Variable response of two tropical phytoplankton species at different salinity and nutrient condition

Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

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Journal of Experimental Marine Biology and Ecology

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Variable response of two tropical phytoplankton species at different salinity andnutrient condition

D. Ganguly ⁎, R.S. Robin, Kanuri Vishnu Vardhan, Pradipta R. Muduli 1, K.R. Abhilash,Sivaji Patra, B.R. SubramanianICMAM Project Directorate, Ministry of Earth Sciences, NIOT Campus, Pallikaranai, Chennai 600 100, India

⁎ Corresponding author. Tel.: +91 9840330227; fax:E-mail address: [email protected] (D.

1 Wetland Research and Training Centre, Chilika DevOdisha 752 030, India.

0022-0981/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.jembe.2013.01.008

a b s t r a c t

Article history:Received 24 March 2012Received in revised form 13 August 2012Accepted 7 January 2013Available online 1 February 2013

Keywords:CarbonChlorophyll aGrowth rateNutrient limitationPhytoplanktonSalinity

The responses of two tropical phytoplankton species (one diatom and one cyanobacterium) at various nutrientconcentrations were studied to determine their optimum growth conditions. Optimum salinities for these twospecies were also determined prior to the nutrient limitation study. As a part of ecosystem monitoring at Asia'slargest brackishwater lagoon (Chilika), axenic cultures of Chaetoceros simplex and Anabaena sp. weremaintainedand nutrient concentrations were regulated according to the in situ conditions. At given pre-defined environ-mental conditions (light, nutrient, temperature, salinity etc.) the maximum growth rate of 0.043 h−1 and0.040 h−1 was recorded for C. simplex and Anabaena sp. at N/P ratios of 12 and 8, respectively. Addition ofammonia significantly stimulated the growth of Anabaena sp., whereas C. simplex did not show such significantincrease. Anabaena sp. showed larger positive response towards the enrichment of phosphate–phosphorous(PO4–P) in terms of specific growth rate compared to that of C. simplex. For both the species, relative decreasein available dissolved inorganic nitrogen (DIN) was indicated by an apparent decrease in chlorophyll-b/a ratios.In in vitro conditions chlorophyll to carbon ratio increases with increasing N concentration, and was found to behigher for C. simplex relative to Anabaena sp. The results suggested that Anabaena sp. is the better competitor atlow nutrient availability and has higher adaptation ability than C. simplex.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Phytoplankton are the major food source for many aquatic organ-isms and the main live feed component for the marine food web. Theability of phytoplankton cells to synthesize new biomass is criticallydepending on their ability to assimilate sufficient carbon, nitrogen andphosphorous, as well as minor nutrients, to ensure replication. Besidesthe essential nutrients, salinity is also an important factor affecting phy-toplankton communities in coastal aquatic ecosystems. Freshwater aswell as marine species suffer severe osmotic stresses (Lobban andHarrison, 1994) even at a salinity change of ~5 psu. This salinity levelforms a lethal barrier for most estuarine planktonic algae (Kies, 1997)even at the nutrient replete conditions. Some phytoplankton areknown to be osmotically adaptive and perform little actual work inadjusting to the external environment, while others are more sensitiveto osmotic changes (Fu and Bell, 2003). From the ecosystem healthpoint of view, therefore it is useful to study the response of somemajor species to the changes in salinity condition.

Among the essential nutrients P has been considered to be the prima-ry nutrient limiting phytoplankton growth in freshwater ecosystems,

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andmanagement efforts have generally focused on controlling P loading(Havens and Walker, 2002). However, other studies suggest that nitro-gen (N) limitation and co-limitation by N and P occur in fresh watermore commonly than previously thought (Maberly et al., 2002).Co-limitation by N and P has commonly been observed in lakes andreservoirs throughout the world, highlighting the importance of both Nand P in regulating freshwater algal growth. As a result, managementefforts targeting P inputs alone will not effectively control nuisancealgal blooms and the associated negative impacts of eutrophication(Dzialowski et al., 2005).

