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Phytoplankton community structure in
Singapore’s coastal waters using HPLC
pigment analysis and flow cytometry
K. Y. H. GIN1,2,*, S. ZHANG1,3 AND Y. K. LEE1,4
1
119223, 2NANYANG TECHNOLOGICAL UNIVERSITY, SCHOOL OF CIVIL AND
50 NANYANG AVENUE, SINGAPORE 639798 3CENTRE FOR MOLECULAR GENETICS, DIVISION OF BIOLOGICAL
4
SCIENCES, UNIVERSITY OF CALIFORNIA, SAN DIEGO, 9500 GILMAN DRIVE, LA JOLLA, CA 92093-0634, USA AND NATIONAL UNIVERSITY OF SINGAPORE,
DEPARTMENT OF MICROBIOLOGY, BLOCK MD4, 5 SCIENCE DRIVE 2, SINGAPORE 117597
TROPICAL MARINE SCIENCE INSTITUTE,
14
KENT RIDGE ROAD, SINGAPORE
ENVIRONMENTAL ENGINEERING, BLOCK N1,
*CORRESPONDING AUTHOR:
[email protected]
To investigate phytoplankton distributions in the coastal waters of Singapore, seawater samples from the
Singapore Strait and the Johor Strait were analysed by high performance liquid chromatography and flow
cytometry. Chlorophyll a (Chl a) concentrations were generally high, accompanied by significant amounts
of fucoxanthin. Chlorophyll b and other major carotenoids, including zeaxanthin and alloxanthin, were
also dominant in the Singapore Strait but were undetectable at most stations in the Johor Strait. Using
ratios of Chl a to pigment that were characteristic of the different algal classes, it was shown that
diatoms comprised 72% of the total Chl a in the Singapore Strait, whereas in the Johor Strait they
comprised 88%. Synechococcus contributed another 18% of total Chl a in the Singapore Strait but was
insignificant in the Johor Strait. Besides providing rapid enumeration of total phytoplankton, flow
cytometric analysis was able to discriminate successfully two different types of Synechococcus (‘bright’
and ‘dim’) in the Singapore Strait based on their different orange fluorescence characteristics under blue
light excitation. However, no ‘bright’ Synechococcus was found in the Johor Strait. Overall, the higher
biodiversity of the phytoplankton community as well as the lower cell concentration and biomass in the
Singapore Strait reflect a more oligotrophic/mesotrophic condition than the Johor Strait.
INTRODUCTION
Early studies exploring the relationship between eutrophication and phytoplankton were generally based on traditional microscope analyses (Shubert, 1984). While detailed
size and species information can be acquired, this method is
very labour intensive. In recent years, pigment analysis
using high performance liquid chromatography (HPLC)
has provided an additional framework for profiling whole
phytoplankton communities in the marine ecosystem
(Mantoura and Llewellyn, 1983; Wright and Shearer,
1984; Everitt et al., 1990; Ondrusek et al., 1991; Barlow
et al., 1997). Not only is chlorophyll a (Chl a) routinely used
to estimate phytoplankton biomass, but other characteristic
pigments are also capable of fingerprinting algal types and
recycling processes, including zeaxanthin, alloxanthin, divinyl Chl a and divinyl Chl b, prasinoxanthin, peridinin and
phaeophytin (Barlow et al., 1997). However, as a bulk
measurement, Chl a gives less information on the physiolo-
gical responses of different phytoplankton taxa within an
assemblage.
Flow cytometry, as a rapid, automated method for
individual particle enumeration and discrimination, provides additional insight into phytoplankton communities.
Based on autofluorescent signals and light-scattering
characteristics, biological oceanographers have been
able typically to divide the phytoplankton community
into Synechococcus, Prochlorococcus, pico-eukaryotic phytoplankton and large eukaryotic phytoplankton (Olson
et al., 1990; Campbell et al., 1994; Partensky et al.,
1996). However, individual species in the eukaryotic
community cannot generally be discerned unless more
specific immunochemical or molecular labelling techniques are involved. Therefore, the best way to analyse
marine phytoplankton entails the combination of a variety of approaches, both for single cells and for bulk
properties of the water.
