Culture Medium Evaluation Using Low-Cost Substrate for Biosurfactants Lipopeptides Production by Bacillus amyloliquefaciens in Pilot Bioreactor

Total biosurfactant production by Bacillus amyloliquefaciens IT45 was evaluated with different substrates concentrations in a culture medium. A central composite design (CCD) was developed to evaluate the influence of variables, including glucose syrup, yeast extract, and calcium chloride, on surface tension (ST), total biosurfactant production, and residual sugar (RS). As a result, the best observed results for ST, RS, and total biosurfactant production were 30 mN m⁻¹, 31%, and 5.5 g L⁻¹, respectively, after 48 h of fermentations carried out in batch operation process. Characterization of the biosurfactant identified the presence of surfactin. To validate the CCD experiments, fermentations were conducted in a 40 L pilot bioreactor. For this fermentation, the cellular growth was 3.0 × 10⁹ CFU mL⁻¹, surfactin production was 0.55 g L⁻¹, and RS was 28%. The results demonstrate that B. amyloliquefaciens IT45 has the potential to produce biosurfactants and does not require high concentrations of carbon and nitrogen sources for its development.     

Sunflower Acid Oil-Based Production of Rhamnolipid Using Pseudomonas aeruginosa and Its Application in Liquid Detergents

Utilization of industrial waste as substrates for the rhamnolipid synthesis by Pseudomonas aeruginosa is a worthy alternative for conventionally used vegetable oils and fatty acids to reduce the production cost of rhamnolipid. Sunflower acid oil (SAO), a by-product of the oil industry, contains 70% 18:0 fatty acid, with oleic acid as a major component. In this scope, production and analysis of rhamnolipid was successfully demonstrated using SAO as a new substrate. Pseudomonas aeruginosa produced rhamnolipid (a glycolipid biosurfactant) at a maximum concentration of 4.9 g L⁻¹ with 60 g L⁻¹ of SAO in the medium. Structural properties of rhamnolipid biosurfactant are confirmed using thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and fourier transformed infrared spectroscopy (FTIR) analysis. Further surface-active properties of the crude rhamnolipid were evaluated by measuring surface tension and emulsification properties. The synthesized rhamnolipid reduced the surface tension of water to 30.12 mN m⁻¹ and interfacial tension (against heptane) to 0.52 mN m⁻¹. Moreover, rhamnolipid shows the highest emulsification index (above 80%) for vegetable oils. This study confirms the use of SAO as a potential substrate for rhamnolipid production. The synthesized rhamnolipid was incorporated in liquid detergent formulation along with alpha olefin sulfonate (AOS) and sodium lauryl ether sulfate (SLES). The performance properties including foaming and cleaning efficiency of liquid detergent were compared.     

Biosurfactant production by <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

A new biosurfactant producer, Bacillus coagulans, was isolated from soil. Its 24-h-old culture broth had a low surface tension (27–29 mN/m). Optimization of cell growth of this bacterium led to maximal biosurfactant production with glucose or starch as the organic carbon source, a pH in the range 4.0–7.5, and incubation temperatures from 20 to 45°C. The crude biosurfactants obtained after neutralization and lyophilization of the acid precipitate yielded a minimal aqueous solution surface tension value of 29 mN/m and an interfacial tension value of 4.5 mN/m against hexadecane. The critical micelle concentration of the crude biosurfactants was 17 mg/L. Addition of NaCl to the aqueous solution of the crude product caused lowering of surface tension at both the aqueous solution-air and aqueous solution-n-hexadecane interfaces. These results indicate that the biosurfactants obtained have potential environmental and industrial applications and may have uses in microbially enhanced oil recovery.     

