Title: Western Region Hazardous Substance Research Center Project 1-OSU-02.
Aerobic Cometabolism of Chlorinated Aliphatic Hydrocarbon Compounds with Butane-Grown Microorganisms:

Investigators: Peter Bottomley, Dan Arp, Lynda Ciuffetti, Mark Dolan, Lewis Semprini, Kenneth Williamson

Institution: Oregon State University

Research Category: Bioremediation, cometabolism

Project Period: January 2002 – December 2005

Objectives: This project was focused on dealing with evaluating how to maximize the chloroethene (CE) and chloroethane (CA) degrading potential of individual strains and mixed communities of butane degrading bacteria and fungi. Specific objectives included identifying growth conditions that maximize reductant flow to cometabolism, and the mechanisms that sustain monooxygenase enzyme activity and minimize cytotoxic damage to the cells; evaluating the potential for the bioaugmentation of a butane culture for in-situ bioremediation of CE and CAs; and to describe the ability of Graphium sp. to degrade a range of CE and CAs volatile organic compounds including chlorinated aliphatic hydrocarbons (CAHs), trichloromethanes and methyl tertiary-butyl ether (MTBE). Based on these objectives the project was divided into three subprojects that are each summarized.

Project 1

Investigators: Peter Bottomley and Dan Arp

Objectives: The project aimed to evaluate how to maximize the chloroethene (CE) degrading potential of individual strains of hydrocarbon degrading bacteria. Specific subobjectives included identifying conditions that maximize reductant flow and the cellular mechanisms that minimize the toxic effects of cometabolism and sustain the process.

Rationale: Studies conducted under laboratory and field conditions have shown that hydrocarbon-oxidizing bacteria cometabolize a wide range of CEs. Nonetheless, there is considerable variability in the properties of cometabolism shown by different types of butane oxidizing bacteria both in terms of the range of CEs degraded and in their transformation capacities. More research was carried out to better understand the microbiological reasons for the range of efficiencies observed, with the goal of using this information to improve the biotechnology of bioremediation by cometabolism.

Summary of Findings: We examined the CE degrading properties of several individual strains of butane-oxidizing bacteria that are genotypically distinct from each other and that possess different butane monooxygenases. We examined the impact of cometabolism of different CEs on monooxygenase activity, and assessed the impact on cell viability. While co-oxidation of trichloroethene (TCE) by Pseudomonas butanovora and Nocardioides CF8 resulted in 96% inactivation of butane monooxygenase, the BMO of Mycobacterium vaccae was found to be more resistant to inactivation by TCE and its respiratory activity was unaffected (Halsey et al. 2005).

Based upon these observations it was proposed that situations might be identified where CE degrading bacterial strains with lower rates of degradation might be more appropriate bioremediatory agents than strains showing higher rates of CE degradation when the latter cannot be sustained. Therefore, we genetically engineered strains of Pseudomonas butanovora by replacing specific amino acids associated with the BMO hydroxylase alpha subunit (Halsey et al. 2006). We examined the CE degrading properties of these mutants and examined the impact of cometabolism of different CEs on monooxygenase activity, and assessed the effect of cometabolism on cell viability (Halsey et al. 2007). As an example, mutant strain G113N was particularly interesting. This strain had an amino acid substitution in, or close to, the catalytic site of the monooxygenase. This strain oxidized butane primarily to 2-butanol rather than to 1-butanol (as also is the case in M. vaccae), but at a slower rate than wild type. Although G113N oxidized TCE and DCEs at slower rates than did wild type, it also liberated less chloride than wild type indicating that the products of oxidation were different from those formed by wild type. In addition, there was no evidence that G113N transformed the epoxide of 1,2 cis DCE that was formed, and, there was no evidence of toxic effects caused by transformation of 1,2 cis-DCE or 1,1DCE in G113N either. The data suggest that a specific amino acid substitution in BMO affected CE turnover-product distribution such that the mutant strain seemed immune to toxicity.

