Fractionation of stable carbon isotopes by marine phytoplankton

Fractionation of stable carbon isotopes by marine pbytoplankton WILLIAM W. WONG and WILLIAM M. SACKETT Department

of Oceanography, (Receioed

14 April

Texas A & M University, College Station. TX 77843. U.S.A 1978; accepted

m recised form

4 August

1978)

Abstract-Stable carbon isotope fractionation by seventeen species of marine phytoplankton, representing the classes of Bacillariophyceae, Chlorophyceae, Prasinophyceae, Chrysophyceae, Haptophyceae and Dinophyceae have been determined in laboratory culture experiments using bicarbonate enriched artificial sea water. The AHCo; values (AHco, = 613C of algae vs HCO;) range from -22.1 to -35.5”&,. Nitzschia clusterium shows the smallest fractionation of -22.1%, and Isochrysls galbana, the greatest of -35.5%,,. Since these algae were cultured under identical laboratory conditions, the wide range ofA Ku; values is seemingly due to the presence of different metabolic pathways within these orgamsms. A temperature dependent fractionation of 0.36:&, per “C with decreasmg temperatures was measured for Skektonema costatum whereas, smaller temperature dependencies of -0.13. +0.15 and -0.07”‘W per “C were observed for Dunaliella sp., Monochrysis lutheri and Glenodinium foliaceum, respectively. The consistency of AHCOTvalues of Skeletonema costatum, Dunaliella sp. and Monochrysis lutheri grown at salinities of 22, 26, 32 and 36& indicates that natural salimty varlatlons have negligible effects on the Isotopic composition of marine phytoplankton.

INTRODUCTION TERRESTRIALplants can be separated into three categories: C3, C, and CAM (Crassulacean Acid Metabolism) plants. C3 plants have 613C values (vs PDB) ranging from -24 to -34”~ and C, plants. -6 to - 19’6, (SMITH and EPSTEIN, 1971). C, plants use the Calvin cycle for COZ fixation (BASSHAMand CALVIN, 1957). Ribulose-1,5-bisphosphate (RuBP) carboxylase, the enzyme which catalyzes the initial carboxylation of RuBP with the formation of two molecules of 3-phosphoglyceric acid (3-PGA) has been shown to be the primary site for stable carbon isotope fractionation in C, plants with an average Ace, (fractionation versus COJ value of -27.7”b0 (CHRISTELLER et al.. 1976; WONG et al., 1977, 1978). C4 plants use the C,-dicarboxylic acid pathway for the assimilation of atmospheric COZ (HATCH and SLACK, 1971). This pathway involves the initial carboxylation of phosphoenolpyruvate (PEP) with the production of a C, acid, oxaloacetic acid (OAA). The OAA is converted either to malate or aspartate and these are subsequently translocated and decarboxylated in the bundle sheath cells for the regeneration of PEP. The CO, released from the decarboxylation is fixed via the Calvin cycle within the bundle sheath cells. The enrichment of 13C in C4 plants has been attributed directly to the small fractionation of stable carbon isotopes by PEP carboxylase (WHELAN et al., 1973; REIBACH and BENEDICT, 1977; REIBACH et al., 1978), the closed system of Kianz anatomy characteristic of C, plants (LAETSCH, 1974), and the depression of RuBP-oxygenase activity by high CO2 concentration within the bundle sheath cells (BOWES et al., 1971). CAM plants have 613C values which fall within the

