Characterization of biosurfactants produced by the oil-degrading bacterium Rhodococcus erythropolis S67 at low temperature.

Production of trehalolipid biosurfactants by Rhodococcus erythropolis S67 depending on the growth temperature was studied. R. erythropolis S67 produced glycolipid biosurfactants such as 2,3,4-succinoyl-octanoyl-decanoyl-2'-decanoyl trehalose and 2,3,4-succinoyl-dioctanoyl-2'-decanoyl trehalose during the growth in n-hexadecane medium at 26 and 10 °C, despite the different aggregate state of the hydrophobic substrate at low temperature. The surface tension of culture medium was found being reduced from 72 to 27 and 45 mN m-1, respectively. Production of trehalolipid biosurfactants by R. erythropolis S67 at low temperature could be useful for the biodegradation of petroleum hydrocarbons at low temperatures by enhancing the bioremediation performance in cold regions.

[Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions].

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45­50°C; their growth optimum (35­37°C) and range (20­53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.

[Oil biodegradation by microbial-plant associations].

The degradation of petroleum hydrocarbons by plant-microbial associations, as well as the peculiarities of the interaction between microorganisms in consortium and the associated plants, have been studied. It was shown that degrader microorganisms that are part of the consortium Rhodococcus erythropolis S26, Acinetobacter baumannii 1 B, Acinetobacter baumannii 7, and Pseudomonas putida F701 were effective in the degradation of oil and were good colonizers of plant roots (barley). The efficiency of oil degradation increases when microorganisms and plants are used together.

[Bioremediation of oil-polluted soils: using the [13C]/[12C] ratio to characterize microbial products of oil hydrocarbon biodegradation].

We compared data on the extent of bioremediation in soils polluted with oil. The data were obtained using conventional methods of hydrocarbon determination: extraction gas chromatography-mass spectrometry, extraction IR spectroscopy, and extraction gravimetry. Due to differences in the relative abundances of the stable carbon isotopes (13C/12C) in oil and in soil organic matter, these ratios could be used as natural isotopic labels of either substance. Extraction gravimetry in combination with characteristics of the carbon isotope composition of organic products in the soil before and after bioremediation was shown to be the most informative approach to an evaluation of soil bioremediation. At present, it is the only method enabling quantification of the total petroleum hydrocarbons in oil-polluted soil, as well as of the amounts of hydrocarbons remaining after bioremediation and those microbially transformed into organic products and biomass.

[Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems].

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10(-5)-10(-4) per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.

[Phenanthrene degradation by bacteria of the genera Pseudomonas and Burkholderia in model soil systems].

Degradation of phenanthrene by strains Pseudomonas putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) was studied in model soil systems. The differences in accumulation and uptake rate of phenanthrene intermediates between the strains under study have been shown, Accumulation of 1-hydroxy-2-naphthoic acid in soil in the course of phenanthrene degradation by strain BS3702 (pBS143) in a model system has been revealed. The efficiency of phenanthrene biodegradation was assessed using the mathematical model proposed previously for assessment of naphthalene degradation efficiency. The efficiency of degradation of both phenanthrene and the intermediate products of its degradation in phenanthrene-contaminated soil is expected to increase with the joint use of strains P. putida BS3701 (pBS1141, pBS1142) and Burkholderia sp. BS3702 (pBS1143).

[Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by bacteria of the genus Pseudomonas].

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24 degrees C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS 1391 bearing plasmid pBS216 was found to be the most effective oil degrader.

[Search for active psychrotrophic microbial oil degraders and their characterization].

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4-6 degrees C and 24 degrees C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce bioemulsifiers, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4-6 degrees C was higher than at 24 degrees C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24 degrees C and from 28 to 47% at 4-6 degrees C. An artificial association of six of the strains degraded the oil by 46% at 24 degrees C.

[Change in the composition of a bacterial association degrading aromatic compounds during oil sludge detoxification in a continuous-flow microbial reactor].

Analysis of oil sludge by direct plating and enrichment cultivation revealed 16 strains degrading aromatic compounds. After 30 days of cultivation in a continuous-flow microbial reactor, 17 more degrader strains were isolated. Genotyping of these strains showed that they were taxonomically diverse, and the range of strains degrading naphthalene, benzene, toluene, ethylbenzene, and xylenes depended on isolation methods. Direct plating yielded more aromatic degraders than enrichment cultivation. A microbial association different from that existing before the enrichment cultivation was obtained in the laboratory continuous-flow reactor.

[The construction and monitoring of genetically marked, plasmid-containing, naphthalene-degrading strains in soil].

A genetically marked, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142::TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain's fate in model soil systems to be monitored, whereas a minitransposon, built in naphthalene biodegradation plasmid pNF142, contains the tetracycline resistance gene and makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene was found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically marked strain is stable and competitive for 40 days. The transfer of marked plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.

[Effect of naphthalene biodegradation plasmids on physiological characteristics of rhizospheric bacteria of the genus Pseudomonas].

