# Photorespiration in continuous of Dunaliella tertiolecta (Chlorophyta) : relationships between serine, glycine, and extracellular glycolate

Christophe Leboulanger, V. Martin-Jézéquel, C. Decolas-Gros, Antoine Sciandra, Henri Jupin

► To cite this version:

Christophe Leboulanger, V. Martin-Jézéquel, C. Decolas-Gros, Antoine Sciandra, Henri Jupin. Photorespiration in continuous of Dunaliella tertiolecta (Chlorophyta) : relationships between serine, glycine, and extracellular glycolate. Journal of Phycology, 1998, 34, pp.651-654. 10.1046/j.1529-8817.1998.340651.x. hal-02696111

HAL Id: hal-02696111

https://hal.inrae.fr/hal-02696111

Submitted on 26 Aug 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
NOTE

PHOTORESPIRATION IN CONTINUOUS CULTURE OF *DUNALIELLA TERTIOLECTA*
(CHLOROPHYTA): RELATIONSHIPS BETWEEN SERINE, GLYCINE, AND
EXTRACELLULAR GLYCOLATE<fnref n="1" />

*Christophe Leboulanger*<fnref n="2" />

Station d'Hydrobiologie Lacustre, INRA BP511, 74203 Thonon-les-Bains, France

Véronique Martin-Jézéquel

UMR CNRS 6539, Laboratoire Flux de Matière et Réponses du Vivant, Université de Bretagne Occidentale, BP 809, 29285, Brest,
France

Chantal Descolas-Gros

UMR CNRS 5556, Laboratoire d'Hydrobiologie Marine et Continentale, Université de Montpellier II 34095 Montpellier, France

Antoine Sciandra

URA 716, Station Zoologique de Villefranche, BP 28, 06230 Villefranche sur Met, France

and

Henri J. Jupin

Laboratoire de Biologie Physico-Chimique, Université de Perpignan, 66860 Perpignan, France

ABSTRACT

The concentrations of extracellular glycolate and intra-
cellular free pools of serine and glycine were monitored in
nitrogen-limited continuous cultures of Dunaliella terti-
olecta (Butcher) UTEX LB999, grown at two different
irradiances on a light:dark cycle. Under steady-state con-
ditions, this microalga excreted into the medium a large
amount of glycolate during the light phase, up to 100
nmol·(10⁶ cells)⁻<fnref n="1" /> for a cell concentration of around 1.5
10⁸ cells·L⁻<fnref n="1" />, but glycolate disappeared from the dissolved
phase in the dark. Cells grown at 70 and those grown at
430 µmol photons·m⁻<fnref n="2" />·s⁻<fnref n="1" /> differed in maximal glycolate
concentration, intracellular serine and glycine concentra-
tions, and serine:glycine ratio. Reversal of these photon
flux densities to which the cultures were exposed caused
rapid modification of the extracellular glycolate and intra-
cellular serine and glycine pools. These results suggest that
photorespiratory metabolism in D. tertiolecta could be ap-
proximately quantified by measuring the changes in dis-
solved glycolate and intracellular serine and glycine con-
centrations, extending previous results from cultured phy-
toplankton and suggesting methods for field studies.

Key index words: Dunaliella tertiolecta; glycine; glycolate; photorespiration; phytoplankton; serine

Abbreviations: CCM, carbon concentrating mechanisms;
CLMW, low molecular weight organic carbon; DFAA, in-
traparticular dissolved free amino; DGA, dissolved glyco-
late; DOC, dissolved organic carbon; exHL and exLL, con-

tinuous cultures HL and LL after light regime inversions;
GLY, glycine; HL, continuous culture under high light
conditions; HPLC, high performance liquid chromatography;
LL, continuous culture under low light conditions; PCOC,
photosynthetic carbon oxidation cycle; PCRC, photosyn-
thetic carbon reduction cycle; RUBISCO, ribulose 1,5-bis-
phosphate carboxylase/oxygenase (E.C. 4.1.1.39); SER,
serine

