Distinct microbial nitrogen cycling processes in the deepest part of the ocean

ABSTRACT The Mariana Trench (MT) is the deepest part of the ocean on Earth. Previous studies have described the microbial community structures and functional potential in the seawater and surface sediment of MT. Still, the metabolic features and adaptation strategies of the microorganisms involved in nitrogen cycling processes are poorly understood. In this study, comparative metagenomic approaches were used to study microbial nitrogen cycling in three MT habitats, including hadal seawater [9,600–10,500 m below sea level (mbsl)], surface sediments [0–46 cm below seafloor (cmbsf) at a water depth between 7,143 and 8,638 mbsl], and deep sediments (200–306 cmbsf at a water depth of 8,300 mbsl). We identified five new nitrite-oxidizing bacteria (NOB) lineages that had adapted to the oligotrophic MT slope sediment, via their CO2 fixation capability through the reductive tricarboxylic acid (rTCA) or Calvin-Benson-Bassham (CBB) cycle; an anammox bacterium might perform aerobic respiration and utilize sedimentary carbohydrates for energy generation because it contains genes encoding type A cytochrome c oxidase and complete glycolysis pathway. In seawater, abundant alkane-oxidizing Ketobacter species can fix inert N2 released from other denitrifying and/or anammox bacteria. This study further expands our understanding of microbial life in the largely unexplored deepest part of the ocean. IMPORTANCE The metabolic features and adaptation strategies of the nitrogen cycling microorganisms in the deepest part of the ocean are largely unknown. This study revealed that anammox bacteria might perform aerobic respiration in response to nutrient limitation or O2 fluctuations in the Mariana Trench sediments. Meanwhile, an abundant alkane-oxidizing Ketobacter species could fix N2 in hadal seawater. This study provides new insights into the roles of hadal microorganisms in global nitrogen biogeochemical cycles. It substantially expands our understanding of the microbial life in the largely unexplored deepest part of the ocean.

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Reviewer #1 (Comments for the Author): I have some editing suggestions, which are shown in the attached file.Please check it out for more details.Keywords: anammox, deep biosphere, hadal trench, metagenomics, nitrite oxidation

