JXB Advance Access originally published online on November 26, 2007
Journal of Experimental Botany 2007 58(15-16):4195-4202; doi:10.1093/jxb/erm276
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© 2007 The Author(s).
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RESEARCH PAPER |
Phosphorus acquisition by Chlamydomonas acidophila under autotrophic and osmo-mixotrophic growth conditions
Department of Ecology and Ecosystem Modelling, University of Potsdam, Am Neuen Palais 10, Potsdam, Germany
* E-mail: spijker{at}rz.uni-potsdam.de
Received 16 August 2007; Revised 11 October 2007 Accepted 15 October 2007
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Abstract |
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Chlamydomonas acidophila Negoro is a green algal species abundant in acidic waters where inorganic phosphorus (Pi) and carbon (CO2) are considered the most important growth-limiting nutrients for the phytoplankton. This paper describes the Pi uptake and growth kinetics under varying carbon supply by cultivating the alga autotrophically, with and without CO2 aeration, and osmo-mixotrophically with glucose under low Pi conditions at pH 2.7. The low minimum cellular phosphorus quota (Q0; ranging from 0.6 to 1.1 mmol P mol–1 C) suggested Pi-limiting conditions under all different modes of carbon supply, and was lowest under CO2-aerated conditions. The threshold Pi concentration for growth did not vary from zero, suggesting no detectable metabolic costs. Maximum Pi-uptake rates (Vmax) were a better indication of Pi limitation when compared with the affinity constant for Pi uptake (Km), as Vmax was only high under Pi-limited conditions whereas Km was low under both Pi-limited and Pi-replete conditions. Osmo-mixotrophic growth conditions did not result in decreased extracellular phosphatase activity, but often resulted in physiological characteristics comparable with CO2-aerated cells, suggesting intracellular CO2 production by glucose respiration. In addition, at low CO2 and in autotrophic conditions, C. acidophila had a higher Q0, lower dissolved organic carbon concentration, lower maximum Pi-uptake rates, and lower phosphatase activity, suggesting that growth was co-limited by CO2 and Pi. Furthermore, cells may respond physiologically to both nutrient limitations simultaneously.
Key words: Acidophilic algae, Chlamydomonas acidophila, CO2, co-limitation, extremophile, glucose, growth, osmo-mixotrophy, phosphatase activity, P limitation
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Introduction |
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Very acidic lakes and rivers with pH values between 2.0 and 3.2 are found all over the world (Doi et al., 2001; Lopez-Archilla et al., 2001; Baffico et al., 2004; Kamjunke et al., 2004). In these waters, Chlamydomonas acidophila is often an abundant species that maintains an optimal growth rate when the external pH is acidic (Nishikawa and Tominaga, 2001; Gerloff-Elias et al., 2005; Spijkerman, 2005). Recent studies unequivocally showed that C. acidophila maintains a neutral intracellular pH (Messerli et al., 2005; Gerloff-Elias et al., 2006), which results in increased metabolic costs (Nishikawa et al., 2006). Increased metabolic costs can result in an increased cellular phosphorus (P) content, as suggested by Nishikawa et al. (2006) and Spijkerman et al. (2007), or increased ATP consumption rates (Messerli et al., 2005), for which proof is still under debate.
Under inorganic phosphorus (Pi)-deprivation, Chlamydomonas reinhardtii increased its maximum Pi-uptake rate (Vmax) and Pi uptake affinity [reflected by a decrease in affinity constant for Pi uptake (Km); Grossman, 2000] and in continuous cultures, these physiological characteristics generally increased with decreasing growth rate (Spijkerman and Coesel, 1996b). These physiological characteristics have not been described for C. acidophila thus far. In acidic lakes, both Pi and CO2 are most likely the growth-limiting nutrients (Tittel et al., 2005; Spijkerman et al., 2007), which makes the interaction between these nutrients interesting to study. It appears likely that the uptake and growth kinetics for Pi and CO2 interact as inorganic carbon acquisition varied with the Pi state of the single-celled chlorophyte Chlorella (Kozlowska-Szerenos et al., 2004; Beardall et al., 2005). The results from both studies on Chlorella were contrasting: in the study of Koslowska-Szerenos et al. (2004), the affinity constant for CO2 uptake decreased with increasing P depletion, whereas the same parameter increased with increasing P depletion in the study of Beardall et al. (2005). In addition, the cellular P quota (Qp) was higher at low CO2 than at high CO2 concentrations; for example, in the marine diatom Skeletonema costatum (Burkhardt and Riebesell, 1997). Under Pi-limited conditions, algal cells normally produce a surplus of organic carbon that is partly excreted (e.g. as glycolate). Therefore, Pi-limited cultures usually contain increased concentrations of dissolved organic carbon (DOC) and Pi-limited algae have an increased C:P ratio and are likely to be heavier.
