International Biodeterioration & Biodegradation xxx (2012) 1e6

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International Biodeterioration & Biodegradation

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Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery
S.N. Al-Bahry a, *, Y.M. Al-Wahaibi b, A.E. Elshae a, A.S. Al-Bemani b, S.J. Joshi a, H.S. Al-Makhmari a, H.S. Al-Sulaimani b
a b

Dean College of Science, Department of Biology, Sultan Qaboos University, Muscat, Oman Department of Petroleum and Chemical Engineering, Sultan Qaboos University, Muscat, Oman

a r t i c l e i n f o
Article history: Received 11 July 2011 Received in revised form 14 January 2012 Accepted 24 January 2012 Available online xxx Keywords: Biosurfactant Date molasses Surface tension Interfacial tension Enhanced oil recovery

a b s t r a c t
This study highlights the use of low-cost agro-industrial raw material e date molasses for fermentative production of biosurfactants. Date palm (Phoenix dactylifera L.) is the primary crop in Oman, which shares 82% of all fruit crops production in the country. When date molasses was used as the sole carbon and energy source for biosurfactant production using Bacillus subtilis B20, a product yield of 2.29 0.38 g/l was obtained. The biosurfactant reduced surface tension and interfacial tension from 60 to 25 mN/m to 27 and 5.02 mN/m respectively. It also showed signicant stability under a wide range of temperatures, pH and salt concentrations. Additional 9.7% oil was recovered through core-ood studies, accessing the potential of biosurfactant to enhance oil recovery under reservoir conditions. Current studies showed date molasses could be suitable for use in the production of biosurfactant and it has the potential for use in enhancing oil recovery. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Industrial demand for biologically produced chemicals is steadily increasing because of its effectiveness, economic concerns and environmental compatibility. Biosurfactants or microbially produced surfactants are used widely in agricultural, cosmetics, food, pharmaceuticals, environmental and petrochemical industries (Desai and Banat, 1997; Makkar and Cameotra, 2002). As biosurfactants are amphipathic in nature, it reduces surface tension (ST) and interfacial tension (IFT) at the interfaces, enhancing mixing of two immiscible phases. Apart from these properties, they are comparatively less toxic and highly biodegradable. They have better foaming properties, and greater stability under harsh environmental conditions, as compared to chemical surfactants; those features make them promising commercial alternatives (Desai and Banat, 1997). Moreover, they are more suited for petrochemical and environmental applications such as, bioremediation of polluted sites, oil spill management and enhanced oil recovery (Banat, 1995; Al-Sulaimani et al., 2011).

* Corresponding author. Tel.: 968 24141401; fax: 968 24143415. E-mail address: snbahry@squ.edu.om (S.N. Al-Bahry). 0964-8305/$ e see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2012.01.006

Members of genus Bacillus are considered a suitable group for industrial synthesis of biosurfactants because the species within this taxon, i.e., Bacillus subtilis and Bacillus licheniformis are wellknown producers of surface active metabolites. They not only produce good biosurfactants but are also capable to grow under facultative or anaerobic conditions, and have also been reported to be non-pathogenic, which permits their use in food and pharmaceutical industries, apart from environmental applications (Neves et al., 2007). Surfactin-cyclic lipopeptide biosurfactant produced by B. subtilis is one of the most effective biosurfactant which can lower the ST and IFT of water and water-n-hexadecane system from 72 to 27 mN/m and 43 to 1 mN/m respectively (Nitschke and Pastore, 2006). Having apparent advantages of biosurfactants over chemical surfactants, it is quite luring to produce them in the large scale for industrial use. However, high production cost and low yield of biosurfactants affect their wide spread applications (Deleu and Paquot, 2004). The success of biosurfactant production at an industrial scale depends on several strategies, which involved the development of cheaper scaled-up processes, strain improvement, optimization of media components and conditions using statistical methods or the use of inexpensive raw materials, which generally account for 10e30% of the overall cost (Nitschke and Pastore, 2004; Joshi et al., 2008a,b). Agro-industrial by-products with high content

Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006

S.N. Al-Bahry et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

of carbohydrates, proteins or lipids meet the criterion for its use as raw material for biosurfactant production (Makkar and Cameotra, 1999). Different agro-industrial wastes are studied and reported to be suitable for biosurfactant production: Molasses (Patel and Desai, 1997; Abdel-Mawgoud et al., 2008; Joshi et al., 2008a,b; Daverey and Pakshirajan, 2009), sludge palm oil (Nawawi et al., 2010), Cashew apple juice (Rocha et al., 2009), Cassava waste water (Nitschke and Pastore, 2006). Date palm (Phoenix dactylifera L.) is the primary crop in Oman occupying 52% of cultivated area and 82% of all fruit crops grown in the country. The global date production is approximately ve millions tons and about 4e5% is produced in Oman (Al-Harthy, 2011). Oman is currently the eighth largest world dates producer having over 250 cultivars and the production of 238,000 metric tons in 2005. Due to the variability in the topographic and climatic growing conditions, date palm harvest season lasts from May to November, giving Oman the longest season of any date-producing country (Al-Yahyai and Al-Khanjari, 2008). Annually more than 22% of the total dates produced are wasted because of inappropriate storage (Al-Harthy, 2011). Date syrup or date molasses is probably the most commonly derived date product, produced in three different ways: (i) as an accidental byproduct in the storage of bagged, humid dates (especially in the Gulf area), (ii) at the home or village level by extraction and boiling down of the juice, and (iii) on a semi- and full industrial scale. Date molasses costs around 9.5 US cents/kg (Forsberg et al., 2002). In this study, date molasses was used as a novel substrate as sole source of carbon for biosurfactant production by B. subtilis 20B. Date molasses was purchased from local market. Produced biosurfactant was checked for stability under different harsh environmental conditions and its potential for enhanced oil recovery was checked using core ooding experiments. 2. Materials and methods

2.3. Media and cultivation techniques LB was used for preparation of the seed inoculum. The culture was grown for 8e10 h at 40  C (OD600 nm 0.9e1.0). The asks containing 50 ml of molasses medium in a 250 ml Erlenmeyer ask were inoculated with 2% (v/v) seed culture. The cultures were incubated on a temperature controlled shaker incubator at 160 rpm. Molasses medium was prepared by addition of 8 g of date molasses in 100 ml basal mineral salt medium. The compositions of different media used in this study are presented in Table 1. The three reported media were used: N (Nazina et al., 2005), C (Cooper et al., 1981) and M (Mukherjee et al., 2009) for biosurfactant production. Media were sterilized by autoclaving at 121  C for 15 min, then sterile trace minerals were added after autoclaving and the pH adjusted to 7.2 by addition of sterile NaOH. Samples were withdrawn every 24 h interval, growth (OD660), pH, biosurfactant production (ST and IFT) and biopolymer production (viscosity) were checked up to 72 h. 2.4. Analytical methods Total soluble carbohydrate in molasses medium was determined by the phenolesulfuric acid method (Dubois et al., 1956), and concentration of carbohydrates was estimated by comparing it with standard sucrose and glucose solutions. Production of biosurfactant was judged on periodic measurement of ST and IFT of examined liquids by means of pedant drop method using the Drop Shape Analyzing system DSA 100 (KRUSS, Germany). For biopolymer production, 1 ml sample were analyzed by viscosity measurement using quartz viscometer (F5 technology, Germany). All measurements were made on cell-free broth after centrifugation (11,292 g for 20 min), and were analyzed at room temperatures. The experiments were performed in duplicate and the results reported are the mean of three independent experiments. 2.5. Scale up studies

