Depth-dependent fate of biologically-consumed dimethylsulfide in the Sargasso Sea

Marine Chemistry 103 (2007) 197 – 208 www.elsevier.com/locate/marchem

Depth-dependent fate of biologically-consumed dimethylsulfide in the Sargasso Sea Daniela A. del Valle a,⁎, David J. Kieber b , Ronald P. Kiene a b

a Department of Marine Sciences, University of South Alabama, Mobile, AL 36688 and Dauphin Island Sea Lab, Dauphin Island, AL 36528, USA State University of New York, College of Environmental Science and Forestry, Chemistry Department, Syracuse, NY 13210, USA

Received 7 March 2006; received in revised form 28 July 2006; accepted 31 July 2006 Available online 14 September 2006

Abstract Biological consumption is a major sink for dimethylsulfide (DMS) in the surface ocean, but the fate of DMS is poorly known. We determined the fate of sulfur from biologically consumed DMS in samples from the upper 60 m of the Sargasso Sea during July 2004. Using tracer levels of 35S-DMS in dark incubations we found that DMS was transformed into three identifiable non-volatile, sulfurcontaining product pools: dimethylsulfoxide (DMSO), sulfate, and particle-associated macromolecules. Together, DMSO and sulfate accounted for most (81–93%) of the non-volatile sulfur products. Only a small fraction (∼ 2%) of the consumed DMS-sulfur was recovered in cellular macromolecules, leaving 5–17% of the metabolic products of DMS consumption unidentified. The relative importance of the two major products varied with depth. DMSO was the main sulfur product (∼ 72%) from DMS metabolism in the surface mixed layer, whereas sulfate was the most important product (∼ 74%) below the mixed layer. Changes in temperature and photosynthetically-active radiation (PAR) did not cause shifts in DMS fate in short term incubations (7–12 h), however these or other factors (e.g., exposure to ultraviolet radiation), operating over longer time scales, could potentially influence the observed pattern of DMS fate with depth. Biological DMSO production rates ranged from 0.07 to 0.33 nM day− 1, with the highest rate found at 30 m, just below the surface mixed layer. With DMSO concentrations ranging from 4.0 to 8.6 nM, turnover times for DMSO were long (15– 61 days) when only the biological production from DMS was considered. Identification of the main sulfur containing products from DMS metabolism improves understanding of this important process in the marine sulfur cycling. Detection and quantification of DMSO production from biological DMS consumption also provides a more complete picture of DMSO biogeochemistry in the ocean. © 2006 Elsevier B.V. All rights reserved. Keywords: Dimethysulfoxide; Sulfate; DMS fate; Sulfur cycle; Sargasso Sea

1. Introduction Dimethylsulfide (DMS) is biologically produced in the ocean from enzymatic cleavage of dimethylsulfoniopro⁎ Corresponding author. Dauphin Island Sea Lab, 101 Bienville Boulevard, Dauphin Island, AL 36528, USA. Tel.: +1 251 861 7525; fax: +1 251 861 7540. E-mail address: [email protected] (D.A. del Valle). 0304-4203/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.marchem.2006.07.005

pionate (DMSP), a compound synthesized by a wide variety of marine phytoplankton (Keller et al., 1989). DMS concentrations are determined by the balance between its production and biological and physical loss mechanisms (Gabric et al., 1999), which are part of a complex web of ecological and biogeochemical processes. DMS is supersaturated in oceanic surface waters and its emission to the atmosphere represents N 90% of the oceanic sulfur flux and N 50% of the global biogenic flux

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(Andreae, 1986). Marine emissions of DMS play an important role as precursors of atmospheric sulfurcontaining aerosols that can influence climate through direct backscatter of solar radiation and through cloud formation (Andreae, 1985; Charlson et al., 1987; Shaw, 1983). This effect of DMS-derived aerosols on climate provides a basis for feedback on the biological communities that are responsible for DMS production (Charlson et al., 1987; Malin et al., 1994; Malin et al., 1992). Oceanic DMS removal occurs through sea–air exchange, photo-oxidation and microbial consumption (e.g., Brimblecombe and Shooter, 1986; Kieber et al., 1996; Kiene and Bates, 1990). Gabric et al. (1999) reported, based on an observation and modeling study, that biological consumption is often the most important loss process for DMS for a range of bloom situations. In addition, a compilation of available data from the literature (Simó, 2004) showed that biological DMS consumption is responsible for the loss of, on average, 50–80% of DMS production. Thus, biological DMS consumption is a process that can significantly control DMS concentrations in surface waters, even though its relative importance may vary spatially and temporally (Kieber et al., 1996; Simó and Pedrós-Alió, 1999). While the importance of biological degradation of DMS in seawater is recognized (Wolfe and Kiene, 1993), and it is of interest with respect to reduced sulfur chemistry (Kiene and Bates, 1990), the metabolic fate of biogenic DMS-sulfur and -carbon in seawater is not well known. Wolfe and Kiene (1993) found that DMScarbon was partially recovered in cellular material and CO2, but other potential products were not quantified. Several recent studies have shown that DMS metabolism results in the formation of dissolved non-volatile (DNV) sulfur compounds (Kiene and Linn, 2000a; Zubkov et al., 2002; Zubkov et al., 2004), but so far, only sulfate has been identified as a specific sulfurcontaining product. Although sulfate was ∼ 80% of the non-volatile products of DMS metabolism in the few samples measured by Kiene and Linn (2000a), it is not known whether sulfate is always a major product or whether other products such as dimethylsulfoxide (DMSO) or dimethylsulfone (DMSO2) are also produced (Andreae, 1980a). DMSO is one of the most abundant forms of methylated sulfur in marine systems, with concentrations often exceeding that of DMS (Hatton et al., 1996; Simó et al., 2000). Known sources of DMSO include photooxidation of DMS (Brimblecombe and Shooter, 1986; Kieber et al., 1996; Toole et al., 2004), atmospheric deposition (Andreae, 1980a) and phytoplankton release (Simó et al., 1998). DMSO has also been shown to be produced in sedimenting

