bRuminococcus albus 7 has played a key role in the development of the concept of interspecies hydrogen transfer. The rumen bacterium ferments glucose to 1.3 acetate, 0.7 ethanol, 2 CO 2 , and 2.6 H 2 when growing in batch culture and to 2 acetate, 2 CO 2 , and 4 H 2 when growing in continuous culture in syntrophic association with H 2 -consuming microorganisms that keep the H 2 partial pressure low. The organism uses NAD ؉ and ferredoxin for glucose oxidation to acetyl coenzyme A (acetyl-CoA) and CO 2 , NADH for the reduction of acetyl-CoA to ethanol, and NADH and reduced ferredoxin for the reduction of protons to H 2 . Of all the enzymes involved, only the enzyme catalyzing the formation of H 2 from NADH remained unknown. Here, we report that R. T he genus Ruminococcus (class Clostridia) consists of species of anaerobic, Gram-positive bacteria. One or more species of this genus are found in significant numbers in the intestines of humans (enterotype 3) (1, 2) and in the rumen and colon of herbivores (3, 4). Ruminococcus flavefaciens and Ruminococcus albus are among the most important plant cell wall-degrading bacteria in the rumen. These two species produce all required enzymes for hydrolyzing the plant cell wall polysaccharides, cellulose and hemicellulose (5, 6).R. albus was isolated in 1957 from the rumen of cattle by Hungate (7,8), who showed that in batch culture the bacterium ferments cellulose, cellobiose, or glucose to acetate, CO 2 , ethanol, and H 2 . Nevertheless, neither H 2 nor ethanol accumulate to high concentrations in the rumen (9, 10). The low H 2 concentrations in the rumen have been explained by the presence of H 2 -consuming microorganisms, such as Methanobrevibacter ruminantium (formerly Methanobacterium ruminantium), that consume H 2 more rapidly than it is formed from cellulose by R. albus (11). The laboratory of Bryant and Wolin then showed in 1973 that the fermentation of glucose by R. albus strain 7 was shifted from 1.3 acetic acid, 0.7 ethanol, 2 CO 2 , and 2.6 H 2 in batch culture to 2 acetic acid, 2 CO 2 , and 4 H 2 in chemostat culture together with Wolinella succinogenes (formerly Vibrio succinogenes), which grew on H 2 and fumarate producing succinate, keeping the H 2 concentration low (12). The interspecies cooperation allowed W. succinogenes to grow at the expense of H 2 produced by R. albus, but it also offered an energetic advantage to R. albus in the form of a higher ATP gain (4 mol of ATP instead of 3.3 mol of glucose fermented) and, consequently, a better growth yield (13). At low H 2 partial pressures, the free energy associated with glucose fermentation is more negative than that at high H 2 partial pressures, which is the thermodynamic basis for the different ATP gains (13). The fermentation of R. albus on glucose has been modeled (14). The literature on interspecies H 2 and formate transfer has been reviewed recently (15).Enzymatic analyses have revealed that R. albus strain 7 grown in batch culture on glucose contains an NAD-specific glyceraldehyde-3-phosphate dehydr...
A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of β-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was 70°C for endoglucanase (0.75 U/ml) and 50°C for β-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to 50°C. At 50°C, endoglucanse, β-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by K+, while K+, Zn+, and tween 20 enhanced β-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of Mn2+ and Cu2+. The broad range of optimum temperatures (20 to 40°C) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.
Background:The thermophilic bacterium Caldanaerobius polysaccharolyticus can utilize mannan polysaccharides found in plant hemicellulose. Results: The mannan degradation gene cluster contains a solute-binding protein with an unexpected tolerance for linear and branched manno-oligosaccharides. Conclusion: Structural studies reveal a binding site optimized for linear and branched mannotriose. Significance: The self-contained mannan-utilizing cluster can be utilized for engineering efforts for the conversion of mannancontaining hemicellulose into cellulosic biofuels.
