• Analysis and comparison of cyanide detoxification methods from spent heaps

      Kroeger, Edwin Bane; Huang, Scott L.; Nelson, Michael G.; Bandopadhyay, Sukumar; Braddock, Joan F.; Chen, Gang; White, Daniel H. (1997)
      This work entailed the testing of cyanide degradation of microorganisms isolated by the U.S. Bureau of Mine's Salt Lake Research Center (SLRC) and native strains isolated from an Alaskan mine, development of conceptual designs for in situ biological detoxification of leached heaps, and comparison of common in situ and ex situ heap detoxification processes using a cost comparison, sensitivity analysis, and Monte Carlo simulation. Microorganisms from an Alaskan mine site and SLRC were tested for cyanide degradation. Testing of samples from the Ryan Lode site indicated that several bacterial colonies could tolerate cyanide, but no degradation was found. Once degradation was confirmed in the SLRC sample, a winter survival rate test and an in situ heap detoxification test were performed. Winter survival experiments indicated approximately 5-20% of the bacterial population in heap agglomerate samples were viable after a winter and subsequent coldroom wait. In addition, bacteria colonized the agglomerate where nutrients were available. During August 1993, a 250 ton test heap was constructed on a heap that was undergoing detoxification using the INCO air-SO$\sb2$ process. The test heap was inoculated with bacteria in October 1993 and September 1994. Unfortunately, the test was terminated at an early stage, limiting the conclusions. The detoxification processes chosen for comparison were in situ biological, peroxide, and chlorination, and ex situ biological, INCO air-SO$\sb2,$ peroxide, and chlorination. To compare the costs, a hypothetical heap of 1 million tons was used. In situ biological detoxification had a cost of $0.41 per ton of ore treated, followed by ex situ biological detoxification, with a cost of \$0.92 per ton. The remaining methods ranged from $1.05 to \$1.35 per ton. The biological detoxification methods were most sensitive to the rinsing rate, labor costs, and capital costs. The chemical detoxification methods were most sensitive to oxidant cost, oxidant to cyanide ratio, and starting cyanide concentration. For the Monte Carlo simulation, in situ biological detoxification had a cost of $0.63 per ton treated, followed by in situ peroxide with a cost of \$1.09 per ton. The remaining methods ranged from $1.11 to \$1.45 per ton.
    • Mechanisms Involved In The Cold Tolerant Trichoderma Atroviride Biocontrol

      Cheng, Mingyuan; McBeath, Jenifer H. (2004)
      Trichoderma atroviride is a cold tolerant fungus that parasitizes a wide range of plant pathogenic fungi. The mechanisms involved in biocontrol by T. atroviride are only partially understood. This research evaluated the effect of four different groups of plant pathogenic fungi (Botrytis cinerea, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum) on enzyme expression at 22�C and 7�C. The enzymes expressed (proteinase and endo-beta-1,3-glucanase) were purified and characterized, and three 73 kDa N-acetyl-beta- D-glucosaminidase genes from three different T. atroviride biotypes were sequenced. The R-1,6-glucanase profiles and the regulation of N-acetyl-beta-D-glucosaminidases by plant pathogenic fungi were also studied. I document the production of N-acetyl-beta-D-glucosaminidase, exochitinase, endochitinase, beta-1,3-glucanase, beta-1,6-glucanase and proteinase by T. atroviride at room temperature. The timing of enzyme expression was pathogen dependent. A high concentration of glucose repressed the expression of glucanases, but had no effect on the expression of N-acetyl-beta-D-glucosaminidase. At 7�C, T. atroviride produced the same enzymes as at room temperature except beta-1,6-glucanase. The total activities of the chitinases increased over a 30 day incubation period while the expression of glucanases and proteinase depended on the inducer. A new 18.8 kDa serine proteinase and a new 77 kDa endo-beta-1,3-glucanase were purified to electrophoretical homogeneity. These two purified enzymes showed strong antifungal activity by inhibiting conidial germination of Botrytis cinerea. Three 73 kDa N-acetyl-beta-D-glucosaminidase genes were sequenced from T. atroviride biotypes 861, 453 and 603. Gene sequences of the enzyme from the T. atroviride biotypes are different from the published gene sequence of T. harzianum . This indicates that the N-acetyl-beta-D-glucosaminidase sequence can be used to differentiate the species and isolates of Trichoderma. The expression of beta-1,6-glucanase is complex and at least three different sizes of beta-1,6-glucanase were detected from T. atroviride. The expression of beta-1,6-glucanase varied with carbon source and pH. Mycelia of plant pathogen regulated the expression of N-acetyl-beta-D-glucosaminidase. Two different sizes of N-acetyl-beta-D-glucosaminidase were detected when T. atroviride was grown with S. sclerotiorum and its filtrates. Only one N-acetyl-beta- D-glucosaminidase was detected with other pathogens, autoclaved mycelia or glucose. The expression of a 73 kDa N-acetyl-beta-D-glucosaminidase was contact-dependent and regulated by an extracellular factor.
    • Microbial ecology and long-term persistence of crude oil in a taiga spruce forest

