Process Stability, Methane Yield, and Microbial Community Structure in Two-Stage Co-Digestion of Plant and Animal Substrates Using Real-World Feedstock from an Agricultural Biogas Plant

Abstract

In the context of the global shift toward sustainable energy systems and circular economy strategies, anaerobic digestion is a well-established biotechnology for the valorisation of organic residues, the production of biogas as a renewable energy carrier, and the generation of nutrient-rich digestate. This study aimed to evaluate the stability and methane yield of a two-stage co-digestion process using a substrate mixture developed based on the actual feedstock composition of a full-scale agricultural biogas plant. The daily feed mixture consisted of maize silage (8.2%), sugar beet pulp (4.9%), cellulose pulp (6.6%), distillery stillage (38.6%), corn syrup (6.4%), cattle slurry (22.5%) and sterilised animal by-products (12.8%). Digestate was recirculated separately as part of the reactor operation. Laboratory-scale experiments were conducted in a two-stage anaerobic digestion system operated at 42 °C and 50 °C. The physicochemical properties of the substrates and digestate were determined, biogas quantity and composition were monitored, and the microbial community structure was assessed using 16S rRNA gene amplicon sequencing. The process remained stable throughout the experimental period, with pH values ranging from 8.23 to 8.53, alkalinity between 2600 and 2940 mg CaCO3/dm3, and a VFAs/alkalinity ratio of 0.17–0.93. Despite ammonium nitrogen concentrations reaching 4346 mg N-NH4+/L, no clear concurrent reduction in gas or methane production was observed. Methane accounted for approximately 70–80% of the biogas produced. The overall specific methane yield reached 346.4 NL CH4 kg−1 VS added. 16S rRNA gene amplicon sequencing revealed a diverse microbial community containing taxa previously associated with hydrolysis, fermentation and syntrophic interactions, including Proteiniphilum and Syntrophaceticus. The results demonstrate stable process performance and methane production in this site-specific laboratory-scale case study based on the feedstock composition and process configuration of a full-scale agricultural biogas plant.