Updated project metadata. Meta-proteomics analysis approach in the application of biogas production from anaerobic digestion has many advantages that has not been fully uncovered yet. This study aims to investigate biogas production from a stable 2-stage chicken manure fermentation system in chemical and biological perspective. The diversity and functional protein changes from the 1st stage to 2nd stage is a good indication to expose the differential metabolic processes in anaerobic digestion. The highlight of identified functional proteins explain the causation of accumulated ammonia and carbon sources for methane production. Due to the ammonia stress and nutrient limitation, the hydrogenotrophic methanogenic pathway is adopted as indicative of meta-proteomics data involving the key methanogenic substrates (formate and acetate). Unlike traditional meta-genomic analysis, this study could provide both species names of microorganism and enzymes to directly point the generation pathway of methane and carbon dioxide in investigating biogas production of chicken manure.