Environ. Sci. Technol. 1998, 32, 2130-2136
DMS Formation by Dimethylsulfoniopropionate Route in Freshwater B. GINZBURG,† I. CHALIFA,† J. GUN,† I . D O R , † O . H A D A S , ‡ A N D O . L E V * ,† Division of Environmental Sciences, Fredy and Nadine Herrmann School of Applied Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Yigal Alon Kinneret Limnological Laboratory, P.O. Box 345, Tiberias 14201, Israel
Dimethyl sulfide, the most important form of sulfur gas, is formed by bacterial degradation of dimethylsulfoniopropionate (DMSP) in the Lake of Galilee. DMSP is believed to be a methionine metabolite produced by marine algae and higher plants as part of their osmoregulatory systems. Until now, this process was found exclusively in saline water and therefore was regarded as insignificant for the formation of DMS in freshwaters. It is hereby demonstrated that the process can be dominant in freshwater systems as well, and its product can even affect the odor quality of some drinking and recreational water systems. Peridinium gatunense, a freshwater dinoflagellate which dominates the phytoplankton population in the lake during the winter-spring season, stores a considerable amount (up to 5.5 pg/cell) of DMSP. P. gatunense growth curves reveal an increased storage of DMSP toward the stationary and declining growth phases. The DMSP undergoes bacterial and chemical degradation to release DMS. Released fluxes of DMS from the Lake of Galilee are estimated to be in the range of 0.1 mmol/m2 month during the late period of the Peridinium bloom season.
Introduction Dimethyl sulfide (DMS) is believed to play an important role in the global sulfur cycle. DMS emissions represent more than 90% of the biogenic sulfur emissions from oceans and approximately half of the total biogenic sulfur emissions to the atmosphere (1, 2). Oxidation products of DMS contribute to the acidity of atmospheric particles and rain, and it was also postulated that DMS plays an important role in marine cloud formation and climate regulation (3). Three dominant processes are responsible for the natural formation of DMS. Methylation of hydrogen sulfide and methylmercaptan (4) is likely to predominate under anoxic conditionssin the hypolimnion and sediments. The process occurs in the presence of methyl donors such as methoxylated aromatic compounds (5). A second possible way is by degradation of sulfur-containing organic compounds such as the cleavage of methylmethionine by C-S lyase enzymes (6, 7), which can take place also under aerobic conditions (8, 9). The most dominant pathway for the production of DMS in saline environment is through the enzymatic cleavage of dimethylsulfoniopropionate (10) (DMSP) to give DMS and acrylic * Corresponding author fax: 972-2-6586155; e-mail: ovadia@ vms.huji.ac.il. † The Hebrew University of Jerusalem. ‡ Yigal Alon Kinneret Limndogical Laboratory. 2130
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acid, the same products formed by base hydrolysis of DMSP. DMSP can also be degraded by alternative pathways that do not lead to DMS production through series demethylation giving 3-methiolpropionate and subsequently methanethiol or 3-mercaptopropionate depending on the position of the C-S cleavage (11, 12). Storage of DMSP by marine algae and its role as a DMS precursor was first noted by Challenger et al. (13). Since then, DMSP was found in the leafs of plants in salt marshes and in numerous unicellular and multicellular algae (see, for example, a review by Keller et al.; 14). Dacey and Wakeman (15) have demonstrated that grazing by zooplankton greatly enhances DMSP degradation and DMS production, and it is likely that disruption of algal cells by physical means also releases DMSP and DMS. The physiological role of DMSP in algae is believed to be either osmoregulation in saline waters or cryoprotection in very low-temperature marine environments and arctic conditions (16). The possibility that DMSP serves as a source for acrylic acidsa known biocideswas also suggested (17), but experiments to confirm this hypothesis were still unfruitful. In fact, Slezak and co-workers found that the biocidal effect is too weak at natural concentrations of acrylic acid and only under specific conditions, when algae form aggregates, could