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International Journal of Biotechnology. Photon 111 (2013) 253-262 https://sites.google.com/site/photonfoundationorganization/home/international-journal-of-biotechnology Original Research Article. ISJN: 3352-7304

International Journal of Biotechnology Ph ton Isolation, identification, and assessment of the ability of loca l Streptomyces isolate from Iraq to utilize crude oil and diesel fuel Rehab S. Ramadhaa, Rebah N. Jabbarb, Nour Abdulatifc* a Applied Biotechnology College, Al-Nahrain University, Iraq b Biotechnology Research Center, Al-Nahrain University, Iraq c College of Science, Al-Nahrain University, Iraq Article history: Received: 20 September, 2013 Accepted: 04 October, 2013 Available online: 22 November, 2013 Keywords: Hydrocarbon utilization, Streptomyces, Crude oil contamination, bioremediation, biosurfactant Corresponding Author: Abdulatif N.* Assistant Lecturer Email: nour_laen@yahoo.com Ramadha R.S. Email: rehabrebah2004@yahoo.com Phone: +9647709401269 Jabbar R.N. Email: rebahalgafari@gmail.com Phone +9647714479000 Abstract Two Streptomyces isolates, were selected that showed the ability to utilize crude oil and diesel fuel in culture media. Both were cultivated on mineral salt medium with both complex hydrocarbons as the sole source of carbon. They were able to grow and differentiate in 3 days of cultivation. Complete

consumption of crude oil and diesel fuel was observed in 3 days of cultivation in YMD medium. Biosurfactant was produced in medium and detected using surface tension as an indicator for this process. CTAB and blood hemolysis test gave positive result for the production of lipase. Gas Chromatography, and FTIR analysis showed that these strains were able to degrade long and complex hydrocarbon chains to smaller ones. Results obtained from Thin Layer Chromatography (TLC) of hydrocarbon hydrolysates showed the presence of polar, non polar, carbohydrates, and amino acids among the mixture. Factors affecting hydrocarbon utilization studied were pH, temperature, salt concentration and agitation showed that, neutral pH, with temperature range of 37° - 45° C, and agitation rate of 150 rpm gave the optimum conditions to expedite crude oil and diesel fuel hydrolysis, while salt concentration had no effect on the process. Citation: Ramadha R.S., Jabbar R.N., Abdulatif N., 2013. Isolation, identification, and assessment of the ability of local Streptomyces isolate from Iraq to utilize crude oil and diesel fuel. International Journal of Biotechnology. Photon 111, 253-262.

1. Introduction

Pollution caused by crude oil and its products is the most prevalent problem in the environment (Caprino and Togna, 1998, Millioli et al., 2009). Various physical, chemical and biological techniques are used to reduce its negative impact on human health, flora and fauna. Bioaugmentation, one of the most efficient biological techniques, is based on remediation of the contaminated environment by adding of hydrocarbon degrading microorganisms (Kosaric, 2001). Unfortunately, biodegradation of hydrocarbons often is limited by bioavailability which is associated with their poor solubility in water and sorption to soil particles. Thus, to enhance hydrocarbon bioavailability, synthetic surfactants and biosurfactants are used. Bacteria play a vital role in degrading aromatic

hydrocarbons into less toxic forms. Catechol (1, 2-dihy-droxybenzene) is a central intermediate in the aerobic bacterial degradation of a wide variety of aromatic compounds, such as benzene, toluene, ethylbenzene, xylene, naphthalene, and phenol (Dagley 1986). The primary way by which bacteria convert catecholic substrates into readily usable Krebs cycle intermediates is via aromatic ring cleavage dioxygenases, of which there are 2 types: intradioldioxygenases cleave ortho to the hydroxyl groups while extradioldioxygenases cleave meta to the hydroxyl groups (Burlage et al. 1989, Assinderand Williams1990). The extent of hydrocarbon biodegradation in contaminated soils is critically dependent upon four factors, namely the creation of optimal environmental

