Introduction

In productive aquatic systems, the remineralization of organic matter regularly leads to oxygen (O2) deficiency and finally to anoxia or even euxinia. This transition is accompanied by the establishment of a pelagic redoxcline that separates the oxic surface from reducing bottom waters. Meromictic lakes are especially prone to such developments but semi-restricted water bodies, such as fjords and brackish/marine and hypersaline basins, are vulnerable as well [1,2,3,4,5]. The Black Sea is the world’s largest semi-enclosed basin and the type-locality for density-stratified aquatic systems, as severe bottom water euxinia is prevalent since ~8000 years [6, 7]. In the Black Sea redoxcline, a layer in which O2 and sulfide (S2−, here defined as the sum of H2S, HS−, and S2−) are virtually absent, referred to as the “suboxic zone,” has been frequently reported since 1989 [1, 6, 8,9,10,11]. This zone may span tens of meters but it is not well-defined, as some descriptions also encompass waters with low concentrations of O2 (<5 µM [8]; <3 µM [12]; <1 µM [13]) and S2− (<0.2 µM [12]; <0.1 µM [13]). In the following, we refer to the zone of transition from oxic to anoxic and euxinic water conditions as the redoxcline.

Due to the separation of O2 and often nitrate (NO3−) and nitrite (NO2−) from S2− in the redoxcline of the Black Sea, the depletion of S2− at the upper boundary of euxinic water (also known as chemocline) has been attributed to lateral intrusions of oxygenated waters [12, 14] or to the intense cycling of the redox-sensitive trace metal manganese (Mn) [10, 15]. In their one-dimensional biogeochemical model, Yakushev et al. [16] reproduced the vertical geochemical water column profile measured in the Black Sea without lateral intrusion, instead identifying reduced and oxidized Mn species as the main drivers of O2 reduction and S2− oxidation in the redoxcline. Although lateral intrusions from the Bosporus plume may affect the redoxcline in the far southwestern part of the Black Sea [14], extensive data gathered over a two-year period by Stanev et al. using Argo floats [11] support this one-dimensional model and the importance of a manganese shuttle.

During Mn-cycling, dissolved Mn2+ and intermediate Mn3+ are transported upwards by diffusion [1, 12, 15, 17, 18], and thus from reducing towards oxygenated waters, where both Mn species are then oxidized [19]. The resulting Mn (IV) oxide particles (here referred to as MnO2) sink back towards the underlying euxinic water [20], where they are reduced to dissolved Mn3+ and Mn2+ by S2− and/or Fe2+ [21]. The chemical oxidation of S2− by MnO2 in laboratory experiments was shown to occur rapidly (within minutes) [22, 23]. However, in those studies, MnO2 was supplied in millimolar concentrations, i.e., several magnitudes larger than the natural levels in the Black Sea [1, 9, 10]. Because the chemical oxidation of S2− by MnO2 follows second-order reaction kinetics, such that the concentrations of both reactants affect the overall speed of the reaction [23], the laboratory results on chemical oxidation may not represent the in situ processes in the Black Sea.

The recent description of S2− oxidation coupled to the reduction of MnO2 by the Black Sea isolate Candidatus Sulfurimonas marisnigri SoZ1 (phylum Campylobacterota, class Campylobacteria [24]), isolated from the upper boundary of euxinic water, supports a biological mode of S2− oxidation in the absence of O2 and NO3− [25]. In laboratory experiments, Ca. S. marisnigri SoZ1 oxidized S2−, elemental sulfur (S0), and thiosulfate (S2O32−) to sulfate (SO42−), with MnO2 as the sole electron acceptor [25, 26]. The reduction of MnO2 proceeded by its conversion to Mn3+ and further to Mn2+, which precipitated as Ca-rich Mn-carbonate [25]. A biological mode of S2− oxidation with MnO2 may therefore account for the observed accumulation of the intermediate Mn3+ in the redoxcline [1, 12], for the chemosynthetic production of Campylobacterota in the absence of O2 and NO3− [27], and for the maintenance of anoxic and non-sulfidic zones in the redoxcline. However, in the absence of data on the abundance, activity, and taxonomy of putative S-oxidizing and Mn-reducing bacteria, the impact of Mn-dependent S oxidation on the water column geochemistry of the Black Sea is unclear and unquantified.

Our study investigated the potential contribution of extent microbial S2− oxidation by Sulfurimonas spp., using MnO2 as terminal electron acceptor, to the overall S2− oxidation in the redoxcline of the Black Sea. For this purpose, cell-specific S2− oxidation rates with MnO2 by the representative isolate Ca. S. marisnigri SoZ1 (=JCM 39139; =DSM 111879) were determined in laboratory experiments together with assessments of the in situ abundances and gene expression of Sulfurimonas spp. across the redoxcline of the Black Sea. The cell-specific oxidation rates and the in situ abundance were then combined to model the S2−-concentration profile in the Black Sea. The results provide strong evidence that the activity of Sulfurimonas spp. contributes significantly to anaerobic sulfide oxidation and to the formation of a sulfide-free, anoxic zone, both in the Black Sea and potentially in other redox-stratified systems.

Material and methods

The materials and methods are described here in brief. A detailed description of the Material and methods can be found in the supplementary information.

Field sampling

Samples were taken during a cruise with the R/V “Maria S. Merian” (MSM33) in November/December 2013 and sampling was performed as described previously [9]. All data presented herein originated from station 32, located in the western central gyre of the Black Sea (43° 31.922′ N, 32° 30.909′ E; water depth 2070 m), with the exception of the high-resolution data on total dissolved Mn (Mndiss) and dissolved reactive Mn (dMnreact; [18]), obtained from station 66 (43° 31.8019′ N, 36° 05.9960′ E; water depth 2177 m). The characteristics of the water columns of stations 32 and 66 were similar, as indicated by the disappearances of O2 at densities (σθ) of 15.93 and 15.94 and of S2− at σθ of 16.15 and 16.16, respectively. The profiles of O2 and S2− at both stations were vertically separated by approx. 15 m thick layer (shown in Fig. 1a for station 32). Water column profiles from successive casts were constructed by aligning the data according to σθ of cast P0014F13 (station 32), as done previously [9].

