Screening and Functional Characterization of Ammonia and Nitrite-Oxidizing Bacteria from Wastewater Sources

IJEP 46(8): 733-741 : Vol. 46 Issue 8 (August 2026)

Full Text

Anant J. Bhende and Hiren B. Soni*

Charutar Vidya Mandal University, Department of Environmental Science and Technology, Institute of Science and Technology for advanced Studies and Research, Vallabh Vidyanagar – 388 120, Gujarat, India

Abstract

Petrochemical effluents present persistent nitrogen and hydrocarbon loads that complicate standard treatment methods. This study isolated, identified and assessed nitrifying bacteria from the IOCL (Vadodara, Gujarat) sewage system for crude oil remediation under controlled conditions. We isolated two strains from refinery wastewater: Nitrosomonas aestuarii (ammonia-oxidizing bacteria (AOB)) and Nitrococcus mobilis (nitrite-oxidizing bacteria (NOB)). We used morphology, biochemical testing and 16S rRNA sequencing to identify them. Factorial single-variable assays measured the effects of pH (5–9), temperature (20–40°C), initial oil load (0–2.0% v/v) and nitrogen source (equimolar nitrogen as NH4NO3, NaNO3, NaNO2, NH4Cl; no-nitrogen control) in a defined mineral medium. We measured growth (OD600) and hydrocarbon removal (gravimetry after n-hexane extraction) over 24-120 hr. We used one-way ANOVA with Tukey post-hoc tests to analyze the data (mean±SD, n=3). The best conditions for removing crude oil were pH 7–8 and 30–35°C, which removed about 75–78% of it. However, pH 5 or 9 and 40°C removed only about 35–40%. At 0.25–1.0% oil (78–83%), there was a high-efficiency plateau, but at 2.0%, it dropped significantly (45–50%). Nitrogen supplementation was necessary: ammonium nitrate removed the most (80–82%), more than nitrate, nitrite and ammonium salts and much more than the no-N control (~45%). These findings establish an operational range (pH 7–8, 30–35°C, 0.75–1.0% oil; NH4NO3 as the preferred nitrogen source) and support the use of N. aestuarii–N. mobilis consortia for bioremediation of petrochemical wastewaters.

Keywords

Nitrosomonas aestuarii, Nitrococcus mobilis, Nitrification, Crude oil biodegradation, Petrochemical wastewater bioremediation

