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1.  Optimization of the Nutritional Parameters for Enhanced Production of B. subtilis SPB1 Biosurfactant in Submerged Culture Using Response Surface Methodology 
Nutritional requirements can contribute considerably to the production cost and the bioprocess economics. Media optimisation using response surface methodology is one of the used methods to ameliorate the bioprocess economics. In the present study, biosurfactant production by Bacillus subtilis SPB1 was effectively enhanced by response surface methodology. A Plackett-Burman-based statistical screening procedure was adopted to determine the most important factor affecting lipopeptide production. Eleven variables are screened and results show that glucose, K2HPO4, and urea concentrations influence the most biosurfactant production. A Central Composite Design was conducted to optimize the three selected factors. Statistical analyses of the data of model fitting were done by using NemrodW. Results show a maximum predicted biosurfactant concentration of 2.93 (±0.32) g/L when using 15 g/L glucose, 6 g/L urea, and 1 g/L K2HPO4. The predicted value is approximately 1.65 much higher than the original production determined by the conventional one-factor-at-a-time optimization method.
PMCID: PMC3357924  PMID: 22649736
2.  In Situ Biosurfactant Production by Bacillus Strains Injected into a Limestone Petroleum Reservoir▿  
Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO2 and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 ± 0.01 h−1), carbon balances (107% ± 34%), biosurfactant production rates (0.02 ± 0.001 h−1), and biosurfactant yields (0.015 ± 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.
PMCID: PMC1828672  PMID: 17172458
3.  Microbial production of surfactants and their commercial potential. 
Many microorganisms, especially bacteria, produce biosurfactants when grown on water-immiscible substrates. Biosurfactants are more effective, selective, environmentally friendly, and stable than many synthetic surfactants. Most common biosurfactants are glycolipids in which carbohydrates are attached to a long-chain aliphatic acid, while others, like lipopeptides, lipoproteins, and heteropolysaccharides, are more complex. Rapid and reliable methods for screening and selection of biosurfactant-producing microorganisms and evaluation of their activity have been developed. Genes involved in rhamnolipid synthesis (rhlAB) and regulation (rhlI and rhlR) in Pseudomonas aeruginosa are characterized, and expression of rhlAB in heterologous hosts is discussed. Genes for surfactin production (sfp, srfA, and comA) in Bacillus spp. are also characterized. Fermentative production of biosurfactants depends primarily on the microbial strain, source of carbon and nitrogen, pH, temperature, and concentration of oxygen and metal ions. Addition of water-immiscible substrates to media and nitrogen and iron limitations in the media result in an overproduction of some biosurfactants. Other important advances are the use of water-soluble substrates and agroindustrial wastes for production, development of continuous recovery processes, and production through biotransformation. Commercialization of biosurfactants in the cosmetic, food, health care, pulp- and paper-processing, coal, ceramic, and metal industries has been proposed. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants. Perspectives for future research and applications are also discussed.
PMCID: PMC232600  PMID: 9106364
4.  Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry of Lipopeptide Biosurfactants in Whole Cells and Culture Filtrates of Bacillus subtilis C-1 Isolated from Petroleum Sludge 
Applied and Environmental Microbiology  2002;68(12):6210-6219.
An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.
PMCID: PMC134408  PMID: 12450846
5.  Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. 
Rsan-ver, a strain of Pseudomonas aeruginosa isolated at this department, was used for the development of a continuous process for biosurfactant production. The active compounds were identified as rhamnolipids. A final medium for production was designed in continuous culture by means of medium shifts, since the formation of surface-active compounds was decisively influenced by the composition and concentration of the medium components. In the presence of yeast extract, biosurfactant production was poor. For the nitrogen-source nitrate, which was superior to ammonium, an optimum carbon-to-nitrogen ratio of ca. 18 existed. The iron concentration needed to be minimized to 27.5 micrograms of FeSO4 X 7H2O per g of glucose. A carbon-to-phosphate ratio below 16 yielded the maximum production of rhamnolipids. The final productivity dilution rate diagram indicated that biosurfactant production was correlated to low growth rates (dilution rate below 0.15 h-1). With a medium containing 18.2 g of glucose liter-1, a biosurfactant concentration (expressed as rhamnolipids) of up to 1.5 g liter-1 was obtained in the cell-free culture liquid.
