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Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico Hussein Al-Nasrawi* Fulbright Visiting Scholar, Florida State University, USA

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Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico Hussein Al-Nasrawi* Fulbright Visiting Scholar, Florida State University, USA t edia ion & em B r i o o i d B e f g o r a Journal of l d a Al-Nasrawi, J Bioremed Biodegrad 2012, 3:4 a n r t i o u n o DOI: 10.4172/2155-6199.1000147 J ISSN: 2155-6199 Bioremediation & Biodegradation Research Article OpOpenen Access Access Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico Hussein Al-Nasrawi* Fulbright visiting scholar, Florida state university, USA Abstract In the present study, sand samples contaminated with oil spill were collected from Pensacola beach (Gulf of Mexico) and tested to isolates fungal diversity associated with beach sands and investigates the ability of isolated fungi for crude oil biodegradation. From sixteen fungal strains, four strains were confirmed for biodegradation ability of crude oil, the isolated fungi belongs to Aspergillus niger with higher activity followed by Penicillium documbens, Cochliobolus lutanus and Fusarium solani. Aspergillus niger recorded the highest weight loss of 8.6%, Penicillium documbens (7.9 %) and Cochliobolus lutanus (4.7%) whereas the lowest weight loss was demonstrated by Fusarium solani strain 421502 (1.9%). Keywords: Biodegradation; Fungi; Crude oil; Gulf of Mexico Several authors have made lists containing bacteria and fungi genera that are able to degrade a wide spectrum of pollutants, proceeding from Introduction marine atmosphere as well as the soil [10-12]. The dominance of petroleum products in the world economy creates Recently, many researchers studied the role of fungi in biodegradation the conditions for distributing large amounts of complex compounds process of petroleum products and the most common fungi which have consist of hundreds of different hydrocarbon molecules, and a huge been recorded as a biodegrades belongs to following genera: Alternaria, volume of oily sludge, a carcinogenic and a potent immunotoxicant Aspergillus, Candida, Cephalosporium, Cladosporium, Fusarium, [1,2]. Oil spillage is the accidental discharge or pouring of crude oil Geotrichum, Gliocladium, Mucor, Paecilomyces, Penicillium, Pleurotus, into the environment. It involves the contamination of any part of Polyporus, Rhizopus, Rhodotolura, Saccharomyces, Talaromyces and the environment with any liquid hydrocarbon. These spills endanger Torulopsis [3,4,13-20]. public health, imperil drinking water, devastate natural resources, and disrupt the economy [3]. Crude oil is a naturally occurring complex The aim of the present study is to isolation fungi from polluted mixture of hydrocarbon and non-hydrocarbon compounds which at beach sand in Gulf of Mexico and test the ability of isolated fungi in appropriate concentration, possesses a measurable toxicity towards biodegradation of crude oil. living systems. The toxicity of crude oil or petroleum products varies widely, depending on their composition, concentration, environmental Materials and Methods factors and on the biological state of the organisms at the time of the Sample collection and culture methods contamination [4]. Soil samples (400 g) from surface soil (0-15 cm depth) were Although oil spills from tankers and pipelines release crude collected from different localities in Pensacola beach (Gulf of Mexico) oil particles to the water surface and move it to the beaches and which was contaminated with crude oil. Sample were made from 3-4 contaminates living and nonliving organisms, microorganisms random locations per plot, mixed and transferred into sterile bottles specially fungi have a higher tolerance to the toxicity of hydrocarbons due to their physiology and adaptation to such variations in the using sterile spatula for microbiological quality determination and environment and have the mechanism for the elimination of spilled oil stored in ice box to a void contamination. In the lab, stones and other from the environment [5,6]. unwanted soil debris were removed by using 2.5 mm sieve, one gram of each sorted soil sample was homogenously mixed with 1 drop (0.1 ml) The effect of oil on microbial populations depends upon the of Tween 80 and a loopful (3 mm) of it was collected and inoculated chemical composition of the oil and on the species of microorganisms by sprinkling method onto SDA and Czapek agar plates, respectively. present. Populations of some microbes increase; typically, such Soil fungi were estimated by soil dilution plate count method. Sodium microbes use the petroleum hydrocarbons as nutrients. The same crude chloride 0.85 % was used as diluent for inoculum preparation. 