Digestion of plant cell wall polysaccharides is important in energy capture in the gastrointestinal tract of many herbivorous and omnivorous mammals, including humans and ruminants. The members of the genus Ruminococcus are found in both the ruminant and human gastrointestinal tract, where they show versatility in degrading both hemicellulose and cellulose. The available genome sequence of Ruminococcus albus 8, a common inhabitant of the cow rumen, alludes to a bacterium well-endowed with genes that target degradation of various plant cell wall components. The mechanisms by which R. albus 8 employs to degrade these recalcitrant materials are, however, not clearly understood. In this report, we demonstrate that R. albus 8 elaborates multiple cellobiohydrolases with multi-modular architectures that overall enhance the catalytic activity and versatility of the enzymes. Furthermore, our analyses show that two cellobiose phosphorylases encoded by R. albus 8 can function synergistically with a cognate cellobiohydrolase and endoglucanase to completely release, from a cellulosic substrate, glucose which can then be fermented by the bacterium for production of energy and cellular building blocks. We further use transcriptomic analysis to confirm the over-expression of the biochemically characterized enzymes during growth of the bacterium on cellulosic substrates compared to cellobiose.
The mutually‐beneficial interdependence of hydrogen‐producing and hydrogen‐utilizing bacteria was discovered by M. P. Bryant, M. J. Wolin and R. S. Wolfe at the University of Illinois in 1967. Based on thermodynamic principles, interspecies hydrogen transfer is a central process in anaerobic environments linking transfer of reducing power from fermentation of organic molecules to inorganic electron acceptors via hydrogen. Interspecies hydrogen transfer is the most significant example of unidirectional substrate supply enabling the syntrophic metabolic association between interacting microbial species and plays a significant role in the global methane cycle. R. albus 7 is a hydrogen‐producing, fermentative bacterium with two known hydrogenase complexes (HydABC and HydA2) as well as a putative hydrogen‐sensing protein, HydS. HydABC is the only chromosomal hydrogenase, while HydA2 and HydS form a transcriptional unit in R. albus 7 on its plasmid pRumal01. The electron‐bifurcating ferredoxin‐ and NAD‐dependent [FeFe]‐hydrogenase, HydABC, couples proton reduction using nicotinamide adenine dinucleotide (NADH) to proton reduction using reduced ferredoxin, producing molecular hydrogen: 3H+ + NADH + Fdred ‐> 2H2 + NAD+ + Fdox. HydA2, a ferredoxin‐dependent [FeFe]‐hydrogenase, reduces protons to molecular hydrogen using only reduced ferredoxin: 2H+ + Fdred ‐> H2 + Fdox. HydS contains a C‐terminal PAS domain, which often are present on sensory proteins. In addition, HydS contains a putative redox‐sensing [4Fe:4S] cluster. We hypothesized that HydS transcriptionally regulates HydA2 in a manner dependent on the presence of a hydrogen‐utilizing skyntroph. To test this hypothesis, we grew R. albus 7 and a hydrogen‐utilizing bacterium, W. succinogenes DSM‐1740, in mono‐ and bi‐culture. We monitored cell growth by optical density (OD600) and quantitative polymerase chain reaction (qPCR), as well as gas and fermentation product production. Lastly, based on qPCR growth data, we determined mid‐log phase (ΔOD~0.20 for R. albus, 0.14 for W. succinogenes, and 0.35 for the bi‐culture) and extracted RNA for sequencing to compare whole genome transcriptomic profiles. In bi‐culture with the hydrogen‐utilizing bacterium, R. albus produced 1.11 moles acetate and 0.03 moles ethanol per mole available glucose. In monoculture, R. albus produced 0.75 moles acetate and 0.30 moles ethanol per mole available glucose. We confirmed that hydrogen accumulated in the R. albus monoculture, but not in the bi‐culture. From RNA‐Seq analysis, we identified that R. albus, in bi‐culture, had a lowered transcript abundance of HydA2 (90‐fold), relative to monoculture. Interestingly, the electron‐bifurcating hydrogenase, HydABC, had a similar transcript abundance in bi‐culture to monoculture (1.2–1.3‐fold change). This suggests that HydS might be sensing hydrogen levels and regulating the transcription of HydA2. These results also suggest the electron‐bifurcating hydrogenase (HydABC) functions in central metabolism regardless of external hydrogen concentration.Support or Funding InformationAgriculture and Food Research Initiative Competitive Grant 2012‐67015‐19451 from the US Department of Agriculture National Institute of Food and Agriculture
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