Microbial communities inCaldicellulosiruptorspp. pretreatment vessels and anaerobic digesters fed dairy manure

The composition of microbial communities was determined in commercial-scale vessels used for 1) hyperthermophilic pretreatment (EBP) of dairy manure with four species ofCaldicellulosiruptorspp at 70–72⁰C, 2) anaerobic digestion of 8% manure in continuously stirred tank reactors (CSTRs) at 35– 37°C, and 3) anaerobic digestion at 35-37⁰C in induced bed reactors (IBRs) fed a mixture of effluents from EBPs and CSTRs. Samples were collected approximately weekly for 60 days. pH, volatile fatty acid anion and bicarbonate concentrations, COD, and methane yield were measured to assess environmental conditions. Microbial community compositions were obtained via 16S rRNA gene sequencing.Caldicellulosiruptorspp. pretreatment altered microbial composition and amplified community connectivity in the IBRs which enhanced syntrophic methanogenesis. Methanobacteriaceae,Methanobrevibacter, andMethanosarcinawere the main methanogenic taxa detected in all reactors. MethanogenicMethanothermobacterand CandidatusMethanoplasmawere uniquely associated with pretreated digestate. Co-occurrence network analysis of pretreated manure in EBP tanks and IBRs revealed dense community interactions, with methylotrophicMethanosphaeraand hydrogenotrophicMethanobrevibacterstrongly connected to syntrophic bacteria capable of H₂production. Raw manure and CSTR community networks were loose and disconnected and exhibited higher modularity. Acetoclastic methanogens did not display co-occurrence relationships with other taxa due to high acetate concentrations in the raw manure and EBP effluent.