Natural and Technical Phytoremediation of Oil-Contaminated Soil.

Natural and technical phytoremediation approaches were compared for their efficacy in decontaminating oil-polluted soil. We examined 20 oil-contaminated sites of 800 to 12,000 m2 each, with different contamination types (fresh or aged) and levels (4.2-27.4 g/kg). The study was conducted on a field scale in the industrial and adjacent areas of a petroleum refinery. Technical remediation with alfalfa (Medicago sativa L.), ryegrass (Lolium perenne L.), nitrogen fertilizer, and soil agrotechnical treatment was used to clean up 10 sites contaminated by oil hydrocarbons (average concentration, 13.7 g/kg). In technical phytoremediation, the per-year decontamination of soil was as high as 72-90%, whereas in natural phytoremediation (natural attenuation with native vegetation) at 10 other oil-contaminated sites, per-year decontamination was as high as that only after 5 years. Rhizodegradation is supposed as the principal mechanisms of both phytoremediation approaches.

Effect of remediation techniques on petroleum removal from and on biological activity of a drought-stressed Kastanozem soil.

Many petroleum extraction and refinement plants are located in arid climates. Therefore, the remediation of petroleum-polluted soils is complicated by the low moisture conditions. We ran a 70-day experiment to test the efficacy of various combining of remediation treatments with sorghum, yellow medick, and biochar to remove petroleum from and change the biological activity of Kastanozem, a soil typical of the dry steppes and semideserts of the temperate zone. At normal moisture, the maximum petroleum-degradation rate (40%) was obtained with sorghum-biochar. At low moisture, the petroleum-degradation rate was 22 and 30% with yellow medick alone and with yellow medick - sorghum, respectively. Biochar and the biochar-plant interaction had little effect on soil remediation. Both plants promoted the numbers of soil microbes in their rhizosphere: yellow medick promoted mostly hydrocarbon-oxidizing microorganisms, whereas sorghum promoted both hydrocarbon-oxidizing and total heterotrophic microorganisms. Low moisture did not limit microbial development. In the rhizosphere of sorghum, dehydrogenase and urease activities were maximal at normal moisture, whereas in the rhizosphere of yellow medick, they were maximal at low moisture. Peroxidase activity was promoted by the plants in unpolluted soil and was close to the control values in polluted soil. Biochar and the biochar-plant interaction did not noticeably affect the biological activity of the soil.

Study of Boraginaceae plants for phytoremediation of oil-contaminated soil.

Long-term field observations of the natural vegetation cover in industrial and adjacent areas has revealed that the Boraginaceae was one of the main plant family representatives of which were noted in oil-contaminated area. In this study against the background of the previously well characterized plant families Poaceae and Fabaceae, the phytoremediation potential of Boraginaceae plants was investigated under the field conditions and described. Among the members of this family, Lithospermum arvense, Nonea pulla, Asperugo procumbens, Lappula myosotis, and Echium vulgare were the most common in oil-contaminated areas. N. pulla was the most tolerant to hydrocarbons and, along with L. arvense and E. vulgare, actively stimulated the soil microorganisms, including hydrocarbon-oxidizing ones, in their rhizosphere. A comparative assay confirmed that the plants of the Fabaceae family as a whole more efficiently enrich the soil both with available nitrogen and with pollutant degradation genes. Nevertheless, the comparatively high ammonium nitrogen content in the rhizosphere of N. pulla and E. vulgare allows these species to be singled out to explain their high rhizosphere effect, and to suggest their remediation potential for oil-contaminated soil.Novelty statement Against the background of the previously well characterized plant families Poaceae and Fabaceae, the remediation potential of Boraginaceae plants was described for the first time. Overall, this study contributes to understanding the differences in remediation potential of plants at the family level and suggests the monitoring pollutant degradation genes as an informative tool to the search for plant promising for use in the cleanup of oil-contaminated soil.

Combined effects of cadmium and oil sludge on sorghum: growth, physiology, and contaminant removal.

