Overlooked role of heterotrophic prokaryotes in sulfur oxidation makes the sediment of the Bohai Sea as a sufficient sink of H2S

doi:10.21203/rs.3.rs-4925125/v1

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Overlooked role of heterotrophic prokaryotes in sulfur oxidation makes the sediment of the Bohai Sea as a sufficient sink of H2S

https://doi.org/10.21203/rs.3.rs-4925125/v1

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Background:

Marine sediments are the active sites for the biogeochemical cycling of sulfur. Sulfate is used as a major terminal electron acceptor for anaerobic oxidation of organic compounds in marine sediments, and the produced sulfide is normally oxidized back to sulfate in upper sediments, mainly considered as a biological process. The low abundance or even absence of autotrophic sulfur oxidizing bacteria in marine sediments challenges this point.

Results:

Here, we used metagenomics, metatranscriptomics, and the testing of sulfide, thiosulfate, and sulfite oxidation in sediment samples to figure out how sulfide is oxidized in the Bohai Sea sediments. Surprisingly, sulfur oxidation is wide-spread in the microbial community (> 85.8%) of mostly heterotrophic prokaryotes across 67 phyla, dominated by Proteobacteria. Known chemolithotrophic sulfur oxidizers were absent. The prevalent sulfur oxidizing pathway was sulfide to zerovalent sulfur, sulfite, and then sulfate. Thiosulfate, which was only slowly oxidized, is not a major metabolic intermediate, contradicting with the dominant thiosulfate shunt in sulfur cycling. Genes encoding sulfide oxidation (sqr and fccAB), zerovalent sulfur oxidation (pdo, rdsrAB, and hdrABC), and sulfite oxidation (aprAB/sat) were abundant and upregulated after adding NaHS.

Conclusions:

The findings reveal how sulfur is oxidized, and the common participation in sulfur oxidation by most heterotrophic prokaryotes ensures that sulfide is effectively oxidized in the surface sediment.

sulfur oxidation

marine sediment

microbial diversity

metagenomics

metatranscriptomics

The biogeochemical sulfur cycle is an essential process consisting of abiotically and biologically mediated reactions in marine sediments. The sulfur cycles interlink with other biogeochemical cycles, such as carbon and nitrogen cycles. On a global scale, sulfate reduction facilitates the oxidation of organic matter from 22 up to 77 Tmol of carbon per year in marine sediments[1, 2], which is associated with the production of a large amount of reduced sulfide. However, over 80% sulfide is eventually reoxidized back to sulfate, while only less than 20% is ultimately buried as metal sulfides, e.g., FeS, FeS₂, or sulfurized organic matter[3, 4]. Sulfide oxidation from the most reduced state (-2, sulfide) to the most oxidized state (+ 6, sulfate) is primarily conducted by microorganisms, such as sulfur-oxidizing bacteria, with a range of intermediates, including zerovalent sulfur, thiosulfate, tetrathionate, and sulfite[5, 6].

Various microorganisms, including autotrophy, heterotrophy, and phototrophy, could oxidize sulfur[7]. However, sulfur oxidation is still considered as a specialized character in the community. Several sulfur oxidation pathways associated with different key genes using different inorganic sulfur compounds as substrates have been identified. Sulfide is oxidized by sulfide:quinone oxidoreductase (SQR)[8] and flavocytochrome c:sulfide dehydrogenase (FCSD) to zerovalent sulfur. Zerovalent sulfur is oxidized to sulfite by oxidative bacterial type dissimilatory sulfite reductase (rDsrAB)[9], persulfide dioxygenase (PDO)[10], and heterodisulfide reductase (Hdr)-like enzyme (HDR) which is homologous to the HdrABC in methanogens[11, 12]. Further, zerovalent sulfur can be disproportionated to sulfide, thiosulfate and sulfite by sulfur oxygenase reductase (SOR)[13, 14]. Sulfite is oxidized to sulfate by sulfite oxidase (SUOX)[15], the periplasmic heterodimeric sulfite dehydrogenase (SorA)[16], membrane-bound sulfite dehydrogenase (SoeABC)[17], and adenylylsulfate reductase (AprAB) and sulfate adenylyltransferase (SAT)[18]. Alternatively, sulfite reacts with a zerovalent sulfur to generate thiosulfate[19]. Thiosulfate is oxidized by the sulfur oxidation system (SoxABCDXYZ)[20] to sulfate, or by thiosulfate dehydrogenase (TsdA) and thiosulfate:quinone dehydrogenase (DoxD) to tetrathionate[21, 22]. Tetrathionate is converted to sulfate, thiosulfate, and zerovalent sulfur by tetrathionate hydrolase[23], of which thiosulfate and zerovalent sulfur can be further metabolized by enzymes mentioned above.

