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Phytoplanktoniccarbonisotopefractionation:equationsaccountingforCO2-concentratingmechanisms.JPlanktonRes

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TakahitoYoshioka

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Journal of Plankton Research Vol.19 no.10 pp.1455-1476, 1997

Phytoplanktonic carbon isotope fractionation: equationsaccounting for CO2-concentrating mechanisms

Takahito Yoshioka

Institute for Hydrospheric-Atmospheric Sciences, Nagoya University, Furo-cho,Chikusa-ku, Nagoya 464-01, Japan

Abstract A model of carbon isotope discrimination by phytoplankton was developed which took intoaccount the occurrence of a carbon-concentrating mechanism (CCM). A simple equation wasobtained for the model involving CO2 active transport. In the case of HCO3~ active transport, anotherequation was developed based on a series of approximations. The former equation was used toanalyse reported and newly obtained data from culture experiments and field observations in bothfreshwater and marine environments. In most cases, a linear relationship between a combined para-meter, (1 -/)Ci, which was made up of the relative contribution of active CO2 uptake to total carbonuptake (/) and the intracellular CO2 concentration (Ci), and CO2 concentration in bulk solution (Ce)was obtained as (1 -/)Ci = aCe - b, with a high correlation coefficient (r2 > 0.9). The slope a is sug-gested as a measure of the ratio of diffusive to total (diffusive + active) CO2 transport, while b/a rep-resents CO2 demand.

Introduction

In studies on the global carbon cycle, the stable carbon isotope natural abundance(813C value) has been proved to be a powerful tool for estimating processes (e.g.Quay et al, 1992). The inverse relationship between [CO2]aq and the 813C oforganic matter produced by phytoplankton has been widely recognized, withvarious equations proposed for expressing such a relationship (e.g. Freeman andHayes, 1992; Rau et al, 1992). For geochemical purposes, [CO2]aq was estimatedusing the regression equations between [CO2]aq or log [CO2]aq and the isotopefractionation factor (ep) (Hollander and McKenzie, 1991; Freeman and Hayes,1992; Goericke and Fry, 1994). Equations based on the fractionation modelduring plant photosynthesis have also been developed (Berry, 1988; Rau et al.,1992; Raven et al., 1993). From the ecophysiological point of view, carbon isotopefractionation by phytoplankton has been used for assessments of growth rate(Takahashi et al, 1991) and CO2 availability (Fogel et al, 1992).

Mechanisms directly associated with the carbon uptake process in phyto-plankton should be considered in developing the fractionation equation. It isrecognized that phytoplankton actively transport CO2 by a carbon-concentratingmechanism (CCM) (Sharkey and Berry, 1985; Burns and Beardall, 1987; Thiel-mann et al., 1990), which affects the 813C value of phytoplankton. Some equationstake the CCM into account (Sharkey and Berry, 1985; Fogel et al, 1992).However, it has been assumed that when the CCM is considered in the equation,it must be in response to the total CO2 uptake. When active transport occurs,the equation using flux (F) is adopted (Berry, 1988; Fogel et al, 1992). In thesecases, only active transport is used in CO2 influx, and its efficiency in respect tocarbon assimilation is represented by 'leakiness'. (3-Carboxylation catalysed by

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phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase alsoaffects the 813C of phytoplankton (Descolas-Gros and Fontugne, 1985; Falkowski,1991). However, culture experiments of marine phytoplankton showed norelationship between p-carboxylation and the 813C value of particulate organiccarbon (POC) (Leboulanger et al, 1995).

Seasonal changes in the 813C value of phytoplankton have been found in fresh-water lakes (Yoshioka et al, 1989; Zohary et al, 1994). A remarkable increase inthe 813C value in the bloom season suggests that phytoplankton photosynthesismay be limited by CO2 depletion (Takahashi et al., 1990). However, since no frac-tionation model seems to be generally agreed, as mentioned above, applicationof the fractionation equation to the field data seems to present difficulties. In thispaper, we briefly review the fractionation equations reported for phytoplanktonphotosynthesis on a physiological basis, and propose a new equation in whichactive transport of inorganic carbon by the CCM is considered. Furthermore, theproposed equation is applied to the analyses of culture experiments and field datafrom marine and freshwater environments.

