Photoautotrophic Suspension Cultures II — Transition from Photoheterotrophic to Photoautotrophic Growth

Photoautotrophic Suspension Cultures II - Transition from Photoheterotrophic to Photoautotrophic Growth T.

HARDY,

D.

CHAUMONT*).

M. E.

WESSINGER,

and P.

BOURNAT

A.R.B.S., Laboratory of Solar Biotechnology C.E.N. Cadarache, Bat. 161, 13108 - Saim Paul Lez Durance, Cedex-Francc Received June 3, 1986 . Accepted March 28 , 1987

Summary Euphorbia characias suspension cultures were used to study the transition from photoheterotrophy to photoautotrophy by in situ measurement of pH, dissolved O 2 and dissolved CO2 . The culture was able to reversibly alter its metabolism depending on culture conditions. Variation in pH was found to be a direct reflection of the culture's photosynthetic capacity. Photoautotrophic cultures grow at a rate six times slower than photoheterotrophic cultures. Possible limiting factors are discussed with emphasis on photorespiration.

Introduction

In the past ten years, numerous workers have reported the ability to grow plant cells photoautotrophically (Yamada et aI., 1978; HUseman and Barz, 1977; Dalton and Peel, 1983; Horn and Widhalm, 1984; Chaumont and Gudin, 1985; Hardy et aI., 1987). This ability to control photosynthesis in in vitro cell culture has potential in the use of cell cultures to select for agronomically or industrially useful traits related to photosynthesis. However, many technical difficulties arise with initiation and maintenance of photoautotrophic cultures, and in some cases it has not been possible to achieve and maintain photoautotrophic growth (Vasil and Hildebrandt, 1966; Neumann and Raafat, 1973; Kumar, 1974; Nato et al., 1983; Laulhere et aI., 1984). In successful studies, the authors describe how photoautotrophic growth can be obtained by reducing the sucrose content while simultaneously increasing light and CO 2 input. Photoheterotrophic, photomixotrophic and photoautotrophic cultures have then been compared with respect to growth rate, chlorophyll content, and CO 2 fixation capacity (La Rosa et aI., 1984; Dalton, 1980). To our knowledge, no one has followed culture parameters in situ during the transition from heterotrophic growth to photosynthetic growth. Such a study should permit a better understanding of this transition, therefore allowing photoautotrophic suspensions to be obtained for a large number of species. In a previous publication (Hardy et aI., 1987) we showed that photoautotrophy can be considered a physiological adaptation of the culture to change in environment (i.e. sugar depletion) under controlled conditions (light, CO" 0,). *) To whom correspondances should be addressed.

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T. HAJlDY, D. CHAUMONT, M. E. WESSINGER, and P. BOUllNAT

In the present repon, we studied the transition to photoautotrophy by measuring the following parameters in situ: dissolved CO" 0" and pH. We then studied the resulting photoautotrophic culture, particularly those factors which may he responsible for limiting growth (limiting light conditions, photorespiration). Materials and Methods 1. Culture conditions and measurements Initiation and maintenance of the photoautotrophic culture of Euphorbia characias L., as well as determination of chlorophyll content, sucrose and cell number were as described previously (Hardy et al., 1987).

2. Culture reactors Two commercial reactors, adapted to follow the in situ changes in pH and dissolved O 2 and CO2, were wed to study the transition from photoheterotrophic to photoautotrophic growth: - A 2.2 liter Biolafitte reactor (Biolafitte, Saint Germain en Laye, Fnmce) equipped with steam sterilizable oxygen and pH electrodes. - A 2 liter Setric reactor (Setric, Toulouse, France) equipped as above and with a CO2 electrode (Ingold) for continuous measurement of dissolved CO2 • The dissolved CO2 electrode was calibrated every two days. This reactor was connected to a Hewlett-Packard data logging system (HP8SF calculator, 30SHDT controller and HP9872C chan recorder). With this program short time plots of culture parameters can be obtained (Fig. 3) as well as continuous plots for the entire month-long culture period (Fig. 7). Both reactors were maintained at 25°C±1 °C unda an 18h photoperiod. Irradiance of l00-DOpE m - 2s - 1 was provided by a mixture (50% of each) of Grolux and Cool White fluorescent Sylvania tubes. Gas flow was maintained at 20 liters/hour with 2 % Cal in air. Cultures were stirred continuously at a rate of 60 rpm. Growth curves were obtained in an airlift reactor (Hardy et 31., 1987).

