Bioremediation of oil-polluted soil with an association including the fungus Pleurotus ostreatus and soil microflora

The possibility of application of the Pleurotus ostreatus D1-soil microflora to bioremediation of oil-polluted soils was studied. The fungus degraded mainly the aromatic fractions, whereas soil microflora intensely degraded paraffin and naphthene oil fractions. Introduction of the fungus Pleurotus ostreatus D1 to soil induces degradation of a wider range of oil hydrocarbons. It is reasonable to further investigate the discovered phenomenon in order to improve procedures of remediation of oil-polluted soils.     

Comparison of efficiencies of 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 multiyear 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.     

Production of lignocellulose-degrading enzymes and changes in soil bacterial communities during the growth ofPleurotus ostreatus in soil with different carbon content

The extracellular enzyme activity and changes in soil bacterial community during the growth of the ligninolytic fungus Pleurotus ostreatus were determined in nonsterile soil with low and high available carbon content. In soil with P. ostreatus, the activity of ligninolytic enzymes laccase and Mn-peroxidase was several orders of magnitude higher than in soil without the fungus. Addition of lignocellulose to soil increased the activity of cellulolytic fungi and the production of Mn-peroxidase by P. ostreatus. The counts of heterotrophic bacteria were more significantly affected by the presence of lignocellulose than by P. ostreatus. The effects of both substrate addition and time (succession) were more significant factors affecting the soil bacterial community than the presence of P. ostreatus. Bacterial community structure was affected by fungal colonization in low carbon soil, where a decrease of diversity and changes in substrate utilization profiles were detected.     

Influence of cadmium and mercury on activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil

The white-rot fungus Pleurotus ostreatus was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[a]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.     

Efficiencies of introduction of an oil-oxidizing <Emphasis Type="Italic">Dietzia maris</Emphasis> 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.     

Development and Application of a Liquid Preparation with Oil-Oxidizing Bacteria

An association of four bacterial strains with high oil-oxidizing and bioemulsifying activities, psychrophilicity, resistance to chemical pollutants, and lack of pathogenicity was selected from a collection of natural oil-oxidizing microorganisms. A new liquid preparation containing stabilizers and preservatives that maintain cell viability and oil-oxidizing activity during long-term storage was developed. A field experiment in oil-polluted sod-podzol and clay sand soils demonstrated that this preparation accelerated the biodegradation of oil and its individual fractions, especially in the presence of mineral and organic fertilizers. Treatment of oil-polluted soil with this preparation and additives decreased the oil-induced suppression of certain groups of soil microflora.     

Creation and use of a liquid substance based on association of petroleum-oxidizing bacteria

An association of four bacterial strains with high oil-oxidizing and bioemulsifying activities, psychrophilicity, resistance to chemical pollutants, and lack of pathogenicity was selected from a collection of natural oil-oxidizing microorganisms. A new liquid preparation containing stabilizers and preservatives that maintain the cell viability and oil-oxidizing activity during long-term storage was developed. A field experiment in oil-polluted sod-podzol and clay sand soils demonstrated that this preparation accelerated the biodegradation of oil and its individual fractions, especially in the presence of mineral and organic fertilizers. Treatment of oil-polluted soil with this preparation and additives decreased the oil-induced suppression of certain groups of soil microflora.     

Mobilizing agents enhance fungal degradation of polycyclic aromatic hydrocarbons and affect diversity of indigenous bacteria in soil

