Rhodococcus rhodochrous IEGM 1360, an Effective Biocatalyst of C3 Oxidative Transformation of Oleanane Triterpenoids.

The optimal conditions for C3 oxidative biotransformation of 1.0 g/L pentacyclic triterpenoids oleanolic (OA) and glycyrrhetinic (GA) acids were determined using the resting cells of Rhodococcus rhodochrous IEGM 1360 from the Regional Specialised Collection of Alkanotrophic Microorganisms. Resting cell suspensions (OD600 2.6, pH 8.0, and OD600 2.2, pH 6.0) showed the highest catalytic activity against OA and GA, resulting in the formation of 61 and 100% of their 3-oxo derivatives, respectively. Using phase contrast, atomic force, and confocal laser scanning microscopy, an adaptive response of rhodococci to the effects of OA and GA was revealed. In silico, the apoptotic activity of 3-oxo-OA and antioxidant activity of 3-oxo-GA have been assumed. In vitro, a pronounced antibacterial activity of 3-oxo-OA against Micrococcus luteus, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was shown. The absence of toxic effects of the above triterpenoids and their 3-oxo derivatives on aquatic objects and plants was demonstrated in silico and in vitro, respectively. Supplementary Information: The online version contains supplementary material available at 10.1134/S0026261722603360.

Ecological criteria for assessing the content of petroleum hydrocarbons in the main soils of coniferous-deciduous forests and forest steppe.

The effect of pollution of Albicluvisols/Retisols, Calcaric Leptosols, Luvic Phaeozems, Greyzamic Phaeozems and Folic Fluvisols with oil (Solovatovsky oil field, Perm region) added in amounts of 1, 2, 3 and 5 g oil/kg of soil on the organisms was studied in a model laboratory experiment. Oil addition showed phytotoxic effects on root length in Triticum aestivum L., Lepidium sativum L., Picea obovata Ledeb. and Pinus sylvestris L. in all soils. However, oil contamination of Calcaric Leptosols and Greyzamic Phaeozems led to growth stimulation in Picea obovata seedlings. A remarkable shift in the diversity and number of colony-forming units of heterotrophic and oil-oxidizing bacteria was detected in all soil types. The maximum decrease in biodiversity (45%) was noted for heterotrophic bacteria in Luvic Phaeozems. Aqueous extracts from all oil-contaminated soils had a toxic effect on Chlorella vulgaris Beijer, causing an increase in biomass by more than 30%, but did not show acute toxicity on Daphnia magna Straus. Oil addition in the range of 1-3 g oil/kg soil posed no environmental risk to human health. However, oil addition at 5 g oil/kg of soil led to an increase in the level of carcinogenic risk to children to the threshold values of acceptable risk and ranged from 0.95 × 10-4 for Greyzamic Phaeozems and Folic Fluvisols to 1.098 × 10-4 for Luvic Phaeozems. Our results suggest that the reaction of test organisms to oil pollution depends on the soil type, and their complex application makes it possible to identify the most sensitive factor and assess the dangerous level of pollution.

Effects of Monoacyltrehalose Fraction of Rhodococcus Biosurfactant on the Innate and Adaptive Immunity Parameters In Vivo.

The biosurfactant monoacyltrehalose fraction isolated from Rhodococcus ruber IEGM 231 actinobacterium suppresses antibody production, bactericidal potential, and production of IL-1ß by mouse peritoneal cells after intraperitoneal and intramuscular injection and stimulates the production of IL-10 after intraperitoneal injection. The data of in vitro experiments attest to an important role of bacterial glycolipids in the regulation of the functions of splenocytes and peritoneal macrophages.

Effects of Glycolipid Rhodococcus Biosurfactant on Innate and Adaptive Immunity Parameters In Vivo.

The glycolipid biosurfactant complex from actinobacterium Rhodococcus ruber IEGM 231 inhibits the innate and adaptive immunity parameters after intraperitoneal and intramuscular injection. Marked suppression of antibody production, bactericidal potential, and production of proinflammatory cytokines by peritoneal macrophages, detected in vivo, do not agree with the previously detected immunostimulatory activity of biosurfactants towards the immunocompetent cell cultures; this fact indicates an important role of the cell environment in the formation of immune response under the effect of bacterial glycolipids.

[Methods of microorganism immobilization for dynamic atomic-force studies (review)].

Atomic-force microscopy (AFM) is an efficient method for studying the surface ultrastructure and nanomechanical properties of biological objects (including microorganisms). A correctly selected method of microorganism immobilization (that provides a strong attachment of cells on the surface of a biologically inert substrate and the preservation of their native properties) is an important condition of AFM scanning in a liquid medium. Comparative characteristics of methods of microorganism immobilization (that are applied in dynamic AFM studies) are given in the review. Technologies of mechanical retention and chemical binding of cells to a substrate, as well as protein and immunospecific adsorption, are considered.

