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The growing industrial and medical use of gold nanoparticles (AuNPs) requires environmentally friendly methods for their production using microbial biosynthesis. The ability of actinobacteria of the genus Rhodococcus to synthesize AuNPs in the presence of chloroauric acid (HAuCl4) was studied. The effect of elevated (0.8-3.2 mM) concentrations of HAuCl4 on bacterial viability, morphology, and intracellular accumulation of AuNPs by different Rhodococcus species was shown. An increase in surface roughness, a shift of the zeta potential to the positive region, and the formation of cell aggregates of R. erythropolis IEGM 766 and R. ruber IEGM 1135 during nanoparticle synthesis were revealed as bacterial adaptations to toxic effects of HAuCl4. The possibility to biosynthesize AuNPs at a five times higher concentration of chloroauric acid compared to chemical synthesis, for example, using the citrate method, suggests greater efficiency of the biological process using Rhodococcus species. The main parameters of biosynthesized AuNPs (size, shape, surface roughness, and surface charge) were characterized using atomic force microscopy, dynamic and electrophoretic light scattering, and also scanning electron microscopy in combination with energy-dispersive spectrometry. Synthesized by R. erythropolis spherical AuNPs have smaller (30-120 nm) dimensions and are positively (12 mV) charged, unlike AuNPs isolated from R. ruber cells (40-200 nm and -22 mV, respectively). Such differences in AuNPs size and surface charge are due to different biomolecules, which originated from Rhodococcus cells and served as capping agents for nanoparticles. Biosynthesized AuNPs showed antimicrobial activity against Gram-positive (Micrococcus luteus) and Gram-negative (Escherichia coli) bacteria. Due to the positive charge and high dispersion, the synthesized by R. erythropolis AuNPs are promising for biomedicine, whereas the AuNPs formed by R. ruber IEGM 1135 are prone to aggregation and can be used for biotechnological enrichment of gold-bearing ores.
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Actinobacteria , Nanopartículas Metálicas , Rhodococcus , Ouro/química , Nanopartículas Metálicas/química , Antibacterianos/química , Escherichia coliRESUMO
This work has a focus on adaptive capabilities of the actinobacterium Rhodococcus ruber IEGM 326 to cope with drotaverine hydrochloride (DH), a known pharmaceutical pollutant. Cultivation of R. ruber in a nitrogen-limited medium with incubation at the ambient temperature resulted in the formation of cyst-like dormant cells (CLDCs). They maintained viability for 2-7 months, possessed the undetectable respiratory activity and elevated resistance to heating, and had a specific morphology. CLDCs are regarded to ensure long-term survival in various habitats and may be used as storage formulations. R. ruber IEGM 326 was tolerant to DH (MIC, 200 mg/l) and displayed different abilities to degrade this compound, depending on inoculum, temperature, and the presence of glucose as co-oxidized substrate. Thus, the loss of DH (20 mg/l) over 48 h at the optimal temperature (27 ± 2 °C) was 5-8 % in the absence of glucose after inoculating with vegetative cells. The addition of glucose (5 g/l) increased DH degradation up to 46 %. Noteworthy, CLDCs as inoculum were advantageous over vegetative cells to degrade DH at the non-optimal temperature (35 ± 2 °C) at reduced bulk respiratory activity. The obtained results are promising to improve the biodegrading capabilities of other Rhodococcus strains.