material due to bacteria-mediated transformation of DMSP, with DMS as a potential intermediate (Hatton, 2002). It has been proposed that bacteria can enzymatically oxidize DMS to DMSO in seawater, based on results from microbial culture experiments (Juliette et al., 1993; Zhang et al., 1991); however, microbial production of DMSO from dissolved DMS has not yet been demonstrated in a natural, aerobic system. In this work, we used 35S-DMS to trace the fate of biologically-consumed DMS-sulfur into macromolecules, sulfate, and DMSO, as a function of depth in the water column of the Sargasso Sea. We also conducted experiments in order to assess the role of temperature and visible light on the metabolic utilization of DMS. 2. Methods 2.1. Hydrographic stations and sampling Sampling was carried out in the Sargasso Sea, in the vicinity of the Bermuda Atlantic Time-Series Study (BATS) station, aboard the RV Seward Johnson during a Lagrangian experiment where an upwelling mesoscale eddy was followed with drifters from July 15 to July 25, 2004 (Fig. 1). Oligotrophic conditions prevailed during the summer in this area with low chlorophyll concentrations (b 0.2 μg l− 1 above 60 m) and low macronutrient concentrations (phosphate b16 nM, nitrate b 7 nM). Clear skies or low cloud cover was experienced during the 11 days of sampling. The attenuation depth (depth to which 1% of surface irradiance penetrated) for 320 and 395 nm radiation, was 43.6 and 106.3 m, respectively, as determined with a Biospherical PUV 511 underwater radiometer. Detailed information about the physicochemical characteristics of the sampling sites will be published elsewhere (Bailey et al., submitted for publication). Water samples were generally collected just before dawn, except when otherwise noted, in 10-l Niskin bottles attached to a CTD rosette. Six depths were sampled in the upper 60 m for DMS-sulfur fate determination, three below and three above the mixed layer depth, which was located at 18.1 ± 1.4 m at the time of sampling. 2.2. Seawater incubations Duplicate or triplicate incubations of seawater samples from each depth were carried out in 20-ml glass serum vials to determine the consumption rate of 35S-DMS, the assimilation of 35S, and the production of dissolved nonvolatile products. Prior to use, the vials were acid cleaned and then rinsed three times with the seawater sample.

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Fig. 1. Cruise track of the RV Seward Johnson from July 15 to July 25, 2004. The ship followed drogues that tracked the upper 10 m of the water column of a cyclonic eddy, named C1. Stations are shown as solid circles. The solid square shows the location of the Bermuda Atlantic Time-Series Study (BATS) station.

Unfiltered seawater samples were carefully pipetted or poured into the vial and the vial sealed with a Teflonfaced butyl rubber stopper (Wheaton), leaving no more than 10% of the vial volume as headspace. No difference was observed in this system between pipetting or pouring the samples into the vials. Gaseous 35S-DMS (specific activity 106 disintegrations per minute (dpm) pmol− 1; generated from 35S-DMSP by alkaline cleavage) was injected by syringe through the stopper yielding additions of 35S of 2000–4000 dpm ml− 1 dissolved in the seawater. This corresponds to a DMS addition of 2–4 pM, which did not perturb the ambient seawater concentration (ca. 1–7 nM). Samples were incubated in the dark in a closed incubator with running surface seawater (ca. 28 °C). Abiotic controls consisting of seawater filtered first through a 0.2 μm Nylon filter to remove bacteria and other particles, then treated with 35S-DMS, were run in parallel to live samples in order to account for any nonbiological loss of the 35S-DMS (e.g., chemical oxidation, adsorption to vial surface). The loss of tracer observed in the controls was always b 0.5% of the total 35S-DMS added.