doi: 10.1093/plankt/fbg112, available online at www.plankt.oupjournals.org
Journal of Plankton Research 25(12), Oxford University Press; all rights reserved
JOURNAL OF PLANKTON RESEARCH
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There are few published reports on phytoplankton
communities in Singapore’s coastal waters. The most
extensive survey of phytoplankton was conducted by
Tham (Tham, 1953) and showed a dominance of
diatoms in surface waters. A later study by Chou
and Chia (Chou and Chia, 1991) also confirmed the
dominance of diatoms in the Singapore and Johor
Straits. In recent years, a baseline monitoring study has
been carried out in which the spatial and temporal
distributions of selected biological, physical and chemical
parameters were measured, including the analysis
of nutrients and size structures of the phytoplankton
community in both the Singapore and the Johor Straits
(Gin et al., 2000). In this paper, we further explore the
phytoplankton populations indigenous to the coastal
waters of Singapore with the aid of flow cytometry,
HPLC and microscopy. The aim of this study is to
characterize the phytoplankton populations according
to their pigment composition and to determine their
numerical abundance and distribution in Singapore’s
marine waters.
METHOD
Field sample collection and preservation
Based on geographical proximity, the stations sampled
(between latitude 1 090 N and 1 290 N and longitude
103 380 E and 104 060 E; Figure 1) were divided into two
103ο40′E
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groups: (i) the Singapore Strait (from July 1999 to November 1999, on a monthly basis), spanning the region from the
south-west islands (station S39) to the east coast of Singapore (station S73); and (ii) the West Johor Strait (from July
1999 to November 1999 on a bi-monthly basis), from the
causeway (station J1) to Tuas buoy (station J8).
For HPLC analysis, 4 l of sea water were collected from
5 m depth and filtered (in duplicate) through GF/F filters
(47 mm). The filters were then wrapped in aluminium foil,
and stored in liquid nitrogen for no more than 1 month
until HPLC analysis. For flow cytometric analysis of picophytoplankton, 2 ml duplicate seawater samples were collected in CryoTube vials (Nunc), fixed with
glutaraldehyde (final concentration 0.1%) and subsequently stored in liquid nitrogen (Vaulot et al., 1989).
Net samples (>10 mm) were collected and concentrated
by a vertical haul using a 10 mm mesh phytoplankton
net. Each haul was taken from a depth of 5 m and
nominally sampled 377 l of water. The samples were
then preserved with formalin (final concentration 4%)
for morphological screening and enumeration analysis
using a Nikon Optiphot-2 microscope. Duplicate net
samples were also fixed with glutaraldehyde (final concentration 0.1%) for flow-cytometric analysis of larger
nano/microphytoplankton. Measurements of selected
water-quality parameters (salinity, temperature, dissolved
oxygen, Chl fluorescence, beam transmittance, Secchi
depth and nutrients) were carried out in a parallel study
(Gin et al., 2000).
103ο50′E
West Johor Strait
j
E8
JOHOR
Causeway
J4
J5
SINGAPORE
J6
Changi
1ο20′N
J7
S73
S103
J8
S89
S59
S43
Pulau
Pawai
1ο10′N 0
1ο20′N
S84
S49
S13
5
10 km
S39
103ο40′E
S71
Singapore Strait
S53
BATAM
S31
103ο50′E
Fig. 1. Geographical locations of sampling sites in the Singapore Strait and Johor Strait.
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Algal cultures and standard pigments for
HPLC
Five batch cultures of species representative of major
algal groups, including Amphidinium carterae, Dunaliella
tertiolecta, Emiliania huxleyi, Phaeodactylum tricornutum,
Synechococcus sp., were selected (Liaaen-Jensen, 1977). All
cultures were grown in 250 ml Duran flasks containing
30 ml of culture medium and transferred every 2 weeks.
Illumination was provided by ‘daylight’ fluorescent tubes
(Phillips) on 12:12 h (light:dark) cycles. Authentic Chl a,
Chl b and b-carotene were obtained from the Sigma
Chemical Company; zeaxanthin, lutein and canthaxanthin were provided by Dr K. Bernhard (Hoffman-La
Roche); and fucoxanthin was separated and purified from
the pigment extracts of P. tricornutum by semi-preparative
HPLC. All these standards were stored in a dry, dark
environment below 0 C.
Cell harvesting and pigment extraction
Algal cultures
Cultured cells were harvested before the end of log phase by
centrifuging a 10 ml cell culture in a bench centrifuge (Sigma
laborzentrifugen 3K15) at 5000 g and 4 C for 10 min. The
precipitate was extracted in 5 ml of 90% cold acetone and
then sonicated for 10 cycles (sonication:break = 10:20 s).
