Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414

ISSN: 2319-7706 Volume 3 Number 4 (2014) pp. 401-414 http://www.ijcmas.com

Original Research Article Assessment Mixed Culture of Actinomyces and Sacchromyces for biodegradation of Complex Mineral Oil hydrocarbon

Ahmad F. Shahaby1,2*

1College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Saudi Arabia, 2Cairo University, College of Agriculture, Department of Microbiology, Cairo, Egypt *Corresponding author

A B S T R A C T

The biodegradation of mineral oil hydrocarbon by mixed culture of hydrocarbon- degrading organisms was investigated. The mixture or consortium of , denoted as AS2 consisted of 2 microorganisms. Microorganisms were Actinomyces octodloyts, Saccharomyces cerevieace. Microorganisms used in this study were K e y w o r d s from the Biotechnology and Genetic Engineering Research Unit (BGERU) Microbial Bank collection strains, at Taif University, KSA and were isolated from Bioremediation hydrocarbon-contaminated samples by enrichment technique on hydrocarbons control; as the sole carbon and energy source. The strains of the mixture were identified as Mixed Actinomyces octodloyts, and Saccharomyces cerevieace, by means of 16S-rRNA cultures; genetic method. The strains were selected based on the criteria that they were able Biosurfactant; to display good growth under complex mineral oil. Each of these strains 16S-rRNA demonstrated a strong ability to grow as a single strain on a hydrocarbon as sole gene. carbon source. Their ability to degrade complex mineral oil hydrocarbon were monitored by gas chromatography (GC) for 6 days. The biodegradation percentage of mineral oil at 3% ml/L in liquid medium was 41.8% after 4 days of growth only. Potential biosurfactant production tested using the two methods named modified drop collapse (MDC) and blue agar plate (BAP) showed that the species are biosurfactant producers. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

Introduction

Hydrocarbon degradation in soil by environment. Bioremediation is a modern microorganisms has undergone renewed method in which the natural ability of emphasis because of the increased microorganisms is employed for the incidence of petroleum-based pollution. reduction of the concentration and/or Knowledge about hydrocarbon toxicity of various chemical substances, degradation is needed to determine how such as petroleum derivatives, aliphatic microorganisms might be utilized in the and aromatic hydrocarbons, industrial removal of the pollutants from the s olvents, pesticides and metals. The class

401 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414 of petroleum products known as mineral members of 6 genera to be able to oils can be generally understood to include effectively degrading crude oil. a variety of products which go by different names such as white oils, lubricating oils, The vast range of substrates and light fuel oils, residual fuel oils, as well as metabolites present in hydrocarbon transformer and cable oils (Gary and impacted surely provides an Handwerk, 2001). Mineral oils refer to all environment for the development of a oils which are made from dewaxed quite complex microbial community paraffin-based crude oils which are (Butier and Mason, 1997). Microbial blended with additives to particular populations that consist of strains that properties for specific uses (Aluyor and belong to various genera have been Ori-jesu 2009). Mineral oils are composed detected in petroleum-contaminated soil or of straight and branched chain paraffinic, water (Sorkhoh et al., 1995, Chikere et naphthenic, and aromatic hydrocarbons al.,2009). This strongly suggests that each with 15 or more carbons in a complex strain or genera have their roles in the mixture (Aluyor and Ori-jesu 2009). hydrocarbon transformation processes. More recently, microbial degradation was Biological treatment most commonly found to be an available alternative involves the breakdown of contamination method over the conventional methods. into nontoxic forms using microbiological Microbial treatment can control processes (Lee et al., 1998). Therefore, contamination of soils or water with crude bioremediation may be defined as the use oil, used or fresh petroleum products by of living organisms to remove reducing the length of the paraffin and oil environmental pollutants from soil, water molecules and by producing by-products and gases (Collin, 2001). The advantages that act as surfactants and paraffin and oil of employing mixed cultures as opposed to solvents. However, information on pure cultures in bioremediation have been numbers and local species of demonstrated. It could be attributed to the microorganisms as well as their efficiency effects of synergistic effects among in degradation of complex mineral oil in members of the consortium. Moreover, Saudi Arabia is scarce. However, the some substances can be decomposed only biodegradation of these compounds using by cometabolism. The mechanisms in mixed cultures or microbial consortia which petroleum degraders benefit from isolated from contaminated sites with synergistic relationships may be complex. petroleum products has not been evaluated It is possible that one species removes the and these microorganisms are adapted to toxic metabolites of the species preceding grow and thrive under environments with it. It is also possible that the second high concentrations of complex mineral species are able to degrade compounds oils, and the effects of environmental that the first are able to only partially factors on the microbial growth are not degrade it (Alexander, 1999). Further known yet. research should be directed towards understanding the roles of individual High molecular weight hydrocarbons are members in influencing the effectiveness highly difficult to degrade. High molecular of a microbial consortium or mixer. weight biosurfactants are highly efficient Rambeloarisoa et. al. (1984) demonstrated emulsifiers that work at low a consortium of 8 strains made up of concentrations and exhibit considerable

