Tropical Natural History 12(1): 55-73, April 2012 2012 by Chulalongkorn University

Temporal and Spatial Variations of Heterotrophic Bacteria, Pico- and Nano-phytoplankton along the Bangpakong Estuary of Thailand

VICHAYA GUNBUA1,2, NITTHARATANA PAPHAVASIT1 AND AJCHARAPORN PIUMSOMBOON1*

1Department of Marine Science, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10330, THAILAND 2Present address: Department of Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131, THAILAND * Corresponding author. E-mail: [email protected] Received: 28 August 2011; Accepted: 4 January 2012

ABSTRACT.– The densities and distribution of heterotrophic bacteria and pico- and nano-phytoplankton were determined along the Bangpakong River during the dry (February and April) and wet (July and September) seasons of 2004. Heterotrophic bacteria, with average densities ranging from 1.38 x 106 to 1.87 x 108 cells/ml, were 10 to 100 times more abundant than autotrophic picoplankton (picophytoplankton). The communities of nanophytoplankton were dominated by nanoflagellates, with average densities ranging from 2.54 x 106 to 1.33 x 109 cells/l. Other nanophytoplankton included diatoms, dinoflagellates, coccolithophorids and cyanobacteria, with average densities ranging from < 1 to 108 cells/l. The abundances of pico- and nano-plankton were higher in the wet season than in the dry season. Higher densities of both groups were also recorded in the upper estuarine region and the densities tended to decrease towards the sea. Heterotrophic bacteria and nanoflagellates dominated the pico- and nano-plankton communities throughout the study periods, while the densities of the other groups showed an increasing trend from the lower estuarine region of the river (salinity > 25 psu) towards the sea. The densities of the pico- and nano- plankton were statistically related to the temperature, salinity and dissolved inorganic nutrients in the Bangpakong River.

KEY WORDS: Picophytoplankton, Nanophytoplankton, heterotrophic bacteria, Bangpakong estuary, Thailand

INTRODUCTION nature of the water bodies, which interferes with the quantitative measurements of either The importance of small-sized phyto- phytoplankton abundance or biomass. In plankton, the pico- (0.2 – 2 µm) and nano- Thai waters, the determination of extracted phytoplankton (2 – 20 µm), as primary chlorophyll-a level has been used as a producers in marine food webs, as well as measure to assess the phytoplankton their roles in biogeochemical cycles of biomass in both coastal and offshore elements, are widely recognized in environments. However, this then places a temperate oceans (Detmer and Bathmann, strong emphasis accordingly on the 1997; Tarran et al., 2001; Not et al., 2002; microphytoplankton (large-sized phyto- Jochem, 2003) but not in the tropical ones plankton of 20 – 200 µm in diameter), that (Nielsen et al., 2004; Pan et al., 2005). The is the diatoms, filamentous cyanobacteria current information on the role of the small- and dinoflagellates (Piumsomboon, 2002). sized phytoplankton in the coastal areas of The measurement of size-fractionated tropical regions is sporadic due to the turbid chlorophyll-a containing organisms was 56 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012 used as a part of the study into the marine are present that consume dissolved organic food web structure in the coastal and matters and serve as a major biomass and estuarine ecosystems to assess the carbon food source among the plankton rehabilitation and/or reforestation of community (Fuhrman et al., 1989; Cho and degraded mangrove ecosystem in the Gulf Azam, 1990). In the microbial loop food of Thailand (Piumsomboon et al., 2001) as web, heterotrophic bacteria play an well as the ecosystem health and stability of important role as an intermediate group the tsunami-disturbed mangrove and coastal between phytoplankton and zooplankton in ecosystems of the Andaman Sea transforming dissolved organic matter into (Piumsomboon et al., 2007). The contri- biomass (Azam et al., 1983; Sherr and butions of phytoplankton of smaller than 20 Sherr, 1994), contributing some 9 – 48% of μm in size (pico- and nano-phytoplankton) the total carbon food source (Jochem, 2003). were estimated to be 41% - 91% of the total The Bangpakong River locates in the chlorophyll-a biomass in mangrove eastern part of the Gulf of Thailand. The estuaries and river mouths in the Gulf of river is strongly influenced by the monsoon Thailand (Piumsomboon, 2002), and this system (Buranapratheprat et al., 2002) as proportion increased to 82% in the less well as by intense human activities, turbid seawater of the near-shore waters of including irrigation systems, industry, the Andaman Sea (Paphavasit et al., 2009). aquaculture, animal farms, municipal supply A high percentage of pico- and nano- and waste water discharge, which control phytoplankton biomass results in a different the variations of the physico-chemical structure of the pelagic food webs than those characteristics of this river, and in particular starting with microphytoplankton as the the salinity and nutrients (Wattayakorn, important producers, and so will alter the 2001; Bordalo et al., 2001). Indeed, the biological productivities of marine fluctuations in the salinity and nutrient ecosystems. concentrations will primarily alter the The variation of water quality, and community structure of microphyto- especially the salinity and nutrient levels, plankton, as has been reported in the eastern may play an important role in affecting the part of the Gulf of Thailand (NRCT-JSPS, distribution and densities of small 1998; Buranapratheprat et al., 2002), and phytoplankton. High densities and biomass hence will affect the higher tropic levels of of picophytoplankton have been found in the food chains including the fish oligotrophic waters of high salinity and low productivity. However, there are no report nutrient levels, whilst lower densities and concerning the community structure of the biomass are observed in coastal waters with small-sized phytoplankton and heterotrophic a low salinity and high nutrient bacteria in this area. concentration. Nanophytoplankton, on the Therefore, this study into the structure of other hand, had abundant densities and the phytoplankton community, and in biomass in coastal waters rather than in the particular the pico- and nano-phytoplankton oligotrophic waters (Sieburth et al., 1978; plus the heterotrophic bacteria was designed Gobler et al., 2005; Pan et al., 2007). to determine the variability in the Besides the small-sized phytoplankton as community of small-sized plankton along important photosynthesis primary producers the Bangpakong estuary, as a baseline data in marine ecosystem, heterotrophic bacteria GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 57

