Regulation of the induction of bicarbonate uptake by dissolved CO2 in the marine diatom, Phaeodactylum tricornutum

Authors


Correspondence: YusukeMatsuda Fax: + 81 798 51 0914; E-mail: yusuke@kwansei.ac.jp

ABSTRACT

Physiological properties of photosynthesis were determined in the marine diatom, Phaeodactylum tricornutum UTEX640, during acclimation from 5% CO2 to air and related to H2CO3 dissociation kinetics and equilibria in artificial seawater. The concentration of dissolved inorganic carbon at half maximum rate of photosynthesis (K0·5[DIC]) value in high CO2-grown cells was 1009 mmol m3 but was reduced three-fold by the addition of bovine carbonic anhydrase (CA), whereas in air-grown cells K0·5[DIC] was 71 mmol m3, irrespective of the presence of CA. The maximum rate of photosynthesis (Pmax) values varied between 300 and 500 μmol O2 mg Chl1 h1 regardless of growth pCO2. Bicarbonate dehydration kinetics in artificial seawater were re-examined to evaluate the direct HCO3 uptake as a substrate for photosynthesis. The uncatalysed CO2 formation rate in artificial seawater of 31·65°/oo of salinity at pH 8·2 and 25 °C was found to be 0·6 mmol m3 min1 at 100 mmol m3 DIC, which is 53·5 and 7·3 times slower than the rates of photosynthesis exhibited in air- and high CO2-grown cells, respectively. These data indicate that even high CO2-grown cells of P. tricornutum can take up both CO2 and HCO3 as substrates for photosynthesis and HCO3 use improves dramatically when the cells are grown in air. Detailed time courses were obtained of changes in affinity for DIC during the acclimation of high CO2-grown cells to air. The development of high-affinity photosynthesis started after a 2–5 h lag period, followed by a steady increase over the next 15 h. This acclimation time course is the slowest to be described so far. High CO2-grown cells were transferred to controlled DIC conditions, at which the concentrations of each DIC species could be defined, and were allowed to acclimate for more than 36 h. The K0·5[DIC] values in acclimated cells appeared to be correlated only with [CO2(aq)] in the medium but not to HCO3, CO32, total [DIC] or the pH of the medium and indicate that the critical signal regulating the affinity of cells for DIC in the marine diatom, P. tricornutum, is [CO2(aq)] in the medium.

INTRODUCTION

Photosynthetic CO2 fixation in algae is known to be efficient, despite the fact that the concentration of substrate – dissolved CO2– is generally low in natural waters and the diffusion resistance in water is extremely high in comparison with that in the atmosphere. Although a number of aquatic photo-autotrophs appear to possess C3-type photosynthesis (Pelroy & Bassham 1972; Colman 1989; Raven 1997), photorespiratory activity exhibited in most of the algae so far studied is much lower than would be expected for a C3 plant under atmospheric [CO2] conditions (Moroney, Wilson & Tolbert 1989; Beardall 1989; Raven 1994). Such suppression of photorespiration is thought to be due to an inorganic-carbon-concentrating mechanism (CCM), by which aquatic photo-autotrophs can concentrate CO2 in close proximity to the active site of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) (Badger 1987; Miller, Espie & Canvin 1990; Raven 1997; Moroney & Somanchi 1999).

The CCM has been studied extensively in freshwater cyanobacteria and green algae (references cited above and therein). In cyanobacteria, a number of studies have shown the existence of Na+-dependent and Na+-independent HCO3 transport and CO2 transport (Espie & Canvin 1987; Espie & Kandasamy 1992; Tyrrell et al. 1996). Using the green alga, Chlamydomonas reinhardtii, Sültemeyer et al. (1989) showed that air-grown cells are able to transport both CO2 and HCO3, but that CO2 is preferentially taken up from the medium. The utilization of both HCO3 and CO2 by air-grown cells has also been reported for Chlorella species in a quantitative manner (Williams & Colman 1995; Matsuda, Williams & Colman 1999).

The marine environment differs from freshwater primarily in salinity and alkalinity, factors that affect the CO2/carbonate equilibrium, and gives rise to CO2 limitation in marine photo-autotrophs, since under these conditions the equilibrium is biased to HCO3 formation. The rate of HCO3 dehydration to form CO2 is therefore extremely low, but by knowing the exact maximum rate of uncatalysed CO2 formation in the bulk medium, HCO3 utilization by cells lacking in external carbonic anhydrase (CA) activity can be determined. Extensive use of HCO3 dehydration kinetics has been made to identify HCO3 uptake in freshwater phototrophs such as Chara, cyanobacteria, green algae and diatoms (Lucas 1975; Birmingham & Colman 1979; Miller & Colman 1980; Gehl, Colman & Sposato 1990) and has contributed to a partial understanding of the CCM.