Method

Lake Kizaki (36°33'N, 137°50'E) is a mesotrophic lake in Honshu Island, Japan.Its surface area is 1.4 km2 with a maximum depth of -29 m. Field observationswere carried out at the centre of Lake Kizaki during April-July 1992. Water tem-perature and photon flux density (PFD) were measured by a thermistor ther-mometer (ET-5, Toho-Dentan, Co., Ltd) and photometer (Model LI-189,LI-COR, Co., Ltd) lowered to the sampling depth (2 m). Samples were taken witha Van Dorn water sampler. The pH value of each sample was measured on boardwith a pH meter (HPH-110, Denki-Kagaku Keisoku, Co., Ltd). Samples for themeasurement of dissolved inorganic carbon (DIC) concentrations were intro-duced into a glass-stoppered bottle and preserved with formalin solution. DICconcentration was measured by an infrared analyser (VIA-300, Horiba, Co., Ltd).A glass cylinder (-200 ml in volume) was used for the 813C measurement of DIC.Sample water was introduced through the Teflon screw stop valve at one end ofthe cylinder. After overfilling water to twice or more than the cylinder volume,saturated CuSO4 solution (2 ml) was added as a preservative, and a syringeseptum of silicone rubber was placed at the other end. The entire sample in thecylinder was introduced into a stripping apparatus (Kroopnick, 1974) by pure N2

gas through a rubber septum. DIC was extracted by acidification and purifiedcryogenically within 12 h after sampling.

Samples for isotope and photosynthetic activity measurements were put intopolyethylene bottles, stored in an insulated box, and brought back to the labora-tory. After samples were filtered through a 334 urn mesh net to eliminate zoo-plankton, suspended particles were collected on a pre-combusted glass fibre filter(Whatman GF type C). Chlorophyll content was measured by the methanolextraction procedure (Marker et al, 1980). For isotope measurement, filters wererinsed with a small amount of dilute HC1 solution (-0.005 N) and distilled water,and dried at 60°C overnight. POC was converted to CO2 gas according to the

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method of Minagawa et al. (1984). In the preparation of CO2 gas, the carboncontent of the POC was manometrically determined.

The carbon isotope ratio was measured with an isotope ratio mass spectrome-ter (delta S or MAT 251, Finnigan MAT Instruments Inc.). The isotope ratio isexpressed as a per mil deviation (813C value) from the PDB standard as follows:

8 1 3 C = ( ^Miiiple _

\ "• standard

where R denotes 13C/12C.Concentrations and 813C values of [CO2]aq were calculated using the dissocia-

tion constants of carbonate (Stumm and Morgan, 1981) and equilibrium isotopefractionation factors (Deines et al., 1974).

Photosynthetic activity was measured by O2 production in the oxygen bottleswhich were placed under various PFDs. During 4-5 h of incubation, the bottleswere kept in a water bath at in situ temperature. After incubation, the dissolvedO2 concentration was measured by the Winkler method. In situ photosyntheticactivity was calculated from the resultant O2 production rate-PFD curve andthe assumption of stoichiometric conversion of CO2 carbon to carbohydratecarbon.

Model description

Basic equation for expressing photosynthetic carbon isotope fractionation

The theoretical basis of photosynthetic carbon isotope fractionation was derivedfrom land C3 plants (e.g. O'Leary, 1981; Farquhar et al., 1989). The notations aredesignated as follows.

The photosynthetic carbon uptake process is:

i 2

CO2out <— CO2in—> organic carbon

where kt is the rate constant for process /. Processes 1 and 3 are the diffusive influxand efflux of CO2, respectively. Process 2 is the carboxylation step by ribulose bis-phosphate carboxylase-oxygenase (RUBISCO).

At steady state, or — = 0, the overall fractionation factor (a) is equatedas follows: &

A*,) i | (1)

where Ce and Ci are the CO2 concentrations in air and at the carboxylation site,respectively, and A/c, = a, - 1. In the equation of O'Leary (1981), subscripts forefflux and carboxylation steps were 2 and 3, respectively, and £, = 1 + AJfc,:

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When a = AAcb b = Afc2 and C02 concentrations in air and intercellular leaf spacesare expressed in partial pressure pt and piy respectively, equation (1) is equatedto Farquhar's equation:

A- -\ - a + (b - a\ (3)

Re-examination of fractionation equation

When only passive diffusion contributes to the inward and outward fluxes of CO2,the fractionation equation for phytoplankton photosynthesis is substantially thesame as that for land C3 plants (Table I), although Ce denotes the CO2

Table L Fractionation equations for land plant and phytoplankton photosynthesis

Literature Fractionation equation

Land plant£12

O'Leary (1981) -^5- (overall) = £,-

Farquhar er al. (1989) A = a + (b - a) —

Phytoplankton

Passive diffusion model

Rau er al. (1992) —- = (613CpUnl - 813Cco2 + d)Kd + f)

Fogel er al. (1992) A = a + ( f t -a)-^-

Francois er aL (1993) ep = e,

Jasper er al. (1994) ep = A

Laws era/. (1995)

Active transport

Berry (1988)

Fogel era/. (1992)

Francois er al. (1993)

A and B in Jasper er al. (1994) are -27%o and -130%<i uM, respectively.|j in Laws et al. (1995) means growth rate, u = K,Ce - A^Ci.Subscripts represent each process in CO2 uptake kinetics and are adjusted as follows: 1, influx of CO2;2, CO2 assimilation by RUBISCO; 3, efflux of CO2. Note that subscripts were changed from the orig-inal ones in O'Leary (1981), Francois er al. (1993) and Laws er al. (1995).

