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Continuous ATP production by spinach thylakoid in a stirred tank reactor
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 73, No. 6, 471-476. 1992
Continuous ATP Production by Spinach Thylakoid in a Stirred Tank Reactor ALEX H. C. YU§ A~D KUNIAKI HOSONO* Fermentation Research Institute, Agency of Industrial Science and Technology, 1-1-3 Higashi, Tsukuba, lbaraki 305, Japan Received 7 October 1991/Accepted 14 March 1992 Spinach thylakoid was immobilized by two different methods for the purpose of retention within a continuous-flow stirred tank reactor (CSTR). The glutaraldehyde crosslinked albumin polymer method completely inactivated the cyclic ATP photophosphorylation of thylakoid. In contrast, agarose-entrapped thylakoid retained about 17Yo of the activity of the cyclic photophosphorylation of non-immobilized thylakoid. This activity declined continuously during ATP production in the CSTR. Fifty percent of the initial activity was lost within about 5.5 h. Aseorbate was found to increase the stability of ATP photophosphorylation; about twice as much ATP was produced at the optimal ascorbate concentration of 5 mM. Under the optimal dilution rate of 2.36 h - l , about 60 gmol of ATP per mg chlorophyll were produced in 20 h by agarose-entrapped thylakoid in the CSTR. These results showed that, compared to non-immobilized thylakoid in batch operation, agarose-entrapped thylakoid produced only a low amount of ATP under continuous operation.
For almost two decades, the development of a cofactor regeneration system has been recognized as a prerequisite to the industrial development of economically viable biosynthetic processes. Since many cofactors (e.g. ATP and NADH) are consumed as co-reactants in biosynthesis reactions, they must be supplied at stoichiometric amounts. The obvious way to lower the high cost of these cofactors in biosynthetic processes is to regenerate and recycle them. The regeneration of ATP by various means (chemical synthesis, cell-free enzymes, isolated organelles, and whole ceils) has been attempted in the past (1-9), and has been reviewed and discussed by Langer et al. (10). The use of photosynthetic organelles to convert light energy into ATP by phosphorylation of ADP is an attractive system, since no expensive substrate is required as compared to ATP regeneration by cell-free enzymes. Chromatophores, isolated membrane vesicles from photosynthetic bacteria, have been extensively studied for this purpose (4, 5, 11-13). Recently, we have carried out studies on the alternative photosynthetic organelle, namely, thylakoid membranes from spinach leaves (7). We have demonstrated that spinach thylakoid is an inexpensive ATP regeneration source which can be coupled to ATPconsuming kinases to produce phosphorylated products at an order of magnitude reduction of the requirement of ATP (7). However, it is known that the photochemical activities of the isolated photosynthetic organelles are very unstable. Under the typical reaction conditions, light and oxygen both play a role in inactivating the photosynthetic activities (14, 15). In the past, immobilization was attempted in order to stabilize the photochemical activity of these organelles (11, 16). The commercial usefulness of the photosynthetic organelles in continuous ATP regeneration in bioreactors will be largely dependent on their operational stability. The present paper reports on one part of our continuing
effort to develop an economical ATP regeneration system; namely, the continuous operation of spinach thylakoid in a continuous stirred tank reactor (CSTR) was studied. We are interested in comparing the capabilities of ATP production by thylakoid carried out under batch and continuous operations. Previously, we have shown that a production rate of 84/~mol A T P / h . m g Chlorophyll (Chl) can be achieved by thylakoid in batch reactors (7). However, the photophosphorylation activity of thylakoid typically ceased after about 2 h (7). In this study, we have investigated the ATP productivities under continuous operation to determine if more ATP can be produced than in batch reactors within the operational life-time of the thylakoid. Isolated spinach thylakoid was immobilized in order to allow for the retention of the organelle within the reactor under continuous operation. The thylakoid membranes were immobilized by two different methods: agarose gel entrapment and glutaraldehyde crosslinked albumin polymer. Attempts were made to improve the operational stability of immobilized thylakoid by using rutin, a scavenger of active oxygen species, and by adjusting the redox potential with ascorbate. Total ATP production was optimized by adjusting the dilution rate of the reactor. MATERIALS AND METHODS Isolation of spinach thylakoid membranes The isolation of thylakoid membranes from spinach leaves was described previously (7). Immobilization of isolated thylakoid