In addition to the basic incubation experiments for depth profile samples outlined above, we also conducted an experiment to determine the effect of temperature on particulate and dissolved non-volatile product yields. For this experiment, whole seawater was collected from 40 m on July 25 and incubated at surface (28 °C) and in situ (24 °C) water temperature in the dark for 12 h. Preparation of the samples was performed as described above. An experiment was also conducted to determine the effect of added unlabeled DMSO on product yields. Whole seawater for this experiment was collected from 10 and 30 m on July 25 at 1200 local time and amended with 75 nM DMSO (+DMSO treatment) prior to the addition of the 35S-DMS. Whole seawater controls, with only 35S-DMS added, were incubated under the same conditions (i.e., in the dark in a surface seawater bath). The natural DMSO concentration in the controls was ∼ 4 nM for both 10 and 30 m samples. A time course experiment was conducted to determine the effect of incubation time on product yields. Unfiltered seawater collected from 10 m on July 24 was divided into

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a series of 25-ml glass serum bottles, which were inoculated with 35S-DMS and placed in a dark incubator with surface running water. Triplicate bottles were analyzed for product yields at each time point (12, 25 and 49.5 h). Finally, we examined the effect of Photosynthetically Active Radiation (PAR) on biological DMS consumption rates and product yields. Unfiltered seawater collected on July 23 from 10 and 40 m (i.e., within and below the mixed layer, respectively) was incubated under PAR and dark conditions for ∼ 7 h. For incubations under PAR, triplicate samples were placed in a neutral density screen bag in order to attenuate 35% of incident solar radiation. The bag was incubated beneath an ultraviolet radiationopaque longpass filter (UF3 Plexiglas) in an incubator kept at a constant temperature with running surface seawater. Samples under the PAR treatment were exposed to a total PAR dose of 27.6 Ein m− 2 during the incubation time. For dark incubations, triplicate samples were covered with aluminum foil and placed in the same water bath with PAR-incubated samples. Radiolabeled 35 S-DMS was added at tracer concentrations (ca. 2–4 pM) immediately before the start of the incubation. Controls with 0.2 μm filtered seawater were incubated under dark and PAR conditions in order to account for any nonbiological loss. All samples were processed immediately after the incubation was finished. 2.3. Analytical methods 2.3.1. DMS and DMSO concentrations DMS concentrations were determined using a purgeand-trap system followed by flame photometric gas chromatography (for a detailed description of the system see Kiene and Service, 1991). Dissolved DMSO was determined by gas chromatography after its quantitative conversion to DMS by chemical reduction with TiCl3 (Fisher Scientific Co.), as described in Kiene and Gerard (1994). 2.3.2. Biological DMS consumption rate After seawater samples were incubated from 10 to 12 h, the Teflon-lined butyl rubber stopper was removed from the serum vials and a 1 ml subsample was pipetted into a scintillation vial containing 5 ml Ecolume scintillation fluid. This subsample accounted for the total activity per ml added to the sample (Atotal). The remaining sample was sparged with N2 for 10 min (flow: 100 ml min− 1) to remove the remaining volatile 35 S-DMS. After sparging, a 1 ml subsample was counted to obtain the dpm ml− 1 present in the nonvolatile products (ANV). Any activity lost due to sparg-

ing was assumed to be from unreacted 35S-DMS. This assumption is based on the good agreement found between DMS loss estimated by gas chromatography and the tracer approach (D.A. del Valle, unpublished data). Moreover, even though H2S and methanethiol have been found to be products or intermediates of DMS metabolism (de Bont et al., 1981; Suylen et al., 1986; Visscher and Taylor, 1993), the high reactivity of these gases in oxic seawater transforms them quickly into nonvolatile (NV) products (Millero, 1986; Kiene, 1996). The consumption rate constant (k) was taken as the absolute value of the slope of a linear regression of the natural log of the fraction of untransformed 35S-DMS (i.e., ln (1 − ANV / Atotal)) plotted as a function of incubation time, assuming first-order uptake kinetics (Kiene and Linn, 2000b). Typically, b20% of the added label was converted to non-volatile products during the incubations. Rates were obtained by multiplying the rate constant by the corresponding DMS concentration ([DMS]) at the depth that the sample was collected: DMS Consumption Rate ¼ k½DMS