FIGURE 1. The 13 sampling stations (ST 1 – ST 13) located within the Bangpakong River (ST 1 – ST 9), estuary (ST 10 and ST 11) and sea in the river mouth (ST 12 and ST 13). to serve for future assessment of the variations in the nutrient concentrations that planktonic food webs in this ecosystem. may affect the phytoplankton biomass in this area (NRCT – JSPS, 1998; Buranapra- MATERIALS AND METHODS theprat et al., 2002). Thirteen sampling stations were set up Study Area and Period.– Bangpakong along the Bangpakong River from Baan River is the most important river in the Srang district in Prachinburi province to eastern part of Thailand with its 122 km Bangpakong district in Chachoengsao length covering a watershed of 7,988 km2 in province, which were comprised of nine, the Prachinburi and Chachoengsao two and two sampling stations along the provinces (NRCT-JSPS, 1998). It is under river, in the estuary and in the sea off the the influence of the monsoon system; the river mouth respectively (Table 1 and Fig. dry northeast (November to March) and the 1). This study was carried out during the wet southwest (May to October) monsoons. daytime in February, April, July and Thus, the discharge controlled by the river September of 2004. The first two sampling displays seasonal variations, including months represented the dry season while 58 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

TABLE 1. Location of each sampling station and any associated human activity in the proximity of that station.

Upstream UTM Activity Station Location Distance Easting Northing (km) ST 1 Amphoe Ban Srang, Prachinburi 732222 1534220 (0 km) Agriculture ST 2 Amphoe Bang Khla, 737194 1518099 (30 km) Urban Chachoengsao ST 3 Lower Dam, Amphoe Mueang, 730404 1516321 (40 km) Animal farming Chachoengsao ST 4 Na Mueang, Amphoe Mueang, 724784 1513547 (50 km) Urban Chachoengsao ST 5 Amphoe Ban Pho, Chachoengsao 724647 1507213 (65 km) Urban ST 6 Amphoe Ban Pho, Chachoengsao 725051 1504103 (70 km) Urban ST 7 Tha Sa-an, Amphoe 716355 1497161 (85 km) Urban Bangpakong, Chachoengsao ST 8 Tha Kham, Amphoe 717000 1491500 (95 km) Urban Bangpakong, Chachoengsao ST 9 Amphoe Bangpakong, 711014 1487408 (105 km) Industry, Chachoengsao Aquaculture ST 10 Bangpakong estuary, Chonburi 708716 1485977 (110 km) Aquaculture ST 11 Bangpakong estuary, Chonburi 705819 1484246 (115 km) Aquaculture ST 12 Bangpakong estuary, Chonburi 700056 1477925 (125 km) Aquaculture ST 13 Bangpakong estuary, Chonburi 698150 1473581 (130 km) Aquaculture other two months represented the wet from algal cells retained on the GF/F filter season. paper was extracted in 90% (v/v) acetone solution and the concentration was Study Methods.– At each station, the pre- measured fluorometrically (Arar and sampling measurement of four physico- Collins, 1992). Filtrates, after passing chemical parameters, the water temperature, through GF/F paper, were also preserved for salinity, dissolved oxygen and pH, were the analyses of the dissolved inorganic + - - 3- carried out 0.5 m below the water surface nutrients (NH4 -N, NO2 -N, NO3 -N, PO4 -P 2- and 1.0 m above the river / estuary bottom. and SiO3 -Si) according to Parsons et al. Note that at all sampling sites the water (1984). depth was greater than 1.5 m. Water In order to estimate the picoplankton samples were collected for subsequent community structure, water samples analysis of the size-fractionated chlorophyll- collected with a cleaned stainless water a biomass. Each sample was separated into sampler were immediately filtered through a three fractions; 20 – 200 µm, 3 – 20 µm and 200 µm-meshed net and then a 20 µm- 0.7 – 3 µm fractions as representing the of meshed net. The filtrate was collected in a micro-, nano- and pico-plankton, 150 ml cleaned bottle and preserved with respectively. These fractions have been buffered formalin to give a final formalin successfully used before in studying the concentration of 2% (v/v). An aliquot of 1 – estuarine plankton in The Gulf of Thailand 10 ml (depending on the turbidity) of each (Piumsomboon et al., 2004). Chlorophyll-a filtrate was then filtered onto a 0.2 µm black GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 59 polycarbonate membrane filter. log C = -0.422+0.758 (log v) for diatoms Picophytoplankton were separated from (Strathmann, 1967), log C= -0.363+0.863 heterotrophic bacteria based on their red (log v) for flagellates and cyanobacteria chlorophyll-fluorescence under blue light (Verity et al., 1992) and log C = - while heterotrophic bacteria exhibited a 0.760+0.819 (log v) for dinoflagellates bright blue color under UV excitation after (Menden-Deuer and Lessard, 2000). DAPI (4’6-diamidino-2-phenylindoli) – staining due to the DNA fluorescence Data Analysis.– The Bray-Curtis similarity (Porter and Feig, 1980). At least 400 cells of coefficient was calculated to explain the each group were counted with an accuracy degree of similarity of plankton assemblages of ± 10% (Venrick, 1978) and the cell on the temporal scale of sampling months. density were calculated (cells/ml). Prior to analyses, the abundance data were The nanophytoplankton community transformed to log (x+1) to normalize the structure was determined by the FTF (filter- distributions and stabilize variances. The transfer-freeze) technique (Hewes and Holm output similarity matrix was subjected to – Hansen, 1983). Between 1 to 10 ml of the cluster analysis (group – average mean filtrate sample was filtered onto a 1.2-µm linkage). An ordination technique, principal polycarbonate membrane filter under low component analysis (PCA) was used to vacuum. The filter containing the explain the spatial differences in plankton nanophytoplankton cells was placed upside communities. These analyses were down onto a clean glass slide with a small performed using the PRIMER computer drop of water. The slide was then frozen by package version 5.0 (Clarke and Warwick, placing onto a cold surface or in the freezer 1994). Correlation analysis was also (-20 °C) for 5 – 10 minutes. After the performed upon the data to analyze the sample was frozen, the membrane filter was relationships between the physico-chemical carefully peeled of leaving the and biological parameters. The test of nanophytoplankton cells on the glass slide. significance of water quality parameters as The slide was mounted with glycerol well as plankton abundances from different solution and examined under a compound stations and seasons were performed with microscope for classification at the higher ANOVA model and Duncan’s MMT for taxonomic level. parametric analysis and two independent Cell volumes of picoplankton and samples T test. nanoplankton were calculated from measuring the cell size using approximate RESULTS cell geometry. Conversion factors from the literatures were used to estimate the carbon Temporal and spatial variations in the biomass of different groups of picoplankton: physico-chemical parameters.– Temporal heterotrophic bacteria: 105 fgC/µm3 (Theil variations in the average physical-chemical – Nielsen and Søndergaard, 1998) parameters along the Bangpakong River cyanobacteria Synechococcus: 205 fgC/cell were clearly noticed with a higher salinity, (Kana and Glibert, 1987). pH and dissolved oxygen (DO) content The nanophytoplankton carbon contents being observed in the dry season than in the were also calculated from their cell volumes wet season (Table 2). Concentrations of using the following empirical relationships; dissolved inorganic nitrogen and 60 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