The impact of several mobilizing agents (MAs) (i.e., soybean oil, Tween-20, Tween-80, olive-oil mill wastewaters, and randomly methylated beta-cyclodextrins) on the degradation performances of the white-rot fungi Irpex lacteus and Pleurotus ostreatus was comparatively assessed in a soil spiked with a mixture of seven polycyclic aromatic hydrocarbons (PAHs). Among the different MAs, soybean oil best supported the growth of both fungi that was twice that observed in soil in the absence of MAs. In addition, soybean oil positively affected PAH degradation by both fungi. In this case, the total weight of organic contaminants (TWOC) was lower than that in the absence of MAs (57.7 vs. 201.3 and 26.3 vs. 160.4 mg kg(-1) with I. lacteus and P. ostreatus, respectively). On the other hand, the number of cultivable heterotrophic bacteria was significantly lower in the soil with soybean oil augmented with either one of the two fungi (5.21 vs. 8.71 and 0.22 vs. 0.51 x 10(7) CFU g(-1) soil with I. lacteus and P. ostreatus, respectively). The effect of soybean oil was confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes that showed a general decrease in biodiversity. The impact of the other MAs on bacterial diversity was either slightly negative or positive in incubation controls. Both richness and Shannon-Weaver index decreased upon treatment with P. ostreatus. Moreover, with this fungus the composition of the indigenous bacteria was not significantly affected by the type of MA used. By contrast, both indices increased in soil with I. lacteus in the presence of randomly methylated beta-cyclodextrins (39 vs. 33 and 1.43 vs. 1.26, respectively) and soybean oil (19 vs. 5 and 1.01 vs. 0.65, respectively).     

Aerobic biodegradation of biphenyl and polychlorinated biphenyls by Arctic soil microorganisms.

We examined the degradation of biphenyl and the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221 by indigenous Arctic soil microorganisms to assess both the response of the soil microflora to PCB pollution and the potential of the microflora for bioremediation. In soil slurries, Arctic soil microflora and temperate-soil microflora had similar potentials to mineralize [14C]biphenyl. Mineralization began sooner and was more extensive in slurries of PCB-contaminated Arctic soils than in slurries of uncontaminated Arctic soils. The maximum mineralization rates at 30 and 7 degrees C were typically 1.2 to 1.4 and 0.52 to 1.0 mg of biphenyl g of dry soil-1 day-1, respectively. Slurries of PCB-contaminated Arctic soils degraded Aroclor 1221 more extensively at 30 degrees C (71 to 76% removal) than at 7 degrees C (14 to 40% removal). We isolated from Arctic soils organisms that were capable of psychrotolerant (growing at 7 to 30 degrees C) or psychrophilic (growing at 7 to 15 degrees C) growth on biphenyl. Two psychrotolerant isolates extensively degraded Aroclor 1221 at 7 degrees C (54 to 60% removal). The soil microflora and psychrotolerant isolates degraded all mono-, most di-, and some trichlorobiphenyl congeners. The results suggest that PCB pollution selected for biphenyl-mineralizing microorganisms in Arctic soils. While low temperatures severely limited Aroclor 1221 removal in slurries of Arctic soils, results with pure cultures suggest that more effective PCB biodegradation is possible under appropriate conditions.     

Extent of humification of anthracene, fluoranthene, and benzo[α]pyrene by Pleurotus ostreatus during growth in PAH-contaminated soils

The white-rot fungus Pleurotus ostreatus catalysed some humification of anthracene, benzo[ a ]pyrene and fluoranthene in two polycyclic aromatic hydrocarbon (PAH)-contaminated soils, one from a former manufactured gas facility and one from an abandoned electric coking plant. However, the extent of humification of PAH observed in these experiments was considerably less than that previously reported for other pollutants, such as chlorophenols. Addition of surfactants and related amendments significantly enhanced PAH removal from both soils by P. ostreatus , although humification of PAH was not always enhanced under these conditions.     

Variations of lignocellulosic activities in dual cultures of Pleurotus ostreatus and Trichoderma longibrachiatum on unsterilized wheat straw

Trichoderma spp., soil filamentous fungi, are antagonists that can cause great losses in mushroom production. We have investigated the influence of T. longibrachiatum on the production of lignocellulolytic enzymes by Pleurotus ostreatus during its vegetative growth on a straw-based cultivation substrate that either had been sterilized, pasteurized or not heat treated. The variations in the lignocellulolytic activities and the electrophoretic patterns in single and dual cultures were used as a tool for perturbation assessment. The various heat treatments of the wheat straw before inoculation affected both the bacterial populations and the abilities of T. longibrachiatum and P. ostreatus to colonize the substrate and to produce extracellar lignocellulolytic enzymes. Interactions between T. longibrachiatum and the microflora of the substrate led to a great decrease of hydrolytic activities due to reduced colonization of the substrate. Pleurotus ostreatus also was affected but it was less sensitive than T. longibrachiatum. As a consequence, in dual cultures with P. ostreatus, the competitive ability of T. longibrachiatum was reduced by bacteria in the substrates. The presence of total microflora or thermotolerant microflora increased the production of phenoloxidase activities by P. ostreatus, despite reduced colonization of the substrate. This contributed to the improvement of the competitive ability of P. ostreatus in the pasteurized substrate. Furthermore, a direct effect of bacteria on T. longibrachiatum also was observed. In sterilized substrate, both laccase and Mn-peroxydase activities were increased dramatically in dual cultures due to a faster production of a laccase isoform, which was stimulated by T. longibrachiatum.     