After a further 10 min of centrifugation (5000 g at 4 C),
the supernatant was retained for reverse-phase HPLC
(RP HPLC) analysis (Rowan, 1989).
Field samples
The frozen filters were removed from liquid nitrogen, cut
into small pieces (several mm2 in area) and then ground in a
motorized grinder with 5 ml of 90% cold acetone. After the
extract had been transferred to a 15 ml centrifuge tube,
sonication was carried out (six cycles, sonication:break =
10:20 s) under the same conditions as for the cultured cells.
This was followed by a further 24 h of extraction in the
dark at 20 C. The clarified pigment extract was harvested after 10 min of centrifugation (5000 g at 4 C). This
crude pigment extract was then concentrated by aeration
with a gentle stream of N2 gas to a final volume of 2 ml
for further quantitative analysis (Rowan, 1989).
RP HPLC was conducted on a Hewlett Packard
HPLC 1100 series. Two reverse-phase columns, a
Hewlett Packard Lichrosorb RP C18 (5 mm particle size,
200 4.6 mm) and a Perkin Elmer RP C18 (3 8 CR,
3 mm particle size), were used in serial connection for a
thorough separation of pigments (Zhang, 2001). Pigments in field samples were identified by comparison
with the retention times of standard pigments. In addition, each eluted peak’s spectroscopic curve (scanning
from wavelength 200 to 900 nm) was acquired to
reconfirm selected peaks in the case of pigment co-elution.
Most peak areas were converted to pigment abundance
using individual calibration curves made using dilution
series in 90% acetone. For those coefficients not available
for standard pigments, the weight of pigment was estimated from an extension of Beer’s Law (Mantoura and
Llewellyn, 1983). The contributions of each algal class to
total Chl a were calculated by multiplying the concentrations of selected marker pigments by an appropriate ratio
of Chl a to pigment (Everitt et al., 1990; Ondrusek et al.,
1991).
Flow cytometry analysis
A Coulter EPICS Elite EPS flow cytometer was
operated with a 100 mm quartz flow cell and a 488 nm
laser. Two main protocols were used according to the
cell size of the phytoplankton: (i) pico-setting, for analysing picophytoplankton and ultraphytoplankton in the
size range from 0.2 to 5.0 mm; and (ii) micro-setting,
for studying large nano- and microphytoplankton from
5 to 60 mm in size (Zhang, 2001). Samples were prescreened through a 60 mm mesh to prevent clogging of
the flow cell and 2 and/or 10 mm calibrated standard
beads (blue-excited) were added for reference.
RESULTS
Pigment and phytoplankton distribution
Almost all major chemotaxonomic pigments, including
fucoxanthin, diadinoxanthin, zeaxanthin, Chl a, Chl b,
alloxanthin, b-carotene, peridinin and phaeopigments,
were readily identified in the field samples. The absolute
concentrations of the pigments at each sampling station
in the Singapore and the Johor Straits are shown in
Figures 2 and 3, along with the nutrient data. (Note
that negligible amounts of ammonia and phosphate
were measured in the Singapore Strait.) The corresponding pigment-derived composition of the phytoplankton communities (by class) in this coastal region is
shown in Table I. Pigment to Chl a ratios for estimating
the relative biomass of the different phytoplankton
groups are referred to in Table II.
In the Singapore Strait, the average Chl a concentration was 0.55 mg l1 and ranged from 0.25 to 1.23 mg l1.
The highest concentrations were always found at stations
73 and 84, located off the east coast. As expected, the
Chl a levels in the more eutrophic Johor Strait were
much higher and varied from 1.55 to 10.11 mg l1,
with an average value of 5.11 mg l1. This is 10 times
that found in the Singapore Strait and is consistent with
nutrient measurements which showed that concentrations
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Fig. 2. Concentration of major pigments and nutrients at each sampling station in the Singapore Strait. (Data were collected in September and
October 1999.)
in the Johor Strait were normally four to eight times those
in the Singapore Strait (Gin et al., 2000). Another common pigment present in phytoplankton cells, b-carotene,
had an average concentration of 0.02 mg l1 in
the Singapore Strait, ranging from 0.01 to 0.05 mg l1.
In contrast, the average value in the Johor Strait
was considerably higher at 0.17 mg l1, with a range
between 0.05 and 0.29 mg l1. In this study, a correlation
of Chl a and b-carotene was obtained ( y = 0.1222x,
r2 = 0.6426).
