Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Viennamycins: Lipopeptides Produced by a Streptomyces sp.

Extracts from Streptomyces sp. S4.7 isolated from the rhizosphere of edelweiss, an alpine medicinal plant, exhibited activity against Gram-positive bacteria. LC-HRMS analyses of the extracts resulted in the detection of two unknown, structurally related lipopeptides that were assumed to be responsible for the antibiotic activity. LC-MS guided isolation and structure elucidation of viennamycins A and B (1 and 2) by HR-MS/MS, 1D and 2D NMR, and Marfey's analyses revealed them to be novel compounds, with viennamycin A containing cysteic acid, a unique feature for lipopeptides. Tests for antibacterial, antifungal, and cytotoxic activities of purified viennamycins, both with and without divalent cations, did not reveal any bioactivity, suggesting that their biological function, which could not be determined in the tests used, is atypical for lipopeptides. The genome of Streptomyces sp. S4.7 was sequenced and analyzed, revealing the viennamycin biosynthetic gene cluster. Detailed bioinformatics-based analysis of the viennamycin gene cluster allowed elucidation of the biosynthetic pathway for these lipopeptides.

A comprehensive genomic and growth proteomic analysis of antitumor lipopeptide bacillomycin Lb biosynthesis in Bacillus amyloliquefaciens X030.

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering).

Production, characterization and biotechnological potential of lipopeptide biosurfactants from a novel marine Bacillus stratosphericus strain FLU5.

This work aimed at studying the potential of a new hydrocarbonoclastic marine bacterium, Bacillus stratosphericus FLU5, to produce an efficient surface-active agent BS-FLU5. Biosurfactant production was examined on different carbon sources; using the surface tension measurement and the oil displacement test. Strain FLU5 showed its capacity to produce biosurfactants from all tested substrates, in particular the residual frying oil, which is a cheap renewable carbon source alternative, thus minimizing the high cost of producing those surfactants. MALDI-TOF MS/MS analysis confirmed the presence of lipopeptides, which are identified as members of surfactin and pumilacidin series. The critical micelle concentration (CMC) of the purified lipopeptides produced by strain FLU5 was 50 mg/l. At this concentration, the surface tension of the water was reduced from 72 to 28 mN/m. Furthermore, the crude lipopeptides showed an interesting stability against a broad range of pH, temperature and salinity. In addition, the application of BS-FLU5 in oil recovery from hydrocarbons-contaminated soil (used motor oil) showed that it was more effective on the hydrocarbon-remobilization than some tested synthetic surfactants. Interestingly, the biosurfactant BS-FLU5 showed a negligible cytotoxic effect against the mammalian cells HEK293. These results highlight the applicability of the lipopeptides BS-FLU5 in different fields, especially in environmental remediation processes.

Utilization of Grape Seed Flour for Antimicrobial Lipopeptide Production by Bacillus amyloliquefaciens C5 Strain.

An endophytic Bacillus amyloliquefaciens strain called C5, able to produce biosurfactant lipopeptides with a broad antibacterial activity spectrum, has been isolated from the roots of olive tree. Optimization of antibacterial activity was undertaken using grape seed flour (GSF) substrate at 0.02, 0.2, and 2% (w/v) in M9 medium. Strain C5 exhibited optimal growth and antimicrobial activity (MIC value of 60 µg/ml) when incubated in the presence of 0.2% GSF while lipopeptide production culminated at 2% GSF. Thin layer chromatography analysis of lipopeptide extract revealed the presence of at least three active spots at Rf 0.35, 0.59, and 0.72 at 0.2% GSF. Data were similar to those obtained in LB-rich medium. MALDI-TOF/MS analysis of lipopeptide extract obtained from 0.2% GSF substrate revealed the presence of surfactin and bacillomycin D. These results show that GSF could be used as a low-cost culture medium supplement for optimizing the production of biosurfactants by strain C5.

New lipopeptide produced by Corynebacterium aquaticum from a low-cost substrate.

