Abstract. Inorganic carbon (Ci) uptake and e‚ux has
been investigated in the marine microalga Nannochlor-
opsis gaditana Lubian by monitoring CO
2
¯uxes in cell
suspensions using mass spectrometry. Addition of
H
13
CO
ÿ
3
to cell suspensions in the dark caused a
transient increase in the CO
2
concentration in the
medium far in excess of the equilibrium CO
2
concen-
tration. The magnitude of this release was dependent on
the length of time the cells had been kept in the dark.
Once equilibrium between the Ci species had been
achieved, a CO
2
e‚ux was observed after saturating
light intensity was applied to the cells. External carbonic
anhydrase (CA) was not detected nor does this species
demonstrate a capacity to take up CO
2
by active
transport. Photosynthetic O
2
evolution and the release
CO
2
in the dark depend on HCO
ÿ
3
uptake since both
were inhibited by the anion exchange inhibitor, 4,4¢-
diisothiocyanatostilbene-2,2¢-disulfonic acid (DIDS).
The bicarbonate uptake mechanism requires light but
can also continue for short periods in the dark.
Ethoxyzolamide, a CA inhibitor, markedly inhibited
CO
2
e‚ux in the dark, indicating that CO
2
e‚ux was
dependent upon the intracellular dehydration of HCO
ÿ
3
.
These results indicate that Nannochloropsis possesses a
bicarbonate uptake system which causes the accumula-
tion of high intracellular Ci levels and an internal CA
which maintains the equilibrium between CO
2
and
HCO
ÿ
3
and thus causes a subsequent release of CO
2
to
the external medium.
Key words: Bicarbonate transport ± Carbonic
anhydrase ± CO
2
e‚ux ± Eustigmatophyceae ± Inhibitor
(DIDS) ± Nannochloropsis ±(HCO
ÿ
3
uptake)
Introduction
The slow di€usion of CO
2
and low conversion rate of
HCO
ÿ
3
to CO
2
in air-equilibrated seawater control the
availability of CO
2
for photosynthetic organisms in the
marine environment. On the other hand, the concentra-
tion of HCO
ÿ
3
is about 200-fold higher than that of CO
2
(Round 1981) within the usual pH range of 8.0±8.3 in
oceanic waters. Under these conditions, marine photo-
trophs have developed mechanisms to enhance the
supply of CO
2
for photosynthesis (Raven and Johnston
1991). Although the mechanisms of inorganic carbon
(Ci) transport are not fully understood, the physiological
gas-exchange characteristics of most algal groups are
due to an inducible CO
2
-concentrating mechanism
(CCM), which increases the concentration of CO
2
at
the active site of ribulose-1,5-bisphosphate carboxylase-
oxygenase (Rubisco). This intracellular accumulation of
inorganic carbon is achieved by the active transport of
HCO
ÿ
3
and/or CO
2
and the action of intracellular
carbonic anhydrase (CA). The increase in the intracel-
lular CO
2
concentration favours the carboxylation
reaction, inhibits the oxygenase activity of the Rubisco
and, thereby increases photosynthesis and reduces
photorespiration (Raven 1997a).