Fucoxanthin, a representative pigment found in diatoms, was widely distributed in both the Singapore and
Johor Straits. Although prymnesiophytes and chrysophytes
are also fucoxanthin-containing species (Everitt et al.,
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β -carotene (µ g/L) Zeaxanthin (µ g/L) Fucoxanthin (µ g/L)
10
8
6
4
2
0
0.3
0.2
0.1
0.0
0.2
0.15
0.1
0.05
Chl b like (µ g/L)
0
2
1.5
1
0.5
0
6
4
2
0
J1
J3
J4
J6
Stations
6
4
2
0
0.8
0.6
0.4
0.2
0
0.3
0.2
0.1
0
0.02
0.01
0
0.3
0.2
0.1
0.0
0.08
(µM)
NO3- & NO2- (µg/L)
8
8
0.06
PO4
Ammonia (µM) Phaeopigment (µ g/L) Diadinoxanthin (µg/L) Alloxanthin(µg/L) Chlorophyll b ( µg/l) Chlorophyll a ( µg/l)
K. Y. H. GIN ETAL.
0.02
0.04
0.00
J8
J1
J3
J4
J6
Stations
J8
Fig. 3. Concentration of major pigments and nutrients at each sampling station in the Johor Strait. (Data were collected in September and
October 1999.)
1990), the lack of both 190 -hexanoyloxyfucoxanthin
(biomarker for prymnesiophytes) and 190 -butanoyloxyfucoxanthin (biomarker for chrysophytes) corroborated the
theory that diatoms were the only significant source of
fucoxanthin in this coastal region (Arpin et al., 1976).
Therefore, fucoxanthin can be used as a direct biomarker
for diatoms in these waters. In the Singapore Strait, the
average fucoxanthin concentration was 0.36 mg l1 and
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Table I: Percentage estimates of the contributions of algal classes to total Chl a in the Singapore and
Johor Straits during September and October 1999
Johor Strait
Stations:
J1
J3
Singapore Strait
J6
J8
S84
S89
S103
S53
S59
S71
S13
12.7
Cryptophytes
ND
ND
2.4
11.3
9.3
6
3.5
9.2
11.5
10
Diatoms
95.2
93.3
81.4
32.2
77.4
67.9
80.1
62.2
85.3
67.3
88
Dinoflagellates
14.9
11.4
<1
ND
ND
ND
ND
ND
ND
ND
ND
Green algae
ND
ND
1.4
3.9
8.3
7.3
5
11
10.7
8.2
9
Prochlorophytesa
ND
ND
ND
ND
ND
ND
3.5
ND
ND
6.8
ND
Synechococcus
ND
ND
ND
46.6
22.2
23.1
7.4
17.9
18.7
17.1
20.3
Total (%)
110
105
87
95
117
104
95
102
126
109
129
ND, non-detectable.
a
Calculated from the data of Chl b-like pigment.
Table II: Marker pigment ratios used to estimate the contribution of phytoplankton classes to Chl a
(Everitt et al., 1990; Ondrusek et al., 1991)
Class
Cryptomonads
Pigment ratio
Values
Chl a:alloxanthin
1.85
b-carotene:alloxanthin
0.0825
Cyanobacteria
Chl a:zeaxanthin
1.7
Diatoms
Chl a:fucoxanthin
1.4
Dinoflagellates
Chl a:peridinin
2.35
Prasinophytes
Chl a:prasinoxanthin
4.2
Other green algae
Prochlorophytes
Prymnesiophytes
Chl b:prasinoxanthin
2.08
Chl a:Chl b
0.75
Zeaxanthin:Chl b
0.02
Divinyl Chl a:b-carotene
4.86
Divinyl Chl b:b-carotene
7.97
Divinyl Chl b:divinyl Chl a
0.931
Zeaxanthin:divinyl Chl b
0.16
Chl a:19’-butanoyloxyfucoxanthin
1.6
Fucoxanthin:19’-butanoyloxyfucoxanthin
0.09
ranged from 0.12 to 0.78 mg l1. In contrast, the average
concentration in the Johor Strait was 2.29 mg l1, and
ranged from 0.62 to 5.72 mg l1. Corresponding diatom
contributions to total Chl a in the Singapore Strait
and Johor Strait were 72 and 88%, respectively.