The physiological and biochemical responses of Sorghum bicolor (L.) Moench. to cadmium (Cd) (30 mg kg-1) and oil sludge (OS) (16 g kg-1) present in soil both separately and as a mixture were studied in pot experiments. The addition of oil sludge as a co-contaminant decreased Cd entry into the plant by almost 80% and simultaneously decreased the stimulation of superoxide dismutase (SOD) and peroxidase. The decrease in glutathione reductase (GR) activity and the increase in glutathione-S-transferase (GST) activity under the influence of oil sludge indicated that its components were detoxified by conjugation with glutathione. Cd additionally activated the antioxidant and detoxifying potential of the plant enzymatic response to stress. This helped to enhance the degradation rate of oil sludge in the rhizosphere, in which the participation of the root-released enzymes in the degradation could be possible. Cd increased the extent of soil clean-up from oil sludge, mainly owing to the elimination of paraffins, naphthenes, and mono- and bicyclic aromatic hydrocarbons. The mutual influence of the pollutants on the biochemical responses of sorghum and on soil clean-up was evaluated. The results are important for understanding the antistress and detoxification responses of the remediating plant to combined environmental pollution.

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

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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 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.

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 fungi's 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.

Dynamics of natural revegetation of hydrocarbon-contaminated soil and remediation potential of indigenous plant species in the steppe zone of the southern Volga Uplands.

The result of monitoring of natural vegetation growing on oil-contaminated (2.0-75.6 g/kg) and uncontaminated (0.04-2.0 g/kg) soils of a petroleum refinery for a period of 13 years is presented. Floristic studies showed that the families Poaceae, Asteraceae, Fabaceae, and eventually Brassicaceae were predominant in the vegetation cover of both types of soils. Over time, the projective vegetation cover of the contaminated sites increased from 46 to 90%; the species diversity increased twofold: in the ecological-cenotic structure of the flora, the number of ruderal plant species decreased; and the number of steppe, i.e., zonal, plant species increased. Using 62 dominant plant species, we conducted a field study of plant characteristics such as resistance to oil pollution, the ability to enrich the rhizosphere soil with microorganisms and bioavailable mineral nitrogen, and reduction of the concentration of petroleum hydrocarbons. The results enable us to characterize the phytoremediation potential (PRP) of the native plants and identify species that, probably, played a key role in the natural restoration of oil-contaminated soils. Statistical analysis showed correlations between the PRP constituents, and the leading role of rhizosphere microorganisms in the rhizodegradation of petroleum hydrocarbons was proven. A conditional value of PRP was proposed which allowed the investigated plants to be ranked in 11 classes. The study of a large sample of plant species showed that some plants held promise for the use in reclamation of soils in arid steppe zone, and that other species can be used for the rehabilitation of saline soils and semideserts.

Peroxidases from root exudates of Medicago sativa and Sorghum bicolor: Catalytic properties and involvement in PAH degradation.

Peroxidases from root exudates of sorghum (Sorghum bicolor L. Moench) and alfalfa (Medicago sativa L.) were purified and characterized, and their ability to oxidize native PAHs and PAH-derivatives was evaluated. The obtained data confirm that peroxidases are involved in the rhizosphere degradation of PAHs. Nondenaturing PAGE showed that the peroxidases of both plants were represented by a range of isoforms/isoenzymes (five to eight). Minor forms were lost during further purification, and as a result, the major anionic form from alfalfa root exudates and the major cationic form from those of sorghum were obtained. Both electrophoretically homogeneous peroxidases were monomeric proteins with a molecular weight of about 46-48 kDa. The pH optima and the main catalytic constants for the test substrates were determined. On the basis of their molecular and catalytic properties, the obtained enzymes were found to be typical plant peroxidases. Derivatives of PAHs and potential products of their microbial degradation (9-phenanthrol and 9,10-phenanthrenequinone), unlike the parent PAH (phenanthrene), inhibited the catalytic activity of the peroxidases, possibly indicating greater availability of the enzymes' active centers to these substances. Peroxidase-catalyzed decreases in the concentrations of a number of PAHs and their derivatives were observed. Sorghum peroxidase oxidized anthracene and phenanthrene, while alfalfa peroxidase oxidized only phenanthrene. 1-Hydroxy-2-naphthoic acid was best oxidized by peroxidase of alfalfa. However, quinone derivatives of PAHs were unavailable to sorghum peroxidase, but were oxidized by alfalfa peroxidase. These results indicate that the major peroxidases from root exudates of alfalfa and sorghum can have a role in the rhizosphere degradation of PAHs.

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.