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Papaverina/análogos & derivados , Rhodococcus/metabolismo , Biodegradação Ambiental , Meios de Cultura , Poluentes Ambientais/metabolismo , Papaverina/metabolismo , Rhodococcus/ultraestrutura , TemperaturaRESUMO
Immobilization of microorganisms on/in insoluble carriers is widely used to stabilize functional activity of microbial cells in industrial biotechnology. We immobilized Rhodococcus ruber, an important hydrocarbon degrader, on biosurfactant-coated sawdust. A biosurfactant produced by R. ruber in the presence of liquid hydrocarbons was found to enhance rhodococcal adhesion to solid surfaces, and thus, it was used as a hydrophobizing agent to improve bacterial attachment to a sawdust carrier. Compared to previously used hydrophobizers (drying oil and n-hexadecane) and emulsifiers (methyl- and carboxymethyl cellulose, poly(vinyl alcohol), and Tween 80), Rhodococcus biosurfactant produced more stable and homogenous coatings on wood surfaces, thus resulting in higher sawdust affinity to hydrocarbons, uniform monolayer distribution of immobilized R. ruber cells (immobilization yield 29-30 mg dry cells/g), and twofold increase in hydrocarbon biooxidation rates compared to free rhodococcal cells. Two physical methods, i.e., high-resolution profilometry and infrared thermography, were applied to examine wood surface characteristics and distribution of immobilized R. ruber cells. Sawdust-immobilized R. ruber can be used as an efficient biocatalyst for hydrocarbon transformation and degradation.
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Células Imobilizadas/metabolismo , Hidrocarbonetos/metabolismo , Rhodococcus/metabolismo , Tensoativos/metabolismo , Madeira/microbiologia , Aderência Bacteriana , Biotecnologia/métodos , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Rhodococcus/química , Rhodococcus/fisiologiaRESUMO
Nickel nanoparticles (NPs) are used for soil remediation and wastewater treatment due to their high adsorption capacity against complex organic pollutants. However, despite the growing use of nickel NPs, their toxicological towards environmental bacteria have not been sufficiently studied. Actinobacteria of the genus Rhodococcus are valuable bioremediation agents degrading a range of harmful and recalcitrant chemicals. Both positive and negative effects of metal ions and NPs on the biodegradation of organic pollutants by Rhodococcus were revealed, however, the mechanisms of such interactions, in addition to direct toxic effects, remain unclear. In the present work, the influence of nickel NPs on the viability, surface topology and nanomechanical properties of Rhodococcus cells have been studied. Bacterial adaptations to high (up to 1.0 g/L) concentrations of nickel NPs during prolonged (24 and 48 h) exposure were detected using combined confocal laser scanning and atomic force microscopy. Incubation with nickel NPs resulted in a 1.25-1.5-fold increase in the relative surface area and roughness, changes in cellular charge and adhesion characteristics, as well as a 2-8-fold decrease in the Young's modulus of Rhodococcus ruber IEGM 231 cells. Presumably, the treatment of rhodococcal cells with sublethal concentrations (0.01-0.1 g/L) of nickel NPs facilitates the colonization of surfaces, which is important in the production of immobilized biocatalysts based on whole bacterial cells adsorbed on solid carriers. Based on the data obtained, cell surface functionalizing with NPs is possible to enhance adhesive and catalytic properties of bacteria suitable for environmental applications.
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Nanoparticles (NPs) of transition metals and their oxides are widely used in industries and exhibit diverse biological activities - from antimicrobial to growth promoting and regulating biofilms. In this study, the concentration-dependent effects of negatively charged metal and metal oxide NPs on the viability and net surface charge of Rhodococcus cells were revealed. Our hypothesis that zeta potential values of bacterial cells approach the zeta potential of NPs with an increase in the concentration of nanoparticles was statistically validated, thus suggesting the accumulation of nanoparticles on the cell surface. Thus, based on the dynamics of zeta potential, it would be possible to predict the accumulation of metal NPs on the cell surface of particular Rhodococcus species. It seemed that more toxic nanometals (e.g. CuO) accumulate more intensively on the bacterial cell wall than less toxic nanometals (Bi, Ni and Co). Physical properties of NPs, such as shape, size, dispersity and zeta potential, were characterized at different nanoparticle concentrations, in order to explain their diverse effects on bacterial viability, cellular charge and adhesion to hydrocarbons. Interestingly, an increase in Rhodococcus adhesion to n-hexadecane was observed in the presence of Cu and CuO NPs, while treatment with Fe3O4 NPs resulted in a decrease in the adhesive activity. The obtained data help to clarify the mechanisms of nano-bio interaction and make it possible to select metal and metal oxide nanoparticles to modify the surface of bacterial cells without toxic effects.
