Maria S. Kuyukina

Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction.

In the present study, we proposed methyl tertiary-butyl ether (MTBE) as a solvent for extraction of biosurfactants from Rhodococcus bacterial cultures. After comparison with other well known solvent systems used for biosurfactant extraction, it was found that MTBE was able to extract crude surfactant material with high product recovery (10 g/l), efficiency (critical micelle concentration (CMC), 130-170 mg/l) and good functional surfactant characteristics (surface and interfacial tensions, 29 and 0.9 mN/m, respectively). The isolated surfactant complex contained 10% polar lipids, mostly glycolipids possessing maximal surface activity. Ultrasonic treatment of the extraction mixture increased the proportion of polar lipids in crude extract, resulting in increasing surfactant efficiency. Due to certain characteristics of MTBE, such as relatively low toxicity, biodegradability, ease of downstream recovery, low flammability and explosion safety, the use of this solvent as an extraction agent in industrial scale biosurfactant production is feasible.

Alkanotrophic Rhodococcus ruber as a biosurfactant producer

Abstract. 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.

Oil desorption from mineral and organic materials using biosurfactant complexes produced by Rhodococcus species

Rhodococcus strains from the culture collection at the Institute of Ecology and Genetics of Microorganisms, Perm, Russia were examined for biosurfactant production during growth on n-alkanes and the ability to remove oil associated with contaminated sands and oil shale cuttings. Members of the genus, particularly R. ruber, were shown to produce low toxicity surfactants effective in removing oil from surfaces. The extent of desorption was inversely related to the concentration of high molecular weight hydrocarbons, namely asphaltenes and resins. In addition, crude surfactant complexes enhanced the degradation of crude oil, in the short term, when added to contaminated agricultural soil during bioremediation studies utilizing biopiling technology.

Bioremediation of Crude Oil-Contaminated Soil Using Slurry-Phase Biological Treatment and Land Farming Techniques

Field-scale experiments on bioremediation of soil heavily contaminated with crude oil were undertaken on the territory of the Kokuyskoye oil field (Perm region, West Urals, Russia) owned by the LUKOIL Company. The pollution consisted of the contents of a oil waste storage pit, which mostly received soils contaminated after accidental oil spills and also the solid n-alkane (paraffin) wastes removed from the surface of drilling equipment. Laboratory analyses of soil samples indicated contamination levels up to 200 g/kg of total recoverable petroleum hydrocarbons (TRPH). Average oil composition consisted of 64% aliphatics, 25% aromatics, 8% heterocyclics, and 3% of tars/asphaltenes. Ex situ bioremediation techniques involved the successive treatment of contaminated soil using a bioslurry reactor and land farming cells. An oleophilic biofertilizer based on Rhodococcus surfactant complexes was used in both treatment systems. An aerobic slurry bioreactor was designed, and the biofertilizer applied weekly. Slurry-phase biotreatment of the contaminated soil resulted in an 88% reduction in oil concentration after 2 months. The resulting reactor product, containing approximately 25 g/kg of TRPH, was then loaded into land farming cells for further decontamination. To enhance bioremediation, different treatments (e.g., soil tilling, bulking with woodchips, watering, and biofertilizer addition) were used. The rates of oil biodegradation were 300 to 600 ppm/day. As a result, contamination levels dropped to 1.0 to 1.5 g/kg of TRPH after 5 to 7 weeks. Tertiary soil management involved phytoremediation where land farming cells were seeded with a mixture of three species of perennial grass. The effect of phytoremediation on the residual decontamination and rehabilitation of soil fertility is being evaluated.

Rhodococcus Biosurfactants: Biosynthesis, Properties, and Potential Applications

Members of the genus Rhodococcus produce biosurfactants in response to the presence of liquid hydrocarbons in the growth medium. These biosurfactants are predominantly cell-bound glycolipids containing trehalose as the carbohydrate. Physiological roles of these glycolipids are diverse and involve participation in the uptake of water-insoluble substrates, promotion of the cell adhesion to hydrophobic surfaces, and increased rhodococcal resistance to physicochemical influences. In terms of surfactant characteristics (e.g., surface and interfacial tension, critical micelle concentration, emulsifying activity), Rhodococcus biosurfactants compete favorably with other microbial and synthetic surfactants. Additionally, biological activities of trehalolipids from rhodococci were revealed, including immunomodulating and antitumor properties. Recently developed optimization procedures for their biosynthesis and recovery would broaden potential applications of Rhodococcus biosurfactants in new advanced technologies, such as environmental bioremediation, improved polymeric material construction, and biomedicine. The present chapter summarizes recent research on Rhodococcus biosurfactants, updating the comprehensive review of Lang and Philp (Antonie van Leeuwenhoek Int J Gen Mol Microbiol 74:59–70, 1998), and focuses on biosynthesis features, physicochemical and bioactive properties, and their application potential.