In addition, Pi-limited algal cells synthesize an extracellular enzyme called alkaline or acid phosphatase (Spijkerman and Coesel, 1998; Beardall et al., 2001; Grossman and Takahashi, 2001). The synthesis of phosphatase enzymes under Pi-deplete, acidic conditions was reported for C. acidophila (Boavida and Heath, 1986; Spijkerman et al., 2007) and for C. reinhardtii (pH 4; Joseph et al., 1995). In phytoplankton from a eutrophic lake and an acid bog, the induction of this enzyme appeared especially important because P acquisition was faster from glucose-6-P than from Pi (Nedoma et al., 2003). The hydrolysis of glucose-6-P, results in glucose and Pi, both of which can be used by C. acidophila (Bissinger et al., 2000). Therefore, phosphatase enzyme activity could relate both to Pi depletion and carbon availability. As the production of Pi is known to inhibit phosphatase activity (O'Brien and Herschlag, 2001), it is possible that the presence of the other end-product, glucose, might also inhibit phosphatase activity.
In this study, the influence of varying carbon sources on Pi physiology and phosphatase activity in C. acidophila was investigated. Specimens were grown in semi-continuous, autotrophic Pi-limited culture conditions, with and without CO2 aeration, and under osmo-mixotrophic Pi-limited conditions. Osmo-mixotrophic growth conditions were obtained by the addition of glucose to the growth medium. The minimum cellular P quota (Q0), threshold Pi concentration for growth (Pi,t), Pi uptake kinetics, and phosphatase activity were obtained at pH 2.7.
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Cultures
Chlamydomonas acidophila Negoro, isolated from Lake 111 (SAG Göttingen, strain no. 2045), was grown in semi-continuous cultures at 19.5±1 °C in Woods Hole (WH) medium (Nichols, 1973), with no buffer, a Pi concentration of 1.6 µmol l–1, and a pH adjusted to 2.7 with HCl. Pi-saturated batch cultures contained 50 µmol Pi l–1 and were harvested in the mid-exponential phase of growth, with a low cell density. Osmo-mixotrophic growth was established by the addition of 1 mmol glucose l–1 in the medium, and cultures were placed in light identical to that used for autotrophic cultures. Dilution rates were 0.1, 0.2, 0.3, 0.4, and 0.6 d–1 in non-aerated cultures and from 0.1, 0.2, 0.4, 0.6, and 0.8 d–1 in cultures aerated with 4.5% CO2 in normal air (v/v) and in osmo-mixotrophic cultures (which were non-aerated). All treatments were performed in duplicate. Aeration did not result in significant mixing of the culture suspension and was
15 ml h–1. All cultures were mixed regularly and a total culture volume of 500 ml in a 1.0 l Erlenmeyer flask provided a large surface area for O2 exchange with the air. Incident light supply was 200 µmol photons m–2 s–1 with a light/dark period of 16/8 h. Daily dilution and harvesting were performed 4–5 h after the onset of light. Average CO2 concentrations in the CO2-aerated cultures were measured as dissolved inorganic carbon using a carbon analyser (HighTOC+N; Elementar) and were 0.33 (±0.05, n=20) mmol C l–1. This rendered CO2-limitation unlikely and this was confirmed by experiments where the CO2 concentration in the medium was increased, but growth rates did not change. Non-aerated cultures had a CO2 concentration below the detection limit of the carbon analyser (<0.04 mmol C l–1) and were likely to be equivalent to equilibrium concentrations with the air (0.02 mmol C l–1). The pH of all cultures was 2.7, independent of aeration with CO2. Average optical density (OD) ranged from 0.01 to 0.13. The OD of each culture was measured before and after dilution at 750 nm (UV1202, Shimadzu, Germany). After the cultures had reached a steady state (remaining at constant OD after an exchange of 3–5 times the culture volume), samples were taken for cell and bacterial counts, chemical analyses, Pi-uptake rates, and phosphatase activity. Cell numbers were determined using an automatic cell counter (CASY 1, Model TT, Schärfe, Reutlingen, Germany). Bacteria were enumerated under an epifluorescence microscope (Axioscop2, Zeiss) after staining with acridine orange on black 0.2 µm Nuclepore filters (Hobbie et al., 1977).
