Screening of Diesel Oil Degrading Species of Aspergillus for the Production of Different Enzymes Using Agar Plate Assay Method


School of Biological Engineering & Life Sciences, Shobhit Institute of Engineering & Technology, (NAAC Accredited Grade “A”, Deemed to-be-University) NH-58, Modipuram, Meerut -250110, India.

Amar P. Garg *

Swami Vivekanand Subharti University, Meerut, India.

*Author to whom correspondence should be addressed.


We have earlier isolated Aspergillus luchuensis, A. violaceofuscus, A. niveus, A. terreus, A. japonicus  and A. ustus from naturally degraded diesel oil in the soils using nutrient rich technique. These species were cultured to produce enzyme complexes on 10% (w/v) diesel oil broth containing 0.5 and 1.0% (w/v) jaggery at 37 and 45℃ temperature  separately for 7, 14 and 21 days. It was observed that all 6 species of Aspergillus produced luxuriant mycelial growth at day-7 on both temperatures, however, at 45℃, the liquid broth dried on 14th day of incubation, and therefore, secretory extracellular enzymes produced at 37℃ were screened to test the activities of amylases, gelatinases, lipases, cellulases, pectinases, proteases and manganese peroxidases using agar plate assay method. These enzymes are responsible for beaking down different bonds of the complex hydrocarbons. It was found that all 6 test species of Aspergillus produced all 7 enzymes at higher concentration at day 21 on diesel oil containing 1.0% jaggery. The production of enzyme increased with increasing period of incubation from day 7 to day 21. A. luchuensis produced highest activities of proteases, A. violacefuscus - amylases, A. niveus - gelatinases, A. terreus and A. ustus - pectinases, and A. japonicus produced highest activities of manganese peroxidases. All these enzymes contribute in the degradation of diesel oil in nature. This study revealed that the production of varying amounts of different enzymes by all these 6 species of Aspergillus has significant ecological and bioremediation potential.

Keywords: Diesel oil degrading fungi, aspergillus, agar plate assay, hydrocarbons, enzymes

How to Cite

Ruchika, & Garg , A. P. (2023). Screening of Diesel Oil Degrading Species of Aspergillus for the Production of Different Enzymes Using Agar Plate Assay Method. International Journal of Environment and Climate Change, 13(11), 1360–1371.


Download data is not yet available.


Mahmud T, Sabo IA, Lambu ZN, Danlami D, & Shehu A A. Hydrocarbon degradation potentials of fungi: A Review. Journal of Environmental Bioremediation and Toxicology, JEBAT. 2022; 5(1):50–56. DOI:

Jacob EL, Mohan AP and Joseph AV. Bioremediation of petroleum-polluted soil using biosurfactant producing bacteria, Pseudomonas sp. Journal of Scientific Research; 2022. DOI: 10.37398/JSR.2022.660124.

Roy A, Sar P, Sarkar J, Dutta A, Sarkar P, Gupta A, Mohapatra B, Pal S, Kazy SK. Petroleum hydrocarbon rich oil refinery sludge of North-East India harbours anaerobic, fermentative, sulfate-reducing, syntrophic and methanogenic microbial populations, BMC Microbiology; 2018. DOI:

Varjani SJ and Upasani VZ. A new look on factors affecting microbial degradation of petroleum hydrocarbon pollutants, International Biodeterioration & Biodegradation, May 2017;120:71-83 DOI:

Oyewole OA, Raji RO, Okeke SK, Musa IO, Chukwuma GN and Yakubu JG. Potential of fungi isolated from diesel contaminated soils to degrade diesel, Tanzania Journal of Science 2021;47(1): 47-56. ISSN 0856-1761, e-ISSN 2507-7961. DOI:

Benguenab A and Chibani A. Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil, Acta Ecologica Sinica. October 2021;41(5):416-423, DOI:

Yanto DHY and Hidayat A. Biodegradation of buried crude oils in soil microcosm by fungal co-culture, IOP Conf.Series: Materials Science and Engineering 2020; 980:012084, DOI:10.1088/1757-899X/980/1/012084.

Ghanem KM, Al-Garni SM, Al-Zahrani MA. Bioremediation of diesel fuel by fungal consortium using statistical experimental designs, PolJ.Environ. Stud. 2016;25(1): 97-106, DOI: 10.15244/pjoes/42493

Chaudhry S, Luhach J, Sharma V, Sharma C. Assessment of diesel degrading potential of fungal isolates from sludge contaminated soils of petroleum refinery, Haryana, Research Journal of Microbiology. 2017;7:182-190. DOI: 10.3923/jm.2012.182.190

Rao MA, Scelza R, Acevedo F, Diez MC, Gianfreda L. Enzymes as useful tools for environmental purposes, Chemosphere. July 2014;107:145-162 DOI:

Yang JS, Yang FL, Yang Y, Xing GL, Deng CP, Shen YT, Luo LQ, Li BZ, Yuan HL.A proposal of core enzymes bio indicator in long-term Pb-Zn ore pollution areas based on top soil properties analysis, Environmental Pollution. 2016;213:760-769 DOI:

Thapa B, Kc A, Ghimire A. A review on bioremediation of petroleum hydrocabon contaminants in soil, Kathmandu University Journal of Science Engineering & Technology 2012;8(I):164-170. DOI:10.03126/kuset.V811.6056.

