Studies on the biosynthesis of surfactin from Bacillus

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The 8th International Chemical Engineering Congress & Exhibition (IChEC 2014) Kish, Iran, 24-27 February, 2014

Studies on the biosynthesis of surfactin from Bacillus subtilis, Effect of vitamin B12 on cell growth and surfactin production H. Hajfarajollah1, B. Mokhtarani*1, K. Akbri Noghabi2 , H. Abbasi3 1

Chemistry and Chemical Engineering Research Center of Iran, P.O. Box 14335-186, Tehran, Iran National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran 3 Department of Chemical Engineering, Jundi-Shapur University of Technology, Dezful, Iran Corresponding author E-mail: [email protected] 2

ABSTRACT

Surfactin is one of the most important kinds of biosurfactants excreted by some species of Bacillus. In the present study, the production of surfactin from Bacillus subtilis DSMZ 3256 was initially investigated on different carbon sources. The best carbon source was selected and the further experiments was conducted in order to verify the role of Vitamin B12 (VB12) on cell growth and surfactin production. Physical properties such as surface tension, oil spreading test and emulsion index were investigated. Thin layer chromatography (TLC) confirmed the lipopeptide structure of the biosurfactant. Glucose appeared to be the best carbon source for production of surfactin. The VB12 with the concentration above 0.1 mg/L inhibited the cell growth, while the concentration of 0.001 was useful on the production of surfactin. Keywords: Surfactin. Lipopeptide, Carbon sources, Vitamin B12

1. Introduction Biosurfactants are produced by many living organisms. They can possess not only the valuable characteristic of the high specific activity of chemical surfactants, but also many advantages, such as a variety of biological activities [1,2]. At present, the use of biosurfactants has been limited by their high production costs and for most applications they do not compete with chemically synthesized surfactants [3]. Some strains of Bacillus subtilis produce a variety of lipopeptides. The pattern of lipopeptides produced is strain-dependant. The strain DSMZ 3256 produces the surfactin lipopeptide [4]. Surfactin is known as a very powerful biosurfactant. It essentially is a heptapeptide (L-Glu-LLeu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu) linked to a β-hydroxy fatty acid comprising mainly 14 or 15 carbon atoms. Surfactin has exceptional surface active power because it can lower the surface tension of water from 72 mN/m to 27 mN/m at a very low concentration. Hence, the

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Studies on the biosynthesis of surfactin from Bacillus subtilis