range of both C3 and C4 plants: - 14 to -34%” (BENDER et al., 1973). These plants fix CO, at night solely through the /3-carboxylation of PEP with an accumulation of malic acid. In the day time. the malic acid is decarboxylated either to pyruvate or PEP. The pyruvate and PEP are subsequently converted to starch whereas atmospheric CO1 and CO, released from the decarboxylation of malic acid are assimilated by RuBP carboxylase through the Calvin cycle. It is the ability of CAM plants to shift the major flow of CO* fixation via PEP or RuBP carboxylase in response to environmental changes and the direct relationship between /I-carboxylation and starch biosynthesis that apparently accounts for these plants having a wide span of 6’ 3C values (BLACK, 1976). In spite of the importance of marine phytoplankton in the marine life cycle, reasons for the IS:;, range in natural oceanic phytoplankton populations are not very well documented with variations being attributed directly to a temperature dependent discrimmation of “C from ’ *C by marine p h y to p lankton during photosynthesis (SACKETT rt al., 1965), a temperature associated carbon dioxide pool effect (DEGENS et al., 1968) and a temperature associated species effect (SACKETI. et a/., 1973). Laboratory studies on stable carbon isotope fractionation by two species of marine diatoms, a single species of green algae. and five species of blue-green algae have indicated that the A values of these marine phytoplankton vary from -% to -23.9” 0. (DEGENS et al., 1968: CALDER and PARKER, 1973; PARDUE et al., 1976). The AcoZ values vary depending on the algal species and culture conditions. The purpose of this study was to examme the effects of species composition, temperature and

1809

1810

W. W WONG and W. M. SACKETT

salinity on the stable carbon isotope compositions of marme phytoplankton under well-defined laboratory conditions. MATERIALS

AND

METHODS

Seventeen species of marine phytoplankton representing the classes of Baclllarlophyccae. Chrysophyceae. Haptophyceae, Chlorophyceae. Dlnophyceae and Praslnophyceae were cultured m one-hter aliquots of an artificial sea water medium with a final bicarbonate concentration about ten times the 2 mM found In natural sea water. The excess bicarbonate minimized lsotoplc changes in the substrate during the experiments and seemingly did not affect the viability of the cultures. These algae were umalgal cultures and were by no means axenic. However. only healthy cultures exammed under the light microscope and freshly sterlhzed media and glassware were used for the experlments. The pHs of the media were adjusted to 7.6 at the beginning and remained within kO.2 unit for the duration of the experiments. The culture vessels consisted of 2-liter Erlenmeyer flasks with stoppers containing two sections of glass tubing: one a screwcap tube to provide easy access to the cultures. the other connected to an ascarlte absorption tube to prevent exchange of CO? between the outside and Inside atmospheres. For all species studies the sahnities of the culture media were adjusted to 26”<,,, and the culture chamber was maintained at l8’C. A constant ~llumination of approx 60 microelnstems/M’/sec was provided by two Sylvania I5 W cool white fluorescent tubes mounted on a reflector. The algae together with the media were harvested from the culture vessels at different mtervals after visible growth had been established. The algae were separated from the media by filtration through two Gelman type A/E glass fiber filters. The algae retained by the filters were stored at -20°C until ready for combustion. The bicarbonate in the filtrates was prepared Immediately for 6°C analysis. The relative growth rate of each organism was determined by measuring the particulate orgamc carbon as a function of time for aliquots of each culture.

For the temperature experiments, the culture chamber was adjusted to 18. 24 or 30°C accordingly. However, for the salinity experiments, the culture chamber was kept at 18°C and the salinities 26. 32 and 36”,,

of the media

were adjusted

to 22,

RESULTS

The organic carbon m algal cells was converted to CO2 by combustion over cupric oxide at 85o’C m an oxygen atmosphere (CRAIG. 1953). Inorganic carbon in the culture media or filtrates was converted to CO1 by aadification with 85’; phosphoric acid after the evacuation of atmospheric gases from the reaction vessels. The purified CO2 was analyzed with a Nier-McKmney type mass spectrometer (MCKINNEY et a/.. 1950) and the results are reported as 6’jC values which are defined as follows.

6°C (“<,,,)=

(WLLC)sample _ 1 x (1’C/‘2C)

standard

1o3

I

sample (CRAIG. peaks. mass 44 tailing. and “0 contribution to mass 45 were made m the manner prescribed by CRAIG (1957). The workmg standards for organic and inorganic samples were Nori; a powdered charcoal sample (613C = -24.8”<, vs PDB), and NBS-Solenhofen limestone No. 20 (613C = - l.l”,,,, vs PDB). respectively. where

