High hydrostatic pressure stimulates microbial nitrate reduction in hadal trench sediments under oxic conditions

Domain: earth_science
Published: 2024-03-19
Editorial outcome: minor_revision

Abstract
Hadal trenches are extreme environments situated over 6000 m below sea surface, where enormous hydrostatic pressure affects the biochemical cycling of elements. Recent studies have indicated that hadal trenches may represent a previously overlooked source of fixed nitrogen loss; however, the mechanisms and role of hydrostatic pressure in this process are still being debated. To this end, we investigate the effects of hydrostatic pressure (0.1 to 115 MPa) on the chemical profile, microbial community structure and functions of surface sediments from the Mariana Trench using a Deep Ocean Experimental Simulator supplied with nitrate and oxygen. We observe enhanced denitrification activity at high hydrostatic pressure under oxic conditions, while the anaerobic ammonium oxidation–a previously recognized dominant nitrogen loss pathway–is not detected. Additionally, we further confirm the simultaneous occurrence of nitrate reduction and aerobic respiration using a metatranscriptomic dataset from in situ RNA-fixed sediments in the Mariana Trench. Taken together, our findings demonstrate that hydrostatic pressure can influence microbial contributions to nitrogen cycling and that the hadal trenches are a potential nitrogen loss hotspot. Knowledge of the influence of hydrostatic pressure on anaerobic processes in oxygenated surface sediments can greatly broaden our understanding of element cycling in hadal trenches.

Introduction
The hadal trenches are deep-sea ecosystems (depth range of 6000–11,000 m) featured by high hydrostatic pressure (≥60 MPa)1,2. Despitetheirextremeconditions,hadaltrenchesarehotspotsofele-mentalcyclingwithhighorganicmatterdepositionrateandmicrobialactivities3–6. Recent investigations, grounded in both experimental evidenceandmodelingresults,haverevealedthatbioavailablenitrogenspecies(suchasNH4+,NO2−,andNO3−)areconvertedintobiologicallyinertN2inhadalsediments7–10.Thiscontinuouslossofnitrogenleads to an elevated carbon-to-nitrogen ratio and even nitrogen-limitation to benthic microorganisms11,12, which subsequently alters the biogeochemical processes in the deep ocean floor. Thus, a comprehensiveunderstanding ofthe mechanismofnitrogenloss isvitalindecipheringthebiogeochemistryofthisuniqueenvironment. Denitrification and anaerobicammonium oxidation (anammox) are the major known microbial processes that produce N2. These processesaregenerallyconsideredanaerobic13,14,andgenesinvolvedintheseprocesseshavebeenidentifiedinvarioustrenchsediments,irrespectiveofoxygenavailability7,15,16.Currently,anammoxisrecognizedasthedominantprocessresponsiblefornitrogenlossinanoxicsediments,contributing significantly more than denitrification. For instance, Thamdrup et al. studied the bottom-axis sediments from Atacama Trench and Kermadec Trench, demonstrating the prominenceofanammox,whiledenitrificationgenerallyoccurredatlowerratesandwaslimitedtothesurfacelayer8. Zhou et al. reported the heterogenous distribution of denitrification and anammox across the Mariana Trench, with the anammox being more intensive in bottom-axissedimentswithstrongeroxygendepletioncomparedtotheslope sites9. However, the contribution of denitrification on nitrogen loss in oxygenated trench surface sediments is yet to be investigated.

Microbialphysiology,activities,andmetabolisminhadaltrenchesareaffectedbyhydrostaticpressure1,17.Underhighhydrostaticpressures, microorganisms prefer anaerobic metabolisms over aerobic respiration. This is explained by the “common adaptation strategy”, whichspeculatesthatanaerobicmetabolismscauselessintracellularoxidative stress than the latter17–19. Furthermore, high hydrostatic pressuresshifttheredoxladder,leadingtoagreaterreactionofGibbs free energy being generated from denitrification compared to that underambientpressure20. Therefore, we propose a hypothesis that in hadaltrenchsedimentswherethehydrostaticpressureisextremelyhigh,denitrificationisanenergeticallyfavorablepathwaycontributingtothenitrogenlossintheoxygenatedupperlayersediment. In this study, we test this hypothesis using a specially-designed Deep Ocean Experimental Simulator to ensure a continuous supply of oxygen, usingtheMarianaTrenchsedimentsastheinoculum (Fig. 1). We then apply microbial activity analysis, metagenomic and metatranscriptomicanalysestodecipherthemechanismsbehindit.

Results
High hydrostatic pressures modified microbial community trajectory
To investigate the influence of hydrostatic pressure on the microbial community and functions, the sediment sample, collected at a water depth of 6002 m in the Mariana Trench, was sequentially incubated at 0.1, 40, 70, 90, and 115 MPa for 15 days each with a continuous supply of nitrate and dissolved oxygen. With the elevated hydrostatic pressures, the cell numbers of both bacteria and archaea declined (except for a slight increase for bacteria at 70 MPa). Nevertheless, the bacterial population consistently outnumbered the archaeal population by two orders of magnitude (Fig. 2A, B and Supplementary Data 1).

The hydrostatic pressure had distinctive effects on bacterial and archaeal community compositions. For the bacterial community, its taxonomic composition dramatically shifted with increasing hydrostatic pressures: under 0.1 MPa and 40 MPa, Halomonadaceae (31.8% and 23.6%, respectively) and Bacillaceae (44.0% and 43.0%, respectively) were the dominant groups; Under hydrostatic pressures of 70, 90 and 115 MPa, Halomonadaceae remained dominant (32.5–37.4%); with the increased relative abundance of Thalassospiraceae (11.9%, 23.1% and 19.3%), Enterobacteriaceae (4.5, 6.6 and 8.2%), and Shewanellaceae (5.4, 9.4 and 9.2%) (Fig. 2C; Supplementary Data 2). In contrast, the archaeal taxonomic composition remained stable under all hydrostatic pressures with the dominance of Nitrosopumilaceae (89.5–96.3%) (Fig. 2D and Supplementary Data 2).

High hydrostatic pressures promote anaerobic nitrate consumption under oxic conditions
We monitored the fluxes of dissolved nitrogen species (NO3−, NO2−, and NH4+) and gaseous nitrogen species (N2O) across the entire incubation. A relatively stable nitrate consumption (38.4–49.5 μmol/day) was observed under a continuous supply of dissolved oxygen (Fig. 3A and Supplementary Data 3). Moreover, because the cell number decreased under elevated hydrostatic pressures, the nitrate consumption rate per cell actually increased (Figs. 2A and 3A). Net nitrite production was also observed, with the rate being significantly higher at 70 MPa (3.00 μmol/d) than those at 0.1 and 40 MPa (one-way ANOVA with Tukey’s multiple comparisons test, all p < 0.05) (Fig. 3B and Supplementary Data 3).

The mechanism of the enhanced nitrate consumption was investigated using metagenomic and metatranscriptomic analyses. We estimated the gene transcription levels based on the percentage of gene transcripts (relative to all nitrogen transformation-related genes) associated with each pathway of nitrogen cycling (Fig. 4A and Supplementary Data 4). The napAB and narGHI genes involved in the first step of dissimilatory nitrate reduction were transcribed more actively at higher hydrostatic pressures. Specifically, the transcriptional activity of nitrate reduction to nitrite pathway accounted for 5.2% of all nitrogen transformation-related genes at 0.1 MPa, then increased to 9.6% at 40 MPa, 21.7% at 70 MPa, 12.8% at 90 MPa, and 13.0% at 115 MPa. In additionally, genes involved in the terminal oxygen reduction were abundant and actively transcribed over the entire incubation period (Supplementary Fig. 1).

