http://www.chemistrymag.org/cji/2003/05c094pe.htm |
Dec. 1, 2003 Vol.5 No.12 P.94 Copyright |
(Key Laboratory of Marine Environmental Science(Xiamen Unversity), Ministry of Education; Environmental Science Research Center, Xiamen University, Xiamen 361005, China) Received Sep. 1, 2003; Supported financially by the National Natural Science Foundation of China (NO.20077022)
Abstract A novel analysis method
combining microwave irradiation and gas chromatography (GC) has been developed for the
determination of DMSP in algae cultural mixture. Microwave irradiation was applied to
speed up the process of transformation of dimethylsulfoniopropionate (DMSP) to dimethyl
sulfide (DMS). The determination of DMS was carried out using GC coupled with pulsed flame
photometry detector (PFPD). The method detection limit was as low as 0.02 ng·mL-1
for DMSP in algae culture mixture, with RSD of 3.77% (n=4). The method has been
successfully applied to the analysis of DMS in culture and DMSP in algae culture mixtures
of Prorocentrum donghai, Scrippsiella trochoidea, Phaeocystis pouchetii and
Alexandrium tamarense, respectively, during their growing periods. The DMSP production
of Phaeocystis pouchetii was found to be about 10 times less than that of other
three algae. The highest release of DMS appeared in the senescent stage.
Keywords DMS, DMSP, microwave irradiation, solid phase microextraction
1 INTRODUCTION
DMS is a volatile sulfur compound mainly released from the oceans. DMS undergoes oxidation
to yield sub-micron size sulphate and sulphonate particles that contribute to the acidity
of aerosols and serve as precursors of tropospheric cloud condensation nuclei. DMSP, the
former form of DMS, is a metabolism product of various oceanic algae. Both DMS and DMSP
are believed to play very important role in the global changing.
In the traditional method of determination of DMSP, high concentration
of NaOH is needed to reach pH¡Ý13 and 6~14 hours are
required to achieve full transformation of DMSP into DMS with normal heating method.
Afterward the determination of DMS is carried out with GC. The method especially the
pretreatment is not only time consuming but also low efficient [1,2].
Since last decade, microwave energy has been investigating and widely
applying in various analytical chemistry areas such as sample digestion, extraction, and
chemical reactions [3-5], to accelerate the process. Through the dipole
rotation and ionic conductance of polar substances or ionic species under the microwave
irradiation, the temperature of system rises rapidly. Based on the preliminary study in
our research group, where microwave assisted extraction and GC-PFPD were adopted for the
determination of DMSP in algae culture mixture [6], this study adopted a new
type of SPME fiber, aimed to further develop a more sensitive, reliable and faster
analytical method, and apply it to practical use. In the study microwave irradiation had
been adopted to accelerate the hydrolysis conversion of DMSP in algae culture mixture to
DMS, and then the determination of DMS was done with GC coupled with either headspace
solid phase microextraction (SPME) or headspace direct injection.
2 EXPERIMENTAL
2.1 Instruments and chemicals
The microwave system used was National NN-8552WF oven from Shanghai National Microwave
Oven Co. (Shanghai, China). GC system was Varian CP-3800 gas chromatography equipped with
pulsed FPD at sulphur mode (Varian Co, USA), DB-5 (30m¡Á0.32mm, 0.25mm coating, J&W Co.,USA), and 0.8mm
SPME injector liner (Varian Co, USA). SPME holder and 75
DMS (analytical grade, Supleco Co. USA) standard stock solution of 845ng·mL-1 was prepared by dissolving 10mL DMS in 10mL methanol (HPLC grade, Tedia Co., USA). The diluted solution for daily use was prepared from the stock solution with methanol. All the solutions should be kept in the refrigerator and taken out only right before use.
Solid NaCl (analytical grade, Shanghai Reagent Co. Shanghai, China) was added into deionized water to obtain a saturated NaCl solution with the concentration about 360mg·mL-1. 1.0 mol·L-1 NaOH solution was prepared by adding appropriate amount of solid NaOH (analytical grade, Shanghai Reagent Co. Shanghai, China) in 180mg·mL-1 NaCl solution.
DMS free seawater was from Xiamen University beach. Beach seawater was collected and filtrated through a 0.45mm HA membrane (Hangzhou Membrane Industry Co. Zhejiang, China), and then bubbled by a nitrogen flow to remove DMS and then kept in dark. This seawater was analyzed with the SPME method and no DMS had been detected.
2.2 Gas chromatographic parameters
The chromatography system was operated at the splitless mode with 1079 split/splitless injector at 300ºC. The gas flows were 16mL·min-1, 17mL·min-1, and 10mL·min-1 for H2, air1 and air2 for the detector, respectively, and 1.0mL·min-1 for carrier gas N2. The detector temperature was 220ºC, and the column temperature was 50ºC, where retention times for DMS of SPME with direct injection were 2.85min and 2.92min, respectively. Desorption time for SPME fiber in the injector port was 2 min. No carryover was found.
