314

J. Phycol.

36,

314–320 (2000)

ACTIVE TRANSPORT OF CO

2

BY THREE SPECIES OF MARINE MICROALGAE

1

I. Emma Huertas

Instituto de Ciéncias Marinas de Andalucía (CSIC), Polígono Río San Pedro s/n, 11510 Puerto Real (Cádiz), Spain

Brian Colman

2

Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada

George S. Espie

Department of Botany, Erindale College, University of Toronto, Mississauga, Ontario, L5L 1C6, Canada

and

Luis M. Lubian

Instituto de Ciéncias Marinas de Andalucía (CSIC), Polígono Río San Pedro s/n, 11510 Puerto Real (Cádiz), Spain

The occurrence of an active CO

2

transport systemand of carbonic anhydrase (CA) has been investi-gated by mass spectrometry in the marine, unicellu-lar rhodophyte

Porphyridium cruentum

(S.F. Gray)Naegeli and two marine chlorophytes

Nannochlorisatomus

Butcher and

Nannochloris maculata

Butcher.

Illumination of darkened cells incubated with 100

m

MH

13

CO

3

2

caused a rapid initial drop, followed by aslower decline in the extracellular CO

2

concentra-tion. Addition of bovine CA to the medium raisedthe CO

2

concentration by restoring the HCO

3

2

–CO

2

equilibrium, indicating that cells were taking up CO

2

and were maintaining the CO

2

concentration in themedium below its equilibrium value during photo-synthesis. Darkening the cell suspensions caused arapid increase in the extracellular CO

2

concentra-tion in all three species, indicating that the cells hadaccumulated an internal pool of unfixed inorganiccarbon. CA activity was detected by monitoring therate of exchange of

18

O from

13

C

18

O

2

into water. Ex-change of

18

O was rapid in darkened cell suspensions,but was not inhibited by 500

m

M acetazolamide, amembrane-impermeable inhibitor of CA, indicatingthat external CA activity was not present in any ofthese species. In all three species, the rate of exchangewas completely inhibited by 500

m

M ethoxyzolamide,a membrane-permeable CA-inhibitor, showing that anintracellular CA was present. These results demon-strate that the three species are capable of CO

2

up-take by active transport for use as a carbon sourcefor photosynthesis.

Key index words:

carbonic anhydrase; CO

2

transport;mass spectrometry;

Nannochloris atomus

;

Nannochlorismaculata

;

Porphyridium cruentum

Abbreviations:

AZ, acetazolamide; CA, carbonic an-hydrase; C

i

, inorganic carbon; EZ, ethoxyzolamide

The dominant dissolved inorganic carbon (C

i

) speciesin air-equilibrated seawater is bicarbonate (1.8 mM at pH8 and 20

8

C), whereas the CO

2

concentration is very low(10 to 12

m

M) and the rate of the uncatalyzed conversionof bicarbonate to CO

2

is very slow at alkaline pH. Thelimitation in the supply of free CO

2

could severely restrictphotosynthesis in marine phototrophs, since CO

2

is thesubstrate of the principal carboxylating enzyme in theseorganisms, ribulose-1,5-bisphosphate carboxylase-oxyge-nase (Rubisco). Some marine phytoplanktonic specieshave been shown to possess mechanisms to increase theCO

2

level in the proximity of the active site of Rubisco(Falkowski and Raven 1997). CO

2

uptake may possiblyoccur by diffusion (Riebesell et al. 1993) or by active up-take from the bulk medium (Rotatore et al. 1995), ormay be derived from HCO

3

2

taken up by active transport(Merrett et al. 1996). Some marine microalgae possessan external carbonic anhydrase (CA), which catalyzes theinterconversion of HCO

3

2

and CO

2

at the plasmalemma(Burns and Beardall 1987, Dionisio-Sese and Miyachi1992, Colman and Rotatore 1995, Nimer et al. 1997, Rob-erts et al. 1997, John-McKay and Colman 1997), andmaintains a constant equilibrium concentration of CO

2

at the site of the CO

2

transporter (Williams and Turpin1987). In addition, the direct uptake of bicarbonate byan active transport system has been found in several spe-cies of marine phytoplankton (Colman and Gehl 1983,Rees 1984, Dixon and Merrett 1988, Colman and Rota-tore 1995, Merrett et al. 1996, Sukenik et al. 1997), andthe uptake of both forms of C

i

has been described in ma-rine diatoms (Colman and Rotatore 1995, Korb et al.1997).