Since aerobic cometabolic biodegradation is often limited by product toxicity in the form of enzyme inactivation or loss of cellular viability, the results obtained in this study indicate that the oxidative pathway favored by mutant strain G113N would promote more sustainable biodegradation of CEs, and that this was likely due to an enzyme mechanism that had been modified to disfavor formation of an unstable epoxide, and also disfavor subsequent attack of the epoxide by BMO. Butane-grown P. butanovora co-oxidizes cis-DCE, 1,2, trans-DCE, and 1,1-dichloroethene (1,1-DCE). When P. butanovora was exposed to each of the three DCEs, and residual BMO activity measured by ethylene-dependent ethylene oxide formation, BMO activity was reduced in a time-dependent manner that varied with the specific DCE. BMO activity decreased by 50% after 15 min exposure to cis-DCE, after 6 min exposure to trans-DCE, and after 30 sec exposure to 1,1-DCE. In addition, cooxidation of the DCEs had different cytotoxic effects on P. butanovora. Although cooxidation of cis-DCE and trans-DCE inactivated the majority of BMO activity, cells retained lactate-dependent O2 consumption but they were unable to grow normally after removal of the DCEs. In contrast, cooxidation of 1,1-DCE caused a rapid decrease in both BMO activity and lactate-dependent O2 consumption within three min of exposure, and cells lysed. Treating cells with acetylene to inactivate BMO eliminated the effects of 1,1-DCE, and lactate-grown cells (in which BMO was not expressed) were also unaffected.

Construction of a LacZ/ BMO reporter strain allowed us to compare the efficiency of induction of BMO gene expression by DCEs with the induction of BMO activity in the wild type parent by the same compounds (see Table 2). The relative induction characteristics of the three DCEs differed from their substrate properties. Trans-DCE induced BMO activity in both the wild type and in the Lac Z reporter strain, while cis-DCE only induced enzyme activity in the wild type. Enzyme activities in wild type cells were induced to£ 25 and 45% of the butane control by cis-DCE and trans-DCE, respectively. LacZ expression was induced to 80% of maximal by trans-DCE in the reporter strain. In the case of trans-DCE, BMO induction could be detected in the reporter strain at lower concentrations (5 to 10mM) than could be detected by ethylene-dependent ethylene oxide formation in the wild type (30mM). the ability of 1,2-trans DCE to induce BMO implies that bioremediatory activity might be activated in a polluted plume regardless of the presence of the natural substrate.

Project 2

Investigators: Lewis Semprini and Mark Dolan

Objectives: Evaluate the potential for bioaugmentation of a butane-utilizing culture that is effective in transforming mixtures of 1,1,1-trichloroethane (1,1,1-TCA), 1,1-dichloroethane (1,1-DCA), and 1,1-dichloroethene. Specific objectives were to evaluate the bioaugmentation potential the butane culture in a continuous flow column study; use molecular tools to track the culture upon its addition in the column study; develop kinetic parameters for butane utilization and CAH transformation for use in a modeling analysis of the column tests; model the results of the column experiment with a numerical model that includes kinetic terms for the microbial processes that have been determined independently in the laboratory; and conduct studies on the potential for a butane culture to transform the epoxide of cis-dichloroethene transformation.

Rationale: Laboratory and microcosm studies have shown different abilities of butane-utilizing microorganisms to cometabolize CAHs. Of particular interest is 1,1,1-TCA, 1,1-DCE, and 1,1-DCA. A Rhodocococcus sp. culture has been sequenced and obtained in pure culture that effectively transforms these compounds. This culture was bioagumented to the subsurface at the Moffett Field tests zone in a collaborative research grant funded through the DoD SERDP program. The Center project conducted continuous flow column experiments, like those performed in the field, for direct comparison with the field results, and to permit more detailed evaluation of the of processes and conditions that can not be performed in the field. Transformation rate parameters for the Rhodocococcus culture, including maximum utilization rate (kmax) and half-saturation coefficients (Ks) values were also determined, and compared with previously determined values obtained with the mixed culture from which the pure culture was derived. Modeling studies were then performed to simulate the results of the column experiments. Studies were also conducted to evaluate the ability of the parent mixed culture to transform cis-1,2-dichloroethylene epoxide that is formed during the aerobic cometabolism of cis-dichloroethylene.