PMCID: PMC241507  PMID: 6435520
6.  Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions 
The lipopeptide, surfactin, is produced by Bacillus subtilis. A study has been made on large-scale production of this surfactant. A good yield was obtained from a glucose substrate fermentation by continuously removing the product by foam fractionation. The surfactin could be easily recovered from the collapsed foam by acid precipitation. The yield was also improved by the addition of either iron or manganese salts. Hydrocarbon addition to the medium, which normally increases biosurfactant production, completely inhibited surfactin production by B. subtilis.
PMCID: PMC244028  PMID: 16345840
7.  Investigation of Antimicrobial Activity and Statistical Optimization of Bacillus subtilis SPB1 Biosurfactant Production in Solid-State Fermentation 
During the last years, several applications of biosurfactants with medical purposes have been reported. Biosurfactants are considered relevant molecules for applications in combating many diseases. However, their use is currently extremely limited due to their high cost in relation to that of chemical surfactants. Use of inexpensive substrates can drastically decrease its production cost. Here, twelve solid substrates were screened for the production of Bacillus subtilis SPB1 biosurfactant and the maximum yield was found with millet. A Plackett-Burman design was then used to evaluate the effects of five variables (temperature, moisture, initial pH, inoculum age, and inoculum size). Statistical analyses showed that temperature, inoculum age, and moisture content had significantly positive effect on SPB1 biosurfactant production. Their values were further optimized using a central composite design and a response surface methodology. The optimal conditions of temperature, inoculum age, and moisture content obtained under the conditions of study were 37°C, 14 h, and 88%, respectively. The evaluation of the antimicrobial activity of this compound was carried out against 11 bacteria and 8 fungi. The results demonstrated that this biosurfactant exhibited an important antimicrobial activity against microorganisms with multidrug-resistant profiles. Its activity was very effective against Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecalis, Klebsiella pneumonia, and so forth.
PMCID: PMC3321739  PMID: 22536017
8.  Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release 
Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach.
Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm.
The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications.
PMCID: PMC3144451  PMID: 21707984
9.  Factorial Design to Optimize Biosurfactant Production by Yarrowia lipolytica 
In order to improve biosurfactant production by Yarrowia lipolytica IMUFRJ 50682, a factorial design was carried out. A 24 full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (ΔST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mN m−1 of ΔST) were obtained in a medium composed of 10 g 1−1 of ammonium sulfate and 0.5 g 1−1 of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and a ΔST of 19.5 mN m−1. The experimental design optimization enhanced ΔEI by 110.7% and ΔST by 108.1% in relation to the standard process.
PMCID: PMC2846354  PMID: 20368788
10.  Biosurfactant Production by Pseudomonas aeruginosa from Renewable Resources 
Indian Journal of Microbiology  2011;51(1):30-36.
This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.
PMCID: PMC3209860  PMID: 22282625
Biodegradation; Bioremediation; Biosurfactant; Emulsification; Lipopeptides
11.  Spray drying as a strategy for biosurfactant recovery, concentration and storage 
SpringerPlus  2014;3:49.
The objective of this study was to analyze the use of Spray Drying for concentration and preservation of biosurfactants produced by Bacillus subtilis LBBMA RI4914 isolated from a heavy oil reservoir. Kaolinite and maltodextrin 10DE or 20DE were tested as drying adjuvants. Surface activity of the biosurfactant was analyzed by preparing dilution x surface activity curves of crude biosurfactant, crude biosurfactant plus adjuvants and of the dried products, after their reconstitution in water. The shelf life of the dried products was also evaluated. Spray drying was effective in the recovery and concentration of biosurfactant, while keeping its surface activity. Drying adjuvants were required to obtain a solid product with the desired characteristics. These compounds did not interfere with tensoactive properties of the biosurfactant molecules. The dehydrated product maintained its surfactant properties during storage at room temperature during the evaluation period (120 days), with no detectable loss of activity.
