Effect of increasing organic loading rate on hydrolytic enzyme activities and microbial communities during anaerobic digestion of energy crops

Introduction Efficiency and stability of anaerobic digestion (AD) processes strongly depend on maintaining a balance between microbial conversion steps. Operational stress can disrupt this balance, leading to shifts in the microbial community, accumulation of intermediate metabolites, and changes in overall process performance. Therefore, the objective of the study was to better understand the role of hydrolysis and its interplay with subsequent conversion steps in the destabilization of the AD process during increasing organic loading rate (OLR). Methods To systematically investigate the response of an AD process to operational stress, a stepwise increase in OLR until 15 g volatile solids (VS)/(L d) along with stepwise decrease of the hydraulic retention time (HRT) was applied in two parallel operated mesophilic lab-scale anaerobic digesters loaded with maize silage, cow manure and coarse-ground grain. The duplicate systems were comprehensively monitored, including key process parameters such as biogas production and composition, pH, buffer capacity, and volatile fatty acid (VFA) concentrations. Hydrolytic enzyme activities (amylase, protease, esterase) were measured to quantify key microbial functions, and microbial communities were analyzed via amplicon sequencing of 16S rRNA and mcrA genes to monitor compositional changes of the bacterial and methanogenic populations, respectively. Results and discussion Both reactors showed similar trends in process performance until OLR was 2.5-fold increased, characterized by increasing hydrolytic enzyme activities and minor VFA accumulation. At higher OLR, VFA accumulation increased, and the process performance and microbial communities of the two reactors diverged. While methane production in one reactor collapsed due to over-acidification and was replaced by chain elongation as the predominant microbial process, the other reactor maintained methanogenic activity until 3-fold increase in OLR. These changes were also reflected in declining enzyme activities, where protease activity decreased first. Bacterial diversity in the reactor shifting to chain elongation decreased dramatically, indicating a loss of AD functionality under elevated OLR. These findings highlight the importance of microbial community composition and diversity for maintaining AD stability under increasing operational stress. Contrasting reactor responses suggest that even small differences in microbial communities or stochastic effects can influence process resilience.