the standard

IS the Chicago

PDB

1953). Corrections for gas mixing, background

DISCUSSION

Data were treated as illustrated by Coscir~odiscus astermophalus in Fig. 1. The figure shows a plot between the S’%Z values for cells of Coscinodiscus and the corresponding 613C values for the dissolved bicarbonate in the filtrates. In order to avoid the time functionality of the 613C values of the algae and the dissolved bicarbonate, the initial fractionations, A HCO The AHcOT value. , values, were calculated. characteristic of each organism under the specific culture conditions. is the difference between the imtial 613C value of algal cell and the appropriate 613C value of bicarbonate in the original culture media. The initial 613C values are the intercepts from linear regression analyses between the S’%I values of algal cells and the corresponding 613C values of bicarbonate in the filtrates as shown in Fig. 1. The intercept for Coscinodiscus asteromphalus is -33.5”~~ and so the AHcO, value is equal to - 33.5”,, minus - 5.3”,, or -28.2”, The value, - 5.3”,,, is the initial isotopic composition of bicarbonate m the culture medium of Coscrnodiscus. Based on the results of six replicate experiments using four different organisms. (1) Skelrfonerna cosfatum (a. -29.1, -28.8; b -31.0, -31.1): (2) Chaetoceros didymus (a. - 29.7, - 28.3: b. - 30.4. - 29.8); (3) Thalassiosira subtilis (- 29.1. - 29.5): and (4) Monochr~sis lutheri (- 36.0. - 34.2); the standard deviation for a given experiment is calculated to be 0.4”I,0 The results for the effect of species composition experiments are summarized in Table 1. The table identifies the algal species and their AHCOi and Ace, values. The AcoZ values are the differences between the initial Si3C values of algal cells and the 613C values of dissolved CO2 in equilibrium with the dissolved HCO;. The 6’ 3C values of the dissolved CO2 were calculated according to the equation of WENDT (1968):

6A-6B= Mass spvcrrornetricana/Jws

AND

10.2-(0.064x

7’)

where 6A and 6B are the 613C values of dissolved HCO; and CO* respectively and Tis the temperature in Celcius. According to the results in Table 1. the A Ha), Or ACO, values vary from species to species and differ by as much as 13.49;,. Nitzschia closterium has the smallest fractionation of -22.1”, and Isochrysis galbana, the largest of -35.5”,. Among the diatoms, values vary from - 22. lob0 (Nitzschia closterium) to -29.9”,, (Skeletonema costatum). With the exception of Cyclotella, the pennate diatoms seem to fractionate less than the centric diatoms. The haptophytes and chrysophyte appear to discriminate more against 13C than any other marine phytoplankton. Of the few plausible explanations for the species related differences in isotope fractionation, variability in growth rates IS an obvious first choice. Since all isotope fractionating processes show decreasing apparent fractionations in the total accumulated product versus the reactant as a particular reaction

Isotopic

composition

of marme

phytopiankton

1811

5 ::

6

e :: 00 -33r is !!I

(

INTERCEPT

t!

a 2

= - 33.5

%.

-34-

0

hm 2 -35 cx) -5.3

8&

8 ,;;C

VALUE

OF HCO;

IN

ORIGINAL

CULTURE

I

I

I

-4.3

-3.3

-2.3

VALUES

OF DISSOLVED

BICARBONATE

IN THE

MEDIUM

-1.3 FILTRATE

(%.)

Fig. I. A plot of the 61,3C values of the cells of Cosc~~od~scus usrrrornphalus vs the corresponding SL3C values for the dissolved bicarbonate in the filtrates. The algae were cultured in an artificial sea water medium with a salinity of 26”,,,, and a temperature of 18’C and under a constant rllumination of 60 mtcroeinsteins/M2/sec.

Table 1. Effect of species composition marine phytoplankton cultured under

on AHCO, and A,-d, values of identical laboratory conditions

BACILLARIOPHYCEAE Centric: Skeletonema -~ costatum Chaetoceros didymus Chaetoceros lorenzianus Thalassiosira -subtllis Thalassiosira pseudonana Coscinodiscus asteromphalus Cyclotellasp.

