A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, a two-step membrane filtration process was developed using a lab scale tangential flow filtration (TFF) unit with 10 kDa MWCO regenerated cellulose (RC) and polyethersulfone (PES)membranes at three different transmembrane pressure (TMP) of 1.5 bar, 2.0 bar and 2.5 bar. Two modes of filtrations were studied, with and without cleaning of membranes prior to UF-2. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effectively retained by the membrane in UF-2. Flux of permeates, rejection coefficient (R) of surfactin and proteinwere measured during the filtrations. Overall the three different TMPs applied have no significant effect in the filtrations and PES is the more suitable membrane to selectively separate surfactin from fermentation broth, achieving high recovery and level of purity. In addition this two-step UF process is scalable for larger volume of samples without affecting the original functionality of surfactin, although membranes permeability can be affected due to exposure to methanolic solution used in UF-2.

Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and it is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, two-step membrane filtration processes were evaluated using centrifugal and stirred cell devices while the mechanisms of separation were investigated by particle size and surface charge measurements. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effective]), retained by the membrane in UF-2. Ultrafiltration was carried out either using centrifugal devices with 30 and 10 kDa MWCO regenerated cellulose membranes, or a stirred cell device with 10 kDa MWCO polyethersulfone (PES) and regenerated cellulose (RC) membranes. Total rejection of surfactin was consistently observed in UF-1, while in UF-2 PES membranes had the lowest rejection coefficient of 0.08 +/- 0.04. It was found that disruption of surfactin micelles, aggregation of protein contaminants and electrostatic interactions in UF-2 can further improve the selectivity of the membrane based purification technique. (C) 2007 Elsevier B.V. All rights reserved.

Surface activity of surfactin recovered and purified from fermentation broth using a two-step ultrafiltration (UF) process

B. subtilis under certain types of media and fermentation conditions can produce surfactin, a biosurfactant which belongs to the lipopeptide class. Surfactin has exceptional surfactant activity, and exhibits some interesting biological characteristics such as antibacterial activity, antitumoral activity against ascites carcinoma cells, and a hypocholesterolemic activity that inhibits cAMP phosphodiesterase, as well as having anti-HIV properties. A cost effective recovery and purification of surfactin from fermentation broth using a two-step ultrafiltration (UF) process has been developed in order to reduce the cost of surfactin production. In this study, competitive adsorption of surfactin and proteins at the air-water interface was studied using surface pressure measurements. Small volumes of bovine serum albumin (BSA) and β-casein solutions were added to the air-water interface on a Langmuir trough and allowed to stabilise before the addition of surfactin to the subphase. Contrasting interfacial behaviour of proteins was observed with β-casein showing faster initial adsorption compared to BSA. On introduction of surfactin both proteins were displaced but a longer time were taken to displace β-casein. Overall the results showed surfactin were highly surface-active by forming a β-sheet structure at the air-water interface after reaching its critical micelle concentration (CMC) and were effective in removing both protein films, which can be explained following the orogenic mechanism. Results showed that the two-step UF process was effective to achieve high purity and fully functional surfactin.

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims: To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes, Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces. Methods and Results: Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10(2) CFU cm(-2). On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion. Conclusions: The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria. Significance and Impact of the Study: This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes, Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria

Biofilms represent a great concern for food industry, since they can be a source of persistent contamination leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, microbial compounds that can modify the physicochemical properties of surfaces changing bacterial interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of L monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% adhesion of L monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids can be explored to control the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens. (C) 2011 Elsevier Ltd. All rights reserved.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

Abstract Background Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. Results A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu. Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. Conclusion AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Surfactina: propriedades químicas, tecnológicas e funcionais para aplicações em alimentos

Surfactin, a lipopeptide produced by strains of Bacillus subtilis, has been proved to be a suitable biosurfactant in several applications. For many years, it has been investigated mainly for oil recovery and environmental usage. Its chemical, technological and functional characteristics turn surfactin into an attractive compound for several utilizations. In this review we emphasize some aspects of surfactin as a new food ingredient and its potential pharmaceutical and health applications.

