Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87

DOI: 10.1007/s13131-010-0054-7

http://www.hyxb.org.cn

E-mail: hyxbe@263.net

Experimental studies on dimethylsulfide (DMS) anddimethylsulfoniopropionate (DMSP) production byfour marine microalgae

LI Chengxuan1, YANG Guipeng1∗, PAN Jinfen2, ZHANG Honghai1

1 Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean

University of China, Qingdao 266100, China

2 College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100,China

Received 23 January 2009; accepted 26 December 2009

©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2010

AbstractThe production of dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) by marine mi-croalgae was investigated to elucidate more on the role of marine phytoplankton in ocean-atmosphereinteractions in the global biogeochemical sulfur cycle. Axenic laboratory cultures of four marinemicroalgae–Isochrysis galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella were tested forDMSP production and conversion into DMS. Among these four microalgae, Isochrysis galbana 8701and Pavlova viridis are two species of Haptophyta, while Chlorella and Platymonas sp. belong toChlorophyta. The results demonstrate that the four algae can produce various amounts of DMS(P),and their DMS(P) production was species specific. With similar cell size, more DMS was releasedby Haptophyta than that by Chlorophyta. DMS and dissolved DMSP (DMSPd) concentrationsin algal cultures varied significantly during their life cycles. The highest release of DMS appearedin the senescent period for all the four algae. Variations in DMSP concentrations were in strongcompliance with variations in algal cell densities during the growing period. A highly significantcorrelation was observed between the DMS and DMSPd concentrations in algal cultures, and therewas a time lag for the variation trend of the DMS concentrations as compared with that of theDMSPd. The consistency of variation patterns of DMS and DMSPd implies that the DMSPd pro-duced by phytoplankton cells has a marked effect on the production of DMS. In the present study,the authors’ results specify the significant contribution of the marine phytoplankton to DMS(P)production and the importance of biological control of DMS concentrations in oceanic water.

Key words: dimethylsulfide (DMS), dimethylsulfoniopropionate (DMSP), marine microalgae,

growth stages, production

1 Introduction

The climatically relevant trace gas dimethylsul-fide (DMS) is one dominant volatile organic biogeniccompound in the surface ocean (Yang et al., 2005;Yang and Tsunogai, 2005; Andreae, 1990; Bates et al.,1987; Andreae and Barnard, 1984; Wakeham et al.,1984; Lovelock et al., 1972). The global flux of DMSfrom the oceans is thought to be 15–33 Tg/a (Kettleand Andreae, 2000), which corresponds to 10%–40% ofthe total flux of gaseous sulfur compounds to the atmo-sphere (Kettle et al., 1999; Bates et al., 1987). Biologi-cal production of DMS in the ocean is the main naturalsource of biogeochemical cycling of sulphur on a global

scale, with important consequences for global climateand environmental regulation, according to CLAW hy-pothesis (Charlson et al., 1987). Many species of ma-rine phytoplankton have been demonstrated to releaseatmospherically reactive trace gas DMS (Keller et al.,1989), which gain much interest with the potential im-pact on global climatic change.

The algal osmolyte dimethylsulfoniopropionate(DMSP), synthesized by phytoplankton as an internalcell component, is the major precursor of marine DMS.Marine DMSP exists in operationally defined particu-late and dissolved fractions. Particulate DMSP (DM-SPp) is often defined as the fraction of DMSP which

Foundation item: The National Natural Science Foundation of China under contract Nos 40525017 and 40476034; the ChangjiangScholars Programme, Ministry of Education of China; the Science and Technology Key Project of Shandong Province under contractNo. 2006GG2205024; the “Taishan Scholar” Special Research Fund of Shandong Province, China.