ð1Þ

2.3.3. Macromolecular sulfur assimilation A 10 ml sub-sample of the incubated seawater sample or control was sparged as described above, and then withdrawn by pipette and transferred to a 10-place Hoefer filtration manifold. Samples were filtered through a 0.2 μm Nylon filter (Whatman) using a gentle vacuum (b 5 cm Hg) and then rinsed with 0.2 μm-filtered seawater (FSW) of the same salinity as the sample. The filters were then treated with 5 ml of 5% trichloroacetic acid (TCA) for 5 min in order to precipitate macromolecules and rinse away soluble cell components (Kirchman et al., 1985). After rinsing the filters with Milli-Q water, the radioactivity in the TCA-rinsed filters was determined in 5 ml Ecolume scintillation fluid (Afilter). The percentage of the consumed DMS sulfur assimilated into macromolecules (Assimilation yield) was calculated using the equation:    Afilter Assimilation yield ð%Þ ¼ =ANV  100% 10 ml ð2Þ Tests showed that nearly all the 35S associated with particles was in the TCA insoluble fraction (i.e., assimilatory uptake equal to total uptake) indicating that the assimilated sulfur was incorporated into macromolecular material and not simply stored or converted into low molecular weight species. Sparging of the water sample before filtration had no effect on the measured assimilation yields (data not shown).

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2.3.4. Dissolved non-volatile products The 0.2 μm filtrate obtained from the assimilation filtration was collected before the 0.2 μm FSW rinse to determine the dissolved non-volatile products. A 1 ml subsample of the filtrate was removed by pipette and counted to quantify the activity associated with dissolved non-volatile (DNV) sulfur containing products per ml (ADNV). The remaining filtrate was used to obtain the 35SDMSO and the 35S-sulfate content in the DNV fraction. 2.3.5. Sulfate yield from DMS The amount of sulfate produced during the incubation was quantified with a BaSO4 precipitation technique described in Kiene and Linn (2000a). Briefly, 1 ml subsamples of the DNV-containing filtrate were pipetted into a duplicate series of microcentrifuge tubes, containing either 0.1 ml of Milli-Q water (non-treated sample) or 0.1 ml of 1 M BaCl2. The tubes were centrifuged in order to sediment the BaSO4 formed. After centrifugation, 1 ml subsamples of supernatant of the Batreated sample (ADNV–SO4) and the non-treated sample (AH2O) were counted separately in 5 ml Ecolume. The sulfate yield (%) in the NV produced from the biologically consumed DMS was calculated using the formula: 

Sulfate yield ð%Þ ¼

  ADNV SO4 1− 100% AH 2 O   100%−Assimilation Yield  100%

ð3Þ

The first factor represents the sulfate yield in the DNV fraction while the second factor is introduced in order to relate the sulfate yield to the total NV fraction (i.e., the total product pool). The second factor had a minimal effect on the calculated sulfate yield because assimilation was usually a minor product pool (∼ 2%, see below). Since barium can form insoluble salts with sulfite as well as sulfate, a similar precipitation method was developed in order to test whether the precipitated 35S was exclusively from sulfate or not. Instead of adding 0.1 ml of 1 M BaCl2, the same volume of a 3.2 M NiCl2 solution was added into the centrifuge tube. At the final concentration used, nickel forms an insoluble salt with sulfite but not with sulfate. Due to the low natural sulfite concentration in seawater, the addition of 0.1 ml of 0.72 M unlabeled Na2SO3 was necessary to induce precipitation. The non-treated sample consisted of a centrifuge tube with 0.1 ml of Milli-Q water and 0.1 ml of the Na2SO3 solution. A 1-ml subsample of the DNV fraction was pipetted into each tube and then treated as explained for the barium precipitation technique. In

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experiments carried out with both coastal and oceanic water, b 3% of the activity precipitated with barium was also precipitated with nickel (presumably sulfite, although some coprecipitation of sulfate was also possible) suggesting that sulfate is indeed the main 35S containing compound precipitated with barium. 2.3.6. DMSO yield from DMS A 3–4 ml subsample of the filtrate was transferred to a 7 ml glass serum vial where approximately 1 mg of cobalt-doped sodium borohydride (Sigma) was added to reduce DMSO to DMS (Riseman and DiTullio, 2004; Simó et al., 1996). After 30 min, the sample was sparged with N2 for 10 min (flow: 100 ml min− 1) to remove the 35 S-DMS, which was formed from the reduction reaction. Once the N2 flow was turned off, it was necessary to wait for 10 min to allow the unreacted NaBH4 solid phase to settle, thereby avoiding problems during liquid scintillation counting associated with scattering. Once the solution was relatively clear, a 1 ml subsample of supernatant was counted in 5 ml Ecolume in order to obtain the dpm ml− 1 present in the 35S-DMSO-free DNV fraction (ADNV–DMSO). It was possible to obtain a clearer supernatant by centrifuging the reaction mixture, however, no significant difference between the results obtained by either method was found. The percentage of DMSO present in the total non-volatile fraction produced from DMS consumption (DMSO yield) was calculated using the following equation: DMSO yield ð%Þ ¼

