Although several enzymes presumed to be part of the delignification/kraft pulp biobleaching system of the white-rot basidiomycete Trametes versicolor have been studied, characterized, and even exploited as pulp treatments, the complete system remains poorly understood. Little is known about which enzymes are essential for delignification, how they are regulated, or whether there remain unknown enzymes essential to wood decay in this system. Auxotrophic mutants of T. versicolor 52J were developed and characterized. Plasmid pUC18 was complemented with the T. versicolor 52J genome to create a gene library. Transformation with this plasmid-gene library converted argB and ade2 auxotrophs of T. versicolor 52J to prototrophy. Attempts to rescue the plasmids responsible were unsuccessful. Several different pre-existing plasmid constructs were examined for their potential as selectable markers on the fungus. One of these, pGPhT, worked well at conferring phleomycin resistance. Given that there are now available partial or complete sequences for four T. versicolor laccases, the single cellobiose dehydrogenase, and a lignin peroxidase isozyme, these genetic tools should be very useful in dissecting the mechanisms of white-rot delignification.

The microbial degradation of pyrene, a 4-ring polycyclic aromatic hydrocarbon (PAH), has been elucidated with the increasing number of pyrene-degrading bacteria that have been isolated in recent years. A pyrene degrading bacterium identified as Mycobacterium sp. strain S65, was isolated from a jet-fuel contaminated site in Sept-Iles, northern Quebec, Canada. S65 utilized pyrene, phenanthrene, and fluoranthene as sole carbon and energy sources, but did not degrade naphthalene, anthracene, and fluorene. Pyrene mineralization was enhanced by adding benz[a]anthracene, benzy[a]pyrene, or phenanthrene as cosubstrates. When added to PAH contaminated soil as a potential bioaugmentation agent, S65 did not appear to survive well, nor was it effective at degrading PAHs under these conditions. / Pyrene catabolic genes in S65 were partially characterized by Southern hybridization using a probe constructed from the naphthalene inducible pyrene dioxygenase gene, nidA, from the pyrene-degrading bacterium, Mycobacterium sp. strain PYR-1.

Soil samples were collected from several corn fields with history of atrazine (herbicide) application. Samples were inoculated into Erlenmeyer flasks each containing 50m1 of minimal basal salts medium amended with 100 ppm atrazine as sole nitrogen source. Flasks were shaken at 200 rpm at ambient temperature and were examined daily for one week for microbial growth and/or disappearance of atrazine. Promising consortia were subcultured for further additional enrichments before characterization of potentially active protein (enzyme)fractions. Proteins from cell-free and cellbound fractions were compared for ability to denature atrazine. Following gel permeation chromatography, isolated protein fractions were examined for atrazinefound in the cell-bound fractions capable degrading degradation. Two were found in the cell-free fractions (approx. Mol. wts. 55kDa and 180kDa) and one (55 kDa) atrazine to hydroxyatrazine. Atrazine and its breakdown products (hydroxyatrazine in particular) were detected via HPLC using C18 and C8 columns with absorbance at 229 nm. / Department of Biology

Six plant species were screened to determine potential suitability for phytoremediation of nitroglycerin (NG), a component in smokeless powders (SP). Seeds of Zea mays (corn), Triticum aestivum (wheat), Medicago sativa (alfalfa), Poa pratensis (Kentucky bluegrass), Trifolium pratense (red clover), and Phaseolus vulgaris (common bean) were sown into greenhouse mesocosms containing commercially prepared soil spiked with 0, 1, and 5% SP (w/w). Soil samples were collected 7, 60, and 90 days after seeding, extracted with ethanol, and analyzed for NG using a gas chromatograph with an electron capture detector. Plant growth observations were recorded using a simple scoring metric at 7, 14, 30, and 60 days after seeding. Soil nitrate and ammonium, potential by-products of NG decomposition, were quantified 90 days after seeding. NG disappearance in plant treatments was markedly, although not significantly (p > 0.05), higher than control at 1% SP, with legumes being the most successful treatment. Nitrate concentrations were significantly (p < 0.05) higher in legume than grass treatments. Soil ammonium was not correlated to any plant or SP treatment. Plant uptake of NG was minimal, suggesting a soil microbial effect in NG disappearance. More extensive screening studies are needed to determine which plants are the most successful remediators of NG. / Department of Natural Resources and Environmental Management