Even though HCO3 dehydration kinetics in seawater were described by Johnson (1982), these data have rarely been used in relation to the biological utilization of inorganic carbon. A number of studies have shown the possible occurrence of HCO3 transport in various marine microalgae (Burns & Beardall 1987; Colman & Rotatore 1995; Nimer, Iglesias-Rodriguez & Merrett 1997; Raven 1997). In the majority of these studies, the utilization of bicarbonate was inferred from O2 evolution kinetics and silicone-oil centrifugation assays where an intracellular accumulation of dissolved inorganic carbon (DIC) up to 10-fold that of the external medium was reported. A similar accumulation of internal DIC was observed by Tortell, Reinfelder & Morel (1997) in a marine diatom mixture from a Californian upwelling using the silicone-oil centrifugation technique and they concluded that HCO3 was the predominant DIC species taken up by this natural diatom population. It should be noted however, that CO2 is the species of DIC that is preferentially taken up from the medium in all the freshwater algae so far studied. In Chlorella species, for instance, the rate constant for CO2 uptake was shown to be 150 times that of HCO3 uptake (Williams & Colman 1995; Matsuda et al. 1999) and these cells can accumulate internal [DIC] to more than 40-fold that of the external medium at alkaline pH without catalysed formation of CO2 in the medium (Matsuda & Colman 1995a); thus, an active CO2 transport system alone may be able to develop an internal [DIC] which is 10-fold that of the bulk medium, even at alkaline pH. In view of the uncertainty inherent in previous results it would seem necessary to re-examine the kinetics of CO2 formation in seawater in order to assess, unequivocally, the capacity for HCO3 utilization in marine photo-autotrophs.

It is well known that expression of the CCM in algae and cyanobacteria is regulated by pCO2 in the air that is passed through the culture medium (Badger 1987; Coleman 1991; Kaplan & Reinhold 1999). Growth of freshwater micro-algae in 5% CO2 represses some or all of the DIC transport systems (Sültemeyer et al. 1989; Price & Badger 1989; Matsuda & Colman 1995a) and transfer of high CO2-grown cells to air causes an induction (or derepression) of DIC transport (Sültemeyer, Fock & Canvin 1991; Matsuda & Colman 1995a). It has been suggested that in freshwater cyanobacteria the total carbon availability might be a critical factor for the regulation of CCM expression (Marcus, Harel & Kaplan 1983; Mayo et al. 1986). In contrast, the green algae, Chlorella species and C. reihardtii have been shown to alter their photosynthetic affinities for DIC in response to external CO2, suggesting the existence of a mechanism which may sense extracellular [CO2] (Matsuda & Colman 1995b; Matsuda et al. 1999; Bozzo & Colman, 2000; Bozzo, Colman & Matsuda, 2000).

There have been few reports on the regulation of the CCM by pCO2 in marine micro-algae. Johnston & Raven (1996) showed that well-aerated (2·0 dm3 min−1) cultures of the marine diatom, Phaeodactylum tricornutum had a photosynthetic affinity for DIC that was only one-tenth of that reported in previous studies of the same species. They also found that by lowering the aeration rate to 0·1 dm3 min−1, the photosynthetic affinity of the cells for DIC was 10 times greater than that observed in well-aerated cultures (Johnston & Raven 1996). This study indicates that the external CO2 concentration may be the controlling factor in the induction of the CCM in this marine diatom. Similarly, extracellular CA, which is thought to be a component of CCM in some algal species (Aizawa & Miyachi 1986), was shown to be regulated by [CO2(aq)] in the medium in the marine diatom Skeletonema costatum (Nimer, Warren & Merrett 1998).

In order to determine which DIC species constitutes the primary signal for the induction of the CCM in a marine photo-autotroph, it is necessary to know the proportions of CO2 and bicarbonate present in seawater under a range of experimental conditions. The apparent dissociation constants of H2CO3 under wide range of conditions of temperature and salinity were determined by Mehrbach et al. (1973) and were further extended by Goyet & Poisson (1989) to develop polynomial functions that allow dissociation constants to be derived for a wide range of seawater conditions. In the present study, the DIC equilibria and the rates of spontaneous dehydration of HCO3 in seawater have been re-examined and related to a physiological analysis of CCM induction in the marine diatom P. tricornutum.

MATERIALS AND METHODS

Algal cells and growth conditions

The marine diatom P. tricornutum (UTEX640) was obtained from the University of Texas Culture Collection at Austin. Cultures were grown axenically in the artificial seawater described by Harrison, Waters & Taylor (1980) with the addition of 0·31% half-strength Guillard’s ‘F’ solution (F/2) (Guillard & Ryther 1962; Guillard 1975). Cultures were grown in 0·5 or 2 dm3 cylindrical glass vessels under continuous illumination with cool-white-fluorescent light at a photon flux density of 40 μmol m−2 s−1 at 20 °C. The culture medium was continuously aerated with either air or 5%-CO2 enriched air at inflow rates of 15 or 600 cm3 min−1, respectively. Alkalinity was controlled, unless otherwise described, at pH 6·8 and 7·1 ± 0·15 for 5% CO2- and air-grown cells, respectively, by the addition of 2N HCl or 1N NaOH. The cells were harvested at the mid-logarithmic phase (OD730 = 0·12–0·18) by centrifugation at 3000 ×g for 15 min.

