Interaction between saline stress and photoinhibition of photosynthesis in the freshwater green algae Chlamydomonas reinhardtii. Implications for glycerol photoproduction

Plant

Physiol.

Biochrm.,

1999,31 (7/8), 623-628 I 0 Elsevier,Paris

Interaction between saline stress and photoinhibition of photosynthesis in the freshwater green algae Chlamydomonas reinhardtii. Implications for glycerol photoproduction Rosa Lebn, Francisco GalvW Departamento de Bioquimica Vegetal y Biologia Molecular, Facultad de Quimica, Apartado 553, Universidad de Sevilla, 41080 Sevilla, Spain * Author to whom correspondence should be addressed (fax +34 954626853; e-mail [email protected]) (Received January 22, 1999; accepted May 10, 1999) Abstract - Light intensity is the main limiting factor for the photosynthetic bioconversion of CO, into glycerol which takes place when Chlamydomonas reinhardtii cells are exposed to saline stressconditions. Although productivity increaseswith light intensity for low irradiances, a strong inhibition is observed for high light intensity values. Saline stressenhances the damage caused by excessof light on the photosynthetic apparatus. The aim of this work is to evaluate the effect of high light intensity and saline stress on photosynthetic activity, cell growth and glycerol photoproduction by C. reinhardtii. The effect of light intensity on C. reinhardtii cells was studied immediately after transfer to a saline medium and after 24 h of adaptation to saline stressconditions. The influence of light intensity on the glycerol production rate was also evaluated for C. reinhardtii cultured in bioreactors of different radius. The factors that significantly affected photoinhibition were light intensity, cell density, radius of the bioreactor and time of exposure to the high light intensity. Our results suggestthat bioreactors with a high surface/volume ratio will enable the achievement of high productivities with relatively low light intensities on the surface and will miminise the photoinhibition effect. 0 Elsevier, Paris Chlamydomonas reinhardtii 1 glycerol photoproduction I phototosynthetic inhibition / saline stress

1. INTRODUCTION

The ability of the freshwater green algae Chlamydomonas reinhardtii to synthesise glycerol as an os-

Photoinhibition of photosynthesis in algae and other photosynthetic systems induced by exposure to strong light intensities has been known for a long time and

constitutes an important limitation for biomass and growth-linked metabolites production in photoautotrophic systems [ 1, 4, 5, 7, 10, 16, 21, 221. The interaction between light-induced inhibition of photosynthesis and other environmental stressing factors, namely osmotic, oxidative and temperature stress, has been described for many vascular plants and algae [20]. Although the exact mechanism for the damage of the photosynthetic apparatus caused by photoinhibition is still not well known, it is generally believed that damage caused by both osmotic stress and high light intensities are implicated in the lesion of the reaction centre of photosystem II [20]. PlantPhysiol.

Biochem.,

0981-9428/99/7-8/O

Elsevier,

Paris

moregulatory metabolite in response to osmotic stress has been previously reported [9, 1 I]. The metabolic pathway for this synthesis and the environmental conditions favouring glycerol production have also been studied [ 121, the availability of CO,, light intensity and saline concentration being the most important limiting factors for glycerol productivity [ 131. The production of glycerol in response to osmotic stress is not unique for C. reinhardtii. One of the microorganisms most studied in this sense is the halotolerant alga Dunaliella [3,8, 181, which has even been proposed as a model system to study the response of plant cells to saline stress [6]. C. reinhardtii, unlike Dunaliella, does not accumulate intracellularly all the glycerol produced but excretes most of it into the culture medium [ 111. This fact justifies the importance of C. reinhardtii for biotechnological production of glycerol.

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300 250 200 150 100 l

I 600 1600

5 pg Chl

A10 . 20

50

2400

”

3200

ml-’

I 4000

Light intensity (pE. mS2. s1 ) Figure

1. Curves of light saturation for photosynthetic activity in Cells were grown in the standard conditions described in Methods (bioreactor of 250 mL and 2-cm radius, light intensity 100 pE.rn-*.s-‘). When the cultures reached cell densities of (0) 5, (A) 10 and (m) 20 pg ChhmL-‘, 2-mL aliquots were withdrawn from each culture, placed in the cuvette of an oxygen electrode and illuminated with white light at the indicated intensities. Time course of oxygen evolution was followed for each light intensity and cell density. C. reinhardtii.

The interaction between osmotic stress and high light-induced photoinhibition of photosynthesis is particularly interesting for the biotechnological photoproduction of glycerol by C. reinhardtii, which is synthesised as a response to osmotic stress and reaches higher productivities at higher light intensities. In this work, we studied this relationship for the freshwater microalga C. reinhardtii and its implications for the photoproduction of glycerol.