2.3 Analytical procedure
2.3.1 Analysis of DMS in culture
DMS can be volatilized into headspace and afterwards adsorbed by SPME fiber. A stirring bar and 10mL algal culture were put into a 45mL glass bottle. The bottle was capped with a silicon seal and well shacked. Headspace SPME was carried out for several minutes according to the concentration of DMS in the culture. The sampled fiber was inserted into GC injector port for chromatographic process.
2.3.2 Analysis of DMSP in algae culture mixture
Algae culture mixture was the mixture of algae and their culture. Since the size of the alga was very small, both filtering and centrifuging could not well work out to separate enough algae from their culture for the experiment. Therefore the algae culture mixture was directly taken as sample and Chlorophyll a was measured to help determination of amount of algae in the mixture. A stirring bar, 1mL algae culture mixture and 19mL 1.0mol·L-1 NaOH solutions were put into a 45mL glass bottle. The bottle was placed into the microwave oven. Microwave irradiation with power of 200W for 5min was applied to hydrolyze DMSP into DMS. According to the different concentration of DMS in the culture, different headspace SPME sampling time was performed. For those with very high DMS concentration direct injection of 250mL headspace gas was applied. Since there was already some DMS existing in the culture before DMSP hydrolysis, the result from the above analytical procedure was the sum of DMS and DMSP.
The concentration of DMSP in the algae culture mixture was calculated based on the following equation: DMSPalga = DMSsum- DMScult, where DMSsum was the total DMS detected after microwave hydrolysis and DMScult was that in culture before hydrolysis.
2.3.3 Analysis of Chlorophyll a
Chlorophyll a, representative of phytoplankton biomass, was determined basically according to spectrometric method [7]. The algae culture mixture was filtered and extracted. The absorbency of the extract solution was measured at 750nm, 664nm, 647nm and 630nm, respectively. And the concentration of chlorophyll a (Chl-a) was calculated.
2.4 Cultivation of algae
Four kinds of algae were selected in this study, namely Prorocentrum donghai, Scrippsiella trochoidea, Phaeocystis pouchetii and Alexandrium tamarense. Among those, Prorocentrum donghai, Scrippsiella trochoidea, and Alexandrium tamarense belong to Pyrrophyta, and Phaeocystis pouchetii belongs to Chrysophyta. f/2 culture recipe was used. All stock solutions of nutrient ingredients were prepared with sterile water. The culture solution was prepared with twice boiled natural seawater and stock nutrient solution. The algae at exponential growth stage were inoculated, and cultured in 5dm3 tanks under designed lighting conditions with average illumination of 5000lx and 12:12 L/D cycle. The culture temperature was kept at 22¡À 2ºC. Every day at 10:00am samples were collected for the analyses of DMS, DMSP and Chlorophyll a.
3 RESULTS AND DISCUSSION
3.1 The effect of microwave irradiation time on alkaline hydrolysis
In this study, the hydrolysis process assisted with microwave was quickened by two factors. First, microwave could speed up the movement of water molecules, and thus rapidly raised the temperature of the reaction system and accelerated the alkaline hydrolysis of DMSP. On the other hand, microwave irradiation could efficiently break down the alga cells in which most DMSP existed and thus increased the dissolving rate of DMSP into the surrounding solvent.
At the stationary growth stage, algae culture mixture contains large amount of DMSP. Therefore the dilution of the mixture with DMS free seawater was necessary to avoid DMS signal out off range during the measurements and guarantee SPME fiber not to be saturated while sampling. The diluted culture mixture was first bubbled with N2 to remove existed DMS.
Usually, microwave irradiation time would affect the degree of hydrolysis, resulting in difference of DMS signal peak area. The study was carried out with both headspace SPME method and the method of direct injection of headspace gas. The results of effect of irradiation time on DMS signal peak area with SPME time of 25min are shown in Fig. 1 in solid line. From the figure, one can see that irradiation time of 5min was found to provide the largest signal peak area. Irradiation longer than 5min did not further increase the signal. It was considered that DMSP in algae culture mixture had been completely transformed into DMS after 5min microwave assisted hydrolysis. In addition, the undiluted algae culture mixture was treated as described in 2.3.2 and 250mL headspace gas was directly injected into GC. The results are shown in Fig. 1 in dotted line. Again irradiation longer than 5min did not increase signal. Therefore, microwave irradiation time of 5min was considered long enough for assisting hydrolysis under the experimental conditions The method was time saving and more efficient compared to traditional heating method.