Although sulfur oxidation is considered as a microbial driven process, the low abundance of autotrophic sulfur oxidizing bacteria disguised the key players of sulfur oxidation and the associated dynamics in marine sediments. Considering the high concentration of organic matter in coastal marine sediments, we hypothesized that sulfur is commonly oxidized by heterotrophic prokaryotes, rather than mainly by the specific group. To test this hypothesis, we selected 15 samples representing three layers of sediments from three sampling stations (M3, M8, and BHB10) in the Bohai Sea, a coastal marine environment with high concentrations of organic matter and active geochemical transformations[24], to untangle the sulfur oxidizing communities with metagenomic sequencing, measurements of inorganic sulfur species, and testing the oxidation rates of these species. Our findings revealed major participants and pathways of sulfur oxidation in the Bohai Sea sediments, and advanced our understanding of sulfur biogeochemical cycling in marine sediments.

Sample collection

Sediment samples were collected from the Bohai Sea with three cruises in 2018, 2022 and 2023 (Supplementary Table 1). DNA was extracted from sediment core samples taken in 2018, representing the surface, middle, and bottom layers in three sites, and subjected to metagenomic sequencing. Porewater were extracted from samples taken in 2022 using Rhizon samplers (pore size of 0.15 µm), and stored at -20 ℃ for the analysis of sulfide, thiosulfate, and sulfite during the cruise in 2022. Sediment core samples for zerovalent sulfur measurement were taken in 2023. Surface sediment samples for incubation in the laboratory were stored at 4°C during the cruise in 2023.

Metagenome assembling and binning

Metagenomes were processed with the same procedure as previously reported[25, 26]. The quality of metagenome-assembled genomes (MAGs) was estimated using CheckM v1.0.5[27] with lineage_wf. Taxonomy of each MAG was assigned using the Genome Taxonomy Database (GTDB)-Tk v1.1.1[28] with release202.

Identification of selected sulfur oxidation genes and autotrophy

Scaffolds ≥ 2,000 bp were submitted to the Integrated Microbial Genomes & Microbiomes (IMG/M) using DOE-JGI Metagenome Annotation Pipeline (MAP v.4) for gene prediction and functional annotation[29]. Sulfur oxidation genes were initially identified based on the KO assignment (Supplementary Table 2) from IMG. Custom databases for SQR, DsrAB, SoxB, PDO, and HDR were used as references to further identify the genes.

Autotrophic pathways, including Wood-Ljungdahl pathway, Calvin–Benson–Bassham, reductive tricarboxylic acid, 3-hydroxypropionate bicycle, 3-hydroxypropionate-4-hydroxybutyrate, dicarboxylate-4-hydroxybutyrate cycles, and reverse glycine cleavage pathway were identified based on the annotation from IMG. The pathway was confirmed by the two standards: the presence of 70% genes or the presence of marker genes plus over 50% accessory genes (Supplementary Table 3).

Phylogeny of selected genes

We selected six sets of protein sequences, i.e., DsrA, DsrB, SoxB, SQR, PDO, and HDR clusters for further phylogenetic classification. Each set of identified sequences was aligned with the corresponding custom databases, and trimmed. All alignments were manually checked to remove the short alignment, and maximum likelihood phylogenetic trees were generated using IQ-TREE v1.6.12[30] with the parameters.

Calculation of the relative abundance of scaffolds, functional genes, and MAGs

The relative abundance of each scaffold was calculated by the number of reads mapped to the scaffold, further normalized by the length of scaffold and the number of reads in each sample, and multiplying 10^9 for readability purposes. The relative abundance of each functional gene was the sum of relative abundance of its corresponding scaffold. Relative abundance of MAGs in each sample was calculated using CoverM v0.6.1 by mapping raw reads to the 5,233 MAGs.

Measurement of sulfide, zerovalent sulfur, thiosulfate, sulfite, and sulfate in sediment

Zerovalent sulfur in sediment samples was analyzed using the methods reported previously[31]. The concentration of sulfide in porewater was measured using the methylene blue method[32]. Sulfite and thiosulfate in porewater were analyzed using HPLC as previously reported[19]. Sulfate was measured using an ICS-1100 Ion Chromatography system (Dionex Corporation, CA, U.S.A.).

Incubation with sulfide, thiosulfate, and sulfite

Sediment samples were incubated with addition of NaHS, Na₂S₂O₃, and Na₂SO₃ solutions. Untreated sediment, autoclaved sediment, and the dilution buffer without sediment was considered as the experimental group, control group, and blank, respectively. Time-series samples were taken to measure the concentration of sulfide, zerovalent sulfur, thiosulfate, and sulfite as described above.

