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.     

Continuous production of biosurfactant with foam fractionation

A glucose‐limited chemostat was used to determine the growth parameters of BBK006 for continuous production of the biosurfactant surfactin. The continuous cultivation exhibited low maintenance metabolism (m = 0.39 mmol_glucose g_bacteria^?1 h^?1) and high molar growth yield ( g_bacteria mol_glucose^?1). It was found that the surfactin production rate in continuous culture was not only a function of dilution rate but also varied with the initial concentration of glucose in the feed. A high steady state concentration of surfactin (18 mg L^?1) was maintained in the culture at a dilution rate of 0.2 h^?1 when glucose concentration in the feed was 0.25 g L^?1. This is the first demonstration of continuous surfactin production and recovery using glucose as a carbon source. The production of surfactin is known to be related to the age of the microorganisms and a simple mathematical model has been constructed to show how the age‐related production can be quantified. Copyright © 2006 Society of Chemical Industry     

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.     

Low-Toxic and Nonirritant Biosurfactant Surfactin and its Performances in Detergent Formulations

Developing eco-friendly, nonirritant, low-toxic, and high-efficient surface active ingredients for detergents is an ongoing challenge in the detergent field. Surfactin is one of the representative lipopeptides produced by microorganisms. In this article, we report the surfactin isolated from cell-free broth of Bacillus subtilis HSO121 and purified by reversed-phase high-performance liquid chromatography for detergent formulations. The biodegradability, acute dermal irritation, acute oral toxicity (LD₅₀ and LC₅₀), surface activity, washing efficiency, and compatibility with hard water of the purified biosurfactant surfactin have been studied to explore the feasibility for applications of the surfactin in detergents. Acute oral toxicity tests (LD₅₀ > 5000 mg kg⁻¹, LC₅₀ > 1000 mg kg⁻¹) and skin irritation tests (PII = 0) indicate that the surfactin is a low-toxic and nonirritant ingredient for detergent formulation. Moreover, the surfactin shows excellent surface and interfacial properties of emulsification and wettability, high compatibility, and stability in a wide range of temperatures, pH, and hard water and acceptable properties in biodegradability and foaming ability, which suggests that the biosurfactant surfactin is a promising ingredient for detergent formations in our daily life and for industrial applications.     

Evaluation of production of lipopeptide biosurfactants and surfactin micelles by native Bacillus of Iran,for a broader application range

Surfactin is one of the most important lipopeptide biosurfactants obtained by biocatalysts of Bacillus subtilis. The aim of this study was to isolate surfactin-producing bacilli from native soils of the country (Iran), investigate their properties, convert surfactin to surfactin micelles, determine the properties of surfactin micelles and investigate the effect of starch-coated Fe⁰ and Fe³⁺ nanoparticles on the production of surfactin from B. subtilis. To do so, 20 bacilli were isolated from the native soil sample by heat shock method and genome sequenced by SrRNA16 method. The samples with strong β-hemolysis activity were screened as surfactant-producing strains. Two species of 61 and 63 (B. subtilis subspecies. Inaquosorum) were selected and examined by quantitative and qualitative screening tests such as hemolysis activity, surfactin production, droplet aggregation, emulsifying activity, and surface tension reduction in MSM medium containing Fe⁰ and Fe³⁺ nanoparticles. Surfactin was converted to surfactin micelles by sonication and confirmed by SEM. The antimicrobial and emulsifying activity and surface tension reduction of surfactin micelles were investigated. According to the results, the surface tension reduction of surfactin micelles was greater than that of surfactin. The strain 61 (99.7%) culture in 5 L bioreactors containing Fe³⁺ nanoparticles produced more surfactin than culture of the same strain without nanoparticles. This study presents an efficient method to increase the biosynthesis of microbial metabolites.     