1 -v-jT T P * - ) ( £ 2 - £3)-

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Table IL List of fractionation factors relating to the photosynthetic carbon assimilation (after Berry,1988)

Symbol

a (land plant)a (phytoplankton) d and EIa,b and/

e,

Fractionation

4.40.7

Negligible27.029.4

1.12.0

-9.0

Process

CO2 diffusion, airCO2 diffusion, aqueous phaseActive transport of HCO3"RUBISCO and 10% PEPCaseRUBISCODissolution of CO2Hydration of CO2 (kinetic)Hydration of CO2 (equilibrium)

concentration in bulk solution, or [CO2]aq, and CO2 diffusion must be consideredin the aqueous phase. Fractionation factors involved in these equations are pre-sented in Table II.

The fractionation equation for passive diffusion-phytoplankton photosyn-thesis is basically the same as equation (1). Rau et al. (1992) introduced the term'CO2 demand = Ce - Ci' into their model (Table I). Francois et al. (1993) analysedthe relationship between the 813C value of paniculate organic matter (POM) and[CO2]aq in the southwestern Indian Ocean using the fractionation equationincluding the (Ce - Ci) term:

ep = e, + 1 - (e2 - Ej) (4)\ t-e /

Data from the SW Indian Ocean fit the model in which (Ce - Ci) was 7-9 uM.Assuming a constant (Ce - Ci) value, Jasper et al. (1994) found a significant cor-relation between ep and Ce in the Pigmy Basin with a = 29.2%o and b = -109%ouM (Table I). When (Ce - Ci) is constant, equation (1) at the infinite Ce is:

a = 1 + Ak2 (5)

This means that overall fractionation reaches the maximum value whichcorresponds to that of RUBISCO (a = 1.029, or Ak2 = 0.029; Roeske and O'Leary,1984) at a high Ce condition. Many authors have used Ak2 = 0.027, taking a 10%contribution of PEPCase to total carbon assimilation into consideration (Far-quhar and Richards, 1984). The fractionation factor must approach 1.027-1.029at high Ce. However, culture experiments conducted by Hinga et al. (1994)showed that fractionation by Skeletonema costatum and Emiliania huxley becamemaximal before reaching these levels. (Ce - Ci) seemed to increase with theincrease in Ce. The authors suggested the possibility of (3-carboxylation at highCe. Indeed, it was found that the activity of the PEPCKase of S.costatumincreased to >50% of RUBISCO activity at the end of growth (Descolas-Grosand Fontugne, 1985, 1990). However, the contention that low fractionation athigh Ce is due to the p-carboxylation has been controversial (Goericke et al.,1994), especially in the case of PEPCKase-mediating (J-carboxylation, whichshows similar discrimination against 13CO2 to that of RUBISCO (Arnelle and

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O'Leary, 1992). Active transport by a CCM may contribute to a fractionation athigh Ce lower than that given by the fractionation equation.

Laws et al. (1995) concluded that passive CO2 diffusion was sufficient to sustainmaximum growth of Phaeodactylum tricornutum and this alga would not needactive transport of inorganic carbon at a [CO2]aq of 10 uM. In their analysis, themaximum growth rate was expected when the CO2 influx was equal to growthrate. However, this situation means Ci = 0, then, growth rate (photosyntheticactivity) = 0, or is even negative, because of oxygenase activity of RUBISCO. Thecontradiction may occur because growth rate is not independent of Ce and Ci.Since CCM requires an energy expenditure (Berry, 1988), one may suppose thatdiffusive transport of CO2, if possible, is operative together with active transport.From the reported equations shown in Table I, it is difficult to identify the rela-tive contribution of active transport to total CO2 influx.

In the derivation of equation (1), it is assumed that the resistance to CO2 dif-fusion is similar in either direction across the cell membrane, or k\ = k3 (Francoisetai, 1993). This assumption originally came from the expectation that resistanceto CO2 diffusion through the stoma of a plant leaf would be the same in bothdirections (O'Leary, 1981). In the case of phytoplankton which may have a CCM,one may expect different values for this resistance (k^ * k3), probably (kt > k3).If that is so, the fractionation equation

M 1 ) ^ - (6)

may provide some measure of the contribution of active transport. Equation (6)is the equation just before assuming fcj = k3 in deriving equation (1). Ifwe assume,as many authors have, that the resistances to CO2 diffusion in both directionsacross the cell membrane are the same (symmetric permeability), a fractionationequation is required to express the decrease in fractionation with the increase inthe relative contribution of active transport (/) as some function off. Practically,/and kx * k3 may have the same meaning for CO2 acquisition by phytoplankton,although their physiological connotations must be determined by further study.