Summary of Findings: The transformation of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethene (1,1-DCE) was evaluated in a continuous flow column reactor after bioaugmentation with the highly enriched Rhodocococcus culture The column was packed with aquifer materials and groundwater obtained from the in situ bioremediation test site at Moffett Field, CA and bioaugmented with 0.9 mg of cells on a dry mass basis. While adding only 1,1,1_TCA at 200 mg/La maximum removal of 84% was achieved 10 days after bioaugmentation and remained fairly constant for a period of 20 days. The influent concentration of 1,1,1-TCA was then doubled, while dissolved oxygen and butane addition was maintained constant. The transformation of 1,1,1-TCA during this period fluctuated between 24%-84%. Upon restoring the 1,1,1-TCA concentration back to 200mg/L the transformation stabilized at 59% removal. In the final phase, 1,1-DCE was injected at 130 ug/L along with 1,1,1-TCA, dissolved butane and oxygen. The butane-utilizing culture transformed 70% of 1,1-DCE; however, the presence of 1,1-DCE inhibited 1,1,1-TCA transformation and approximately 50% of the butane injected was not consumed. The concentration of dissolved oxygen in the column also increased, which also indicated that 1,1-DCE transformation inhibited butane and dissolved oxygen utilization and 1,1,1-TCA transformation. Real-time PCR analysis conducted indicated that during periods of low biotransformation of 1,1,1-TCA, bioaugmented cell densities observed in the column effluent was high. This corresponded to a period of anoxic conditions, which may have caused cell detachment from the aquifer solids.

The column reactor results were simulated using a combined biotransformation-transport model that uses Monod/Michaelis-Menten kinetics along with first-order sorption kinetics, to predict substrate utilization and chlorinated solvent transformation. The culture parameter values used to simulate biotransformation in the model were obtained from laboratory culture experiments described below. Transport parameters (dispersion coefficient, porosity) were determined from modeling breakthrough test data with the CXTFIT2 transport model prior to bioaugmentation and biostimulation. Simulations of the column data using the transport and biotransformation parameters demonstrated that the model was able to simulate biotransformation of 1,1,1-TCA fairly well. The model also indicated that 1,1-DCE transformation was toxic to the butane-utilizing culture and predicted the decreases in consumption of butane, and dissolved oxygen and in 1,1,1-TCA transformation.

This study showed that column experiments conducted on a small scale in a laboratory were of value in determining the biotransformation capabilities of bioaugmented microorganisms. The results suggest that the butane-utilizing culture could be successfully used in situ for bioremediation, but transformation of mixtures of 1,1-DCE and 1,1,1-TCA could prove difficult. Similar extends of 1,1,1-TCA transformation were observed in the Moffett field tests when only 1,1,1-TCA was added. 1,1-DCE transformation in the field was also observed to be toxic to the butane utilizers and to decrease 1,1,1-TCA transformation.

Single compound tests were performed with the butane grown Rhodococcus culture to determine the Monod kinetic parameters. The cells were added to a series of liquid and gas phase microcosms; the maximum degradation rates (kmax) and the half saturation coefficients (Ks) of butane, 1,1,1-TCA and 1,1-DCA, and the cell yield (Y) for growth on butane were determined. The purity of the culture was determined by PCR and TRFLP analysis. The transformation capacity of the two CAHs was evaluated through experiments performed both in the presence and in the absence of butane. 1,1,1-TCA was found to have a transformation capacity 50 times lower than 1,1-DCA. The reciprocal degradation inhibition of butane and each of the solvents was also studied. 1,1,1-TCA transformation was found to be more inhibited by the presence of butane than 1,1-DCA. The overall transformation performance of the isolate culture was better than that of the source culture due to the lower degree of inactivation as a result of transformation product toxicity.