Conventional biosurfactants have high production costs. Therefore, the use of low-cost carbon sources for their production is attractive for industry. The ability to remain stable under various environmental conditions further extends industrial application. Here we aimed to evaluate the stability of a new lipopeptide produced by Corynebacterium aquaticum using fish residue as an unconventional energy source. The biosurfactant was produced using 3% fish residue, 2% of the microorganism, and mineral medium. Biosurfactant characterization was performed by thin layer chromatography (TLC), as well as by testing its infrared, surface tension, emulsifying activity, and ionic character. The stability of the biosurfactant was evaluated by testing its surface tension at a range of temperatures, pH, and saline concentrations, as well as after 6 months of storage. The biosurfactant was characterized as a lipopeptide due to its retention time, which was coincident with the amino acid and lipid chains obtained in the TLC analysis, being confirmed by some regions of absorption verified in the infrared analysis. The surface tension and emulsifying activity of the biosurfactant were 27.8 mN/m and 87.6%, respectively, and showed anionic character. The biosurfactant was stable at temperatures of 20 to 121 °C, in saline concentrations of 1 to 7%, and at pH close to neutrality. Based on our findings, it is possible to use unconventional sources of energy to produce a lipopeptide biosurfactant that can act under various environments.

Cyclic lipopeptide signature as fingerprinting for the screening of halotolerant Bacillus strains towards microbial enhanced oil recovery.

Cyclic lipopeptides (CLPs) are non-ribosomal biosurfactants produced by Bacillus species that exhibit outstanding interfacial activity. The synthesis of CLPs is under genetic and environmental influence, and representatives from different families are generally co-produced, generating isoforms that differ in chemical structure and biological activities. This study to evaluate the effect of low and high NaCl concentrations on the composition and surface activity of CLPs produced by Bacillus strains TIM27, TIM49, TIM68, and ICA13 towards microbial enhanced oil recovery (MEOR). The strains were evaluated in mineral medium containing NaCl 2.7, 66, or 100 g L-1 and growth, surface tension and emulsification activity were monitored. Based on the analysis of 16S rDNA, gyrB and rpoB sequences TIM27 and TIM49 were assigned to Bacillus subtilis, TIM68 to Bacillus vallismortis, and ICA13 to Bacillus amyloliquefaciens. All strains tolerated up to 100-g L-1 NaCl, but only TIM49 and TIM68 were able to reduce surface tension at this concentration. TIM49 also showed emulsification activity at concentrations up to 66-g L-1 NaCl. ESI-MS analysis showed that the strains produced a mixture of CLPs, which presented distinct CLP profiles at low and high NaCl concentrations. High NaCl concentration favored the synthesis of surfactins and/or fengycins that correlated with the surface activities of TIM49 and TIM68, whereas low concentration favored the synthesis of iturins. Taken together, these findings suggest that the determination of CLP signatures under the expected condition of oil reservoirs can be useful in the guidance for choosing well-suited strains to MEOR.

Biosurfactant-biopolymer driven microbial enhanced oil recovery (MEOR) and its optimization by an ANN-GA hybrid technique.

A lipopeptide biosurfactant produced by marine Bacillus megaterium and a biopolymer produced by thermophilic Bacillus licheniformis were tested for their application potential in the enhanced oil recovery. The crude biosurfactant obtained after acid precipitation effectively reduced the surface tension of deionized water from 70.5 to 28.25mN/m and the interfacial tension between lube oil and water from 18.6 to 1.5mN/m at a concentration of 250mgL-1. The biosurfactant exhibited a maximum emulsification activity (E24) of 81.66% against lube oil. The lipopeptide micelles were stabilized by addition of Ca2+ ions to the biosurfactant solution. The oil recovery efficiency of Ca2+ conditioned lipopeptide solution from a sand-packed column was optimized by using artificial neural network (ANN) modelling coupled with genetic algorithm (GA) optimization. Three important parameters namely lipopeptide concentration, Ca2+ concentration and solution pH were considered for optimization studies. In order to further improve the recovery efficiency, a water soluble biopolymer produced by Bacillus licheniformis was used as a flooding agent after biosurfactant incubation. Upon ANN-GA optimization, 45% tertiary oil recovery was achieved, when biopolymer at a concentration of 3gL-1 was used as a flooding agent. Oil recovery was only 29% at optimal conditions predicted by ANN-GA, when only water was used as flooding solution. The important characteristics of biopolymers such as its viscosity, pore plugging capabilities and bio-cementing ability have also been tested. Thus, as a result of biosurfactant incubation and biopolymer flooding under the optimal process conditions, a maximum oil recovery of 45% was achieved. Therefore, this study is novel, timely and interesting for it showed the combined influence of biosurfactant and biopolymer on solubilisation and mobilization of oil from the soil.

