Eduardo J. Gudiña

Isolation and functional characterization of a biosurfactant produced by Lactobacillus paracasei

In this study, the crude biosurfactant produced by a Lactobacillus paracasei strain isolated in a Portuguese dairy industry was characterized. The minimum surface tension (41.8mN/m) and the critical micelle concentration (2.5mg/ml) obtained were found to be similar to the values previously reported for biosurfactants isolated from other lactobacilli. The biosurfactant was found to be stable to pH changes over a range from 6 to 10, being more effective at pH 7, and showed no loss of surface activity after incubation at 60 degrees C for 120h. Although the biosurfactant chemical composition has not been determined yet, a fraction was isolated through acidic precipitation, which exhibited higher surface activity as compared with the crude biosurfactant. Furthermore, this isolated biosurfactant showed antimicrobial and anti-adhesive activities against several pathogenic microorganisms. In addition, L. paracasei exhibited a strong autoaggregating phenotype, which was maintained after washing and resuspending the cells in PBS, meaning that this attribute must be related to cell surface components and not to excreted factors. The autoaggregation ability exhibited by this strain, together with the antimicrobial and anti-adhesive properties observed for this biosurfactant opens the possibility for its use as an effective probiotic strain.

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.

Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20

Aims:  The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro‐organisms, including Gram‐positive and Gram‐negative bacteria, yeasts and filamentous fungi.

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.

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.

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.

Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium.

In this work, biosurfactant production by Bacillus subtilis #573 was evaluated using corn steep liquor (CSL) as culture medium. The best results were obtained in a culture medium consisting of 10% (v/v) of CSL, with a biosurfactant production of about 1.3 g/l. To the best of our knowledge, this is the first report describing biosurfactant production by B. subtilis using CSL as culture medium. Subsequently, the effect of different metals (iron, manganese, and magnesium) on biosurfactant production was evaluated using the medium CSL 10%. It was found that for all the metals tested, the biosurfactant production was increased (up to 4.1, 4.4, and 3.5 g/l for iron, manganese, and magnesium, respectively). When the culture medium was supplemented with the optimum concentration of the three metals simultaneously, the biosurfactant production was increased up to 4.8 g/l. Furthermore, the biosurfactant exhibited a good performance in oil recovery assays when compared with chemical surfactants, which suggests its possible application in microbial enhanced oil recovery or bioremediation.

Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.

Partial Characterization of Biosurfactant from Lactobacillus pentosus and Comparison with Sodium Dodecyl Sulphate for the Bioremediation of Hydrocarbon Contaminated Soil

The capability of a cell bound biosurfactant produced by Lactobacillus pentosus, to accelerate the bioremediation of a hydrocarbon-contaminated soil, was compared with a synthetic anionic surfactant (sodium dodecyl sulphate SDS-). The biosurfactant produced by the bacteria was analyzed by Fourier transform infrared spectroscopy (FTIR) that clearly indicates the presence of OH and NH groups, C=O stretching of carbonyl groups and NH nebding (peptide linkage), as well as CH2–CH3 and C–O stretching, with similar FTIR spectra than other biosurfactants obtained from lactic acid bacteria. After the characterization of biosurfactant by FTIR, soil contaminated with 7,000 mg Kg−1 of octane was treated with biosurfactant from L. pentosus or SDS. Treatment of soil for 15 days with the biosurfactant produced by L. pentosus led to a 65.1% reduction in the hydrocarbon concentration, whereas SDS reduced the octane concentration to 37.2% compared with a 2.2% reduction in the soil contaminated with octane in absence of biosurfactant used as control. Besides, after 30 days of incubation soil with SDS or biosurfactant gave percentages of bioremediation around 90% in both cases. Thus, it can be concluded that biosurfactant produced by L. pentosus accelerates the bioremediation of octane-contaminated soil by improving the solubilisation of octane in the water phase of soil, achieving even better results than those reached with SDS after 15-day treatment.

Biosurfactant-Producing Lactobacilli: Screening, Production Profiles, and Effect of Medium Composition

Biosurfactant production was screened in four lactobacilli strains. The highest biosurfactant production (excreted and cell-bound biosurfactants) was achieved with Lactobacillus paracasei ssp. paracasei A20, a strain isolated from a Portuguese dairy plant, with a decrease in the surface tension of 6.4 mN m−1 and 22.0 mN m−1, respectively. Biosurfactant production by this strain was evaluated under different culture broth compositions. The use of different nitrogen sources revealed that yeast extract is essential for bacterial growth, while peptone is crucial for biosurfactant synthesis. For biosurfactant production, the use of peptone and meat extract yielded a higher production when compared to the standard medium, with a surface tension reduction of 24.5 mN m−1 Furthermore, experiments were also conducted in a reactor with pH and temperature control. Biomass and biosurfactant production in bioreactor was higher comparing with the experiments conducted in shake flaks. The optimization procedure adopted in the current work was found to improve the biosurfactant production and opened new perspectives for the use of L. paracasei ssp. paracasei A20 as a promising biosurfactant-producer.