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conditions to stimulate biodegradative activity, the predominant petroleum hydrocarbon types in the contaminated matrix and the bioavailavility of the contaminants to microorganisms. The petroleum hydrocarbon degradation is also affected by the molecular composition of the hydrocarbons, characteristic which is directly related with the bioavailability of these compounds, and as a consequence, the biodegradation rate may be altered (Huesemann, 1995). Microorganisms with the ability to degrade crude oil are ubiquitously distributed in soil and marine environments, (Facundo et al., 2001). Actinobacteria is gram positive bacteria, aerobic and widely spread in nature. Actinobacterial DNA contains high G+C content about 57-75%. They are predominant in dry alkaline soil. There was at least report on biosurfactant production from marine actinobacteria. There was a chance for the presence of new bioactive compounds in these microorganisms (Shubhrasekhar et al., 2013). Although enormous literature appears on bioemulsifier production by bacteria like Acinetobacter, Bacillus, Pseudomonas, reports on production by actinomycetes are rare (Maniyar et al., 2011). For the last few decades actinobacteria have been categorized as extremely fascinating organisms with unexhausted reserve of bioactive compounds that are being exploited for various commercial applications. Among the diverse microbial communities, actinobacteria have occupied a prominent and significant position as potential producers of secondary metabolite (Battershill et al., 2005, Simmons et al., 2005). During the last decade, Streptomyces caught a lot of attention for its abundance among soil bacteria and for being biologically active bacteria. About two third of antibiotic production comes from this species, and addition it is a safe organism to be used in nature. Our work concentrated on 16 local Streptomyces isolates for their ability to utilize crude oil and diesel from contaminated soils, that may make as a candidate as a bio – remediation in converting contaminated soils with oil spills and diesel to a suitable one for agricultural use. 2. Materials and Methods 2.1 Bacterial isolates About 16 Streptomyce isolates were tested for hydrocarbon degradation trait. These were isolated from different farming soils, cow dung, and areas surrounding local generators that are contaminated with diesel, engine oils, and cooling water of these generators. Bacterial

strains were grown on modified Bennett media (Sigma Co.) composed per liter (1 gm beef extract, 1 gm yeast extract, 2 gm casein digest, 20 gm glucose solution (25% w/v), 20 ml maltose solution (25% w/v), 2 gm KH2PO4, 0.1 g asparagin, 0.1 gm MgSO4.7H2O, 1 gm FeSO4. 7H2O, 1 gm NaCl, 20 ml nystatin solution (0.25% w/v), 20 ml Penicillin G solution (0.30 w/v), Agar 20 gm).PHwas adjusted to 7.4 before autoclave. All solution were sterilized separately by 0.22µm Ø sterile membrane and added after autoclave. Streptomyce isolates wereidentified morphologically and by microscope. 2.2 Hydrocarbon degradation ability The ability of these local bacterial isolates to utilize crude oil and diesel was tested by cultivating each one on mineral salt medium composed per liter (1 gm NaCl, 1 gm KH2PO4, 1 gm Na2HPO4, 0.5 gm NH4NO3, 0.5 gm (NH4)2SO4, 0.2 gm MgSO4.7H2O) (Atlas, 2005). The only sole of carbon source was crude oil, and diesel 5 ml/L. The growth of bacterial isolates was observed after 3, 7, and 14 days of incubation at 30° C. 2.3 Assessment of Crude oil and diesel utilization Bacterial strains selected for hydrocarbon utilization were cultivated on modified YMD broth (Maniyar et al., 2011) composed per liter (10 gm yeast extract, 20 gmbacto peptone, 20 gm Malt extract, and 20 gm dextrose). 5 ml of crude oil and diesel was added to each flask separately, and was observed daily for consumption using the method developed by Hanson et al., 1993 (Utilization of different hydrocarbon components and surface tension). Two bacterial strains were selected which showed the highest ability to utilize crude oil and diesel for further test regarding others hydrocarbon compounds. These were toluene, sodium benzoate, naphthalene, hexadecane, tetrahydrofuran, cyclohexan, and anthracene. 2.4 Measurement of surface tension Samples of the culture media of each selected strain were centrifuged at 8000 g for 20 min. Surface tension (ST) of the supernatant fluid of the culture was measured by the Wilhelmy method with filter paper as a sensing element on a surface tensiometer model (KSV model 307 D Finland). Before the measurements calibration was done with clean water. The emulsifying activity of the culture supernatant was estimated by adding 0.5 ml of sample fluid and 0.5 ml of both crude oil and diesel to 4.0 ml of distilled water. The tube was vortexed for

