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Populations of d~methylsulfoniopropionate (DMSP) degrading bacteria were enumerated in Caribbean Sea waters using Most Probable Number (MPN) methodology. DMSP and 3-methiolpropionate (MMPA), a n intermediate in the demethylation pathway of DMSP degradation, were used as enrichment substrates. Two metabolically distinct populations were obtained: DMSP cleavers, which produced dimethyl sulfide (DMS) from DMSP, and demethylators of DMSP, which produced 3-mercaptopropionate (MPA) and/or methanethiol (MSH) from DMSP or MMPA. Demethylating populations were distinct from DMS producers and formed 5 to 66 % of total DMSP degraders. DMSP utilizers compr~zed about 10 % of the total bacteria (MPNs on glucose-acetate) ind~cating the potential of DMSP as a carbon source for bacteria in the euphot~c zone. demethylated to 3-methiolpropionate (MMPA) and thence to 3-mercaptopropionate (MPA) (Fig. 1) (Kiene & Taylor 1988a, b). A marine bacterium, isolated from a culture of the coccolithophore Emiliania huxleyi, aerobically demethylated DMSP to MMPA, which was mainly demethiolated to yield methanethiol but also demethylated MMPA to MPA (Fig. 1) (Taylor & Gilchrist 1991). In the present study, w e used DMSP and MMPA as selective growth substrates in Most Probable Number 1 dirnethylsulfoniopropionate DMSP Dimethylsulfoniopropionate (DMSP) is synthesized by certain phytoplankton, most notably by some species of dinoflagellates and coccolithophores in which it functions as an osmolyte (Keller et al. 1989). There is interest in the environmental fate of DMSP because it is the principal precursor of dimethyl sulfide (DMS). Quantitatively, DMS is the most important volatile sulfur compound entering the marine atmosphere (Andreae 1990). Evidence from culture studies suggest that DMSP might be released from phytoplankton into seawater by a variety of mechanisms (Dacey & Wakeham 1986) where it is enzymatically cleaved to DMS and acrylate by bacteria (Dacey & Blough 1987, Kiene 1992). Comparisons of the lunetics of DMSP and DMS (CHd2S dimethyl sulfide turnover in surface oceanic and estuarine waters DMS have indicated alternative fates for DMSP other than DMS production (Kiene & Service 19911 Kiene 1992). Fig. 1. Pathways of DMSP metabolism in the marine environIndeed, alternative catabolic routes for DMSP are ment. Shaded boxes indicate products measured in the known. In anoxic coastal sediments DMSP was present study O Inter-Research 1992 Mar. Ecol. Prog. Ser. 89: 293-296, 1992 (MPN) enumerations. We found evidence for 2 distinct bacterial populations involved in DMSP degradation in oceanic waters based on the detection of key metabolic intermediates from either DMSP or MMPA. Materials and methods. MPN incubations were carried out with samples collected during a cruise aboard the RV 'Columbus Iselin' in the Caribbean Sea. Samples were taken west of Montserrat, Leeward Islands (16'45' N, 62'30' W; Stn 1) and southeast of Puerto Rico (18'66' N, 65'45' W; Stn 2). Surface samples were collected by bucket (acid and ethanol rinsed) and samples from the chlorophyll (chl a) maximum and below were taken during CTD hydrocast with Niskin bottles. The MPN medium consisted of a mixture (1 : 1 v/v) of filtered and autoclaved aged-seawater and a carbonate-buffered mineral medium (Visscher et al. 1991), 3 m1 of which was dispensed in 6 m1 test tubes, which were closed off by a n over-sized cap, allowing gas exchange. Substrates tested were DMSP, MMPA, both at a final concentration of 360 pM, and glucose plus acetate added to a final concentration of 180 yM each. Incubations (3 replicates per treatment) were carried out in the dark and the tubes were vortexed 3 times per day to replenish the medium with oxygen. Controls either contained no substrate and an inoculum, or no inoculum and substrate. Heterotrophic growth on glucose plus acetate was checked by turbidity, whlle growth on sulfur compounds was followed by production of the volatile gases DMS, MSH and dimethyldisulfide (DMDS). A headspace sample was removed aseptically from each tube and analyzed for these gases with a Shimadzu GC-Mini 2 gas chromatograph with flame