High hydrostatic pressure stimulates denitrification but suppresses the DNRA pathway
The NO2− produced from NO3− reduction has two alternative transformation pathways. It can either be further reduced to NO, N2O, and finally to N2 through denitrification, or to NH4+ through the dissimilatory nitrate reduction to ammonium (DNRA). The transcription level of all genes involved in denitrification (nirS, nirK, norBC, and nosZ) was up-regulated under high hydrostatic pressures (Fig. 4A and Supplementary Fig. 2 and Supplementary Data 4). Specifically, the percentage of nirS and nirK gene transcripts (NO2− → NO) increased from 2.3% at 0.1 MPa to 15.3% and 12.9% at 90 MPa and 115 MPa, respectively (Fig. 4A). For norBC genes (NO → N2O), the transcripts increased from 4.0% at 0.1 MPa to 9.9% at 40 MPa, 20.2% at 70 MPa, 28.6% at 90 MPa, but then dropped to 8.7% at 115 MPa. For the nosZ gene (N2O → N2), the transcripts increased from 3.1% at 0.1 MPa to 19.3% at 90 MPa and 36.8% at 115 MPa, respectively (Fig. 4A). The enhanced denitrification activity coincided with the strengthened net N2O production rates at high hydrostatic pressures. Specifically, the N2O production rate was significantly higher at 70 MPa (0.287 μmol/d), 90 MPa (0.313 μmol/d), and 115 MPa (0.284 μmol/d) than that at 0.1 MPa (0.189 μmol/d) (one-way ANOVA with Tukey’s multiple comparisons test, P < 0.05) (Fig. 3C and Supplementary Data 3).

As for the DNRA pathway, the chemical profile analysis showed that the average net ammonium production rate at 0.1 MPa was higher than those under high hydrostatic pressures (such as 90 MPa and 115 MPa), although the difference was not significant (one-way ANOVA with Tukey’s multiple comparisons test, p = 0.2629 (Fig. 3D). The metatranscriptomic analysis showed that the transcription level for the key genes (especially nirBD) in DNRA pathway was the highest (80.2%) under 0.1 MPa, which was then halved to 40.9% at 40 MPa and 37.1% at 70 MPa, and then further decreased to 20.7% and 24.5% when the hydrostatic pressure rose to 90 and 115 MPa, respectively (Fig. 4A). Hence, this implies that the activity of DNRA pathway declined with the elevated hydrostatic pressures when the other environmental parameters were kept unchanged.

Sec-dependent N2O reductase is selected over Tat-dependent N2O reductase under high hydrostatic pressures
The transcriptional activity of the nosZ gene, which catalyzes the last step of denitrification leading to nitrogen loss through N2, was elevated under 115 MPa compared with any other hydrostatic pressure used in this study (Supplementary Data 4; Supplementary Fig. 2). Based on the phylogenetic analysis, we identified two types of nosZ gene (i.e., Sec-dependent and Tat-dependent, Fig. 5A) in this flowing incubation system. Most Sec-dependent nosZ gene sequences were assigned to Flavobacteriale, whereas the Tat-dependent nosZ genes mainly belonged to the Alphaproteobacteria and Gammaproteobacteria (Supplementary Data 5). The metatranscriptomic analysis found a higher fold change with elevated hydrostatic pressures in the transcripts of the Sec-dependent nosZ than those of the Tat-dependent nosZ (especially at 115 MPa, Sec vs. Tat = 245 vs. 45, Supplementary Fig. 3).

To confirm the preference of Sec-dependent over Tat-dependent nosZ genes under high hydrostatic pressures, both genes were synthesized and cloned into the piezotolerant Shewanella piezotolerans WP3NR (Fig. 1D). We then performed heterologous gene expression and activity assays of nitrous oxide reductase by quantifying the consumption of the supplied N2O under various hydrostatic pressures. Our results showed that S. piezotolerans WP3NR with the Sec-dependent nosZ gene exhibited a significantly higher N2O consumption rate than that with the Tat-dependent nosZ gene (multiple t-tests, p < 0.0001) (Fig. 5B; Supplementary Data 6a). Additionally, after incubating at 0.1, 20, and 40 MPa for 24 h, the amount of N2O consumed by the S. piezotolerans WP3NR with Sec-dependent nosZ gene gradually increased with the elevated hydrostatic pressures, which was the highest at 40 MPa (56.08 μmol). Transcriptomic analysis consistently showed that the Sec-dependent nosZ gene was more actively transcribed with increasing hydrostatic pressure, being significantly higher than the Tat-dependent nosZ gene (multiple t-tests, p < 0.0001) (Fig. 5C and Supplementary Data 6b). These results confirm that microorganisms prefer to use Sec-dependent N2O reductase to reduce N2O and to produce N2 under high hydrostatic pressures.

The active microbial groups involved in denitrification during incubation
A total of 63 metagenome-assembled-genomes (MAGs) with completeness >50% and contamination <10% were obtained from the six metagenomic datasets derived from the inoculum and flowing incubation sediment samples (Supplementary Data 7). These retrieved MAGs were taxonomically annotated with the GTDB-Tk tool. They were mainly affiliated with Proteobacteria (43 MAGs), Bacteroidota (9 MAGs), Actinobacteriota (4 MAGs), Firmicutes (2 MAGs) and Thermoproteota (2 MAGs) (Supplementary Data 7). The normalized abundance (RPKG) of each recovered MAG is shown in Supplementary Fig. 4 and Supplementary Data 7.

We then identified the microorganisms that can participate in the denitrification pathway at different hydrostatic pressures based on the presence of genes associated with denitrification. Among the 63 MAGs, 40 MAGs contain these genes (Supplementary Data 8). We further mapped the metatranscriptomic reads to these 40 MAGs, and found that 31 MAGs were actively involved in the denitrification process under at least one hydrostatic pressure condition (Supplementary Data 8). Furthermore, the results showed that different microbial groups were dominant drivers of denitrification under various hydrostatic pressures (Fig. 4B). Specifically, Halomonas titanicae was the most abundant denitrifier across the entire incubation period, and its denitrification-related genes (narGHI, norBC, and nosZ) were more actively transcribed under higher hydrostatic pressures, especially under 70, 90 and 115 MPa. Marinobacter hydrocarbonoclasticus was dominant at 40 MPa with its abundance decreased under increased pressures, but the transcriptional level of its denitrification-related genes (nirS, norBC, and nosZ) increased under the elevated pressures. The abundance of Thalassospira xiamenensis increased as pressures increased, but its genes associated with denitrification (napAB, nirS, norBC, and nosZ) were only active under 70, 90, and 115 MPa. Idiomarina loihiensis also participated in denitrification, in which the nirK and norB genes were actively transcribed during incubation, however, its abundance decreased with the elevated pressures. Lastly, the abundance of Aequorivita vladivostokensis remained similar during incubation, but its denitrification-related genes (nirK, norB, and nosZ) were transcribed only under 115 MPa (Supplementary Data 7, 8).

