A field study to determine the ability of selected lignindegrading fungi to remediate soil contam... more A field study to determine the ability of selected lignindegrading fungi to remediate soil contaminated with creosote was performed at a wood-treating facility in south central Mississippi in the autumn of 1991. The effects of solid-phase bioremediation with Phanerochaete sordida and of two control treatments on soil concentrations of 14 priority pollutant polycyclic aromatic hydrocarbon (PAH) components of creosote were followed for 56 days. PAH analytes containing > 5 rings persisted at their original concentrations in all treatments. However, depletion of 3-ring (85-95%) and 4-ring (24-72%) analytes after 56 days was greater in the fungal treatment than in control treatments in all cases. This finding demonstrates the potential of lignin-degrading fungi in the solid-phase bioremediation of creosote-contaminated soils. However, the persistence of the larger analytes represents a significant challenge to this developing technology.
Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental too... more Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for the quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely related lignin peroxidase transcripts were detected in soil. The pattern of lignin peroxidase gene expression was unexpected; certain transcripts abundant in defined cultures were not detected in soil cultures. Transcripts encoding cellobiohydrolases and -tubulin were also detected. The method will aid in defining the roles of specific genes in complex biological processes such as organopollutant degradation, developing strategies for strain improvement, and identifying specific fungi in environmental samples.
mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined... more mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of the 10 known lignin peroxidase (lip) genes in anthracene-transforming soil cultures of Phanerochaete chrysosporium. Levels of extractable lipA transcript and protein (LiP H8) were well correlated, although they were separated by a 2-day lag period. The patterns of transcript abundance over time in soil-grown P. chrysosporium varied among the nine lip mRNAs detected; comparison with lip gene expression under different liquid culture conditions suggested an early phase of carbon limitation for the cultures as a whole, which was followed by a transition to nitrogen starvation. Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons.
U RG OS A ND M. T IE N. 1999. The white-rot fungus Pleurotus ostreatus catalysed some humificatio... more U RG OS A ND M. T IE N. 1999. 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.
The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin pe... more The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese, unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhihited by the free-radical scavenger butylated hydroxytohiene but not by the lignin peroxidase inhibitor NaV0 3 . Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.
The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs)... more The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogenlimited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn 2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz [a] anthracene, and benzo [a] pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn 2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz [a] anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.
The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese per... more The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese peroxidasemediated lipid peroxidation reactions to degrade the polycyclic aromatic hydrocarbons (PAHs) found in creosote were studied. The disappearance of 12 three-to six-ring PAHs occurred in both systems. Both in vivo and in vitro, the disappearance of all PAHs was found to be very strongly correlated with ionization potential. This was true even for compounds beyond the ionization potential thresholds of lignin peroxidase and Mn 3؉ . Deviations from this correlation were seen in the cases of PAHs which are susceptible to radical addition reactions. These results thus begin to clarify the mechanisms of non-lignin peroxidase-labile PAH degradation in the manganese peroxidase-lipid peroxidation system and provide further evidence for the ability of this system to explain the in vivo oxidation of these compounds.
A field study to determine the ability of selected lignindegrading fungi to remediate soil contam... more A field study to determine the ability of selected lignindegrading fungi to remediate soil contaminated with creosote was performed at a wood-treating facility in south central Mississippi in the autumn of 1991. The effects of solid-phase bioremediation with Phanerochaete sordida and of two control treatments on soil concentrations of 14 priority pollutant polycyclic aromatic hydrocarbon (PAH) components of creosote were followed for 56 days. PAH analytes containing > 5 rings persisted at their original concentrations in all treatments. However, depletion of 3-ring (85-95%) and 4-ring (24-72%) analytes after 56 days was greater in the fungal treatment than in control treatments in all cases. This finding demonstrates the potential of lignin-degrading fungi in the solid-phase bioremediation of creosote-contaminated soils. However, the persistence of the larger analytes represents a significant challenge to this developing technology.
Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental too... more Thorough analysis of fungi in complex substrates has been hampered by inadequate experimental tools for assessing physiological activity and estimating biomass. We report a method for the quantitative assessment of specific fungal mRNAs in soil. The method was applied to complex gene families of Phanerochaete chrysosporium, a white-rot fungus widely used in studies of organopollutant degradation. Among the genes implicated in pollutant degradation, two closely related lignin peroxidase transcripts were detected in soil. The pattern of lignin peroxidase gene expression was unexpected; certain transcripts abundant in defined cultures were not detected in soil cultures. Transcripts encoding cellobiohydrolases and -tubulin were also detected. The method will aid in defining the roles of specific genes in complex biological processes such as organopollutant degradation, developing strategies for strain improvement, and identifying specific fungi in environmental samples.
mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined... more mRNA extraction from soil and quantitation by competitive reverse transcription-PCR were combined to study the expression of the 10 known lignin peroxidase (lip) genes in anthracene-transforming soil cultures of Phanerochaete chrysosporium. Levels of extractable lipA transcript and protein (LiP H8) were well correlated, although they were separated by a 2-day lag period. The patterns of transcript abundance over time in soil-grown P. chrysosporium varied among the nine lip mRNAs detected; comparison with lip gene expression under different liquid culture conditions suggested an early phase of carbon limitation for the cultures as a whole, which was followed by a transition to nitrogen starvation. Anthracene transformation occurred throughout the 25-day course of the experiment and, therefore, likely involves mechanisms distinct from those involved in oxidation of non-LiP substrate polycyclic aromatic hydrocarbons.
U RG OS A ND M. T IE N. 1999. The white-rot fungus Pleurotus ostreatus catalysed some humificatio... more U RG OS A ND M. T IE N. 1999. 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.
The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin pe... more The oxidation of fluorene, a polycyclic hydrocarbon which is not a substrate for fungal lignin peroxidase, was studied in liquid cultures of Phanerochaete chrysosporium and in vitro with P. chrysosporium extracellular enzymes. Intact fungal cultures metabolized fluorene to 9-hydroxyfluorene via 9-fluorenone. Some conversion to more-polar products was also observed. Oxidation of fluorene to 9-fluorenone was also obtained in vitro in a system that contained manganese, unsaturated fatty acid, and either crude P. chrysosporium peroxidases or purified recombinant manganese peroxidase. The oxidation of fluorene in vitro was inhihited by the free-radical scavenger butylated hydroxytohiene but not by the lignin peroxidase inhibitor NaV0 3 . Manganese(III)-malonic acid complexes could not oxidize fluorene. These results indicate that fluorene oxidation in vitro was a consequence of lipid peroxidation mediated by P. chrysosporium manganese peroxidase. The rates of fluorene and diphenylmethane disappearance in vitro were significantly faster than those of true polycyclic aromatic hydrocarbons or fluoranthenes, whose rates of disappearance were ionization potential dependent. This result indicates that the initial oxidation of fluorene proceeds by mechanisms other than electron abstraction and that benzylic hydrogen abstraction is probably the route for oxidation.
The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs)... more The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogenlimited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn 2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz [a] anthracene, and benzo [a] pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn 2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz [a] anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.
The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese per... more The abilities of whole cultures of Phanerochaete chrysosporium and P. chrysosporium manganese peroxidasemediated lipid peroxidation reactions to degrade the polycyclic aromatic hydrocarbons (PAHs) found in creosote were studied. The disappearance of 12 three-to six-ring PAHs occurred in both systems. Both in vivo and in vitro, the disappearance of all PAHs was found to be very strongly correlated with ionization potential. This was true even for compounds beyond the ionization potential thresholds of lignin peroxidase and Mn 3؉ . Deviations from this correlation were seen in the cases of PAHs which are susceptible to radical addition reactions. These results thus begin to clarify the mechanisms of non-lignin peroxidase-labile PAH degradation in the manganese peroxidase-lipid peroxidation system and provide further evidence for the ability of this system to explain the in vivo oxidation of these compounds.
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Papers by Richard Lamar