Ogujiofor IF¹, Mbachu IAC⁷, Oghonim PAN⁹, Egurefa SO³, Anieto EC⁴, Igwilo CQ¹, Okey-Ndeche NF⁵, Umeh JE⁶, Orjiude EJ⁸, Uwanta LI¹ and Agu KC¹*

¹Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria
²Microbiology Department, Biological Sciences, University of Delta, Agbor P.M.B 2090, Agbor, Delta State, Nigeria
³Department of Science Laboratory Technology, Southern Delta University, PMB 05, Ozoro, Delta State, Nigeria
⁴Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, University on the Niger, KM-13, Onitsha - Enugu Express Way Umunya, Nigeria
⁵Veritas University Abuja, Area Council, Bwari 901101, Federal Capital Territory, Nigeria
⁶Department of Pharmaceutical Microbiology and Biotechnology, University of Port-Harcourt, Nigeria
⁷Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University Uli, Anambra State, Nigeria
⁸Department of Microbiology, Ahmadu Bello University, Zaria, Nigeria
⁹Microbiology Department, Biological Sciences, University of Delta, Agbor P.M.B 2090, Agbor, Delta State, Nigeria

*Corresponding Author: Agu KC, Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria.

Received: October 21, 2025; Published: November 07, 2025

Abstract

Petroleum refinery effluents are characterized by the presence of pollutants such as hydrocarbons as such could be of serious environmental consequence if discharged into receiving sites without proper treatment to remove the pollutants. This study assessed the capacity of wild and mutant strains of Bacillus subtilis and Pseudomonas putida isolated from refinery effluent in the degradation of hydrocarbons present in the effluent. The physicochemical parameters of the raw and treated effluent samples collected from Kaduna Refinery and Petrochemical Company (KRPC) were determined using standard guidelines. With the exception of turbidity (35.3 and 18.2 NTU), BOD (190 and 29.6 mg/L), COD (351.2 and 78.1 mg/L) and Oil and Grease (45.2 and 17.9 mg/L) for the raw and treated effluent respectively, and conductivity (695 μS/cm) for the raw effluent, all other parameters were within the permissible limits set by FMENV. Six (6) and eleven (11) isolates of Bacillus subtilis and Pseudomonas putida respectively were isolated from the effluent and screened for capacity to utilize and grow on mineral medium containing different concentrations (0.5%, 1%, 1.55% and 2%) of crude oil as the sole source of carbon. Isolates TE8 (B. subtilis) and TEC10 (P. putida) had luxuriant growth across the first three concentrations of crude oil and medium growth on medium containing 2% crude oil. Both isolates were treated with nitrous acid and UV- irradiation to generate mutants. Death rate of 59.67% (40.33% survival) and 66.33% (33.67% survival) were observed for Bacillus subtilis and Pseudomonas putida respectively on treatment with nitrous acid. Also death rate of 51.67% (48.33% survival) and 40% (60%) were observed for Bacillus subtilis and Pseudomonas putida respectively on exposure to UV- irradiation. The relative efficiency of hydrocarbon degradation by the wild and mutant strains was assessed by evaluating the Hydrocarbon utilizing bacterial (HUB) counts and changes in concentration of oil and grease at intervals of 3 days for 15 days. The UV-mutant strains had slightly higher HUB counts (2.73×108, 1.5×108 and 2.391×108) than the wild strains (2.73×108, 1.32×108 and 2.39×108) while the nitrous acid mutant strains had the lowest HUB counts (8.35×107, 8.13×107 and 5.07×107) for P. putida, B. subtilis and their co-culture respectively. Also, higher oil and grease degradation was observed in the UV-mutant strains with 96.23%, 92.60% and 99.38%, followed by the wild strains with 87.25%, 80.25% and 88.89% and the nitrous acid mutant strains with 80.25%, 68.52% and 81.48% oil and grease degradation for P. putida, B. subtilis and their co-culture respectively. It was therefore concluded that while wild strains of B. subtilis and P. putida are good hydrocarbon degrading agents, irradiation with UV results in increased efficiency and the co-culture of the bacteria were more efficient than the individual culture.