ionization detector and Carbopak BHT column (Supelco, Bellefonte, PA, USA). The column temperature was 100 "C and carrier gas flow was 60 m1 min-l. Detection limits were 3 nmol (1 FM) for DMS and MSH and 30 nmol (10 FM) for DMDS. Production of the gases was followed over time by sampling at 4 to 24 h intervals. Positive tubes were removed to avoid carry-over of gases to neighbouring tubes, and kept for microscopic observation and consecutive transfer to fresh medium. The final population size was determined after 14 to 21 d of incubation according to De Man (1975). The production of MPA was measured in the laboratory in subsamples of all of the tubes with either DMSP or MMPA media after reduction of its chemical oxidation product, 3,3'-dithiodipropionic acid. MPA was measured on reverse-phase HPLC after derivatizing to a.n isolndole (Mopper & Delmas 1984, Kiene & Taylor 198813). Subsamples (0.5 ml) were treated with 10 p1 tributylphosphine to cleave disulfide bonds prior to derivatizing (Mopper & Taylor 1986). The detection limit for MPA was 0.2 nmol (67 nM). All tubes were examined microscopically and the highest positives for all treatments were transferred into fresh medium to confirm growth. Subsequently, samples of all tubes displaying growth at the highest dilutions on DMSP were incubated in a medium containing MMPA as the only substrate. Likewise, all of the positive MMPA tubes were transferred into DMSPcontaining medium. Growth was checked for 3 d by analyzing the headspace for MSH, DMS and DMDS, and the culture liquid for MPA after which the population was evaluated as described above. Counts of bacterial cells in water samples were made using epifluorescence microscopy. Slides were prepared aboard ship, after fixing samples (5 ml) in formaldehyde (final concentration 2 % v/v). Cells were stained with acridine orange (final concentration 0.01 % v/v), filtered onto 0.2 pm black-stained Nuclepore filters (Hobbie et al. 1977), and kept refrigerated until counted. DMSP was either obtained through Research Plus (Bayonne, N J , USA), or synthesized (Chambers et al. 1987) and MMPA was obtained by alkaline hydrolysis of its methyl ester supplied by Aldrich (Milwaukee, WI, USA). Results and discussion. In shipboard incubations, production of sulfur gases, which was used as a qualitative measure for bacterial metabolism, was observed within 24 h in lower dilutions (102 to 103) of all samples. Typically, the DMSP-cleaving population took 10 to 14 d to fully develop, while the MMPA utilizing population grew more slowly and peaked after 16 to 20 d. The dominant volatile sulfur species detected from MMPA was MSH, while its oxidation product DMDS was only found at much lower concentrations. The glucose-acetate population fully developed within 4 to 6 d. At lower dilutions both DMSP and MMPA media supported growth, with DMS and MPA production in DMSP media contrasted by MSH and MPA production in MMPA media. The results indicate the presence of mixed populations carrying out cleavage, demethylation and demethiolation. At higher dilutions the products were either DMS or MSH and MPA indlcating the dominance of either cleavers or demethylators/demethiolators. The results of MPN (Table 1) showed no difference in population size between DMSP (2.0 X 104 to 2.0 X 105 cells ml-l) and MMPA utilizers (4.0 X 10' to 5.0 X 104 cells ml-') based on 95 % confidence intervals, although on average the DMS producers seemed to be present in slightly higher numbers. Except for MMPA incubations from Stn 2 at the chl a maximum, the MPAproducing populations in DMSP and MMPA media were smaller than the DMS and MSH-producing population, respectively (Table 1). Vlsscher et al.: Dimethylsulfoniopropionate deg rad~ng bacterla in the Caribbean 295 Table 1. Viable count (cells ml-') of DMSP-utiliz~ng bacteria and MMPA-utllizlng bacterla (95 % confidence interval in parentheses). DMSP: dimethylsulfonioprop~onate; MMPA: 3-methiolpropionate; DMS. dimethyl su l f~de; MPA: 3-mercdptopropionate, nd: not de t e rm~ned Locat~on DMS from MPA from MSH from MPA from DhdSP DMSP MMPA MMPA -

Enumeration of bacteria which cleave or demethylate dimethylsulfoniopropionate in the Caribbean Sea