2.1. Chemicals All major chemicals (NH4NO3, KH2PO4, K2HPO4, Na2HPO4, NaCl) and trace elements used were purchased from SigmaeAldrich, Co. USA. Date molasses was purchased from local market, Sultanate of Oman, and its compositions were (as reported by manufacturer, for each 100 ml): Moisture, 13e16%; Protein, 1.0e2.3%; Ash, 1.4e1.8%; Total sugar, 75e76% (Glucose: 37%; Fructose: 35%); e Pectin, 0.1e0.2%; Tannin, 0.2e0.3%; pH, 4.3e5.2; TDS, 76e78%. Fat and Fiber were undetectable. 2.2. Source of microorganism The biosurfactant producing strains were isolated from petroleum contaminated garage site using oil spreading method as described by Youssef et al. (2007). Those isolates which showed bigger zone of clearance on oil layer were selected and used for further experiments at shake ask level in Luria-Bertani (LB) broth. Isolates which reduced surface tension (ST) below 35 mN/m were selected further as described by Joshi et al. (2008a). One strain e B20 was selected for further screening, and was identied as B. subtilis by morphological and biochemical tests (data not shown) and 16S rRNA gene sequencing (GenBank accession no. DQ922951). The isolate was maintained aerobically on LB agar plates and was regularly subcultured into fresh LB medium for short-term storage. Stock cultures of all pure isolates were prepared in 25% glycerol and stored below 60  C, for long-term maintenance. Scale up studies was carried out in 5 L capacity fermentor (New Brunswick Scientic BIOFLO 110), containing 3 L production medium M. Seed (1.0 OD; 2% (v/v)) was transferred to the fermentor aseptically. The broth culture was cultivated under controlled conditions: aeration rate 0.5 vvm and an agitation speed of 200 rpm at 40  C. The pH and dissolved oxygen (DO%) were

Table 1 Production media composition for biosurfactant production. Composition (g/l) Date molasses NH4NO3 K2HPO4 KH2PO4 Na2HPO4 MgSO4$7H2O FeSO4$7H2O MgCl2 CaCl2 NaHCO3 NaCl NH4Cl Trace elements N* 80.0 e 0.09 0.21 e e e 0.2 0.05 2.2 7.0 0.8 1 mla C* 80.0 4 e 4.08 7.12 0.2 e e e e e e 1 mlb M* 80.0 3.3 2.2 0.14 e 0.6 0.2 e 0.04 e 0.01 e 0.5 mlc

N: Nazina et al., 2005; C: Cooper et al., 1981; M: Mukherjee et al., 2009. a Trace elements (g/l): ZnSO4$7H2O, 0.16; FeCl3$6H2O, 0.27; MnSO4$H2O, 0.017; MgSO4$7H2O, 0.4. b FeSO4$7H2O, 0.0011; MnSO4$4H2O, 0.00067, CaCl2, 0.00077; Na-EDTA, 0.00148. c ZnSO4$7H2O, 2.32; MnSO4$4H2O, 1.78; H3BO3, 0.56; CuSO4$5H2O, 1.0; Na2MoO4$2H2O, 0.39; CoCl2$6H2O, 0.42; Na-EDTA, 1.0; NiCl2$6H2O, 0.004; KI, 0.66.

Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006

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monitored continuously. Samples (10 ml) were collected intermittently to check growth (OD660), biosurfactant production (ST and IFT) and biopolymer production (Viscosity). 2.6. Stability studies Biosurfactant produced by B. subtilis B20 in medium M (after 10e12 h incubation at 40  C in fermentor) was used for stability studies. Cell-free broth (10 ml each) was checked for stability of surface activity under different environmental conditions: high temperatures (40, 60, 70, 80, 90 and 100  C for 60 min and at 121  C for 20 min), different salt concentrations (NaCl: 0, 1, 2, 4, 6, 8, 10, 15 and 20%, w/v) and a wide range of pH (2, 4, 6, 7, 8, 10 and 12). After incubation, ST and IFT were determined at room temperature. 2.7. Biosurfactant recovery Culture broth was centrifuged at 11,292 g for 20 min to get cellfree broth. Biosurfactant was precipitated by adjusting the pH of the cell-free broth to 2.0 using 6N HCl and keeping it at 4  C overnight. The precipitate was collected by centrifugation at 11,292 g for 30 min, dried and resuspended in alkaline distilled water (pH 8.0), and lyophilized to get crude biosurfactant as light brown powder. 2.8. Core ooding experiments A 1.5 inch 3 inch sandstone core plug with porosity of 28% and permeability of 300 mD was obtained from an Omani oil eld and used for the core ooding. The core was initially cleaned using the Soxhlet extraction method, where chloroform and methanol were the solvents used as an azeotropic mixture in the proportion of 75%e25% respectively. These solvents are constantly evaporated and condensed. The condensed solvent passes through the core sample removing all the oil and any other material from the core before returning back for another cycle. This process is repeated until a clear color solvent is observed (Almehaideb and Zekri, 2002). The core was dried at 65  C for 24 h before usage. Next, the core was saturated with ltered formation brine using vacuum desiccators for 24 h and Pore Volume (PV) was determined using the dry and wet volumes of the core. The core was then ooded with crude oil (API gravity, 35.51; viscosity, 1.77 cp; density, 0.838 g/cm3) at 6 cm3/h until no more water was produced and the oil initially in place (OIIP) was determined which was indicated by the volume of water displaced. After that, the core was subjected to water ooding at 30 cm3/h until no further oil was produced. The residual oil was then calculated by measuring the amount of oil produced from the water ood. Finally, 4 PV of the cell-free broth was injected at 30 cm3/h as a tertiary recovery stage and extra oil recovery was determined. All core oods were conducted at 60  C to mimic the average reservoir temperature of the eld of interest. 3. Results and discussion Oil production in the Sultanate of Oman decreased sharply during the last few years even when enhanced oil recovery (EOR) techniques were applied in which chemical, thermal and steam injections were used. These techniques are quite expensive and they require several complicated processes for operation. Microbial enhanced oil recovery (MEOR) is a process that utilizes the ability of microorganisms and their products to improve oil production. This study is the rst to isolate and identify potential bio-products produced by microorganisms from date molasses, which can be utilized for establishing MEOR technology in the Omani oil elds. The microorganisms were isolated from different sources and were checked for their potential for biosurfactant production (data is not
Fig. 1. ST of B. subtilis B20 in N, C, and M media with date molasses after 24, 48 and 72 h incubation.

shown). B. subtilis B20 was found to be the best biosurfactant producer, amongst other isolates using LB medium (data is not shown). Considering economic concerns, three reported minimal media with date molasses as a sole carbon and energy source were analyzed for biosurfactant production. Molasses, byproduct from processing of sugar-rich crops, is one of the most promising substrates for ex-situ biosurfactant production in favor of EOR applications. Different types of molasses are used widely for microbial growth and industrial fermentation, as it is generally cheaper, and apart from carbohydrates, it also contains several minerals, vitamins and organic acids. Dates are major crop of the Sultanate and are easily available throughout the year. Date molasses composition was as reported by manufacturer, and total carbohydrate estimated by phenolesulfuric acid method was found to be 67e70%. Isolate B20 showed a different behavior in the 3 media. The minimum ST was after 24 h incubation (Fig. 1). Similarly, IFT dropped signicantly in media C and M, after 24 h incubation (Fig. 2). Viscosity of B20 in 3 media did not vary signicantly after 48 h of incubation (Fig. 3). The growth of B20 in medium N was lower as compared to media C and M. Microbial density in medium N remained constant after 72 h incubation (Fig. 4). Out of three media, medium M gave better biosurfactant production reducing ST and IFT within 24 h, from 58 and 23 mN/m to 27 and 5.02 mN/m, at shake ask level. B. subtilis B20 was used for scale up studies in fermentor, containing 3.0 L of medium M, where pH and dissolved oxygen concentrations were monitored as a function of time. Air ow (0.5 vvm), agitation (200 rpm) and temperature (40  C) were monitored and maintained at constant values. Heavy foaming was

Fig. 2. IFT of B. subtilis B20 in N, C, and M media with date molasses after 24, 48 and 72 h incubation.

Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006

S.N. Al-Bahry et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

Fig. 3. Viscosity prole of B. subtilis B20 in N, C, and M media with date molasses after 24, 48 and 72 h incubation.

observed from initial log hours, i.e., 7 h, and foam was collected after 9 h, during batch under sterile conditions. Sterile bottles were transferred aseptically for foam collection during the run. Batch was run for 30 h. The minimum IFT observed was 12.9 mN/m within 9 h of fermentation, whereas, IFT was 1.02 mN/m, for collapsed foam. Possible explanation for the lower IFT observed in collapsed foam is because of concentrated biosurfactant in collected foam. The pH was almost stable (6.5e7.5) during the batch, but DO dropped to almost zero after 3e4 h, where cells entered the log phase and maximum growth was observed. The batch prole is shown in Fig. 5(AeB). After 9e10 h, crude biosurfactant was extracted from the cell-free broth by acid-precipitation method. Light brown powder was obtained after lyophilization of neutralized biosurfactant solution. Acidic precipitation of bioproduct allowed recovering 2.29 0.38 g of crude biosurfactant from 1 L of cell-free supernatant. Thus extracted bioproduct was resuspended in 1 L distilled water and it showed ST and IFT values 27 mN/m and 5.02 mN/m respectively, which are similar to cell-free broth before extraction. To check the stability of biosurfactant, cell-free broth subjected to different conditions. It was observed to be stable over wide range of temperatures (40  Ce100  C for 60 min and 121  C for 20 min), but ST and IFT did not changed much (Fig. 6). Several studies conrm the stability of biosurfactants under extreme conditions of temperature. Joshi et al. (2008a,b) reported that biosurfactant produced by four different Bacilli isolates, were stable for nine days at 80  C. Desai and Banat (1997) observed that heat treatment on some biosurfactants caused no appreciable change in their properties even after autoclaving at 120  C for 15 min. Similarly,

Fig. 5. AeB: The batch fermentation prole of B. subtilis B20 in medium M with date molasses.

Borodoli and Konwar (2008) exposed the biosurfactant produced by Pseudomonas aeruginosa strains to temperature of 100  C for different time periods of 5e60 min and found that the biosurfactant activity remained unaffected with respect to ST changes.

Fig. 4. Growth prole of B. subtilis B20 in N, C, and M media with date molasses after 24, 48 and 72 h incubation.

Fig. 6. Effect of temperature on the activity of biosurfactant produced by B. subtilis B20.

Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006

S.N. Al-Bahry et al. / International Biodeterioration & Biodegradation xxx (2012) 1e6

Fig. 7. Effect of different salt (NaCl) concentration on the activity of biosurfactant produced by B. subtilis B20.

Fig. 9. Cumulative oil recovery as a function of pore volume injected (PVI) of water (-,-) and biosurfactant (-:-).

