Isolation and Characterization of Hydrocarbon Biodegrading Fungi from oil contaminated soils in Thika, Kenya

Abstract

Spillage and extensive exploration of petroleum products results in pollution of the environment. Bioremediation of the oil contaminated sites could be achieved by fungal biodegradation; however, the specific fungi involved have not been determined in Kenya. In this study fungal isolates from Thika in central Kenya were screened for biodegradation of engine oil. There sample size was four garages that were more than 10 years. Soil was homogenously mixed for each of the twenty seven soil samples. The initial isolation from the oil contaminated soil was done using potato dextrose agar at a temperature of 30°C for seven days. The biodegradation incubation was done using Bacto Bushnell – Haas broth at 30°C for twenty one days. Colonies were observed using a light microscope at a magnification of x1000 and characterized morphologically. The 18S rRNA genome was amplified, sequenced and the sequences used for phylogenetic analysis. The size of the amplicon targeted was 700bp in approximation. The isolates were grown at varied temperatures and pH, and screened for enzymatic activities. Analysis for the biodegraded oil was done by gas chromatography- mass spectrometry (GCHRGC 400B - MSQ12 Konic-Spain). Eight fungal isolates were recovered from polluted soils namely, Trichoderma viride, Trichoderma spirale, Neosartorya pseudofischeri, Neosartorya aureola, Aspergillus flavus, Aspergillus terreus, Penicillium griseofulvum and Trichoderma longibrachiatum. Comparison of the 18S rRNA gene sequences to known fungal sequences in the Genbank database using BLAST analysis indicated similarity of more than 97%. The percentage similarity for isolates that biodegraded oil namely, Penicillium griseofulvum and Aspergillus flavus was 99% and 100%. The eight fungi were characterized morphologically and were different from each other through different parameters like colour, elevation and margin among others. Out of eight fungal isolates recovered from contaminating soil, only Aspergillus flavus and Penicillium griseofulvum biodegraded seven and seventeen oil compounds respectively. However, some compounds could not be fully biodegraded by Aspergillus flavus namely, decane, undecane and tridecane from a concentration of 5.48 to 0.23, 18.14 to 0.13 and 14.22 to 0.11 mg/l respectively. Penicillium griseofulvum could not fully biodegrade 1-Ethylidene-1–Indene from a concentration of 0.29 to 0.17mg/l. The optimum growth temperature range for the eight fungi was 30oC and 40oC. There was no growth at 50oC for all isolates except some slight growth by Aspergillus flavus. Optimum growth at pH 7 and pH 9 and poor growth at pH 5 was noted. The eight fungi produced amylase, protease, lipase/esterase and cellulase enzymes. This study will contribute to the database on locally available fungal diversity and their ecology. This will also increase knowledge of the fungi involved in biodegradation of oil in Kenya. Moreover Kenya’s Turkana has recently discovered oil and so this could be of great help in dealing with events of oil spill so as to conserve our precious environment.