Fig. 1 Effect of microwave irradiation time on DMSP (DMS) signal
3.2 The effect of headspace SPME sampling
time
The time of headspace SPME extraction is mainly depended on the equilibrium rate of
analyte among the phases of SPME polymeric fiber, sample matrix and headspace gas in the
sample bottle. SPME sampling time had certain effects on signal peak area. Longer time
obviously would benefit the equilibrium, on the other hand increase the analysis time.
Diluted samples treated as described in 2.3.2 were tested for the study of headspace SPME
sampling time. From the results shown in Fig. 2, 30min sampling was found to be optimum,
at which DMS on the fiber had reached the equilibrium. However, if the concentration of
DMS in the sample was high enough, SPME sampling time less than 30minutes was optional.
Fig. 2 Effect of SPME sampling time on DMSP (DMS)
signal
3.3 Linear range, relative standard deviation
and detection limit
The tests of linear range and detection limit were done using a matrix solution of 1mL DMS
free seawater diluted with 19mL 1.0mol·L-1 NaOH. The matrix solution was
spiked with different amount DMS standard solution to obtain a series working solutions.
It was found that DMS concentrations and square roots of the peak areas had good linear
correlation. The correlation coefficient R2 was over 0.999, and the linear
range was 0.01-0.5ng·mL-1. Detection limit was down to 0.001ng·mL-1
for DMS and method determination limit was as low as 0.02ng·mL-1 for
DMSP. Diluted algae culture mixture was used to test the relative standard deviations
(RSD) of DMSP analysis, and 3.77% (n=4) was obtained. Blank peak area was zero.
3.4 Biomass, DMSP and DMS productions of four algae during growth period
To determine the growing rates and biomass productions of the
selected four algae, Chlorophyll a served as the representative to quantitate the algal
biomass. Generally in laboratory the growth of cultured alga batch shows "S" type curve for biomass
production. The growth period includes four stages, namely small growth lag stage,
exponential growth stage, stationary growth stage and senescent stage. In this study the
algal density was relatively high at the beginning of the experiment, therefore for all
four algae the small growth stage did not apparently occur but the exponential growth
stage quickly appeared. The proposed detection technique was applied to determine DMSP in
algae culture mixture and DMS in its corresponding culture during whole lifetime period.
At the same time the content of Chlorophyll a was also determined. From the results shown
Fig. 3, Prorocentrum donghai, Scrippsiella trochoidea, Phaeocystis pouchetii and
Alexandrium tamarense reached the largest biomass at 9th, 7th, 8th,
and 10th day, respectively, after being inoculated. However, the amount of
biomass produced by the four algae was very different from each other. The cell of Phaeocystis
pouchetii was the tiniest one among the four algae, and the density was high in
culture solution. Its biomass production was over10-102 times higher than other
three algae.
Fig. 3 DMSP, DMS, and Chl-a concentration
in algae culture mixture
a: Prorocentrum donghai; b: Scrippsiella trochoidea;c: Phaeocystis pouchetii; d:
Alexandrium tamarense
DMSP in the alga cells can be enzymatically cleaved to DMS. The enzyme involved in the reaction is DMSP lyase. During the growing period of algae, the cells are not so easily crashed due to the function of osmoregulatory played by DMSP. Therefore less amount of DMSP is released out off the cells, resulting in less production of DMS. On the other hand during the senescent period of algae, a lot of senile cells are crashed and large amount of DMSP should be released. Under the effect of DMSP lyase DMSP is degenerated into DMS, thus DMS concentration in the culture is increased. Fig. 3 shows that high release of DMS appeared at the senescent stage of all four kinds of algae. However, production curve of DMS of different algae was rather different. The release period of DMS for Prorocentrum donghai and Alexandrium tamarense was short, DMS production dropped to primary level in only one day. DMS release rate is defined as the ratio of mol concentrations of DMS to that of DMSP. For these two algae DMS release rates were as high as 0.34 and 0.31, respectively. DMS release period for Scrippsiella trochoidea and Phaeocystis pouchetii lasted for several days, and DMS release rates were about 0.03-0.08.
In the laboratory batch study, the release rate of DMS was not as high as expected if compared to the content of DMSP in the algae. However in the ocean environment, there are many factors affecting the DMS release, such as salinity, physical disturbing, macrozooplankton grazing, bacterial conversion and so on, the release rate of DMS may be much different from that in the laboratory.
4 CONCLUSIONS
A novel analysis method combining microwave irradiation and GC-PFPD has been developed for
the determination of DMSP in algae culture mixture. The method has the advantages of being
simple, fast, high efficient, sensitive and reliable, compared with the conventional
methods.
The DMSP production of different algae was various. Prorocentrum
donghai, Scrippsiella trochoidea and Alexandrium tamarense belonging to Dinophytes
had the similar production. While the DMSP production of Phaeocystis pouchetii of Haptophytes
was about 10 times less.
DMS production was different in each physiological growing period of
algae. The highest release of DMS appeared in the senescent period.
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