-29.9 -29.0 -26.7 -29.3 -26.7 -28.3 -25.4

-20.9 -20.0 -17.7 -20.1 -17.6 -19.2 -16.4

-22.1 -24.2 -26.0

-13.0 -15.1 -16.9

-31.9 -29.1

-22.9 -20.0

-28.9

-19.8

-35.5 -33.0

-26.4 -23.9

-35.1

-26.1

-27.2

-18.2

Pennate:

CHLOROPHYCEAE Dunaliella sp. Chlorococcum sp. PRASINOPHYCEAE Platymonas sp. HAPT0PHYCEJ.E Isochrysis galbana Coccolithus pelaglcus CHRYSOPHYCEAE Xonochrysis lutheri DINOPHYCEAE Glenodinium faliaceum

W. W. WONG and W. M.

1812

Thalassiosiro (-26.7 %,)

SACRETT

pseudonana

f FChoetoceros

0

2

4

6

a

AGE FIN

2. Relatwe

growth

curves of diatoms

53

I

Chlorococcum

sp

,-&F/4- ,Monochrvs~s

AGE,

DAYS

Fig 3. Relative growth curves of Dunaliella sp.,

Chlorococ-

cum sp., Monochrysis

lutheri, Coccohthus pelagicus, Isochrysis galbana, Glenodinium foliaceum and Platymonas sp.

The number inside the parentheses

are the AHCoI values.

12

14

(DAYS)

The numbers

g"r

IO

didymus

inslde the parentheses are the AHCO,

approaches completion, a fast growmg organism will show a smaller apparent fractionation relative to a slow grower if they both have the same mechanisms for CO, fixation, e.g. diffusion of HCO; 01 COz across a membrane followed by enzymatic fixation of transfered COz. In view of this rationale, the growth rates of each organism were determined by measuring the particulate organic carbon as a function of time for aliquots of each culture. As can be seen in Figures 2 and 3, there are marked differences in growth rates among these seventeen species of marine phytoplankton. However, there seems to be no correlation between the slopes of experimental curves and the measured fractionations, indicating that growth rates do not appear to affect the stable carbon isotope fractionation in our cultures. Nevertheless. it is Important for the readers to realize that different species of marine phytoplankton do have different optimal growth conditions. The culture conditions as described previously obviously do not represent the optimal growth conditions for all of the seventeen species of marine microalgae examined in these experiments. However, in order to arrive at comparable fractionation values exhibited by various species of marine phytoplankton, it is necessary to grow these algae under identical laboratory conditions such that the effects of envlronmental factors (temperature,

Isotopic composition of marine phytoplankton salinity, light intensity, carbon dioxide pool size or degree of aeration) on stable isotope fractionation during photosynthesis may be eliminated. Another plausible explanation for species related differences in isotope fractionation is the presence of different photosynthetic pathways in one group relative to another group of marine phytoplankton. BEARDALL et al. (1976) have examined the L4C-labeling patterns in five marine phytoplankton. Their results indicated that 3-PGA was the first metabolic product in the green flagellate, Dunaliella tertiolecta, in shortterm experiments. For the two diatoms, Skeletonema costatum and Phaeodactylum tricornutum (= Nitzschia closterium), the majority of 14C fixed was found in amino acids. The amount of labeling in C, acids varies from 22.2% in S. costatum to only 4.1% in N. closterium. However, 3.69; of the total 14C fixed was recovered in 3-PGA within 2 set m S. costatum and none in N. closterium within 10 sec. A haptophyte, Isochrysis galbana, and a dinoflagellate, Gonyaulax tamarensts, seem to have r4C-labeling patterns intermediary between the diatoms and the green alga. Since HCO; is the active species of ‘CO,’ utilized by PEP carboxylase (COOPERet al., 1968; REIBACH and BENEDICT,1977), the amount of isotope fractionation for a typical C4 plant should be around -5 to - 10°O,(REIBACHand BENEDICT,1977; REIBACHet al., 1978). However, the AHCO, values for the seventeen species of marine phytoplankton listed in Table 1 vary from -22.1”,,,, for Nitzschia closterium to -35.5?&, for Isochrysis galbana. This range of AHCW values definitely does not agree with the expected AHCoYvalues of - 5 to - lo”,,, for C4 plants. Carbon dioxide has been shown to be the active species of ‘CO,’ utilized by RuBP carboxylase (COOPERand FXMER, 1969). The amount of fractionation (AC03 by RuBP carboxylase averages -27.77, (CHRISTELLER et al., 1976; WONG et al., 1977, 1978). With the exception of the haptophytes, chrysophyte and the green alga, Dunaliella sp., the Acol values of other marine phytoplankton fall below the amount of isotope fractionation by RuBP carboxylase measured in vitro. In order to explain the isotope fractionation exhibited by the marine phytoplankton, a re-examination of the mechanism for isotope fractionation in terrestrial C4 plants is in order. In C4 plants, ‘CO*’ in the form of HCO; is fixed initially by PEP carboxylase in the mesophyll cells. The first photosynthetic product, malic acid or aspartic acid, is translocated from the mesophyll cells to the bundle sheath cells and is decarboxylated to regenerate PEP. The COZ released from the decarboxylation is assimilated by RuBP carboxylase in the bundle sheath cells via the Calvin cycle. Since isotope fractionation between products and reactants in all fractionating processes diminishes and becomes negligible as all the reactants are being converted to the products, the compartmentation of RuBP carboxylase in the bundle sheath cells and the PEP carboxylase in the mesophyll cells sur-