Incubation for metatranscriptomic analysis and metatranscriptomic data analysis

Five grams of sediment samples were diluted with sterilized seawater containing DTPA. The experimental group was supplied with NaHS solutions five times with an interval of 30 minutes. Sediment samples were then frozen in liquid nitrogen and stored at -80°C for RNA extraction. Libraries were prepared after rRNA removal. Metatranscriptomic data were sequenced on an Illumina Novaseq X Plus platform. rRNA in raw data from the control and experimental samples were further filtered using SortMeRNA v4.3.6[33] against rRNA database release 4.3.4. Cleaned data were mapped to the predicted protein sequences from all 15 assemblies for the estimation of relative abundance.

Overview of sulfur oxidation pathways in the Bohai Sea sediments

We sequenced metagenomes from 15 sediment samples with an average of 932.08 ± 51.51 million reads per sample (Supplementary Table 4). We further recovered 5,233 MAGs with the completeness over 50% and contamination less than 10%. Sulfur oxidation genes involved in different processes were prevalent across all samples (Fig. 1a and Supplementary Table 2). Genes involved in different pathways for sulfur oxidation showed different compositions in surface, middle, and bottom layers (Fig. 1b). The compositions of the sulfur oxidation genes in 0–2 cm samples (M3-2, M8-2, and BHB10-2) were distinct from the rest samples (Fig. 1b). Thus, M3-2, M8-2, and BHB10-2 were classified as surface layer samples. Samples from 8–30 cm and below 30 cm were classified as middle and bottom layer samples, respectively. Sulfide-, zerovalent sulfur-, and thiosulfate-oxidizing genes were significantly more abundant in the surface layer than in the bottom layer (p < 0.05), while sulfite oxidation genes didn’t change significantly (p > 0.05) (Fig. 1c). sqr, pdo, and soxABCXYZ were the dominant genes for sulfide, zerovalent sulfur, and thiosulfate oxidation, respectively, with a tendency of decreased relative abundance along with depths (Supplementary Fig. 1). The soxD gene was absent in our samples. The Sox system without SoxD only partially oxidizes thiosulfate, producing sulfate and zerovalent sulfur[34]. The relative abundance of tsdA[22], which oxidizes thiosulfate to tetrathionate, tends to increase from the surface to the bottom. The indirect AMP-dependent sulfite oxidation pathway, consisting of aprAB and sat[18], was the most abundant among all sulfur-oxidizing genes. The relative abundance of aprAB and sat, bidirectionally catalyzing the oxidation of sulfite and reduction of sulfate[18], changed differently along with depths (Supplementary Fig. 1). The relative abundance of aprAB genes increased, while sat increased along depths. Surprisingly, the direct sulfite-oxidizing enzymes were scarcely present: genes encoding SUOX and the periplasmic heterodimeric SorA had a low abundance in most samples, and the membrane-bound SoeABC was not detected in all the samples.

Heterotrophy dominates sulfur oxidation in the Bohai Sea sediments

Sulfur-oxidizing genes were prevalent in these 5,233 MAGs. Over 85.8% recovered MAGs (4,490 of 5,233), occupying 87.2% abundance of the recovered community, contain at least one gene related to inorganic sulfur oxidation (sulfide, zerovalent sulfur, thiosulfate, and sulfite) (Fig. 2 and Supplementary Fig. 2). Specifically, 43.2% recovered MAGs (2,263 MAGs) contained sqr and fccAB for sulfide oxidation; 38.8% had pdo (1,818 MAGs), rdsrAB (365 MAGs), hdrABC (329 MAGs), and sor (39 MAGs) for the oxidation of zerovalent sulfur; 45.4% carried the sox (473 MAGs), tsdA (1,229 MAGs), and doxD (786 MAGs) for thiosulfate metabolism, and 24.7% possessed sat-aprAB (1,284 MAGs), sorA (37 MAGs), and SOUX (32 MAGs) for potential sulfite oxidation. These data suggest that biological sulfur oxidation is prevalent in marine sediments[6]. These 4,490 MAGs were assigned into 48 bacterial and 6 archaeal phyla, dominated by Proteobacteria, Desulfobacterota, Gemmatimonadota, Myxococcota, and Thermoproteota (Fig. 2).

Heterotrophic and mixotrophic metabolisms dominate the microbial community in the Bohai Sea sediments. We identified 755 MAGs with autotrophic metabolisms. They mostly belong to Proteobacteria (264 MAGs), Desulfobacterota (125 MAGs), and Nitrospirota (72 MAGs). The remaining 4,478 MAGs, occupying 85.57% of the entire community, likely rely on heterotrophic and mixotrophic metabolisms. They mainly belong to Proteobacteria (1,108 MAGs), Desulfobacterota (432 MAGs), Gemmatimonadota (425 MAGs), and Myxococcota (378 MAGs). 3,807 of the 4,478 MAGs with heterotrophic/mixotrophic metabolism are capable of oxidizing different types of sulfur as evidenced by possessing different sulfur oxidation genes. For example, 1,581 MAGs contain SQR (1,220 MAGs) or FCSD (655 MAGs). 1,702 MAGs encode different enzymes, including PDO (1,571 MAGs), rDsrB (253 MAGs), HDR (259 MAGs), and SOR (15 MAGs) that oxidize zerovalent sulfur. Among 1,785 MAGs which oxidize thiosulfate, 1,005 and 740 MAGs oxidize thiosulfate to tetrathionate with TsdA and DoxD, respectively, and 436 MAGs oxidize thiosulfate to sulfate and zerovalent sulfur with the Sox system lacking SoxD. 943 MAGs carry Sat-AprAB that oxidize sulfite to sulfate.