Glycolate, which is produced during photorespi-
ration in photosynthetic metabolism, has often been
shown to be one of the organic compounds released
in the culture medium of actively photosynthesizing
algae (e.g. Hellebust 1965). This pattern was ob-
served even in species where PCOC, or alternative
pathways (Winkler and Stabenau 1995), were able
to reincorporate glycolate carbon into the PCRC,
involving GLY and SER. We hypothesize that, even
if CCM are able to limit phosphoglycolate synthesis
or PCOC is able to divert phosphoglycolate, limiting
further release of glycolate, there could exist a mea-
surable DGA release in phytoplankton cultures, as
demonstrated in the field (e.g. Al-Hasan and Fogg
1987, Leboulanger et al. 1997).

We measured the dissolved glycolate and the in-
tracellular pools of free SER and GLY in two contin-
uous cultures of *Dunaliella tertiolecta* (Butcher), to-
gether with a large suite of variables for several
weeks during a multiparametric experiment de-
scribed elsewhere (Sciandra et al. 1997). Growth of
these cultures was limited by nitrogen availability,
and biomasses were comparable from one to the

<footnote n="1">¹ Received 28 January 1997. Accepted 6 March 1998.</footnote>

<footnote n="2">² Author for reprint requests; e-mail leboulan@thonon.inra.fr.</footnote>
<figure><img src="image_2.png" /><figcaption>FIG. 1. Variations of dissolved glycolic acid (DGA, black squares, solid line) and intraparticular amino acids (SER, open diamonds; GLY, open circles) in the cultures of *Dunaliella tertiolecta* strain UTEX LB999. All data are expressed as nmol·(10⁶ cells)⁻¹. Upper panel (A, B, C): High light initial conditions, cyclostat HL. Maximal errors (SE) for this set are ±4 nmol·(10⁶ cells)⁻¹ for DGA (n = 6), ±0.14 nmol·(10⁶ cells)⁻¹ for SER, and ±0.62 nmol·(10⁶ cells)⁻¹ for GLY (replicates). Lower panel (D, E, F): Low light initial conditions, cyclostat LL. Maximal errors (SE) for this set are ±7.5 nmol·(10⁶ cells)⁻¹ for DGA, ±0.03 nmol·(10⁶ cells)⁻¹ for SER, and ±0.11 nmol·(10⁶ cells)⁻¹ for GLY. Dark periods are represented by shaded areas. The two left figures (A, D) show the steady-state condition, and the two middle figures (B, E) show the rapid variations of the three parameters. The two right figures (C, F) show the evolution when the light was inverted between HL and LL, at t = 136 h, each cyclostat becoming exHL and exLL, respectively.</figcaption></figure>

other, the only difference between them being light availability. The aim of this work was to provide evidence for field ecologists that healthy cells were able to release glycolate into their environment, depending, for example, on carbon, light, and nitrogen resources.

*Cyclostats and cultures.* A precise description of protocol and material was published in Malara and Sciandra (1991) and the main results of experiment for the present paper in Sciandra et al. (1997). *Dunaliella tertiolecta* (strain UTEX LB999) was cultivated on f/4 medium according to Sciandra et al. (1997). Fresh medium was supplied at 2.5 L·day⁻¹, giving a dilution rate of 0.5 day⁻¹. No combined nitrogen was detected in the effluent, and cultures were clearly limited by nitrogen. For the first 5 days, the cell density in the HL cyclostat remained stable, with a growth rate of 0.5 day⁻¹. In contrast, the cell density in LL decreased regularly, and light deficiency induced a supplementary growth limitation at 0.45 day⁻¹. Total dissolved inorganic carbon concentrations were similar in both cultures, with *pCO₂* oscillating between 150 and 350 ppm as a function of diel light cycles, compared to surface water *pCO₂* in the oceans of 355 ppm (Riebesell et al. 1993). In the middle of the light phase of day 5 after the beginning of the experiment, the irradiances of the

cyclostats HL and LL were inverted. Cyclostats were then named exHL and exLL, respectively.

*Intracellular amino acids determination.* Amino acids determination was performed by HPLC according to the method of Lindroth and Mopper (1979) and Martin-Jézéquel et al. (1988). The absolute error on culture samples never exceeded 3.5% for SER and 12% for GLY determinations (based on replicate sampling and replicate measurements).