Keywords: Biodegradation; Pseudomonas Putida; Bacillus subtilis

References

1. Adams GO., et al. “Bioremediation of spent oil contaminated soils using poultry litter”. Research Journal of Environmental Sciences2 (2015): 81-90.
2. Adebusoye SA., et al. “Microbial degradation of petroleum hydrocarbons in a polluted tropical stream”. World Journal of Microbiology and Biotechnology 8 (2007): 1149-1159.
3. Agudosi SO., et al. “Monoculture degradation of Aspergillus flavus and Penicillium chrysogenum of varying concentration of spent engine oil (spent motor oil) in Oba, Anambra State-Nigeria”. International Journal of Research Publication and Reviews8 (2023): 747-754.
4. Agu KC., et al. “Isolation and characterization of microorganisms from oil polluted soil in Kwata, Awka South, Nigeria”. American Journal of Current Microbiology 3 (2015): 46-59.
5. Agu KC., et al. “Isolation and characterization of halotolerant bacteria from Ezzu River Amansea, Awka, Anambra State”. Bioengineering and Bioscience4 (2017): 86-90.
6. Agu KC and Odibo FJC. “Biodegradation potentials of Aspergillus flavipes isolated from Uburu and Okposi salt lakes”. International Journal of Trend in Scientific Research and Development5 (2021): 1160-1170.
7. Agu KC., et al. “Lipase production by fungal isolates from palm oil-contaminated soil in Awka Anambra State, Nigeria”. International Journal of Agriculture and Biosciences6 (2013): 386-390.
8. Agu KC., et al. “Production of mannanase enzyme using Aspergillus isolated from decaying palm press cake”. Scholars Academic Journal of Biosciences 2.12A (2014): 863-870.
9. Agu KC., et al. “Hydrocarbon biodegradation potential of cyanobacteria in oil polluted soil”. International Journal of Trend in Scientific Research and Development7 (2022): 733-737.
10. Agu KC., et al. “Influence of solid waste dumps leachate on bacteriological and heavy metals contamination of ground water in Awka”. American Journal of Life Sciences4 (2014): 450-457.
11. Agu KC., et al. “Isolation and characterization of proteolytic enzyme produced from fungi”. Cognizance Journal of Multidisciplinary Studies 6 (2023): 485-493.
12. Ajao AT., et al. “Biodegradation of petroleum hydrocarbons in petroleum contaminated soil by Pseudomonas species”. Journal of Environmental Science and Technology 2 (2013): 95-105.
13. Ajao AT., et al. “Enhanced biodegradation of crude oil in soil by a microbial consortium. Int J Sci Res Publ 4.7 (2014): 1-7.
14. American Public Health Association. “Standard methods for the examination of water and wastewater”. 19^th Washington, D.C.: APHA; (1995).
15. Anaukwu CG., et al. “Influence of anionic, cationic and non-ionic surfactants on growth of hydrocarbon utilizing bacteria”. American Journal of Current Microbiology 4 (2016): 10-16.
16. Anaukwu CG., et al. “Effect of biosurfactant produced by Citrobacter murliniae AF025369 and a synthetic surfactant on degradation of crude oil”. Edorium™ Journal of Microbiology 2 (2016): 1-6.
17. Anupama M and Padma S. “Biodegradation of petroleum hydrocarbons by Pseudomonas isolated from petroleum contaminated soil”. Journal of Environmental Biology 30.5 (2009): 825-830.
18. Asadirad MH., et al. “Biodegradation of heavy crude oil by bacterial strains isolated from petroleum contaminated soils”. Petroleum Science and Technology 6 (2016): 543-549.
19. Atlas RM. “Microbial degradation of petroleum hydrocarbons: an environmental perspective”. Microbiology Review1 (1981): 180-209.
20. Attiogbe FK., et al. “Correlating biochemical and chemical oxygen demand of effluents—a case study of some selected industries in Kumasi, Ghana”. West African Journal of Applied Ecology 1 (2007): 1-10.
21. Awari VG., et al. “The ubiquity, importance and harmful effects of microorganisms: an environmental and public health perspective”. International Journal of Progressive Research in Engineering Management and Science12 (2023): 1-10.
22. Bako SP., et al. “Bioremediation of refinery effluents by strains of Pseudomonas and Bacillus species”. African Journal of Biotechnology18 (2008): 3349-53.