Effect of glycolipid Rhodococcus biosurfactant on secretory activity of neutrophils in vitro.

Glycolipid biosurfactant synthesized by nonpathogenic strain Rhodococcus ruber IEGM231 modulated the production of ROS and IL-8 by peripheral blood neutrophils in spontaneous and stimulated cultures. Secretion of IL-1ß Ð¸ TNF-α by neutrophils in the presence of biosurfactant changed insignificantly.

[Adaptation of coimmobilized Rhodococcus cells to oil hydrocarbons in a column bioreactor].

The possible adaptation of the association of Rhodococcus ruber and Rhodococcus opacus strains immobilized on modified sawdust to oil hydrocarbons in a column bioreactor was investigated. In the bioreactor, the bacterial population showed higher hydrocarbon and antibiotic resistance accompanied by the changes in cell surface properties (hydrophobicity, electrokinetic potential) and in the content of cellular lipids and biosurfactants. The possibility of using adapted Rhodococcus strains for the purification of oil-polluted water in the bioreactor was demonstrated.

[Effect of cultivation conditions on the adhesive activity of rhodococci towards n-hexadecane].

The effect of cultivation conditions (the composition, acidity, and salinity of the cultivation medium; temperature; and the hydrodynamic conditions of cultivation) on the adhesion of actinobacteria of the genus Rhodococcus to n-hexadecane has been investigated. A study performed showed that the adhesive activity of rhodococci depends on the composition of the cultivation medium and on the cultivation temperature. The possible mechanisms underlying the effect of growth conditions on the adhesion ofrhodococci to liquid hydrocarbons and involving changes in the cell lipid content or the zeta potential of cells are addressed. Rhodococcal strains displaying high adhesive activity (80-90%) at a low temperature (18 degrees C), high salinity (5.0% NaCl), and acidity (pH 6.0) of the cultivation medium have been selected as a result of the present work; these strains have a considerable potential for use in bioremediation of soil and water contaminated by hydrocarbons.

Biodegradation of drotaverine hydrochloride by free and immobilized cells of Rhodococcus rhodochrous IEGM 608.

Drotaverine [1-(3,4-diethoxybenzylidene)-6,7-diethoxy-1,2,3,4-tetrahydroisoquinoline] hydrochloride, an antispasmodic drug derived from benzylisoquinoline was evaluated for its biodegradability using a bacterial strain Rhodococcus rhodochrous IEGM 608. The experiments were performed under aerobic conditions with rhodococci cultures able to degrade drotaverine. In the presence of glucose, the removal efficiency of drotaverine by free Rhodoccocus cells pre-grown with isoquinoline was above 80 % (200 mg/l, initial concentration) after 25 days. Rhodococcus immobilization on hydrophobized sawdust enhanced the biodegradation process, with the most marked drotaverine loss being observed during the first 5 days of fermentation. High metabolic activity of rhodococcal cells towards drotaverine was confirmed respirometrically. GC-MS analysis of transformation products resulting from drotaverine biodegradation revealed 3,4-diethoxybenzoic acid, 3,4-diethoxybenzaldehyde and 3,4-diethoxybenzoic acid ethyl ester which were detected in the culture medium until drotaverine completely disappeared. Based on these major and other minor metabolites, putative pathways for drotaverine biodegradation were proposed. The obtained data broadened the spectrum of organic xenobiotics oxidized by Rhodoccocus bacteria and proved their potential in decontamination of natural ecosystems from pharma pollutants.

[Adsorptive immobilization of rhodococcal cells in hydrophobized derivatives of wide-pore polyacrylamide cryogel].

Adsorption of Rhodococcus ruber cells on columns with polyacrylamide cryogel (CryoPAAG) partially hydrophobized by different quantities (0.2, 1, and 5 mol %) of chemically grafted n-dodecane residues has been studied. The adsorption capacity (1.1 x 10(9) cells/g) of gel carrier for rhodococcal cells and the optimal content (1 mol %) of hydrophobizing groups were determined. The respirometric method showed the high catalytic activity and functional stability of immobilized bacterial cells. Respiratory activity of immobilized rhodococci in the presence of a model mixture of oil hydrocarbons exceeded the respective parameter for free cells by 12-17%. Viability of rhodococcal cells adsorptionally fixed in hydrophobized cryoPAAG was maintained at a level of 93-95% after a half-year period of storage. The results may be used for development of immobilized biocatalyst for directed transformation of hydrocarbon compounds and biological purification of oil-polluted water.

[Biocatalytic synthesis of pharmacology perspective stigmast-4-en-3-one using Rhodococci cells].