Fig. 1: Vertical water-column profile of the Black Sea redoxcline at station 32.
figure 1

Horizontal dashed lines indicate the approximate boundaries of the redoxcline. a Oxygen (O2) and sulfide (S2−). b nitrate (NO3−), nitrite (NO2−), and ammonia (NH4+). c particulate manganese (Mnpart.), total dissolved manganese (Mndiss.), and dissolved reactive manganese (Mnreact ≃ Mn3+ [1, 12, 18]). d Cellular abundance of cells positively stained with CARD-FISH probes EPSY914 (Campylobacterota) and EUB338 (Bacteria) and with the DNA dye DAPI (total cells). The relative abundance of EPSY914-positive cells vs. total DAPI-stained cells is expressed as a percent. e Total transcript abundance of the sulfide:quinone oxidoreductase gene (sqr). The relative abundance of sqr transcripts expressed by Sulfurimonas spp. is expressed as a percent. The data shown in a and b are from Schulz-Vogt et al. [9]. In c, the larger black-enclosed circles and solid lines represent data from bottle casts at station 32; the small non-enclosed circles represent data from high-resolution pump CTD profiles taken at station 66 and aligned according to density.

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Gases, nutrients, and metals

Data for the water column profiles of dissolved gases (O2, H2S) and nutrients (NO3−, NO2−, NH4+) were taken from Schulz-Vogt et al. [9]. The concentrations of Mndiss and dMnreact as well as those of particulate Mn (Mnpart) were measured by inductively coupled plasma optical emission spectrometry, according to the protocols reported in [18] and [10].

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH)

CARD-FISH was carried out following the protocols of Pernthaler et al. [28] and Sekar et al. [29], modified as described in [30]. For the enumeration of Bacteria, a mixture of horseradish-peroxidase-labeled oligonucleotide probes (EUB338, EUB338-II, EUB338-III [31]) was used. Campylobacterota were enumerated using probe EPSY914 [27]. Non-specific binding was determined using the NonEUB probe [32]. Filter sections were counterstained with DAPI (1 mg ml−1) and inspected using an epifluorescence microscope (Axioscope, Carl Zeiss) together with filter sets 01 (DAPI) and 10 (Alexa 488).

Sampling, processing, and data analysis of metagenomic and metatranscriptomic data

Amplicon sequencing of the 16S rRNA gene and 16S rRNA

Samples for 16S rRNA gene and 16S rRNA amplicon sequencing were extracted using the AllPrep DNA/RNA kit (Qiagen; Hilden, Germany). DNA extracts were stored directly; from the extracted RNA, a maximum of 100 ng was DNase-treated using the Turbo DNA-free kit (Thermo Fisher Scientific; Waltham, MA, USA). The DNase-treated RNA (max. 20 ng) was reverse transcribed using Multiscribe RT (Thermo Fisher Scientific). All samples of DNA and treated RNA, including additional Mock Community samples from Zymo Research (Freiburg, Germany) as controls, were sent to LGC Genomics (Berlin, Germany) for sequencing with MiSeq (Illumina). The resulting sequences were analyzed using the SILVA_NGS pipeline (release 138.1) [33, 34], with settings and OTU clustering based on 97% similarity, as described previously [35].

Metagenomic and metatranscriptomic analyses

Sampling and processing were done as described [9]. Genes annotated as Campylobacterota were extracted and sulfur oxidation genes were identified based on functional annotation, sequence similarity, and gene synteny (detailed description in the supplementary information). The identified genes were taxonomically annotated using CAT (v5.2.3), based on the Diamond aligner (v2.0.8.147 [36]) in blastp mode against the NCBI’s NR database (as of July 1, 2021), with the r-parameter set to 3 [37].

Estimation of the rates of S2− oxidation with MnO2 by Ca. S. marisnigri SoZ1

The rates of biological S2− oxidation with MnO2 by the isolate Ca. S. marisnigri SoZ1 were determined by spiking anoxic, constantly stirred medium containing MnO2 and Ca. S. marisnigri SoZ1 or no bacteria either five (sterile controls) or eight (with Ca. S. marisnigri SoZ1) times with Na2S to obtain S2− concentrations of 20–30 µM. The decline in the S2− concentration was documented in three replicates for each treatment using an H2S and pH microsensor (Unisense, Aarhus, Denmark). The reactions in the replicates were then inhibited either by the addition of sodium azide (not shown) or by pasteurization, followed by another three additions of Na2S. The H2S concentrations measured in the experiment were corrected for temperature, salinity, and pH to obtain the S2− values.

The overall reaction can be described by Eq. 1, which can be transposed to Eq. 2 by normalizing the S2− concentration at time t to the S2− concentration at time t0.

$$\left[ {{{{{\rm{S}}}}}^{2 - }} \right]_t = \left[ {{{{{\rm{S}}}}}^{2 - }} \right]_0e^{\left( {a + kt} \right)}$$
(1)
$$\frac{{\left[ {{{{{\rm{S}}}}}^{2 - }} \right]_t}}{{\left[ {{{{{\rm{S}}}}}^{2 - }} \right]_0}} = e^{\left( {a + kt} \right)}$$
(2)

where [S2−]t is the concentration of S2− at time t; [S2−]0 is the initial concentration of S2−, a as a correction variable for the y-intercept; k is the reaction rate coefficient (s−1); and t is the time in seconds.