402 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414 substrate specificity, they are produced by recently recorded that suitable sequence a large number of bacteria and they are differences in the 16S- rRNA gene could composed of polysaccharides, proteins, be used for bacterial identification (Sacchi lipopolysaccharides, lipoproteins etc. et al. 2002) and for subtyping and (Banat et al., 2000, Zhang et al., 2012). In identifying hyper virulent bacterial clones general microorganisms produce (Nilsson et al. 2003). biosurfactants to increase their interfacial area for contact to give improved uptake This work represents a continuation of our of hydrophobic substrates. However, it has research in the area of hydrocarbon been observed that the exopolymers biodegradation technology. The present synthesized by these strains in media with study aims to characterize isolates using glucose as carbon and energy source, had 16S-rRNA gene technique, to produce a remarkable capacity of emulsifying biosurfactant and to degrade complex hydrocarbon compounds (Martinez-Checa mineral oil as sole carbon substrate source et al. 2002, Zhang et al., 2012). Microbial in pure culture or mixed culture treatment can control hydrocarbons efficiently. pollution by reducing the length of the hydrocarbon molecules and by producing by-products that act as biosurfactants and Materials and Methods solvents (Banat, 1995 and Wolicka et al., 2009). Microbial strains

The phylogenetic diversity of microbial Microorganisms were Actinomyces communities can be tested by molecular octodloyts AF104, Saccharomyces methods, like fingerprinting or cloning and cerevieace AF203. Microorganisms used sequencing of PCR-amplified rRNA in this study were from the Biotechnology genes. These techniques have been and Genetic Engineering Research Unit employed to isolates and consortia (BGERU) Microbial Bank collection of enriched from natural environments. The strains, at Taif University, KSA. Strains 16S-rRNAmethod was also used to were local isolated strains from mechanic monitor the microbial diversity in workshops and gas stations contaminated environments and to follow transitions in soils by enrichment culture technique in community structure upon seasonal our previous work in our laboratory. The changes or along spatial gradients in two isolates showed good growth on salinity, temperature, and availability of Bushnell- Haas enrichment mineral substrates and minerals (Kleinsteuber et. medium(BHM) amended with al. 2006). The use of 16S- rRNA gene hydrocarbon and were selected based on sequencing to examine genetic relatedness the growth and degradation ability. of prokaryotic species is well established and has led to increased availability of Growth potential of hydrocarbon- 16S- rRNA databases. The convergence of utilizing microorganisms these technical and computational advances has also enhanced the Inocula were routinely grown in Luria application of 16S- rRNA gene sequence Bertani (LB) broth medium (g L 1): analysis to bacterial identification peptone, 10.0; yeast extract, 5.0; NaCl, 5.0 (Rantakokko-Jalava et al. 2000). It was (Miller, 2007). Media were autoclaved at

