AQUATIC MICROBIAL ECOLOGY Published June 18 Aquat Microb Ecol

Tetrathionate production by oxidizing and the role of in the sulfur cycle of Baltic Sea sediments

Lilijana Podgorsek, Johannes F. Imhoff*

Institut fiir Meereskunde, Abteilung Marine Mikrobiologie, Diisternbrooker Weg 20, D-24105 Kiel, Germany

ABSTRACT: The role of tetrathionate in the sulfur cycle of Baltic Sea sediments was investigated in dif- ferent habitats and under a variety of environmental conditions. Sediment profiles were recorded with regard to numbers of thiosulfate oxidizing bacteria, concentrations of sulfur compounds, and potential rates of thiosulfate oxidation. Products of thiosulfate oxidation were quantified in incubated sediment samples and in pure cultures. Evidence was found that tetrathionate is formed within these sediments, that sulfur oxidizing bacteria are present in considerable numbers, that these bacteria are of major importance in the oxidation of reduced sulfur compounds in their habitat, and that tetrathionate is an important oxidation product of these bacteria. Thiosulfate is oxldized by bacteria isolated from these sediments to varying proportions of tetrathionate, , and also elemental sulfur. In highly sulfidic sediments and in the presence of large amounts of organic matter, tetrathionate was present in sedi- ment horizons in which thiosulfate and elemental sulfur also accumulated. A tetrathionate cycle is pro- posed to be active in natural marine and brackish water sediments in which, due to combined bacter- ial action and chemical reactions, a net oxidation of sulfide to elemental sulfur occurs in the presence of catalytic amounts of thiosulfate and tetrathionate.

KEYWORDS: Tetrathionate . Thiosulfate . Sulfur oxidation . Sulfur oxidizing bacteria . Tetrathionate cycle . Baltic Sea

INTRODUCTION ria from freshwater and marine sources (Fitz & Cypi- onka 1990, Sass et al. 1992).Tetrathionate is also well The roles of sulfate, elemental sulfur and sulfide in known as an intermediate in the oxidation of reduced biological and geochemical cycles are well character- sulfur compounds to sulfate by tepidarius, ized (e.g. Jergensen 1988, Kelly 1988). Thiosulfate has Thiobacillus intermedius and other thiobacilli (Wood & also been recognized as a central intermediate in sul- Kelly 1986, Lu & Kelly 1988, Kuenen et al. 1992, fur cycling of marine sediments (Jsrgensen 1990, Jsr- Wentzien et al. 1994). In addition, tetrathionate forma- gensen & Bak 1991). In recent years an increasing tion is a property of many chemoheterotrophic bacteria amount of information has been accumulated on bac- (Tuttle et al. 1974, Mason & Kelly 1988, Sorokin 1993, terial transformations of slilf~irrnmpounds of interme- 1996, Sorokin et a!. 1996). diate level (elemental sulfur, thiosulfate, sulfite) However, the role of tetrathionate in the sulfur cycle in oxidative and reductive processes, including reac- of marine sediments is poorly understood. Although tions of disproportionation (Jergensen & Bak 1991, from pure culture studies tetrathionate is known as an Thamdrup et al. 1993, Vedenina & Sorokin 1993, Can- oxidation product of thiosulfate in several chemo- field & Thamdrup 1996, Sorokin et al. 1996). Thiosul- heterotrophic bacteria from different marine environ- fate and trithionate, and to a lesser extent also tetra- ments (Tuttle et al. 1974, Sorokin 1992, 1993, Durand thionate, have been found as intermediates during et al. 1994), the importance of these bacteria in the nat- sulfate and sulfite reduction by sulfate reducing bacte- ural environment has so far not been estimated. We have found evidence that tetrathionate is formed 'Addressee for correspondence. within the investigated Baltic Sea sediments, that it is E-mail: [email protected] an important oxidation product of sulfur oxidizing bac-