The non-linear least-squares fit of Eq. 2 was performed with R (version 3.5.1) for individual spikes of Na2S to determine the overall reaction rate coefficient k. The biological reaction rate (kbio) was calculated by subtracting the value of k after pasteurization from the overall reaction rate coefficient k before pasteurization. The value of kbio was divided by the cellular abundance of Ca. S. marisnigri SoZ1 to obtain a cell-specific reaction rate coefficient (kcell), with the mean value (kcell = −1.05 × 10−13 l cell−1 s−1) used in subsequent numerical modeling.

Modeling the S2− concentration profile of the Black Sea

The impact of S2− oxidation activity by Sulfurimonas spp. on the geochemical water column profile in the Black Sea was estimated by combining data on the abundance of Sulfurimonas spp. in the Black Sea and the cell-specific S2− oxidation rates of Ca. S. marisnigri SoZ1 in a numerical model. In the modeling approach of [38], the differential equation for diffusive transport is described by Eq. 3:

$$\delta c/\delta t = D\delta ^2c/\delta x^2$$
(3)

where D is the diffusion coefficient; c is the concentration; t is the time; and x is the distance-coordinate. The equation is solved using the explicit numerical solution obtained with Eq. 4, adapted from [9]:

$$C_{S^{2 - }(x,t + \Delta t)} = {C_{S^{2 - }\left( {x,t} \right)} + \frac{{\Delta t \times D_x \times \left( {C_{S^{2 - }\left( {x + \Delta x,t} \right)} - 2 \times C_{S^{2 - }\left( {x,t} \right)} + C_{S^{2 - }\left( {x - \Delta x,t} \right)}} \right)}}{{\Delta x^2}} + }$$
(4)
$$\Delta t \times k_{{{{{\rm{cell}}}}}} \times CA_{{{{{\rm{EPSY914}}}}}\left( x \right)} \times C_{S^{2 - }\left( {x,t} \right)} \times F_{{{{{\rm{Sulfurimonas}}}}}\,{{{{\rm{in}}}}}\,{{{{\rm{Campylobacterota}}}}}}$$
(4.1)
$$\Delta t \times k_{{{{{\rm{chem}}}}}} \times C_{S^{2 - }(x,t)}$$
(4.2)

where CS2− is the concentration of S2− at a given water depth x and time t, Dx is the diapycnal diffusivity at water depth x, as determined in [17] (we set 4 × 10−6 or 1 × 10−6 m2 s−1 as the upper and lower limits of diapycnal diffusivity). The consumption of S2− during biological or chemical oxidation was taken into account in Eq. 4, by adding either Eq. 4.1 or Eq. 4.2, respectively. The rate of biological S2− oxidation was calculated by multiplying the cell-specific reaction rate coefficient of Ca. S. marisnigri SoZ1 (kcell = −1.05 × 10−13 l cell−1 s−1) by the cellular abundance of Sulfurimonas spp. and the local S2− concentration. The chemical S2− oxidation rate was calculated using a chemical reaction rate coefficient (kchem = −9.53 × 10−8 s−1 [23] based on a constant MnO2 concentration of 10 nM, pH 7, and 10 °C) and the local S2− concentration. The starting condition of the model was 10 µM S2− at 120-m water depth. Oxidation at the upper boundary of euxinic water (106 m water depth) and above was assumed based on the vertical concentration profile of S2− (see the “Discussion” for details). The Excel-based spreadsheet of the model is provided in the Supplementary Material.

Growth of Ca. S. marisnigri SoZ1 with S2− and MnO2 in semi-continuous culture

The semi-continuous culture experiment was performed by [25] to identify the reaction end-products of MnO2 and S2− under sterile conditions and in the presence of Ca. S. marisnigri SoZ1. The method is described in the supplement. In the experiment, the cellular abundance of Ca. S. marisnigri SoZ1 was proportional to the applied S2− flux, and the S2− flux per cell was calculated to allow comparisons with the S2− fluxes in the upper boundary of euxinic water in the Black Sea redoxcline.

Results

Geochemical structure of the pelagic redoxcline of the Black Sea

In the central western gyre of the Black Sea, the zone where O2 and S2− became undetectable (<0.2 µM O2, <1 µM S2−) was located roughly between 90 and 105 m water depth (Fig. 1a). Concentrations of O2 above and S2− below this zone increased steadily. Analyses conducted using an ultra-low switchable trace-oxygen sensor (STOX) [41], with a detection limit of <10 nM, did not detect O2 traces below the NO2− peak at 92 m water depth. Thus, at the time of sampling the redoxcline below 92 m was defined as anoxic. The concentration of NO3− decreased continuously from 80 m to 90 m water depth, followed by a steeper slope from 90 m, where the O2 concentration fell to < 0.5 µM, to 95 m, where NO3− was depleted (Fig. 1b). The decrease in NO3− between 90 and 95 m water depth coincided with the marked production of NO2−. Ammonia (NH4+) decreased linearly from the euxinic water towards 95 m water depth, where it became undetectable (Fig. 1b).

Particulate Mn concentrations were highest (50 nM) in the upper part of the redoxcline, where O2 became depleted, with a second peak (16 nM) at ~104 m water depth (Fig. 1c). With the exception of the second peak, the concentration of Mnpart. generally decreased with increasing water depth. By contrast, Mndiss. increased continuously beginning at ~95 m water depth and continuing into euxinic water, reaching 6 µM at 110 m water depth. Since high-resolution data for Mndiss and dMnreact were not available for the station in the central western gyre, data from the central eastern gyre (2177 m water depth) were aligned according to density and shown instead. In agreement with Trouwborst et al. [12] and Dellwig et al. [1], Mndiss. within the redoxcline was almost entirely composed of the intermediate Mn3+, measured in this study indirectly as dMnreact. [18]. The concentration of dMnreact. peaked at the upper boundary of euxinic water at ~105 m water depth and then decreased sharply below, that is, in the first few meters of euxinic water.

