Bibliography: leaves 139-167 The aim of this project was to identify an enzyme responsible for the metabolism of oxalate which would be suitable for degrading oxalate in the rumen, and clone and characterise that gene.

Call number: LD2668 .T4 1961 H39

Call number: LD2668 .T4 1955 H36 / Master of Science

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A procedure was developed for the quantitative separation of major fermentation products of uniformly labeled glucose-C¹⁴ produced by bovine rumen microorganisms in vitro. After 45 min, the fermentation mixture was fractionated into (a) one control subsample, and duplicate fractions of (b) solid matter “precipitate“, (c) ether extract, (d) “amino acid“, (e) “sugar“, (f) CO₂, and (g) CH₄. Similar fractionation of an unfermented control sample was made. A portion of the fermentation ether extract was subjected to column chromatography to resolve (a) C₁, (b) C₂, (c) C₃, (d) C₄, and (e) C₅ fatty acids, (f) succinic, and (g) lactic acids. Each fraction was analyzed in triplicate for C¹⁴ by a direct plating technique. Corrections for geometry, self absorption, and efficiency were made by direct plating additional triplicate fraction subsamples, each containing a uniformly labeled glucose-C¹⁴ internal standard. The data were expressed as per cent recovery of added C1u. The results indicated that glucose was rapidly fermented with most of the C¹⁴ found in the ether extractable fraction as acetic acid. Significant levels of C¹⁴ were found in the “precipitate“ fractions. The data were compatible with evidence that CH₄ was derived from CO₂. The results of 6 trials indicated that there was no significant difference in the distribution of products resulting from the in vitro fermentation of uniformly labeled glucose-C¹⁴ between animals, between days within animals, or between times within days. / Ph. D.

Two media were adapted to the culture of proteolytic bacteria from the bovine rumen. Modifications were made in the double indicator dairy medium of Donovan and Vincent (SRBP) and in the medium of Hungate for rumen bacteria (SRP). Modifications included use of plant protein suspensions, casein, and skim milk as the nitrogen sources of which skim milk was the most suitable, producing a uniformly opaque medium. Proteolytic colonies were characterized by clear zones in the medium. A third medium containing the artificial sheep-saliva salts mixture of McDougall was developed but was found unsatisfactory for the study of proteolytic organisms. Dilutions of rumen contents to 1 x 10⁻⁸/ml were made in anaerobic dilution fluids. Cultures were grown in roll tubes or bottles containing CO₂ atmosphere. All of the media used in this study repeatedly produced an average count of 40 colonies per tube with 10⁻⁸ dilutions of comminuted whole rumen ingesta as inocula. The average ratio of proteolytic to total colonies was found to be 1 to 5. Each of the media was compared for its ability to support the proteolytic isolates from the other. Minor differences in the specificity of the media were found to exist. Colonial and morphological studies of the proteolytic isolates were made. / Master of Science

One or more factors which occur in bovine rumen fluid stimulate the growth of a strain of Butyrivibrio. The stimulating material is heat stable, organic in nature and non-dialyzable. It cannot be extracted from rumen fluid with lipid solvents and is retained in part on anion and cation exchange resins. It can be eluted from the resins with strong acid. It is stable to enzymatic hydrolysis by trypsin. Granular mucin or bovine saliva will partially replace the stimulatory activity. The part of the material which was not replaced by mucin did not appear to be any compound that is commonly used to stimulate bacterial growth. The presence of a possible inhibitor for the growth of a strain of Butyrivibrio was demonstrated. / Master of Science

Ten strains of anaerobic, gram negative, monotrichous, butyric acid-producing curved rods have been isolated from ingesta of the bovine rumen. These 10 strains of butyrivibrio represented 1/5 of all isolates at 1 x 10⁻⁸ dilutions. Morphological and physiological characteristics of the 10 strains and a strain isolated by gill and king (1958) have also been studied. No two of the isolates were identical in all reactions. Most of the organisms produced a large amount of butyric and some lactic, formic, propionic and succinic acids with the utilization of acetic acid in a rumen fluid glucose medium. The fermentation carried on by these organisms was sensitive to most tested environmental changes. Studies with buffered rumen fluid-glucose media demonstrated a shift of the fermentation products with pH. Addition of fatty acids to this medium indicated that these organisms were active in the conversion of acetate and possibly propionate to butyrate. Two strains apparently had the ability to produce propionate at the expense of lactate. The results of the fermentation tests in 98 per cent rumen fluid medium showed that the tested strains used acetic (plus formic) or lactic (plus succinic) to produce butyric or propionic acid, and produced higher concentrations of fatty acids under a carbon dioxide atmosphere than under nitrogen. When rumen fluid and acetic acid were absent all strains had the ability to produce either formic or acetic acid. / Master of Science