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A comparative study of phytoplankton physiological indicators
J. Exp. Mar. Biol. Ecol., 158 (1992) 149-165 0 1992 Elsevier Science Publishers BV. All rights reserved 0022-0981/92/$05.00
149
JEMBE 01758
A comparative study of phytoplankton indicators
physiological
Iosu de Madariaga and Ian Joint NaturalEnvironmentalResearch Council, PlymouthMarine Laboratory, The Hoe, Plymouth, UK (Received 6 December
1991; accepted 9 January
1992)
Abstract: Pavlovalutheri(Droop) Green was grown in chemostat culture; the three different N sources used were ammonium, nitrate or glycine and the cultures were also grown under N and P limitation. A number of biochemical and physiological measurements were made on these cultures growing at high and low dilution rates. Elemental composition qf the cells remained relatively constant, C content increasing only under conditions of phosphate limitation, while N and P content decreased under their respective limiting conditions. DNA content showed little variation but RNA content declined significantly under both N and P limitation. The largest effect of nutrient limitation to the biochemical parameters measured were to carbohydrate and lipid concentrations which increased significantly. Amino acid concentrations increased more than six times when the cultures were phosphate-starved. Dilution rate, ,ss well as nutrient status, affected the physiological measurements; assimilation numbers were highest at hi;h dilution rates and were reduced under phosphate limitation and when glycine was the sole N source. Photosynthetic efficiency was similar for all growth conditions, except N limitation when values increased. Under both N and phosphate limitation, the proportion of i4C label in low molecular weight intermediates increased, with a decrease of the label in protein. There were significant variations in Chl a, c,c2 and chlorop:iyllide a under nutrient limitation but carotenoid pigm&..ts showed less variation. A multiple intercompariron was carried out by principal component analysis, which indicated that the biochemical composition measurements tended to be related to the growth-limiting factor, whereas the physiological measurements zere related to growth rate. Key words: Biochemical composition; Photosynthetic parameter
Chemostats;
C metabolism;
Nutrient
limitation;
Pavlova lutheri;
INTRODUCTION
A wide range of Gfferent algal properties have been used to estimate the physiological state of phytoplankton. Among them, cell activities, biochemical composition and morphological characteristics have proved to be useful physiological indicators, even when species-specific variability is considered (Zevenboom, 1986). The most commonly investigated physiological processes are photosynthesis, C and N metabolism, nutrient uptake and enzymatic activities (Platt, 198 1); biochemical studies generally include determinations of the elemental, macromolecular and pigment composition (Sakshaug et al., 1983; Roy, 1988). Visual indicators are strictly qualitatiqle and time-consuming Correspondence address: 1. de Madariaga, Ekologi Laborategia, Landare-Biologia Euskal Herriko Unibertsitatea, 644 PK, 48080 Bilbao, Spain.
eta Ekologia Saila,
150
I. DE MADARIAGA AND I. JOINT
and, consequently, they have not been applied widely in research on natural populations. However, these two classes of measurement, physiological and biochemical, are indicators of processes with different time scales. As discussed by Morris (198 l), physiological measurements of cellular activity must be interpreted with respect to the immediate cell environment, and they will indicate potential future changes in cell biochemical composition. In contrast, the actual biochemical composition reflects the physiological history of the cell. Since physiological properties of phytoplankton are influenced by both prevailing and former variations in the conditions for growth, no Gonstant relationships can be assumed a priori, unless steady-state conditions are achieved for an appreciable period of time. Continuous culture techniques offer considerable advantages in those studies which seek to establish the physiological status of algal cells because the cultures are at steady state, growing under controlled environmental conditions (Rhee, 1979). It is to be hoped that data obtained under these constant conditions should aid interpretation of physiological and biochemical measurements made on heterogeneous natural phytoplankton assemblages, which are growing at dirTerent rates. In this work, a comparative study of a number of indicators for the physiological state of phytoplankton was carried out using continuous cultures of the haptophyte Puvlova (formerly Monochqm’s) lutheri (Droop) Green. The aim of the research was to apply those techniques, which are commonly used in studies of natural assemblages, to laboratory cultures grown under defined conditions; continuous cultures were chosen to provide phytoplankton with unifom physiological condition, and were not used to investigate any theoretical aspects of P. hrtheri growth dynamics, such as the work of Droop et al. (1982) and Chalup & Laws (1990). Few attempts have so far been made to systematise those indicators which are normally used with natural assemblages and algal cultures. The rather scattered evidence published to date indicates that some parameters reflect mainly changes in the nutritional state of phytoplankton, i.e. its growth in relative terms, whereas other indicators are related to the growth rate in absolute terms, since all sources of variation are reflected in them (Sakshaug, 1980). The aim of this study is to attempt to establish quantitative relationships between general intrinsic algal properties and growth rate, as well as distinguishing those features that are characteristic of different types of growth limitation.
METHODS CHEMOSTATS P. lutheri was grown
in unialgal continuous culture using axenic techniques described by Wymer & Thake (1980). The clone (CCAP 93111) was obtained from the NERC Culture Collection, Oban, Scotland, UK. A bank of three, replicate, 2 1 chemostats was used throughout the study. Mixing was accomplished by the combination of aeration and magnetic bar stirring. Temperature was maintained at 20 k 0.5 “C and the con-
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151
tinuous incident radiation from four fluorescent tubes (Thorn EM1 Kolor-rite 75W) was 150~E*m-2s-1 at the surface of each culture (measured with a Crump quantum radiometer photometer model 550). The growth medium used was S88 (Turrer, 1979), modified in some cases so that N or P concentrations were limiting. Samples were withdrawn from the cultures every 2 days, and cell numbers were estimated either with a Coulter multisizer, with 50-pm orifice tube for routine counting, or with an improved Neubauer haemocytometer for accurate determination of cell density. In our experiments, steady state was considered to be attained when cell numbers remained constant for 5 consecutive days. 10 triplicate steady states, grouped into five series, were achieved in this study. Luring the first and second series, nutrient-suficient conditions (N : P = 17) were maintained using either ammonium (AS) or nitrate (NS) as N sources. Turner (1979) has shown that glycine is effective as sole N source for the growth of P. lutheri and for the third series (GS), all N sources except i;lycine were removed from the growth medium. In the fourth and fifth series, the cultures were either N-limited (NL) or P-limited (PL). Extreme N : P supply ratios (0.1 and 495, respectively) were kept to ensure severe nutrient limitation. Nutrient levels were measured with a Technicon autoanalyser, following the Strickland & Parsons methods (1972). Chemostats were run at high (H = 0.44 d - * ) and low (L = 0.10 d - ’ ) dilution rates; at each steady state, samples were taken for the different analyses described below and cell numbers and light levels within the culture chambers were determined. l
BIOCHEMICAL
l
ANALYSES
At each steady state, lo-20-ml samples were filtered onto 25.mm pre-ashed GF/F Whatman filters and stored at c - 20 “C until biochemical analyses were performed. Chl CIwas measured in 90% acetone extracts on a Perkin-Elmer spectrophotometer, according to the method described by Strickland & Parsons (1972). Total C and N were determined with a Carlo-Erba elemental analyser, using acetanilide as standard. For lotal P, the persulphate oxidation method of Valderrana (198 1) was used. Proteins and amino acids were analysed following the Clayton et al. method (1988); the procedure involved initial homogenisation in a TCA solution, followed by centrifugation to separate protein and free amino acid fractiors. Proteins were then analysed by a modification of the Lowry et al. procedure ( 1951) ,\nd amino acids by a fluorescamine procedure. Total carbohydrate was determined by ;he phenol-sulphuric acid inethod (Kochert, 1978) and total lipids by the phophosulphovanillin method (Barnes & Blackstock, 1973). Measurements of nucleic acids were made according to Berdalet & Dortch ( 1991‘1;si+ies were homogenized in Tris - Ca2 + buffer and total RNA and DNA were de$ermined by using two fluorochromes: Hoechst 33258, which specifically reacts with DNA, and Thiazole Orange, which allowed total nucleic acid estimation. RNA was estimated from the difference between these two measurements.