However, unlike the Johor Strait, the samples in the
relatively oligotrophic Singapore Strait showed the presence of several other accessory pigments (e.g. zeaxanthin, alloxanthin, Chl b-like pigment), suggesting a
more diverse near-surface phytoplankton community
(Gin et al., 1999).
The average concentration of zeaxanthin measured in
the Singapore Strait was 0.05 mg l1, and ranged from
0.02 to 0.10 mg l1. Synechococcus, an important primary
producer in oceanic waters, was the main source of
zeaxanthin in this region, with only a minor contribution
from green algae (<2.5%). The results showed that
Synechococcus had a relatively stable population in the
Singapore Strait, typically comprising 18% of the
total Chl a. When added to the contributions by other
phytoplankton in the pico- and ultra-plankton range (i.e.
small green algae and cryptophytes), the results were
consistent with our earlier studies on size-fractionated
chlorophyll in that the <5 mm size fraction contributed
20–30% of total Chl in the relatively oligotrophic
Singapore Strait (Gin et al., 2000). Zeaxanthin was
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detected in the Johor Strait at only one station (station
J8, located at the mouth of the Johor Strait, September
1999), where a high concentration of 0.74 mg l1 was
observed (Figure 3).
Traditionally, Chl b has been used as an indicator of
green algae, since these organisms are hard to distinguish because of the absence of morphological features
( Jeffrey, 1974). In the Singapore Strait, the contribution
of green algae to total Chl a was quite stable (8.6%). The
average Chl b concentration was 0.04 mg l1 and ranged
from 0.02 to 0.07 mg l1. In contrast, Chl b concentrations in the Johor Strait fluctuated dramatically from
undetectable (at station J1) to 0.27 mg l1 (at station J4),
without a regular pattern. However, other chlorophyte
markers, such as lutein, were not detected in these
coastal waters, nor were prasinoxanthin and violaxanthin positively identified as being present. It seems
likely that most Chl b was derived from prasinophytes
that lacked significant amounts of prasinoxanthin (Foss
et al., 1986; Everitt et al., 1990; Barlow et al., 1997). In
addition, the finding of Chl b-like pigments (presumably
divinyl Chl b) at stations S103 and S71 suggests that
prochlorophytes may survive in some parts of these
nutrient-poor tropical coastal waters. Nevertheless,
based on their minor contribution to total Chl a (0–5%),
the function of prochlorophytes in Singapore’s coastal
waters is not as important as in the open oceans, where
they can contribute up to 50% of the primary production
(Campbell et al., 1994).
Alloxanthin is an unequivocal marker for only one
group of algae, the cryptophytes. In the Singapore Strait,
its concentration averaged 0.03 mg l1, and varied from
0.01 to 0.06 mg l1. As another contributor to primary
production in this region, cryptophytes on average comprised 8.5% of total Chl a in the Singapore Strait.
However, alloxanthin was undetectable in the Johor
Strait, except in the mouth where a high concentration
(0.17 mg l1) was found at station J8, accompanied by very
high levels of zeaxanthin, as discussed earlier.
Flow cytometry analysis
The average concentration of total pico- and ultraphytoplankton measured in the Singapore Strait was
8.17 104 cells ml1, and ranged from 5.36 104 to
11.52 104 cells ml1 (Figure 4A). More than 70%
of such cells were comprised of zeaxanthin-containing
Synechococcus (5.63 104 cells ml1), the major species
in this size range (Figure 5). Using the results derived
from HPLC, the cellular concentration of zeaxanthin for
the Synechococcus found in Singapore coastal waters was
only 0.84 fg cell1, lower than the 1.8 fg cell1 reported
by Kana et al. (Kana et al., 1988) based on laboratory
cultures. This may result from the high light irradiance
in tropical waters. Cryptophyte, a small eukaryotic species
(‡3 mm), which is generally prominent in oligotrophic
waters, was also detected by its orange fluorescence and
larger cell size (forward scatter) (Figure 6), consistent with
the measurement of alloxanthin by HPLC. However, the
abundance of this population was much lower than that of
Synechococcus (4489 cells ml1).