   ADNV DMSO 1− 100% ADNV   100%−Assimilation Yield  100%

ð4Þ

The first factor represents the DMSO yield in the DNV fraction while the second factor is introduced in order to relate the DMSO yield to the total NV fraction. The DMSO yield was calculated with this approach in order to be consistent with the way the sulfate yield was calculated. Other more direct calculations relating the activity detected from DMSO (ADNV − ADNV–DMSO) to the total non-volatile products pool (ANV) yielded essentially the same results as did Eq. (4). Previous tests have shown that the reagent, NaBH4, does not produce volatile sulfur-containing products from DMSO2, and sulfur containing amino acids and derivatives (Andreae, 1980b). Nanomolar levels of methane sulphinic acid (MSNA) and methane sulphonic acid (MSA) also tested negative for production of volatile compounds when reacted with NaBH4 (data not shown). The only reported DMS interference with NaBH4 is

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DMSP (Simó et al., 1996), which is not likely to be a product of 35S-DMS metabolism in our experiments. 3. Results 3.1. DMS-sulfur fate The fraction of DMS-sulfur assimilated into macromolecules was low and nearly constant throughout the water column, with values ranging from 1.8% to 3.3% of the consumed DMS (Fig. 2). It was found that the majority of the consumed 35S-DMS, ∼ 98%, was converted into dissolved non-volatile products. Within the DNV pool, which included DMSO, sulfate, and unidentified compounds, the metabolic fate of DMSsulfur varied with depth in the water column (Fig. 2). The trend shown in Fig. 2 was a consistent feature of the upwelling eddy in the Sargasso Sea, since it was also observed on two other days (July 19 and 23) and for seawater samples collected at different times of the day (0500 and 1700 local time; data not shown). In the surface mixed layer, DMSO was the main nonvolatile product produced from the biologically consumed DMS. In this layer, the DMSO yield did not vary appreciably with depth and averaged 72% of the NV sulfur containing product pool. At greater depths, the DMSO yield gradually decreased to reach the minimum yield of ∼ 16% at 40–60 m deep. The sulfate yield from

Fig. 3. Time course of DMSO, sulfate and assimilation yields for a single water sample obtained from 10 m depth on July 24, 2004. Triplicate samples were processed after dark incubation for 12, 25 and 49.5 h. Error bars indicate the standard deviation of the mean.

DMS followed an opposite pattern from the DMSO yield (Fig. 2), with low and uniform values (11 ± 1%) in the surface mixed layer and a gradual increase at depth, becoming the most important product below the mixed layer (74%). DMSO and sulfate together accounted for 81–93% of the total NV products at all depths. Longer incubation times produced a significant decrease in the DMSO yield (p b 0.05, One Way ANOVA) from 78% to 65% (Fig. 3); however, that change was not reflected in a significant increase in the sulfate yield or the assimilation yield (p N 0.05, One Way ANOVA), suggesting that some other non-volatile sulfur-containing products were formed from DMSO. All sulfate yields were plotted against the corresponding biological DMS consumption rates and rate constants, as well as against the DMSO yields (Fig. 4). The sulfate yield was positively correlated to the DMS consumption rate (r2 = 0.86, p b 0.001, Fig. 4a), although that relationship was mainly driven by the correlation to the DMS consumption rate constant (r 2 = 0.87, p b 0.001, Fig. 4b), since no significant correlation was found to DMS concentration (r2 = 0.10, p = 0.23, data not shown). A negative correlation between the sulfate and DMSO yields was found (r2 = 0.93, p b 0.001, Fig. 4c). The sulfate yield was not correlated to leucine incorporation (r2 = 0.10, p = 0.32, data not shown), a general indicator of bacterial production (Kirchman et al., 1985). 3.2. Biological DMSO production from DMS

Fig. 2. Vertical profile of the yield of sulfur-containing non-volatile products (assimilation, DMSO or sulfate) from biologically consumed DMS in the Sargasso Sea on July 25, 2004. Error bars indicate the range of duplicate determinations. The broken and dotted lines show the temperature and density profile, respectively. The mixed layer depth was at 18 m.

The biological DMSO production rate from biologically consumed DMS (Fig. 5a) was obtained as the product of the DMSO yield (Fig. 2) and the biological DMS consumption rate (Fig. 5b). The maximum DMSO

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DMSO was present in the mixed layer with a relatively uniform concentration of 4.1 nM (Fig. 5c). Below the mixed layer the DMSO concentration increased with depth to reach a maximum (8.6 nM) at 40 m. No correlation between biological DMSO production and in situ DMSO concentration was found in this limited data

Fig. 4. Sulfate yield as a function of (a) biological DMS consumption rate, (b) biological DMS consumption rate constant (k) and (c) DMSO yield. Linear regression lines are shown with equation and statistics.

production rate occurred at 30 m (0.33 nM day− 1), and below this depth the DMSO production rate decreased due to the lower DMSO yield, despite the higher biological DMS consumption rates. The depth-integrated biological DMS consumption rate from 0–60 m was 28.5 μmol m− 2 day− 1, whereas the integrated DMSO production was 9.24 μmol m− 2 day− 1, with only 17% of this production taking place in the surface mixed layer. Over the water column, a net 32% of the DMS that was biologically consumed was transformed to DMSO.