3. Measurement ofphotosynthetic capacity (Compensation Ratio) In situ measurements of respiration and photosynthesis were achieved as follows: a) Aeration was discontinued and gas inlet and outlet clamped with rubber tube clips. Mechanical agitation was maintained to assure homogeneity of the suspension. b) The light was switched off and oxygen consumption was measured by oxygen electrode and recorded. at 30 second intervals. c) The light was switched on, in most cases when a dissolved oxygen level of 100~ (40% saturated by air) was reached.. Oxygen evolution was then determined as described above. Compensation Ratio was calculated according to Dalton and Street (1977): C R = 1 _ light rate of O 2 evolution . . dark rate of O 2 consumption This ratio represents the actual photosynthetic capacity of the swpension measured under the selected culture conditions. Because oxygen evolution in the light is dependent on the initial concentration of dissolved oxygen (Sato et al., 1979), the results obtained for C.R. may increase up to 50 % if the light is switched on when the dissolved O 2 concentration has fallen to 12.5 ~ (data not shown), in accordance with the Warburg effect of photosynthesis (Gibbs et al., 1968).

4. Mass spectroscopic methods Photorespiration was measured according to the method of Peltier and Thibault (1985).

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Results 1. Transition in the Biolafitte reactor We recently reported that under our conditions, batch cultures of Euphorbi4 char· acias maintained in closed 250 ml erlenmeyer flasks became potentially photoautotrophic at the beginning of the stationary growth phase as a result of the depletion of sucrose (Hardy et aI., 1987). True photoautotrophic growth can then be obtained if the suspension is transferred to a reactor in which CO 2 is supplied as an inorganic carbon source, If the suspension is then subcultured in a flask with sucrose as a carbon source, it returns to photomixotrophy until the sucrose is depleted and a stationary phase is reestablished. We have shown that transition from one mode of growth to the other can be visualized by measuring the consumption of sucrose, increase in

chlorophyll content or modification of the gas phase. In the first experiment described here, we followed the transition from photohete-

rotrophy to photoautotrophy by following the evolution of pH and dissolved 0, in situ. For this experiment, cells of several flasks were subcultured in fresh liquid culture medium containing 5 g ·1- I sucrose in a Biolafitte reactor. The suspension (2 liters) was then continued in batch culture. The sole difference between the two systems of batch culture (erlenmeyer flasks and reactor) was the constant aeration in the Biolafitte reactor. Fig. 1 shows the changes in sucrose content, pH and photosynthetic capacity during the culture period. Sugar was entirely depleted within nine days, which was faster than in erlenmeyer flasks, but the initial concentration of sugars was lower in the reactor. The Compensation Ratios show clearly the evolution of photosynthetic capacity and confirm the results described previously concerning the successive phases in the

Fig. t: pH, total sugar content and compensation ratio of Euphorbia characias during transition to photoautotrophy in a biolafitte cultivator.

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354

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T. fuR.oY, D.

CHAUMONT,

M. E. WESSINGEJl, and P. BoURNAT

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Fig. 2: Oxygen evolution of photoaurotrophic Euphorbia characias suspensions measured by

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batch cultures (Hardy et al., 1987). At the beginning of the culture period, the C.R. was very high as the cells were subcultured at the beginning of the stationary phase and were potentially photoautotrophic. As sugar was consumed, C.R. decreased

rapidly to zero. Photosynthesis gradually recovered after sugar depletion and C.R. stabilized around \.5. The pattern of pH change was very similar to that of the C.R. After 16 days of culture, the suspension could be transferred to the different reactors used in our laboratory (Hardy et al., 1987) and cultivated under photoautotrophic conditions. Fig. 2 illustrates the orygen evolution of these chlorophyllous cells at the beginning of the stationary growth phase, as measured by Clark electrode.