Sulfide oxidation

2,594 of 3,474 SQR homologous sequences, annotated by the combination of IMG/JGI MAP and diamond search, were confirmed as SQR, and the remaining 880 SQR homologues were confirmed as the catalytic subunit of FCSD (FccB) by their phylogeny[35] (Supplementary Fig. 3). Phylogenetic analyses suggest that most of these SQR sequences belong to the membrane-bound type III (1,814), type II (379), and type I (296) SQRs, and few of them belong to type IV (9) and type VI (83) SQRs[35]. Over half of SQRs (1,707/2,594) were assigned in 1,507 MAGs, most of which belong to Proteobacteria (288 MAGs), Gemmatimonadota (239 MAGs), Desulfobacterota (190 MAGs), Chloroflexota (154 MAGs), and Bacteroidota (151 MAGs). FCSDs were mainly distributed in Proteobacteria (304 MAGs), Gemmatimonadota (99 MAGs), Chloroflexota (99 MAGs), and Thermoproteota (58 MAGs). The significantly higher abundance of SQR than FccB in middle and bottom layers (p < 0.001, t-test) suggests that SQR was the dominant enzyme oxidizing sulfide in Bohai Sea sediments (Supplementary Fig. 1).

Zerovalent sulfur oxidation

PDOs were the most abundant, followed by HDRs and rDsrABs for zerovalent sulfur oxidation in the sediments. 3,055 PDO sequences were identified from 15 assemblies. A maximum likelihood phylogenetic tree showed that 1,091, 1,150, and 814 PDO sequences were classified as type I, type II, and type III[36, 37], respectively (Supplementary Fig. 4). 2,047 of 3,055 identified PDO sequences were binned in 1,818 MAGs. Type I PDOs were mainly identified in Proteobacteria (469 MAGs; 295 Gammaproteobacteria MAGs and 174 Alphaproteobacteria MAGs) and Myxococcota (178 MAGs). Few type I PDOs were annotated in Acidobacteriota (36 MAGs), Planctomycetota (3 MAGs), Desulfobacterota (2 MAGs), SAR324 (2 MAGs), Gemmatimonadota (1 MAG), DSWW01 (1 MAG), and Cyanobacteria (1 MAG). Type II PDOs were identified mainly in Proteobacteria (653 MAGs), mostly Gammaproteobacteria (579 MAGs) and Alphaproteobacteria (74 MAGs). Few type II PDOs were annotated in Myxococcota (54 MAGs), Acidobacteriota (22 MAGs), Desulfobacterota (5 MAGs), and Gemmatimonadota (2 MAGs). Type III PDOs were distributed in more phyla (19 phyla) than Type I and TypeII PDOs, mainly in Gemmatimonadota (191 MAGs), Bacteroidota (114 MAGs), Chloroflexota (32 MAGs), Nitrospirota (28 MAGs), Planctomycetota (23 MAGs), and Actinobacteriota (17 MAGs). Types I and II PDOs are present only in Gram-negative bacteria, while type III PDOs were identified in both Gram- negative and positive bacteria but mainly in Gram positive bacteria[36–38]. Type II PDOs were commonly distributed in the surface, while types I and III PDOs were often identified in the bottom layer (Supplementary Fig. 4).

We identified 2,621 α subunit (DsrA) and 2,543 β subunit (DsrB) sequences of dissimilatory sulfite reductase (Dsr), which could be assigned in 1,514 MAGs, in the Bohai Sea sediment samples. The phylogenetic tree showed that 601 DsrAB sequences are the oxidative bacterial type (rDsrAB) oxidizing zerovalent sulfur[9] (Supplementary Fig. 5). rDsrAB sequences were mainly distributed in Gammaproteobacteria (291 MAGs) and Alphaproteobacteria (59 MAGs). Bacterial phyla CG2-30-53-67 (9 MAGs) and Desulfobacterota (6 MAGs), which are mostly considered as sulfate reducers, also have rDsrAB. The rDsrAB sequences were mainly identified in the surface, while the reductive bacterial type DsrAB sequences were mostly distributed in the bottom layer in the Bohai Sea sediment (Supplementary Fig. 5).