Phosphatase activity
Phosphatase activity was measured using difMUP (6,8-difluoro-4-methylumbelliferyl phosphate; Molecular Probes, Leiden, The Netherlands) on a fluorometer (Turner TD-700, ex: 365 nm, em: 410–610 nm; GAT Bremerhaven). The reaction solution contained 20 mmol Na-acetate l–1, 20 mmol HEPES [4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid] l–1, and 2 mmol MgCl2 l–1. This solution was brought to pH 2.7, 6.0, and 9.0, to measure the activity at the culturing pH and at two previously determined optima (Spijkerman et al., 2007). Activity was measured in suspensions of Pi-limited and Pi-replete cultures, and in the supernatant of these cultures after centrifugation (2500 g, 5 min). The cellular activity was calculated by subtraction of these two fractions. Acid phosphatase from potato (EC 3.1.3.2; Sigma) and alkaline phosphatase from Escherichia coli (EC 3.1.3.1; Sigma) were similarly treated and served as standards. The increase in fluorescence was measured over a 3 min period after mixing 0.5 ml of 25 µmol difMUP l–1, 0.5 ml of sample, or standard and 1.5 ml of buffer. Fluorescence changes over time and fluorescence intensity after 3 min were calibrated to the acid phosphatase at pH 2.7 and pH 6.0 and to alkaline phosphatase at pH 9.0. All measurements were performed in triplicate.
Pi-uptake kinetics
Cultures were centrifuged (1500 g, 5 min) and the pellet was resuspended in WH medium without Pi and iron-EDTA at a pH of 2.7. Final densities were between 1.1x105 and 4.4x105 cells ml–1. The culture was placed in the light (90 µmol m–2 s–1 inside the flask) for about 15–30 min. Over a period of 1 min, 33P uptake was measured by addition of H333PO4 (3000 Ci mmol–1 specific activity; Amersham Biosciences, Freiburg) diluted in stock solutions of 50 or 500 µmol K2HPO4 l–1. Uptake was terminated by filtration on 1.2 µm pore-size cellulose acetate filters and subsequent rinsing with 0.2 mol LiCl l–1. The filters were embedded in Ultima Gold (Packard) and counted in a liquid scintillation analyser (2300 TR Packard). The Pi-uptake rate (V) was measured at eight concentrations ranging from 0 to 10 µmol Pi l–1 using six time points each. From these rates, maximum uptake rates (Vmax) and the affinity for uptake (Km) were estimated by fitting the data using SPSS software (version 11.5) to the Michaelis–Menten equation:
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Chemical analyses
Total P content of the cell suspension was determined on culture samples heated to 100 °C for 1 h with K2S2O8 and 0.5 mol H2SO4 l–1. For soluble reactive P (SRP), 12 ml culture samples were centrifuged at 2500 g for 5 min, and 10 ml of supernatant was taken for analysis. Measurements of total P and SRP were performed spectrophotometrically using molybdate and ascorbic acid (Murphy and Riley, 1962).