*Dissolved glycolate analysis.* Analysis was performed by HPLC according to Leboulanger et al. (1994), after standardization with 10–1000 µg·L⁻¹ glycolate in 25 mL f/4 media. The precision of the analysis was always better than 20% absolute error (with n = 6–9), except for values below 100 µg·L⁻¹, where absolute error could equal the mean measurement.

*Steady state.* The steady-state condition was monitored for 2 days (from day 1, 7 h, to day 3, 8 h), and results are presented in Figure 1A, D (HL and LL). In each culture, a large amount of DGA appeared in the medium soon after transfer to light (time scale of less than 1 h for a detectable change in DGA), and greater maximum levels were found in LL (more than 100 nmol [106 cells]⁻¹) relative to HL (around 35 nmol·[10⁶ cells]⁻¹). DGA concentration changes in each culture showed a trend related to the light cycle: When the light was switched on (8 h), the compound
<table><caption>TABLE 1. Compiled data for glycolate release and physiological state of D. tertiolecta cells (data from Sciandra et al. 1997). Modeled values are calculated according to Yokota et al. (1987) for Chlamydomonas reinhardtii. Errors are given as ±SD (pseudoreplicates, n = 3).</caption><thead><tr><th>Culture</th><th>Glycolate release<br>(nmol·h<sup>-1</sup>·10<sup>6</sup> cell<sup>-1</sup>)</th><th>Carbon content<br>(pmol·C·cell<sup>-1</sup>)</th><th>Growth rate<br>(d<sup>-1</sup>)</th><th>Chl a per cell volume<br>(µg·mm<sup>-3</sup>)</th><th>Cell volume<br>(mm<sup>3</sup>·L<sup>-1</sup>)</th><th>Cell count<br>(cell·L<sup>-1</sup>)</th><th>DIC<br>(nmol·L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>HL</td><td>14 (±2)</td><td>3 (±0.3)</td><td>1.3</td><td>5 (±1.5)</td><td>14 (±3)</td><td>2 × 10<sup>8</sup></td><td>200–220</td></tr><tr><td>LL</td><td>30 (±4)</td><td>2.2 (±0.3)</td><td>1.65</td><td>15 (±3)</td><td>8 (±3)</td><td>1.6 × 10<sup>8</sup></td><td>200–220</td></tr><tr><td></td><td></td><td>CO<sub>2</sub> fixation<br>(nmol·h<sup>-1</sup>·10<sup>6</sup> cell<sup>-1</sup>)</td><td></td><td>Modeled CO<sub>2</sub> fixation<br>(nmol·h<sup>-1</sup>·10<sup>6</sup> cell<sup>-1</sup>)</td><td></td><td>Modeled glycolate release<br>(nmol·h<sup>-1</sup>·10<sup>6</sup> cell<sup>-1</sup>)</td><td></td></tr><tr><td>HL</td><td></td><td>75 (±9)</td><td></td><td>52</td><td></td><td>2</td><td></td></tr><tr><td>LL</td><td></td><td>117 (±11)</td><td></td><td>116</td><td></td><td>4</td><td></td></tr></tbody></table>

appeared rapidly, and it decreased as early as 17 h. During the dark phase the DGA concentration was mostly undetectable. Glycolate contributed from 4% to 7% of DOC in HL and from 4% to 12.5% in LL (B. Avril, pers. comm.).

Intracellular free SER and GLY mean concentrations were relatively higher in LL cultures (values mostly 0.4–1.4 nmol·[10⁶ cells]⁻¹) than in cells from HL (values mostly 0.2–0.6 nmol·[10⁶ cells]⁻¹). During steady-state condition, the mean ratio SER:GLY was around 0.6. The concentrations of both amino acids increased at the beginning of each dark phase in the two cultures.

