Isolation, Molecular Identification, and Characterization of Extracellular Lipolytic Activity of Bacillus Strain from Petroleum-contaminated Soil

Document Type : Research Article

Authors

Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran

Abstract

Lipases are versatile industrial enzymes with broad applications in the food, pharmaceutical, detergent, and biofuel industries. Petroleum-contaminated soils are promising sources of microorganisms adapted to hydrocarbon-rich environments and may harbor bacteria capable of producing extracellular enzymes with valuable biochemical properties. This study aimed to isolate and screen lipase-producing bacteria from petroleum-contaminated soils and characterize the extracellular lipolytic activity of the most active isolate. Five soil samples were collected from petroleum product-contaminated sites in Mahan and Kerman, Iran. Bacterial isolates were screened using Rhodamine B-olive oil agar, and extracellular lipase activity was quantified using p-nitrophenyl palmitate. The effects of nitrogen and carbon sources, edible oils, pH, temperature, and incubation time on extracellular lipolytic activity were evaluated, along with thermal stability. Molecular identification was performed using 16S rRNA gene sequencing and phylogenetic analysis. Among the isolates, strain S4, obtained from gasoline-contaminated soil, exhibited the highest lipolytic activity and was selected for further characterization. Peptone was the most effective nitrogen source, while olive oil produced the highest activity among the tested carbon sources (0.098 U/mL). Castor oil further increased extracellular lipolytic activity to 0.134 U/mL. Maximum activity was observed at 30°C after 48 h of incubation and at pH 9.0 (0.154- 0.159 U/mL). The extracellular lipolytic activity retained notable activity at 52.7°C for 40 min but showed rapid inactivation at 60°C, indicating moderate thermal stability. 16S rRNA sequencing identified strain S4 as a Bacillus strain with 99.8% similarity to the Bacillus cereus group (GenBank accession PP092157). Overall, the isolated strain represents a promising source of extracellular alkaline lipolytic activity with moderate thermal stability and potentially useful biochemical characteristics for further investigation and optimization in industrial biotechnology.