Measurements of photosynthetic parameters

Harvested cells were washed twice with 350 mol m−3 NaCl buffered with 10 mol m−3 Tris-HCl (pH 6·8) and resuspended in CO2-free F/2-enriched artificial seawater (F/2AW) buffered with 10 mol m−3 Tris-HCl (pH 8·2) under N2 at a chlorophyll a concentration of 10 μg cm−3. The rate of photosynthesis was measured with a Clark-type oxygen electrode with or without the addition of bovine CA (Sigma-Aldrich Japan, Tokyo, Japan) as described previously (Matsuda & Colman 1995a). The apparent [DIC] at half maximum rate of photosynthesis (K0·5[DIC]) was determined as described by Rotatore & Colman (1991) with and without bovine CA (10 μg cm−3). The CO2-compensation concentration was determined by a gas chromatographic (Shimadzu GC-8A; Shimadzu Co., Kyoto, Japan) method (Birmingham & Colman 1979). Cell suspension (1·5 cm3) was placed in the O2-electrode chamber and illuminated with a fibre illuminator (Megalight100; Hoya-Schott Co., Tokyo, Japan) at photon flux density of 2600 μmol m−2 s−1 until cells reached CO2-compensation concentration. The photon flux density was then increased to 6400 μmol m−2 s−1 and aliquots of KHCO3 were added sequentially to the cell suspension in order to create increasing DIC concentrations. Chlorophyll a concentration was determined by the spectrophotometric method described by Jeffrey & Humphrey (1975).

Measurement of CA activity

Extracellular CA activity was measured by the potentiometric method described by Wilbur & Anderson (1948). Harvested cells from 450 cm3 of culture at mid-logarithmic phase were resuspended in 1 cm3 of a buffer containing 50 mol m−3 Bicine-NaOH (pH 8·3), 1 mol m−3 EDTA, and 10 mol m−3 NaCl. A 100 mm3 aliquot was added to 1·4 cm3 of 20 mol m−3 veronal-NaOH buffer (pH 8·4) in a water-jacketed acrylic chamber maintained at 4 °C. The CA activity was assayed by measuring the time required for acidification of the medium from pH 8·3–8·0 resulting from the formation of protons on the addition of 0·5 cm3 of ice-cold CO2-saturated water.

Determination of the maximum rate of CO2 formation in F/2AW

To estimate the maximum rate of spontaneous CO2 formation from HCO3 in F/2AW, DIC-depletion kinetics were determined under conditions where N2 replaces CO2 in the medium at 25 °C. Seven hundred cubic centimetres of F/2AW was placed in a water-jacketed cylindrical glass vessel (60 mm diameter × 350 mm high) and N2, which was passed through soda lime (Kishida-Kagaku Co., Osaka, Japan) to remove any contaminating CO2, was passed through the medium at a rate of 2 dm3 min−1 using two or three glass-fritted gas dispersion tubes. CO2 was not detectable by gas chromatography in the inflow gas. The medium was buffered with 10 mol m−3 Tris-HCl and maintained at pH 8·2 throughout the experiment. Five cubic millimetres of medium were taken every 5–10 min and [DIC] was measured by gas chromatography as described above. The rates of spontaneous CO2 formation at several specific DIC concentrations were calculated as rates of total [DIC] decrease in the medium. These values were further compared with measured photosynthetic rates at the corresponding [DIC] level to assess the utilization of HCO3 as a substrate for photosynthesis.

The rate constants for the spontaneous HCO3 dehydration to form CO2 at 25 °C and 31·65°/oo salinity were estimated according to Miller & Colman (1980). The rate of spontaneous formation of CO2 from HCO3 can be described by Equation 1.

image(1)

where k1 and k3 are the rate constants of the following reactions, respectively.

image((Reaction 1))
image((Reaction 2))

Johnson (1982) reported that k1 was approximately 1·17 × 10−4 s−1 at 25 °C and 33·77°/oo of salinity. K1 is the product of equilibrium constants for the dissociation equilibria of carbonic acid. KH2CO3 and K2, respectively, are the dissociation constants of the following two reactions:

image((Reaction 3))
image((Reaction 4))

and the KH2CO3 value at 25 °C ranges from 1·58 × 10−4−3·98 × 10−4 (Stumm & Morgan 1995). K1 and K2 were derived by the following equations described by Goyet & Poisson (1989).

image(2)
image(3)

where T and S are the absolute temperature and salinity, respectively. The salinity of the F/2AW used in this study was calculated to be 31·65°/oo and the dissociation constants are expressed in moles per kilogram of solution. K1 and K2 were calculated to be 1·22 × 10−6 and 9·0 × 10−10, respectively. A value for k3 was derived using the values described above and the CO2 formation rates determined in this study.

Acclimation of 5% CO2-grown cells to air or defined CO2 concentrations

Cells grown to the mid-logarithmic phase on 5% CO2 were harvested by centrifugation at 2000 ×g for 5 min and resuspended in 50 cm3 of sterilized F/2AW. Cell suspensions were transferred to new cylindrical glass vessels and diluted with sterilized F/2AW to an OD730 of 0·12–0·18. All these procedures were carried out under sterile conditions. Immediately after transferring the cultures, they were aerated with air or with a defined CO2 concentration in air. Samples of air-acclimating cells were harvested every hour and photosynthetic rates at 100 mmol m−3 of KHCO3, with and without CA, were determined as described above.

