Single-Stage vs. Two-Stage Anaerobic Digestion of Molasses in a Circular Economy Context: Energy Recovery and Digestate Fertilizer Value

Anaerobic digestion (AD) of waste biomass is primarily regarded as a technology for gaseous biofuel production. Depending on process configuration, AD enables the generation of biohydrogen, biomethane, and digestate with potential fertilization value. Although digestate from conventional single-stage (1SD) AD systems has been extensively investigated, digestate derived from two-stage (2SD) configurations remains largely overlooked and is commonly assumed to be equivalent. Here, we present the first interdisciplinary comparison of 1SD and 2SD molasses AD systems, integrating process performance, digestate chemistry, microbial composition, and fertilization response assessed in pot experiments.Operation across a range of hydraulic retention times showed that stages separation restructures carbon conversion pathways. The 2SD system achieved approximately 27% higher total energy recovery than the 1SD configuration and maintained stable operation at short HRTs inaccessible to 1SD system. Chemical analyses demonstrated that the 2SD system produced more mature digestates, characterized by low residual chemical oxygen demand, limited accumulation of fermentation intermediates and long-chain fatty acids, elevated pH, and a pronounced shift toward plant-available mineral nitrogen, whereas digestates from the 1SD system indicated ongoing fermentative processes. Metagenomic analysis revealed that 2SD digestates supported higher microbial diversity, greater archaeal abundance, and dominance of acetoclastic Methanosaetaceae, alongside enrichment of amino acid–degrading consortia, plant growth–promoting taxa (e.g., Alcaligenes and Liquorilactobacillus), and indole-derived auxin precursors. Pot experiments confirmed these differences, showing consistently higher plant biomass following fertilization with 2SD digestates.The two-stage AD enables integrated recovery of renewable energy and agronomically enhanced nutrients from carbon-rich wastes, supporting SDG goals.