FIGURE 2. Spatial variations in the physical parameters of temperature, salinity, dissolved oxygen and pH for the dry and wet seasons. The sampling sites (ST 1 – ST 13), shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively. Data are shown as the mean ± 1 SE. Means with a different lowercase letter are significantly different (p < 0.05; Duncan’s MMT). phosphorus (DIN and DIP) in the dry season at the river sampling sites 1 – 8 (Fig. 2). The were also higher than those in wet season, amount of DO was above 4 mg/l in either although, in contrast, the silicate-silicon upstream stations or in the lower estuary in concentrations were significantly higher in the dry season, but levels were lower in the the wet season. Likewise, in the wet season, wet season, when they were regularly lower the phytoplankton biomass, in terms of total than 4 mg/l at all sample sites and with the chlorophyll-a, was also significantly higher lowest values in the downstream stations of than in the dry season (Table 2). The the dam, some 60 - 70 km from the temperature, however, showed no uppermost sampling station. In contrast, the significant differences. temperature was not significantly different The water salinity and pH showed clear at any sampling station in both seasons. spatial variations with the salinity (Fig. 2). (especially) and the pH trending to increase The maximum value of chlorophyll-a towards the sea with significant and strong biomass was recorded from the uppermost differences between the seasons for the sampling station in the Ban Srang district, salinity, being higher at all sampling Prachinburi province, in the dry season (Fig. stations in the dry season, and for the pH 3), which was accompanied by a bloom of values, which were lower in the wet season the diatom, Cylindrotheca sp. (not shown), GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 61

FIGURE 3. Spatial variations in the concentrations of chlorophyll-a and dissolved inorganic nutrients in the Bangpakong River estuary in the dry and wet seasons. The sampling sites (ST 1 – ST 13), shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively. Data are shown as the mean ± 1 SE. Means with a different lowercase letter are significantly different (p < 0.05; Duncan’s MMT). the highest DO content (Fig. 2) and the phosphate levels upstream and the high lowest silicate-silicon concentration (Fig. 3). concentrations of ammonium and nitrite In addition, spatial variations in the downstream in the estuarine area of the river concentration of the other dissolved mouth (Fig. 3). These appear to correlate inorganic nitrogen and phosphorus were with the land use of farming and industry at observed, such as the high nitrate and site 9. 62 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

TABLE 2. Temporal variations in the average physico-chemical parameters across the 13 sampling sites in the Bangpakong River in both seasons.

Dry season Wet season Parameters Mean ± SE Range Mean ± SE Range Temperature (°C) 30.02 ± 0.36a 28.35 – 32.28 30.36 ± 0.15a 29.78 – 31.95 Salinity (psu) 23.01 ± 1.91a 8.80 – 30.90 7.47 ± 2.41b 0.15 – 25.10 Dissolved oxygen (mg/l) 4.84 ± 0.23a 3.54 – 6.55 3.26 ± 0.15b 2.33 – 4.21 pH 7.43 ± 0.08a 7.03 – 8.12 7.17 ± 0.15b 6.61 – 8.23 Total chlorophyll-a (mg/m3) 1.67 ± 0.50a 0.56 – 8.16 3.21 ± 0.36b 1.71 – 6.66 Ammonium-nitrogen (µM) 3.49 ± 0.82a N.D. – 9.14 2.39 ± 0.39b 0.61 – 5.52 Nitrate-nitrogen (µM) 52.30 ± 6.64a 1.01 – 79.23 14.99 ± 2.17b 3.05 – 25.13 Nitrite-nitrogen (µM) 1.92 ± 0.59a N.D. – 7.68 1.04 ± 0.16b 0.40 – 2.78 Phosphate-phosphorus (µM) 2.28 ± 0.25a 0.27 – 3.60 1.50 ± 0.16b 0.76 – 3.00 Silicate-silicon (µM) 60.78 ± 7.25a 3.89 – 93.04 95.23 ± 8.64b 31.71 – 137.36 Data are shown as the mean ± 1 SE. Means within a row that are followed by a different letter are significantly different (p < 0.05; Two Independent samples T Test). N.D. = non detected.

Temporal and spatial variations in the distance of 110 km from the inner most plankton abundances.– The communities upstream station (Fig. 4). of picophytoplankton in the Bangpakong The communities of nanophytoplankton River and estuary were composed solely of were composed of nanoflagellates, diatoms, cyanobacteria cells of 0.83 – 1.77 µm in dinoflagellates, coccolithophorids and diameter, recognized as Synechococcus cyanobacteria. Nanophytoplankton densities type-cells due to their fluorescent were higher in the wet season, 6.55 x 107 – characteristic. Densities of this picophyto- 1.33 x 109 cells/l, than in the dry season, plankton were in the range of 104 - 105 2.54 x 106 – 1.78 x 108 cells/l (Fig. 5), and cells/ml with the peak abundance in the wet were dominated by nanoflagellates and season (1.48 x 105 cells/ml) being some cyanobacteria, while dinoflagellates, five-fold higher than that in dry season (2.06 coccolithophorids and diatoms made up x 104 cells/ml). High densities of only small fractions. The average densities picophytoplankton were noticed in the peak of nanophytoplankton were highest (8.95 x of the rainy season (July), decreased in the 108 cells/l) at the peak of the rainy season late rainy season (September) and then (July), the same period as the picophyto- increased slightly again in the dry season plankton peak, and lowest in the dry season (February) during the northeast monsoon (February). season before falling to the lowest level in High densities of nanoflagellates were April (Fig. 4). Spatially, the picophyto- usually found in the upstream area and plankton tended to be more abundant in the ranged from 106 – 108 cells/l, and were the river mouth and in the sea (St. 9 – 13) in main component of the nanophytoplankton both seasons (although far more abundant in community along the Bangpakong River in the wet season) than in the upstream summer (April). In contrast, cyanobacteria stations, with the highest densities at a were the major constituent of the nanophytoplankton communities in the wet GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 63