Availability of nutrient, especially nitrogen and phosphorus, isfrequently referred as key factor regulating phytoplankton growth,biomass and species composition (Domingues et al., 2011; Roelke etal., 1999). Therefore, the role of these two nutrients as limiting factorsto phytoplankton growth is an important aspect for eutrophicationmitigation and management (Domingues et al., 2011). Knowledgeof how nutrients interact to limit growth is essential in understandingthe causes of variation in phytoplankton stoichiometry (Goldman et al.,1979; Rhee, 1978), the identity of the nutrient (s) limiting biomass andprimary production (Smith, 1982), and the effect of resource competitionon community structure (Tilman, 1982).

This present study was aimed to derive various ecosystem modelcoefficients associated with phytoplankton growth in the tropicalcoastal waters, similar to Chilika lagoon. Earlier the lagoon wasreported as nitrogen limited (with respect to the Redfield ratio)

Table 1Various concentrations of DIN and DIP used for phytoplankton growth study.

DIN DIP N/P ratio

At constant P and varying N (μM)0.5 0.5 11 0.5 22 0.5 44 0.5 86 0.5 128 0.5 16

At constant N and varying P (μM)6 6 16 3 26 1.5 46 0.75 86 0.5 126 0.38 16

245D. Ganguly et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

system on the basis of N/P ratios, in terms of phytoplankton productiv-ity (Panigrahi et al., 2007). Amongst several diatom and Cyanophyceaespecies available in Chilika waters, Chaetoceros simplex and Anabaenasp. were the two important species found almost throughout the yearwith relatively higher abundance (Raman et al., 2009–'10). Size frac-tionated study further showed dominance of pico/nano phytoplankton(75–80%) in total water column chlorophyll in the lagoon. These twoselected species were well inside the size range (b20 μm) and theirgrowth rate was considered as representative of the respective classes.

Variation in temperature and light intensity throughout the yearwas considered to be less compared to that of macronutrients. Bothspatial and temporal variations in nutrient concentrations recorded inthe Chilika Lagoon often determine water column productivity. There-fore it could be useful to determine optimum conditions for primaryproducers persisting in the lagoon in terms of nutrients. Till date veryless information is available on the optimum growth condition withrespect to macro-nutrients for the tropical phytoplankton. Raghavanet al. (2008) made a study on temperature, salinity and CO2 dependentgrowth rate of tropical marine diatom. This present work is aimed tostudy the differential growth rates of two different phytoplanktongroups under various nutrient and salinity conditions that could helpto maintain the water quality of the tropical lagoon. Cellular pigmentcomposition and their relative contents have often been used to de-scribe nutrient limitation in phytoplankton (Schlüter et al., 1997).Henriksen et al. (2002) previously studied minor but significant varia-tion in phytoplankton chlorophyll b/a along with other pigment ratioswith bio-available N/P ratios in batch culture. In this present study theinfluence of varying N and P relative to each other on chlorophyll b/aand c/a ratios was tried to be determined for the two selected speciesto find out any indicative information on nutrient limiting growthunder controlled environmental conditions. Phytoplankton chlorophylla to carbon ratios under different nutrient conditions was also deter-mined to understand and compare chlorophyll a specific carbon assim-ilation by these two species.

2. Materials and methods

As a part of ecosystem modeling at the Chilika lagoon, axenic cul-tures of a diatom species (C. simplex) and a Cyanophyceae species(Anabaena sp.) were maintained and nutrient concentrations wereregulated according to the in situ conditions to derive various modelcoefficients.

2.1. Phytoplankton culture and growth rate

Natural phytoplankton C. simplex (diameter 5 to 9 μm) and Anabaenasp. (diameter 3 to 6 μm) were isolated from tropical water of North EastCoast of India. In axenic algal cultures, precise measurement of thecellular growth rate is possible after steady state is achieved (Hecky andKilham, 1988). Both the isolated axenic cultures were allowed to achievethe steady state at a high density in the laboratory condition at fixedsalinity of 20 and temperature (25 °C). The goal of our experiments wasto evaluate the influence of changes in salinity and then nutrient environ-ment on growth of selected phytoplankton species.

