Organic carbon in anoxic ecosystems flows in a cascade from complex plant material to more labile sugars, and ultimately to short-chain fatty acids (SCFA) and gasses like carbon dioxide and methane. Microbial communities, groups of microorganisms that interact with one another, facilitate this process. Microbial anaerobic carbon degradation is exemplified in ruminants. These animals harness energy from plant material using the power of interacting microorganisms, which break down plant carbon into SCFA under largely anoxic conditions in the rumen. Because microbial SCFA can provide up to 80% of the animal’s energy, understanding microbial carbon degradation mechanisms in the rumen is important for many agricultural industries including the production of meat, milk, leather, and wool. Beyond domesticated ruminants, there are over 75 million wild ruminants that are fundamental members in ecosystems from Alaska to Australia. Furthermore, the microbial enzymes that break down plant material in the rumen have industrial applications for modifying enzymatic cocktails in biofuel production.
The research presented here uses cultivation-independent and laboratory approaches to assign carbon degradation capabilities to specific members of the microbial community in the moose rumen. Moose, animals that naturally forage on woody biomass, were selected to provide access to natural rumen microbial communities that are especially adapted to a high lignocellulose diet. We sampled rumen fluid from moose in the spring, summer, and winter, along a seasonal gradient in lignocellulose. Rumen fluid was sampled via the rumen cannula, offering access into the active microbial interactions mediating complex carbon degradation. From these rumen fluid samples, we performed high-throughput shotgun metagenomics and metaproteomics, coupled to multiple methods for metabolite quantification (1H NMR, sequential fiber analyses, and carbohydrate microarray polymer profiling (CoMPP)). We binned hundreds of genomes, resulting in 77 unique (~>80% complete) genomes. A majority of these genomes (71%) belong to novel genera, families, and orders. Five of these genomes belong to an uncultivated, Bacteroidetes family, the BS11, which represent the first ever genomic representatives from this family. A newly resolved genus in this family was the most enriched member on a high lignocellulose diet and was found to ferment hemicellulose sugars.
To uncover interactions between these microorganisms and determine their functional role, we mapped metaproteomics data to the unique genome data. This revealed most of the carbon degradation enzymes were encoded within polysaccharide utilization loci from uncultivated Bacteroidetes genomes. We then characterized the carbon metabolite chemistry focusing primarily on carbohydrate polymers and sugars of using CoMPP and 1H NMR. Linking our proteomes to metabolomics, we discovered that proteins from only seven Bacteroidetes genomes were processing all plant polymers detected, suggesting that a few generalist microorganisms are responsible for most of the carbon degradation in the rumen. One of these highly active Bacteroidetes genomes contained protospacer linkages to viral genomes, indicating that immunity against viral predation that may be required for some organisms to sustain carbon degradation in the rumen.
Finally, winter rumen fluid with elevated condensed tannins was used to enrich for tannin-degrading microorganisms. From these reactors, we isolated a Streptococcus sp. that can degrade Sorghum condensed tannins (CT) in the presence of glucose. Label-free proteomics was performed to evaluate the dynamic proteome when the isolate was grown in the presence or absence of CT. CT lead to the enrichment of many proteins annotated as tannase enzyme, transcriptional regulators for phenolic metabolism, putative enzymes involved in phenolic metabolism, stress response proteins, and proteins originating from prophage. Cumulatively, this dissertation research examines the rumen on multiple scales, to identify microbial community and viral interactions, microorganism physiology, and the putative enzymes that facilitate how carbon flows through the rumen.