Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis

Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-)(1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+). Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.     

Single-gene regulated non-spore-forming Bacillus subtilis: Construction,transcriptome responses,and applications for producing enzymes and surfactin

Bacillus subtilis, a spore-forming industrial bacterium, is widely used for production of enzymes and valuable chemicals. The spore-formation, however, always results in remarkably reduced cell-density, thereby reducing product yield. Here, we constructed different non-spore-forming B. subtilis mutants via single-gene regulation. During the three spore-forming stages: signal sensing, transduction, and sporulation, we found that deleting only a single gene of sporulation, i.e. spo0A, spoIIIE, and spoIVB, can completely block the spore generation. Interestingly, the engineered non-sporulating mutants exhibited physiological heterogeneity and distinct synthetic capabilities. The spo0A-null spore-free mutant displayed remarkably high enzyme production capacity, such as 194% enhance amylase production. However, the spoIVB-null non-spore-forming mutant was especially efficient in producing secondary metabolites, such as surfactin; its flask titer increased significantly to 16.7 g/L, with the overexpression and Leu addition strategy. Our results offer a new strategy for re-modeling B. subtilis to further improve its fermentation efficiency and application.     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332

In this work, optimizing trace element composition was attempted as a primary strategy to improve surfactin production from Bacillus subtilis ATCC 21332. Statistical experimental design (Taguchi method) was applied for the purpose of identifying optimal trace element composition in the medium. Of the five trace elements examined, Mg²⁺, K⁺, Mn²⁺, and Fe²⁺ were found to be more significant factors affecting surfactin production by the B. subtilis strain. In the absence of Mg²⁺ or K⁺, surfactin yield decreased to 0.4 g/l, which was only 25% of the value obtained from the control run. When Mn²⁺ and Fe²⁺ were both absent, the production yield also dropped to ca. 0.6 g/l, approximately one-third of the control value. However, when only one of the two metal ions (Fe²⁺ or Mn²⁺) was missing, the B. subtilis ATCC 21332 strain was able to remain over 80% of original surfactin productivity, suggesting that some interactive correlations among the selected metal ions may involve. Taguchi method was thus applied to reveal the interactive effects of Mg²⁺, K⁺, Mn²⁺, Fe²⁺ on surfactin production. The results show that interaction of Mg²⁺ and K⁺ reached significant level. By further optimizing Mg²⁺ and K⁺ concentrations in the medium, the surfactin production was boosted to 3.34 g/l, which nearly doubled the yield obtained from the original control.     

Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry

The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The 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 resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the C₁₄/Leu₇ surfactin was 70 μM, with emulsification efficiency after 24 h (E₂₄) of 67.6% against crude oil. 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.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers

Addition of a small quantity of solid porous carriers (e.g., activated carbon or expanded clay) into fermentation broth significantly increased surfactin production with Bacillus subtilis ATCC 21332. Culture medium containing 25 g L(-1) of activated carbon gave an optimal surfactin yield of 3600 mg L(-1), which was approximately 36-fold higher than that obtained from carrier-free liquid culture. The marked increase in surfactin production was primarily attributed to stimulation of cell growth due to the presence of activated carbon carriers. Concentration of limiting carbon substrate (glucose) is also an important factor affecting the production of surfactin, as an initial glucose concentration of 40 g L(-1) resulted in optimal surfactin production. An appropriate agitation rate also benefited surfactin production, as the best yield appeared at an agitation rate of 200 rpm. Surfactin was purified from fermentation broth via a series of acidic precipitation and solvent extraction. The resulting product was nearly 90% pure with a recovery efficiency of ca. 72%. The purified surfactin reduced the surface tension of water from 72 to 27 mN m(-1) with a critical micelle concentration of ca. 10 mg L(-1). The surfactin product also attained an emulsion index of 70% for kerosene and diesel at a low concentration of 100 and 600 mg L(-1), respectively.     