Thus, we may expect that the active transport of inorganic carbon by CCM maybe dealt with as a homologue of the asymmetric permeability of the cell mem-brane for CO2.

Deviation of fractionation equations involving active transport

In this section, we develop fractionation equations expressing the active trans-port of inorganic carbon. A wide range of phytoplankton can actively transportCO2 and HCO3" (Burns and Beardall, 1987). However, the presence of internaland external carbonic anhydrase (CA), which catalyses the equilibrium betweenCO2 and HC03", complicates the determination of the inorganic carbon speciescrossing the cell membrane. Concerning the isotope ratio of the substrate forphotosynthesis, the difference in inorganic carbon species makes for a consider-able variation in the fractionation factor, because fractionation between dissolved

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CO2 ([CO2]aq) and HCO3~ is as hifih a s 10%° m b° t n equilibrium and CA-catalysed reactions (Deines et ai, 1974; Paneth and O'Leary, 1985). Fractionationequations will be developed below for two cases, in which transported carbon hasthe 813C value of either bulk [CO2]aq or HCO3~. Isotope fractionation in the activetransport step is not considered.

(i) Active transport of CO2. The 813C value of actively transported inorganiccarbon is assumed to be the same as that of Ce (Figure 1). Extracellular CA maycontribute to the conversion of HCO3" t o CO2

a t t n e ce^ surface.At steady state:

^ = fcjCe + F4 - (k2 + A:3)Ci = 0 (7)

The relative contribution of active transport (/) is denned by:

'=WTTK <8)

If 0 <>f< 1, equation (7) can be rewritten as:

^ - ^-f fcjCe - (k2 + A:3)Ci = 0 (9)

Overall fractionation is:

a = 1 +A*! + (A/c2 - A/:,)(l - / § - (10)

assuming the same/value for 12CO2 and 13CO2, and Afcj = A/:3 (see Appendix 1).

Equation (10) is the same as equation (6) when —-?is substituted for (1 - / ) . This

supports the expectation that active transport might be dealt with as a homologueof the asymmetric permeability of the cell membrane for CO2. Leakiness, X (theratio of efflux to influx of DIC; Berry, 1988), is expressed as follows:

(Ce) k, f (Ci) k, 1CO, « I ' CO, • Org. C I

tC°1* Phytoplanktoncell /

Fig. L Scheme of active transport of CO?. The 813C of actively transported carbon (CO2*) is assumedto be the same as that of CO2 in medium (Ce).

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When all carbon is transported by active transport (/= 1), kxCt would be zero.In this case, one cannot substitute / = 1 in equation (10), because the denomina-tor in equation (9) becomes zero. Then, a becomes:

fair- _ Mr. lr-O\a= 1 + -

+ 1 F4

- A * i ) ^ - (12)

X is not zero, but

X ^ (13)

(ii) Active transport of HCOf. In the scheme shown in Figure 2, transportedcarbon has the same 813C value as HCO3~. Although the steady state for Ci isexpressed by the same equation as equation (7), the overall fractionation equa-tion is quite different from equation (10):

- X) + (Afr + 1)(M2 + 1)X

where Afc4 denotes the fractionation in the CO2-HCO3" dissociation process (seeAppendix 2). Definitions of /and X are the same as those in the active transportof CO2.

Assuming that the second- and third-order terms of A/c, are negligible, and thatonce again AAj = AJt3, then a is approximated as follows:

a = 1 + A*,( l- / ) + (A*2- A*!)(l - / ^ M 4 / (15)

Org. C

Phytoplankton cell /

Fig. 2. Scheme of active transport of HCO3-. The B13C of actively transported carbon (HCO3-*) isassumed to be the same as that of HCO3" in medium.

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Phytoplanktonk carbon isotope tractionatktn

W h e n / = 1, a becomes:

a = 1 + (AA:2 - A f c O ^ - - AA:4 (16)PA

From equation (15), it was deduced that all fractionation steps including overallfractionation would be affected by /. The difference between equations (10) and(15), or (A&i + A&4)/, corresponds to the difference in 813C values between CO2

and HCO3~. Equation (16) implies that overall fractionation decreases by (A£j +A/c4), in comparison with the passive diffusion model, equation (6), when allcarbon derives from the active transport of HCO3~ (/= 1). Therefore, it was sug-gested that the approximations in this model did not invalidate the scheme ofcarbon assimilation in Figure 2. These equations indicate that overall fractiona-tion from [CO2]aq to organic carbon may be less than unity under some con-ditions.

