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.     

Rotating discs bioreactor,a new tool for lipopeptides production

Microbial production of two biosurfactants, fengycin and surfactin, by Bacillus subtilis ATCC 21332 in a rotating discs bioreactor was studied. Simultaneous production of these lipopeptides was performed by free and cells immobilized on the surfaces of rotating discs. The aeration applied on surface allowed a non-foaming fermentation process and an important production of lipopeptides for low microbial growth in the culture medium. It was demonstrated that the selectivity of lipopeptides synthesis could be modified varying operating conditions and that the cells immobilization improved greatly fengycin synthesis. The maximal concentration of fengycin and surfactin obtained were 838 mg L^?1 and 212 mg L^?1, respectively. The development of this bubble-less process could advance the scale-up of the fermenters for production of biosurfactants.     

Exploring the use of biosurfactants from Bacillus subtilis in bionanotechnology: A potential dispersing agent for carbon nanotube ecotoxicological studies

In this work, we evaluate the efficiency of biosurfactants produced by Bacillus subtilis LSFM-05 for the dispersion of acid-treated multi-walled carbon nanotubes (CNT-LQES₁) and the effect of dispersion on toxicity testing with Daphnia similis. Carbon nanotubes are very hydrophobic materials and they readily agglomerate in mineral water. As a result, in order to determine their toxicity it is critical to evaluate methods to disperse these nanomaterials in a biologically compatible manner. The biosurfactant used in this work, termed BioS, which is a mixture of the lipopeptides (surfactin and fengycin), was found to be non-toxic to D. similis in an acute toxicity test (48 h) and it was an excellent dispersing agent for CNT-LQES₁ in reconstituted mineral water. Monitoring in real-time using the nanoparticle tracking analysis (NTA) showed that the colloidal stability of the CNT-LQES₁ suspension dispersed with BioS was highly stable. These findings are encouraging for the application of biosurfactants as nontoxic dispersion agents in the emerging fields of bionanotechnology and nanotoxicology.     

Selective fengycin production in a modified rotating discs bioreactor

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k _L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L^?1), but also better selectivity of the bioreaction towards fengycin (up to 88 %) and better yields of fengycin per glucose (up to 62.9 mg g^?1), lipopeptides per glucose (up to 71.5 mg g^?1), fengycin per biomass (up to 309 mg g^?1) and lipopeptides per biomass (up to 396 mg g^?1) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min^?1. The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.     

Chitin and chitosan preparation from shrimp shells using optimized enzymatic deproteinization

Different crude microbial proteases were applied for chitin extraction from shrimp shells. A Box–Behnken design with three variables and three levels was applied in order to approach the prediction of optimal enzyme/substrate ratio, temperature and incubation time on the deproteinization degree with Bacillus mojavensis A21 crude protease. These optimal conditions were: an enzyme/substrate ratio of 7.75 U/mg, a temperature of 60 °C and an incubation time of 6 h allowing to predict 94 ± 4% deproteinization. Experimentally, in these optimized conditions, a deproteinization degree of 88 ± 5% was obtained in good agreement with the prediction and larger than values generally given in literature. The deproteinized shells were then demineralized to obtain chitin which was converted to chitosan by deacetylation and its antibacterial activity against different bacteria was investigated. Results showed that chitosan dissolved at 50 mg/ml markedly inhibited the growth of most Gram-negative and Gram-positive bacteria tested.     

