Influences of layer inoculation on biogas production and microbial community in solid-state anaerobic fermentation of pig manure

China is a large agricultural country, livestock manure is both agricultural waste and resources. Among the many treatment processes, Solid-State Anaerobic Digestion to produce biogas is one of the effective measures to realize its resource utilization and solve environmental pollution. While high Total Solid (TS) content causing a reduction of methane yields or failure of digestion process because the accumulation of Volatile Fatty Acids (VFAs) resulted in the inhibition of methanogens. At present, the research on VFAs mainly focuses on the aspects of co-substrate for digestion, leachate recirculation and external buffer material to increase biogas production. The inoculum has an important impact on the tolerance of intermediate metabolites and the start-up time of anaerobic fermentation. Previous research mainly focuses on microbial acclimation and feedstock/inoculum ratio under the premixing inoculation method (fermentation after mixing the inoculum and substrate), while the exploration of different inoculation methods for SS-AD and the correlation between the dynamic changes of microbial community structure and metabolites (such as VFAs) need to be studied in depth. In this paper, the biogas production performance and micro-ecological succession law of pig manure and maize straw in layer inoculation and premixing inoculation fermentation were compared. A pilot-scale laboratory experiment was performed in a self-made vertical plexiglass reactor with a total volume of 11 L under feedstock/inoculum ratio=25% in SS-AD process (TS=20%). The result showed that, the first peak of TVFAs mass concentration in layer inoculation system reached 33.0 mg/g on the day 15, and had a greater decline until the end of fermentation. The mass concentration of TVFAs in premixing inoculation system was varied in the range of 29.2-38.5 mg/g on days 15-78. The cumulative Specific Methane Yield of layer inoculation fermentation reached 211.5 mL/g is the highest. CH4 yield in layer inoculation system was mucher higher than in premixing inoculation even with the same substrate, which indicated that layer inoculation can lead to the comsuption of VFAs over time, make the process run effectively, and reduce the startup time. The SMY of premixing inoculation was under 1.0 mL/g-VS during the whole experiment. In layer inoculation system, the biogas CH4 content ranged from 54.0% to 66.0% after day 20. The CH4 content in premixing inoculation fermentation showed a rapid increase after 52 days of digestion and reached 58.1% on day 78. High-throughput sequencing results showed that hydrogenotrophic methanogen was the dominant methane production pathway during SS-AD of different inoculation methods. Layer inoculation can increase the richness and diversity of microorganisms in the fermentation system, and the community structure is more stable than premixing inoculation. The results of cluster analysis on microbial diversity showed that the difference both layer inoculation and premixing inoculation system showed good similarity in the bacteria and archaea samples of stage I~II and stage III~IV. Further analysis showed that the main environmental factor affecting layer inoculation and premixing inoculation are acetic acid and pH value, respectively. The results of this study provide a scientific basis for alleviating the VFAs inhibition of SS-AD of livestock and poultry farming waste and increasing methane yield.