PMCID: PMC3930801  PMID: 24570847
Biosurfactant; Spray drying; Maltodextrin; Clay; Environmental biotechnology; Surfactin
12.  Biofilm Formation by Bacillus cereus Is Influenced by PlcR, a Pleiotropic Regulator 
The ΔplcR mutant of Bacillus cereus strain ATCC 14579 developed significantly more biofilm than the wild type and produced increased amounts of biosurfactant. Biosurfactant production is required for biofilm formation and may be directly or indirectly repressed by PlcR, a pleiotropic regulator. Coating polystyrene plates with surfactin, a biosurfactant from Bacillus subtilis, rescued the deficiency in biofilm formation by the wild type.
PMCID: PMC1489338  PMID: 16820512
13.  Distribution of Biosurfactant-Producing Bacteria in Undisturbed and Contaminated Arid Southwestern Soils 
Biosurfactants are a unique class of compounds that have been shown to have a variety of potential applications in the remediation of organic- and metal-contaminated sites, in the enhanced transport of bacteria, in enhanced oil recovery, as cosmetic additives, and in biological control. However, little is known about the distribution of biosurfactant-producing bacteria in the environment. The goal of this study was to determine how common culturable surfactant-producing bacteria are in undisturbed and contaminated sites. A series of 20 contaminated (i.e., with metals and/or hydrocarbons) and undisturbed soils were collected and plated on R2A agar. The 1,305 colonies obtained were screened for biosurfactant production in mineral salts medium containing 2% glucose. Forty-five of the isolates were positive for biosurfactant production, representing most of the soils tested. The 45 isolates were grouped by using repetitive extragenic palindromic (REP)-PCR analysis, which yielded 16 unique isolates. Phylogenetic relationships were determined by comparing the 16S rRNA gene sequence of each unique isolate with known sequences, revealing one new biosurfactant-producing microbe, a Flavobacterium sp. Sequencing results indicated only 10 unique isolates (in comparison to the REP analysis, which indicated 16 unique isolates). Surface tension results demonstrated that isolates that were similar according to sequence analysis but unique according to REP analysis in fact produced different surfactant mixtures under identical growth conditions. These results suggest that the 16S rRNA gene database commonly used for determining phylogenetic relationships may miss diversity in microbial products (e.g., biosurfactants and antibiotics) that are made by closely related isolates. In summary, biosurfactant-producing microorganisms were found in most soils even by using a relatively limited screening assay. Distribution was dependent on soil conditions, with gram-positive biosurfactant-producing isolates tending to be from heavy metal-contaminated or uncontaminated soils and gram-negative isolates tending to be from hydrocarbon-contaminated or cocontaminated soils.
PMCID: PMC161513  PMID: 12788727
14.  Importance of 3-Hydroxy Fatty Acid Composition of Lipopeptides for Biosurfactant Activity 
Applied and Environmental Microbiology  2005;71(12):7690-7695.
Biosurfactant production may be an economic approach to improving oil recovery. To obtain candidates most suitable for oil recovery, 207 strains, mostly belonging to the genus Bacillus, were tested for growth and biosurfactant production in medium with 5% NaCl under aerobic and anaerobic conditions. All strains grew aerobically with 5% NaCl, and 147 strains produced a biosurfactant. Thirty-five strains grew anaerobically with 5% NaCl, and two produced a biosurfactant. In order to relate structural differences to activity, eight lipopeptide biosurfactants with different specific activities produced by various Bacillus species were purified by a new protocol. The amino acid compositions of the eight lipopeptides were the same (Glu/Gln:Asp/Asn:Val:Leu, 1:1:1:4), but the fatty acid compositions differed. Multiple regression analysis showed that the specific biosurfactant activity depended on the ratios of both iso to normal even-numbered fatty acids and anteiso to iso odd-numbered fatty acids. A multiple regression model accurately predicted the specific biosurfactant activities of four newly purified biosurfactants (r2 = 0.91). The fatty acid composition of the biosurfactant produced by Bacillus subtilis subsp. subtilis strain T89-42 was altered by the addition of branched-chain amino acids to the growth medium. The specific activities of biosurfactants produced in cultures with different amino acid additions were accurately predicted by the multiple regression model derived from the fatty acid compositions (r2 = 0.95). Our work shows that many strains of Bacillus mojavensis and Bacillus subtilis produce biosurfactants and that the fatty acid composition is important for biosurfactant activity.