2. RESULTS

AND DISCUSSION

2.1. Effect of light intensity on photosynthetic activity of C. reinhardtii cells C. reinhardtii cells were grown in the standard conditions described in Methods (bioreactor of 250 mL and 2-cm radius, light intensity 100 pE.rn-*s-‘). When the cultures reached the indicated cell densities, 2-mL aliquots were withdrawn from each culture, placed in the cuvette of an oxygen electrode and illuminated with white light of different intensities. Initial photosynthetic rate was measured for the indicated cell densities and light intensities @gure I). Like many other enzymatic or physiological activities, which increase when the limiting substrate is increased, photosynthetic activity of C. reinhardtii Plant

Physiol.

Biochem.

cells increases linearly with light intensity and reaches the maximum at a light intensity of about 1 600 uE.m-*+-I. Above this value, an important light-induced inhibition of photosynthesis was observed for low cell densities, while practically no inhibition was shown for the culture of 20 ug ChlmL-‘. In addition, the initial slope of the rate of photosynthesis versus irradiance was higher for lower cell densities. These two facts, lower photosynthesis-irradiance slope and absence of photoinhibition at the studied light intensities for cultures with a cell density of 20 pg Chl.mL-‘, can be explained on the basis of the mutual shading effect. Light is partially absorbed across the bioreactor and consequently the effective light, which is the real light received by cells, is much lower than the light on the surface of the bioreactor. For cultures with a cell density of 20 ug Chl.mL-‘, the shading effect is very high. For this cell density, light intensity at 2 cm from the surface of the bioreactor is about 30 9%of the light at the surface. The effective light obtained even for the maximum irradiances studied is not enough to cause photoinhibition. Probably photoinhibition could be observed light intensities higher than for 4 000 pE.m2s’. It is also necessary to consider the different cellular age of the cultures of different cell densities. Although all the cultures were in the exponential phase of growth, cells of higher cell densities were older and received lower effective light during growth. This explains the lower maximum of photosynthetic rate reached for cultures with higher cell densities. It is necessary to point out that for these measurements, cells were only exposed to such high intensities for only a few minutes. For longer exposure periods, more dramatic effects were observed (data not shown). 2.2. Effect of saline stress on C. reinhardtii

cells

Transfer of C. reinhardtii cells to a saline medium produces a quick inhibition of photosynthetic activity, which is higher for higher osmotic shocks (table 0. This is only a temporal inhibition since after some time photosynthetic activity starts to recover, reaching normal levels after several hours. Inhibition of photosynthetic activity immediately after the osmotic shock has already been described for C. reinhardtii [ 171, but not the recovery of normal photosynthetic levels several hours after the osmotic shock. This recovery is essential for the viability of a continuous biotechnological process for glycerol photoproduction by C. reinhardtii based on its osmoregulatory response to

Photosynthetic

Table I. Main responses with the indicated NaCl

of C. reinhardtii concentrations.

NaCl (mW

0 50 100 150 200

inhibition

to saline stress. Cells cultured Photosynthetic activity, growth

Photosynthetic (pmol O,h’.mg-’

activity Chl)

by saline stress in Chlamydomonas reinhardtii

in normal conditions were resuspended in fresh medium (20 pg Chl,mLrate, and excreted and accumulated glycerol were determined. Glycerol produced (pmol.mg-‘Chl)

Growth rate at 24 h (ug Chlh’.mL-‘)

Ih

24 h

195 215 I40 95 51

192 195 188 190 190

saline stress. C. reinhardtii cells were also transferred to saline media with 2 pg.mL-’ cycloheximide. Under these conditions, no recovery of photosynthetic activity was observed (data not shown). Growth rate is also partially inhibited by the addition of salt to the culture medium. In this case, the growth rate during the first hours after transfer to the saline medium is almost zero, and only after several hours of adaptation to the saline stress, does the growth rate capacity of C. reinhardtii start to recover. The final growth rate reached depends on the saline concentration of the culture medium as can be seen in table I after 24 h of adaptation to saline stress. The other important effect of saline stress on C. reinhardtii is the synthesis of glycerol. Both intracellular and extracellular glycerol concentrations after 24 h of photoproduction increase linearly with the salt concentration of the culture medium. Glycerol photoproduction by C. reinhardtii cells cultured in bioreactors of different sizes and illuminated with different light intensities on the surfaces was evaluated after 24 h of photoproduction in saline medium (figure 2). Under low light intensity, glycerol photoproduction increases in a linear fashion with the light intensity, confirming that the synthesis of glycerol in C. reinhardtii, in response to osmotic stress, is a photosynthetic light-dependent process. But above a certain light intensity, glycerol photoproduction starts to decrease. The inhibition of glycerol photoproduction depends not only on the light intensity but also on the radius of the bioreactor, cell density of the culture and the time of exposure to the high irradiance. Similar results were observed by other authors for glutamate [ 151 and biomass [ 141 production by photoautotrophic cultures. For a cell density of 25 ug Chl.mL-’ and a bioreactor radius of 2 cm, the maximum rate of glycerol photoproduction was observed for light intensities of 200 pE.mP2.s-‘. For bioreactors of 4.5- and S-cm radius, maximum inhibi-