During the day-to-night transition (day 3, 20 h), DGA disappeared completely within 1 h (day 3, 21 h) in both HL and LL, with a rate of decrease of about 400 µg glycolate·L⁻¹·h⁻¹, which was much greater than the rate of medium dilution of 0.5 d⁻¹ (Fig. 1B, E). The decrease of DGA was not induced but only accelerated by darkness. During the night-to-day transition (day 4, 8 h), DGA appeared rapidly (after 15 min) and then increased at the same rate in HL and LL, at about 350 µg glycolate·L⁻¹·h⁻¹. We observed a higher cell-specific rate of DGA excretion in LL (i.e. 15 nmol·h⁻¹·[10⁶ cells]⁻¹) than in HL (8 nmol·h⁻¹·[10⁶ cells]⁻¹). In the dark, cell-specific disappearance of glycolate was also faster in LL than in HL (respectively, 33 and 26 nmol·h⁻¹·[10⁶ cells]⁻¹).

In both cultures, the decrease of DGA in the beginning of the dark phase coincided with a transient increase of SER and GLY. As in the steady state of the same experiment, SER in HL (0.6 ± 0.02 nmol·[10⁶ cells]⁻¹) was lower than in LL (1.5 ± 0.18 nmol·[10⁶ cells]⁻¹), although transitory peaks of SER were similar (2.3–2.9 nmol·[10⁶ cells]⁻¹, produced in 15 min).

*Effects of light regime inversions.* Irradiance was changed at 14 h on day 6; a strong perturbation in DGA concentration occurred during the hours following the perturbation (Fig. 1C, F). Forty-five minutes after light inversion, DGA concentration fell to undetectable levels during the light phase (day 6, 14 h 45 min). Then a rapid rise occurred until day 6, 18 h, followed by a decrease to undetectable levels in the beginning of the dark phase, in the same way as during the steady state. During stabilization of the two cultures (day 6), the importance of DGA excre-

tion seemed to be minimal relative to previous days, with midday peaks reaching only 38 ± 9 nmol·[10⁶ cells]⁻¹ for exLL and 26 ± 8 nmol·[10⁶ cells]⁻¹ for exHL. The difference in maximal concentrations between the two cultures was smaller than in the steady state. The SER concentration decreased in exLL, falling to the level of HL under high light (<1 nmol·[10⁶ cells]⁻¹). In exHL, SER concentration did not rise after the perturbation but closely followed the change in DGA.

The maximal contribution of DGA to DOC in the culture supernatant never exceeded 12.5%, but this organic pool was very variable and was readily usable for heterotrophic growth in marine waters (Edenborn and Litchfield 1987, Gomes et al. 1991, Jones and Cannon 1986). We hypothesize that the disappearance of DGA in the culture media during the night is due mainly to reassimilation and metabolism by *D. tertiolecta*, as previously demonstrated in *Euglena* (Yokota and Kitaoka 1987, Yokota et al. 1990) and *Chlamydomonas reinhardtii* (Yokota et al. 1987). The observed release of DGA in this work may be compared to calculated predictions. By using the equations and estimates of Bjornsen (1988), with a permeability coefficient of 10⁻⁹ cm·s⁻¹ for glycolate (arbitrary value) and a surface:volume ratio around 0.7 µm⁻¹ for this organism (Mullin et al. 1966), we obtain a theoretical mean daily release of 60% of the intracellular low molecular weight carbon. In our experiment, the total intracellular carbon stock varied between 2.3 and 4.41 pmol C·cells⁻¹ (Sciandra et al. 1997), which was assumed to contain 10%–20% of C<sub>LMW</sub> (Bjornsen 1988). We obtain a theoretical range for the daily total release of C<sub>LMW</sub> from 140 to 500 nmol C<sub>LMW</sub> (10⁶ cells)⁻¹·d⁻¹. In results presented here, DGA amounts are up to 100 nmol·(10⁶ cells)⁻¹·d⁻¹, which equals 200 nmol C<sub>LMW</sub>·(10⁶ cells)⁻¹·d⁻¹. In such a case, and considering the above hypothesis, glycolate could be considered the major C<sub>LMW</sub> compound released by *D. tertiolecta*.