Crosslinked albumin polymer method This immobilization procedure is based on the method described by Coequempot et al. (16) with modifications as follows. Thylakoid suspension (0.32 ml) corresponding to approximately 0.9 mg of chlorophyll was added to 1.68 ml of a solution containing 8.7 mM potassium phosphate, pH 6.8, 6.5% bovine serum albumin (BSA) and 0.360/~ glutaraldehyde. The resulting mixture was deposited as semispherical microdroplets of about 15-20 ~I volume by a micropipette onto a piece of Parafilm®. The Parafilm®
* Corresponding author. § Present address: Pulp and Paper Research Institute of Canada, 3800 Wesbrook Mall, Vancouver, Canada. 471
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YU AND H O S O N O
with the thylakoid/albumin microdroplets was put into a light-tight box and was kept at - 2 0 ° C for 2.5 h. The crosslinked albumin/thylakoid microbeads were thawed and then washed with distilled water. Agarose gel entrapment method This immobilization procedure is based on the method described by Weaver et al. (17) with modification as follows. Thylakoid suspension (0.2 ml) corresponding to 0.85-0.94 mg of chlorophyll was mixed with 1.8 ml of 4% agarose solution at 300C. The agarose solution was prepared by dissolving ultra-low gelling temperature agarose (type IX; Sigma) in a pH 7.6 solution containing 50raM Hepes, 5 mM NaC1, 5 mM MgCI2. The agarose-thylakoid mixture was added dropwise by a 1-ml pipette into a 100-ml beaker containing 50 ml of mineral oil at 25-30°C under constant stirring by a magnetic bar. The speed of stirring was adjusted so as to disperse the agarose-thylakoid droplets into spherical microdroplets of 100 pro-l,000 pm in diameter. The beaker was then transferred to an ice-water bath. After 20 min, the solidified agarose-thylakoid beads were washed with a pH 7.6 solution containing 0.1 M sorbitol, 5 mM MgCI2, 5 mM K2HPO4, 50 mM Hepes to remove the mineral oil. The washed agarose-entrapped beads were then size-fractionated with a steel mesh (360/zm x 400/~m). Determination of initial rate of cyclic photophosphorylation Cyclic photophosphorylation mediated by phenazinc methosulfate (PMS) was carried out as described previously (7). In the case of immobilized thylakoid, the reaction solution was directly used for ATP determination without the trichloroacetic acid treatment that was done in the case of non-immobilized thylakoid. For reaction with agarose-entrapped thylakoid, the amount of thylakoid used in the reaction was determined by recovering all the thylakoid beads after the reaction. The agarose-cntrapped thylakoid beads were melted at 60°C and the chlorophyll content was determined as previously described (7). For reaction with crosslinked albumin-thylakoid beads, the amount of thylakoid used in the reaction was determined by the weight of the beads since the recovery of thylakoid in this immobilized method was almost quantitative. Continuous ATP production in CSTR The cyclic ATP photophosphorylation was carried out in a temperature-controlled stirred tank reactor of 7 ml at 15°C. A simplified diagram of the experimental set-up is shown in the insert of Fig. 1. Saturating light (60 m W / c m 2) was provided by a 250-W light bulb placed 17 cm from the reactor. To absorb the heat from the light source, water at 15°C was circulated through a bottle placed in front of the reactor. Immobilized thylakoid, placed inside the reactor, was agitated by a magnetic stir bar. Substrate solution (0.1 M sorbitol, 5 mM K2HPO4, 0.4raM ADP, 5 m M MgC12, 5 mM NaCI, 0.1 mM PMS and 50ram Hepes, pH 7.6) in a light-tight container was continuously fed into the reactor by a peristaltic pump at a fixed speed. Another channel of the same pump was used to remove the product from the reactor. The pore size of the outlet tube was small relative to the bead size of the immobilized thylakoid, so that immobilized thylakoid was not removed from the reactor. Fractions of the outlet stream were collected. Selected fractions were used for the determination of ATP by Luciferin/Luciferase assay, as previously described (7). The continuous operation of this process was carried out in a cold room at 4°C.
