Characterization of levan produced by a Paenibacillus sp. isolated from Brazilian crude oil.

A levan-type fructooligosaccharide was produced by a Paenibacillus strain isolated from Brazilian crude oil, the purity of which was 98.5% after precipitation with ethanol and dialysis. Characterization by FTIR, NMR spectroscopy, GC-FID and ESI-MS revealed that it is a mixture of linear ß(2 â†’ 6) fructosyl polymers with average degree of polymerization (DP) of 18 and branching ratio of 20. Morphological structure and physicochemical properties were investigated to assess levan microstructure, degradation temperature and thermomechanical features. Thermal Gravimetric Analysis highlighted degradation temperature of 218 °C, Differential Scanning Calorimetry (DSC) glass transition at 81.47 °C, and Dynamic Mechanical Analysis three frequency-dependent transition peaks. These peaks, corresponding to a first thermomechanical transition event at 86.60 °C related to the DSC endothermic event, a second at 170.9 °C and a third at 185.2 °C, were attributed to different glass transition temperatures of oligo and polyfructans with different DP. Levan showed high morphological versatility and technological potential for the food, nutraceutical, and pharmaceutical industries.

Rhamnolipids inhibit aflatoxins production in Aspergillus flavus by causing structural damages in the fungal hyphae and down-regulating the expression of their biosynthetic genes.

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 23-40% and the production of aflatoxins by 93.9-99.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action.

Microbial Surfactants: Alternative to Vegetable Oil Surfactants.

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.

Metal-Biosurfactant Complexes Characterization: Binding, Self-Assembly and Interaction with Bovine Serum Albumin.

Studies on the specific and nonspecific interactions of biosurfactants with proteins are broadly relevant given the potential applications of biosurfactant/protein systems in pharmaceutics and cosmetics. The aim of this study was to evaluate the interactions of divalent counterions with the biomolecular anionic biosurfactant surfactin-C15 through molecular modeling, surface tension and dynamic light scattering (DLS), with a specific focus on its effects on biotherapeutic formulations. The conformational analysis based on a semi-empirical approach revealed that Cu2+ ions can be coordinated by three amide nitrogens belonging to the surfactin-C15 cycle and one oxygen atom of the aspartic acid from the side chain of the lipopeptide. Backbone oxygen atoms mainly involve Zn2+, Ca2+ and Mg2+. Subsequently, the interactions between metal-coordinated lipopeptide complexes and bovine serum albumin (BSA) were extensively investigated by fluorescence spectroscopy and molecular docking analysis. Fluorescence results showed that metal-lipopeptide complexes interact with BSA through a static quenching mechanism. Molecular docking results indicate that the metal-lipopeptide complexes are stabilized by hydrogen bonding and van der Waals forces. The biosurfactant-protein interaction properties herein described are of significance for metal-based drug discovery hypothesizing that the association of divalent metal ions with surfactin allows its interaction with bacteria, fungi and cancer cell membranes with effects that are similar to those of the cationic peptide antibiotics.

The biopolymer produced by Rhizobium viscosum CECT 908 is a promising agent for application in microbial enhanced oil recovery.

Polymer flooding is one of the most promising techniques used to increase the productivity of mature oil reservoirs. Polymers reduce the mobility ratio of the injected water relative to the crude oil, improving the displacement of the entrapped oil and consequently, increasing oil recovery. Biopolymers such as xanthan gum have emerged as environmentally friendly alternatives to the chemical polymers commonly employed by the oil industry. However, in order to seek more efficient biomolecules, alternative biopolymers must be studied. Here, the applicability of a biopolymer produced by Rhizobium viscosum CECT 908 in Microbial Enhanced Oil Recovery (MEOR) was evaluated. This biopolymer exhibited better rheological properties (including higher viscosity) when compared with xanthan gum. Its stability at high shear rates (up to 300 s-1), temperatures (up to 80 °C) and salinities (up to 200 g/L of NaCl) was also demonstrated. The biopolymer exhibited better performance than xanthan gum in oil recovery assays performed with a heavy crude oil, achieving 25.7 ± 0.5% of additional recovery. Thus the R. viscosum CECT 908 biopolymer is a promising candidate for application in MEOR.