∗Corresponding author, E-mail: gpyang@mail.ouc.edu.cn

LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87 79

is caught on filters such as Whatman GF/F (0.7 µmnominal retention) while dissolved DMSP (DMSPd)is that which passes through GF/F filters (Kiene andSlezak, 2006; Dacey et al., 1998; Matrai and Keller,1993). The production of DMSP is almost exclu-sively confined to a few classes of marine micro- andmacroalgae (Karsten et al., 1991), notably Prymesio-phytes and Dinoflaellates, including bloom formingtaxa like Phaecystis (Gibson et al., 1990), Emiliania,and Alexandrium (Blunden et al., 1992; Keller et al.,1989; Reed, 1983). It is reported that there is no lin-ear relationship between the concentration of DMSin ocean surface waters and the cell density of phy-toplankton, because different phytoplankton produceDMSP in varying amounts and not all species have theenzyme DMSP-lyase (the enzyme that cleaves DMSPinto DMS and acrylate) (Keller et al., 1989). There areaccumulating evidences showing that most of the DMSis produced from DMSP after cell lysis as a result ofa number of biochemical and trophic processes in thewater column, such as viral mediated lysis (Kiene etal., 2000; Malin et al., 1998; Bratbak et al., 1995) orzooplankton grazing (Levasseur et al., 1996; Wolfe andSteinke, 1996; Colebrook, 1985).

Currently, most researches have focused on fieldstudies concerning the spatial and temporal distribu-tion of DMS(P), DMS photochemical oxidation andsea-to-air flux (Yang et al., 2007; Toole et al., 2006;Kiene and Linn, 2000). However, there are still gapsin our understanding of the DMS(P) production byphytoplankton at different growth stages. Keller etal. (1989) investigated 123 phytoplankton clones andfound a good correlation between taxa and DMSPcontent. Stefels and van Boekel (1993) reported thatthe DMSP content in Phaeocystis cultures increasedfrom 71 to 161 mmol/dm3 during transition from log-arithmic growth phase to stationary phase of growth.Barnard et al. (1984) found a strong correlation be-tween cell density of Phaeocystis pouchetii and DMSconcentration. The study made by van Rijssel andGieskes (2002) illustrated that both light and temper-ature affected growth rate and cell size of Emilianiahuxleyi, but the concentration of DMSP in the cellswas dependent only on temperature: at low tempera-ture DMSP accumulated.

To date, basic information regarding the produc-tion of DMS(P) from four algae species–Isochrysisgalbana 8701, Pavlova viridis, Platymonas sp. andChlorella, which are the predominant species in Qing-dao near-shore waters, is quite limited. They are uni-

cellular algae and contain green photosynthetic pig-ments chlorophyll a and b in their chloroplast. In ourwork we investigated the DMS and DMSP productionduring a batch culture study of the four microalgae,which could synthesize DMSP but differ in their abil-ities to convert it to DMS, in order to gain insightinto the mechanism and function of DMS formationby phytoplankton in the marine environment which isessential to determine the role of DMS in the globalsulfur cycle.

2 Materials and methods

2.1 Alga laboratory cultures

All the experiments were conducted withbacteria-free cultures of four taxonomically differentunicellular microalgal species: Isochrysis galbana 8701,Pavlova viridis, Platymonas sp. and Chlorella, whichwere obtained from microalgal laboratory of FisheriesCollege, Ocean University of China.

Each strain used in the experiments was inocu-lated into 1 dm3 sterile conical flasks covered withsterile filter paper to prevent contamination but per-mit gas exchange with the atmosphere and filled withthe autoclaved natural seawater (Bohai Sea) amendedwith f/2 media of nitrogen, phosphorus, trace metals,and vitamins (McLachlan, 1973) in batch cultures un-der identical conditions. One-liter flasks were filled toa working volume of 600 ml, and each flask was in-oculated with an equal inoculum (105 cells/cm3) ofrespective species from stock cultures in late exponen-tial phase. All cultures (including stock cultures) werekept at (20±2) ◦C with a L:D 12:12 cycle and averagefluorescent illumination of 74 µmol photons m−2 s−1

provided by an illumination incubator (GXZ-380B,Ningbo Jiangnan Instrument Factory) and measuredwith a Li-Cor photometer equipped with a Quantumcosine collector). Before experiments, strains weregrown exponentially by repeated (at least 3X) inocula-tion into fresh medium. To avoid precipitation duringautoclaving, the medium was filter-sterilized by pass-ing through Millipore membrane filter (0.45 µm) usingGelman Sciences filter before supplemented to the ex-perimental systems. All glassware used were soakedfor at least 24 h in HCl and then thoroughly rinsedwith ultrapure water. All of the cultures used for thepresent study were unialgal, axenic and checked regu-larly for contamination with bacteria by fluorescencemicroscope (DM4000, Leica) using DAPI dye (Atlas,1995). The growth rates of all cultures were calculated

80 LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87

using the expression (Phatarpekar et al., 2000):

K = lnNt − lnN0/t, (1)

where Nt is cell count at time t, N0 is initial cell countat time 0 and t is time (d).

