The hadal zone, encompassing oceanic regions from 6 km below sea level (b.s.l.) to the ultimate depth of 11 km at the Challenger Deep in the Mariana Trench, represents one of Earth's most extreme and least explored environments. In this study, we employed an in situ filtration apparatus aboard human-occupied vehicle (HOV) for microbial collection in the hadal zone, filtering high-volume (>100 Liters per sample) seawater during 12 dives (~6-11 km b.s.l.), thus providing sufficient microbial biomass for subsequent analyses.
Based on these samples, we developed a framework integrating DNA-protein co-extraction and metagenome-guided metaproteome analysis for efficient protein identification. This workflow identified 135,313 non-redundant expressed hadal proteins, with 95% being hadal-specific, highlighting microbial activities distinct from those in shallower waters. Specialized hadal active processes in refractory carbon utilization (e.g., aromatic and halogenated compounds) and diverse energy conversion (e.g., heavy metals, thiosulphate, CO oxidation) suggested adaptative strategies to nutrient-limited conditions under high hydrostatic pressures, fundamentally different in element cycling from upper ocean. We then observed significant correlations between relative abundances in metaproteome and metagenome across taxonomic ranks from phylum to species level, and further categorized microbes into high-active and low-active groups according to the slope of linear fits. High-active taxa, largely unique to the hadal zone, display versatile metabolic capabilities, driving processes like heavy metal detoxification and carbon utilization.
These results provide the first proteome-level evidence of unique hadal microbial activities necessitating specific consideration when evaluating global ocean biogeochemical cycles, and underscores the need to integrate hadal microbial contributions into oceanographic models, enhancing our understanding of Earth's deepest waters.

hadal zone,metaproteome,metagenome,microbial metabolism,active function,in situ filtration