It was observed that salinity affects the biosurfactant activity when addition of 4% or more NaCl caused the IFT to increase (Fig. 7). The IFT continued to increase with rising salt concentration but then stabilized at concentrations higher than 8%. However, even at the highest salt concentrations, the biosurfactant could still retain almost 60% of its surface activity. The biosurfactant showed high stability in pH values ranging from 6.0 to 12.0, where maximum activity was observed at pH of 7.0 (Fig. 8). At pH 2.0e4.0, we observed much higher ST and IFT, since the biosurfactant is not soluble at such acidic conditions, it tends to precipitate. Thus precipitated and structurally distorted biosurfactant losses its capability of reducing surface tension. This higher instability of biosurfactants produced from some lactobacilli in acidic conditions was described by some researchers to be related to the presence of negative charged groups at the polar ends of the molecules (Gudina et al., 2010). Several reports conrm the stability of biosurfactant at different pH values mostly in the alkaline medium (Batista et al., 2005; Ghojavand et al., 2008; Joshi et al., 2008a,b; Gudina et al., 2010). We also observed similar stability trend in our biosurfactant containing cell-free broth at pH range of 6e12. Oil reservoirs are one of the harsh environments, where temperature can range from 20 to 90  C, normal salinity to hypersalinity and pH over a wide range. So, the suitable candidate for EOR studies has to be stable over those criteria. The bioproduct produced by B. subtilis B20 meets those criteria and showed stability over a wide range of environmental factors, while retaining its surface activity. To check the effect of biosurfactant on enhanced oil recovery, core-ood experiments were carried out. The pore volume and

initial oil saturation (Soi) of the core sample was calculated to be 24.2 cm3 and 67% after oil ooding. It was found that after injecting 10 PV of formation water, no more oil was produced and residual oil saturation (Sor) was 38%. Extra oil recovery (9.7% of Sor) occurred after injecting 4 PV of biosurfactant injection. Fig. 9 shows a summary of the core ooding results where the number of PV of all uids injected was plotted against cumulative oil recovery. Thomas et al. (1993), reported enhancement of oil recovery from Berea sandstone core treated with cell-free metabolites from surfactant producing strain Bacillus sp., JF-2. Apart from some EOR activities during in-situ experiments, no oil mobilization with the addition of puried biosurfactants in sandstone cores were observed by Yakimov et al. (1997). They have reported that this failure of ex-situ EOR might be due to complete adsorption of biosurfactant applied in insufcient amounts to the surfaces of core material. Therefore, loss of their interfacial activity and/or a necessity for co-surfactants, such as short chain alcohols and acids was concluded. This would potentiate the oil mobilizing activities of biosurfactants and that may be present in culture supernatant uid but not in puried surfactant preparations. Therefore, in this study core ooding experiments were carried out using cores which were inoculated with cell-free biosurfactant. As mentioned above, 9.7% increase in oil recovery in ex-situ MEOR experiments was detected following biosurfactant injection. This is similar to reports published by other researchers (McInerney et al., 1985; Yakimov et al., 1997; Almeida et al., 2004; Youssef et al., 2007; Joshi et al., 2008a,b), where they also observed positive effect of microbial bioproduct under laboratory conditions (using sand-pack columns or core ood experiments) and eld conditions. 4. Conclusion The excellent ST and IFT reducing characteristic of biosurfactant produced by B. subtilis B20 grown in a low-cost date molasses based mineral media and its stability over a wide range of temperature; pH and different salt concentrations imply the possibility of using this biosurfactant at a commercial level. Moreover, the encouraging results were observed in the enhanced oil recovery experiments at high temperature and salt concentrations, prevailing in Oman reservoirs. This indicates that the biosurfactant produced using date molasses is suitable for use in the petroleum industry, such as enhanced oil recovery. Further studies to analyze the chemical structure of biosurfactant and to understand its interaction with oilerock interfaces in oil elds will be studied for its application in oil reservoirs in Oman.

Fig. 8. Effect of pH change on the activity of biosurfactant produced by B. subtilis B20.

Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006

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Acknowledgments Authors acknowledge His Majesty Research Funds (SR/SCI/BIOL/ 08/01) and Petroleum Development Oman (PDO) for funding this project. Also, special thanks to Ms. Wafa Al-Alawi, Ms. Ratiba AlMaaini, Mr. Ike and Mr. Nayyer for their technical help.

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Please cite this article in press as: Al-Bahry, S.N., et al., Biosurfactant production by Bacillus subtilis B20 using date molasses and its possible application in enhanced oil recovery, International Biodeterioration & Biodegradation (2012), doi:10.1016/j.ibiod.2012.01.006