Most studies of the CCM have been carried out with
freshwater microalgae and cyanobacteria. These studies
have shown that several species are able to use both CO
2
and HCO
ÿ
3
as substrates for the CCM (Badger and Price
1992) and that more than one mode of Ci utilization
may be involved in the process (Espie et al. 1991). The
utilization of both forms of Ci has also been reported in
marine diatoms (Colman and Rotatore 1995; Rotatore
et al. 1995; Korb et al. 1997). In some microalgae,
external CA may be an accessory system to active
transport by maintaining the HCO
ÿ
3
±CO
2
equilibrium
close to the cell surface (Burns and Beardall 1987;
Dioniso-Sese and Miyachi 1992; Colman and Rotatore
1995; Roberts et al. 1997; John-Mackay and Colman
1997). This may also maintain a supply of CO
2
which
enters the cell either passively (Riebesell et al. 1993) or
by active transport across the plasma membrane
Abbreviations: CA = carbonic anhydrase; Ci = inorganic car-
bon; DIDS = 4,4¢-diisothiocyanatostilbene-2,2¢-disulfonic acid;
EZ = ethoxyzolamide
Correspondence to: B. Colman;
E-mail: colman@yorku.ca; Fax: +1-416-736-5698
Planta (2000) 211: 43±49
Light-dependent bicarbonate uptake and CO
2
e‚ux
in the marine microalga Nannochloropsis gaditana
I. Emma Huertas
1,3
, George S. Espie
2
, Brian Colman
3
, Luis M. Lubian
1
1
Instituto de Ciencias Marinas de Andalucia (CSIC), Poligono Rio San Pedro s/n, 11510 Puerto Real (Ca diz), Spain
2
Department of Botany, Erindale College, University of Toronto at Mississauga, Mississauga, Ontario L5L 1C6, Canada
3
Department of Biology, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada
Received: 20 September 1999 /Accepted: 25 October 1999

(Rotatore et al. 1995). In the absence of this enzyme,
however, the existence of a transport system causing a
direct uptake of HCO
ÿ
3
across the plasma membrane has
been also described in marine phytoplankton species
(Colman and Gehl 1983; Rees 1984; Dixon and Merrett
1988; Colman and Rotatore 1995; Merrett et al. 1996).
Little is known about Ci acquisition in marine
eustigmatophyte algae although they are known to be
major primary producers in pelagic ecosystems (Van den
Hoek et al. 1995). Species of the genus Nannochloropsis
have been the only members of this group to be studied
in any detail; they have been shown to lack external CA
and it has been inferred that they have the capacity to
take up HCO
ÿ
3
from their low K
1/2
(Ci) at alkaline pH
and from the inhibition of photosynthesis by the anion-
exchange inhibitor 4,4¢-diisothiocyanatostilbene-2,2¢-
disulfonic acid (DIDS) (Mun
Ä
oz and Merrett 1989;
Merrett et al. 1996; Sukenik et al. 1997). However, active
uptake of HCO
ÿ
3
has been clearly demonstrated by
a kinetic method in N. gaditana and N. oculata (Huertas
and Lubian 1998). In addition, one species has been
reported to lack internal CA and a lack of CO
2
transport
in this alga was inferred from its high K
1/2
(CO
2
)over
a pH range of 5.0±8.3 (Mun
Ä
oz and Merrett 1989).
A number of marine phytoplanktonic species have
been shown to be net producers of CO
2
at high light
intensities, one of them being a species of Nannochlor-
opsis (Sukenik et al. 1997; Tchernov et al. 1997). In
Nannochloropsis the rate of Ci uptake apparently
exceeded the rate of CO
2
®xation and resulted in an
e‚ux of CO
2
(Sukenik et al. 1997). It was inferred from
the relatively high anity for Ci at alkaline pH, that
HCO
ÿ
3
was the Ci species taken up and, because
photosynthesis was inhibited by the permeable CA
inhibitor ethoxyzolamide (EZ), it was inferred that
CO
2
was formed intracellularly by the action of an
internal CA (Sukenik et al. 1997).
The present study examines the Ci ¯uxes in the
eustigmatophyte alga Nannochloropsis gaditana using
mass spectrometry, and analyses the mechanisms by
which Ci is taken up and CO
2
is formed in these cells.
Materials and methods
Growth conditions. Nannochloropsis gaditana Lubian (strain B3)
was obtained from the culture collection of the Instituto de
Ciencias Marinas de Andalucia (CSIC, Spain). Cells were grown in
arti®cial seawater (Harrison et al. 1980) supplemented with F/2
medium (Guillard and Ryther 1962) modi®ed with double nitrate
and phosphate concentrations to avoid nutrient limitation. Cul-
tures were bubbled with sterile air containing 330 lLL
)1
(v/v) CO
2
at a rate of 60 mL min
)1
and maintained at a temperature of 20 °C
and at a photon ¯ux density of 75 lmol m
)2
s
)1
. Illumination was
provided by a combination of cool-white and Gro-lux ¯uorescent
lamps (Sylvania).