J. FERMENT. BIOENO.,
RESULTS AND DISCUSSION Immobilization of spinach thylakoid In order to retain the thylakoid membranes within a CSTR, the soluble thylakoid membranes needed to be immobilized. Immobilization often inadvertently affects the activity of the catalyst; different procedures cause different degrees of inactivation. Therefore, two defferent methods were used to immobilize the thylakoid in this study. The first method, utilizing glutaraldehyde to crosslink albumin and thylakoid into a foam structure, was described by Larreta-Garde et al. in the immobilization of bacterial chromatophores (11) and lettuce thylakoid (16). The second method, utilizing low-temperature gelling agarose to entrap thylakoid into spherical microdroplets, was a modification of the method described by Weaver et al. in the immobilization of microorganisms and mammalian cells (17, 18). As shown in Table 1, the initial rate of cyclic ATP photophosphorylation of thylakoid after immobilization were greatly affected. In the case of the albuminthylakoid foam method, essentially complete inactivation of the cyclic photophosphorylation of the immobilized thylakoid was observed. The inactivation observed was different from the previous reports (19) where 9.5% of cyclic ATP photophosphorylation was recovered after immobilization. It was also reported that 85-90% of oxygen production activity (i.e. electron transport chain activity) of non-immobilized thylakoid were obtained for thylakoid immobilized by this albumin foam method (16). Thus, this suggests the likelihood that the inactivation observed is due to the uncoupling of ATP phosphorylation from electron transport and/or damage to the components of the ATPase. For agarose-entrapped thylakoid, only 17% of the cyclic ATP phosphorylation activity was recovered after immobilization. The physical recovery of thylakoid membranes in this immobilization procedure was found to be about 65%. Therefore, the total activity of ATP photophosphorylation recovered in terms of initial thylakoid used for immobilization was only about 11%. In this immobilization method, the thylakoid was briefly exposed ( ~ 2 m i n ) to a temperature of 30°C when liquid agarose was mixed with the thylakoid suspension. We have observed in other experiments that 80% of the cyclic photophosphorylation activity were lost after pre-incubation of the thylakoid at 25°C in the dark for 1 h. It has also been reported that heat treatment at 50°C for 3 min is sufficient to almost completely inactivate the electron transfer between water and photosystem II (20). Thus, the observed inactivation with this immobilization method is likely to be due to the heat inactivation of some components of the electron transfer chain. As expected, immobilization of the thylakoid by either TABLE 1. Recovery of the activity of cyclic ATP photophosphorylation after thylakoid immobilization Thylakoid Free Agarose entrapped BSA foam structure
Chlorophyll recovery
Initial rate of cyclic Total recovery of photophosp.horylation activity after
(°A)
(%)
immobilization (0A)
100 65
100 17
100 11
~95
~0.05
~0.05
VOL.73, 1992
CONTINUOUS OPERATION OF SPINACH THYLAKOID 473 0.4 •
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FIG. 1. ContinuousATP production by agarose-entrapped thylakoid in a CSTR. CyclicATP photophosphorylationwas carried out in the absence of ascorbate (o) or in the presence of 5 mM ascorbate (•). Other reaction conditions were as described in Materials and Methods. The insert shows a simplified diagram of the experimental set-up. of these two methods resulted in the inactivation of the cyclic ATP photophosphorylation, albeit to a different extent in each case. It is not the objective of this study to try to find the best immobilization method which produces the least inactivation, or extends the life-time of the photophosphorylation activity. Rather, immobilization was used here to allow for the retention of thylakoid in a CSTR for the study of continuous ATP production. Hence, agarose-entrapped thylakoid was used in the following studies of continuous ATP production. Continuous production of ATP by immobilized thylakoid Continuous ATP production was carried out in a CSTR containing agarose-entrapped thylakoid. Throughout the operation, the CSTR was exposed to light, and substrate solution was continuously fed into the reactor. The reactor temperature of 15°C, the pH of the reaction solution and the inlet ADP concentration of 0.4 mM were chosen according to the results of our previous batch experiments (7), so as to give the maximum rate of cyclic photophosphorylation. A representative result of the continuous ATP production is shown in Fig. 1 (line with open circles). The activity of the cyclic ATP photophosphorylation decreased continuously during operation. Inactivation of this activity was very rapid for the first 8 h of continuous operation under saturating light; only about 30% of the initial activity remained. This was followed by a slower decline in the next 15 h. The residual activity (about 10% of initial activity) was fairly constant for at least another 48 h. Since the inactivation did not follow first order decay, as often observed for cell-free enzymes, no attempt was made to model this observed decay. For the purpose of comparing the stability of the photophosphorylation activity under different operation conditions, the time taken for the reactor to lose 50% of its initial activity (T~0) was used as a crude parameter of decay. The maximum conversion of input ADP, usually achieved after I-2 h from the start of operation, was between 75-I00% in the CSTR. The variations of the percent ADP conversion in repetitive experiments might be a reflection of the variations in the recovery of thylakoid activity
