Production and Characterization of Rhamnolipids Produced by Pseudomonas aeruginosa DBM 3774: Response Surface Methodology Approach.

Rhamnolipids are extensively studied biosurfactants due to their potential in many industrial applications, eco-friendly production and properties. However, their availability for broader application is severely limited by their production costs, therefore the optimization of efficacy of their cultivation gains significance as well as the information regarding the physio-chemical properties of rhamnolipids resulting from various cultivation strategies. In this work, the bioprocess design focused on optimization of the rhamnolipid yield of Pseudomonas aeruginosa DBM 3774 utilizing the response surface methodology (RSM). Six carbon sources were investigated for their effect on the rhamnolipid production. The RSM prediction improved the total rhamnolipid yield from 2.2 to 13.5 g/L and the rhamnolipid productivity from 11.6 to 45.3 mg/L/h. A significant effect of the carbon source type, concentration and the C/N ratio on the composition of the rhamnolipid congeners has been demonstrated for cultivation of P. aeruginosa DBM 3774 in batch cultivation. Especially, changes in presence of saturated fatty acid in the rhamnolipid congeners, ranging from 18.8% of unsaturated fatty acids (carbon source glycerol; 40 g/L) to 0% (sodium citrate 20 g/L) were observed. This demonstrates possibilities of model based systems as basis in cultivation of industrially important compounds like biosurfactants rhamnolipids and the importance of detailed study of interconnection between cultivation conditions and rhamnolipid mixture composition and properties.

Aspergillus fumigatus DBM 4057 biofilm formation is inhibited by chitosan, in contrast to baicalein and rhamnolipid.

The biofilms of filamentous-forming fungi are a novel and still insufficiently understood research topic. We have studied Aspergillus fumigatus, an ubiquitous opportunistic pathogenic fungus, as a representative model for a study of biofilm formation by filamentous fungi and for assessing the potential anti-biofilm activity of natural substances. The activity of antibiotic amphotericin B and selected natural substances: baicalein, chitosan and rhamnolipid was studied. The minimum suspension inhibitory concentrations (MIC) were determined and the biofilm susceptibility was investigated by determining the metabolic activity of sessile cells (XTT assay) and total biofilm biomass (crystal violet staining). Significant time-dependent differences in substances' anti-biofilm activity were observed. Images of A. fumigatus biofilm were obtained by Cellavista automatic light microscope and spinning disc confocal microscopy. Baicalein and rhamnolipid were not found as suitable substances for inhibition of the A. fumigatus biofilm formation, as neither of the substances inhibited the sessile cells metabolic activity or the total biofilm biomass even at tenfold MIC after 48 h. In contrast, chitosan at 10 × MIC (25 µg mL-1), suppressed the biofilm metabolic activity by 90 % and the total biofilm biomass by 80 % even after 72 h of cultivation. Amphotericin B inhibited only 14 % of total biofilm biomass (crystal violet staining) and 35 % of metabolic activity (XTT assay) of adherent cells under the same conditions. Our results therefore suggest chitosan as potential alternative for treating A. fumigatus biofilm-associated infections.

Multicomponent biosurfactants--A "Green Toolbox" extension.

The unflagging interest in the surfactants of biological origin, representing ecological alternatives to their synthetic counterparts, has enhanced R&D effort both to produce their new types and to resolve the bottlenecks of their commercialization. In this context, the rhamnolipids, offering a relatively large scale of potential applications, variety of congeners, low toxicity as well as stability towards the extremes of environment, logically attract attention. In this connection, the current state of knowledge concerning these compound exploitation, biosynthesis control and non-genetic factors affecting both production yield and final rhamnolipid product is surveyed.

Structural and physiochemical characterization of rhamnolipids produced by Acinetobacter calcoaceticus, Enterobacter asburiae and Pseudomonas aeruginosa in single strain and mixed cultures.

Rhamnolipids are naturally occurring biosurfactants with a wide range of potential commercial applications. As naturally derived products they present an ecological alternative to synthetic surfactants. The majority of described rhamnolipid productions are single strain Pseudomonas spp. cultivations. Here we report rhamnolipids producing bacteria Acinetobacter calcoaceticus, Enterobacter asburiae and Pseudomonas aeruginosa that were cultivated separately and as mixed populations. The ratio and composition of rhamnolipid congeners was determined by tandem mass spectrometry with negative electrospray ionization. Mono-rhamnolipid and di-rhamnolipid homologues containing one or two saturated or monounsaturated 3-hydroxy fatty acids were found in all strains. Physiochemical characterization of rhamnolipids was evaluated by the critical micelle concentration determination, the emulsification test, oil displacement test and phenanthrene solubilization. Critical micelle concentrations of rhamnolipids produced by both single strain and mixed cultures were found to be very low (10-63 mg/l) and to correspond with saturated/unsaturated fatty acid content of rhamnolipid homologues. The rhamnolipids produced by all strains effectively emulsified crude petroleum in comparison with synthetic surfactants Tween 80 and sodium dodecyl sulfate (SDS). Good performance of phenanthrene solubilization was exhibited by rhamnolipids from E. asburiae. The single strain and co-cultures cultivations were proposed as a possible way to produce rhamnolipid mixtures with a specific composition and different physiochemical properties, which could be exploited in bioremediation of various hydrophobic contaminants.

Characterization of rhamnolipids produced by non-pathogenic Acinetobacter and Enterobacter bacteria.

Rhamnolipid production by two non-pathogenic bacterial strains Acinetobacter calcoaceticus and Enterobacter asburiae, and established rhamnolipid producer Pseudomonas aeruginosa was investigated. Rhamnolipids were separated from supernatant and further purified by thin-layer chromatography. Mass spectrometry with negative electrospray ionization revealed rhamnolipid homologues varying in chain length and unsaturation. Tandem mass spectrometry identified mono-rhamnolipid and di-rhamnolipid homologues containing one or two 3-hydroxy fatty acids. Several media differing in carbon (sunflower oil, glycerol and sodium citrate), nitrogen (ammonium ions, nitrate) and phosphorus (total content) source, respectively, were tested to obtain enhanced rhamnolipid production. The best production (0.56g/l) was obtained when nitrate was used as a nitrogen source. Both strains produced rhamnolipids that exhibited excellent emulsification activity with aromatic and aliphatic hydrocarbons and several plant oils. Unlike P. aeruginosa the two strains, i.e. Acinetobacter and Enterobacter, are not pathogenic to humans.