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10 s, held stationary for 1 min, and then visually examined for turbidity of a stable emulsion. Emulsification activity was measured by vortexing equal volumes of the centrifuged culture with both test substances for 1 min and determining the percentage of volume occupied by the emulsion. The mixture was allowed to settle for 24 h and the height of the emulsion was measured (Tuleva et al., 2002). 2.5 CTAB method Blue agar plates containing cetyltrimethylammonium bromide (CTAB) (0.2 mg / ml Sigma Chemical Co) and methylene blue (5 mg / ml) were used to detect extracellular glycolipid production. Biosurfactantswere observed by the formation of dark blue halos around the colonies (Gunther et al., 2005). 2.6 Effect of different factors on hydrocarbon utilization Different factors were tested for optimization of hydrocarbon utilization. These were pH, NaCl concentration, cultivation temperature and agitation. At each factor, time of crude oil and diesel consumption was recorded (Satpute et al., 2008). 2.7 Extraction of hydrocarbon crude oil and diesel compound Media used in crude oil and diesel hydrolysis were extracted with equal volume of ethyl acetate to be subjected to further analysis. 2.8 Thin layer chromatography (TLC) A thin layer chromatography system for neutral lipids, polar lipids, carbohydrate, and amino acid was construed (Hanson et al., 1993) respectively. The TLC plates were spotted with biosurfactant extracts and developed with the following: solvent 1, petroleum ether - diethyl ether-acetic acid (80:20:1) for neutral lipids, solvent 2, chloroform methanol-water (65:25:4) for polar lipids, solvent 3, n-buthanol-acetic acid-water (4:1:1) for amino acids and solvent 4, ethyl acetate-acetic acid-methanol water (12:3:3:2) for carbohydrate compounds. After developing, the spots were visualized with standard reagents. The lipid components were detected as yellow spots after placing the plates in a closed jar saturated with iodine vapours. Using the ninhydrin solution followed by heating at 90° C for 5 minutes, generated a red or purple color when the compound had an amine function. Carbohydrate components were detected as red spots on the plates after spraying with an alpha- naphthol solution followed by concentrated sulphuric acid. Rf

values and photos were taken after process. TLC plates type silica G were processed in a closed TLC jar and visualized under U.V. transilluminator type Flowgen / U.K. 2.9 FTIR analysis For crude oil and diesel,thehydrolysatewas extracted from the supernatant fluid with equal volume of ethylacetate, dried with Na2SO4 and vaporated on a rotary evaporator. In order to avoid band saturation spectra were obtained with the ATR technique. The IR spectra were recorded on the Shimadzu Affinity- 1 FTIR spectrometer, in the 500 - 4000 cm-1 spectral region at a resolution 50 cm-1, using a 0.23 mm KBr liquid cell. 2.10 Gas chromatography (GC) Hydrolysateswere subjected for analysis. Biodegradation of hydrocarbons was quantified by GC type Shimadzu, Japanquantitative gas chromatographic analysis equipped with an SE-54 fused silica capillary column (25 m × 0.32 mm i.d.; 0.45 cm film thickness) and a flame ionization detector. Hydrogen (1 kg/cm) was used as the carrier gas. The temperature program consisted of an initial oven temperature of 50° C for 5 min increased at a rate of 10° C/min to 280° C for 10 min and then isothermal for 10 min. Injector and detector temperatures were maintained at 280° C. The splitting ratio was 1:60 (Domenico et al., 2004). 3. Results Bacterial strains identification Among 16 isolates of Streptomyces, two isolates were found to give quick and high consumption of crude oil and diesel in medium. These were designated as No. 7 and No. 14. Recommended biochemical tests were used to identify both of them (Williams et al., 1981). 3.1 Morphology Isolates No. 7 and No. 14 were examined for morphology. The shape of sporophores respectively spira, Retinaculiaperti, smooth and spiny spores, grey color colonies, creamy substrate mycelia, with red diffusible pigments, No. 14 can produce melanin, and can grow in range from 28° C - 45° C. 3.2 Biochemical characteristics The ability to utilize different types of sugars was tested for both isolates. They were found to utilize glucose, arabinose, xylose, sucrose, and mannose, while isolate No. 14 was able to utilize, in addition to the mentioned sugars,