Structural characterization of a biosurfactant produced by Bacillus subtilis at 45°C

Structural and biochemical characterization of a biosurfactant produced by Bacillus subtilis under thermophilic conditions was performed. Preliminary structural determination of CHCl₃/CH₃OH (65∶15) extracts by thin-layer chromatographic reagents showed it to be identical to surfactin. Also, the infrared, ¹H nuclear magnetic resonance, and mass spectroscopy analysis confirmed it to be identical to surfactin. Biochemically, the surfactant was a lipopeptide-containing lipid (17.05%) and protein (13.2%). The surfactant yielded a minimal aqueous surface-tension value of 28 dyne/cm and an interfacial tension value at 0.1% concentration of 0.2 dyne/cm against diesel oil. The critical micelle concentration of the surfactant was 35 mg/L. The biosurfactant exhibited an emulsification value (E ₂₄) of 90 against diesel oil and a sand-pack oil recovery of 62%. It has potential application in microbial-enhanced oil recovery in thermophilic, alkaline, acidic, and halophilic 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     

The effects of a biosurfactant on oxygen transfer in a cyclone column reactor

A laboratory-scale cyclone column reactor was tested to determine how its oxygen transfer characteristics were affected by surfactants in the liquid medium. The volumetric oxygen transfer coefficient was greatly decreased by small quantities of the synthetic surfactants dodecyltrimethylammonium bromide and sodium dodecylsulfate, and the biosurfactant surfactin produced by Bacillus subtilis (ATCC 21332). Since the gas holdup fraction was generally increased due to foaming, the effectiveness of the surfactants was probably due to an increase in the interfacial film resistance. B. subtilis was grown in the cyclone column to 0.6 g dm^?3 with a significant level of surfactin produced while maintaining at least 75% oxygen saturation in the broth. Process optimization and scale-up of surfactin production will have to consider oxygen transfer as a key parameter.     

Performance evaluation of batch and unsteady state fed‐batch reactor operations for the production of a marine microbial surfactant

BACKGROUND: Biosurfactants are microbially derived surface‐active and amphipathic molecules produced by various microorganisms. These versatile biomolecules can find potential applications in food, cosmetics, petroleum recovery and biopharmaceutical industries. However, their commercial use is impeded by low yields and productivities in fermentation processes. Thus, an attempt was made to enhance product yield and process productivity by designing a fed‐batch mode reactor strategy. RESULTS: Biosurfactant (BS) production by a marine bacterium was performed in batch and fed‐batch modes of reactor operation in a 3.7 L fermenter. BS concentration of 4.61 ± 0.07 g L^?1 was achieved in batch mode after 22 h with minimum power input of 33.87 × 10³ W, resulting in maximum mixing efficiency. The volumetric oxygen flow rate (K_La) of the marine culture was about 0.08 s^?1. BS production was growth‐associated, as evident from fitting growth kinetics data into the Luedeking‐Piret model. An unsteady state fed batch (USFB) strategy was employed to enhance BS production. Glucose feeding was done at different flow rates ranging from 3.7 mL min^?1 (USFB‐I) to 10 mL min^?1 (USFB‐II). USFB‐I strategy resulted in a maximum biosurfactant yield of 6.2 g l^?1 with an increment of 35% of batch data. The kinetic parameters of USFB‐I were better than those from batch and USFB‐II. CONCLUSION: Comparative performance evaluation of batch and semi‐continuous reactor operations was accomplished. USFB‐I operation improved biosurfactant production by about 35% over batch mode. USFB‐I strategy was more kinetically favorable than batch and USFB‐II. © 2012 Society of Chemical Industry     

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.     

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.     

Surfactin from Bacillus velezensis H2O-1: Production and Physicochemical Characterization for Postsalt Applications

The use of surfactin, a powerful biosurfactant, is generally hampered by poor production yield. Consequently, identification of new producers and the study of operational parameters are essential. We identify Bacillus sp. H2O-1 as Bacillus velezensis, a species previously not investigated for its biosurfactant production. Among the nitrogen sources we tested, (NH₄)₂SO₄ and NH₄NO₃ were the most appropriate for surfactin production, reaching 608.5 and 659.5 mg L⁻¹, respectively. Only temperature affected the production, whereas rotation and the C/N ratio did not. Biosurfactants can be used in enhanced oil recovery (EOR) in reservoirs located in the presalt and postsalt layers, where conditions of temperature, pressure, and salinity are quite varied, requiring a study of the stability of these molecules under these conditions. We found the surfactin produced by B. velezensis to be stable at different temperatures, pH, and ionic strengths. We evaluated the concurrent effects of different salinity, temperature, and pressure conditions on surface and interfacial activities of this surfactin. Overall, we found the surfactin produced by B. velezensis H2O-1 to have considerable potential for industrial applications, mainly due to the stability of its physical and chemical characteristics when subjected to different temperatures, pressures, and salinities, in addition to its low toxicity.     