O Inter-Research 1999 256 Aquat Microb Ec

teria isolated from these sediments, and that these bac- basins of the Baltic Sea. Depending on irregular intru- teria are present in considerable numbers within the sions of salt water into the Baltic Sea, transient oxic sediments. We therefore conclude that tetrathionate conditions occur in the water column and at the sedi- plays a significant role within the sulfur cycle of marine ment surfaces of the Gotland Deep. Samples were and brackish water sediments. Apparently, its forma- taken at 241 m depth in the central part of the Basin tion is favored in highly sulfidic sediments and in the (57"18.511N, 20°06.88'E) in June 1994, after oxic water presence of large amounts of organic matter, as indi- had completely replaced the sulfidic deep water, and cated by experimental manipulation of the sediments. in October 1995, when sulfide concentrations m the A preliminary discussion on the role of tetrathionate in upper sediment layers had increased again and sulfide the sulfur cycle of marine sediments and on the impor- had already penetrated into the water column (Piker et tance of possible interactions with sulfide to form ele- al. 1998). mental sulfur and thiosulfate has been presented ear- In coastal sediments cores were taken with 15 to lier (Imhoff 1996). 30 cm long cylinders of plexiglas, in the Gotland Basin with a gravity corer (Rumohr-Lot) fitted with 1 m long liners. Necessary storage was at in situ temperatures MATERIALS AND METHODS (experimental sediment system 15"C, Hiddensee 10°C, Gotland Basin 4°C). Cores were cut into 0.1 to 2.0 cm Habitats and sampling. The present studies were slices and, depending on the heterogeneity of the habi- confined to habitats oi the Baltic Sea. The principal tats, corresponding slices from 3 to 5 cores were mixed part of these investigations was made within sedi- under a nitrogen atmosphere and used for further ments of 2 coastal locations. In addition a few support- analyses. ing experiments and measurements were done in the Bacteria used during this study. As reference strains Gotland Basin. Paracoccus versutus (formerly Thiobacillus versutus, Hiddensee: One of the locations was at the shore of Katayama et al. 1995) DSM 582T, Thiobacillus thio- the 'Fahrinsel' close to Hiddensee, Germany (site Hid- parus DSM 505T and DSM 65T densee K). An area of approx. 15 m2 was covered with were obtained from the Deutsche Sammlung von a plastic sheet from June to November 1993 to produce Mikroorganismen und Zellkulturen (DSMZ, Braun- stable anoxic conditions. A comparison between an schweig, Germany). uncovered control area and the covered area was Determination of viable cell numbers. Aliquots of made in November immediately after removal of the the sediment samples were diluted in artificial sea cover sheet. water mineral salts medium (MSM) (8.2 g NaCl, 0.4 g Bottsand: A second location was at the Bottsand MgCl2.6H20, 0.35 g MgSO4.7H20, 0.35 g KCI, 0.2 g close to Kiel, Germany. It was used to set up an exper- CaCl,.2H20, and 0.03 g KBr I-'), which was adjusted to imental sediment system (1.20 X 2.80 m in size) under 15 PSU salinity. From the appropriate dilutions 0.1 m1 controlled conditions in the lab to study the effect of was streaked onto agar plates with TSB (trypticase different environmental parameters on processes of soya broth, Becton Dickinson, Cockeysville, MD, USA) the sulfur cycle. Sediment was taken from the environ- and THSTh (synthetic medium for thiobacilli) media ment in layers that were homogenized and reassem- and incubated at 15°C under aerobic conditions in the bled in the experimental system. Temperature, salinity dark. and flow rate of the supernatant water were controlled The numbers of bacteria on TSB agar (TSB 10 g I-', and together with pH-value and redox potential of the NaCl 4.5 g I-', Biomatik 'type BRC AB' agar, 18 g I-', sediment profiles continuously recorded. Four com- pH 7.2) were determined after 2 wk of incubation. partments were distinguished by different amounts of Numbers of sulfur oxidizing bacteria were estimated natural organic matter (finely ground seaweed and by using the synthetic THSTh medium that also proved ;!gal m;:cri-;n!) addcd 2t 2 depth sf 2 tc 3 err- intc the tc he uitah!~for the ri~ltivatinnof Pararoccus ver.su- sediment. The untreated compartment (compartment tus, P. denifrificans and Thiobacillus thioparus (in this K)and the compartment with the highest load of added case without acetate). Colonies on this medium were algal biomass (compartment A, with 0.3 g dry weight counted after 3 to 4 wk of incubation. Purification and of algal biomass per cm2 sediment) were selected for isolation of sulfur oxidizing bacteria was done by the present study. Measurements were mad.e in the repeated dilution on agar plates with the THSTh recovered sediment after incubation periods of 6 wk at medium. 4°C and for an additional 4 wk at 15°C and at intermit- In a final volume of 1 l, THSTh medium contained tent 1ight:dark cycles of 8:16 h. the following components that were sterilized sepa- Gotland Basin: Prolonged stagnation periods with rately and mixed after cooling (the final pH was ad- sulfidic conditions are characteristic for the deeper justed to 7.2): Solution A, 250 m1 4-fold concentrated Podgorsek & Imhoff: Tetrathionate in Baltic Sea sediments 257

