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Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high-biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment.

Hadal biosphere represents the deepest part of the ocean with water depth >6,000 m. Accumulating evidence suggests the existence of unique microbial communities dominated by heterotrophic processes in this environment. However, investigations of the microbial diversity and their metabolic potentials are limited because of technical constraints for sample collection. Here, we provide a detailed metagenomic analysis of three seawater samples at water depths 5,000–6,000 m below sea level (mbsl) and three surface sediment samples at water depths 4,435–6,578 mbsl at the Yap Trench of the western Pacific. Distinct microbial community compositions were observed with the dominance of Gammaproteobacteria in seawater and Thaumarchaeota in surface sediment. Comparative analysis of the genes involved in carbon, nitrogen and sulfur metabolisms revealed that heterotrophic processes (i.e., degradation of carbohydrates, hydrocarbons, and aromatics) are the most common microbial metabolisms in the seawater, while chemolithoautotrophic metabolisms such as ammonia oxidation with the HP/HB cycle for CO2 fixation probably dominated the surface sediment communities of the Yap Trench. Furthermore, abundant genes involved in stress response and metal resistance were both detected in the seawater and sediments, thus the enrichment of metal resistance genes is further hypothesized to be characteristic of the hadal microbial communities. Overall, this study sheds light on the metabolic versatility of microorganisms in the Yap Trench, their roles in carbon, nitrogen, and sulfur biogeochemical cycles, and how they have adapted to this unique hadal environment.

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Deep-sea oceanic crust constitutes the largest region of the earth’s surface. Accumulating evidence suggests that unique microbial communities are supported by iron cycling processes, particularly in the young (<10 million-year old), cool (<25°C) subsurface oceanic crust. To test this hypothesis, we investigated the microbial abundance, diversity, and metabolic potentials in the sediment-buried crust from “North Pond” on western flank of the Mid-Atlantic Ridge. Three lithologic units along basement Hole U1383C were found, which typically hosted ∼104 cells cm-3 of basaltic rock, with higher cell densities occurring between 115 and 145 m below seafloor. Similar bacterial community structures, which are dominated by Gammaproteobacterial and Sphingobacterial species closely related to iron oxidizers, were detected regardless of variations in sampling depth. The metabolic potentials of the crust microbiota were assayed by metagenomic analysis of two basalt enrichments which showed similar bacterial structure with the original sample. Genes coding for energy metabolism involved in hydrocarbon degradation, dissimilatory nitrate reduction to ammonium, denitrification and hydrogen oxidation were identified. Compared with other marine environments, the metagenomes from the basalt-hosted environments were enriched in pathways for Fe3+ uptake, siderophore synthesis and uptake, and Fe transport, suggesting that iron metabolism is an important energy production and conservation mechanism in this system. Overall, we provide evidence that the North Pond crustal biosphere is dominated by unique bacterial groups with the potential for iron-related biogeochemical cycles.

Symbiotic microorganisms have been found in the hemolymph (blood) of many aquatic invertebrates, such as crabs, shrimps and oysters. Hemolymph is a critical site in host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies using selective media (e.g., TCBS, 2216E, and LB) for enumerating and isolating microbial cells. However, doubts remain about the "true" representation of the microbial abundance and diversity of symbiotic microorganisms in hemolymph, particularly for uncultivable microorganisms which are believed to be more abundant than the cultured. To explore this, we developed a culture-independent cell extraction method for separating microbial cells from the hemolymph of three aquatic invertebrates (, , and) involving filtration through a 5-μm mesh filter membrane (the Filtration Method). A combination of the Filtration Method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. More than 2.6 × 10 cells/mL of microbial cells dominated by and, and, and and, were detected in the hemolymph of ,, and , respectively. A parallel study for investigating the hemolymph microbiome by comparing the Filtration Method and a culture-dependent method (the Plate Count Method), showed significantly higher microbial abundances (between 26 and 369-folds difference; P<0.05) and less biased community structures of the Filtration Method than those of the Plate Count Method. Furthermore, hemolymph of the three invertebrates harbored many potential pathogens, including ,, and Finally, the Filtration Method provides a solution that improves understanding of the metabolic functions of uncultivable hemolymph microorganisms (e.g., metagenomics) devoid of host hemocytes contamination. Microorganisms are found in invertebrates' hemolymph, a critical site in host immune response. Currently, studies on hemolymph microorganisms are mostly performed with culture-dependent strategies. However, doubts remain about the "true" representation of hemolymph microbiome. This study developed a culture-independent cell extraction method that could separate microbial cells from the hemolymph of three aquatic invertebrates (, , and) based on filtration through a 5-μm mesh filter membrane (the Filtration Method). A combination of the Filtration Method with fluorescence microscopy and high-throughput sequencing technique provides insight into the abundances and diversity of the total microbiota in the hemolymph of these three invertebrates. Our results demonstrate that the hemolymph of aquatic invertebrates harbors a much higher microbial abundance and distinct microbial community composition than previously estimated. Furthermore, this work provides a less biased solution for studying the metabolic functions of uncultivable hemolymph microbiota devoid of host hemocytes contamination.