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.

One advantage of Integrated Multi-Trophic Aquaculture (IMTA) is the potential for bioremediation by organic-extractive organisms. In British Columbia, a number of marine invertebrate species are being considered for use in open-water IMTA with sablefish (Anoplopoma fimbria). These include both filter-feeding bivalves (e.g. cockles, mussels, oysters, scallops) which would consume the finer suspended particulates from the finfish culture component and deposit/detrital feeders (e.g. sea cucumbers, sea urchins, prawns) which would feed on the heavier-settleable solids. The following candidate species were tested for their ability to consume sablefish faeces and uneaten sablefish feed in laboratory feeding trials: green sea urchin (Strongylocentrotus droebachiensis), basket cockle (Clinocardium nuttallii), blue mussel (Mytilus edulis), spot prawn (Pandalus platyceros), and California sea cucumber (Parastichopus californicus). Whether they can remove organic material from aquaculture wastes was tested by measuring ingestion rate or clearance rate and absorption efficiency when they were fed a diet of sablefish waste, relative to those fed a natural control diet. Egestion rates in the candidate species were quantified to estimate the potential amount of waste that may be lost from the organic-extractive component. Biophysical properties including shape, size, and settling velocity were measured in faecal pellets egested by the candidate species to provide input data for models to assess dispersal of faeces from IMTA sites. Results from the laboratory feeding trials demonstrate that all candidate species are capable of consuming wastes from sablefish aquaculture and absorbing the organic material. The relative merits and drawbacks of each candidate species are discussed with respect to the results and within the broader context of IMTA. The general conclusion is that, in order to achieve efficient removal of organic material and successful bioremediation, deposit feeders should be included in the organic-extractive component, whether alone or in conjunction with suspension feeders. / Graduate

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.

Perchloroethene and trichloroethene are among the most persistent groundwater pollutants, and Dehalococcoides is the only known species that can degrade these compounds completely to non-toxic ethene. Characterization of the reductive dehalogenase (RDase) enzymes responsible for dechlorination is important to understanding this process. A series of dechlorination assays were performed with whole cell suspensions and cell-free extracts of three Dehalococcoides-containing mixed microbial consortia to compare dechlorination kinetics and to characterize co-contaminant inhibition. Michaelis-Menten kinetic parameters Vmax and Km, as well as non-competitive inhibition coefficients for 1,1,1-trichloroethane and 1,1-dichloroethane inhibitors are reported. Secondly, blue native gel electrophoresis was developed as a method to isolate active protein complexes containing RDases. Thirdly, sources of variability in the isotopic fractionation of vinyl chloride to ethene reaction step were examined using cell-free extracts and whole-cell suspensions. Understanding the function and range of RDases are goals towards the successful application of Dehalococcoides-containing cultures to remediate contaminated sites.

The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental context for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the expected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation.

The enhancement of bioremediation by bioaugmentation in soil contaminated with diesel fuel No. 2 and No. 6 (Bunker C) is uncertain. A clayey soil was contaminated with 6,000 mg of either diesel fuel per kg of soil and seeded (5 x 10-7 cells/g of soil) with a Hawaii soil bacterium (UH138) known to utilize several hydrocarbons. The soil was limed, fertilized, and incubated in jars at 30°C for several months. The concentrations of total petroleum hydrocarbons (TPH) and of polycyclic aromatic hydrocarbons (PAH) in soil were measured by gravimetry and immunoassay, respectively. Poisoned controls (0.6% HgCl2) were used to determine the extent of hydrocarbon degradation due to microbial activity. A rapid first order biodegradation of TPH (84% in 23 days) occurred in soil contaminated with diesel fuel No. 2, regardless of bacterial seeding. Biodegradation of PAH was linear and reached 84% by day 98 in both seeded and unseeded treatments. Bioaugmentation had no effect on bioremediation of diesel fuel No.2. The decrease in TPH and PAH was paralleled by an increase in populations of total bacteria, phenanthrene-degrading bacteria and microorganisms capable of utilizing hexadecane and diesel fuel No. 2 as well as by an enhancement in CO2 evolution by the soil. Indigenous Zygomycetes grew profusely in diesel fuel No. 2 contaminated soil. Cunninghamella echinulata var. echinulata was isolated from the soil and was shown to be able to utilize several hydrocarbons. Thus, Zygomycetes may have contributed to the rapid decrease in contaminant. In soil contaminated with diesel fuel No. 6, the measurements of TPH and PAH were more variable due to the uneven distribution of the product. No biodegradation of the contaminant occurred over a period of 138 days. The growth of Zygomycetes was scant. The counts of total bacteria remained unchanged after the addition of diesel fuel No. 6. However, counts of the indigenous phenanthrene-degrading bacteria increases dramatically ( 4 log units) during the first 54 days whereas the level of the seeded bacteria remained stable. The counts of mineral oil degraders decrease by 2 log units after day 2. Co2 evolution from the soil confirmed that diesel fuel No. 6 was not degraded by either the indigenous microflora or the seeded bacterium. Thus, diesel fuel No. 2 was highly degradable by the indigenous population, however, diesel fuel No. 6 was recalcitrant. / Thesis (M.S.)--University of Hawaii at Manoa, 1998. / Includes bibliographical references (leaves 106-117). / Available also on microfiche. / Department of the Interior; U.S Geological Survey via Water Resources Research Center

Thesis (M.S.)--University of Wyoming, 2007. / Title from PDF title page (viewed on Nov. 3, 2008). Includes bibliographical references (p. 49-55).

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "December, 2007." Includes bibliographical references (leaves 152-157). Online version available on the World Wide Web.

Thesis (M.S.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 49-52). Also available on the World Wide Web.