Structural and functional analysis of the active cow rumen’s microbial community provides a catalogue of genes and microbes participating in the deconstruction of cardoon biomass
Authors: Firrincieli Andrea , Minuti Andrea , Cappelletti Martina , Ferilli Marco , Ajmone-Marsan Paolo , Bani Paolo , Petruccioli Maurizio , Harfouche Antoine L. 
Published: April 2024

Abstract
Background
Ruminal microbial communities enriched on lignocellulosic biomass have shown considerable promise for the discovery of microorganisms and enzymes involved in digesting cell wall compounds, a key bottleneck in the development of second-generation biofuels and bioproducts, enabling a circular bioeconomy. Cardoon (Cynara cardunculus) is a promising inedible energy crop for current and future cellulosic biorefineries and the emerging bioenergy and bioproducts industries. The rumen microbiome can be considered an anaerobic “bioreactor”, where the resident microbiota carry out the depolymerization and hydrolysis of plant cell wall polysaccharides (PCWPs) through the catalytic action of fibrolytic enzymes. In this context, the rumen microbiota represents a potential source of microbes and fibrolytic enzymes suitable for biofuel production from feedstocks. In this study, metatranscriptomic and 16S rRNA sequencing were used to profile the microbiome and to investigate the genetic features within the microbial community adherent to the fiber fractions of the rumen content and to the residue of cardoon biomass incubated in the rumen of cannulated cows.

Results
The metatranscriptome of the cardoon and rumen fibre-adherent microbial communities were dissected in their functional and taxonomic components. From a functional point of view, transcripts involved in the methanogenesis from CO2 and H2, and from methanol were over-represented in the cardoon-adherent microbial community and were affiliated with the Methanobrevibacter and Methanosphaera of the Euryarchaeota phylum. Transcripts encoding glycoside hydrolases (GHs), carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), and glycoside transferases (GTs) accounted for 1.5% (6,957) of the total RNA coding transcripts and were taxonomically affiliated to major rumen fibrolytic microbes, such as Oscillospiraceae, Fibrobacteraceae, Neocallimastigaceae, Prevotellaceae, Lachnospiraceae, and Treponemataceae. The comparison of the expression profile between cardoon and rumen fiber-adherent microbial communities highlighted that specific fibrolytic enzymes were potentially responsible for the breakdown of cardoon PCWPs, which was driven by specific taxa, mainly Ruminococcus, Treponema, and Neocallimastigaceae.

Conclusions
Analysis of 16S rRNA and metatranscriptomic sequencing data revealed that the cow rumen microbiome harbors a repertoire of new enzymes capable of degrading PCWPs. Our results demonstrate the feasibility of using metatranscriptomics of enriched microbial RNA as a potential approach for accelerating the discovery of novel cellulolytic enzymes that could be harnessed for biotechnology. This research contributes a relevant perspective towards degrading cellulosic biomass and providing an economical route to the production of advanced biofuels and high-value bioproducts.