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Bioremediation represents a sustainable approach to remediating petroleum hydrocarbon contaminated soils. One aspect of sustainability includes the sourcing of nutrients used to stimulate hydrocarbon-degrading microbial populations. Organic nutrients such as animal manure and sewage sludge may be perceived as more sustainable than conventional inorganic fertilizers. However, organic nutrients often contain antibiotic residues and resistant bacteria (along with resistance genes and mobile genetic elements). This is further exacerbated since antibiotic resistant bacteria may become more abundant in contaminated soils due to co-selection pressures from pollutants such as metals and hydrocarbons. We review the issues surrounding bioremediation of petroleum-hydrocarbon contaminated soils, as an example, and consider the potential human-health risks from antibiotic resistant bacteria. While awareness is coming to light, the relationship between contaminated land and antibiotic resistance remains largely under-explored. The risk of horizontal gene transfer between soil microorganisms, commensal bacteria and/or human pathogens needs to be further elucidated, and the environmental triggers for gene transfer need to be better understood. Findings of antibiotic resistance from animal manures are emerging, but even fewer bioremediation studies using sewage sludge have made any reference to antibiotic resistance. Resistance mechanisms, including those to antibiotics, have been considered by some authors to be a positive trait associated with resilience in strains intended for bioremediation. Nevertheless, recognition of the potential risks associated with antibiotic resistant bacteria and genes in contaminated soils appears to be increasing and requires further investigation. Careful selection of bacterial candidates for bioremediation possessing minimal antibiotic resistance as well as pre-treatment of organic wastes to reduce selective pressures (e.g., antibiotic residues) are suggested to prevent environmental contamination with antibiotic-resistant bacteria and genes.
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Antibacterianos , Farmacorresistência Bacteriana , Petróleo , Poluentes do Solo , Bactérias , Biodegradação Ambiental , Farmacorresistência Bacteriana/genética , Hidrocarbonetos , Solo , Microbiologia do SoloRESUMO
Pine sawdust treated by a series of hydrophobising agents (drying oil, organosilicon emulsion, n-hexadecane and paraffin) was examined as carrier for adsorption immobilisation of hydrocarbon-oxidizing bacterial cells Rhodococcus ruber. It was shown that hydrophobising agents based on drying oil turned out to be optimal (among the other modifiers examined) for the preparation of sawdust carriers suitable for the efficient immobilisation. The results obtained demonstrate promising possibilities in developing a wide range of available and cheap, biodegradable cellulose-containing carriers that possess varying surface hydrophobicity.
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Biotecnologia/métodos , Hidrocarbonetos/química , Oxigênio/química , Rhodococcus/metabolismo , Madeira , Alcanos/química , Biodegradação Ambiental , Catálise , Celulose/química , Concentração de Íons de Hidrogênio , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Modelos Químicos , Óleos , PinusRESUMO
An efficient scheme to synthesize novel ring-A fused heterocyclic derivatives of betulin was developed. The starting reaction of this synthesis was one-pot selective bacterial oxidation of betulin to betulone used as the key compound to synthesize the substituted azoles such as C(2)-C(3)-fused 1,2,3-triazoles, oxazoles and 1,2,4-triazine, as well as C(1)-C(2)-fused isoxazoles. The semi-synthetic compounds were screened for their cytotoxic activity against human cancer cell lines A549, HCT 116, HEp-2, MS and RD TE32 with use of the photometric MTT assays. Among the tested compounds, N-acetyltriazole of betulin (10) displayed impressive cytotoxic activity with IC50 2.3-7.5 µM against HCT 116, HEp-2, MS and RD TE32 cell lines as well as 3-methyl-4-oxido-1,2,4-triazine-derivative of betulonic acid (12) that was active against HCT 116 and HEp-2 cell lines with IC50 1.4 and 1.5 µM, respectively. Comparative experiments showed triazole (10) to have a lower cytotoxicity to normal epithelial cells, in comparison with compound (12). In accord with the in vivo acute toxicity test, the LD50 of triazole (10) exceeded 600 mg/kg. The ability of the most potent active triazole (10) to trigger apoptotic cell death was explored in the Annexin V-FITC test and by analyzing of caspase activity and morphological alterations in mitochondria and nuclei of HCT 116 cells.