acrylic acid play a role in reducing bacterial metabolism (18). On the other hand, the osmoregulatory function is supported by numerous observations of increase in DMSP storage in algal cells when the salinity is increased (19). It was also found that under nitrogen-deficient conditions, when the production of quaternary ammonium osmoregulators such as proline and glycinebetaine are hindered, DMSP production is increased (20). Until now, DMSP was found exclusively in saline aquatic systems, i.e., salt marshes, marine algae, and dissolved in ocean waters (21). As far as we know, this is the first report on the production of DMSP by a freshwater algae and on the dominance of this pathway in DMS discharge from any terrestrial aquatic system. We became interested in the study of the occurrence of DMS during the course of the investigation of the source of mild odors that can be noticed in the Lake of Galilee. A mild aroma that can be sensed only by rather sensitive individuals is emitted every spring from the lake. DMS as well as other dimethyl sulfides (dimethyl disulfide, dimethyl trisulfide, and related oxygenated compounds such as methyl, methane thiosulfonate) were identified to be abundant in the lake during this period (22). Two separate mechanisms were found to be responsible for the occurrence of dimethyl sulfide and the dimethyl oligosulfides. The current report delineates the mechanism of formation of the DMS alone, and a subsequent paper will describe our views on the formation of dimethyl oligosulfides under oxic conditions. The Lake of Galilee (also known as Sea of Ginosar or Lake Kinneret) is an old tertiary lake having its basin formed as a part of the Syrian-African Rift Valley, created by the Pliocene-Pleistocene tectonic movements. The lake is located ca. 210 m below sea level; its area is 168 km2; its average and maximum depth are 23 and 42 m, respectively. The lake is warm and monomictic; it is stratified between May and December, during which the thermocline is located at 15-17 m. The lake collects the inflowing Jordan River as well as winter surface floods. Lake of Galilee supplies half of the drinking water of the state of Israel through a national water carrier that is pumped from its northern section. The lake’s natural outlet is the Jordan River, which flows from its southern side to the Dead Sea, a hypersaline lake that has no outlet to the ocean. Seasonal winter-spring bloom of S0013-936X(97)00907-3 CCC: $15.00
1998 American Chemical Society Published on Web 06/09/1998
the dinoflagellate Peridinium gatunense appears every year (23, 33). The first reports on the algal bloom in the Kinneret date back to the last century. During the peak of the Peridinium bloom, the total suspended solids in the lake is more than doubled (reaching approximately 9-10 mg/L TOC), and the Peridinium algae reach from 600 to 4000 cells/ mL in large patches of algae. The Peridinium biomass constitutes more than 90% of the phytoplankton mass in the lake during the bloom. In the last 2 years, the algal bloom was much less intense, reaching only ca. 20-30% of the average cell count reported during the previous twenty years. Peridinium gatunense, formerly known as Peridinium cinctum var. westii, is a rather ubiquitous unicellular organism reported in numerous freshwater rivers and lakes throughout the world, including Faylor Lake in Pennsylvania (24), the lowland courses of the River Plate basin in Argentina (25), and the Ob River in western Siberia (26). The size of the alga varies from 30 to 70 µm; the wet and dry weight are 0.075 and 0.03 µg, respectively; its body is surrounded by theca, which consists of a noncellulosic glucan. The alga has two flagella that impart mobility and phototaxis; the flagella however are vulnerable and sensitive to mild shear stresses. P. gatunense is phylogenetically related to the marine dinoflagellates. However, the dinoflagellates constitute a very broad family including, for example, Amphidinium and Prorocentrum in addition to the Peridinium strain. However, sulfur-containing volatile compounds or DMS emission from P. gatunense or any other Peridinium strain were never reported before, as far as we know. There are numerous reports on emissions of DMS by marine dinoflagellates (27).