1.DE MADARIAGA
152 PHYSIOLOGICAL
AND 1.JOINT
ANALYSES
Samples from the chemostats were diluted lo-fold for the determination of primary production, and the incorporation patterns of C into different cell constituents. Samples were incubated in 60-ml tissue-culture bottles with 370 kBq (10 FCi) NaH 14C03for 4 h, maintained under the same light and temperature conditions as the chemostats. Photosynthesis-irradiance parameters were also measured on lo-fold dilutions from the chemostat and followed the procedures described by Joint & Pomroy (1986). Samples were removed from the chemostat, diluted and incubated for 4-5 h in a light gradient, cooled with circrilating water at 20 OC; the incubation period was kept as short as practicable to reduce the photoadaptation which would occur in the light gradient. All incubations were terminated by gentle filtration through l-pm Nuclepore membrane litters, which were dried and counted in a liquid scintillation counter. The distribution of 14C in low molecular weight metabolites (LMW), lipids, polysaccharides and proteins was determined using the cellular fractionation procedure of Li et al. (1980), as described in detail by Madariaga & Fernandez (1990). Recovery values of 91-107:;, (11= 30) were found when the sum of the radioactivity in all fractions was compared with unfractionated samples. PIGMENT
ANALYSIS
Sample litters were extracted in 2-6 ml 900/, acetone using sonication and further grinding by hand using a glass rod. Extracts were centrifuged to remove filter and cell residues and a 50-~1 aliquot mixed with 50 ~1 ammonium acetate was injected into a Shimadzu HPLC system with a 3-pm Percosphere column. Pigments were separated by a modification of the reverse phase method of Mantoura & Llewellyn (1983) as detailed by Barlow et al. (1991). Chloropl~ylls and carotenoids were detected spectrophoton~ctrically at 440 nm. whereas dctcction of phacopigments was done fluorometricatty with excitation set at 400 nm and emission > 600 nm. The output was monitored on a Dell PC, where peak areas and pigment concentration calculations were performed using the Philips PU6000 software. Pigment identification and quantification for the HPLC system used here was previously established by Barlow et al. (1991). STATISTICAL
ANALYSES
All statistical analyses were performed on a IBM 9370 computer using various SAS procedures (SAS institute, 1985). RESULTS BIOCHEMICAL
CtiMPOSITION
The results of the biochemical analyses are >hown in Figs. 1 and 2 and the data and statistics given in Table I. The C content cell - ’ (Fig. 1) remained relatively constant, l
PHYTOPLANKTON
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153
25
T- 20 z x
15
10
5
0 Carbon
25
Nitrogen
Protein
Amino
Lipid
Acids
LOW
Dilution Rate T= 20 $ x 15
II
10
5
0 Carbon
Nitrogen
ei
Amino Acids
Lipi
Fig. 1. C, N, protein, amino acid and lipid composition (pg acell - I ) of P. lutherigrown at high dilution rate (H) and low dilution rate (15.)in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (GS); results are also shown for N-limited (NL) and phosphate-limited (PL) cultures.
I. DE MADARIAGA
154
AND I. JOINT
1
0
Chlorophyll
5
Phosphorus
RNA
DNA
Carbohydrate
Phosphorus
RNA
DNA
Carbohydrate
Low Dilution
&_4 b E
3
2
1
0 Chlorophyll
Fig. 2. Chlorophyll, P, RNA, DNA and carbohydrate composition (pg - cell - ’ ) of P. lutheri grown at high dilution rate (H) and low dilution rate (L) in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (C S); results are also shown for N-limited (NL) and phosphate-limited (PL) cultures.
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TABLE I
Elemental and biochemical composition of P. lutherigrown at high dilution rate (H) and low di!ution rate (L) in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (GS); results are also shown for N limited (NL) and phosphate-limited (PL) cultures. Data are pg - cell - ’ and values in parentheses are f 1 SE. Biochemical parameter
Dilution rate
AS
NS
GS
NL
0. I68 (0.003) 0. I05 (0.004) 10.965 (0.161) IO.176 (0.622) I.991 (0.041) I.532 (0.122) 0.180 (0.005) 0.202 (0.038) 12.508 (0.463) 10.550 (0.559) 3.187 (0.357) 2.121 (0.336) 3.043 (0.058) 3.547 (0.303) 7.715 (0.863) 5.430 (0.614) 0.164 (0.013) 0.166 (0.015) I .074 (0.235) 0.639 (0.052)
0.076 (0.004) 0.078 (0.004) 10.420 (0.763) 12.332 (0.192) 0.714 (0.010) 0.838 (0.058) 0.253 (0.043) 0. I I6 (0.020) 8.831 (1.133) 7.588 (0.081) 0.532 (0.130) 0.5 19 (0.049) 4.3 10 (0.207) 4.182 (0.316) 19.141 (1.162) 19.460 (2.316) 0.150 (0.015) 0.120 (0.008) 0.2 I I (0.020) 0.128 (0.012)
PL
-Chlorophyll
H L
C
H L
H L
P
H L
H L
Amillo acids
H
Carbohydrate
H
Lipid
H
DNA
H
RNA
x
L
L L I
L
0.23 I (0.037) 0.2 I4 (0.025) 10.075 (0 151) 9.918 (11.630) 2.089 (0.156) I.601 (!I.OlI) 0.06 I (0.004) 0.066 (0.002) IO.523 (0.010) 10.922 (0.137) 3.275 (0.050) I .794 (0.075) I .777 (0.008) 2.398 (0.480) 4.44 I (0.468) 5.576 (0.459) 0. I53 (0.004) 0.162 (0.009) I.190 (0.109) I .068 (0.022)
0. I52 O.l90 X.81I 8.382 I .706 I .353 0. I I4 0.072 9.915 8. I54 I .460 1.076 1.736 1.522 10.493 5.429 0.149 0. I28 I.123 0.952
(0.002) (0.013) (0.128) (0.287) (0.024) (0.033) (0.002) (0.00I ) (0.256) (0.570) (0.034) (0.167) (0.006) (0.138) (0.455) (0.725) (0.005) (0.004) (0.048) (0.076)
0.088 0.072 16.066 15.476 4.096 3.47 I 0.014 0.006 9.3 13 7.780 2 I .706 16.777 4.837 4.668 19.460 17.678 0.127 0.126 0.179 0.163
(0.002) (0.001) ( 1.330) (0.448) (0.401) (0.060) (0.002) (0.004) (0.570) (0.166) (0.922) (2.302) (0.109) (0.172) (1.564) ( 2 950) (0.036) (0.009) (0.048) (0.012)
increasing only under phosphate limitation; similarly, N content was constant in cells grown with excess of each of the three N sources, but it decreased under N limitation and doubled under phosphate limitation. Protein cell - ’ showed relatively small changes in concentration but there were much more significant variations in amino acid concentration, wV:h increased over six-fold when the cullrlres were P-limited. Lipid concentration showed similar responses to both N and P limitation and increased by more than three times. The RNA : DNA ratio decreased under both N and phosphate limitation, as a result of the decline in RNA content cell- ’ (Fig. 2). In contrast, DNA concentration remained relatively constant. The polysaccharide content increased under N and phosphate limitation, but was also significantly higher in the cultures grown with glycine as sole N source. Chlorophyll content, as determined spectrophotometrically (Table I), was very sensitive to nutrient limitation, showing maxima under nutrient-sufficient conditions, i.e., when light was limiting due to the high attenuation caused by selfshading. P content increased when glycine was the N source and under N limitation, but was greatly reduced under phosphate 1mitation. C : P ratios (by atoms) of > 2000 and C : N ratios of > 15 were found under P and N starvation, respectively. l
l
I. DE MADARIAGA AND I. JOINT
156
Generally, there were very few differences between the results obtained with cultures growing at high or low dilution rates. The largest difference was an increase in the lipid content of P. hrtheri growing at high dilution rate with nitrate and glycine as N source. GROWTH
AND
PHOTOSYNTHESIS
Under nutrient-sufficient conditions, C-specific growth rates were significantly higher than steady-state growth rates (Table II); this has been reported previously for P. lutheri (Li & Goldman, 198l)? and has been related to disturbances of the steady state when samples are removed from the chemostat for determination of primary production. In our experiments, self-shading (and consequently light limitation) was significantly reduced when samples were diluted, growth conditions being thus optimized during the incubations. Growth was consistently lower under low dilution rates, but nutrient limitation had a more marked effect. 14Cfixation rates, determined at irradiance values equivalent to those at the surface of the chemostat cultures, and normalized to Chl a (P:B) was generally high, and paralleled changes in growth. TABLE
II
Physiological parameters of P. lutheri obtained with chemostat cultures at high dilution rate (H) and low dilution rate (L); tic C-specific growth rate (h - i), P/B production normalized to chlorophyll [PgC- (pg chl)- ’ h - ‘1, Pfl maximum chlorophyll-specific rate of i4C fixation [PgC (pg chI)- ’ -h- I], aB photosynthetic efftciency [PgC (pg chl) h - i/pE m - 2. s - ‘1, percentage of i4C incorporation into low molecular weight metabolites ( y0 LMW), lipids (“/, lipid), proteins (Y, protein) and polysaccharides ( y-,carb). Nutrient conditions in chemostats are same as in Table I and values in parentheses are + I SE. l
l
l
AS
NS
_.____-I-_-_---H
0. I40 (0.028)
L
0. I I6 (0.035)
H L
p!,
H L H L
“,, LMW
H L
u,, lipid
H
“,, protein
H
‘,, Garb
H
L L L
7.320 (0.90 I )
5.329 5. I32 3.599 0.035 0.02 I 16.25 24.16 21.15 17.98 5 1.37 49.22 Il.23 8.64
(0.X30) (0.406) (0.638) (0.004) (0.004) (2.58) (3.68) (3.48) (1.27) (4.50) (5.90) (1.83) (0.95)
GS
--P_
(0.000) 0.I 2’1 (0.000) x.22I (0.006) 0. I 32
6.258 6.670 4.815 0.026 0.024 17.27 15.57 22.24 29.55 49.83 37.92 10.66 16.96
0.059 0.038 3.940 ( I mO74) 3.245 (0.449) 3.225 (0.621) 2.506 (0.002) 0.02 I (0.002) 0.027 (0.18) 20.47 (2.41) 31.00 (2.40) 33.15 (2.94) 14.17 (3.56) 22.73 (0.29) 27.90 (0.97) 23.65 (0.25) 26.93
NL
PL
--
(0.004) (0.003 ) (0.214) (0.394) (0.176) (0.055) (0.002) (0.001j (0.71) (2.67) (0.88) (1.63) (3.10) (4.45) (2.66) (3.48)
0.037 0.020 5.184 3.272 6.069 3.469 0.062 0.052 33.41 40.83 34.64 17.01 18.45 12.62 13.50 29.54
(0.002) (0.003) (0.254) (0.485) (0.459) (0.465) (0.007) (0.007) (1.50) ( I .08) (2.77) (1.24) (2.41) (0.56) (1.41) (1.87)
0.016 0.009 2.853 2.047 3.556 3.536 0.017 0.020 31.31 36.87 31.55 38.47 26.76 17.81 10.38 6.85
(0.003) (0.00 I ) (0.385) (0.280) (0.997) (0.106) (0.005) (0.004) (0.94) ( I .24) (0.59) (0.97) (0.51) (0.94) (0.20) (0.33)
Fig. 3 shows typical examples of the curves fitting to the data for the photosynthesisirradiance relationship under different nutrient conditions. Maximum specific photosynthetic rate (Pf,) was highest in the culture with high dilution rate and were reduced
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0
400
PHYSIOLOGICAL
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157
4
600
1200
1600
PLH
Fig. 3. Typical photosynthesis-irradiance (P-I) curves of P. lutheri for different nutritional conditions: NSH, N-sufficient culture, growing at high dilution rate; NLH, N-limited, growing at high dilution rate; PLH, P-limited, growing at high dilution rate.