Application of Rhodococcus in Bioremediation of Contaminated Environments

Environmental pollution with anthropogenic organic compounds is the global problem of our planet. Bioremediation has a great potential to effectively restore polluted environments by using biodegradative activities of microorganisms. The genus Rhodococcus is a promising group of bacteria suitable for biodegradation of recalcitrant contaminants, such as petroleum hydrocarbons, chlorinated, nitrogenated, and other complex organics. Rhodococcus species are ubiquitous in pristine and contaminated environments, survive under harsh environmental conditions, compete successfully in complex bacterial populations, and therefore could be efficiently used in bioremediation applications. Some success in bioremediation of contaminated soils, waters, and air has been achieved using rhodococci either as bioaugmentation agents or members of indigenous microbial communities stimulated by nutrient and oxygen amendments. Laboratory and field-scale studies on Rhodococcus application in cleanup technologies are reviewed relating to in-situ subsurface and groundwater remediation, on site treatments of contaminated soils, sludges, wastewaters, and waste gases.

Identification and environmental detection of Rhodococcus species by 16S rDNA‐targeted PCR

Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. There is a need for improved tests to identify Rhodococcus species. PCR‐based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species‐specific sequences in the 16S rRNA gene, were successfully developed for R. globerulus, R. erythropolis, R. opacus and R. ruber. These tests gave positive results with all or most strains of target species but did not generally cross‐react with other species. Cases of apparent cross‐reaction were shown to be due to prior misclassification of strains of R. opacus as R. erythropolis and of strains of R. ruber as R. rhodochrous. A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R. erythropolis in an environmental sample. Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze‐thaw cycles. Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL‐6B in a spun‐column format. Extracts prepared using a tris‐EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds. A detection limit of 104 cfu g−1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g−1.

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-1β and TNF-α cytokines without modifying the production of IL-6. In the mononuclear fraction, the glycolipid biosurfactant exhibited no effects on the production of IL-1β, TNF-α, and IL-6. These results indicate good prospects for further studies of immunomodulating and antitumor activities of biosurfactant drug.

Selective adsorption of hydrocarbon-oxidizing Rhodococcus cells in a column with hydrophobized poly(acrylamide) cryogel

A method for selective adsorption of Rhodococcus cells in the column with hydrophobized poly(acrylamide) cryogel (cryoPAAG) was developed that allowed rhodococci separation from mixed bacterial populations and their effective concentration within a sponge-like gel matrix. Hydrophobization of cryoPAAG using the n-dodecane graft (C12) was performed to enhance the adhesion of Rhodococcus cells to the cryogel; this was suggested by our finding that alkanotrophic rhodococci possess high adhesive activity (91-98%) towards n-alkanes, whereas other Gram-positive and Gram-negative bacteria tested did not adhere strongly to hydrocarbons. The selective index of the hydrophobic C12-cryoPAAG column for Rhodococcus cells was 72% that ensured their separation from complex bacterial cultures. Respirometry results using the Columbus Micro-Oxymax respirometer showed that the maximal respiratory activity of C12-cryoPAAG-immobilized Rhodococcus cells incubated with petroleum hydrocarbons was 1.6-1.8 times higher than that of freely suspended cells, and this correlated with the largest immobilized cell number. Moreover, high respiration rates were maintained over 3 weeks of incubation, indicating a considerable functional stability of the cryoPAAG-immobilized biocatalyst developed.

In vitro cytokine stimulation assay for glycolipid biosurfactant from Rhodococcus ruber: role of monocyte adhesion

Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1β and IL-6 production when applied as an ultrasonic emulsion to the adherent human peripheral blood monocyte culture. However, a lack of cytokine-stimulating activity was registered with the GLB applied as a hydrophobic film coating in 24-well culture plates, indicating that it may have been due to its inhibitory effect on monocyte adhesion. The mode of GLB application may therefore play an important role in in vitro assay of immunostimulatory activity of this compound as well as other bacterial glycolipids. Additionally, GLB from R. ruber displayed no cytotoxicity against human lymphocytes and therefore could be proposed as a potential immunomodulating and antitumor agent.