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rhamnose, and inositol. Both isolates gave positive catalase reaction, urea utilization and were able to grow on Czapeck’s medium and No. 14 can produce organic acids. Blood hemolysis is an important criterion to be tested. Isolate No. 14 was found to show complete hemolysis of blood β while No. 7 was of γ type, which is partial hemolysis. Carrillo et al. (1996) and Youssef et al. (2004) found an association between hemolytic activity and surfactant production, and they recommended the use of blood agar lysis as a primary method to screen for biosurfactant activity. 3.3 Colony morphology Colony morphology was determined for both isolates and listed in table (1). Table 1: Colony morphology of isolates No. 7 and No. 14 Both isolates were grown on YMD for 14 days at 37° C

Streptomyces isolate

Description

14 Colonies are small in diameter (3 – 5) mm, discrete and butyrous. Initially relatively smooth surfaced; lately grow to velvety aerial mycelia. Color of aerial mycelia is white on most media.

7 Colonies are medium in diameter (5-7) mm, discrete and butyrous. Initially relatively smooth surfaced; lately grow to velvety aerial mycelia. Color of aerial mycelia is white on most media.

3.4 Crude oil, diesel and other hydrocarbon consumption Preliminary detection of complex hydrocarbon utilization was performed on mineral salt medium supplemented with crude oil and diesel as the sole of carbon. Two isolates grew during three days of cultivation and differentiate in to distinct colonies. These were selected as for complex hydrocarbon utilization. The YMD helped these isolates to grow heavily, andconsumption of complex hydrocarbons was observed after 5 days of incubation. The indicator for this was the disappearance of crude oil and diesel from the medium and measurement of surface tension. CTAB plates showed an obvious activity to consume different types of hydrocarbons by forming clear zone around plugs transferred from well grown colony plates. 3.5 Consumption of hydrocarbons Different types of hydrocarbons other than crude oil and diesel were used in this study.

Bacterial isolates were grown on broth medium in which these compounds were added. Different consumption patterns and surface tension was measured during this experiment by comparing data obtained after 5 days of incubation listed in table (2). Table 2: Consumption of different hydrocarbon sources; regard to the surface tension measurement. The surface tension of the control was 70

S. No Hydrocarbon compound

Bacterial strain

ST

1 Toluene 14 Non 7 48.84

2 Sodium benzoate 14 49.88 7 58.43

3 Naphthalene 14 Non 7 57.00

4 Hexadecane 14 79.30 7 31.48

5 THF 14 Non 7 43.03

6 Cyclohexane 14 Non 7 43.8

7 Anthracine 14 Non 7 51.18

ST = surface tension Non= no consumption of the hydrocarbon in medium 3.6 Effect of different factors on bacterial consumption of complex hydrocarbons Different factors suggested by Kokare et al., 2007 were tested to influence hydrocarbon utilization. These were pH, salt concentration of NaCl, temperature, and agitation. Figures (1), (2), (3), and (4) represent results obtained from this test. Figure 1: Effect of pH on hydrocarbon consumption by isolate No. 7 and No. 14. Legend on the right gives gradient of pH used beginning with 5 and ends with 9