平菇(Pleurotus ostreatus)对稻草中木质素的生物降解及降解产物分析

变色圈试验证明平菇可以选择性优先降解稻草中的木质素,培养15d后,平菇对稻草中的木质素降解率为17.86%,对综纤维素降解率为2.44%,选择性指数为9.79。生料栽培平菇后,稻草中的木质素被降解50.24%。用气—质色谱(GC/MS)和红外光谱(IR)对木质素降解产物分析结果表明,平菇对稻草中木质素的降解效果十分明显,降解产物中检测出了大量含有苯环的小分子,证明木质素聚合体的降解首先发生在单体的侧链及单体间的连键上,发生Cα-Cβ、β-O-4等断裂,形成了单体。在进一步的降解过程中,平菇表现了其自身特有的降解机制,取代苯环单体上的甲氧基为甲基,而后发生苯环的开裂,这与报道的白腐菌降解过程有所不同。红外光谱分析中,平菇对木质素的降解明显,降解产物中含有很多木质素单体所特有的基团,如紫丁香基、愈创木基等,说明木质素的降解首先发生的是侧链的氧化反应。     

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microflora and combinations of white-rot fungi in a coal-tar contaminated soil

Four white-rot fungi (Phanerochaete chrysosporium IMI 232175, Pleurotus ostreatus from the University of Alberta Microfungus Collection IMI 341687, Coriolus versicolor IMI 210866 and Wye isolate #7) and all possible combinations of two or more of these fungi, were incubated in microcosms containing wheat straw and non-sterile coal-tar contaminated soil to determine their potential to degrade polycyclic aromatic hydrocarbons (PAHs). Biotic and abiotic controls were prepared similarly and PAH concentrations remaining in each microcosm were determined after 8, 16 and 32 weeks by GC-MS following extraction with dichloromethane. The greatest PAH losses were in the biotic control, compared to small or negligible differences in microcosms inoculated with one or more fungi. These results suggest that in the biotic control native microorganisms colonised the straw added as organic substrate and degraded PAH as an indirect consequence of their metabolism. By contrast, in other microcosms, colonisation of straw by the natural microflora was inhibited because the straw was previously inoculated with fungi. Soil cultures prepared at the end of the experiment showed that though introduced fungi were still alive, they were unable to thrive and degrade PAH in such a highly contaminated soil and remained in a metabolically inactive form.     

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 molecular weight 64 kDa. Its absorption spectrum lacks the maximum at 610 nm, characteristic of fungal laccases and corresponding to type I copper atom. The optimum pH values for the enzyme were determined. They proved 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 were determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme was studied. It was shown that yellow laccase from Pleurotus ostreatus D1 oxidized anthracene to anthraquinone by 95% without any mediator.     

Utilization of fat and degradation of cholesterol by pleurotus spp.

Plant oil was found to stimulate the formation of biomass inPleurotus ostreatus, P. ostreatus formFlorida andP. cornucopiae. The highest increase of the delipidized dry substance was found inP. ostreatus. The supernatant after submerged fermentation contained active emulsifiers. Fruiting bodies and mycelium ofP. ostreatus did not contain cholesterol. Cholesterol added to homogenates of the fruiting bodies was degraded in a temperature-dependent manner. Degradation of cholesterol in the fermentation medium was slower.     

