No description available.

No description available.

A detailed kinetic analysis was made of growth of Nitrosomonas europaea and Nitrobacter sp. in chemostat culture. Steady states were established at a number of dilution rates as measured by substrate concentration and cell number. Biomass concentration was also estimated at each dilution rate and these data were used to evaluate the kinetic growth constants; maximum specific growth rate (m), saturation constant (Ks), true growth yield (Yg) and maintenance energy coefficient (me). The transient response to dilution rate changes was monitored and step increases in dilution rate always resulted in substrate overshoots which were mirrored by cell number undershoots. Step reductions in dilution rate resulted in monotonic changes in substrate concentration to lower steady state levels. Nitrobacter exhibited different growth characteristics when cultured continuously in an airlift column fermenter. This was considered to result from biomass settling within the column, resulting from inadequate mixing. Kinetic growth constants for culture of Nitrobacter in the airlift column fermenter were determined. The optimum pH for nitrite oxidation in batch culture was pH 7.5 with no growth at pH values less than 6.0. Nitrite steady states were established in continuous culture at pH 8.0, 6.0 and 5.5, with washout occurring at pH 5.0 and transient nitrite undershoots were observed following pH changes imposed. Surface growth of Nitrobacter was investigated in batch and continuous culture. Attachment and growth on glass and anion exchange resin surfaces resulted in the development of attached microcolonies. Biofilm development on anion exchange resin surfaces was facilitated by slime production which may assist in the irreversible attachment of cells. The specific rate of nitrite oxidation of cells attached to glass surfaces in batch culture was 20-25% greater than that of freely suspended cells. The enhanced activity of attached cells was independent of pH and pH-activity curves of free and attached cells were similar. Similarly cells attached to anion exchange resins in continuous culture exhibited increased oxidation rates per cell, compared to free cells cultured in the same system. Attachment to surfaces significantly lowered the minimum pH for nitrite oxidation and increased protection against low pH, Reduction in pH to 3.5 prevented nitrite oxidation, however cells remained viable and a steady state was subsequently established at pH 4.5.

by Tat-ming Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1981. / Bibliography: leaves 288-329.

vii, 194 leaves : ill. ; 26 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 Biochemistry, 1974

In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.

Bacillus subtilis and Micrococcus lysodeikticus grew poorly when subjected to conditions of limiting iron nutrition after inocula were washed. Growth of both organisms was stimulated by ferrichrome and various phenolic acids. The chelating agents ethylene-diamine tetraacetic acid and nitrilotriacetic acid stimulated growth of B. subtilis but not of M. lysodeikticus . The effect of these compounds as antagonists to the action of the peptide antibiotic albomycin depended on growth conditions. Phenolic acid production by B. subtilis required relatively high levels of a carbon source, with glucose serving as the most effective substrate of those tested. B. subti lis WB746 produced only 2,3-dihydroxybenzoic acid whereas strain B1471 produced an unidentified phenolic acid early in log phase and 2,3-dihydroxy-benzoylglycine in the late log and early stationary phases of growth, under iron deficient conditions. Mutant strains of B. subtilis produced phenolic acids in the absence or presence of iron in the growth medium. DHB synthesizing enzymes were repressed by growth of B. subtilis in the presence of iron, ferrichrome or the aromatic amino acids. Active dihydroxybenzoic acid synthetase was not affected by these compounds. The DHB synthetase system from B. subtilis was partially purified by DEAE-cellulose column chromatography and sucrose gradient centrifugation; the result suggested the existence of a multienzyme complex. Iron uptake by B. subtilis and typhimurium was shown to be energy-dependent and repressible by growth in the presence of adequate iron. The iron uptake capacity of M. lysodei kticus appeared to be inducible, with a chelating agent or an Fe: chelate complex serving as the inducer. Ferrichrome and dihydroxybenzoic acid appeared to serve as iron transport factors for all three organisms, whereas ethylenediaminetetraacetic acid inhibited iron uptake. 3-F1uorobenzoic acid, a dihydroxybenzoic acid analog, had little effect on growth, but did reduce both phenolic acid production by, and iron uptake capacity of, Bacillus subtilis. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

LD2668 .T4 1966 D38 / Master of Science

No description available.

Pseudomonas aeruginosa ATCC 9027 grew in a chemically defined medium with n-hexadecane or glucose, 0.5% (v/v or w/v, respectively), as the sole carbon source, and the K$ sb2$HPO$ sb4$, concentration was either 0.6 mM or 2.3 mM with both carbon sources. Only cells grown on n-hexadecane and 0.6 mM K$ sb2$HPO$ sb4$ produced pyocyanine. The variable lag period associated with cells grown on n-hexadecane was regulated by the state of the inoculating culture, grown on glucose and 2.3 mM K$ sb2$HPO$ sb4$. It was found that organic phosphate was more prevalent in cells grown on both phosphate concentrations with glucose and the high phosphate concentration with n-hexadecane, than it was for the low phosphate, n-hexadecane grown cells. The inorganic phosphate levels remained low under all conditions, decreasing as the cell culture became older. The inorganic polyphosphate level remained stable for all conditions, except in the high phosphate, n-hexadecane grown cells, where there was an increase.