Specific growth rate, duration of the lag phase, stability of plasmids, and activities of the key enzymes involved in naphthalene biodegradation were studied in rhizospheric pseudomonades carrying structurally similar plasmids pOV17 and pBS216. It was demonstrated that these plasmids determined various levels of catechol 2,3-dioxygenase activities. The structural rearrangements in the plasmid pBS216 could "switch off" the genes of catechol oxidation meta-pathway. It was shown that certain combinations of biodegradation plasmids and bacterial hosts, such as Pseudomonas chlororaphis PCL1391(pBS216), P. chlororaphis PCL1391(pOV17), and P. putida 53a(pOV17), were considerably more efficient than natural variants in their growth characteristics and stability of the biodegradation activity, having a potential for bioremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs).

[Effect of transposons on expression of genes for naphthalene biodegradation in Pseudomonas putida BS202(NPL-1) and derivative strains].

NPL-1 and its derivative plasmid pBS106, which control the degradation of naphthalene and salicylate, were found to contain class II transposons of the Tn3 family. These transposons are involved in intraplasmid rearrangements, such as deletions and inversions, and can influence the expression of the catabolic and regulatory genes borne by biodegradation plasmids. The formation of a strong NahR-independent constitutive promoter by the inversion of a DNA fragment may be responsible for changing the character of naphthalene dioxygenase synthesis from inducible (in the case of plasmid NPL-1) to constitutive (in the case of plasmid NPL-41). The stability of plasmids NPL-1 and NPL-41 in the Pseudomonas putida strains grown on different substrates depends on the expression of the nah and tnp genes.

[The use of the [13C]/[12C] ratio for the assay of the microbial oxidation of hydrocarbons]. / Ispol'zovanie otnoshenii [13C]/[12C] dlia opredeleniia mikrobnogo okislenia uglevodorodov.

The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12C and 13C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (delta 13C = -44.6 +/- 0.2@1000) were characterized by the isotope effects delta 13CCO2 = -50.2 +/- 0.4@1000, delta 13Cbiom = -46.6 +/- 0.4@1000 and delta 13Cexo = -41.5 +/- 0.4@1000, respectively. The isotope composition of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (delta 13C = -21 +/- 0.4@1000) were characterized by the isotope effects delta 13CCO2 = -24.1 +/- 0.4@1000, delta 13Cbiom = -19.2 +/- 0.4@1000 and delta 13Cexo = -19.1 +/- 0.4@1000, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the enviroment is discussed.

[Bacteria--degraders of polycyclic aromatic hydrocarbons, isolated from soil and bottom sediments in salt-mining areas]. / Bakterii--destruktory politsiklicheskikh aromaticheskikh uglevodorodov, vydelennye iz pochv i donnykh otlozhenii raiona solerazrabotok.

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical and salt producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1-1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.

[Degradation of phenanthrene by mutant strains--naphthalene degraders]. / Degradatsiia fenantrena mutantnymi shtammami--destruktorami naftalina.

Five naphthalene- and salicylate-utilizing Pseudomonas putida strains cultivated for a long time on phenanthrene produced mutants capable of growing on this substrate and 1-hydroxy-2-naphthoate as the sole sources of carbon and energy. The mutants catabolize phenanthrene with the formation of 1-hydroxy-2-naphthoate, 2-hydroxy-1-naphthoate, salicylate, and catechol. The latter products are further metabolized by the meta- and ortho-cleavage pathways. In all five mutants, naphthalene and phenanthrene are utilized with the involvement of plasmid-borne genes. The acquired ability of naphthalene-degrading strains to grow on phenanthrene is explained by the fact that the inducible character of the synthesis of naphthalene dioxygenase, the key enzyme of naphthalene and phenanthrene degradation, becomes constitutive.

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture.

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D = 0.05 h-1) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive.

[Genetic control of naphthalene biodegradation by a strain of Pseudomonas sp. 8909N]. / Geneticheskii kontrol' biodegradatsii naftalina shtammom Pseudomonas sp. 8909N.

The ability of Pseudomonas sp. 8909N to grow using Naphthalene and salicylate as the sole source of carbon and energy is mediated by the presence of an 80-kb conjugative pBS1145 plasmid in this strain. Structural genes for naphthalene degradation in pBS1145 plasmid are homologous to those in the known NAH7 plasmid. Conjugational transfer of pBS1145 from the original strain is accompanied by a deletion of a plasmid DNA fragment that does not affect the Nah+Sal+ phenotype. Plasmid pBS1145 specifies a low constitutive level of catechol-2,3-dioxygenase, the key enzyme of the metha-pathway of catechol degradation. Activity of this enzyme is induced in the presence of salicylate. Enzymes of both the metha and ortho-pathway of catechol degradation (catechol-2-3-dioxygenase) were shown to operate in the process of naphthalene degradation in Pseudomonas sp. 8909N. The ability of this strain to bring about transformation of polycyclic aromatic hydrocarbon phenanthrene is also controlled by pBS1145 plasmid.