HDR is encoded by a hdrC1B1A-hyp-hdrC2B2 cluster acting as a sulfur-oxidizing entity[11, 12]. In total, 406 HDRs were annotated, and 334 of them were assigned in 329 Proteobacteria MAGs (Supplementary Fig. 6), belonging to Gammaproteobacteria (296 MAGs) and Alphaproteobacteria (33 MAGs). Several orders, including Woeseiales, Kilonielales, UBA8366, and SMXQ01, could oxidize zerovalent sulfur by using HDR. Gammaproteobacteria with HDR were mainly identified in the surface sediment, while Alphaproteobacteria with HDR were mostly distributed in the bottom layer (Supplementary Fig. 6).

Only 105 SOR sequences were identified, and 50 of them were assigned in 39 MAGs from 14 samples. 33 of 39 MAGs were classified as Desulfobacterota, which is known for dissimilatory sulfate reduction[39]. The narrow distribution of SOR suggests that it is a conserved gene within a small community with rare horizontal gene transfer across different phyla, and its rare presence suggests that it is probably not a key enzyme for zerovalent sulfur oxidation in Bohai Sea sediments.

Thiosulfate oxidation

SoxABCXYZ, TsdA, and DoxD[21] were annotated for thiosulfate oxidation in all sediment samples. However, the absence of subunits SoxD in the Sox system suggests the incomplete thiosulfate oxidation to sulfate and zerovalent sulfur[34]. 473 SoxABXYZ complexes were exclusively distributed in Alphaproteobacteria (93 MAGs) and Gammaproteobacteria (380 MAGs), mainly in the orders Woeseiales (137 MAGs), GCA-001735895 (56 MAGs), Thiohalobacterales (43 MAGs), Xanthomonadales (40 MAGs), Kiloniellales (37 MAGs), and UBA8366 (33 MAGs). Among 473 MAGs containing Sox complex, 460 MAGs also have at least one pathway for zerovalent sulfur oxidation (Supplementary Fig. 7), suggesting a potential high turnover rate of zerovalent sulfur and low chance of accumulating zerovalent sulfur during the incomplete oxidation of thiosulfate. 1,017 catalytic subunits of Sox complex (SoxB) were annotated, and 695 were assigned in 608 MAGs mainly belonging to Proteobacteria (607 MAGs). Gammaproteobacteria with SoxB were mostly distributed in the surface, and Alphaproteobacteria with SoxB were mainly identified in the bottom layer (Supplementary Fig. 8). 2,606 TsdA and 1,514 DoxD sequences were identified for thiosulfate oxidation to tetrathionate. TsdAs were mainly distributed in Proteobacteria (436 MAGs), Chloroflexota (156 MAGs), Nitrospirota (143 MAGs), and Desulfobacterota (134 MAGs). DoxDs were mainly identified in Gemmatimonadota (252 MAGs), Proteobacteria (167 MAGs), Chloroflexota (159 MAGs) and Bacteroidota (62 MAGs).

Sulfite oxidation

SAT and AprAB catalyzing both sulfite oxidation and sulfate reduction are abundant in the sediments (Fig. 2), suggesting the sediments are an active site for sulfate reduction or sulfite oxidation[40]. We identified 7,916 SAT and 2,918 AprAB sequences. 1,464 of 1,737 MAGs encode AprAB, which are mainly Proteobacteria (567 MAGs), Desulfobacterota (315 MAGs), and Nitrospirota (157 MAGs). 3,282 of 4,655 MAGs encode SAT, which are mainly Proteobacteria (1,081 MAGs), Desulfobacterota (426 MAGs), Myxococcota (258 MAGs), Acidobacteriota (247 MAGs), and Gemmatimonadota (236 MAGs). 151 MAGs classified into 15 phyla, such as Thermoplasmatota (53 MAGs) and Actinobacteriota (30 MAGs), only encode SAT without AprAB. The high abundance of AprAB and SAT in surface sediment suggests the SAT-AprAB pathway oxidizes sulfite and sulfate reducers are tolerant to oxygen in surface sediment[41, 42]. We further identified 56 SorA sequences oxidizing sulfite, and 37 SorAs were assigned exclusively in 37 Myxococcota MAGs. Among 66 identified SOUX sequences, 34 sequences were assigned in 32 MAGs, mainly Gemmatimonadota (18 MAGs), which may involve sulfite oxidation.