Other nitrogen transformation processes occurred in this flowing incubation system
The nifDHK genes involved in nitrogen fixation were detected in samples incubated at 40, 70, and 115 MPa, with the highest transcriptional activity being detected at 40 MPa (Fig. 4A and Supplementary Fig. 2). The nasAB and narB genes involved in assimilatory nitrate reduction to nitrite were also identified and were most actively transcribed at 70 MPa (Fig. 4A and Supplementary Fig. 2). The nirA gene involved in assimilatory nitrite reduction to ammonia was identified only in the metagenome of the inoculum sediment sample, but its RNA transcript was not identified during the incubation (Supplementary Data 4). Additionally, we did not identify any genes associated with anammox (i.e., hzs and hdh) during the incubation (Supplementary Data 4). Ammonia monooxygenase gene (amoABC) and hydroxylamine dehydrogenase gene (hao) involved in the nitrification pathway were not detected during the entire flowing incubation period (Supplementary Data 4). This seemed to contradict the dominance of ammonia-oxidizing archaea Nitrosopumilaceae in the archaeal community obtained by 16S rRNA gene sequencing (Fig. 2B). However, considering that the cell number of archaea, which consistently decreased with the elevated hydrostatic pressures, was two orders of magnitude lower than that of bacteria, the absence of ammonia monooxygenase gene in subsequent metagenome and metatranscriptome may be explained by their low abundance and insufficient sequencing depth.

The simultaneous denitrification and aerobic respiration under high hydrostatic pressure
The simultaneous denitrification and aerobic respiration in trench microorganisms under high hydrostatic pressure was verified in both in vitro and in situ surface sediment samples. In the flowing incubation experiments, the key genes (e.g., Cyo, Cyd, Cco, and Cox) involved in terminal oxygen reduction were abundant and actively transcribed under all incubation pressures (Supplementary Fig. 1), with the denitrification activity being enhanced with increased pressure (Fig. 4A). In addition, we further analyzed the transcription of genes associated with these pathways at the MAGs level. Our results showed that aerobic respiration-related genes encoding terminal oxidases and denitrification-related genes were actively transcribed concurrently in many taxonomic groups, such as Halomonas titanicae, Marinobacter hydrocarbonoclasticus, and Thalassospira xiamenensis (Supplementary Data 9).

Furthermore, to confirm this occurrence is happening in natural trench environments, we analyzed the metatranscriptomic dataset of in situ fixed surface sediments (0–10 cm) from the Mariana Trench. These sediment samples were immediately fixed with RNALater after being collected at the trench bottom (see “Methods”), therefore the metatranscriptomic analysis on these samples could reveal the in situ microbial activities. Our results showed that genes related to aerobic and anaerobic energy metabolic pathways, such as respiratory electron transport chains, denitrification, and TCA cycle were actively transcribed simultaneously (Fig. 6A and Supplementary Data 10). Additionally, these genes were mainly assigned to Proteobacteria, Actinobacteriota, Chloroflexota, Gemmatimonadota, Planctomycetota, and Thermoproteota (Fig. 6A). Thus, Proteobacteria and Actinobacteriota are concurrently capable of aerobic oxidation of organic matter and denitrification pathway, which is consistent with the flowing incubation experiments (Supplementary Data 9).

Methods
Samples collection and continuous flowing incubation at different hydrostatic pressures under oxic conditions
The sediment sample for high-pressure incubation was obtained by the JiaoLong Human Occupied Vehicle (HOV) from the northern slope of the Mariana Trench (142.2516°E, 11.6639°N) at Dive 119 station with a water depth of 6002 m during the cruise on R/V Xiangyanghong 09 Cruise DY37-II in June 2016. The collected sediments were sliced into 4 cm interval layers and were immediately preserved in multiple 70 MPa (approximately the pressure at the sampling site) pressure-retaining vessels on shipboard and stored at 4°C until used for subsequent experiments. In addition, we collected surface sediment at the Mariana Trench at water depths of 8207–10,898 m during the R/V Tansuoyihao Cruise TS-21 from August to December 2021 (142.5947°E, 11.3649°N; 142.5926°E, 11.3867 N; 142.5869°E, 11.3740°N; 142.5602°E, 11.3619°N; 142.5602°E, 11.3619°N; 142.1562°E, 11.1615°N; 142.1572°E, 11.1590°N; 142.3429°E, 11.1970°N; 142.3429°E, 11.1970°N; 142.2038°E, 11.3393°N; 142.2166°E, 11.3350°N). These sediments were collected and immediately fixed with RNALater at in situ condition via a specially designed in situ fixation sampler, and used for metatranscriptomic analysis. Sample collection and transportation have been permitted by the Federated States of Micronesia, with the permit number FM-XXRS-23522. The research complies with all relevant ethical regulations.

The above 20 cm layers of sediments were used as the inoculum for the incubation (hereafter referred to as inoculum sediments) (Fig. 1A). The concentration of NO3− (NO3− and NO2−) in the overlying water sample was 213.7 μg/L (1.98 μmol/L) as shown in the previous study39. During incubation, the inoculum sediment was suspended in the chemically defined marine medium which was modified from Widdel and Bak40 and contained (/L): NaCl (Cat. No. 10019318, Sinopharm Chemical Reagent, China) 26.0 g, MgCl2·6H2O (Cat. No. 10012818, Sinopharm Chemical Reagent, China) 5.0 g, CaCl2·2H2O (Cat. No. 20011160, Sinopharm Chemical Reagent, China) 1.4 g, Na2SO4 (Cat. No. 10020518, Sinopharm Chemical Reagent, China) 4.0 g, KH2PO4 (Cat. No. 10017618, Sinopharm Chemical Reagent, China) 0.1 g, KCl (Cat. No. 10016318, Sinopharm Chemical Reagent, China) 0.5 g, D-(+)-glucose (Cat. No. DX0145, Sigma-Aldrich, Germany) 5.4 g, NaNO3 (Cat. No. 10019918, Sinopharm Chemical Reagent, China) 100 μmol (as sole nitrogen source), bicarbonate solution 30 mL, trace element mixtures 1 mL, vitamin mixture 1 mL, thiamine solution 1 mL, and vitamin B12 solution 1 mL. Glucose was provided as the organic carbon source and electron acceptor to investigate the microbial nitrate reduction process, according to previous studies41,42. The homogenized slurry was purged with continuous argon flushing to achieve a dissolved oxygen concentration of 100 μmol/L (this value is based on the oxygen concentration of ~100 μmol/L in the 0–20 cm sediment layers at ~6000 m deep site3). Then, it was transferred into the Deep Ocean Experimental Simulator (DOES, which is specially designed to construct an environment in the laboratory that is similar to the deep biosphere, in terms of temperature, hydrostatic pressure, flow rate, pH, nutrient availability, etc.19) and incubated in a flow-through mode at a flow rate of 0.3 mL/min (Fig. 1B). The incubation was performed at 4°C, and the incubation hydrostatic pressures (0.1 MPa, 40 MPa, 70 MPa, 90 MPa and 115 MPa) were changed every 15 days.

Biochemical analysis
To monitor the metabolic activities, the consumption of nitrate, as well as the production of ammonium, nitrite, and nitrous oxide were analyzed. Sampling of the above chemical parameters was conducted every 3 days without depressurization throughout the incubation experiments. The concentrations of dissolved inorganic nitrogen species (NO3−, NO2−, and NH4+) were quantified using an AA3 Auto-Analyzer system (Seal Analytical, UK). The concentration of gas N2O was measured by an Agilent 6890N Gas Chromatograph (Agilent Technologies, USA). The initial concentration of dissolved oxygen was measured using the Microx 4 oxygen meter (PreSens, Germany). The concentration difference between the inlet and outlet of this DOES system was multiplied by the flow rate (0.3 mL/min) to calculate the corresponding consumption and production rates of these nitrogenous compounds (nitrate and nitrite, ammonium, nitrous oxide). We further estimated the nitrogen loss rate (N–N2O, nmol N/day/mL) due to denitrification-derived N2O alone under 70 MPa using 125 mL of Mariana Trench sediment as the inoculum. The corresponding calculation formula is as follows: net nitrous oxide production rate*2*1000/125 mL.

