Journal of Environmental Sciences 2011, 23(Supplement) S63–S65

Production of surfactin using pentose carbohydrate by Bacillus subtilis Abdul Wahab Khan1,3 , Mohammad Shahedur Rahman1,3 , Umme Salma Zohora1,3 , Masahiro Okanami2 , Takashi Ano1,2,∗ 1. Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan. E-mail: [email protected] 2. Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, 930 Nishimitani, Kinokawa-city, Wakayama, 649-6493, Japan 3. Institute of Biological Resources, Anise Corporation, N605 Building #C, 1-12 Minamiwatarida-cho, Kawasaki-ku, Kawasaki-shi 210-0855, Japan

Abstract Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, mass production possibility, selectivity, performance under extreme conditions and potential applications in environmental protection. In this study two pentose sugars (xylose and arabinose) were investigated for the submerged fermentation (SmF) of Bacillus subtilis in surfactant production medium for bio-surfactant surfactin production. An excellent vegetative growth of B. subtilis (× 1010 CFU/mL) was observed for xylose and arabinose containing medium which were comparable to glucose supplemented medium. Low growth (× 108 CFU/mL) was found when medium was not supplemented with any of the sugars. Surfactin production in xylose, arabinose and glucose containing medium was 2700, 2600 and 2000 mg/L, respectively, whereas, medium without any sugar showed low surfactin (700 mg/L) production. These results clearly indicate the effect of pentose sugars on production of surfactin. Gradual depletion of the xylose and arabinose were confirmed by HPLC analysis during the growth phase of the strain that ultimately produced the surfactin. Key words: xylose, arabinose; Bacillus subtilis; surfactin, submerged fermentation

Introduction Surfactants are surface active agents. They are amphipathic molecules with both hydrophilic and hydrophobic (generally hydrocarbon) moieties. For this reason, surfactants partition preferentially at the interface between fluid phases with different degrees of polarity and hydrogen bonding such as oil-water or air-water interfaces. These characteristics confer surfactant to use as an excellent detergent, emulsifying agent, foaming agent, and dispersing agent, which make it some of the most versatile process chemicals. Almost all surfactants currently in use are chemically derived from petroleum; however, interest in microbial surfactants has been steadily increasing in recent years due to their diversity, environmentally friendly nature, the possibility of their production through fermentation, and their potential applications in the environmental protection, crude oil recovery, health care, and food-processing industries (Banat, 1995; Fiechter, 1992; Mulligan, 2005). Usually chemically defined medium and hexose are used as carbon source for biosurfactin production. The demand for hexose carbon source is increasing gradually even for ethanol production. Naturally we have abundant hemi-cellulose resources. Hemi-cellulose is mainly composed of pentose sugars like xylose and arabinose. Escherichia coli (Lawlis et al., 1984) and Bacillus * Corresponding author. E-mail: [email protected]

(Rygus et al., 1991) are already reported for utilization of xylose. B. subtilis MI113 (pC115) is a recombinant of plasmid pC112 containing the Ipa-14 gene related to surfactin production from B. subtilis RB14 (Ohno et al., 1995). Due to the presence of lpa-14, B. subtilis MI113 is capable of producing bio-surfactant surfactin. This study was conducted to investigate the suitability of pentose sugar, xylose and arabinose as carbon source in SmF of B. subtilis MI113 for surfactin production for the first time.

1 Materials and methods 1.1 Experimental setup B. subtilis MI113 (pC115) strain was transfer red from culture stock into 5 mL of modified L-medium (1% Polypepton (Nippon Pharmaceuticals Co., Tokyo), 0.5% yeast extract (Oriental Yeast Co., Tokyo), 0.5% NaCl, pH 7.0) and incubated overnight with 120 strokes per minute (spm) at 37°C to prepare the preculture. SmF was conducted in 200 mL Erlenmeyer flasks which were containing xylose or arabinose as pentose sugar in surfactin production medium (8% Polypepton S (Nippon Pharmaceuticals Co., Tokyo), 6.7% xylose or arabinose (Kanto Chemical Co., Tokyo), 0.5% K2 HPO4 (Kanto Chemical Co., Tokyo), 0.05% MgSO4 ·7H2 O (Kanto Chemical Co., Tokyo), 25

Journal of Environmental Sciences 2011, 23(Supplement) S63–S65 / Abdul Wahab Khan et al.

1.2 Determination of microbial growth, pH and surfactin concentration Samples were collected from the culture mix at specific intervals to analyze. Samples were serially diluted and spread on agar plate for total CFU count. At the same time samples were heated at 80°C for 10 min before spreading for spore count. For iturin A extraction, 100 μL of sample was added into 900 μL of surfactin extraction buffer (acetonitrile: 3.8 mmol/L trifluoroacetic acid (TFA) (80:20; V/V)). The mixture was vortexed at room temperature for 20 min and then centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was filtrated through 0.20 μm polytetrafluoroethylene (PTFE) membrane filter (Advantec, Tokyo, Japan) and 20 μL of the filtrate was injected into HPLC column for surfactin ditection. A mixture of acetonitrile and 3.8 mmol/L trifluoroacetic acid (80:20, V/V) was used in mobile phase through ODS column (GL science ODS-2 4.6 mm φ × 250 mm) at a flow rate of 1.5 mL/min at 30°C. 1.3 Determination of carbon depletion rate in medium For the determination of residual sugar concentration, 100 μL of sample was diluted in 900 μL of buffer (CH3 CN: 3.8 mmol/L trifluoroacetic acid (TFA) solution = 4:1 (V/V)). The mixture was vortexed at room temperature for 20 min and then centrifuged at 15,000 × g for 10 min at 4°C. After centrifugation, the supernatant was filtered by 0.20 μm PTFE membrane filters. The filtrate of 20 μL was injected into HPLC column for determination of residual sugar concentration. A mixture of 10 mmol/L CH3 CN: H2 O = 3:1 (V/V) was used in mobile phase through ODS column (column: Chromolith Performance RP-18eb (MERCK) 4.6 mm φ × 100 mm) at a flow rate of 1 mL/min at 40°C.