In contrast, the average concentration of total picoand ultraphytoplankton in the more eutrophic Johor
Strait (except station J8) was 2.71 104 cells ml1,
only about one-third of that in the Singapore Strait
(Figure 4B). Earlier studies on size-fractionated chlorophyll showed that cells in this size range contributed
<5% to the total biomass, compared with 30% in the
Singapore Strait (Gin et al., 2000). Furthermore, the abundance of Synechococcus at all stations in the Johor Strait
(excluding J8) was quite low (1.24 104 cells ml1,
only 22% of that in the Singapore Strait), consistent
with levels of zeaxanthin at these stations that were undetectable by HPLC. In sharp contrast is the high abundance
of total pico- and ultraphytoplankton at station J8 (September 1999) where the concentration was as high as 1.29 106 cells ml1, most of which was contributed by a dense
Synechococcus population (1.13 106 cells ml1) (Figures 4C
and 5). This location lies at the junction of the west Johor
Strait and Singapore Strait.
In the Singapore Strait, two populations of Synechococcus were distinguished based on their fluorescence
signatures. It is possible that these were two different
strains were defined by their phycoerythrin chromophore composition because Synechococcus with a high
ratio of phycourobilin (PUB) to phycoerythrin (PEB)
(‘bright’) emit a brighter orange fluorescence compared
with those with a low PUB:PEB ratio (‘dim’), since the
488 nm excitation is absorbed by PUB (maximal absorbance at 490 nm) more efficiently than PEB (maximal
absorbance at 550 nm) (Olson et al., 1988, 1990). The
mean orange fluorescence for the high PUB:PEB strain
was 0.0333 relative bead units (rbu, in this case 2 mm
beads) whilst that for the low PUB:PEB strain was
0.0101 rbu, which is distinct enough for their discrimination (Figure 6A). However, the difference in red
fluorescence (i.e. relative Chl a cellular concentration)
between the two strains is not as distinguishable as the
orange fluorescence (0.214 rbu versus 0.160 rbu). In
addition, the mean forward scatters for both types were
roughly the same (0.24 rbu), indicating their similar cell
size despite the dissimilarity in pigment content. None of
the ‘bright’ Synechococcus were found in the Johor Strait.
The relative mean orange fluorescence for ‘dim’ Synechococcus in the Johor Strait is only 0.0045 rbu, much lower
than its counterpart in the Singapore Strait (Figure 6C).
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Synechococcus
(X 10-4 cells/mL)
Fig. 4. Bivariate plots showing flow cytometric signatures of picophytoplankton and ultraphytoplankton at different locations analysed on the
‘pico’ setting (gated for total pico- and ultraphytoplankton counting): (A) station S89 in the Singapore Strait (November 24, 1999); (B) station J3 in
the west Johor Strait (September 9, 1999); (C) station J8 in the west Johor Strait (September 9, 1999); and (D) 0.2 mm filtered sea water as blank
control. Standard beads (2 mm) were used as reference and internal standards.
Stations
Fig. 5. Concentrations of Synechococcus at each sampling station in the
Singapore Strait, including station J8. Value for J8 is 1.13 106 cells
ml1. An asterisk indicates that the concentration is off the scale.
For net samples running under the ‘micro’ setting (i.e.
10–60 mm), individual species could not be distinguished
because those commonly encountered large eukaryotic
phytoplankton (e.g. diatoms and dinoflagellates) absorb
in similar regions of the spectrum and all emit
fluorescence from Chl a. However, their total abundance
could be determined. In the Singapore Strait, the average concentration of larger nano- and microphytoplankton was 2.1 104 cells l1 whilst that in the Johor
Strait was 1.5 105 cells l1, showing the greater significance of larger nano- and microphytoplankton in the
eutrophic Johor Strait. Moreover, the mean value of
forward scatter for the phytoplankton population in the
Johor Strait [0.996 rbu (with 10 mm beads)] was considerably higher than that in the Singapore Strait (0.686 rbu)
(Figure 7). This reflects the larger size of cells in the
eutrophic waters of the Johor Strait. In addition, stations
S73 and S84 were distinct from other stations in the
Singapore Strait in terms of their higher forward scatter
mean value (0.92 rbu) and higher red fluorescence,
indicating the dominance of large microphytoplankton
(Figure 8). Gin et al. (Gin et al., 2000) verified that for
these two stations, microphytoplankton contributed 65%
of total Chl a, whereas the contribution at other stations
generally ranged from 14 to 48% (P < 0.05).
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Fig. 6. Bivariate plots showing flow cytometric signatures of Synechococcus and cryptophytes for samples at different locations analysed on the ‘pico’
setting: (A and B) S89 station in the Singapore Strait (November 24, 1999); (C and D) J8 station in the west Johor Strait (September 9, 1999); (E and F)
J3 station in the West Johor Strait (September 9, 1999). Two populations (‘bright’ and ‘dim’) of Synechococcus as well as cryptophytes were discriminated
in the red fluorescence versus orange fluorescence cytogram (A). A significant ‘dim’ population of Synechococcus was observed (C), while smaller
concentrations of either ‘bright’ or ‘dim’ Synechococcus were discriminated (E). Standard beads (2 mm) were used as reference and internal standards.