625

Intracellular 1.50 1.65 0.80 0.95 0.45

tion

at 24 h Extracellular

0.14 0.50 I .45 5.52 10.30

was observed .

for

‘)

0 7 17 35 55

light

intensities

around

400 yE.m-*s’

2.3. Saline stress and photosynthetic photoinhibition of C. reinhardtii cells Inhibition of photosynthetic activity of C. reinhardtii cells exposed to saline stress and enhancement of light-induced photoinhibition of photosynthesis were previously reported [9, 171. We have investigated the influence of light intensity not only on the photosynthetic activity of stressed cells that have recovered their normal photosynthetic level (figure 3), but also on the recovery of the photosynthetic activity of the cells immediately after the osmotic shock (figure 4). In the first case, standard grown cells of C. reinhardtii cells were transferred to a saline medium

100

200

300

400

500

Light intensity (pE. m-* s-l ) Figure 2. Influence

of light intensity and bioreactor radius on glycerol photoproduction rate by C. reinhardtii cells. Cells (25 ug Chl.mL-I) were cultured in bubble columns (250 mL, 2 and 5 L) of (0) 2-, (A) 4.5- and (W) g-cm radius, respectively, and illuminated with white light at the indicated intensities, in standard medium supplemented with 200 mM NaCI, as described in Methods. Glycerol excretion was followed for 24 h and glycerol excretion rate measured for each radius and light intensity. vol. 37 (7/8)

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3000 pE m-2. s -1

80 CONTROL

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200 mMNaC

0

0

IO

20

30

40

0

Time

(min)

10

20

30

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Figure 3. Effect of strong light on photosynthetic activity of stressed C. reinhardtii cells. Cells (25 pg Chl,mL-‘) were cultured in a liquid medium supplemented with 200 mM NaCl at normal irradiance (100 pE.rn-‘.s-’ on the surface of a cylindrical flask of 50 mL and l-cm radius) during 24 h. Then stressed cells (0) that had already recovered their normal photosynthetic activity, which corresponds to 200 kmol O,.h-‘.mg-’ Chl (100 %), were exposed to strong light of 2 000 (A) and 3 000 (B) ftE.m-‘.s-’ on the surface. The controls (W) were cultures grown without the addition of NaCl and exposed to the same high irradiances.

80 60 40 20 n

0

50

100

Time

150

200

(min)

Figure 4. Recuperation of photosynthetic activity of stressed C. r&ha&i. Cells grown in the standard conditions indicated in Methods were harvested at the exponential phase of growth, resuspended in liquid medium (25 pg Chl.mL-‘) supplemented with 200 mM NaCl and distributed into four cylindrical flasks of 50 mL and 1-cm radius, and illuminated with white ligth of (0) 100, (m) 500, (A) 2 000 and (0) 3 000 pE.rn-‘.s-’ on the surface. At the times indicated, samples of the culture were withdrawn and light saturated photosynthetic activity was measured as described in Methods (100 % corresponding to 200 pmol O,h’.mg-’ Chl).

(200 mM NaCl) and grown for 24 h under normal irradiance (100 pE.rn-*.s-‘). Then the saline adapted Plant

Physiol.

Binchem.

cells, that had already recovered their normal photosynthetic activity (200 pmol O,.h-‘.mg-’ Chl), were exposed to light intensities of 2 000 uE.m-*s’ @gwe 3 A) and 3 000 pE.m-*.s-’ @gum 3 B). In both cases, oxygen evolution was compared to non-stressed cells cultured at the same irradiances (control). It can be observed that high light intensities cause a quick photoinhibition that is much stronger for cells exposed to saline stress, even when they have had an adaptation period and have already recovered their normal photosynthetic level. For light intensities of 200 and 500 pE.mp2.ss’, saline-stressed cells had a similar behaviour to control non-stressed cells, with no decrease in photosynthetic activity during the first hour (data not shown). After 30 min, osmotic-stressed cells exposed to irradiances of 2 000 and 3 000 pE.m-2s’ showed 30 and 10 % of their initial photosynthetic activity, respectively, while control cells exposed to the same irradiances lost only 20 % of their photosynthetic activity during the same period. Susceptibility to photoinhibition for saline-adapted C. reinhardtii cells is higher than for control non-saline-adapted cells. The inhibition measured here is stronger than that reported by Neale and Melis [17]. But these authors did not specify the cell density of the culture and the geometry of the reactor which also influence the photoinhibition degree, as previously shown (figure 2). In the second case, C. reinhardtii cells were harvested at the beginning of the exponential phase of growth and resuspended in fresh culture medium supplemented with 200 mM NaCl. Cells were distributed into four cylindrical flasks and grown at 25 “C under identical conditions, except that they were illuminated with different light intensities (figure 4). In this figure, it can be clearly observed that immediately after the transfer to a saline medium, photosynthetic activity decays to zero and the recovery of the initial activity is not possible at light intensities as high as 500 pE.rnp2s’. Although the underlying causes of this water stressinduced susceptibility to photoinhibition are unknown, it has been reported that damage by both excess light and saline stress are associated to damage to the reaction centre (P680) of PSI1 [20]. The damage to the photosynthetic apparatus occurs when it absorbs light in excess of its capacity for energy dissipation by useful photochemistry reactions and/or protective quenching mechanisms [17]. In C. reinhurdtii, not only have some authors observed the loss of Dl protein of PSI1 when exposed to high light intensity, but they have demonstrated that de novo synthesis of the protein is necessary to recover the photosynthetic