Nucleic acid extraction and metagenomic/metatranscriptomic sequencing projects
Biomass sampling was performed when switching incubation pressure (Fig. 1C). Total microbial genomic DNA was extracted and purified using the modified SDS-based method described by Natarajan et al.43, and stored at −20°C before further assessment. The quantity and quality of extracted DNA were measured using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and agarose gel electrophoresis, respectively. The extracted microbial DNA was processed to construct metagenome shotgun sequencing libraries with insert sizes of 350 bp following the standard Illumina TruSeq DNA Sample Preparation Guide. Each library was sequenced by Illumina NovaSeq 6000 platform (Illumina, USA) with PE150 strategy at Shanghai Personal Biotechnology (Shanghai, China). The extraction of RNA from sediment samples was carried out using the RNeasy® PowerSoil® Total RNA Kit (Cat. No. 12866-25, Qiagen, Germany) according to the manufacturer’s instructions, then quantified using a Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). To ensure DNA removal, the RNA extracts were treated with TURBO DNase (Cat. No. AM2238, Invitrogen, Waltham, MA, USA) as directed by the manufacturer. The purified RNA was converted to cDNA, then the metatranscriptomic library was constructed by using Illumina TruSeq Stranded mRNA LT Sample Prep Kit, and subsequent sequencing as described above.

16S rRNA gene sequencing and quantitative PCR (qPCR) analysis
The V4–V5 region and V4 region of archaeal and bacterial 16S rRNA genes were amplified by polymerase chain reaction (PCR) with the primer pair Arch516F (5’-TGYCAGCCGCCGCGGTAAHACCVGC-3’)/Arch855R (5’-TCCCCCGCCAATTCCTTTAA-3’), and the primer pair Bac533F (5’-TGCCAGCAGCCGCGGTAA-3’)/Bac806R (5’-GGACTACCAGGGTATCTAATCCTGTT-3’), respectively. The PCR amplification procedure was previously described44. Subsequently, the PCR products were purified using an EZNA Gel Extraction Kit (Cat. No. D2500-01, Omega Bio-Tek, Norcross, GA, USA). The purified DNA was sequenced on the Illumina NovaSeq platform by Shanghai Personal Biotechnology (Shanghai, China). Microbiome analysis was performed with QIIME2 (v.2020.8)45. The paired-end raw sequenced data containing the forward or reverse reads for each sample were demultiplexed and quality-filtered using the q2‐demux plugin. The paired-end demultiplexed sequences were denoised by DADA246 (via q2‐dada2). In DADA2, the sequence bases with quality score greater than 20 (i.e., <1% error rate) were kept based on the resulting interactive quality plot of the previous step, and these command parameters (i.e., --p-trunc-len-f, --p-trunc-len-r, --p-trim-left-f, --p-trim-left-r, --p-chimera-method) were applied to truncate and trim the read sequences and remove chimeras. The taxonomy classification and taxonomic analysis were assigned to the ASVs by comparing the query sequences to a reference SILVA 138 database using the q2-feature-classifier47 classify-sklearn Naive Bayes taxonomy classifier.

The quantification of archaeal and bacterial 16S rRNA genes was respectively conducted using the primer pair Bac341F (5’-CCTACGGGWGGCWGCA-3’)/Bac519R (5’-TTACCGCGGCKGCTG-3’), and the primer pair Uni519F (5’-GCMGCCGCGGTAA-3’)/Arch908R (5’-CCCGCCAATTCCTTTAAGTT-3’). The 7500 Real-Time PCR System and Power-UpTM SYBRTM Green Master Mix (2X) (Cat. No. A25741, Applied Biosystems, Foster City, CA, USA) were used for all qPCR analysis according to the manufacturer’s instructions. The archaeal and bacterial cell counts were calculated using archaeal and bacterial 16S rRNA gene copy numbers divided by 1.7 and 5.2 (mean 16S rRNA operon copy number)48, respectively.

Metagenomic and metatranscriptomic analysis
The omics analysis was performed on both gene-centric strategy and genome-centric strategy. Briefly, the 150 bp paired-end raw reads were first trimmed by BBDuk tool (v.38.96) (https://sourceforge.net/projects/bbmap/) with a sequence quality score of >20 and a final minimum length of >90 bp. Obtained clean reads were assembled by SPAdes (v.3.12.0)49 with “--meta --only-assembler -k 65, 75, 96, 115, 127”. The assembly was filtered for a minimum length of 500 bp using a custom Python script50. All metatranscriptomic reads were first filtered by BBDuk tool (v.38.96) and then aligned to a combined rRNA database from SILVA and Rfam51 using Bowtie2 (v.2.4.1)52. The unaligned mRNA reads were collected for quantification of gene expression.

For the assembled contigs, genes were predicted by Prodigal (v.2.6.3)53 for the filtered assembly and those with lengths smaller than 100 bp were discarded. The modified genes set was functionally annotated with an integrated result, with the following priorities: GhostKOALA (v.2.2)54 > emapper (v.2.0.1) against the EggNOG database (v.5.0)55,56 > KofamKOALA (v.1.0.3)57. Clean metagenomic and metatranscriptomic reads from each incubation pressure were mapped to the assembly by BBMap (v.38.24) with “k=13 minid=0.95 pairlen=350 rescccuedist=650”. The mapped file in SAM format was converted to BAM format and sorted by SAMtools (v.1.15.1)58. For metagenomic datasets, the depth of each scaffold in every incubation was determined by the script “jgi_summarize_bam_contig_depths” from MetaBAT2 (v.2.15)59 with the default parameters. FeatureCounts (v.1.5.3)60 was used to count the read number of each gene, and the transcripts per million (TPM) value was calculated with a custom Python script50.

Three binning software programs were used to obtain the primary MAGs, as described in the previous study61. For MetaBAT2 (v.2.15), different sensitivities (--maxP 60, 75, and 90) and specificities (--minS 60, 75, and 90) were combined. The two marker gene sets (40 and 107) were analyzed by MaxBin (v.2.2.6)62. CONCOCT (v.1.0.0) analysis63 was also carried out. Then DAS tools (v.1.2.2)64 was used to integrate the results to calculate an optimized, non-redundant set of MAGs for each incubation. Quality and taxonomy were determined by CheckM (v.1.1.5)65 and GTDB-Tk tools (v.2.3.2)66 with the GTDB r20267 respectively. MAGs with completeness >50% and contamination <10% were dereplicated by dRep (v.3.4.2)68 with 95% average nucleotide identity (ANI) to obtain a non-redundant MAG set for the whole incubations. Clean metagenomic and metatranscriptomic reads from each incubation pressure were mapped to the MAG set for gene quantification with the methods described above. The normalized abundance of recovered MAGs was evaluated by the RPKG (reads recruited per kilobase of genome per gigabase of metagenome) values for the comparison among different genomes and metagenomes, as described by Liu et al.69.