Fig. 5. (a) Vertical profile of DMSO production rate obtained on July 25 as the product of the DMSO yield and the biological DMS consumption rate. The bars represent the propagated error from the product of the biological DMS consumption rate and the DMSO yield. (b) Vertical profiles of biological DMS consumption rate and (c) of DMSO concentration. Values are the mean of duplicate determinations with the bars indicating the range for both (b) and (c).

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set (r2 = 0.057; p = 0.65), presumably due to the slow turnover of DMSO and its low biological production rate in the upper 20 m. 3.3. Experimental tests 3.3.1. DMSO addition In order to test if sulfate was a product of the subsequent oxidation of the DMSO produced from DMS, we incubated 10 and 30 m seawater with 35SDMS tracer and 75 nM unlabeled DMSO (+DMSO treatment) and compared the 35SO42− formed in these to controls receiving 35S-DMS only. Any 35S-DMSO formed from 35S-DMS would be diluted by the unlabeled DMSO, which would slow down any conversion of 35SDMSO to 35SO42−, thereby decreasing the sulfate yield from 35S-DMS. We found, however, that the fate of the consumed DMS was not affected by the addition of unlabeled DMSO in either surface (10 m) or deeper (30 m) water (Fig. 6). No significant change in DMSO yield (p = 0.30 and p = 0.33, t-test, respectively) or sulfate yield (p = 0.31 and p = 0.18, t-test, respectively) was observed when comparing the +DMSO and control treatments.

35

Fig. 6. DMSO and sulfate yield from S-DMS measured in control and +DMSO (+75 nM) treatment in (a) 10 and (b) 40 m samples. Error bars denote the standard deviation of the mean.

Fig. 7. Yields obtained for assimilation, DMSO and sulfate when 40 m water was incubated in the dark with 35S-DMS at in situ and surface temperature for 12 h. Error bars indicate the standard deviation of the mean.

3.3.2. Incubation temperature Since incubations were carried out in a water bath with running surface seawater, the samples collected from below the surface mixed layer (40 m) were incubated at a temperature slightly higher than the in situ temperature (28 instead of 24 °C). In order to determine

Fig. 8. Assimilation, DMSO and sulfate yields obtained from 35S-DMS in (a) 10 and (b) 40 m samples incubated under 65% solar incident PAR and in the dark for ∼ 7 h. Error bars denote the standard deviation of the mean.

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if the difference in the metabolic yield of biologically consumed DMS that we observed with depth (Fig. 2) was partly due to a change in the temperature for the deeper samples, the fate of 35S-DMS in 40 m water was determined in samples incubated at the in situ temperature at 40 m, 24 °C, and at the surface incubator temperature, 28 °C (Fig. 7). No significant difference between the two treatments was found for any of the product yields (sulfate, p = 0.31; DMSO, p = 0.37; assimilation of sulfur into macromolecules, p = 0.51, t-test). 3.3.3. PAR exposure Since planktonic communities present in the surface mixed layer of the Sargasso Sea may experience high levels of PAR during the day, an experiment was undertaken to test whether exposure to relatively high levels of PAR (65% incident solar radiation; UV excluded) would affect the metabolic fate of DMS in samples collected from above and below the mixed layer (10 and 40 m). No significant differences in the DMSO and sulfate yields (Fig. 8) were observed between the two treatments for either surface or deep water (p N 0.1, t-test). Abiotic controls consisting of 0.2 μm filtered seawater with 35S-DMS showed no abiological loss of DMS under the same incubation conditions as the experimental samples. 4. Discussion This study shows that the fate of the biologically consumed DMS varies with depth in the water column of the Sargasso Sea. We quantified the DMSO and sulfate production from the biologically consumed DMS, which allowed the characterization of most of the NV pool; only 7–19% of the NV fraction was unidentified. Other potential sulfur-containing products of DMS metabolism could include DMSO2, sulfide, or methanesulfonate, but we have no information on whether these compounds were produced, due to lack of specific methods for their radiochemical analysis. The microbial community metabolized most of the DMS to DMSO in the surface mixed layer, whereas the microbial community present below the mixed layer transformed the DMS mostly to sulfate. Sulfur assimilation was uniformly low (∼ 2% of DMS consumed) at all depths examined. DMS-sulfur assimilation contributed only 0.7–4.2% of the calculated bacterial sulfur assimilation based on measured leucine incorporation rates and an assumed C:S ratio of 248. These low values of DMSsulfur assimilation agree with results obtained in other oceanic waters, where DMSP instead of DMS satisfied most of the bacterial sulfur needs (Kiene and Linn, 2000a; Zubkov et al., 2002).