Experimental Section Chromatographic Methods. DMS was determined on a HP5890 gas chromatograph equipped with an on-column injector, EI (electron ionization) mass selective detector (HP5971 MSD), and P&T preconcentrator (Tekmar LSC-2). Purge time was 20 min, gas flow was 25 mL/min., and sample volume was 10 mL. Aqueous solution of d6-DMS internal standard was added to the sample before analysis. After desorption at 200 °C for 10 min, the gases were directed to the on-column injector of the gas chromatograph. A 30 m × 0.32 mm i.d. Poraplot Q column (Chrompack) was used. The following temperature program was employed: 1 min, 3 °C/ min ramp to 140 °C, 30 °C/min to 250° C followed by 3 min final hold time. DMS was analyzed in the selected ion monitoring (GC-MSD-SIM) mode. Analysis of the filtered algal cultures was performed by introducing the sample to the purge-and-trap apparatus by attaching a syringe equipped with syringe filter (FP 030/3, 0.2 µm, Schleicher & Schuell) to the inlet port of the purgeand-trap and filtering the sample directly into the purgeand-trap vessel. This way is preferable to a vacuum filtration because it minimizes losses of volatile compounds. Determination of acrylic acid was performed using the following procedure: a 1 L sample was stirred for 1 h with 10 mL of 20% NaOH. Then 200 g of NaCl and excess of 20% HCl were added to reach pH 2. A solution of internal standard (decanone-2) in methanol was added to the sample in an extraction funnel. Three 25-mL portions of methyl tert-butyl ether were used for extraction, and then the solvent layers were combined, dried over MgSO4, and evaporated to 0.2 mL. GC/MS equipped with a split-splitless injector was used, and the analysis was conducted in SIM mode. A 25 m, 0.32 mm i.d., cross-linked FFAP-coated (Hewlett-Packard), 0.52 mm film thickness capillary column was used. The following chromatographic program was used: first hold time at 45 °C for 1 min 5 °C/min ramp to 135 °C, 50 °C/min second ramp to 220 °C, and 10 min final hold time. Determination of Chlorophyll a. Determination of chlorophyll a content in the cells was performed according
TABLE 1. Concentration of Major Chemical Species of Lake of Galilee during the Algal Bloom Period (April) parameter
value (mg/L)
parameter
value (mg/L)
pH O2 H2S Clorganic N ammonia NO3-
8.69 10.8 0 213 0.827 0.029 0.11
total P dissolved P alkalinity Ca2+ SO42conductivity (Ω/cm)
0.041 0.008 106 40 52 968
to Talling and Driver method (28). The o.d. of the centrifuged methanol extract was determined by Cary 1E UV/vis spectrophotometer at 665 nm and corrected for residual turbidity. Determination of Extracellular and Intracellular DMS. The 10-mL samples were collected by a syringe from the well-mixed culture, 10 µL of internal standard (d6-DMS) water solution was added, and the mixture was transferred into the purging vessel of the purge-and-trap for GC analysis. For determination of the extracellular DMS, the samples were filtered on 0.2 mm HPLC syringe filters and the filtrate was analyzed by the previous protocol. Determination of DMSP. The 10-mL samples were collected by a syringe from the well-mixed culture; the samples were treated with 0.1 mL of 20% NaOH for 0.5 h; then 10 mL of internal standard (d6-DMS) solution was added; and the mixture was transferred into the purging vessel of the purge-and-trap for GC analysis. DMSP was calculated by subtraction of the DMS concentration from the result. For determination of the extracellular DMSP, 10-mL samples were filtered on 0.2 mm HPLC syringe filters, and the filtrate was analyzed by the previous protocol. Synthesis of d6-DMSP:d6-DMSP bromide was used as an analytical standard and as a substrate in cases that biogenic DMSP is produced and can interfere. The synthesis procedure of Dickson and co-workers (19) for DMSP was adopted. Briefly, 3-bromopropionic acid and an excess of d6-DMS were allowed to react overnight, and