Introduction
The hadal biosphere refers to a trench area with water depths greater than 6,000 m, accounting for 45% of the vertical depth of the ocean.This habitat is characterized by high hydrostatic pressure (>60 MPa), low temperature (<4 o C), and relatively isolated environments [1][2][3].In general, the topography of a trench is shaped like a funnel consisting of slope and axis area.Importantly, the high carbon turnover rates were measured in the sediments, which contribute to the accumulation of organic carbon and nitrogen compounds (e.g.nitrate and amino acids) [4][5][6].However, compared to the trench axis, the trench slope is steeper with a more complex topography (e.g.horst, graben, seamount, and ridges), and it covers a much wider area [7,8].Sinking organic material that is unevenly distributed along the slope can also slide from the top to the trench bottom via ocean crust movements (such as earthquakes) and seawater circulation.Therefore, the sediment layer of the slope is generally thinner than that at the trench bottom and thus has a lower content of organic matter and microorganisms [4,6,9].
Previous studies have shown that diverse bacterial and archaeal communities have been detected in the MT.For example, Proteobacteria, Bacteroidota, Marinisomatota and Thaumarchaeota were dominant groups in the hadal seawater [10].The common microorganisms in surface sediment were Proteobacteria, Chloroflexota, Planctomycetes, Thaumarchaeota and Nanoarchaeota [9,11].
Recently, the metabolic features and adaptation strategies of the hadal microbiome have been uncovered using several techniques, such as genomics (metagenomics and metatranscriptomics), in situ measurements, and laboratory incubations.For example, Thaumarchaeota in MT seawater possessed two distinct sets of adenosine-triphosphate (ATP) synthases (binding Na + or H + ) in response to the extreme hadal environment [12].Chloroflexi in the sediments of MT can utilize a wide range of organic matter, and these bacteria responded to poor nutrition and a periodically fluctuating environment with a "feast-or-famine" metabolic strategy [13].
Labrenzia aggregate strains isolated from the MT (0-9,600 m) had an increased number of plasmidic genes along with the isolation depth and obtained the additional genetic potential mainly through plasmid exchange to withstand the extremely high hydrostatic pressure [14].Dermacozines, a new secondary metabolite produced by the bacterial strain Dermacoccus abyssi MT1.1 T (isolated from MT sediment at 10,898 m), has multiple activities such as free radical scavenging and cytotoxicity [15].Some Alphaproteobacteria isolates (e.g.Sagittula stellata, Labrenzia aggregate, Pelagibaca bermudensis) produce dimethylsulfoniopropionate, which protects bacteria from high hydrostatic pressure in the aphotic deep sea [16].Taken together, the adaptation of microbial communities to the hadal environment may contribute to their metabolic activities and important roles in the global biogeochemical cycles of carbon and nitrogen.
Nitrogen is a fundamental nutrient element in the environment.The microbial nitrogen cycle is a basic metabolic process for the synthesis of biological molecules (e.g.amino acids, proteins and nucleic acids) during microbial growth and reproduction.However, bioavailable nitrogen (e.g.nitrate and ammonia) is scarce on Earth, and microbially mediated nitrogen cycling processes contribute a significant portion of bioavailable nitrogen in many environments [17,18].In general, the nitrogen compounds in hadal environments consist of NO were detected in the surface sediment (<50 cmbsf) [6], but these nitrogen compounds showed the opposite trends in the deep sediment [21].Few studies have reported microbial nitrogen metabolism in MT surface sediment and seawater.For example, multiple microbial groups (e.g.Proteobacteria, Planctomycetota and Bacteroidota) are involved in NO 3 -reduction (dissimilatory nitrate reduction to ammonium (DNRA) and denitrification) [6,22,23].In the surface sediment of the Atacama Trench (5-10 cm at 8,085 m) and the Kermadec Trench (15-20 cm at 10,010 m), about 67% and 91% of the N 2 was derived from the anammox process, respectively [24].Furthermore, some anammox bacterial MAGs (metagenome-assembled genomes) were retrieved from the MT surface sediments (18-21 cm at 10,840 m) [6].The nitrogen fixation gene nifH has been detected in MT seawater (>9,600 m) [25].For the nitrification process, ammonia-oxidizing archaea (AOA), which are dominated by ammonium oxidation [12].However, the metabolic features and adaption strategies of these anammox bacteria, other types of nitrifying bacteria (e.g.NOB), and N 2 fixers have not yet been discovered in the hadal biosphere.
To address these knowledge gaps, we used comparative metagenomic approaches to uncover the unique features of microbial nitrogen cycling processes in three different hadal habitats, including hadal seawater (9,600-10,500 mbsl), surface sediment (0-46 cmbsf at a water depth between 7,143 and 8,638 mbsl), and deep sediment (200-306 cmbsf at a water depth of 8,300 mbsl) (Table S1).

Sample collection, DNA extraction, and metagenome sequencing
The nine deep sediment samples were collected from the MT slope in the western Pacific Ocean during a cruise in 2019 using the research vessel Haida (11.13N, 142.33 E).The water depth was 8,300 mbsl (Figure S1).One sediment core was collected by gravity corer, and the subsamples of 200, 213, 229, 242, 250, 260, 272, 290, and 306 cmbsf were used in this study.The samples were stored at -80 o C immediately after recovery onboard.DNA was extracted from 0.5 g of each sediment using a DNeasy PowerSoil Pro Kit (Qiagen, Germany) according to the manufacturer's instructions, and only the interior sections of the sediment was subsampled with a flame-sterilized scoop for DNA extraction.In parallel, blank controls for all sampling and DNA extractions were prepared using Milli-Q water (18.2MΩ; Millipore, USA) filtered through the 0.22-m mesh membrane.
The six surface sediment samples were obtained from the MT slope at water depths between 7,143 and 8,638 mbsl as described elsewhere [6].All intact core samples were immediately subsampled onboard and then frozen at -80 °C until use.
DNA was extracted from 10 g sediment of each sample using PowerMax ® Soil DNA Isolation Kit (MoBio, Germany) according to the manufacturer's protocol.The three seawater samples were collected from the MT Challenger Deep at water depths between 9,600 and 10,500 mbsl as described elsewhere [26].About 50L of each seawater was filtered onto a 0.22-μm polycarbonate membrane (GTTP, 142 mm, Millipore, USA).DNA was extracted using the SDS method as described previously [26].Sequencing was performed on Illumina HiSeq X-Ten platform using 2 × 150 bp paired-end technology.

