Bio24332 Vol. 24 No. 1 (2025)

Vol. 24, No. 1 (2025), Bio24332 https://doi.org/10.24275/rmiq/Bio24332

Biodegradation of crude oil by Aspergillus tubingensis: biosurfactant-producing strain

Authors

D.L. Peraza-Liñan, B.A. Ayíl-Gutiérrez, A.A. Urías-Salazar, J.A. López-Santillán, B. Estrada-Drouaillet, M.T.J. Segura-Martínez, W.A. Poot-Poot

Abstract

Crude oil is one of the most demanded fuels which causes an increase in environmental pollution due to spills. Bioremediation by hydrocarbonoclast microorganisms can be an alternative for hydrocarbon degradation through biochemical processes involving enzymes and biosurfactants. The objective of this work was to evaluate the capacity of oil biodegradation by a hydrocarbonoclast strain through growth kinetics and biosurfactant production. The strain was inoculated in flasks with mineral medium (MM) supplemented with oil (0, 20, 40, 60, and 80 %) and grown for 20 days. The variables evaluated were oil degradation by Soxhlet extraction, quantification of extracellular proteins, proteolytic activity, biosurfactant production by lysis in blood agar and emulsification capacity (E24). The results allow concluding that the strain identified as Aspergillus tubingensis was able to biodegrade 78.38 % of the crude oil at the 80 % concentration and maintain a biomass production of 55.12 g/L, a low proteolytic activity of 0.0015 U/mL, as well as biosurfactant production and an E24 of 65.16 %. Therefore, A. tubingensis can be used in bioremediation works to recover oil contaminated sites.

Keywords

Bioremediation, Hydrocarbons, Filamentous Fungi, Biosurfactants, Growth Kinetics.

