Danka Galabova

Rhamnolipid–Biosurfactant Permeabilizing Effects on Gram-Positive and Gram-Negative Bacterial Strains

The potential of biosurfactant PS to permeabilize bacterial cells of Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis on growing (in vivo) and resting (in vitro) cells was studied. Biosurfactant was shown to have a neutral or detrimental effect on the growth of Gram-positive strains, and this was dependent on the surfactant concentration. The growth of Gram-negative strains was not influenced by the presence of biosurfactant in the media. Cell permeabilization with biosurfactant PS was shown to be more effective with B. subtilis resting cells than with Pseudomonas aeruginosa. Scanning-electron microscopy observations showed that the biosurfactant PS did not exert a disruptive action on resting cells such that it was detrimental to the effect on growing cells of B. subtilis. Low critical micelle concentrations, tender action on nongrowing cells, and neutral effects on the growth of microbial strains at low surfactant concentrations make biosurfactant PS a potential candidate for application in different industrial fields, in environmental bioremediation, and in biomedicine.

Permeabilization of Yarrowia lipolytica cells by triton X-100

We established a positive effect of the nonionic surfactant Triton X-100 on the permeability of Yarrowia lipolytica cells to p-nitrophenyl phosphate, a substrate of acid and alkaline phosphatases. Maximal permeabilization of the cells was achieved with 0.1–0.2% of Triton X-100. The enhanced cell permeability depended to a greater extent on the detergent concentration than on the ratio of cells to the detergent. The lack of proteolytic activity released from the vacuoles, the relatively low amount of phosphatases present in the supernatant after treatment with the detergent, as well as electron microscopy observations showed that the permeabilization of yeast cells with Triton X-100 is a mild process. The procedure is simple and rapid, and may be used for the assay of enzyme activities associated with surface structures.

A simple and rapid test for differentiation of aerobic from anaerobic bacteria

A rapid qualitative test is proposed for bacterial respiratory type based on 24 h culturing of bacteria in liquid medium supplemented with a redox indicator: methylene blue or resazurin. Five reference bacterial strains with definite respiratory type as well as nine bacterial isolates from a laboratory digester for methane fermentation were used. Results obtained showed that both indicators can be used for distinction of strict aerobes from other bacterial representatives with definite respiration. In addition, the resazurin is able to differentiate strict anaerobes from microaerophiles and other anaerobes. The main advantages of the methylene blue is that it is a cheap, easily accessible dye, wide-used in microbiological practice and the results obtained with it are more stable over time. It was also noticed that the test with both indicators gave reliable results for the bacterial respiration only when an inoculum up to 48 h old was used.

The Effect of Rhamnolipid Biosurfactant Produced by Pseudomonas fluorescens on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.

The Importance of Rhamnolipid-Biosurfactant-Induced Changes in Bacterial Membrane Lipids of Bacillus subtilis for the Antimicrobial Activity of Thiosulfonates

The antimicrobial properties of methyl (MTS) and ethyl (ETS) esters of thiosulfonic acid alone and in combination with rhamnolipid-biosurfactant (RL) have been characterized for their ability to disrupt the normal physiological functions of living pathogens. Bactericidal and fungicidal activities of MTS and ETS and their combination with rhamnolipid were demonstrated on strains of Pseudomonas aeruginosa, Bacillus subtilis, Alcaligenes faecalis, and Rhizopus ngtricans. It was found that the combination of rhamnolipid and thiosulfonic esters has a synergistic effect leading to decreasing of bactericidal and fungicidal concentrations of MTS and ETS. More extensively was studied the effect of rhamnolipid on the lipid composition of B. subtilis bacterial membrane. To our knowledge, in this article is reported for the first time a remarkable increase of negatively charged phospholipid cardiolipin in the presence of rhamnolipid. The capacity of RL as a surface-active substance was confirmed by scanning electron microscopy (SEM). The occurrence of surface infolds and blebs on B. subtilis shown by SEM, was not accompanied by changes in membrane permeability tested by a live/dead viability staining for fluorescence microscopy. When RL was applied in combination with MTS, a dramatic permeability shift for propidium iodide was observed in vegetative cells.