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In the work presented by Kiene and Linn (2000a), sulfate accumulation from DMS lagged DNV accumulation in time course experiments, but it eventually became the most important DNV product. From these results, the authors suggested that one or more extracellular dissolved intermediate compounds were involved in the biologically mediated transformation of DMS to sulfate. The higher DMSO yield in the surface layer reported in this work could have been due to a short incubation time, with DMSO and the unidentified fraction of the DNV pool acting as possible intermediates in the net transformation of DMS to sulfate. This hypothesis is supported by the correlation between the sulfate yield and the biological DMS consumption rate (Fig. 4a), where a less active bacterial community in terms of DMS consumption would probably require a longer time to complete the oxidation of DMS to sulfate. However, this was not the case for the water from the surface layer, since prolonged incubations (49.5 h) did not show a significant increase in sulfate yield, although there was a small decrease in DMSO yield (12%) (Fig. 3). DMSO was transformed into an unknown compound, increasing the fraction of the NV pool that was not identified. Moreover, since DMSO and sulfate accounted for most of the DNV product pool, in order to significantly increase the sulfate yield if a longer incubation was performed, DMSO should have been the compound acting as the extracellular intermediate. The fact that the sulfate yield was unaffected by the addition of unlabeled DMSO, gives further evidence against DMSO being the extracellular intermediate in the oxidation of DMS to sulfate at this study site. The differences in fate of the biologically consumed DMS-sulfur in the top 60 m of the water column of the Sargasso Sea might have been due to different regulatory responses of the bacteria responsible for DMS consumption to the environmental conditions experienced above and below the mixed layer. A direct or indirect relationship with mixing seems apparent due to the uniform values found in the mixed layer and the gradual change with depth in the layer below it. We ruled out PAR exposure (Fig. 8) and temperature (Fig. 7) as possible factors driving a short-term (7–12 h) regulatory change in DMS metabolism; moreover, macronutrient concentrations were uniformly low throughout the studied depths. Other variables that were not tested or measured in the water column that could have been involved in regulating the metabolic use of DMS include micronutrient concentrations, organic matter availability, and UV exposure. It is also possible that the difference in metabolic utilization of DMS-sulfur in the two layers could have been related to the presence of different microbial communities, with different metabolisms. Several studies

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have shown depth-specific distributions of both autotrophic and heterotrophic bacteria (DuRand et al., 2001; Field et al., 1997; González et al., 2000), as well as archaea (Massana et al., 1997). Bacteria are the most likely consumers of dissolved DMS, although there is little evidence to support a strictly bacterial role at present. It has been shown that ultraviolet radiation has the potential to produce changes in the natural marine bacterial community composition (Winter et al., 2001). Relatively shallow mixed layers, combined with deep light penetration, could have driven a selection for UV-resistant DMS consuming bacteria in the mixed layer, which can potentially have a different metabolism than DMS consuming bacteria present below the mixed layer. In experiments carried out in the Ross Sea, Antarctica, 5 m whole water exposed to PAR for ∼ 72 h showed no significant change in DMSO yields compared to the initial seawater, in agreement with the present results from the Sargasso Sea. On the other hand, in unfiltered Ross Sea water exposed to UV for 72 h, the DMSO yield increased significantly from the initial value of 27% to 54% (D.A. del Valle, unpublished results). However, no significant changes in the metabolic fate of DMS were observed in the Ross Sea among samples exposed to UV, PAR, and dark treatments for short incubation times (b 10 h). Thus, it seems that UV might be a factor controlling DMS fate in the upper layers of the ocean, probably by driving bacterial community changes. Vila-Costa et al. (in press) reported changes in the metabolic fate of DMS when Sargasso Sea water (collected during this same cruise) was enriched with different carbon sources. In the initial seawater, 70.1% of the consumed DMS was transformed to DMSO. At the end of the experiment (12 days), a similar DMSO yield was found in the glucose enrichment (88.3%), while the treatments amended with micromolar concentrations of DMS and DMSO presented DMSO yields of only 12.3% and 9.9%, respectively. In the work of Vila-Costa et al. (in press), a change in the metabolic use of DMS was seen when bacterial community composition shifted due to a change in the available carbon source. DMSO is present in seawater at concentrations comparable to, or often higher than DMS (Hatton et al., 1996; Simó et al., 2000), yet relatively little progress has been made regarding the role of this compound in the sulfur cycle. Although it has been speculated upon, up to now there has been no direct evidence of biologically-mediated DMSO production from dissolved DMS. Lack of information in this regard may be due to methodological shortcomings. Current gas chromatographic methods for DMSO are not sensitive or precise enough to detect the small changes in the concentration of DMSO that are caused by its dark