      Lindstrom, Jon Eric; Braddock, Joan F. (1997)
      The microbial ecology of a 1976 experimental crude oil spill in an Alaskan taiga black spruce forest was investigated in this study. Substantial oil residue remained in the soil, and several microbial parameters showed evidence of long-term oiling effects. Overall, the data suggest that the surviving community in the oiled plot has shifted toward using oil C for growth. Numbers of hydrocarbon degrading microbes, and specific hydrocarbon mineralization potentials, were significantly elevated in the oiled (OIL) plot compared to an adjacent oil-free, reference (REF) plot. Glutamate mineralization potentials and soil C mineralization, on the other hand, were not different between treatments, suggesting that OIL plot heterotrophs were well-acclimated to the oil. Despite little difference between OTL and REF soils in total C mineralized in vitro, net N mineralized was lower and net nitrification was absent in OIL soils. Analysis of the residual oil indicated minimal amounts of N were added with the spilled oil. Biomasses of total fungi and bacteria, and numbers of protozoa, showed no consistent effects due to oiling, but metabolically active fungal and bacterial biomasses were uniformally lower in OTL samples. Community-level multiple substrate metabolism (Biolog) was assessed using a new technique for extracting kinetic data from the microplates. This analysis suggested that the microbial population diversity in the OIL soils was lower than in REF soils. Further, these data indicated that the surviving populations in the OIL plot may be considered metabolic generalists. Some evidence of crude oil biodegradation was seen in the chemistry data, but enrichment of the oil residue in higher molecular weight components, duration of contact with soil organic material, and slow rates of C mineralization indicate the crude oil will persist at this site for decades. Contamination of Alaskan taiga soil at this site has yielded observable long-term microbial community effects with larger-scale consequences for ecosystem function.
    • Seasonal Fungal Biomass Dynamics In An Interior Alaskan Paper Birch (Betula Papyrifera Marsh) And Quaking Aspen (Populus Tremuloides Michx.) Stand And Effects Of Long-Term Fertilization (Fungi, Mycology, Mushrooms, Ecosystem(S))

      Moore, Terry A. (1985)
      Standing crop fungal biomass was measured at bi-weekly intervals for two successive field seasons in contiguous, 50 year old stands of quaking aspen (Populus tremuloides Michx.) and paper birch (Betula papyrifera Marsh) and in contiguous stands of aspen and birch undergoing long-term fertilization by yearly application of inorganic nitrogen, phosphorus and potassium fertilizers. Soil temperature and moisture were monitored throughout the study. Principal goals were: (1) to delineate seasonal fluctuations in fungal biomass in the forest floor and mineral soils of aspen and birch vegetation sites considered representative of upland, interior Alaskan hardwood taiga; (2) to determine if biomass fluctuations were correlated with fluctuations in soil microclimate; (3) to determine if differences in fungal biomass were correlated with dominant overstory vegetation; i.e. differences in primary or secondary site substrate (resource) quality; (4) to determine if long-term (nine years) application of inorganic fertilizers altered overall standing crop fungal biomass in the two vegetation types studied; and (5) to determine if soil bulk density or microclimate were influenced by vegetation type or fertilization. Results show that seasonal biomass for both control and fertilized sites was closely correlated with soil moisture and exhibited little or negative correlation with soil temperature. Unamended aspen soils supported significantly greater fungal biomass than birch soils due to increased soil moisture, a more favorable chemical environment and production of organic matter more conducive to growth of soil fungi. Fertilization significantly decreased fungal biomass in aspen soils indicating the long-term treatment with inorganic fertilizers could be detrimental to mineral cycling in this forest type. Fertilization significantly increased fungal biomass in birch soils due to increased soil organic matter content and increased soil moisture. Hyphae of basidiomycetes was significantly decreased by fertilization in both vegetation types suggesting that basidiomycetes involved in saprotrophic decomposition and/or mycorrhizal associations were adversely affected by fertilization. The effects of vegetation type and fertilization on soil temperature, moisture and bulk density are discussed.
    • Soil consumption of atmospheric methane: Importance of microbial physiology and diversity