Microbial network for sulfur oxidation

The dominant phyla involved in sulfur oxidation included Proteobacteria (1,342/1,372 MAGs), Desulfobacterota (483/564 MAGs), Gemmatimonadota (435/438 MAGs), Myxococcota (364/390 MAGs), Acidobacteriota (278/296 MAGs), Chloroflexota (256/312 MAGs), Thermoproteota (175/209 MAGs), Nitrospirota (228/239 MAGs), Bacteroidota (181/190 MAGs), and Planctomycetota (161/293 MAGs). Abundant lineages participated in the oxidation of multiple sulfur species, while rare taxa tend to only oxidize one species (Supplementary Fig. 9). Proteobacteria were the dominant group that oxidized different inorganic sulfur species across depths (Fig. 3). However, more phyla were involved in sulfur oxidation in the deeper sediments than in the shallower sediments, and the portion of Proteobacteria for sulfur oxidation decreased along the depth (Fig. 3). Desulfobacterota, previously known for dissimilatory sulfate reduction[39] as part of Deltaproteobacteria within the phylum Proteobacteria but recently renamed as a new phylum, also had diverse sulfur oxidation genes. Most Desulfobacterota used SQR for sulfide oxidation and AprAB and SAT for sulfite oxidation, but many of them lacked the genes for zerovalent sulfur oxidation (Fig. 2). Interestingly, Nitrospirota, known as a nitrifying phylum[43–45], also had diverse enzymes for sulfur oxidation, including SQR for sulfide oxidation, rDsrAB, PDO, and SOR for the oxidation of zerovalent sulfur, AprAB and SAT for sulfite oxidation, and TsdA for thiosulfate oxidation. Most Myxococcota (216/390 MAGs), another new phylum originally assigned to Deltaproteobacteria[46, 47], also were abundant in Bohai Sea sediments, and they contained mainly PDO for zerovalent sulfur oxidation. The less abundant enzymes oxidizing sulfite were conservatively distributed in specific lineages, such as Sor sequences were only present in Myxococcota and SUOX sequences were mostly identified in Gemmatimonadota.

Sulfur species in the sediments

The sulfate concentrations were around 20–25 mmol/L in sediment porewater from 0–60 cm at stations M3, M8, and BHB10[24]. The concentration of thiosulfate, sulfide, and sulfite were extremely low (< 5 nmol/g sediment) in situ in the sediment porewater at station BHB10 (Supplementary Fig. 10). The maximal concentration of zerovalent sulfur was around 2 µmol/g wet sediment at station M8, and mostly below 1 µmol/g sediment at three stations (~ 2 mM zerovalent sulfur, Supplementary Fig. 11). The low concentration of sulfide (< 5 µM) and ~ 2 mM zerovalent sulfur in sediments suggests a high rate of sulfur oxidation in sediments. Alternatively, sulfate is likely reduced by sulfur reducing bacteria to zero valent sulfur instead of sulfide[48].

Estimation of the maximal potential of sulfur oxidation in the sediments

Three types of reduced sulfur species (sulfide, thiosulfate, and sulfite) were added to sediment samples to estimate the maximal rates of their oxidation under oxic conditions (Fig. 4). When NaHS was added, sulfide was quickly consumed with maximal rates at 140.3 ± 11.6 and 76.4 ± 10.8 µmol/h/g sediment for biological and chemical consumption, respectively (Fig. 4a). Chemical consumption was estimated by using heat-killed sediments. The maximal production rates of zerovalent sulfur, sulfite, and thiosulfate reached 154.8 ± 3.1, 25.2 ± 2.7, and 10.2 ± 1.1 µmol/h/g sediment during the incubation (Fig. 4b, 4d, and 4c). When sulfite was the substrate, the maximal rates of its chemical and biological oxidation were 1.3 ± 0.1 and 12.8 ± 1.0 µmol/h/g sediment (Fig. 4i and 4h). We washed the sulfate in the sediment before adding thiosulfate to determine the product of thiosulfate oxidation under oxic conditions. When thiosulfate was the substrate, the maximal rates of its chemical and biological oxidation were 0.01 ± 0.01 and 0.05 ± 0.01 µmol/h/g sediment (Fig. 4e). Zerovalent sulfur was not directly tested as the substrate due to its low solubility in water; its maximal rate of oxidation was estimated from sulfite production during sulfide oxidation (Fig. 4l), which was the sum of net sulfite production (25.2 ± 2.7 µmol/h/g sediment, Fig. 4d) and sulfite consumption (thiosulfate production and sulfate production, 10.2 ± 1.1 and 12.8 ± 1.0 µmol/h/g sediment, respectively) at about 48.2 µmol/h/g sediment (Fig. 4c, 4h and 4l). Since the maximal rate of thiosulfate oxidation is significantly lower than other rates, thiosulfate oxidation is unlikely to be a dominant reaction in the sediment.