Heterologous expression and activity assay of N2O reductase genes
The phylogenetic tree of NosZ amino-acid sequences was constructed together with the reference sequences from a previous study70 by IQ-TREE tool (v.2.1.2)71 with LG+F+R9 model using 1000 ultrafast bootstrap replicates and 1000 bootstrap replicates for SH-aLRT. The nosZ gene clusters, including Tat-dependent (clade I) and Sec-dependent (clade II) clades belonging to Halomonadaceae and Flavobacteriaceae families respectively, were synthesized by Shanghai Saiheng Biotechnology Co., Ltd (Shanghai, China). The two gene clusters were subsequently cloned into the pSW2 expression vector between the sites BamHI and SalI72. These three plasmids (pSW2-Tat, pSW2-Sec and pSW2) were heterologously expressed in deep-sea model bacterium Shewanella piezotolerans WP3NR (a piezotolerant strain with the pressure range of 0.1–50 MPa)73 by conjugal transfer using E. coli WM3064 strain (Fig. 1D). Three transconjugants (WP3NR-pSW2-Tat, WP3NR-pSW2-Sec and WP3NR-pSW2) were selected by chloramphenicol resistance and were verified via colony PCR for subsequent experiments. The obtained strains were cultured in modified marine 2216E medium (5 g/L tryptone (Cat. No. LP0042, Thermo Scientific™ Oxoid™, USA), 1 g/L yeast extract (Cat. No. LP0021, Thermo Scientific™ Oxoid™, USA), 34 g/L NaCl (Cat. No. 10019318, Sinopharm Chemical Reagent, China)) to activate cells. At the early stationary phase (OD600 = 1), cells were washed with culture salt solutions to remove the residual organic matter and then transferred into the defined LMO-812 minimal medium74. The inoculum was then purged with argon gas to ensure anoxic conditions. To determine N2O reduction activity, 15 mL inoculum was amended with 500 μmol acetate and 2 mL (~83 μmol) of N2O gas (99.999%), then was incubated under 0.1, 20, and 40 MPa for 24 h at 15°C. The N2O concentrations were determined by an Aligent 6890N gas chromatograph (Agilent Technologies, USA). After the incubation, total RNA was extracted with the common TRIzol method27, and RNA samples in triplicate were subjected to quality control and sequencing on the Illumina platform at Shanghai Personal Biotechnology (Shanghai, China). The subsequent transcriptomic analysis was performed as described previously22.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Yang et. al collected sediment samples from hadal zone of Mariana Trench and incubated in a chemostat with increasing pressure condition to explore the denitrification process mediated by hadal microbial communities. This study presents one of the few datasets that have examined nitrogen cycles in deep trenches, and the combination of omics tools, biochemistry measurement and microbiology experiment provide new insights on how high hydrostatic pressure (HHP) will induce denitrification in the trench sediments. While the paper does report novel results, I found that the authors did not successfully convey the clarity of their results. The authors can add more details to this manuscript and fully explain their findings if the article type allows.\nSpecific comments:\nL69-75: the sediment sample were collected at 6000m but pre-incubated under 70Mpa before transferred to the chemostat, it is no longer ‘in-situ’ condition.\nL74-75: ‘better growth’ better than which condition? Or does here mean, in the chemostat the microbes grew better under 70MPa than the pre-incubation (which is also 70MPa)? This brings out another question, before transferring to the chemostat, there were de-pressurizing process and nutrient supplement, drastic changes were found in taxonomic compositions, cell abundances and N compounds consumption/production (Fig 2-3), how would this pre-processing affect the conclusion that sediment microbes grew ‘better’ under 70 MPa?\nL99-110, L112-134 here the omics data were used to support the idea that HHP drove the denitrification of sediment microbial community even under oxic conditions. The conclusion heavily relies on the transcriptis abundance of targeted genes (nar, nap, nir and nos) but the taxonomic affiliation of those gene/transcripts are missing. I can get some clue from Extended data fig 2 but the direct link is missing. For example, the gene abundance of nirB and nosZ are relatively stable in the cheomstats despite pressure changes, is it related to Halomonadaceae, which also didn’t change so much? This is important because the community shifted from pre-incubation to chemostat incubation, one may wonder how the total community(16S, metagenome) and active community (metatranscriptome) will change accordingly.\nAlso I can see ammonia oxidation in the pre-incubation sample, but the gene/transcripts were completely gone after transfer into chemostat(Fig 4, table S4), how do the authors explain this data with the dominance of Nitrosopumilaceae from all conditions? Does this mean the amoABC are from AOB not AOA? I would suggest the author carry out in-depth analysis with the omics dataset to pinpoint the key species responding to HHP induced denitrification and discuss the differences between pre-incubation and chemostat conditions. MAG analysis can provide species level resolution especially for the chemostat based experiments and both metagenome and metatranscriptome will help identify the physiologic responses to HHP.\nL137-158: The data here nicely showed HHP can induce the expression and functioning of nosZ. As far as I can see, the metageneome were assembled separately, with 59 TAT-nosZ and 27 Sec nosZ, what is the similarity for each group of nosZ, are they redundant (both occurrence and gene abundance) when the pressure changes? Nevertheless, this question is minor, the recombinant experiment provides direct evidence for the nosZ activity under HHP.Fig.5A is hard to read, I would suggest separate the transcripts abundance data from the tree.\nL173: O2 consumption data is missing. Or is this sentence related to Extended fig1?\nL180-181: this sentence is misleading. Does this mean the denitrifiers can also use O2 or denitirifier can live under oxic conditions while others are using O2?\nL222: 30.3 nmol N /day cm3. How is this value calculated? From table S3, the N2O production rate is 0.287 umol/day under 70MPa, 0.287*2*1000/30.3 =18cm3(ml), 18ml/0.3ml/min=60min (1H), I hope my calculation is wrong, otherwise the N2 production is equivalent to Atacama Trench.\nL 247-249: contextual data for sampling site is vague. What is the NO3 concentration of ambient water, how deep was the sediment sample collected, top 5cm?\nOverall I would suggest the authors focus more on the physiological response of denitrifiers to HHP rather than getting back to ‘in-situ’ biogeochemical mechanisms as the pre- and after- incubations drives too far away from real scenarios.", "reviewer_2": "The manuscript entitled “High hydrostatic pressure stimulates microbial nitrate reduction in hadal trench sediments under oxic condition” by Yang et al. investigated the microbiology and omics of a marine sediment sample collected at a depth of over 6,000 m. This work demonstrated that denitrification is a major metabolic process in the nitrogen cycle under aerobic conditions and high hydrostatic pressure. The results are interesting, well presented and documented. However, this work lacks control experiments (e.g. in the absence of oxygen) in my view, and the experimental design is inadequate, as samples pressurized and maintained at in situ hydrostatic pressure during collection and storage, were directly incubated first at atmospheric pressure and then successively at increasing pressures applied during incubation. The enrichment of microorganisms at atmospheric pressure undoubtedly led to growth and an increase in the ratio of non-piezophilic or piezotolerant microorganisms, which became dominant during incubation compared with microorganisms adapted to in situ pressure conditions. Metagenomic and metatranscriptomic analysis lacks analysis of gene diversity and binning in relation to the dominant taxonomic groups present. These results do not support the authors' conclusions regarding active and major metabolisms in situ.\nSpecific comments:\n- Lines 86-88: Under your experimental conditions, can you tell us what role Thaumarchaea play in the nitrogen cycle?\n- Lines 112-125: Could you indicate to which taxonomic group the genes described in this paragraph can be assigned?\n- Line 142: Similarly, to which taxonomic group does the selected nosZ gene refer?\n- Lines 180-181: this conclusion is not supported by the results obtained, i.e. what is the diversity of respiratory chains present and active, for example?