Metagenomic assembly and binning
All the metagenomes used in this study were assembled and binned using the same method.Briefly, the raw metagenomic reads were trimmed using Trimmomatic (v.0.38) [27] with default parameters.All clean reads from the six surface sediments were pooled together before de novo assemble to one coassembly.Meanwhile, all clean reads from the three seawater samples were pooled together before de novo assemble to one coassembly.The clean reads were then assembled into contigs using SPAdes (v.3.15.0) [28] with the parameters: --meta -k 21,29,39,59,79,99.

Taxonomic classification and relative abundance
Taxonomic assignments of the MAGs were performed using the "classify_wf" workflow in the GTDB-Tk software (v.2.1.0)[33].[3].Statistical significance of the relative abundance between two samples was analyzed using Mann-Whitney U test in SPSS (v.22.0), and differences were considered significant when P<0.05 as described elsewhere [35,36].

Retrieval of anammox, NOB, and Ketobacter reference genomes
The collection of anammox, NOB, and Ketobacter reference genomes were obtained from three sources, which included the MAGs recovered from the metagenomes of this study, the published genomes downloaded from the NCBI GenBank database [37] (August 16th, 2022), and those from the genomic catalog of Earth's microbiomes [38].

Phylogenetic analysis and HGT gene identification
The phylogenetic trees of nxrA, aclA, aclB, rbcL, nifH, and cytochrome c oxidase genes were constructed using IQ-TREE (v.1.6.3)[53] with ModelFinder [54], and ultrafast bootstrapping was used to estimate the reliability of each branch with 1,000 times resampling.The reference sequences with BLASTP identity >30% to the target genes were retrieved from the NCBI nr database [37] and the UniProt database [55].
The phylogenomic tree of anammox bacteria was constructed using IQ-TREE with ModelFinder, based on the 120 conserved single-copy ubiquitous bacterial genes [33].
The trees were visualized using the iTOL online tool (v.6) [56].The HGT genes were predicted by HGTector tool (v.2.0b3) [57] and the genes encoding type A cytochrome c oxidase in D200.bin4.133was analyzed by constructing a phylogenetic tree using IQ-TREE.

Microbial community structures in the MT biosphere
A detailed survey based on the 16S rRNA gene sequences retrieved from the clean reads of the metagenome revealed 348, 222, and 139 microbial classes in the deep sediments, surface sediments and seawater of the MT, respectively (Figure 1; Table S2).The three habitats all showed a dominance of bacteria over archaea (>84.5% bacteria in relative abundance).Proteobacteria, Chloroflexota, Planctomycetota and Thermoproteota (represented by Nitrososphaeria) were common microbial groups in the MT sediments and seawater.However, distinct dominant microbial taxa were observed in the three habitats (Figure 1; Table S2 reads per kilobase per million sequenced reads (RPKM)] in the denitrification pathway (nirK, norBC) were higher in the surface sediments than in the deep sediments, and an abundance of genes in the DNRA or denitrification process (napA/narG, nrfA) were higher in the surface sediments than in the seawater (Figure 2; Table S3; Mann-Whitney U test, P<0.05).The MT seawater metagenomes also showed higher abundances of nirK and norB genes, as compared with those in the deep sediments (Mann-Whitney U test, P<0.05).However, the abundance of nosZ gene in the deep sediments was 8-folds higher than that in seawater (Mann-Whitney U test, P<0.05).Notably, unique microbial nitrogen cycling genes were detected in each of the three habitats, including nitrite oxidation and anammox in the sediments, and nitrogen fixation in the seawater.