Metatranscripts during the incubation with sulfide

With the supplements of NaHS, the transcripts mapped to genes for sulfur oxidation were mostly enriched in the whole community (Fig. 4j). The sqr transcripts were enriched with supplements of NaHS (Fig. 4j). The abundance of fccB (catalytic subunit of FCSD) and sqr genes were similar in the surface sediment at station BHB10 (Supplementary Fig. 1); however, the transcripts of sqr were much more abundant than fccB under both control and experimental samples, suggesting the important role of SQR for sulfide oxidation in surface sediments. The upregulated sqr transcripts were mainly from Proteobacteria, Actinobacteriota, Acidobacteriota, Myxococcota, Bacteroidota, and Verrucomicrobiota (Fig. 4k).

The expression of all genes responsible for zerovalent sulfur oxidation, including rdsrAB, pdo, hdr, and sor, were highly upregulated during the incubation (Fig. 4j). The upregulated expression of rdsrAB was in a candidate phylum CG2-30-53-67 that consists of known sulfate reducers[9] and different orders belonging to the classes Alphaproteobacteria and Gammaproteobacteria (Supplementary Fig. 12), including GCA-001735895, Thiohalobacterales, SZUA-229, etc. (Supplementary Fig. 13). The abundance of pdo transcripts increased over 76 times in Planctomycetota after adding NaHS (Fig. 4k), suggesting the key role of pdo for sulfur oxidation. Twenty phyla, including Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Nitrospirota, Gemmatimonadota, Bacteroidota, etc. (Fig. 4k and Supplementary Fig. 12), increased the gene expression of pdo with the supplements of NaHS, suggesting the oxidation of zerovalent sulfur is a common function in microbial communities.

The transcripts of the genes encoding the SOX system were slightly enriched during the incubation, mainly due to their expression in Proteobacteria and Campylobacterota (Supplementary Fig. 14), a chemolithoautotrophic phylum for disproportionation of sulfur previously reported in deep-sea environments[49, 50]. Transcripts for sulfite oxidation did not increase, except for sat transcripts. The total transcripts of aprA or aprB did not change with the supplements of NaHS. However, their proportion in different phyla changed, such as the transcripts in Acidobacteria, Planctomycetota, Verrucomicrobiota, Desulfobacterota, and Gemmatimonadota decreased, while increased in Proteobacteria in experimental samples (Supplementary Fig. 14). The shifts of aprAB transcripts among different phyla suggests different responses to NaHS for sulfite oxidation in sediments.

The Bohai Sea is a semi-enclosed continental shelf sea with many aquacultures and a large amount of terrestrial inputs, including nutrients and organic matter[51–53]. The organic rich sediments provide an active site for microbes to participate in biogeochemical cycling, in which sulfur-oxidizing genes are prevalent in heterotrophy, suggesting they significantly contribute to sulfur oxidation in sediments. Proteobacteria, especially the classes Alphaproteobacteria and Gammaproteobacteria, is the dominant lineage for sulfur oxidation both in relative abundance of functional genes and at transcriptomic levels. Most of these bacteria are heterotrophic bacteria, including the Roseobacter clade belonging to Alphaproteobacteria. The prevalence of Proteobacteria among sulfur oxidation communities decreases along the depths, with increased microbial taxa involved in sulfur oxidation, including rare taxa, which often contain genes for specific steps of sulfur oxidation. The incomplete pathway for sulfur oxidation from sulfide to sulfate within a single genome supports the common handoff of sulfur oxidation within the microbial community and shortcuts in the sulfur cycling[54, 55]. The increased microbial interactions are also supported based on the co-occurrence analysis of 16S rRNA genes in the same region[24].

Thiosulfate is unlikely a major intermediate during sulfide cycling in the Bohai Sea sediments. The maximal oxidation rate of added thiosulfate in our sediment samples under oxic conditions is very slow (~ 0.06 ± 0.01 µmol/h/g sediment, Fig. 4e) compared to the oxidation rates of sulfide, zerovalent sulfur, and sulfite. We further estimated the maximal consumption rates of thiosulfate under oxic conditions without washing remaining sulfate in the sediment, the maximal consumption rates of thiosulfate did not increase (~ 0.06 µmol/h/g sediment, Supplementary Fig. 15). Although the Sox system that oxidizes thiosulfate is present, the soxD gene is missing in all the samples. The incomplete Sox system will only partially oxidize thiosulfate to sulfate and zerovalent sulfur instead of two sulfate molecules[12]. The absence of the complete Sox systems may be partly responsible for the slow thiosulfate oxidation in the Bohai Sea sediments. The addition of NaHS to the sediments did not significantly upregulate the expression of sox genes and other thiosulfate-metabolizing genes. This observation is a surprise, as a major portion of sulfide oxidation through thiosulfate has been reported in other marine sediments[54, 56]. Thiosulfate could be reduced by thiosulfate reductase (PhsA) to sulfide by sulfur reducers, and in the absence of additional electron donors sulfur reducers may oxidize sulfide to zerovalent sulfur, which couples with thiosulfate reduction[57]. We identified 197 MAGs, mainly Desulfobacterota (107 MAGs) and Gemmatimonadota (33 MAGs), containing both DsrAB and PhsA, and they are likely sulfur reducers. However, the estimated maximal thiosulfate consumption rate under anoxic conditions was about 0.06 µmol/h/g sediment, similar to the consumption rate under oxic conditions (Supplementary Fig. 15). As we did not detect accumulated thiosulfate in situ, the production rate of thiosulfate was likely overestimated with the spiking of ample sulfide in the sediments (Supplementary Fig. 10). The importance of thiosulfate shunt in sulfur cycling may be overlooked as the estimated maximal potential of sulfur oxidation is higher than the in situ rate[58]. Nonetheless, the microbial community in the Bohai Sea sediments has limited thiosulfate-oxidizing genes.