23. Bay S., et al. “Impacts of stormwater discharge on southern California coastal waters”. Environmental Science and Technology 16 (2003): 361A-8A.
24. Beg KR., et al. “Chemical and biological assessment of petroleum refinery effluent”. Bulletin of Environmental Contamination and Toxicology 1 (2001): 87-95.
25. Chen J., et al. “Effect of oil and grease on the activated sludge process”. Water Research11 (1999): 2635-2641.
26. Chikere CB and Ekwuabu CB. “Molecular characterization of autochthonous hydrocarbon-utilizing bacteria in oil-polluted sites”. British Microbiology Research Journal12 (2014): 1305-20.
27. Cowan ST and Steel KJ. “Manual for the identification of medical bacteria”. 3^rd Cambridge: Cambridge University Press (2003).
28. Darsa KV., et al. “Biodegradation of petroleum compound using the bacterium Bacillus subtilis”. Scientific International1 (2014): 20-25.
29. Das N and Chandran P. “Microbial degradation of petroleum hydrocarbon contaminants: an overview”. Biotechnology Research International 2011 (2011): 941810.
30. Egurefa SO., et al. “Isolation and identification of fungal species in pesticide-contaminated soil from Abo-Obosi, Anambra”. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences10 (2024): 1-12.
31. Egurefa SO., et al. “Assessment of the bacteriological and heavy metal contamination in drinking water from borehole sites in Enugu metropolis”. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences5 (2024): 13-26.
32. Ekhaise FO and Nkwelle J. “Microbiological and physicochemical analyses of oil contaminated soil from major motor mechanic workshops in Benin City, Nigeria”. Journal of Applied Sciences and Environmental Management 4 (2011): 597-600.
33. Eniola KI., et al. “Biodegradation of crude oil by Bacillus and Pseudomonas species isolated from hydrocarbon contaminated soil”. Ife Journal of Science2 (2014): 217-225.
34. Esedafe WK., et al. “Biodegradation of Bonny light crude oil by bacteria isolated from contaminated soil”. International Journal of Environmental Bioremediation and Biodegradation1 (2015): 28-34.
35. Ezeokoli CM., et al. “Bacteriological evaluation of Kwata abattoir waste water, Awka, Nigeria”. Innovare Journal of Life Sciences 11 (2023): 1-4.
36. Ezenwelu CO., et al. “Ecological significance of dynamism of physiologic variance on properties of palm oil mill effluents”. Asian Journal of Microbiology and Biotechnology 1 (2024): 60-67.
37. Hamza AA., et al. “Biodegradation of petroleum hydrocarbons in the environment: a review”. Journal of Environmental Science and Technology 4 (2012): 217-228.
38. Idise OE., et al. “Biodegradation of petroleum by bacteria isolated from hydrocarbon polluted soil”. African Journal of Environmental Science and Technology5 (2010): 271-278.
39. Ifediegwu MC., et al. “Isolation, growth and identification of chlorpyrifos degrading bacteria from agricultural soil in Anambra State, Nigeria”. Universal Journal of Microbiology Research4 (2015): 46-52.
40. Ijah UJJ. “Studies on relative capabilities of bacterial and yeast isolates from tropical soil in degrading crude oil”. Waste Management5 (1998): 293-299.
41. Ikram A., et al. “Mutation induced biosynthesis of polygalacturonase by Bacillus subtilis”. Pakistan Journal of Botany6 (2009): 3259-3268.
42. Joel OF and Amajuoyi CA. “Physicochemical characteristics of refinery effluents from Kaduna refinery and petrochemical company, Nigeria”. Journal of Applied Sciences and Environmental Management 4 (2009): 65-69.
43. Kayode-Ishola TM., et al. “Microbial degradation of petroleum hydrocarbons in a polluted tropical stream”. Journal of Applied Sciences and Environmental Management 2 (2008): 83-89.
44. Kiran MG. “Bioremediation of petroleum hydrocarbons in soil”. International Journal of Environmental Biology 1 (2013): 1-8.
45. Leahy JG and Colwell RR. “Microbial degradation of hydrocarbons in the environment”. Microbiology Review3 (1990): 305-315.
46. Liyanage GY and Manage PM. “Biodegradation of petroleum refinery wastewater by a bacterial consortium”. Journal of the National Science Foundation of Sri Lanka 4 (2016): 397-404.