Biosurfactant-producing Bacillus are present in produced brines from Oklahoma oil reservoirs with a wide range of salinities

Nine wells producing from six different reservoirs with salinities ranging from 2.1% to 15.9% were surveyed for presence of surface-active compounds and biosurfactant-producing microbes. Degenerate primers were designed to detect the presence of the surfactin/lichenysin (srfA3/licA3) gene involved in lipopeptide biosurfactant production in members of Bacillus subtilis/licheniformis group and the rhlR gene involved in regulation of rhamnolipid production in pseudomonads. Polymerase chain reaction amplification, cloning, and sequencing confirmed the presence of the srfA3/licA3 genes in brines collected from all nine wells. The presence of B. subtilis/licheniformis strains was confirmed by sequencing two other genes commonly used for taxonomic identification of bacteria, gyrA (gyrase A) and the 16S rRNA gene. Neither rhlR nor 16S rRNA gene related to pseudomonads was detected in any of the brines. Intrinsic levels of surface-active compounds in brines were low or not detected, but biosurfactant production could be stimulated by nutrient addition. Supplementation with a known biosurfactant-producing Bacillus strain together with nutrients increased biosurfactant production. The genetic potential to produce lipopeptide biosurfactants (e.g., srfA3/licA3 gene) is prevalent, and nutrient addition stimulated biosurfactant production in brines from diverse reservoirs, suggesting that a biostimulation approach for biosurfactant-mediated oil recovery may be technically feasible.     

Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M. A., & Zuber, P. (1988) J. Bacteriol. 170, 5662-5668]. Cell material was disintegrated by treatment with lysozyme and French press. A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP. Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester. D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process. The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin. This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages. The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride. De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety.     

Production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation using a medium based on okara with sugarcane bagasse as a bulking agent

We studied the production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation (SSF), using a medium based on okara with the addition of sugarcane bagasse as a bulking agent. The optimum proportions of okara and sugarcane bagasse were 50% each, by mass. Due to the relatively high production of proteases during SSF, pre-hydrolysis of the okara with a protease did not improve surfactin levels. The optimum temperature for surfactin production was 37 °C, with the incubation temperature affecting the ratios of the various surfactin homologues produced. Cultivation in column bioreactors with forced aeration under optimized conditions gave a surfactin level of 809 mg L^?1 of impregnating solution, which corresponds to 3.3 g kg-dry-solids^?1. This is the highest surfactin level that has been produced to date in SSF with a non-recombinant microorganism. The peak O₂ uptake rate was 20 mmol min^?1 kg-initial-dry-solids^?1, corresponding to metabolic waste heat production rate of 182 W kg-initial-dry-solids^?1. The tensioactive properties of the surfactin were similar to those reported in the literature for surfactin produced by submerged fermentation. These results suggest that it might be feasible to use SSF to produce surfactin but at large scale special attention will need to be given to heat removal.     

Second stage production of iturin A by induced germination of Bacillus subtilis RB14

Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.     

Recovery and purification of the lipopeptide biosurfactant of Bacillus subtilis by ultrafiltration

Surfactin, a lipopeptide biosurfactant produced as micelles by Bacillus subtilis, was recovered from the fermentation broth by ultrafiltration with a 30 kDa MWCO membrane. The retained surfactin micelles were then ruptured and collected in the permeate by adding methanol to 50% (v/v). The final yield of surfactin was 95%.     