FIGURE 4. Temporal and spatial variations in the picophytoplankton densities. The sampling sites, shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively. Data are shown as the mean ± 1 SE. Means with a different lowercase letter are significantly different (p < 0.05; Duncan’s MMT).

FIGURE 5. Temporal and spatial variations in the nanophytoplankton densities. The sampling sites, shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively. Data are shown as the mean ± 1 SE. Means with a different lowercase letter are significantly different (p < 0.05; Duncan’s MMT).

FIGURE 6. Temporal and spatial variations in the heterotrophic bacterial densities. The sampling sites, shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively. Data are shown as the mean ± 1 SE. Means with a different lowercase letter are significantly different (p < 0.05; Duncan’s MMT).

64 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

the early and late wet season (Fig. 7). A PCA of the abundance of picoplankton and nanophytoplankton communities (Figs 8 and 9, respectively) indicated the separation between communities in the sampling stations along the river to the upper estuary (stations 1 - 9) with those of the lower estuary (stations 10 and 11) and the sea (stations 12 & 13) due to the differences in

FIGURE 7. Dendrogram of the similarity index from salinity (Table 3). The abundance of phytoplankton communities in the wet and dry heterotrophic bacteria tended to be higher in seasons. the river and upper estuarine regions rather than the lower estuary and the sea without season, where they were comprised of any significant difference between seasons cyanobacteria at 86% to 97% of the total (Fig. 8). This result was in agreement with nanophytoplankton along the river. the significant negative relationships Heterotrophic picoplankton (heterotro- between heterotrophic bacteria and salinity phic bacteria or bacterioplankton), with an as well as the positive relationships of average density of 106 – 108 cells/ml, were heterotrophic bacteria to temperature and 10 – 100 times more abundant than phosphate concentrations. The communities picophytoplankton. Their densities ranged of picophytoplankton with a higher from 1.38 x 106 - 1.31 x 108 and 3.29 x 106 - abundance in the sea and in the lower 1.87 x 108 cells/ml in the dry and wet estuary than in the upper estuary and river seasons, respectively (Fig. 6), although this showed a high positive correlation with the is some 10-fold lower than that for salinity level but an inverse relationship nanophytoplankton. High densities of with the temperature, nitrate and phosphate bacteria were found in the upper estuarine levels (Figs 8 and 9; Table 4). areas in the dry, and especially in the wet On the other hand, nanoplanktonic- seasons. The communities of bacterio- diatoms, which exhibited the same plankton in the lower estuary were always distribution pattern as the picophyto- lower than those of the upstream areas (Fig. plankton, showed a significant relationship 6). Note that although the highest levels of only with the salinity (Table 4). The heterotrophic bacteria were seen in the peak distribution patterns of nanophytoplankton of the wet season (July), as per the nano- showed a spatial variation between the river and pico-plankton, in contrast it did not and upper estuarine, lower estuary and the decline much in the latter part of the wet sea without any significant difference season (September). between seasons (Fig. 9). Nanoflagellates Communities of pico- and nano-plankton densities also indicated an inverse in the Bangpakong River showed temporal relationship with the salinity level, while variation between the wet and dry seasons their densities correlated well with the with a similarity of 80%. The communities temperature and, ammonia, nitrate and in the wet season showed a higher phosphate concentrations (Table 4). similarity, of approximately 88%, between

GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 65

TABLE 3. Average physico-chemical parameters from the different zones in the Bangpakong River and estuary.

Zone I (ST 1 – 9) Zone II (ST 10 – 11) Zone III (ST 12 – 13) Parameters Dry Wet Dry Wet Dry Wet Temperature Range 28.35-32.28 29.98-31.95 28.55-28.95 30.13-30.23 28.40-30.50 29.78-29.85 (°C) Mean 30.48 ± 0.26a 29.46 ± 0.42a 29.63 ± 0.44a Range 8.80-27.65 0.15-9.28 28.88-29.43 16.15-16.55 29.45-30.90 24.03-25.10 Salinity (psu) Mean 10.88 ± 2.58a 22.75 ± 3.70b 27.37 ± 1.66c Dissolved oxygen Range 3.54-5.84 2.33-4.21 5.05-5.15 3.39-4.16 6.27-6.55 2.92-4.05 (mg/l) Mean 3.76 ± 0.21a 4.44 ± 0.41b 4.94 ± 0.88b Range 7.03-7.45 6.61-7.39 7.59-7.61 7.72-7.82 8.07-8.12 8.05-8.13 pH Mean 7.03 ± 0.07a 7.68 ± 0.05b 8.12 ± 0.04c Data are shown as the mean ± 1 SE. Means within a row that are followed by a different letter are significantly different (p < 0.05; Duncan’s MMT). Sampling sites (ST 1 – ST 13) are as described and shown in Table 1 and Figure 1, respectively).

TABLE 4. Correlations of the plankton community with the physico-chemical parameters.

Heterotrophic Pico phytoplankton Nanoflagellates Cyanobacteria picoplankton (log10x+1) (log10x+1) (log10x+1) (log10x+1) Temperature -.462** .389** .419** ns Salinity .501** -.717** -.354** -.597** Ammonia ns ns .428** ns Nitrate -.274* ns .597** -.581** Nitrite ns ns ns ns Phosphate -.310* .337** .567** -.293* * Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed). ns = not significant.