Cells of P. tricornutum grown on 5%-CO2 were allowed to acclimate for at least 36 h in defined DIC conditions: cells suspended in F/2AW of five different pHs, 6·5, 7·5, 8·2, 8·5 and 8·7 were aerated with air (0·032% CO2); and cells suspended in F/2AW of four different pHs, 6·5, 7·5, 9·0, and 9·4 were aerated with nitrogen (0·0006% CO2; Neriki Gas Co., Osaka, Japan) passed through glass-fritted gas dispersion tubes as described above. The inflow rate was 2 dm3 min−1. The equilibrium between gaseous and dissolved CO2 was confirmed in that the total DIC concentration measured by gas chromatography agreed with the calculated DIC concentration. The theoretical total DIC concentration was calculated according to Stumm & Morgan (1995) using the apparent dissociation constants of carbonic acid (K1 and K2) in seawater described by Goyet & Poisson (1989).

Acclimated cells were harvested and the apparent K0·5[DIC] values were determined in the absence of bovine CA as described by Rotatore & Colman (1991).

RESULTS

Characterization of photosynthesis in P. tricornutum grown in 5% or air level CO2

The average doubling rate of 5% CO2-grown cells and air-grown cells were 1·7 and 0·93 times d−1, respectively.

Cells grown in 5% CO2 exhibited anomalous behaviour at the apparent CO2-compensation point, where O2 evolution ceases due to the depletion of external DIC. At the apparent compensation point, the signal voltage of the O2 electrode increased at a constant rate (data not shown) although the DIC concentration in the bulk medium, as measured by gas chromatography, was found to be fairly constant. The [DIC] at the compensation point was constant at about 80 mmol m−3 for more than 1 h and therefore the K0·5[DIC] and O2 evolution rate of 5% CO2-grown cells were determined at this [DIC]. In contrast to the 5% CO2-grown cells, stable CO2-compensation points were found in cells grown in air and found to range from 5 to 10 mmol m−3. [DIC] in the medium was 21 000 and 3 mmol m−3 for 5% and air cultures, respectively.

The measured value of K0·5 clearly showed that 5% CO2-grown cells possess much lower affinities than those observed in air-grown cells (Fig. 1). Five percent CO2-grown cells exhibited K0·5[DIC] values of 1009 and 318 mmol m−3 without and with added CA, respectively, whereas those of air-grown cells were found to be 71 mmol m−3 irrespective of the presence of bovine CA (Table 1). External CA activity was not detected either in 5% CO2-grown or air-grown cells. These data suggest that the marine diatom P. tricornutum induces (and/or de-represses) high-affinity photosynthesis in response to DIC limitation. The Pmax values varied between 300 and 500 μmol mg−1 Chl h−1 whereas K0·5[DIC] values were maintained relatively constant irrespective of variation in Pmax (Fig. 1 & Table 1).

Figure 1.

Rate of photosynthetic O2 evolution at various DIC concentrations in the marine diatom P. tricornutum UTEX640 at pH 8·2 and 25 °C. Plots are shown for high CO2-grown cells with (bsl00084) and without (bsl00066) added CA and air-grown cells with (bsl00000) and without (▪) added CA. The inset shows rates of O2 evolution at DIC concentrations less than 0·2 mol m−3.

Table 1.  Photosynthetic parameters in 5% CO2- or air-grown cells of P. tricornutum measured in pH 8·2 at 25 °C
Growth conditionK0·5[DIC]
(mmol m−3)
Pmax
(μmol O2 mg−1 Chl h−1)
  1. aValues are the means ± SE of three separate experiments. bValues are the means ± SE of two separate experiments.

5% CO2
without CA1009 ± 82a479 ± 49a
with CA318 ± 55a475 ± 105b
Air
without CA71 ± 18a440 ± 67a
with CA71 ± 12a433 ± 128b

Assessment of the capacity for HCO3 uptake

The availability of CO2 for photosynthesis in the marine environment is restricted by the low formation rate of CO2 from its hydrated form due to high pH (8·2) and salinity (more than 30°/oo). However, the rate of photosynthetic O2 evolution by P. tricornutum was significantly higher than that of CO2 formation in the bulk medium, and in the absence of external CA, implies that HCO3 is taken up by the cells as a substrate for photosynthesis.