By using the model of Yokota et al. (1987) established for *Chlamydomonas reinhardtii* and the data set for our experiment (Table 1), we can compare the predicted and the observed glycolate release and inorganic carbon net fixation. The estimated DGA release for the *Chlamydomonas* model was, respectively, 2
and 4 nmol C·h⁻¹·(10⁶ cells)⁻¹ in HL and LL che-
mostat. In our experiment, *D. tertiolecta* released 14
and 30 nmol C·h⁻¹·(10⁶ cells)⁻¹ in HL and LL che-
mostat, values that correspond to about seven times
the predictions in both cultures. Adding the C lost as
glycolate to the C increase in particulate organic mat-
ter yields a loss of glycolate around 16 for HL to 20%
for LL of the total C fixation rate by *D. tertiolecta*.

Part of this released DGA seems to be reassimat-
ed, as previously demonstrated in other species in
laboratory cultures (e.g. Yokota et al. 1990 for Eu-
glena gracilis), and up to 16% of added dissolved gly-
colate has been found to be incorporated into stable
products of *D. tertiolecta* cells in batch cultures (Le-
boulanger et al. 1995). Dissolved free amino acids—
SER and GLY—are higher in cells from LL cyclostat
than in cells from HL, possibly because of the rela-
tive nitrogen depletion in HL, where growth rate
and average cell density are higher than in LL. Ni-
trate limitation in HL appears to enhance the car-
bon concentrating mechanism (Beardall et al.
1991), and the subsequent increase of the intracel-
lular partial pressure of CO₂ represses the oxygenase
activity of RUBISCO, so that glycolate is produced
at a lower rate than under LL conditions.

*Dunaliella tertiolecta* cells allocate up to 20% of total reduced CO₂ into extracellular glycolate, even in active growth. This apparent release, a possible source of error in estimating primary production, should be considered the result of conditions in which cells have grown. Photorespiration has been considered as O₂ uptake and CO₂ and NH₃ evolution associated with the intracellular metabolism of phosphoglycolate, but this function needs to be explored in terms of energy and matter fluxes, for example, by CO₂ exchange and fluorescence measurements (review for higher plants in Leegood et al. 1995). As with phosphate depletion in cultured *Chlorella* (Kozlowska and Maleszewski 1994), inadequate nitrate and light supplies enhance the release of glycolate by *D. tertiolecta* in our experiment. Better estimates of photorespiratory losses must employ simultaneous DGA and intraparticular free SER and GLY measurements to allow a better estimation of C and N fluxes though the photorespiratory pathway in microalgae. In such a case, the use of tracers, especially ¹⁸O, could achieve quantification of total carbon fluxes through photorespiration and glycolate release (Raven 1990). A precise understanding of such fluxes and regulations is needed to ensure a better understanding of the results obtained on natural phytoplankton communities.

We thank Dr. Michel Denis and Dr. Jacques Gostan for helpful
discussion about this experiment. Dr. Bernard Avril gave us DOC
concentrations, and Anne Cueff performed amino acid analysis.
We are grateful to Dr. Yves Collos, Prof. John A. Raven, Dr. M.J.
Dring, and an anonymous referee for comments, corrections, and
suggestions that improved this note. This article is an APPA
(JGOFS-France) publication supported by CNRS grants.

Al-Hasan, R. & Fogg, G. E. 1987. Glycollate concentrations in relation to hydrography in the Liverpool Bay. *Mar. Ecol. Prog. Ser.* **37**:305–7.

Beardall, J., Roberts, S. & Millhouse, J. 1991. Effects of nitrogen limitation on uptake of inorganic carbon and specific activity of ribulose 1,5-bisphosphate carboxylase oxygenase in green microalgae. *Can. J. Bot.* **69**:1146–50.

Bjornsen, P. K. 1988. Phytoplankton exudation of organic matter: why do healthy cells do it? *Limnol. Oceanogr.* **33**:151–4.

Edenborn, H. M. & Litchfield, C. D. 1987. Glycolate turnover in the water column of the New York Bight apex. *Mar. Biol.* **95**: 459–67.

Gomes, H. R., Pant, A., Goes, J. I. & Parulekar, A. H. 1991. Heterotrophic utilization of extracellular products of phytoplankton in a tropical estuary. *J. Plankton Res.* **13**:487–98.

