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ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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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
Corresponding Author : Dr. Hussein Al-Nasrawi
Post doctoral fellow, Fulbright visiting scholar
Florida state university, USA
E-mail: [email protected], [email protected]
Received April 06, 2012; Accepted April 20, 2012; Published April 22, 2012
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
Copyright: © 2012 Al-Nasrawi H, et al. This is an open-a ccess article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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
Introduction
The dominance of petroleum products in the world economy creates the conditions for distributing large amounts of complex compounds consist of hundreds of different hydrocarbon molecules, and a huge volume of oily sludge, a carcinogenic and a potent immunotoxicant [1,2]. Oil spillage is the accidental discharge or pouring of crude oil into the environment. It involves the contamination of any part of the environment with any liquid hydrocarbon. These spills endanger public health, imperil drinking water, devastate natural resources, and disrupt the economy [3]. Crude oil is a naturally occurring complex mixture of hydrocarbon and non-hydrocarbon compounds which at appropriate concentration, possesses a measurable toxicity towards living systems. The toxicity of crude oil or petroleum products varies widely, depending on their composition, concentration, environmental factors and on the biological state of the organisms at the time of the contamination [4].
Although oil spills from tankers and pipelines release crude oil particles to the water surface and move it to the beaches and contaminates living and nonliving organisms, microorganisms specially fungi have a higher tolerance to the toxicity of hydrocarbons due to their physiology and adaptation to such variations in the environment and have the mechanism for the elimination of spilled oil from the environment [5,6].
The effect of oil on microbial populations depends upon the chemical composition of the oil and on the species of microorganisms present. Populations of some microbes increase; typically, such microbes use the petroleum hydrocarbons as nutrients. The same crude oil can favor different genera at different temperatures [7].
In the aquatic ecosystems, fungi plays an important role during 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 such substrate and use carbon source from hydrocarbons in polluted sediment particles to biodegrade crude oil from the sediments in the beaches. Fungi have been found to be better degraders of petroleum than traditional bioremediation techniques including bacteria, and although hydrocarbon degraders may be expected to be readily isolated from a petroleum oil- associated environment, the same degree of expectation may be anticipated for microorganisms isolated from a totally unrelated environment [8,9].
Several authors have made lists containing bacteria and fungi genera that are able to degrade a wide spectrum of pollutants, proceeding from marine atmosphere as well as the soil [10-12].
Recently, many researchers studied the role of fungi in biodegradation process of petroleum products and the most common fungi which have been recorded as a biodegrades belongs to following genera: Alternaria, Aspergillus, Candida, Cephalosporium, Cladosporium, Fusarium, Geotrichum, Gliocladium, Mucor, Paecilomyces, Penicillium, Pleurotus, Polyporus, Rhizopus, Rhodotolura, Saccharomyces, Talaromyces and Torulopsis [3,4,13-20].
The aim of the present study is to isolation fungi from polluted beach sand in Gulf of Mexico and test the ability of isolated fungi in biodegradation of crude oil.
Materials and Methods
Sample collection and culture methods
Soil samples (400 g) from surface soil (0-15 cm depth) were collected from different localities in Pensacola beach (Gulf of Mexico) which was contaminated with crude oil. Sample were made from 3-4 random locations per plot, mixed and transferred into sterile bottles using sterile spatula for microbiological quality determination and stored in ice box to a void contamination. In the lab, stones and other 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) of Tween 80 and a loopful (3 mm) of it was collected and inoculated by sprinkling method onto SDA and Czapek agar plates, respectively. Soil fungi were estimated by soil dilution plate count method. Sodium chloride 0.85 % was used as diluent for inoculum preparation. 1.0 g of homogenized, 2 mm sieved soil sample was aseptically transferred, using a flame-sterilized steel spatula, into a sterile test tube containing 9.0 mL of the diluent. This gave 10-1 dilution. Subsequently, three-fold (103) serial solutions were prepared from the 10-1 dilution. 1 ml of dilution was poured on Czapak Dox Agar (CDA) plates (30 g Powder of Czapak Dox Agar (CDA) and 49.5 gm of Malt Extract Agar (MEA) were added to 1000 ml of distilled water respectively. Streptomycin (500 mg/l) as antibiotic inhibit bacterial growth was added to the media after sterilization process.