Formulation of crude oil spill dispersants based on the HLD concept and using a lipopeptide biosurfactant.

Solvent-free dispersants for crude oil spills were formulated based on the hydrophilic-lipophilic deviation (HLD) concept and using lipopeptides from Bacillus sp. GY19. The lipopeptides were recovered and concentrated from cell-free broth by foam fractionation and freeze-drying. They had good surface activity under varying temperatures, pH and NaCl levels. Moreover, the lipopeptides had low toxicity to copepods (LC50 1174mg/L) and whiteleg shrimp (LC50 1050mg/L). The characteristic curvature (Cc) of the lipopeptides showed that they were more hydrophobic (Cc 4.93) than sodium dihexyl sulfosuccinate (SDHS, Cc -0.92). The HLD equation was used to calculate the lipopeptide and the SDHS fractions in the dispersant formulations according to the equivalent alkane carbon number (EACN) of hydrocarbons and seawater salinity. The molar fraction of lipopeptides increased with increasing EACN. The lipopeptide-SDHS mixtures formed microemulsion Type III with specific hydrocarbons and crude oils. Oil displacement and baffled flask tests showed that the formulations reduced the interfacial tension and solubilized crude oil in the water column at higher efficiency than commercial dispersants or lipopeptides alone. In summary, the effectiveness of the lipopeptide-based dispersant corresponded to the optimal HLD.

Pyrene biodegradation enhancement potential of lipopeptide biosurfactant produced by Paenibacillus dendritiformis CN5 strain.

Effect of biosurfactant on biodegradation of pyrene was studied using a microbial consortium predominantly composed of Pseudomonas viridiflava (49.5%) and Pseudomonas nitroreducens (32.5%) in a batch experiment containing lipopeptidic biosurfactant, produced by Paenibacillus dendritiformis CN5 strain, and mineral salt medium. The results showed that the lipopeptide at 600 and 300mgL-1 enhanced pyrene degradation to 83.5% and 67% respectively in 24days compared to 16% degradation in its absence. However degradation of pyrene was reduced to 57% as the lipopeptide supplementation was raised to 900mgL-1. This demonstrates that the biodegradation of pyrene was found to increase with an increase in the lipopeptide concentration up to a threshold level. The experimental data were fitted to the logistic kinetic model which provided best fit with a coefficient of determination (R2) values≥0.97. Maximum specific growth rate, µmax of 0.97 and 0.69d-1 were achieved in the 600 and 300mgL-1 lipopeptide amendments in comparison to 0.54d-1 in the unamended one. The carrying capacity, Xmax increased 4.4-fold in 600mgL-1 lipopeptide supplemented samples in comparison to its absence. Generally the lipopeptide showed potential application in improving bioremediation of polycyclic aromatic hydrocarbons contaminated environmental media.

Antifungal Lipopeptides Produced by Bacillus sp. FJAT-14262 Isolated from Rhizosphere Soil of the Medicinal Plant Anoectochilus roxburghii.

This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7 mN m-1. Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.

Production of lipopeptide biosurfactants by Bacillus atrophaeus 5-2a and their potential use in microbial enhanced oil recovery.