2.2. Experimental design

Prior to start each experiment, culturesweremaintained at the in vitroconditions for an adaptation period of approximately five generations.The cultures were started by inoculating ~10% of the total volume. Thedense culturewas then diluted to a desired population by using sterilizedsynthetic seawater (Harrison et al., 1980) of the same salinity. For estima-tion of specific growth rate at various nutrient conditions both thealgae were allowed to grow in 2 l glass bottle (in triplicate) containing1 l synthetic sea-water medium at 12:12 light:dark incubation period.The light illumination was maintained at 5000 lux. Experiments were

conducted in batch cultures (continued up to 10 days), which weregrown until either late exponential phase or early stationary phase.

All the culture conditions including the nutrient concentrationsweremaintained taking into account of themean environmental condi-tions found at Chilikawhere the salinity varies between a large range of0 and 30 (Rajakumari et al., 2010). Considering this, at first the cultureexperiment was carried out at six different salinities (0, 6, 12, 18, 24,and 30) at constant N/P ratio of 16 (Redfield ratio) (DIN=8 μM asNO3–N, DIP=0.5 μM as PO4–P) and growth rates for both the specieswere determined to obtain optimum condition. Desired salinities wereobtained by diluting synthetic sea water (Harrison et al., 1980) withsterilized ultra-distilled water. Each alternate day the DIN and DIPconcentrations were measured and the initial N/P ratio was maintainedby adding sterilized stock solution of the respective nutrients.

After obtaining the optimum salinity conditions, themacronutrientsconcentrations were maintained considering the annual mean values(b6 and 2 μM for DIN and DIP, respectively) found at Chilika lagoon(Panigrahi et al., 2007, 2009). Inorganic nitrogen concentrations weremaintained between 0.5 and 8 μM (i.e. 0.5, 1, 2, 4, 6, 8 μM) and phos-phate concentrations were maintained between 0.38 and 6 μM withsix sets of different N/P ratios (Table 1). NaNO3, NH4Cl and KH2PO4

were used as the source of nitrate-nitrogen, ammonia-nitrogen andphosphate–phosphorous, respectively. Si was never found to be alimiting nutrient at the Chilika waters (Raman et al., 2009-‘10).Thus, Si was added as silicate (Na2SiO3 9H2O) at a concentration of8 μM (for C. simplex) and the concentration was maintained tillthe end of each experiment by adding autoclaved stock solution ofsodium silicate on each alternate day. Trace element solutions andvitamins were added in equivalent amount that present in F/2(Guillard, 1973) medium. The glassware used for the experimentswas rinsed twice with Milli-Q water and sterilized in an autoclavebefore use.

2.3. Analysis of the sample

Samples were removed aseptically for cell number determinationat intervals of 24 h. Cells were immobilized by the addition of a dropof iodine solution (2 g KI, lg I in 100 ml water) to the sample of cellsuspension. Cell enumeration was made, after suitable dilution,using a compound microscope and Sedgewick–Rafter counting slideor a haemo-cytometer.

Growth rate of phytoplankton is expressed in terms of doublingtime (d) in hours and specific growth constant (μ, h−1) (Guillard,1973).

μ ¼ ln N2=N1ð Þ= t2−t1ð Þ

0

0.01

0.02

0.03

0.04

0.05

0 5 10 15 20 25 30 35

Spec

ific

gro

wth

rat

e (h

-1)

Salinity

C. simplexAnabaena sp.

Fig. 1. Variation of specific growth rate with respect to salinity at N:P=16 and T=25 °C.

0

0.01

0.02

0.03

0.04

0.05

0 2 4 6 8 10 12 14 16 18

Spec

ific

gro

wth

rat

e (h

-1)

N/P ratio

C. simplex

Anabaena sp.

