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Biodegradation of Petroleum Hydrocarbon by indigenous Fungi isolated from Ship breaking yards of Bangladesh Dhar K., Dutta S. and Anwar M.N. Microbiology Research Laboratory, Department of Microbiology, University of Chittagong, Chittagong-4331, BANGLADESHAvailable online at: 
www.isca.in, www.isca.me
Received 21st March 2014, revised 5th May 2014, accepted 26th June 2014Abstract  Ship breaking activities along some coastal areas of Bangladesh by traditional beaching method release a huge amount of petroleum hydrocarbons, for example, diesel, petrol, anthracene, phenanthrene cause severe environmental pollution and endanger the entire ecosystem. As an attempt to clean up such petroleum hydrocarbons, a well-timed process bioremediation that involves microbes and plants, offers an efficient, cost-effective, easy-to-use and eco-friendly alternative over physical and chemical treatment approaches. The involvement of naturally-occurring or indigenous fungi has been reported to grow on diverse organic pollutants, and their survival in even extreme environmental conditions make them promising bioremediating agent. The present study aims to isolate and identify petroleum hydrocarbon degrading indigenous fungi from ship breaking yards in order to facilitate the candidacy of the fungi as bioremediating agents. Five out of 14 indigenous fungal isolates were initially screened as petroleum hydrocarbon degraders, which exhibited utilization of petroleum hydrocarbon-rich crude oil in mineral salt medium. The concerned 5 isolates were subsequently validated as the degraders of the same of crude oil in Bushnell Hass medium, exhibiting decolorization of a redox indicator 2,4-dichlorophenol indophenol. The degrader isolates were identified as Cladosporium tenuissium, Fusarium moniliforme, Penicillium corylophilum, Trichoderma koningii and Aspergillus niger after critical analysis of their morphological and cultural characteristics on Potato Dextrose Agar and Czapek Dox Agar media. However, the candidacy of the fungi as bioremediating agent was evaluated by estimating the rate of degradation of common petroleum fuels, kerosene, diesel and octane. All the species degraded octane the most efficiently, followed by diesel and kerosene. Though Fusarium moniliforme caused the maximum degradation of octane (58%) and diesel (56%), Penicillium corylophilum caused the same of kerosene (40%). Hence, this study reveals that the indigenous fungi of the yards spontaneously play a pivotal in restoring a petroleum hydrocarbon-free ecosystem through bioremediation.  Keywords: Environmental pollution, petroleum hydrocarbon, biodegradation, bioremediation, fungi.Introduction The age of ship breaking industry in Bangladesh is about three decades. However, within this short period, Bangladesh has established itself as a leading ship breaking state. Gradually, more than 100 locally registered ship recycling yards have grown along the coastal belt of Bangladesh, from Bhatiary to Barawlia at Sitakund, some 20 kilometer southwest of Chittagong divisional city, between latitude 22°25 and 22°28N and longitude 91°42and 91°45. The industry is indeed fulfilling a crucial need of steel around the country, and generates revenues for government authorities. On the other hand, the ship is destined for ship breaking by old-fashioned beaching method, improper storage and disposal of scrap wastes release significant quantities of toxic and hazardous materials like engine oil, bilge oil, hydraulic oil, lubricant oil, grease, polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), tribtylin (TBT) and heavy metals, and cause severe damage to the ecosystem. The petroleum hydrocarbons are hazardous to various forms of terrestrial and aquatic life like fish, bird and human, and are also carcinogenic, mutagenic and potentially immunotoxigenic. The traditional physical and chemical treatment approaches to clean-up the petroleum hydrocarbons are expensive and appear ineffectual as they do not lead to complete mineralization, and awfully can produce toxic byproducts or residues. In contrast, as an innovative and eco-friendly strategy, bioremediation involving microbial agents, such as protozoa, bacteria, fungi, plants offers successful alternatives to clean-up the petroleum pollutionAmong fungal bioremediating agents, mold species of Aspergillus, Penicillium, Fusarium, Amorphoteca, Paecilomyces, and Talaromyces, andyeast species of Candida, Yarrowia, and Pichia have been recognized in hydrocarbon degradation and its derivatives4-9Fungal bioremediation technology is highly efficient, versatile and cost-effective, compared to that of protozoan or bacterial type. Fungi usually grow on a wide variety of organic materials, survive under environmental conditions of stress like nutrient deficiency and high salinity, and can spread their mycelium wider and deeper than others. In recent days, a significant number of researches have been carried out in characterizing the functional variations of petroleum hydrocarbon degrading microorganisms toward bioremediation of oil spills, reasonably a little incentive has been devoted to promote or facilitate bioremediation of petroleum hydrocarbon pollutants associated with ship breaking industry. The present study aims to explore the candidacy of indigenous fungi as potential bioremediating agents in the ship breaking yards. Following 