artificial sea water MSM; Solution B, 0.1 g NH,Cl, 0.7 g Measurements of thiosulfate oxidation. The poten- sodium acetate, 0.15 g KH2P0,, 0.8 g K2HP04 in 250 m1 tial of thiosulfate transformations within the sediments aqua dest; Solution C, 20 g Biomatik 'type BRC AB' was measured under standard conditions. An aliquot agar in 400 m1 aqua dest. of 1 cm3 sediment was suspended in 50 m1 THSTh The following components were prepared separately medium (containing MSM with MgS0,.?H20 re- as stock solutions, sterilized by filtration and added to placed by equimolar amounts of MgC12.6H20) and a final volume of 1 1 medium: 5 m1 0.2% FeC13.6H20, preincubated for 12 h under aerobic conditions on a 5 m1 0.35% MnC12.4H20, 50 m1 10% Na2S203.5H20, rotary shaker (100 rpm) prior to the addition of acetate 10 m1 0.12% Na2S.9H20, 1 m1 0.15% yeast extract (0.25 mM) and thiosulfate (1 mM). Thereafter the mix- (Merck), 1 m1 trace element solution TET2, 1 m1 vita- tures were incubated for another 24 h (samples from min solution VA (Imhoff 1988), and l m1 stock solution Hiddensee at 20°C and samples from the experimental of vitamin BI2(containing 15 pg I-'). sediment at 15"C), then filtered through 0.2 pm pore TET2 contained the following components: 50.0 g size cellulose acetate filters and frozen at -20°C until EDTA, 0.4 g ZnC12, 3.0 g MnC12.4H20, 4.0 g FeC12. use for chemical analysis. The turnover rates were 4H20, 10.0 g FeC13.6H20, 0.4 g Na2Mo04-2H20, expressed as pm01 thiosulfate transformed per hour 0.04 g CuC12.2H20, 0.24 g CoC12-6H20, 0.4 g H3B03, and cm3 of sediment. 0.01 g NiC12.6H20, 0.001 g Na2Se03-5H20 per liter Oxidation rates of thiosulfate by pure cultures were aqua dest. determined in THSTh medium containing 1 mM thio- Chemical analyses. The content of organic material sulfate and 0.25 mM acetate. After 24 h incubation at was determined as percentage of the dry weight of the 20°C aliquots were taken for analyses of elemental sul- sediment by heating at 550°C for 5 h. fur and chromatographic analysis of sulfur com- Chemical parameters were determined in sediment pounds. The latter were frozen at -20°C after sterile pore water, which was recovered after centrifugation filtration. at 4000 U min-l (approx. 1700 X g) in a Sigma 3K30 Fatty acid analysis. Fatty acid analysis was made centrifuge under an atmosphere of argon or dinitro- essentially as outlined in the standardized procedures gen. For the determination of sulfide, samples were of the Microbial Identification System (MIS, MIDI taken immediately after centrifugation and fixed in Incorp., Newark, Delaware, USA). The growth condi- gas-tight tubes containing 5 m1 2 % Zn acetate. tions were changed slightly. Agar plates contained Sulfide was determined photometrically as methyl- 0.3% TSB, 5 g Na2S203.5H20, 1 m1 VA (Imhoff 1988), ene blue (absorbance at 668 nm) according to Pach- 1 m1 TET2 and 15 g agar in artificial sea water of 5 PSU mayr (1960). For the determination of sulfate, sulfite, salinity. Final pH was 7.2. Plates were incubated at thiosulfate and tetrathionate, pore water and culture 28°C for 7 d. Harvesting of cells, extraction procedures fluids were filtered through a 0.2 pm pore size cellu- and gas chromatographic analysis were made as lose acetate filter (Sartorius, Gijttingen, Germany). described by Thiemann & Imhoff (1996). Sulfate, sulfite and thiosulfate were separated on an anion exchange column (Ionpack-AS4A, Dionex, ldstein, Germany) and eluted with 1.7 mM NaHC03 + RESULTS 1.8 mM Na2C03 (flow rate was 2 m1 min-l). Thiosulfate and tetrathionate were separated by ion-pair chroma- Sites of investigations tography (Dionex ionpack-NS1 column) and eluted with 2 mM tetrabutylammonium hydroxide, 0.88 mM Sediments of the Baltic Sea investigated during this Na2C03, 30% acetonitrile. Quantitative analyses were study were rich in organic matter. They were known to made using suppressed conductivity and UV- frequently develop massive blooms of phototrophic absorbance at 215 and 254 nm, respectively, for detec- andlor chemotrophic su!fur bacteria. Oxygen pene- tion. Tetrathionate standards were stable during the trated only into the uppermost few millimeters of these analytical procedure and did not give indication of sediments and sulfidic conditions regularly were found degradation during storage at -20°C for up to a week. close to the sediment surface. Often sulfide penetrated Elemental sulfur was determined according to Chan & into the water above the sediment. Two of the sites Suzuki (1993).Samples of 1 cm3 sediment were mixed (Hiddensee and Bottsand) were coastal habitats, one with 1 m1 artificial seawater (MSM) and extracted with was a deep basin sediment (Gotland Deep). 2 rnl petrol ether. An aliquot of the petrol ether phase The sediment of Hiddensee consisted of a fine silty (0.5 ml) was mixed with 1 m1 NaCN solution (0.075 % material mixed with large amounts of decaying sea- in 95% acetone) and 1 m1 95% acetone. After 2 min, weed in the top 10 cm overlying a coarse sandy sedi- 1 m1 FeC13 solution (2 % in 95 % acetone) was added ment. The content of organic matter was 2 to 10% of and the extinction measured at 464 nm within 10 min. the sediment dry weight. During the investigations in 258 Aquat ~MicrobEcol 17: 255-265. 1999

vlable cell wunts/cm3 105 1v I 0' 10 L 5 ~'~'',I0 i ,'.'#'l ' 8~~m~~l formation of SO:. and s,o," (90) SO,'., S,O$ (pm01 h" cmJ) Fig. 1. Untreated natural sed- iment at Hiddensee K (con- trol). (a) Viable cell counts of sulfur oxidizing bacteria, transformation rates of thio- sulfate (Sz03'-) and forma- tion rates of sulfate (SO,'-) 4 and tetrathionate (S40sZ-). (b) Oxidation of thiosulfate to sulfate and tetrathionate in 6 0 25 75 50 100 percent transformed thiosul- El fate sulfur. (c) Depth profiles of sulfide (S2-) and sulfate 0 sulfate (SO,'-) in the pore water; S'' (PM) m tetrathionate thiosulfate and tetrathionate sulfide A sulfate were not detected a sulfur-oxidizingbacteria thiosulfate A sulfate o tetrathionate

viable cell wunts/cm3 1o5 ID 10' 1O8 I # 0 ).)..*I . n ,,,,,,I a ~~~~'dformation of SO," and S.0:- (%l SO,^.. (pm01 h.' cm') 0 2 4 6

