Published on International Journal of Biology, Physics & Matematics
Publication Date: March 22, 2019
Ahonsi, C.O. & Emoghene A.O.
Nigeria Natural Medicine Development Agency, Federal Ministry of Science and Technology
Department of Microbiology, Faculty of Life Sciences, University of Benin, Ugbowo Campus
Crude oil pollution is a wide spread environmental problem of major concern, oil spill due to oil pipeline rupture, tank failures various pollution and storage problem, and transportation accident are the major causes, This study was aimed at determining the physico-chemical, Microbiological potential of fungi isolated from soil polluted with crude oil.Crude oil impacted soil from Nigeria Petroleum Distribution Company (NPDC) exploration site in Ologbo, Edo State was analyzed for Microbiological profiles using standard methods. Physicochemical properties which include, electrical conductivity, pH, carbon, phosphorus and nitrogen were analyzed using standard methods, Total Petroleum hydrocarbon (TPH) and Polyaromatic hydrocarbon (PAH) was elucidated using gas chromatography (GC). Identification of fungi isolates with 18S rRNA and phylogenetic analysis were elucidated. There was no significant change in the pH of the soils, however the electrical conductivity was higher (0.4 to 1.11 µs/cm) in contaminated soil samples. Carbon content in all the contaminated soil samples was significantly higher than normal soil and ranged from 2.67 to 7.33%. The TPH contents in the contaminated soils were found to be in the range of 684.86 mg/kg to 5,991.69mg/kg of soil compared with the control 37.8mg/kg. The PAH content in the contaminated soil ranged from 1.841mg/kg to 13.913mg/kg of soil with the control being 0.059mg/kg. A total of 15 fungi were isolated from crude polluted soil 18S rRNA revealed the following genera of Fungi viz : Scedosporium, Penicillium, Candida, Aspergillus, Pseudalescheria, and Byssochlamys.
Keywords: Phylogenome, Physicochemical, Gas-chromatography, Polyaromatic hydrocarbon PAH & Total petroleum hydrocarbon.
Crude Oil product are continually used as a main source of energy in industry and daily life (Kvenvolden and cooper 2003). Crude oil is transported via pipelines road ships and rail posing great danger to the environment in case of spills. This necessitate the need for developing techniques that are eco-friendly to clean up oil spill one of such method is the use of biological agent due to its efficiency and cost effectiveness, compared to physicochemical methods, Ojo 2006.
Crude oil consists of naturally occurring hydrocarbon that are considered environmental pollutants, its recalcitrance and abundance has been reported in most polluted sites. The degradability of individual hydrocarbon component is influenced by the quality of the hydrocarbon content present in the crude oil. Oil containing large amount of high –molecular weight compounds is difficult to degrade biologically because of the complexity of their structure Guo-Lin et al 2005. The chemical composition of oil influence the growth of microbial population. Microbes that use hydrocarbon as a source of energy thrive under high temperature and salinity conditions Westlake et al 1974. Fungi are known to play a significant role in eliminating hazardous compounds from soil and water contaminated with oil spill as they inhabit such substrate and utilize hydrocarbons as a source of carbon.
Scientists all over the world are searching for inexpensive and environmentally safe approaches to remediation of crude oil polluted soils. Microbes naturally selected in sols impacted by crude oil spills are increasingly being tested for their use in bioremediation purposes (Amirlatifi et al 2013). We believe that indigenous microbes selected in soils impacted by crude will be most suitable for application in bioremediation in the Niger Delta region of Nigeria. This report represents our initial study to determine the physicochemical parameters, microbial diversity and isolation of candidate fungi capable of using crude oil as sole carbon source.
Fungi are known to be one of the best oil degrading organism (Ojo, 2006; Batelle 2000). Different studies have identified numerous fungi genera capable of utilizing crude oil as source of carbon and energy this fungi include Cephalosporium, Rhizopus, Paecilomyces, Alternaria, Mucur Talaromyces, Gliocladium, Aspergillus, Penicilium, Torulopsis and Pleurotus (Ameen et al 2016; Dawoodi et al 2015; Zhang et al 2016). The objectives of this work was to investigate population of fungi from crude oil polluted soil in Ologbo, Edo State Niger Delta Region Of Nigeria.
2. Materials and Methods
2.1 Study Area and Geo-mapping of the Study Area
NPDC exploration site is situated at Ologbo (host) community, Ikpoba – Okha Local Government Area of Edo State South – South Nigeria. the communities play host to oil fields and flow stations as well. The vegetation is typical of the rainforest except for drainage streams where swampy areas exists NPDC geographical location is at 50 39’ 17” E . 60 3’ 30” N located within 60 20’ 0” – 60 4’ 30” N and 50 39’ 0” – 50 41’ 0” E with elevation below sea level.
2.2 Sample Collection
Crude oil contaminated soil where obtained from the Nigeria petroleum Distribution company (NPDC with exploration activities for 16 years) Exploration site in Ologbo Using a soil Auger at 15cm and 30cm depth into sterile polyethene bags, thereafter they were transported to the laboratory for Microbiological Examination.