The involvement of a CO

2

transporter in C

i

-accu-mulating mechanisms has been demonstrated by massspectrometry in cyanobacteria (Badger and Andrews1982, Miller and Canvin 1985, Miller et al. 1988),green algae (Sültemeyer et al. 1989, Rotatore and Col-man 1991, Rotatore et al. 1992), and freshwater andmarine diatoms (Rotatore and Colman 1992, Rota-tore et al. 1995). However, relatively few marine phy-toplankton species have been examined, and the oc-currence of active CO

2

uptake has not been reported

1

Received 9 August 1999. Accepted 20 December 1999.

2

Author for reprint requests; e-mail colman@yorku.ca.

CO

2

TRANSPORT IN MARINE MICROALGAE

315

in many marine microalgae. In order to gain insightsinto C

i

acquisition mechanisms in these organisms wehave studied CO

2

uptake in three species of oceanicphytoplankton.

Porphyridium cruentum

is a unicellular red alga thatcan occur in a wide spectrum of habitats; it has beenisolated from freshwater, brackish, and marine envi-ronments, as well as from the surface of moist soils.Several studies of this species have shown that all theCA is localized within the cell (Dixon et al. 1987, Ya-gawa et al. 1987a), with almost all the activity detectedin the chloroplast (Yagawa et al. 1987b); in contrast,Burns and Beardall (1987) found an appreciable ex-ternal CA activity in the same species. Lack of externalCA would indicate that the major C

i

flux across theplasma membrane is bicarbonate while its presencemight indicate that CO

2

is the principal species takenup. However, these discrepancies regarding the oc-currence of external CA between isolates belongingto the same species may be due either to differentmethods of growing the cultures or to phenotypicand/or genetic differences ( John-McKay and Colman1997). Strong evidence for the existence of a bicar-bonate transport system has been obtained by someauthors (Colman and Gehl 1983, Dixon et al. 1987,Burns and Beardall 1987), but there are no reports ofactive CO

2

uptake in this species.Studies on the physiology of inorganic carbon up-

take in the genus

Nannochloris

are scarce, and little isknown about the characteristics of C

i

assimilation. Ithas been shown that

N. atomus

and

N. maculata

lackexternal CA and have no capacity for active bicarbon-ate uptake but do have a high affinity for CO

2

and usecarbon dioxide as the preferred inorganic carbonsource for photosynthesis, although the mechanismof inorganic carbon uptake is unknown (Huertas andLubian 1998).

The aim of this work was to study the possible occur-rence of an active CO

2

transport system in three speciesof marine microalgae by monitoring CO

2

fluxes in cellsuspensions using the mass spectrometric technique.

materials and methods

Organisms and growth conditions. Porphyridium cruentum

(S.F.Gray) Naegeli

, Nannochloris atomus

Butcher, and

Nannochlorismaculata

Butcher were obtained from the ICMAN Culture Col-lection of Marine Microalgae and were grown in artificial sea-water (Harrison et al. 1980) supplemented with F/2 medium(Guillard and Ryther 1962) modified with double nitrate andphosphate concentrations. Cultures were grown in 500-mL sep-aratory funnels at 20

8

C and maintained in suspension by aera-tion (350

m

L

?

L

2

1

CO

2

in air) at a rate of 60 mL

?

min

2

1

. Suspen-sions were continuously illuminated at a photon flux density of75

m

mol

?

m

2

2

?

s

2

1

provided by a combination of cool-white andGro-lux fluorescent lamps (Sylvania).

Experimental procedure.

Cells were harvested by centrifugation(12,500

g

, 5 min), then washed twice with and resuspended inC

i

-free medium containing NaCl (450 mM), MgCl

2

(40 mM),KCl (10 mM), Na

2

SO

4

(10 mM), and CaCl

2

(5 mM) buffered atpH 8.0 with 25 mM TRIZMA base. Cells were concentrated to2

3

10

8

cells

?

mL

2

1

for

Nannochloris

species and to 4

3

10

7

cells

?

mL

2

1

for

P. cruentum.

Cell numbers were determined us-ing a hemocytometer (Neubauer). Cell suspensions were placed

in a thermostatted glass reaction vessel at 25

8

C and illuminatedat a photon flux density of 1000

m

mol

?

m

2

2

?

s

2

1

. Subsequenttreatments were always applied after the CO

2

compensationpoint had been reached.