under phosphate limitation; values were significantly lower at both dilution rates when glycine was the sole N source. Photosynthetic eficiency (orB)estimates were generally similar for all growth conditions except N limitation, when higher values were found. 111some cases, P:B estimates appear to be higher than PK; however, the differences are no? statistically significant and, in any case, the two parameters were determined under different experimental conditions. The caveat applies that the physiological state of the
C : Chl a ratio
C : N ratio C : P ratio Carbohydrate : Protein ratio Lipid : Protein ratio Amino acid : Protein ratio
RNA : DNA ratio C-specific growth rate Production/Biomass Maximum chlorophyll-specific “C fixation rate Photosynthetic efficiency Protein :toLMW synthesis storage productsratiosynthesis ratio
Chlorophyllide a : Chl a ratio Chl c,cz : Chl a ratio Fucoxanthin : Chl a ratio Diadinoxanthin : Chl a ratio /?-Carotene : Chl a ratio Dilution rate Light within chemostats
C:N C:P @a: Pr Li : Pr Aa: Pr
R:D k PIB p: xB pr/lmw pr/l + s
Chide ChlC Fuc Diad B car Dr Light
Definition
(h-l) (pE.m-2*s-‘)
(li- ‘) [j.q$(pg Chl) - ’ - h - ’ ] [(pgChl)-r-h-r] [pgC(pg Chl)-‘ah-r/pE
Units
m-2.s-‘]
0.013 0.29 1 0.857 0.858 0.550 0.075 0.092
- 0.122 - 0.696 C.380 0.269 0.699 - ii.158 0.812
0.849 - 0.846 -0.115 0.028 - 0.814
0.367 0.370 0.85 1 0,808 0.433
0.006 0.069 0.273 0.403 0.810 -0.088 -0.147
- 0.33 1
0.895
0.908 0.934 0.743 0.374 0.285 - 0.795 0.919
-
PC2
coefficient
PC1
Correlation
and culture conditions included in principal component analysis (PCA) matrix and their respective units, and correlation coeffkients obtained with first two components in analysis.
C:Chl
Parameter
Physiological parameters
TABLE IV
5
Fj
g 2 u
5 Fj
g
S
2 0 z
5
2 =s 0 p
. DE MADARIAGA
162
AND I. JOINT
C-specific growth rates (& were significantly different to steady-state growth rates (p) pc generally exceeding p by a factor between 1 and 20, depending on the growth conditions. The divergence appeared to increase with increasing Pi, and could be related to methodological limitations and perturbations from steady state, as discussed in Results. However, similar data have been related by Reynolds et al. (1985) to physiological voiding of excess C by respiration or excretion processes. These authors also showed that loss rates varied in direct proportion to pc. The growth rate dependence of respiration is well-known (Langdon, 1988) and, as discussed above, it can be explained by the onset of photosynthetic N assimilation into protein under nutrientsufficient conditions, which stimulates respiratory C flow (Turpin, 1991). Thus, C fixation rates should be viewed as a measure of capacity for cellular increase (Talling, 1984) rather than actual growth. Similarly, calculated C-specific growth rates should be considered as potential maximum growth rates which, owing to other limitations upon growth, are seldom reached (Reynolds et al., 1985). In an attempt to summarize the results obtained in this study, a multiple intercomparison of the various physiological indicators (Table IV) was carried out by performing a principal component analysis. The projection of 10 steady states on the plane of the two axes (Fig. 5) accounted for 70% of the variability, 46 and 24% for the
NbH N:‘
1
,
NStJ
NzL
C&L 1
2
3
4
’ 5
PC2
-1
-2
-3
-4
P:H PLL
-5
Fig. 5. Representation of first (PCl) and second (PC2) principal components determined by principal component analysis of steady-state culture conditions described in Table IV; error bars are + SE.
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163
first and second principal components. PC 1 was controlled by growth indicators, while PC2 indicated N/P limitation (Table IV). Significant correlations with the second axis (PC2) and elemental composition ratios, carotenoids to Chl F ratios, an, and the amino acid to protein ratio indicate that these biochemical indicators provide qualitative information on the growth-limiting factor, rather than the actual growth rate. Therefore, it is important to consider this source of variation when establishing quantitative relationships between physiological estimators and growth rate, because, in some cases, these relationships are valid only under a particular kind of limitation, and they must be used with caution in field work.
TABLE V
Regression equations of growth rate (p,-) and physiological indicators. Model types: 1, linear (y = a + bx); 2, exponential (;, = aehV). Significant at P < 0.0001 Independent variable (X )
Model
Parameters a (+2SE)
C:Chl Ca: Pr Li : Pr P/B RNA : DNA Pr/LM W Pr/L + S Chl cIc, Dependent variable J’ = pc
2 2 2 1 1 1 1 2 (h - ’ )
0.188 0.154 n.lO1 - 0.843 - O.Oi3 0.00 1 0.00 1 0.00 I
(0.018) (0.043) (0.019) (0.023) (0.013) (0.009) (0.020) (O.Otl)
fl 6(+2SE)
- 0.014 (0.003) - 3.750 ( 1.336) - 0.702 (0.005) 0.023 (0.005) 0.019 (0.003) 0.046 (0.005) 0.077 (0.02 1) 145.6 (48.56)
0.809 0.539 0.466 0.772 0.862 0.920 0.660 0.571
With the exception of the chlorophyllidc N : Chl CIratio, and the dilution rate, which did not show significant correlations with either PC1 or PC2, the other variables appeared to be related to phytoplankton growth, and therefore their relationship could be quantified. Table V shows the equations, model type, i.e., linear or exponential, and significance level of such relationships. Biochemical indicators seemed to follow exponential relationships with growth, whereas cell activities followed a linear relationship. The protein to LM W synthesis ratio resulted the most accurate estimator of growth rate, which is consistent with previous findings working with natural populations (Madariaga & Fernandez, 1990). other indicators like DNA : RNA (Dortch et al., 1983) and C : Chl (Geider, 1987) also showed good agreement with phytoplankton growth rate. In general, our results are coincident with the patterns of variation of physiological i. 4 :ators under different growth conditions that Zevenboom (1986) summarized based on literature. Thus, we believe that the trends observed in this study are not sptik:ies-specific but, most likely, general for phytoplankton.
164
I. DE MADARIAGA
AND I. JOINT
ACKNOWLEDGEMENTS
This work formed part of Laboratory Research Project 2 of the Plymouth Marine Laboratory, a component of the UK Natural Environmental Research Council. I. de Madariaga acknowledges the receipt of a sectoral grant from the Commission of the European Communities. We thank M. Carr for statistical advice.