Characterization and optimization of biosurfactants produced by Acinetobacter baylyi ZJ2 isolated from crude oil-contaminated soil sample toward microbial enhanced oil recovery applications

The present work aims to investigate the surface activity of the biosurfactant produced by Acinetobacter baylyi ZJ2 isolated from crude oil-contaminated soil sample in China and evaluate its potential application in microbial enhanced oil recovery. The biosurfactant produced by A. baylyi ZJ2 was identified as lipopeptide based on thin-layer chromatography, Fourier transform infrared spectroscopy and nuclear magnetic resonance techniques. This biosurfactant could reduce the surface tension of water from 65 mN/m to 35 mN/m, and interfacial tension against oil from 45 mN/m to 15 mN/m. Moreover, surface activity stability results showed that this biosurfactant was effective when the salinity was lower than 8% and the pH value was 4–9, and it was especially stable when the salinity was lower than 4% and pH was 6–7. Based on the result of gas chromatography, there was a decrease in heavy components and an increase in light components, which indicated that A. baylyi ZJ2 exhibited the biodegradability on the heavy components of crude oil. Furthermore, the ability of recovering oil from oil-saturated core showed that nearly 28% additional residual oil was displaced after water flooding. The lipopeptide biosurfactant produced by A. baylyi ZJ2 presented a great potential application in microbial enhanced oil recovery process, owing its good surface activity and satisfying degradation ability to crude oil.     

A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization

An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 °C. The thermostability of the enzyme was significantly increased by the addition of CaCl₂. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease.The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease.The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H₂O₂ and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 °C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents.     

Comparison of lipopeptide biosurfactants production by Bacillus subtilis strains in submerged and solid state fermentation systems using a cheap carbon source: Some industrial applications of biosurfactants

Biosurfactants, in general has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high production and purification costs limit its use in these high-volume applications. In the present study, the efficiency of two Bacillus subtilis strains viz., DM-03 and DM-04 for the production of biosurfactants in two fermentation systems viz., solid state fermentation (SSF) and submerged fermentation (SmF) was compared. Both the B. subtilis strains produced appreciable and equal amount of crude lipopeptide biosurfactants (B. subtilis DM-03: 80.0 ± 9 mg/gds in SmF and 67.0 ± 6 mg/gds in SSF; B. subtilis DM-04: 23.0 ± 5.0 mg/gds in SmF and 20.0 ± 2.5 mg/gds in SSF) in the two different fermentation systems using potato peels as cheap carbon source. These thermostable lipopeptide biosurfactants produced by B. subtilis strains either in SSF or in SmF, exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand pack column. Further, it was shown by biochemical analysis, RP-HPLC profile and IR spectra that there is no qualitative and qualitative differences in the composition of crude biosurfactants produced either in SmF or in SSF system.     

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.     

New integrated bioprocess for the continuous production,extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor

Recently, a bubbleless membrane bioreactor (BMBR) has been successfully developed for biosurfactant production by Bacillus subtilis [1]. In this study, for the first time, continuous culture were carried out for the production of surfactin in a BMBR, both with or without a coupled microfiltration membrane. Results from continuous culture showed that a significant part of biomass was immobilized onto the air/liquid membrane contactor. Immobilized biomass activity onto the air/liquid membrane contactor was monitored using a respirometric analysis. Kinetics of growth, surfactin and primary metabolites production were investigated. Planktonic biomass, immobilized biomass and surfactin production and productivity obtained in batch culture (3 L) of 1.5 days of culture were 4.5 g DW, 1.3 g DW, 1.8 g and 17.4 mg L^?1 h^?1, respectively. In continuous culture without total cell recycling (TCR), the planktonic biomass was leached, but immobilized biomass reached a steady state at an estimated 6.6 g DW. 11.5 g of surfactin was produced after 3 days of culture, this gave an average surfactin productivity of 54.7 mg L^?1 h^?1 for the continuous culture, which presented a surfactin productivity of 30 mg L^?1 h^?1 at the steady state. TCR was then investigated for the continuous production, extraction and purification of surfactin using a coupled ultrafiltration step. In continuous culture with TCR at a dilution rate of 0.1 h^?1, planktonic biomass, immobilized biomass, surfactin production and productivity reached 7.5 g DW, 5.5 g DW, 7.1 g and 41.6 mg L^?1 h^?1 respectively, after 2 days of culture. After this time, biomass and surfactin productions stopped. Increasing dilution rate to 0.2 h^?1 led to the resumption of biomass and surfactin production and these values reached 11.1 g DW, 10.5 g DW, 7.9 g and 110.1 mg L^?1 h^?1, respectively, after 3 days of culture. This study has therefore shown that with this new integrated bioprocess, it was possible to continuously extract and purify several grams of biosurfactant, with purity up to 95%.     

Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: A biocontrol antagonist of Fusarium head blight

The complete genome of the biocontrol antagonist Bacillus amyloliquefaciens AS 43.3 is reported. B. amyloliquefaciens AS 43.3 has previously been shown to be effective in reducing Fusarium head blight in wheat. The 3.9 Mbp genome was sequenced, assembled, and annotated. Genomic analysis of the strain identified 9 biosynthetic gene clusters encoding secondary metabolites associated with biocontrol activity. The analysis identified five non-ribosomal peptide synthetase clusters encoding three lipopeptides (surfactin, iturin, and fengycin), a siderophore (bacillibactin), and the antibiotic dipeptide bacilysin. In addition, three polyketide synthetase clusters were identified which encoded for the antibacterials: bacillaene, difficidin, and macrolactin. In addition to the non-ribosomal mediated biosynthetic clusters discovered, we identified a ribosomally encoded biosynthetic cluster that produces the antibiotic plantazolicin. To confirm the gene clusters were functional, cell-free culture supernatant was analyzed using LC–MS/MS. The technique confirmed the presence of all nine metabolites or their derivatives. The study suggests the strain is most likely a member of the B. amyloliquefaciens subsp. plantarium clade. Comparative genomics of eight completed genomes of B. amyloliquefaciens identify the core and pan-genomes for the species, including identifying genes unique to the biocontrol strains. This study demonstrates the growing importance of applying genomic-based studies to biocontrol organisms of plant pathogens which can enable the rapid identification of bioactive metabolites produced by a prospective biological control organism. In addition, this work provides a foundation for a mechanistic understanding of the B. amyloliquefaciens AS 43.3/Fusarium head blight biocontrol interaction.     

Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor

Surfactin and fengycin are lipopeptide biosurfactants produced by Bacillus subtilis. This work describes for the first time the use of bubbleless bioreactors for the production of these lipopeptides by B. subtilis ATCC 21332 with aeration by a hollow fiber membrane air–liquid contactor to prevent foam formation. Three different configurations were tested: external aeration module made from either polyethersulfone (reactor BB1) or polypropylene (reactor BB2) and a submerged module in polypropylene (reactor BB3). Bacterial growth, glucose consumption, lipopeptide production, and oxygen uptake rate were monitored during the culture in the bioreactors. For all the tested membranes, the bioreactors were of satisfactory bacterial growth and lipopeptide production. In the three configurations, surfactin production related to the culture volume was in the same range: 242, 230, and 188 mg l⁻¹ for BB1, BB2, and BB3, respectively. Interestingly, high differences were observed for fengycin production: 47 mg l⁻¹ for BB1, 207 mg l⁻¹ for BB2, and 393 mg l⁻¹ for BB3. A significant proportion of surfactin was adsorbed on the membranes and reduced the volumetric oxygen mass transfer coefficient. The degree of adsorption depended on both the material and the structure of the membrane and was higher with the submerged polypropylene membrane.     

Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m⁻¹. The microorganism produced 1.64 ± 0.1 g l⁻¹ of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l⁻¹ respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC₅₀ 80 μg ml⁻¹) and HT-29 (IC₅₀ 120 μg ml⁻¹).     