2.2 Analytical procedures

To allow for any possible diurnal rhythm in celldivision time, samples of experimental batch cultureswere taken aseptically and periodically (every day at9:00 am) until cultures reached senescent phase (Day15) for the analysis of cell abundances and DMS(P).All experiments were performed in triplicate. Aliquotsof 5 ml were preserved with 0.15 ml Lugol’s iodine andsettled in 0.1 mm haemocytometer for direct counts ofcells of four microalgae with an inverted microscope.Cell volumes were measured by flow cytometer using aCoulter Epics XL MCL (Beckman Coulter, USA) withtriggering set on red fluorescence.2.2.1 DMS determination

The samples for DMS analysis were gravity-filtered through Whatman GF/F filters and were thenimmediately analyzed using the purge-and-trap tech-nique of Andreae and Barnard (1983). In brief, analiquot of 10 ml of the sample filtrate was directlyinjected into a glass bubbling chamber, the DMS init was stripped out with high purity nitrogen, driedthrough a glass tube containing K2CO3 and thentrapped in an inert stainless steel U-shaped tubepacked with Tenax-TA at the liquid nitrogen temper-ature (–78 ◦C). The trapped DMS was desorbed at90 ◦C with hot water and finally analyzed using a Shi-madzu GC-14B gas chromatograph equipped with aflame photometric detector after separation using a2-m glass column packed with 10% DEGS on Chro-mosorb W-AWDMCS. Standard curve was preparedby injecting known amounts of DMS in ethylene gly-col solution into the bubbling chamber and subjectingthem to the same manipulations as the samples. Theanalytical precision was generally better than 5% andthe absolute detection limit was 1 pmol of DMS.2.2.2 DMSP determination

DMSP concentrations were analyzed after alka-line cleavage to DMS in 1:1 stoichiometry. For theDMSPd determination, an aliquot of 2 ml seawatersample was filtered through a Whatman GF/F filterunder low vacuum (<5 mm Hg). The filtrate was col-lected directly into a 40 ml glass vial containing 2 mlof 10 mol/dm3 NaOH solution and 36 ml of distilled

water. Then the vial was immediately sealed with agastight seal, leaving no headspace. To determine theDMSPp concentration, the Whatman GF/F filter wasquickly placed in a 40 ml vial with 38 ml of distilledwater and 2 ml of 10 mol/dm3 NaOH, then the vialwas immediately sealed. The samples were incubatedin the dark at 4 ◦C for at least 24 h. The algal tis-sue was digested in alkaline solution and DMSP wascleaved to DMS. The DMS generated by the break-down of DMSP was analyzed as described above. TheDMS concentration measured in the filtered seawateris the sum of original DMS and that generated fromDMSPd, thus we can figure out DMSPd by subtract-ing the original DMS from this sum. Detection limitfor DMSP was between 0.3 and 0.5 nmol/dm3 for thealgal samples processed.

3 Results and discussion

3.1 Algal growth curves

The variations in cell densities of four culturesduring the experimental period (15 d) in natural sea-water with a salinity of 35 are shown in Fig.1. Allspecies exhibited an “S” type curve growth pattern:small growth lag phase, exponential growth phase, sta-tionary growth phase and senescent phase. From theresults shown in Table 1 and Fig. 1, a small amountof lag time was observed in the culture growth for allthe four algae because of enough nutrients in freshmedium. Isochrysis galbana 8701, Pavlova viridis,Platymonas sp. and Chlorella reached the largestabundance on Day 9, 9, 7 and 8, respectively, afterbeing inoculated.

The growth rates of the cultures are depicted inFig. 2. The average growth rates of Isochrysis galbana8701, Pavlova viridis, Platymonas sp. and Chlorellawere 0.209, 0.147, 0.111 and 0.196 d−1, respectively.The monoculture of Pavlova viridis and Platymonassp. revealed a relatively slow and steady growth, whileChlorella and Isochrysis galbana 8701 displayed thehigher growth rates. The differences in growth ratesobserved may be attributable to the size differencesof the four species (Isochrysis galbana 8701—3 µm di-ameter; Pavlova viridi—8 µm diameter; Platymonassp.—12 µm diameter; Chlorella—3 µm diameter). Ithas been reported that smaller size species grow fasterthan larger ones (Fogg, 1965). This is because greatersurface/volume ratio of smaller sized cells facilitatesassimilation of nutrients at a relatively faster rate.

LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87 81

Fig.1. Cell abundances (mean±SD of triplicates) of Isochrysis galbana 8701, Pavlova viridis, Platymonassp. and Chlorella at different life stages.

Fig.2. Growth rates (mean±SD of triplicates) of Isochrysis galbana 8701, Pavlova viridis, Platymonas sp.and Chlorella in batch cultures.

Furthermore, a close inspection of our data in-dicates that abundances of the four algae were verydifferent from each other. The cells of Chlorella andIsochrysis galbana 8701 were tinier than those of theother two, and hence their densities were higher inculture solution. A comparison of Fig. 1a to Fig.1b shows that the abundance productions of Chlorellaand Isochrysis galbana 8701 were over ten times higherthan those of Pavlova viridis and Platymonas sp. Thesudden collapse in growth rate of Chlorella on Day 8and Isochrysis galbana 8701 on Day 7 could perhapsbe attributed to the initial rapid growth which mightresult in the depletion of the nutrient to a limitingconcentration for the algae growth. Many previousstudies suggested cell-cycle dependence for nutrientuptake in algae cultures, and phytoplankton growthrates were controlled by the one nutrient in short-est supply (Brzezinski et al., 1990; Brown and Har-ris, 1978; Droop, 1975). In contrast, Pavlova viridisand Platymonas sp. had a relatively slow and almost asteady growth rate during the exponential phase. This

characteristic may be attributed to its comparativelylarge size which slows down the nutrient assimilationcapability (Fogg, 1965).

3.2 DMSP (DMSPd and DMSPp) concentra-

tions at different growth stages

Tables 1 and 2 show the DMSPd concentrationsin the cultures of Isochrysis galbana 8701, Pavlovaviridis, Platymonas sp. and Chlorella increased in par-allel with cell densities during exponential growth, butthe levels continued to rise slowly up to a plateau of17.5, 1.95, 0.24 and 5.36×10−5 µmol/dm3 on Day 12,11, 10 and 10 during the stationary phase of growth,respectively. Thereafter, the concentrations of DM-SPd decreased considerably. The high productionamounts of DMSPd observed at the onset of the sta-tionary phase of four algae cultures suggest that cellautolysis might account for most of the DMSP produc-tion. Metabolism associated with active cell growthis another possible mechanism as rapid generation ofDMSPd occurred during the same phase.

82 LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87

Table 1. Variation of cell abundances, DMSPp and DMS concentrations per cell in algal cultures at different growth

stages of Isochrysis galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella

Day No. Cell abundance/×107cell·dm−3 DMS/×1016 mol per cell DMSPp/×1016 mol per cell

A1) B2) C3) D4) A B C D (10−5) A B C D (10−4)

1 46 16 18 84 3.31 6.10 0.13 2.55 34.6 41.2 0.72 2.07

2 61 20 25 141 3.12 5.05 0.29 3.37 35.8 49.6 0.82 2.24

3 96 28 36 245 1.86 6.26 0.28 0.23 41.7 47.0 1.25 2.26

4 157 40 58 603 1.74 5.63 0.24 0.31 31.7 43.5 1.78 1.48

5 403 58 89 1 225 1.24 3.72 0.29 0.61 35.0 53.4 2.29 2.54

6 770 80 115 1 557 0.81 5.32 0.29 1.02 35.2 59.5 2.70 3.81

7 1 014 113 136 1 910 1.74 5.63 0.46 1.00 41.1 58.6 2.39 2.88

8 1 186 119 116 1 921 2.86 5.72 0.64 1.18 44.7 58.3 3.36 2.47

9 1 436 139 109 1 708 4.32 5.46 0.70 1.89 42.3 52.4 2.76 2.93

10 1 373 130 101 1 704 5.26 5.78 0.89 2.37 47.4 59.9 3.10 2.97

11 1 271 122 100 1 691 5.42 7.56 0.85 2.89 55.1 51.7 2.53 2.1

12 1 020 134 100 1 260 7.97 6.86 0.93 2.07 53.6 45.5 2.72 3.08

13 1 045 136 110 1 360 8.38 6.91 0.57 1.92 48.0 43.6 2.22 2.78

14 962 133 106 1 348 7.10 7.30 0.57 1.27 46.9 42.4 2.70 2.66

15 867 125 103 1 300 7.23 6.69 0.61 1.21 50.6 42.7 2.68 3.07

Average5) 780±46 93±7 88±5 1 204±50 4.16±0.32 6.00±0.72 0.52±0.19 1.59±0.26 42.9±1.32 50.0±0.67 2.27±0.39 2.62±0.37

Notes: 1)The concentrations of Isochrysis galbana 8701. 2)The concentrations of Pavlova viridis. 3)The concentrations of

Platymonas sp. 4)The concentrations of Chlorella. 5)Mean±mean standard deviation.