In situ bioremediation of hydrocarbon in soil

Laboratory and field experiments were carried out for bioremediation of soils contaminated by fuel oil and motor oil. Bioventing was combined with the application of selected bacteria and dissolved nutrients. In the field experiments, soil gas was evacuated by air pumps from the permeable boreholes. The process was followed by both soil and gas analysis. Biodegradation of oil contamination and the microbial activity was measured by the oil and cell concentration in the soil. In 2 months, the oil content decreased considerably, and the cell number increased by one order of magnitude or more. The evacuated gas was tested for CO₂, O₂ and volatilized hydrocarbon content. The CO₂ level proves the presence of biodegradation: a permanent high value about ten times higher than normal, could be measured for 2 months, followed by a slow decrease in the third month. Volatilized hydrocarbon content was the highest in the first 2 d. After a continuous decrease, it dropped under the threshold of measurability for the third month. Selective biodegradation of hydrocarbon mixtures (oily wastes) was investigated as well: gas Chromatographic oil analysis showed the changes in the oil composition. The appropriate microflora was working in an ideal commensalism, and as a result, all of the hydrocarbon components were degraded nearly to the same extent.     

Liquid chromatographic and electrophoretic characterisation of extracellular beta-glucosidase of Pleurotus ostreatus grown in organic waste

The production of beta-glucosidase by the ligninolytic fungus Pleurotus ostreatus has been studied in different culture media containing agro-industrial wastes. The enzyme is purified by anion-exchange chromatography, the molecular mass and isoelectric point of purified beta-glucosidase are measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing and the stability and kinetic parameters of the enzyme assessed by spectrophotometry. It has been established that the retention time, molecular mass and isoelectric point of the enzyme depend on the composition of the culture media while the activity and stability of beta-glucosidases of different origin were very similar. The combined chromatographic and electrophoretic methods have proved to be suitable techniques for the purification and characterisation of the beta-glucosidases produced by the ligninolytic fungus Pleurotus ostreatus in different culture media.     

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81–87%, 84–93% and 41–64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.     

Respiration and microflora of the rhizosphere soil of wheat influenced by foliar application of urea

The numbers of micromycetes and bacteria were investigated with respect to oxygen consumption in the rhizosphere soil of wheat and in non-rhizosphere soil. Plants after foliar application of urea (2 % solution) and non-treated plants were cultivated in degraded chernozem and garden soil in a green-house. Changes in oxygen consumption by the suspensions of rhizosphere and non-rhizosphere soils corresponded to changes in the number of bacteria designated as the rhizosphere effect (R/S). Values of R/S depended on the presence of organic substrates. Changes in oxygen consumption by the soil suspension from the rhizosphere of wheat occurring due to foliar application of urea corresponded to changes in the amount of microflora. The results obtained are discussed with respect to a possible utilization of the data to follow metabolic activity of soils in a natural environment (in situ) determined according to oxygen consumption by a soil suspension, and to assess changes in the microflora of rhizosphere and non-rhizosphere soil.     

Use of bacterial acc deaminase to increase oil (especially poly aromatic hydrocarbons) phytoremediation efficiency for maize (zea mays) seedlings

Oil presence in soil, as a stressor, reduces phytoremediation efficiency through an increase in the plant stress ethylene. Bacterial 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, as a plant stress ethylene reducer, was employed to increase oil phytoremediation efficiency. For this purpose, the ability of ACC deaminase-producing Pseudomonas strains to grow in oil-polluted culture media and withstand various concentrations of oil and also their ability to reduce plant stress ethylene and enhance some growth characteristics of maize and finally their effects on increasing phytoremediation efficiency of poly aromatic hydrocarbons (PAHs) in soil were investigated. Based on the results, of tested strains just P9 and P12 were able to perform oil degradation. Increasing oil concentration from 0 to 10% augmented these two strains population, 15.7% and 12.9%, respectively. The maximum increase in maize growth was observed in presence of P12 strain. Results of high-performance liquid chromatography (HPLC) revealed that PAHs phytoremediation efficiency was higher for inoculated seeds than uninoculated. The highest plant growth and PAHs removal percentage (74.9%) from oil-polluted soil was observed in maize inoculated with P12. These results indicate the significance of ACC deaminase producing bacteria in alleviation of plant stress ethylene in oil-polluted soils and increasing phytoremediation efficiency of such soils.