Mass spectrometry.

CO

2

(

m

/

z

5

44 or 45) uptake and O

2

(

m

/

z

5

32) evolution by cell suspensions were measured using an aque-ous inlet mass spectrometer as described by Miller et al. (1988).Briefly, washed cell suspensions (6 mL) were placed in a glassreaction vessel (25

8

C), containing a magnetic stirrer, and thechamber was closed with a plexiglass stopper leaving no headspace. The stopper contained a capillary bore to allow for addi-tions during the experiments. The reaction chamber was con-nected to the ion source of the mass spectrometer by an inletcovered with a dimethyl silicone membrane that allowed dis-solved gases to pass freely, but not ionic species such as HCO

3

2

.The illuminated cells were allowed to consume residual C

i

inthe medium before experiments commenced. Inorganic C forexperiments was supplied to the cells from stock solutions ofK

213

CO

3

(300 mM), K

213

C

18

O

3

(300 mM), or

12

CO2 (4.3 mM) toyield the desired final concentration.

The ability of cells to transport CO2 actively was tested in twoways. The first method used the disappearance of 13CO2 fromthe medium upon illumination to determine whether a chemi-cal disequilibrium was created, as indicated by a rise in CO2 sub-sequent to the addition of 40 mg?mL21 bovine CA (Miller et al.1988). A second method utilized the disappearance of 12CO2from the cell suspension in the light and dark after the additionof a pulse of pure 12CO2 to yield an initial concentration of 4.3 mM.The difference between the light and dark control disappear-ance curves was taken as a measure of CO2 transport (Espie etal. 1989). Pure aqueous CO2 was generated by bubbling 45 mLice-cold acidified water (pH 2.0) with 5% v/v CO2 in N2 Themass spectrometer was calibrated for 13CO2, 12CO2, and O2 con-centration as described by Espie et al. 1989.

Carbonic anhydrase assay. The activity of CA was measuredmass spectrometrically using the 18O exchange procedure de-scribed by Miller et al. (1997). In aqueous solution, there is aloss of 18O from 13C18O18O caused by the repeated hydrationand dehydration of the inorganic carbon species. The ex-change of 18O from 13C18O18O (m/z 5 49) with 16O in H2O givesrise to transient amounts of 13C 18O 16O (m/z 5 47) and finallyyields 13C16O16O (m/z 5 45). Catalysis by CA accelerates the iso-topic exchange and is reflected in the time course of appear-ance and disappearance of the different 13CO2 species relative toa buffer control. CA assays were initiated by the addition 200 mM13C18O-labeled Ci to darkened cell suspensions and the timecourse of changes in m/z 49, 47 and 45 was determined with themass spectrometer. In some experiments, the effect of the CA-inhibitors acetazolamide (AZ, 500 mM) and ethoxyzolamide(EZ, 500 mM) on 18O exchange was also determined. Cells wereincubated in the dark at the reaction temperature with theinhibitor for 5 to 10 min before 200 mM 13C18O-labeled Ci wasadded. The presence of an extracellular CA was also inferredfrom the ability of an illuminated cell suspension to generateand maintain a chemical disequilibrium between CO2 andHCO3

2 during photosynthesis. Evidence for a disequilibriumwas indicated if the extracellular CO2 concentration rose after40 mg?mL21 bovine CA (Sigma) was added to the illuminatedcell suspension (Miller et al. 1988).

resultsCO2 uptake. The time course of CO2 uptake and O2

evolution by cells of P. cruentum suspended in me-dium at pH 8.0 was monitored over short periods ofphotosynthesis. The cells were allowed to reach CO2compensation point in the light; then the light wasswitched off and 100 mM Ci was added to the cell sus-pension. Upon subsequent illumination, there was arapid drop in the extracellular CO2 concentration,

316 I. EMMA HUERTAS ET AL.

which occurred before O2 evolution started (Fig. 1A).After about 2 minutes, the CO2 concentration de-clined gradually until the CO2 compensation pointwas reached. Upon darkening the cell suspension, theCO2 concentration in the medium rose at a rate toofast to be accounted for by the uncatalyzed dehydra-tion of HCO3