Fig. 2. Sediment at Hidden- see K after 5 mo of artifi.cia1 anoxic conditions. (a) Viable cell counts of sulfur oxidizing bacteria, transformation rates of thiosulfate (S203'-) and for- mation rates of sulfate (SO4'-) and tetrathionate (s4o6'-). (b) Oxidation of thiosulfate to a sulfate and tetrathionate in percent transformed thio- sulfate sulfur. (c) Depth pro- D sulfate files of sulfide (S"), sulfate m tetrathionate (SO,'-), thiosulfate (S2Os2-) sulfide a sulfate 0 sulfur-oxidizing bacteria and tetrathionate (S4062-)in thiosulfate o tetrathionate the pore water thiosulfate A sulfate tetrathionate

November 1993 the redox potential was +210 mV at (THSTh). This medium was suitable to cultivate re- the sediment surface of the uncovered area compared presentative chemotrophic sulfur bacteria such as to -200 mV at the covered area. The sediment of the Thiobacillus thioparus and Paracoccus versutus, which Bottsand had a high content of fine and coarse sandy were used as reference organisms during this study. All material and contained up to 4% (0.1 to 4.0%) of of the investigated isolates, which represented the most organic matter by sediment dry weight. The silty sedi- numerous bacteria growing under these conditions, zent ef the C,~l!!a_n_rl_ Deep w;lr rharact~rixed by very were able to oxidize thiosulfate. For simplicity, viable high contents of water (95% at 10 cm depth) and or- numbers obtained on THSTh medium will be called ganic matter (15 to 30% of the sediment dry weight) sulfur oxidizing bacteria in the following. Primarily within the top 20 cm. chemoheterotrophic (and possibly facultative chemo- autotrophic), but not obligately chemolithoautotrophic sulfur oxidizing bacteria were ob- tained with this Bacterial cell counts within the sediments medium, as was concluded from the properties of the isolated bacteria (see below). Numbers of these bacte- The numbers of sulfur oxidizing bacteria were deter- ria were compared to those of bacteria growing on a mined by using a mineral salts medium with additions complex standard medium (TSB), in the following of thiosulfate and acetate as energy and carbon sources called chemoorganotrophic bacteria for simplicity. Podgorsek & Imhoff: Tetrathionate in Baltic Sea sediments 259

v~ablecell counts/cm3 transformat~onrate (pmol cm-' h-') forrnat~onof products (%)

105 io6 107 10' io8 0 1 2 3 0 50 100 150 Fig. 3. Transformation of thio- sulfate in the untreated ex- perimental sediment system (compartment K) (a) Viable cell counts of sulfur oxidizing bacterla, ratio of sulfur 0x1- d~zingbacteria and chemo- organotrophlc bacteria and the content of organic matter (b) Rates of thiosulfate oxida- tion (S2032-)and formation of sulfate (SOA2-) and tetra- 01234 thionate (S4O6'-). (c) Oxida- rat10bactena thiosulfate 0 sulfate tion of thiosulfate to sulfate and tetrathionate In percent organic matter (%) A sulfate m tetrathionate of transformed thiosulfate D tetrathlonate sulfur 0 organlc matter sulfur-ox~diz~ngbactena

0 rat10sulfur-oxidtz~ng bactenal heterotroph~cbacteria

3 v~ablecell countslcm transformat~onrate (pmol cm3 ' h-') formation of products (%) 105 io6 107 iod 1oS 0.0 0 2 0.4 0 6 0 8 1.0 0 50 100 150

Fig. 4. Transformation of thiosulfate in the experimen- tal sediment system with high orgamc content (com- partment A). (a) V~ablecell counts of sulfur oxid~zing bacteria, ratio of sulfur oxi- dizing bacteria and chemo- organotrophic bacterla and the content of organic matter (b) Rates of thiosulfate oxida- 01234 t~on(S,032 ) end formation of ratlo bactena thiosulfate 0 sulfate sulfate (SO,') and tetra- A sulfate thionate (S,ObL ) (c) Oxida- organic matter (%) tetrathlonate t~onof thiosulfate to sulfate o tetrathlonate and tetrathionate In percent 0 organnl matter of transformed thiosulfate sulfur-ox~d~z~ngbactena sulfur 0 ratlo sulfur-ox~d~z~ngbactenal heterotrophic bactena