Biosurfactant Production by Bacillus tequilensis ZSB10: Structural Characterization,Physicochemical, and Antifungal Properties

A new biosurfactant was obtained from a moderately halophilic bacterium identified as Bacillus tequilensis ZSB10 that was isolated from a saline water pond located in Tehuacan-Cuicatlan valley, Mexico. A kinetic analysis of the bacterial growth of the ZSB10 strain showed a maximum growth at 24 h regardless of the initial pH (5, 7.4, and 9). The best results were found at pH = 7.4 in terms of bacterial growth, besides which the produced biosurfactant showed emulsifying and surfactant properties with an emulsification index (E₂₄) and surface tension change (ΔST) of 54 ± 0% and 26 mN m⁻¹, respectively. Extracted ZSB10 crude biosurfactant had a yield of 106 ± 6 mg L⁻¹, an E₂₄ = 58.4 ± 0.2%, and a ΔST = 26 mN m⁻¹ with a critical micelle concentration (CMC) of 44.82 mg L⁻¹. Also, its structure was characterized by MALDI-TOF mass spectrometry as a surfactin, iturin A, and fengycin mixture whose main isoform was leu/ile-7 C₁₅ surfactin [M + Na]⁺. Finally, the ZSB10 crude biosurfactant showed antifungal activity against Helminthosporium sp., with a 79.3% growth inhibition and an IC₅₀ of 1.37 mg per disc. Therefore, this biosurfactant could be used as biopesticide.     

Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions

Traditionally, biosurfactants have been produced from hydrocarbons. Some possible substitutes for microbial growth and biosurfactant production include urban wastes, peat hydrolysate, and agro-industrial by-products. Molasses, a nonconventional substrate (agro-industrial by-product) can also be used for biosurfactant production. It has been utilized by two strains of Bacillus subtilis (MTCC 2423 and MTCC1427) for biosurfactant production and growth at 45°C. As a result of biosurfactant accumulation, the surface tension of the medium was lowered to 29 and 31 dynes/cm by the two strains, respectively. This is the first report of biosurfactant production by strains of B. subtilis at 45°C. Potential application of the biosurfactant in microbial enhanced oil recovery is also presented.     

Studies on Biosurfactants Produced using Bacillus cereus Isolated from Seawater with Biotechnological Potential for Marine Oil-Spill Bioremediation

With the aim of producing a biotensioactive material for use in the remediation of marine environments, screening for biosurfactant-producing bacteria was conducted with strains isolated from seawater contaminated with petroleum derivatives. Gene sequencing revealed that all four promising biosurfactant-producing isolates belonged to the same genus and species, namely Bacillus cereus. The biosurfactant-producing bacteria were cultivated with different carbon (glucose, soybean oil, and waste frying soybean oil) and nitrogen (ammonium chloride, sodium nitrate, urea, and peptone) sources. B. cereus strain BCS0 was chosen as the best biosurfactant producer in a mineral medium with 2% frying oil and 0.12% peptone. Following the optimization of agitation and cultivation time, an agitation rate of 250 rpm and 48 h of cultivation were selected. Under these conditions, the surface tension was reduced to 27 mN m⁻¹ and the biosurfactant concentration was 3.5 g L⁻¹. The critical micelle concentration (CMC) of the biosurfactant was defined as 500 mg L⁻¹. The biosurfactant remained stable within large ranges of pH (2–10), salinity (2–10%), and temperature (5–120 °C). Under these conditions, motor oil emulsification rates were greater than 90%. Moreover, the biosurfactant properties remained unaltered after heating at 90 °C for 120 min. The biosurfactant enhanced the degradation of motor oil up to 96% in 27 days and exhibited considerable motor oil displacement capacity. Thus, the biosurfactant has potential in the application of remediation processes in marine environments.     

Effect of surfactants on α‐amylase production in a solid substrate fermentation process

Solid substrate fermentation (SSF) is a process where the substrate is a moist solid, which is insoluble in water but not suspended in water. In this study SSF of Bacillus subtilis (ATCC 21556) was used to produce an enzyme of commercial importance, α‐amylase, using as a substrate potato peel. To enhance the production of this enzyme, two nonionic synthetic surfactants were used, Tween 80 and Tween 20, one anionic surfactant, SDS at concentrations of 0.05% and 0.10% (v/w) and a biosurfactant produced by Bacillus subtilis (ATCC 21332), known as surfactin, at concentrations of 0.003%, 0.007%, 0.013% and 0.03% (w/w). The results have shown that surfactants significantly increase the production of α‐amylase. Tween 80 at 0.10% and surfactin at 0.013% provided the highest enzyme activity when compared with the control. © 1999 Society of Chemical Industry     