      Gulledge, Jay Michael (1996)
      Recently, atmospheric CH$\sb4$ concentration has risen dramatically, apparently due to human activities. Since is CH$\sb4$ is involved in several atmospheric processes that regulate Earth's climate, it is important that we understand the factors that control its atmospheric concentration. One such factor is biological CH$\sb4$ consumption in well-drained soils. Although this sink may comprise nearly one-tenth of the annual destruction of atmospheric CH$\sb4$, We know relatively little about it. I conducted a research project to investigate the influences of CH$\sb4$ supply, soil moisture, dissolved salts, and NH$\sb4\sp+$-fertilizer on the activity of soil CH$\sb4$ oxidizers. When starved of CH$\sb4$, two upland taiga soils gradually lost their capacities to oxidize CH$\sb4$, indicating that the process was not merely fortuitous, and that the organisms involved were truly methanotrophic. The relationship between soil moisture and CH$\sb4$ consumption was parabolic, with maximum oxidation occurring at a moisture level that achieved the maximum possible CH$\sb4$ diffusion rate, while minimizing water stress on the methanotrophs. Optimal soil moisture occurred in a relatively narrow range among an array of physically dissimilar soils, providing that moisture content was expressed as a percentage of the water holding capacity fo a particular soil, rather than as absolute water content. In recent years, one of the most intensely investigated controls on soil CH$\sb4$ consumption has been its inhibition by NH$\sb4\sp+$-fertilizer. In addition to NH$\sb4\sp+,$ however, I found that other ions inhibited CH$\sb4$ oxidation. In some soils non-NH$\sb4\sp+$ ions were so toxic that they completely masked the NH$\sb4\sp+$ effect. It is crucial, therefore, to control for salt effects when investigating NH$\sb4\sp+$-inhibition. In both field and laboratory experiments, CH$\sb4$ consumption in a birch soil was sensitive to NH$\sb4\sp+$, whereas a spruce soil was unaffected. In the birch soil, NH$\sb4\sp+$ apparently inhibited methanotroph growth, rather than enzymatic CH$\sb4$ oxidation, whereas methanotrophs in the spruce soil were apparently insensitive to NH$\sb4\sp+$. These results suggest that the primary landscape-level control over the response of soil CH$\sb4$ consumption to NH$\sb4\sp+$-fertilization is the cross-site distribution of physiologically distinct CH$\sb4$ oxidizers.
    • The lectin-like properties of the extracellular protein produced by Pseudomonas aeruginosa during hexadecane degradation

      Smith, Richard Leland; Braddock, Joan (1996)
      Numerous microorganisms can degrade hydrocarbons and many produce extracellular compounds. These compounds are generally thought to emulsify hydrocarbons making them more available to the microorganism and stimulating growth. Pseudomonas aeruginosa produces both a rhamnolipid surfactant and an extracellular protein during growth on n-hexadecane (C$\rm\sb{16}H\sb{34}).$ The protein has been hypothesized to stimulate growth by emulsifying hexadecane. However, it has never been shown that the protein has hydrophobic properties characteristic of emulsifiers. An isolation procedure was developed in this study that produces 20-30 mg of very pure protein per 500 ml culture of strain ATCC 17423. The protein is monomeric with a molecular weight of approximately 14,500 determined by SDS-PAGE. Structurally the protein is similar to two other proteins from strains S7B1 and PG210. The proteins from strain S7B1 and 17423 stimulate growth of P. aeruginosa on hexadecane. The hydropathic index of the protein from strain PG201 shows no strong hydrophobic regions in the amino acid sequence. Also the isolated protein from strain 17423 will not bind during hydrophobic chromatography and always acts as a monomer in solution even at concentrations which should cause hydrophobic proteins to aggregate. These results indicate that the protein is not hydrophobic and therefore does not have surfactant-like properties. A surprising result from this investigation is that the protein from strain 17423 has lectin-like (carbohydrate binding) qualities and agglutinates P. aeruginosa, Escherichia coli, human type O, and horse red blood cells. The agglutination is inhibited by EDTA, glucose, mannose and rhamnose. The exact carbohydrate(s) the protein binds has not been determined but evidence suggests that it may bind the carbohydrate portion of the rhamnolipid surfactant. A new model is presented describing the function of the protein. In this model the extracellular protein functions by binding the emulsified hydrocarbon to the outer membrane of the bacterium by both the carbohydrate on the glycolipid surfactant and the lipopolysaccharide of the bacterium. This binding of the hydrocarbon stimulates growth of the bacterium on hexadecane.
    • Thermally enhanced bioventing of petroleum hydrocarbons in cold regions