Aerobic cometabolism of cis-1,2-dichloroethylene (c-DCE) by a butane-grown mixed culture was evaluated in batch kinetic tests. The transformation of c-DCE resulted in the coincident generation of c-DCE epoxide. Chloride release studies showed ~75% oxidative dechlorination of c-DCE. Mass spectrometry confirmed the presence of a compound with mass-to-charge-fragment ratios of 112, 83, 48, and 35. These values are in agreement with the spectra of chemically synthesized c-DCE epoxide. The transformation of c-DCE required O2, was inhibited by butane and was inactivated by acetylene (a known monooxygenase inactivator), indicating that a butane monooxygenase enzyme was likely involved in the transformation of c-DCE. This study as showed c-DCE epoxide was biologically transformed, likely by a butane monooxygenase enzyme. c-DCE epoxide transformation was inhibited by both acetylene and c-DCE indicating an monooxygenase enzyme was involved. The epoxide transformation was also stopped when mercuric chloride (HgCl2) was added as a biological inhibitor, further support a biological transformation. To our knowledge this is the first report of the biological transform c-DCE epoxide by a butane-grown culture.


Project 3

Investigators: Ken Williamson and Lynda Ciuffetti

Objectives: This project had two main goals. The primary objective of this project was to describe the ability of Graphium sp. to degrade a range of volatile organic compounds including chlorinated aliphatic hydrocarbons (CAHs), trichloromethanes and methyl tertiary-butyl ether (MBTE). The study also aimed to demonstrate that these reactions are catalyzed by an alkane inducible cytochrome P450 monooxygenase through heterologous expression assays with yeast.

3mRationale: Volatile organic compounds including trichloroethylene (TCE), 1,1-dicloroethylene (1,1-DCE), 1,2-dichloroethylene (1,2-DCE), carbon tetrachloride (CT) and chloroform (CF), a trichloromethane, are important soil and groundwater contaminants. The ability of microorganisms to degrade these compounds represents a promising avenue for the attenuation of polluted sites.

Summary of Findings: Graphium sp., a filamentous fungus, is one of the few eukaryotes known to grow on gaseous n-alkanes. The initial enzymatic step by which Graphium sp. oxidizes n-alkanes for energy and growth is initiated by a highly nonspecific and alkane-inducible cytochrome P450 monooxygenase. Previous studies have suggested that this enzyme also enables Graphium sp. to cometabolically degrade CAHs, trihalomethanes, and PAHs. More specifically, evidence suggests that Graphium sp. can degrade numerous CAHs including all 4 trihalomethanes, chloromethane, dichloromethane, chloroethane, 1,2-DCE and 1,1,2,2-tetrachloroethane. This fungus can also reductively dechlorinated CT to CF in the absence of oxygen and then consume CF when aerobic conditions are reestablished. However, neither the substrate range nor the rates of these Graphium sp. mediated reactions have been determined. The primary aim of this project was to more quantitatively describe both the substrate range and the rate of these reactions.

Although preliminary evidence suggests that a cytochrome P-450 monooxygenase catalyzes the initial steps of these reactions, the role of this enzyme has not been conclusively established. The study also aimed to demonstrate the role of this enzyme in cometabolic degradation of environmentally significant pollutants.

Graphium sp cultures were grown on a variety of environmentally relevant compounds. These assays indicated that Graphium sp. is able to utilize a broader range of alkanes than originally thought and has an alkane substrate range that extends beyond ethane, propane and butane to include isobutane, isopentane, and pentane. Although our results indicated that Graphium sp. hydroxylates alkanes at both terminal and subterminal carbons, in the case of isobutane oxidation, Graphium sp. is only able to use the immediate downstream intermediates that result from primary oxidation of isobutane. However, it does grow on both the subterminal and the terminal oxidation products of isopentane and straight-chain alkane oxidation. In addition, our investigations demonstrated that Graphium sp. utilizes the cyclic ether, tetrahydrofuran, as a growth substrate. Tetrahydrofuran (THF) is metabolized via the pathway that was previously described in some bacteria, including Rhodococcus ruber and two Pseudonocardia strains. Likewise, Graphium sp. grows on the concomitant metabolic products of THF oxidation and a spectrum of related compounds. Although these assays showed that Graphium was unable to grow on another cyclic ether, 1,4-dioxane, Graphium sp. was able to cometabolize this environmentally relevant ether after growth on propane or THF. We also observed that cometabolism of MTBE by this fungus results in the incomplete digestion of MTBE oxidation products, and therefore led to an accumulation of the MTBE metabolite, tert butyl alcohol. These investigations also determined that Graphium-mediated MTBE oxidation is not subject to the regulatory effects produced by MTBE metabolites that have been previously described in an MTBE-utilizing bacterium, Mycobacterium austroafricanum.