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isolation and identification of a number of petroleum hydrocarbon-degrading fungi, the extent of degradation of multiple hydrocarbons by the isolates reveals their potentials in alleviating the devastating pollution through bioremediation. Material and MethodsSamples: Petroleum hydrocarbon-contaminated soil samples were collected from a total of 20 distinct places, wet to semi-dry, from 5 ship breaking yards situated along the coastal area of Sitakunda, Chittagong, Bangladesh. While collecting each sample, a 5 cm depth of surface soil was dug with a sterile scrape, and an approximate of 50g soil resided beneath the dug surface soil was collected into a sterile container, placed in a cooler box and immediately transported to the laboratory. Materials: Potato Dextrose Agar (PDA) and Czapek Dox Agar (CDA) media [HiMedia Laboratories] were used for isolation, enumeration, cultural and morphological characterization of fungi. Oil Agar (OA) medium was used for preliminary screening of petroleum hydrocarbon degrading fungi. The OA medium was prepared by adding 1% crude oil to Mineral Salt Medium (MSM) [v/v] of Mills et al.10 as modified by Okpokwasili and Okorie11. The composition of MSM was NaCl (10.0g), MgSO.7HO (0.42g), KCl (0.29g), KHPO (0.83g), NaHPO (1.25g), NaNO(0.42g), agar (20g), distilled water (1L) and pH 7.2. Bushnell-Haas (BH) broth12 supplemented with 2% 2,4-dichlorophenol indophenol (2,4-DCPIP) [w/v], 0.1% Tween 80 [v/v] and 1% crude oil [v/v] was used for confirmatory screening of petroleum hydrocarbon degrading fungi. The composition of BH broth was MgSO.7HO (0.2g), CaCl (0.02g), KHPO (1g), KHPO (1g), FeCl (0.05g), NHNO (1g) and distilled water (1L).  Enumeration and isolation of indigenous fungi from soil samples: After careful sorting of stones and debris using 2 mm sieve, serially diluted soil samples were plated onto PDA medium supplemented with 0.1% tetracycline13, and total heterotrophic fungi were enumerated by dilution plate count method14Distinct colonies were isolated through repeated passages.  Screening of petroleum hydrocarbon degrading fungi: For preliminary screening of petroleum hydrocarbon degrading fungi, OA plate was inoculated with spore suspension of each isolate, incubated at 26°C, and the appearance of substantial growth was monitored daily up to 7 days. For confirmatory screening, BH brothsupplemented with a redox indicator, an emulsifier and crude oil was inoculated with agar block of each isolate, incubated with agitation at 26°C, and monitored daily for color change from deep blue to colorless15. Identification of petroleum hydrocarbon degrading fungi: The indigenous fungi exhibiting degradation of petroleum hydrocarbon were grown on PDA and CDA to examine morphology, viz size, mycelia and sporulation, and culture, viz color, texture, substrate color and colonial appearances. The observed characteristics of the isolates were recorded and compared with the established identification key given in the “Manual of Soil Fungi”16. Estimation of petroleum hydrocarbon degradation: The efficiency of the isolates in degrading petroleum hydrocarbon was evaluated for some common petroleum fuels, kerosene, diesel and octane, where the rate of degradation is expressed as percent degradation as described by Juwarkar and Khirsagar17. Briefly, the isolates were cultured in MSM supplemented with 0.2% each of kerosene, diesel and octane. Natural seawater, instead of water, was also used in the preparation of the medium. Following 7 days incubation at 26° C with constant shaking, the petroleum fraction of cell free culture broth was extracted with three volumes of toluene. The optical density (OD) was measured at 420 nm by Visible-UV spectrophotometer (UV-VIS RS Spectrophotometer, LaboMed). The extent of degradation was calculated using the following equation: Degradation (%) = (C-C)/C×100, where Cand C refers to initial and final concentration, respectively. The assay was carried out in triplicate. Results and DiscussionShip breaking yards inhabit indigenous filamentous mycobiota: A heterotrophic and filamentous type of mycobiota was found to reside in the petroleum hydrocarbon contaminated soil of the ship breaking yards, utilizing the naturally-occurring organic substances in soil, and the count ranged from 2.5×10 to 4.0×10 CFU/mL as determined by soil dilution plate count method. Indeed, the presence of such a significant number of fungi in the yards reflects their adaptive ability to survive even in incidents of deliberating continuous and prolonged discharges of various petroleum products. Notably, a total of 14 indigenous fungal species was isolated based on the distinct cultural characteristics of each on PDA medium.  Not all species of indigenous mycobiota degrade petroleum hydrocarbon: In order to carry out the preliminary screening of the petroleum hydrocarbon degrading fungi, each isolate was plated onto OA medium where crude oil serves as the sole source of carbon and energy. Only 5 out of the 14 indigenous isolates were found to utilize petroleum hydrocarbon-rich crude oil for cellular biosynthesis, thereby develop substantial colonies. However, the confirmatory screening of petroleum hydrocarbon degradation by the fungi was emphasized by culturing the isolates in BH broth incorporated with the crude oil, an emulsifier Tween 80 and a deep-blue colored redox indicator 2,4-DCPIP. Tween 80, through its emulsifying property, facilitates active contact between petroleum hydrocarbon and the isolate.  Petroleum hydrocarbon-degrading mycobiota: The petroleum hydrocarbon degrading fungi were grown on PDA and CDA, and their cultural and morphological characteristics are summarized in table 1. The fungi were identified up to the species as Cladosporium tenuissium (figure- 1A), Fusarium moniliforme (figure- 1B), Penicillium corylophilum (figure- 1C), Trichoderma koningii (figure- 1D)and Aspergillus niger (figure- 1E)while compared with the standard key given in the “Manual of Soil Fungi”16.  