Experimental procedure. Cells were washed and resuspended in a
Ci-free medium containing NaCl (450 mM), MgCl
2
(40 mM), KCl
(10 mM), Na
2
SO
4
(10 mM) and CaCl
2
(5 mM) bu€ered at pH 8.0
with 25 mM TRIZMA-Base. The cell suspension was subsequently
placed in a thermostatted (25 °C) glass reaction vessel and purged
with a stream of N
2
in order to reduce the O
2
concentration. The
chamber was then closed and the cells allowed to reach the CO
2
compensation point before starting any treatment. The cell
suspension was continously stirred with a magnetic stirrer during
experiments. Light was provided by a tungsten-halogen projector
lamp with 1000 lmol photons m
)2
s
)1
incident upon the front
surface of the reaction vessel. The ®nal cell density was 2 ´ 10
8
cells mL
)1
which corresponded to 30 lg chlorophyll mL
)1
. Cell
numbers were determined using a haemocytometer (Neubauer).
Photosynthetic O
2
evolution was measured in a Clark-type O
2
electrode as described previously (Colman and Rotatore 1995).
Mass spectrometry. The concentration of dissolved gases in cell
suspensions was measured in a closed reaction vessel with a
magnetic sector mass spectrometer (model MM 14-80 SC; VG Gas
Analysis, Middlewich, UK) equipped with a membrane inlet. The
mass spectrometer was calibrated for CO
2
and O
2
as described
previously (Miller et al. 1988).
Extracellular CA assay. Carbonic anhydrase activity in cell
suspensions was assayed using the
18
O exchange method (Silver-
man 1982). The irreversible loss of
18
O from
13
C
18
O
18
OtoH
2
Oin
aqueous solution is caused by the repeated hydration and dehy-
dration of Ci species. These reactions are catalyzed by CA . The
loss of
18
O from
13
C
18
O
18
O (m/z = 49) results in the transient
formation of
13
C
18
O
16
O (m/z = 47) and ultimately yields
13
C
16
O
16
O (m/z = 45). The time courses of the disappearance/
appearance of the various
13
CO
2
species in solution are predictable
and are governed largely by the rate and equilibrium constants of
the reactions, the pH and ionic strength of the solution and the
temperature. Catalysis by CA accelerates the isotopic depletion of
18
O from
13
C
18
O
18
O and this will be re¯ected in the time courses of
the appearance and disappearance of the various
13
CO
2
species.
Transport of CO
2
. In order to determine whether cells of
N. gaditana had the ability to transport CO
2
actively, the disap-
pearance of
12
CO
2
from cell suspensions in the light and dark
following the addition of a pulse of CO
2
was measured. The
di€erence between the light and dark control disappearance curves
was taken as a measure of CO
2
transport (Espie et al. 1989).
Aqueous solutions of CO
2
were prepared by equilibrating acidi®ed
ice-cold water (pH 2.0) with 5% (v/v) CO
2
in N
2
.
Chemicals. The TRIZMA-Base, CA, DIDS and EZ were obtained
from Sigma Chemical Co. Synthesis of K
2
13
C
18
O
3
(99 atom %
13
C)
was as described by Miller et al. (1997). Stocks of
12
Ci and
13
Ci
were made by dissolving a measured quantity of K
2
12
CO
3
or
K
2
13
CO
3
in distilled H
2
O.
Results
Extracellular CA. Catalysis of CO
2
/HCO
ÿ
3
interconver-
sion at the cell surface has been reported to occur in
some algal species but not in others. Since extracellular
CA activity will in¯uence Ci acquisition, we tested for
the occurrence of CA in N. gaditana using a mass-
spectrometric assay (Fig. 1). In these experiments, the
cells were placed in the reaction cuvette in light and
allowed to ®x any residual CO
2
(m/z = 44) in the bu€er.
The suspension was darkened and the reaction was
started by the addition of 200 lMK
2
13
C
18
O
3
. In control
experiments lacking cells the m/z 49 signal initially
rose rapidly as the
13
C
18
O
2
/H
13
C
18
O
ÿ
3
/
13
C
18
O
2ÿ
3
system
approached chemical equilibrium (Fig. 1A). Subse-
quently, m/z 49 began to decline slowly as
18
O was lost
from
13
C
18
O
2
to H
2
O during non-enzymic hydration-
dehydration cycles. These reactions also led to the
gradual appearance of the m/z 47 and m/z 45 species of
44 I. E. Huertas et al.: CO
2
e‚ux in Nannochloropsis gaditana