2, which indicates that CO2 was releasedby the cells (Fig. 1A). This post-illumination release ofCO2 indicates the presence of an intracellular pool ofunfixed Ci. The rate and magnitude of CO2 uptake byilluminated cells indicate that they created a chemicaldisequilibrium between CO2 and HCO3

2 in the me-dium; this is supported by the finding that the CO2

concentration sharply increased when bovine CA wasadded to the cell suspension (Fig. 1A). The rise inCO2 following CA addition also indicates that the cellslacked an extracellular CA capable of maintaining theCO2–HCO3

2 equilibrium.Changes in the concentrations of CO2 and O2 in

cell suspensions of N. atomus and N. maculata duringphotosynthesis were also monitored (Fig. 1B and 1C,respectively). The pattern of CO2 utilization by thesespecies was similar to that obtained in P. cruentum; inboth species there was a rapid decrease in the CO2concentration when photosynthesis was initiated byilluminating cells that had been incubated with 100mM Ci in the dark. This initial uptake was followed bythe same gradual decline observed in cells of P.cruentum, and both species accumulated an internalpool of Ci, as indicated by the rapid increase in theCO2 concentration in the medium when cells weredarkened. This post-illumination release was, there-fore, due to the efflux of CO2 from the unfixed intra-cellular Ci pool and declined over a 3–5 minute pe-riod as the pool was depleted.

The capacity of the cells to remove CO2 selectivelyfrom the medium was also established using a CO2pulsing procedure. The rise in the CO2 concentrationin the medium was monitored when 10 mL of CO2-saturated water (a CO2 pulse) was added to illumi-nated cells of P. cruentum (Curve b, Fig. 2) and com-pared with that occurring when CO2-saturated waterwas added to a buffer solution at the same pH (Curvea, Fig. 2). The disappearance of CO2 in the presence

FIG. 1. Simultaneous measurement of photosynthetic oxygenevolution (dashed line) and CO2 uptake (solid line) in suspen-sions of (A) Porphyridium cruentum, (B) Nannochloris atomus, and(C) Nannochloris maculata. Cells at the CO2 compensation pointwere provided with inorganic 13C at a final concentration of 100mM and CO2 uptake and photosynthesis were initiated by turningon the light. The same procedure was repeated once the inor-ganic C (Ci) was consumed in photosynthesis, but after 30 s of il-lumination CA was added at a final concentration of 40 mg?mL21.

FIG. 2. Measurement of transient CO2 uptake following aCO2 pulse. Buffer solution (a), illuminated cells of Porphyridiumcruentum (b), and darkened cells of the same species (c) at pH8.0 were pulsed with CO2 by the addition of 10 mL of waterequilibrated with a gas phase containing 5% CO2.

CO2 TRANSPORT IN MARINE MICROALGAE 317

of cells was more rapid than that in buffer, where thedecline in CO2 concentration is due to the formationof bicarbonate. These results show that the decrease inCO2 concentration was due to a selective removal ofCO2 from the medium. Furthermore, the selective up-take of CO2 ceased when cells were darkened (Curvec, Fig. 2). Similar results were obtained with both Nan-nochloris species (not shown).

CA activity. A qualitative mass spectrometric assaywas used to check for the presence of CA activity in in-tact cells of the three species. In control experiments,inorganic carbon initially in the form of K2

13C18O3 (200mM) was injected into buffer solution (pH 5 8) andthe time course of the spontaneous conversion of13C18O18O (m/z 5 49) to 13C16O18O (m/z 5 47) and13C16O16O (m/z 5 45) was monitored by mass spec-trometry (Fig. 3). In this system without cells, the m/z49 signal rose rapidly as the H13C18O18O2–13C18O18Oreached chemical equilibrium. This was followed bythe slow decline of the m/z 49 signal as 18O was lostfrom 13C18O to H2O during hydration–dehydration cy-cles, giving rise to the gradual appearance of the m/z47 and m/z 45 species of CO2. Comparisons were madebetween control rates and the rates of exchange of 18O-labeled species obtained with algal cells resuspended inthe same buffer but maintained in the dark. In P. cruen-tum cells (Fig. 4A), the interconversion of the differentCi species was accelerated: there was a rapid rise in them/z 47 and 45 signals and a lower than expected rise inthe m/z 49 signal as this species was the source of m/z47 and 45. This accelerated isotopic exchange indi-cated the presence of CA in the cell suspension, butthe production of m/z 47 and 45 was not sustained aswould be expected if an external CA were acting on