1813

rounding the bundle sheath cells in C, plants mimics a ‘closed system’ for COZ fixation, i.e. all the CO, fixed by PEP carboxylase in the form of C, acids are quantitatively translocated, decarboxylated, and re-fixed by RuBP carboxylase in the bundle sheath cells. The effect of ‘closed system’ on carbon isotope fractionation by Atriplex rosea, a C4 plant, and Antriplex patula sp. hastata, a C3 plant, has been demonstrated by BERRYand TROUGHTON(1973). In an open atmosphere, the Atriplex patula and Atriplex rosea have 6l 3C values of - 21.5 and - S.O& respectively, typical of C3 and C, plants. However, the 613C values of these plants become equal, approx O.O:&, in a closed atmosphere. Therefore the amount of isotope fractionation in C4 plants is determined primarily by the kinetic effect during the entrance of atmospheric CO2 into the leaves (PARK and EPSTEIN,1960) and by the enzymatic fractionation associated wtth PEP carboxylase (WHELANet al., 1973; REIBACHand BENEDICT,1977; REIBACHet al., 1978). It is rather obvious that the Kranz anatomy characteristic of terrestrial C4 plants does not exist in unicellular microalgae. Therefore the ‘closed system’ for CO2 fixation is not operable in marine phytoplankton even if they can use both PEP and RuBP carboxylases for CO* fixation. Consequently, the amount of isotope fractionation will depend on the magnitude of b-carboxylation and the amount of fractionation associated with RuBP carboxylase within the mdividual organism. Therefore, the Ace, or AHcOT values in Table 1 indicate that the Calvin cycle must be operative in these microalgae for the net synthesis of metabolites during photosynthesis. The small Ace, values in the pennate diatoms, Nitzschia closterium, Nttzschia frustulum, and Nitzschia curvilineata as well as the centric diatom, Cyclotella sp., and others, can now be rationalized as a result of extensive /?-carboxylation in these microalgae during CO* fixation. Therefore the amount of stable carbon isotope fractionation during photosynthesis in unicellular microalgae capable of using both PEP and RuBP carboxylase will depend on the isotope fractionation during the uptake of HCO; or CO, across the cellular membrane (PARK and EPSTEIN,1960), discrimination against 13C by PEP carboxylase (WHELANer al., 1973; REIBACH and BENEDICT,1977; REIBACHet al., 1978) and RuBP carboxylase (CHRISTELLER et al., 1976; WONG et al., 1977, 1978), and isotope fractionation associated with lipid synthesis (DENIROand EPSTEIN,1977). The 6°C values of lipids and fatty acids isolated from different organisms have been determined by PARKER (1962, 1964). His results indicate that the lipids or fatty acids are always more depleted in i3C (-4 to - 15x,) than the whole organisms. Therefore, it is possible that the variation in AHcoi or AC0 values among the seventeen species of marine phytof plankton as shown in Table 1 could be the result of an accumulation of 13C-depleted lipids in one organism as compared to another. Just for comparison, the chrysophyte (Monochrysis lutheri) has a

1814

W. W. WONGand W. M.

SACKETT

Table 2. Effect of temperature on the AHMj values of Skeletonema costatum. Dunalie/la sp.. Monochrpis lutherI and Glenodrnium foliaceum ORGANISM

TEMPERATURE OC

'HCO; "I..