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Azóis/síntese química , Azóis/toxicidade , Triterpenos/química , Azóis/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Dose Letal Mediana , Estrutura Molecular , Triazinas/química , Triazinas/toxicidade , Triterpenos/toxicidadeRESUMO
A simple biosurfactant-based hydrophobization procedure for poly(vinyl alcohol) (PVA) cryogels was developed allowing effective immobilization of hydrocarbon-oxidizing bacteria. The resulting partially hydrophobized PVA cryogel granules (granule volume 5 microl) contained sufficient number (6.5 x 10(3)) of viable bacterial cells per granule, possessed high mechanical strength and spontaneously located at the interface in water-hydrocarbon system. Such interfacial location of PVA granules allowed high contact of immobilized biocatalyst with hydrophobic substrate and water phase, thus providing bacterial cells with mineral and organic nutrients. As a result, n-hexadecane oxidation efficiency of 51% after 10-day incubation was achieved using immobilized biocatalyst. PVA cryogels with increased hydrophobicity can be used for immobilization of bacterial cultures performing oxidative transformations of water-immiscible organic compounds. Immobilization of in situ biosurfactant producing Rhodococcus bacteria into PVA cryogel is discussed. PVA cryogel granules with entrapped alkanotrophic rhodococcal cells were stable after 10-month storage at room temperature.
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Proteínas Sanguíneas , Fibronectinas , Hidrocarbonetos/metabolismo , Álcool de Polivinil , Rhodococcus/fisiologia , Tensoativos/metabolismo , Alcanos/metabolismo , Biodegradação Ambiental , Proteínas Sanguíneas/química , Células Imobilizadas , Criogéis , Criopreservação/métodos , Fibronectinas/química , Hidrogéis , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Polissorbatos/metabolismo , Rhodococcus/crescimento & desenvolvimento , Rhodococcus/metabolismoRESUMO
This study evaluated the effects of a trehalolipid biosurfactant produced by Rhodococcus ruber IEGM 231 on the bacterial adhesion and biofilm formation on the surface of polystyrene microplates. The adhesion of Gram-positive (Arthrobacter simplex, Bacillus subtilis, Brevibacterium linens, Corynebacterium glutamicum, Micrococcus luteus) and Gram-negative (Escherichia coli, Pseudomonas fluorescencens) bacteria correlated differently with the cell hydrophobicity and surface charge. In particular, exponentially growing bacterial cells with increased hydrophobicities adhered stronger to polystyrene compared to more hydrophilic stationary phase cells. Also, a moderate correlation (0.56) was found between zeta potential and adhesion values of actively growing bacteria, suggesting that less negatively charged cells adhered stronger to polystyrene. Efficient biosurfactant concentrations (10-100 mg/L) were determined, which selectively inhibited (up to 76 %) the adhesion of tested bacterial cultures, however without inhibiting their growth. The biosurfactant was more active against growing bacteria rather than resting cells, thus showing high biofilm-preventing properties. Contact angle measurements revealed more hydrophilic surface of the biosurfactant-covered polystyrene compared to bare polystyrene, which allowed less adhesion of hydrophobic bacteria. Furthermore, surface free-energy calculations showed a decrease in the Wan der Waals (γ(LW)) component and an increase in the acid-based (γ(AB)) component caused by the biosurfactant coating of polysterene. However, our results suggested that the biosurfactant inhibited the adhesion of bacteria independently on their surface charges. AFM scanning revealed three-type biosurfactant structures (micelles, cord-like assemblies and large vesicles) formed on glass, depending on concentrations used, that could lead to diverse anti-adhesive effects against different bacterial species.