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120 °C for 20 min. Cultures were grown Biodegradation of hydrocarbons overnight first on LB medium without hydrocarbon addition. Then, grown on Strains were incubated overnight in 50 ml Bushnell- Haas enrichment mineral LB broth medium in triplicate, pH 7.5 at medium (BHM) containing (g/l): 30oC (shaken culture: 150rpm). Cells were MgSO4.7H2O, 0.2; K2HPO4, 1.0; centrifuged and washed twice with the KH2PO4, 1.0; FeCl3, 0.05; NH4NO3, 1.0; liquid inorganic salts BHM. The pellet was CaCl2, 0.02; pH to 7.2 and sterilized at suspended in 5ml BHM and inoculated 121oC for 15 min. Bacteria were grown in into 250 ml of BHM in 3 flasks 250 ml Erlenmeyer flasks for one week in supplemented with 3 concentrations of a rotary shaker. Flasks were amended with mineral oil: 1, 3 and 5%(v/v) as sole mineral oil 1, 3, and 5 % (v/v) for each carbon and energy sources. organism. The pH of media was adjusted to 7. One ml was taken to measure Biodegradation Efficiency turbidity at 595 nm with spectrophotometer. Growth on mineral oil Samples taken from the flasks were mixed was monitored by measuring the optical with equal volumes of hexane and shaken density (O.D.) at 595 nm in 2 ml cuvettes to extract mineral oil. Residual mineral oil using a spectrophotometer (Biophotometer was monitored using the method used by plus, Eppendorf). Martinez-Checa et. al. (2002).

The net dry weight for the biomass was Biosurfactant production screening determined simultaneously. A 1 mL of using the modified drop collapse culture was centrifuged at 1500 rpm for 10 method (MDC) min, washed twice with distilled water, poured into a pre-weighed container, dried To prepare the assay, three plates were overnight at 90 °C to constant weight and rinsed successively with hot water, 75% cooled for reweighing. Mineral oil adapted ethanol, distilled water and dried with air. cells were harvested and washed twice After preparation, plates were equilibrated with BHM and the pellet suspended in and coated with a thin layer of crude oil. 0.1M phosphate buffer at pH 7.0. Cells The preparation was left for 24 h to ensure were harvested by centrifugation for 5 min a uniform oil coating. Bacterial or yeast at 3,000 x g at room temperature. The suspensions of all isolated strains were growth rates of cultures in exponential prepared and OD (595 nm) was adjusted to phase were determined from linear 0.8 for each strain. A volume of 0.5 l of regressions of log10 absorbency vs. time, each organism suspension was transferred calculating a least squares fit of data from on the thin oil layer.The shape of the drop the exponential growth phase, and was inspected after 1 min; if the drop determining the slope of this line. The remained beaded, the result was scored as instantaneous growth rate ( ) was negative (-). If the drop collapsed, the determined from the slope of this line x result was scored as positive (+) (Bodour ln10; had the dimensions h-1 (Koch, and Miller-Maier, 1998). 1984).

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Biosurfactant production screening of each primer and 12.5 l of 2xSuperHot using the blue agar plate method (BAP) PCR Master Mix (Bioron, Ludwigshafen, Germany) mixed with 50 to 100 ng of This method shows an ionic biosurfactant DNA template. Sterile d. H2O was added producing strains by color reaction. to a final volume of 25 l. Thermal cycler Mineral salts agar medium (MSA) (Uno II, Biometra, Germany) with the (Siegmund and Wagner, 1991) following thermal profile: 94 °C for 4 supplemented with carbon sources min., 94 °C for 1 min., 55 °C for 1 min., (glycerol), 2%, cetyltrimethylammonium 72 °C for 1.5 min, the number of cycles bromide (CTAB) 0.5 mg/ml), methylene was 35 cycle and the post PCR reaction blue (0.2 mg/ml) were prepared. Three time was 72°C for 5 min. Plates was streaked with organism of interest and incubated at 30°C for 24 h. A Analysis of the PCR products dark blue halo around the culture was considered as positive for biosurfactant The PCR reaction products were production. Sodium dodecyl sulfate electrophoresed with 100 bp ladder marker (SDS), 1 mg/l and sterile distilled water (Fermentas, Germany) on 10 x 14 cm were used respectively as positive and 1.5%- agarose gel (Bioshop, Canada) for negative controls. 30 min using Tris-borate- EDTA Buffer. The gels were stained with 0.5ug/ml of DNA extraction, PCR, and sequence ethidium bromide, visualized under the analysis UV light (Watanabe et al., 2001) and documented using a GeneSnap 4.00- Gene DNA Extraction Genius Bio Imaging System (Syngene, Frederick, Maryland, USA). The genomic DNA of Actinomyces sp. sample was extracted using a bacteria Sequencing of 16S-rRNA gene DNA Preparation Kit and Yeast DNA Preparation kit (Jena Bioscience, Jena, The PCR-products of each organism was Germany) according to the manufacturer s purified from excess primers and instructions (www.jenabioscience.com). nucleotides by the use of AxyPrep PCR Clean-up kit (AXYGEN Biosciences, PCR amplification of 16S-rRNA gene Union City, California, USA) and directly sequenced using the same primers as Primer sequences used to amplify the 16S- described for the amplification process. rRNA gene fragment were: primers The microorganisms DNA sequences were forward fD1 (5'- determined with the chain-termination CCGAATTCGTCGACAACAGAGTTTG method on an ABI 3730 DNA sequencer ATCCTGG CTCAG-3') and reverse rD1 by a commercial service (Seoul, Korea). (5'-CCCGGGATCCAAGCTGGAGGT G Sequences were aligned in the GenBank ATCCAG CC-3') for Actinomycetes and database using the BLASTN program at primers P1 (ATCAATAAGCG the National Center for Biotechnology GAGGAAAAG and P2 Information (NCBI), and percent CTCTGGCTTCACCCTATTC for yeast homology scores were obtained to identify as described by Ren et al. (2007). The microorganisms. PCR reaction mixture contained 10 Pmol