application potentials of surfactin in cosmetic, pharmaceutical, food, and petroleum refinery industries as well as in environmental engineering have been presented [5-7]. Small amounts of growth factors are required by cells because of the specific roles they have in biosynthesis. The need for a growth factor results from either a blocked or missing metabolic pathway in the cells. Growth factors can be categorized into three classes. Purines and pyrimidines, amino acids and vitamins. Vitamins are needed as coenzymes and functional groups of certain enzymes. Some bacteria (e.g. E. coli) do not require any growth factors: they can synthesize all essential purines, pyrimidines, amino acids and vitamins, starting with their carbon source, as part of their own intermediary metabolism. Certain other bacteria (e.g. Lactobacillus) require purines, pyrimidines, vitamins and several amino acids in order to grow. These compounds must be added in advance to culture media that are used to grow these bacteria. However, in many cases, vitamins in a certain amount can inhibit the cell growth and in this case they can be used for cancer therapy by inhibiting the cancer cells growth [11]. In the present study, the production of surfactin over some carbon sources was investigated. After choosing the best carbon source, the effect of VB12 on the cell growth and surfactin production was studied for the first time. Physical properties of the surfactin were evaluated in terms of surface tension, oil spreading test and emulsion index. Chemical characterization was studied by thin layer chromatography. 2. Materials and Methods 2.1 Chemicals and microorganism All chemicals were obtained from Merck (Germany) unless otherwise stated. Bacillus Subtilis DSMZ 3256 was purchased from German Culture Collection in lyophilized state and stored at 70 °C in glycerol stocks. 2.2 Media and Cultivation B. subtilis was cultured in shake flasks. The preculture medium was performed in LB at 200 rpm, 30 ˚C for 16 h. After primary cultivation, 4% inoculation was done into the production medium. The production medium is based on optimized medium by Sen [8] and consisted of 50 mM NH4NO3, 30 mM KH2PO4, 30 mM Na2HPO4, 7 µM CaCl2, 4 µM Sodium EDTA, 0.8 mM MgSO4, 14.5 µM FeSO4, 1.63 mM MnSO4. The amount of carbon source was used according to experiments. 2.3 Extraction and Analysis The fermentation broth was centrifuged at 7000 rpm for 15 min to remove biomass and other impurities. The resulting supernatant was adjusted to be a pH around 2 by adding 3M HCl; it was then precipitated over night for 16 h. The precipitate (crude powder) was obtained by centrifuging at 10,000 rpm, 4 ˚C for 17 min and oven drying at 37 ˚C for two days. Thin layer chromatography was performed on silica gel plate using developing solvent system of chloroform: methanol: water (65:15:2). After development, the plate sprayed evenly with the ninhydrin reagent (0.5 g ninhydrin (Sigma-Aldrich) in 100 ml anhydrous acetone) and placed in an oven at 110 °C for 10 min to detect the presence of lipopeptides as red spots [9]. Three methods including surface tension (ST), oil spreading test (OST) and emulsification activity (E24%) were employed to indirect analysis of this biosurfactant. Surface tension was determined with a Data Physics Contact Angle system (OCA20, Germany) using the pendent drop method. Oil spreading test is a method used to find biosurfactant activity. It is also sometimes used as a reliable way to measure the concentration of biosurfactants [10]. The standard procedure of oil spreading test was done as described elsewhere [11]. The emulsification activity of produced biosurfactant was measured according to Cooper and 2

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The 8th International Chemical Engineering Congress & Exhibition (IChEC 2014) Kish, Iran, 24-27 February, 2014

Goldenberg [12]. The emulsification index (E24) is the height of the emulsion layer divided by the total height that is measured after 24 hr and expressed as percentage. Cell growth was obtained by optical density (OD) measurement using UV spectrophotometer (PerkinElmer, USA). For all samples, the cultivated broth was diluted to give values less than (1 OD600) for better accuracy. The OD of culture was converted to cell dry weight (CDW) through a linear correlation standard curve. Based on standard curve of this strain, One OD600 was almost equivalent to 0.29 g/L. 2.4. Experimental procedure Five carbon sources including glucose, lactose, starch, kerosene and sunflower oil were used to evaluate the production of biosurfactant in terms of ST reduction, OST and E24. The best carbon source was selected to evaluate the effect of VB12 on cell growth and surfactin production. VB12 in five different concentrations of 0.001 to 0.2 mg/liter were used, and the surfactin production as well as CDW changes were studied. 3. Results and discussion 3.1 Carbon source evaluation Five different carbon sources from various origins (hydrocarbon, carbohydrate and vegetable oil) were used to investigate surfactin production by B .subtilis in terms of OST, ST and E24. Fig. 1 shows the results. The overall results of this study indicated that glucose was the best carbon source. The best emulsion index was achieved on glucose while for other carbon sources this index is generally low. ST reduction on glucose media was also more than other sources. However, the results of OST for the culture grown on kerosene is a little better than others. It’s because kerosene itself is able to spread the oil in OST. Therefore, glucose was chosen for further experiments. After the extraction of biosurfactant from culture broth of glucose media, thin layer chromatography confirmed the lipopeptide structure of the biosurfactant by appearing red spot. 90 80 OST (mm) ST (mN/m) E24 %