Results

Field observations

POC at a 2 m depth in Lake Kizaki increased during April-May 1992, thendecreased (Table III). Peridinium bipes predominated in the biomass throughoutthe observation period. The in situ gross production rate (Pg) and chlorophyll(Chi) a content changed with POC, although Pg was mainly affected by lightintensity. Pg per unit of Chi a ranged from 0.03 to 0.19 mol C g"1 Chi a tr1.

The DIC concentration gradually decreased from 300 to 160 uM. Because ofan increase in pH of the lake water, the fraction of [CO2]aq in DIC decreasedmarkedly by early May (Table III). On June 3, 10 and 23, and July 6, pH valuesmeasured by a pH meter were unavailable. However, the measurements bycolour indicator showed little change in the pH value (9.0-9.4) on those days.Furthermore, pH meter measurements made on May 25, June 15 and July 21 alsoshowed little change during June-July (Table III). Judging from these obser-vations, it was estimated that the pH value remained constant at 9.4 from June 3to July 6. The [CO2]aq concentration was extremely low (<0.2 uM) after May 25.

The 813C value of POC increased to -18.5%o during April-June, although themaximum levels of 813C were l-2%o lower than those found in previous years(Yoshioka et at., 1989; T.Yoshioka, unpublished data). After June, the 813C ofPOC maintained a constant level as high as -18.5%o. Since Pg varied with PFD onsampling dates, the temporal change in the 813C of POC did not follow that in Pg.The 813C of POC seemed to change with DIC and [CO2]*, (Table III).

A clear hyperbolic relationship was found between [CO2]aq concentration (Ce)and the 813C of POC (Figure 3). It seemed that carbon isotope fractionation mightbe controlled by [CO2]aq during the observation period. The fractionation factor(a), calculated from 813C values of [CO2]aq and POC, ranged from 1.013 to 1.002(Table III). In general, a was calculated from the carbon isotope ratio of POMand [CO2]aq on the sampling data. It should be noted, however, that the frac-tionation equation assumes a steady state, while the environmental conditions for

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Table i n . Summary of field observation in Lake Kizaki in 1992

Date

April 9April 24MaylMay 6May 12May 16May 25June 3June 10June 15June 23July 6July 21

WT(°C)

8.409.18

10.1210.4612.04143014.5916.6118.0418.3818.2220.3221.49

pH

7.688.45

8.989.289.199.559.4>9.4"9.449.4'9.4*9.39

Chlo(MgH)

17.426.018319.523.152.682.174.920.1

47.760.0153

PFD(uE nr2 s-')

24.236.1

145234

6.1165183390

65.7150162

P g

(MM h-1)

1.51.12.12.01.55.46.33.8

1.52.01.9

DIC

334300298268270252213191202194178157171

COz*

19.93.11

0.780.370.410.140.180.180.160.160.140.15

, POC

(MM)

no178108128144297467408125

26819789

DIC

-7.1-5.8-6.0-6.1-45

-5.8-5.1-6.3

-6.4-7.4

co^[813C

-16.5-15.6

-15.9-14.1

-15.1-14.3-15.3

-15.4-16.2

POC

-28.7-24.3-23.5-22.1-20.6-20.0-19.2-18.6-18.6

-18.3-18.5-193

a

1.012591.00884

1.006351.00660

1.004201.004421.00334

1.002951.00233

Blank, not determined.•Estimated pH values from the measurements by colour indicator.

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-15.0

-20.0

-2S.0

-30.00 5 10 15

[COJ.,(nM)

Fig. 3. Relationship between 813C of POC and [COJ,, concentration in Lake Kizaki.

phytoplankton are not always at a steady state in nature. The variation in suchconditions during the growing season may lead to an overestimation of a. Never-theless, calculated a values in Lake Kizaki were quite low, particularly in Juneand July, which suggested that photosynthetic carbon uptake would be limited bythe CO2 supply.

Application of the fractionation equation

When a and Ce are known, kt Ci and (1 -/)Ci are analytically estimated as com-bined parameters, from equation (6) and equation (10), respectively. However,algebraic analysis using equation (15) is difficult, because Ci and/appear in someterms in the equation. Other independent data on Ci or/are required to use equa-tion (15), although such data are usually difficult to obtain from the natural eco-system. When HCO3" is actively transported, overall fractionation apparentlydecreases, compared with CO2 active transport. If HCO3" transport occurs, thecontribution of CCM may be smaller than that estimated by equation (10).However, it is difficult to estimate the carbon acquisition mechanism of naturalphytoplankton over relatively long periods, such as a week and a month.

The physiological implications of carbon isotope fractionation by phyto-plankton calculated with equation (10) are discussed, using data from cultureexperiments by Hinga et al. (1994) and from the field observations. We used thefollowing values for fractionation factors of diffusion, Akx = 0.0007, and car-boxylation, Mc2 = 0.029.