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Statistical analysis Psedomonas aeruginosa, subtilis and Halomonaseurihalina (moderately Statistical analysis was performed using halophilic bacterium) species were the SPSS 10.0 software. Data underwent a effective bacteria in the biodegradation of one-way ANOVA test, and means were heavy hydrocarbons (mineral oil) and n- compared using Duncan s multiple range tetradecane (Martinez-Checa et. al., 2002, tests at 5% significance level. Sadeghazad and Ghaemi, 2003, Shahaby and El-Tarras, 2011, and El- Tarras et al., Results and Discussion 2012, Shahaby et al., 2013). of alkane degrading microorganisms from oil Identification of microbial candidates contaminated soil has been reported by several researchers. Nazina et al (2005) Mixtures of organisms were obtained have obtained hydrocarbon oxidizing during enrichment using 0.1% yeast Geobacilli strains from formation waters extract in BHM. Screening on an agar of oil fields. plate containing mineral oil resulted in isolation of several candidates from the Nucleotide sequence accession numbers contaminated sites. Two isolates of Actinomycessp. and three isolates of The partial 16S-rRNA gene sequences that Saccharomyces sp. were identified and were determined have been deposited in characterized. Strains AF104 and AF203 the GenBank, EMBL, and DDBJ were local isolates isolated by enrichment nucleotide sequence databases under technique and deposited in our microbial accession no. NJ700209 for bank at Taif University during our Saccharomyces cerevieceae AF203 and previous work (Shahaby and El-Tarras, NJ700210 for Actinomyces 2011) in our laboratory. The isolates were odontolyticus AF104. identified on the basis of their cultural and biochemical characteristics according to Growth rates and biomass of the Bergey's Manual of Determinative microbial candidate's mixture Bacteriology (9th edition) (Holt et al., 1994) and Apikit profiles (2009). Optical density and biomass were Phenotypic examination of the recovered determined simultaneously. The linear microorganisms revealed that they belong relation between OD595 and dry mass was to the genera of Actinomyces, and obtained during growth on 1% of mineral Saccharomyces. Two isolates AF104 and oil. The specific growth rate and net dry AF203 showed good growthon BHM weight of the two isolates were determined amended with complex mineral oil and and illustrated in Table 1. These figures were selected based on the growth and indicate the effluence of the specific degradation ability. Strains AF104 and growth rate and biomass precipitation on a o AF203 showed optimal growth at 30 C. period of bacterial cultivation in BHM The results of 16S-rDNA sequence containing 1 % (v/v) hydrocarbon. Little alignment analysis revealed that 16S- adaptation occurred at 5% mineral oil, rDNA sequence of strain AF104 and indicating that the highest hydrocarbon AF204 were more than 98% identical to exceeded the strains capability to that of Actinomyces octodloyts, and adapt.Table (1) summarize the results of Saccharomyces cerevieace, respectively. growth rates, and biomass content during