For carbon analyses, culture samples were filtered on pre-combusted, pre-weighed QF20 (Schleicher and Schuell) or GF/F filters. The filter was used for particulate and the filtrate for dissolved organic carbon (DOC) determination in the carbon analyser (HighTOC+N; Elementar). Before measuring particulate organic carbon, filters were dried for 1 week at 30 °C and the dry weight of the algae determined.
Calculation of Pi
Because SRP concentrations in the culture vessel were very low and their values would not equal Pi (Rigler, 1968), Pi was calculated by coupling the kinetics of steady-state nutrient uptake (V, 
equation 1) with the kinetics of growth (µ; Spijkerman and Coesel, 1996b). Growth rates were related to the cellular P quota (Qp) by Droop's equation (Droop, 1973):
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. These Pi concentrations were used for the determination of maximal growth rate (µmax), the affinity constant for growth (Ks), and the threshold Pi concentration for growth (Pi,t, i.e. Pi at µ=0) according to the Monod equation (modified by Tilman and Kilham, 1976):
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Results |
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The growth rates of C. acidophila decreased with decreasing cellular quota and were significantly different under CO2-aerated, non-aerated, and osmo-mixotrophic growth conditions (ANCOVA, P <0.005; Fig. 1A). Estimation of the minimum Qp (Q0) using
equation 2 (Droop equation) showed that Q0 is lowest in CO2-aerated cells (+CO2), intermediate for the osmo-mixotrophic cells (+glucose), and highest for the non-aerated cells (–CO2). Estimated Q0 values from the Droop equation are provided in Table 1.
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Growth rates decreased with decreasing residual Pi concentrations in the medium, which allowed the Monod equation (equation 4; Fig. 1B) to be fitted. The estimated threshold Pi concentration for growth (Pi,t) of C. acidophila was not significantly different from zero and not significantly different under the various culture conditions (Table 1). Pi,t concentrations were on average 0.023 nmol Pi l–1. The largest difference among the various culture conditions was detected in the estimated maximum growth rate. The µmax was about 1.5-fold greater in the CO2-aerated and 2.5-fold greater in the osmo-mixotrophic cultures than in the non-aerated cultures. There was no significant difference in Ks between cultures due to the high standard error, although the estimated value of the autotrophic cultures appeared lower than that of the osmo-mixotrophic cultures (Table 1). The conductance to Pi (µmax/Ks) was highest in the CO2-aerated cells, intermediate in the osmo-mixotrophs, and lowest in the non-aerated cells. This implies that at the lower Pi concentrations, CO2-aerated cells can achieve the highest growth rates.
SRP concentrations at the lowest growth rates (
0.4 d–1) were averaged and considered to be the threshold SRP concentration for growth (SRPt). The SRPt concentrations were calculated to be 0.12±0.03, 0.12±0.02, and 0.10±0.02 µmol P l–1 (mean ±standard error) with and without CO2 aeration and with glucose, respectively. These values were not significantly different and were >1000-fold higher than the Pi,t concentrations.
The phosphatase activity of C. acidophila was highest at pH 6, whereas both at pH 2.7 and pH 9.0 very low activities were determined (ANCOVA, P <0.001; Fig. 2). Phosphatase activities increased with decreasing growth rates once the effect of the C-source was accounted for (ANCOVA, P <0.05). In the cultures at low growth rates (i.e. µ=0.1 and 0.2 d–1), phosphatase activities were lower in the non-aerated than in CO2-aerated and osmo-mixotrophic cultures (Friedman test paired over the measurements at all three pH values, n=6, df=2, P <0.05).
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Maximum Pi-uptake rates (Vmax) were independent of growth rate in all different Pi-limited cultures (Pearson, P >0.05) and were only significantly higher in the high CO2 than in the low CO2 cultures (ANOVA, P <0.05; Fig. 3). In the P-saturated cultures, Vmax was 1.3 µmol P 10–9 cells h–1, this being
60-fold lower than in Pi-limited cultures. The affinity constant for uptake (Km) was also independent of growth rate (Pearson, P >0.05) and did not differ significantly between the application of different C-sources (ANOVA, P >0.05). Remarkably, in P-saturated cultures, Km was significantly lower than in the Pi-limited cultures independent of the application of different C-sources (ANOVA, P <0.05).