Fig. 2. Variation in specific growth rate at different N:P conditions.

246 D. Ganguly et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

where N2 and N1 are the phytoplankton number produced duringtime t1 and t2.

Subsamples for determining chlorophyll a, b, c and phaeopigments(pp) concentration were collected after every 24 h cycle and weredetermined spectrophotometrically after extraction in 90% acetone.Pigment concentrations in the acetone extracts were then calculatedusing the equations of Parsons et al. (1984).

Duplicate water samples from each culture bottle were collectedon each alternate day for analysis of dissolved inorganic nutrients(NH4

+, NO3− and PO4

3−). Samples were filtered and analyzed for ammo-nium (Solorzano, 1969) and phosphate (Strickland and Parsons, 1968).Sub-samples for nitrate analysis were frozen for later analysis usingspectrophotometric method (Grasshoff et al., 1999).

A linear transform of the Michaelis Menten equation (Eppley et al.,1969):

S ¼ Vm S=vð Þ–Ks;

was used to calculate Ks and Vm and their 95% confidence limits by aregression analysis. In this transform, Ks is given as the Y intercept andVm as the slope of the regression equation of S on (S/v), where S=substrate concentration (nutrient), v=rate of nutrient consumption,Vm=maximum nutrient consumption rate (slope), and Ks=half satura-tion constant (intercept). Phytoplankton organic carbon (de-carbonatedby HCl fumes) content was measured (filtering 20 ml of culture withpre-combusted (at 450° for 4 h) 25 mm Whatman GF/F filter (0.7 μm))by elemental analyzer at each alternate day (Thermo Finnigan, FlashEA1112) (Redalje, 1983). L-Cystine was used as standard and precisionof analysis was checked against NIST 1941b and found to be at ±0.1%.

2.4. Statistical analysis

Analysis of variance (ANOVA) was employed to find any signifi-cant variation in phytoplankton growth rate with respect to salinity.Regression analysis was performed to derive the relative dependenceof phytoplankton Chl-a to carbon ratio on the N/P ratios. All the statis-tical analysis was done by using Minitab 13 software.

3. Results

All the measurements were carried out during the healthy expo-nential growth phase of the phytoplankton life cycle. Abundance ofC. simplex varied between 4.73 and 10.23×104 cells ml−1, whereasfor Anabaena sp., it varied between 3.68 and 8.78×104 cells ml−1

during all the growth rate studies under different salinity and nutri-ent conditions.

3.1. Effect of salinity and nutrients

Significant variations (pb0.001 at 95% confidence limit) in the growthrate of both C. simplex and Anabaena sp. were observed with variation insalinity between the range of 0 and 30. However, variation in maximumspecific growth ratewith respect to salinitywas recorded relatively lesserfor C. simplex than that of Anabaena sp. Highest growth rate for C. simplexwas found at salinity 18, whereas the same for Anabaena sp. was found atthe salinity 6 (Fig. 1). Considering these highest growth rate values, nutri-ent dependent growth experiment for these two species was carried outunder the respective optimum salinity condition of 18 and 6. At thepre-defined physico-chemical conditions (constant temperature, salinity,light intensity etc.), phytoplankton showed variable responses to thevariations in the N and P concentrations and their ratios, in terms oftheir specific growth rates.