PMCID: PMC1317328  PMID: 16332741
15.  Biosurfactant Production by a Soil Pseudomonas Strain Growing on Polycyclic Aromatic Hydrocarbons 
The capacity of polycyclic aromatic hydrocarbon (PAH)-utilizing bacteria to produce biosurfactants was investigated. Twenty-three bacteria isolated from a soil contaminated with petroleum wastes were able to form clearing zones on mineral salt agar plates sprayed with solutions of PAHs. Naphthalene and phenanthrene were utilized as sole substrates. Biosurfactant production was detected by surface tension lowering and emulsifying activities from 10 of these strains grown in an iron-limited salt medium supplemented with high concentrations of dextrose or mannitol, as well as with naphthalene or phenanthrene. Glycolipid determinations showed that in cultures of Pseudomonas aeruginosa 19SJ on naphthalene, the maximal productivity of biosurfactants was delayed compared with that in cultures grown on mannitol. However, when small amounts of biosurfactants and naphthalene degradation intermediates were present at the onset of the cultivation, the delay was markedly shortened. Production of biosurfactants was accompanied by an increase in the aqueous concentration of naphthalene, indicating that the microorganism was promoting the solubility of its substrate. Detectable amounts of glycolipids were also produced on phenanthrene. This is the first report of biosurfactant production resulting from PAH metabolism.
PMCID: PMC1388868  PMID: 16535330
16.  Contribution of Surfactin and SwrA to Flagellin Expression, Swimming, and Surface Motility in Bacillus subtilis 
Applied and Environmental Microbiology  2012;78(18):6540-6544.
Multicellular communities produced by Bacillus subtilis can adopt sliding or swarming to translocate over surfaces. While sliding is a flagellum-independent motility produced by the expansive forces in a growing colony, swarming requires flagellar functionality and is characterized by the appearance of hyperflagellated swarm cells that associate in bundles or rafts during movement. Previous work has shown that swarming by undomesticated B. subtilis strains requires swrA, a gene that upregulates the expression of flagellar genes and increases swimming motility, and surfactin, a lipopeptide biosurfactant that also facilitates sliding. Through an analysis of swrA+ and swrA mutant laboratory strains with or without a mutation in sfp (a gene involved in surfactin production), we show that both swrA and surfactin upregulate the transcription of the flagellin gene and increase bacterial swimming. Surfactin also allows the nonswarming swrA mutant strain to efficiently colonize moist surfaces by sliding. Finally, we reconfirm the essential role of swrA in swarming and show that surfactin, which increases surface wettability, allows swrA+ strains to produce swarm cells on media at low humidity.
PMCID: PMC3426715  PMID: 22773650
17.  Biosurfactants production by yeasts using soybean oil and glycerol as low cost substrate 
Brazilian Journal of Microbiology  2012;43(1):116-125.
Biosurfactants are bioactive agents that can be produced by many different microorganisms. Among those, special attention is given to yeasts, since they can produce many types of biosurfactants in large scale, using several kinds of substrates, justifying its use for industrial production of those products. For this production to be economically viable, the use of residual carbon sources is recommended. The present study isolated yeasts from soil contaminated with petroleum oil hydrocarbons and assessed their capacity for producing biosurfactants in low cost substrates. From a microbial consortium enriched, seven yeasts were isolated, all showing potential for producing biosurfactants in soybean oil. The isolate LBPF 3, characterized as Candida antarctica, obtained the highest levels of production - with a final production of 13.86 g/L. The isolate LBPF 9, using glycerol carbon source, obtained the highest reduction in surface tension in the growth medium: approximately 43% of reduction after 24 hours of incubation. The products obtained by the isolates presented surfactant activity, which reduced water surface tension to values that varied from 34 mN/m, obtained from the product of isolates LBPF 3 and 16 LBPF 7 (respectively characterized as Candida antarctica and Candida albicans) to 43 mN/m from the isolate LPPF 9, using glycerol as substrate. The assessed isolates all showed potential for the production of biosurfactants in conventional sources of carbon as well as in agroindustrial residue, especially in glycerol.