References
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Hydrocarbon biodegradation and transcriptome responses of cellulase, peroxidase, and laccase encoding genes inhabiting rhizospheric fungal isolates. Saudi Journal of Biological Sciences 28, 2083-2090. https://doi.org/10.1016/j.sjbs.2021.01.009 Arregui, L., Ayala, M., Gómez-Gil, X., Gutiérrez-Soto, G., Hernández-Luna, C.E., De Los Santos, M., Levin, L., Roja-Domínguez, A., Romero-Martínez, D., Saparrat, M.C.N., Trujillo-Roldán, M.A., and Valdez-Cruz, N.A. (2019). Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories, 18, 1-33. https://doi.org/10.1186/s12934-019-1248-0 Atakpa, O.E., Zhou, H., Jiang, L., Ma, Y., Liang, Y., Li, Y., Zhang, D. and Zhang, C. (2022). Improved degradation of petroleum hydrocarbons by co-culture of fungi and biosurfactant-producing bacteria. Chemosphere 290, 1-10. https://doi.org/10.1016/j.chemosphere.2021.133337 Balaji, V., Arulazhagan, P. and Ebenezer, P. (2014). Enzymatic bioremediation of polyaromatic hydrocarbons by fungal consortia enriched from petroleum contaminated soil and oil seed. Journal of Environmental Biology 35, 1-9. Barrios San Martín, Y. (2011). Biorremediación: una herramienta para el saneamiento de ecosistemas marinos contaminados con petróleo. Biotecnología Aplicada 28, 60-68. Benguenab, A. and Chibani, A. (2021). Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil. Acta Ecologica Sinica 41, 416-423. https://doi.org/10.1016/j.chnaes.2020.10.008 Binazadeh, M., Li, Z., Karimi, I.A. (2020). Optimization of biodegradation of long chain n- Alkanes by Rhodococcus sp. Moj-3449 using response surface methodology. Physical Chemistry Research 8, 45-59. https://doi.org/10.22036/pcr.2019.204077.1682 Cáceres-Zambrano, J.Z., Rodríguez-Córdova, L.A., Sáez-Navarrete, C.A. and Coca Rives Y. (2024). 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Environment: Science and Policy for Sustainable Development 56, 16-31. http://dx.doi.org/10.1080/00139157.2014.922382 Jha, A., Barsola, B., Pathania, D., Sonu, Raizada, P., Thakur, Singh, P., Rustagi, S., Khosla, A., and Chaudhary, V. (2024). Nano-biogenic heavy metals adsorptive remediation for enhanced soil health and sustainable agricultural production. Environmental Research 252, 1-18. https://doi.org/10.1016/j.envres.2024.118926 Hkiri, N., Olicón-Hernández, D.R., Pozo, C., Chouchani, C., Asses, N. and Aranda, E. (2023). Simultaneous heavy metal-polycyclic aromatic hydrocarbon removal by native tunisian fungal species. Journal of Fungi 9, 299. https://doi.org/10.3390/jof9030299 Kebria, D.Y., Khodadadi, A., Ganjidoust, H., Badkoubi, A. and Amoozegar, M.A. (2009). Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel. International Journal of Environmental Science & Technology 6, 435-442. https://doi.org/10.1007/bf03326082 Khan, S., Nadir, S., Shah, Z. U., Shah, A.A., Karunarathna, S.C., Xu, J., Khan, A., Munir, S and Hasan, F. (2017). Biodegradation of polyester polyurethane by Aspergillus tubingensis. Environmental Pollution 225, 469-480. http://dx.doi.org/10.1016/j.envpol.2017.03.012 Khandelwal, A., Singh, S.B., Sharma, A., Nain, L., Varghese, E. and Singh, N. (2021). Effect of surfactant on degradation of Aspergillus sp. and Trichoderma sp. mediated crude oil. International Journal of Environmental Analytical Chemistry 103, 1667-1680. https://doi.org/10.1080/03067319.2021.1879800 Kunitz, M. (1946). Crystalline soybean trypsin inhibitor. The Journal of General Physiology 29, 149-154. https://doi.org/10.1085/jgp.29.3.149 Lowry, O., Rosebrough, N., Farr, A.L. and Randall, R. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265-275. https://doi.org/10.1016/s0021-9258(19)52451-6 Lu, M., Zhang, Z., Sun, S., Wang, Q. and Zhong, W. (2009). Enhanced degradation of bioremediation residues in petroleum-contaminated soil using a two-liquid-phase bioslurry reactor. Chemosphere 77, 161-168. http://dx.doi.org/10.1016/j.chemosphere.2009.08.001 Mahjoubi, M., Jaouani, A., Guesmi, A., Ben Amor, S., Jouini, A., Cherif, H., Najjari, A., Boudabous, A., Koubaa, N and Cherif, A. (2013). Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential. New Biotechnology 30, 723-733. http://dx.doi.org/10.1016/j.nbt.2013.03.004 Menezes Bento, F., de Oliveira Camargo, F.A., Okeke, B.C. and Frankenberger, W.T. (2005). Diversity of biosurfactant producing microorganisms isolated from soils contaminated with diesel oil. Microbiological Research 160, 249-255. https://doi.org/10.1016/j.micres.2004.08.005 Mishra, S. and Singh, S.N. (2012). Microbial degradation of n-hexadecane in mineral salt medium as mediated by degradative enzymes. Bioresource Technology 111, 148-154. http://dx.doi.org/10.1016/j.biortech.2012.02.049 Nadumane, V.K., Venkatachalam, P. and Gajaraj, B. (2016). Aspergillus Applications in Cancer Research. In: New and Future Developments in Microbial Biotechnology and Bioengineering. (V.K. Nadumane, P. Venkatachalam and B, Gajaraj, eds.), Pp. 243-255. Elsevier, India. https://doi.org/10.1016/b978-0-444-63505-1.00020-8 Perera, M., Wijesundera, S., Wijayarathna C.D., Seneviratne, G. and Jayasena, S. (2019). Identification of long-chain alkane-degrading (LadA) monooxygenases in Aspergillus flavus via in silico analyses. Frontiers in Microbiology 13, 1-14. https://doi.org/10.3389/fmicb.2022.898456 Pérez-Montiel, G., Torres-García, J.L., Juarez-Santacruz, L., Cortés-Espinosa, D.V., Rubio-Piña, J. and Ahuactzin-Pérez, M. (2021). Crecimiento y producción de enzimas lacasas de Pleurotus ostreatus durante el proceso de degradación de bisfenol a. Biotecnia, 23, 39-46. https://doi.org/10.18633/biotecnia.v23i2.1357 Santos, E.O., Rosa, C.F.C., Passos, C.T., Sanzo, A.V.L., Burkert, J.F.M., Kalil, SJ. and Burkert, G.A.V. (2008). Pre-screening of filamentous fungi isolated from a contaminated site in Southern Brazil for bioaugmentation purposes. African Journal of Biotechnology 7, 1314-1317. Sayed, K., Baloo, L. and Kumar S., N. (2021). Bioremediation of total petroleum hydrocarbons (TPH) by bioaugmentation and biostimulation in water with floating oil spill containment booms as bioreactor basin. International Journal of Environment Research and Public Health 18, 1-26. https://dx.doi.org/10.3390/ijerph18052226 Sharma, A., Singh, S.B., Sharma, R., Chaudhary, P., Pandey, A.K., Ansari, R., Vasudevana, V., Arora, A., Singh, S., Saha, S. and Nain, L. (2016). Enhanced biodegradation of PAHs by microbial consortium with different amendment and their fate in-situ condition. Journal of Environmental Management 181, 728736. https://doi.org/10.1016/j.jenvman.2016.08.024 Ye, J.-S., Yin, H., Qiang, J., Peng, H., Qin, H.-M., Zhang, N. and He, B.-Y. (2011). Biodegradation of anthracene by Aspergillus fumigatus. Journal of Hazardous Materials 85, 174-181. https://doi.org/10.1016/j.jhazmat.2010.09.015 Youssef, N.H., Duncan, K.E., Nagle, D.P., Savage, K.N., Knapp, R.M. and McInerney, M.J. (2004). Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods 56, 339-347. https://doi.org/10.1016/j.mimet.2003.11.001 Yuniati, M.D. (2018). Bioremediation of petroleum-contaminated soil: a review. IOP Conference Series: Earth and Environmental Science 118, 1-8. https://doi.org/10.1088/1755-1315/118/1/012063 Zhang, B., Matchinski, E. J., Chen, B., Ye, X., Jing, L. and Lee, K. (2019). World Seas: An Environmental Evaluation. In: Marine Oil Spills-Oil Pollution, Sources and Effects. (B. Zhang, E. J. Matchinski, B. Chen, X. Ye, L. Jing and K. Lee, eds.), Pp. 391-406. https://dx.doi.org/10.1016/b978-0-12-805052-1.00024-3 Zhang, J.-H., Xue, Q.-H., Gao, H., Ma, X. and Wang, P. (2015). Degradation of crude oil by fungal enzyme preparations from Aspergillus spp. for potential use in enhanced oil recovery. Journal of Chemical Technology & Biotechnology 91, 865-875. https://doi.org/10.1002/jctb.4650

