A bacterial isolate from the Black Sea oxidizes sulfide with manganese(IV) oxide

Mn is one of the most abundant redox-sensitive metals on earth. Some microorganisms are known to use Mn(IV) oxide (MnO2) as electron acceptor for the oxidation of organic compounds or hydrogen (H2), but so far the use of sulfide (H2S) has been suggested but not proven. Here we report on a bacterial isolate which grows autotrophically and couples the reduction of MnO2 to the oxidation of H2S or thiosulfate (S2O32−) for energy generation. The isolate, originating from the Black Sea, is a species within the genus Sulfurimonas, which typically occurs with high cell numbers in the vicinity of sulfidic environments [Y. Han, M. Perner, Front. Microbiol. 6, 989 (2015)]. H2S and S2O32− are oxidized completely to sulfate (SO42−) without the accumulation of intermediates. In the culture, Mn(IV) reduction proceeds via Mn(III) and finally precipitation of Ca-rich Mn(II) carbonate [Mn(Ca)CO3]. In contrast to Mn-reducing bacteria, which use organic electron donors or H2, Fe oxides are not observed to support growth, which may either indicate an incomplete gene set or a different pathway for extracellular electron transfer.

I n stratified basins, for example the Black Sea, in between the oxygenated surface waters and sulfidic bottom waters a suboxic zone lacking oxygen (O 2 ), H 2 S, and mostly also nitrate (NO 3 − ) has been frequently reported (1). Despite the absence of electron acceptors, high bacterial CO 2 fixation rates at the border with sulfidic waters were measured, without a known energy metabolism which could fuel growth under these environmental conditions (2,3). Thermodynamically, a suitable electron acceptor for H 2 S oxidation at this depth would be Mn. Even though Mn concentrations are low (Fig. 1A), the oxidized form MnO 2 is particulate and is therefore transported much faster than dissolved electron acceptors to the sulfidic waters by gravitational sinking (4). Nevertheless, so far all attempts to cultivate microorganisms which oxidize H 2 S with MnO 2 have failed.
During an expedition on the research vessel Maria S. Merian in November 2013 we sampled the water column of the Black Sea, focusing on the suboxic zone. We took a water sample at the depth of highest abundance of Epsilonbacteraeota [12 to 15% (2)] and transferred it into a gas-tight serum bottle containing MnO 2 (Fig. 1A, red arrow). At this depth H 2 S was detectable and neither O 2 nor NO 3 − was present. After a first enrichment with daily additions of H 2 S resulting in ∼10 to 20 µM concentrations, we transferred a small volume into an artificial medium. From here on we used mainly S 2 O 3 2− for cultivation instead of H 2 S as it is a more convenient electron donor, nontoxic even in higher concentrations, and nonreactive with the MnO 2 used in our study. A single strain was isolated by repeated series of dilutions-to-extinction transfers.
The isolate belongs to the genus Sulfurimonas and its closest cultured relative is Sulfurimonas gotlandica, known for the oxidation of H 2 S with NO 3 − in the pelagic redoxcline of the Baltic Sea (5) with a 3% difference in the full 16S rRNA gene sequence. We propose calling the strain 'Sulfurimonas marisnigri,' in reference to the Latin notation of Pontus Euxīnus, meaning Sulfurimonas from the Black Sea. The cells are slightly curved, with lengths of 1 to 4 µm and widths of 200 to 300 nm (Fig. 1B). 'S. marisnigri' grows autotrophically with doubling times of 9 to 13 h during the exponential growth phase and reaches a final cell density of 3 to 6 × 10 7 cells per mL after 7 to 10 d ( Fig. 2A). Toward the end of the growth phase, the medium turns from black to brownish-gray ( Fig. 1 C and D, Inserts), due to the reduction of MnO 2 and precipitation of Mn(Ca)CO 3 (Fig. 2B). Even though this may be an artifact due to the cultivation conditions, this particular mineral phase was reported in exceptional amounts from the anoxic basins of the Baltic Sea, but the mechanism of its formation is still under debate (6). Cultivation of 'S. marisnigri' with NO 3 − and successive additions of H 2 S resulted in growth and undetectable H 2 S levels in the culture, indicating a principal ability to use H 2 S directly as electron donor. In contrast to Shewanella oneidensis and Geobacter metallireducens, attempts to cultivate 'S. marisnigri' with amorphous FeOOH, goethite (α-FeOOH), Fe 2 O 3 , and FeCl 3 were unsuccessful, leading to the conclusion that Fe(III) was not a viable electron acceptor under these conditions. This may be due to the absence of a critical protein component for the reduction of Fe oxides or could indicate that extracellular electron transfer onto MnO 2 might function in a different manner.
Growth of 'S. marisnigri' with MnO 2 and a constant supply of H 2 S resulted in accumulation of SO 4 2− as cell numbers increased, and negligible concentrations of elemental sulfur (S 0 ). In the sterile control, however, S 0 accumulated and SO 4 2− increased just slightly (Fig. 2C). Likewise, in the central gyres of the Black Sea S 2 O 3 2− and sulfite (SO 3 2− ) were undetectable (7) and S 0 occurred in nanomolar concentrations (8). Growth with MnO 2 and S 2 O 3 2− was accompanied by the complete oxidation of S 2 O 3 2− to SO 4 2− with a stoichiometry of 1:2 ( Fig. 2A) and following Eq. 1:

