A. E. Filonov

Bacterial Degraders of Polycyclic Aromatic Hydrocarbons Isolated from Salt-Contaminated Soils and Bottom Sediments in Salt Mining Areas

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.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.

The construction and monitoring of genetically tagged, plasmid-containing, naphthalene-degrading strains in soil

A genetically tagged, 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, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, 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 is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.

The efficiency of salicylate utilization by Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways.

Abstract Parameters of growth and substrate consumption by plasmid-harbouring Pseudomonas putida strains catabolizing naphthalene via different biochemical pathways under batch cultivation on salicylate were estimated. The values of a specific growth rate, μ , and the yield coefficient, Y , for natural isolates with the functioning genes of the ortho-pathway and silent genes of meta cleavage of catechol oxidation were less than those for isogenous strains with the functioning genes of the meta-pathway. All strains under study with functioning genes of the meta-pathway were shown to possess the activities of the enzymes both of ortho- and meta pathway of catechol oxidation: catechol-1,2-dioxygenase and catechol-2,3-dioxygenase. The comparison of the functioning of the genes of the ortho- and meta-pathways was carried out in an isogenous host background. An approach to estimate the efficiency of the biodegradation process using the kinetics of microbial growth and substrate consumption under batch cultivation was proposed.

Growth and survival of Pseudomonas putida strains degrading naphthalene in soil model systems with different moisture levels

The effect of different moisture levels (from 20 to 70%) on the growth and survival of Pseudomonas putida strains G7 and BS3701 degrading naphthalene was studied in soil model systems. P.putida G7 contains plasmid NAH7 and P.putida BS3701 harbours plasmids pBS1141 and pBS1142. A mathematical model is proposed to describe the observed dynamics of the number of viable bacterial cells. Naphthalene and soil organic matter were considered as substrates available to bacteria. Data fitting allowed the estimation of model parameters characterizing microbial growth rate, utilization rate of substrates, specific maintenance rate and yield coefficient. Both the maximum bacterial concentration and the highest yield coefficient were observed at a soil moisture level of 40%. This optimal moisture level is close to but less than the water capacity (48%) of the soil used.

Pseudomonas putida as a receptor element of microbial sensor for naphthalene detection

Abstract Pseudomonas putida BS238 carrying the naphthalene degradative plasmid pBS2 was immobilized by adsorption on chromatographic paper and fixed on the surface of a Clark electrode. Naphthalene activated the respiratory activity of the cells, which was the basis for determining its concentration in one-component aqueous media. The lower limit of naphthalene detection was in the region of 0·9 μ m . The maximal responses of the sensor were observed at pH 6·0–6·2, 20–25°C and a salt concentration 2·9–3·2 g ion/litre. The sensor responded weakly to the presence of biphenyl, monocyclic aromatic compounds, ethanol, carbohydrates and organic acids in the medium. A sensor based on an isogenic strain without plasmid pBS2 had no response to naphthalene. The possibility of improving the selectivity of naphthalene detection using a pair of sensors based on the original and isogenic strains is discussed.

Degradation of Phenanthrene by Mutant Naphthalene-Degrading Pseudomonas putida Strains

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-born 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.

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 Pseudomona, Moscow, KMK, 2004simova, I.A. and Chernov, I.s putida BS3701 (pBS1141, pBS1142), Pseudomonas putida BS3745 (pBS216), and Burkholderia sp. BS3702 (pBS1143) were 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 (pBS1143) 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 Pseudomonas bacteria

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24°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 BS1391 bearing plasmid pBS216 was found to be the most effective oil degrader.

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.