Table 2. Variation of DMSPd, DMSPp and DMS concentrations in algal cultures at different growth stages of Isochrysis

galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella

Day No. DMS/µmol·dm−3 DMSPd/µmol·dm−3 DMSPp/µmol·dm−3

A1) B2) C3) D(10−5)4) A B C D(10−5) A B C D(10−4)

1 0.152 0.098 0.002 0.214 0.265 0.448 0.006 0.079 1.59 0.660 0.013 0.174

2 0.191 0.101 0.007 0.475 0.398 0.345 0.007 1.03 2.19 0.992 0.021 0.315

3 0.179 0.175 0.010 0.056 0.546 0.475 0.016 0.979 4.00 1.32 0.045 0.554

4 0.273 0.225 0.014 0.190 1.00 0.343 0.076 2.98 4.98 1.74 0.103 0.893

5 0.500 0.216 0.025 0.746 1.25 0.775 0.081 0.937 14.1 3.10 0.204 3.11

6 0.622 0.426 0.033 1.60 6.32 0.637 0.172 3.43 27.1 4.76 0.310 5.93

7 1.76 0.637 0.063 1.90 5.78 1.09 0.214 2.95 41.7 6.63 0.326 5.50

8 3.39 0.680 0.074 2.27 12.4 1.26 0.207 3.65 53.0 6.94 0.390 4.74

9 6.21 0.759 0.076 3.23 15.3 1.44 0.205 4.24 60.7 7.28 0.301 5.00

10 7.23 0.751 0.090 4.04 14.2 1.82 0.244 5.36 65.0 7.79 0.313 5.07

11 6.89 0.922 0.085 4.89 16.3 1.95 0.215 3.96 70.1 6.31 0.253 3.56

12 8.13 0.919 0.093 2.61 17.5 1.43 0.181 3.12 54.7 6.10 0.272 3.89

13 8.76 0.940 0.062 2.61 13.7 1.38 0.181 3.30 50.2 5.93 0.244 3.79

14 6.83 0.971 0.060 1.71 12.5 1.38 0.180 2.94 45.2 5.63 0.287 3.59

15 6.26 0.836 0.063 1.58 11.3 1.21 0.168 2.64 43.9 5.34 0.276 3.99

Average5)3.83±0.720.58±0.290.050±0.121.88±0.458.58±0.921.06±0.130.144±0.092.77±0.2235.9±1.264.70±0.720.224±0.083.34±0.21

Notes: 1)The concentrations of Isochrysis galbana 8701. 2)The concentrations of Pavlova viridis. 3)The concentrations of

Platymonas sp. 4)The concentrations of Chlorella. 5)Mean±mean standard deviation.

DMSPp concentrations of four microalgae dur-ing the experimental period are presented in Table2. The change trend of DMSPp concentrations fol-lowed that of cell abundances (Tables 1 and 2). Inthe algal culture time course, rapid DMSP accu-mulation was observed in their exponential growthphase. DMSPp concentrations for Isochrysis galbana8701, Pavlova viridis, Platymonas sp. and Chlorellaincreased to a maximum of 70.1, 7.79, 4.61 and

5.07×10−4 µmol/dm3 on Day 11, 10, 8 and 10, respec-tively, but dropped down abruptly upon entering thelate stationary growth phase and the senescent phase.In comparison with DMSPd concentrations, DMSPpconcentrations were highly correlated with cell num-bers (p <0.001, Fig. 3), indicating that DMSPp pro-duction was in proportion to the amount of algal abun-dances in the cultures. Recently, Zhang et al. (2009)pointed out that the concentrations of DMSPp and

LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87 83

Fig.3. Variations of DMSPd, DMSPp concentrations in algal cultures at different growth stages of Isochrysis

galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella with their cell abundances.