      Filler, Dennis M.; Carlson, Robert F. (1997)
      Petroleum-based contamination of the environment has and will likely continue to be a problem as long as oil and natural gas supply much of the world energy demands. In cold regions, where vast quantities of these fuels are extracted and used, climate and frozen soils limit remedial efforts to a few technologies. Bioventing has shown promise as a viable method for the remediation of spilled petroleum-based fuels in cold regions. An in situ study of bioventing with soil warming was conducted at a Fairbanks, Alaska site. The main purpose of this research effort was to compare the effectiveness of thermal enhancement techniques applied to bioventing. Objectives included (1) developing a suitable thermal insulation system(s) that would provide year-round bioventing of petroleum contaminated soils, (2) modeling of the thermal regime below three treatment areas, (3) relating monitoring and testing data to thermally enhanced biodegradation, and (4) presenting the information in a way that is useful to engineers, biologists and environmental scientists. Active soil warming with electrical heat tape beneath polystyrene insulation and sand and gravel overburden raised subsurface soil temperatures from the ground surface to the water table by as much as 15$\sp\circ$F. The actively warmed test plot was successfully heated year-round, preventing soil freezing and enhancing microbial activity. Soil gas, microbiological, and geochemical sampling data evidenced correlation between increased bioactivity and soil warming. Passively treated soils evidenced some winter increase in temperatures, although some periodic soil freezing did occur. Overall, biodegradation within both passively treated and untreated contaminated test plots was noticeably slower than within the actively warmed plot. Thermally enhanced bioventing successfully remediated hydrocarbon contamination in vadose zone soils at a subarctic site within two years. After oxygen, temperature appears to be the most important factor affecting microbial activity and biodegradation. Variable and low moisture contents did not seem detrimental to bioactivity.
    • Wild-type and mutant 2,2-dialkylglycine decarboxylases: The catalytic role of active site glutamine 52 investigated by site-directed mutagenesis and computer analysis

      Woon, See-Tarn (1998)
      The ability of pyridoxal 5$\sp\prime$-phosphate (PLP)-dependent 2,2-dialkylglycine decarboxylase (DGD) to catalyze decarboxylation and transamination of amino acids at a single active site depends on the subsite within the active site that cleaves $\alpha$-H and $\alpha$-COO$\sp-$ bonds. As observed in the crystal structure, the strategic position of glutamine 52 at the active site suggests a role in enhancing decarboxylation via formation of a hydrogen bond to the substrate carboxyl group. Supporting evidence for this hypothesis is provided by studies with glutamine 52 active site mutants, computer modeling and protein sequence analyses. Ten mutant DGDs containing alanine, asparagine, aspartate, arginine, glutamate, glycine, histidine, leucine, lysine, and tryptophan at position 52 were produced. All, except the histidine mutant, exhibited decreased rates of decarboxylation compared to wild-type. Histidine and asparagine mutants showed measurable decarboxylation rates. These results and that of wild-type DGD suggest that hydrogen bonding with the substrate is required for decarboxylation. Mutants incapable of hydrogen bonding to the substrate, such as alanine, leucine and tryptophan mutants, showed negligible decarboxylation reactions. Transamination rates increased for some mutants and decreased for others. These data imply that the DGD subsite is influenced by the presence of glutamine 52. Furthermore, there is evidence showing that the subsite environment of wild-type DGD, the histidine and the glutamate mutants are different; the three DGD forms exhibited different chromophores at around $\rm\lambda\sb{max}$ of 500 nm when treated with 2-methylalanine or L-alanine in the presence of 3% glycerol. These results have important implications for other PLP-dependent enzymes, such as ornithine aminotransferase and $\gamma$-aminobutyrate aminotransferase. Since protein sequence alignment indicates DGD is homologous to the two aminotransferases, mutations at amino acid position corresponding to glutamine 52 of DGD at the active sites of these aminotransferases could disrupt the functionality of the enzymes. Protein sequence alignment showed that all but one of the PLP-dependent aminotransferases lack residues at position 52 capable of hydrogen bonding with the substrate carboxyl group, further re-affirming the role of glutamine 52 in decarboxylation.