70

OST, ST, E24

60 50 40 30 20 10 0

glucose

lactose

starch

kerosen

sunflower oil

Carbon source

Fig 1. Evaluation of carbon sources for surfactin production

3.2 Effect of VB12 on the growth of B. subtilis and surfactin production To investigate the effect of VB12 (cobalamin) concentrations on the growth of B subtilis and surfactin production, a range of concentrations of vitamin B12 (0.001 to 0.2 mg/liter) was examined. Cultures containing 0.001 mg/liter of VB12 reduced the surface tension of broth more

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Studies on the biosynthesis of surfactin from Bacillus subtilis

than control, as shown in Fig. 2. However, with increasing VB12 concentration it can be seen that the ST was increased, and E24 and OST were decreased. These demonstrate that higher concentrations of vitamin B12 provided no additional benefit. Increasing VB12 concentration not only has no positive effect but also the growth of bacterial cells was completely inhibited for concentrations of VB12 more than 0.1 mg/L. 100 OST (mm)×10 ST (mN/m) E24 % CDW(g/L)×10

OST×10, ST, E24, CDW×10

80

60

40

20

0

control

0.001

0.025

0.05

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Fig 2. Effect of VB12 concentration on surfactin production

4. Conclusion The production of surfactin on various carbon sources was evaluated. The results showed that the production of biosurfactant on glucose is much better than on other carbon sources. With using glucose as a carbon source, the effect of VB12 on the biosurfactant production and cell growth was studied. VB12 concentration of 0.001 mg/L was effective for biosurfactant production. However, the concentration of more than 0.1 mg/L completely inhibited cell growth as well as surfactin production. References [1] J.D. Desai, I.M. Banat, Microbial production of surfactants and their commercial potential, Microbiol. Mol. Biol. Rev. 61 (1997) 47–64. [2] G. Georgiou, S. C. Lin, M. M. Sharma, Surface-active compounds from microorganisms. Biotechnology 10 (1992)60–65. [3] C. Syldatk, F. Wagner, Production of biosurfactants, in Biosurfactants and Biotechnology, ed. by N. Kosaric,W.L. Cairns, N.C.C. Gray, Marcel Dekker, New York, pp 89–120, 1987. [4] S.C. Lin, Biosurfaces: recent advances, J. Chem. Technol. Biotechnol. 6 (1996) 109–120. [5] F. Peypoux, J.M. Bonmatin, J. Wallach, Recent trends in the biochemistry of surfactin, Appl. Microbiol. Biotechnol. 51 (1999) 553–563. [6] H.L. Chen, R.S. Juang, Recovery and separation of surfactin from pretreated fermentation broths by physical and chemical extraction, Biochem. Eng. J. 38 (2008) 39–46.

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[7] R. Sen, T. Swaminathan, Characterization of concentration and purification parameters and operating conditions for the smallscale recovery of surfactin, Process Biochem. 40 (2005) 2953– 2958. [8] R. Sen, Response Surface Optimization of the Critical Media Components for the Production of Surfactin, J. Chem. T ech. Biotechnol. 68 (1997) 263-270 [9] H. Yin, J. Qiang, Y. Jia, J. Ye, H. Peng, H. Qin, N. Zhang, B. He, Characteristics of biosurfactant produced by Pseudomonas aeruginosa S6 isolated from oil-containing wastewater, Process Biochem. 44 (2009) 302–308. [10] M. Morikawa, Y. Hirata, T. Imanaka, A study on the structure– function relationship of the lipopeptide biosurfactants, Biochim. Biophys. Acta. 1488 (2000) 211 – 218. [11] T. Blair, H. A. Miller, Effect of Vitamin K1 on Cell Growth Inhibition and Apoptosis on the U937 Cell Line, Journal of Cancer Therapy, 3 (2012) 167-172 [12] D.G. Cooper, B.G. Goldenberg, Surface-active agents from two Bacillus species, Appl. Environ. Microbiol. 53 (1987) 224

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