References

Ahuactzin-Pérez, M., Tlecuitl-Beristain, S., García-Dávila, J., González-Pérez, M., Gutiérrez-Ruíz, M.C. and Sánchez, C. (2016). Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum : Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling. Science of The Total Environment 566-567, 1186-1193. https://doi.org/10.1016/j.scitotenv.2016.05.169

Al-Hawash, A.B., Zhang, J., Li, S., Liu, J., Ghalib, H.B., Zhang, X. and Ma, F. (2018a). Biodegradation of n-hexadecane by Aspergillus sp. RFC-1 and its mechanism. Ecotoxicology and Environmental Safety 164, 398-408. https://doi.org/10.1016/j.ecoenv.2018.08.049

Al-Hawash, A.B., Zhang, X. and Ma, F. (2018b). Removal and biodegradation of different petroleum hydrocarbons using the filamentous fungus Aspergillus sp. RFC-1. MicrobiologyOpen e00619. https://doi.org/10.1002/mbo3.619

Al-Zaban, M.I., AlHarbi, M.A. and Mahmoud, M.A. (2021). Hydrocarbon biodegradation and transcriptome responses of cellulase, peroxidase, and laccase encoding genes inhabiting rhizospheric fungal isolates. Saudi Journal of Biological Sciences 28, 2083-2090. https://doi.org/10.1016/j.sjbs.2021.01.009

Arregui, L., Ayala, M., Gómez-Gil, X., Gutiérrez-Soto, G., Hernández-Luna, C.E., De Los Santos, M., Levin, L., Roja-Domínguez, A., Romero-Martínez, D., Saparrat, M.C.N., Trujillo-Roldán, M.A., and Valdez-Cruz, N.A. (2019). Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories, 18, 1-33. https://doi.org/10.1186/s12934-019-1248-0
Atakpa, O.E., Zhou, H., Jiang, L., Ma, Y., Liang, Y., Li, Y., Zhang, D. and Zhang, C. (2022). Improved degradation of petroleum hydrocarbons by co-culture of fungi and biosurfactant-producing bacteria. Chemosphere 290, 1-10. https://doi.org/10.1016/j.chemosphere.2021.133337

Balaji, V., Arulazhagan, P. and Ebenezer, P. (2014). Enzymatic bioremediation of polyaromatic hydrocarbons by fungal consortia enriched from petroleum contaminated soil and oil seed. Journal of Environmental Biology 35, 1-9.

Barrios San Martín, Y. (2011). Biorremediación: una herramienta para el saneamiento de ecosistemas marinos contaminados con petróleo. Biotecnología Aplicada 28, 60-68.
Benguenab, A. and Chibani, A. (2021). Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil. Acta Ecologica Sinica 41, 416-423. https://doi.org/10.1016/j.chnaes.2020.10.008
Binazadeh, M., Li, Z., Karimi, I.A. (2020). Optimization of biodegradation of long chain n- Alkanes by Rhodococcus sp. Moj-3449 using response surface methodology. Physical Chemistry Research 8, 45-59. https://doi.org/10.22036/pcr.2019.204077.1682
Cáceres-Zambrano, J.Z., Rodríguez-Córdova, L.A., Sáez-Navarrete, C.A. and Coca Rives Y. (2024). Biodegradation capabilities of filamentous fungi in high-concentration heavy crude oil environments. Archives of Microbiology 206, 123. https://doi.org/10.1007/s00203-024-03835-6
Canul-Chan, M., Rodas-Junco, B.A., Uribe-Riestra, E. and Houbron, E. (2023). Biodegradation of crude oil present in wastewaters: evaluation of biosurfactant production and catechol 2,3 dioxygenase activity. Revista Mexicana de Ingeniería Química 22, 1-9. https://doi.org/10.24275/rmiq/Bio2932

Carrillo, P.G., Mardaraz, C., Pitta-Alvarez, S.I. and Giulietti, A.M. (1996). Isolation and selection of biosurfactant-producing bacteria. World Journal of Microbiology and Biotechnology 12, 82-84. https://doi.org/10.1007/bf00327807

Czarny, J., Staninska-Pieta, J., Piotrowska-Cyplik, A., Juzwa, W., Wolniewicz, A., Marecik, R., Ławniczak, Ł. and Chrzanowski, Ł. (2020). Acinetobacter sp. as the key player in diesel oil degrading community exposed to PAHs and heavy metals. Journal of Hazardous Materials 383, 1-10. https://doi.org/10.1016/j.jhazmat.2019.121168

