The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo / universal&rdquo / archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo / universal&rdquo / archaeal primers A571F (5&rsquo / -GCY TAA AGS RIC CGT AGC-3&rsquo / ) and UA1204R (5&rsquo / -TTM GGG GCA TRC IKA CCT-3&rsquo / ) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.

Potential thermophiles for ethanol production at elevated temperature were compared based on their kinetic performances. T. thermophilus HB27 demonstrated superior kinetics and its growth was not greatly affected when containing the recombinant plasmid pTEV231. Detailed kinetic studies demonstrated that it could utilize glucose and xylose in medium containing 0.5 % (w/v) yeast extract, and could produce low levels of L-lactate, acetate and ethanol. Kinetic evaluation of the newly isolated G. thermoglucosidasius M10EXG showed it could grow on fully defined media and coferment glucose and xylose. G. thermoglucosidasius M10EXG produced higher levels of products (acetate, and L-lactate) at about 2 g/l each, compared to T. thermophilus HB27 although ethanol levels were only 0.1-0.2 g/l in shake flask fermentations under partially aerobic conditions. Improved conditions for natural transformation of T. thermophilus HB27 were determined. Optimal conditions for electroporation of were: Milli-Q water washing of cells rather than with 10% (v/v) glycerol; an electrical field of 5 kV/cm; cell concentration of 1.4x1010 cells/ml; and a DNA concentration of 500 ng in 40 Fl (125 Fg/ml) which achieved a transformation efficiency of 3x103 transformants /Fg DNA. The chloramphenicol resistance (cat) and green fluorescence protein (gfp) genes from pCJS10 were cloned into an E. coli -- T. thermophilus shuttle vector (pMK18) as possible selection markers at elevated temperatures. Both genes were expressed in E. coli DH5H and it was demonstrated that gfp expression in E. coli DH5H decreased as temperature increased to 45oC. However following transformation of T. thermophilus HB27, no evidence of expression were found. The pet operon containing adhT (with its promoter) from G.thermoglucosidasius M10EXG and pdc from Z. mobilis Zm4, was cloned into pMK18 and low level expression in E. coli JM109 occurred with some increase in ethanol production. However the pet operon was not expressed in T. thermophilus with pMK18. For further gene expression studies, a new shuttle vector, pPOPTE (4-5 kb), based on the T. thermophilus plasmid pTEV231 (containing thermostable kanamycin resistance gene) was constructed. pPOPTE was capable of multiplying in both E. coli and T. thermophilus HB27 and demonstrated higher stability and transformation efficiency compared to pMK18.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 147-155). Also available for download via the World Wide Web; free to University of Oregon users.

The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with &ldquo / universal&rdquo / archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of &ldquo / universal&rdquo / archaeal primers A571F (5&rsquo / -GCY TAA AGS RIC CGT AGC-3&rsquo / ) and UA1204R (5&rsquo / -TTM GGG GCA TRC IKA CCT-3&rsquo / ) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermal systems.

Potential thermophiles for ethanol production at elevated temperature were compared based on their kinetic performances. T. thermophilus HB27 demonstrated superior kinetics and its growth was not greatly affected when containing the recombinant plasmid pTEV231. Detailed kinetic studies demonstrated that it could utilize glucose and xylose in medium containing 0.5 % (w/v) yeast extract, and could produce low levels of L-lactate, acetate and ethanol. Kinetic evaluation of the newly isolated G. thermoglucosidasius M10EXG showed it could grow on fully defined media and coferment glucose and xylose. G. thermoglucosidasius M10EXG produced higher levels of products (acetate, and L-lactate) at about 2 g/l each, compared to T. thermophilus HB27 although ethanol levels were only 0.1-0.2 g/l in shake flask fermentations under partially aerobic conditions. Improved conditions for natural transformation of T. thermophilus HB27 were determined. Optimal conditions for electroporation of were: Milli-Q water washing of cells rather than with 10% (v/v) glycerol; an electrical field of 5 kV/cm; cell concentration of 1.4x1010 cells/ml; and a DNA concentration of 500 ng in 40 Fl (125 Fg/ml) which achieved a transformation efficiency of 3x103 transformants /Fg DNA. The chloramphenicol resistance (cat) and green fluorescence protein (gfp) genes from pCJS10 were cloned into an E. coli -- T. thermophilus shuttle vector (pMK18) as possible selection markers at elevated temperatures. Both genes were expressed in E. coli DH5H and it was demonstrated that gfp expression in E. coli DH5H decreased as temperature increased to 45oC. However following transformation of T. thermophilus HB27, no evidence of expression were found. The pet operon containing adhT (with its promoter) from G.thermoglucosidasius M10EXG and pdc from Z. mobilis Zm4, was cloned into pMK18 and low level expression in E. coli JM109 occurred with some increase in ethanol production. However the pet operon was not expressed in T. thermophilus with pMK18. For further gene expression studies, a new shuttle vector, pPOPTE (4-5 kb), based on the T. thermophilus plasmid pTEV231 (containing thermostable kanamycin resistance gene) was constructed. pPOPTE was capable of multiplying in both E. coli and T. thermophilus HB27 and demonstrated higher stability and transformation efficiency compared to pMK18.

Thesis (Ph. D.)--Catholic University of America, 1926. / Vitae. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 44-46).

Thesis (Ph. D.)--Catholic University of America, 1926. / Vitae. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (p. 44-46).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1941. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [55-62]).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 158-185).

Anaerobic fermentation has been an established technology ever since man started treating sewage. Recently this process has received increased attention because of its inherent ability to produce methane gas, which apart from solar energy, is the cleanest, most non-polluting source of energy. Methanobacterium thermoautotrophicum, a thermophilic bacterium, grows on CO(,2) as a source of carbon as well as electron acceptor, using hydrogen as an electron donor. Labeling studies carried out with ('14)C have shown a presence of partial reductive TCA cycle. In this work, the enzyme fumarate reductase, which belongs to this cycle, has been purified to homogeneity using various separation techniques. In keeping with the thermophilic character of the organism, fumarate reductase is extremely heat resistant. Incubation at 75(DEGREES)C for 24 hours led to an increase in purification. In contrast, the enzyme was found to be very sensitive to oxygen. The crude extract, when exposed to air, lost half of its activity within 20 minutes. Reducing agents were helpful in protecting against loss of enzymatic activity provided that a strict anaerobic atmosphere was maintained. For this reason, the entire purification was performed inside a Freter-type anaerobic chamber using reducing agents. The molecular weight of the native fumarate reductase, as determined by Sephacryl S-300 gel exclusion chromatography, was found to be approximately 80,000. SDS polyacrylamide gel electrophoresis data suggested that the enzyme is a tetramer. Treatment with sulfhydyl reagents as well as Cu('++) caused loss in fumarate reductase activity, indicating that the enzyme contains at least one sulfhydryl group which is important to its activity. The UV/Visible spectrum of fumarate reductase did not reveal the presence of a flavin moiety as a cofactor. Both UV/Visible and fluorescence spectra of fumarate reductase from M. thermoautotrophicum instead, indicated the presence of an unusual cofactor, which could be similar to either tetrahydromethanopterin or F(,420).