BACKGROUND: Lipopeptides are known as promising microbial surfactants and have been successfully used in enhancing oil recovery in extreme environmental conditions. A biosurfactant-producing strain, Bacillus atrophaeus 5-2a, was recently isolated from an oil-contaminated soil in the Ansai oilfield, Northwest China. In this study, we evaluated the crude oil removal efficiency of lipopeptide biosurfactants produced by B. atrophaeus 5-2a and their feasibility for use in microbial enhanced oil recovery. RESULTS: The production of biosurfactants by B. atrophaeus 5-2a was tested in culture media containing eight carbon sources and nitrogen sources. The production of a crude biosurfactant was 0.77 g L-1 and its surface tension was 26.52 ± 0.057 mN m-1 in a basal medium containing brown sugar (carbon source) and urea (nitrogen source). The biosurfactants produced by the strain 5-2a demonstrated excellent oil spreading activity and created a stable emulsion with paraffin oil. The stability of the biosurfactants was assessed under a wide range of environmental conditions, including temperature (up to 120 °C), pH (2-13), and salinity (0-50 %, w/v). The biosurfactants were found to retain surface-active properties under the extreme conditions. Additionally, the biosurfactants were successful in a test to simulate microbial enhanced oil recovery, removing 90.0 and 93.9 % of crude oil adsorbed on sand and filter paper, respectively. Fourier transform infrared spectroscopy showed that the biosurfactants were a mixture of lipopeptides, which are powerful biosurfactants commonly produced by Bacillus species. CONCLUSIONS: The study highlights the usefulness of optimization of carbon and nitrogen sources and their effects on the biosurfactants production and further emphasizes on the potential of lipopeptide biosurfactants produced by B. atrophaeus 5-2a for crude oil removal. The favorable properties of the lipopeptide biosurfactants make them good candidates for application in the bioremediation of oil-contaminated sites and microbial enhanced oil recovery process.

Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria.

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased.

Burkholderia genome mining for nonribosomal peptide synthetases reveals a great potential for novel siderophores and lipopeptides synthesis.

Burkholderia is an important genus encompassing a variety of species, including pathogenic strains as well as strains that promote plant growth. We have carried out a global strategy, which combined two complementary approaches. The first one is genome guided with deep analysis of genome sequences and the second one is assay guided with experiments to support the predictions obtained in silico. This efficient screening for new secondary metabolites, performed on 48 gapless genomes of Burkholderia species, revealed a total of 161 clusters containing nonribosomal peptide synthetases (NRPSs), with the potential to synthesize at least 11 novel products. Most of them are siderophores or lipopeptides, two classes of products with potential application in biocontrol. The strategy led to the identification, for the first time, of the cluster for cepaciachelin biosynthesis in the genome of Burkholderia ambifaria AMMD and a cluster corresponding to a new malleobactin-like siderophore, called phymabactin, was identified in Burkholderia phymatum STM815 genome. In both cases, the siderophore was produced when the strain was grown in iron-limited conditions. Elsewhere, the cluster for the antifungal burkholdin was detected in the genome of B. ambifaria AMMD and also Burkholderia sp. KJ006. Burkholderia pseudomallei strains harbor the genetic potential to produce a novel lipopeptide called burkhomycin, containing a peptidyl moiety of 12 monomers. A mixture of lipopeptides produced by Burkholderia rhizoxinica lowered the surface tension of the supernatant from 70 to 27 mN·m(-1) . The production of nonribosomal secondary metabolites seems related to the three phylogenetic groups obtained from 16S rRNA sequences. Moreover, the genome-mining approach gave new insights into the nonribosomal synthesis exemplified by the identification of dual C/E domains in lipopeptide NRPSs, up to now essentially found in Pseudomonas strains.

[Construction and evaluation of an engineered bacterial strain for producing lipopeptide under anoxic conditions].

Biosurfactant-facilitated oil recovery is one of the most important aspects of microbial enhanced oil recovery (MEOR). However, the biosurfactant production by biosurfactant-producing microorganisms, most of which are aerobes, is severely suppressed due to the in-situ anoxic conditions within oil reservoirs. In this research, we successfully engineered a strain JD-3, which could grow rapidly and produce lipopeptide under anoxic conditions, by protoplast confusion using a Bacillus amyloliquefaciens strain BQ-2 which produces biosurfactant aerobically, and a facultative anaerobic Pseudomonas stutzeri strain DQ-1 as parent strains. The alignment of 16S rDNA sequence (99% similarity) and comparisons of cell colony morphology showed that fusant JD-3 was closer to the parental strain B. amyloliquefaciens BQ-2. The surface tension of culture broth of fusant JD-3, after 36-hour cultivation under anaerobic conditions, decreased from initially 63.0 to 32.5 mN · m(-1). The results of thin layer chromatography and infrared spectrum analysis demonstrated that the biosurfactant produced by JD-3 was lipopeptide. The surface-active lipopeptide had a low critical micelle concentration (CMC) of 90 mg · L(-1) and presented a good ability to emulsify various hydrocarbons such as crude oil, liquid paraffin, and kerosene. Strain JD-3 could utilize peptone as nitrogen source and sucrose, glucose, glycerin or other common organics as carbon sources for anaerobic lipopeptide synthesis. The subculture of fusant JD-3 showed a stable lipopeptide-producing ability even after ten serial passages. All these results indicated that fusant JD-3 holds a great potential to microbially enhance oil recovery under anoxic conditions.