Bushnell-Haas broth medium was used for the screening test which composed of: MgSO4 (0.2 g/l), CaCl2 (0.02 g/l), KH2PO4 (1 g/l), KH2PO4 (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 incorporated into the broth.
Identification of fungal isolates
Fungal genera were identified according to morphology characters and classified according to taxonomy keys in many literatures [21-28].
Species were identified by using DNA sequence method. A suitable mass of inoculum of fungal isolate was prepared with carefully removing the upper surface of the isolate without agar medium, the DNA extraction technique used to remove inhibitory materials, i.e. polysaccharides, proteins, mineral salts, etc., which limit the sensitivity of the different reactions in which isolated DNA is applied [29].
Genomic DNA was extracted from fungal isolates using a Mo-Bio Power Soil DNA extraction kit following manufacturer’s protocol (Mo- Bio, Carlsbad CA, USA). Approximately 0.5 g of fungal hyphae were scraped off Petri dishes and transferred to bead tubes provided in the kit. Mechanical lysis was enhanced using a Talboys High Throughput Homogenizer (Troemner, Thorofare, NJ, USA) at 1600 rpm for 3 minutes. DNA extracts were assessed using a Nano drop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Fungal 18S rRNA genes were PCR amplified using NS-1 [5’-GTA GTC ATA TGC TTG TCT-3’] and FR-1 [AIC CAT TCA ATC GGT AIT]. Reactions were performed in 50 μl volumes containing 0.5 mM dNTPS, 0.5 μM of each primer, 1X DreamTaqTM Green Buffer, 1.5 U DreamTaqTM polymerase, and 10 ng template DNA. Thermo cycling conditions consisted of an initial denaturation stage of 95°C for 5 minutes followed by 35 cycles of 95°C for 45 seconds, 55°C for 90 seconds, and 72°C for 90 seconds, and a final stage of 72°C for 10 minutes.
Amplicons were cleaned using a Mo-Bio Ultra Clean® PCR Cleanup Kit and sent for sequencing on an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems Inc., Foster City CA, USA). Raw sequence data were processed to remove low quality sequence data using the software package Sequencer (Gene Codes, Ann Arbor, MI, USA). The basic local alignment search tool-BLAST was used to classify and identify closely related fungal sequences.
Screening procedure
The biodegradability of isolated fungi was verified using the modified technique based on the redox indicator 2, 6-dichlorophenol indophenol (DCPIP) [30]. From 7 days fungal isolate old two plugs ( 1 cm2 for each plug ) were picked from the peripheral area of Petri dish and transferred carefully to inoculate into 50 ml Bacto Bushnell - Haas broth medium using 250 ml Conical flask. 0.1% (v/v) Tween 80 and 1% (v/v) crude oil and 0.008 mg/50 ml of redox indicator as a powder were added to the Bacto Bushnell-Haas broth medium. All flasks incubated in room temperature using a shaker with 180 rev/min for seven days. Changing in color of inoculated media in the flasks from deep blue to colorless indicates the ability of fungi to biodegradation of crude oil.
Inoculum of 0.2 ml of fungal isolate was added to essay tubes (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/ ml. The tubes were kept under agitation 60 rpm at 28.0 ± 1.0°C. Biodegradation activity of fungi observed during the change of blue color of DCPIP to colorless.
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 oil broth was prepared in a test tube and inoculated with 0.1 ml of filtrate from a soil sample. The test tube was then incubated at 35 ± 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 funnel, and weighed. The percentage degradation of the crude oil was then calculated as described by Ijah and Ukpe [32].
 
Statistical analyses
The present study conducted an ANOVA (analysis of variance), which was performed on all the treatments and done using the SPSS (version 10.0) package to determine whether or not, a significance difference exists between the weight loss of degraded crude oil by the microorganisms over time. There is a significant effect of time (percent weight loss of crude oil increases over time) and there is also a species*time interaction (the percent weight loss of crude oil differs by fungal species) as shown in table 1.
Results and Discussion
Results of this study revealed that sixteen fungal strains were isolated from beach sand in Gulf of Mexico area, as shown in Table 2, the mentioned fungal isolates were tested for insure their ability to biodegrade crude oil, three fungal strains demonstrated perfect biodegradation ability, Aspergillus niger, Cochliobolus lutanus and Penicillium documbens as shown in Figure 1 and 2.