(i) Culture experiments (Hinga et al, 1994). Hinga et al. (1994) have presented asystematic data set on carbon isotope fractionation by phytoplankton culture.Each plot in Hinga's Figure 4, which showed the relationship between ep and Ce

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in the culture experiments, was read using digital slide calipers. Before analysis,the value of ep was transformed to a value for a.

From equation (10), we obtained the following relationship between (1 - /)CiandCe:

(17)

The data analysis showed that there were intercepts (/ 0) for linear regression for(1 -/)Ci and Ce, as follows:

At 9°C, (1 -/)Ci - 0.69Ce - 2.6, r2 = 1.000At 15°C, (1 - /)Ci = 0.60Ce - 2.1, r2 = 0.992At 25°C, (1 - /)Ci = 0.69Ce - 3.8, r2 = 0.999

Fractionation factors for each temperature were obtained as follows:

Ce-3.8At 9°C, a = 1.00O7 + 0.0283 X 0.69 X

At 15°C, a = 1.0007 + 0.0283 X 0.60 X

At 25°C, a = 1.0007 + 0.0283 X 0.69 X

CeCe-3.6

Ci

Ce-5.6

(18)(19)(20)

(21)

(22)

(23)

Hinga's data fairly matched with these fractionation equations (Figure 4).These equations suggested that the isotope fractionation due to carboxylation by

120

^ Relationship between fractionation factor (a) and [CC^],,, concentration vi the culture experi-ments of S.costatum (Hinga el aL, 1994). Symbols indicate culture temperatuie: open circle, 9°C;closed triangle, 15°C; closed circle, 25°C. lines denote the fractionation equations for each tempera-ture; 9°C, dotted line equation (21); 15°C, dashed line equation (22); 25°C, solid line equation (23),see the text

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1.015

1.010

1.005

1.0005 10 15

[CO.Ml'M)

20

Fig. 5. Relationship between the fractionation factor (a) and [CC^]^ in Lake Kizaki. The solid linedenotes equation (24). The broken line denotes equation (25).

RUBISCO might not occur below a certain low Ce level, such as 3.8 uM at 9°C,3.6 uM at 15°C and 5.6 uM at 25°C.

(ii) Field observation (Lake Kizaki). Equation (10) was also applied to the dataset from Lake Kizaki, and the following equation was obtained:

a = 1.0007 + 0.0283 X 0.42 XCe - 0.26

Ce(24)

The estimate of a from equation (24) does not agree with the a at low Ce con-centrations (Figure 5). Based on the data from April 24 to July 6, we obtained thenext equation:

a = 1.0007 + 0.0283 X 0.29 XCe-0.13

Ce(25)

In both cases, it suggested that the fractionation in carboxylation step did notoccur under a low Ce concentration of <0.3 uM.

Discussion

Implication of combined parameter, (1 -f)Ci

Hinga et al. (1994) analysed their data using Rau's model (Rau et ai, 1992), andfound that fractionation data distributed across the contour of CO2 demand(Ce - Ci). They suggested that p-carboxylation was expected at a high Ce con-dition, because there was no reason why CO2 demand should increase with anincrease in Ce. According to Rau's model, a linear relationship with the slope ofunity will be expected between Ce and Ci, assuming constant CO2 demand

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constant CO, demand

variable CO, demand

m tCO, demand

Ce

Fig. 6. Schematic diagram showing the relationship between Ce and Ci. Solid lines show the constantCO2 demands. The broken line shows the variable CO2 demand with Ce.

(Figure 6). In our analysis using Hinga's data, even if / = 0, the slopes of Ce-Cirelationships do not change from equations (18), (19) and (20), and are less thanunity. One may conclude that CO2 demand changes with Ce in a linear fashion.However, the mechanism is not suggested by the diffusion model.

The CO2 demand, according to the definition by Rau et al. (1992), correspondsto the Ce level below which the isotope fractionation derived from carboxylationby RUBISCO does not occur. Therefore, it is suggested that the subtrahends (3.8,3.6, 5.6, 0.26 and 0.13) in the numerators in equations (21)-(25) represent CO2

demand. The coefficients of numerics (0.69, 0.60, 0.69, 0.42 and 0.29), except for0.0283, may indicate the relative contribution of diffusive transport of CO2, or(1 - / ) . Thus, the apparently variable CO2 demand with changing Ce (Figure 6)can be expressed by (1 -f) < 1 for some CO2 demand. Although the physiologi-cal judgements of these considerations may be very difficult, characterization ofphotosynthesis by culture and natural phytoplankton population may beachieved by the combined parameter, or CO2 demand and /.