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Figure 2: Effect of salt concentration on hydrocarbon consumption by isolate No. 7 and No. 14. Legend on the right gives concentrations of salts used beginning with 1% and ends with 5%

Figure 3: Effect of temperature on hydrocarbon consumption by isolate No. 7 and No. 14. Legend on the right gives temperature range used beginning with 15° C and ends with 45° C

Figure 4: Effect of agitation speed on hydrocarbon consumption by isolate No. 7 and No. 14. Legend on the right gives agitation speed used beginning with 50 rpm and ends with 200 rpm

3.7 Thin layer Chromatography Hydrolysates from crude oil and diesel were extracted with ethyl ether and subject to TLC to analyze the types of component might be produced by such process. Lysates were found to be polar and non-polar lipids, proteins, and carbohydrates. These were found after 5 days of cultivation in YMD medium. The following figures show TLC of hydrolysates. Figure 5: Polar lipids chromatography for hydrolysates produce in culture media by the two Streptomyces isolates 7 and 14. The first two spots are hydrolysates of isolate No. 7, while the others are from isolate No. 14

Figure 6: Non-Polar lipids chromatography for hydrolysates produce in culture media by the two Streptomyces isolates 7 and 14. The first two spots are hydrolysates of isolate No. 7, while the others are from isolate No. 14

Figure 7: Carbohydrate chromatography for hydrolysates produce in culture media by the two Streptomyces isolates 7 and 14. The first two spots are hydrolysates of isolate No. 7, while the others are from isolate No. 14

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3.8 Gas Chromatography Gas chromatography was done to identify the ability to these local isolate to degrade diesel and crude oil. Charts obtained showed a significant change upon both compound

structure. Most of long and complex carbon chains were broken into simpler type, and disappearance of some compounds was observed during the process. As shown in figures below.

Figure 8: Gas chromatography analysis of hydrolysate produce by isolate No. 7 grown on crude oil

Figure 9: Gas chromatography analysis of hydrolysate produce by isolate No. 7 grown on diesel oil

Figure 10: Gas chromatography analysis of hydrolysate produce by isolate No. 14 grown on crude oil

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Figure 11: Gas chromatography analysis of hydrolysate produce by isolate No. 14 grown on diesel oil

3.9 FTIR analysis The range of FTIR spectrum from 500 – 7000 nm was used to determine the rate of change upon both diesel and crude oil. The charts obtained showed an obvious change on specific regions referring to complex chains

and aromatic rings in both compounds. Widening areas were observed at these regions indicating the increase of these compounds concentrations after bacterial hydrolysis for crude and diesel oil. These are listed in figures below.

Figure 12: FTIR analysis of hydrolysate produce by isolate No. 7 grown on crude oil

Figure 13: FTIR analysis of hydrolysate produce by isolate No. 7 grown on diesel oil

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Figure 14: FTIR analysis of hydrolysate produce by isolate No. 14 grown on crude oil