3.1.1. C. simplexAmong the entire experimental conditions, the highest specific

growth rate (μmax) of 0.043 h−1 for C. simplex was recorded at N/P of

12 when nitrate was used as the lone source of dissolved inorganicnitrogen (DIN). Decrease of inorganic nitrogen relative to inorganicphosphorous (N limitation w.r.t. the Redfield ratio) caused significantdecrease in their specific growth rate with least values (0.012 h−1)obtained at N/P=1 (Fig. 2 and Table 2). When ammonia was usedinstead of nitrate-nitrogen as the lone source of DIN no significantvariation (ANOVA, p>0.05) was observed in growth rate. The growthpattern followed a similar trend as NO3–N although the maximum spe-cific growth rate was lowered by ~0.006 units (i.e. μmax=0.037 h−1)compared to that in presence of nitrate-nitrogen and was recorded onfirst 24th h of growth with doubling time of 18.5 h. The half saturationconstant (Ks) which is regarded as the affinity of phytoplankton interms of nutrients, for NO3–N uptake and NH3–N uptake were foundto be 0.004 mg N l−1 and 0.012 mg N l−1, respectively. Comparisonbetween the responses of C. simplex in presence of NO3–N and NH3–Nrevealed differential coupling between growth rate and nutrient uptake(Sciandra, 1991) under different nutrient conditions.

At constant N (6 μM) and varying PO4–P concentration (Table 1),the maximum growth rate of 0.038 h−1 with doubling time 18.24 hwas observed at N/P Redfield ratio 12 (6:0.5) on 2nd day of culture.No significant variation (p>0.05, n=12) in C. simplex growth ratewas observed with increase in PO4–P concentration from 0.38 to6 μM. Various phyto-pigments including phaeopigment (pp) weremeasured at the exponential phase of the C. simplex life cycle to deter-mine the influence of nutrient limitation. Chlorophyll a values variedfrom 7.86 to 13.79 mg m−3 in the culture with higher values observedat higher N/P ratios up to 16. Phytoplankton pigment ratios wereconsidered to understand the changes in response to varying nutrientconditions for the two species. With decreasing dissolved N, pigment ad-aptation (especially for Chl-b/a ratios) was prominent for both the twostudied species. For C. simplex the mean b/a, c/a and pp/a ratios were

Table 2Various parameters related to phytoplankton growth during the exponential phase oftwo tropical species.

C. simplexMaximum specific growth rate (h−1) 0.043±0.002Doubling time (h) 22.7Half‐saturation constant for NO3–N uptake 0.004 mg N l−1 (0.286 μm)Half‐saturation constant for NH4–N uptake 0.011 mg N l−1 (0.786 μm)Half‐saturation constant for PO4–P uptake 0.0037 mg P l−1 (0.119 μm)Chl-a:C, mg Chl-a (mg C) −1 0.0212±0.007 (0.0172–0.0284)Chl-b:Chl-a 0.293±0.09Chl-c:Chl-a 0.497±0.36Pheaopigment:Chl-a 0.006±0.001

Anabaena sp.Maximum Specific Growth rate (h−1) 0.040±0.001Doubling time (h) 25Half‐saturation constant for NO3–N uptake 0.0026 mg N l−1 (0.186 μm)Half‐saturation constant for NH4–N uptake 0.002 mg N l−1 (0.143 μm)Half‐saturation constant for PO4–P uptake 0.0014 mg P l−1 (0.045 μm)Chl-a:C, mg Chl-a (mg C) −1 0.0132±0.001 (0.0126–0.0145)Chl-b:Chl-a 0.170±0.09Chl-c:Chl-a 0.021±0.19Pheaopigment:Chl-a 0.006±0.004

247D. Ganguly et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

found to be 0.293±0.09, 0.497±0.36 and 0.006±0.001. Chlorophyll-b/aratio varied from 0.198 to 0.264 during the healthy exponentialgrowth phase. Increase in N/P ratios showed a positive influence onchlorophyll-b/a ratios for C. simplex (Fig. 3). In contrary, no such signifi-cant relation was observed (p>0.05) between N/P and chlorophyll-c/aratios.

3.1.2. Anabaena sp.For Anabaena sp. the maximum growth rate of 0.040 h−1 was

observed when the N/P ratio was 8, during the exponential phase ofgrowth with doubling time of 17 h. An increase of PO4–P from 0.5 to0.66 μM significantly stimulated (pb0.001) the growth of Anabaenasp. Unlike C. simplex, presence of NH4

+–N instead of NO3–N significantlyincreased Anabaena growth rate. In presence of NH3–N the maximumgrowth rate of 0.052 h−1 was observed at N/P=8, on first 48 h ofgrowth with doubling time of 11.06 h.