Keywords

Main Subjects

References

Abro, A. A., Qureshi, A. S., Naqvi, M., Khushk, I., Jatt, A. N., Ali, C. H., … & Shafaq, U. (2024). Lipase production from alkalophilic-thermophilic-ionic liquid tolerant Bacillus cereus using agricultural residues for its applications in biodiesel and detergents. Industrial crops and products, 220, 119208. https://doi.org/10.1016/j.indcrop.2024.119208
Abubakar, A., Abioye, O. P., Aransiola, S. A., Maddela, N. R., & Prasad, R. (2024). Crude oil biodegradation potential of lipase produced by Bacillus subtilis and Pseudomonas aeruginosa isolated from hydrocarbon contaminated soil. Environmental Chemistry and Ecotoxicology, 6, 26-32. https://doi.org/10.1016/j.enceco.2023.12.001
Akhter, K., Karim, I., Aziz, B., Bibi, A., Khan, J., & Akhtar, T. (2022). Optimization and characterization of alkaliphilic lipase from a novel Bacillus cereus NC7401 strain isolated from diesel fuel polluted soil. PLOS One, 17(8), e0273368. https://doi.org/10.1371/journal.pone.0273368
Ali, S., Khan, S. A., Hamayun, M., & Lee, I.-J. (2023). The recent advances in the utility of microbial lipases: A review. Microorganisms, 11(2), 510. https://doi.org/10.3390/microorganisms11020510
Borrelli, G. M., & Trono, D. (2015). Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. International Journal of Molecular Sciences, 16(9), 20774-20840. https://doi.org/10.3390/ijms160920774
Casas-Godoy, L., Gasteazoro, F., Duquesne, S., Bordes, F., & Marty, A. (2018). Lipases: An overview. Lipases and Phospholipases: Methods and Protocols, 1835, 3-38. https://doi.org/10.1007/978-1-4939-8672-9_1
Chouhan, R., Lapaka, S., Alpula, N., & Podeti, S. (2020). Optimization and molecular characterization of lipase producing Bacillus subtilis strain RCPS3 and Bacillus fumarioli strain RCPS4 from oil-contaminated soils of Warangal. Journal of Pure and Applied Microbiology, 14(3), 2063-2070. https://doi.org/10.22207/JPAM.14.3.47
Das, N., & Chandran, P. (2011). Microbial degradation of petroleum hydrocarbon contaminants: An overview. Biotechnology Research International, 2011, 941810. https://doi.org/10.4061/2011/941810
Dasetty, S., Blenner, M. A., & Sarupria, S. (2017). Engineering lipases: Walking the fine line between activity and stability. Materials Research Express, 4(11), 114008. https://doi.org/10.4061/2011/941810114008. 10.1088/2053-1591/aa9946
Demirkan, E., Aybey Çetinkaya, A., & Abdou, M. (2021). Lipase from new isolate Bacillus cereus ATA179: Optimization of production conditions, partial purification, characterization and its potential in the detergent industry. Turkish Journal of Biology, 45(3), 287-300. https://doi.org/10.3906/biy-2101-22
Devi, T., Sistla, S., Khan, R. T., Kailoo, S., Bhardwaj, M., & Rasool, S. (2025). Purification and characterization of detergent stable alkaline lipase from Bacillus safensis TKW3 isolated from Tso Kar brackish water lake. PeerJ, 13, e18921. https://doi.org/10.7717/peerj.18921
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
Guncheva, M., & Zhiryakova, D. (2011). Catalytic properties and potential applications of Bacillus lipases. Journal of Molecular Catalysis B: Enzymatic, 68(1), 1-21. https://doi.org/10.1016/j.molcatb.2010.09.002
Gupta, R., Beg, Q. K., & Lorenz, P. (2002). Bacterial alkaline proteases: Molecular approaches and industrial applications. Applied Microbiology and Biotechnology, 59(1), 15-32. https://doi.org/10.1007/s00253-002-0975-y
Gürkök, S. (2021). Statistical optimization of extracellular thermo-alkaline lipase production from Aeromonas caviae LipT51 with response surface methodology. Journal of the Institute of Science and Technology, 11(3), 1770-1780. https://doi.org/10.21597/jist.872699
He, B., Li, N., Qin, Y., Xian, L., Zhou, J., Liu, S., … & Liang, X. (2025). Gene cloning, purification, and characterization of a cold-active alkaline lipase from Bacillus cereus U2. Fermentation, 11(7), 365. https://doi.org/10.3390/fermentation11070365
Ilesanmi, O. I., Adekunle, A. E., Omolaiye, J. A., Olorode, E. M., & Ogunkanmi, A. L. (2020). Isolation, optimization and molecular characterization of lipase producing bacteria from contaminated soil. Scientific African, 8, e00279. https://doi.org/10.1016/j.sciaf.2020.e00279
Irianto, V. S., Demirkan, E., & Çetinkaya, A. A. (2024). UV mutagenesis for lipase overproduction from Bacillus cereus ATA179, nutritional optimization, characterization and its usability in the detergent industry. Preparative Biochemistry and Biotechnology, 54(7), 918-931. https://doi.org/10.1080/10826068.2023.2299441