Cellular abundance and S2− oxidation activity in the redoxcline of the Black Sea

Total cellular abundance at station 32 increased from ~2 × 108 cells l−1 above and below the redoxcline to ~3 × 108 cells l−1 in the intervening layer (Fig. 1d). Likewise, total bacterial cell counts determined using the FISH probe EUB338[I-III] were elevated within the redoxcline and accounted for 50 to 89% of the total cell counts, depending on the water depth. The abundance of Campylobacterota increased steadily towards the euxinic water, from ~2 × 106 cells l−1 in oxic water to ~4 × 107 cells l−1 at the upper boundary of euxinic water, with the highest abundance (6.5 × 107 cells l−1) occurring at 100 m water depth (Fig. 1d). The relative abundance of Campylobacterota (% of DAPI counts) increased as well, from >1% at the upper boundary of the redoxcline at 89 m to ~15% at the upper boundary of euxinic water at 106 m water depth (Fig. 1d).

Transcription of the sulfide:quinone oxidoreductase gene (sqr), a key enzyme in the oxidation of S2− to S0 and polysulfides [42], was used as a molecular marker of bacterial S2− oxidation across the redoxcline. The expression of sqr was detectable at all sampled water depths and increased across the redoxcline by more than two orders of magnitude, from 2.6 × 105 transcripts l−1 in oxic water to 3.8 × 107 transcripts l−1 at the upper boundary of euxinic water (~105 m), before undergoing a steep decline (by 93%) below (~110 m) (Fig. 1e). Because we chose a conservative method for the taxonomic annotation of sqr and other sulfur oxidation genes (CAT with r-parameter set to 3), the taxonomic annotation of these genes was restricted to the family level (Helicobacteraceae), but with high confidence. However, since Sulfurimonas spp. comprised 99.9–100% of 16S rRNA gene reads related to the family Helicobacteraceae, transcription of sqr could be assigned exclusively to the genus Sulfurimonas. The relative transcription level of sqr by Sulfurimonas spp. for all detected sqr transcripts increased with water depth, from 29.6% at ~86 m to 92.9% at ~110 m. At ~105 m, Sulfurimonas spp. expressed 89.2% of the total sqr transcripts.

Sequence-based abundance and the taxonomic structure of the phylum Campylobacterota

Relative 16S rRNA gene and 16S rRNA abundances of Camplyobacterota increased steadily across the redoxcline (Fig. 2), in line with the results of microscopic counting using the CARD-FISH probe EPSY914, targeting the phylum Campylobacterota (Fig. 1d). The relative abundance of Campylobacterota-related 16S rRNA gene and 16S rRNA reads increased over a water depth of ~82–110 m, from ~0.1% to ~ 15% and from ~2% to ~35%, respectively (Fig. 2, Tables S1 and S2). Within Campylobacterota-specific reads, those belonging to Sulfurimonas spp. dominated, accounting for ~70% (16S rRNA gene) and >95% (16S rRNA) at 82 m water depth and >96% (16S rRNA gene) and >99% (16S rRNA) at the upper boundary of euxinic water (Tables S3 and S4). For Sulfurimonas spp., relative 16S rRNA abundance was 2.5 times higher than the corresponding relative abundance of the 16S rRNA gene in the same sample (R2 = 0.92). According to these findings, Sulfurimonas spp. accounted for almost all of the phylum Campylobacterota and was metabolically active across the redoxcline.

Fig. 2: Taxonomic composition of the phylum Campylobacterota in the redoxcline of the Black Sea.
figure 2

Relative 16S rRNA gene (a) and 16S rRNA (b) abundance of the phylum Campylobacterota across the redoxcline at station 32 from three density-aligned consecutive CTD casts shown at 1-m resolution. The relative abundances of the 16S rRNA gene and of Campylobacterota-related 16S rRNA gene reads increased across the redoxcline towards euxinic bottom waters. Relative abundances of the 16S rRNA gene and of 16S rRNA of Sulfurimonas spp. (blue) increased with depth, from < 1% to ~14% and from ~2% to > 35%, respectively (Tables S1 and S2). The relative read abundance of Sulfurimonas spp. within Campylobacterota increased from ~70% to ~99% across the redoxcline, based on 16S rRNA gene abundance; 16S rRNA abundance was always > 95% (Tables S3 and S4). Relative bacterial abundance of the family Arcobacteraceae (red) and the remaining Campylobacterota-related reads (others, green) were low (Arcobacteraceae) or too low to be visible in the figure (others).

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Sulfur oxidation activity of Sulfurimonas spp. in the Black Sea redoxcline

The analysis of the S oxidation genes expressed by Sulfurimonas spp. revealed, besides sqr, two sulfur oxidation (sox) clusters (soxXYZAB and soxCDYZH), polysulfide reductase (psrABC), and sulfite:cytochrome c oxidoreductase (sorAB) (Fig. 3). In agreement with the water column profile of S2−, the expression of these genes was highest at the upper boundary of euxinic water (105 m water depth; Fig. 3a–f). The expression of soxCDYHZH was approximately two orders of magnitude higher than that of soxXYZAB (Fig. 3a, b). Transcripts of soxY and soxZ were also detected on small contigs that could not be assigned to cluster soxXYZAB or soxCDYZH, respectively (uncertain loci, Fig. 3c, i). The transcript abundance and vertical expression pattern of soxCDYZH were similar to those of sqr (Fig. 3b, d), suggesting that S2− oxidation by sqr continued via soxCDYZH. The cellular-abundance-related transcription of Sulfurimonas spp.-annotated genes was calculated by dividing the respective transcript numbers by the cellular abundance of Sulfurimonas spp., determined by multiplying the relative 16S rRNA gene abundance of Sulfurimonas by the total cellular abundance of DAPI-stained cells. Cellular-abundance-related (normalized) transcript numbers indicated that sqr and soxCDYZH were equally expressed also in the upper part of the redoxcline towards oxic water (Fig. 3h, j). The normalized expression patterns of psrABC (Fig. 3k) and sorAB (Fig. 3l) did not resemble or were far less obvious than those of sqr and soxCDYZH, suggesting that psrABC and sorAB activities were not directly coupled to the activities of sqr and soxCDYZH.

