Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling

Abstract

The rumen microbial ecosystem is known for its biomass-degrading and methane-producing phenotype. Fermentation of recalcitrant plant material, comprised of a multitude of interwoven fibers, necessitates the synergistic activity of diverse microbial taxonomic groups that inhabit the anaerobic rumen ecosystem. Although interspecies hydrogen (H2) transfer, a process during which bacterially generated H2 is transferred to methanogenic Archaea, has obtained significant attention over the last decades, the temporal variation of the different taxa involved in in situ biomass-degradation, H2 transfer and the methanogenesis process remains to be established. Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing. Switchgrass filled nylon bags were placed in the rumen of a cannulated cow and collected at nine time points for DNA extraction and 16S pyrotag profiling. The microbial community colonizing the air-dried and non-incubated (0 h) switchgrass was dominated by members of the Bacilli (recruiting 63% of the pyrotag reads). During in situ incubation of the switchgrass, two major shifts in the community composition were observed: Bacilli were replaced within 30 min by members belonging to the Bacteroidia and Clostridia, which recruited 34 and 25% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 16 h of rumen incubation, when members of the Spirochaetes and Fibrobacteria classes became more abundant in the fiber-adherent community. During the first 30 min of rumen incubation ~13% of the switchgrass dry matter was degraded, whereas little biomass degradation appeared to have occurred between 30 min and 4 h after the switchgrass was placed in the rumen. Interestingly, methanogenic members of the Euryarchaeota (i.e., Methanobacteria) increased up to 3-fold during this period of reduced biomass-degradation, with peak abundance just before rates of dry matter degradation increased again. We hypothesize that during this period microbial-mediated fibrolysis was temporarily inhibited until H2 was metabolized into CH4 by methanogens. Collectively, our results demonstrate the importance of inter-species interactions for the biomass-degrading and methane-producing phenotype of the rumen microbiome-both microbially facilitated processes with global significance.

Keywords: cellulolytic bacteria; interspecies H2 transfer; methanogenic archaea; microbe-microbe interactions; rumen microbiology.

Figure 1

Rarefaction curves calculated from 16S rRNA pyrotag data of switchgrass-adherent microbiomes after rumen-incubation. 0 h indicates the non-incubated switchgrass sample (control).

Figure 2

Principal coordinate analysis of switchgrass-adherent microbiomes. Each point corresponds to one of nine rumen-incubated samples (0.5, 1, 2, 4, 6, 16, 24, 48, and 72 h) and one control sample (0 h). The percentage of variation explained by the plotted principal coordinates is indicated on the axes.

Figure 3

Effect of rumen incubation on relative abundance of bacterial and archaeal phyla of switchgrass-adherent microbiome. Relative OTU abundance of rarefied 16S rRNA gene 454-pyrotag data. Taxonomy was assigned based on Greengenes (McDonald et al., 2012).

Figure 4

Microbial succession and biomass-degradation during rumen-incubation. Heat map show succession of genera recruiting >0.5% of the generated sequences. Line graphs show the relative dry mater change during rumen-incubation. (A) Upper panel shows genera with >10% relative abundance, lower panel shows genera with a relative abundance between 2 and 10%; (B) Upper panel shows genera with relative abundance between 1 and 2%, lower panel shows genera with a relative abundance between 0.5 and 1%.

Figure 5

Scanning Electron Microscopy of air-dried and rumen-incubated switchgrass samples and adherent microorganisms. Scale bars indicate 0.5 μm.