The rate of occurrence among the fungi isolates obtained from the soil samples is shown in Table 3. Aspergillus niger had the highest rate of occurrence, being 23.5 % while Cordyceps sinensis and Pleospora herbarum had the least occurrence rate of 1.9 %.
Table 4 shows the ability of fungal isolates in biodegradation of crude oil, by flask and test tube experiment.
The screening method used in the present study depends on changing in the color of fungal isolates treated with redox indicator technique, so the fungal isolates which has the ability to degrade crude oil in the presence of redox indicator, confirmed the ability of the three fungi, Cochliobolus lutanus, Aspergillus niger and Penicillium documbens to biodegrade crude oil whereas low ability was observed by Fusarium solani strain 421502. Although Aspergillus and Penicillium species were recorded in former studies as crude oil biodegrades, the present study confirmed that the fungus Cochliobolus lutanus demonstrated as a new record fungus in biodegradation of crude oil.
The ability to analyses crude oil compounds to its components leads to oxidation of the carbon source in the crude oil components. There are three indicators leads to the ability of these fungi in biodegradation process, the first one is changes in color of culture media from blue to colorless, the second is disappearance of crude oil from the medium and the third is developing a mass of fungal growth in the bottom of the culture medium. Mechanism of biodegradation of crude oil occurred by incorporating an electron acceptor such as DCPIP to the culture medium, it is possible to ascertain the ability of the fungi to utilize the substrate by observing the color change of DCPIP from blue (oxidized) to colorless (reduced) [30].
 
In the present work Aspergillus niger and Penicillium documbens were the perfect fungal isolates demonstrated active ability to biodegrades crude oil, this result agree with results of Gesinde et al. [3] who indicated that Aspergillus niger have very active degradation capabilities of four kinds of oil compounds, Durb oil, Escravos light, Arabian light and Bonny light. Furthermore, in comparison with eight other genera, Aspergillus, Penicillium and Fusarium species were the most efficient metabolizers of hydrocarbons [4,33].
In a previous review, Bartha and Atlas [34] listed 14 genera of fungi isolated from an aquatic environment which had been demonstrated to contain members which utilize petroleum hydrocarbon. The evolution of the hydrocarbon mixture depends on the nature of the oil, microbial community, and environmental factors which impact microbial activities.
An interesting demonstration generated in this work which shows an increase in rates of fungal growth in the media containing crude oil compared with inoculated media without crude oil, this might be due to the fact that fungi use crude oil as a substrate for their survival growth using extra cellular enzymes to break down the recalcitrant hydrocarbon molecules, by dismantling the long chains of hydrogen and carbon, thereby, converting petroleum into simpler forms or products that can be absorbed for the growth and nutrition of the fungi [13].
According to Table 5, there are different weight losses of fungal strains after 3 weeks of incubation. Aspergillus niger was demonstrated the highest weight loss (8.6% ) with Penicillium documbens (7.9%) and Cochliobolus lutanus (4.7%) whereas the lowest weight loss was demonstrated by Fusarium solani strain 421502 (1.9% ), this result agree with results of Gesinde et al. [3] who confirmed the ability of Aspergillus niger to biodegrade crude oil with 18% weight loss and penicillium notatum with 11.2% weight lost, this indicates that these isolates have the potential to utilize crude oil as a carbon source. Various workers have reported the similar results by micoorganisms utilize crude oil [34-38]. The significant difference in the weights of the crude oil samples before and after degradation by the fungal isolates confirms their performances, statistical analysis revealed a significant difference at 95% confidence level between weights of crude oil samples before and after exposure to fungal isolates.
Conclusions
Oil spills consider one of the critical problems faces global nature due to impact of discharge pollutants which cause decline of environment health. Currently the nature became more familiar with biological control solutions to remove hazardous from the environment. Although disposal methods can become prohibitively expensive when the amounts of pollutants are huge, using microbial remediation process is successful and safe way to enhance environment health in particular with low cost, technique and high public acceptance to cleaning up aquatic ecosystems from oil spills.
Acknowledgements
I would like to thank the Kostka lab at FSU for their assistance with fungal isolation and taxonomy. This work was supported by a Fulbright scholarship to H. A-N.
 
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