Fig. 3: Transcription of the S oxidation genes across the redoxcline with taxonomic annotation to the genus Sulfurimonas.
figure 3

a–f Total number of transcripts expressed by Sulfurimonas spp.; g–l transcript abundance, normalized to the cellular abundance of Sulfurimonas spp. determined in Campylobacterota by CARD-FISH (probe EPSY914, Fig. 1d), multiplied by the relative 16S rRNA gene abundance of Sulfurimonas spp. within Camplyobacterota-specific reads (Table S1). Sampled depths are displayed in (a) and (g).

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Rates of manganese-oxide-mediated sulfide oxidation by Ca. S. marisnigri SoZ1 in lab experiments

The slope and linear shape of the vertical concentration profile of S2− (Fig. 1a) indicated high S2− oxidation rates at the upper boundary of euxinic water (~105 m) in the absence of O2 and NO3− (Fig. 1a, b) but in the presence of MnO2 as an available electron acceptor for S2− oxidation (Fig. 1c). The potential impact of biological S2− oxidation on total S2− oxidation was quantified in a lab experiment in which the rate of S2− oxidation by Ca. S. marisnigri SoZ1 using MnO2 as the electron acceptor was determined (Fig. 4). In biological treatments containing ~5 × 107 Ca. S. marisnigri SoZ1 cells ml−1, ~30 µM S2− was removed within 10 min, compared to ~45 min in the abiotic controls (Fig. 4a). After thermal inhibition by pasteurization (Fig. 4a, red arrow), the speed of S2− removal in the biological treatments was indistinguishable from that in the abiotic treatments. The reaction rate coefficient k for the consumption of each S2− spike was calculated individually (Fig. 4b) and showed that pasteurization lowered the reaction rate to the chemical background level (Fig. 4c). The biological reaction rate coefficient (kbio) was calculated by subtracting the S2− removal rate after pasteurization from the overall rate before pasteurization; dividing the result by the cellular abundance of Ca. S. marisnigri SoZ1 in the respective replicate yielded the cell-specific kinetic rate coefficient (kcell). The mean value of kcell (−1.05 × 10−13 l cell−1 s−1) was used in downstream numerical modeling. To illustrate, with 9 × 105 Sulfurimonas spp. cells l−1, the biological S2− oxidation rate in the model would be equal to that of chemical oxidation (kchem), which is described below.

Fig. 4: Rates of MnO2-mediated sulfide oxidation by Ca. S. marisnigri SoZ1.
figure 4

a Consumption of S2− in medium containing MnO2 and Ca. S. marisnigri SoZ1, was recorded using H2S microsensors. After pasteurization of the samples (red arrow), S2− removal was considerably slower. b Calculation of the reaction rate coefficient k (s−1) in response to individual S2− spikes and based on the normalized S2− concentrations with a non-linear least-squares fit using the displayed equation. c Reaction rate coefficients for the biological treatment with Ca. S. marisnigri SoZ1 and the sterile control, before (blue) and after (red) sample pasteurization (mean ± SD). N refers to the total number of Na2S spikes as recorded in three replicates for each biological treatment and sterile control.

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Modeling sulfide oxidation in the Black Sea redoxcline

The potential impact of S2− oxidation by Sulfurimonas spp. on the vertical concentration profile of S2− (Fig. 1a) was estimated by assuming either biological or chemical S2− oxidation on the diffusive supply of S2− from euxinic water at the upper boundary of those waters and above (Fig. 5). For the biological model, the cell-specific S2− oxidation rate kinetics (kcell) estimated with Ca. S. marisnigri SoZ1 (Fig. 4) were combined with the natural abundance of Sulfurimonas spp. (Fig. 5a). Chemical oxidation was considered as constant, with kchem = −9.53 × 10−8 s−1 (calculated after [23] with 10 nM MnO2, pH 7, and 10 °C).

Fig. 5: Modeling the S2− concentration profile across the redoxcline of the Black Sea.
figure 5

Results of the modeling of the S2− concentration profile in the Black Sea based on biological S2− oxidation catalyzed by Sulfurimonas spp. a, b or chemical oxidation with MnO2 (c, d) with a diapycnal diffusivity coefficient Dx of either 1 × 10−6 m2 s−1 (violet) or 4 × 10−6 m2 s−1 (dark green). The in situ S2− concentration profile (gray dots) was taken from Schulz-Vogt et al. [9], using data originating from the same sampling campaign and same station. Modeled data of the S2− concentration (a, c) and S2− oxidation rate (b, d) are shown. The abundance of Sulfurimonas spp. (black diamonds) and the interpolated abundance (blue) in the biological model were calculated as the product of EPSY914-positive cell counts and the relative 16S rRNA gene abundance of Sulfurimonas spp. within the total reads annotated as Campylobacterota.

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A steady state was reached in both the chemical and the biological models, but the resulting S2− concentration profiles differed (Fig. 5a, c). In the biological model (Fig. 5a, b), 95% of the modeled S2− oxidation activity across the redoxcline occurred over a narrow depth horizon, between ~105 m and ~106 m (Dx = 1 × 10−6 m2 s−1; Fig. 5b, violet) or ~103 m and ~106 m (Dx = 4 × 10−6 m2 s−1; Fig. 5b, dark green) water depth, due to the in situ distribution of Sulfurimonas. This led to the almost linear shape of the modeled S2− concentration profile (Fig. 5a), which was similar to the observed one (gray dots). The expression of sqr by Sulfurimonas spp. across the redoxcline was in line with the modeled S2− oxidation rate (Fig. 5b, black triangles). The modeled S2−-concentration profile based on a purely abiotic reaction deviated from the measured data (Fig. 5c). In particular, abiotic S2− oxidation rates (Fig. 5d) at the upper boundary of euxinic water were at least one order of magnitude lower than the biological S2− oxidation rates (Fig. 5b) and too low to counterbalance S2− fluxes from euxinic water. In the model based on chemical oxidation, 95% of S2− oxidation across the redoxcline occurred over a broad depth horizon, between ~97 m and ~106 m (Dx = 1 × 10−6 m2 s−1; Fig. 5d, violet) or ~90 m and 106 m (Dx = 4 × 10−6 m2 s−1; Fig. 5d, dark green) water depth, which resulted in a large curvature in the S2− profile (Fig. 5c).