Most microbial activity in nature takes place at interfaces where redox discontinuities are present. Organic pollutants in groundwater encounter oxic/anoxic interfaces when they emerge to surface water bodies or volatilize above the plume. Such oxic/anoxic interfaces are key habitats for aerobic bacteria and are in turn created by the bacteria that degrade organic electron donors. In the absence of biodegradation, synthetic pollutants can migrate from the plume and impact a variety of receptors. The aims of our study were to determine whether microbes at oxic/anoxic interfaces can use synthetic chemicals as electron donors and protect the overlying vadose zone or surface water from groundwater pollutants. The approach was to design columns representing the interfaces and measure activities of the microbial communities responsible for the biodegradation of synthetic compounds.Taken together the above studies established clearly that contaminants recalcitrant under anaerobic conditions but degradable under aerobic conditions can be biodegraded at the narrow oxic/anoxic interface resulting in the protection of the overlying soil or water. The findings provide the basis for new approaches to natural attenuation that can serve to dramatically reduce the cost of bioremediation actions. Synthetic chemicals are widespread in the environment because of their extensive use in industry. These chemicals were recalcitrant until their microbial degradation pathways evolved. Currently the biodegradation pathways of many synthetic chemicals are known and serve as the basis for bioremediation strategies. The second part of the research described here involved discovery of the aerobic degradation pathway of a dye additive: 4-nitroaniline (4NA). Annotation of the whole genome sequence coupled with assays and supported with cloned enzymes revealed that the 4NA biodegradation pathway contains two monooxygenase steps prior to ring cleavage. Because nitroaniline degradation was not previously understood our work advanced the understanding of metabolic diversity in degradation of amino and nitro compounds by providing enzymes with unique activities.

The purpose of this report was to investigate how the abiotic factors (temperature, pH, nutrients, oxygen levels and water content) would affect the decomposition of oil-contaminated soil and how a variance in structured soil would affect the decomposition-rate. A minor field-study was made and soil where structured in five different ways (one was a default pile and was used as a reference). The study took place over 137 days and three samples were taken (start, middle, end) and analyzed for aromatic and aliphatic compounds. The result of this study shows that there is a significant difference between all methods for the aliphatic compounds and no significant difference for the aromatic compounds. The abiotic factors that affect decomposition vary from organism to organism, depending on what type of environment it will function the best. In this type of environment it could be necessary to change temperature interval, nutrient availability or pH to optimize the conditions for the microorganisms. The conclusion of this report is that the structured of the soil-piles will affect decomposition rate, but it is unsure how much. Further research is needed to conclude how the soil should be structured to maximize the decomposition-rate.

Sugar maple foliage litters from fertilized and unfertilized plots on a base-poor site and from a naturally base-rich site were incubated in litterbags of 1 and 3 mm mesh on fertilized and unfertilized plots at the base-poor site. Mass loss of unfertilized litter was slower in fertilized than unfertilized plots, suggesting a negative effect of fertilization on decomposers. Mass loss of fertilized litter was faster than unfertilized litter in the same plots, indicating that changes in litter quality due to fertilization enhanced microbial decomposition. Mass loss was higher in large mesh than in small mesh size bags, suggesting that larger soil fauna played a significant role in litter decomposition. / Potassium appeared to be rapidly leached, whereas Ca and Mg were released at rates more closely related to litter mass loss. Nitrogen was mineralized from N-rich Arboretum litter only; all other litters immobilized N. Release of Ca and Mg was reduced significantly on fertilized plots. Large soil fauna enhanced Ca release, while they delayed N-mineralization in Arboretum litter.

Graduation date: 1992

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1978. / Bibliography: leaves 193-205. / Microfiche. / xv, 205 leaves ill. 28 cm

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references. Also available via the Internet.