2 Results and discussions 2.1 Pentose sugar as carbon source for B. subtilis MI113 growth Xylose, arabinose and glucose containing medium showed similar vegetative growths which were 100 times higher compared to without any sugar containing surfactin production medium during their four days of cultivation (Fig. 1). When additional sugar was not added, the inherent carbon content of the Polypepton S supported vegetative growth of this strain. The growth difference between the cultures with or without sugar clearly shows the effect

Total cell in: Glucose Spore in Glucose Residual sugar Glucose 11

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mg/L FeSO4 ·7H2 O (Kanto Chemical Co., Tokyo), 22 mg/L MnSO4 ·7H2 O (Kanto Chemical Co., Tokyo) and 184 mg/L CaCl2 (Kanto Chemical Co., Tokyo)). Whereas glucose containing medium and without any sugar containing medium were used as positive control and negative control respectively. All the flasks were autoclaved at 121°C for 20 min. After sterilization, medium was cooled at room temperature and the preculture (1%, V/V) was then added. The flasks were incubated at 37°C at 120 r/min for four days.

Colony forming units (mL)

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0 2 3 4 Cultivation time (day) Fig. 1 B. subtilis MI113 growth and residual sugar concentration in medium. 0

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of different sugars. On the other hand, similar vegetative growths in arabinose, xylose and glucose attribute that B. subtilis MI113 (pC115) can utilize pentose sugars arabinose and xylose. The residual sugar concentration in the medium decreased with respect to cultivation time for all culture medium. Among the three sugars (glucose, xylose and arabinose), arabinose was found to be easily and rapidly assimilated by Bacillus, whereas, assimilation of xylose was slow (Fig. 1). Bacillus took almost the double time for complete depletion of xylose compared to arabinose and glucose. The depletion rate of carbon source also affects the Bacillus spore formation in the medium (Fig. 1). During the fermentation medium pH was observed. The glucose, xylose, arabinose and without sugar containing medium showed their maximum pH on their 4th, 3rd, 3rd and 2nd day of cultivation which were 8, 7.5, 8 and 9 respectively (Fig. 2). 2.2 Pentose sugar as carbon source in surfactin production medium A number of carbon substrates have been used by many researchers for biosurfactant production. The nature of carbon substrate influence and affect the quantity and quality of the biosurfactant production (Raza et al., 2007). The hexose carbon source glucose and the pentose carbon source xylose and arabinose also produce different amount of biosurfactant. In this investigation the maximum biosurfactant production was observed on day 4 of cultivation which were 2000, 2700, 2600 and 700 mg/L (Fig. 3) for glucose, xylose, arabinose and no sugar containing medium, respectively. Lee and Kim (1993) reported that in batch culture and in fed-batch culture of Torulopsis bombicola about 37% and 60% of the carbon input was incorporated into biosurfactant. Thus the low production in medium without any sugar was due to the lack of carbon source in medium. Differences in biosurfactant

Production of surfactin using pentose carbohydrate by Bacillus subtilis

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production. Therefore it can be said that pentose sugars are more suitable for B. subtilis MI113 (pC115) for surfactin production.

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3 Conclusions 8.5

The depletion of pentose sugar in the medium attributes the utilization of pentose sugar by B. subtilis MI113 (pC115). The remarkable enhanced production of surfactin confers that hemicellulose originated pentose sugar xylose and arabinose might be a good alternative of hexose sugar like glucose. Further experiments are required to optimize the pentose sugar content in medium and fermentation parameters for successful utilization of pentose sugar.

pH

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2 3 4 Cultivation time (day) Fig. 2 pH profile of different medium during submerged fermentation. 3000

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0 Glucose Xylose Arabinose No sugar Fig. 3 Surfactin production in different sugar containing medium by B. subtilis MI113.

productions between hexose and pentose are very clear whereas two pentose carbon sources showed similar

Banat I M, 1995. Biosurfactants production and possible uses in microbial enhanced oil recovery and oil pollution remediation: a review. Bioresource Technology, 51: 1–12. Fiechter A, 1992. Biosurfactants: moving towards industrial application. Trends Biotechnology, 10: 208–217. Lawlis V B, Dennis M S, Chen E Y, Smith D H, Henner D J, 1984. Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli. Applied and Environmental Microbiology, 47: 15–21. Lee L H, Kim J H, 1993. Distribution of substrate carbon in sophorose lipid production by Torulopsis bombicola. Biotechnology Letter, 15: 263–266. Mulligan C N, 2005. Environmental applications for biosurfactants. Environmental Pollution, 133(2): 183–198. Ohno A, Ano T, Shoda M, 1995. Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation. Biotechnology and Bioengineering, 47: 209–214. Rygus T, Scheler A, Allmansberger R, Hillen W, 1991. Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization. Archives of Microbiology, 155: 535– 542. Raza Z A, Rehman A, Khan M S, Khalid Z M, 2007. Improved production of Biosurfactant by Pseudomonas aeruginosa mutant using vegetable oil refinery wastes. Biodegradation, 18(1): 115–121.