The half saturation constants (Ks) for NO3–N uptake and NH4+–N

uptake were found to be 0.0026 mg N l−1 and 0.002 mg N l−1,respectively, whereas the same for PO4–P was calculated to be0.0014 mg P l−1. In the present study, the Ks value for NO3–N wasfound higher (0.001 mg N l−1) than that reported for the Cyanobacteria

0.18

0.2

0.22

0.24

0.26

0.28

0 2 4 6 8 10 12 14 16 18

Chl

-b/a

rat

io

N/P

Chaetoceros simplex

Anabaena sp.

Fig. 3. Variation of Chl-b/a ratios with respect to N/P ratio at the exponential growthphase.

at Smith Lake (temperate waters), whereas the same for NH4+–N was

found significantly less than that of the same lake (0.30 mg N l−1 )(Binhe and Alexander, 1993). Lower Ks values for NH4

+–N than NO3–Nimplied that at low concentrations, phytoplankton have a higher affinityfor NH4 and are able to use it more efficiently than its NO3 counterpart(Berman et al., 1984). Chlorophyll a values for Anabaena sp. variedfrom 7.14 to 11.51 mg m−3 in the culture with higher values observedat higher N/P ratios. For Anabaena sp. the mean chlorophyll b/a, c/a andpp/a ratios were found to be 0.170±0.09, 0.021±0.19 and 0.006±0.004, respectively. Like Chaetoceros sp., Anabaena sp. also showed asignificant positive correlation between the N/P and chlorophyll b/aratios (Fig. 3).

3.2. Chlorophyll-a to carbon (C) ratio

In the present experimental conditions, Chl-a:C ratio showedlower values under nitrogen limiting condition (N/Pb16) for boththe species and C. simplex showed greater variations than Anabaenasp. with the changes in N/P ratio. For C. simplex, the Chl-a:C ratios var-ied between 0.0172 and 0.0284 (mean 0.0212 ), whereas the same forAnabaena sp. varied between 0.0129 and 0.0145 (mean 0.0132) underconstant light conditions. Variation of Chl-a to C ratio with respect toN/P values at the exponential growth phase was given in Fig. 4. Earliermeasurements with phytoplankton, grown in various culture showedthat Chl-a:C is highly variable, ranging from 0.003 (F'alkowskpi et al.,1985) to >0.1 mg Chl-a (mg C) −1 (Geider, 1987).

For C. simplex (Eq. (1), r2=0.73, p=0.011, n=18) and Anabaena sp.(Eq. (2), r2=0.76, p=0.014, n=18) the following linear regressionequations were derived that explains the N dependent variation in theChl-a:C.

Log Chl� a : Cð Þ ¼ 0:1593Log N=Pð Þ−1:7847 ð1Þ

Log Chl� a : Cð Þ ¼ 0:0125Log N=Pð Þ−1:885 ð2Þ

For both the species, it was evident that a relative decrease in nitro-gen (w.r.t. P) concentration could cause a decrease in chlorophyll-a tocarbon ratio under the given environmental condition (i.e. fixed lightintensity, temperature etc.).

The relationship between Chl-a to C ratio and the specific growthrate for both the species revealed a cellular level regulation of funda-mental metabolic pathways in response to nutrient limitation. Resultshowed that under N-deficiency, cellular Chl-a increased with μ,while cellular C and volume decreased with μ (Halsey et al., 2010).In present study, Chl-a:C was strongly and linearly correlated with μ(C. simplex; r2=0.838, pb0.001 and Anabaena sp.; r2=0.759, p=

0.000

0.004

0.008

0.012

0.016

0.020

0.024

0.028

0.032

1 2 4 8 12 16

Chl

-a :

C r

atio

N/P ratio

Chaetoceros sp.

Anabaena sp.

Fig. 4. Variation of Chl-a to C ratio with respect to N/P ratio at the exponential growthphase.

248 D. Ganguly et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

0.009)), with a regression-based intercept of 0.012 for both thespecies.