REFERENCES Barlow, R.G., M. A. Gough, R. F.C. Mantoura & T. W. Fileman, 1992. Pigment signatures of the phytoplankton composition in the North Eastern Atlantic during the 1990 spring bloom. Deep Sea Res., submitted. Barnes, H. & J. Blackstock, 1973. Estimation of lipids in marine animals and tissues; detailed investigation of the sulphophosphovanillin method for “total” lipids. J. Exp. Mar. Biol. Ecol., Vol. 12, pp. 103-I 18. Berdalet, E. & Q. Dortch, 1991. New double-staining technique for RNA and DNA measurement in marine phytoplankton. Mar. Ecol. Prog. Ser., Vol. 73, pp. 295-305. Chalup, M. S. & E. A. Laws, 1990. A test of the assumptions and predictions of recent microalgal growth models with the marine phytoplankter Pavlovulutheri. Lirnnol. Oceunogr., Vol. 35, pp. 583496. Clayton, J. R., Jr., Q. Dortch, S. S. Thoresen & S. I. Ahmed, 1988. Evaluation of methods for separation and analysis of proteins and free amino acids in phytoplankton samples. J. PlanktonRes., Vol. 10, pp. 341-358. Dortch, Q., 1982. Effect of growth conditions on accumulation of internal nitrate, ammonium, amino acids, and protein in three marine diatoms. J. Exp. Mar. Biol. Ecol., Vol. 61, pp. 243-264. Dortch, Q,, T. L. Robers, J. R. Clayton, Jr. & S. I. Ahmed, 1983. RNA/DNA ratios and DNA concentrations as indicators of growth rate and biomass in planktonic marine organisms. Mar. Ecol. Prog. Ser., Vol. 13, pp. 61-71. Droop, M. R., M. J. Mickelson, J. M. Scott & M. F. Turner, 1982. Light and nutrient status of algal cells. J. Mar. Biol, Assoc. U.K., Vol. 62, pp. 403-434. Elrifi, I. R, & D. H. Turpin, 1987. The path of cal bon flow during NO, induced photosynthetic suppression in N-limited Selenastrw nlintrtum,Plant, Physiol., Vol. 83, pp. 97-104. Falkowski, P. G., A. Sukenik & R. Herzig, 1989. Nitrogen limitation in Isochrysisgulim=r (Haptophyceae). II. Relative abundance of chloroplast proteins. J. Phycol., Vol. 25, pp. 471-478. Furnas, M. J., 1990. frr situgrowth rates of marine phyt~plnnkton: approaches to measurement, community and species growth rates. J. PlanktollRex, Vol. 12, pp. 1117-l 15 1. Geider, R. J., 1987. Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: implications for physiology and growth of phytoplankton. Wew Phytol., Vol. 106, pp. l-34. Jeffrey, S. W., 1980. Algal pigment synthesis. In, Primaqlproductivityin the sea, edited by P.G. Falkowski, Plenum Press, New York, pp. 33-58. Joint, I. R. & A. J. Ponxoy, 1986. Photosynthetic characteristics of nanoplankton and picoplankton from the surface mixed layer. Mar. Biol., Vol. 92, pp. 465-474. Klein, B., 1988. Variations of pigment content in two benthic diatoms during growth in batch cultures. J. E-VP.Mar. Biol. Ecol., Vol. 115, pp. 237-248. Kochert, G., 1978. Carbohydrate determination by the phenol-sulphuric acid method. In, Handbook of pt!!~*cological methods,edited by J. A. Hellebust & J. S. Craigie, Cambridge University Press, Cambridge, pp. 95-98. Langdon. C., 1988. ,On the causes of interspecific differences in the growth-irradiance relationship for phytoplankton. l$&general revie>. J. PlarzktonRes., Vol. 10, pp. 1291-1312. Larsson, C. M. & Mr.L&sb;i;a; 1!#7. Regulation of nitrate utilisation in green algae. In, Inorganic nitrogen metabolism?, edited by W. lJllrich et al., Springer-Verlag, New York, pp. 203-207. Li, W. K. W., I-I. E. Glover $r I. Morris, 1980. Physiology ofcarbon photoassimilation by Oscillutoriathiebautii in the Caribbean Sea. Limol. Ocearlogr.,Vol. 25, pp. 447-456.
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PHYSIOLOGICAL
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Li, W. K. W. & J. C. Goldman, 198 1. Problems in estimating growth rates of marine phytoplankton from short-term “C assays. Microb. Ecof., Vol. 7, pp. 113-121. Lowry, 0. H., N. J. Rosebrough, A. L. Farr & R. J. Randall, 195 1. Protein measurement with the folin phenol reagent. J. Biol. Chem., Vol. 193, pp. 265-280. Madariaga, I. de & E. Fernandez, 1990. Photosynthetic carbon metabolism of size-fractionated phytoplankton during an experimental bloom in marine microcosms. J. Mar. Biof. Assoc. U.K., Vol. 70, pp. 53 l-543. Mantoura, R. F. C. & C. A. Llewellyn, 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high performance liquid chromatography. Anal. Chim. Ada, Vol. 151, pp. 279-314. Morris, I., 1981. Photosynthesis products, physiological state, and phytoplankton growth. Can. Bull. Fish_ Aqua?. Sci., Vol. 210, pp. 83-102. Platt, T., 1981. Physiological bases of phytoplankton ecology. Can. Bull. Fish. Aqlxzt. Sci., Vol. 210, pp. l-346. Prezelin, B. B., 1981. Light reactions in photosynthesis. Can. Bull. Fish. Aquat. Sci., Vol. 210, pp. l-43. Reynolds, C. S., G. P. Harris & D. N. Gouldney, 1985. Comparison of carbon-specific growth rates of cellular increase of phytoplankton in large limnetic enclosures. J. PlanktonRes., Vol. 7, pp. 791-820. Rhee, G.-Y., 1979. Continuous culture in phytoplankton ecology. In, Advances in aquaticmicrobiology,vol. 2, edited by M. R. Droop & H. W. Jannasch, Academic Press, London, pp. 151-203. Rhiel, E., E. Morschel & W. Wehrmeyer, 1985. Correlation of pigment deprivation and ultrastructural organization of thylakoid membranes in Cryptomonasmaculatafollowing nutrient deficiency. Protoplasma, Vol. 129, pp. 62-73. Roy, S., 1988. Effects of changes in physiological condition on HPLC-defined chloropigment composition of Phaeoductylumzricornutum(Bohlin) in batch and turbidostat cultures. J. Exp. Mar. Biol. Ecol., Vol. 118, pp. 137-149. Sakshaug, E., 1980. Problems in the methodology of studying phytoplankton. In, The physiologicalecology of phytoplankton,edited by I. Morris, University of California Press, Berkeley, California, pp. 57-89. Sakshaug, E., K. Andresen, S. Myklestad & Y. Olsen, 1983. Nutrient status of phytoplankton communities in Norwegian waters (marine, brackish, and fresh) as revealed by their c ;aemical composition. J. Plankton Res., Vol. 5, pp. 175-196. Sakshaug, E. & 0. Holm-Hansen, 1977. Chemical composition of Skeletonemu costutum(Grev.) Cleve and Puvlova(Monochrysis) lutheri (Droop) Green as a function of nitrate-, phosphate-, and iron-limited growth. J. Exp. Mar. Biol. Ecol., Vol. 29, pp. l-34. SAS Institute, 1985. SAS user’s guide: statistics.SAS InstitutL . Cary, North Carolina. Version 5 Edition. USA. Strickland, J. D. H. & T. R. Parsons, 1972. A practical handbook of seawater analysis, 2nd edition. Bull. Fish. Res. Borrrd Can., Vol. 167, pp. l-3 10. Talling, J. F., 1984. Past and contemporary trends and attitudes in work on primary productivity. J. Plankton Res., Vol. 6, pp. 203-2 17. Turner, M. F., 1979. Nutrition of some marine microalgae with special reference to vitamin requirements and utilization of nitrogen and carbon sources. J. Mar. Biof. Assoc. U.K., Vol. 59, pp. 535-552. Turpin, D. H., I. R. Elrifi, D.G. Birch, H.G. Weger & J. J. Holmes, 1988. Interactions between photosynthesis, respiration, and nitrogen assimilation in microalgae. Can. J. Bot., Vol. 66, pp. 2083-2097. Turpin, D. H., 199 1. Effects of inorganic N availability on alga1 photosynthesis and carbon metabolism. J. Phycof., Vol. 27, pp. 14-20. Valderrama, J. C., 1981. The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem., Vol. 10, pp. 109-122. Wymer, P. E.O. & B. Thake, 1980. The importance of phosphorus in microalgal growth and species composition in mixed populations: experiments and simulations. Proc. R. Sot. Land., Vol. 209, pp. 333-353. Zevenboom, W., 1986. Ecophysiology of nutrient uptake, photosynthesis and growth. Can. Bd!. Fish. Aquat. Sci., Vol. 214, pp. 39 1-422.
