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    Growth of Streptococcus bovis and a Butyrivibrio in batch and continuous culture and the relationship of molar growth yield to intermicrobial competition : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology.
    (Massey University, 1974) Asmundson, Roderick Vincent
    Cell growth yield of Streptococcus bovis and Butyrivibrio were determined in batch cultures where growth was separately limited by glucose, CO2 and trypticase. With bovis, glucose limited growth and a Yg of 39.6 g / M in the presence of excess CO2 was determined. S. bovis grew in the absence of CO2 , but the Yg was reduced to 16.5 g / M. In the presence of excess CO2 the Yg determined for Butyrivibrio was 55 g / M. Butyrivibrio was strictly limited by CO2 and the YCO2 was equal to Yg. This led to the suggestion that CO2 metabolism allows the generation of at least two additional ATP when combined with glucose metabolism for both organisms. Monod growth constants were determined for both organisms in continuous culture under glucose limitation. Ks and μmax for S. bovis were 0.429 mM / 1 and 2.47 hr-1 , respectively. For Butyrivibrio, Ks and μmax were 0.332 mM / 1 and 0.704 hr-1 , respectively. The cell growth yields for S. bovis and Butyrivibrio were determined to be 39.6 g / M and 69.1 g / M, respectively. At growth rates less than 0.2 hr-1 colony forming units and total cell counts of S. bovis decreased, but cell yield did not. Colony forming units, total counts and cell growth yield of Butyrivibrio did not decrease at low growth rates. When S. bovis and Butyrivibrio were grown in continuous mixed culture, Butyrivibrio dominated at growth rates below 0.5 hr-1 and growth of S. bovis was strongly depressed. That Butyrivibrio dominated mixed cultures supports the proposition that an organism deriving more ATP per mole of substrate that another will dominate in environments comparable with continuous culture. The roles of maintenance energy, Ks and μmax and cell yield in competition are considered.
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    Comparative genomics of Butyrivibrio and Pseudobutyrivibrio from the rumen : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Palevich, Nikola
    Determining the role of rumen microbes in plant polysaccharide breakdown is fundamental to understanding digestion, and maximising productivity, in ruminant animals. Rumen bacterial species belonging to the genera Butyrivibrio and Pseudobutyrivibrio are important degraders of plant hemicellulose, an abundant heterogeneous, branched polymer, involved in crosslinking cellulose microfibrils to lignin. To investigate their genes required for hemicellulose degradation, the genomes of 40 Butyrivibrio and 6 Pseudobutyrivibrio strains isolated from the plant-adherent microbiome of New Zealand bovine ruminants, were sequenced, and their CAZyme-encoding genes compared. Within the Butyrivibrio and Pseudobutyrivibrio pan-genomes, respectively, there were a total of 4,421 and 441 glycoside hydrolases, as well as 1,283 and 122 carbohydrate esterases with predicted activities involved in the degradation of the insoluble plant polysaccharides such as xylan and pectin. To examine species differences, the genes of the previously characterised bacterium B. proteoclasticus B316 were compared in detail with those from the newly sequenced B. hungatei MB2003. B316 was found to encode a much more developed polysaccharide-degrading repertoire and it was thus hypothesised that B316 would out-compete MB2003 when grown in co-culture on the insoluble hemicellulose substrate, xylan. To test this hypothesis, the two strains were grown on xylan and pectin, either alone in mono-cultures, or in direct competition in a co-culture. The results showed that MB2003 had little ability to utilise xylan or pectin alone, but was capable of significant growth when co-cultured with B316. This indicates a commensalistic interaction between these species, in which B316 initiates the primary attack on the insoluble substrate, while MB2003 has a secondary role, competing for the released soluble sugars. This work provides the first systematic phenotypic, comparative genomic and functional analysis of ruminal Butyrivibrio and Pseudobutyrivibrio species, which not only defines their conserved features involved in hemicellulose degradation, but is also beginning to differentiate their unique gene complements and growth characteristics that separate them as discrete species.