Newly-identified NOB and anammox bacteria in the MT sediment
Nitrite oxidation was a significant microbial nitrogen process in the MT sediment compared with seawater, because the metagenomes in the surface and deep sediments contained the hallmark gene nitrite oxidoreductase (nxr) (average RPKM value of 149 and 60, respectively; Figures 2 and S3).Of note, seven nitrite oxidoreductase-containing genomes were recovered from the metagenomes of the MT deep sediment.These MAGs were assigned to taxa including Desulfobacterota, Latescibacterota, Omnitrophota, Planctomycetota, and unclassified bacteria JABMQX01 according to the taxonomy of Genome Taxonomy Database (GTDB) (Table S4), which are distinct from the common NOB taxa (e.g.Chloroflexota, Proteobacteria, Nitrospinota, and Nitrospirota) as described in previous studies [58].
Furthermore, these NOB could potentially fix inorganic carbon because the complete gene sets in the rTCA or CBB cycles were identified in the NOB genomes, including the key genes encoding ATP citrate lyase (acl) or ribulose-bisphosphate carboxylase (rbc), respectively (Figures 3, S4, S5 and S6; Table S5).
Furthermore, anammox, determined by the presence of the hydrazine synthase gene (hzs), only occurred in the MT sediment (Figure 2).S6).To reveal the characteristics of anammox bacteria in the MT deep sediment, we performed the comparative genomic analysis of 76 anammox bacteria genomes (>50% completeness and <5% contamination), including 3 MAGs retrieved from this study [59,60], 69 MAGs from the NCBI Assembly database [37], and 4 MAGs from the genomic catalog of Earth's microbiomes [38].Interestingly, some genomes from Bathyanammoxibiaceae, Scalinduaceae and Brocadiaceae contained genes encoding cytochrome c oxidases (Figure S7).A phylogenetic analysis of the cytochrome c oxidase large subunit genes showed that D.200.bin4.133belonged to type A cytochrome c oxidase (aa3), and that genes in the other anammox bacteria were type C cytochrome c oxidase (cbb3) (Figure S8).In addition, complete gene sets of the glycolysis pathway were detected in the three anammox MAGs from the MT deep sediments, including those encoding enzymes that oxidize multiple carbohydrates such as glucose, lactose, starch/glycogen, and oligosaccharides (Figure 4; Table S6).

N 2 Fixers in the MT Seawater
The key gene nifH, which encodes nitrogenase, was detected exclusively in the MT seawater, suggesting the existence of microorganisms that convert inert N 2 to bioavailable ammonia.Furthermore, the metagenomic binning of the three seawater samples recovered a nifH-containing MAG W.bin6.184.The MAG was assigned to the genus Ketobacter of the family Alcanivoracaceae, and its average relative abundance was 4.0% in the MT seawater (Figures 2c and S9; Table S4).Meanwhile, all of the protein-coding genes in the nifH-containing contig of W.bin6.184 were assigned to Ketobacter (Table S7).To assess the metabolic features of W.bin6.184, we performed a comparative genomic analysis of 29 Ketobacter genomes (>50% completeness and <5% contamination) retrieved from this study (W.bin6.184,W.bin6.104), the NCBI Assembly database (20 MAGs) [37], and the genomic catalog of Earth's microbiomes (7 MAGs) [38].Notably, a key gene alkane 1-monooxygenase (alkB), which is involved in the first step of medium-chain alkane oxidation, was detected in the two Ketobacter MAGs in this study (Figure 5).In addition, complete gene sets involved in β-oxidation, the tricarboxylic acid cycle, and cytochrome c oxidases were present in the MAGs of W.bin6.184 (completeness 98.89%, contamination 2.39%) and W.bin6.104 (completeness 80.90%, contamination 0.65%) (Figure 5; Table S8).

Distinct microbial communities exist the different MT environments
Due to the funneling effect of the hadal trench, the abundant organic matter accumulates in the MT trench, which harbors abundant microorganisms [6].
Proteobacteria (represented by Gammaproteobacteria and Alphaproteobacteria) and Thaumarchaeota (represented by Nitrososphaeria) were the most abundant bacterial and archaeal groups in the MT seawater and surface sediments, respectively (Figure 1) [6,25,26].Dehalococcoidia and JS1 (Atribacterota) showed the highest relative abundances in the deep MT sediments compared to those in seawater and surface sediments (Figure 1; Mann-Whitney U test, P<0.05).These results were consistent with the deep sediments of the eastern equatorial Pacific and the North Pacific Gyre, indicating that these bacterial groups might degrade halogenated organic compounds and aromatics present in the deep MT sediments [13,61,62].