"I.0 pc (High versus low temperature)

Skeletonema costatum

18 24 30

-29.9 -28.8 -25.6

+0.36

Dunaliella sp.

18 24 30

-31.9 -32.0 -33.5

-0.13

Monochrysis lutheri

18 24

-34.4 -33.5

+0.15

Glenodinium foliaceum

18 24

-27.2 -27.6

-0.07

A“co, value of - 35.1”(,,,;whereas the pennate diatom (Nitzschia closterium), -22.1?&,. If the lower AHcOi value of -35.1?&, is an indication of excess accumulation of lipids in M. lutheri, the diatom must have a lipid composition quite different from that of the chrysophyte. However, an examination of the pigment and fat contents of these marine algae (PARSONSet al., 1961) shows that the chrysophyte has less fat or pigment per carbon as compared to the diatoms. Interestingly, the fatty acid compositions between these two classes of marine algae are quite similar (WOOD, 1974). Therefore, the variation in AHcOTvalues among these marine phytoplankton cannot be accounted for simply by their lipid contents. This is inconsistent with the observation made by SACKETTet al. (1965) that excess lipids cannot explain the “C enrichment in natural plankton samples collected from water of about 0°C (-27.97~) as compared to those collected from water of about 25°C (-21.77,). Nevertheless, this does not eliminate the possibility that these microalgae have different routes for biosynthesis of lipids. The effect of temperature on the AHCOYvalues of four species of marine phytoplankton is summarized in Table 2. Only Skeletonema costatum shows a decrease in fractionation of approximately 0.36”, per “C with increasing temperatures. The other three species, Monochrysis lutheri. Dunaliella sp. and Glenodiniutnfoliaceum show much smaller effects with the latter two apparently showing a slight reversal. SACKETT et al. (1965) have measured the 6i3C values of natural oceanic plankton samples collected at various latitudes. Their results indicated that there is a decrease in isotope fractionation of 0.23”, per “C with increasing temperature. An increase in AHTO_values of 0.3”“,, per “C has also been observed by ‘DEGENS et al. (1968) in Cyclotella nana and Skeletonema costaturn bubbled with air (0.03% CO,). However, these authors did not detect any temperature effect on the AHcoi values of Cyclotella nana cultured in media bubbled with 5% CO2 or supplied with excess NaHCO,. A comparison of the AHCoYvalues of our

sp. cultured at 18°C (-25.4”J with their nana cultured at 20°C in a medium with 20 mM NaHCO, (- 26.0%,) indicates that our results are compatible wtth theirs. Considering that our standard deviation for a given experiment is 0.4”6, only one of the four species of phytoplankton tested here showed a significant temperature effect. Thus, on the basis of the experiments reported here and in DECENS et al. (1968) carbon isotope temperature effects during marine photosynthesis would seem to be minimal. The results for the effect of salinity on the stable carbon isotope fractionation of Skeletonema costatum, Dunaliella sp., and Monochrysis lutheri are compiled in Table 3. Since phytoplankton are passive drifters in the ocean, encounters with water masses of somewhat different salinities are not unusual. The effect of such changes on the isotopic composition of marine phytoplankton has not yet been examined. However, according to the results in Table 3, changes in salinity from 22 to 36”,, do not seem to have any effect on the AHCoFvalues of the three phytoplankton which were investigated. In conclusion. the 15”,,, range in 613C values observed in natural plankton samples can now be accounted for in terms of species composition, and the CO* pool size effect as observed by DEGENSet Cyclotella