Throughout studies that investigated the substrate range of Graphium sp., trends were observed that indicate that the alkane and ether metabolic pathways are superimposed on each other. At least five separate physiological observations support this hypothesis. First, the oxidation of THF and propane are fully inhibited by the same alkenes and alkynes. Second, both compounds appear to behave as mutually competitive substrates. Third, propane-grown cells oxidize THF without a lag phase or the accumulation of THF metabolic intermediates, indicating an apparent ability of propane-grown mycelia to concurrently consume THF and THF-derived oxidation products. Fourth, the rates of THF oxidation by propane-grown mycelia are equivalent to the rates observed when THF-oxidizing activity is fully induced in PDB-grown mycelia. Last, comparable rates of MTBE and 1,4-dioxane were observed when propane- and THF-grown mycelia were assayed in short-term initial rate experiments. These observations indicate that THF and alkane oxidation are mediated by the same cytochrome P450 hydroxylase and suggest a greater degree of overlap between the remaining enzymes in the alkane and ether oxidation pathways. Because monooxygenase-catalyzed substrate activation is both the first and the rate-determining step of these pathways, the gene encoding the alkane monooxygenase from this Graphium sp. was characterized.

We used a strategy that was developed in long-chain alkane-utilizing yeasts to identify and clone a cytochrome P450 alkane monooxygenase from Graphium sp. This gene was designated CYP52L1, which encodes the cytochrome P450 protein GSPALK1. Although CYP52L1 shares some sequence similarity with other yeast alkane hydroxylases from the CYP52 subfamily, unlike cytochrome P450s from yeast, our analyses indicate that CYP52L1 is not closely related to any known CYP52 member. Likewise, CYP52L1 is present in a single copy, whereas alkane hydroxylases from yeast often belong to multi-gene families that encode proteins with overlapping function. The differences between CYP52L1 and its CYP52 relatives are not surprising given that no other CYP52 member is known to oxidize gaseous n-alkanes.

Initial experiments that attempted to express CYP52L1 in yeast and in an alternate filamentous fungus were unsuccessful. Unsuccessful forward characterization of CYP52L1 was most likely due to the lack of a corresponding NADPH oxidioreductase in the heterologous hosts. Therefore, we used a reverse genetics approach to characterize CYP52L1. GSPALK1 was functionally characterized by introducing a construct that causes the fungus to express a double-stranded (ds)- CYP52L1 transcript. Expression of the ds- CYP52L1 transcript triggered endogenous post-transcriptional gene silencing machinery that degraded the native transcript, and therefore abolished expression of this gene in a sequence specific manner. The diminishment of CYP52L1 expression was associated with a loss of function phenotype and disabled alkane and ether oxidation. We observed that although the transformed fungi are no longer able to grow on alkanes or ethers, the ability of the transformants to grow on the downstream metabolites of alkane and ether oxidation (propanol, isobutanol or g-butyrolactone) is not affected by post-transcriptional gene silencing of CYP52L1. This observation therefore indicates that only the first step in each of these pathways are affected by CYP52L1 silencing, and thus correlate alkane and ether oxidation to CYP52L1 expression.

Another filamentous fungus that is able to grow on gaseous n-alkanes, Graphium cuneiferum, also harbors a significantly similar (>99.6% amino acid identity) coding sequence, indicating that GSPALK1-like sequences are present in other eukaryotic alkanotrophs. During our investigations, a CYP52L1 silencing vector was produced. This vector provides a tool for post-transcriptional gene silencing of CYP52L1-like genes in other fungi. Because the substrate range of these fungi differ from the one characterized here, it would be interesting to compare the primary protein structure of CYP52 members from these fungi to further our understanding of the molecular determinants of both substrate range and cometabolism.