the product was triply recrystallized from ethanol. TSS and VSS. Total suspended solids and volatile suspended solids were determined according to Standard Methods (29). Algal Cultures. “Medium x6” proposed by Lindstrom (31) was used for cultivation, and the culture was subjected to periodic (day/night) illumination at constant temperature (18 °C). The chemical composition of the medium before and after cultivation was determined (Table 2). P. gatunense is rather delicate alga, sensitive to water quality, and axenic cultures were never cultivated by us or other investigators despite numerous repeated attempts (32). Cell Counts. Enumeration of the Peridinium cells in the lake water was conducted by microscope-assisted cell count (hemocytometer) of a centrifuge (3000-4000 rpm, Safeguard, USA) concentrated samples, according to the Standard Methods procedure (29). Each sample was measured approximately 20 times, and the mean and relative standard deviation were determined. Heterotrophic bacteria cell count was performed according to reported Standard Method (29). Sampling. Water samples were collected periodically throughout the year. Intensive sampling efforts were concentrated during the Peridinium bloom seasons of 1996. Depth profiles of the lake were taken from point A, located in the deepest point of the Kinneret. The Yigal Alon Limnological laboratory maintains a continuous monitoring station and intensive sampling activities at this point; therefore, it was selected as a sampling point for this study. The samples were stored in dark glass bottles equipped with Teflon seal with no headspace and transported in an ice box VOL. 32, NO. 14, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Composition of Cultivation Media during Peridinium gatunense Growth Cycle nutrients concentrations in medium nutrient
before cultivation (mg/L)
after cultivation (mg/L)
ClNO3SO42PO43Mg2+ Ca2+ Fe3+ Mo6+ Zn2+ Mn2+ Co2+ conductivity (µS/cm) alkalinity (mequiv/L) pH
32.6 48.8 27.5 4.0 7.0 19.5 0.120 0.120 0.140 0.020 0.020 255 1.6 8.4
39.3 0.6 26 0.5 6 13 0.035 0.120 0.075 0.004 0.020 1.6 8.45
FIGURE 1. Peridinium gatunense cell count (A) and DMS concentration (B) in the Lake of Galilee. to a refrigerated room. The samples were analyzed within 24 h of sampling. Autoclave treatments were performed in Tuttnauer 3870 E autoclave at 120 °C for 25 min.
Results To determine the DMS in the lake and its relationship to the algal population, we have conducted both field sampling throughout 1996 and laboratory studies on unialgal Peridinium cultures. Field Studies. Periodic depth sampling at levels 0, 5, 20, 30, and 40 m below water surface level were conducted at the deepest point of the lake (point A). Results of the analysis of DMS are shown in Figure 1 along with the corresponding changes in the population of the algal cells at that point. The bloom started in February and continued until August. It was accompanied by the appearance of dissolved DMS in the lake water. Before and after the bloom season, the concentration of the dissolved DMS was below the detection limit (approximately 0.1 µg/L). The spatio-temporal distribution of Peridinium and DMS were rather similar (the correlation coefficient, R, for the data of Figure 1 ) 0.73) although the spatial distribution of DMS was broader as 2132
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compared to the Peridinium distribution. We have never observed DMS in the hypolimnion or under anoxic conditions, showing that DMS is originated in the oxygen-rich epilimnion. The broader vertical distribution of the DMS as compared to the alga illuminates the difference between the deterministic phototaxis of the living cells and the random dispersion of the chemical species. The Peridinium spreads almost over the whole water column during the night and moves upward during the daytime. Since sampling was conducted always at daytime, Peridinium