Design and Operation of a Continuous Flow Bioreactor to Determine the Effect of Growth Conditions on Monooxygenase Expression by Methanotrophic Bacteria. CHUCK PEPE-RANNEY (Colorado School of Mines Golden, CO 80401) DEBORAH NEWBY (Idaho National Laboratory, Idaho Falls, ID, 83415)

Methane monooxygenase (MMO) shows promise for use in biocatalysis because of its broad substrate range. MMO is produced by methanotrophic bacteria and allows the bacteria to utilize methane as both a carbon and energy source. This research project involved designing and operating a continuous flow bioreactor to determine the effect of growth conditions on monooxygenase expression by methanotrophic bacteria. Understanding MMO expression under various growth conditions will allow for more efficient biocatalysis with MMO. A bioreactor consisting of a 1 L vessel, mixing impeller, sparger, media inlet and outlet, and gas outlet was assembled. The bioreactor was continuously sparged with 25% methane in air at approximately 350 mL/min. Within the reactor, Methylosinus trichosporium OB3b was grown to log phase under batch conditions in a total volume of 500 mL. Once the culture reached log phase, the bioreactor was converted to continuous flow and brought to steady state. Copper-free nitrate mineral salts medium was pumped into the reactor while reactor contents were pumped from the vessel at the same flowrate. Biomass was monitored via optical density measurements to verify steady state was achieved and sustained. The bioreactor maintained constant biomass when the influent and effluent flowrates were approximately 13 mL/hour. Samples were periodically collected and analyzed for enzymatic activity, however, not enough data has been collected to report any meaningful enzymatic activity results. Future research will involve changing growth conditions and monitoring the enzymatic response as well as monitoring MMO gene expression using molecular techniques. Research done with this bioreactor will ultimately aid in the operation of a methanotrophic bioreactor for the production of value added chemicals.

Isolation and Characterization of a Novel Enzyme from a Thermoacidophilic Organism. MORGAN BRUNO (University of Idaho Moscow, ID 83844) VICKI THOMPSON (Idaho National Laboratory, Idaho Falls, ID, 83415)

Extremophilic organisms live and thrive at extremes of temperature, pH, salt and pressure. The enzymes produced by these organisms share their extreme nature and operate under conditions that normal enzymes can not. These enzymes are valuable industrially where conditions are often very extreme. In this study, an enzyme from a thermoacidophilic organism was produced, purified and characterized. The organism was grown in liquid media pH 3.5 and 60ºC. After growing up to 1L, the cultures were harvested. Centrifugation at eight thousand rpm yielded supernatant that was adjusted to pH 3.0 and vacuum filtered with a 0.22µL filter. Cation exchange HPLC was performed on the supernatant to purify the enzyme. The column solution was desalted, concentrated 3X, and assayed for protein content and activity. Based upon the results of the fraction activity assay, select fractions were run in an SDS-PAGE gel and Silver stained for visualization. The fractions showing the isolated enzyme were pooled together. The enzyme was concentrated 10X via ultrafiltration. Activity assays were performed on the purified enzyme over a temperature range of 20 to 90 C with 10-degree increments to determine the optimum temperature of the enzyme. Activity assays were also performed on the purified solution over a pH range of 2.0 to 8.0 with 1.0-pH unit increments to determine the optimum pH of the enzyme. These activity assays showed that this novel enzyme follows the expected activity trends for a thermoacidophilic enzyme.

Trichloroethylene Co-metabolism by Methylosinus trichosporium OB3b at Maintenance Level Activity. ALICIA KRUPP (Brigham Young University Provo, UT 84602) FREDERICK COLWELL (Idaho National Laboratory, Idaho Falls, ID, 83415)

Methanotrophs are aerobic microorganisms that metabolize methane. Some methanotrophs have the ability to aerobically co-metabolize trichloroethylene (TCE), a contaminant in the Snake River Plain Aquifer (SRPA) through the action of the enzyme soluble methane monooxygenase (sMMO). The methanotroph Methylosinus trichosporium OB3b was grown in a biomass recycle reactor (BRR), simulating groundwater conditions, to determine rates of methane oxidation and TCE co-metabolism. sMMO activity was measured in the presence and absence of TCE, using the coumarin oxidation assay, which monitors the oxidation of coumarin (in place of methane) to a fluorescent product, 7-hydroxycoumarin. Quantitative real-time PCR and acridine orange direct counts (AODC) were used to determine cell numbers of M. trichosporium in the BRR. Data suggests that starved cells exposed to TCE have lower rates of sMMO activity. From this data and from current literature, we infer that cells under stressed conditions may be less affected by TCE compared to cells under ideal growth conditions. The rates of methane oxidation and TCE co-metabolism measured in M. trichosporium will assist the development of models that describe natural attenuation of TCE in the SRPA.