H13C18O18O18O2 in the suspending medium. This con-clusion was supported by the finding that the additionof 500 mM AZ, a weakly membrane-permeable CA-inhibitor, to the cell suspension did not affect the ratesof CO2 formation (Fig. 4B). Identical results were ob-tained with the other two algal species: an enhance-ment of isotope exchange was found in both N. atomus(Fig. 5A) and N. maculata (Fig. 6A), unaffected by AZ(Figs. 5B and 6B).

FIG. 3. Time course of changes in the concentration of13C18O18O (m/z 5 49), 13C16O18O (m/z 5 47) and 13C16O16O(m/z 5 45) after the addition of inorganic C in the form ofKH13C18O3 to a buffer solution, pH 8.0, at a final concentrationof 200 mM.

FIG. 4. Time course of the extracellular concentrations of13C18O18O (m/z 5 49), 13C16O18O (m/z 5 47) and 13C16O16O(m/z 5 45) in cells of Porphyridium cruentum after supplying in-organic C in the form of KH13C18O3 (200 mM) to (A) cell sus-pension, (B) cells in the presence of 500 mM AZ, and (C) cellstreated with 500 mM EZ.

318 I. EMMA HUERTAS ET AL.

In contrast, when the same procedure was followedafter providing 500 mM EZ to the cell suspension (Fig.4C), there was no initial rapid formation of m/z 45and the rate of appearance of other labeled specieswas not enhanced. Since EZ is known to permeate theplasma membrane relatively easily, this observationclearly indicates that EZ caused the inhibition of in-ternal CA activity. Consequently, the rates of 18O ex-

change were similar to the uncatalyzed rates in buffersolution (Fig. 3). Identical results were obtained withboth N. atomus (Fig. 5C) and N. maculata (Fig. 6C).

discussionThere appear to be a variety of strategies for the acqui-

sition of Ci by marine phytoplankton. Some are assumed

FIG. 5. Time course of the extracellular concentrations of13C18O18O (m/z 5 49), 13C16O18O (m/z 5 47) and 13C16O16O(m/z 5 45) in cells of Nannochloris atomus after supplying inor-ganic C in the form of KH13C18O3 (200 mM) to (A) cell suspen-sion, (B) cells in the presence of 500 mM AZ, and (C) cellstreated with 500 mM EZ.

FIG. 6. Time course of the extracellular concentrations of13C18O18O (m/z 5 49), 13C16O18O (m/z 5 47) and 13C16O16O(m/z 5 45) in cells of Nannochloris maculata after supplying in-organic C in the form of KH13C18O3 (200 mM) to (A) cell sus-pension, (B) cells in the presence of 500 mM AZ, and (C) cellstreated with 500 mM EZ.

CO2 TRANSPORT IN MARINE MICROALGAE 319

to take up CO2 by diffusion (Riebesell et al. 1993), othershave been shown to take up CO2 derived from HCO3

2 bythe action of an external CA (Williams and Turpin 1987),some appear to use only HCO3

2 (Huertas and Lubian1998), while other use both CO2 and HCO3

2 (Colmanand Rotatore 1995, Rotatore et al. 1995, Korb et al. 1997).At present, however, there is limited information on theability of marine autotrophs to acquire Ci, and reports ofthe active uptake of CO2 are limited to a cyanobacterium(Badger and Andrews 1982), a green alga (Amoroso et al.1998), and two diatoms (Rotatore et al. 1995).

The results presented here indicate clearly that P.cruentum, N. maculata, and N. atomus have the capacity totake up CO2 by active transport: all three species rapidlydepleted the external medium of free CO2 by a light-dependent process and maintained the concentrationbelow the expected equilibrium value. With the uptakeof CO2 there was a stoichiometric release of O2, and inthe two Nannochloris species (which have no capacity forHCO3

2 uptake and no external CA) the rate of O2 re-lease could be supported by the uncatalyzed productionof CO2 from HCO3

2 in the suspending medium. For ex-ample, in the case of N. maculata (Fig. 1C), the initialrate of O2 production was 6.0 nmol?mL21?min21, whilethe calculated rate of uncatalyzed CO2 production at100 mM Ci in seawater at 258 C is 8.6 nmol?mL?min21.