Numbers of sulfur oxidizing bacteria growing on in compartment A (Fig. 4a). In the most active top lay- THSTh medium varied from 105to 10' cells cm-3 in the ers of the sediments their numbers exceeded those of investigated sediments. In correlation with the poten- the bacteria growing on TSB medium (see Figs. 3a & 4a). tial oxidation rates of thiosulfate, maxima of sulfur oxi- dizing bactena were always found at or close to the sediment surface (see also Imhoff et al. 1995).Numbers Concentration profiles of sulfur compounds of sulfur oxidizing bacteria at Hiddensee decreased from approx. 107 close to the sediment surface to Concentration profiles of sulfur compounds were approx. 5 X 105 cm-3 at 6 cm depth (Figs. la & 2a). The highly variable and dependent on the environmental protection of the sediment by a plastic cover sheet had conditions. Limitation of oxygen penetration into the no significant influence on these numbers. In the ex- sediment and increased supply of degradable organic perimental sediment system numbers of sulfur oxidiz- matter favored active sulfate reduction. Zones of in- ing bactena were similar in both areas, with 108 cells creased sulfide concentrations indicated short-term cm-3 at the sediment surface (Figs. 3a & 4a) and activation of sulfate reduction, while zones of sulfate approx. 107 cells cm-3 at 5 cm depth, with a minimum depletion were due to highly active sulfate reduction at the depth where the organic matter had been added for prolonged time periods. 260 Aquat Microb Ecol l?: 255-265, 1999

The most obvious difference between the 2 sediment Thiosulfate oxidation within the sediments compartments of Hiddensee was the rather stable con- centration of sulfate (8 mM) throughout the sediment In all investigated sediments significant rates of thio- depth in the control area and its strong depletion to sulfate transformation were found. approx. 6 mM at the surface and to concentrations of less than 1 mM below 3 cm sediment depth in the cov- ered sediment (Fig. 2c). This depletion was correlated Sediment at Hiddensee with strongly increased concentrations of sulfide (up to 1.25 mM) and thiosulfate (up to 250 PM) and the accu- The profiles of potential thiosulfate oxidation corre- mulation of significant amounts of tetrathionate (up to lated well with those of cell numbers of sulfur oxidizing 6 PM) (Fig. 2c). In the uncovered area sulfide was pre- bacteria. In the uncovered sediment the highest rates sent at low concentrations throughout the sediment of thiosulfate oxidation (2.8 pm01 h-' together profile (10 to 25 FM, Fig. lc) and concentrations of both with those of sulfate and tetrathionate formation, were thiosulfate and tetrathionate were below the detection found at 1 to 2 cm depth (Fig. la). In the covered sedi- limit of 0.5 and 1.0 pM, respectively. ment area, maximum values of these activities moved The massive inflow of oxic bottom water into the to the sediment surface and a second strong maxirrlllnl Gotland Basin and the following depletion of oxygen at of tetrathionate formation was found at approx. 3 cm the sediment surface and in the water caused dynamic depth (Fig. 2a). Sulfate was the major oxidation prod- changes in the chemistry and the profiles of sulfur uct of thiosulfate in all sediment layers ot both areas. compounds within the upper sediment layers (Piker et However, the proportion of tetrathionate formed was al. 1998). Compared to June 1994, concentrations of higher in the covered sediment than in the uncovered sulfide and elemental sulfur had increased dramati- control sediment. It reached up to 4.1 % of the total cally in October 1995, and at that time elemental sulfur transformed thiosulfate sulfur in the former compared represented a major fraction of the total sulfur of these to 1.2% in the latter (Table 1, Figs. lb & 2b). sediments. In addition, significant concentrations of thiosulfate and tetrathionate were present (Fig. 5). Experimental sediment system tetrathionate (nmo~cmj 0123450 Rates of potential thiosulfate oxidation were highest 0 o tetrathionate at or close to the sediment surface in both compart- (June 1994) - 5 ments of this sediment system. In the control compart- 5 tetrathionate ment a sharp peak of potential thiosulfate oxidation E 10 (October 1995) close to the sediment surface correlated strongly with a 'aQ 5 15 0 elemental sulfur maximum of sulfate formation. Sulfate was the pre- (June 1994) E dominant or sole oxidation product in the top part of 3 20 elemental sulfur this sediment, while below 1 cm depth considerable (October 1995) 25 proportions of tetrathionate were formed (Fig. 3c). In 0123456 sediments of the treated compartment, tetrathionate elemental sulfur (prno!Jcm3) was the major product of thiosulfate oxidation in the surface layers as well as in deeper parts (Fig. 4c, Table Fig. 5. Depth profiles of the concentrations of tetrathionate and elemental sulfur in June 1994 during oxic conditions and 1). However, the overall potential of thiosulfate oxida- in October 1995 during anoxic conditions in the Gotland tion was significantly lower in this compartment Basin (Figs. 3b & 4b).