The yeast-like fungus Aureobasidium thailandense LB01 produces a new biosurfactant using olive oil mill wastewater as an inducer.

In this study, the biosurfactant production by an Aureobasidium thailandense LB01 was reported for the first time. Different agro-industrial by-products (corn steep liquor, sugarcane molasses, and olive oil mill wastewater) were evaluated as alternative low-cost substrates. The composition of the culture medium was optimized through response surface methodology. The highest biosurfactant production (139±16mg/L) was achieved using a culture medium containing yeast extract (2g/L); olive oil mill wastewater (1.5%, w/w); glucose (6g/L) and KH2PO4 (1g/L) after 48h of fermentation. The partially purified biosurfactant exhibited a critical micelle concentration of 550mg/L, reducing the surface tension of water up to 31.2mN/m. Its molecular structure was found to be similar to a lauric acid ester. The biosurfactant exhibited a better performance than the chemical surfactant sodium dodecyl sulfate (SDS) in oil dispersion assays, thus suggesting its potential application in bioremediation.

Biosurfactants Produced by Marine Microorganisms with Therapeutic Applications.

Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to the discovery of novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

Novel bioemulsifier produced by a Paenibacillus strain isolated from crude oil.

BACKGROUND: Surface active compounds produced by microorganisms are attracting a pronounced interest due to their potential advantages over their synthetic counterparts, and to the fact that they could replace some of the synthetics in many environmental and industrial applications. RESULTS: Bioemulsifier production by a Paenibacillus sp. strain isolated from crude oil was studied. The bioemulsifier was produced using sucrose with and without adding hydrocarbons (paraffin or crude oil) under aerobic and anaerobic conditions at 40°C. It formed stable emulsions with several hydrocarbons and its emulsifying ability was not affected by exposure to high salinities (up to 300 g/l), high temperatures (100°C-121°C) or a wide range of pH values (2-13). In addition, it presented low toxicity and high biodegradability when compared with chemical surfactants. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that the bioemulsifier is a low molecular weight oligosaccharide-lipid complex. CONCLUSION: The production of a low molecular weight bioemulsifier by a novel Paenibacillus strain isolated from crude oil was reported. To the best of our knowledge, bioemulsifier production by Paenibacillus strains has not been previously reported. The features of this novel bioemulsifier make it an interesting biotechnological product for many environmental and industrial applications. Graphical Abstract Novel bioemulsifier from Paenibacillus sp.

Bioconversion of agro-industrial by-products in rhamnolipids toward applications in enhanced oil recovery and bioremediation.

In this work, biosurfactant production by a Pseudomonas aeruginosa strain was optimized using low-cost substrates. The highest biosurfactant production (3.2 g/l) was obtained using a culture medium containing corn steep liquor (10% (v/v)) and molasses (10% (w/v)). The biosurfactant reduced the surface tension of water up to 30 mN/m, and exhibited a high emulsifying activity (E24=60%), with a critical micelle concentration as low as 50 mg/l. The biosurfactant produced in this alternative medium was characterized as a mixture of eight different rhamnolipid congeners, being the most abundant the mono-rhamnolipid Rha-C10-C10. However, using LB medium, nine different rhamnolipid congeners were identified, being the most abundant the di-rhamnolipid Rha-Rha-C10-C10. The rhamnolipid mixture produced in the alternative medium exhibited a better performance in removing oil from contaminated sand when compared with two chemical surfactants, suggesting its potential use as an alternative to traditional chemical surfactants in enhanced oil recovery or bioremediation.

Effects of biosurfactants on the viability and proliferation of human breast cancer cells.

Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l(-1) BioEG for 48 h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.

Potential therapeutic applications of biosurfactants.