After a period of CO2 uptake in the light, there was arapid rise in the CO2 concentration in the dark. This in-crease occurred at a rate too fast to be the result of theuncatalyzed dehydration of HCO3

2 in the medium andindicates that CO2 was released from an intracellularpool of unfixed Ci. This rapid and selective uptake ofCO2 against a concentration gradient demonstrates thatCO2 was taken up by active transport and was not the re-sult of diffusive uptake.

The mass spectrometric measurement of the uptakeof CO2 by all three species indicated clearly that the cellswere capable of maintaining the CO2 concentration inthe medium out of chemical equilibrium with HCO3

2.This also demonstrated the lack of an external CA, whichwould have maintained an equilibrium CO2 concentra-tion. A confirmation of the absence of external CA is thelack of any effect of the weakly permeable CA-inhibitorAZ, which did not affect the rate of interconversion of Cispecies in the dark, where the rapid formation of m/z 47and 45 (Figs. 4B, 5B, and 6B) was essentially the same asin the controls (Figs. 4A, 5A, and 6A). The minor effectof AZ on the rate of appearance of m/z 47 could havebeen due, in all cases, to some penetration of the cells bythe inhibitor and its action on internal CA as has beensuggested by Williams and Turpin (1987). These findingsare in agreement with previous observations that exter-nal CA could not be detected, using conventional poten-tiometric methods, in P. cruentum (Dixon et al. 1987,Yagawa et al. 1987a) or in the two Nannochloris species(Huertas and Lubian 1998). Appreciable external CA ac-tivity has been detected in one isolate of P. cruentum(Burns and Beardall 1987), but it is not unknown for dif-ferent isolates of a species to differ in their expression ofexternal CA ( John-McKay and Colman 1997).

Although there is no evidence of external CA inthese species, the intact cells were found to catalyze arelatively rapid interconversion of Ci species in the dark(Figs. 4A, 5A, and 6A). In the dark there would be noactive uptake of CO2 or HCO3

2, but there would be adiffusive exchange of labeled Ci species outside the cellwith unlabeled species inside the cell, and therefore therapid isotopic equilibration could be explained by thepresence of an internal CA. Treatment of the cells of allthree species with the membrane-permeable CA-inhibi-tor EZ markedly reduced the rate of formation of la-beled CO2 of m/z 45 and 47 (Figs. 4C, 5C, and 6C) andthe rates of interconversion of Ci species were reducedto rates similar to the uncatalyzed rates in buffer solu-tion (Fig. 3). These results clearly demonstrate that allthree species possess internal CA. The inability to detectinternal CA in cell extracts of the same Nannochloris spe-cies by the potentiometric method (Huertas and Lu-bian 1998) may have been a result of the severity of theprocedure needed to break these very small cells.

It has previously been shown that the red microalgaP. cruentum is capable of active HCO3

2 transport (Col-man and Gehl 1983; Dixon and Merrett 1988) and thepresent results demonstrate that it can also take upCO2 by active transport. However, although it has thecapacity to take up both Ci species, the uptake of CO2is so rapid that CO2 would be quickly depleted in theregion round the cell and it is probable that the majorflux of Ci across the plasma membrane is HCO3

2.The two Nannochloris species examined in this study

have been shown to have no capacity for HCO32 trans-

port (Huertas and Lubian 1998), and their growth isstimulated by a supply of supplementary CO2 (Huertas1998). The present results demonstrate that they havean effective CO2 transport system which allows them toreduce the ambient dissolved CO2 in the surroundingmedium to very low levels, but, considering the low rateof HCO3

2 dehydration and low rate of diffusion of CO2in seawater and the absence of an external CA, it seemsthat these species may function under severe Ci stress inthe marine environment. Growth could be sustained ifNannochloris occupied niche habitats rich in CO2. Sus-tained CO2 evolution by a light-dependent process hasbeen reported in some autotrophic marine algae andcyanobacteria (Sukenik et al. 1997; Tchernov et al.1998), which might provide a specialized niche for spe-cies, such as Nannochloris, that have developed an effi-cient CO2 uptake system but lack HCO3

2 transport. It isinteresting to note that Nannochloris species are often re-ported to occur in populations with Nannochloropsis andSynechococcus spp. (Lewin and Cheng 1989, Lubian et al.1992), both of which are CO2 producers (Tchernov etal. 1998).

