The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite’s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities