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 (GO) 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 <= G(0) <= 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 (G(0) >= 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. (C) 2008 Society of Chemical Industry

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

Characterization of lipopeptides from Paenibacillus sp (IIRAC30) suppressing Rhizoctonia solani

The main aim was to identify the active compound against Rhizoctonia solani produced by the cassava endophyte Paenibacillus sp. IIRAC-30. The compounds produced were extracted with ethyl acetate and purified by Sephadex column prior to analysis by Q-TOF mass spectrometry. A C(15)-lipopeptide with an estimated molecular weight of 1036 Da and homologues were identified. The lipopeptide had a cyclic structure, which was deduced by interpreting the ESI-MS/MS spectra of main protonated homologues containing 15:0 FA, and the amino acid composition was Glu-Leu-Leu-Val-Asp-Leu-Leu. Therefore, the lipopeptides produced by isolate IIRAC-30 was characterized as a surfactin series. Thus, the main mechanism used by Paenibacillus sp. IIRAC-30 to suppress R. solani was elucidated. Furthermore, because lipopeptides active against phytopathogens generally show low toxicity to humans and the environment, the positive findings presented here suggest that the isolate IIRAC-30 could be a possible candidate for biocontrol of R. solani.

Antimicrobial peptide genes in Bacillus strains from plant environments

The presence of the antimicrobial peptide (AMP) biosynthetic genes srfAA (surfactin), bacA (bacylisin), fenD (fengycin), bmyB (bacyllomicin), spaS (subtilin), and ituC (iturin) was examined in 184 isolates of Bacillus spp. obtained from plant environments (aerial, rhizosphere, soil) in the Mediterranean land area of Spain. Most strains had between two and four AMP genes whereas strains with five genes were seldom detected and none of the strains had six genes. The most frequent AMP gene markers were srfAA, bacA, bmyB, and fenD, and the most frequent genotypes srfAA-bacA-bmyB and srfAAbacA-bmyB-fenD. The dominance of these particular genes in Bacillus strains associated with plants reinforces the competitive role of surfactin, bacyllomicin, fengycin, and bacilysin in the fitness of strains in natural environments. The use of these AMP gene markers may assist in the selection of putative biological control agents of plant pathogens

Surfactina: propriedades químicas, tecnológicas e funcionais para aplicações em alimentos

Surfactin, a lipopeptide produced by strains of Bacillus subtilis, has been proved to be a suitable biosurfactant in several applications. For many years, it has been investigated mainly for oil recovery and environmental usage. Its chemical, technological and functional characteristics turn surfactin into an attractive compound for several utilizations. In this review we emphasize some aspects of surfactin as a new food ingredient and its potential pharmaceutical and health applications.

Microbial Biosurfactants – Review

Biosurfactants are surface active compounds released by microorganisms. They are biodegradable non-toxic and eco-friendly materials. In this review we have updated the information about different microbial surfactants. The biosurfactant production depends on the fermentation conditions, environmental factors and nutrient availability. The extraction of the biosurfactants from the cell-free supernatant using the solvent extraction procedure and the qualitative and quantitative analysis has been discussed with appropriate equipment details. The application of the biosurfactant includes biomedical, cosmetic and bioremediation. The type of microbial biosurfactants include trehalose lipids, rhamnolipids, sophorolipids, glycolipids, cellobiose lipids, polyol lipids, diglycosyl diglycerides, lipoloysaccharides, arthrofactin, lichensyn A and B, surfactin, viscosin, phospholipids, sulphonyl lipids and fatty acids. Rhamnolipid biosurfactants produced by Pseudomonas aeruginosa DS10-129 showed significant applications in the bioremediation of hydrocarbons in gasoline spilled soil and petroleum oily sludge. Rhamnolipid biosurfactant enhanced the bioremediation process by releasing the weathered oil from the soil matrices and enhanced the bioavailability of hydrocarbons for microbial degradation. It is having potential applications in the remediation of hydrocarbon contaminated sites. Biosurfactants from marine microorganisms also offer great potential in bioremediation of oil contaminated oceanic environments

Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process

The aim of this work was to investigate the lipopeptides aggregation behavior in single and mixed solutions in a wide range of concentrations, in order to optimize their separation and purification following the two-step ultrafiltration process and using large pore size membranes (up to MWCO = 300 kDa). Micelle size was determined by dynamic light scattering. In single solutions of lipopeptide both surfactin and mycosubtilin formed micelles of different size depending on their concentration, micelles of average diameter = 5–105 nm for surfactin and 8–18 nm for mycosubtilin. However when the lipopeptides were in the same solution they formed mixed micelles of different size (d = 8 nm) and probably conformation to that formed by the individual lipopeptide, this prevents their separation according to size. These lipopeptides were purified from fermentation culture by the two-step ultrafiltration process using different MWCO membranes ranging from 10 to 300 kDa. This led to their effective rejection in the first ultrafiltration step by membranes with MCWO = 10–100 kDa but poor rejection by the 300 KDa membrane. The lipopeptides were recovered at 90% purity (in relation to protein) and with 2.34 enrichment in the permeate of the second ultrafiltration step with the 100 KDa membrane upon addition of 75% ethanol.