Characterization of phytoplanktonic photosynthesis using CO2 demand and fvalue

From equations (21)-(23), it was suggested that carbon assimilation by S.costa-tum might operate under almost constant CO2 demand (-4-6 uM), and that theactive transport of CO2 contributed -30-40% of the total carbon influx. If 10%of the total carbon uptake were mediated by p-carboxylation (A&2 = 0.027), therelative contribution of active transport would reach 25-35% without any changein CO2 demand.

In Lake Kizaki, since P.bipes predominated during the observation period(data not shown), equation (25) may reflect the physiology of this alga. The CO2demand is considerably lower than that of S.costatum. The relative contributionof CCM (J) was estimated to be 60-70% of total carbon uptake. Berman-Frank

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et al. (1994,1995) reported that the CA activity of Peridinium gatunense in LakeKinneret was stimulated by low [CO2]aq concentrations (1-10 uM). Although thespecies of Peridinium and the level of alkalinity (or DIC concentration) are differ-ent from those in Lake Kinneret, the CA activity of P.bipes in Lake Kizaki mayalso increase under low [CO2]aq concentrations. The high value of / in LakeKizaki might reflect an intense CA activity of P.bipes.

The CO2 demand and/were calculated using the published data (Table IV).While /values in freshwater lakes were significantly higher than those in marineenvironments (r-test, P < 0.05), CO2 demands were significantly lower (P < 0.01).Negative CO2 demand estimated for Mohonk Lake may indicate the predomin-ance of CCM, or may be caused by species succession and physiological changesin phytoplankton attributable to a significant increase in water temperature(11.1-25.6°C) and pH values (6.92-9.50). In a similar context to the latter case,the organic matter of diatom and other algae on April 9 in Lake Kizaki mighthave caused a discordance in the ot-Ce relationship, as shown in Figure 5.Alternatively, CA activity (or CCM) of the algal community might have changedduring April 9 and April 24.

Carbon isotope fractionation of phytoplankton is thought to be affected byvarious factors other than [CO2]aq. The relationship between carbon isotope dis-crimination and the growth rate of phytoplankton was found in both culture andenclosure experiments (Fry and Wainwright, 1991; Takahashi et al, 1991; Laws etal, 1995). Since the growth rate of phytoplankton depends, in general, on tem-perature, nutrient concentration and light intensity, discrimination also dependson these physicochemical parameters. The difference in the 813C values bothwithin and between species of phytoplankton may indicate the metabolic per-formance among phytoplankton (Fry, 19%). Therefore, it has been recognizedthat the 813C value of organic matter derived from phytoplankton may not becontrolled solely by [CO2]aq concentration (Thompson and Calvert, 1994; Lawset al, 1995; Johnston, 1996).

The fractionation equation itself, based on a physiological model, does notimply that [COJaq controls the fractionation factor, but rather that it is deter-mined either by the relative ratio of CO2 concentration at the carboxylation siteto ambient CO2 concentration, or by the efflux-influx ratio of CO2. Factors suchas growth rate, temperature and light intensity can be considered to modify theseratios. Since there are interspecies differences in such factors as growth rate,photosynthetic activity and CA activity, the discrimination between [COJaq andPOC may vary from season to season and from place to place, independently of[COJaq concentration. The [COJaq may not be estimated by the POC and sedi-ment carbon isotope analysis (Francois et al, 1993). However, when a high cor-relation between the combined parameter (1 - /)Ci and [CO2]aq concentration isobserved, these factors seem to remain constant or have little effect on carbonisotope fractionation in comparison with [COJaq concentration. If such situationsare to be expected at specific times and places in aquatic environments, the com-bined parameter (1 -/)Ci, proposed in this paper, would prove its usefulness incharacterizing phytoplankton photosynthesis and in estimating [COJaq.When thecorrelation is low, species succession among phytoplankton with distinct

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TaMe IV. Comparison of calculated /and CO2 demand using equation (10)

Marine

Skeletoncma costatumb

Delaware Bay

Southwest Indian Ocean

Freshwater

Mohonk LakeLake Suwac

9°C15°C25°CSpringSummer

July 6-September 12,1986June 19-August 18,1987

Lake KizakiApril 9-July 21,1992April 24-July 21,1992

[COJ^ (uM)

-5-50-5-110

-10-12014.2-19.411.8-22.79.8-21.6

0.5-35

0.13-2.20.15-3.6

0.14-200.14-3.1

1.0000.9920.9990.3320.9410.962Average ± 1 cr

0.981

0.9900.941

0.9980.997Average ± 1 a

f

0.310.400.310.650.280.24037 ± 0.14

0.42

0.860.85

0.580.710.67 ± 0.19

CO2 demand (uM)

3.83.65.63.44.05.24.4 ± 0.90

-0.54"

0320.64

0.260.130.29 ± 0.21

Remarks

Hinga etal, 1994

Fogel etai, 1992

Francois etal., 1993

Herczeg and Fairbanks, 1987Takahashiefa/.,1990Microcyslis spp.Microcystis spp.This studyDiatom and P.bipesP.bipes dominate

"Correlation coefficient between Ce and (1 - / )Ci .bData were estimated from Figure 4 in Hinga et al. (1994).c[CO2]»q was recalculated from original data because the [CO?]*, concentration was not shown in Takahashi etal. (1990). Mass balance consideration was notapplied for the calculation of the parameters.dData were not used for calculating the average.