concentrations reflect the daytime position of the cells while DMS concentration are influenced also by the trail of the alga. Moreover, some of the observed DMS in the upper few meters is due to lysis of alga and hydrolysis of DMSP in the sample. The Peridinium bloom of 1996 was much less intense than previous years. Since the sampling was conducted at a stationary point, regardless of the algal distribution in the lake, the maximal cell count observed in our studies was only ca. 150 cells/mL, which is 5-25% of the peak alga concentration found in algal patches during regular blooms in the lake. The reason for the lower algal bloom this year is still unclear. Nevertheless, DMS concentrations were rather high (as compared to 0.32-11 nmol for freshwater Canadian lakes and 2.6 nmol mean open ocean value (30)) often exceeding 1 µg/L (16 nmol) during the bloom. The chemical water quality of the lake is very relevant to the current discussion. Table 1 depicts typical concentration ranges of major chemical species in the Lake of Galilee (data after ref 35). Depth profiles of the temperature and key chemical species that help understand the stratification and the redox conditions in the lake are depicted in Figure 2. The depth profiles were taken by the Kinneret Limnological Laboratory at the location of the DMS sampling. The stratification of the lake begins in April-May long after the onset of the Peridinium bloom and the appearance of DMS (Figure 1). The epilimnion is always rich or even oversaturated in dissolved oxygen. Anoxic conditions in the hypolimnion start only in July when the Peridinium peak and the corresponding DMS level are already decreasing. A similar trend can be observed for hydrogen sulfide and ammonium. Again, it can be clearly observed that the first occurrence of hydrogen sulfide and the increase in the level of ammonia in the hypolimnion of the Lake of Galilee occur only in midJune long after the DMS and Peridinium profiles develop. The water of the Lake of Galilee is relatively alkaline (Figure 2C); the pH ranges between 8 and 9 during most of the year, peaking during the Peridinium bloom season. The pH of the upper 10 m of the lake often exceeds pH 9 during the bloom, and even higher values are encountered in the algal patches during the daytime. Figure 2D depicts the sum of concentration of all other oligosulfides (namely, dimethyl disulfide, dimethyl trisulfide, dimethyl tetrasulfide, and methyl methanthiosulfonate (22)). These concentrations correlate even better with the P. gatunense level (correlation coefficient, R, for 1995 and 1996 period is 0.81). Studies on Peridinium Cultures. To elucidate the mechanism of formation of DMS, we have conducted experiments with P. gatunense algal cultures. Despite considerable efforts of the Kinneret Limnological Laboratory and our group to cultivate axenic cultures, we were unsuccessful in achieving truly axenic cultures. Therefore, the algal growth cycle experiments were conducted using unialgal cultures that were not treated by any antibiotics. The growth medium proposed by Lindstrom (31) was used in these tests. Two sets of experiments were conducted in order to establish the dependence of DMS production on the algal population and in order to confirm that the Peridinium does indeed produce DMSP. One series of tests was performed using a single beaker that was periodically sampled and analyzed. A subsequent set of experiments was performed
FIGURE 2. Depth profiles: A, temperature, °C; B, pH; C, oxygen, mg/L; D, NH4+, mg/L; E, H2S, mg/L; F, sum of dimethyloligosulfides, ng/L. VOL. 32, NO. 14, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Comparison of single and separate beakers growth curves of Peridinium gatunense. Chlorophyll a in a single beaker test (1), chlorophyll a in the separate beakers test (2), algal cells in a single beaker test (3), and algal cells in the separate beakers test (4).
