Culture-independent discovery of a novel thermotolerant lipase and its producer from mesophilic anaerobic digestion sludge.

Anaerobic digestion of lipid-rich wastes holds significant potential for enhanced biomethane production, due to the high energy density of lipids. However, to fully harness this potential, a deeper understanding of lipolytic microorganisms is essential, as key microbial players involved in lipid hydrolysis remain largely unidentified. In this study, we employed an integrated approach combining zymography, metaproteomics, and metagenomics to identify the lipolytic microorganisms from anaerobic digester sludge. This activity-based strategy identified a novel lipase distantly related to known lipases. Besides, although this lipase originates from a mesophilic environment, it exhibited unexpected extremophilic-like properties, with maximal activity at 97.5 °C and pH 11. We further reconstructed a metagenome-assembled genome encoding this lipase and demonstrated that it likely represents a novel genus closely related to Candidatus Scatomorpha. Metabolic reconstruction suggested that this bacterium hydrolyzes extracellular lipids and utilizes the resulting hydrolysate, glycerol, to produce lactate and ethanol. Habitat analysis revealed that this bacterium is specifically detected in anaerobic digesters, particularly those processing lipid-rich waste. These findings highlight the pivotal role of this bacterium in anaerobic lipid degradation.