O. V. Turkovskaya

Phytoremediation of oil-sludge-contaminated soil.

The aim of this research was to select plant species that could be effective in the phytoremediation of a former oil-sludge pit. Seven crop plants (Triticum aestivum L., Secale cereale L., Avena sativa L., Hordeum vulgare, Sorghum bicolor L. Moench, Panicum miliaceum L., and Zea mays L.), five wild grasses (Lolium perenne L., Bromopsis inermis, Agropyron cristatum L., Agropyrum tenerum L., and Festuca pratensis Huds.), and three legumes (Medicago sativa L., Trifolium pratense L., and Onobrychis antasiatica Khin.) were screened for phytotoxicity, including the assessment of germination, shoot biomass, and root biomass, in a pot experiment. The estimation of oil-sludge degradation in the root zone of the tested plants showed that rye accelerated cleanup most effectively, degrading all of the main contaminant fractions in the oil sludge by a total of 52%. Although alfalfa had a lower phytoremediation potential than did rye, it maintained large numbers of soil microorganisms, including polycyclic aromatic hydrocarbon degraders, in its rhizosphere. Rye and alfalfa were chosen for a large-scale study to remediate an oil-sludge pit on the grounds of a petroleum refinery. Remediation monitoring confirmed the effectiveness of rye: the oil-sludge content decreased consistently for 3 years and remained low in comparison with the results from other plant species.

Plant--rhizosphere-microflora association during phytoremediation of PAH-contaminated soil.

The capability of plants to promote the microbial degradation of pollutants in rhizosphere soil is a principal mechanism of phytoremediation of PAH-contaminated soil. The formation of a specific rhizosphere microbocenosis with a high degradative potential toward contaminants is largely determined by plant species. The comparative PAH-degradation in unplanted soil and in soil planted with reed (Phragmites australis) and alfalfa (Medicago sativa) was studied in pot experiments during 2 years. Both alfalfa and reed successfully remediated contaminated soil by degrading 74.5 and 68.7% of PAHs, respectively. The study of the rhizosphere, rhizoplane, and unplanted-soil microflora in experimental pots showed that alfalfa stimulated the rhizosphere microflora of PAH-contaminated soil more effectively than did reed. Alfalfa clearly enhanced both the total number of microorganisms (1.3 times, according to fluorescence microscopy data) and the rate of the PAH-degrading population (almost seven times, according to plate counting). The degradative potential of its rhizosphere microflora toward PAHs was higher than the degradative activity of the reed rhizosphere. This study provides relevant information for the successful application of alfalfa to phytoremediate PAH-contaminated soil.

Rhizosphere microflora of plants used for the phytoremediation of bitumen-contaminated soil.

The microbial communities and their degradative potential in rhizospheres of alfalfa (Medicago sativa) and reed (Phragmites australis) and in unplanted soil in response to bitumen contamination of soil were studied in pot experiments. According to the results of fluorescence microscopy, over a period of 27 months, bitumen contamination of soil reduced the total number of microorganisms more significantly (by 75%) in unplanted than in rhizosphere soil (by 42% and 7% for reed and alfalfa, respectively) and had various effects on some important physiological groups of microorganisms such as actinomycetes as well as nitrogen-fixing, nitrifying, denitrifying, ammonifying, phosphate-solubilizing, sulphur-oxidizing, cellulolytic and hydrocarbon-degrading microorganisms. The changes in the physiological structure of the microbial community under bitumen contamination were found to hinge on not merely the presence of plants but also their type. It was noted that the rhizosphere microflora of alfalfa was less inhibited by hydrocarbon pollution and had a higher degradative potential than the rhizosphere microflora of reed.