Five new NOB lineages fix CO 2 in the MT sediment
Compared with sediment from the adjacent trench bottom and abyssal plain, the sediment layer of the MT slope is generally thinner and has a lower organic carbon content [4,6].Such an environment might favor the growth of chemolithoautotrophic microorganisms, such as AOA and NOB.In general, AOA is a dominant group of archaea in many marine sedimentary environments [63].However, NOB lineages are often overlooked because of the low NO 2 -concentration and their rapid turnover rate in the sediments [61,64,65].Previous studies reported that NOBs have a very high affinity for NO 2 -and that they are the predominant fixers of dissolved inorganic carbon in the dark ocean [64,66].In the sediment of the MT slope, the average gene abundance of nxrA was comparable to that of norBZ (Figure 2), suggesting that nitrite oxidation might be an important process that has similar activity to NO reduction in the dominant denitrification pathway.Nitrogen retention via nitrite oxidation ) reduces nitrogen loss through denitrification (NO 2 -→NO) [67].
However, the source of NO 2 -in the MT sediments is unknown because the NO 2 concentration was below the detection limit (<0.1 μM) [10].A recent study showed that the microbial nitrate reduction process was stimulated in the MT sediments [68], suggesting that NO 2 -is supplied to the sediment through the microbial nitrate reduction process or from the environment [58,69].
Seven NOB genomes were recovered from the metagenomes of the MT deep sediments (Figures 3 and S3).These MAGs were identified as new NOB lineages that are not classified into the common NOB taxa Nitrospinota, Nitrospirota, Chloroflexota, or Proteobacteria.Notably, each of the seven new NOB was able to fix inorganic carbon via the rTCA or CBB cycle (Figure 3) [70].However, the O 2 content in the MT sediment would be much lower than that in the surrounding seawater, indicating that these hadal NOB had a microaerophilic lifestyle and entered the hadal sediment through physical conditions or biological processes, similar to the NOB from Nitrospira [58,71].As a result, the identification of these new NOBs not only expands their phylogenetic diversity, but also reveals that nitrite oxidation might be an important but overlooked microbial carbon fixation process in hadal sediment [71].

Anammox bacteria perform aerobic respiration in the MT sediment
Identification of the hzs gene and recovery of three anammox MAGs in the MT sediment confirm previous results that anammox is an important nitrogen removal process in hadal sediment environments [6,24,61].Notably, the anammox bacterium (D.200.bin4.133)was able to perform aerobic respiration because it contained a gene encoding type A cytochrome c oxidase (Figure S7).There are three types of cytochrome c oxidases (types A, B, and C), of which type A cytochrome c oxidase mainly functions in high oxygen concentration environments compared to the other two types [72].Type A cytochrome c oxidase is also the terminal oxidase with the highest efficiency in energy generation [73].In some bacterial strains containing type A and type C cytochrome c oxidases (e.g.Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1), type A is highly expressed, while type C remains unchanged under nutrient-limiting conditions [74,75].This implies that type A Furthermore, reconstruction of the metabolic pathway of the anammox bacterium (D.200.bin4.133)showed that this strain can obtain energy through the utilization of several carbohydrates (glucose, lactose, starch/glycogen, or oligosaccharides) (Figure 4), which would provide more ATPs than anaerobic metabolism when O 2 is the terminal electron acceptor [76,77].Anammox bacteria have demonstrated their ability to live in oxygenated environments.For example, "Candidatus Brocadia caroliniensis" and "Candidatus Kuenenia stuttgartiensis" were observed to tolerate oxygen concentrations of up to 120 μM and 200 μM, respectively [78].The defense mechanisms against oxygen in these anammox bacteria may be attributed to the expression of enzymes such as bilirubin oxidase, cytochrome c oxidase and bifunctional catalase-peroxidase [79].Similarly, the gene encoding type A cytochrome c oxidase in the genome of D.200.bin4.133might act as an oxygen scavenger to maintain the anammox process in an anaerobic environment, which confers a selective advantage to D.200.bin4.133that faces O 2 concentration fluctuations in MT [4,80,81].The O 2 content in the MT seawater below 6,000 mbsl was constantly within the range between 156 μM and 188 μM [10].The O 2 source in the MT sediment may be derived from the overlying seawater intruding through the seawater-sediment interface [6,82].Moreover, the abundant AOA species in the MT sediment might also produce a small amount of O 2 , as recently reported [83,84], and the O 2 could be rapidly utilized by other aerobic microorganisms such as anammox bacterium (D.200.bin4.133).However, further evidence is needed to test the aerobic respiration activity of MT anammox bacteria, for example through metatranscriptomics, metaproteomics and rate measurements.