149
JEMBE 01758
A comparative study of phytoplankton indicators
physiological
Iosu de Madariaga and Ian Joint NaturalEnvironmentalResearch Council, PlymouthMarine Laboratory, The Hoe, Plymouth, UK (Received 6 December
1991; accepted 9 January
1992)
Abstract: Pavlovalutheri(Droop) Green was grown in chemostat culture; the three different N sources used were ammonium, nitrate or glycine and the cultures were also grown under N and P limitation. A number of biochemical and physiological measurements were made on these cultures growing at high and low dilution rates. Elemental composition qf the cells remained relatively constant, C content increasing only under conditions of phosphate limitation, while N and P content decreased under their respective limiting conditions. DNA content showed little variation but RNA content declined significantly under both N and P limitation. The largest effect of nutrient limitation to the biochemical parameters measured were to carbohydrate and lipid concentrations which increased significantly. Amino acid concentrations increased more than six times when the cultures were phosphate-starved. Dilution rate, ,ss well as nutrient status, affected the physiological measurements; assimilation numbers were highest at hi;h dilution rates and were reduced under phosphate limitation and when glycine was the sole N source. Photosynthetic efficiency was similar for all growth conditions, except N limitation when values increased. Under both N and phosphate limitation, the proportion of i4C label in low molecular weight intermediates increased, with a decrease of the label in protein. There were significant variations in Chl a, c,c2 and chlorop:iyllide a under nutrient limitation but carotenoid pigm&..ts showed less variation. A multiple intercompariron was carried out by principal component analysis, which indicated that the biochemical composition measurements tended to be related to the growth-limiting factor, whereas the physiological measurements zere related to growth rate. Key words: Biochemical composition; Photosynthetic parameter
Chemostats;
C metabolism;
Nutrient
limitation;
Pavlova lutheri;
INTRODUCTION
A wide range of Gfferent algal properties have been used to estimate the physiological state of phytoplankton. Among them, cell activities, biochemical composition and morphological characteristics have proved to be useful physiological indicators, even when species-specific variability is considered (Zevenboom, 1986). The most commonly investigated physiological processes are photosynthesis, C and N metabolism, nutrient uptake and enzymatic activities (Platt, 198 1); biochemical studies generally include determinations of the elemental, macromolecular and pigment composition (Sakshaug et al., 1983; Roy, 1988). Visual indicators are strictly qualitatiqle and time-consuming Correspondence address: 1. de Madariaga, Ekologi Laborategia, Landare-Biologia Euskal Herriko Unibertsitatea, 644 PK, 48080 Bilbao, Spain.
eta Ekologia Saila,
150
I. DE MADARIAGA AND I. JOINT
and, consequently, they have not been applied widely in research on natural populations. However, these two classes of measurement, physiological and biochemical, are indicators of processes with different time scales. As discussed by Morris (198 l), physiological measurements of cellular activity must be interpreted with respect to the immediate cell environment, and they will indicate potential future changes in cell biochemical composition. In contrast, the actual biochemical composition reflects the physiological history of the cell. Since physiological properties of phytoplankton are influenced by both prevailing and former variations in the conditions for growth, no Gonstant relationships can be assumed a priori, unless steady-state conditions are achieved for an appreciable period of time. Continuous culture techniques offer considerable advantages in those studies which seek to establish the physiological status of algal cells because the cultures are at steady state, growing under controlled environmental conditions (Rhee, 1979). It is to be hoped that data obtained under these constant conditions should aid interpretation of physiological and biochemical measurements made on heterogeneous natural phytoplankton assemblages, which are growing at dirTerent rates. In this work, a comparative study of a number of indicators for the physiological state of phytoplankton was carried out using continuous cultures of the haptophyte Puvlova (formerly Monochqm’s) lutheri (Droop) Green. The aim of the research was to apply those techniques, which are commonly used in studies of natural assemblages, to laboratory cultures grown under defined conditions; continuous cultures were chosen to provide phytoplankton with unifom physiological condition, and were not used to investigate any theoretical aspects of P. hrtheri growth dynamics, such as the work of Droop et al. (1982) and Chalup & Laws (1990). Few attempts have so far been made to systematise those indicators which are normally used with natural assemblages and algal cultures. The rather scattered evidence published to date indicates that some parameters reflect mainly changes in the nutritional state of phytoplankton, i.e. its growth in relative terms, whereas other indicators are related to the growth rate in absolute terms, since all sources of variation are reflected in them (Sakshaug, 1980). The aim of this study is to attempt to establish quantitative relationships between general intrinsic algal properties and growth rate, as well as distinguishing those features that are characteristic of different types of growth limitation.
METHODS CHEMOSTATS P. lutheri was grown
in unialgal continuous culture using axenic techniques described by Wymer & Thake (1980). The clone (CCAP 93111) was obtained from the NERC Culture Collection, Oban, Scotland, UK. A bank of three, replicate, 2 1 chemostats was used throughout the study. Mixing was accomplished by the combination of aeration and magnetic bar stirring. Temperature was maintained at 20 k 0.5 “C and the con-
PHYTOPLANKTON
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151
tinuous incident radiation from four fluorescent tubes (Thorn EM1 Kolor-rite 75W) was 150~E*m-2s-1 at the surface of each culture (measured with a Crump quantum radiometer photometer model 550). The growth medium used was S88 (Turrer, 1979), modified in some cases so that N or P concentrations were limiting. Samples were withdrawn from the cultures every 2 days, and cell numbers were estimated either with a Coulter multisizer, with 50-pm orifice tube for routine counting, or with an improved Neubauer haemocytometer for accurate determination of cell density. In our experiments, steady state was considered to be attained when cell numbers remained constant for 5 consecutive days. 10 triplicate steady states, grouped into five series, were achieved in this study. Luring the first and second series, nutrient-suficient conditions (N : P = 17) were maintained using either ammonium (AS) or nitrate (NS) as N sources. Turner (1979) has shown that glycine is effective as sole N source for the growth of P. lutheri and for the third series (GS), all N sources except i;lycine were removed from the growth medium. In the fourth and fifth series, the cultures were either N-limited (NL) or P-limited (PL). Extreme N : P supply ratios (0.1 and 495, respectively) were kept to ensure severe nutrient limitation. Nutrient levels were measured with a Technicon autoanalyser, following the Strickland & Parsons methods (1972). Chemostats were run at high (H = 0.44 d - * ) and low (L = 0.10 d - ’ ) dilution rates; at each steady state, samples were taken for the different analyses described below and cell numbers and light levels within the culture chambers were determined. l
BIOCHEMICAL
l
ANALYSES
At each steady state, lo-20-ml samples were filtered onto 25.mm pre-ashed GF/F Whatman filters and stored at c - 20 “C until biochemical analyses were performed. Chl CIwas measured in 90% acetone extracts on a Perkin-Elmer spectrophotometer, according to the method described by Strickland & Parsons (1972). Total C and N were determined with a Carlo-Erba elemental analyser, using acetanilide as standard. For lotal P, the persulphate oxidation method of Valderrana (198 1) was used. Proteins and amino acids were analysed following the Clayton et al. method (1988); the procedure involved initial homogenisation in a TCA solution, followed by centrifugation to separate protein and free amino acid fractiors. Proteins were then analysed by a modification of the Lowry et al. procedure ( 1951) ,\nd amino acids by a fluorescamine procedure. Total carbohydrate was determined by ;he phenol-sulphuric acid inethod (Kochert, 1978) and total lipids by the phophosulphovanillin method (Barnes & Blackstock, 1973). Measurements of nucleic acids were made according to Berdalet & Dortch ( 1991‘1;si+ies were homogenized in Tris - Ca2 + buffer and total RNA and DNA were de$ermined by using two fluorochromes: Hoechst 33258, which specifically reacts with DNA, and Thiazole Orange, which allowed total nucleic acid estimation. RNA was estimated from the difference between these two measurements.