Inhibitory activity of surfactin,produced by different Bacillus subtilis subsp. subtilis strains,against Listeria monocytogenes sensitive and bacteriocin-resistant strains

Three surfactin-producing Bacillus subtilis strains, C4, M1 and G2III, previously isolated from honey and intestines from the Apis mellifera L. bee, were phylogenetically characterized at sub-species level as B. subtilis subsp. subtilis using gyrA gene sequencing. The antagonistic effect of surfactin was studied against seven different Listeria monocytogenes strains, 6 of which were resistant to bacteriocins. Surfactin showed anti-Listeria activity against all 7 strains and a dose of 0.125 mg/mL of surfactin was enough to inhibit this pathogen. Surfactin sintetized by B. subtilis subsp. subtilis C4 inhibited the pathogen in lower concentrations, 0.125 mg/mL, followed by G2III and M1 with 0.25 and 1 mg/mL, respectively. In particular, a dose of 0.125 mg/mL reduced the viability of L. monocytogenes 99/287 RB6, a bacteriocin-resistant strain, to 5 log orders. Surfactin assayed maintained anti-Listeria activity within a pH range of between 2 and 10, after heat treatment (boiling for 10 min and autoclaving at 121 °C for 15 min) and after treatment with proteolytic enzymes. These results suggest that surfactin can be used as a new tool for prevention and the control of L. monocytogenes in different environments, for example, in the food industry.     

An efficient method for separation of surfactin from Bacillus amyloliquefaciens fmb50 broth by flocculation

An efficient method for removing microbial cells and macromolecular impurities and purification of surfaction from fermentation broth produced by Bacillus amyloliquefaciens fmb50 was carried out in this study. Among three inorganic flocculants and a macromolecular flocculants, the combination use of CaCl₂ and Na₂HPO₄ was the most effective separation process. Addition of 50% ethanol into fermentation broth could not only disrupt the surfactin micells, but also promote the permeating of surfactin in filtration. The flocculation condition was optimized by an L₉ (3⁴) orthogonal design. The light transmittance, surfactin recovery rate, protein removal rate and filtration flux could reach to 96.3%, 95.31%, 56.59% and 3204.41 L m⁻² h⁻¹ respectively, the surfactin purity reached to 79.5% and the residual protein was 8.1% in separated product under the optimal flocculation condition (flocculants dosage 0.5%, pH 5.0, and temperature 35 °C). Validation test also demonstrated stable results under the optimal conditions. Due to higher efficiency, lower cost and scale-up more easily of flocculation and filtration processes, it is feasible to separate surfactin from fermentation broth.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Leishmanicidal activity of the crude extract,fractions and major piperidine alkaloids from the flowers of Senna spectabilis

Senna spectabilis (sin. Cassia excelsa, C. spectabilis) is an endemic tree of South America and Africa, very common in Brazil, where it is known as “canafistula-de-besouro” and “cassia-do-nordeste”. In folk medicine, this plant is indicated for the treatment of constipation, insomnia, anxiety, epilepsy, malaria, dysentery and headache. Phytopharmacological studies have also confirmed anticonvulsive, sedative, anti-malarial, antimicrobial and cytotoxic properties of many parts of S. spectabilis. In this communication, we present a comparative study of the leishmanicidal activity of the crude ethanolic extract, its fractions and also the two major alkaloidal metabolites (−)-cassine/(−)-spectaline, trying to establish a relationship between the presence of piperidine alkaloidal constituents and leishmanicidal activity. The growth inhibitory effect of promastigote forms of Leishmania major was determined for the crude extract, fractions of the flowers of S. spectabilis and a mixture of (−)-cassine/(−)-spectaline in comparison to pentamidine used as standard drug. The cytotoxic effects were assessed on macrophage strain J774 by lactate dehydrogenase assay. Fractions dichloromethane (FL-DCM) and n-butanol (FL-Bu) and a mixture of (−)-cassine/(−)-spectaline (∼7:3) exhibited significant activity against the parasite Leishmania major (IC₅₀ values of 0.6 ± 0.1 μg/ml, 1.6 ± 0.9 μg/ml and 24.9 ± 1.4 μg/ml, respectively), without toxic effects on murine macrophages. Due to the promising results elicited, further studies in vivo need to be performed to confirm the therapeutic potential of Senna spectabilis.     

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.     

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55 mg/mL) and savory (0.32 mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40 mg/mL for thyme oil and 0.21 mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).