1 INTRDUCTION
Phaeocystis globosa belongs to the class of the Haptophytes or Prymnesiophytes, which has a worldwide distribution ^[1,2]. P. globosa has a complex polymorphic life cycle involving in the form of different types of free-living cells (vegetative non-motile (6-8 um diameter), vegetative flagellate(4-5 um diameter) and microzoospore) and gelatinous colonies^[3] usually reaching several mm up to 3 cm ^[4]. Colonies have a spherical shape and numerous derived forms with 10³-10⁴ cells embedded in a transparent mucous matrix ^[5]. Van Rijssel et al^[6] considered colonies as hollow structures built with small amount of polysaccharides.
    P. globosa has a world-wide distribution. In recent years, blooms of p. globosa have caused large-scale red tides frequently, which produce potent toxin. P. globosa blooms appeared each year along the Dutch coast, including the shellfish cultivation areas of the Wadden Sea and the Oosterschelde estuary since the early 1970s ^[7,8] and in the central Arabian sea in 1996^[9]. A dense bloom of p. globosa first occurred in the coastal waters of southeast China in the autumn of 1997 and reoccurred in the summer of 1999^[4] and 2005. These episodes posed a serious threat to commercial fisheries and aquaculture, human health and also coastal tourism through the odorous foams on the beaches during the wane. Furthermore, P. globosa produces dimethylsulfoniopropionate (DMSP) and converts it enzymatically into dimethylsulfide (DMS) and acrylate (Veronique et al., 2005; Noordkamp et al, 2000). The oxidation products of DMS have an impact on the chemical quality of the atmosphere and give rise to cloud condensation nuclei that affect cloud albedo. The consequent higher ocean emissions of DMS would counteract global warming ^[3,10]. So it is important to monitor and study p. globosa. P. globosa biomass is usually determined by spectrophotometer that measures optical density(OD) on algae suspensions. Chlorophyll of the algae has absorbance at 680 nm, but cell density could not be reflected accurately by OD when algae biomass is great. Determination of chlorophyll in phytoplankton has been based on extraction into a solvent followed by fluorometry, more recently, by high performance liquid chromatography (HPLC). Fluorometric measurement has higher sensitivity and accuracy than spectrophotometric analysis, moreover, the synchronous fluorescence spectra has higher selectivity. HPLC allows the separation of pigments that may interfere with chlorophyll a.
    In this paper, pigments of p. globosa were analyzed respectively by using fluorometric and HPLC methods. The objective of the study was to find some composition of the pigments and appropriate analysis methods in order to monitor p. globosa biomass.

3 RESULTS AND DISCUSSION
3.1 Fluorometric pigment analysis
Fig.1 shows that the fluorescence emission spectrum excited at 446 nm, exhibited two peaks at 670 nm and 650 nm. Chlorophyll a (chl a) fluorescence peak is at 670 nm, and the peak of 640 nm is related to chlorophyll c (chl c). F₆₅₀ is higher than F₆₇₀.The relative height of F₆₄₀ and F₆₇₀ chlorophyll fluorescence emission band depend upon chlorophyll of pigments. In excitation spectra measured respectively at 670 nm and 640 nm and showed in Fig. 2, the shape and intensity were different. Three peaks at 431nm,590nm and 621nm were found in the fluorescence excitation spectra with emission at 670 nm, and the excitation spectra measured at 640 nm had two peaks of 446 nm, 590 nm. The fluorescence spectra with excitation of 431 nm had a peak at 670 nm and the peak of 640 nm disappeared. The pigment composition of the cultured strain of  P. globosa examined in fluorescent measurement is characterized by the presence of chl a and chl c. Fig.2 presents the intensity of excitation spectra at 670 nm were higher than that of excitation spectra at 640 nm. From Fig. 1, it was found that Chl a and chl c could be detected by using ordinary fluorescence method, but their peaks were not separated completely.

Fig.1 Fluorescence emission spectra of pigments of P. globosa with excitation at 446nm

Fig.2 Fluorescence excitation spectra of pigments of P. globosa with emission at 670(A) and 640nm(B), respectively

    Synchronous scanning fluorescence spectroscopy is another technique of great potential in analysis of mixture of fluorescent compounds. As a result, the selectivity for individual components is considerably improved; much more information on mixtures of fluorescent compounds is gained^[12]).
    Fig. 3 shows the synchronous fluorescence spectra depends on the difference between the excitation and emission wavelengths, Dl. For Dl=193 nm, a maximum at 640 nm in the synchronous spectrum ascribed chl c, the spectra with the peak of 670 nm at Dl=258 nm is the spectrum of chl a. As it is evident from Fig. 3, the main fluorescent components identified are the same with those identified by ordinary fluorescence method. Compared to the emission band, the bandwidths reduced, and the intensity improved. Moreover chl a and chl c were separated completely and did not interfere with each other.

Fig.3 Synchronous fluorescence spectra of pigments of P. globosa
A: Dl=193 nm B: Dl=258 nm

3.2 HPLC pigment signature
Absorption chromatogram of P. globosa strain is showed in Fig. 4. The pigments contained chl a, chl c, b-carotene and fucoxanthin and they were separated completely. Chl a and fucoxanthin were the dominant pigments. Compared to fluorescence method, HPLC could measure pigments accurately and separate more effectively, although the analysis operation time was longer. Information on the density of P. globosa populations is central to the study and surveillance of red tide. Pigments are used as reliable indicator of biomass by pigment analysis. Pigment concentration is correlated with the density of P. globosa. Accurate quantification of chl a is often used in phytoplankton biomass in both marine and freshwater environments^[13]. Moreover among the extracted pigments from P. globosa strain, chl a concentrations are the highest, so it can be used to monitor P. globosa biomass by determining chl a concentrations with fluorometric and HPLC pigment methods.

Fig.4 HPLC absorpbance chromatogram of P. globosa strain Peak identification:
1= chl a, 2= fucoxanthin, 3= chl c, 4= b-carotene fucoxanthin.

球形棕囊藻色素的分析方法研究
王 梅，尹平河, 赵 玲，欧云付，晏荣军
（暨南大学生命科学技术学院，广州510632）
摘要  球形棕囊藻是一种海洋赤潮藻，对其监测和研究有着重要意义。分别运用普通荧光、同步荧光法以及HPLC三种方法，对球形棕囊藻的色素进行分析。结果表明，普通荧光和同步荧光法可检测到叶绿素a和c,方法简便、快速，并且同步荧光法可使叶绿素a和c荧光峰互不干扰，灵敏度高。经HPLC分析，球形棕囊藻色素由叶绿素a、叶绿素c、b－胡萝卜素和岩藻黄质组成，其中叶绿素a和岩藻黄质主要色素。与两种荧光方法相比较，HPLC能更准确的分析色素组成。
关键词 球形棕囊藻；普通荧光；同步荧光；HPLC