The time course of changes in [DIC] in the bulk medium were determined in DIC depletion experiments (Fig. 2) and the rate of DIC loss with time illustrates the pseudo first-order nature of the reaction (Fig. 2, inset). The rate of decrease of [DIC] in the bulk medium at an inflow rate of CO2-free N2 below 0·5 dm3 min−1 was found to be much slower than that observed under the inflow rate at 2 dm3 min−1 (data not shown). The rate of DIC depletion however, was found to be fairly constant at N2 inflow rates above 1·0 dm3 min−1 (Fig. 2). The measured rates of spontaneous dehydration of HCO3 to form CO2 decreased from 6·4 ± 0·5 to 0·6 ± 0·3 mmol m−3 min−1 (n = 2) as the initial [DIC] in the medium was reduced from 0·8 to 0·1 mmol m−3 (Table 2). The measured CO2 formation rates at various [DIC] and the absolute rates of photosynthetic O2 evolution in high CO2 and air-grown cells of P. tricornutum at corresponding [DIC] are summarized in Table 2. The rates of O2 evolution in both high CO2-grown and air-grown cells were significantly higher than the spontaneous CO2 formation rates at external [DIC] over the range of 100 to 600 mmol m−3 (Table 2). In air-grown cells, O2 evolution rate was about 54-fold the spontaneous CO2 formation rate whereas that in high CO2-grown cells was found to be seven-fold the CO2 formation rate.

Figure 2.

Changes with time in total DIC concentration under conditions in which CO2-free N2 replaces CO2 in F/2AW medium at pH 8·2 and 25 °C. The two symbols represent values from two independent measurements. The inset is a plot of log[DIC] remaining in the medium over the initial [DIC] multiplied by the negative of gas constant was plotted against time.

Table 2.  Measured rates of spontaneous CO2 formation compared with rates of photosynthesis in high CO2-and air-grown cells of P. tricornutum at various [DIC] in the bulk medium at pH 8·2 and 25 °C
 The rate (mmol m−3 min−1) of
  O2 evolution
Initial [DIC]
(mmol m−3)
Spontaneous
CO2
formation
Air-grown
cells
High
CO2-grown
cells
  1. aThe number of replicates.

1000·6 ± 0·3 (2)a32·1 ± 7·6 (5)a 4·4 ± 0·9 (6)a
2001·6 ± 0·1 (2)a40·7 ± 6·7 (5)a 9·1 ± 0·9 (4)a
4003·2 ± 0·3 (2)a50·0 ± 4·9 (5)a16·3 ± 3·2 (4)a
6004·8 ± 0·4 (2)a61·2 ± 4·1 (5)a21·9 ± 4·7 (4)a

According to Johnson (1982), the rate constant for the direct formation of CO2 via HCO3 dehydration (k1) was estimated at 1·17 × 10−4 s−1 at 25 °C and 33·77°/oo of salinity. Given that the k1 value and dissociation constant (KH2CO3) of true H2CO3, which ranges from 1·58 × 10−4 to 3·98 × 10−4 (Stumm & Morgan 1995), the rate constant (k3) of the second reaction giving rise to indirect HCO3 dehydration via H2CO3 at 25 °C and 31·65°/oo of salinity was calculated to be 1·32 to 3·31 s−1 from Equation 1. It should be noted that k1 is the published value at a salinity 33·77 °/oo whereas other constants were calculated using a salinity of 31·65 °/oo. This difference however, is negligible and will not affect the values of the estimated rates.

Acclimation of 5% CO2-grown cells to air

When cells of P. tricornutum, grown on 5% CO2, were transferred to air, the rate of photosynthesis at limited [DIC] (100 mmol m−3) remained constant at about 30 μmol O2 mg−1 Chl h−1 for an initial 3 h but then increased steadily to a level of about 250 μmol O2 mg−1 Chl h−1 over the next 15 h (Fig. 3). This steady increase in photosynthetic rate was followed by a gradual increase after 18 h of acclimation (Fig. 3). The photosynthetic rate at 100 mmol m−3 DIC was stimulated to about two-fold the original level by the addition of bovine CA during the initial 6 h of acclimation (Fig. 3). This stimulation however, diminished as the acclimation proceeded and disappeared after 20 h of acclimation to air (Fig. 3). External CA activity was not detected at any stage of acclimation (data not shown). The maximum rate of photosynthesis was found to be stable in a range 450 to 530 μmol O2 mg−1 Chl h−1 throughout the acclimation period (Fig. 3).

Figure 3.

Changes in photosynthetic O2 evolution rate in high CO2-grown cells during acclimation to air determined at [DIC] of 100 mmol m−3 and 5 mol m−3, pH 8·2 and 25 °C. (▪), O2 evolution rate without added CA; (bsl00000), with added CA; (bsl00066), the maximum rate of photosynthesis at [DIC] of 5 mol m−3. First (time 0) and last (after 30 h) points indicate high CO2-grown cells and air-grown cells, respectively. Each point represents the averaged value ± SE of three separate experiments.