Carbon biomass of the plankton DISCUSSION community.– The carbon biomass, calculated from the biovolumes of the Structure of plankton communities in the phytoplankton and heterotrophic bacteria, in Bangpakong River estuary.– The the Bangpakong River and estuary varied communities of picoplankton in the from 17.1 – 1,870.7 µgC/l in the dry season Bangpakong River estuary showed a broad to 157.9 – 4,132.8 µgC/l in the wet season resemblance to that seen in the Klongkone (Fig. 10). Nanophytoplankton dominated the mangrove creek in the Upper Gulf of plankton communities of Bangpakong Thailand, particularly in terms of estuary in terms of the proportion of the picophytoplankton densities (Tarangkoon, carbon biomass ranging from 72.3% to 2002). However, the Bangpakong River and 98.0% of the total carbon biomass in the dry estuary communities were dominated by season and 58.7% to 90.0% in the wet heterotrophic bacteria rather than by the season. Heterotrophic bacteria contributed autotrophic picoplankton found in up to 28% of the total carbon biomass Klongkone mangrove creek of Samut throughout the study period, while carbon Songkram province. The abundance of derived from picophytoplankton was almost heterotrophic bacteria from our study was negligible, except in the river mouth area. 100-fold higher than that that reported for the Klongkone creek (Tarangkoon, 2002), a 66 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

FIGURE 8. Principal component analysis (PCA) of the log (x+1) transformed density of picoplankton. Sampling sites (ST 1 – ST 13) are as detailed and shown in Table 1 and Figure 1, respectively. difference that may due to the significant and the St. Lucie river estuary in Florida, basin characteristics and activities of the USA (Table 5). In contrast, the abundance Bangpakong River in comparison to the of local picophytoplankton at less than Klongkone creek (Tarangkoon, 2002; 50x104 cells/ml was in the same range as Paphavasit et al., 2005). For example, the that previously reported in the above concentrations of dissolved organic carbon estuaries (Table 5). were in the range of 2 - 4 mg/l in the The abundance of nanophytoplankton in Bangpakong River and river mouth the Bangpakong River / estuary found in (Paphavasit et al., 2005), levels that can this study was 10-fold higher than the support the bacterial production in this communities reported in the Tha Chin estuary. estuary in the western part and Pak Poon The dominance of heterotrophic bacteria estuary in the southern part of the Gulf of over picophytoplankton has also been Thailand (Table 5), although the reported before in tropical and temperate nanophytoplankton community structure estuaries (Garrison et al., 2000; Hare et al., (mostly composed of nanoflagellates and 2005; Pan et al., 2006, 2007). However, the cyanobacteria with diatoms, dinoflagellates, abundance of heterotrophic bacteria in the and coccolithophorids as minor Bangpakong River / estuary was much constituents) was similar to that reported for higher than those previously reported in the the Thachin estuary and the Pak Poon temperate regions of Asia and America, estuary in the Gulf of Thailand such as in the Hiroshima, Mutsu and Isu (Phromthong, 1999; Piumsomboon et al., Bays in Japan, the Changjiang estuary in 2000). Thus, the nanophytoplankton China, the Arabian Sea in the middle-east community structure is supported by the GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 67

FIGURE 9. Principal component analysis (PCA) of the log (x+1) transformed density of nanophytoplankton. Sampling sites (ST 1 – ST 13) are as detailed and shown in Table 1 and Figure 1, respectively.

68 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

TABLE 5. Reported abundances and biomass of heterotrophic bacteria, pico- and nanophytoplankton in estuaries and coastal areas of the tropical-subtropical regions.

Organisms/Abundance Environment, Biomass Heterotrophic Pico- Nano- Methods References location, area (µgC/l) bacteria phytoplankton phytoplankton (x106 cells/ml) (x104 cells/ml) (x106 cells/l) Bangpakong estuary 1.4 – 187 0 – 14.8 2.54 – 1,330 11 – 4,132 LM This study Gulf of Thailand Tha Chin estuary, Phromthong - - 3.41 – 24.80 - LM Samut Sakhon (1999) Pak Poon estuary, Piumsomboon Nakorn Si - - 1 – 100 - LM et al. (2000) Thammarat Klong Kone Tarangkoon mangrove swamp, 1 – 1.8 6.4 – 21.8 - - LM (2002) Samut Songkhram Andaman Sea The Andaman Sea, Nielsen et al. Indian Ocean, 0.2 – 0.3 - - 20 – 150 FM (2004) Thailand Other locations Hiroshima Bay - 0.15 – 2 0.35 – 150 - Nishitani et al. Mutsu Bay - 0.3 – 0.9 0.10 – 2.10 - FM (2005) Ise Bay - 0.02 – 9 0.23 – 710 - The Changjiang 0.2 – 27 (Syn) Pan et al. estuary and adjacent 0.4 – 2.5 0 – 21 (Pro) 0.01 – 21.40 - FCM (2007) coastal regions 0.01 – 0.9 (Euk) The northern Arabian Garrison et al. - - 0.02 – 120 0.02 – 11.6 - Sea (1998) 0.2 – 34.7 3 – 17 (Pro) 0.0 – 47.2 Garrison et al. The Arabian Sea 0.7 – 1 3 – 9.7 (Syn) 0.30 – 7.60 0.0 – 112.9 FCM (2000) 0.1 – 0.2 (Euk) 0.0 – 5.8 0.1 – 36.3 The northern Gulf of 0.9 – 1.3 - - - FCM Jochem (2001) Mexico The Mississippi river plume 0.1 – 2 - - - FCM Jochem (2003) 5.1 – 28.3 0.1 – 4.3 The Mississippi River 1 - 0 – 6.1 (Syn) FCM Jochem (2003) (Syn and Pro) 0.1 – 0.8 (Pro) The North Fork of the St. Lucie river 0.7 – 42.5 Millie et al. - - - FM estuary, Florida (Syn and Synt) (2004) (USA) FM: Fluorescence microscopy, FCM: Flow cytometry, LM: Light microscopy, Syn: Synecococcus, Synt: Synechocystis, Pro: Prochorococcus, Euk: Eukaryotic forms. results of other studies in the subtropical aquatic environments has been attributed to northwestern Philippine Sea (Tsuji and efficient light harvesting mechanisms by Adachi, 1979) and the Arabian Sea pigments, adaptations and mechanisms to (Garrison et al., 1998), and so may be a live in diverse habitats, and their efficiency more general ecosystem characteristic. In in nutrient uptake (Sigee, 2005). general, the successful distribution of nanoflagellates and cyanobacteria in various GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 69

FIGURE 10. Temporal and spatial variations of the carbon biomass in the plankton community. The sampling sites, shown as the distance upstream from the sea, are as detailed and shown in Table 1 and Figure 1, respectively.

