published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

This thesis concerns the mechanism of oxidative phosphorylation, with special reference to the bacterium Paracoccus denitrificans. A new method using affinity chromatography is described for the separation of vesicles of different membrane orientation. The working hypothesis is the chemisosmotic theory of Mitchell with its emphasis on the transmembrane proton motive force made up of a membrane potential and a transmembrane pH gradient. The 3 experimental systems used ape bovine heart submitochondrial particles, chromatophores from the photosynthetic bacterium Rhodospirillum rubrum and phosphorylating vesicles from P. denitrificans. From the uncoupling obtained with various ions added to the P. denitrificans particles it is concluded that the transmembrane distribution of weak bases and permeant anions permits a determination of the protonmotive force. A flow-dialysis method has been developed to measure the steady-state transmembrane distribution of weak bases and anions in a determination of the protonmotive force. This method successfully overcomes problems which arise with the techniques previously used for these measurements. Under the experimental conditions used to determine the extent of oxidative phosphorylation (the phosphorylation potential) no transmembrane pH gradient was detectable, and the protonmotive force could be accounted for completely by the membrane potential. A method using ion-selective electrodes was developed to provide a continuous determination of this membrane potential. In all 3 systems the proton motive force determined by the above methods was found to be significantly lower than that required by the chemiosmotic hypothesis. A simple modification of the chemiosmotic hypothesis is proposed. This new concept is based on electrodic theory. It is shown to be useful in explaining results reported in the literature (from both photosynthetic and respiratory systems) which appeared to be at odds with the chemiosmotic theory in its usual form.

This study involves the development of a biofilm reactor that supports growth of a deep biofilm on a gas permeable membrane. The reactor solution is not aerated, and oxygen is supplied through the membrane. The reactor is termed a substratum-aerated-biofilm reactor or SAB. With adequate concentrations of electron-donors and electron-acceptors, a deep biofilm grows on the membrane and is comprised of different layers of bacterial activity. The aerobic layers are near the membrane support, while the anaerobic layers are near the biofilm-liquid boundary. In the SAB, the substrate diffuses from the bulk liquid into the biofilm to react. Oxygen diffuses through the membrane into the biofilm. All products likewise are transported by molecular diffusion through the biofilm and into the bulk liquid. The reactors consisted of a reactor wall made of a plexiglass cylinder with the gas permeable membrane supported on a shallow rotating cup. The cup was designed so that the cup and the membrane function as a flat plate. The flat plate was utilized for support of the biological growth, transfer of oxygen, and mixing of the bulk liquid and the gas phase. The experiments were conducted in completely mixed, continuous-flow reactors maintained at 25°C with a hydraulic detention of 8 hours. Pure oxygen was delivered to a gas compartment under the membrane. All reactors were fed a synthetic waste buffered to pH 7.0. The background solution for the feed solution was made from distilled water combined with adequate inorganic nutrients and vitamins. The background solution was supplemented with acetate and ammonia to obtain the desired substrate compositions. Combined nitrification and heterotrophic oxidation activity resulted when the SABs where fed 5 or 10 mg/1 acetate, and 10 mg-N/1 ammonia. Combined nitrification, heterotrophic oxidation, and denitrification resulted with acetate concentration of 20, 40, and 100 mg/1, and 10 mg- N/1 ammonia. Combined heterotrophic oxidation and fermentation resulted with acetate concentration of 800 mg/1, and 10 mg-N/1 ammonia. A series of mass balances were developed to determine the fate of the nitrogen compounds and acetate. These results showed that the flux values for carbon oxidation, nitrification, denitrification, and fermentation are higher than those reported for competing technologies such as rotating biological contactors. / Graduation date: 1990

Includes bibliographical references (p. 62-73).

Thesis (M.S.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2010. Title from PDF t.p. Includes bibliographical references.

Symbiotic interaction of Rhizobium with roots of legume plants leads to the formation of root nodules that are capable of reducing atmospheric nitrogen. In nodule cells, bacteria are always enclosed inside the peribacteroid membrane (pbm) which is derived from the plant cell plasma membrane. The pbm mediates all the molecular exchanges between the host plant and bacteria. The pbm of soybean (Glycine max) nodules induced by Bradyrhizobium japonicum was characterized for the presence of nodule-specific proteins (nodulins), which may be involved in some of the nodule-specific roles the pbm plays. The pbm was found to contain a number of nodulins, and most of them were shown to be of plant origin. The peribacteroid fluid (pbf) was also purified and shown to contain nodulins. Some of the pbf proteins were highly glycosylated. Immunological localization at the electron microscopic level has shown that pbm nodulins are inserted specifically in the pbm; almost none are detected in the plasma membrane (pm) of infected cells even though the pbm is derived from it. The amino acid sequences of three different pbm nodulins (nodulin-23, -24 and -26) do not share significant primary structure homology, neither do their tentative secondary structures. Our results do not preclude targeting based on pbm-nodulin-specific feature(s) of the proteins; but it is possible that pbm-specific localization is achieved by differential rates of membrane synthesis, or turnover, in pbm as compared to the plasma membrane.

This study investigated possible reasons for the failure of the acetylene inhibition assay of denitrification in highly reducing environments and resulted in the isolation of a denitrifying Cytophaga from soil enriched by anaerobic incubation with glucose, nitrous oxide (N(,2)O), acetylene (C(,2)H(,2)), and sulfide (S('2-)). Such soil enrichments and pure cultures of the isolated Cytophaga (Is-11) reduced N(,2)O rapidly even in the presence of a normally inhibitory concentration of C(,2)H(,2) (4 kPa), providing S('2-) was present 8.0 umol (g soil)('-1) or 0.4 umol (mL culture)('-1) . The observed reaction appears to be unique to this soil Cytophaga since other organisms tested, using the same or similar experimental conditions, failed to show this response. / The isolate was characterized, after extensive comparative studies with five Cytophaga johnsonae strains, as a pigmented, Gram-negative, non-motile, gliding filamentous bacillus. Although these features established a taxonomic link with the family Cytophagaceae, the inability of Is-11 to digest chitin and DNA base composition of about 42.5 mol% (G+C) make the organism's specific affiliation uncertain.

xxix, 283 leaves : ill. ; 25 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agricultural Biochemistry and Soil Science, 1974

Thesis (Ph.D.) -- University of Adelaide, Dept. of Agricultural Biochemistry and Soil Science, 1974.

Thesis (Ph.D)--Murdoch University, 2008. / Thesis submitted to the Faculty of Sustainability, Environmental and Life Sciences. Includes bibliographical references (p. 213-220).