Adaptive mechanism of effluent recirculation in driving methanogenesis of hybrid Pennisetum.

Effluent recirculation can stabilize anaerobic digestion (AD), but the microbial and functional basis of its long-term effects during the mono-digestion of high-carbon-to-nitrogen ratio lignocellulosic feedstocks remains unclear. This study investigated the effects of 90 days of effluent recirculation on the AD of hybrid Pennisetum. Effluent recirculation increased the specific methane yield to 123 ± 34 mL/g VS, 129% higher than that of the control, while reducing volatile fatty acids (VFAs) accumulation by 99% and maintaining the pH between 6.87 and 7.49. Rather than uniformly enhancing hydrolysis, effluent recirculation redistributed CAZyme functional potential: selected families involved in oligosaccharide processing increased, whereas several families associated with terminal hydrolysis and debranching decreased, indicating moderated release of fermentable sugars. This functional redistribution was accompanied by the enrichment of syntrophic bacteria, including unclassified Syntrophaceae and Syntrophorhabdus, and complementary methanogenic populations. The relative abundances of acetoclastic Methanothrix and hydrogenotrophic Methanoculleus increased by 100% and 130%, respectively. Genes associated with fatty-acid β-oxidation, propionate conversion, acetate/C1 metabolism, and terminal methanogenesis were also enriched, indicating stronger coupling between VFAs oxidation and the methanogenic removal of acetate and H2/formate. Taxon-resolved analysis further linked key CAZymes and methanogenesis genes to their microbial carriers. These findings indicate that long-term effluent recirculation improved process stability by coordinating moderated hydrolysis, syntrophic VFAs conversion, and complementary methanogenic substrate utilization.