Rhamnolipid and surfactin production from olive oil mill waste as sole carbon source.

Olive mill waste (OMW) creates a major environmental problem due to the difficulty of further waste processing. In this work we present an approach to give OMW added value by using it for the production of biosurfactants. Two bacterial species, Pseudomonas aeruginosa and Bacillus subtilis, were grown with OMW as the sole carbon source. Glycerol and waste frying oil were used as comparative carbon sources. B. subtilis produced surfactin (a lipopeptide) at a maximum concentration of 3.12 mg/L with 2% w/v of OMW in the medium, dropping to 0.57 mg/L with 10% w/v of OMW. In contrast, P. aeruginosa produced 8.78 mg/L of rhamnolipid with 2% w/v OMW increasing to 191.46 mg/L with 10% w/v OMW. The use of solvent-extracted OMW reduced the biosurfactant production by 70.8% and 88.3% for B. subtilis and P. aeruginosa respectively. These results confirm that OMW is a potential substrate for biosurfactant production.

Pectin Enhances Bio-Control Efficacy by Inducing Colonization and Secretion of Secondary Metabolites by Bacillus amyloliquefaciens SQY 162 in the Rhizosphere of Tobacco.

Bacillus amyloliquefaciens is a plant-beneficial Gram-positive bacterium involved in suppressing soil-borne pathogens through the secretion of secondary metabolites and high rhizosphere competence. Biofilm formation is regarded as a prerequisite for high rhizosphere competence. In this work, we show that plant extracts affect the chemotaxis and biofilm formation of B. amyloliquefaciens SQY 162 (SQY 162). All carbohydrates tested induced the chemotaxis and biofilm formation of the SQY 162 strain; however, the bacterial growth rate was not influenced by the addition of carbohydrates. A strong chemotactic response and biofilm formation of SQY 162 were both induced by pectin through stimulation of surfactin synthesis and transcriptional expression of biofilm formation related matrix genes. These results suggested that pectin might serve as an environmental factor in the stimulation of the biofilm formation of SQY 162. Furthermore, in pot experiments the surfactin production and the population of SQY 162 in the rhizosphere significantly increased with the addition of sucrose or pectin, whereas the abundance of the bacterial pathogen Ralstonia decreased. With increased production of secondary metabolites in the rhizosphere of tobacco by SQY 162 and improved colonization density of SQY 162 in the pectin treatment, the disease incidences of bacterial wilt were efficiently suppressed. The present study revealed that certain plant extracts might serve as energy sources or environmental cues for SQY 162 to enhance the population density on tobacco root and bio-control efficacy of tobacco bacterial wilt.

Production and properties of a surface-active lipopeptide produced by a new marine Brevibacterium luteolum strain.

Microbial-derived surfactants are molecules of great interest due to their environmentally friendly nature and low toxicity; however, their production cost is not competitive when compared to synthetics. Marine microorganisms are exposed to extremes of pressure, temperature, and salinity; hence, they can produce stable compounds under such conditions that are useful for industrial applications. A screening program to select marine bacteria able to produce biosurfactant using low-cost substrates (mineral oil, sucrose, soybean oil, and glycerol) was conducted. The selected bacterial strain showed potential to synthesize biosurfactants using mineral oil as carbon source and was identified as Brevibacterium luteolum. The surface-active compound reduced the surface tension of water to 27 mN m(-1) and the interfacial tension (water/hexadecane) to 0.84 mN m(-1) and showed a critical micelle concentration of 40 mg L(-1). The biosurfactant was stable over a range of temperature, pH, and salt concentration and the emulsification index (E24) with different hydrocarbons ranging from 60 to 79 %. Structural characterization revealed that the biosurfactant has a lipopeptide nature. Sand washing removed 83 % of crude oil demonstrating the potential of the biosurfactants (BS) for bioremediation purposes. The new marine B. luteolum strain showed potential to produce high surface-active and stable molecule using a low-cost substrate.

The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.