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Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414 growth at 1% (v/v) concentration of mineral oil as sole carbon source however, complex mineral oil for a week under the efficiency of utilization varied among laboratory conditions. The specific strains, with AF203 being the best (40.2% growth rates of the two isolates on mineral removal within 5 days) at 5% hydrocarbon oil showed that strain AF203 was faster concentration.AF104 was able to degrade than strain AF104in growth in mineral mineral oil by more than 38.3%, whereas salts medium BHM containing 1 % (v/v) the mixture of AF104 and AF203 named hydrocarbon. Most growth occurred in the (AS2) could degrade about 41.8% after 4 first2 days for the two strains resulting in days only at 3% concentration of good biomass production (Table 1). hydrocarbon. Two reference strains Maximum specific growth rates ( max) for Actinomyces, Saccharomyces used as AF104 and AF203 strains were occurred negative control could only degrade after two and three days of growth being approximately 9 and 7% of mineral oil, 0.073 h 1 and 0.064 h 1, respectively. respectively under the same conditions Strains were also grown on 3% and 5% (data not shown). The results on the hydrocarbon (data not shown). However, assessment for degradation capacity were growth on 1% hydrocarbon was better similar to those of growth (Fig. 1). In than 3 and 5 % (v/v) concentrations. mineral oil 5% -supplemented BHM, a Moreover, isolate AF203 produced more small degree of degradation was observed biomass from hydrocarbon being 3.85 g only in cases of using consortium of cells l-1mineral oil hydrocarbon after six AF104 and AF203 strains (AS2) being days of growth. No change was observed 32.3 % after 2 days only. The consortium in pH during the first seven days of degrades 39.4% of hydrocarbon after 2 incubation for strains. days only at concentration 3%. Mineral oil was utilized to the extent of 38.3% for The reduction in heavy hydrocarbon AF104 after 6 days at 5% concentration, fractions by biodegradation of paraffinic 40.2% after 5 days at 5% concentration hydrocarbons using Pseudomonas and and 39.4% for consortium of both AF104 Actinomyces species was noticed (Etoumi, and AF203 (AS2) after 2 days only of 2007). It was mentioned that the lower the growth. Strain AF203 was more efficient concentration of hydrocarbons the higher than strain AF104 being 40.2% and 38.3% was the utilization. Similar growth rates at 5% concentration, respectively. One and biomass on hydrocarbon were might thus expect that incubation at 3% or obtained (Etoumi, 2007, Shahaby and El- 5% hydrocarbon beyond6 days would Tarras, 2011). have resulted in even more mineral oil degradation than the 41.8% observed. Degradation capacity of hydrocarbon Interestingly, CFU and total cell counts at by isolates 3% and 5% concentration were even higher than those in the maximally active The hydrocarbon degradation capacity of incubation at 1% hydrocarbon selected isolates in BHM supplemented concentration (data not shown). This with complex mineral oil are illustrated in indicates reduced hydrocarbon degradation Fig. 1-3. No evaporation of hydrocarbon per cell at the higher mineral oil from the screw-capped flasks was concentration, which goes along with observed throughout the experiments. All lower protein content per cell. The isolates showed the ability to utilize maximally active incubation at 1%