2.3 Physicochemical Analysis
The physicochemical analysis of the samples were carried out. The following parameters were analyzed: pH, Nitrogen, phosphate, Electrical conductivity, Total organic carbon, total petroleum hydrocarbon, Polyaromatic hydrocarbon.
2.4 Total Petroleum Hydrocarbon/Polyaromatic Hydrocarbon in Soil samples
Soil samples were weighed (2gm) into a clean extraction tube. 10ml of extraction solvent (Hexane) was weighed added into the samples and mixed thoroughly and allowed to settle. The mixture was carefully filtered into clean solvent rinsed extraction bottle using filter paper fitted into Buchner funnels. The extracts were concentrated to 2ml then transferred for clean up/ separation
2.4.2 Clean up/ Separation
A 1cm of moderately packed glass wool was placed at the bottom of 10mm ID×250mm long chromatographic column, at the top of the column was added 0.5cm of sodium sulphate. The column was rinsed with additional 10ml of methylene chloride. The column was pre-eluted with 20ml of hexane this was allowed to flow through the column at a rate of about two minutes until the liquid in the column was just above the sulphate layer. Immediately 1ml of the extracted sample was transferred into the column. The extraction bottle was rinsed with 1mlof hexane and added to the column as well. The stop clock of the column was opened and the eluent was collected with a 10ml graduated cylinder. Just prior to exposure of the sodium sulphate layer to air, hexane was added to the column 1-2ml increment. Accurately measured volume of 8-10ml of the eluent was collected and was labeled aliphatic (API 1994).
2.4.3 Gas Chromatographic Analysis
The concentrated aliphatic fraction were transferred into labeled glass vials with Teflon or rubber crimp caps for GC analysis,1µl of the concentrated sample was injected by means of hypodermic syringe through a rubber septum into the column. Separation occur as the vapour constituent partition between the gas and liquid phases. The samples were automatically detected as it emerges from the column (at a constant flow rate) by the FID detector whose response is dependent upon the composition of the vapour. (INRCC 1997).
2.4.4 Cultivation counting and isolation of indigenous fungi
The enumeration of fungi was done using the dilution plate technique Potato dextrose agar (PDA) medium was used for isolating and enumerating heterotrophic soil fungi and Mineral Salt Medium (MSM) supplemented with 1% crude oil was used for isolation, enumeration and preliminary identification of fungi isolates from soil. mineral salt medium (MSM) which contain per liter 5.0g NaCl; 1.0gK2HP04; 5.0gKH2P04; 1.0gNaNO3; 0.25g Mgso4 7H20; 0.02gCaCl2. 2H20; 0.02gFeCl3 5g of yeast extract and 10g of Agar in 500ml of deionized water. Streptomycine antibiotics was added to the media to prevent bacteria growth. A 0.1ml aliquot of appropriate dilutions of sample was inoculated into three replicate. The plates were incubated for 2-3days at room temperature and colonies formed were counted, the mean was taken and expressed in cfu/g.
2.4.5 DNA Extraction for Fungi isolates
Genomic DNA for Fungi isolate was done using CTab, 25mg of growth culture was transferred into 1.5ml eppendof tube and stored overnight at -80OC. add 300µl of CTab solution into tube and crush sample with pistol, add an additional 300µl of CTab (CTab 600µl). Add 60µl of 20% SDS (sodium dodecyl sulphate) mix gently thereafter place on a water bath at 60OC for 1h. Add 660µlof phenol chloroform, isoamyl alcohol (25:24:1) invert to mix, centrifuge at 11500 ×g for 20mins.transfer 450µl of supernatant to a new tube, add 450µl of chloroform, isoamyl alcohol (24:1). Centrifuge at 11500×g for 15mins, transfer 300µl to a new tube add 0.6ml (300×0.6) i.e 180µl of ice cold isopropanol and invert to mix. Centrifuge at 12000×g for 10mins, decant supernatant add 300µl of ice cold 70% ethanol and centrifuge at 12000×g for 5mins decant supernatant, centrifuge at 12000×g for 1mins to remove the remaining ethanol. Air dry tubes for 30-40mins. Add 40µl (0.1MTE) to elute DNA and store at -20OC freezer for downstream application.
2.4.6 Purification of Cultured Fungi Isolates
GeneJET PCR purification kit (Thermo scientific) was used to purify post pcr proct of bacteria and fungi isolates. 40µl of Binding buffer was added to 40µl of post PCR product and mix by pipetting,(1:1 of binding buffer and PCR mixture). Add 40µl of isopropanol and mix (1:2 i.e 40µl of isopropanol in 80µl in step 1). Place column and load the entire solution into the column and spin at 12000× g for 1mins. Add 700µl of wash buffer (9ml of concentrated wash buffer and 45ml of Ethanol) spin at 12000×g for one minutes and transfer column to a clean 1.5ml microcentrifuge tube. Add 50µl of Elution buffer to the center of the GeneJET purification column membrane and centrifuge for 1min. Discard the GeneJET purification column and store the purified DNA at -20OC.