, and mixtures of talc with bagasse or sawdust (1:1, w/w). Among these, talc powder and the sawdust-talc mixture were the most effective carriers, maintaining ammonium uptake activity and viable cell numbers reaching above 106 CFU/g after 180 days of storage. The addition of vitamin B12 to the bacterial suspension did not significantly enhance bacterial viability or activity. Bioformulations retained their ammonium removal capability in both mineral medium and a lab-scale domestic wastewater treatment model. These findings highlighted P. protegens WV5.7 as a promising candidate for the development of microbial products to treat ammonium-rich wastewater.
Downloads
References
An D. H., Khoa N. D., Oanh N. T. P., 2017. Selection of carrier materials for the antagonistic Bacillus aerophilus against rice bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae. Can Tho University Journal of Science, 52: 8–15. https://doi.org/10.22144/ctu.jvn.2017.118. (In Vietnamese with English summary).
Bensidhoum L., Nabti E., Tabli N., Kupferschmied P., Weiss A., Rothballer M., Schmid M., Keel C., Hartmann A., 2016. Heavy metal tolerant Pseudomonas protegens isolates from agricultural well water in northeastern Algeria with plant growth promoting, insecticidal and antifungal activities. European Journal of Soil Biology, 75: 38–46. https://doi.org/ 10.1016/j.ejsobi.2016.04.006
Blazej R. G., Kumaresan P., Mathies R. A., 2006. Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proceedings of the National Academy of Sciences, 103(19): 7240–7245. https://doi.org/10.1073/pnas.060247 6103
Breugelmans P., 2005. The book of protocols for general and molecular microbiology. Division Soil and Water Management, KU Leuven, Belgium.
Camargo J. A., Alonso Á., 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems. Environment International, 32(6): 831–849.
Cao X., Hu C., Sun X., Zhang L., Wang H., Dong L. & Li S., 2023. Long-term stability of reactor microbiome through bioaugmentation with Alcaligenes aquatilis AS1 promotes nitrogen removal of piggery wastewater. Journal of Environmental Management, 330: 117146. https://doi.org/ 10.1016/j.jenvman.2022.117146
Dong N. H., Viet N. T., Hang D. T. T., Hung P. H., Lich N. Q., Duan T. H., 2019. Nitrite oxidation capability of Pseudomonas aeruginosa HT1 isolated from biogas effluent of a pig farm in Ha Tinh province. Hue University Journal of Science, 128: 119–132. (In Vietnamese with English summary).
Fan H., Zhang Y., Liu J., Wang Y., Zhao X., 2024. Salinity stress results in ammonium and nitrite accumulation during the elemental sulfur-driven autotrophic denitrification process. Environmental Technology & Innovation, 34: 103235.
Felsenstein J., 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39: 783–791. https://doi.org/10.1111/j.1558-5646.1985. tb00420.x
Fewtrell L., 2004. Drinking-water nitrate, methemoglobinemia, and global burden of disease: A discussion. Environmental Health Perspectives, 112(14): 1371–1374.
Freyschmidt A., Beier M., 2023. Activity of ammonium oxidizing bacteria - An essential parameter for model-based N2O mitigation control strategies for biofilms. Water, 15(13): 2389.
Funge-Smith S. J., Briggs M. R. P., 1998. Nutrient budgets in intensive shrimp ponds: Implications for sustainability. Aquaculture, 164(1–4): 117–133.
Han F., Zhou W., 2022. Nitrogen recovery from wastewater by microbial assimilation: A review. Bioresource Technology, 363: 127933. https://doi.org/10.1016/j.biortech. 2022.127933
Keeney D. R., Nelson D. W., 1982. Nitrogen - inorganic forms. Methods of Soil Analysis Agronomy Monograph 9, Part 2 (2nd ed., pp. 643-698). Madison, WI: ASA, SSSA https://doi.org/10.2134/agronmonogr9.2.2ed.c33
Kumar S., Stecher G., Suleski M., Sanderford M., Sharma S., Tamura K., 2024. Mega12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Molecular Biology and Evolution, 41(12): msae263. https://doi.org/ 10.1093/molbev/msae263
Lang E., Burghartz M., Spring S., Swiderski J., Spröer C., 2010. Pseudomonas benzenivorans sp. nov. and Pseudomonas saponiphila sp. nov., represented by xenobiotics degrading type strains. Current Microbiology, 60(1): 85–91. https://doi.org/ 10.1007/s00284-009-9501-0
Madhaiyan M., Poonguzhali S., Saravanan V. S., Selvapravin K., Duraipandiyan V., Al-Dhabi N. A., 2017. Pseudomonas sesami sp. nov., a plant growth-promoting Gammaproteobacteria isolated from the rhizosphere of Sesamum indicum L. Antonie van Leeuwenhoek, 110(7): 843–852. https://doi.org/10.1007/s10482-017-0859-x
Madigan M. T., Bender K. S., Buckley D. H., Sattley W. M., Stahl D. A., 2018. Brock Biology of microorganisms (15th ed.). Pearson Education, New York, 173−208.