2. Transition in the Setric reactor

In a second experiment, we were able to follow in situ changes in dissolved CO, in addition to pH and dissolved 0,. In this case, the suspension used was already photoautotrophic, and was transferred to the Setric reactor containing the same medium as used in the first experiment (5g·1- 1 sugar). We expected two transitions: one from photoautotrophy to photomixotrophy while the sugars are preferentially utilized,

followed by a return to photoautotrophic growth after sugar depletion. Fig. 3 (a and b) shows the levels of dissolved 0, and CO" during in situ measurement of photosynthesis and respiration as described in materials and methods 3. The j. PLmt PlrysioL Vol. 130. pp. 351 - 361 (1987)

Photoautotrophic cultures of Euphorbia characias

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Fig. 3: Transition to photoautotrophy of Euphorbia characias suspensions in a setric reactor: culture parameters after 4 days (a) and after 15 days (b) (d - dark, I-light).

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Fig. 4: Dissolved oxygen and CO2 content and pH of Euphorbia characias suspensions during transition to photoautotrophy in a setric reactor_

plots obtained four days after inoculation (Fig. 3 a) show the first transition from photoautotrophy to pholOmixotrophy. Fig. 3 b, obtained 15 days after inoculation

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Plant P/ry>iol. Vol. 130. pp. 351-361 (1987)

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T. H.uoY, D.

CHAUMONT,

M. E. WESSINGEll, and P. BOURNAT

shows the return to photoautotrophic growth. In both cases (a and b) pH was measured and total carbon content (CO,+HCO,) was calculated using the HendersonHasselbach equation (Umbreit et al., 1972) to be certain that the rapid modification of CO2 content was due to cellular metabolism and was not a result of modification

of the ratio of HCO,/CO, due to the pH variation. Between day 4 and day 15, during the return to photoautotrophy: - the pH increased from 5 to 6 as shown in Fig. 7, - dissolved O 2 increased to the saturation point, as a result of more active photosynthesis, - dissolved CO, diminished, essentially due to pH variation as total carbon re-

mained nearly stable. Evolution of photosynthetic capacity (C.R.) during the entire culture period, as well as variations in measured parameters is shown in Fig. 4. The variation of pH, dis-

solved 0, and C.R. are similar: after decreasing until the tenth day during sugar consumption, the three parameters increase, corresponding to a transition towards photo-

autotrophic growth. An increase in dissolved CO, was observed during the first day (transition to photomixotrophy), after which there is a drop which coincides with the increase in C.R. (return to photoautotrophy). These slow variations in the level of dissolved CO, are a result of the modifications of the equilibrium balance (HCO,/CO,) due to pH variation.

3. Growth ofphotoautotrophic cells Growth of cells of E. characias suspensions maintained under photoautotrophic conditions is very different from growth under photomixotrophic conditions

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Fig.6: Mass spectroscopy measurements of photoresEiration in photoautotrophic Euphorbia characiAs suspensions. a) Total O 2 consumption with 1 O 2• b) Net photosynthesis with 160 2 .

