Ekaterina Dubrovskaya

The coupling of the plant and microbial catabolisms of phenanthrene in the rhizosphere of Medicago sativa.

We studied the catabolism of the polycyclic aromatic hydrocarbon phenanthrene by four rhizobacterial strains and the possibility of enzymatic oxidation of this compound and its microbial metabolites by the root exudates of alfalfa (Medicago sativa L.) in order to detect the possible coupling of the plant and microbial metabolisms under the rhizospheric degradation of the organic pollutant. A comparative study of phenanthrene degradation pathways in the PAH-degrading rhizobacteria Ensifer meliloti, Pseudomonas kunmingensis, Rhizobium petrolearium, and Stenotrophomonas sp. allowed us to identify the key metabolites from the microbial transformation of phenanthrene, including 9,10-phenanthrenequinone, 2-carboxybenzaldehyde, and 1-hydroxy-2-naphthoic, salicylic, and o-phthalic acids. Sterile alfalfa plants were grown in the presence and absence of phenanthrene (0.03 g kg(-1)) in quartz sand under controlled environmental conditions to obtain plant root exudates. The root exudates were collected, concentrated by ultrafiltration, and the activity of oxidoreductases was detected spectrophotometrically by the oxidation rate for various substrates. The most marked activity was that of peroxidase, whereas the presence of oxidase and tyrosinase was detected on the verge of the assay sensitivity. Using alfalfa root exudates as a crude enzyme preparation, we found that in the presence of the synthetic mediator, the plant peroxidase could oxidize phenanthrene and its microbial metabolites. The results indicate the possibility of active participation of plants in the rhizospheric degradation of polycyclic aromatic hydrocarbons and their microbial metabolites, which makes it possible to speak about the coupling of the plant and microbial catabolisms of these contaminants in the rhizosphere.

Efficiencies of introduction of an oil-oxidizing Dietzia maris strain and stimulation of natural microbial communities in remediation of polluted soil

Two approaches to bioremediation of oil-polluted soils are compared: use of active degrader strain Dietzia maris AM3 and stimulation of natural microflora. Introduction of D. maris AM3 to soil freshly polluted with oil accelerated its remediation twofold within the first month in comparison with the stimulation. After three months, the purification degrees were approximately equal. By the end of bioremediation, the soil with the introduced strain had higher dehydrogenase and catalase activities. In soil with aged pollution, introduced strain D. maris AM3 did not affect the rate of oil product degradation, and no significant differences between the two bioremediation methods were detected in purification degree and biological activity of soil after three months.

Changes in physiological, biochemical, and growth parameters of sorghum in the presence of phenanthrene

Physiological, biochemical, and growth parameters of sorghum (Sorghum bicolor (L.) Moench) plants grown in the presence of phenantrene (10 and 100 mg/kg soil) were examined. Activities of intracellular tyrosinases, peroxidases, and laccase-like oxidases were analyzed in 1 and 2 months after planting. The tyrosinase activity in root and leaf tissues correlated positively throughout the experiment with the level of soil pollution. The oxidase activity was apparent only in the first month; it also correlated positively with the concentration of phenanthrene. Intracellular peroxidases exhibited the highest activity; positive correlation of this activity with the level of soil contamination was observed in the first period of observations. The soil pollutant had a negative impact on growth characteristics (germination capacity, survival rate, and accumulation of plant biomass). In addition, soil contamination with phenanthrene reduced the total content of photosynthetic pigments and changed their ratio. The maximum extent of phenanthrene elimination in soil was found to occur in the root zone of sorghum plants at high-level contamination, which indicates a significant contribution of plants to the decomposition (binding) of this xenobiotic.

Changes in phytotoxicity of polycyclic aromatic hydrocarbons in the course of microbial degradation

Phytotoxicity of six polycyclic aromatic hydrocarbons (PAHs) and their 16 oxidized derivatives that may be microbial metabolites arising in the course of PAH degradation was determined using an express test with the seedlings of sorghum (Sorghum bicolor L. Moench) and alfalfa (Medicago sativa L.). It was shown that germinating capacity is the least informative characteristic and the most useful parameter is development of seedlings during 3 days in the presence of compounds under investigation. Among unsubstituted compounds, toxicity in respect to seedlings decreased in the series fluorene > phenanthrene > anthracene. Chrysene, fluoranthene, and pyrene stimulated shoot development. It was found that some of the metabolites produced as a result of microbial degradation of phenanthrene (9,10-phenanthrenequinone, 1-hydroxy-2-naphthoic and benzoic acids) are more toxic for plants than starting PAH molecules. The obtained results are important for understanding rhizosphere processes associated with phytoremediation technique.