The conditions for a directed biocatalytic oxidation of beta-sitosterol to a pharmacologically promising stigmast-4-en-3-one using Rhodococcus actinobacteria were selected. It was shown that palmitic acid induced the cholesterol oxidase reaction and allowed for the decrease in the bioconversion process duration from 7 to 5 days. The maximum level ofstigmast-4-ene-3-one formation was achieved using n-hexadecane as an additional growth substrate. With increased concentrations of beta-sitosterol (up to 2 g/L) an effective target product formation (80%) was achieved in the presence of Tween-80 and beta-cyclodextrine. R. erythropolis strains were 1.5-2 times more active than R. ruber strains in catalyzing the beta-sitosterol biotransformation process.

Modulation of cytokine secretion and oxidative metabolism of innate immune effectors by Rhodococcus biosurfactant.

The glycolipid biosurfactant complex from Rhodococcus ruber IEGM 231 had a stimulatory effect on the production of IL-12, IL-18, and reactive oxygen species by cells of the innate immunity. This effect depended on the composition of cell cultures and presence of LPS. It was primarily observed in non-stimulated cultures. The glycolipid biosurfactant complex had little effect on IL-10 secretion by monocytes and mononuclear cells.

[Oxidative biotransformation of thioanisole by Rhodococcus rhodochrous IEGM 66 cells].

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0-1.5 g/l) concentrations of sulfide substrate.

In vitro immunomodulating activity of biosurfactant glycolipid complex from Rhodococcus ruber.

The biosurfactant glycolipid complex synthesized by Rhodococcus ruber actinobacteria is not toxic and exhibits no appreciable effect on proliferative activity of peripheral blood leukocytes. In the monocyte fraction, the biosurfactant activates the production of IL-1beta and TNF-alpha cytokines without modifying the production of IL-6. In the mononuclear fraction, the glycolipid biosurfactant exhibited no effects on the production of IL-1beta, TNF-alpha, and IL-6. These results indicate good prospects for further studies of immunomodulating and antitumor activities of biosurfactant drug.

[Catalysis of the biodegradation of unusable medicines by alkanotrophic rhodococci].

Experiments on the biodegradation of unusable medicines containing a phenolic hydroxy group by actinobacteria of the genus Rhodococcus were performed. Six species and sixty-four strains were tested. It was found that rhodococci could degrade paracetamol, and some R. ruber strains showed high levels of its degradation. An efficient method for the identification and quantification of paracetamol and the products of its conversion (p-aminophenol, pyrocatechol, and hydroquinone) immediately in the culture liquid was developed. Conditions for the complete biodegradation of paracetamol dosage forms (pills) were optimized. The experimental results can be applied for the development of biotechnological methods for degrading medicines: faked, rejected, or those that are expired.

[Bioconversion of beta-sitosterol and its complex esters by Rhodococcus actinobacteria].

The ability of pure cultures of Rhodococcus actinobacteria from the Ural specialized collection of alkanotrophic microorganisms (World Federation for Culture Collections accession number 768; http://www.ecology.psu.ru/iegmcol) to convert beta-sitosterol (BSS) and its 3beta-acylated derivatives was studied. Rhodococcus strains with pronounced cholesterol oxidase activity, capable of converting BSS to stigmat-4-ene-3-one in the reaction of cooxidation with n-hexadecane, were selected. The dependence of the activity of cholesterol oxidase of rhodococci on the length of the acyl group in BSS esters was studied. Conditions under which Rhodococcus cells convert BSS to 17beta-hydroxyandrost-4-ene-3-one (testosterone), commonly used in pharmacology, were determined.

[The efficient accumulation of cesium ions by Rhodococcus cells]. / Effektivnoe izvlechenie tseziia kletkami bakterii roda Rhodococcus.

Bacteria of the genus Rhodococcus were found to be able to accumulate cesium by means of active transport and nonspecific sorption on the cell surface structures. The maximum removal (up to 97%) of cesium from a medium with ammonium acetate was observed at 28 degrees C, pH 7.8-8.6, and an equimolar content (0.2 mM) of potassium and cesium ions in the medium. The most active cesium-accumulating Rhodococcus sp. strains can be used for purification of industrial wastewaters contaminated with radionuclides.

Alkanotrophic Rhodococcus ruber as a biosurfactant producer.

In this report we examined the structure and properties of surface-active lipids of Rhodococcus ruber. Most historical interest has been in the glycolipids of Rhodococcus erythropolis, which have been extensively characterised. R. erythropolis has been of interest due to its great metabolic diversity. Only recently has the metabolic potential of R. ruber begun to be explored. One major difference in the two species is that most R. ruber strains are able to oxidise the gaseous alkanes propane and butane. In preparation for investigation of the effects of gas metabolism on biosurfactant production, we set out to characterise the biosurfactants produced during growth on liquid n-alkanes and to compare these with R. erythropolis glycolipids.