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Production and Structural Characterization of Surfactin (C14/Leu7) Produced by Bacillus Subtilis Isolate LSFM-05 Grown on Raw Glycerol from the Biodiesel Industry

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Production and Structural Characterization of Surfactin (C14/Leu7) Produced by Bacillus Subtilis Isolate LSFM-05 Grown on Raw Glycerol from the Biodiesel Industry Process Biochemistry 46 (2011) 1951–1957 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Contents lists available at ScienceDirect provided by Elsevier - Publisher Connector Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry Andreia Fonseca de Faria a,∗, Diego Stéfani Teodoro-Martinez b, Geraldo Nazareno de Oliveira Barbosa a, Boniek Gontijo Vaz b, Ísis Serrano Silva a, Jerusa Simone Garcia b, Marcos Rogério Tótola c, Marcos N. Eberlin b, Matthew Grossman d, Oswaldo Luiz Alves b, Lucia Regina Durrant a a Food Science Department, Food Engineering Faculty, University of Campinas, Rua Monteiro Lobato 80, Cidade Universitária Zeferino Vaz, 13083-862 Campinas, São Paulo, Brazil b Chemistry Institute, University of Campinas, Post Box 6154, Cidade Universitária Zeferino Vaz, 13083-970 Campinas, São Paulo, Brazil c Microbiology Department, Federal University of Vic¸osa, Av. PH Rolfs - Campus Universitário, 36570 000 Vic¸osa, Minas Gerais, Brazil d BioSage, 807 Eagles Chase Drive, Lawrenceville, NJ 08648, United States article info abstract Article history: The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained Received 3 February 2011 from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was Received in revised form 24 June 2011 carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The Accepted 1 July 2011 crude surfactant was purified by silica gel column chromatography with a yield of 230 mg of the purified biosurfactant per liter of foam. TLC, IR spectroscopy, 1H and 13C NMR and Fourier transform ion cyclotron Keywords: resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the B. subtilis purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified Biosurfactant Surfactin the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety Raw glycerol contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the Biodiesel C14/Leu7 surfactin was 70 ␮M, with emulsification efficiency after 24 h (E24) of 67.6% against crude oil. Mass spectrometry Raw glycerol represents an abundant and renewable carbon source and provides an opportunity for reducing the cost of biosurfactant production and may add value to biodiesel production by creating new commercial applications for this by-product. © 2011 Elsevier Ltd. Open access under the Elsevier OA license. 1. Introduction order to solve these problems, many studies have been car- ried out using low-cost feedstock or agricultural byproducts as Biosurfactants or microbial surfactants are surface active substrates for biosurfactant production. Low-cost carbon sources molecules that are produced by a variety of microorganisms that have been used for biosurfactant production by microor- including bacteria, yeast and filamentous fungus. Due to their ganisms include sludge palm oil [4], cassava wastewater [5], amphipathic nature, these biomolecules reduce the interfacial vegetable oil refinery waste [6], molasses [7] and raw glycerol tension between an aqueous phase and hydrophobic molecules, [8]. Raw glycerol is a by-product of biodiesel production [9]. thereby enhancing the solubility and bioavailability of hydropho- Biodiesel is produced by transesterification of fatty acids derived bic organic compounds [1]. In comparison to synthetic chemical from vegetable oils and animal fats and the raw glycerol by- surfactants, biosurfactants are of interest due to their high level product represents 10% (v/v) of the total biodiesel production of activity, specific activity at extreme temperatures, pH and [9,10]. salinity, ability to be produced from renewable feedstock and The European Union has been the global leader in biodiesel pro- high degree of biodegradability [2]. However, biosurfactants have duction [11]. According to the European Biodiesel Board [12], the not yet been commercialized extensively due to low produc- overall biodiesel production in the EU has increased from 9.0 mil- tion yields and high feedstock and purification costs [2,3].In lion tonnes in 2009 to nearly 21.9 million tonnes in 2010. Brazil is also among the largest producers and consumers of biodiesel in the world with an annual production in 2010 of 2.4 billion liters and ∗ an installed capacity in the same year of 5.8 billion liters, accord- Corresponding author. Tel.: +55 19 35212173; fax: +55 19 35212173. E-mail address: [email protected] (A.F. de Faria). ing to the Brazilian National Agency for Petroleum, Natural Gas and 1359-5113 © 2011 Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.procbio.2011.07.001 