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Table-1 Cultural and morphological characteristics of the isolates on Czapek Dox agar (CDA)Isolate Cultural Characteristics Morphological Characteristics 
  Mycellial Morphology Sporulation Characteristics 
Cladosporium tenuissium Rapidly growing, thick hairy, dark black colony; substrate color black Branched, brown to dark brown, septate Conidiophores thread like, septate, mid to dark brown, slightly curved, smooth, occasionally swollen; Conidia lemoniform or fusiform; hyaline, aseptate when young but became pale colored mono-septate with aging Conidiophore: 60-94 m × 2.5-6.0 m Conidia: 12-18 m ×1.7-3.5 m 
Fusarium moniliforme Rapidly growing, wooly, white colony; substrate color pale yellow Branched, hyaline, septate Conidiophores thread like, septate, hyaline; both microconidia and macroconidia were present; macroconidia multi-septate, ranging from 2-5 septa; macroconidia usually sickle shaped, but bi-septate conidia were spindle; microconidia single celled, egg shaped; chlamydospores present 3 septate Macroconidia: 10.5-12 m × 1.7-2.5 m 
Penicillum corylophilum Rapidly growing, wooly or cottony, greenish white colony; substrate color yellow Branched, hyaline, septate Conidiophores were long, smooth, erect, hyaline, branched, biverticilliate, frequently branched below the level of metulae; phialides borne in one series bearing chain of conidia forming a brush like cluster; conidia elliptical, almost hyaline Conidiophore: 90-165 m Phialides: 6-10.5×1.5-3.5 m Individual conidia: 2.6-3.5 m 
Trichoderma koningii Wooly, greenish white colony, mature fruiting area green in color; substrate color unchanged Branched, hyaline and septate Conidiophores long, rough walled, erect; fruiting heads arised from alternate side branching bear conidia at apex; conidia small, globose to ovate Conidiophore: 50-75 m×2.5-3.5 m Fruiting Head: 3.5-10 m Conidia: 2.5-4 m in diameter. 