This work was financed by the Ministry of Education and Sci-ence of Spain, Project CICYT AMB 94-0684-CO2-O1 and bygrants from the Natural Sciences and Engineering ResearchCouncil of Canada to B. C. and G. S. E.

320 I. EMMA HUERTAS ET AL.

Amoroso, G., Sültemeyer, D., Thyssen, C. & Fock, H. P. 1998. Up-take of HCO3

2 and CO2 in cells and chloroplasts from the mi-croalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta.Plant Physiol. 116:193–201.

Badger, M. R. & Andrews, T. J. 1982. Photosynthesis and inorganiccarbon usage by the marine cyanobacterium Synechococcus sp.Plant Physiol. 70:517–23.

Burns, B. D. & Beardall, J. 1987. Utilisation of inorganic carbon bymarine microalgae. J. Exp. Mar. Biol. Ecol. 107:75–86.

Colman, B. & Gehl, K. 1983. Physiological characteristics of photo-synthesis in Porphyridium cruentum: evidence for bicarbonatetransport in a unicellular red alga. J. Phycol. 19:216–9.

Colman, B. & Rotatore, C. 1995. Photosynthetic inorganic carbonuptake and accumulation in two marine diatoms. Plant Cell En-viron. 18:919–24.

Dionisio-Sese, M. L. & Miyachi, S. 1992. The effect of sodium chlo-ride on carbonic anhydrase activity in marine microalgae. J.Phycol. 28:619–24.

Dixon, G. K. & Merrett, M. J. 1988. Bicarbonate utilization by themarine diatom Phaeodactylum tricornutum Bohlin. New Phytol.109:47–51.

Dixon, G. K., Patel, B. N. & Merrett, M. J. 1987. Role of intracellularcarbonic anhydrase in inorganic-carbon assimilation by Porphy-ridium purpureum. Planta 172:508–13.

Espie, G. S., Miller, A. G. & Canvin, D. T. 1989. Selective and revers-ible inhibition of active CO2 transport by hydrogen sulfide in acyanobacterium. Plant Physiol. 91:387–94.

Falkowski, P. G. & Raven, J. A. 1997. Aquatic Photosynthesis. 1st ed.Blackwell Science Co., Mississauga, Ontario, Canada, pp. 128–62.

Guillard, R. R. L. & Ryther, J. H. 1962. Studies on marine plank-tonic diatoms. I. Cyclotella nana Hustedt and Denotula confer-vaceae (Cleve) Gran. Can. J. Microbiol. 8:229–39.

Harrison, J. H., Rosemary, E. W. & Taylor, F. J. R. 1980. A broadspectrum artificial seawater medium for coastal and openocean phytoplankton. J. Phycol. 16:28–35.

Huertas, I. E. 1998. Mecanismos de incorporación de carbono in-orgánico disuelto en microalgas marinas en cultivo. Tipifi-cación de la respuesta fisiológica y de crecimiento ante varia-ciones en la concentración de CO2 en el medio. Ph.D. thesis.University of Cádiz, Spain.

Huertas, I. E. & Lubian, L. M. 1998. Comparative study of dissolvedinorganic carbon utilization and photosynthetic responses inNannochloris (Chlorophyceae) and Nannochloropsis (Eustig-matophyceae) species. Can. J. Bot. 76:1104–8.

John-McKay, M. & Colman, B. 1997. Variation in the occurrence of ex-ternal carbonic anhydrase among strains of the marine diatomPhaeodactylum tricornutum (Bacillariophyceae). J. Phycol. 33:988–90.

Korb, R. E., Saville, P. J., Johnston, A. M. & Raven, J. A. 1997.Sources of inorganic carbon for photosynthesis by three spe-cies of marine diatoms. J. Phycol. 33:433–40.

Lewin, R. A. & Cheng, L. 1989. Some lipogenic, eukaryotic, pico-pleuston algae from the Caribbean region. Phycologia 28:96–108.