DMS were significantly correlated with the levelsof chlorophyll a, demonstrating that phytoplanktonbiomass might play an important role in controllingthe distributions of biogenic sulfurs in Qingdao near-shore waters.

The increase in DMSPd production of Isochry-sis galbana 8701 was relatively rapid and the averageconcentration was 8.58 µmol/dm3 during the exper-imental period. In contrast, the DMSPd productionin the cultures of Pavlova viridis and Platymonas sp.was relatively lower (mean: 1.06, 0.144 µmol/dm3)during the corresponding period. In addition, theDMSPd concentration level of Chlorella during cul-ture period was substantially less than others, rang-ing from 0.079×10−5 to 5.36×10−5 µmol/dm3 withan average of 2.73×10−5 µmol/dm3. The average DM-SPp production amounts for Isochrysis galbana 8701,Pavlova viridis, Platymonas sp. and Chlorella were

35.9, 4.70, 0.224 and 3.34×10−4 µmol/dm3, respec-tively. In the present study, it was found that bothDMSPd and DMSPp production amounts of Hapto-phyta were higher than those of Chlorophyta in thecondition of the similar cell size. Moreover, Isochry-sis galbana 8701 grew faster than Pavlova viridis andPlatymonas sp. and had correspondingly higher DM-SPd and DMSPp concentrations. The discrepanciesin DMSPd and DMSPp production observed may bemostly due to cell size differences of the four species.

To investigate the DMSPp production ability percell, we calculated cell abundance normalized DMSPpconcentrations. Table 1 showed that the average con-centrations of DMSPp per cell were 0.042, 0.050, 0.002and 2.6×10−7 µmol/dm3, respectively. Our resultsabout levels of DMSPp in the Haptophyta Isochrysisgalbana 8701 were in good agreement with those previ-ously reported by Niki et al. (2000) who found a value

84 LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87

of 4×10−15 mol per cell. Levels in the Chlorella were alittle greater than previously reported, at 2.62×10−20

mol per cell in this study, as opposed to those notdetected (Keller et al., 1989). Previous experimentswere conducted with a 14 h light:10 h dark cycle andwere not 12:12, factors which may have altered DMSPconcentrations in the present study.

DMSP is found in various groups of marine algaeand DMSPd production by different algae has largediversities. High concentrations of DMSP were foundin Isochrysis galbana 8701 and Pavlova viridis of Hap-tophyt, while Platymonas sp. and Chlorella belongingto Chlorophyta contained minor amounts of DMSP.Some species of phytoplankton produce more DMSP.It is generally thought that the main producers ofDMSP are confined to a few algal classes, mostly Dino-phyceae and Prymnesiophyceae (Keller et al., 1989).However, other classes, including the Chlorophyta andCryptophyceae, are minor producers. This conclusionis consistent with the findings of Turner et al. (1988)and Andreae (1990) that phytoplankton speciation ismore important than algal biomass or productivity incontrolling DMS levels in seawater.

From Tables 1 and 2, it is clear that the curve pat-terns of both cell densities and DMSP (DMSPd andDMSPp) concentrations are similar for the same alga,implying the dependence of DMSP (DMSPd and DM-SPp) concentration upon the algal cell density. Thelinear regression analysis was applied to evaluate thestatistical relationships among cell abundances, DM-SPd and DMSPp. Consequently, significant correla-tion between population densities and DMSPp con-centrations was visualized in the monocultures of fourmicroalgae during exponential and early stationarygrowth phase (Fig. 3), suggesting that the concen-tration of DMSPp was directly related to cell num-bers. Moreover, there was time lag between the maxi-mum of abundances and DMSPd concentrations. Thisphenomenon may be due to the time needed for thesynthesis of DMSPd. Algal cells incept sulphur fromsurroundings and synthesize dissoluble cysteine, thenmethionine is formed via cystine and homocystine, andDMSP is synthesized through methylation of methio-nine (Stefels, 2000).