1.DE MADARIAGA
152 PHYSIOLOGICAL
AND 1.JOINT
ANALYSES
Samples from the chemostats were diluted lo-fold for the determination of primary production, and the incorporation patterns of C into different cell constituents. Samples were incubated in 60-ml tissue-culture bottles with 370 kBq (10 FCi) NaH 14C03for 4 h, maintained under the same light and temperature conditions as the chemostats. Photosynthesis-irradiance parameters were also measured on lo-fold dilutions from the chemostat and followed the procedures described by Joint & Pomroy (1986). Samples were removed from the chemostat, diluted and incubated for 4-5 h in a light gradient, cooled with circrilating water at 20 OC; the incubation period was kept as short as practicable to reduce the photoadaptation which would occur in the light gradient. All incubations were terminated by gentle filtration through l-pm Nuclepore membrane litters, which were dried and counted in a liquid scintillation counter. The distribution of 14C in low molecular weight metabolites (LMW), lipids, polysaccharides and proteins was determined using the cellular fractionation procedure of Li et al. (1980), as described in detail by Madariaga & Fernandez (1990). Recovery values of 91-107:;, (11= 30) were found when the sum of the radioactivity in all fractions was compared with unfractionated samples. PIGMENT
ANALYSIS
Sample litters were extracted in 2-6 ml 900/, acetone using sonication and further grinding by hand using a glass rod. Extracts were centrifuged to remove filter and cell residues and a 50-~1 aliquot mixed with 50 ~1 ammonium acetate was injected into a Shimadzu HPLC system with a 3-pm Percosphere column. Pigments were separated by a modification of the reverse phase method of Mantoura & Llewellyn (1983) as detailed by Barlow et al. (1991). Chloropl~ylls and carotenoids were detected spectrophoton~ctrically at 440 nm. whereas dctcction of phacopigments was done fluorometricatty with excitation set at 400 nm and emission > 600 nm. The output was monitored on a Dell PC, where peak areas and pigment concentration calculations were performed using the Philips PU6000 software. Pigment identification and quantification for the HPLC system used here was previously established by Barlow et al. (1991). STATISTICAL
ANALYSES
All statistical analyses were performed on a IBM 9370 computer using various SAS procedures (SAS institute, 1985). RESULTS BIOCHEMICAL
CtiMPOSITION
The results of the biochemical analyses are >hown in Figs. 1 and 2 and the data and statistics given in Table I. The C content cell - ’ (Fig. 1) remained relatively constant, l
PHYTOPLANKTON
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INDICATORS
153
25
T- 20 z x
15
10
5
0 Carbon
25
Nitrogen
Protein
Amino
Lipid
Acids
LOW
Dilution Rate T= 20 $ x 15
II
10
5
0 Carbon
Nitrogen
ei
Amino Acids
Lipi
Fig. 1. C, N, protein, amino acid and lipid composition (pg acell - I ) of P. lutherigrown at high dilution rate (H) and low dilution rate (15.)in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (GS); results are also shown for N-limited (NL) and phosphate-limited (PL) cultures.
I. DE MADARIAGA
154
AND I. JOINT
1
0
Chlorophyll
5
Phosphorus
RNA
DNA
Carbohydrate
Phosphorus
RNA
DNA
Carbohydrate
Low Dilution
&_4 b E
3
2
1
0 Chlorophyll
Fig. 2. Chlorophyll, P, RNA, DNA and carbohydrate composition (pg - cell - ’ ) of P. lutheri grown at high dilution rate (H) and low dilution rate (L) in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (C S); results are also shown for N-limited (NL) and phosphate-limited (PL) cultures.
PHYTOPLANKTON
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155
TABLE I
Elemental and biochemical composition of P. lutherigrown at high dilution rate (H) and low di!ution rate (L) in chemostat culture with N sources of ammonia (AS), nitrate (NS) and glycine (GS); results are also shown for N limited (NL) and phosphate-limited (PL) cultures. Data are pg - cell - ’ and values in parentheses are f 1 SE. Biochemical parameter
Dilution rate
AS
NS
GS
NL
0. I68 (0.003) 0. I05 (0.004) 10.965 (0.161) IO.176 (0.622) I.991 (0.041) I.532 (0.122) 0.180 (0.005) 0.202 (0.038) 12.508 (0.463) 10.550 (0.559) 3.187 (0.357) 2.121 (0.336) 3.043 (0.058) 3.547 (0.303) 7.715 (0.863) 5.430 (0.614) 0.164 (0.013) 0.166 (0.015) I .074 (0.235) 0.639 (0.052)
0.076 (0.004) 0.078 (0.004) 10.420 (0.763) 12.332 (0.192) 0.714 (0.010) 0.838 (0.058) 0.253 (0.043) 0. I I6 (0.020) 8.831 (1.133) 7.588 (0.081) 0.532 (0.130) 0.5 19 (0.049) 4.3 10 (0.207) 4.182 (0.316) 19.141 (1.162) 19.460 (2.316) 0.150 (0.015) 0.120 (0.008) 0.2 I I (0.020) 0.128 (0.012)
PL
-Chlorophyll
H L
C
H L
H L
P
H L
H L
Amillo acids
H
Carbohydrate
H
Lipid
H
DNA
H
RNA
x
L
L L I
L
0.23 I (0.037) 0.2 I4 (0.025) 10.075 (0 151) 9.918 (11.630) 2.089 (0.156) I.601 (!I.OlI) 0.06 I (0.004) 0.066 (0.002) IO.523 (0.010) 10.922 (0.137) 3.275 (0.050) I .794 (0.075) I .777 (0.008) 2.398 (0.480) 4.44 I (0.468) 5.576 (0.459) 0. I53 (0.004) 0.162 (0.009) I.190 (0.109) I .068 (0.022)
0. I52 O.l90 X.81I 8.382 I .706 I .353 0. I I4 0.072 9.915 8. I54 I .460 1.076 1.736 1.522 10.493 5.429 0.149 0. I28 I.123 0.952
(0.002) (0.013) (0.128) (0.287) (0.024) (0.033) (0.002) (0.00I ) (0.256) (0.570) (0.034) (0.167) (0.006) (0.138) (0.455) (0.725) (0.005) (0.004) (0.048) (0.076)
0.088 0.072 16.066 15.476 4.096 3.47 I 0.014 0.006 9.3 13 7.780 2 I .706 16.777 4.837 4.668 19.460 17.678 0.127 0.126 0.179 0.163
(0.002) (0.001) ( 1.330) (0.448) (0.401) (0.060) (0.002) (0.004) (0.570) (0.166) (0.922) (2.302) (0.109) (0.172) (1.564) ( 2 950) (0.036) (0.009) (0.048) (0.012)
increasing only under phosphate limitation; similarly, N content was constant in cells grown with excess of each of the three N sources, but it decreased under N limitation and doubled under phosphate limitation. Protein cell - ’ showed relatively small changes in concentration but there were much more significant variations in amino acid concentration, wV:h increased over six-fold when the cullrlres were P-limited. Lipid concentration showed similar responses to both N and P limitation and increased by more than three times. The RNA : DNA ratio decreased under both N and phosphate limitation, as a result of the decline in RNA content cell- ’ (Fig. 2). In contrast, DNA concentration remained relatively constant. The polysaccharide content increased under N and phosphate limitation, but was also significantly higher in the cultures grown with glycine as sole N source. Chlorophyll content, as determined spectrophotometrically (Table I), was very sensitive to nutrient limitation, showing maxima under nutrient-sufficient conditions, i.e., when light was limiting due to the high attenuation caused by selfshading. P content increased when glycine was the N source and under N limitation, but was greatly reduced under phosphate 1mitation. C : P ratios (by atoms) of > 2000 and C : N ratios of > 15 were found under P and N starvation, respectively. l
l
I. DE MADARIAGA AND I. JOINT
156
Generally, there were very few differences between the results obtained with cultures growing at high or low dilution rates. The largest difference was an increase in the lipid content of P. hrtheri growing at high dilution rate with nitrate and glycine as N source. GROWTH
AND
PHOTOSYNTHESIS
Under nutrient-sufficient conditions, C-specific growth rates were significantly higher than steady-state growth rates (Table II); this has been reported previously for P. lutheri (Li & Goldman, 198l)? and has been related to disturbances of the steady state when samples are removed from the chemostat for determination of primary production. In our experiments, self-shading (and consequently light limitation) was significantly reduced when samples were diluted, growth conditions being thus optimized during the incubations. Growth was consistently lower under low dilution rates, but nutrient limitation had a more marked effect. 14Cfixation rates, determined at irradiance values equivalent to those at the surface of the chemostat cultures, and normalized to Chl a (P:B) was generally high, and paralleled changes in growth. TABLE
II
Physiological parameters of P. lutheri obtained with chemostat cultures at high dilution rate (H) and low dilution rate (L); tic C-specific growth rate (h - i), P/B production normalized to chlorophyll [PgC- (pg chl)- ’ h - ‘1, Pfl maximum chlorophyll-specific rate of i4C fixation [PgC (pg chI)- ’ -h- I], aB photosynthetic efftciency [PgC (pg chl) h - i/pE m - 2. s - ‘1, percentage of i4C incorporation into low molecular weight metabolites ( y0 LMW), lipids (“/, lipid), proteins (Y, protein) and polysaccharides ( y-,carb). Nutrient conditions in chemostats are same as in Table I and values in parentheses are + I SE. l
l
l
AS
NS
_.____-I-_-_---H
0. I40 (0.028)
L
0. I I6 (0.035)
H L
p!,
H L H L
“,, LMW
H L
u,, lipid
H
“,, protein
H
‘,, Garb
H
L L L
7.320 (0.90 I )
5.329 5. I32 3.599 0.035 0.02 I 16.25 24.16 21.15 17.98 5 1.37 49.22 Il.23 8.64
(0.X30) (0.406) (0.638) (0.004) (0.004) (2.58) (3.68) (3.48) (1.27) (4.50) (5.90) (1.83) (0.95)
GS
--P_
(0.000) 0.I 2’1 (0.000) x.22I (0.006) 0. I 32
6.258 6.670 4.815 0.026 0.024 17.27 15.57 22.24 29.55 49.83 37.92 10.66 16.96
0.059 0.038 3.940 ( I mO74) 3.245 (0.449) 3.225 (0.621) 2.506 (0.002) 0.02 I (0.002) 0.027 (0.18) 20.47 (2.41) 31.00 (2.40) 33.15 (2.94) 14.17 (3.56) 22.73 (0.29) 27.90 (0.97) 23.65 (0.25) 26.93
NL
PL
--
(0.004) (0.003 ) (0.214) (0.394) (0.176) (0.055) (0.002) (0.001j (0.71) (2.67) (0.88) (1.63) (3.10) (4.45) (2.66) (3.48)
0.037 0.020 5.184 3.272 6.069 3.469 0.062 0.052 33.41 40.83 34.64 17.01 18.45 12.62 13.50 29.54
(0.002) (0.003) (0.254) (0.485) (0.459) (0.465) (0.007) (0.007) (1.50) ( I .08) (2.77) (1.24) (2.41) (0.56) (1.41) (1.87)
0.016 0.009 2.853 2.047 3.556 3.536 0.017 0.020 31.31 36.87 31.55 38.47 26.76 17.81 10.38 6.85
(0.003) (0.00 I ) (0.385) (0.280) (0.997) (0.106) (0.005) (0.004) (0.94) ( I .24) (0.59) (0.97) (0.51) (0.94) (0.20) (0.33)
Fig. 3 shows typical examples of the curves fitting to the data for the photosynthesisirradiance relationship under different nutrient conditions. Maximum specific photosynthetic rate (Pf,) was highest in the culture with high dilution rate and were reduced
PHYTOPLANKTON
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400
PHYSIOLOGICAL
INDICATORS
157
4
600
1200
1600
PLH
Fig. 3. Typical photosynthesis-irradiance (P-I) curves of P. lutheri for different nutritional conditions: NSH, N-sufficient culture, growing at high dilution rate; NLH, N-limited, growing at high dilution rate; PLH, P-limited, growing at high dilution rate.