References

  1. Malmasi, S., Jozi, S., Monavari, S. and Jafarian, M. 2010. Ecological impact analysis on Mahshahr petrochemical industries using analytic hierarchy process method. Int. J. Env. Res., 4: 725-734.
  2. Nadal, M., Schuhmacher, M. and Domingo, J. L. 2011. Long-term environmental monitoring of persistent organic pollutants and metals in a chemical/petrochemical area: Human health risks. Env. poll., 159: 1769-1777. DOI: 10.1016/j.envpol.20 11.04.007.
  3. Abouzari, M., Pahlavani, P., Izaditame, F. and Bigdeli, B. 2021. Estimating the chemical oxygen demand of petrochemical wastewater treatment plants using linear and non-linear statistical models–A case study. Chemosphere. 270: 129465. DOI: 10.1016/j.chemosphere.2020.1294 65.
  4. Carey, R.O. and Migliaccio, K.W. 2009. Contribution of wastewater treatment plant effluents to nutrient dynamics in aquatic systems: A review. Env. manage., 44: 205-217. DOI: 10.1007/s00 267-009-9309-5.
  5. Kubsad, V., Gupta, S. and Chaudhari, S. 2005. Treatment of petrochemical wastewater by rotating biological contactor. Env. tech., 26: 1317-1326. DOI: 10.1080/09593332608618614.
  6. Thorat, B.N. and Sonwani, R.K. 2022. Current technologies and future perspectives for the treatment of complex petroleum refinery wastewater: A review. Bioresour. Tech., 355: 127263. DOI: 10.101 6/j.biortech.2022.127263.
  7. Jafarinejad, S. and Jiang, S.C. 2019. Current technologies and future directions for treating petroleum refineries and petrochemical plants (PRPP) wastewaters. J. Env. Chem. Eng., 7: 103326. DOI: 10.1016/j.jece.2019.103326.
  8. Dey, S., Das, A., Mallick, K., Sahu, A. and Das, A. P. 2024. Environmental petroleum waste: Pollution, toxicity, sustainable remediation (chapter 7). In Impact of petroleum waste on environmental pollution and its sustainable management through circular economy. Springer Nature Switzerland. DOI: 10.1007/978-3-031-48220-5.
  9. Kolawole, A.S. and Iyiola, A.O. 2023. Environmental pollution: threats, impact on biodiversity and protection strategies. In Sustainable utilization and conservation of Africa’s biological resources and environment. Ed Izah, S.C. and Ogwu, M.C. Springer, Singapore. pp 377–409. DOI: 10.1007/978-981-19-6974-4_14.
  10. Daims, H., Lücker, S. and Wagner, M. 2016. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends microbiol., 24: 699-712. DOI: 10.1016/j.tim.2016.05.004.
  11. Hayatsu, M., Tago, K. and Saito, M. 2008. Various players in the nitrogen cycle: Diversity and functions of the micro-organisms involved in nitrification and denitrification. Soil Sci. Plant Nutrition. 54: 33-45. DOI: 10.1111/j.1747-0765.2007.00195.x.
  12. Soliman, M. and Eldyasti, A. 2018. Ammonia-oxidizing bacteria (AOB): Opportunities and applications—A review. Reviews Env. Sci. Biotech., 17: 285-321. DOI: 10.1007/s11157-018-9463-4.
  13. Ravi, A., Ravuri, M., Krishnan, R., Narenkumar, J., Anu, K., Alsalhi, M.S., Devanesan, S., Kamala-Kannan, S. and Rajasekar, A. 2022. Characterization of petroleum degrading bacteria and its optimization conditions on effective utilization of petroleum hydrocarbons. Microbiol. Res., 265: 127184. DOI: 10.1016/j.micres.2022.127184.
  14. Figuerola, E.L. and Erijman, L. 2010. Diversity of nitrifying bacteria in a full-scale petroleum refinery wastewater treatment plant experiencing unstable nitrification. J. hazard. mater., 181: 281-288. DOI: 10.1016/j.jhazmat.2010.05.009.
  15. Semenova, E., Ershov, A., Sokolova, D.S., Tourova, T. and Nazina, T. 2020. Diversity and biotechnological potential of nitrate-reducing bacteria from heavy-oil reservoirs (Russia). Microbiol., 89: 685-696. DOI: 10.1134/S0026261720060168.
  16. Gevertz, D., Telang, A.J., Voordouw, G. and Jenne-man, G.E. 2000. Isolation and characterization of strains CVO and FWKO B, two novel nitrate-reducing, sulphide-oxidizing bacteria isolated from oil field brine. Appl. Env. Microbiol., 66: 2491-2501. DOI: 10.1128/AEM. 66.6.2491-2501.2000.
  17. Grigoryan, A.A., Cornish, S.L., Buziak, B., Lin, S., Cavallaro, A., Arensdorf, J.J. and Voordouw, G. 2008. Competitive oxidation of volatile fatty acids by sulphate-and nitrate-reducing bacteria from an oil field in Argentina. Appl. Env. Microbiol., 74: 4324-4335. DOI: 10.1128/AEM.00419-08.
  18. Nemati, M., Jenneman, G.E. and Voordouw, G. 2001. Impact of nitrate-mediated microbial control of souring in oil reservoirs on the extent of corrosion. Biotech. progress. 17: 852-859. DOI: 10.1021/bp010084v.
  19. Simon, J. 2002. Enzymology and bioenergetics of respiratory nitrite ammonification. FEMS microbiol. reviews. 26: 285-309. DOI: 10.1111/j.1574-6976. 2002.tb00616.x.
  20. Adedeji, J.A., Tetteh, E.K., Opoku Amankwa, M., Asante-Sackey, D., Ofori-Frimpong, S., Armah, E. K., Rathilal, S., Mohammadi, A.H. and Chetty, M. 2022. Microbial bioremediation and biodegradation of petroleum products—A mini review. Appl. Sci., 12: 12212. DOI: 10.3390/app 122312212.