Cyclotella

Table 3. Effect of salinity on AHCoT values of Skeletonema costatum. Dunaliella sp. and Monochrysis lutheri

Isotopic

composition

1815

of marine phytoplankton

al. (1968). Of course. the readers should be aware that the algal species we have used in our experiments are algae that have been adapted to laboratory conditions and can usually tolerate a wide range of salinity and temperature without any effect on their physiology or metabolism. In the natural environmentmany phytoplankton are stenothermal and stenohahne. If these organisms are transported to environments different from their optimal growth conditions, they would begin to deteriorate and of course, their normal metabolism would be altered. The effect of such drastic changes might have tremendous impact on the stable carbon isotope composition of marme phytoplankton and should not be overlooked.

of carbon

dloxrde.

Gro&rt%

C~~.s~?i~lch~})~Acta

12.

133-149.

DEGENSE. T.. GUILL~RD R. R. L., SACKETTW M. and HELLEFKJST J. A. (1968) Metabohc fractionation of carbon isotopes in marine plankton-l. Temperature and resptration experiments. i&y-Sea Re.5 15, l-9. DENIROM. J and EPSTEIN S. (1977) Mechamsm of carbon isotope fractlonatlon associated with hpld synthesis SLICIIC~~ 197, 261-263. HATCHM. D and SLACKC. R (1971) PhotosyntheticCO,fixatron pathways Atrn. Rec. P/ant Phptot. 21, 141-162. LAETS~H W. M. (1974) The Co syndrome: a structural analysts. Am. Rw. PIanr Physrol. 25, 27-52. MCKINIYEY. C. R., MCCR~A J. M., EPSTEINS.. ALLEN H A and UREY H. C (1950) Improvements in mass spectrometers for the measurements of small differences in lsotoprc abundance ratios. Rev. Sci. Instr. 21, 724-730. PARDUEJ. W.. SCALANR. S., VAN BAALENC. and PARKER Ack,toM’/udye,,lrrlts-We wish to thank GRETA A. FRYXELL P. L. (1976) Maximum carbon isotope fractionation m and E R COX for provrding the algal cultures. photosynthesls by blue-green algae and a green algae. The manuscript was crlttcally reviewed by W. L. ORR, Geochim. Cosmochf~~. Actu 40, 309-3 I2 S. R. SILVERMAX and B. N. SMITH.Thts work was supPARK R. and EPSTEINS. (1960) Carbon lsotopc fractionported by Natlonnl Science Foundation Grants GA41077 ation during photosynthesis. Geochlm Cosmochim. Acta and OCE75-13299 and the Robert A Welch Foundation. 21. 110-126 PARKERP. L. (1962) The isotopic composition of the carbon of fatty acids. Carnegie Inst. Wash. Yearb. 61, 187-190. PARKER P.

REFERENCES BASSHAM J. A. and CALVIN M. (1957) The Path of Curhon m Fhoto.s~~t~resis. Prenttce Hall BEARDALLJ.. MUKERJI D.. CLOVERH E. and MORRIS I (1976) The path of carbon m photosynthesis by marine phytoplankton. J. Phq.col 12. 409-417. BENDERM. M., ROUHANII., VINESH. M. and BLACKC. C. (1973) 13C/‘2C ratio changes in Crassulacean acid metabohsm plants. Plant Physioi. 52, 427-430. BERRYJ. A. and TROUGHTON J. H. (1973) Carbon isotope fractionation by C, and C, plants in ‘closed’ and ‘open’ atmosoheres. Carneate Inst. Wash. Yeurb. 73. 785-790. BLACK6. C (1976) Fr~ctiona~on of stable carbon isotopes during Crassulacean Acid Metabolism and the presentatlon of a unified concept of diurnal metabohsm in CAM plants. In The Fractionation of Stable Carbon lsotapes by Plants (ed. C. R. Benedict), pp. 51-73. Proceedings of a syrn~slurn held at Tulane University, New Orleans, Louisiana, 2 June 1976. Sponsored by the Southern Section of the American Society of Plant Physiologists and Cotton Incorporated. BOWESG.. OCREN W. L. and HAGEMANR. H. (1971) PhosphogIycoIate production catalyzed by ribulose drphosphate carboxylase. Blochem. Erophys. Res. Comm. 45, 716-722. CALDERJ. A. and PARKERP. L. (1973) Geochemlcal Imphcations of Induced changes m 13C fractionation by bluegreen algae. G~uc~I~~?~ Cosmoch~~~r. Acta 37, I33- 140. CHRISTELLER J. T.. LAING W A and TROUGHTO~J. H (1976) Isotope dlscrrmmatlon by rlbulose-l.5.-dlphosphate carboxylase. Plum Physlol. 57. 580-582 COOPERT. G. and FILMER D. (I 969) The active species of XYO,’ utihzed by ribulose d~phosphate carboxylase. J Brol. Cltem. 244. 1081-1083. COOPERT. C.. TCHEY T. T.. WOOD M. G and BENEDICT C R. (1968) The carboxylation of phasphoenolpyrurare and pyruvate. The active species of ‘COI’ uttlized by phospb~nolpyruvate carboxlklnase, carboxytransphosphorylase, and pyruvate carboxyiase. .!. Bioi. Chem. 243, 3857-3863