EL-Hanafy, A.A.-E.-M., Anwar, Y., Sabir, J.S., Mohamed, S.A., Al-Garni, S.M., Zinadah, O.A.A. and Ahmed, M.M. (2016). Characterization of native fungi responsible for degrading crude oil from the coastal area of Yanbu, Saudi Arabia. Biotechnology & Biotechnological Equipment 31, 105-111. http://doi.org/10.1080/13102818.2016.1249407
Espinosa-Ortiz, E.J., Rene, E.R., and Gerlach, R. (2021). Potential use of fungi-bacterial co-cultures for the removal of organic pollutants. Critical Reviews in Biotechnology 42, 361-383. https://doi.org/10.1080/07388551.2021.1940831
Environmental Protection Agency (EPA). (1998). Method 9071B n-hexane extractable material (hem) for sludge, sediment, and solid samples. Environmental Protection Agency (EPA) 1-13.
Farrington, J.W. (2014). Oil Pollution in the Marine Environment II: Fates and Effects of Oil Spills. Environment: Science and Policy for Sustainable Development 56, 16-31. http://dx.doi.org/10.1080/00139157.2014.922382
Jha, A., Barsola, B., Pathania, D., Sonu, Raizada, P., Thakur, Singh, P., Rustagi, S., Khosla, A., and Chaudhary, V. (2024). Nano-biogenic heavy metals adsorptive remediation for enhanced soil health and sustainable agricultural production. Environmental Research 252, 1-18. https://doi.org/10.1016/j.envres.2024.118926
Hkiri, N., Olicón-Hernández, D.R., Pozo, C., Chouchani, C., Asses, N. and Aranda, E. (2023). Simultaneous heavy metal-polycyclic aromatic hydrocarbon removal by native tunisian fungal species. Journal of Fungi 9, 299. https://doi.org/10.3390/jof9030299
Kebria, D.Y., Khodadadi, A., Ganjidoust, H., Badkoubi, A. and Amoozegar, M.A. (2009). Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel. International Journal of Environmental Science & Technology 6, 435-442. https://doi.org/10.1007/bf03326082
Khan, S., Nadir, S., Shah, Z. U., Shah, A.A., Karunarathna, S.C., Xu, J., Khan, A., Munir, S and Hasan, F. (2017). Biodegradation of polyester polyurethane by Aspergillus tubingensis. Environmental Pollution 225, 469-480. http://dx.doi.org/10.1016/j.envpol.2017.03.012
Khandelwal, A., Singh, S.B., Sharma, A., Nain, L., Varghese, E. and Singh, N. (2021). Effect of surfactant on degradation of Aspergillus sp. and Trichoderma sp. mediated crude oil. International Journal of Environmental Analytical Chemistry 103, 1667-1680. https://doi.org/10.1080/03067319.2021.1879800
Kunitz, M. (1946). Crystalline soybean trypsin inhibitor. The Journal of General Physiology 29, 149-154. https://doi.org/10.1085/jgp.29.3.149
Lowry, O., Rosebrough, N., Farr, A.L. and Randall, R. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265-275. https://doi.org/10.1016/s0021-9258(19)52451-6
Lu, M., Zhang, Z., Sun, S., Wang, Q. and Zhong, W. (2009). Enhanced degradation of bioremediation residues in petroleum-contaminated soil using a two-liquid-phase bioslurry reactor. Chemosphere 77, 161-168. http://dx.doi.org/10.1016/j.chemosphere.2009.08.001
Mahjoubi, M., Jaouani, A., Guesmi, A., Ben Amor, S., Jouini, A., Cherif, H., Najjari, A., Boudabous, A., Koubaa, N and Cherif, A. (2013). Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential. New Biotechnology 30, 723-733. http://dx.doi.org/10.1016/j.nbt.2013.03.004
Menezes Bento, F., de Oliveira Camargo, F.A., Okeke, B.C. and Frankenberger, W.T. (2005). Diversity of biosurfactant producing microorganisms isolated from soils contaminated with diesel oil. Microbiological Research 160, 249-255. https://doi.org/10.1016/j.micres.2004.08.005
Mishra, S. and Singh, S.N. (2012). Microbial degradation of n-hexadecane in mineral salt medium as mediated by degradative enzymes. Bioresource Technology 111, 148-154. http://dx.doi.org/10.1016/j.biortech.2012.02.049