Alkane-degrading
Ketobacter fix N 2 in the MT seawater N 2 production through microbial denitrification and anammox processes forms the largest nitrogen sink in the ocean [24,85].Recent studies revealed that most of N 2 is produced via the anammox process in the hadal sediments of the Atacama Trench (~67%) and the Kermadec Trench (>90%) [6,24].In the MT seawater, however, the denitrification process is responsible for the majority of N 2 production, as nitrate (~36 μM) and denitrifying microorganisms are abundant (Figure 2) [10,25].The accumulated N 2 is then used by an abundant nitrogenase-containing Ketobacter strain W.bin6.184 (Figure 5).However, N 2 fixation is an extremely high energy-consuming process (16 ATPs per mole of N 2 fixed) [86,87].Various organic matters could serve as energy sources for nitrogen fixation, such as cellulose, chitin, glucan, pectin, polyphenols, starch, and alkane [88,89].To meet this large energy requirement, Ketobacter (W.bin6.184)might have the potential to perform aerobic degradation of medium-chain alkanes to produce acetyl-CoA (>100 ATPs per mole of oxidized medium-chain alkane) [90] due to the presence of complete gene sets for alkane oxidation (alk and β-oxidation) (Figure 5).This is consistent with the features of most genera of Alcanivoracaceae, a well-known aerobic hydrocarbon-degrading bacterial family [91,92].Of note, the alkB gene was detected in most of the Ketobacter genomes (Table S8), and the utilization of n-alkane by a pure Ketobacter strain was demonstrated by incubation experiments [91], suggesting that Ketobacter W.bin6.184 could obtain energy from alkane degradation.The concentration of n-alkanes was 23.5 μg/gdw in the MT seawater as previously measured [26,93], and the alkane-degrading bacteria including the members of Alcanivoracaceae were abundant in the MT seawater (Figure 1).Possible sources of the alkanes in the MT might include a mixture of biological processes (through rotting organisms) and geological processes (through water-rock reactions) [94,95].The slow degradation of alkanes may spread over long distances due to the effects of hadal seawater currents [94].