3.3 DMS concentrations at different growth

stages

DMS productions in different cultures were shownin Tables 1 and 2. Combining these values with celldensities, it is obvious that the DMS concentrations

showed marked differences among the four microal-gae as well as among the different phases of growth.The DMS values increased in parallel with the cellgrowth during lifetime, until the end of the station-ary phase. DMS in cultures was most efficiently pro-duced in the stationary phase, while little formationof the compound was found at the beginning of al-gae culturing. The initial slow production observedin the cultures might be due to the limited amountof DMSP produced by algae as well as the negligi-ble release of DMSP with the osmoregulatory func-tion played by DMSP in cells. Therefore, only smallamount of DMSP was released out off the cells, re-sulting in little production of DMS. DMS productionwas different in each physiological growing period ofalgae. Significant DMS generation was observed inthe initial lag phases of cultures, and it seemed thatthese increases were caused by algal leakage and dis-ruption. The highest release of DMS appeared in thesenescent period for all cultures. In most phytoplank-ton species the production of DMS from intracellularDMSP by healthy cells is relatively limited (Keller etal., 1989; Turner et al., 1988). Natural senescence maystimulate the liberation of DMSP from cells into thewater where it is partly broken down to DMS, becausethe cells were subjected to physiological function thatcaused cell lysis (Sunda et al., 2002).

In this present study, DMS concentrations var-ied concomitantly with DMSPd concentrations (Fig.4), but there was 1- or 2-day time lag between thepeaks of DMS and DMSPd concentrations (Tables 1and 2). There was no significant correlation betweenDMS and DMSPp concentrations at exponential andearly stationary growth stages for these four microal-gae (Fig. 4), suggesting that maybe DMSPd was morerelated to DMS conversion in the seawater. Therefore,it is possible the DMS was also formed from the algalDMSP released into the seawater by the cells. DM-SPd in the seawater can be enzymatically cleaved toDMS (Steinke and Kirst, 1996; Stefels and van Boekel,1993) under the effect of DMSP lyase.

It is noteworthy that the production curves ofDMS in different algal cultures were rather different.On the average, DMS release amounts in the cul-tures of Isochrysis galbana 8701 was as high as 3.83µmol/dm3. In contrast, the low DMS release amountswere observed in the cultures of Pavlova viridis andPlatymonas sp., with average values of 0.58 and 0.05µmol/dm3, respectively. The data obtained in thisstudy clearly demonstrated that DMS release amounts

LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87 85

Fig.4. Variations of DMSPd, DMSPp concentrations in algal cultures at different growth stages of Isochrysis

galbana 8701, Pavlova viridis, Platymonas sp. and Chlorella with their DMS concentrations.

might be related to the size of microalgae species.Additionally, it was interesting to note that DMS re-lease amounts of Haptophyta were higher than thoseof Chlorophyta in the condition of the similar cell size.It is clear from Table 1 that the average levels of DMSper cell were the highest in the Isochrysis galbana 8701cultures and the lowest in the Chlorella cultures. Thishints that the cell size is not an only factor influencingthe DMS production. The algal species might morestrongly influence DMS release capacities than algalcell size. Turner et al. (1988) and Andreae (1990)found that phytoplankton speciation is more impor-tant than algal biomass or productivity in controllingDMS levels in seawater.

4 Summary and conclusions

In the present study, the concentrations of DMS

and DMSP (DMSPd and DMSPp) in the labora-tory culturing of four microalgae (Isochrysis galbana8701, Pavlova viridis, Platymonas sp. and Chlorella)were investigated. The observed DMS and DMSP(DMSPd and DMSPp) concentrations showed markeddifferences among four cultures as well as in thedifferent stages of growth. We interestingly notedthat Isochrysis galbana 8701, which grew faster thanPavlova viridis and Platymonas sp., produced moreDMSP (DMSPd and DMSPp). Moreover, DMS re-lease amounts of Haptophyta were higher than those ofChlorophyta, even though their cell sizes were similar.These results suggested that the taxa and sizes of phy-toplankton might play an important role in determin-ing the production of DMS(P) in seawater. The un-coupling relation between DMS and DMSPp concen-trations of some algae species implied that the DMSwas mainly formed from the algal DMSP released into

86 LI Chengxuan et al. Acta Oceanol. Sin., 2010, Vol. 29, No. 4, P. 78-87

the seawater, and the enzymatic cleavage of DMSPdto DMS is the primary control of DMS formation inseawater. Overall, we conclude that there is com-pelling observational evidence to indicate that marinemicroalgae participate significantly in the processes ofthe production of the DMS(P) and the conversion ofDMSP-to-DMS, thus play great role in the marine sul-phur cycle and the global climate change. As yet thereare many facets of DMS production in marine phyto-plankton that are not understood, but that requiresattention if the processes and mechanisms of biogenicformation and consumption of DMS(P) in a marineenvironment are to be detailed.

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