  21. Chekroud, Z., Gouda, M. and Houhamdi, M. 2011. Biodegradation of crude oil in marine medium. Proteomics Bioinfor., 4: 231-237. DOI: 10.4172/jpb.1000194.
  22. Boszczyk-Maleszak, H., Zabost, A., Wolicka, D. and Kacieszczenko, J. 2006. Effectiveness of biodegradation of petroleum products by mixed bacterial populations in liquid medium at different pH values. Polish j. microbiol., 55: 69-73.
  23. Al-Hoqani, U.H., Azmi, S.N.H., Al-Habsi, H.S., Reddy, S.H., Sah, P., Al-Harthi, J.S., Saad, Z.N.B., Al-Nabhani, Z.S., Ahmed, Q.U. and Mia, M.A.R. 2025. A prospective study on the biodegradation of petroleum hydrocarbons mediated by selected marine bacterial isolates. Regional Studies Marine Sci., 85: 104143. DOI: 10.1016/j.rsma.2025.104 143.
  24. Bao, M.T., Wang, L.N., Sun, P.Y., Cao, L.X., Zou, J. and Li, Y.M. 2012. Biodegradation of crude oil using an efficient microbial consortium in a simulated marine environment. Marine poll. bulletin. 64: 1177-1185. DOI: 10.1016/j.marpolbul.2012.03. 020.
  25. El-Liethy, M., El-Noubi, M., Abia, A., El-Malky, M., Hashem, A. I. and El-Taweel, G. 2022. Eco-friendly bioremediation approach for crude oil-polluted soils using a novel and biostimulated Enterobacter hormaechei ODB H32 strain. Int. J. Env. Sci. Tech., 19: 10577-10588. DOI: 10.10 07/s13762-021-03885-z.
  26. Abed, R.M., Al-Kharusi, S. and Al-Hinai, M. 2015. Effect of biostimulation, temperature and salinity on respiration activities and bacterial community composition in an oil polluted desert soil. Int. Biodeterioration Biodegradation. 98: 43-52. DOI: 10.1016/j.ibiod.2014.11.018.
  27. Ibrahim, S.Y., Abdelhamid, E.A., El-Hagrassi, A.M. and Kamal, N.M. 2025. Statistical optimization of crude oil bioremediation using Streptomyces auran-tiogriseus isolated from Egypt’s western desert. Biodegradation. 36: 1-25. DOI: 10.1007/s10532-025-10154-0.
  28. Shen, T., Pi, Y., Bao, M., Xu, N., Li, Y. and Lu, J. 2015. Biodegradation of different petroleum hydrocarbons by free and immobilized microbial consortia. Env. Sci.: Processes Impacts. 17: 2022-2033. DOI: 10.1039/c5em00318k.
  29. Okuda, T., Alcántara-Garduño, M.E., Suzuki, M., Matsui, C., Kose, T., Nishijima, W. and Okada, M. 2007. Enhancement of biodegradation of oil adsorbed on fine soils in a bioslurry reactor. Chemosphere. 68: 281-286. DOI: 10.1016/j.chemosphere. 2006.12.081.
  30. Wang, Z., Sun, X., Song, K., Lu, J. and Wu, M. 2025. Combined optimization of petroleum hydrocarbon degradation rate prediction model using response surface methodology and artificial neural networks. J. Indian Chem. Soc., 1102(9): 101937. DOI: 10.1016/j.jics.2025.101937.
  31. Graham, D., Smith, V., Cleland, D. and Law, K. 1999. Effects of nitrogen and phosphorus supply on hexadecane biodegradation in soil systems. Water, air soil poll., 111: 1-18. DOI: 10.1023/A: 1005091427942.
  32. Obinna, O.A., Chibuike, U.S. and Onwurah, I. 2015. Variation in the carbon (C), phosphorus (P) and nitrogen (N) utilization during the biodegradation of crude oil in soil. J. Petroleum Env. Biotech., 6(2): 1000206. DOI: 10.4172/2157-7463.1000206.
  33. Ball, J.S., Whisman, M.L. and Wenger, W.J. 1951. Nitrogen content of crude petroleums. Ind. Eng. Chem., 43: 2577-2581.
  34. Ning, Z., Liang, J., Ti, J., Zhang, M. and Cai, C. 2025. Enhanced natural attenuation of gasoline contaminants in groundwater: Applications and challenges of nitrate-stimulating substances. Microorganisms. 13(7): 1575. DOI: 10.3390/microorganisms13071575.
  35. Youssef, N., Elshahed, M.S. and McInerney, M.J. 2009. Microbial processes in oil fields: Culprits, problems and opportunities. Adv. appl. microbiol., 66: 141-251. DOI: 10.1016/S0065-2164(08) 00806-X.
  36. Foght, J., Semple, K., Gauthier, C., Westlake, D., Blenkinsopp, S., Sergy, G., Wang, Z. and Fingas, M. 1999. Effect of nitrogen source on biodegradation of crude oil by a defined bacterial consortium incubated under cold, marine conditions. Env. Tech., 20: 839-849. DOI:10.1080/095 93332008616879.
  37. Wrenn, B.A., Haines, J.R., Venosa, A.D., Kadkho-dayan, M. and Suidan, M.T. 1994. Effects of nitrogen source on crude oil biodegradation. J. Ind. Microbiol., 13: 279-286. DOI: 10.1007/BF0 1569 729.
  38. Gouda, M.K., Omar, S.H., Chekroud, Z.A. and Eldin, H.M.N. 2007. Bioremediation of kerosene I: A case study in liquid media. Chemosphere. 69: 1807-1814. DOI: 10.1016/j.chemosphere.2007. 05.079.
  39. Deni, J. and Penninckx, M.J. 1999. Nitrification and autotrophic nitrifying bacteria in a hydrocarbon-polluted soil. Appl. Env. Microbiol., 65: 4008-4013. DOI: 10.1128/AEM.65.9.4008-4013.1999.
  40. Sharma, S. and Pant, A. 2000. Biodegradation and conversion of alkanes and crude oil by a marine Rhodococcus. Biodegradation. 11: 289-294. DOI: 10.1023/a:1011185806974.