The ability of a cellulolytic bacterium Cellvibrio gilvus, to absorb and utilize members of the cellulose oligosaccharide series was investigated. Resting cell suspensions prepared from 24-hour cultures were incubated with the cellulodextrins. At various times samples were removed from the incubation flasks and filtered. The filtrates were analyzed for sugar concentration by a modified phenol-sulfuric acid procedure and degree of polymerization (D.P.) by a borohydride reduction phenol-sulfuric acid method. The rate of disappearance of the oligosaccharides from the supernatant of the resting cell suspensions was linear with respect to time indicating an active absorption mechanism. The conclusion that the rate of absorption was controlled by the respiration of the cell was based on the observation that independent of the D.P. of the cellulodextrin, the number of glucose molecules removed per cell per minute was approximately the same. The D.P. of the substrates remained constant over the experimental period with the cells suggesting that the molecules were removed intact. No effect on sugar concentration or D.P. was observed when the oligosaccharides were incubated with the filtrates which confirm these data. It was not possible from these results to determine if the oligosaccharides entered the cell or were metabolized at the cell wall. / Master of Science

A cellulose-decomposing bacterium isolated from bovine feces was purified, identified as a member of the genus, Cellvibrio, and the new species name gilvus was proposed. Cellulose decomposition was demonstrated and cellobiose was the only hydrolytic sugar product. Excellent growth was obtained on mineral salts medium containing cellobiose, a vitamin mixture, and organic nitrogen (casein hydrolyzate) which was required for growth. Volatile and non-volatile acids, volatile neutral compounds, and carbon dioxide constituted the fermentation products. A preference for cellobiose was shown by a 30 to 46% greater growth rate than resulted on glucose. An investigation was conducted to explain this disaccharide preference. Intact cells oxidized glucose and cellobiose immediately and the rate of glucose oxidation was 10% less than obtained from cellobiose. Thus, hypotheses that adaptive hexokinase, hexokinase deficiency or impermeability to glucose could explain the preference were dispelled. Oxygen assimilation ratios of equivalent amounts of glucose, cellobiose and a mixture of these were 100:110:140. Alternate metabolic pathways were indicated. Resting cells grown on cellobiose esterified inorganic phosphate in the presence of glucose or cellobiose showed that these sugars were metabolized to phosphate esters. Relative rates of utilization of both sugars revealed that 130% more cellobiose was used at pH 6.5 and 590% more at pH 7.0. On a molar basis 30% more acid was formed from glucose than from cellobiose. A phosphorylase was indicated by the stimulation of cellobiose respiration by inorganic phosphate. Disaccharide preference was associated with the intracellular enzymes because soluble enzymes utilized 7.25 uM of cellobiose but only 2.8 uM of glucose. The adenosine triphosphate requirement for glucose utilization indicated hexokinase activity. Inorganic phosphate increased cellobiose utilization two-fold and was accompanied by esterification. Soluble enzymes from glucose-grown cells produced a constitutive cellobiose enzyme. Phosphate depressed glucose utilization while adenosine triphosphate depressed cellobiose utilization. Fructose-6-phosphate was the only ester detected in cells grown on either sugar. Cellobiose-grown cells contained 6.5 mg total P/2 g cells whereas glucose-grown cells contained only 2.1 mg. Glucose was converted to glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-diphosphate by cell-free enzymes in the presence of adenosine triphosphate. Fructose-1,6-diphosphate was metabolized to pyruvic acid. This was evidence for an Embden-Meyerhof pathway. A cellobiose phosphorylase was demonstrated as a reversible reaction producing glucose and alpha-D-glucose-1-phosphate. Conversion of glucose-1-phosphate to glucose-6-phosphate was not obtained but fructose-6-phosphate was formed. Ground cells oxidized fructose-1,6-diphosphate and fructose-6-phosphate in the presence of diphosphopyridine nucleotide, coenzyme-A and methylene blue. These preparations in which oxidizing systems were saturated with fructose-1,6-diphosphate and cofactors oxidized glucose and cellobiose at greatly increased rates above that obtained from the ester. Gluconic acid was detected in the final reaction mixtures. An explanation of the disaccharide preference resides in several factors. The very active direct phosphorylation of cellobiose yielding a hexose phosphate at less expense energy-wise than the adenosine triphosphate-requiring phosphorylation of glucose showed cellobiose to be a more efficient energy-yielding substrate. The weak hexokinase would limit the formation of hexose phosphates which in turn could impede the energy obtained as high energy phosphate compounds necessary for growth and reproduction. A portion of available glucose was probably wasted in the direct oxidation to gluconic acid. Finally, a proposed scheme for the metabolic pathways of glucose and cellobiose in Cellvibrio gilvus was presented. / Ph. D.