Factors influencing temporal and spatial overcome the problem of the changing variations in plankton communities.– salinity in the wet season and hence Communities of pico- and nano-plankton in supports the high tolerance of cyanobacteria the Bangpakong River were characterized in an extreme environment (Sigee, 2005). by the dominance of heterotrophic bacteria The situation in the dry season where the over autotrophic picoplankton and by the concentrations of nutrients are scarce, cyanobacteria dominated nanophyto- therefore, favors the nanoflagellates with plankton communities in the water with a their smaller sizes and high surface area: salinity range from 0.15 to 25.1 psu. The volume ratio which offers a high nutrient resemblance between the plankton uptake rate (Safi and Hall, 1997) in communities in the early and late dry season comparison to larger sized phytoplankton. was about 85% (Fig. 7) with the average Our result also indicates that salinity is salinity ranged from 8.80 to 30.90 psu. the major environmental factor shaping the These communities also had heterotrophic plankton community structure in the bacteria dominating the picoplankton Bangpakong River and estuary, with the community but the nanophytoplankton one concentration of dissolved inorganic was dominated by nanoflagellates rather nutrients as the minor controlling factors. than by cyanobacteria. Cyanobacteria may The distribution pattern of diatoms reflected take advantage of their thick cell wall or the dominance of different species inhibited mucilaginous sheath (Jeffrey et al., 1997) to the river and upper estuary in comparison to 70 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012 the species in the lower upper estuary and study period with a density of about 10-fold the sea. The importance of salinity and to 100-fold higher than that for dissolved inorganic nutrients as factors picophytoplankton, which were composed controlling picoplankton abundance in the of prokaryotic coccoid cyanobacteria or Bangpakong River are also in agreement of Synechococcus-like species. The nanophyto- the results reported for the Arabian sea plankton were mostly comprised of (Brown et al., 1999), equatorial Pacific nanoflagellates and cyanobacteria as the (Blanchot et al., 2001), Gulf of Venice dominant groups, together with diatoms, (Aubry et al., 2006), Andean-Patagonian dinoflagellates and coccolithophorids. High lakes (Callieri et al., 2007), subtropical densities of heterotrophic picoplankton and coastal lagoons of Uruguay (Vidal et al., nanophytoplankton were recorded in 2007) and South Australian coastal lagoons upstream regions (upper and lower estuary) (Schapira et al., 2010). while picophytoplankton dominated in the seaward area. Nanophytoplankton accounts Carbon biomass of plankton for a large proportion of the biomass in community.– The estimated carbon bio- terms of chlorophyll-a and make up an mass levels in this study at the Bangpakong important contribution to the primary River and estuary are higher than those production in the Bangpakong River and reported from the temperate bays and estuary. This also suggested further estuaries (Table 5). The high carbon investigation on the structure of the food biomass of nanophytoplankton may due to chain/ food web based on small-size the application of the microphytoplankton producers in this area. carbon-biovolume conversion values to calculate the carbon biomass of nanophyto- ACKNOWLEDGEMENTS plankton, since there is currently no conversion value specific for nanophyto- We are grateful to members of the plankton. Therefore, the biomass for Marine Ecology Laboratory, Department of nanophytoplankton is one to two orders of Marine Science for help in the field-work. magnitude higher in the upstream regions Financial support for this research was than the sea, while pico-phytoplankton provided by the Department of Marine and biomass increases in the sea, indicating their Coastal Resources, the Ministry of Natural different responses to these different Resources and Environment. Partial changing environments. Nanophytoplankton financial support from The Graduate seemingly make a higher contribution to the School, Chulalongkorn University, to the phytoplankton biomass in the upstream first author’s dissertation is also appreciated. regions while picophytoplankton become The authors also thank the anonymous more important in the sea. reviewers for their fruitful suggestion.

CONCLUSION LITERATURE CITED

Among the communities of picoplankton Arar, E.J. and Collins, G.B. 1992. Methods 445.0: In in the Bangpakong River and estuary, vitro determination of chlorophyll a and phaeophytin a in marine and freshwater algae by heterotrphic picoplankton (bacteria) were fluorescence. In USEPA methods for the the most abundant group throughout the determination of chemical substances in Marine GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 71