407 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414 hydrocarbon preserved the highest Saadoun (1997) determined ability of metabolic versatility. Given that the two Rhodococcus erythropolis to degrade strains were capable of strong growth in mineral oil and other hydrocarbons by both solid and liquid media at 30°C and using a Warburg constant volume 3% or 5% of hydrocarbon as sole carbon respirometer. Results of oxygen uptake source, we tested the performance of each indicated that hexane and tetrade cane of the two strains separately via rotary were more degradable than mineral oil and shaker under these conditions Table (2) decane. R. erythropolis exhibited highest and Fig. 1-3.However, these percentages QO2 values (2.9 and 2.8) when exposed to also demonstrated a moderate level of tetradecane and hexane, respectively. mineral oil biodegradability by each of Mineral oil and decane were degraded these two strains performed separately at more slowly with QO2 values 0.87 and 5% concentration. 0.94, respectively. Alvarez et al. (2011) evaluated the effectiveness of monitored Zhao et al. (2011) selected consortium by natural attenuation, bioenrichment, and positive end dilution method. The bioaugmentation using a consortium of consortium consisted of Rhizobiales sp., three actinomycetes strains in remediating Pseudomonas sp., Brucella sp., Bacillus two distinct typical Brazilian soils that sp., Rhodococcus sp., Microbacterium sp. were contaminated with crude oil, with or and Roseomonas sp. and removed nearly without the addition of NaCl. Microcosms 52.1% of crude oil at initial concentration were used to simulate bioremediation of 10,000 mg l 1 at 30 °C within 7 days. treatments over a 120-day period. During The strains Pseudomonas sp., Brucella sp. this period, they monitored total petroleum and Rhodococcus sp. likely played a key hydrocarbons (TPHs) and n-alkanes role in the crude oil degradation(Abdel- degradation and changes in bacterial Megeed et al., 2012 ).The mixed communities. Over time, they found the populations of Pseudomonas, degradation rate of n-alkanes was higher Rhodococcusand Bacillus are capable of than TPH in both soils, independent of the degrading mineral oil of up to 120 ppm. treatment used. Suggesting that the total However, biodegradation ofmineral oil bacterial community in the soils was was fast by the mixed culture comparing mainly affected by the experimental period to the biodegradtion of each strain of time, while the type of bioremediation separately. treatment used was the main factor influencing the actinomycetes populations Lazar et al. (1999), Pokethitiyook et al. in both soils.Growth on mineral oil before (2002), and Sadeghazad and Ghaemi exposure to specific hydrocarbon (2003) reported that, Pseudomonas and compounds was intended as a pre- Bacilli species were the most effective enrichment step, similar to the initial microorganisms in the biodegradation of enrichment of polychlorinated biphenyl heavy hydrocarbons. Lazar et al. (1999) (PCB) degraders on biphenyl (Bedard et also, stated that microorganisms involved al.,1986,1987) or methylcyclohexane for with the microbial treatment of crude oil, bacteria able to grow on a wide range of are generally live, naturally occurring, and alicyclic compounds (Trudgill, 1984).The are mainly facultative anaerobic, reduction in heavy hydrocarbon fractions pathogenic, contain no sulphate-reducing by biodegradation of paraffinic bacteria or slime-forming bacteria and are hydrocarbons using Pseudomonas and environmentally safe. On the other hand, 408

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Table.1 Growth rates, biomass yield and biosurfactant production (using MDC and BAP techniques) of two strains AF104and AF203grown in Bushnel-Hass medium amended with 1% of mineral oil hydrocarbon.

Strains Biomass yield Growth rate Biosurfactant (g cells /Lmineral oil) (µ) (h-1) production Actinomyces(AF104) 3.58 0.071 Sacchromyces(AF203) 3.85 0.062

Table.2 Biodegradation performance of complex mineral oil hydrocarbon by aconsortium of Actinomyces sp.AF104 and Saccharomycessp.AF203 (AS2)

Culture Name Hydrocarbon Incubation time Biodegradation % concentration% (days) Sacharomycessp. 3 6 39.5 Actinomycetssp. 5 6 38.3 Mixture of Actinomycessp. and 3 4 41.8 Saccharomyces sp. (AS2)

Fig.1 Performance of ActinomycesAF104 in various concentration of hydrocarbon and incubation time at 30 oC. M. O., mineral oil content.

M.O 1 %

M.O 3 %

M.O 5 %

Fig.2 Performance of Saccharomyces AF203 in various concentration of hydrocarbon and incubation time at 30 oC. M. O., mineral oil content.

M.O 1 % M.O 3 %

M.O 5 %

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Fig.3 Performance of ActinomycesAF104 and Saccharomyces AF203 consortium (AS2) under various concentration of hydrocarbon and incubation time at 30oC. M. O., mineral oil content