Photosynthetic inhibition by saline stress in Chlamydomonas

activity [ 191, in agreement with our observation that saline-stressed cells are not able to recover their photosynthetic activity in the presence of cycloheximide. If cells are exposed to photoinhibition conditions for a long time, a continuous replacement of damaged protein is necessary and when the degradation rate overcomes the repair rate, total destruction of the photosynthetic apparatus occurs.

3. CONCLUSION Glycerol photoproduction by C. reinhardtii is a light-dependent process. Under normal culture conditions, glycerol productivity is limited by light intensity. Nevertheless, light intensity cannot be increased above certain values without the risk of photoinhibition. Susceptibility to photoinhibition is enhanced by saline stress. Interaction between osmotic stress and high light-induced photoinhibition of photosynthesis is particularly interesting for the biotechnological production of glycerol by C. reinhardtii, which is synthesised as a response to osmotic stress and reaches higher productivities at higher light intensities. We have also observed that together with light intensity on the surface of the bioreactor, there are several factors, such as cell density, size of the bioreactor or time of exposure to high h-radiances, that influence both the productivity and the photoinhibition. Adequate bioreactor design for maximising the surface/volume ratio would allow the obtention of a high effective intensity light inside the bioreactor by applying only a relatively low light intensity on the surface, thus minimising the photoinhibition effect. On the basis of these results, a small bioreactor with a 2-cm radius illuminated with white light at 200 pE.rn-*.s-’ and a NaCl concentration in the culture medium of 200 mM were the conditions chosen to obtain continuous glycerol photoproduction. The design of alternative bioreactor configurations allowing better distribution of light, such as a bioreactor with internal light sources, is now being studied.

4.2. Microorganism

627

reinhardtii

and culture conditions

Chlamydomonas reinhardtii, wild strain 21 gr from Dr R.. Sager (Sidney Farber Cancer Center, Boston, MA), ‘Were grown at 25 “C in 15 mM K-phosphate (pH 7.0) buffered medium [23] containing 10 mM KNO, as nitrogen source. The standard cultures were carried out, unless indicated otherwise, in a cylindrical bioreactor of 250 mL and 2-cm radius, bubbled with air containing 5 % (v/v) CO, and continuously illuminated with white light of 100 pE.rn-*+-’ in intensity. For the photoproduction of glycerol, the medium was supplemented with 200 mM NaCl.

4.3. Measurements

of photosynthetic

activity

Photosynthetic activity was determined using a Clark-type electrode to measure the light-dependent O,-evolution from 2 mL cell suspension in the corresponding buffered culture medium. Measurements were made at 25 “C under saturating white light, except when other values were indicated. 4.4. Analytical

determinations

Chlorophyll was determined by heating and extracting with acetone, using an absorbance coefficient at 652 nm of 34.5 mg-‘.mLcm-’ [2]. Glycerol was determined enzymatically by the UVmethod of Wieland [24], using glycerokinase and glycerol-3-phosphate dehydrogenase. Extracellular glycerol was determined in 2 mL aliquots of cell free medium. Intracellular glycerol was determined from cells exhaustively washed with isotonic medium, followed by boiling for 10 min to extract the accumulated glycerol.

Acknowledgments This work was supported by set-up funds provided by the Junta de Andalucia (Spain) to our group (No. CVI-0163) and by research project PB96-1358 from DGICYT (Spain).

4. METHODS 4.1. Chemicals

Glycerokinase and glycerol-3-phosphate dehydrogenase were obtained from Boehringer Mannheim, Germany. All other reagents were supplied by Merck, Darmstadt, Germany.

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