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oai:figshare.com:article/32826248 2026-10-01T16:09:34Z portal_1172 item_type_8 month_year_10_2026

Isolation, identification, and phenotyping of novel rumen bacteria Theano Stoikidou (18868724) PUREID: 672237250 Rumen microbiome unculturable bacteria culturomics microbial isolation dilution to extinction novel rumen bacteria MALDI-TOF MS microbial identification high-throughput identification rumen methanogens identification custom database development database validation microbial phenotyping carbohydrate utilisation rumen bacterial profiling rumen fermentation The rumen microbiome enables ruminants to make energy stored in plant material metabolically available. Current understanding of the rumen microbiome is impeded by poor culture collections and a reduced effort placed on culturing following an explosion in ‘omic’ technologies in recent years; leading to the assumption that many microbes are unculturable. This thesis addresses a critical gap in rumen microbiome research: how to improve the limited availability of cultured isolates to facilitate functional research. It is structured around three synergistic pillars: (1) the isolation and identification of rumen bacteria, (2) development of a custom MALDI-TOF MS database to facilitate their identification, and (3) phenotypic characterisation of novel isolates. Using direct plating and dilution-to-extinction approaches with diverse culture media, 354 rumen bacterial strains spanning nine phyla encompassing 59 species were isolated. Identification based on nearly full-length 16S rRNA gene sequences revealed 62 isolates with <97% sequence identity to known taxa, strongly suggesting they represent novel species that remain to be described and validly named. In addition, a MALDI-TOF MS database extension specific to the rumen microbiome was developed, covering 222 new bacterial strains and six archaeal species absent from commercial libraries. This resource substantially improved identification accuracy, enhancing the efficacy of isolation campaigns. Finally, sixteen phylogenetically diverse isolates were profiled on 182 carbohydrate substrates using the Biolog technique. This revealed both conserved lineage-level metabolic traits and strain-specific differences, providing valuable metabolic information about important rumen bacteria. This work expands our understanding of cultured diversity in the rumen microbiome and provides a valuable resource, the rumen-specific MALDI-TOF MS database, to improve rumen microbial identification. Finally, our results may also enable the biotechnological exploitation of the rumen microbiome by providing potential direct-fed microbials (DFMs) that will allow re-direction of hydrogen produced during fermentation away from methane biochemical pathways, potentially enhancing efficiency and sustainability of ruminant production.<br><br><i>Thesis is embargoed until 31 July 2028.</i><br> 2026-10-01T16:09:34Z Text Thesis 10.17034/32826248.v1 https://figshare.com/articles/thesis/Isolation_identification_and_phenotyping_of_novel_rumen_bacteria/32826248 All Rights Reserved Open Access after 2028-07-31