Aspergillus niger Rapidly growing,  black color colony; substrate color pale yellow Well branched, compact, septate, hyaline Conidiophore mostly arise directly from the substratum, conidiophores were long, smooth, unbranched, light brown to colorless, double layered with down thick walled foot cell; conidiophores terminated at round vesicle; Conidial head radiate; Sterigmata borne in two series bearing conidial chains;  Conidial chain thickly covered the head; Conidia round, dark brown to black Conidiophore: 150-210 m × 3.5-7 m Vesicle: 40-65 m Primary Sterigmata: 28-40 m Individual conidia: 3-4 m 
Indigenous mycobiota degrades octane the most, followed by diesel and kerosene: The efficiency of the indigenous fungal isolates was examined by co-incubating the fungal isolates in MSM along with common petroleum fuels, 0.2% each of kerosene, diesel and octane. After 7 days of incubation in MSM, the isolates were found to deplete more than 26% kerosene (figure- 2), 41% diesel (figure- 3) and 45% octane (figure- 4) from the medium individually. For any given isolate, the efficiency of degradation of the fuels follows the order:  octane � diesel � kerosene  
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Figure- 1(A) Figure- 1(B) 
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Figure- 1(C) Figure- 1(D) 
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Figure- 1(E) Legend of Figure-1 Photomicrographs of the isolates at 400 magnifications. (A) conidiophores and conidia of Cladosporium tenuissium; (B) macroconidia of Fusarium moniliforme; (C) biverticilliate conidiophores, phialides and conidia of Penicillum corylophilum; (D) alternate side branching of conidiophores  as well as conidia of Trichoderma koningii; (E) conidiophores, conidial heads and conidia  Aspergillus niger The above phenomenon could be indicative of the effect of compositional and structural complexity on biodegradability of petroleum derivatives. Octane fuel has the simplest atomic structure and has the least amount of -C-C- bonds as compared to diesel and kerosene, thus doesn’t resist microbial attack18,19. After seven days of incubation, both Fusarium moniliforme and Aspergillus niger caused the highest (58%) octane degradation, while other isolates degraded a significant amount of octane too (figure- 4). Fusarium moniliforme showed the highest (56%) extent of diesel degradation followed by Cladosporium tenuissium (51%) (figure- 3). It was observed that the isolates utilized comparatively less amount of kerosene from the media. The percentage of kerosene degradation was limited in the range of 26% to 41%. The highest (41%) extent of kerosene degradation was recorded for Penicillium  corylophilum (figure- 2).  Kerosene and diesel fuel are mixture of carbon chains containing 6-16 and 8-21 carbon atoms per molecule, respectively. Octane (2, 2, 4-trimethylpentane or isooctane) is a common component of gasoline and other petroleum products. Simpler linear hydrocarbon fractions do not resist attacks by microorganisms and comparatively better degraded by microflora. The extent of degradation supports the fact that the isolated fungi are efficient in degrading simpler, linear, nC – nC21 containing petroleum hydrocarbons. Besides aliphatic constituents, commercial kerosene and diesel fuel also contain naphthalene, alkyl benzene and similar derivatives. As the isolated fungi degrade the above-mentioned commercial fuel oil, there is a possibility that these fungi could also degrade aromatic hydrocarbon derivatives. The indigenous mycobiota utilize hydrocarbons in extreme habitats: It has been observed that the indigenous fungi were found to survive and degrade petroleum hydrocarbons receiving only mineral nutrients and supplied hydrocarbons from MSM. Given that the indigenous fungi utilize petroleum hydrocarbons in the presence of fixed form of minerals. Lack of mineral nutrients is one of the common environmental limitations to biodegradation. Several authors reported that the available concentration of fixed form of nitrogen and phosphorus severely limits petroleum hydrocarbon biodegradation in a coastal marine environment 20,21,22. Venosa et al.23 demonstrated that the addition of nutrients addition was the critical factor in stimulating biodegradation in real petroleum contaminated sites. A fortunate fact is that fungi are capable to grow under 
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environmental conditions of stress, e.g.: environment with low pH values or poor in nutrients. In accordance with above stated experimental facts, this study suggests inorganic nutrient amendment to enhance biodegradation of petroleum hydrocarbon pollutants of ship breaking yards and thereby cleaning-up the pollutants. In the course of evaluation of biodegradation, natural seawater was used as a solvent of media to mimic the real salinity condition of the coastal area. As seen in figure- 2, 3 and 4, the indigenous fungi showed significant degradation of petroleum hydrocarbon in the extreme salinity condition in vitro, which postulates high possibility of biodegradation of petroleum pollutants in situ by the isolatesFigure-2 Estimated efficiency of kerosene degradation by the fungal isolates. The biodegradation assay was performed by incubating Cladosporium tenuissium Fusarium moniliformePenicillium corylophilum Trichoderma koningii andAspergillus niger in MSM with 0.2% kerosene. After seven days of incubation, the petroleum fraction was extracted with toluene followed by measurement of OD420. The extent of degradation was calculated from the following equation, Degradation (%) = (C-C)/C×100, where C and C refers to initial concentration and final concentration respectively.The data are representatives of three independent experiments. Here, * = P 0.05, ** = P 0.01 and *** = P 0.001, P = significance level Figure-3 Estimated efficiency of diesel degradation by the fungal isolates Cladosporium tenuissium Fusarium moniliformePenicillium corylophilum Trichoderma koningii andAspergillus niger.  The biodegradation assay was performed by following the similar method described in figure- 2. The data are representatives of three independent experiments. Here, * = P 0.05, ** = P 0.01 and *** = P 0.001, P = significance level Conclusion This study has revealed that the indigenous fungi of the ship breaking yards have the potentiality in degrading petroleum hydrocarbon pollutants like kerosene, diesel and octane which is a very promising approach to remediate contaminated soil and water. In order to implement the isolates or their products, e.g., enzymes as bioremediating agents, thorough investigations on optimal growth conditions, genetic diversities and degradation mechanisms are necessary. 
