Detoxifying and depolymerizing microorganisms reveal intertwined guild collaborations in the gut microbiome of a generalist macro-algivorous fish

The biotransformation of macroalgal biomass represents a major catabolic challenge due to its structurally diverse polysaccharides and inhibitory polyphenols. Unlike terrestrial lignocellulosic substrates, macroalgae polysaccharides contain multiple monomer types, branching patterns, and sulfation states. Additionally, macroalgae polyphenols have been shown to inhibit both microbial growth and their catalytic enzymes. While herbivorous fishes have evolved specialized gut microbiota to process these substrates, the enzymatic pathways remain poorly characterized, with few experimentally validated polysaccharide utilization loci or biochemically defined marine sulfatases, and limited understanding of polyphenol degradation. Here, we developed in vitro microcosms, based on the gut microbiome of the generalist macro-algivorous fish Kyphosus cinerascens, to temporally resolve the activity of the microbial guilds involved in macroalgal polysaccharide and polyphenol transformation. First, parallel cDNA/DNA amplicon sequencing were employed to distinguish the natural active fraction from transient gut microbiome taxa. Four media combinations were able to propagate between 96% to 99% of the active hindgut microbial families, reproducing the cooperative degradation dynamics observed in vivo. Metagenomic and metatranscriptomic profiling of these four optimized in vitro microcosms served as models to assess the stepwise functional successions occurring in the natural gut microbiome. Early Gammaproteobacteria expressed enzymes linked to polyphenol detoxification and alginate degradation, followed by Bacillota, Bacteroidota, and Verrucomicrobiota guilds targeting more recalcitrant sulfated polysaccharides and polyphenols. Together, these results identified temporal and taxonomic coordination as key features of macroalgal biomass deconstruction, providing an experimentally tractable model for discovering novel carbohydrate-active enzymes and elucidating poorly understood pathways of marine polyphenol degradation. IMPORTANCESeaweed represents a source of sustainable biomass for various applications, but scalable industrial methods struggle to break down seaweed biomass into intermediate products due to the complexity of its constituents. Fish of the genus Kyphosus feed on different seaweed types by leveraging gastrointestinal bacteria to neutralize inhibitory polyphenols and convert their polysaccharides into simple sugars. This study identifies microbial groups that are transcriptionally active in natural fish hindgut microbiomes to propagate these active microbial communities in vitro. This enabled assessing how distinct microbial guilds act in succession to transform complex polysaccharides and polyphenols. Notably, this is the first study to assess the biotransformation capacities of macroalgal polyphenols by complex in vitro hindgut microbiomes of a generalist herbivorous fish. These findings advance our ecological understanding of cooperative degradation in marine gut symbioses and establish a tractable platform for discovering new enzymes and pathways with potential applications in algal biomass utilization.