DISCUSSION
The HPLC results confirmed our previous conclusion,
based on microscopic observation, that the phytoplankton community in the Singapore Strait was more diverse
than that in the west Johor Strait (Zhang, 2001). It also
demonstrated the general pattern of the spatial distribution of Chl a in the Singapore Strait. In general, the Chl a
concentration increased from the south-west to the east
coast of Singapore (stations S73 and S84), despite slight
seasonal variation as a result of different monsoon seasons (Gin et al., 2000). The discharge of treated domestic
effluent and urban storm run-off may be the main reason
for the relatively higher Chl a concentrations along the
east coast of the Singapore Strait, consistent with the
generally higher nutrient measurements at these two eastern stations (Gin et al., 2000). For the more eutrophic
stations in the west Johor Strait, higher anthropogenic
inputs coupled with relatively shallow, less well-mixed
waters could have contributed to the much higher Chl a
concentration and generally large phytoplankton size.
The dominance of diatoms in coastal environments
has been well established. Apart from their high
efficiency of nutrient uptake in nutrient-rich environments, diatoms may also have another advantage
because of their high fucoxanthin content. In coastal
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Fig. 7. Bivariate plots showing characteristic flow cytometric signatures of microphytoplankton and large nanophytoplankton (10–60 mm)
analysed on the ‘micro’ setting: (A) station J4 in the West Johor Strait (September 9, 1999); and (B) station S39 in the Singapore Strait (October
12, 1999). Standard beads (10 mm) were used as references.
Fig. 8. Bivariate plots and histogram showing characteristic flow cytometric signatures of microphytoplankton and large nanophytoplankton
(10–60 mm) analysed on the ‘micro’ setting: (A) S73 in the Singapore Strait (September 11, 99); (B) S84 in the Singapore Strait (October 11, 1999);
(C) S49 in the Singapore Strait (October 12, 1999). (D) Comparison of forward scatter values (relative to 10 mm beads) for the main populations
at station S49 (bold line) (October 12, 1999) and S73 (dotted line) (October 11, 1999). Standard beads (10 mm) were used as references.
waters where particulate and dissolved organic matter
are in high concentrations, blue light is rapidly attenuated with preferential transmission of the green-to-yellow
wavelengths for photosynthesis. Since fucoxanthin is the
most efficient photosynthetic carotenoid absorbing light
in the green waveband (Ondrusek et al., 1991), this
places fucoxanthin-containing diatoms at a competitive
advantage to other species. Strong correlations between
fucoxanthin and total Chl a were observed for both
stations in the Singapore Strait (R2 = 0.902) and the
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west Johor Strait (R2 = 0.964) (September 1999), excluding
station J8.
This is the first time that abundant populations of
Synechococcus have been detected in tropical waters around
Singapore. These results complement the observation that
this species is ubiquitous in many areas of the world’s
oceans. For example, Synechococcus populations have been
measured in oceanic surface waters off Japan [5.5–15 103 cells ml1 (Kudoh et al., 1990)], the Arabian Sea
[5.3 105 cells ml1 (Campbell et al., 1998)], the north
Pacific Ocean (Iturriaga and Mitchell, 1986), the north
Atlantic Ocean (Olson et al., 1990) and in the waters off
Hawaii (Landry et al., 1984). In the Singapore Strait, the
abundance of Synechococcus populations was generally high
and fairly uniform. Presumably, its small size not only
minimizes its sinking velocity but also gives it a high surface
area to volume ratio, thereby optimizing light and nutrient
absorption efficiencies (Chisholm, 1992). Moreover, Ikeya
et al. (Ikeya et al., 1997) verified that the rapid growth of
Synechococcus is supported by the development of a highaffinity nutrient-uptake system (e.g. phosphate uptake),
with a similar mechanism to some heterotrophic Gramnegative eubacterial cells (Scanlan et al., 1993). These
results are consistent with the relatively low phosphate
concentration (average 0.29 mM) and nitrate plus nitrite
concentration (average 2.57 mM) observed in the Singapore Strait, compared to the Johor Strait (averages 1.29
and 7.14 mM, respectively).