Hydrolytic Enzymes and Surfactants of Bacterial Isolates from Lubricant- Contaminated Wastewater

Fifteen bacterial monocultures were isolated from lubricant-contaminated wastewater of an electric power station in Sofia. Six isolates showed best growth in liquid media with 1.5% hexadecane, and on mineral salt agar plates supplemented with one of the following hydrocarbons: n-hexadecane, paraffin, kerosene and samples of wastewater. The ability of all isolates to produce extracellular hydrolytic enzymes and surface-active glycolipids was assessed on the basis of their growth on hydrocarbons. The study of this relatively closed micro-ecosystem revealed the existence of well-balanced microbial consortium where different members have their own role and support each other. On this basis, an alternative approach is proposed for bioaugmented clean up of wastewater contaminated with hydrocarbons and organic polymers using a mixed culture of indigenous bacteria that combines the best producers of glycolipids and hydrolytic enzymes

Anti-herpesvirus activities of Pseudomonas sp. S-17 rhamnolipid and its complex with alginate.

The rhamnolipid biosurfactant PS-17 and its complex with the polysaccharide alginate, both produced by the Pseudomonas sp. S-17 strain, were studied for their antiviral activity against herpes simplex virus (HSV) types 1 and 2. They significantly inhibited the herpesvirus cytopathic effect (CPE) in the Madin-Darby bovine kidney (MDBK) cell line. The investigations were carried out according to the CPE inhibition assay protocol. The suppressive effect of the compounds on HSV replication was dose-dependent and occurred at concentrations lower than the critical micelle concentration of the surfactant. The 50% inhibitory concentration (IC50) of rhamnolipid PS-17 was 14.5 μg/ml against HSV-1 and 13 μg/ml against HSV-2. The IC50 values of the complex were 435 μg/ml for HSV-1 and 482 μg/ml for HSV-2. The inhibitory effects of the substances were confirmed by measuring the infectious virus yields with the multicycle virus growth experimental design as well: ∆log CCID50 of 1.84−2.0 against the two types of herpes simplex viruses by rhamnolipid PS-17 (20 μg/ml), and a strong reduction of the HSV-2 virus yield under the effect of the alginate complex at a concentration of 450 μg/ml. The results indicate that rhamnolipid PS-17 and its alginate complex may be considered as promising substances for the development of anti-herpetic compounds.

Biosurfactant–rhamnolipid effects on yeast cells

Aim: The aim of this work was to study the effect of the novel surfactant PS from Pseudomonas sp. S‐17 on Saccharomyces cerevisiae 83–20 yeast cells and to compare it with the effect of the well known surfactant Triton X‐100.

Phosphatase activity and copper uptake during growth of Aspergillus niger

Abstract The fungus Aspergillus niger produced extracellular and cellular acid phosphatase activities during growth in the presence or absences of copper ions in the medium. The levels of both phosphatase activities depended on the copper concentrations in the medium. Enzyme activities were maximal at the mid-exponential growth phase, as well as at the higher copper concentration and decreased as growth time increased from 12 to 48 h. The total uptake of copper (II) by mycelia, growing at the presence of copper was highest when the levels of enzyme activities were maximal. Between 10 and 20% copper (II) was not removed by washing in 0.1 M H2SO4, suggesting that this copper (II) was bound intracellularly by mycelia of different age. On the other hand, copper ions slightly inhibited cellular acid phosphatase activity of Aspergillus niger with a Ki of 0.89 mM. Acid phosphatase overproduction, as well as copper uptake of mycelia indicated a possible participation of acid phosphatases in the resistance of the fungal strain to the copper toxicity.

Evaluation of Different Carbon Sources for Growth and Biosurfactant Production by Pseudomonas fluorescens Isolated from Wastewaters

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m-1 with phenanthrene to 49.6 mN m-1 with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m-1. The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a signifi cant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0 - 7.2 and temperature of (28 ± 2) °C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.