47. Mbachu AE., et al. “Hydrocarbon degrading potentials of indigenous bacteria isolated from auto-mechanic workshops at Mgbuka-Nkpor, Nigeria”. Journal of Global Biosciences1 (2014): 321-326.
48. Majid A., et al. “Biodegradation of crude oil by Bacillus and Pseudomonas species isolated from hydrocarbon contaminated soil”. Nigerian Journal of Microbiology1 (2008): 1773-1780.
49. Marcus AC and Ekpete OA. “Physicochemical characteristics of refinery effluents from Port Harcourt refinery company, Nigeria”. Journal of Applied Chemistry8 (2014): 15-20.
50. Moneke AN and Nwangwu CO. “Biodegradation of crude oil by Bacillus and Pseudomonas species isolated from oil contaminated soil”. Nature Science11 (2011): 60-67.
51. Musa JJ., et al. “Biodegradation of petroleum hydrocarbons in soil by microbial consortium”. International Journal of Scientific Engineering and Research4 (2015): 1234-1240.
52. Naga SR., et al. “Isolation and identification of hydrocarbon-degrading bacteria from oil-contaminated soil”. Journal of Basic and Applied Sciences 12 (2016): 260-268.
53. Nwaichi EO., et al. “Distribution of selected carcinogens in refined petroleum products and oil spill sites”. Journal of Environmental Chemistry and Ecotoxicology8 (2013): 216-221.
54. Nwanyanwu CE and Abu GO. “Biodegradation of petroleum hydrocarbons by Pseudomonas and Bacillus species isolated from oil contaminated soil”. Journal of Microbiology and Biotechnology Research 2 (2012): 298-307.
55. Oaikhena EE., et al. “Physicochemical and microbiological characteristics of refinery effluent from Kaduna refinery and petrochemical company, Nigeria”. International Journal of Science Engineering and Technology5 (2016): 239-245.
56. Obot EA., et al. “Environmental impact assessment of refinery effluents on water quality in the Niger Delta region of Nigeria”. Journal of Applied Science8 (2007): 1173-1179.
57. Ojiagu KD., et al. “Biosorption of hexavalent chromium by Pseudomonas aeruginosa strain ANSC: equilibria isothermic, kinetic and thermodynamic studies”. Bioengineering and Bioscience 1 (2018): 1-10.
58. Ojo OA. “Petroleum hydrocarbon utilization by native bacterial population from a wastewater canal Southwest Nigeria”. African Journal of Biotechnology 5 (4 (2006): 333-337.
59. Okafor UC., et al. “Bioremediation of crude oil-polluted soil using broiler-chicken droppings”. Applied and Environmental Microbiology Journal 4 (2016): 75-84.
60. Okafor UC., et al. “Effect of chicken droppings amendment on bioremediation of crude oil polluted soil”. European Journal of Experimental Biology4 (2016): 62-68.
61. Okerentugba PO and Ezeronye OU. “Petroleum degrading potentials of single and mixed microbial cultures isolated from rivers and refinery effluent in Nigeria”. African Journal of Biotechnology9 (2003): 288-292.
62. Okoh AI., et al. “Biodegradation of Nigerian crude oil in seawater by microorganisms”. Journal of Scientific Research and Development 6 (2003): 1-8.
63. Okeke BC., et al. “Monoculture solid phase degradative potential of Congo red by Aspergillus niger and Aspergillus flavus”. International Journal of Trend in Scientific Research and Development3 (2023): 684-688.
64. Okonkwo NN., et al. “Identification of indigenous bacteria from soil contaminated with cassava mill effluents”. International Journal of Progressive Research in Engineering Management and Science 9 (2023): 393-399.
65. Onuoha SC., et al. “Petroleum hydrocarbon degradation by mixed culture of Bacillus and Pseudomonas species”. Journal of Microbiology and Antimicrobials 7 (2011): 186-191.
66. Oparaji EH., et al. “Studies on kinetic properties of Aspergillus producing peroxidase from petroleum hydrocarbon spilled soil”. Rafidain Journal of Science 1 (2024): 49-67.
67. Orji MU., et al. “Bioremediation of glyphosate polluted soil using fungal species”. International Journal of Trend in Scientific Research and Development 7 (2022): 726-732.
68. Orji MU., et al. “Harnessing the antifungal properties of Ocimum gratissimum for sustainable water-based paint formulations”. International Journal of Innovative and Applied Research 1 (2025): 12-29.