Studies of the Efficacy of Alfalfa and Reed in the Phytoremediation of Hydrocarbon-Polluted Soil

The efficacy of plants as means of decontaminating hydrocarbon-polluted soil has been studied. Ditch reed (Phragmites australis) and alfalfa (Medicago sativa) markedly intensified processes of pollutant destruction, the effect being particularly pronounced in the case of polycyclic aromatic hydrocarbons. Comparative analysis of microflora in soils (including those devoid of plants and rhizosphere) demonstrated that, in addition to preventing a pollutant-induced decrease in the amount of heterotrophic microorganisms, the plants stimulated their development, significantly increasing the population of degraders. Effects of plants on major physiological groups of soil microorganisms under conditions of pollution were ambiguous. The rhizosphere microbial consortium of alfalfa was less susceptible to effects of pollutants than that of reed.

[Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum].

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indole-3-acetic acid. The synthesis of indole-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.

Yellow Laccase from the Fungus Pleurotus ostreatus D1: Purification and Characterization

Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with a molecular weight of 64 kDa. Its lacks an absorption spectrum maximum at 610 nm, a result which is characteristic of fungal laccases and corresponds to the presence of type I copper atoms. The optimum pH values for the enzyme are determined. They prove to be 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2′-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6-dimethoxyphenol. Kinetic parameters (Km and Vmax) for oxidation of these substrates are determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme is studied. It is shown that yellow laccase from Pleurotus ostreatus D1 in the absence of a mediator oxidizes anthracene to anthraquinone to 95%.

Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains--Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7--possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas-liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C-P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.

Oxidoreductase activity of sorghum root exudates in a phenanthrene-contaminated environment.

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene on the enzymatic activity of root exudates of the phytoremediating plant Sorghum bicolor (L.) Moench was studied. Analysis of sorghum root exudates allowed us to reveal the activities of oxidase, peroxidase, and tyrosinase. The activities of these enzymes were progressive as the soil phenanthrene concentration increased. Using lyophilized samples, we found that as a result of the enzymatic activity of the root exudates, some of the PAHs and products of PAH degradation were oxidized in the reaction mixture supplemented with the mediating agents (ABTS or DL-DOPA) but that no oxidation was observed in the reaction mixtures without the mediators. The revealed enzymatic activity of the sorghum root exudates may indicate the involvement of the root-released oxidoreductases in rhizospheric degradation of PAHs and/or their derivatives. In addition, from the data obtained, the coupling of plant and microbial metabolisms of PAHs in the rhizosphere may be surmised.

Emulsifying Agent Production During PAHs Degradation by the White Rot Fungus Pleurotus Ostreatus D1

For the first time the production of an emulsifying agent during phthalic, 2,2′-diphenic and α-hydroxy-β-naphthoic acids, phenanthrene, anthracene, fluorene, pyrene, fluoranthene, and chrysene degradation by white rot fungus Pleurotus ostreatus was found. The emulsifying activity of the cultivation medium after degradation of these compounds was assessed. Maximal activities were found in the presence of chrysene (48.4%) and α-hydroxy-β-naphthoic acid (52.2%). Emulsifying activity inversely dependent on the water solubility of the compounds used. Versatile peroxidase was produced concurrently with the emulsifying agent.

Degradation of phenanthrene by the rhizobacterium Ensifer meliloti.

The biodegradation of the polycyclic aromatic hydrocarbon phenantherene by the rhizobacterial strain Ensifer meliloti P221, isolated from the root zone of plant grown in PAH-contaminated soil was studied. Bacterial growth and phenanthrene degradation under the influence of root-exuded organic acids were also investigated. Analysis of the metabolites produced by the strain by using thin-layer chromatography, gas chromatography, high-pressure liquid chromatography, and mass-spectrometry revealed that phenanthrene is bioconverted via two parallel pathways. The first, major pathway is through terminal aromatic ring cleavage (presumably at the C3–C4 bond) producing benzocoumarin and 1-hydroxy-2-naphthoic acid, whose further degradation with the formation of salicylic acid is difficult or is very slow. The second pathway is through the oxidation of the central aromatic ring at the C9–C10 bond, producing 9,10-dihydro-9,10-dihydroxyphenanthrene, 9,10-phenanthrenequinone, and 2,2′-diphenic acid. This is the first time that the dioxygenation of phenanthrene at the C9 and C10 atoms, proven by identification of characteristic metabolites, has been reported for a bacterium of the Ensifer genus.