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The DOC concentrations in CO2-aerated cultures were negatively correlated to the growth rate (Pearson, P <0.005), whereas a correlation was not found in the non-aerated cultures (Table 2; the presence of glucose prevented the possibility of determining DOC in the osmo-mixotrophic cultures). In addition, DOC concentrations were significantly higher in CO2-aerated than in non-aerated cultures once the effect of growth rate was accounted for (ANCOVA, P <0.05; Table 2). The relative carbon content of C. acidophila (calculated as mg carbon per mg dry weight) did not correlate with growth rate nor differ with C-source (ANCOVA, all P >0.05; Table 2). In CO2-aerated and osmo-mixotrophic cultures, cell volume had a negative correlation with growth rate (Pearson, P <0.05), whereas in non-aerated cultures, this correlation was only found at growth rates between 0.1 d–1 and 0.5 d–1 (Table 2). The cell volume was largest in CO2-aerated, intermediate in size in osmo-mixotrophic, and smallest in non-aerated cells once the effect of growth rate was accounted for (ANCOVA, P <0.05; Table 2). Consequently, the cellular carbon content and cellular dry weight were highest in CO2-aerated, intermediate in osmo-mixotrophic, and smallest in non-aerated cells (results not shown). Bacterial densities were independent of growth rate, DOC, and CO2 aeration (not shown), and were considered too low (max. 1x109 bacteria l–1) to influence algal physiology.
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Discussion |
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Chlamydomonas acidophila had unconventional adaptations to Pi-limiting conditions, as Km was low under both Pi-limited and Pi-replete conditions. The presence of different C-sources had a clear influence on the Pi physiology of C. acidophila, and both Pi and CO2 most likely limited growth under autotrophic, non-aerated Pi-limited conditions.
Growth and minimal Pi requirements
In accordance with previous studies, maximum growth rate of C. acidophila was increased by both CO2 aeration and glucose (Tittel et al., 2005). The extent of the stimulation of µmax by CO2 was comparable under the Pi-replete (Tittel et al., 2005) and Pi-limiting conditions (this study) and indeed comparable CO2 concentrations were applied in both studies. The µmax in the osmo-mixotrophic (i.e. in the presence of glucose) cultures in this study was higher than that reported for Pi-replete cultures (Tittel et al., 2005), and was likely to be the result of the higher glucose concentrations used in this study (1 mmol l–1 in this study compared with 0.4 mmol l–1 in Tittel et al., 2005).
The calculated Monod relationshops revealed that there was no detectable threshold concentration of Pi for growth (Pi,t) in C. acidophila. The Pi,t was similar in all cultures and was estimated to be 0.023 nmol Pi l–1 on average, but this was not significantly different from zero. A Pi,t was described in the green algae Cosmarium abbreviatum, Staurastrum pingue, and Staurastrum chaetoceras (Spijkerman and Coesel, 1996a) but the Pi,t of these desmids was 1000-fold higher (ranging from 17 to 43 nmol Pi l–1) than the Pi,t described here, underlining the non-significance of the Pi,t concentration in this study. The absence of a Pi,t in C. acidophila suggests that all Pi can be directly converted into biomass and therefore indicates no measurable increased metabolic costs. This does not exclude the metabolic costs suggested for an isolate of C. acidophila from the Rio Tinto because these costs consisted of only slightly increased ATP consumption rates that cannot be measured with the methods used in this study (Messerli et al., 2005).
In an earlier study of Pi limitation in C. acidophila using a complex medium containing high concentrations of iron, aluminium, and zinc (the composition of this medium is described in Spijkerman et al., 2007), threshold SRP values for growth (SRPt) were determined from the SRP concentrations at the lowest growth rates (0.4 d–1; Spijkerman et al., 2007). In the metal-rich medium, SRPt was estimated as 0.19 and 0.22 µmol P l–1 with and without CO2 aeration, respectively. In the present study, using WH medium that contains low concentrations of iron and zinc and no aluminium, SRPt values ranged from 0.10 to 0.12 µmol P l–1. These 2-fold lower concentrations can be explained by increased Fe–P complexation in the metal-rich medium that is obviously unavailable for growth of C. acidophila, but is detected in the SRP fraction. Most of the acidic lakes are rich in Fe, suggesting therefore that the SRP measured is not 100% available for algal growth.
