27. Studying Electron Bifurcation With Ruminococcus albus by Observing Cellulose Degradation Levels in Controlled Environments

Abstract

Studying Electron Bifurcation with Ruminococcus albus by Observing Cellulose Degradation Levels in Controlled Environments

Electron bifurcation is a recently recognized third mechanism of biological energy conservation in addition to the mechanisms of substrate level phosphorylation and electron transport phosphorylation. Bifurcating enzymes simultaneously couple exergonic and endergonic oxidation-reduction reactions to overcome thermodynamic barriers and more efficiently utilize free energy. The bifurcating hydrogenase enzyme plays an important ecological role in the global carbon cycle by producing H₂ with the reducing equivalent generated by sugar fermentation. Ruminococcus albus is found in the rumen of cattle where it degrades cellulosic material, allowing its host the to digest high cellulose feedstocks such as grass. This is an ideal organism for the study of bifurcation because R. albus is easy to culture and it is non-pathogenic. This anaerobic bacterium converts glucose obtained from cellulose to 4 H₂, 2 CO₂ and 2 acetate. In the rumen, the H₂ is typically converted to methane by methanogens, a type of archaea, keeping H₂ concentrations low. The equilibrium is shifted in the presence of high H₂ concentrations. In this case R. albus implements ethanol fermentation with less H₂ production and at the cost of lower ATP yields. In this ongoing research, we are culturing R. albus anaerobically with various amounts of filter paper as cellulose carbon source. Culture groups are divided into low local H₂ (shaken) and high local H₂ (not shaken) environments. By shaking bottles in the low H₂ group, H₂ can diffuse out of solution more quickly. Because of the bifurcating hydrogenase the ATP yields are higher and more efficient cellulose degradation is predicted in the shaken bottles. Initial experiments show that cellulose is a tough substrate to support growth, and R. albus cellulose cultures grow much slower as compared to the soluble disaccharide cellobiose cultures. Growth rates on cellulose also increased significantly with the addition of short chain fatty acids to the media and phenyl acetate and phenyl propionate appear required for efficient cellulose degradation. In the first experiments, there was no difference in cellulose degradation between shaken and not shaken bottles. We believe that there was too much headspace in the bottles allowing H₂ gas to diffuse out of solution even in the not shaken bottles. In future experiments, we plan to “prime” the R. albus with small amounts of cellobiose to encourage cellulose degradation and to decrease the headspace in the bottles to increase the difference in local H₂ concentrations.