Table 1 Oxidation rates of thiosulfate (TS; pm01 h-' cm-') and formation ot the products suiiate (SA) and ieiraihiondie jTTj ill percent of transformed thiosulfatc sulfur in the experimental sediment system (control compartment K with 0.9% organic matter of the dry weight and compartment A with organic matter enriched to 2.2%)and in coastal sediments of Hiddensee (untreated area in comparison to an artificial anoxic area). Rates were integrated over 2 depths of the sedlments

Experimental sedlment Sediment at Hiddensee Compartment K Compartment A Untreated Anoxlc TS SA lT TS SA TT TS SA TT TS SA TT Podgorsek & Irnhoff: Tetrathlo mate in Balt~cSea sediments 26 1

Aerobic sulfur oxidizing bacteria isolated from the investigated sediments Thb.hp.

Par.vrs. Sulfur oxidizing bacteria were isolated from sedi- ments of different locations at the shoreline and in cen- straln HK9b tral parts of the Baltic Sea. It became quite obvious tetrathionate strain B17 during isolation and cultivation experiments that some elemental sulfur of these bacteria occurred frequently and were present strain BS9 in different sediments. straln BS8 Using the database of the MID1 system, including reference strains of Paracoccus denitrificans, P, versu- tus and Shewanella putrefaciens, representative iso- lates of thiosulfate oxidizing bacteria were character- ized by their fatty acid composition. Accordingly, Fig. 6. Product formation during the oxidation of thiosulfate strains B3, B17 and B11 formed a cluster similar to P. by Thiobacillus thioparus (Thb. thp.), Paracoccus versutus denitrificans and P. versutus (data not shown). Total (Par. vrs.) and a few selected sulfur-oxidizing bacteria isolated viable numbers of bacteria represented by strains such from Baltic Sea sediments; sulfite was not detected as B3 were approx. 2 X 107 ml-' in the Bottsand sedi- ment (0 to 1 cm depth), correlating to 18% of total sul- erable amounts of unidentified oxidation products fur oxidizing bacteria at this particular place. Another were missing; from observations with cultures and group of bacteria with transparent beige-colored microscopic examinations elemental sulfur can be ex- colonies containing small rod-shaped cells (strains cluded as one of the major missing products of these 2 BSI, BS8 and BS9) always represented a major fraction strains. Although an appropriate standard of trithio- of viable cell counts of sulfur oxidizing bacteria and nate was not available, significant amounts of this was common to different sediments. According to their component would have been detected in the elution fatty acid composition, these bacteria were most profile of the used chromatographic column. Therefore similar to Pseudornonas stutzeri. The identification of trithionate quite unlikely is contributing to the missing strain BS8 was substantiated by 16s rDNA analyses. balance of oxidation products. Similarly sulfite would Although the fatty acid composition of some additional have been detected, if present. Two isolates of Pseudo- strains was moderately similar to that of S. putrefa- monas stutzeri (strains BS8 and BS9) did not form sul- ciens, the composition of a larger number of other iso- fate but instead tetrathionate (40 to 50% of the thio- lates was not particularly similar to any other bacteria sulfate sulfur) as the major product, together with of the database. According to the analyses, these appreciable amounts of elemental sulfur (15 to 30 %). strains belonged to the gamma- and to the Gram-positive bacteria, respectively (data not shown). DISCUSSION

General aspects of the sulfur cycle Thiosulfate oxidation by isolated sulfur oxidizing bacteria Discussions on the biogeochemistry of sulfur are governed by the role and interactions of sulfide, sulfate Thiosulfate oxidation was exhibited by all bacteria and elemental sulfur (Jsrgensen 1988, Kelly 1988, selected from dilution plates of THSTh medium. How- Ehrlich 1990). This is reflected in our knowledge of ever, in contrast to the oxidation reactions of Paracoc- transformations of snlft~rcompounds by various bacte- cus versutus and Thiobacillus thioparus, which form ria. We understand very well the processes of sulfate sulfate as the major and final oxidation product, iso- reduction to sulfide by eubacterial but also archaebac- lates selected during this study oxidized thiosulfate to terial sulfate reducers (Widdel 1988) and the oxidation varying amounts of tetrathionate, elemental sulfur and reactions by phototrophic bacteria and chemolitho- sulfate (Fig. 6). The products and their proportions var- trophic sulfur oxidizing bacteria (e.g. Triiper 1975, ied greatly between the different isolates. Strain B17, Kuenen et al. 1992). In many cases elemental sulfur is which was similar to P. versutus and P. denitrificans an intermediate in the oxidation of sulfide to sulfate according to the fatty acid composition, only formed either inside the cells (e.g. Chromatiaceae, Beggiaioa tetrathionate, while the unidentified strain HK9b spp.) or outside (Chlorobium spp., Thiobacillus spp.). nearly exclusively produced sulfate. In the mass bal- These are the most impressive examples of sulfur bac- ances of the sulfur compounds of both strains, consid- teria and therefore have been most intensively studied. 262 Aquat MicrobEcol 17: 255-265,1999