The difference between the two straits in terms of
Synechococcus populations is noticeable. In the Johor Strait
where nutrients are higher, a lower abundance of Synechococcus was observed. Studies have shown that Synechococcus is
generally a poor competitor with the larger algal cells in
eutrophic ecosystems, whereas it usually thrives in nutrientdepleted environments because of its higher surface area to
volume ratio as well as its lower subsistence quota and high
growth rate (Ondrusek et al., 1991; Chisholm, 1992; Gin,
1996). Conversely, micro- and large nanophytoplankton
(e.g. diatoms) are generally dominant when nutrients are
abundant. Graneli et al. (Graneli et al., 1993) also showed
that the phytoplankton community shifted towards picoplankton in nutrient-poor incubations and, conversely, to
larger nanoplankton in enriched environments.
The ‘bright’ and ‘dim’ strains of Synechococcus observed in
this study are similar to the dual populations observed in
studies of the north Atlantic and Pacific Oceans (Olson
et al., 1990) and the Arabian Sea (Campbell et al., 1998). One
possible explanation for the existence of ‘bright’ Synechococcus
in the Singapore Strait is the less turbid environment, which
is a result of better through-flow, compared with the Johor
Strait. In clearer waters, with less suspended particulate and
dissolved matter, blue light is able to penetrate more deeply.
Consequently, the ‘bright’ strain with a high PUB:PEB ratio
Fig. 9. Concentrations of NO3– plus NO2– and PO43– at stations (i.e.
J1, J3, J4, J8) in the Johor Strait, September 1999. The average
concentrations of NO3– plus NO2– and of PO43– in the Singapore
Strait (SG) are shown for comparison.
may be better able to absorb blue light, and hence outcompete the ‘dim’ strain. In contrast, the non-existence of
‘bright’ Synechococcus in the Johor Strait could be ascribed to
the turbid and productive waters, which are a result of
higher anthropogenic inputs. In addition, a causeway
located mid-way in this narrow strait hinders the mixing
and dilution of its waters. As a result of higher turbidity and
dissolved substances, blue light is likely to be attenuated
rapidly, with a preferential transmission of green-to-yellow
light (Olson et al., 1991). At the same time, larger species
containing pigments that efficiently absorb light in the green
wave band (e.g. diatoms with fucoxanthin) will probably
thrive, which further increases the turbidity of the surface
water. Hence, it is possible that only ‘dim’ Synechococcus with
low PUB:PEB ratios would be able to sustain growth by
absorbing the green light.
Overall, it is assumed that the ratio of ‘bright’ to ‘dim’
Synechococcus strains is inversely correlated with the
eutrophic level of coastal waters, presumably because
blue light attenuation is often concurrent with high
nutrient loads. This assumption is partly supported by
the correlation between total nitrogen concentration and
the ‘bright’:‘dim’ Synechococcus ratio (R2 = 0.8024), indicating the potential use of this ratio as an indicator for
assessing trophic states in coastal regions, which range
from mesotrophic to eutrophic environments.
The extremely high concentration of Synechococcus
observed at station J8 in September 1999 (as shown by
both HPLC and flow cytometric analyses) coincided
with a higher N:P nutrient ratio compared to other
stations. Nutrient analysis revealed that at station J8,
inorganic phosphate was almost undetectable, but nitrite
plus nitrate was at average levels (3.5 mM). Consequently,
the N:P ratio was as high as 21, whilst that of other
stations ranged from 5 to 8.9 (Figure 9). This suggested
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that the growth of the larger eukaryotic cells, e.g.
diatoms, which are able to thrive in nutrient-replete
waters, could have been hindered when phosphate
was depleted (Donald et al., 1997). In contrast, the
phosphate-uptake rate of Synechococcus can be markedly
enhanced as a result of the involvement of a periplasmic
phosphate-binding protein (Ikeya et al., 1997). Rapid cell
growth is thus possible for Synechococcus even if phosphate
concentrations are extremely low, as long as nitrogen is
not a limiting factor. This physiological characteristic
may be one of the reasons for the wide distribution of
Synechococcus in oligotrophic environments.
ACKNOWLEDGEMENTS
This study was funded by the National University of
Singapore and the National Science and Technology
Board, Singapore. Vessel support for the research cruises
was provided by the Maritime and Port Authority of
Singapore.
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Received on November 6, 2002; accepted on September 15, 2003
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