The minimal cell quota (Q0) represents the amount of phosphate associated with the structural and metabolic components that are essential for cellular integrity and viability (Droop, 1974). In this study, using WH medium that contains NO3– as the sole N-source, Q0 was higher in non-aerated than in CO2-aerated cultures (1.14±0.14 and 0.59±0.06 mmol P mol–1 C, respectively). In a previous study, using a metal-rich medium that contained NH4+ as the primary N-source, a non-significant trend towards a similar difference was found (0.81±0.04 and 0.77±0.04 mmol P mol–1 C in non-aerated and CO2-aerated cultures, respectively; Spijkerman et al., 2007). The Q0 in CO2-aerated cells was not significantly different in WH medium or metal-rich medium (ANCOVA; df=24, 2; F=11; P >0.05). The cellular carbon content and cell volume determinations support the similar Q0 in CO2-aerated cells cultured in both media as these cellular characteristics are the same in the cells grown in WH medium as those in metal-rich medium (compare 4.1 pmol C cell–1 and 111 µm3 in WH medium and 3.7 pmol C cell–1 and 106 µm3 in metal-rich medium, both grown at 0.2 d–1). By contrast, the Q0 in non-aerated cells cultured in WH medium was significantly higher than cells cultured in the metal-rich medium (ANCOVA; df=18, 2; F=25; P <0.05), which might be a consequence of increased metabolic costs resulting from the reduction of NO3– to NH4+ before the incorporation of N into amino acids. The reduction of NO3– to NH4+ requires electrons delivered from photosynthesis (Toepel et al., 2004) thereby increasing the ATP and NADPH demand compared with a situation where NH4+ is the acquired N-species. As a consequence µmax was 1.5-fold higher in Dunaliella saline when NH4+, rather than NO3–, was the N-source used (Giordano, 2001). In addition, the affinity for CO2 uptake was higher in cells of Dunaliella salina and D. parva grown in NH4+-N than in NO3–-N medium (reviewed in Giordano et al., 2005). To summarize, algae grown in medium containing NH4+-N might have a lower cellular P and a higher cellular C content than cells in NO3–-N medium.
Phosphatase activity
In this study, very little extracellular phosphatase activity was found in measurements performed at pH 2.7 and pH 9.0. The latter is in accordance with a previous study on the induction of phosphatase enzymes in C. acidophila (Boavida and Heath, 1986). The present results at pH 9.0, however, contrast with a previous study (Spijkerman et al., 2007) where inducible alkaline phosphatase activity was found in C. acidophila. This alkaline phosphatase activity was detected in natural phytoplankton as well as in cultures grown in metal-rich medium reflecting the chemical composition of the lake water. Some components of the mining lake water, such as high concentrations of sulphur, iron, or other metal ions, might possibly stimulate the induction of alkaline phosphatase enzymes (Sabater et al., 2003). However, the alkaline phosphatase activity was only detected when the algae were grown in Pi-deplete, ion-rich medium, and consequently high concentrations of ions alone do not induce these enzymes. At a growth rate of 0.1 d–1, phosphatase activity of C. acidophila measured at pH 6.0 was about 2-fold lower in this study using WH medium, than when cultured in metal-rich medium (Spijkerman et al., 2007). Therefore, the activity at pH 6.0 might also have been increased by the high concentrations of ions in the metal-rich medium. The negative correlation between phosphatase activity and dilution rates is in accordance with findings from studies of other algae, for example, C. reinhardtii (Olsen et al., 1983).
The phosphatase activity was lowest in the non-aerated cultures, which suggests that growth of C. acidophila in the non-aerated cultures was not Pi-limited to the same extent as CO2-aerated or osmo-mixotrophic cultures. Presumably, growth of C. acidophila was co-limited by Pi and CO2 in the non-aerated cultures (also see below).