M.O 1 %

M.O 3 %

M.O 5 %

Actinomyces species was noticed (Etoumi, consortium of 8 strains made up of 2007). It was mentioned that the lower the members of 6 genera to be able to concentration of hydrocarbons the higher effectively degrading crude oil was the utilization (El_Tarras et al., 2012, (Rambeloarisoa et al. 1984). Interestingly, Shahaby et. al., 2013). Biodegradation only 5 of these strains were able to grow in would be required if the contaminated site pure cultures using a variety of lacked proper microorganisms (Kauppi et hydrocarbons. However, when the other 3 al., 2011). Therefore, isolation of pure strains were removed from the consortium, microbial strains or enrichment of the effectiveness of the mixed culture was microbial consortium from contaminated remarkably reduced. These further support sites for biodegradation of hydrocarbons is the theory that each member in a microbial considered as an approach to provide community has a significant role and may inocula for bioaugmentation (Liu et al., need to depend on the presence of other 2011 and Kauppi et al., 2011). Due to species or strains to be able to survive microbial complexity and diversity, a microbial consortium could work better Production of biosurfactant and more stable than pure culture for the bioremediation of crude oil-contaminated Biosurfactant are produced by many soil. Mainly because that crude oil is a bacterial strains that can degrade or complex mixture consisting of aliphatics, transform the components of petroleum aromatics, resins and asphaltenes (van products. They are non-toxic, non- Hamme et al., 2003 and Malina and hazardous, biodegradable and Zawierucha, 2007). Although, a crude oil- environmentally friendly compounds degrading consortium could be made up (Banat et al., 2000).Using the two by combining a number of individual qualitative methods (MDC and BAP), microbial strains, the bioremediation results demonstrated that Actinomyces sp. performance of the combined bacteria AF104 and Saccharomyces sp.AF203 usually is not satisfactory (Komukai- strains used in this study were Nakamura et al., 1996 and Ko and biosurfactant producers (Table 1). Lebeault, 1999). Demonstrated a

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This can explain the mineral oil the controls. The amount of mineral oil degradation performance obtained with removed vs. mineral oil content suggested these two strains. Knowing that probable first order reaction kinetics for biodegradation depends strongly on the mineral oil removal in vitro and higher hydrocarbon emulsion, the use of rate was achieved with bioremediation.In biosurfactant producer strains like general, microorganisms produce Actinomyces, and Saccharomyces species biosurfactants to increase their interfacial in bioremediation technology seems to area for contact to give improved uptake offer more potential than chemical of hydrophobic substrates. Both surfactant, due to their structural diversity, microorganisms of consortium AS2 biodegradability and biocompatibility produced biosurfactants and this improved relative to synthetic surfactant (Abalos et breakdown of complex mineral oil al., 2004, Amaral et al., 2010a, 2010b, hydrocarbon. The highest rate of Konishi, 2010, Shubhrasekhar et al., hydrocarbon degradation occurred when 2013). Some yeasts are preferred to member of consortium are biosurfecant bacteria as sources for biosurfactants, producers. These two strains were able to mainly due to their GRAS status for produce biosurfactants. Therefore, the environmental and health safety reasons main phase of mineral oil removal by the (Campos-Takaki et al., 2010).These results two strains was achieved in the first four mean that the bacterial consortium days of treatment. These percentages also obtained is capable of using the complex demonstrated a moderate level of mineral mineral oil as a carbon and energy source oil biodegradability by each of these two under these conditions. strains performed separately. However, the effect of different environmental factors In conclusion complex mineral oil onthe growth of the stabilized microbial hydrocarbons are highly difficult to consortium might affect the performance degrade. A functional bacterial consortium of the consortium.The results revealed the consisting of Actinomyces and possibility touse these microbes for the Saccharomyces was selected based on the reduction of complex mineral oil in growth and degradation ability. Strains ecosystems where they accumulate and AF104 and AF203 showed optimal growth cause pollution problems. Furthermore, at 30oC.The results mean that the bacterial results indicated that the microbial consortium obtained is capable of using consortium AS2 had a promising the complex mineral oil as a carbon and application in bioremediation of oil- energy source in different environmental contaminated environments and could be conditions. After 4 days incubation, this potentially used in microbial enhanced oil consortium removed nearly 42% of 3% recovery (MEOR). hydrocarbon content. The strains Actinomyces sp. and Saccharomyces sp. References likely played a key role in the mineral oil degradation. The study of complex Abalos A, Vinas M, Sabaté J, Manresa MA, mineral oil degradation demonstrated that Solanas AM 2004. Enhanced the selected functional consortium biodegradation of Casablanca crude oil significantly enhanced the mineral oil by a microbial consortium in presence of removal efficiency up to 38 42% over a 6 a rhamnolipid produced by day period in comparison with 7- 9% in Pseudomonas aeruginosaAT10. Biodegradation 15:249-260. 411 Int.J.Curr.Microbiol.App.Sci (2014) 3(4): 401-414

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