Lubian, L. M., Blasco, J. & Establier, R. 1992. A comparative studyof acid and alkaline phosphatase activities in several strains ofNannochloris (Chlorophyceae) and Nannochloropsis (Eustig-matophyceae). Br. Phycol. J. 27:119–30.

Merrett, M. J., Nimer, N. A. & Dong, L. F. 1996. The utilization ofbicarbonate ions by the marine microalga Nannochloropsis ocu-lata (Droop) Hibberd. Plant Cell Environ. 19:478–84.

Miller, A. G. & Canvin, D. T. 1985. Distinction between HCO32 and

CO22dependent photosynthesis in the cyanobacterium Syn-echococcus leopoliensis based on the selective response of HCO3

2

transport to Na1. FEBS Lett. 187:29–32.Miller, A. G., Espie, G. S. & Canvin, D. T. 1988. Active transport of

CO2 by the cyanobacterium Synechococcus UTEX 625. Measure-ment by mass spectrometry. Plant Physiol. 86:677–83.

Miller, A. G., Salon, C., Espie, G. S. & Canvin, D. T. 1997. Measure-ment of the amount and isotopic composition of the CO2 re-leased from the cyanobacterium Synechococcus UTEX 625 afterrapid quenching of the active CO2 transport system. Can. J. Bot.75:981–97.

Nimer, N. A., Iglesias-Rodriguez, M. D. & Merrett, M. J. 1997. Bicar-bonate utilization by marine phytoplankton species. J. Phycol.33:625–31.

Rees, T. A. V. 1984. Sodium-dependent photosynthetic oxygen evo-lution in a marine diatom. J. Exp. Bot. 35:332–7.

Riebesell, U., Wolf-Gladrow, D. & Smetacek, V. 1993. Carbon dioxidelimitation of phytoplankton growth rates. Nature 361:249–51.

Roberts, S. B., Lane, T. W. & Morel, F. M. M. 1997. Carbonic anhy-drase in the marine diatom Thalassiosira weissflogii (Bacillario-phyceae). J. Phycol. 33:845–50.

Rotatore, C. & Colman, B. 1991. The active uptake of carbon diox-ide by the unicellular green algae Chlorella saccharophila and C.ellipsoidea. Plant Cell Environ. 14:371–5.

——— 1992. The active uptake of CO2 by the diatom Navicula pelli-culosa. J. Exp. Bot. 43:571–6.

Rotatore, C., Lew, R. R. & Colman, B. 1992. Active uptake of CO2during photosynthesis in the green alga Eremosphaera viridis ismediated by a CO2-ATPase. Planta 188:539–45.

Rotatore, C., Colman, B. & Kuzma, M. 1995. The active uptake ofcarbon dioxide by the marine diatoms Phaeodactylum tricornu-tum and Cyclotella sp. Plant Cell Environ. 18:913–8.

Sukenik, A., Tchernov, D., Kaplan, A., Huertas, I. E., Lubian, L. M.& Livne, A. 1997. Uptake, efflux and photosynthetic utilizationof inorganic carbon by the marine eustigmatophyte Nannochlo-ropsis sp. J. Phycol. 33:969–74.

Sültemeyer, D. F., Miller, A. G., Espie, G. S., Fock, H. P. & Canvin,D. T. 1989. Active CO2 transport by the green alga Chlamydomo-nas reinhardtii. Plant Physiol. 89:1213–9.

Tchernov, D., Hassidim, M., Vardi, A., Luz, B., Sukenik, A., Rein-hold, L. & Kaplan, A. 1998. Photosynthesizing marine microor-ganisms can constitute a source of CO2 rather than a sink. Can.J. Bot. 76:949–53.

Williams, T. G. & Turpin, D. M. 1987. The role of external carbonicanhydrase in inorganic carbon acquisition by Chlamydomonasreinhardtii. Plant Physiol. 80:92–6.

Yagawa, Y., Shoshi, M. & Miyachi, S. 1987a. Carbonic anhydrase of aunicellular red alga Porphyridium cruentum R-1. I. Purificationand properties of the enzyme. Plant Cell Physiol. 28:1253–62.

———1987b. Carbonic anhydrase of a unicellular red alga Porphy-ridium cruentum R-1. II. Distribution and role in photosynthesis.Plant Cell Physiol. 28:1509–16.