Production of Ligninolytic Enzymes by White-rot Fungi during Bioremediation of Oil-contaminated Soil

A number of white-rot fungi, including strains of Pleurotus ostreatus, Lentinus edodes, Coriolus sp., and Agaricus sp., were tested for the production of ligninolytic enzymes during decontamination of old oil-polluted industrial soil. The solid-state cultures of all studied fungi were able to grow and produce ligninolytic enzymes in the soil. The growth rates, the mycelium densities, the production of ligninolytic enzymes, and the degradation of old oil contamination decreased in the order Agaricus sp. > P. ostreatus > L. edodes > Coriolus sp. The most active producers of laccase and peroxidase were the strains of P. ostreatus and Agaricus sp. The production of peroxidase by the strains of L. edodes and Coriolus sp. was not found under the conditions used. The fungi that actively colonized the soil and produced ligninolytic enzymes also actively degraded old oil contamination in the soil.

The degradative activity and adaptation potential of the litter-decomposing fungus Stropharia rugosoannulata

The ability of the litter-decomposing basidiomycete Stropharia rugosoannulata DSM 11372 to degrade a wide range of structurally different environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs: phenanthrene, anthracene, fluorene, pyrene, and fluoranthene), synthetic anthraquinone dyes containing condensed aromatic rings, environmentally relevant alkylphenol and oxyethylated alkylphenol representatives, and oil was demonstrated within the present study. 9,10-Anthraquinone, phenanthrene-9,10-quinone, and 9-fluorenone were identified as products of anthracene, phenanthrene, and fluorene degradation, respectively. Fungal degradation was accompanied by the production of the ligninolytic enzymes: laccase and Mn peroxidase, suggesting their involvement in pollutant degradation. Extracellular polysaccharide(s) (EPS) and emulsifying compound(s) were concomitantly produced. EPS composed of mannose, glucose, and galactose was isolated from the cultivation medium, and its effects on catalytic properties of purified laccase from S. rugosoannulata (the dominating ligninolytic enzyme under the applied conditions) were studied. A simultaneous decrease of KM and Vmax values observed for the enzymatic oxidation of non-phenolic (2,2-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt; ABTS) and phenolic compounds (2,6-dimethoxyphenol) in presence of EPS suggest an interaction of EPS and laccase resulting in a modulation of the catalytic performance of the enzyme, which has, to the best of our knowledge, not been reported before. In line with such a modulation, the laccase-catalyzed oxidation of natural aromatic compounds (veratryl alcohol, adlerol) and environmental pollutants (the alkylphenol representative nonylphenol, the diphenylmethane derivative bisphenol A, and the PAH representative anthracene) was found to be enhanced in presence of EPS. The relevance of such effects for real environmental processes and their implications remain to be investigated.Graphical abstract

Degradative properties of two newly isolated strains of the ascomycetes Fusarium oxysporum and Lecanicillium aphanocladii

Two ascomycete strains were isolated from creosote-contaminated railway sleeper wood. By using a polyphasic approach combining morpho-physiological observations of colonies with molecular tools, the strains were identified as Fusarium oxysporum Schltdl. (IBPPM 543, MUT 4558; GenBank accession no. MG593980) and Lecanicillium aphanocladii Zare & W. Gams (IBPPM 542, MUT 242; GenBank accession no. MG593981). Both strains degraded hazardous pollutants, including polycyclic aromatic hydrocarbons, anthraquinone-type dyes, and oil. Oil was better degraded by F. oxysporum, but the aromatic compounds were better degraded by L. aphanocladii. With both strains, the degradation products of anthracene, phenanthrene, and fluorene were 9,10-anthraquinone, 9,10-phenanthrenequinone, and 9-fluorenone, respectively. During pollutant degradation, F. oxysporum and L. aphanocladii produced an emulsifying compound(s). Both fungi produced extracellular Mn-peroxidases, enzymes possibly involved in the fungal degradation of the pollutants. This is the first report on the ability of L. aphanocladii to degrade four-ring PAHs, anthraquinone-type dyes, and oil, with the simultaneous production of an extracellular Mn-peroxidase.

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization. A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungis affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.

Peroxidases from alfalfa roots: Catalytic properties and participation in degradation of polycyclic aromatic hydrocarbons

From the roots and root exudates of 3-week-old plants of alfalfa (Medicago sativa L.), anionic and cationic peroxidases differing in principal physicochemical and catalytic properties were isolated and purified. Main features of anionic peroxidases detected in the roots and root exudates were identical. Phenanthrene present in the soil used for alfalfa growing influenced the number of forms and activity of peroxidases in crude enzyme preparations but did not affect the properties of pure enzymes. In the presence of a synthetic mediator, purified peroxidases can oxidize phenanthrene and its derivatives, including potential microbial metabolites of polycyclic aromatic hydrocarbons (PAH). The fact that the enzymes excreted in root exudates in a purified form can oxidase PAH proves their participation in degradation of PAH and their microbial metabolites in alfalfa rhizosphere. These new data indicate that the processes of plant and microbial degradation of pollutants in the rhizosphere are coupled; they are relevant to understanding the molecular mechanisms of degradation of persistent pollutants by plant-microbial complexes.