Jaeger, K.-E., & Eggert, T. (2002). Lipases for biotechnology. Current Opinion in Biotechnology, 13(4), 390-397. https://doi.org/10.1016/S0958-1669(02)00341-5
Kattatheyil, H., Sajeela, V., Kabeer, S. S., Ezhuthanikkunnel, A. P., Padinchati, K. K., & Ammanamveetil, M. H. (2024). Screening, optimization, and molecular characterization of cold-active lipase producing Bacillus cereus I13 from Arctic sediments. Biologia, 79(3), 1041-1055. https://doi.org/10.1007/s11756-024-01610-y
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16(2), 111-120. https://doi.org/10.1007/BF01731581
Koshlaf, E., & Ball, A. S. (2017). Soil bioremediation approaches for petroleum hydrocarbon polluted environments: A review. Advances in Intelligent Systems and Computing Microbiol, 3(1), 25-49. https://doi.org/10.3934/microbiol.2017.1.25
Srinivas Kumar, D., Prasad, R. M. V., Raja Kishore, K., & Raghava Rao, E. (2012). Effect of Azolla (Azolla pinnata) based concentrate mixture on nutrient utilization in buffalo bulls. Indian Journal of Animal Research, 46(3), 268-271. https://indianjournals.com/article/ijar1-46-3-011
Liu, C.-H., Chen, W.-M., & Chang, J.-S. (2007). Methods for rapid screening and isolation of bacteria producing acidic lipase: Feasibility studies and novel activity assay protocols. World Journal of Microbiology and Biotechnology, 23(5), 633-640. https://doi.org/10.1007/s11274-006-9272-8
McDonald, A. G., Boyce, S., & Tipton, K. F. (2015). Enzyme classification and nomenclature. Encyclopedia of Life Sciences, 1-11. https://doi.org/10.1002/9780470015902.a0000710.pub3
McDonald, A. G., & Tipton, K. F. (2023). Enzyme nomenclature and classification: The state of the art. Federation of European Biochemical Societies Journal, 290(9), 2214-2231. https://doi.org/10.1111/febs.16274
Mobarak-Qamsari, E., Kasra-Kermanshahi, R., & Moosavi-Nejad, Z. (2011). Isolation and identification of a novel, lipase-producing bacterium, Pseudomonas aeruginosa KM110. Iranian Journal of Microbiology, 3(2), 92-98.
Mostafa, N., Maher, A., & Abdelmoez, W. (2013). Production of mono-, di-, and triglycerides from waste fatty acids through esterification with glycerol. Advances in Bioscience and Biotechnology, 4(9), 900-907. https://doi.org/10.4236%2Fabb.2013.49118
Saitou, N., & Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4(4), 406-425. https://doi.org/10.1093/oxfordjournals.molbev.a040454
Sari, N. N., Nurhasanah, & Haryati, T. (2026). Partial purification and characterization acidophilic lipase from Bacillus cereus ALP E1. Protein Expression and Purification, 239, 106870. https://doi.org/10.1016/j.pep.2025.106870
Sharma, A., Bardhan, D., & Patel, R. (2009). Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490. Indian Journal of Biochemistry and Biophysics, 46(2), 178-183. https://pubmed.ncbi.nlm.nih.gov/19517996/
Sharma, S., & Kanwar, S. S. (2014). Organic solvent tolerant lipases and applications. The Scientific World Journal, 2014, 625258. https://doi.org/10.1155/2014/625258
Sharma, R., & Sharma, N. (2018). Microbial lipase mediated by health beneficial modification of cholesterol and flavors in food products: A review. Recent Patents on Biotechnology, 12(2), 81-91. https://doi.org/10.2174/1872208311666170615094552
Singh, M. G., Chandraveer, T. A., & Tripathi, A. (2017). Isolation and screening of lipase producing microorganisms from natural sources. Indian Journal of Ecology, 44(1), 19-23.
Sohail, A. (2017). Optimized production of lipase from Bacillus subtilis PCSIRNL-39. African Journal of Biotechnology. 16(19), 1106-1115. https://doi.org/10.5897/AJB2017.15924
Ul ain Rana, Q., Rehman, M. U., Irfan, M., Ahmed, S., Hasan, F., Shah, A. A., … & Badshah, M. (2019). Lipolytic bacterial strains mediated transesterification of non-edible plant oils for generation of high quality biodiesel. Journal of Bioscience and Bioengineering, 127(5), 609-617. https://doi.org/10.1016/j.jbiosc.2018.11.001
Ulwiyyah, N. H., Saidi, N., & Iqbalsyah, T. M. (2019). Screening and production of lipase from a thermo-halophilic bacterial isolate of Pria Laot Sabang 80 isolated from under water hot spring. KnE Engineering, 105-115. https://doi.org/10.18502/keg.v1i2.4436 
Journal of Genetic Resources
Volume 12, Issue 2
2026
Pages 245-254

Files

Share

How to cite

Statistics