\n- Lines 247—249: What are the physicochemical characteristics of the sediment sample used in this study?\n- Line 254: Why was glucose used as a source of carbon and energy?"}, "author_response": "Response to the reviewers’ comments:\nReviewer #1 (Remarks to the Author):\nYang et. al collected sediment samples from hadal zone of Mariana Trench and incubated in a chemostat with increasing pressure condition to explore the denitrification process mediated by hadal microbial communities. This study presents one of the few datasets that have examined nitrogen cycles in deep trenches, and the combination of omics tools, biochemistry measurement and microbiology experiment provide new insights on how high hydrostatic pressure (HHP) will induce denitrification in the trench sediments. While the paper does report novel results, I found that the authors did not successfully convey the clarity of their results. The authors can add more details to this manuscript and fully explain their findings if the article type allows.\nResponse: We are glad to see that the reviewer appreciates the novelty of our research. The manuscript has been modified according to the reviewers’ comments. Briefly, we have included a detailed description and discussion on the taxonomy of denitrifiers to clarify the key players. Moreover, we have added the metatranscriptomic data of the in situ fixed samples to demonstrate what we found in the lab also occurs in natural conditions.\nSpecific comments:\nL69-75: the sediment sample were collected at 6000m but pre-incubated under 70Mpa before transferred to the chemostat, it is no longer ‘in-situ’ condition.\nResponse: The sediments were collected at 6002 m in the Mariana Trench and immediately preserved at 70 MPa (approximately the pressure at the sampling site) using a pressure-retaining vessel. We agreed that it was no longer an ‘in-situ’ condition, as the microbial community would have changed during the storage. Thus, we deleted these inappropriate descriptions and the modified contents as follows:\nTo investigate the influence of hydrostatic pressure on the microbial community and functions, the sediment sample, collected at a water depth of 6002 m in the Mariana Trench, was sequentially incubated at 0.1 MPa, 40 MPa, 70 MPa, 90 MPa, and 115 MPa for 15 days each with a continuous supply of nitrate and dissolved oxygen. With the elevated hydrostatic pressures, the cell numbers of both bacteria and archaea declined (except for a slight increase for bacteria at 70 MPa). Nevertheless, the bacterial population consistently outnumbered the archaeal population by two orders of magnitude (Fig. 2A, B; Supplementary Table 1).\nThese modified contents were in the revised manuscript L68-75.\nL74-75: ‘better growth’ better than which condition? Or does here mean, in the chemostat the microbes grew better under 70MPa than the pre-incubation (which is also 70MPa)? This brings out another question, before transferring to the chemostat, there were de-pressurizing process and nutrient supplement, drastic changes were found in taxonomic compositions, cell abundances and N compounds consumption/production (Fig 2-3), how would this pre-processing affect the conclusion that sediment microbes grew ‘better’ under 70 MPa?\nResponse: What we meant was that the microbes exhibited better growth than they were at the lower hydrostatic pressures (i.e., 0.1 MPa, 40 MPa). However, as the difference was not significant, we agree that this statement is not accurate. We have rephrased the sentence as “With the elevated hydrostatic pressures, the cell numbers of both bacteria and archaea declined (except for a slight increase for bacteria at 70 MPa).” in L71-73. Regarding the reviewer’s second concern, we agree that the pre-processing would affect the microbes. Therefore, we have only focused on the effect of pressure on the inoculum after being transferred into the flowing incubation system in the revised manuscript.\nL99-110, L112-134 here the omics data were used to support the idea that HHP drove the denitrification of sediment microbial community even under oxic conditions. The conclusion heavily relies on the transcript’s abundance of targeted genes (nar, nap, nir and nos) but the taxonomic affiliation of those gene/transcripts are missing. I can get some clue from Extended data fig 2 but the direct link is missing. For example, the gene abundance of nirB and nosZ are relatively stable in the cheomstats despite pressure changes, is it related to Halomonadaceae, which also didn’t change so much? This is important because the community shifted from pre-incubation to chemostat incubation, one may wonder how the total community (16S, metagenome) and active community (metatranscriptome) will change accordingly.\nResponse: We have added the taxonomic affiliation of these actively transcribed genes at the MAGs level. In brief, we obtained 40 MAGs that contain nitrogen-cycling associated genes. Additionally, these nitrogen-cycling associated genes were actively transcribed in 31 MAGs. These 31 MAGs were mainly affiliated with Proteobacteria (28 MAGs), Bacteroidota (8 MAGs), Actinobacteriota (3 MAGs) and Thermoproteota (1 MAG). Regarding the nosZ and nirB genes, most of them were identified from Halomonas titanicae (Halomonadaceae), which remained dominant under every incubation pressure. We have added a new descriptive paragraph (L167-196), new figures (Fig. 4B and Extended Data Fig. 4), added Supplementary Tables 7 and 8 in the revised manuscript. These results are also discussed in the revised manuscript L251-255.\nAlso, I can see ammonia oxidation in the pre-incubation sample, but the gene/transcripts were completely gone after transfer into chemostat (Fig 4, table S4), how do the authors explain this data with the dominance of Nitrosopumilaceae from all conditions? Does this mean the amoABC are from AOB not AOA?\nResponse: The amoABC genes in the inoculum sediment were affiliated with both AOB (Nitrosomonadaceae) and AOA (Nitrosopumilaceae). The disappearance of the amoABC genes may be because the sequencing depth was not high enough to identify low-abundance genes. Our results showed that the relative abundance of AOB (Nitrosomonadaceae) was extremely low (~0.03%) based on the amplicon sequencing results; whereas the AOA (Nitrosopumilaceae) was two orders of magnitude lower than bacteria (Fig. 2B). Therefore, the ammonia oxidizers are present throughout the incubation but was not detected in the metagenome. More sensitive methods such as amplicon sequencing targeting the amoABC genes may be required for the detection. Nevertheless, as these ammonia oxidizers are consistently present, ammonia oxidation activity is expected to present, but may be at a very low level given the low abundance of AOA and AOB.\nThe corresponding content was modified in the revised manuscript L207-216.\nI would suggest the author carry out in-depth analysis with the omics dataset to pinpoint the key species responding to HHP induced denitrification and discuss the differences between pre-incubation and chemostat conditions. MAG analysis can provide species level resolution especially for the chemostat based experiments and both metagenome and metatranscriptome will help identify the physiologic responses to HHP.\nResponse: We carried out additional genome-centric approaches at the MAGs level. We found that the taxonomy of dominant microbial groups driving denitrification changed under various hydrostatic pressures (Fig. 4B). Specifically, Marinobacter hydrocarbonoclasticus was dominant at 40 MPa with its abundance decreased under increased pressures, but the transcriptional level of its denitrification-related genes (nirS, norBC, and nosZ) increased under the elevated pressures. The abundance of Thalassospira xiamenensis increased as pressures increased, but its genes associated with denitrification (napAB, nirS, norBC, and nosZ) were only detected as being active under 70, 90, and 115 MPa. Idiomarina loihiensis also participated in denitrification, in which the nirK and norB genes were actively transcribed during incubation, however, its abundance decreased with the elevated pressures. The abundance of Aequorivita vladivostokensis remained stable during incubation, but its denitrification-related genes (nirK, norB,and nosZ) were transcribed only under 115 MPa. Lastly, Halomonas titanicae was the most abundant denitrifier across the entire incubation period, and its denitrification-related genes (narGHI, norBC, and nosZ) were more actively transcribed under high hydrostatic pressures, especially under 70, 90 and 115 MPa.