Anaerobic digestion of microalgae: microbial response and recovery after organic loading disturbances.

Unlabelled: Industrial anaerobic digestion (AD) represents a relevant energy source beyond today's fossil fuels, wherein organic matter is recycled to methane gas via an intricate and complex microbial food web. Despite its potential, anaerobic reactors often undergo process instability over time, which is frequently caused by substrate composition perturbations, making the system unreliable for stable energy production. To ensure the reliability of AD technologies, it is crucial to identify microbial and system responses to better understand the effect of such perturbations and ultimately detect signatures indicative of process failure. Here, we investigate the effect of the microalgal organic loading rate (OLR) on the fermentation product profile, microbiome dynamics, and disruption/recovery of major microbial metabolisms. Reactors subjected to low- and high-OLR disturbances were operated and monitored for fermentation products and biogas production over time, while microbial responses were investigated via 16S rRNA gene amplicon data, shotgun metagenomics, and metagenome-centric metaproteomics. Both low- and high-ORL fed systems encountered a sudden decline in methane production during OLR disturbances, followed by a recovery of the methanogenic activity within the microbiome. In the high-OLR disturbances, system failure triggered an upregulation of hydrolytic enzymes, an accumulation of fermentation products, and a shift in the methanogenic population from hydrogenotrophic to acetoclastic methanogens, with the latter being essential for recovery of the system after collapse.

Importance: Anaerobic digestion (AD) with microalgae holds great potential for sustainable energy production, but process instability caused by substrate disturbances remains a significant barrier. This study highlights the importance of understanding the microbial dynamics and system responses during organic loading rate perturbations. By identifying key shifts in microbial populations and enzyme activity, particularly the transition from hydrogenotrophic to acetoclastic methanogens during recovery, this research provides critical insights for improving AD system stability and can contribute to optimizing microalgae-based AD processes for more reliable and efficient methane production.