(Fig.5). [n the first case, the time necessary for cellular doubling is about 6 times greater than under photomixotrophic conditions. In photoautotrophy, a latent phase of about 7 days is observed during which the chlorophyll content diminishes. This is followed by a period of slow growth during which the chlorophyll content is can· stant, and finally a period of more rapid growth accompanied by synthesis of large amounts of chlorophyll during the last two weeks of culture. Measurements of photorespiration were obtained with these photoautotrophic

cells. This study could not be conducted under actual culture conditions due to technical limitations, but was conducted in a special reactor described by Peltier and Thibault (1985). This experiment also reveals a high value for the compensation point (500 ppm). In this experiment, photorespiration (the level of 0, uptake sensitive to CO,) accounts for 40% of the total 0, uptake in the light (Fig. 6). Discussion and conclusion

The results reponed here on the in situ measurement of pH, dissolved 0, and dis· solved CO, confirm that photoautotrophy is a physiological adaptation of cultured cells which modify their metabolism according to culture conditions (Hardy et al., 1987). The ability of the cells to rapidly and reversibly pass from heterotrophy to autotrophy indicates that photoautotrophy is adaptive rather than selective. When sucrose is present in the growth medium, photosynthetic activity diminishes as the cells preferentially utilize this carhon source. When sucrose is no longer available,

photosynthetic carbon fixation is reestablished. This may be followed by 0, produc.

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T. HARDY, D.

CHAUMONT,

M. E. WESSINGER, and P.

BOURNAT

tion and C.R. augmentation (Figs. 1 and 4). In the case illustrated in Fig. 4, the suspension subcultured into the reactor was already photoautotrophic, which may explain the difference in base level of C.R. between Figs. 1 and 4. To determine photosynthetic activity, most workers use cultures under optimal

conditions of pH, light, and bicarbonate (Sato et al., 1979; Yasuda et al., 1980; Horn et al., 1983). In our case, we obtained photosynthetic oxygen production by cells under normal growth conditions (Fig. 3 b). In order to obtain a photoautotrophic suspension, it is essential to control the gas phase, which is facilitated by electrodes which measure the in situ dissolved gas content. In situ pH measurement is very useful, as the pattern of pH variation is very

similar to that of photosynthetic capacity (Figs. 1 and 4), diminishing with C.R. during the transition to photomixotrophy, then increasing as photoautotrophy is

reestablished. In addition, minor variations in pH synchronized with the photoperiod may be observed (Fig. 7). Thus, culture pH, a parameter which is easily measured in situ on a continuous basis, yet is seldom mentioned in cell culture studies, appears

to be a good indication of the metabolic and physiological state of the cells. What governs these variations of pH is not well known but may be explained as follows:

- During the transition from photoautotrophy to photomixotrophy, the existence of H + I sucrose antiport and the liberation of CO, by the cells growing in the presence of sucrose may explain the acidification of the medium.

- During the return to photoautotrophy following sucrose depletion, HeO,/OHantiport and HCO,/H+ cotransport may drive the alkalinization of the medium. Photoautotrophic growth of E. characias cells shows several characteristics com-

mon to most photoautotrophic suspensions. Growth is much slower than under photoheterotrophic conditions and we do not obtain the typical .S. shaped growth curve (Fig. 5). In Fig. 5, we obtain a drop in the chlorophyll content, followed by a

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Fig. 7: Variation in pH of Eupharbia characias suspension during transition [0 photoautotrophy in a setric cultivator (d - dark period, I -light period).

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stationary period indicating that the accumulation of chlorophyll occurs at a slower

rate than cellular division. The same depigmentation was observed by Dalton and Peel (1983). While cases of photoautotrophic cell suspensions with high growth rates have been described {peel, 1982; Horn et al., 1983; Chaumont and Gudin, 1985), most photoautotrophic cultures have a much longer doubling time than photomixotrophic or heterotrophic cultures. In our case, there are severa] possible explanations for this limitation: a) Photoautotrophic cells in culture may need a different growth medium than that formulated for heterotrophic cultures. More precisely, one must consider

hormonal balance (HUsemann and Barz, 1977; Sato et al., 1981) and phosphate limitation (Sato et al., 1981; Dalton, 1983). We hope that the optimization of the culture medium will improve our culture. b) Under our conditions, light is probably a limiting factor. Increasing the irradiance

increases the photosynthetic capacity of the cells (data not shown). In fact, light has frequently been shown to be a limiting factor in the growth of photoautotrophic cultures (Peel, 1982; Dalton and Peel, 1983). In addition, the 18 h photoperiod under which our cultures were maintained may amplify this limitation. c) Photorespiration may also be a factor in limitation of growth. At present, most workers believe that cells cultivated under photoautotrophic conditions do not perform photorespiration, although they possess the genetic information to do so