The Sat-AprAB pathway is mainly responsible for sulfite oxidation in the Bohai Sea sediment. Sulfite oxidation by the Sat-AprAB pathway has been reported with pure bacteria[59]. Several lines of our evidence indicate the pathway can also be functional in the Bohai Sea sediments. The genes encoding the Sat-AprAB pathway are abundant, and other well-known enzymes for sulfite oxidation, such as the sulfite dehydrogenases (SoeABC and SorAB)[60, 61] and sulfite oxidase (SUOX)[62], are rare or absent. The overall transcripts of aprAB and sat were high, and sulfide spike induced the differential regulation of aprAB transcripts from different taxa. The upregulated aprAB transcripts are likely in the organisms involved in sulfite oxidation. The high expression of the genes encoding the Sat-AprAB pathway is likely associated with the high biological sulfite oxidation rate in the sediments samples (Fig. 4j, 4k, and 4h).

The estimated sulfide oxidation rate in the undiluted marine sediment was 150 nmol/cm^− 3/day in Aarhus Bay[58], which is four orders of magnitude lower than our estimated maximal sulfur oxidation rate, suggesting a high potential of sulfur oxidation in Bohai Sea sediments. This also supports that sulfur oxidation is a general metabolic process in the coastal sediments. In contrast, sulfate reduction is a more specialized function driven by sulfate reducers. Thus, the rate-limiting step of sulfur oxidation is the sulfate reduction, which is the main source of sulfide in marine sediments.

The integrated analysis of metagenomic data, metatranscriptomic data, physiological experiments, and in situ biogeochemical characters showed the complex pathways and diverse players for sulfur oxidation in coastal marine sediments. Our data extend the diversity of sulfur oxidation lineages. Most microorganisms, mainly heterotrophy, participate in the oxidation instead of limited chemolithotrophs. The common participation in sulfur oxidation by most heterotrophic prokaryotes ensures the surface sediment, especially the organic rich sediment as a sink for sulfide. Sulfur oxidation may vary in other marine sediments, further studies are necessary to gain a better understanding of sulfur oxidation in marine sediments.

Data availability

All sequences and annotations of scaffolds are available at IMG/JGI under GOLD Study ID: Gs0149406.

Acknowledgments:

We would like to thank Prof. Bo Barker Jørgensen for constructive comments that improved this manuscript. We thank the captain and crew of the R/V Chuangxin Yi for the help in sample collection.

Funding:

This work was supported by the National Natural Science Foundation of China (Grant numbers 92351302) and Shandong University Foundation for Future Scholar Plan.

Authors’ contributions:

Conceptualization: YX, LX, and XG

Data curation: ZC and XG

Funding acquisition: LX and XG

Investigation: ZC and XG

Methodology: ZC, YX, LX, and XG

Project administration: LX and XG

Resources: LX and XG

Supervision: LX and XG

Visualization: ZC and XG

Writing – original draft: ZC and XG

Writing – review & editing: ZC, YX, LX, and XG

Competing interests: The authors declare that they have no competing interests.

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

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Overlooked role of heterotrophic prokaryotes in sulfur oxidation makes the sediment of the Bohai Sea as a sufficient sink of H2S

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Abstract

Background:

Results:

Conclusions:

Figures

Introduction

Materials and methods

Sample collection

Metagenome assembling and binning

Identification of selected sulfur oxidation genes and autotrophy

Phylogeny of selected genes

Calculation of the relative abundance of scaffolds, functional genes, and MAGs

Measurement of sulfide, zerovalent sulfur, thiosulfate, sulfite, and sulfate in sediment

Incubation with sulfide, thiosulfate, and sulfite

Incubation for metatranscriptomic analysis and metatranscriptomic data analysis

Results

Overview of sulfur oxidation pathways in the Bohai Sea sediments

Heterotrophy dominates sulfur oxidation in the Bohai Sea sediments

Microbial network for sulfur oxidation

Sulfur species in the sediments

Estimation of the maximal potential of sulfur oxidation in the sediments

Metatranscripts during the incubation with sulfide

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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