Carbohydrate-active enzyme architectures reflect distinct fungal and bacterial strategies for lignocellulose conversion to volatile fatty acids.

Anaerobic fermentation with microbial communities is an emerging platform for converting lignocellulosic biomass to biofuels and bioproducts. The process relies on diverse anaerobic microbes that interact to deconstruct and convert lignocellulosic biomass into a range of products, such as volatile fatty acids (VFAs), through arresting methanogenesis during fermentation. However, defining the distinct functional roles played by various fungi and bacteria during anaerobic biodegradation remains poorly understood. Here, we performed parallel enrichment experiments from cow feces, goat feces, and anaerobic digester sludge, selecting for fungal- or bacterial-dominated communities that convert sorghum biomass into VFAs. Subsequently, we reconstructed metabolic networks across these enrichments using recovered bacterial metagenome-assembled genomes (MAGs), fungal isolate genomes, and genome-centric metatranscriptomics. This enabled direct comparison of the carbohydrate-active enzymes and fermentation pathways expressed by bacterial and fungal communities during lignocellulose conversion to VFAs. Our findings implicate diverse bacteria affiliated with the Bacteroidales and Lachnospiraceae in the direct conversion of lignocellulosic biomass to propionate and butyrate, respectively, whereas Neocallimastix-dominated fungal enrichments converted lignocellulose to lactate, acetate, and formate. Analysis of carbohydrate-active enzymes revealed that bacterial and fungal communities primarily targeted the same dominant lignocellulosic substrates while employing distinct enzyme repertoires and multidomain carbohydrate-active enzyme architectures, highlighting complementary enzymatic strategies for accessing and deconstructing lignocellulosic biomass. We anticipate that these findings will help inform efforts to develop synthetic consortia with tailored functionality for low-cost conversion of lignocellulosic biomass to fuels and bio-based chemicals.