In other bacteria, the intermediate formation of ele- pounds that undergo gradual degradation to elemental mental sulfur was not detected and sulfide is oxidized sulfur, sulfurous acid and sulfuric acid under acidic to sulfate without stable intermediates, as in Rhodo- conditions and to thiosulfate and sulfite under alkaline vulum sulfidophilum (Neutzling et al. 1985) or in many conditions (Hollemann & Wiberg 1971). Tetrathionate thiobacilli, which form various mixtures of polythio- reacts with sulfite readily to produce thiosulfate and nates as intermediates in the oxidation of reduced sul- tnthionate (Hollemann & Wiberg 1971). Under alkaline fur compounds (Kuenen et al. 1992). conditions tetrathionate may also undergo asymmetri- Many of the more recent findings on sulfur transfor- cal hydrolysis to thiosulfate, elemental sulfur and sul- mations by pure cultures and in the natural environ- fate according to S40s2- + OH- + S2O3'- + So + HSO4- ment have extended this view. Most important was the (Roy & Trudinger 1970). Also the enzymatically cata- recognition of the central role of thiosulfate in the lyzed reaction S40s2-+ H20+ 2 HS3O3- + SOq2-+ H'is sediment biogeochemistry by extended studies of J0r- favored under alkaline conditions (Steudel et al. 1987). gensen and coworkers (e.g. J~rgensen1990, J~rgen- This reaction may explain the formation of tetrathio- sen & Bak 1991) that shed light on the role of thio- nate and elemental sulfur from thiosulfate by the sulfate in oxidative, reductive and disproportionation strains of Pseudomonas stutzeri in this study, because processes. The ability of bacteria to gain energy out of the intermediate disulfane-monosulfonate is highly the disproportionation not only of thiosulfate but also unstable and readily forms elemental sulfur (Wentzien of other sulfur compounds of intermediate redox state, et al. 1994). The presence of this reaction sequence such as sulfite and elemental sulfur, enlarges the pos- and the participating enzymes in P. stutzeri still have to sibilities of transformations in the natural environment be proven. and of possible interactions between 'sulfur bacteria' Also, the chemical reaction of tetrathionate with sul- (Bak & Pfennig 1987, Canfield & Thamdrup 1996). fide to form elemental sulfur and thiosulfate has been More than any other findings, these results have known for a long time (Roy & Trudinger 1970). The demonstrated the participation of sulfur compounds of impact of this reaction on product formation by bacte- intermediate redox state in oxidative as well as in rial cultures oxidizing sulfide to tetrathionate was first reductive processes. Not only can these compounds recognized by Hansen (1974), working with the photo- undergo oxidative as well as reductive reaction se- trophic purple nonsulfur bacterium Rhodomicrobium quences, but they also may be produced during dis- vannielii. This bacterium oxidizes sulfide to tetrathio- similatory processes as oxidation products or as reduc- nate as final product (Hansen 1974). However, the tion products. In fact, thiosulfate and tnthionate, and to stoichiometric transformation was observed only in a lesser extent also tetrathionate, have been found as chemostat cultures with limiting sulfide concentra- intermediates during sulfate and sulfite reduction by tions. When grown in batch cultures with higher con- cultures of sulfate reducing bacteria from freshwater centrations of sulfide, elemental sulfur and thiosulfate and marine sources (Fitz & Cypionka 1990, Sass et al. were formed as products of the reaction of sulfide with 1992).Tetrathionate also can be reduced by a number the tetrathionate formed by R. vannielii according to of bacteria that are not sulfate reducers (Barrett & S4Oe2-+ S*- + 2S2032-+ So (Hansen 1974). Clark 1987). On the other hand, tetrathionate (and Reports in the literature indicate the exclusive for- other thionates) is also well known as an intermediate mation of tetrathionate from thiosulfate by a number of in the oxidation of reduced sulfur compounds to sulfate chemoheterotrophic bacteria (Mason & Kelly 1988), by Thiobacillus tepidarius, T. interrnedius and other but also for the production of tetrathionate and sulfate thiobacilli (Wood & Kelly 1986, Lu & Kelly 1988, Kue- by others (Durand et al. 1994). The simple oxidation of nen et al. 1992, Wentzien et al. 1994). thi.osulfate to tetrathionate appears to be widespread among bacteria and has been found in Klebsiella aero- genes, Bacillus globigii, B. rnegaterium, Pseudomonas Rac!~riz! and chemical formatinn and reartions putida, P. fluorescens and P. aeruginosa (Mason & of tetrathionate Kelly 1988), but also several marine bacteria (Tuttle & Jann.asch 1973, Sorokin 1992, 1993, 1996, Sorokin et al. With the recognition of the central role of thiosulfate 1996). Catenococcus thiocyclus is able to oxidize sul- in the sulfur cycle of marine sediments (Jsrgensen fide and thiosulfate to tetrathionate, but not to sulfate 1990), tetrathionate should also receive more attention, (Sorokin 1992, Sorokin et al. 1996). When cultures of because both compounds are readily transformed into this bacterium were grown with thiosulfate and sulfide each other by a number of chemical and biological was added in addition, growth stimulation occurred reactions. and was considered to be due to the sulfide mediated Textbooks of inorganic chemistry characterize poly- reduction of tetrathionate, which regenerates the elec- thionates like tetrathionate as rather instable com- tron donor thiosulfate (Sorokin et al. 1996). These Podgorsek & Imhoff: Tetrath~oinate in Baltic Sea sediments 263

