Browsing by Author "Steven Peretti, Committee Member"

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Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5
(2009-04-20) House, Alan; James Brown, Committee Member; Jonathan Olson, Committee Member; Steven Peretti, Committee Member; Michael Hyman, Committee Chair
Characterizing MTBE Cometabolism and Propane Metabolism by Mycobacterium austroafricanum JOB5. (Under the direction of Michael R. Hyman.) Cometabolic transformations are unable to support cell growth. This process is often catalyzed by, and superimposed upon, enzyme systems expressed to catalyze carbon- or energy-yielding reactions. Biodegradation of the gasoline additive methyl tertiary butyl ether (MTBE) is known to be superimposed upon a propane-oxidizing system in the aerobic bacterium Mycobacterium austroafricanum (vaccae) JOB5. Taking a whole-cell approach, we investigated the physiology of propane metabolism and MTBE cometabolism in this strain. Multiple major gasoline components are frequent co-contaminants with MTBE in the environment, and we determined the impacts of these hydrocarbons on the cometabolism of both MTBE and its commonly encountered metabolite, tertiary butyl alcohol (TBA). Most of the hydrocarbons tested supported cell growth and concurrent MTBE and TBA oxidation occurred without affecting final culture optical density. Results suggest hydrocarbon-grown cells simultaneously expressed more than one alkane-oxidizing enzyme system. Nuclear magnetic resonance spectroscopy (NMR) was used to study the pathway of MTBE oxidation in propane-grown cells of strain JOB5. We confirmed the existence of predicted intermediates, including a hemiacetal, formate and formaldehyde. Hydroxyisobutyraldehyde, a predicted intermediate in MTBE oxidation by some bacterial strains was not detected, despite attempts to promote its accumulation. As the pathway of MTBE oxidation progressed, the rate of daughter product oxidation decreased, which may be preventing MTBE-dependent cell growth in strain JOB5. Propane metabolism was examined using a series of growth experiments and substrate oxidation assays. We observed the simultaneous production, and later consumption of both 1- and 2-propanol during cell growth. This divergent oxidation of propane was apparently followed by the divergent oxidation of propionate and the divergent oxidation of acetone. Our results suggest at least two CO2-fixation steps are involved in propane metabolism in strain JOB5. Finally, we used NMR to contribute to several studies that characterized the pathway of (i) MTBE oxidation by Nitrosomonas europaea or (ii) bacterial oxidation of a fluorinated analog of TBA. The later study identified a compound that may serve as a tracer for TBA degradation in situ.
Glycerol Combustion
(2007-11-06) Metzger, Brian; Steven Peretti, Committee Member; William L. Roberts, Committee Chair; Kevin Lyons, Committee Member
As worldwide production of biodiesel fuel increases, one of the largest concerns is the abundance of waste glycerol. The price of crude glycerol has fallen drastically and many large biodiesel producers are currently paying to landfill this large waste stream. In the search to find a value added alternative, glycerol combustion may be one of the simplest solutions. Heat recovered from glycerol oxidation could easily be used to reduce heating costs inherent to large-scale biodiesel production. It has been stated "Combustion of glycerol would be an elegant solution, if it worked". Clean combustion of glycerol is difficult due to its high viscosity, high auto ignition temperature, and concerns of hazardous emissions. In particular, most in the biodiesel producing community share a fear that burning glycerol could produce acrolein, an aldehyde which is a thermal decomposition product of glycerol and is toxic at very low concentrations. This report will detail the design of a burner that can safely and easily burn crude glycerol for process heating. Emissions testing in the burner using glycerol sources of varying quality confirm that this burner design completely oxidizes the glycerol into CO2 and H2O with very low levels of pollutants, typical of other hydrocarbon fuels. These results show that safe, clean, and efficient combustion of a wide range of glycerol purities is possible with a properly designed burner.
Transcriptional analysis of biofilm formation and stress response in hyperthermophilic microorganisms.
(2004-03-24) Pysz, Marybeth Anne; David Olllis, Committee Member; Robert Kelly, Committee Chair; James Brown, Committee Member; Steven Peretti, Committee Member
The significance of surface colonization and changing thermal conditions in hydrothermal environments motivated examination of biofilm formation and thermal stress response in the model heterotrophic hyperthermophilic microorganisms, Thermotoga maritima and Pyrococcus furiosus. Continuous culture, using maltose-based media and anaerobic conditions at 80°C for T. maritima and 95°C for P. furiosus, was used to generate dense biofilms on nylon mesh and polycarbonate filters; significant amounts of wall growth were observed in the chemostats for both organisms. Transcriptional analysis of biofilm- bound cells showed that genetic mechanisms observed for biofilm formation in less thermophilic bacteria applied to T. maritima. L-lactate dehydrogenase (TM1867), NADH oxidase (TM0379), sensor histidine kinase (TM0187), and TetR family transcriptional regulator (TM0823) were among the genes induced in T. maritima biofilms with mesophilic counterparts. Also consistent with cells in mesophilic biofilms was the differential expression of stress-related genes. Thermal stress genes, hrcA (TM0850), grpE (TM0851), and dnaK (TM0373) were up-regulated, indicating that elements of stress response are operational in hyperthermophilic biofilm environments. Expression of stress-related genes in the T. maritima biofilm prompted a study of stress response during heat shock at 90°C. A 407-gene targeted cDNA microarray was used to study the genetic differences between cells at 80°C and cells at 90°C after 0, 5, 10, 20, 30, 60, and 90 minutes. The two major heat shock operons dnaJ-grpE-hrcA (TM0849-TM0850-TM0851) and groEL-groES (TM0505-TM0506), as well as the genes encoding DnaK (TM0373) and heat shock protein class I (TM0374), exhibited maximal induction at early times (~5 minutes), subsequently decreasing to a steady-state level. This expression pattern has also been observed during heat shock of the mesophilic bacteria Escherichia coli and Bacillus subtilis. Also observed was the stress-related response of the SOS regulon involving usrB (TM1761) and recA (TM1859), and the down-regulation of this operon’s repressor lexA (TM1082). Atypical of heat shock response, the majority of genes encoding ATP-dependent proteases, including ClpP (TM0695), ClpQ (TM0521), ClpY (TM0522), LonA (TM1633), and LonB (TM1869), were down-regulated. ATPase Clp C subunits 1 (TM0198) and 2 (TM0873) were both up-regulated, along with ClpX (TM0146) and FtsH (TM0580). The ATP-independent heat shock serine protease HtrA (TM0571) was also induced. A number of other genes not related to stress response also showed significant changes in expression levels. These include transcriptional regulators, genes within the gluconate metabolic pathway, sugar transporters and glycosidases, and sigma factors. Homologs to E and A were induced during heat shock at 90°C, and suggesting that they are implicated in stress response regulation in T. maritima, although they have not been characterized to date. This work led to the development of chemostat-based methods for generating RNA from hyperthermophiles embedded in anaerobic biofilms that could be used for transcriptional analysis. Such analysis indicated possible connections between the genetic response of biofilm-bound cells and thermal stress response. The results here point to the significance of surface colonization and modification of cellular function arising from thermal changes in the microbial ecology of hydrothermal environments.