4. Discussion

The present study has demonstrated that both salinity and nutri-ent are very important parameters in controlling the growth of thetwo studied algal species. C. simplex showed higher growth rate to-ward higher salinity compare to Anabaena sp. Growth of C. simplexwas found to be relatively less dependent on phosphorous concentra-tion than that of Anabaena sp. at 25 °C temperature and respectiveoptimum salinity. Similar observations with relatively more probabil-ity of N-limitation than P-limitation were reported for Chaetocerosgracilis from the temperate water (western Mediterranean) by Kutluet al. (2012).

At this defined nutrient conditions C. simplex found to be moreproductive (in terms of specific growth rate) than Anabaena sp.Under these environmental conditions the half saturation constants(Ks) for both NO3–N and NH4–Nwere found to be higher for C. simplexcompared to that of Anabaena sp. The Ks values for C. simplex werefound to be well comparable with the values reported for the marinediatom by Sunlu and Baha (2006) (NO3–N=0.007 mg N l−1 andNH4–N=0.012 mg N l−1) and Eppley et al. (1969) (NO3–N=0.004 mg N l−1 and NH4–N=0.01 mg N l−1) but relatively lesserthan that reported by Sunlu et al. (2010) for C. gracilis at the optimumtemperature (28 °C). These results depicted that under nutrientstressed condition C. simplex will be limited by dissolved inorganicnitrogen earlier than Anabaena sp. KNO3 for Anabaena sp. was greaterthanKNH3, whereas for C. simplex this trendwas reversed. This indicatedthat the former species have more preference for NH4–N relative toNO3–N, whereas for the latter, it was reverse.

ANOVA revealed that in presence of NO3–N as the lone source ofDIN, specific growth rate of C. simplex was little but significantlyhigher than Anabaena sp.(pb0.05, n=17). This observation deviatedfrom the normal concept that fast-growing species tend to havelower Ks, values than slow growers. This anomaly could be explainedby the differential rates of carbon (including other nutrients) assimi-lation with respect to phytoplankton chlorophyll-a. Presence ofdissolved NH4–N in water stimulated the growth of Anabaena sp.compared to that of C. simplex. On the other hand, higher Ks valuesof PO4–P uptake for C. simplex than that of Anabaena sp. indicatedthat the latter will be less easily limited by the low concentration ofPO4–P than the former.

Between C. simplex and Anabaena sp., the former showed approxi-mately 1.6 times higher Chl-a:C values than that of the latter. This resultindicated higher degree of carbon assimilation by Anabaena sp. per unitweight of chlorophyll-a (Chl-a specific carbon assimilation) comparedto that of C. simplex. In contrary, nutrient uptake rate for Anabaena sp.was found lower (lower Ks value) than C. simplex, which could be attrib-uted to the species specific elemental stoichiometry of C:N and C:P atthe maximum growth rate (Klausmeier and Litchman, 2004). Thestrong positive relationship between Chl-a:C ratios and N/P ratioswere well in agreement with earlier in situ observation by Finenko etal. (2003). Li et al. (2010) reported an increase in the Chl-a:C ratiodue to acclimation to low light and high nutrients from in situ observa-tion at California coastal and pelagic ecosystems. Arin et al. (2002)reported similar observation with lower Chl-a:C ratio under nutrientpoor condition and explained this by reporting a decrease in phyto-plankton cell size. Although the increase or decrease in cell size of thephytoplanktonwas not examined in the present study, relatively higherC concentration (with respect to Chl-a) under low nutrient concentra-tion was evident compared to that of nutrient richer condition. Thepresent results also support the fact that the relative chlorophyll a con-tent increases with a decrease in nutrient limitation (Geider, 1993).Under nutrient depleting conditions the cells could decrease metabolicactivities while photosynthesis (i.e. carbon assimilation) continues.

