Hythane and methane production in a multistage anaerobic hythane reactor (MAHR): Stage separation, functional microbe selection, thermodynamics, and kinetics

The establishment of a multistage anaerobic hythane reactor (MAHR) that integrates biohythane and biomethane production into a single reactor demonstrates promising potential for simultaneously producing hythane and methane. However, separating hythane and methane production in a single reactor remains a challenge, and stages separation in MAHR system is not well understood. Moreover, the necessity to heating pretreatment of the inoculum for acidogenesis and hythane production is impractical for the full-scale application of MAHR due to the extra economic and energy input. Therefore, heating preselected and not preselected inocula were adopted in this study to reveal the biofuel production performance and the mechanisms of stage separation in MAHRs with different inoculum strategies. Results indicated that stable hythane and methane production of MAHR without inoculum preselection could be achieved and maintained after a longer start-up period compared to the preselected inoculum condition. A comparable hythane and methane production and organics conversion performance were observed in the two MAHRs during stable period, in which hythane with a suitable hydrogen concentration (5%-25%) and methane with a high concentration (57%-75%) were produced. Supported by the more stable microbial correlation networks, MAHR without inoculum preselection suggested a higher methane production rate (13.5 ± 2.8 L/L/d) and was resistant to organic loading shock (80 g COD/L/d). Furthermore, microbial diversity, thermodynamics, and kinetic analysis revealed that the stage separation was highly dependent on microbial dynamics, metabolism characteristics, and reactor configurations. Acetogens (Desulfovibrio and Syntrophobacter) and aceticlastic methanogens (Methanosaeta and Methanosarcina) were more likely to wash out form the hythane production zone (Mh) and be enriched in the methane production zone (Mm) compared to acidogens (Streptococcus, Rikenellaceae_RC9_gut_group, Bacteroides, etc.) and hydrogenotrophic methanogens (Methanobacterium and Methanobrevibacter). The results from this study deepened the understanding of functional microbial community dynamics and stage separation mechanisms, which provided insights into the design, start-up, and full-scale operation of MAHR.