These investigations evidence that extends the substrate range of this fungus to include a spectrum of straight chain and branched alkanes, cyclic ethers, lactones, diols, and acids. They also refined the pathway and the metabolic interactions that were thought to regulate Graphium-mediated MTBE cooxidation. We also showed that the oxidation of alkanes and ethers is linked through a common catalyst, a cytochrome P450 alkane monooxygenase that mediates the first step of these pathways. This enzyme, designated GSPALK1, was further characterized through molecular genetic and biochemical analyses. The characterization of GSPALK1 and the gene encoding it, CYP52L1, is the first description of a cytochrome P450 involved in the terminal oxidation of gaseous n-alkanes and cyclic ethers as well as the first description of a cytochrome P450 involved in MTBE and 1,4-dioxane cometabolism.


Journal Articles

Doughty D.M., Sayavedra-Soto L.A., Arp D.J., and Bottomley P.J. (2005).  Effects of dichloroethene isomers on the induction and activity of butane monooxygenase in the alkane-oxidizing bacterium, Pseudomonas butanovora, Applied and Environmental Microbiology, 71(10): 6054-6059.

Goltz, M.N. and K.J. Williamson (2002). Transfer and Commercialisation of Contaminated Groundwater Remediation Technologies. International Journal of Technology Transfer and Commercialisation, 1(4):329-346.

Frascari, D., Y. Kim, et al. (2003). A Kinetic Study of Aerobic Propane Uptake and Cometabolic Degradation of Chloroform, cis-Dichloroethylene and Trichloroethylene in Microcosms with Groundwater and Aquifer Solids. Water, Air, & Soil Pollution: Focus Vol. 3 (3) 285-298.

Halsey K.H., Sayavedra-Soto L.A., Bottomley P.J., Arp D.J. (2005). Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects. Applied Microbiology and Biotechnology, 68(6): 794-801.  

Halsey, K.H., L.A. Sayavedra-Soto, P.J. Bottomley, and D.J. Arp (2006). Site-directed amino acid substitutions in the hydroxylase α subunit of butane monooxygenase from Pseudomonas butanovora: implications for substrates knocking at the gate. J. Bacteriol. 188: 4962-4969.

Halsey, K.H., D.M. Doughty, L.A. Sayavedra-Soto, P.J. Bottomley, and D.J. Arp (2007). Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase α-subunit of butane monooxygenase. J. Bacteriol. 189: 5068-5074.

Kim, Y. and L. Semprini (2005). Cometabolic Transformation of cis-1,2-dichloroethylene and cis-1,2-dichloroethylene epoxide by a butane-grown mixed culture. Water Science & Technology Vol 52 (8): 125-131.

Semprini, L., M. E. Dolan, M. A. Mathias, G. D. Hopkins and P. L. McCarty (2007). Laboratory, field and modeling studies of bioaugmentation of butane-utilizing microorganisms for the in situ cometabolic treatment of 1,1-dichloroethene, 1,1-dichloroethane, and 1,1,1-trichloroethane. Advances in Water Resources, 30 1528-1546.

Semprini, L., M.E. Dolan, M.A. Mathias, G.D. Hopkins, and P. L. McCarty (2007).Bioaugmentation of Butane-Utilizing Microorganisms for the In-situ Cometabolic Treatment of 1,1-Dichloroethene, 1,1-Dichloroethane, and 1,1,1-Trichloroethane. European Journal of Soil Biology. 43 322-327.

Skinner, K.M., A. Martinez-Prado, K.J. Williamson, and L.M. Ciuffetti. Pathway, Inhibition and Regulation of MTBE Oxidation by a Filamentous Fungus, a Graphium sp..Applied Microbiology and Biotechnology (in press 2007).

Skinner, K.M., L.M. Ciuffetti, and M.R. Hyman. Metabolism and Cometabolism of Cyclic Ethers by a Filamentous Fungus, a Graphium sp.. Applied and Environmental Microbiology (manuscript under review 2007).


Abstracts and Posters

Doughty, D.M., L.A. Sayavdero-Soto, P.J. Bottomley, and D.J. Arp (2004). Metabolism of dichloroethenes by Pseudomonas butanovora. Annu Mtg. Amer. Soc. Microbiol., New Orleans.

