Enriching ruminal polysaccharide-degrading consortia via co-inoculation with methanogenic sludge and microbial mechanisms of acidification across lignocellulose loading gradients.

Using lignocellulosic materials as substrates, ruminal microbiota were co-inoculated with anaerobic sludge at different loading rates (LR) to study the microbial community in the semi-continuous mode. The results indicated that the highest CH4 yield reached 0.22 L/g volatile solid at LR of 4 g/L/day, which obtained 56-58% of the theoretical value. In the steady stage with LR of 2-4 g/L/day and slurry recirculation, copies of total archaea increased. Especially the Methanobacteriales increased significantly (p < 0.05) to 3.30 × 108 copies/mL. The microbial communities were examined by MiSeq 16S rRNA sequencing. Enriched hydrolytic bacteria mainly belonged to Clostridiales, including Ruminococcus, Ruminiclostridium, and Ruminofilibacter settled in the rumen. High-active cellulase and xylanase were excreted in the co-inoculated system. Acid-producing bacteria by fermentation were affiliated with Lachnospiraceae and Bacteroidales. The acidogen members were mainly Spirochaetaceae and Clostridiales. Syntrophic oxidation bacteria mainly consisted of Synergistetes, propionate oxidizers (Syntrophobacter and Pelotomaculum), and butyrate oxidizers (Syntrophus and Syntrophomonas). There had no volatile fatty acid (VFA) accumulation and the pH values varied between 6.94 and 7.35. At LR of 6 g/L/day and a recirculation ratio of 1:1, the hardly degradable components and total VFA concentrations obviously increased. The total archaea and Methanobacteriales then deceased significantly to 8.56 × 105 copies/mL and 4.14 × 103 copies/mL respectively (p < 0.05), which resulted in the inhibition of methanogenic activities. Subsequently, microbial diversity dropped, and the hydrolytic bacteria and syntrophic oxidizers obviously decreased. In contrast, the abundances of Bacteroidales increased significantly (p < 0.05). Acetate, propionate, and butyrate concentrations reached 2.02, 6.54, and 0.53 g/L, respectively, which indicated "acidification" in the anaerobic reactor. Our study illustrated that co-inoculated anaerobic sludge enriched the ruminal function consortia and hydrogenotrophic methanogens played an important role in anaerobic digestion of lignocelluloses.