1952 A.F. de Faria et al. / Process Biochemistry 46 (2011) 1951–1957 Biofuel (ANP) [13]. Considering the increasing demand for biodiesel comparing the profiles of the fatty acid methyl esters to those in the Aerobe reference production, the production of raw glycerol will also increase, result- library database (TSBA, version 4.0). ing in a further decrease in its price [10]. In contrast to the relatively expensive purified glycerol, which is an important ingredient in 2.2. Media and culture conditions food, drug, cosmetic and chemical production, raw glycerol con- B. subtilis LSFM-05 was grown on nutrient agar (Difco) plates at 30 ◦C for 24 h tains impurities, such as salts and other organic compounds, and is a and transferred to 200 mL conical flasks containing 50 mL of Nutrient Broth (Difco), potentially inexpensive carbon source for the microbial production followed by incubation at 32 ◦C and 150 rpm for 48 h. The inoculum was prepared of chemicals [10,14]. by centrifuging the culture for 10 min at 18,000 × g in a HITACHI-CR-21 centrifuge, The use of raw glycerol as a feedstock for the production of and the cells were washed twice with a sterile NaCl solution 0.85% (w/v) and re- suspended in the basal salts medium containing (per liter): 3 g of NaNO ;1gof chemicals could provide the biodiesel industry with an added value 3 KH2PO4; 0.1 g of NaCl; 0.5 of MgSO4·7H2O; 1 mL of vitamin stock solution (0.002 g/L outlet for the large quantities of raw glycerol by-product. One folic acid, 0.010 g/L pyridoxine, 0.005 g/L riboflavin, 0.005 g/L thiamine, 0.005 g/L of the possible applications of raw glycerol is its use as a car- nicotinic acid, 0.005 g/L pantothenic acid, 0.001 g/L cyanocobalamin, 0.005 g/L ␳- bon and energy source for biosurfactant production by Bacillus aminobenzoic acid, 0.005 g/L thioctic acid and 0.002 g/L biotin), at pH 6.8. The and other microorganisms. Rooney et al. [15] previously isolated fermentation was conducted using a 15-L bench-top fermentor (Bioflo 3000 New Brunswick Scientific) with 10 L working volume. The organic substrate was added rhamnolipid-producing bacteria from soils at a biodiesel facility, to basal salt medium in the concentration of 5% (v/v) as the sole carbon source. The on the basis of their ability to grow on glycerol as the sole carbon glycerol sample was donated by the biodiesel industry Granol, Anápolis, Brazil and source, and Morita et al. [8] described the use of waste glycerol stored at 4 ◦C. According to the Granol industry, the raw glycerol sample contained from biodiesel in the production of mannosylerythritol lipids by 80% glycerol, 5–8% water, 6% mineral salts, 5% unidentified organic compounds Pseudozyma antarctica JCM 10317. Das et al. [16] demonstrated other than glycerol and 0.3% methanol. The culture medium was inoculated with 106 CFU/mL giving an initial optical density at 600 nm of 0.1. The fermentation was the production of crude biosurfactants by a marine microorgan- carried out at 32 ◦C for 72 h, with agitation at 250 rpm, oxygenation at 0.5 vvm in ism using glycerol as substrates. However, to our knowledge this the absence of a chemical antifoaming agent. The pH was not controlled during is the first report on the production of surfactin by a Bacillus sub- the fermentation. The fermentative process was carried out in three independent tilis strain using raw glycerol (instead of the more costly purified replicates. The glycerol consumption and microbial growth were determined from samples collected at 12-h intervals following inoculation. To remove the bacterial glycerol) obtained from a biodiesel factory as feedstock. cells, the samples were centrifuged at 18,000 × g for 10 min at 4 ◦C. The glycerol B. subtilis strains produce a spectrum of lipopeptides that are concentration was determined by high performance liquid chromatography (HPLC) powerful biosurfactants, and also have potent antimicrobial, antivi- using a Shimadzu Chromatograph model CR-21 equipped with a Supercogel C-610H ◦ ral and antitumor activities [17]. The most studied lipopeptides column conditioned at 75 C and an isocratic mobile phase of 0.1% H2SO4 (98% purity, family is the surfactins, which may also be the most powerful Synth, Brazil) at a flow rate of 1.0 mL/min [24]. To determine cellular growth the samples were submitted to serial dilutions in saline solution (0.85%, w/v) and viable ␤ biosurfactants known, and contain heptapeptides attached to a - counts were performed by the spread plate technique. The results are expressed as hydroxy fatty acid, with carbon numbers in the range from 13 to colony-forming units per milliliter (CFU/mL). The pH of the culture was measured 15 [18,19]. during the fermentation using a pH probe inserted into the fermentation broth. The present work describes the production of surfactin by the B.
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