Cyclic Lipopeptide Biosynthetic Genes and Products,and Inhibitory Activity of Plant-Associated Bacillus against Phytopathogenic Bacteria

The antibacterial activity against bacterial plant pathogens and its relationships with the presence of the cyclic lipopeptide (cLP) biosynthetic genes ituC (iturin), bmyB (bacillomycin), fenD (fengycin) and srfAA (surfactin), and their corresponding antimicrobial peptide products have been studied in a collection of 64 strains of Bacillus spp. isolated from plant environments. The most frequent antimicrobial peptide (AMP) genes were bmyB, srfAA and fenD (34-50% of isolates). Most isolates (98.4%) produced surfactin isoforms, 90.6% iturins and 79.7% fengycins. The antibacterial activity was very frequent and generally intense among the collection of strains because 75% of the isolates were active against at least 6 of the 8 bacterial plant pathogens tested. Hierarchical and correspondence analysis confirmed the presence of two clearly differentiated groups. One group consisted of Bacillus strains that showed a strong antibacterial activity, presented several cLPs genes and produced several isoforms of cLPs simultaneously, mainly composed of B. subtilis and B. amyloliquefaciens, although the last one was exclusive to this group. Another group was characterized by strains with very low or none antibacterial activity, that showed one or none of the cLP genes and produced a few or none of the corresponding cLPs, and was the most heterogenous group including B. subtilis, B. licheniformis, B. megaterium, B. pumilus, B. cereus and B. thuringiensis, although the last two were exclusive to this group. This work demonstrated that the antagonistic capacity of plant-associated Bacillus against plant pathogenic bacteria is related to the presence of cLP genes and to the production of the corresponding cLPs, and it is mainly associated to the species B. subtilis and B. amyloliquefaciens. Our findings would help to increase the yield and efficiency of screening methods to obtain candidate strains to biocontrol agents with a mechanism of action relaying on the production of antimicrobial cLPs.     

Isolation of new variants of surfactin by a recombinant Bacillus subtilis

A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized. Received: 18 December 1996 / Received revision: 20 February 1997 / Accepted: 28 February 1997     

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

Biosurfactants production by immobilized cells of Bacillus subtilis ATCC 21332 and their recovery by pertraction

Microbial production and isolation of biosurfactants was studied. The production of lipopeptides surfactin and fengycin was performed by free and immobilized aerobic cells of Bacillus subtilis ATCC 21332. After preliminary tests with 5 polymer materials, the particles of polypropylene foamed with powder activated carbon (PPch) were selected for lipopeptides production for their thermal and mechanical stability and for the high colonizing effect. To avoid foaming during biosurfactant production, biofilm grown on solid floating support was aerated by air injected over the surface of cultural medium. The synthesis of both lipopeptides and especially of the fengycin was greatly enhanced by the immobilization. The relationship between support wettability, colonization of the cells, and lipopeptide production was discussed. Extraction behaviour of the lipopeptides into alkanes was studied. The distribution ratio of surfactin was found to be higher than this of fengycin at the same conditions and the n-heptane was more efficient solvent for both lipopeptides. Kinetics of surfactin recovery from fermentation broth applying batch pertraction in a rotating discs contactor was studied. Lipopeptide was successfully extracted (more than 75% in the first hour) using n-heptane as liquid membrane and a 0.2 mol L⁻¹ phosphate buffer solution (pH ∼ 7.3) as receiving solution. However, the stripping of the organic liquid and surfactin accumulation into the receiving phase were less efficient.     

Enhancement of surfactin production in iron-enriched media by bacillus subtilis ATCC 21332

Production of a lipopeptide surfactant, surfactin, by Bacillus subtilis ATCC 21332 was highly enhanced when iron concentration in the medium was raised from 4 μ to the few m level. This iron enrichment of the medium also resulted in increased biomass concentration. With 1.7 m iron sulfate added to the media of different initial iron concentrations or at different stages of growth, surfactin concentration can be raised to levels of several hundred mg l⁻¹ in general. Acidification of the broth was usually observed following the iron addition, and surfactin in the broth disappeared rapidly due to precipitate as soon as the pH level dropped below 5.0; however, if this acidification phenomenon was delayed, avoided (for reasons still unknown), or reversed (by alkaline addition), surfactin production was highly enhanced to as high as 3,500 mg l⁻¹ which was almost ten times over the previously reported level for this strain and higher than most reported values for genetically improved strains.