FIGURE 4. (A) Changes of algal cell count (1) and heterotrophic bacteria plate count (2) during Peridinum gatunense growth curve. (B) pH (3), TSS (4), and VSS (5) evolution during the growth curve. starting with a culture solution that was divided into separate beakers, each opened and consumed after a different time interval. The two growth curves were quite similar as can be seen in Figure 3, although the stationary (i.e., late exponential-early decline) phase in the separate beakers tests was longer, probably because the Peridinium cultures were less disturbed by the sampling action. Therefore, the results are described for the separate beakers test, although all the trends discussed in this paper were similar for the two experimental setups. Figure 4 describes the alga growth curve obtained for P. gatunense in the separate beaker mode along with a number of relevant indicators that were monitored during the growth curve. The enumeration of the Peridinium cells was complicated by the accumulation of empty theca that interfered with the viable cell count. This is apparent from the rather large standard deviation bars in Figure 4A. The concentration 2134
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FIGURE 5. Concentration of DMS and DMSP in Peridinium gatunense culture. DMS in culture (1), DMS in filtrate (2), DMSP in culture (3), and DMSP in filtrate (4). of Peridinium cells grew exponentially and reached 65 000 ( 20 000 cells/mL. The maximum growth rate was observed after ca. 40 days, and it corresponded to 0.073 and 0.11 day-1 for the separate and single beaker tests, respectively. These values are comparable to the average division rate reported by Pollinger (34) for the algae during the peak of the bloom season in the lake. Nitrate deficiency is encountered toward the declining growth phase as can be seen in Table 2. The evolution of chlorophyll a during the growth cycle precedes by a small time span the growth curve determined by the cell count, which indicates that there is some overestimate of the viable cell count by the microscopic cell count technique. However, the chlorophyll a curve is also not an ideal indicator of the viable cell concentration because old cells are usually smaller and produce less chlorophyll a as compared to young ones. Change of pH during the growth cycle is shown in Figure 4B. Despite the carbonate buffer in the cultivation cultures, there is a substantial increase of pH during the exponential growth phase due to the photosynthetic activity of the Peridinium cells. The pH restabilizes at its initial value due to the increased respiration activity of the larger population of bacteria and alga. Figure 4A shows that the increase in the Peridinium cell count is accompanied by a corresponding increase in the total bacterial cell count. During the exponential growth phase, the ratio of bacteria/Peridinium cells is ca. 3.5, and it increased up to approximately 8 in the declining growth phase. This is to be expected since Peridinium lysis releases nutrients that stimulate bacterial growth. TSS and VSS increased steadily during the growth curve (at least during the first 80 days, Figure 4B). The absence of a maximum in the VSS and TSS can be explained by the rather slow degradation of the poliglucan theca. Indeed, degraded theca can be observed weeks after cell death. To verify that the algal cells and not the bacteria or their exerted chemical species in the solution are responsible for DMSP and DMS formation, we conducted a set of tests to distinguish the DMSP in the supernatant and the cells. Figure 5, based on the single beaker set of experiments, describes the algal growth and the evolution of DMS and DMSP in the Peridinium culture and in filtered samples as a function of the culture age. The distinction between intra- and extracellular species is always a problem in algal research due to lysis and exertion of the intracellular material during analysis. To avoid exertion of DMS into supernatant during the vacuum filtration step, we used the pressure filtration technique described in the Experimental Section. Another problem can arise from the disintegration of Peridinium cells in the purge-and-trap vessel. The purge-and-trap operates under very mild agitation conditions for only 10 min and the surrounding theca of the Peridinium provides significant protection against shear forces, but a certain degree of lysis
FIGURE 7. Comparison between chemical and bacterial d6-DMSP decomposition. 1, autoclaved lake water, pH adjusted to 9.5 (axis ×1); 2, lake water, pH ) 8.5 (axis ×1); 3, Peridinium culture filtrate (axis ×2); 4, Peridinium culture untreated (axis ×2).