1.0 g oil can favor different genera at different temperatures [7]. of homogenized, 2 mm sieved soil sample was aseptically transferred, In the aquatic ecosystems, fungi plays an important role during using a flame-sterilized steel spatula, into a sterile test tube containing their ability in removing hazardous compounds from the water, whereas sediment particles contaminated with crude oil from oil spills is one of the desired ecological niche to fungi which inhabits *Corresponding author: Hussein Al-Nasrawi, Post doctoral fellow, Fulbright visiting scholar, Florida state university, USA, E-mail: [email protected], such substrate and use carbon source from hydrocarbons in polluted [email protected] sediment particles to biodegrade crude oil from the sediments in the Received April 06, 2012; Accepted April 20, 2012; Published April 22, 2012 beaches. Fungi have been found to be better degraders of petroleum than traditional bioremediation techniques including bacteria, and Citation: Al-Nasrawi H (2012) Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico. J Bioremed Biodegrad 3:147. doi:10.4172/2155-6199.1000147 although hydrocarbon degraders may be expected to be readily isolated from a petroleum oil- associated environment, the same degree of Copyright: © 2012 Al-Nasrawi H, et al. This is an open-a ccess article distributed expectation may be anticipated for microorganisms isolated from a under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the totally unrelated environment [8,9]. original author and source are credited. J Bioremed Biodegrad ISSN: 2155-6199 JBRBD, an open access journal Volume 3 • Issue 4 • 1000147 Citation: Al-Nasrawi H (2012) Biodegradation of Crude Oil by Fungi Isolated from Gulf of Mexico. J Bioremed Biodegrad 3:147. doi:10.4172/2155- 6199.1000147 Page 2 of 6 9.0 mL of the diluent. This gave 10-1 dilution. Subsequently, three-fold added to the Bacto Bushnell-Haas broth medium. All flasks incubated (103) serial solutions were prepared from the 10-1 dilution. 1 ml of in room temperature using a shaker with 180 rev/min for seven days. dilution was poured on Czapak Dox Agar (CDA) plates (30 g Powder Changing in color of inoculated media in the flasks from deep blue to of Czapak Dox Agar (CDA) and 49.5 gm of Malt Extract Agar (MEA) colorless indicates the ability of fungi to biodegradation of crude oil. were added to 1000 ml of distilled water respectively. Streptomycin (500 mg/l) as antibiotic inhibit bacterial growth was added to the media Inoculum of 0.2 ml of fungal isolate was added to essay tubes after sterilization process. (triplicates) that contained 10 ml sterile Bushnell-Hass (BH) medium and 1% v/v of crude oil. The concentration of DCPIP was 0.16 mg/ Bushnell-Haas broth medium was used for the screening test which ml. The tubes were kept under agitation 60 rpm at 28.0 ± 1.0°C. composed of: MgSO (0.2 g/l), CaCl (0.02 g/l), KH PO (1 g/l), K HPO 4 2 2 4 2 4 Biodegradation activity of fungi observed during the change of blue (1 g/l), FeCl2 (0.05 g/l) and NH4NO3 (1 g/l). Tween 80 (0.1%), redox reagent (2% 2, 6-dichlorophenol indophenols) and crude oil (1%) were color of DCPIP to colorless. incorporated into the broth. To measure weight loss of fungal strains, weight loss method of Bartha and Bossert [31] was used in the present study. 10 ml of crude Identification of fungal isolates oil broth was prepared in a test tube and inoculated with 0.1 ml of Fungal genera were identified according to morphology characters filtrate from a soil sample. The test tube was then incubated at 35 ± and classified according to taxonomy keys in many literatures [21-28]. 2°C being shaken on a mechanical shaker for 10 minutes at 200 rpm. After 48 hours, the remaining crude oil was separated using a separator Species were identified by using DNA sequence method. A suitable mass of inoculum of fungal isolate was prepared with carefully N 32 removing the upper surface of the isolate without agar medium, the DFE 16 DNA extraction technique used to remove inhibitory materials, i.e. All Between polysaccharides, proteins, mineral salts, etc., which limit the sensitivity Test Value Exact F NumDF DenDF Prob>F of the different reactions in which isolated DNA is applied [29]. F Test 22590.437 24096.467 15 16 <.0001 Intercept Genomic DNA was extracted from fungal isolates using a Mo-Bio Test Value Exact F NumDF DenDF Prob>F Power Soil DNA extraction kit following manufacturer’s protocol (Mo- F Test 5890.5625 94249 1 16 <.0001 Bio, Carlsbad CA, USA). Approximately 0.5 g of fungal hyphae were Species scraped off Petri dishes and transferred to bead tubes provided in the Test Value Exact F NumDF DenDF Prob>F kit. Mechanical lysis was enhanced using a Talboys High Throughput F Test 22590.437 24096.467 15 16 <.0001 Homogenizer (Troemner, Thorofare, NJ, USA) at 1600 rpm for 3 Within Subjects minutes. DNA extracts were assessed using a Nano drop ND-1000 Contrast Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). All With in Interactions Test Value Approx. F NumDF DenDF Prob>F Fungal 18S rRNA genes were PCR amplified using NS-1 [5’-GTA Wilks’ Lambda 1.05e-7 3078.4312 30 30 <.0001 GTC ATA TGC TTG TCT-3’] and FR-1 [AIC CAT TCA ATC GGT Time AIT].
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