[1]
As with H 2 S, no detectable accumulation of S 0 and SO 3 2− was observed with S 2 O 3 2− as electron donor. Growth was observed concurrent with the reduction of Mn(IV) to Mn(III) from day 3 to 6 and continued with the reduction of Mn(III) to Mn(II), leading to the precipitation of Ca-rich particles ( Fig. 2 A and B), in The isolate belongs to the group of Epsilonbacteraeota which is reported to be highly abundant in the redox trasition zones of, for example, the Black Sea (12% of the total bacterial community), the Baltic Sea (21%), and the Cariaco Basin (27%) (2,10). In these systems, Epsilonbacteraeota can be responsible for up to 100% of the dark CO 2  In addition to its presence in pelagic environments, the genus Sulfurimonas is globally abundant in redox transition environments such as hydrothermal vents and marine sediments (12). In sediments, the addition of MnO 2 is thought to promote the production of SO 4 2− , apparently depending on microbial activity and leading to the precipitation of Mn(Ca)CO 3 (13). In those experiments, addition of FeOOH did not stimulate SO 4 2− production. Those findings fit remarkably well to our observations in pure culture, suggesting that an organism with a physiology similar to that of 'S. marisnigri' may have been responsible for the observed activity.
A further indication that bacterial H 2 S oxidation with MnO 2 may be of more general importance is the formation of intermediate Mn 3+ in our cultures, which we detected indirectly as dissolved reactive Mn (14) (Fig. 2B). Mn 3+ was reported to be a major constituent of the marine Mn cycle both in sediments (15) and in the water column of the Black Sea (16) in the absence of O 2 and H 2 S (17). So far, known processes mediating Mn 3+ formation are the oxidation of organic matter with MnO 2 reduction, enzymatic oxidation of Mn 2+ , and the abiotic reaction of MnO 2 with Fe(II) and H 2 S (15,18). Our study adds another biologically mediated process via lithotrophic MnO 2 reduction, which can promote the buildup of Mn 3+ , as observed both in marine sediments and across pelagic redoxclines. In conclusion, we suggest that this bacterial metabolism, which we prove here in pure culture, may be widespread in pelagic redoxclines and to a minor extent in marine sediments where H 2 S is produced and Mn is present in sufficient amounts with important consequences for Mn and S cycling.

Materials and Methods
Gases and Nutrients. Gases and nutrients in the water column were measured as reported in Schulz-Vogt et al. (19).
Cultivation. Water samples were taken at 44 • −16.7586 N and 36 • −18.9567 E at the depth indicated in Fig. 1. Culture purity was ensured by sequencing, lack of growth in organic-rich media, and by microscopy. Medium for cultivation and experiments was prepared anaerobically following the technique described by Widdel (14)] were measured by ICP-OES (iCAP 7400 Duo; Thermo Fisher Scientific) using an external calibration and Sc as internal standard. Precision and accuracy were checked by international reference materials (SGR-1b for the part. and SLEW-3 for the diss. fraction) and were below 2%.

SEM-EDX.
A Zeiss Merlin Compact SEM (variable pressure, in-lens SE and BSE detector) equipped with EDX (Oxford Instruments) was used to identify Mn(Ca)CO 3 precipitates and to directly quantify the Ca-to-Mn ratios.
The sample preparation was done as described elsewhere (19). Reduced Mn in the part. phase was calculated with Eq. 2: part.red.Mn = part. Mn * total part. Ca/Mn by ICP − OES spot Ca/Mn by SEM − EDX in Mn(Ca)CO 3 . [2] TIC. Dried material was treated with 40% H 3 PO 4 and the released CO 2 was analyzed by an IR detector (multi-EA 4000; Analytic Jena). Pure standard CaCO 3 (12.0% TIC) was used for calibration.