Isolation and Identification of Current Biosurfactant-Producing Microbacterium maritypicum ABR5 as a Candidate for Oily Sludge Recovery

Biosurfactants have a wide range of applications in different areas, including petroleum microbiology and environmental biotechnology. In this study, removing and recovering oil from oily sludge using microbactan-producing bacteria have been investigated. The best biosurfactant-producing isolate was obtained from a petroleum reservoir and was identified by 16S rDNA analysis as Microbacterium maritypicum ABR5. Its 16S rDNA sequence was deposited in GenBank, NCBI under the accession number MK100468. Chemical analysis using thin-layer chromatography and Fourier Transform Infrared confirmed that the produced biosurfactant was glycolipoprotein. The strain reduced surface tension from 72 to 34.6 mN m⁻¹. The addition of 5 mg L ZnO nanoparticles to the biosurfactant-producing medium showed no bacterial toxicity effect and raised the emulsification index to 25.7%. Higher concentrations of ZnO nanoparticles, such as 10 and 100 mg L, decreased the bacterial growth rate and biosurfactant production. The mixing of M. maritypicum ABR5 culture medium and oily sludge increased the oil recovery from oily sludge by up to 70% after 5 days of incubation. This is the first report of biosurfactant production by a newly identified strain, M. maritypicum ABR5, isolated from a petroleum reservoir. We proposed that the isolated biosurfactant-producing strain could be considered an economical asset for oil recovery from oily sludge in the petroleum industry and environmental biotechnology.     

Chicken Tallow,a Renewable Source for the Production of Biosurfactant by Yarrowia lipolytica MTCC9520, and its Application in Silver Nanoparticle Synthesis

The present study is focused on the production of a biosurfactant using Yarrowia lipolytica MTCC 9520 by employing inexpensive lipid waste, chicken tallow from slaughterhouses. Plackett–Burman and Box–Behnken Design analyses were adopted for preliminary screening of medium variables and further optimization. The maximal yield of 4.4 g L⁻¹ of the biosurfactant was obtained from the optimized medium. The highest emulsification activity was found to be 55%, and the surface tension decreased to 37 mN m⁻¹ at the end of 96 h. The critical micelle concentration of the biosurfactant was calculated as 1.2%. The produced biosurfactant was characterized as cationic lipoprotein in type, and the proteins present in the biosurfactant were observed to have molecular weights between 75 and 100 kDa. The fatty acids composition of the biosurfactant was detected by gas chromatography–mass spectrometry (GC–MS) analysis. Fourier transform infra red (FTIR) and nuclear magnetic resonance (NMR) analysis confirmed the lipoprotein nature of the extracted biosurfactant. Thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis revealed the thermostable nature of the extracted biosurfactant. Surface plasmon resonance vibration peak at 421 nm was observed for the surfactant-stabilized silver nanoparticles (AgNP) through UV–Vis spectrophotometry. The average particle size of the synthesized AgNP was calculated as 7.2 ± 0.4 nm from transmission electron microscopy (TEM) analysis. Energy dispersive x-ray (EDX) spectroscopy exhibited the presence of silver in the synthesized nanoparticles. The zeta potential value of the synthesized AgNP was measured as −22.2 mV, and the polydispersity index was found as 2.3 through dynamic light scattering (DLS) analysis.     

Isolation,characterization, and antifungal application of a biosurfactant produced by Enterobacter sp. MS16

Isolate MS16 obtained from diesel contaminated soil, identified as Enterobacter sp. using 16S rRNA gene analysis produced biosurfactant when grown on unconventional substrates like groundnut oil cake, sunflower oil, and molasses. Of these carbon substrates used, sunflower oil cake showed highest biosurfactant production (1.5 g/L) and reduction in surface tension (68%). The biosurfactant produced by MS16 efficiently emulsified various hydrocarbons. The carbohydrates and fatty acids of the biosurfactants were studied using TLC, FTIR, NMR, and GC‐MS. The carbohydrate composition as determined by GC‐MS of their alditol acetate derivatives showed the predominance of glucose, galactose and arabinose, and hydroxyl fatty acids of chain length of C₁₆ and C₁₈ on the basis of FAMEs analysis. Biosurfactant showed antifungal activity and inhibited the fungal spore germination. Practical applications : Enterobacter sp., MS16 produces a biosurfactant composed of carbohydrates and fatty acids which exhibits excellent surface active properties. Use of industrial wastes for biosurfactant production is economical and facilitates the industrial production of this biosurfactant which has potential antifungal activity.     