Conclusion
This study provides new insights into the unique features of microbial nitrogen cycling processes in the deepest part of the ocean.The distinct dominant microbial taxa were observed in the different MT habitats.The identification of five new NOB lineages in the MT sediment has uncovered an overlooked process of inorganic carbon fixation.Meanwhile, anammox bacteria might perform aerobic respiration in response to nutrient limitations or O 2 fluctuations in the sediment.In the MT seawater, an abundant Ketobacter strain might obtain energy during alkane degradation, and then fix N 2 released by sedimentary denitrifiers and anammox bacteria (Figure 6).
Integrating a multi-omics strategy that combines metagenomics with metatranscriptomics, metaproteomics or metabolomics will provide more comprehensive understanding of hadal microbial communities in future studies.
Meanwhile, laboratory incubation experiments and in situ activity tests are also needed to verify the contribution of hadal microbial communities to global biogeochemical nitrogen cycles.S2.S3.S5.S6.S8.S4.
Huang, a,b Xinxu Zhang, a,b Yu Xin, c Jiwei Tian, d Meng Li a,b,# a Archaeal Biology Center, Shenzhen Key Laboratory of Marine Microbiome Engineering, Institute for Advanced Study, Shenzhen University, Shenzhen 518060, PR China b Synthetic Biology Research Center, Institute for Advanced Study, Shenzhen University, Shenzhen 518060, PR China c Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Institute for Advanced Ocean Study, Ocean University of China, Qingdao, Shandong, China d MOE Key Laboratory of Physical Oceanography, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Ocean University of China, Qingdao, China Running Head: Microbial nitrogen cycling in the Mariana Trench #Address correspondence to Meng Li, limeng848@szu.edu.cn(Meng Li) Yuhan Huang and Xinxu Zhang contributed equally to this work.Author order was determined by drawing straws.Abstract The Mariana Trench (MT) is the deepest part of the ocean on Earth.Previous studies have described the microbial community structures and functional potential in the seawater and surface sediment of MT, but the metabolic features and adaptation strategies of the microorganisms involved in nitrogen cycling processes are poorly understood.In this study, comparative metagenomic approaches were used to study microbial nitrogen cycling in three MT habitats, including hadal seawater (9,600-10,500 m below sea level (mbsl)), surface sediments (0-46 cm below seafloor (cmbsf) at a water depth between 7,143 and 8,638 mbsl), and deep sediments (200-306 cmbsf at a water depth of 8,300 mbsl).We identified five new lineages of nitrite-oxidizing bacteria (NOB) that had adapted to the oligotrophic MT slope sediment, via their ability to fix CO 2 through the reductive tricarboxylic acid (rTCA) or Calvin-Benson-Bassham (CBB) cycle; An anammox bacterium could perform aerobic respiration and utilize sedimentary carbohydrates for energy generation because it contains genes encoding type A cytochrome c oxidase and the full glycolysis pathway.In seawater, abundant alkane-oxidizing Ketobacter species can fix inert N 2 released by other denitrifying and/or anammox bacteria.This study expands our understanding of microbial life in the largely unexplored deepest part of the ocean.Importance The metabolic features and adaptation strategies of nitrogen cycling microorganisms in the deepest part of the ocean are largely unknown.This study revealed that anammox bacteria might perform aerobic respiration in response to nutrient deficiencies or O 2 fluctuations in the Mariana Trench sediments.Meanwhile, an abundant alkane-oxidizing Ketobacter species could fix N 2 in hadal seawater.This study provides new insights into the role of hadal microorganisms in global biogeochemical nitrogen cycles and significantly expands our understanding of microbial life in the largely unexplored deepest part of the ocean.
cytochrome c oxidase is better adapted to oligotrophic environments such as the MT deep sediment compared to type C cytochrome c oxidase.In addition, a phylogenetic tree containing a collection of 229 type A cytochrome c oxidase reference genes revealed that D.200.bin4.133might have acquired the type A cytochrome c oxidase gene by horizontal gene transfer from Desulfobacterota (Figure S10).

Figure 1 .
Figure 1.Relative abundances of dominant microbial groups based on 16S rRNA gene sequences retrieved from the clean reads of each metagenome.A relative abundance less than 2% was grouped into others.Detailed values are provided in TableS2.

Figure 3 .
Figure 3. Reconstructed metabolic pathways of seven NOB MAGs in the MT deep sediment.Each filled or hollow circle indicates that the gene is present or absent in the MAG, respectively.A full list of genes labeled with different letters is provided in TableS5.

Figure 4 .
Figure 4. Reconstructed metabolic pathways of three anammox bacteria MAGs in the MT deep sediment.Each filled or hollow circle indicates that the gene is present or absent in the MAG, respectively.A full list of genes labeled with different numbers is provided in TableS6.

Figure 5 .
Figure 5. Reconstructed metabolic pathways of two Ketobacter MAGs in the MT

Figure 6 .
Figure 6.Schematic representation of the microbial nitrogen cycling processes in the MT sediment and seawater.OC, organic carbon.Detailed information on the MAGs belonging to AOA, NOB, anammox bacteria, N 2 fixers, and denitrifying microbes is provided in TableS4.