authors proposed that the chemical reaction of sulfide of thiosulfate and tetrathionate and the elemental sul- with tetrathionate led to the accumulation of elemental fur accumulation within Baltic Sea sediments has been sulfur and formation of thiosulfate, similar to the situa- given before (Imhoff 1996). tion described for Rhodomicrobium vannielii (Hansen Due to the high rates of tetrathionate formation from 1974). thiosulfate in sediments (max. 0.1 pm01 h-' cm-3) as well as by isolated sulfur oxidizing bacteria (e.g. 0.8 pm01 h-' mg-' protein by Pseudomonas stutzeri strain BS8), Role of tetrathionate in the sediment one could easily be misled into expecting much higher concentrations of tetrathionate in the pore water of Up to now all discussion on ecological aspects of the these sediments than were actually measured. How- role of tetrathionate in the sulfur cycle of the sediments ever, accumulation of tetrathionate to high concentra- is based either on the analytical detection of tetra- tions is naturally excluded because of its high reactiv- thionate in the environment or on transformations ity not only by biological but also in particular by with pure cultures Like Catenococcus thiocyclus (e.g. chemical reactions, as outlined above. Most signifi- Sorokin et al. 1996). The presence of tetrathionate in cantly, in the presence of sulfide it readily reacts to the natural environment has been reported in a few elemental sulfur and thiosulfate as products (Roy & cases only (Luther et al. 1986, Henneke et al. 1997). Trudinger 1970). With seasonal dependence, extraordinarily high con- Thus, in sulfidic marine environments tetrathionate centrations of up to 300 pM tetrathionate were de- is an unstable component that does not easily accumu- tected in pore waters of the Great Marsh, Delaware late to detectable quantities unless continued new for- (Luther et al. 1986). In the Tyro and Bennock Basins mation is fast enough to overcome depositional pro- of the Mediterranean Sea concentrations of 11.1 and cesses. In no way is the size of the tetrathionate pool an 20.9 PM, respectively, were measured (Henneke et al. indicator of the turnover through the pool. In fact, low 1997). concentrations of thiosulfate and tetrathionate are suf- Our results demonstrate that depending on the envi- ficient to promote the large-scale oxidation of sulfide to ronmental conditions tetrathionate and sulfate are pro- elemental sulfur by coupled bacterial and chemical duced as oxidation products of thiosulfate in different reactions. Thereby, bacteria that are unable to utilize proportions in brackish water sediments. Concentra- sulfide by themselves may be able to gain energy out tions of tetrathionate of up to 21.6 yM in the pore of the net oxidation of sulfide to elemental sulfur. The waters of sediments at Hiddensee and up to 7.5 pM in chemical reaction of tetrathionate with sulfide (Roy & sediments of the Gotland Basin were found. Depth pro- Trudinger 1970, Hansen 1974) to produce elemental files of the distribution of sulfur oxidizing bacteria sulfur and 2 molecules of thiosulfate forms the oxida- revealed a strong correlation between viable cell tion product of the overall reaction (elemental sulfur) counts of these bacteria and potential rates of thiosul- and reconstitutes the substrate for the bacteria on a fate oxidation to tetrathionate and sulfate within the stoichiometric basis. Thus, the combined action of bac- Baltic Sea sediments. In addition, pure culture studies terial tetrathionate formation from thiosulfate and the with representative isolates proved the ability of the chemical reaction with sulfide results in a cyclic turn- bacteria to oxidize thiosulfate and to form tetrathionate over of thiosulfate and tetrathionate and in the net and also sulfate as oxidation products. A preliminary accumulation of elemental sulfur as the final oxidation report of this work including a discussion of the cycling product, with thiosulfate and tetrathionate acting as a catalytic couple (Fig. 7). Therefore, bacterial tetrathio- nate formation under sulfidic conditions can account very well for the accumulation of elemental sulfur (but not tetrathionate) in natural marine sedinlents as pre- sented in the reaction scheme of the 'tetrathionate cycle'. In the naturally alkaline marine environment tetrathionate also may be hydrolyzed chemically or enzymatically to form elemental sulfur, thiosulfate and Fig. 7. The tetrathionate cycle. The combined action of bacte- rial thiosulfate oxidation to tetrathionate and the chemical sulfate (Roy & Trudinger 1970). Although the accumu- reactions between sulfide and tetrathionate lead to a cyclic lation of large amounts of elemental sulfur has been reaction sequence with a net formation of elemental sulfur observed in several basin sediments of the Baltic Sea (Roy & Trudinger 1970, Hansen 1974). On the basis of these during the past years (e.g. Fig. 5, Podgorsek 1998), at reactions elemental sulfur may accumulate in marine sedi- ments. Such a cycle has been previously proposed on the present we cannot give any quantitative estimation of basis of pure culture studies with Catenococcus thiocyclusby the proportion of the elemental sulfur that has passed Sorokin et al. (1996) through the tetrathionate pool. 264 Aquat Microb Ecol 17: 255-265, 1999

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Editorial respondbility: Tom Fenchel, Submitted. February 2, 1998; Accepted. November 2, 1998 Hels~ng~r,Denmark Proofs received from a uthor(s):June 1, 1999