CRAIG H. (1953) The geochemistry of the stable carbon

isotopes.

Geochim.

Cosmochun.

Ana

3, 53-92.

CRAIGH. (I 957) Isotopic standards for carbon and oxygen and correction factors for mass-spectrometric analysis

L. (1964) The blogeochemistry of the stable Isotopes of carbon in a marine bay. Geochim. C’osmochlm. Acta 28, 1155-l 164. PARSONS T. R., STEPHENSK. and STRICKLANDJ. D. H. (1961) On the chemrcal composition of eleven species of marine phytoplankters. J. Fisheries Rea. Bourd Gun. 18, 100-1016. REXBACHP. H.

and BENEDICTC. R. (19773 Fractionation of stable carbon isotopes by phosphoenolp~ruvate carboxylase from Cr plants. Plant Physiol. 59, 564-568. REIBACHP. H., WONG W. W. and BENEDICTC R. (1978) The enzymatic fractionatron of H”CO; - H13CO; by phosph~nolpyruvate carboxylase. Plant Physlol. Suppl. 61, 110 (Abstract). SACUTT W. M .EADIEB. J and EXNFRM. E. (1973) Stable Isotope cornposItIon of organic carbon m recent Antarctic sediments. In Adrome. in Orytmrc Geochrmrsrry pp 661.-671. SACKETT W. M.. ECKELMANN W. R.. BEND&RM. L. and BE A. W. H. (1965) Temperature dependence of carbon isotope compositlon in marme plankton sediments. Scwncr 148, 235-237 SMITH B. N. and EPSTEIX S. (I 971) Two categorres of ‘3C,“ZC ratios for higher plants P/ant Ph~.~~o~. 47, 380-383. WENUT1. (1968) Fractlonatton of carbon Isotopes and its temperature dependence m the system CO,-gas-CO, m solution and HCO;-CO2 m solutton. Earrh Pkmet. Sci. Lrtters 4, 64-68. WHELANT., SACK~Z:~W. M. and BENEDICTC. R. (1973) Enzymatic fractionation of carbon isotopes by phosphoenolpyruvate carboxylase from C4 plants. PIant Physiol. 51, 1051-1054. WONG W. W. L.. BENEDKT C. R., MCGRATH T., and KOHEL R. J (1977) The fractionation of stable carbon isotopes by RuBP carboxylase. PIant Physiol. Suppl 59, 42 (Abstract). WANG W

W , BENEDICTC. R. and KOHEL R. J (1978)

The enzymatic fraction of ‘2COZ-‘3C02 by rtbulose-ISbisphosphate carboxyiase. Plant Phvsrol Suppl. 61, 109 (Abstract). WOOD B J. B. (1974) Fatty acids and saponifiable liprds. In Alyal Physiology and Brochemisrry (Ed. W. D. P. Stewart). Ch. 8. pp 236-265. Universrty of Cahfornia Press.