Phosphatase activities in osmo-mixotrophic cultures were similar to those in CO2-aerated cultures, although they were also non-aerated. Therefore, the presence of glucose did not inhibit enzyme activity. Possibly, CO2 was produced intracellularly by the respiration of the glucose (Chen and Gibbs, 1991; Villarejo et al., 1995), resulting in high CO2 conditions.
Pi uptake kinetics
By contrast to expectations based on other studies (Fu et al., 2006), the maximum uptake rate of Pi was independent of the growth rate, although the cellular P quota increased at least 2-fold with increasing growth rate. Although maximum Pi-uptake rates (Vmax) are inhibited by an increased Qp (Nieuwenhuis and Borst-Pauwels, 1984), only under Pi-replete conditions, when Qp was 10-fold higher than under Pi-limited conditions (54±32 mmol P mol–1 C), was Vmax 30-fold lower. Similarly, Vmax and Km in the green alga Cosmarium abbreviatium remained stable over a 2-fold change in Qp and only changed when cells were nearly flushed out of the continuous cultures by high dilution rates (Spijkerman and Coesel, 1996b).
Chlamydomonas acidophila had a high affinity uptake system for Pi, independent of aeration with CO2, availability of glucose, or Pi saturation (Fig. 3). With a Km value varying between 0.1 and 1.2 µmol Pi l–1, this high affinity uptake system is comparable with that described in a broad range of other green algal species (Rhee, 1973; Gotham and Rhee, 1981; Healey and Hendzel, 1988; Jansson, 1993; Spijkerman and Coesel, 1996b; Grossman, 2000).
The Vmax of Pi uptake by C. acidophila under non-aerated conditions was lower than that under CO2-aerated growth conditions, which together with the higher Q0, lower DOC, and lower phosphatase activity indicated that the non-aerated cells were less stringently Pi-limited than the CO2-aerated or osmo-mixotrophic cells. Presumably, non-aerated cells were co-limited by CO2 and Pi under low CO2 and low Pi conditions, and invested metabolic energy in physiological responses related to both nutrient limitations. Co-limiting conditions for Pi and NO3– in phytoplankton growth have often been described (Elser et al., 1990; Davies et al., 2003), but not many studies describe the regulatory influence of both resource limitations on the physiological acclimation. From the few studies on the physiological acclimation to low CO2 and low Pi concentrations, a down-regulation of the inorganic carbon uptake was shown under Pi-limited conditions in Chlorella emersonii (Beardall et al., 2005). Inorganic carbon acquisition under low CO2 conditions is an active process in most micro-algae, requiring ATP, and it might therefore be expected that Pi limitation could have a direct regulatory influence on carbon acquisition. At present, it is not clear if active CO2 uptake is an important process compared with CO2 diffusion in C. acidophila, and this requires further investigation. A recent paper on CO2 acquisition in an acid-tolerant Chlamydomonas suggests solely diffusive uptake (Balkos and Colman, 2007), whereas an energy-demanding carbon-concentrating mechanism was found in C. acidophila under low CO2 conditions (Spijkerman, 2005). Future work will report on the effect of different C-sources on CO2-acquisition during Pi-limitation in C. acidophila. This study, however, provides the first indications of a co-limitation for CO2 and Pi in C. acidophila and the possible regulation of CO2 limitation on Pi acquisition.
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Acknowledgements |
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This work was supported by the German research foundation (DFG, SP695/2).
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Abbreviations |
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DOC, dissolved organic carbon; Km, affinity constant for Pi uptake; Ks, affinity constant for growth; µ, growth rate; µmax, maximum growth rate; µ'max, apparent maximal growth rate that would occur if Qp became infinite; OD, optical density; Pi, inorganic phosphorus; Pi,t, threshold Pi concentration for growth; Q0, minimum cellular P quota; Qp, cellular P quota; SRP, soluble reactive P; SRPt, threshold SRP value for growth; V, Pi-uptake rate; Vmax, maximum Pi-uptake rate; WH medium, Woods Hole medium.
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