Determination of the DIC species critical for CCM regulation

High CO2-grown cells were allowed to acclimate to different equilibrium concentrations of DIC. To obtain defined DIC conditions, the equilibrium between gaseous and dissolved CO2 was confirmed by comparing the total [DIC] in the medium with the calculated [DIC]. Two dissociation constants of H2CO3, K1 and K2 (see MATERIALS AND METHODS), were used to derive the [DIC] in the bulk medium. Values of calculated and measured [DIC] at various pHs and at two different pCO2 (0·032 and 0·0006%) in inflow air were compared (Table 3); the values of the calculated [DIC] were in good agreement with the actual [DIC] found in the medium, except that [DIC] in the culture medium at pH 9·4 contained a lower [DIC] than the calculated amount (Table 3). Values of K0·5[DIC] and the calculated concentrations of each DIC species present in the growth media are summarized in Table 4. Photosynthetic affinities for DIC (K0·5[DIC]) of cells grown in defined [DIC] at various pHs from 6·5 to 9·4 did not exhibit any correlation with either pH of the medium or with the [DIC] in the growth media (Table 4). The K0·5[DIC] values, in contrast, appeared to be determined by [CO2] in the growth media and were found to be about 1000 mmol m−3 and 300 mmol m−3 when cells were grown in 0·032 and 0·0006%pCO2-equilibrated conditions, respectively (Table 4).

Table 3.  Calculated and measured [DIC] at various pHs in the bulk medium during acclimation of P. tricornutum to 0·032% or 0·0006% CO2
Growth conditions[DIC] in the medium
(mmol m−3)
pCO2 (%)pHCalculatedMeasureda
  1. aValues ± SD of 10 separate measurements.

0·0326·56055 ± 10
 7·5502440 ± 20
 8·227413400 ± 200
 8·561325000 ± 300
 8·7109689900 ± 300
0·00067·5105 ± 3
 9·0644680 ± 10
 9·423431000 ± 150
Table 4. K0·5[DIC] values exhibited in cells grown under various defined DIC conditions
Growth conditions[DIC] in the medium (mmol m−3)
pCO2 (%)pH[CO2][HCO3][CO32−]K0·5[DIC]
(mmol m−3)
0·0326·511·3 ± 2·0 43·6 ± 7·90·125 ± 0·021180
 7·510·8 ± 0·5417·2 ± 19·0 11·9 ± 0·541160
 8·215·3 ± 0·92970 ± 174421·7 ± 24·81140
 8·510·0 ± 0·63880 ± 2331110 ± 66·51020
 8·711·1 ± 0·36810 ± 2063070 ± 93·01160
0·00066·5 did not grow
 7·5 0·113 ± 0·074·47 ± 0·080·127 ± 0·08250
 9·0 0·239 ± 0·004298 ± 4·4268 ± 3·9310
 9·4 0·096 ± 0·014303 ± 4·5684 ± 103260

DISCUSSION

The photosynthetic affinity of algal cells for DIC is known to represent the capacity of the cells to provide CO2 to Rubisco. In most of the algae studied so far, it has been shown that air-grown cells possess a significantly higher affinity than that of Rubisco (Badger et al. 1998) and that affinity diminishes when cells are grown in CO2-enriched conditions (Sültemeyer et al. 1989; Price & Badger 1989; Matsuda & Colman 1995a). However, few reports have described affinities for DIC in cells of marine phototrophs, grown in air and high CO2 except for a detailed report on P. tricornutum by Johnston & Raven (1996).

The two conditions employed in the present study provided contrasting conditions with respect to DIC availability for the cells. Photosynthetic affinity for DIC was shown to change in P. tricornutum in response to DIC concentration in the medium as observed in many freshwater photo-autotrophs (Badger 1987; Coleman 1991; Johnston & Raven 1996; Kaplan & Reinhold 1999). The photosynthetic affinity for DIC in high CO2-grown cells increased almost three-fold on the addition of 10 μg cm−3 of bovine CA; this indicates that there is CO2 transport in high CO2-grown cells which is not saturated by the uncatalysed formation of CO2 in the medium. However, an increase in photosynthetic affinity for DIC by the addition of CA was not observed in air-grown cells. Since extracellular CA activity was not observed in air-grown cells (data not shown), consistent with the results of John-Mckay & Colman (1997), it is probable that efficient utilization of HCO3 occurs in air-grown cells at a rate which may be comparable to CO2 uptake even in the presence of bovine CA.

Photosynthetic affinities of P. tricornutum cells for DIC obtained in this study indicate that high CO2-grown cells without and with added CA, and air-grown cells exhibited affinities for CO2, respectively, 4·53, 1·42 and 0·32 mmol m−3. Studies have shown that the Km[CO2] of Rubisco from a diatom was 31 mmol m−3 (Read & Tabita 1994) and that of P. tricornutum Rubisco was reported to be 41 mmol m−3 (Badger et al. 1998). The present data show that the affinities for CO2, in even high CO2-grown cells, were seven- to nine-fold and 22- to 29-fold the Km[CO2] of Rubisco, without and with added bovine CA, respectively, and this further increased to about 97- to 128-fold the Rubisco Km[CO2] after acclimation to air. These data indicate that P. tricornutum cells can concentrate CO2 internally to supply substrate to Rubisco and that this mechanism appears to occur, at reduced levels, even in high CO2-grown cells.