Biosurfactants have recently emerged as promising molecules for their structural novelty, versatility, and diverse properties that are potentially useful for many therapeutic applications. Mainly due to their surface activity, these molecules interact with cell membranes of several organisms and/or with the surrounding environments, and thus can be viewed as potential cancer therapeutics or as constituents of drug delivery systems. Some types of microbial surfactants, such as lipopeptides and glycolipids, have been shown to selectively inhibit the proliferation of cancer cells and to disrupt cell membranes causing their lysis through apoptosis pathways. Moreover, biosurfactants as drug delivery vehicles offer commercially attractive and scientifically novel applications. This review covers the current state-of-the-art in biosurfactant research for therapeutic purposes, providing new directions towards the discovery and development of molecules with novel structures and diverse functions for advanced applications.

Biosurfactant-producing and oil-degrading Bacillus subtilis strains enhance oil recovery in laboratory sand-pack columns.

Microbial Enhanced Oil Recovery (MEOR) technology uses microorganisms and their metabolites to retrieve unrecoverable oil from mature reservoirs. In situ stimulation of biosurfactant-producing and oil-degrading microorganisms reduces the capillary forces retaining the oil inside the reservoir and decreases its viscosity, thus promoting oil flow and consequently production. In this work, a sand-pack column model was designed to simulate oil recovery operations and evaluate mobilization of residual oil by the selected microorganisms. Four different hydrocarbon mixtures and three Bacillus subtilis strains isolated from crude oil samples were used. Additional oil recoveries ranged from 6 to 24% depending on the hydrocarbon mixture and microorganism used. Biosurfactant production was observed with all the microorganisms and hydrocarbon mixtures studied. The oils recovered after incubation with B. subtilis isolates showed a reduction in the percentage of long-chain n-alkanes and lower viscosity when compared with the original oils. The results obtained suggest that stimulation of the selected B. subtilis strains in situ can contribute to mobilize entrapped oil in mature reservoirs.

Characterization by electrospray ionization and tandem mass spectrometry of rhamnolipids produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oil.

In this work, biosurfactants produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oils were identified by proton nuclear magnetic resonance ((1)H NMR) and further characterized by mass spectrometry (MS) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) analysis in positive mode and their surface activities evaluated. Mono-rhamnolipids and di-rhamnolipids were identified for both isolates, but the most abundant were found to be mono-rhamnolipids. The similarity of rhamnolipids produced by the two strains was in good agreement with their surface activities. Both biosurfactants exhibited similar aqueous solution surface tensions, high emulsification indexes and critical micelle concentration values. The results obtained show that ESI-MS and MS/MS analysis alone provide a fast and highly specific characterization of biosurfactants produced by microbial strains.

Performance of a biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants.

In this work, the biosurfactant produced by a Bacillus subtilis strain isolated from crude oil samples was characterized and its properties compared with commercially available chemical surfactants. The purified biosurfactant production yield (Y(P/X)) was 0.20 g/gcell dry weight. The surface tension (29.0 mN/m) and critical micelle concentration (40 mg/l) were found to be similar to the values previously reported for surfactin. Temperature and pH stability of the biosurfactant were also evaluated. The biosurfactant was exposed to different temperatures (20, 37 and 46°C) during two weeks and was found to be as stable as the commercial chemical surfactants Glucopone(®)215, Glucopone(®)650, Findet(®)1214N/23 and linear alkylbenzene sulfonates (LAS). Moreover, the biosurfactant subjected to 121°C for 20 min did not exhibit a significant loss of surface activity. Contrary to the commercial chemical surfactants that were found to be stable over a wide range of pH (3.0-10.0), the biosurfactant was unstable precipitating at pH values below 5.0. The emulsification indexes showed that the biosurfactant possesses equal or superior capacity to form emulsions with n-hexadecane as compared to the commercial chemical surfactants. Moreover, the anti-adhesive activity of the biosurfactant and commercial chemical surfactants was evaluated. The biosurfactant showed some activity against Staphylococcus aureus and Escherichia coli. Nevertheless, no particular trend or special effect could be assigned to the use of commercial chemical surfactants as anti-adhesives. Results gathered in this work suggest that the biosurfactant recovered from B. subtilis EG1 constitutes an interesting alternative to the commercial chemical surfactants with potential use in several industries.