biological production in the Sargasso Sea (e.g.,b0.33 nM DMSO produced day− 1, Fig. 5a). Moreover, DMSO can potentially be released from phytoplankton cells (Simó et al., 1998) masking the microbial production from dissolved DMS. The use of radiolabeled 35S-DMS permitted the detection of 35S-DMSO production and allowed the measurement of DMSO production from dissolved DMS to be made without being affected by any other potential source. The sensitivity of this method is related to the fraction of DMS consumed and not to the absolute amount consumed, and it can be improved by increasing the amount of radioactivity added without significantly affecting the in situ DMS concentration. Using this approach, low DMSO production rates were measured over the water column with values ranging from 0.07 to 0.33 nM day− 1. If we assume no DMSO losses and that the biologically consumed DMS was the only source of DMSO, it would have taken 15–61 days to build up the measured DMSO pool in the water column (0–60 m). These data suggest that either DMSO turns over very slowly, or that production of DMSO from the biologically consumed DMS is not a main source of DMSO in oceanic waters. The fact that there was not a significant relationship between DMSO production rates and DMSO concentration (r2 = 0.057; p = 0.65), supports the latter view. Other sources of DMSO to surface seawater include photochemical oxidation of seawater DMS (Brimblecombe and Shooter, 1986; Kieber et al., 1996), direct release from phytoplankton (Simó et al., 1998) and deposition from the atmosphere (Andreae, 1980b). The integrated (0–60 m) DMSO production rate from photolysis, obtained using average photolysis rate constants for the eddy (Bailey et al., submitted for publication), DMS concentrations from July 25, and an average photolysis DMSO yield of 68% for the water column (as obtained on July 16, data not shown), was 5.68 μmol m− 2 day− 1, which constitutes only 61% of the DMSO production calculated from biological DMS consumption. However, in the surface mixed layer, DMSO production from DMS photolysis averaged 2.95 μmol m− 2 day− 1, approximately 1.6 times the integrated DMSO production rate obtained from DMS consumption. These estimates suggest that photolysis is a more important source of DMSO in the surface mixed layer of the Sargasso Sea; however, when the whole water column is considered, biological DMS consumption becomes a more important DMSO source. Overall, DMS photolysis and DMS biological consumption seem to be comparable sources of DMSO in the water column of the Sargasso Sea. In summary, this study has shown that biologically consumed sulfur from DMS can be traced mainly into two major product pools, DMSO and sulfate, and that

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the relative proportion of each product can vary even within the water column. The factors driving the different fates of biologically consumed DMS sulfur in the Sargasso Sea remain largely unknown but do not appear to include PAR or temperature. Moreover, we have shown direct evidence of DMSO production from biological DMS consumption and quantified the process. Further understanding of the metabolisms involved in the consumption of DMS, including identification and characterization of the organisms involved in the process, will help to provide a better picture of the DMS cycle and will also help understanding the factors that can control this process. Acknowledgements Funding for this research was provided by grants from the National Science Foundation Biocomplexity in the Environment Program (OPP-0221748; P. Matrai, P.I) and the NSF Office of Polar Programs (OPP-0230497, R.P. Kiene; OPP-0230499, D.J. Kieber). We greatly appreciate the help of George Westby who collected the DMS concentration data, and of Doris Slezak, who provided the leucine incorporation data. Patricia Matrai, and the other members of the DMS Biocomplexity project are thanked for encouragement, excellent planning, and logistical support. D. McGillicuddy provided satellite altimetry images during the cruise and guidance in following the C1 eddy. We thank the captain and crew of the RV Seward Johnson and especially Ray Najjar, Kathleen Bailey and Karen Tinklepaugh for aid in collecting samples. References Andreae, M.O., 1980a. The production of methylated sulfur compounds by marine phytoplankton. In: Trudinger, P., Walter, M. (Eds.), Biogeochemistry of Ancient and Modern Environments. Springer-Verlag, Berlin, pp. 253–259. Andreae, M.O., 1980b. Determination of trace quantities of dimethylsulfoxide in aqueous solutions. Anal. Chem. 52, 150–153. Andreae, M.O., 1985. The emission of sulfur to the remote atmosphere: background paper. In: Galoway, J.N., Charlson, R.J., Andreae, M.O., Rodhe, H. (Eds.), The Biogeochemical Cycling of Sulfur and Nitrogen in the Remote Atmosphere. D. Reidel Publishing Co, Boston, Mass. USA, pp. 5–25. Andreae, M.O., 1986. The ocean as a source of atmospheric sulfur compounds. In: Buat-Menard, P. (Ed.), The Role of Air–Sea Exchange in Geochemical Cycling. D. Reidel Publishing Co, pp. 331–362. Bailey, K.E., Toole, D.A., Blomquist, B., Najjar, R.G., Huebert, B., Kieber, D.J., Kiene, R.P., Matrai, P., Westby, G.R. del Valle, D.A. (submitted for publication). Estimation of dimethylsulfide production in Sargasso Sea Eddies. Deep-Sea Res. Brimblecombe, P., Shooter, D., 1986. Photo-oxidation of dimethylsulphide in aqueous solution. Mar. Chem. 19, 343–353.

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