El-Aziz ARMA, Al-Othman MR, Hisham SM, Shehata SM. Evaluation of crude oil biodegradation using mixed fungal cultures. PLos ONE. 2021;16(8): e0256376. DOI:

Al Otibi F, Al Zahrani RM & Marraiki N.The crude oil biodegradation activity of Candida strains isolated from oil reservoirs soils in Saudi Arabia, Sci Rep. 2022 Jun 23;12(1): 10708. DOI:

Garg AP, Sharma PD. Ecology of phylloplane and leaf-litter fungi of Cyamopsis tetragonoloba (L.) Taub. Rev. Eco. Sol.1985;22:35-55.

Malik ZA, Ahmed S. Degradation of petroleum hydrocarbons by oil field isolated bacterial consortium. Afr. J. Biotechnol. 2012;11(3):650. DOI:10.5897/AJB11.036

Sahrawat R and Garg AP. Rice Straw Degradation with mixed cultures of microfungi and their enzmyes, International Journal of Environment and Climate Change. 2023;13(8). DOI: 10.9734/ijecc/2023/v13i821167.

Robledo-Mahón T, Calvo C and Aranda E. Enzymatic potential of bacteria and fungi isolates from the sewage sludge composting process. Appl. Sci. 2020;10: 7763. DOI:10.3390/app10217763

Ruchika and Garg AP. Isolation and identification from diesel oil contaminated soils and their physico-chemical analysis, International Journal of Environment and Climate Change. 2023;13(10):3476-3487.

Article no. IJECC. 106072.

Raper KB and Fennell DI. The Genus Aspergillus, Published by Baltimore, Williams & Wilkins; 1965.

Gams W, Christensen M, Onions AH, Pitt JI, Samson RA. Infrageneric Taxa of Aspergillus. In: Samson RA , Pitt JI (eds) Advances in Penicillum and Aspergillus Systematics. NATO ASI Series. Springer, Boston, MA. 1986; 102. DOI:

Bisht N, Garg AP. Isolation, characterization and probiotic value of lactic acid bacteria from milk and milk products, Biotech Today : 2019;9(2):54-63. Doi:10.5958/2322-0996.2019.00022.X.

Saadoun I, Alawawdeh M, Jaradat Z, Ababneh Q, Al-Joubori BM & Elsheikh EAE. Characterization of diesel-degrading, hydrolytic enzymes - producing Streptomyces spp. isolated from fuel-oil polluted soils, Arab Journal of Basic and Applied Sciences, 2023;30(1):248-255 DOI: 10.1080/25765299.2023.2196110

Ali S, Khan SA, Hamayun M and Lee IJ. The recent advances in the utility of microbial lipases: A Review. Microorganism. 2023 Feb; 11(2): 510. DOI:10.3390/microorganisms11020510.

Correa-García S, Pande P, Séguin A, StArnaud M and Yergeau E. Rhizoremediation of petroleum hydrocarbons: a model system for plant microbiome manipulation. Microbial Biotechnology. 2018;11(5):819-832. DOI: 10.1111/1751-7915.13303.

Al-Jawhari IF. Ability of sediments fungi in biodegradation of diesel fuel. Int. J. Pure Appl. Biosci. 2016; 4(2): 27-37. DOI: 2275. 2016;23(2):211-218

Oluwafemi J, Olukanni D, and Justin LD. Improper waste disposal in Ota, Ogun State-a proposed waste segregation approach, Journal of Physics: Conference Series, International Conference on Recent Trends in Applied Research (ICoRTAR). 15 August 2020:1734;14. DOI:10.1088/1742-6596/1734/1/012038.

Ghanem KM, Al-Garni SM, Al-Zahrani MA. Bioremediations of diesel fuel by fungal consortium using statistical experimental designs, Pol. J. Environ. Stud. 2016;25(1): 97-106, DOI: 10.15244/pjoes/42493

EL-Hanafy AAEM, Anwar Y, Sabir JSM, Mohamed SA, Al-Garni SMS, Zinadah OAA and Ahmed MM. Characterization of native fungi responsible for degrading crude oil from the coastal area of Yanbu, Saudi Arabia, Biotechnology & Biotechnological Equipment, 2017;31(1): 105-111. DOI:10.1080/13102818.2016.1249407.

Manjunatha BK and Praveen KSV. Isolation of diesel, engine oil and crudeoil degrading fungi from oil contaminated soil for their bioremediation potential, Journal of Agriculture, Food and Natural Resources, J. Agric. Food Nat. Resour. 2017;1(1):65-70. Available:

Asemoloye MD, Tosi S, Dacco C, wang X, Xu S, Marchisio MA, Gao W, Jonathan SG, Pecoraro L. Hydrocarbon degradation and enzyme activities of Aspergillus oryzae and Mucor irregularis isolated from Nigerian crude oil-polluted sites, Microorganis. 2020 Nov 30;8(12):1912. Doi:10.3390/microorganisms8121912

Al-Hawash AB, Dragh MA, Li S, Alhujaily A, Abbood HA, Zhang X, Ma F.Principle of Microbial degradation of petroleum hydrocarbons in the environment, The Egyptian Journal of Aquatic Research. 2018;44 (2):71-76. DOI:

Ameen F, Moslem M, Hadi S, Al-Sabri AE. Biodegradation of diesel fuel hydrocarbons by mangrove fungi from Red Sea Coast of Saudi Arabia. Saudi J. Biol. Sci. 23:211–218. Saudi J Biol Sci. 2016 Mar;23(2):211-8. DOI: 10.1016/j.sjbs.2015.04.005.

Novianty R, Saryono, Awaluddin A, Pratiwi NW, Hidayah A, Juliantari E.The diversity of fungi consortium isolated from polluted soil for degrading petroleum hydrocarbon, Biodiversitas, ISSN: 1412-033X, E-ISSN: 2085-4722; 2021. DOI:10.1305/biodiv/d221145.