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    Genome sequencing of rumen bacteria involved in lignocellulose digestion : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in the Institute of Molecular Biosciences at Massey University, Palmerston North, New Zealand
    (Massey University, 2011) Palevich, Nikola; Palevich, Nikola
    Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. In order to learn more about lignocellulose degradation in pasture-grazed dairy cows under NZ conditions, twenty representative strains from five major phylotype clusters (Butyrivibrio fibrisolvens/hungatei cluster 383, Pseudobutyrivibrio xylanivorans clusters 247 and 245, Selenomonas ruminantium cluster 212, and Lachnospiraceae cluster 121), cultivated directly from the fibre-adherent rumen microbial fraction of dairy cows were selected. Genotypic and phenotypic analysis of these strains led to identification of Butyrivibrio sp. MB2003 that adheres to and efficiently degrades the plant fibre. The 3.3 Mb MB2003 genome was sequenced and annotated and found to consist of four replicons: a chromosome (7 contigs, in 1 super scaffold), a chromid (Bhu II), a megaplasmid (pNP144) and a small plasmid (pNP6). A novel feature of the MB2003 genome is the presence of a chromid (Bhu II) which is now the smallest chromid reported for all bacteria. The MB2003 polysaccharide-degrading enzymes, surface structures and predicted strategy for attachment to, and degradation of, complex polysaccharides was found to be comparable to that of the fibrolytic bacterium Butyrivibrio proteoclasticus B316. Both MB2003 and B316 are non-motile, despite the presence of flagellar gene clusters, and utilise a range of insoluble plant polysaccharides, but not cellulose. Xylan is the preferred insoluble substrate of MB2003 and its genome encodes a large repertoire of enzymes predicted to metabolise this complex polysaccharide. The MB2003 draft genome produced in this work is the first opportunity to conduct comparative analysis of two rumen bacteria belonging to the same genus. Although both MB2003 and B316 have similar phenotypic characteristics and occupy the same habitat, the genome of MB2003 is much smaller and contains fewer extracellular polysaccharide degrading enzymes. From this comparison it can be concluded that MB2003 is a secondary hemicellulose degrader, offering an alternate view of the genes required for a xylanolytic lifestyle in the rumen, and posing an interesting question about the purpose of the wider range of polysaccharide degrading enzymes found in B316.
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    The auxiliary replicons of Butyrivibrio proteoclasticus : a thesis presented in fulfilment of the Doctorate of Philosophy degree at Massey University, Palmerston North, New Zealand
    (Massey University, 2009) Yeoman, Carl
    Butyrivibrio proteoclasticus B316T is the most recently described species of the Butyrivibrio / Pseudobutyrivibrio assemblage and now the first to have its genome sequenced. The genome of this organism was found to be spread across four replicons: a 3.5 Mb major chromosome and three additional large replicons: 186, 302 and 361 Kb in size. This thesis describes the sequencing, analysis, annotation and initial characterisation of all three B. proteoclasticus auxiliary replicons. Most significantly, these analyses revealed that the 302-Kb replicon is a second chromosome. This small chromosome, named BPc2, encodes essential systems for the uptake and/or biosynthesis of biotin and nicotinamide adenine mononucleotide, as well as the enzymes required for utilisation of fumarate as the terminal electron acceptor during anaerobic respiration, none of which are found on the main chromosome. In addition, BPc2 contains two complete rRNA operons, a large number of enzymes involved in the metabolism of carbohydrates, nitrogen and fatty acids. In contrast to BPc2, both megaplasmids appear largely cryptic, collectively encoding 421 genes not previously described in public databases. Nevertheless, only the 186-Kb, but not 361-Kb megaplasmid, could be cured from Butyrivibrio proteoclasticus B316T. The largest megaplasmid has a copy number of 5, while all other replicons are present at a copy number of 1. %GC content and codon usage analyses strongly suggests that all three auxiliary replicons have co-resided with the major chromosome for a significant evolutionary period. Moreover, the replication machineries of these three replicons are conserved. Interestingly, a survey of a number of Butyrivibrio / Pseudobutyrivibrio species revealed that the megaplasmids are widespread in this assemblage, however these other large plasmids do not show concordance with their 16S rRNA phylogeny and appear distinct to those of B. proteoclasticus B316T. A microarray analysis of gene expression in a co-culture experiment between B. proteoclasticus and the important ruminal methanogen, Methanobrevibacter ruminantium M1, revealed a potentially mutualistic interspecies interaction. In this relationship M. ruminantium appears to provide B. proteoclasticus with glutamate, essential to the final step of NAD+ biosynthesis, while B. proteoclasticus appears to provide M. ruminantium with formate, hydrogen and carbon dioxide, each important substrate for methanogenesis.