Discussion

Field-based indications of S oxidation with MnO2 catalyzed by Sulfurimonas spp. in the Black Sea redoxcline

In line with earlier research, an anoxic zone free of O2 and S2− spanning ~15 m was detected within the pelagic redoxcline of the western central gyre (Fig. 1a) [10, 11, 43]. Putative O2 contamination was avoided during sampling by measuring O2 directly in the outflow of the pump-CTD system using custom-made micro-sensors within glass tubes. O2 traces below 92 m water depth could not be detected, even by the ultra-sensitive O2 STOX sensors [41] (Fig. 1a). Thus, the redoxcline below 92 m water depth was considered to be completely anoxic. Vertical profiles of dissolved and particulate Mn species indicated the active shuttling of redox equivalents across the redoxcline (Fig. 1c), thereby connecting oxic and euxinic water as previously shown [1, 10, 15, 21]. Based on the S2− concentration profile, S2− oxidation was presumed to proceed at the upper boundary of euxinic water and above, because the linear shape of the profile indicated diffusive transport of S2− towards the redoxcline, with no net production or consumption [44]. Across the redoxcline, Mndiss. mainly consisted of Mn3+ (Fig. 1c), in agreement with previous results [1, 10, 12]. Whether Mn3+ was generated via biological or chemical reduction could not be determined. However, the reduction of MnO2 with S2O32− by Ca. S. marisnigri SoZ1 in laboratory experiments resulted in the accumulation of Mn3+ before Mn(II) precipitated as Ca-rich Mn-carbonate [25], suggesting that Mn3+ is generated biologically by MnO2 reduction with S2−. The Ca-rich Mn-carbonate that formed in culture [25] may also have been the source of the often-observed second peak of Mnpart occurring at the lower boundary of the redoxcline [10, 45, 46], since Mn3+/2+ oxidation to particulate MnO2 would have been unlikely given the absence of oxidants.

In line with the model’s results, sqr transcription, as an indicator of biological oxidation, peaked at the upper boundary of euxinic water, where the bulk of S2− oxidation activity would be expected, and dropped sharply within euxinic water (Fig. 1e). The sox gene cluster soxCDYZH [47,48,49] was highly expressed as well (Fig. 3b, d), indicating its functional interaction with sqr. A previous study reported up-regulation of the soxCDYZH cluster under S2− and S0 oxidizing conditions [47]. The tetrameric complex Sox(CD)2 oxidizes the sulfane sulfur bound to SoxYZ to sulfone sulfur, which is hydrolytically released as SO42− by SoxB [42, 50]. However, soxB was hardly expressed (Fig. 3a), such that SoxH in the cluster soxCDYZH likely acted as a hydrolase homolog of SoxB, as suggested earlier [47]. The low-level expression of cluster soxXYZAB (Fig. 3a) indicated that S2O32− was not an important intermediate for Sulfurimonas spp. at the upper boundary of euxinic water in the Black Sea water column [47, 51,52,53]. The expression of psrABC indicated S0 reduction [54], possibly using the S0 formed, for example, by the abiotic oxidation of S2− with MnO2 [22, 55, 56] prior to Sqr- and SoxCDYZH-mediated oxidation. In summary, the pattern of gene expression by Sulfurimonas spp. was consistent with the complete oxidation of S2− and S-intermediates to SO42− at the upper boundary of euxinic water and in the absence of O2, NO3−, and NO2−.

Model-derived indication for S2− oxidation with MnO2 catalyzed by Sulfurimonas spp. in the Black Sea redoxcline

The cell-specific S2− oxidation rates of Ca. S. marisnigri SoZ1 obtained in the lab experiments were fast enough to account for the S2− concentration profile in the Black Sea obtained in a one-dimensional numerical model based on the in situ abundance of Sulfurimonas spp. (Fig. 5a, b). By contrast, the chemical oxidation of S2− with MnO2 was about one order of magnitude slower (Fig. 5b, d), which resulted in a S2− concentration profile with a large curvature (Fig. 5c). Therefore, S2− oxidation was most likely biological, as also suggested by Mn3+ formation (Fig. 1c; [12]), the low concentrations of S-compounds in intermediate oxidation states [57, 58], and the chemosynthetic activity of Campylobacterota in the absence of O2 and NO3− [27].

Nonetheless, our results need to be interpreted cautiously, because the underlying model is a simplification based on several assumptions. First, S2− oxidation was assumed to occur at the upper boundary of euxinic water and above, based on the measured linear concentration profile of S2−, which indicated a diffusive flux towards the redoxcline without net production or consumption below [44]. The gross oxidation of S2− within and below the redoxcline may in fact be higher, with a rate equal to that of S2− production via SO42− reduction [13]. This would have led to a cryptic cycle not represented by the model. However, the finding that sqr expression fell off sharply below the redoxcline (110 m water depth), indicating negligible biological S2− oxidation activity via the Sqr pathway, supports the assumption that S2− oxidation proceeds at the upper boundary of euxinic water and above (Fig. 1e). Second, for the chemical oxidation, a realistic MnO2 concentration of 10 nM was assumed in the abiotic model. However, even a ten-fold higher concentration was still too low to reproduce the observed profile. The latter therefore implied rapid S2− oxidation and thus a biologically catalyzed process. Lastly, other potential oxidants than MnO2 have been excluded in the laboratory experiments but might be present in undetectable concentrations in the Black Sea. However, this is rather unlikely based on the data presented in Fig. 1.