Biosurfactants Production Using Permeate from Whey Ultrafiltration and Bioproduct Recovery by Membrane Separation Process

The management of whey is a challenge for dairy products where the volume produced is remarkable. This problem is minimized through membrane separation processes (MSP) to obtain whey protein concentrate, which has high added value. However, a permeate effluent stream is still generated that is composed of lactose, vitamins, and minerals, which can serve as raw material for the production of biotechnological compounds. Thus, this study aimed to produce biosurfactants using the permeate from whey ultrafiltration as part of the culture media of the bioprocess, to recover the biosurfactant produced using MSP, and to identify the biocompound. The production was carried out using Bacillus methylotrophicus and Bacillus pumilus. The variables nitrogen source (urea or ammonium sulfate), nitrogen source concentration (0.5% or 1.0%), inducer (soybean oil or biodiesel), inducer concentration (1% or 2%), and the addition of micronutrients (with our without) were studied using a fractional factorial experimental design 2^5-1_IV. In the fermentation processes, it was possible to verify the biosurfactant production through the reduction of surface tension, obtaining a minimum value of 35.07 mN/m for B. methylotrophicus and 26.02 mN/m for B. pumilus. Recovery via MSP was an efficient strategy for biosurfactant purification, which was concentrated in the fraction of the retentate. We produced a high-value-added biocompound identified as surfactin, valuing the permeate residue from whey ultrafiltration.     

Surfactant Effects on Aeration Performance of Stirred Tank Reactors

The effect of surfactants on aeration performance in stirred tank reactors (STR) at high rates of foaming is studied. The volumetric oxygen transfer coefficient (k_La) and foaming activity estimated as foaming height (H_f) were determined. Biotechnology of lipopeptide biosurfactants from aerobic organisms, e.g., Bacillus subtilis were addressed. Using model solutions of known foam‐generating properties, high‐molecular weight surfactin and low‐molecular weight sodium dodecyl sulphate (SDS), as well as impellers of different types, with flat and fluid‐foil blades, clues on the concentration dependence of STR oxygen transfer and foaming as well as options for foam reduction in the presence of biosurfactant were sought. In response to a two‐fold decrease of surface tension by surfactin, k_La values decreased up to 30 % but remained within the range expected for the mixing system in water; the experiments with SDS showing stronger dependence on surfactant concentration and surface tension. Mixing of surfactant media by a standard six‐blade disc turbine (RT) imposed rate limitations on gassing. A low‐shear impeller Narcissus (NS) could be used to avoid bulk foam outflow, while preserving k_La values that remained unchanged. The ‘power per unit volume' correlation of k_La in stirred tanks is tested in the presence of surfactin.     

A new application of biosurfactant for the preparation of polycaprolactone/layered silicate nanocomposites

In this study, by using the biosurfactant as the clay modifier, one first attempted to prepare polycaprolactone (PCL)/montmorillonite nanocomposites via a melt‐blending method. The production of biosurfactant (surfactin) and the modification of clay proceeded simultaneously by the incubation of Bacillus subtilis CWS1. The evidence for the formation of intercalated/exfoliated nanocomposites was assessed by X‐ray diffraction spectroscopy, scanning electron microscopy, and transmission electron microscopy observations. The conclusions were also supported by the result of dynamic mechanical thermal analysis, thermogravimetric analyzer, and differential scanning calorimetry. As a result, the modified clay can be well dispersed into the PCL matrix in nanoscale sizes, because the biosurfactant is partially compatible with PCL chains intercalating into clay layers. As for mechanical properties, a marked increase in tensile strength and Young's modulus can be observed when the biosurfactant‐pretreated clay was used to replace the neat clay for the preparation of nanocomposites. Based on the considerations of thermal and mechanical properties, it was also found that 10 wt% of clay content was optimal for the preparation of nanocomposite. POLYM. ENG. SCI., 2008. © 2008 Society of Plastics Engineers     

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.     