Halsey, K.H., L.A. Sayavedra-Soto, P.J. Bottomley, and D.J. Arp (2004). Kinetics of TCE transformation and comparisons of TCE transformation-dependent toxicities in butane-oxidizing bacteria. Annu. Mtg. Amer. Soc. Microbiol, New Orleans.

Martinez-Prado A., Skinner K., Ciuffetti L.M., Williamson K.J. (2002). MTBE kinetics by n-alkane-grown Mycobacterium vaccae and Graphium sp.. In: Batelle (ed) Third International Conference on Remediation of chlorinated and recalcitrant compounds, Session E9: MTBE characterization and treatment. Batelle, Monterey, CA, pp 20–23.

Martinez-Prado, A., K.J. Williamson, L. M. Ciuffetti, and R. Guenther (2003) Modeling of MTBE Kinetics by Alkane Grown Mycobacterium vaccae. The Seventh International Symposium on In Situ and On-Site Bioremediation, Orlando, FL, (June 2-5).

Semprini, L, M. E. Dolan, M. Mathias, G. D. Hopkins, and P.L. McCarty (2003).

Laboratory, Field, and Modeling Studies of Aerobic Cometabolism of CAHs by Butane-Utilizing Microorganisms. The Seventh International Symposium on In Situ and On-Site Bioremediation, Orlando FL (June, 2-5).

Semprini, L., M.E. Dolan. M. Mathias, G.D. Hopkins, and P.L. McCarty (2005). Laboratory and Field Studies of Bioaugmentation of Butane-Utilizing Microorganisms for the In-situ Cometabolic Treatment of Treatment of 1,1-Dichloroethene, 1,1-Dichloroethane, and 1,1,1-Trichloroethane. Proceedings of the Third European Conference on Bioremediation, Crete (July 3-5).

Razzetti, C. M.E. Dolan, and L. Semprini (2004). Cometabolic degradation of 1,1,1-trichloroethane and 1,1-dichloroethane by a Rhodococcus species. SETAC Annual Meeting, Portland, OR.

Razzetti, C., M.E. Dolan, and L. Semprini (2005). Cometabolic degradation of 1,1,1-trichloroethane and 1,1-dichloroethane by a Rhodococcus species in a soil column reactor, 10th EuCheMS Conference on Chemistry and the Environment, Rimini (September).

Skinner, K.M. (2004). Groundwater Remediation through Environmental Biotechnology: Transgenic Phytoremediation of Methyl tertiary Butyl Ether. EPA STAR Fellowship Conference, Washington D.C..

Skinner, K.M. and L.M. Ciuffetti (2005). Characterization of a fungal gene results in the development of technologies for transgenic bioremediation and biosensing of environmental pollutants. 23rd Annual Fungal Genetics and Biology Conference, Pacific Grove, CA.

Skinner, K.M., M.R. Hyman and L.M. Ciuffetti (2006). Pathway, Inhibition and Regulation of MTBE Oxidation by a Filamentous Fungus, a Graphium sp.. 106th General Meeting of the American Society for Microbiology, Orlando, FL.



Doughty, D.M. (2004). Metabolism of dichloroethenes by the butane-oxidizing bacterium, Pseudomonas butanovora. M.S., Oregon State University.

Maremanda, B. (2004). Aerobic cometabolism of 1, 1, 1-Trichloroethane and 1, 1-Dichloroethene by a bioaugmented butane-utilizing culture in a continuous flow column. M.S., Oregon State University.

Razzetti, C. (2005). Cometabolic degradation of 1,1,1-trichloroethane and 1,1-dichloroethane by a butane grown Rhodococcus species: kinetic studies, reactor operation and modeling. Ph.D., University of Bologna.

Sattayatewa, C. (2004). Growth Characteristics and Chlorinated Hydrocarbon Transformation Ability of a Rhodococcus sp. Isolate. M.S., Oregon State University.

Skinner, K.M. (2007). Characterization of the Molecular Foundations and Biochemistry of Alkane and Ether Oxidation in a Filamentous Fungus, a Graphium species. Ph.D., Oregon State University.


Supplemental Keywords: Groundwater, remediation, biotransformation, bioremediation