PMCID: PMC3768975  PMID: 24031810
yeast; Biosurfactants; Glycerol; Soybean oil
18.  Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2. 
Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.
PMCID: PMC201265  PMID: 8117083
19.  Application of extracellular lipopeptide biosurfactant produced by endophytic Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) in microbially enhanced oil recovery (MEOR) 
3 Biotech  2013;4(1):41-48.
Bacillus subtilis K1 isolated from aerial roots of banyan tree secreted mixture of surfactins, iturins and fengycins with high degree of heterogeneity. The extracellular extract consisting of mixture of these cyclic lipopeptides exhibited very good emulsification activity as well as excellent emulsion stability. The culture accumulated maximum surfactant up to 48 h of growth during batch fermentation in Luria broth. The emulsion of hexane, heptane and octane prepared using 48-h-old culture supernatant of B. subtilis K1 remained stable up to 2 days while emulsion of four stroke engine oil remained stable for more than a year. The critical micelle concentration of crude lipopeptide biosurfactant extracted by acid precipitation from 48-h-old fermentation broth of B. subtilis K1 was found to be 20.5 μg/mL. The biosurfactant activity was found to be stable at 100 °C for 2 h, over a pH range of 6–12 h and over an NaCl concentration up to 10 % (w/v). The application of biosurfactant on laboratory scale sand pack column saturated with four stroke engine oil resulted in ~43 % enhanced oil recovery, suggesting its suitability in microbially enhanced oil recovery.
PMCID: PMC3909566
Bacillus subtilis; Lipopeptide biosurfactants; Emulsifying activity; Critical micelle concentration; MEOR
20.  Production of surface-active lipids by Corynebacterium lepus. 
Corynebacterium lepus was grown in 20-liter batch fermentations with kerosene as the sole carbon source. Critical micelle concentration measurements indicated the production of appreciable quantities of biosurfactants. This surface activity of the culture medium was due to lipids, which were extracted and identified. Samples of C. lepus whole broth were taken during a fermentation and monitored for surface tension, amount of surfactant present, and lipid content. The changes in the surfactant measured correlated with concentration changes of several surface-active lipids. An early dramatic increase in surfactant concentration was attributed to the production of a mixture of corynomycolic acids (beta-hydroxy alpha-branched fatty acids). Surface activity at the end of the fermentation was due to a lipopeptide containing corynomycolic acids plus small amounts of several phospholipids and neutral lipids which were identified by thin-layer chromatography.
PMCID: PMC243394  PMID: 760639
21.  Utilization of oleo-chemical industry by-products for biosurfactant production 
AMB Express  2013;3:68.
Biosurfactants are the surface active compounds produced by micro-organisms. The eco-friendly and biodegradable nature of biosurfactants makes their usage more advantageous over chemical surfactants. Biosurfactants encompass the properties of dropping surface tension, stabilizing emulsions, promoting foaming and are usually non- toxic and biodegradable. Biosurfactants offer advantages over their synthetic counterparts in many applications ranging from environmental, food, and biomedical, cosmetic and pharmaceutical industries. The important environmental applications of biosurfactants include bioremediation and dispersion of oil spills, enhanced oil recovery and transfer of crude oil. The emphasis of present review shall be with reference to the commercial production, current developments and future perspectives of a variety of approaches of biosurfactant production from the micro-organisms isolated from various oil- contaminated sites and from the by-products of oleo-chemical industry wastes/ by-products viz. used edible oil, industrial residues, acid oil, deodorizer distillate, soap-stock etc.
PMCID: PMC3923564  PMID: 24262384
Biosurfactants; Agro- chemical waste; Rhamnolipid; Oil industry
22.  Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. 