Over consumption of dissolved inorganic carbon under low nitrate con-centration could also be responsible for elevated carbon concentrationin the phytoplankton cell relative to N. In N limiting conditions, increasein carbohydrate concentration inside the diatom cells was reported byThomas et al. (1999).

The intra-species variability in observed Chl-a:C ratio includes adap-tive responses to ambient light, temperature, and nutrient conditions.Although Chl-a:C is a sensitive indicator of algal physiological conditionand growth rate, there is no unique relationship between growth rateand Chl-a:C. For example, Laws and Bannister (1980) demonstrat-ed that different functional relationships exist between Chl-a:Cand growth rate (y), depending on whether phytoplankton aregrown under conditions of nutrient limitation (Chl-a:C increaseswith y) or light limitation (Chl-a:C decreases with y). Hein andRiemann (1995) earlier reported a constant specific communitygrowth rate (chlorophyll a specific), irrespective of marked in-creases in the carbon biomass.

Although at low nutrient conditions reduced growth rate wasrecorded in the present study, the chlorophyll specific C assimilationrate (inverse of Chl-a to C ratio) was relatively high compared to a richernutrient environment. Our observations were closely resembling withthe work by Perry (1976) which emphasized the relation between theinfluence of nutrient deficiency on the one hand and decreasing growthrate and increasing C:Chl-a ratio on the other hand. Thus an increasingratio level could indicate a decreasing growth rate due to any kind ofnutrient limitation (Jonge, 1980).

The Chl-a:C ratio could be an important variable in the model ofmicroalgae growth as it provides a link between phytoplankton growthand chlorophyll a specific photosynthesis rate (Finenko et al., 2003;Shuter, 1979). The developed equations in the present study, depictingthe relationship between Chl-a:C and dissolved inorganic N/P ratioscould be useful to model the phytoplankton carbon biomass underdifferent nutrient conditions in the tropical waters.

Higher values of chlorophyll b/a ratio coincided with higherphytoplankton growth rate corresponding to a higher production byboth C. simplex and Anabaena sp. The results indicated that cell Chl-aand Chl-b content decreased under N deficiency but the loss of Chl-bwas more pronounced than that of Chl-a in both the cases. This resultwas very similar to that of Jiménez and Niell (2003) depicting a de-crease in pigment content and a looser packing of the pigmentswith de-crease in dissolved inorganic N concentrations which in turn led to anincrease of the effective rate of light harvesting per unit chlorophyll ain Dunaliella viridis. These observations were very similar to thatreported for marine phytoplankton by Henriksen et al. (2002).

Chl-b/a ratios were found higher for Anabaena sp. at lower N/P thanC. simplex, whereas this relationship was reversed at higher N/P ratios(Fig. 3). This result demonstrated that depletion in N availability cancause significant decrease in Chl-a in Anabaena sp. relative to C. simplex.This also indicates that both enrichment and depletion of N have con-siderably higher impact on Chl-b/a ratios in C. simplex compare toAnabaena sp.

5. Conclusion

Specific growth rate of both the species varied significantly with thesalinity and Anabaena sp. showed poorer adaptability than C. simplexwith salinity change. These two species, representing two differentgroups, have differing values for both μmax and Ks and the competitiveadvantage shifts between the species, as the nutrient concentrationchanges. At lower nutrient concentrations, Anabaena sp. will dominate,as it grows faster; but at higher nutrient concentrations C. simplex be-comes dominant, as it achieves a higher maximum rate of growth. De-rived information on μmax, Ks and Chl-a:C ratio could be useful tomodel the coastal water ecosystems in the tropical environment. Signif-icant positive correlation between N/P ratio and chlorophyll-b/a ratio

249D. Ganguly et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 244–249

could also be suitable as a tracer of anynutrient limitation for both thesetwo tropical species.

Acknowledgment

The authors are grateful for Dr. Shailesh Nayak, Secretary, Ministryof Earth Sciences, Government of India for continuous encouragement.This study is a part of MoES–ICMAM's comprehensive project “Ecosys-tem Modelling for Chilika Lagoon.” [RH]

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