Mohn W. W., Wilson A. E., Bicho P., Moore E. R. B., 1999. Physiological and phylogenetic diversity of bacteria growing on resin acids. Systematic and Applied Microbiology, 22(1): 68–78. https://doi.org/10.1016/ S0723-2020(99)80029-0
Oanh N. T. P., Tai V. P., Man N. N., Qui N. V., Uyen C. T., Khoa N. H., Khoa N. D., 2022. Selection of carriers for preservation of Rhodococcus sp. XL6.2 capable of degrading benzene, toluene, and xylene. Can Tho University Journal of Science, 58(4A): 62–70. (In Vietnamese with English summary).
Perry J. J., Staley J. T., Lory S., 2002. Chapter 5: Isolation, nutrition, and cultivation of microorganisms. In Microbial Life. Sinauer Associates Incorporated, pp. 117–124.
Ramette A., Frapolli M., Fischer-Le Saux M., Gruffaz C., Meyer J.-M., Défago G., Sutra L., Moënne-Loccoz Y., 2011. Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin. Systematic and Applied Microbiology, 34(3): 180–188. https://doi.org/10.1016/j.syapm.2010.10.005
Rittmann B. E., McCarty P. L., 2001. Environmental Biotechnology: Principles and Applications. McGraw-Hill.
Saitou N., Nei M., 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406–425. https://doi.org/ 10.1093/oxfordjournals.molbev.a040454
Sambrook J., Fritsch E. F., Maniatis T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press.
Singh, J., Singh, A. V., Upadhayay, V. K., & Khan, A., 2020. Comparative evaluation of developed carrier based bioformulations bearing multifarious PGP properties and their effect on shelf life under different storage conditions. Environment and Ecology, 38 (1): 96–103.
Takeuchi K., Someya N., Morohoshi T., 2023. Genome-mining approaches for the evaluation of Pseudomonas protegens and related strains isolated from the rhizosphere in Japan. Physiological and Molecular Plant Pathology, 125: 101981. https://doi.org/ 10.1016/j.pmpp.2023.101981
Tamura K., Nei M., Kumar S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences USA, 101(30): 11030–11035. https://doi.org/ 10.1073/pnas.0404206101
Thandar S. M., Ushiki N., Fujitani H., Sekiguchi Y., Tsuneda S., 2016. Ecophysiology and comparative genomics of Nitrosomonas mobilis Ms1 isolated from autotrophic nitrifying granules of wastewater treatment bioreactor. Frontiers in Microbiology, 7: 1869. https://doi.org/10.3389/fmicb.2016. 01869
Wang X., Wei L., Kotra L. P., 2007. Cyanocobalamin (vitamin B12) conjugates with enhanced solubility. Bioorganic and Medicinal Chemistry, 15(4): 1780–1787. https://doi.org/10.1016/j.bmc.2006.11.036
Xu M., Chen L., Xin Y., Wang X., Wang Z., Meng X., Zhang W., Sun H., Li Y., Zhang W., et al., 2025. Characteristics and mechanism of ammonia nitrogen removal by heterotrophic nitrification bacterium Klebsiella pneumoniae LCU1 and its application in wastewater treatment. Microorganisms, 13(2): 297. https://doi.org/ 10.3390/microorganisms13020297
Zhang T., Shao M. F., Ye L., 2012. 454 Pyrosequencing reveals bacterial diversity of activated sludge from 14 sewage treatment plants. The ISME Journal, 6(6): 1137–1147. https://doi.org/10.1038/ismej. 2011.188
Zhao B., He Y. L., Hughes J., Zhang X. F., 2010. Heterotrophic nitrogen removal by a newly isolated Acinetobacter calcoaceticus HNR. Bioresource Technology, 101(14): 5194–5200. https://doi.org/10.1016/j.bior tech.2010.02.015
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Cong Phu Pham, Vu Luan Truong, Phat Tai Vo, Kim Toan Diep, Thi Phi Oanh Nguyen
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Academia Journal of Biology (AJB) is an open-access and peer-reviewed journal. The articles published in the AJB are licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0), which permits for immediate free access to the articles to read, download, copy, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited (with a link to the formal publication through the relevant DOI), and without subscription charges or registration barriers. The full details of the CC BY-NC-ND 4.0 License are available at https://creativecommons.org/licenses/by-nc-nd/4.0/.
