Engineering robustness in hyperthermophilic acidification reactor through adaptive laboratory evolution of dairy...

Hyperthermophilic anaerobic acidification (HTA) of dairy manure (DM) enabled volatile fatty acid (VFA) production from lignocellulose-rich substrates, but stable operation at short hydraulic retention time (HRT) requires microbial adaptation under sustained selection pressure. In this study, a 50 L anaerobic acidification reactor (AAR) treating DM was operated at 70 °C, and HRT was progressively reduced from 8 days to 6 days and then to 5 days to evaluate process performance together with microbial community succession. Shortening HRT increased VFA productivity from 0.29 ± 0.07 g/L/d to 1.22 ± 0.19 g/L/d, while stabilized 5-day operation maintained an average total VFA concentration of 20.83 ± 1.50 g/L. Acetic acid became increasingly dominant as operation progressed, indicating a stable carbohydrate fermentation route under intensified conditions. A linear mixed model confirmed a significant HRT effect on total VFA concentration. Improved reactor performance at short HRT coincided with enrichment of thermophilic fermenters associated with polymer breakdown and carbohydrate fermentation, particularly Clostridiaceae and Ruminococcaceae, followed by reestablishment of a resilient hyperthermophilic core dominated by Caldicoprobacteraceae, Thermodesulfobiaceae, and Clostridiaceae. Diversity analyses further supported structured community reassembly during stabilized 5-day operation. These findings showed that stepwise HRT reduction at 70 °C selected a resilient microbiome that sustained stable, high-rate VFA production from dairy manure and established an operational strategy for hyperthermophilic acidification of lignocellulosic manure for downstream bioprocess integration.