Assessing rumen microbial composition and fibre attachment in dairy cows

Enteric fermentation in ruminants produces methane (CH4) which is considered as a  greenhouse gas (GHG). CH4 that comes from enteric and manure fermentation, accounts for 106 Tg CH4 per year from anthropogenic sources. Rumen micro-organisms  (bacteria, anaerobic fungi and protozoa) break down complex compounds in the feed by hydrolysis to produce monomers which are fermented to volatile fatty acids (VFA), mainly acetate, propionate and butyrate in addition to other products like formate, hydrogen (H2) and carbon dioxide (CO2). Methanogenic archaea in the rumen utilize H2 and CO2 and other methyl containing compounds to produce CH4. To increase understanding of  ruminal methanogenesis, it is important to gain information on the initial colonizers of fibrous dietary components that are crucial for the attachment of the fibre associated microbiota to the feed and fibre  degradation in the rumen. CH4 is the end-product of feed degradation and that is  not used by  the ruminant itself but mainly eructated into the atmosphere. As this is posing a negative  impact on the climate, a broad range of strategies like use of direct fed  microbial  and  inhibitors of CH4 production have been assessed with the aim of CH4 mitigation. The main objective of this PhD project was to apply integrated cultivation based and molecular approaches to assess the microbial community composition and fibre attachment in relation to CH4 emission.

In Chapter 1 of the thesis, I summarize the currently available background knowledge  on the role of microbiota in rumen feed degradation with a particular emphasis on CH4 is presented. Furthermore, the composition and function of the different microbial communities associated with rumen fluid (RF) and the fibrous fraction (FC), the pros and  the cons  of  culture dependent and culture independent methods are discussed. Lastly the current understanding of experimental, dietary and animal associated factors that influence rumen microbiota, fermentation patterns and enteric methanogenesis are reviewed. From the experimental factors affecting the rumen microbial composition discussed, Chapter 2  addresses the potential bias of DNA extraction methods on the picture we can obtain of RF-  and FC-associated microbial communities, using next generation sequencing based profiling    of the bacterial, archaeal and fungal composition (16S rRNA gene and fungal ITS region). In addition, bacterial and archaeal numbers were assessed by 16S rRNA based qPCR. Most importantly, DNA extraction methods have an impact on the outcome of the downstream microbial community analysis, resulting in differences in absolute abundance, and relative abundances of specific community members.  DNA extracted using the PBB method resulted  in higher relative abundance of Ruminococcaceae than the FDSS method, whereas relative abundance of Fibrobacteraceae was lower compared to the RBB method. Whilst the effect of DNA extraction method was limited compared to that of rumen fraction, differences due to  both DNA extraction method and fraction were observed for certain taxa.

Following this experiment, in Chapter 3 we explore the rumen microbiome in terms of  its bacterial and archaeal composition and concentrations, in order to characterize the communities associated with different ratios of grass and maize silage rations diets  and  identify key microbial players associated with CH4 measurements previously reported by van Gastelen et al. (2015). As these analyses focused on rumen fluid, anaerobic fungi were not measured. The changes in the rumen microbiota in response to dietary treatments having different grass/maize silage ratios (GS100, GS67, GS33, and GS0 wherein GS100 indicates 100% grass silage) after 10 and 17 days of feeding were assessed. The bacterial and archaeal composition changes were used to help understand ruminal VFA profiles and CH4 measurements. In terms of ruminal VFA, no significant diet effects were found but the molar proportions of isovalerate were affected by time, being lower on day 17 than day 10. Diet affected bacterial concentrations, which were lower for the GS0 diet compared with the other three diets. There was no diet effect on archaeal concentrations. Bacterial and archaeal concentrations significantly increased from day 10 to day 17. This observed increase in  bacterial concentrations was suggestive of an increase in fermentation, while the biological significance of increased archaeal concentrations with time could not be elucidated, as CH4 emissions were only measured from days 12 to 17 in the study of van Gastelen et al. (2015). Several bacterial and archaeal genera could be associated with diet, but not with time. The bacterial families Succinivibrionaceae and Ruminococcaceae were associated with the maize silage diets, indicating their role in the lower CH4 emissions observed before (van Gastelen et al., 2015).

Another research objective of this thesis was to explore the fibre associated microbiota   as many are still uncultured and unidentified. To this end, in Chapter 4  rumen  fibre  associated microbes were enriched from grass silage (GS) or maize silage (MS) fibres  recovered from the bovine rumen. Enrichments used pre-autoclaved ruminal silage fibres (GS or MS, respectively, for each isolation source) as an attachment matrix in bottles containing different fibre related components (cellulose, amylopectin or xylan). All enrichments were incubated at 39 oC for a 14 day period, with storage at 4 ºC for 3-4 weeks between transfers. Although overall activity in all the enrichments was low, fibre-degrading bacteria were considered to be enriched as visual inspection of PE bottles from both the MS and GS enrichment series showed initial fragmentation or breaking up of the fibre structure.  Sequencing analysis revealed that members of the genus Ruminofilibacter dominated all the  MS SE5 bottles, whereas all GS SE5 bottles contained a more diverse community  predominated by members of Prevotella 1 and Bacteroides). The in vitro experimental  approach of biweekly serial transfer seems likely to be important to enrich for fibre associated bacteria that are not favoured by shorter incubation times, such as e.g. Ruminofilibacter.

Chapter 5 describes a novel propionate producing bacterium, Propionibacterium ruminifibrarum strain JV5T, isolated from the rumen fibrous content of a Holstein Friesian  dairy cow. Characterization of this strain at genomic, biochemical and physiological level provided insights into the metabolic capacity of Propionibacterium ruminifibrarum. This species was able to utilize several sugars and sugar alcohols D-adonitol, galactose, glucose, inositol, DL-lactate, mannose, meso-erythritol, ribose and sorbitol mainly converting them to propionate and acetate, and succinate and/or  formate in some cases.  Furthermore, the ability  of strain JV5T to degrade representative plant carbon sources,  i.e. cellulose, xylan or starch,  was tested and we observed that strain JV5T could not directly use plant  polymeric  components, but probably can associate with fibres to use the compounds that are released by primary degraders.

Finally, Chapter 6  provides an integrated overview and discussion of results obtained   in the research described in this thesis as well as findings from other studies. In this context, preliminary results regarding the ability of the novel isolate strain JV5T to attach onto silage derived grass fibres are presented and discussed in light of the potential for this isolate to be developed into a probiotic feed supplement. In addition, this  chapter  provides  future  directions of research on rumen microbial management to mitigate CH4 and improve ruminal fermentation.