under phosphate limitation; values were significantly lower at both dilution rates when glycine was the sole N source. Photosynthetic eficiency (orB)estimates were generally similar for all growth conditions except N limitation, when higher values were found. 111some cases, P:B estimates appear to be higher than PK; however, the differences are no? statistically significant and, in any case, the two parameters were determined under different experimental conditions. The caveat applies that the physiological state of the
C : Chl a ratio
C : N ratio C : P ratio Carbohydrate : Protein ratio Lipid : Protein ratio Amino acid : Protein ratio
RNA : DNA ratio C-specific growth rate Production/Biomass Maximum chlorophyll-specific “C fixation rate Photosynthetic efficiency Protein :toLMW synthesis storage productsratiosynthesis ratio
Chlorophyllide a : Chl a ratio Chl c,cz : Chl a ratio Fucoxanthin : Chl a ratio Diadinoxanthin : Chl a ratio /?-Carotene : Chl a ratio Dilution rate Light within chemostats
C:N C:P @a: Pr Li : Pr Aa: Pr
R:D k PIB p: xB pr/lmw pr/l + s
Chide ChlC Fuc Diad B car Dr Light
Definition
(h-l) (pE.m-2*s-‘)
(li- ‘) [j.q$(pg Chl) - ’ - h - ’ ] [(pgChl)-r-h-r] [pgC(pg Chl)-‘ah-r/pE
Units
m-2.s-‘]
0.013 0.29 1 0.857 0.858 0.550 0.075 0.092
- 0.122 - 0.696 C.380 0.269 0.699 - ii.158 0.812
0.849 - 0.846 -0.115 0.028 - 0.814
0.367 0.370 0.85 1 0,808 0.433
0.006 0.069 0.273 0.403 0.810 -0.088 -0.147
- 0.33 1
0.895
0.908 0.934 0.743 0.374 0.285 - 0.795 0.919
-
PC2
coefficient
PC1
Correlation
and culture conditions included in principal component analysis (PCA) matrix and their respective units, and correlation coeffkients obtained with first two components in analysis.
C:Chl
Parameter
Physiological parameters
TABLE IV
5
Fj
g 2 u
5 Fj
g
S
2 0 z
5
2 =s 0 p
. DE MADARIAGA
162
AND I. JOINT
C-specific growth rates (& were significantly different to steady-state growth rates (p) pc generally exceeding p by a factor between 1 and 20, depending on the growth conditions. The divergence appeared to increase with increasing Pi, and could be related to methodological limitations and perturbations from steady state, as discussed in Results. However, similar data have been related by Reynolds et al. (1985) to physiological voiding of excess C by respiration or excretion processes. These authors also showed that loss rates varied in direct proportion to pc. The growth rate dependence of respiration is well-known (Langdon, 1988) and, as discussed above, it can be explained by the onset of photosynthetic N assimilation into protein under nutrientsufficient conditions, which stimulates respiratory C flow (Turpin, 1991). Thus, C fixation rates should be viewed as a measure of capacity for cellular increase (Talling, 1984) rather than actual growth. Similarly, calculated C-specific growth rates should be considered as potential maximum growth rates which, owing to other limitations upon growth, are seldom reached (Reynolds et al., 1985). In an attempt to summarize the results obtained in this study, a multiple intercomparison of the various physiological indicators (Table IV) was carried out by performing a principal component analysis. The projection of 10 steady states on the plane of the two axes (Fig. 5) accounted for 70% of the variability, 46 and 24% for the
NbH N:‘
1
,
NStJ
NzL
C&L 1
2
3
4
’ 5
PC2
-1
-2
-3
-4
P:H PLL
-5
Fig. 5. Representation of first (PCl) and second (PC2) principal components determined by principal component analysis of steady-state culture conditions described in Table IV; error bars are + SE.
PHYTOPLANKTON
PHYSIOLOGICAL
INDICATORS
163
first and second principal components. PC 1 was controlled by growth indicators, while PC2 indicated N/P limitation (Table IV). Significant correlations with the second axis (PC2) and elemental composition ratios, carotenoids to Chl F ratios, an, and the amino acid to protein ratio indicate that these biochemical indicators provide qualitative information on the growth-limiting factor, rather than the actual growth rate. Therefore, it is important to consider this source of variation when establishing quantitative relationships between physiological estimators and growth rate, because, in some cases, these relationships are valid only under a particular kind of limitation, and they must be used with caution in field work.
TABLE V
Regression equations of growth rate (p,-) and physiological indicators. Model types: 1, linear (y = a + bx); 2, exponential (;, = aehV). Significant at P < 0.0001 Independent variable (X )
Model
Parameters a (+2SE)
C:Chl Ca: Pr Li : Pr P/B RNA : DNA Pr/LM W Pr/L + S Chl cIc, Dependent variable J’ = pc
2 2 2 1 1 1 1 2 (h - ’ )
0.188 0.154 n.lO1 - 0.843 - O.Oi3 0.00 1 0.00 1 0.00 I
(0.018) (0.043) (0.019) (0.023) (0.013) (0.009) (0.020) (O.Otl)
fl 6(+2SE)
- 0.014 (0.003) - 3.750 ( 1.336) - 0.702 (0.005) 0.023 (0.005) 0.019 (0.003) 0.046 (0.005) 0.077 (0.02 1) 145.6 (48.56)
0.809 0.539 0.466 0.772 0.862 0.920 0.660 0.571
With the exception of the chlorophyllidc N : Chl CIratio, and the dilution rate, which did not show significant correlations with either PC1 or PC2, the other variables appeared to be related to phytoplankton growth, and therefore their relationship could be quantified. Table V shows the equations, model type, i.e., linear or exponential, and significance level of such relationships. Biochemical indicators seemed to follow exponential relationships with growth, whereas cell activities followed a linear relationship. The protein to LM W synthesis ratio resulted the most accurate estimator of growth rate, which is consistent with previous findings working with natural populations (Madariaga & Fernandez, 1990). other indicators like DNA : RNA (Dortch et al., 1983) and C : Chl (Geider, 1987) also showed good agreement with phytoplankton growth rate. In general, our results are coincident with the patterns of variation of physiological i. 4 :ators under different growth conditions that Zevenboom (1986) summarized based on literature. Thus, we believe that the trends observed in this study are not sptik:ies-specific but, most likely, general for phytoplankton.
164
I. DE MADARIAGA
AND I. JOINT
ACKNOWLEDGEMENTS
This work formed part of Laboratory Research Project 2 of the Plymouth Marine Laboratory, a component of the UK Natural Environmental Research Council. I. de Madariaga acknowledges the receipt of a sectoral grant from the Commission of the European Communities. We thank M. Carr for statistical advice.