One of the primary objectives of this study was to re-examine HCO3 dehydration kinetics in seawater in order to relate the kinetics to a physiological analysis of steady-state photosynthesis under DIC-limiting condition. CO2 depletion kinetics in F/2-enriched artificial seawater were first analysed using gas chromatography as described by Birmingham & Colman (1979) (Fig. 2). The dehydration rate calculated at 100 mmol m−3 HCO3 and pH 8·2 was about 0·6 mmol m−3 min−1 which is about 8·4 times slower than that in freshwater as calculated by the method of Miller & Colman (1980). The resulting estimate for the rate constant of H2CO3 dissociation into H2O and CO2 (k3), based upon published values of k1 and KH2CO3, was found, maximally, to be 3·31 s−1 which is about 5·4 times lower in value than that in freshwater (Pocker & Bjorkquist 1977). The rate of aeration of medium with N2 clearly affected the CO2 depletion kinetics presumably because of changing the diffusion rate of CO2 out of the medium. With the aeration rate more than 1·0 dm3 min−1, however, the CO2 diffusion rate appeared to be constant. There are other data available which can be used to assess the uncatalysed CO2 formation rate in medium of 32°/oo salinity at pH 7·5, namely, Rotatore, Colman & Kuzma (1995) monitored, mass-spectrophotometrically, the formation of CO2 in 25 mol m−3 1,3 Bis[tris(hydroxymethyl)methylamino]-propane (BTP) buffer with a salinity of 32°/oo upon the addition of 100 mmol m−3 of HCO3 to the medium and, from the published data and taking into account the dissociation equilibria described by Goyet & Poisson (1989), it can be calculated that the final concentration of HCO3 immediately after the addition of HCO3 was about 132 mmol m−3 (Rotatore et al. 1995). The rate of CO2 formation at 100 mmol m−3 HCO3 calculated from their data was 3·7 to 3·8 mmol m−3 min−1. The CO2 formation rate calculated as described above, using KH2CO3 of 3·98 × 10−4 and k3 of 3·31 s−1, is 2·2 mmol m−3 min−1 at a salinity of 31·65°/oo, a [DIC] of 100 mmol m−3 and pH 7·5. This value is in reasonable agreement with the value obtained from mass-spectrometry described above. Under natural seawater of about 2 mol m−3 [DIC], CO2 formation rate could be calculated to be about 16 mmol m−3 min−1 at 25 °C whereas diatom culture of 10 μg Chl a cm−3, could photosynthesize maximally at the rate 50–83 mmol m−3 min−1. If cells take up only CO2, the capacity of seawater to supply substrate for photosynthesis can limit the rate of photosynthesis.

Comparison of the O2 evolution rate with the spontaneous CO2 formation rate clearly indicates that photosynthetic rates at defined DIC concentrations are much higher than the rate of CO2 supply in the medium. The photosynthetic rate reached 53·5 times the calculated supply rate at 100 mmol m−3 DIC in air-grown cells and 7·3 times in high CO2-grown cells, unequivocally demonstrating that HCO3 in the medium is taken up directly by this alga, whether grown in air or high CO2. These data indicate that both CO2 and HCO3 are taken up as substrates for photosynthesis in high CO2-grown cells of P. tricornutum. It is not clear from the present data whether CO2 is taken up actively in air-grown cells since no increase in photosynthetic affinity for DIC was detected by the addition of bovine CA, but in P. tricornutum strain UTEX642, it was clearly shown by mass-spectrometric assay that air-grown cells take up CO2 rapidly from the medium before steady-state photosynthesis is established (Rotatore et al. 1995). These data suggest that P. tricornutum used in this study may use both CO2 and HCO3 actively as substrates for photosynthesis. Considering that the rate of O2 evolution greatly exceeds the maximum CO2 supply rate in the medium, it appears that the capacity of air-grown P. tricornutum to take up HCO3 is extremely high and may be comparable to that of CO2 uptake. This conclusion is supported by the finding that cells could be grown on 0·0006% CO2 at alkaline pH but not at pH 6·5. As the concentration of CO2(aq) is constant, independent of pH of the bulk medium under equilibrium condition, the growth of cells seems to rely largely on the use of HCO3.

High CO2 concentrations partially suppressed DIC uptake but complete repression of bicarbonate transport or CO2 transport was not observed. The rate of photosynthesis in high CO2-grown cells was seven times the spontaneous supply rate of CO2 and there was an active uptake of CO2. The rate of O2 evolution at 100 mmol m−3 DIC was comparable to that reported in Chlorella sp. at the same [DIC] but lower pH (7·8) (Matsuda & Colman 1995a). As the [CO2] available in seawater at pH 8·2 is about 8·5 times lower than that in freshwater at pH 7·8 and the CO2 formation rate is also lower by a factor of about 18, cells may still require HCO3 from the medium to compensate for the severe CO2 limitation in seawater. Nevertheless, the utilization of CO2 was very clearly demonstrated in high CO2-grown cells of P. tricornutum. However, as acclimation proceeds it is apparent that bicarbonate transport activity increased and by the time acclimation was complete CO2 transport was less important than bicarbonate transport, that is, the addition of bovine CA did not increase the photosynthetic rate. These observations are in sharp contrast with those of the acclimation of the green alga C. ellipsoidea to air (Matsuda & Colman 1995a) in that, both HCO3 and CO2 transport increased as acclimation proceeded and HCO3 was never the major DIC species taken up, presumably because enough CO2 was still available for cells even at pH 8·0.