4. Discussion Streptomyces bacterium is considered one of the most important organisms in industry, and ecosystem. All of these bacteria are filamentous type rich with enzymes, and can perform many functions in nature. The interference of Streptomyces in hydrocarbon utilization was reported in few literatures (Javed et al., 2011) whereas most literatures found that Pseudomonas is widely abundant in such contaminated areas. However, the importance of streptomycetes in bio - remediation comes from that it is low requirements of nutrition, non - pathogenicity, and the ability to released organic compounds into the medium, which provided the culture broth with emulsifying activities and foaming properties. Surface tension as indicator for complex hydrocarbon hydrolysis gave a reasonable results to identify which isolate is capable of such function, even they were grown on minimal salt medium and using diesel oil and crude oil as a source of carbon. Physical and chemical factors had an obvious effect on bacterial ability to degrade complex hydrocarbons. Our finding was that at neutral pH, temperature range 37 - 45° C, the bacteria was able to perform their highest ability of hydrocarbon degradation, whereas salt did not affect these criteria. Agitation rate increased the ability of the bacteria to assimilate complex hydrocarbon. The optimum agitation rate was 150 rpm gave the bacteria the ability to attach to hydrocarbon molecules which was hydrolyzed with good supplementation of oxygen. An important point was referred to by some authors is that hydrocarbon utilizing bacteria show complete blood hemolysis (Javed et al., 2011), where as our two bacteria did not show this criteria completely since only one of them did that which shows better

performance on crude oil utilization, while the other one can partially utilize blood. The increase in finger print areas shown in FTIR spectrum resulted from the increase of these compound concentrations as a result of degradation of long chains of carbon. This was confirmed with gas chromatography regarding hydrolysates extracted from the medium. According to Rehm and Reiff (1981) and Finnerty (1992), a well-known property of Gram-positive bacteria, is the capability of degrading alkanes, which is often accompanied, by the ability to produce biosurfactants. The possession of cell-surface hydrophobicity and biosurfactant production by microorganisms creates chemical and physical compatibility between the organisms and the hydrophobic substrates, thus resulting in enhanced interaction between them (Stelmack et al., 1999). Many bacteria are known to degrade liquid hydrocarbons only after adherence to the substrate (Ganesh and Lin 2009). However, results obtained from this research are promising in future project to use this bacteria as a biological agent to remediate soils contaminated with oil spills, and diesel oil. Limitations Some limitations faced this research especially those that requires security clearance to collect data and samples from refineries and oil extraction well. Moreover, some sites that may be useful for sampling in this research were hard to get to. These areas are near oil pipes in the desert, or near military operation areas or camps.

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Recommendations We recommend the use of non – pathogenic, soil bacteria especially genera with high enzymatic activity as an agent for bioremediation and soil detoxification. Conclusion Our main conclusion is that even with partial ability for blood hemolysis Streptomyces bacteria can be able to degrade complex hydrocarbons and make it a perfect candidate in bioremediation. Author’s Contribution and Competing Interests Biotechnologist with major of molecular biology, microbial technology, and fermentation technology. Head and team leader of three major project from Ministry of Higher Education and Scientific Research. References Battershill C., Jaspars M., Long P., 2005. Marine Biodiscovery: New Drugs from the Ocean Depths. Biologist, 52: 107-114. Tulevaa B.K., Ivanov G.R., Christova N.E., 2002. Biosurfactant Production by a New Pseudomonas putida Strain. Z. Naturforsch. 57c, 356-360 Burlage R.S., Hooper S.W., Sayler G.S., 1989. The TOL (pWWO) catabolic plasmid. Applied and Environmental Microbiology 551323-1328 Caprino, L., Togna G.J., 1998. Potential health effects of gasoline and its constituents. Environmental Health Perspectives. V.106, p. 115-125. Carrillo P.G., Mardaraz C., Pitta-Alvarez S.J., Giulietti A.M., 1996. Isolation and selection of biosurfactant-producing bacteria. World Journal of Microbiology and Biotechnology 12, 82-84 Dagley S., 1986. Biochemistry of aromatic hydrocarbon degradation in Pseudomonads. In: Sokatch JR (ed) The bacteria, Vol 10. The biology of Pseudomonas. Academic Press, London, p 527-556 Facundo J., Marquez-Rocha, Vanessa Hernandez-Rodreguez M.A., 2001. Teresa Lamela. Biodegradation of Diesel Oil in Soil by A Microbial Consortium. Water, Air, and Soil Pollution 128: 313–320, Finnerty W.R., 1992. The biology and genetics of the genus Rhodococcus. Annual. Review in Microbiology. 46, 193-218.

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