FIGURE 6. (A) Concentrations of DMS (1) and DMSP (2) in the culture. (B) Concentrations of DMS (3) and DMSP (4) per algal cell during a growth curve. is still possible. Figure 5 shows that the concentration of DMS in the filtered water increased monotonically, although its level remained very low (in the several ppb range, much smaller as compared to the in-culture DMS). It is not clear for us to what extent the DMS in culture reflects contribution of hydrolysis of DMSP exerted from the cells during the analysis. The kinetic experiments of the DMSP cleavage described below show that this cannot be ruled out. Similar to the DMS, the level of DMSP in the filtrate remained very low and in fact did not follow any deterministic trend during the growth cycle. On the basis of these observations, we did not conduct tests of filtered samples in the separate beaker tests. Figure 6A depicts the in-culture concentration of DMS and DMSP in the Peridinium culture. Figure 6A shows that both in-culture DMS and DMSP were increased with culture age (a similar trend is observed in Figure 5). In fact, most of the increase occurred after 70 days in the stationary growth phase. Moreover, the values of curves 1 and 2 divided by the number of cells show that the specific storage of DMSP is increased in aged cells. A similar trend of increase in DMSP storage for aged cells of marine dinoflagellates was observed by Matrai and co-workers (36). Table 2 shows that nitrogen becomes a limiting growth nutrient in declining growth phase. So, the observation of the increased accumulation of DMSP in aged cells can be explained by nitrogen deficiency, driving the cells to rely on sulfonium instead of ammonium osmoregulation. This shift in the osmoprotection mechanism was proposed by Grone and Kirst (20) for Tetraselmis subcordiformis alga cultivated under nitrogen-deficient conditions. The increase in DMS content per cell is much less pronounced and can be attributed to an external adsorption (e.g., dissolution in the theca) of DMS on the viable cells glucan or on empty theca rather than internal accumulation of DMS. Unfortunately, we do not have means to differentiate between externally adsorbed DMS and DMS inside the Peridinium cells. On the basis of Figures 5 and 6, it can be concluded that most of the DMS and DMSP is located inside or attached to the cells and is not dissolved in the supernatant. This is especially significant for DMSP. The reason for this phenomenon is that exerted sulfur species are not conserved in the solution. The DMS evaporates, and the released DMSP
is soon degraded after cell lysis by the high concentration of bacteria in the culture. The specific content of DMSP reaches up to 0.04 pmol/cell. This value falls in the broad range of values reported for marine dinoflagellates that range between 0.001 and 35.5 pmol/cell (14). To confirm that Challengher’s cold alkali test indeed quantifies DMSP only, we have checked the concentration of acrylic acid after hydrolysis, which confirmed the quantitative and qualitative determinations based on the cold alkali tests. The concentration of acrylic acid in the lake was below our detection limit (∼10 µg/L), which can also be predicted by the low level of DMS found in the lake. The pH level of the epilimnion of the Lake of Galilee during the peak of the Peridinium bloom is rather high, often exceeding pH 9 in the Peridinium patches. It was therefore postulated that chemical hydrolysis plays a significant role in the base hydrolysis of DMSP and DMS production. It was also postulated that the Peridinium alga contains (or is capable of developing) C-S lyase enzymes (10) that are capable of cleavage of DMSP and release of acrylic acid biocide. Figure 7 demonstrates the results of a set of experiments aimed at investigating these possibilities. Water samples taken from the lake at the end of the Peridinium bloom, autoclaved lake water samples, and filtered and nonfiltered water samples from a unialgal Peridinium culture were spiked with d6-DMSP, and the cleavage rate was followed by gas chromatography. The cleavage rate of d6-DMSP by the lake water (pH ) 9.5) followed first-order kinetics, k ) 0.005 L/h. It was only 5 times lower than the cleavage rate in raw lake water (containing bacteria) k ) 0.026 L/h. The cleavage of DMSP by the bacteria in the Peridinium culture was faster by approximately 2 orders of magnitude (k ) 2.3 vs 0.026 L/h) as compared to its rate in the lake water. Indeed, the bacteria population (manifested in Heterotrophic plate count) was 2 orders of magnitude larger in the culture