Optimization and Characterization of Biosurfactant Rhamnolipid Production by Pseudomonas aeruginosa Isolated from an Artificially Contaminated Soil

Biosurfactants are surfactants biologically produced by microorganisms, presenting several advantages when compared to synthetic surfactants. Pseudomonas aeruginosa is known for producing rhamnolipids, considered one of the most interesting types of biosurfactants due to their high yields, when compared to other types. In this work, the production of rhamnolipid from P. aeruginosa was optimized. At first, the Plackett–Burman design was used to select most significant variables affecting the biosurfactant production yield among nine variables—carbon–nitrogen ratio, carbon concentration, nitrogen source, pH, cultivation time, potassium and magnesium concentrations, agitation, and temperature. Then, using main variables, a central point experimental design aiming to optimize rhamnolipid production was performed. The maximum biosurfactant concentration obtained was 0.877 mg L⁻¹. The rhamnolipid also displayed a great emulsification rate, reaching approximately 67%, and the ability to reduce water surface tension from 72.02 to 35.26 mN m⁻¹ at a critical micelle concentration (CMC) of 127 mg L⁻¹, in addition to presenting a good stability when exposed to wide pH and salinity ranges. The results suggest that rhamnolipids are promising substitutes for synthetic surfactants, especially due to lower impacts on the environment.     

Synthesis of enhanced biosurfactant by Bacillus subtilis MTCC 2423 at 45°C by foam fractionation

Production of a lipopeptide surfactant in a 6.5-L batch fermentor was carried out using Bacillus subtilis MTCC 2423 at 45°C. A good yield was obtained from sucrose (2%) substrate fermentation by continuous removal of the product by foam fractionation. The biosurfactant was recovered from collapsed foam by acid precipitation. The biosurfactant yield (4.5 g/L) was about 4.5 times higher than the yield (ca. 1 g/L) obtained by shake-flask fermentation. Surface activity of the collapsed foam was very high, and total surface activity was observed in the collapsed foam. The structural characterization of this biosurfactant produced at 45°C by the strain used in this study was recently reported. The biosurfactant was analyzed by thin-layer chromatography, infrared, ¹H nuclear magnetic resonance, and mass spectroscopy and was found to be identical to surfactin, a lipopeptide surfactant.     

Production and stability studies of the biosurfactant isolated from marine Nocardiopsis sp. B4

A potential biosurfactant producing strain, marine Nocardiopsis B4 was isolated from the West coast of India. Culture conditions involving variations in carbon and nitrogen sources were examined at constant pH, temperature and revolutions per min (rpm), with the aim of increasing productivity in the process. The biosurfactant production was followed by measuring the surface tension, emulsification assay and emulsifying index E24. Enhanced biosurfactant production was carried out using olive oil as the carbon source and phenyl alanine as the nitrogen source. The maximum production of the biosurfactant by Nocardiopsis occurred at a C/N ratio of 2:1 and the optimized bioprocess condition was pH 7.0, temperature 30° C and salt concentration 3%. The production of the biosurfactant was growth dependent. The surface tension was reduced up to 29 mN/m as well as the emulsification index E24 was 80% in 6 to 9 days. Properties of the biosurfactant that was separated by acid precipitation were investigated. The biosurfactant activity was stable at high temperature, a wide range of pH and salt concentrations thus, indicating its application in bioremediation, food, pharmaceutical and cosmetics industries.     