Estimation of the contribution of Mn-dependent S2− oxidation to the total oxidation of S2− requires a quantification of the downward-directed flux of MnO2 particles and the upward-directed flux of S2−. The downward-directed flux of particulate MnO2 should be equal to the upward-directed flux of Mndiss., because once the latter is oxidized to particulate Mn(IV), its movement is unidirectional, in the form of gravitational sinking [15, 20, 21]. For a Mndiss. concentration gradient of 0.32 mmol m−3 m−1, the corresponding flux would be in the range of 3.1 × 10−7–1.2 × 10−6 mmol Mndiss. m−2 s−1 (Dx of 1 or 4 × 10−6 m−2 s−1 [17]), although due to riverine inputs of Mn the real flux might be slightly higher [59]. The S2− flux of 6.7 × 10−7–2.68 × 10−6 mmol S2− m−2 s−1, based on a S2− gradient of 0.67 mmol m−3 m−1 [9], is in agreement with the values reported by Brewer and Spencer (2.31 × 10−6 mmol S2− m−2 s−1; [60]), Jørgensen et al. (5.1 × 10−7–3.3 × 10−6 mmol S2− m−2 s−1; [58], calculated based on a diapycnal diffusivity coefficient of 1 or 4 × 10−6 m−2 s−1), and by Fuchsman et al. (1.24 × 10−6 mmol S2− m−2 s−1; [61]). Assuming both the complete reduction of MnO2 to Mn(II) and the oxidation of S2− to SO42− (and no quantitative importance of MnO2 reduction by Fe2+ [21]), roughly 25% of the total S2− oxidation can be explained by MnO2. However, some of the electrons derived from S2− oxidation are needed for chemosynthesis by Sulfurimonas. Based on the Mn(IV)/Mn(II) to S2O32−/SO42− ratio of 3.7 determined in growth experiments with Ca. S. marisnigri SoZ1 [25], 7.5% of the electrons from S are used for CO2 reduction to support autotrophic growth, although larger proportions, up to 20%, have been reported [62]. Chemoautotrophic growth by Mn-reducing chemolithoautotrophic bacteria could therefore account for another 1–11% of the oxidation of the S2− flux, depending on the assumed Mn flux and whether 7.5% or 20% of the electrons derived from S2− are used for CO2 reduction. In summary, as much as one-third of the total S2− flux may be utilized by chemolithoautotrophic Mn-dependent S2− oxidation by Sulfurimonas.

Lab-experiment-derived indications of S2− oxidation with MnO2 by Sulfurimonas spp. in the Black Sea redoxcline

Sulfurimonas spp.-mediated S2− oxidation with MnO2 in the Black Sea is further supported by the experimental data of Henkel et al. [25], obtained in an experiment in which the continuous addition of S2− to MnO2-spiked medium inoculated with Ca. S. marisnigri SoZ1 resulted in bacterial growth and the accumulation of SO42−. With Na2S addition reproducing a flux of 1 × 10−3 mmol S2− m−2 s−1 or 2.5 × 10−3 mmol Na2S m−2 s−1, Ca. S. marisnigri SoZ1 abundance plateaued at 1.1 × 1013 or 2.2 × 1013 cells m−3, the equivalent of 3.4 × 10−17 or 11.6 × 10−17 mmol S2− cell−1 m−2 s−1, respectively. In the Black Sea, the S2− flux of 6.7 × 10−7–2.68 × 10−6 mmol S2− m−2 s−1 from euxinic water feeds the S2−-oxidizing community at the upper boundary of euxinic water. At a cell density of 4 × 1010 Sulfurimonas spp. cells m−3 (Fig. 5a), a single Sulfurimonas spp. cell is fed by a S2− flux of 1.68 to 6.70 × 10−17 mmol S2− m−2 s−1. The overall order of magnitude of the S2− flux needed to feed a single cell in lab experiments and in the Black Sea was surprisingly similar, indicating that the natural abundance of Sulfurimonas spp. is consistent with the observed input flux of S2− and MnO2.

Elevated abundance of Sulfurimonas spp. as an indicator of the quantitative importance of Mn-dependent S2− oxidation in the Black Sea redoxcline

The present study identified Sulfurimonas spp. as a key player in S2− oxidation in the redoxcline of the Black Sea, but their activity may not explain total S2− consumption. Along with Campylobacterota or Sulfurimonas spp., gammaproteobacterial sulfur oxidizers (GSO) of clades SUP05 and BS-GSO2 may contribute quantitatively to S2− oxidation in the Black Sea [13, 27, 61, 63, 64]. In the isolate Candidatus Thioglobus autotrophicus EF1 (clade SUP05), S oxidation is coupled to the incomplete reduction of NO3− to NO2− [65], in agreement with the lack of nitrite reductases in its genome. Those genes are also absent in the metagenome-assembled-genome (MAG) of Ca. Thioglobus pontius (SUP05 from the Black Sea redoxcline) [13]. The Black Sea MAG of Ca. Thioponita autotrophica of clade BS-GSO2 indicates the genetic potential to reduce NO3− and NO2− based on the presence of nitrate reductase (narGHI) and nitrite reductase (nirBD/K) genes [13]. However, whether SUP05 or BS-GSO2 also utilizes MnO2 as a terminal electron acceptor is unknown.