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Microbially produced biosurfactants were studied to enhance crude oil desorption and mobilization in model soil column systems. The ability of biosurfactants from Rhodococcus ruber to remove the oil from the soil core was 1.4-2.3 times greater than that of a synthetic surfactant of suitable properties, Tween 60. Biosurfactant-enhanced oil mobilization was temperature-related, and it was slower at 15 degrees C than at 22-28 degrees C. Mathematical modelling using a one-dimensional filtration model was applied to simulate the process of oil penetration through a soil column in the presence of (bio)surfactants. A strong positive correlation (R(2)=0.99) was found between surfactant penetration through oil-contaminated soil and oil removal activity. Biosurfactant was less adsorbed to soil components than synthetic surfactant, thus rapidly penetrating through the soil column and effectively removing 65-82% of crude oil. Chemical analysis showed that crude oil removed by biosurfactant contained a lower proportion of high-molecular-weight paraffins and asphaltenes, the most nonbiodegradable compounds, compared to initial oil composition. This result suggests that oil mobilized by biosurfactants could be easily biodegraded by soil bacteria. Rhodococcus biosurfactants can be used for in situ remediation of oil-contaminated soils.
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Modelos Teóricos , Petróleo/metabolismo , Poluentes do Solo/análise , Tensoativos/química , Cinética , Rhodococcus/química , TemperaturaRESUMO
Actinobacteria of the genus Rhodococcus produce trehalolipid biosurfactants with versatile biochemical properties and low toxicity. In recent years, these biosurfactants are increasingly studied as possible biomedical agents with expressed immunological activities. Applications of trehalolipids from Rhodococcus, predominantly cell-bound, in biomedicine are also attractive because their cost drawback could be less significant for high-value products. The review summarizes recent findings in immunomodulatory activities of trehalolipid biosurfactants from nonpathogenic Rhodococcus and related actinobacteria and compares their biomedical potential with well-known immunomodifying properties of trehalose dimycolates from Mycobacterium tuberculosis. Molecular mechanisms of trehalolipid interactions with immunocompetent cells are also discussed.
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Fatores Imunológicos/biossíntese , Fatores Imunológicos/imunologia , Lipídeos/imunologia , Rhodococcus/classificação , Rhodococcus/metabolismo , Tensoativos/metabolismo , Animais , Camundongos , Rhodococcus/imunologia , Especificidade da EspécieRESUMO
Crude oil and petroleum products are widespread water and soil pollutants resulting from marine and terrestrial spillages. International statistics of oil spill sizes for all incidents indicate that the majority of oil spills are small (less than 7 tonnes). The major accidents that happen in the oil industry contribute only a small fraction of the total oil which enters the environment. However, the nature of accidental releases is that they highly pollute small areas and have the potential to devastate the biota locally. There are several routes by which oil can get back to humans from accidental spills, e.g. through accumulation in fish and shellfish, through consumption of contaminated groundwater. Although advances have been made in the prevention of accidents, this does not apply in all countries, and by the random nature of oil spill events, total prevention is not feasible. Therefore, considerable world-wide effort has gone into strategies for minimising accidental spills and the design of new remedial technologies. This paper summarizes new knowledge as well as research and technology gaps essential for developing appropriate decision-making tools in actual spill scenarios. Since oil exploration is being driven into deeper waters and more remote, fragile environments, the risk of future accidents becomes much higher. The innovative safety and accident prevention approaches summarized in this paper are currently important for a range of stakeholders, including the oil industry, the scientific community and the public. Ultimately an integrated approach to prevention and remediation that accelerates an early warning protocol in the event of a spill would get the most appropriate technology selected and implemented as early as possible - the first few hours after a spill are crucial to the outcome of the remedial effort. A particular focus is made on bioremediation as environmentally harmless, cost-effective and relatively inexpensive technology. Greater penetration into the remedial technologies market depends on the harmonization of environment legislation and the application of modern laboratory techniques, e.g. ecogenomics, to improve the predictability of bioremediation.