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Figure-4 Estimated efficiency of octane degradation by the fungal isolates Cladosporium tenuissium Fusarium moniliformePenicillium corylophilum Trichoderma koningii andAspergillus niger.  The biodegradation assay was performed by following the similar method described in figure- 2. The data are representatives of three independent experiments. Here, * = P 0.05, ** = P 0.01 and *** = P 0.001, P = significance level. References 1.Zakaria N.M.G., Ali M.T. and Hossain K.A., Underlying problems of ship recycling industries in Bangladesh and way forward, J. Nav. Archit. Mar. Eng.99, 1-102 (2012)2.Alexander M., Biodegradation and Bioremediation, Academic Press, New York, 692 (1994)3.Bento F.M., Camargo F.A.O., Okeke B.C. and Frankenberger W.T., Comparative bioremediation of soils contaminated with diesel oil bynatural attenuation, bioestimulation and bioaugmentation, Bioresour. Technol.,96, 1049-1055 (2005)4.Chaillan F., Flèche A.L., Bury E., Phantavong Y., Grimont P., Saliot A. and Oudot J. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms, Res. Microbiol., 155, 587-595 (2004)5.Potin O., Rafin C. and Veignie E. Bioremediation of an aged polycyclic aromatic hydrocarbons (PAHs) –contaminated soil by filamentous fungi isolated from the soil, Int. Biodeterior., Biodegradation., 54, 45-52 (2004)6.Romero M.C., Salvioli M.L., Cazau M.C. and Arambarri A.M. Pyrene degradation by yeasts and filamentous fungi, Environ. Pollut.,117, 159-163 (2002)7.Santos V.L. and Linardi V.R. Biodegradation of phenol by a filamentous fungi isolated from industrial effluents - identification and degradation potential, Process Biochem., 39, 1001-1006 (2004)8.Garon D., Krivobok S. and Seigle-Murandi F., Fungal degradation of fluorine, Chemosphere,40, 91-97 (2000)9.George-Okafor U., Tasie F. and Muotoe-Okafor F., Hydrocarbon degradation potentials of indigenous fungal isolates from petroleum contaminated soils, J. Phy. Nat. Sci.3(1), 1-6 (2009)10.Mills A.L., Breuil C. and Colwell R.R., Enumeration of petroleum-degrading marine and estuarine microorganisms by the most-probable number, Can. J. Microbiol., 24, 552-557 (1978)11.Okpokwasili G.C. and Okorie B.B. Biodeterioration potentials of microorganisms isolated from car engine lubricating oil, Tribol. Int., 21(4), 215–220 (1988)12.Bushnell L.D. and Haas H.F., The utilization of certain hydrocarbons by microorganisms, J. Bacteriol.,  41, 653 (1940)13. Walker J.D. and Colwell R.R. Enumeration of petroleum-degrading microorganisms, Appl. Environ.  Microbiol., 31,198-207, (1976)14.Waksman S.A. A method for counting the number of fungi in soil, J. Bact., , 339-341 (1922)15. Desai A., Jitendra J., Desai D. and Hanson K.G., A rapid and simple screening technique for potential crude oil degrading microorganisms, Biotechnol. Tech., 7(10), 745-748 (1993)16. Gilman J.C. Manual of soil fungi, 2nd Edition, The Iowa State College Press-Ames, Iowa, U.S.A. (1957)17.Juwarkar A. and Khirsagar D.G., The utilization of certain hydrocarbon by microorganisms, J. Bacteriol.,41: 653-673 (1991)18.Atlas R.M., Bioremediation of petroleum pollutants, Int. Biodeterioration. Biodegrad., 35, 317-327 (1995)19.Jones J., Knight M. and  Byron J.A., Effect of gross population by kerosene hydrocarbons on the microflora of a moorland soil, Nature,  227, 1166 20.Atlas R.M., Microbial hydrocarbon degradation-bioremediation of oil spill, J. Chem. Tech. Biotechnol.,52,149 (1991)21.Prince R.C., Petroleum bioremediation in marine environment, Crit. Rev. Microbiol., 19, 217-242 (1993)
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22.Swannell R.P.J., Lee K. and Mc Donagh M., Field evaluation of marine oil spill bioremediation, Microbiol.  Rev.,60, 342-365 (1996)23.Venosa A.D., Suidan M.T., Wrenn B.A., Strohmeier K.L., Haines J.R., Eberhart B.L., King D. and Holder, E. Bioremediation of an experimental oil spill on the shoreline of Delaware Bay, Environ.  Sci.  Technol., 30,1764-1776 (1996)