Changes in Microbial Communities in Industrial Anaerobic Digestion of Dairy Manure Caused by Caldicellulosiruptor Pretreatment

Abstract Hyperthermophilic pretreatment with Caldicellulosiruptor species (EBP) increases substrate availability in anaerobic digestion, but the effect on downstream microbial community composition in industrial systems is not characterized. Changes in microbial communities were determined at an industrial facility processing dairy manure in a modified split-stream system with three reactor types: 1) EBP tanks at 70–72°C, 2) mesophilic Continuously Stirred Tank Reactors (CSTRs), and 3) mesophilic Induced Bed Reactors (IBRs) receiving combined CSTR and EBP effluent. All reactors had a two-day hydraulic retention time. Samples were collected weekly for 60 days. pH, volatile fatty acid and bicarbonate concentrations, COD, and methane yield were measured to assess tank environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing. EBP pretreatment increased acetate availability but led to a decline in the relative abundance of acetoclastic Methanosarcina species in downstream IBRs. Rather, syntrophic methanogens, e.g., Methanobrevibacter species, increased in relative abundance and became central to microbial co-occurrence networks, particularly in association with hydrogen-producing bacteria. Network analysis also demonstrated that these syntrophic relationships were tightly coordinated in pretreated digestate but absent in the untreated CSTRs. By promoting syntrophic methanogenesis while increasing acetate concentrations, EBP pretreatment requires system configurations that enable acetoclast retention to prevent acetate underutilization and maximize methane yields. Importance Hyperthermophilic Caldicellulosiruptor pretreatment in anaerobic digestion increased acetate availability while suppressing acetoclastic methanogens and promoting robust syntrophic methanogenic networks. The results offer engineers microbial guidance essential for designing effective multi-stage industrial anaerobic digestion facilities employing EBP methods. Competing Interest Statement The authors have declared no competing interest. Footnotes This revision presents a clearer interpretation of the results, emphasizing their practical significance for anaerobic digestion system design. We clarify how changes in microbial activity due to Caldicellulosiruptor pretreatment can inform the design of multi-stage anaerobic digestion plants implementing EBP techniques. In addition, the methods section was revised to improve clarity and support reproducibility. Abbreviations - RP - Reception Pit - CSTR - Continuously Stirred Tank Reactor - EBP - Extremophilic Biological Process - IBR - Induced Bed Reactor - AD - Anaerobic Digestion - VFA - Volatile Fatty Acid - VFAA - Volatile Fatty Acid Anion - COD - Chemical Oxygen Demand - TDS - Total Dissolved Solids - RA - Relative Abundance - HRT - Hydraulic Retention Time