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Vazamento de Resíduos Químicos/prevenção & controle , Recuperação e Remediação Ambiental/métodos , Poluição por Petróleo , Biodegradação Ambiental , Conservação dos Recursos Naturais , Petróleo , Medição de RiscoRESUMO
Investigations into bacterial responses to vanadium are rare, and in this study were initiated by isolating cultures from crude oil contaminated soil from Russia and Saudi Arabia. Addition of vanadyl sulphate and vanadium pentoxide created acid conditions in the media whilst sodium metavanadate and sodium orthovanadate produced neutral and alkaline effects, respectively. Buffers were introduced for wider comparison of the sample set treatments and to distinguish between the effects of pH and compound toxicity. This study has resulted in the creation of protocols for the pH stabilisation of media containing vanadium compounds and revealed that, although vanadium salts demonstrated some toxic effects, as revealed by MIC and bioluminescence decay tests, the effects were mainly due to pH rather than inherent toxicity of the metal. Capacity for sorption of vanadium to biomass was also investigated.
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Bactérias/efeitos dos fármacos , Petróleo , Microbiologia do Solo , Poluentes do Solo , Vanádio/farmacologia , Bactérias/isolamento & purificação , Contagem de Colônia Microbiana , Concentração de Íons de Hidrogênio , Medições Luminescentes , OxirreduçãoRESUMO
Using combined confocal laser scanning and atomic force microscopy (CLSM/AFM), bacterial viability under organic solvent stress was assessed at single cell level. Solvent-exposed bacteria stained with the LIVE/DEAD BacLight fluoresced green or red, allowing viable and dead cell discrimination. However, with toluene, butanol and acetonitrile, dually fluorescent cells appeared having compromised cell membranes. Changes in size, surface/volume ratio and roughness were revealed as possible resistance mechanisms.
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Bactérias/efeitos dos fármacos , Microscopia de Força Atômica , Microscopia Confocal , Compostos Orgânicos/farmacologia , Solventes/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodosRESUMO
We report a draft genome sequence of Rhodococcus ruber IEGM 231, isolated from a water spring near an oil-extracting enterprise (Perm region, Russian Federation). This sequence provides important insights into the genetic mechanisms of propane and n-butane metabolism, organic sulfide and beta-sitosterol biotransformation, glycolipid biosurfactant production, and heavy metal resistance in actinobacteria.
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Specialized nonmedical microbial culture collections contain unique bioresources that could be useful for biotechnology companies. Cooperation between collections and companies has suffered from shortcomings in infrastructure and legislation, hindering access to holdings. These challenges may be overcome by the transformation of collections into national bioresource centers and integration into international microbial resource networks.
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Bancos de Espécimes Biológicos , Biotecnologia/métodos , Federação RussaRESUMO
A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems.
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Hidrocarbonetos/farmacologia , Petróleo/metabolismo , Reação em Cadeia da Polimerase/métodos , Rhodococcus/crescimento & desenvolvimento , Rhodococcus/isolamento & purificação , Células Imobilizadas/química , Células Imobilizadas/classificação , Células Imobilizadas/efeitos dos fármacos , Hidrocarbonetos/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Álcool de Polivinil/química , Rhodococcus/efeitos dos fármacos , Rhodococcus/genética , Especificidade da EspécieRESUMO
Glycolipid biosurfactant (GLB) from Rhodococcus ruber IEGM 231 was found to stimulate tumor necrosis factor-α (TNF-α), interleukin (IL) -1ß 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.
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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.