Hellebust, J. A. 1965. Excretion of some organic compounds by marine phytoplankton. *Limnol. Oceanogr.* **10**:192–206.

Jones, A. K. & Cannon, R. C. 1986. The release of micro-algal photosynthate and associated bacterial uptake and heterotrophic growth. *Br. Phycol. J.* **21**:341–58.

Kozlowska, B. & Maleszewski, S. 1994. Low level of inorganic orthophosphate in growth medium increases metabolism and excretion of glycolate by *Chlorella vulgaris* cells cultivated under air conditions. *Plant Physiol. Biochem.* **32**:717–21.

Leboulanger, C., Descolas-Gros, C. & Jupin, H. 1994. HPLC determination of glycolate in seawater. *J. Plankton Res.* **7**:897–903.

——. 1995. Some ecological and physiological implications of
photorespiration in marine phytoplankton. *In* Mathis, P.
[Ed.]* Photosynthesis: From Light to Biosphere*, vol. 5. Kluwer Academie Publishers, Amsterdam, pp. 869–72.

Leboulanger, C., Oriol, L., Jupin, H. & Descolas-Gros, C. 1997. Diel variability of glycolate in the eastern tropical Atlantic Ocean. *Deep-Sea Res.* **44**:2131–9.

Leegood, R. C., Lea, P. J., Adcock, M. D. & Hausler, R. E. 1995. The regulation and control of photorespiration. *J. Exp. Bot.* **46**:1397–1414.

Lindroth, P. & Mopper, K. 1979. High performance liquid chro-
matography determination of subpicomole amount of ami-
no-acids by precolumn fluorescence derivatization with o-
phthalaldialdehyde. *Anal. Chem.* **51**:1667–74.

Malara, G. & Sciandra, A. 1991. A multiparameter phytoplank-
tonic culture system driven by microcomputer. *J. Appl. Phycol.*
**3**:235–41.

Martin-Jézéquel, V., Poulet, S. A., Harris, R. P., Moal, J. & Samain, J. F. 1988. Interspecific and intraspecific variation of free amino acids in marine phytoplankton. *Mar. Ecol. Prog. Ser.* **44**: 303–13.

Mullin, M. M., Sloan, P. R. & Eppley, R. W. 1966. Relationship between carbon content, cell volume, and area in phytoplankton. *Limnol. Oceanogr.* **11**:307–11.

Raven, J. A. 1990. Use of isotopes in estimating respiration and photorespiration in microalgae. *Mar. Microbial Food Webs* **4**: 59–86.

Riebesell, U., Wolf-Gladrow, D. A. & Smetaceck, V. 1993. Carbon dioxide limitation of marine phytoplankton growth rates. *Nature* **361**:249–51.

Sciandra, A., Gostan, J., Collos, Y., Descolas-Gros, C., Leboulanger, C., Martin-Jézéquel, V., Denis, M., Lefevre, D., Copin, C. & Avril, B. 1997. Growth compensating phenomena in continuous cultures of *Dunaliella tertiolecta* limited simultaneously by light and nitrate. *Limnol. Oceanogr.* **42**:1325–39.

Winkler, U. & Stabenau, H. 1995. Isolation and characterization of peroxisomes from diatoms. *Planta* **195**:403–7.

Yokota, A., Asama, K. & Kitaoka, S. 1990. Evidence of reentrance of glycolate carbon into the photosynthetic carbon reduction cycle in photosynthesizing *Euglena gracilis* Z. *Plant Physiol.* **94**:388–91.

Yokota, A., Iwaki, T., Miura, K., Wadano, A. & Kitaoka, S. 1987. Model for the relationships between CO₂ concentration mechanism, CO₂ fixation, and glycolate synthesis during photosynthesis in *Chlamydomonas reinhardtii*. *Plant Cell Physiol.* **28**: 1363–76.

Yokota, A. & Kitaoka, S. 1987. Rates of glycolate synthesis and metabolism during photosynthesis of *Euglena* and microalgae grown on low CO₂. *Planta* **170**:181–9.