Strain BAS50, isolated from a petroleum reservoir at a depth of 1,500 m and identified as Bacillus licheniformis, grew and produced a lipopeptide surfactant when cultured on a variety of substrates at salinities of up to 13% NaCl. Surfactant production occurred both aerobically and anaerobically and was optimal at 5% NaCl and temperatures between 35 and 45 degrees C. The biosurfactant, termed lichenysin A, was purified and chemically characterized. A tentative structure and composition for the surfactant are described. Lichenysin A is a mixture of lipopeptides, with the major components ranging in size from 1,006 to 1,034 Da. The lipid moiety contains a mixture of 14 linear and branched beta-hydroxy fatty acids ranging in size from C12 to C17. There are seven amino acids per molecule. The peptide moiety is composed of the following amino acids: glutamic acid as the N-terminal amino acid, asparagine, valine, leucine, and isoleucine as the C-terminal amino acid, at a ratio of 1.1:1.1:1.0:2.8:1.0, respectively. Purified lichenysin A decreases the surface tension of water from 72 mN/m to 28 mN/m and achieves the critical micelle concentration with as little as 12 mg/liter, characterizing the product as a powerful surface-active agent that compares favorably to others surfactants. The antibacterial activity of lichenysin A has been demonstrated.
PMCID: PMC167432  PMID: 7646007
23.  Gene yerP, Involved in Surfactin Self-Resistance in Bacillus subtilis 
Antimicrobial Agents and Chemotherapy  2001;45(12):3566-3573.
Surfactin is a cyclic lipopeptide biosurfactant. Transposon mutagenesis was performed in Bacillus subtilis strain 168, and a surfactin-susceptible mutant, strain 801, was isolated. Analysis of the region of insertion revealed that yerP was the determinant of surfactin self-resistance. YerP had homology with the resistance, nodulation, and cell division (RND) family proton motive force-dependent efflux pumps only characterized in gram-negative strains. The yerP-deficient strain 802, in which the internal region of the yerP gene of B. subtilis strain 168 was deleted, showed susceptibility to acriflavine and ethidium bromide. When strain 802 was converted to a surfactin producer by introducing a functional sfp which encodes a 4′-phosphopantetheinyl transferase and is mutated in B. subtilis strain 168, this yerP-deficient strain produced surfactin, although surfactin production was significantly reduced. The expression of yerP was at its maximum at the end of the logarithmic growth phase and was not induced by surfactin. yerP is the first RND-like gene characterized in gram-positive strains and is supposed to be involved in the efflux of surfactin.
PMCID: PMC90870  PMID: 11709341
24.  Production of a Lipopeptide Biosurfactant by a Novel Bacillus sp. and Its Applicability to Enhanced Oil Recovery 
ISRN Microbiology  2013;2013:621519.
Biosurfactants are surface-active compounds derived from varied microbial sources including bacteria and fungi. They are secreted extracellularly and have a wide range of exciting properties for bioremediation purposes. They also have vast applications in the food and medicine industry. With an objective of isolating microorganisms for enhanced oil recovery (EOR) operations, the study involved screening of organisms from an oil-contaminated site. Morphological, biochemical, and 16S rRNA analysis of the most promising candidate revealed it to be Bacillus siamensis, which has been associated with biosurfactant production, for the first time. Initial fermentation studies using mineral salt medium supplemented with crude oil resulted in a maximum biosurfactant yield of 0.64 g/L and reduction of surface tension to 36.1 mN/m at 96 h. Characterization studies were done using thin layer chromatography and Fourier transform infrared spectroscopy. FTIR spectra indicated the presence of carbonyl groups, alkyl bonds, and C–H and N–H stretching vibrations, typical of peptides. The extracted biosurfactant was stable at extreme temperatures, pH, and salinity. Its applicability to EOR was further verified by conducting sand pack column studies that yielded up to 60% oil recovery.
PMCID: PMC3800565  PMID: 24205445
25.  Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). 
Applied and Environmental Microbiology  1992;58(10):3276-3282.
A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.
PMCID: PMC183091  PMID: 1444363

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