SUP05 are considered to be non-motile [66], evidenced by microscopic inspection and a lack of genes encoding flagella; however, this evidence is inconclusive (Robert Morris, personal communication) and no data are available for clade BS-GSO2. Our results lead to the hypothesis that Sulfurimonas spp. dominates the S-oxidizing community in the Black Sea when a stable Mn cycle has developed that separates O2, NO3−, and NO2− from S2−. A larger role for Sulfurimonas spp. than for BS-GSO2 and SUP05 would then be based on the ability to perform active movement and reduce MnO2, which would favor the success of these bacteria in the presence of a well-developed Mn cycle. Such conditions might be reflected in the accumulation of Mnreact. (Mn3+) at pelagic redoxclines, the formation of which requires a hydrographically stable water column without major disturbances such as by lateral intrusions [1]. Whether the remaining S2− oxidation can be explained by the activity of phototrophs [67], the vertical migration of magnetotactic bacteria with internal vacuoles [9], or by as-yet-unidentified mechanisms is unknown.

General implications for geochemical cycles and microbial communities in redoxclines

Microbially catalyzed Mn-dependent S2− oxidation affects the depth of the S2− interface (Fig. 5), thereby fostering the separation of S2− from O2, NO3− and NO2−. This separation has consequences for element cycling, e.g., the N-loss processes of denitrification and anammox. In the central Black Sea, where heterotrophic denitrification was generally undetectable within the redoxcline and denitrification via S2− oxidation is excluded due to a missing interface between S2− and NO3−, anammox was the main N-loss process [68].

Although the Black Sea redoxcline is the most prominent system with a zone that simultaneously lacks O2 and S2−, other examples from euxinic systems have been reported. In the central Baltic Sea, Hannig et al. [69] showed a shift in the N-loss process from S2−-coupled denitrification to anammox in the redoxcline after saltwater inflows in 2002 and 2003. In 2005, after re-establishment of the redoxcline, a Black Sea-like separation of O2, NO3−, and NO2− from S2− was detectable that was accompanied by a 3- to 13-fold increase in the maximum concentrations of reduced and oxidized Mn within the redoxcline. Under these conditions, anammox instead of denitrification was the main N-loss process. The authors suggested that Mn-dependent S2− oxidation, with the subsequent vertical separation of NO3− and S2−, supported anammox rather than denitrification as the dominant N-loss process. Similar indication came from marine sediments. Engström et al. [70] found that the relative contribution of anammox to total N2 production increased with Mn-oxide content to up to 80%, indicating that Mn-oxides compete with denitrification for substrates, thereby favoring anammox activity. It can therefore be suggested that Mn-cycling eliminates the inhibitory effect of S2− on anammox [71].

Pelagic redoxclines have been reported from stratified systems worldwide, ranging from meromictic and seasonally anoxic lakes (e.g., Fayetteville Green Lake and Lake Dagow [1, 72]) to brackish (e.g., Baltic and Black Seas [10]), marine (e.g., Cariaco basin and anoxic fjords [2, 3]), and hypersaline (Orca basin [4]) environments. Despite fundamental differences in the general conditions and patterns of their chemical redox profiles [73], those systems have substantial similarities with respect to the biogeochemical processes at their pelagic redoxclines. A prominent example is the cycling of dissolved and particulate Mn species in the presence of sulfide, which suggests that those redoxclines host microbial communities with a similar niche as reported here for the Black Sea genus Sulfurimonas. Thus, chemosynthesis in the absence of NO3− and O2 was already reported, with microbial S2− oxidation coupled to Mn reduction proposed in the Cariaco Basin and in the Baltic Sea [2, 74]. It is therefore not surprising that another Mn-reducing and S2− oxidizing species, Ca. Sulfurimonas baltica GD2, could be isolated from the bottom waters of the Gotland Basin in the Baltic Sea [26].

Conclusions

In this study, the geochemistry of the pelagic redoxcline of the Black Sea was characterized at high resolution. The local abundance and gene expression of Sulfurimonas spp. were determined as well, together with laboratory-based assessments on Mn-dependent S2− oxidation rates by Ca. S. marisnigri SoZ1. Sulfurimonas spp. were highly abundant across the redoxcline, where O2, NO3−, and NO2− were absent. The expression of sqr, soxCDYZH, soxXYZAB, psrABC, and sorAB at the upper boundary of euxinic water indicated the complete oxidation of S2− and S-intermediates to SO42− by Sulfurimonas spp., without an important role for S2O32−. The cellular abundance of Sulfurimonas spp. was consistent with the availability of MnO2 and with S2− fluxes and suggested the coupling of S2− oxidation to MnO2 by this genus. The rapid oxidation of S2− at the upper boundary of euxinic water was crucial in explaining the observed S2− concentration profile. Abiotic oxidation of S2− with MnO2 was too slow to counterbalance S2− fluxes from euxinic water. By contrast, the rate of biologically catalyzed oxidation was sufficiently high and consistent with the expression of the sqr, which Sulfurimonas spp. dominated by about 90 percent at the boundary to euxinic water. Biological S2− oxidation with MnO2 can explain the accumulation of Mn3+ and offers a plausible explanation for the chemosynthetic activities of Campylobacterota in the Black Sea in the absence of O2, NO3−, and NO2− [27]. Our study therefore suggests that, by catalyzing the oxidation of S2− with MnO2, Sulfurimonas spp. in the Black Sea redoxcline play a crucial role in the establishment and maintenance of a broad anoxic and non-sulfidic zone separating oxic and euxinic water. This may in turn create ecological niches for other important functional groups of prokaryotes, such as anammox bacteria, that thrive in these zones.

Because the modern Black Sea is a prime example of a redox-stratified aquatic ecosystem, we are confident that our findings are of relevance for other stratified settings worldwide, including lakes, fjords, and semi-restricted brackish/marine basins. As the first indication for biological Mn-dependent S2− oxidation came from marine sediments [75, 76], the results of the present work may also be relevant for marine sediments, where rapid biological Mn-dependent S2− oxidation could prevent the escape of sulfide into bottom waters, thereby counteracting the spread of hypoxia.