Multifactorial Approach to Biosurfactant Production by Adaptive Strain Candida tropicalis MTCC 230 in the Presence of Hydrocarbons

In this study, Candida tropicalis MTCC 230 was used to adapted in hydrocarbon along with glucose for biosurfactant production, showing diauxic growth during the production. Biosurfactant was characterized through TLC and FTIR analysis as surfactin, a lipopeptide. Process parameters were optimized one factor at a time, showing the highest emulsification index (%E₂₄) at 54 %. The production of biosurfactant was enhanced by using biostatistically based experimental design with the interactive effect of different parameters. On the basis of Placket–Burman design, four factors, hydrocarbon, ammonium chloride, microelements and temperature are found to be significant (P < 0.05) for the production of biosurfactant. A second order polynomial regression model in central composite design estimated the maximum biosurfactant production in terms of the emulsification index (%E₂₄). The optimum combination of different parameters for the biosurfactant production, obtained for hydrocarbon, ammonium chloride, microelements and temperature are 81.41 %, 1.63 (g/l), 1.69 (g/l) and 35.25 °C, respectively. The biosurfactant production was increased twofold after optimization and selection of interactive parameters by response surface methodology.     

A mathematical model of a fluidized bed reactor for the continuous production of solvents by immobilized Clostridium acetobutylicum

A fluidized bed reactor has been employed for the continuous production of solvents from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar. Substrate diffusion equations have been developed for the bioparticles, and a mathematical model has been advanced to describe the operation of the reactor. The model was fitted to the experimental data using the concept that not all of the biomass within the reactor was active in solvent production. On this basis, less than 5% was ‘active’ biomass.     

Batch production of biosurfactant with foam fractionation

Methods of producing the biosurfactant surfactin from cultures of Bacillus subtilis (BBK006) have been investigated. A reactor with integrated foam fractionation was designed and used in batch mode, and the performance compared with that of the same culture in shaken flasks. In the batch reactor, significant foaming occurred between 12.5 h and 14.5 h of culture time. During this period, the foam was routed through the foam fractionation column to a mechanical foam breaker, and a biosurfactant‐enriched foamate was collected. Concentration of surfactin in the foamate product was around 50 times greater than that in the culture medium. Using the integrated reactor, 136 mg L^?1 of surfactin was produced, significantly more than was achieved in shaken flasks (92 mg L^?1). The foam fractionation method allowed a real‐time measurement of the rate of surfactin production during growth. This showed that the maximum rate of production occurred at the interphase between log and stationary modes of growth, in contrast to previous work showing that surfactin is exclusively a secondary metabolite. The high value of surfactin yield in relation to biomass (Y_P/x = 0.262) indicated that surfactin was produced very efficiently by Bacillus subtilis (BBK006) in this integrated bioreactor. Copyright © 2006 Society of Chemical Industry     

Effects of various nutritional supplements on biosurfactant production by a strain of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> at 45°C

Effects of various factors on growth and biosurfactant production by Bacillus subtilis MTCC 2423 were studied. Sucrose (2%) and potassium nitrate (0.3%) were the best carbon and nitrogen sources. The addition of various metal supplements (magnesium, calcium, iron, and trace elements) greatly affected growth and biosurfactant production. The effect of the metal cations, used together, is greater than when they are used individually. The biosurfactant production increased considerably (almost double) by addition of metal supplements. Very high concentrations of metal supplements, however, inhibited biosurfactant production. Amino acids such as aspartic acid, asparagine, glutamic acid, valine, and lysine increased the final yield of biosurfactant by about 60%. The organism could produce biosurfactant at 45°C and within the pH range of 4.5–10.5. The biosurfactant was thermostable and pH stable (from 4.0 to 12.0). The capability of the organism to produce biosurfactant under thermophilic, alkaliphilic, and halophilic conditions makes it a suitable candidate for field applications. Infrared, nuclear magnetic resonance, and mass spectroscopy studies showed the surfactant to be identical to surfactin.     