Removal of mercury by foam fractionation using surfactin, a biosurfactant

The separation of mercury ions from artificially contaminated water by the foam fractionation process using a biosurfactant (surfactin) and chemical surfactants (SDS and Tween-80) was investigated in this study. Parameters such as surfactant and mercury concentration, pH, foam volume, and digestion time were varied and their effects on the efficiency of mercury removal were investigated. The recovery efficiency of mercury ions was highly sensitive to the concentration of the surfactant. The highest mercury ion recovery by surfactin was obtained using a surfactin concentration of 10 × CMC, while recovery using SDS required < 10 × CMC and Tween-80 >10 × CMC. However, the enrichment of mercury ions in the foam was superior with surfactin, the mercury enrichment value corresponding to the highest metal recovery (10.4%) by surfactin being 1.53. Dilute solutions (2-mg L(-1) Hg(2+)) resulted in better separation (36.4%), while concentrated solutions (100 mg L(-1)) enabled only a 2.3% recovery using surfactin. An increase in the digestion time of the metal solution with surfactin yielded better separation as compared with a freshly-prepared solution, and an increase in the airflow rate increased bubble production, resulting in higher metal recovery but low enrichment. Basic solutions yielded higher mercury separation as compared with acidic solutions due to the precipitation of surfactin under acidic conditions.     

Biomonitoring of biosurfactant production by green fluorescent protein‐marked Bacillus subtilis W1012

BACKGROUND: Biosurfactant production was investigated using two strains of Bacillus subtilis, one being a reference strain (B. subtilis 1012) and the other a recombinant of this (B. subtilis W1012) made able to produce the green fluorescent protein (GFP). RESULTS: Batch cultivations carried out at different initial levels of glucose (G₀) in the presence of 10 g L^?1 casein demonstrated that the reference strain was able to release higher levels of biosurfactants in the medium at 5.0≤G₀≤10 g L^?1 (B_max = 104–110 mg L^?1). The recombinant strain exhibited slightly lower levels of biosurfactants (B_max = 90–104 mg L^?1) but only at higher glucose concentrations (G₀ ≥ 20 g L^?1). Under these nutritional conditions, the fluorescence intensity linked to the production of GFP was shown to be associated with the cell concentration even after achievement of the stationary phase. CONCLUSION: The ability of the genetically‐modified strain to simultaneously overproduce biosurfactant and GFP even at low biomass concentration makes it an interesting candidate for use as a biological indicator to monitor indirectly the biosurfactant production in bioremediation treatments. Copyright © 2008 Society of Chemical Industry     

Production of protease by bacillus subtilis using simultaneous control of glucose and ammonium concentrations

Batch, ammonium-controlled and simultaneous glucose and ammonium controlled fermentations were compared for the production of protease by Bacillus subtilis NCIB 8054. During fermentations of B. subtilis, maximum protease production was obtained in the stationary phase. Protease production in fermentations controlled at 5 mmol dm^?3 ammonium was 1.5 times greater than in uncontrolled batch fermentations. Simultaneous control of ammonium at 5 mmol dm^?3 and glucose at 0.15 g dm^?3 using controllers based on an ammonium electrode and an oxygen electrode, doubled protease production compared with fermentations having only an ammonium control and tripled protease production compared with uncontrolled batch fermentations. The protease yield on glucose and protease yield on ammonium was increased in fermentations with simultaneous glucose and ammonium control.     

Modification of biologically active peptides: production of a novel lipohexapeptide after engineering of Bacillus subtilis surfactin synthetase

The Bacillus subtilis strain ATCC 21332 produces the lipoheptapeptidesurfactin, a highly potent biosurfactant synthesized by a largemultimodular peptide synthetase. We report the genetic engineeringof the surfactin biosynthesis resulting in the production ofa novel lipohexapeptide with altered antimicrobial activities.A combination of in vitro and in vivo recombination approacheswas used to construct a modified peptide synthetase by eliminatinga large internal region of the enzyme containing a completeamino acid incorporating module. The remaining modules adjacentto the deletion were recombined at different highly conservedsequence motifs characteristic of amino acid incorporating modulesof peptide synthetases. The primary goal of this work was toidentify permissive fusion sites suitable for the engineeringof peptide synthetase genes by genetic recombination. Analysisof the rearranged enzymes after purification from B.subtilisand from the heterologous host Escherichia coli revealed thatthe selection of the recombination site is of crucial importancefor a successful engineering. Only the recombination at a specificHHIIxDGVS sequence motif resulted in an active peptide synthetase.The expected lipohexapeptide was produced in vivo and firstevidence of a reduced toxicity against erythrocytes and an enhancedlysis of Bacillus licheniformis cells was shown.