REFERENCES Barlow, R.G., M. A. Gough, R. F.C. Mantoura & T. W. Fileman, 1992. Pigment signatures of the phytoplankton composition in the North Eastern Atlantic during the 1990 spring bloom. Deep Sea Res., submitted. Barnes, H. & J. Blackstock, 1973. Estimation of lipids in marine animals and tissues; detailed investigation of the sulphophosphovanillin method for “total” lipids. J. Exp. Mar. Biol. Ecol., Vol. 12, pp. 103-I 18. Berdalet, E. & Q. Dortch, 1991. New double-staining technique for RNA and DNA measurement in marine phytoplankton. Mar. Ecol. Prog. Ser., Vol. 73, pp. 295-305. Chalup, M. S. & E. A. Laws, 1990. A test of the assumptions and predictions of recent microalgal growth models with the marine phytoplankter Pavlovulutheri. Lirnnol. Oceunogr., Vol. 35, pp. 583496. Clayton, J. R., Jr., Q. Dortch, S. S. Thoresen & S. I. Ahmed, 1988. Evaluation of methods for separation and analysis of proteins and free amino acids in phytoplankton samples. J. PlanktonRes., Vol. 10, pp. 341-358. Dortch, Q., 1982. Effect of growth conditions on accumulation of internal nitrate, ammonium, amino acids, and protein in three marine diatoms. J. Exp. Mar. Biol. Ecol., Vol. 61, pp. 243-264. Dortch, Q,, T. L. Robers, J. R. Clayton, Jr. & S. I. Ahmed, 1983. RNA/DNA ratios and DNA concentrations as indicators of growth rate and biomass in planktonic marine organisms. Mar. Ecol. Prog. Ser., Vol. 13, pp. 61-71. Droop, M. R., M. J. Mickelson, J. M. Scott & M. F. Turner, 1982. Light and nutrient status of algal cells. J. Mar. Biol, Assoc. U.K., Vol. 62, pp. 403-434. Elrifi, I. R, & D. H. Turpin, 1987. The path of cal bon flow during NO, induced photosynthetic suppression in N-limited Selenastrw nlintrtum,Plant, Physiol., Vol. 83, pp. 97-104. Falkowski, P. G., A. Sukenik & R. Herzig, 1989. Nitrogen limitation in Isochrysisgulim=r (Haptophyceae). II. Relative abundance of chloroplast proteins. J. Phycol., Vol. 25, pp. 471-478. Furnas, M. J., 1990. frr situgrowth rates of marine phyt~plnnkton: approaches to measurement, community and species growth rates. J. PlanktollRex, Vol. 12, pp. 1117-l 15 1. Geider, R. J., 1987. Light and temperature dependence of the carbon to chlorophyll a ratio in microalgae and cyanobacteria: implications for physiology and growth of phytoplankton. Wew Phytol., Vol. 106, pp. l-34. Jeffrey, S. W., 1980. Algal pigment synthesis. In, Primaqlproductivityin the sea, edited by P.G. Falkowski, Plenum Press, New York, pp. 33-58. Joint, I. R. & A. J. Ponxoy, 1986. Photosynthetic characteristics of nanoplankton and picoplankton from the surface mixed layer. Mar. Biol., Vol. 92, pp. 465-474. Klein, B., 1988. Variations of pigment content in two benthic diatoms during growth in batch cultures. J. E-VP.Mar. Biol. Ecol., Vol. 115, pp. 237-248. Kochert, G., 1978. Carbohydrate determination by the phenol-sulphuric acid method. In, Handbook of pt!!~*cological methods,edited by J. A. Hellebust & J. S. Craigie, Cambridge University Press, Cambridge, pp. 95-98. Langdon. C., 1988. ,On the causes of interspecific differences in the growth-irradiance relationship for phytoplankton. l$&general revie>. J. PlarzktonRes., Vol. 10, pp. 1291-1312. Larsson, C. M. & Mr.L&sb;i;a; 1!#7. Regulation of nitrate utilisation in green algae. In, Inorganic nitrogen metabolism?, edited by W. lJllrich et al., Springer-Verlag, New York, pp. 203-207. Li, W. K. W., I-I. E. Glover $r I. Morris, 1980. Physiology ofcarbon photoassimilation by Oscillutoriathiebautii in the Caribbean Sea. Limol. Ocearlogr.,Vol. 25, pp. 447-456.
PHYTOPLANKTON
PHYSIOLOGICAL
INDICATORS
165
Li, W. K. W. & J. C. Goldman, 198 1. Problems in estimating growth rates of marine phytoplankton from short-term “C assays. Microb. Ecof., Vol. 7, pp. 113-121. Lowry, 0. H., N. J. Rosebrough, A. L. Farr & R. J. Randall, 195 1. Protein measurement with the folin phenol reagent. J. Biol. Chem., Vol. 193, pp. 265-280. Madariaga, I. de & E. Fernandez, 1990. Photosynthetic carbon metabolism of size-fractionated phytoplankton during an experimental bloom in marine microcosms. J. Mar. Biof. Assoc. U.K., Vol. 70, pp. 53 l-543. Mantoura, R. F. C. & C. A. Llewellyn, 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high performance liquid chromatography. Anal. Chim. Ada, Vol. 151, pp. 279-314. Morris, I., 1981. Photosynthesis products, physiological state, and phytoplankton growth. Can. Bull. Fish_ Aqua?. Sci., Vol. 210, pp. 83-102. Platt, T., 1981. Physiological bases of phytoplankton ecology. Can. Bull. Fish. Aqlxzt. Sci., Vol. 210, pp. l-346. Prezelin, B. B., 1981. Light reactions in photosynthesis. Can. Bull. Fish. Aquat. Sci., Vol. 210, pp. l-43. Reynolds, C. S., G. P. Harris & D. N. Gouldney, 1985. Comparison of carbon-specific growth rates of cellular increase of phytoplankton in large limnetic enclosures. J. PlanktonRes., Vol. 7, pp. 791-820. Rhee, G.-Y., 1979. Continuous culture in phytoplankton ecology. In, Advances in aquaticmicrobiology,vol. 2, edited by M. R. Droop & H. W. Jannasch, Academic Press, London, pp. 151-203. Rhiel, E., E. Morschel & W. Wehrmeyer, 1985. Correlation of pigment deprivation and ultrastructural organization of thylakoid membranes in Cryptomonasmaculatafollowing nutrient deficiency. Protoplasma, Vol. 129, pp. 62-73. Roy, S., 1988. Effects of changes in physiological condition on HPLC-defined chloropigment composition of Phaeoductylumzricornutum(Bohlin) in batch and turbidostat cultures. J. Exp. Mar. Biol. Ecol., Vol. 118, pp. 137-149. Sakshaug, E., 1980. Problems in the methodology of studying phytoplankton. In, The physiologicalecology of phytoplankton,edited by I. Morris, University of California Press, Berkeley, California, pp. 57-89. Sakshaug, E., K. Andresen, S. Myklestad & Y. Olsen, 1983. Nutrient status of phytoplankton communities in Norwegian waters (marine, brackish, and fresh) as revealed by their c ;aemical composition. J. Plankton Res., Vol. 5, pp. 175-196. Sakshaug, E. & 0. Holm-Hansen, 1977. Chemical composition of Skeletonemu costutum(Grev.) Cleve and Puvlova(Monochrysis) lutheri (Droop) Green as a function of nitrate-, phosphate-, and iron-limited growth. J. Exp. Mar. Biol. Ecol., Vol. 29, pp. l-34. SAS Institute, 1985. SAS user’s guide: statistics.SAS InstitutL . Cary, North Carolina. Version 5 Edition. USA. Strickland, J. D. H. & T. R. Parsons, 1972. A practical handbook of seawater analysis, 2nd edition. Bull. Fish. Res. Borrrd Can., Vol. 167, pp. l-3 10. Talling, J. F., 1984. Past and contemporary trends and attitudes in work on primary productivity. J. Plankton Res., Vol. 6, pp. 203-2 17. Turner, M. F., 1979. Nutrition of some marine microalgae with special reference to vitamin requirements and utilization of nitrogen and carbon sources. J. Mar. Biof. Assoc. U.K., Vol. 59, pp. 535-552. Turpin, D. H., I. R. Elrifi, D.G. Birch, H.G. Weger & J. J. Holmes, 1988. Interactions between photosynthesis, respiration, and nitrogen assimilation in microalgae. Can. J. Bot., Vol. 66, pp. 2083-2097. Turpin, D. H., 199 1. Effects of inorganic N availability on alga1 photosynthesis and carbon metabolism. J. Phycof., Vol. 27, pp. 14-20. Valderrama, J. C., 1981. The simultaneous analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem., Vol. 10, pp. 109-122. Wymer, P. E.O. & B. Thake, 1980. The importance of phosphorus in microalgal growth and species composition in mixed populations: experiments and simulations. Proc. R. Sot. Land., Vol. 209, pp. 333-353. Zevenboom, W., 1986. Ecophysiology of nutrient uptake, photosynthesis and growth. Can. Bd!. Fish. Aquat. Sci., Vol. 214, pp. 39 1-422.