and estuarine environmental samples. EPA/600/R- Fuhrman, J.A., Sleeter, T.D., Carlson, C.A., and 92/121. U.S. Environmental protection Agency. Proctor, L.M. 1989. Dominance of bacterial Ohio. biomass in the Sargasso Sea and its ecological Aubry, F.B., Acri, F., Bastianini, M., Pugnetti, A. and significance. Marine Ecology Progress Series, 57: Socal, G. 2006. Picophytoplankton contribution to 207-217. phytoplankton community structure in the Gulf of Garrison, D.L., Gowing, M.M. and Hughes, M.P. 1998. Venice (NW Adriatic Sea). International Review of Nano- and microplankton in the northern Arabian Hydrobiology, 91: 51-70. Sea during the southwest monsoon, August- Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyer- September 1995, A US-JGOFS study. Deep-Sea Reil, L.A. and Thingstad, F. 1983. The ecological Research II, 45: 2269-2299. role of water-colume microbes in the sea. Marine Garrison, D.L., Gowing, M.M., Hughes, M.P., Ecology Progress Series, 10: 257-263. Campbell, L., Caron, D.A., Dennett, M.R., Shalap- Blanchot, J., Andre, J.M., Navarette, C., Neveux, J. and yonok, A., Olson, R.J., Landry, M.R., Brown, S.L., Radenac, M.H. 2001. Picophytoplankton in the Liu, H., Azam, F., Steward, G.F., Ducklow, H.W. equatorial Pacific vertical distributions in the warm and Smith, D.C. 2000. Microbial food web pool and in the high nutrient low chlorophyll structure in the Arabian Sea: a US JGOFS study. conditions. Deep-Sea Research I, 48: 297-314. Deep-Sea Research II, 47: 1387-1422. Bordalo, A.A., Nilsumranchit, W. and Chalermwat, K. Gobler, C.J., Cullison, L.A., Koch, F., Harder, T.M. and 2001. Water quality and uses of the Bangpakong Krause, J.W. 2005. Influence of freshwater flow, river (eastern Thailand). Water Research, 35: 3635- ocean exchange, and seasonal cycles on 3642. phytoplankton - nutrient dynamics in a temporarily Buranapratheprat, A., Yanagi., T., Boonphakdee T. and open estuary. Estuarine, Coastal and Shelf Science, Sawangwong, P. 2002. Seasonal variations in 65: 275-288. inorganic nutrient budgets of the Bangpakong Hare, C.E., DiTullio, G.R., Trick, C.G., Wihelm, S.W., estuary, Thailand. Journal of Ocenaography, 58: Bruland, K.W., Rue, E.L. and Hutchins, D.A. 2005. 557-564. Phytoplankton community structure changes Brown, S.L., Landry, M.R., Barber, R.T., Campbell, L., following simulated upwelled iron inputs in the Garrison, D.L. and Gowing, M.M. 1999. Peru upwelling region. Aquatic Microbial Ecology, Picophytoplankton dynamics and production in the 38: 269-282. Arabian sea during the 1995 southwest monsoon. Hewes, C.D. and Holm-Hansen, O. 1983. A method for Deep-Sea Research II, 46: 1745-1768. recovering nanoplankton from filters for Callieri, C., Modenutti, B., Queimalinos, C., Bertoni, R. identification with the microscope: The filter- and Balseiro, E. 2007. Production and biomass of transfer-freeze (FTF) technique. Limnology picophytoplankton and larger autotrophs in Andean Oceanography, 28: 389-394. ultraoligotrophic lakes: differences in light Jeffrey, S.W., Mantoura, R.F.C. and Wright, S.W. harvesting efficiency in deep layers. Aquatic 1997. Phytoplankton pigments in oceanography. Ecology, 41: 511-543. UNESCO, 661 pp. Cho, B.C. and Azam, F. 1990. Biogeochemical Jochem, F.J. 2001. Morphology and DNA content of significance of bacterial biomass in the ocean's bacterioplankton in the northern Gulf of Mexico: euphotic zone. Marine Ecology Progress Series, 63: analysis by epifluorescence microscopy and flow 253-259. cytometry. Aquatic Microbial Ecology, 25: 179- Clarke, K.R. and Warwick, R.M. 1994. Change in 194. marine communities approach to statistical analysis Jochem, F.J. 2003. Photo- and heterotrophic pico- and and interpretation. Plymouth Marine Laboratory, nanoplankton in the Mississippi River plume and UK, 144 pp. distribution and grazing activity. Journal of Detmer, A.E. and Bathmann, U.V. 1997. Distribution Plankton Research, 25: 1201-1214. patterns of autotrophic pico- and nanoplankton and Kana, T.M. and Glibert, P.M. 1987. Effect of their relative contribution to algae biomass during irradiances up to 2000 Em-2 s-1 on marine spring in the Atlantic sector of the southern ocean. Synechococcus WH7803.1. Growth, pigmentation, Deep-Sea Research II, 44: 299-320. and cell composition. Deep-Sea Research, 34: 479- 495.