\nWe have added a new descriptive paragraph (L167-196), new figures (Fig. 4B and Extended Data Fig. 4), added Supplementary Tables 7 and 8 in the revised manuscript.\nL137-158: The data here nicely showed HHP can induce the expression and functioning of nosZ. As far as I can see, the metageneome were assembled separately, with 59 TAT-nosZ and 27 Sec nosZ, what is the similarity for each group of nosZ, are they redundant (both occurrence and gene abundance) when the pressure changes? Nevertheless, this question is minor, the recombinant experiment provides direct evidence for the nosZ activity under HHP. Fig.5A is hard to read, I would suggest separate the transcripts abundance data from the tree.\nResponse: We checked the presence of gene redundancy by dereplicating the sequences at 95% identity using the mmseqs2 tool. This ended with nine Sec-nosZ and 22 Tat-nosZ clusters, therefore confirming the presence of gene redundancy. The results showed that sequence redundancy is more prevalent in Tat-nosZ, with up to six different Tat-nosZ clusters being active at the same time under 90 MPa, more than any other pressures. These clusters also belong to different microbial groups. Additionally, gene redundancy in Sec-nosZ was also observed with three different clusters being active at the same time under 115 MPa.\nWe have modified Fig. 5A and separated the transcripts abundance data from the phylogenetic tree. The detailed results (TPM) are now present in the modified Supplementary Table 5.\nL173: O2 consumption data is missing. Or is this sentence related to Extended fig1?\nResponse: The oxygen was continuously supplied at the same concentration to the flowing incubation system (DOES), thus was not quantified. The conclusion was based on the expression of genes associated with the aerobic respiration chain but not the dissolved oxygen concentration rate. We have replaced “O consumption” with “aerobic respiration” to better describe our results in the revised manuscript L244.\nL180-181: this sentence is misleading. Does this mean the denitrifiers can also use O2 or denitirifier can live under oxic conditions while others are using O2?\nResponse: By genome-centric analysis, our results showed that aerobic respiration-related genes and denitrification-related genes within the same MAG were transcribed concurrently under higher pressures. Thus, we proposed that denitrifiers (such as Halomonas titanicae, Marinobacter hydrocarbonoclasticus and Thalassospira xiamenensis) could simultaneously use both oxygen and nitrate as electron acceptors. This is revised as in L251-255.\nL222: 30.3 nmol N /day cm3. How is this value calculated? From table S3, the N2O production rate is 0.287 umol/day under 70MPa, 0.287*2*1000/30.3 =18cm3(ml), 18ml/0.3ml/min=60min (1H), I hope my calculation is wrong, otherwise the N2 production is equivalent to Atacama Trench.\nResponse: It is difficult to estimate the nitrogen loss rate as the N production was not 2 quantified. Therefore, we have used the calculation suggested by the reviewer and used the gaseous N O production rate to estimate the nitrogen loss potentials of the sediment. 2 We rephrased the sentence as “Under laboratory conditions, we observed that the rate of nitrogen loss (N-N O) caused by denitrification-derived N O alone was approximately ~ 2 2 4.6 nmol N/day/mL under 70 MPa with 125 mL of Mariana Trench sediment as the initial inoculum (see Methods).” in the revised version L301-306. The corresponding calculation method was added in the revised manuscript L358-365 and the modified Supplementary Table 3.\nL 247-249: contextual data for sampling site is vague. What is the NO3 concentration of ambient water, how deep was the sediment sample collected, top 5cm?\nResponse: This information is added in the revised manuscript L330-333. The above 20-cm layers of sediments were used as the inoculum for the incubation. The concentration of NO − (NO − and NO −) in the overlying water sample was 213.7 μg/L x 2 3 (1.98 μmol/L) (referenced from Jing et al. 2018, doi:10.3389/fmicb.2018.02821).\nOverall, I would suggest the authors focus more on the physiological response of denitrifiers to HHP rather than getting back to ‘in-situ’ biogeochemical mechanisms as the pre- and after- incubations drives too far away from real scenarios.\nResponse: We agree. The revised manuscript focuses on the physiological response of denitrifiers to HHP, instead of an in situ simulation as proposed in the previous version.\nReviewer #2 (Remarks to the Author):\nThe manuscript entitled “High hydrostatic pressure stimulates microbial nitrate reduction in hadal trench sediments under oxic condition” by Yang et al. investigated the microbiology and omics of a marine sediment sample collected at a depth of over 6,000 m. This work demonstrated that denitrification is a major metabolic process in the nitrogen cycle under aerobic conditions and high hydrostatic pressure. The results are interesting, well presented and documented. However, this work lacks control experiments (e.g. in the absence of oxygen) in my view. The experimental design is inadequate, as samples pressurized and maintained at in situ hydrostatic pressure during collection and storage, were directly incubated first at atmospheric pressure and then successively at increasing pressures applied during incubation. The enrichment of microorganisms at atmospheric pressure undoubtedly led to growth and an increase in the ratio of non-piezophilic or piezotolerant microorganisms, which became dominant during incubation compared with microorganisms adapted to in situ pressure conditions.\nResponse: We are glad to see that the reviewer appreciates the presentation of our results. The manuscript has been modified according to the reviewers’ comments.\nThis study aims to investigate the effect of increased hydrostatic pressures on the microbial nitrate reduction pathway under oxygenic conditions. We don’t think the anaerobic control is required since the surface sediment in the hadal trenches is typically aerobic. Having the incubation performed at anaerobic conditions may further enhance the denitrification process, but this is beyond the scope of the study. We agree that the microbial community structure changed with the sequential incubation experiment. However, due to the relatively short incubation time (15 days) at each pressure, the relative abundance of non-piezotolerant microorganisms enriched at low pressure was generally low. More importantly, the dominant denitrifier Halomonadaceae was relatively stable with a high abundance (23.6–37.4%) throughout the flowing incubation period (Fig. 2C and Supplementary Table 2). Thus, we believe that our experiment was sufficient to support our two main findings, which were 1) aerobic respiration and anaerobic metabolic pathways (denitrification) could occur simultaneously, and 2) microbial nitrogen metabolism route changed with increasing hydrostatic pressure.\nMetagenomic and metatranscriptomic analysis lacks analysis of gene diversity and binning in relation to the dominant taxonomic groups present. These results do not support the authors' conclusions regarding active and major metabolisms in situ.\nResponse: We have performed additional genome-centric approaches and added the in-depth analysis at the MAGs level. This added content describes the dominant taxonomic groups that were actively involved in the denitrification pathway. This includes a new descriptive paragraph (L167-196), new figures (Fig. 4B and Extended Data Fig. 4), and Supplementary Tables 7 and 8 in the revised manuscript.\nIn addition, we also added the metatranscriptomic analysis on in situ RNA-fixed surface sediments from the Mariana Trench, which further supported the conclusion of simultaneous denitrification and aerobic respiration under high hydrostatic pressure in natural environments. This content is now shown as a new descriptive paragraph in the revised manuscript L217-242.\nSpecific comments:\n- Lines 86-88: Under your experimental conditions, can you tell us what role Thaumarchaea play in the nitrogen cycle?\nResponse: The 16S rRNA genes of Thaumarchaea were detected in amplicon sequencing results. However, the marker gene of ammonium oxidation (amoABC gene) was not identified in the metagenome nor the metatranscriptome. We propose that this may be due to insufficient sequencing depth. As the cell number of archaea is two orders of magnitude lower than bacteria, the amoABC gene may fail to be detected (Fig. 2B). Therefore, we acknowledge the presence of Thaumarchaea, but their ammonia-oxidizing activity may be neglectable in our setup.