Rumen Metaproteomics Highlight the Unique Contributions of Microbe-Derived Extracellular and Intracellular Proteins for In Vitro Ruminal Fermentation

Abstract

Rumen microorganisms can be used in in vitro anaerobic fermentation to encourage the sustainable exploitation of agricultural wastes. However, the understanding of active microbiota under in vitro ruminal fermentation conditions is still insufficient. To investigate how rumen microbes actively participate in the fermentation process in vitro, we resolved the metaproteome generated from ruminal fermentation broth after seven days of in vitro incubation. Herein, the sample-specific database for metaproteomic analysis was constructed according to the metagenomic data of in vitro ruminal fermentation. Based on the sample-specific database, we found in the metaproteome that Bacteroidetes and Firmicutes_A were the most active in protein expression, and over 50% of these proteins were assigned to gene categories involved in energy conversion and basic structures. On the other hand, a variety of bacteria-derived extracellular proteins, which contained carbohydrate-active enzyme domains, were found in the extracellular proteome of fermentation broth. Additionally, the bacterial intracellular/surface moonlighting proteins (ISMPs) and proteins of outer membrane vesicles were detected in the extracellular proteome, and these ISMPs were involved in maintaining microbial population size through potential adherence to substrates. The metaproteomic characterizations of microbial intracellular/extracellular proteins provide new insights into the ability of the rumen microbiome to maintain in vitro ruminal fermentation.