72 TROPICAL NATURAL HISTORY. 12(1), APRIL 2012

Menden-Deuer, S. and Lessard, E.J. 2000. Carbon to Paphavasit, N, Aksornkoae, S. and Silva, J.D. 2009. volume relationships for dinoflagellates, diatom, Tsunami impact on mangrove ecosystem. Thailand and other protest plankton. Limnology Environment Institute. Nonthaburi, Thailand. Oceanography, 45: 569-579. Parsons, T.R., Maita, R.Y. and Lalli, C.M. 1984. A Millie, D.F., Carrick, H.J., Doering, P.H. and manual of chemical and biological methods for Steidinger, K.A. 2004. Intra-annual variability of seawater analysis. Pergamon Press, Oxford, water quality and phytoplankton in the North Fork England. of the St. Lucie River estuary, Florida (USA): a Phromthong, I. 1999. Dynamics and diversity of quantitative assessment. Eatuarine, Coastal and phytoplankton in Tha Chin estuary, Samut Sakhon Shelf Science, 61: 137-149. province. Master thesis, Department of Marine Nielsen, T.G., BjØrnsen, P.K., Boonruang, P., Fryd, Science, Graduate school, Chulalongkorn M., Hansen, P.J., Janekarn, V., Limtrakulvong, V., University (in Thai). Munk, P., Hansen, O.S., Satapoomin, S., Piumsomboon, A., Paphavasit, N., Soonsawad, N., Sawangarreruks, S., Thomsen, H.A. and Sikhantakasamit, B. and Phromthong, I. 2000. Østergaard, J.B. 2004. Hydrography, bacteria and Plankton communities in Pak Poon estuary, protist communities across the continental shelf and Nakhon Si Thammarat, Southern Thailand. In: shelf slope of the Andaman Sea (NE Indian Ocean). Annual report 1999 (second year : May 1999 – Marine Ecology Progress Series, 274: 69-86. March 2000) on Green Carpet Project in Nakhon Si Nishitani, G., Yamaguchi, M., Ishikawa, A., Yanagiya, Thammarat, Thailand. KEIDANREN Nature S., Mitsuya, T. and Imai, I. 2005. Relationships Conservation Fund (KNCF) and Japan Fund for between occurences of toxic Dinophsis species Environment Conservation (JEC), 45-62 p. (Dinophyceae) and small phytoplanktons in Piumsomboon, A., Paphavasit, N., Phromthong, I. and Japanese coastal waters. Harmful Algae, 4: 755- Tarangkoon, W. 2001. Effects of changes in 762. phytoplankton size fraction on the energy transfer Not, F., Simon, N., Biegala, I.C. and Vaulot, D. 2002. in the coastal ecosystems. In: Symposium on Application of fluorescent in situ hybridization aquatic resources and environmental: integrated coupled with tyramide signal amplification (FISH- management and utilization. Aquatic resource TSA) to assess eukaryotic picoplankton composi- institute, 6-8 December, Chaing Mai province. tion. Aquatic Microbial Ecology, 28: 157-166. Piumsomboon, A. 2002. The study of marine plankton NRCT-JSPS. 1998. An integrated study on physical, in Thailand. Journal of Scientific Research, chemical and biological characteristics of the Chulalongkorn University (section T), 1: 275-290. Bangpakong estuary. Final report cooperative Piumsomboon, A., Tarangkoon, W., Sao-sii, P., research NRCT-JSPS, the National Research Sikhanthakasamit, B., Punnarak, P., Paphavasit, N. Council of Thailand (NRCT) and the Japan Society and Sivaipram, I. 2004. Diversity and plankton for the Promotion of Science (JSPS). 127 pp. production in mangrove plantation and pakphanang Pan, L.A., Zhang, L.H., Zhang, J., Gasol, J.M. and estuary, Nakorn si Thammarat province. In Chao, M. 2005. On-board flow cytometric Integrated Management of Mangrove Plantations observation of picoplankton community structure in for Development of Coastal Resources and the East China Sea during the fall of different years. Environment of Thailand. The Thailand Research FEMS Microbiology Ecology, 52: 243-253. Fund (TRF). Pan, L.A., Zhang, J., Chen, Q. and Deng, B. 2006. Piumsomboon, A., Mongkongsangsuree, N., Papha- Picoplankton community structure at a coastal front vasit, N. and Punnarak, P. 2007. Assessment of region in the northern part of the South China Sea. Marine Ecosystem Stability: A Case Study of Journal of Plankton Research, 28: 337-343. Mangrove Ecosystems of the Andaman Sea. Pan, L.A., Zhang, J. and Zhang, L.H. 2007. Picophyto- Proceedings of the National Conference on plankton, nanophytoplankton, heterotrophic Mangrove Ecosystem “Mangroves: Sustainable bacteria and viruses in the Changjiang Estuary and economics of coastal communities”. Department of adjacent coastal waters. Journal of Plankton Coastal and Marine Resources and Thailand Research, 29: 187-197. Environment Institute, 12-14 September, 2007, pp: Paphavasit, N., Wattayakorn, G., Piumsomboon, A. and 398-411. Sivaipram, I. 2005. The ecosystem of the Bangpa- Porter, K.G. and Feig, Y.S. 1980. The use of DAPI for kong River estuary. Bangkok, Chulalongkorn identifying and counting aquatic microflora. University. Limnology Oceanography, 25: 943-948. GUNBUA ET AL. –VARIATIONS OF PICO- AND NANO-PHYTOPLANKTON 73

Safi, K.A.and Hall, J.A. 1997. Factors influencing Tarran, G.A., Zubkov, M.V., Sleigh, M.A., Burkill, autotrophic and heterotrophic nanoflagellate P.H. and Yallop, M. 2001. Microbial community abundance in five water masses surrounding New structure and standing stocks in the NE Atlantic in Zealand. New Zealand Journal of Marine and June and July of 1996. Deep-Sea Research II, 48: Freshwater Research, 31: 51-60. 963-985. Schapira, M., Buscot, M.J., Pollet, T., Leterme, S.C. Theil-Nielsen, J. and SØndergaard, M. 1998. Bacterial and Seuront, L. 2010. Distribution of picophyto- carbon biomass calculated from biovolumes. plankton communities from brackish to hypersaline Archiv für Hydrobiologie, 141: 195-207. waters in a South Australian coastal lagoon. Saline Tsuji, T. and Adachi, R. 1979. Distribution of nano- Systems, 6: 1-15. phytoplankton including fragile flagellates in the Sherr, E.B., and Sherr, B.F. 1994. Bacterivory and subtropical northwestern Philippine Sea. Journal of herbivory: Key roles of phagotrophic protists in the Oceanographical Society of Japan, 35: 173-178. pelagic food webs. Microbial Ecology, 28: 223- Venrick, E.L. 1978. Estimating cell numbers: How 235. many cells to count?, pp. 167–189. In Sournia, A Sieburth, J.M., Smetacek, V. and Lenz, J. 1978. Pelagic (ed.), Phytoplankton Manual. UNESCO, Paris, 337 ecosystem structure: Heterotrophic compartments pp. of the plankton and their relationship to plankton Verity, P.G., Robertson, C.Y., Tronzo, G.R., Andrews, size fractions. Limnology and Oceanography, 23: M.G., Nelson, J.R. and Sieracki, M.F. 1992. 1256-1263. Relationships between cell volume and carbon and Sigee, D.C. 2005. Freshwater microbiology: biodiver- nitrogen content of marine photosynthetic sity and dynamic interactions of microorganisms in nanoplankton. Limnology Oceanography, 37: 1434- the aquatic environment. John Wiley & Sons, Ltd, 1446. UK, 537 pp. Vidal, L., Rodrίguez-Gallego, L., Conde, D., Martίnez- Strathmann, R.R. 1967. Estimating the organic carbon López, W. and Bonilla, Sylvia. 2007. Biomass of content of phytoplankton from cell volume or autotrophic picoplankton in subtropical coastal plasma volume. Limnology Oceanography, 12: lagoons: Is it relevant? Limnetica, 26: 441-452. 411-418. Wattayakorn, G., Prapong, P. and Noichareon, D. 2001. Tarangkoon. W. 2002. Annual variation in abundance Biogeochemical budgets and processes in Bandon and biomass of picoplankton in Klong Kone Bay, Suratthani, Thailand. Journal of Sea Research, mangrove swamp. Samut Songkhram province. 46: 133-142. Master thesis, Department of Marine Science, Graduate school, Chulalongkorn University (in Thai).