(Horn and Widholm, 1984). This theory is based on the fact that photoautotrophic cells require a substantial supply of CO, (2 % in air) which seems to be incompatible with the oxygenase function of Rubisco. However, we cannot be certain of the gaseous environment with respect to Rubisco because the CO2 introduced must surmount several barriers before entering the cell. In addition, at

the cellular level, anhydrase activity may limit the amount of CO, readily accessible to Rubisco (Tsuzuki et al., 1981). In the absence of more precise information, it is not certain that the active site of Rubisco is saturated with CO 2 , On the other hand, our studies attempt to show that cultured photoautotrophic cells may undergo photorespiration. Our cells are in fact capable of photorespiration (Fig. 6) and under our conditions, the C.R. values rise as the O 2 content drops, suggesting

that photorespiration may occur at higher oxygen levels (Sato et al., 1979). These high levels of O 2 are a characteristic of our conditions, as shown by our in situ

measurements of dissolved A, (Fig. 3 b, Fig. 4). With this culture and our conditions, low oxygen supply is not essential for photoautotrophic growth, in contrast

to other reports (Yamada et al., 1981; Dalton and Peel, 1983). The compensation point is quite high {Fig. 6), but similar results have been obtained by Tsuzuki et a1. (1981), and may be the result of a low level of carbonic anhydrase activity, a high level of respiration, or the heterogeneity of the cultured cells. The application of continuous and semicontinuous culture techniques may be

uscful in the selection of pure autotrophic strains (Chaumont ct Gudin, 1985). This selection procedure could be used in identification of mutants with low photorespiratory activity. Only through experimentation under actual culture conditions with

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0 2 mass spectroscopy and demonstration of photorespiratory metabolite flux can unambiguous conclusions be drawn from the O 2 exchange information. Acknowledgements The authors wish to thank H. CIlFSTIN (Orstom, Abidjan) for critical reading of the manuscript and F. RafJUE and G. PElTIn (DB/SRA, CEN Cadarache) for their mass spectrometric analysis.

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SATO, F., N. NAkAGAWA, T. TANIO, and Y. YAMADA: An improved medium for the photoautotrophic culture of Cytisus scoparius Link cells. Agric. BioI. Chem. 45, 2463-2467 {1981}. TsuzlJJJ, M., S. MrvACHI, F. SATO, and Y. YAMADA: Photosynthetic characteristics and carbonic anhydnse activity in cells cultured photoautotrophically and mixotrophically cells isolated from leaves. Plant and Cell. Physiol. 22, 51-57 (1981). UMBJlEJT, W. W., R. H. BullJUs, and J. F. STAUFFER: Manometric and biochemical techniques. Burgress Publishing Company, Mine'polis, Minnesota (1972). VASIL,!. K. and A. C. HILDEBRANDT: Gro'Wth and chlorophyll production in plant callus tissues grown in vitro. Planta, 68, 69-82 (1966). YAMADA, Y., T. KUBOI, and F. SATO: Cell differentiation. [n Proc. Symp. Plant Tissue Culture, May 25- 30, 1978, Peking, Science Press, 371- 389 (1978). YAMADA, Y., K. lMAIZUMI, F. SATO, and T. YASUDA: Photoautotrophic and photomixotrophic culture of green tobacco cells in a jar·fermenter. Plant and Cell Physiol. 22, 917 -922 (1981). YASUDA, T., T. HASHIMOTO, F. SATO, and Y. YAMADA: An efficient method of selecting photoautotrophic cells from cultured heterogeneous cells. Plant and Cell Physiol. 21, 929-932 (1980).

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