Utilization of Oil Industry Residues for the Production of Rhamnolipids by Pseudomonas indica

In the present work, a new strain Pseudomonas indica MTCC 3714 was studied for the production of biosurfactants using various rice‐bran oil industry residues viz. rice‐bran, de‐oiled rice‐bran, fatty acids and waxes. Among all the carbon sources, a maximum reduction in surface tension (26.4 mN/m) was observed when the media were supplemented with rice‐bran and the biosurfactant was recovered using the ultrasonication technique as one of the steps in the extraction process. Biosurfactants were obtained in yields of about 9.6 g/L using rice‐bran as the carbon source. The structure of the biosurfactants as characterized by FT‐IR, NMR (¹H and ¹³C) and LC–MS analysis revealed that the majority of the biosurfactants were di‐rhamnolipids. The biosurfactants produced were able to emulsify various hydrocarbons and showed excellent potential in microbial enhanced oil recovery, as it was able to recover kerosene up to 70 % in a sandpack test.     

Characterization of Biosurfactant Produced by a Novel Strain of Pseudomonas aeruginosa,Isolate ADMT1

Several biosurfactant‐producing bacterial strains were isolated from petroleum‐contaminated soil. The isolate ADMT1, identified as a new strain of Pseudomonas aeruginosa, was selected for further studies on the basis of oil displacement test and emulsification index (E24). The optimal parameters for production, determined by employing Box–Behnken design, were temperature 36.5 °C and pH 7. The environmental isolate ADMT1 produced significant amount of biosurfactant (1.7 g L^?1 in 72 h) in minimal salt medium (MSM) using dextrose as the sole carbon source. The E24 value and critical micelle concentration (CMC) of the biosurfactant was 100% and 150 mg L^?1, respectively. At CMC, the surface tension of water was reduced to 28.4 mN m^?1. The biosurfactant exhibited hemolytic activity and antibacterial activity against 8 reference strains of pathogenic bacteria, including 2 methicillin‐resistant Staphylococcus aureus strains (MRSA ATCC 562 and MRSA ATCC 43300), with minimum inhibitory concentration (MIC) of 0.4 and 0.2 mg mL^?1, respectively. The structure of biosurfactant was characterized by FTIR, ¹H, and ¹³C NMR spectroscopy. 7 di‐rhamnolipid (RL) congeners were identified in the biosurfactant by ultraperformance liquid chromatography–mass spectrometry analysis. The major congeners, which constituted 67% of the RL mixture, included Rha‐Rha‐C₁₀‐C₁₀, Rha‐Rha‐C₁₂‐C₁₀, and Rha‐Rha‐C_12:1‐C₁₀. The minor congeners were Rha‐Rha‐C₁₀‐C₈, Rha‐Rha‐C_10:1‐C₁₀, Rha‐Rha‐C₁₀‐C_14:1, and Rha‐Rha‐C₁₀‐C₁₄. The congener Rha‐Rha‐C₁₀‐C₁₄ is being reported for the first time from any species of Pseudomonas. The high surface activity and E24 value make the ADMT1‐RL a potential candidate for its use in detergents, environmental bioremediation, and as an emulsifier in the food industry.     

Economic production of Bacillus subtilis SPB1 biosurfactant using local agro‐industrial wastes and its application in enhancing solubility of diesel

BACKGROUND: The present work aimed to optimize a new economic medium for lipopeptide biosurfactant production by Bacillus subtilis SPB1 for application in the environmental field as an enhancer of diesel solubility. Statistical experimental designs and response surface methodology were employed to optimize the medium components. RESULTS: A central composite design was applied to increase the production yield and predict the optimal values of the selected factors. An optimal medium, for biosurfactant production of about 4.5 g L^?1, was found to be composed of sesame peel flour (33 g L^?1) and diluted tuna fish cooking residue (40%) with an inoculum size of 0.22. Increased inoculum size (final OD₆₀₀) significantly improved the production yield. The emulsifier produced was demonstrated to be an alternative to chemically synthesized surfactants since it shows high solubilization efficiency towards diesel oil in comparison with SDS and Tween 80. CONCLUSION: Optimization studies led to a strong improvement in production yield. The emulsifier produced, owing its high solubilization capacity and its large tolerance to acidic and alkaline pH values and salinity, shows great potential for use in bioremediation processes to enhance the solubility of hydrophobic compounds. © 2012 Society of Chemical Industry