Nadumane, V.K., Venkatachalam, P. and Gajaraj, B. (2016). Aspergillus Applications in Cancer Research. In: New and Future Developments in Microbial Biotechnology and Bioengineering. (V.K. Nadumane, P. Venkatachalam and B, Gajaraj, eds.), Pp. 243-255. Elsevier, India. https://doi.org/10.1016/b978-0-444-63505-1.00020-8
Perera, M., Wijesundera, S., Wijayarathna C.D., Seneviratne, G. and Jayasena, S. (2019). Identification of long-chain alkane-degrading (LadA) monooxygenases in Aspergillus flavus via in silico analyses. Frontiers in Microbiology 13, 1-14. https://doi.org/10.3389/fmicb.2022.898456
Pérez-Montiel, G., Torres-García, J.L., Juarez-Santacruz, L., Cortés-Espinosa, D.V., Rubio-Piña, J. and Ahuactzin-Pérez, M. (2021). Crecimiento y producción de enzimas lacasas de Pleurotus ostreatus durante el proceso de degradación de bisfenol a. Biotecnia, 23, 39-46. https://doi.org/10.18633/biotecnia.v23i2.1357
Santos, E.O., Rosa, C.F.C., Passos, C.T., Sanzo, A.V.L., Burkert, J.F.M., Kalil, SJ. and Burkert, G.A.V. (2008). Pre-screening of filamentous fungi isolated from a contaminated site in Southern Brazil for bioaugmentation purposes. African Journal of Biotechnology 7, 1314-1317.

Sayed, K., Baloo, L. and Kumar S., N. (2021). Bioremediation of total petroleum hydrocarbons (TPH) by bioaugmentation and biostimulation in water with floating oil spill containment booms as bioreactor basin. International Journal of Environment Research and Public Health 18, 1-26. https://dx.doi.org/10.3390/ijerph18052226
Sharma, A., Singh, S.B., Sharma, R., Chaudhary, P., Pandey, A.K., Ansari, R., Vasudevana, V., Arora, A., Singh, S., Saha, S. and Nain, L. (2016). Enhanced biodegradation of PAHs by microbial consortium with different amendment and their fate in-situ condition. Journal of Environmental Management 181, 728736. https://doi.org/10.1016/j.jenvman.2016.08.024
Ye, J.-S., Yin, H., Qiang, J., Peng, H., Qin, H.-M., Zhang, N. and He, B.-Y. (2011). Biodegradation of anthracene by Aspergillus fumigatus. Journal of Hazardous Materials 85, 174-181. https://doi.org/10.1016/j.jhazmat.2010.09.015
Youssef, N.H., Duncan, K.E., Nagle, D.P., Savage, K.N., Knapp, R.M. and McInerney, M.J. (2004). Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods 56, 339-347. https://doi.org/10.1016/j.mimet.2003.11.001
Yuniati, M.D. (2018). Bioremediation of petroleum-contaminated soil: a review. IOP Conference Series: Earth and Environmental Science 118, 1-8. https://doi.org/10.1088/1755-1315/118/1/012063
Zhang, B., Matchinski, E. J., Chen, B., Ye, X., Jing, L. and Lee, K. (2019). World Seas: An Environmental Evaluation. In: Marine Oil Spills-Oil Pollution, Sources and Effects. (B. Zhang, E. J. Matchinski, B. Chen, X. Ye, L. Jing and K. Lee, eds.), Pp. 391-406. https://dx.doi.org/10.1016/b978-0-12-805052-1.00024-3
Zhang, J.-H., Xue, Q.-H., Gao, H., Ma, X. and Wang, P. (2015). Degradation of crude oil by fungal enzyme preparations from Aspergillus spp. for potential use in enhanced oil recovery. Journal of Chemical Technology & Biotechnology 91, 865-875. https://doi.org/10.1002/jctb.4650