as compared to the lake water, i.e., 104 ( 105 in the culture as compared to 850 ( 100 colonies/mL in the lake water used for construction of Figure 7. Thus, it can be concluded that both chemical and bacterial processes are responsible for the DMSP cleavage, although the bioprocess is more dominant. Visscher and van Gemerden (37) studied the relative rates of biological and chemical degradation of DMSP in Microcoleus chthonoplastes-dominated marine microbial mats. They concluded that even at pH 10 the chemical hydrolysis of DMSP is insignificant as compared to the biological cleavage pathway. Our observations show that at VOL. 32, NO. 14, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Effect of Salinity on DMS and DMSP Content in Peridinium gatunense Culture 8 days NaCl (mg/L) DMS (µmol/L) DMSP (mmol/L) algal cells/mL SD DMSP (pmol/cell) SD
30 0.026 0.005 290 90 0.018 0.9
120 0.027 0.009 310 95 0.029 1.4
19 days 310 0.024 0.007 285 85 0.025 1.2
30 0.081 0.019 1000 100 0.019 0.3
least for freshwaters the chemical hydrolysis process cannot be neglected. So, our observations revealing a large concentration of DMSP in nonsaline freshwater were unexpected in view of unanimous consensus regarding the osmoregulatory role of DMSP. Also, we have not found any substantial evidence to support the role of DMSP as a precursor of an acrylic acid biocide for protection of the Peridinium cells against bacteria. The bacterial growth was not affected by the release of acrylic acid during the late exponential growth phase (Figure 4A). We have conducted tests to verify the dependence of DMSP accumulation on the salinity of the medium. Cells from the same culture were divided into five beakers containing the ×6 Lindstrom medium (Table 2). Different concentrations of NaCl were added to the beakers. In all cases, the Peridinium growth rate was higher in the less saline beakers (Table 3), and NaCl concentrations larger than 1000 mg/L proved lethal to the culture, as expected for a freshwater alga. During the first 8 days, there was no significant increase in the DMSP/cell content (Table 3), showing that the DMSP production is not a rapid shock regulator for P. gatunense. However, after 19 days, a significant increase in the level of DMSP/cell was observed in the more saline cultures. The increase in DMSP occurs even before nitrogen deficiency becomes the rate-limiting nutrient (e.g., the level of nitrate after 35 days was 7.1, 1.2, and 0.4 µg/L for 310, 120, and 30 mg/L NaCl, respectively). So, it seems likely that DMSP functions as an osmoregulator even for freshwater alga. This part of the research concentrated on processes that take place in the water side; however, the impact of the biogenic processes on air quality can also be roughly estimated. For that, we have conducted tests to verify that DMS is not appreciably consumed in the lake water column. The Lake of Galilee water that was collected during June (at the end of the Peridinium bloom season) was spiked with d6-DMS and the remaining concentration of d6-DMS in the water was monitored. No decrease of d6-DMS, was reported in a 5-day test. Since the mean distance of the DMS to the surface is much smaller than the distance to the anaerobic sediment and since the kinetic barrier in the gas side is practically irreversible (because of the large dispersion coefficient in air), it can be concluded that most of the DMS is lost by liquid to gas transfer and not by the bacterial degradation processes in the sediment. Since DMSP was found to constitute some 5.5 pg/cell (i.e., 0.036 pmol/cell) or 0.02% of the cell mass and since during a regular bloom the total mass of Peridinium cells reaches some 200 g/m2, then one can readily calculate that the flux of DMS during the relevant period is approximately 6 mg of DMS m-2 month-1 (3 mg of S m-2 month-1) as compared to a value of 8.3 mg of S m-2 month-1 from the ocean (1). The total emission of DMS from the Lake of Galilee during a regular year can then be calculated to be 0.97 ton month-1.
Literature Cited (1) Andreae, M. O.; Raemdonck, H. Science 1983, 221, 744-745. (2) Dacey, J. W. H.; King, G. M.; Wakeham, S. M. Nature 1987, 330, 643-645. 2136
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120 0.069 0.018 720 85 0.025 0.4
35 days 310 0.047 0.019 550 100 0.034 0.9
30 0.155 0.381 17500 7200 0.022 1.4
120 0.126 0.268 10300 4700 0.026 1.8
310 0.077 0.403 8400 2400 0.048 2.1
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Received for review October 17, 1997. Revised manuscript received April 1, 1998. Accepted April 24, 1998. ES9709076