69. Orji MU., et al. “Utilization of used Global System for Mobile Communication (GSM) recharge cards for cellulase production by Penicillium species and Aspergillus species”. American Journal of Life Sciences4 (2014): 548-556.
70. Otukunefor TV and Obiukwu CO. “Impact of refinery effluent on the physicochemical properties of a water body in the Niger Delta”. Applied Ecology and Environmental Research 3 (1 (2005): 61-72.
71. Prakash AA., et al. “Biodegradation of petroleum hydrocarbons by Bacillus and Pseudomonas species”. International Journal of ChemTech Research 4 (2014): 2431-2435.
72. Radojavic M and Bashkin VN. “Practical environmental analysis”. Cambridge: Royal Society of Chemistry; (1999).
73. Rahman KSM., et al. “Towards efficient crude oil degradation by a mixed bacterial consortium”. Bioresource Technology3 (2002): 543-549.
74. Salam LB and Obayori OS. “Biodegradation of bonny light crude oil by Bacillus and Micrococcus species isolated from contaminated soils”. International Journal of Environmental Bioremediation and Biodegradation1 (2013): 14-21.
75. Salam LB., et al. “Biodegradation of crude oil by Bacillus and Pseudomonas species isolated from oil contaminated soil”. Nigerian Journal of Microbiology 25 (2011): 2327-2335.
76. Salleh AB., et al. “Bioremediation of petroleum hydrocarbon pollution”. Indian Journal of Biotechnology3 (2003): 411-25.
77. Sepahi AA., et al. “Isolation and characterization of crude oil degrading Bacillus spp”. Journal of Environmental Health Science and Engineering3 (2008): 149-154.
78. Singh H., et al. “Biodegradation of petroleum hydrocarbons by Bacillus and Pseudomonas species”. International Journal of Advanced Research6 (2013): 1-5.
79. Stanley HO., et al. “Bioremediation of crude oil polluted soil using microbial consortium”. J Nat Sci Res 5.8 (2015): 122-129.
80. Umanu G., et al. “Microbial degradation and its kinetics on crude oil polluted soil”. 2.1 (2013): 1-9.
81. Umeoduagu ND., et al. “Randomized genetically modified monoculture degradation by Aspergillus niger and Penicillium chrysogenum on varying concentration of spent engine oil”. International Journal of Research6 (2024): 162-173.
82. Unimke AA., et al. “Microbial degradation of petroleum hydrocarbons: a review”. International Journal of Environmental Sciences and Natural Resources3 (2018): 1-7.
83. Uwanta LI., et al. “Comparative mechanical properties of cassava (Manihot esculenta) and potato (Ipomoea batatas) peel starch biodegradable plastics in food packaging”. International Journal of Research Publication and Reviews4 (2024): 4376-4379.
84. Uwanta LI., et al. “Pseudomonas aeruginosa isolated from vermicompost in the degradation of varying concentration of polyvinyl chloride (PVC) and polyvinyl alcohol (PVA)”. International Journal of Research Publication and Reviews8 (2023): 547-553.
85. Uzoekwe NS and Oghosanine FA. “The effect of refinery and petrochemical effluent on water quality of Ubeji creek Warri, Southern Nigeria”. Ethiopian Journal of Environmental Studies and Management2 (2011): 107-116.
86. Victor-Aduloju AT., et al. “Comparative analysis of microbial load of water in selected hostels in Ifite, Awka”. International Journal of Progressive Research in Engineering Management and Science9 (2023): 400-408.
87. Vinothini C., et al. “Biodegradation of petroleum and crude oil by Pseudomonas putida and Bacillus cereus”. International Journal of Current Microbiology and Applied Sciences 1 (2015): 318-327.
88. Xu X and Zhu W. “Treatment of refectory oily wastewater by electro-coagulation process”. Chemosphere 10 (2004): 889-894.
89. Anene BO and Chika EC. “Comparative study of the biodegradation of crude oil by Bacillus subtilis and Pseudomonas aeruginosa”. Journal of Research in Biology8 (2011): 556-561.

Citation

Citation: Agu KC., et al. “Comparative Hydrocarbon-Degrading Efficiency of Wild and Mutant Strains of Bacillus subtilis and Pseudomonas putida Isolated from Refinery Effluents".Acta Scientific Microbiology 8.12 (2025): 14-38.

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Copyright: © 2025 Agu KC., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.