The time required to fully express DIC transport has been found to be 3–6 h in freshwater algae depending on the species (Shiraiwa & Miyachi 1985; Sültemeyer et al. 1991; Matsuda & Colman 1995a) whereas it was about two to three times longer in P. tricornutum (Fig. 3) so that high CO2-grown cells were fully acclimated to air after 24 h. Few reports are available on time course of acclimation of high CO2-grown marine photo-autotrophs to air. The finding that the induction of DIC transport in this diatom has a 2–4 h lag period and is relatively slow in developing high-affinity photosynthesis on exposure to CO2-limiting conditions is noteworthy although the reason for such slow acclimation is unclear.

Changes in pCO2 in the inflow air and the photosynthetic activity of the cells change the concentrations of all DIC species in the medium and hence it is difficult to determine exactly which DIC species is the critical species determining CCM expression. In the present study, defined conditions of DIC were created as described by Mayo et al. (1986) and Matsuda & Colman (1995b) by using air of two pCO2 and seven different pHs. The [DIC], calculated according to polynomial function described by Goyet & Poisson (1989) gave values about 1·3 times those derived according to Mehrbach et al. (1973) (Table 3). The actual concentration of DIC in the culture media at the corresponding conditions of pH and pCO2 agreed closely with calculated values, clearly indicating that the species of DIC, CO2, HCO3 and CO32−, are maintained at equilibrium and hence their concentrations can be determined from the total measured [DIC]. The affinity of the cells acclimated to defined DIC conditions exhibited a clear correlation with CO2(aq) in the media but did not change in response to changes in [HCO3], [CO32−] or total [DIC].

The K0·5[DIC] values observed in cells acclimated to 0·0006%pCO2 were about three times that of air-grown cells (Fig. 1, Table 1). It is probable that very slow aeration created extremely low [CO2] condition. In fact, the measured value of [DIC] in air-grown cells was 3 mmol m−3 in which the maximum calculated [CO2] is 0·07 mmol m−3. Thus, cells acclimated to 0·0006% equilibrium condition probably attained an intermediate level of photosynthetic affinity for DIC. Supporting this interpretation, cells acclimated to less than 0·0002% CO2-equilibrated medium exhibited a K0·5[DIC] of less than 80 mmol m−3 (data not shown). DIC in the medium did not reach equilibrium by aeration with 0·0006% CO2 at pH 9·4 (Table 3) presumably due to the rapid use of DIC by the cells. However, it is evident that high-affinity photosynthesis in P. tricornutum is expressed independent of either HCO3 or CO32− since it is fully repressed under 40 and 0·13 mmol m−3 HCO3 and CO32− at pH 6·5, respectively, whereas it is fully expressed under 300 and 270 mmol m−3 of HCO3 and CO32− at pH 9·0, respectively (Table 4). This clearly indicates that the primary signal which triggers the development of high-affinity photosynthesis and determines the extent of acclimation in P. tricornutum is the [CO2(aq)] in the medium. This agrees with the results obtained with green algae (Matsuda & Colman 1995b; Bozzo et al. 2000) but are in sharp contrast to those reported for a cyanobacterium by Mayo et al. (1986) where the extent of acclimation was found to be determined by total [DIC] in the medium rather than [CO2(aq)].

The concentration of CO2(aq) functioning as a signal in P. tricornutum is below air level (12·3 mmol m−3). It was similarly shown by Johnston & Raven (1996) that air equilibrium conditions resembled high-CO2 conditions in terms of regulating the CCM in P. tricornutum CCAP1052/1 A. Similar results were also obtained in the green algae, C. reinhardtii (Bozzo & Colman 2000) and Chlorella kessleri (Bozzo et al. 2000). In the green alga, C. ellipsoidea, however, the critical CO2 concentration was more than three times air level, i.e. 40–120 mmol m−3 (Matsuda & Colman 1995b).

The sensitivity of aquatic photo-autotrophs to CO2(aq) certainly varies with species and the mechanisms by which cells perceive changes in the concentration of DIC may also vary depending on species. The inorganic carbon signal may be HCO3 (Mayo et al. 1986; Sültemeyer et al. 1998; Chen et al. 2000) or CO2 (Matsuda & Colman 1995b; Bozzo et al. 2000; Bozzo & Colman, 2000) and thus aquatic photo-autotrophs may regulate the CCM by means of a CO2-sensing mechanism or a HCO3 sensing mechanism. The type of sensing mechanism may be an adaptation to a particular habitat; in the case of neretic species, where the environment changes markedly in CO2 concentration and photon flux density but where the bicarbonate concentration remains relatively constant, it may be advantageous to the survival of an alga to respond to changes in CO2 concentration. The marine diatom, P. tricornutum, from the data in this study, is clearly a CO2-sensing type of alga.

ACKNOWLEDGMENTS

This work was supported in part by a Grant from Invitation for RITE (Research Institute of Innovative Technology for the Earth) Research Proposals; and in part by Kwansei-Gakuin University through a Special Grant for Individual Researchers to Y.M and a Visiting Professorship to B.C.

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