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photosynthetic characters, and input of organic carbon with different 813C value,may have occurred. Therefore, the isotopic analyses of individual species andspecific biomarkers for phytoplankton would be important.

Analysis using the new fractionation equation should improve our under-standing of phytoplankton ecophysiology and the biogeochemical carbon cycle inaquatic environments, although the physiological meanings of the combinedparameter must be rigorously checked in future studies.

Acknowledgements

I am grateful to Prof. H.Hayashi and Prof. E.Wada for their encouragement. I wishto thank the many students of the Faculty of Science of Shinshu University, especi-ally Mr K.Matsushima, for their assistance in the field survey. I am also grateful toProf. Y.Saijo for allowing the use of his facilities in Lake Kizaki. This study wassupported by a Grant-in-Aid for Encouragement of Young Scientists, nos 01740378and 02854079, from the Ministry of Education, Science, Sports and Culture, Japan.

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and the annual dinoflagellate bloom in Lake Kinneret. LimnoL Oceanogr., 39,1822-1834.Berman-Frank.I., Kaplan,A-> Zohary.T. and Dubinsky,Z. (1995) Carbonic anhydrase activity in the

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Received on April 1,1997; accepted on June 3, 1997

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Appendix 1: Derivation of equation (10)

From equation (9):

ratio of Ci is:

k2 + k

k\

Ce'3 ' ( 1 - / 0

Ce (ii)

(I"/)

where a prime (') means the term for 13C.Assuming f = f, then:

kV Ce'

13C/12C ratio of Ce isCe

k2 + k3

k\' \ k2

, k3' k3

where A/c; = —- - 1 or AA:, = a, - 1.

k2' Ci' 1^C/iZC of organic product is R- = _,. = —r R Q (V)

From equations (iv) and (v), the overall fractionation factor a is as follows:

(vi)

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T.Yosfaioka

Assuming A/CJ = Ak3, then:

Since (k2 + k3)Ci = ^Ce + F4,

/* 4(vii)

Leakiness, — - 2 — _ = (1 _ / ) - £ _ _ (vm)K]Ce + r4 «! Ce

From equations (vii) and (viii):

. , , . - k* Ci (ix)

If the resistances to CO2 diffusion are the same in both directions across the cellmembrane (k\ = k3), equation (ix) becomes as follows [equation (10) in the text]:

Ak1)(l-f)^ (x)

Appendix 2: Derivation of equation (15)

At steady state:

^ - = klCe + F4-(k2 + k3)Ci = 0 (i)

~ Jt2' + k3'

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Ptaytopbuiktoiiic carbon isotope fractionatkm

k2' k2 k£k£_ k3 yfaCe ACe F± , , .k2 k2 + k3 k3 k2 + k3) {k^Ce k^Ce + F4 F4 k}Ce + F

k Ci k-Equation (iii) is substituted by leakiness X = 3 and Ak, = —^ - 1.

k2d + K3Q Ac,-

Then:

From equations (iv) and (v):

(M3 + 1)(1 - / ) + (M, + 1)(M3 + 1)(M4 + 1)/

Equation (vi) is the same as equation (14) in the text.Assuming Ak^kj = 0 and AkjAkjAkk » 0:

_ 1 + Mj + Ak3 + (Ak2 - Ak3)XAk3 + (A*, + Ak4)f

Aki 4- (Ak2 - MC3)*- (Akt + Ak.,)/

1 M (M, + Ak4)f

(AJt2-AJt3);r-

3 (v)"4

-X) + (Ak1 + 1)(M2 + 1)X(vi)

(Akl + Ak4)f

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XYoshioka

I

I

1a « 1 + AA:j + (Ak2 - Ak3)X - (Akx + Ak4)f

From Akx = Ak3:

a * 1 + Mj + (A£2 - A^^Z - (AA:j + Ak^f (vii)

t CiSince X = 3 (1 - / ) , then equation (vii) becomes as follows:

+(AJt2 - M j ) ^- (l-f)Q—(Aki + Ak4)f

^ ^ (viii)

If kx = k3, then:

Equation (ix) is equation (15) in the text.

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