\n- Lines 112-125: Could you indicate to which taxonomic group the genes described in this paragraph can be assigned?\nResponse: We have carried out additional genome-centric analysis. The results showed that denitrification-associated genes were identified from 40 MAGs, and actively transcribed in 31 MAGs under different pressures. These MAGs mainly belonged to Proteobacteria (28 MAGs), Bacteroidota (8 MAGs), Actinobacteriota (3 MAGs) and Thermoproteota (1 MAG). We found that the relative abundance of denitrifies changed under various hydrostatic pressures (Fig. 4B). Specifically, Marinobacter hydrocarbonoclasticus was dominant at 40 MPa with its abundance decreased under increased pressures, but the transcriptional level of its denitrification-related genes (nirS, norBC, and nosZ) increased under the elevated pressures. The abundance of Thalassospira xiamenensis increased as pressures increased, and its genes associated with denitrification (napAB, nirS, norBC, and nosZ) were only detected as being active under 70, 90 and 115 MPa. Idiomarina loihiensis also participated in denitrification, in which the nirK and norB genes were actively transcribed during incubation, however, its abundance decreased with the elevated pressures. The relative abundance of Aequorivita vladivostokensis remained stable during incubation, but its denitrification-related genes (nirK, norB, and nosZ) were transcribed only under 115 MPa (Supplementary Tables 7 and 8). Lastly, Halomonas titanicae was the most abundant denitrifier across the entire incubation period, and its denitrification-related genes (narGHI, norBC and nosZ) were more actively transcribed under higher hydrostatic pressures, especially under 70, 90 and 115 MPa.\nThis content to describe the key taxonomic groups that are actively involved in the denitrification pathway under different hydrostatic pressures, has been as a new descriptive paragraph (L167-196), new figures (Fig. 4B and Extended Data Fig. 4), added Supplementary Tables 7 and 8 in the revised manuscript.\n- Line 142: Similarly, to which taxonomic group does the selected nosZ gene refer?\nResponse: Based on the taxonomic annotation, we found that most Sec-dependent nosZ genes were affiliated with the Flavobacteriales, whereas the Tat-dependent nosZ genes were mainly affiliated with Alphaproteobacteria and Gammaproteobacteria.\nThis content has been added to the revised manuscript L145-147 and the modified Supplementary Table5.\nFurthermore, we also analyzed the MAGs for actively transcribing the nosZ genes in this incubation system. The results showed that the Sec-dependent nosZ gene mainly belonged to Aequorivita vladivostokensis (Flavobacteriales) and Muricauda sp002167435 (Flavobacteriales). The Tat-dependent nosZ gene mainly belonged to Halomonas titanicae (Gammaproteobacteria), Marinobacter hydrocarbonoclasticus (Gammaproteobacteria) and Thalassospira xiamenensis (Alphaproteobacteria).\nThis content has been shown in added Supplementary Table 8.\n- Lines 180-181: this conclusion is not supported by the results obtained, i.e., what is the diversity of respiratory chains present and active, for example?\nResponse: This sentence is revised as “By genome-centric analysis, our results showed that aerobic respiration-related genes and denitrification-related genes within the same denitrifier MAGs, such as Halomonas titanicae, Marinobacter hydrocarbonoclasticus and Thalassospira xiamenensis, were transcribed concurrently under higher pressures (Fig. 6A).” as revised in L251-255.\nIn the revised manuscript, we additionally analyzed the MAGs that are involved in the denitrification and/or aerobic respiration pathways. The results showed that both aerobic respiration-related genes (e.g., Cyo, Cyd, Cco, and Cox) and denitrification-related genes in the dominant taxa Halomonas titanicae and Marinobacter hydrocarbonoclasticus concurrently transcribed actively under higher hydrostatic pressures (newly added Supplementary Table 9).\nMoreover, we also added the metatranscriptomic data from in situ RNA-fixed sediments at the Mariana Trench. The results showed that the related genes (e.g., Cyo, Cyd, Cco, and Cox) encoding terminal oxidases and the key genes involved in denitrification co-occurred and both were active transcriptions (newly added Supplementary Table 10).\nThis content has been added to the revised manuscript L217-242.\n- Lines 247—249: What are the physicochemical characteristics of the sediment sample used in this study?\nResponse: The amount of sediment we received in this study was insufficient to quantify the physicochemical characteristics. However, we have obtained physicochemical parameters (temperature, salinity, and nutrients) in the overlying water, which was collected tens of centimeters above this sediment surface (Jing et al. 2018). The concentration of NO − (NO − and NO −) was 213.7 μg/L (1.98 μmol/L), the concentration of x 2 3 NH + was 101.31 μg/L (5.63 μmol/L), the concentration of PO 3− was 82.71 μg/L (0.87 4 4 μmol/L), the temperature was 1.59°C, the salinity was 34.69 psu (Jing et al. 2018).\nThus, we have added this information in the revised manuscript L332-333 with an appropriate citation.\nIn addition, the physicochemical characteristics of sediment from the Mariana Trench in previous literature are: the NO − concentration was approximately 30–40 μmol/L in the 3 0–20 cm layer of sediment (Nunoura et al. 2018). The O concentration was greater than 2 100 μmol/L in the 0–20 cm layer of 6018 m sediment (Glud et al. 2013).\n- Line 254: Why was glucose used as a source of carbon and energy?\nResponse: We followed the protocol of Zhong & Jia, 2013; Qin et al. 2017, in which glucose was used as an organic carbon source and electron acceptor to study the nitrate reduction process of microorganisms. This has been added to the revised manuscript L338-340."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my questions and the manuscript has been improved. The following aspects could be considered for better accuracy:\nLine 23-24 ‘our findings demonstrate that hydrostatic pressure can restructure the chemical zonation in hadal trenches’, this is from the discussion in line 275-290, no direct evidence to support ‘restricting’ in the hadal sediment , consider rephrasing and tone down.\nLine 316-317 “High hydrostatic pressures promote denitrification activity under oxic conditions” not for all denitrifiers, but only for those survived in the depressurizing-repressurizing cycle in the chemostat. The chemostat incubation helps to recover a group of denitrifiers that can use both O2 and nitrate but their contribution to ‘in-situ’ denitrification activity is undetermined."}, "author_response": "Response to the reviewers’ comments:\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all my questions and the manuscript has been improved. The following aspects could be considered for better accuracy:\nLine 23-24 ‘our findings demonstrate that hydrostatic pressure can restructure the chemical zonation in hadal trenches’, this is from the discussion in line 275-290, no direct evidence to support ‘restricting’ in the hadal sediment, consider rephrasing and tone down.\nResponse: Thanks for your suggestion. We have rephrased and toned down this sentence, as follows: ‘Taken together, our findings demonstrate that hydrostatic pressure can influence microbial contributions to nitrogen cycling and the hadal trenches are a potential nitrogen loss hotspot’, in Line 28-30.\nLine 316-317 “High hydrostatic pressures promote denitrification activity under oxic conditions” not for all denitrifiers, but only for those survived in the depressurizing-repressurizing cycle in the chemostat. The chemostat incubation helps to recover a group of denitrifiers that can use both O2 and nitrate but their contribution to ‘in-situ’ denitrification activity is undetermined.\nResponse: Thanks for your suggestion. We have revised the sentence as “We observed that high hydrostatic pressure promotes denitrification activity even under oxic conditions, presenting an indication that hydrostatic pressure has the potential to modify the niche breadth and the activity of microorganisms. This modification, in turn, influences their contributions to the elemental cycling processes within hadal trenches”, in Line 315-319."}]

Decision letter