after the immobilization procedure. Since immobilized thylakoid was prepared on the same day as the continuous ATP production experiment, the total amount of photophosphorylation activity recovered and subsequently used in each experiment would be slightly different. To test the possibility that the decay in the photophosphorylation activity might be due to the instability of some components of the substrate solution (e.g. PMS), fresh substrate solution was fed to the CSTR after 18 h of continuous operation. It was found that the photophosphorylation activity was not resurrected to the initial level. The ATP production remained unchanged upon the feeding of fresh substrate solution. This suggests that the decay in ATP production was due to the inactivation of the thylakoid. The declining activity of ATP production by immobilized thylakoid during continuous operation is most likely due to the light-induced, chlorophyll-sensitized oxidation of electron-rich components of the thylakoid membranes (14, 15). The photooxidation of the double bonds in membranes could have a detrimental effect on the structure and function of the thylakoid. The protective mechanism, which involves the carotenoid pigments and operated normally in intact chloroplast, might not be functioning in the isolated thylakoid. Carotenoids have the ability to quench excited chlorophyll molecules and to scavenger potentially toxic singlet oxygen. Rutin, a flavonoid which has a chemical structure that could act as a scavenger of active oxygen species, has been shown to retard pigment loss and inhibit lipid peroxidation that are associated with photooxidation of thylakoid membranes (21). Therefore, we had included 0.2 mM rutin in some of the continuous ATP production experiments. But rutin was found not to have any effect on the operational stability of the immobilized thylakoid in the CSTR. It has been established that there is an optimum redox potential for photophosphorylation in bacterial ehromatophores (22). Previously, Pace et aL had found that an ascorbate concentration of 17 mM under anaerobic condition was optimum for photophosphorylation by chromatophores in a batch reactor (4). In order to test whether
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YU AND HOSONO
J. FERMENT.BmEr~G.,
ascorbate will increase the stability of photophosphorylation by thylakoid, continuous A T P production was carried out with 5 mM ascorbate. The result is shown in Fig. 1 (line with full circles). It can be seen that the decay of the cyclic photophosphorylation activity was slowed down significantly in the first 14 h when 5 mM ascorbate was included in the substrate solution. The Ts0 for the continuous reaction with ascorbate was about 10 h compared to a Tso of about 5.5 h in the absence of ascorbate. To optimize this effect of ascorbate on the Ts0 of A T P production, different concentrations of ascorbate were included in the substrate solution for the continuous A T P production. As shown in Fig. 2, continuous operation of the immobilized thylakoid reactor with 5 mM ascorbate had the longest Ts0 ( ~ 11 h). At 0, 20 and 50 mM ascorbate, the T50 was between 4.5-5.6 h. Furthermore, the use of 20 and 50raM ascorbate in the experiments had a deleterious effect on the rate of A T P photophosphorylation. This was indicated by the drop in the maximum ADP conversion at the beginning of the experiments, as shown in the insert of Fig. 2. These results suggested that an increase of A T P production by a factor of 2 can be obtained in the immobilized thylakoid reactor by including 5 mM ascorbate in the substrate solution (see later and Table 2). The optimal concentration of ascorbate for the continuous A T P production in immobilized thylakoid CSTR is lower than the optimal concentration (17 mM) of ascorbate for chromatophores found previously by Pace et aL in a batch reactor (4). The mechanism by which ascorbate affects the stability of cyclic photophosphorylation of thylakoid is u n k n o w n at present. It could be due to the reduction of some components of the photosystems. To optimize the production of A T P , continuous operation of the immobilized thylakoid reactor was carried out at different dilution rates. The Ts0 of A T P production was affected by the dilution rate used as shown in Fig. 3. The Ts0 values for dilution rates of 0.73 h -~, 1.42 h -~, 2.36 h -~ and 3.73 h -~ were 12.1___1.7 h, 8.6+--0.0 h, 9.6+-0.5 h and 5.17___0.6 h, respectively. The maximum A D P conversions
oo \\ 01\
at the beginning of the experiments with respect to dilution rates are shown in the insert of Fig. 3. The initial A D P conversions were about the same (between 75-100%) for dilution rates of 0.73h -t, 1.42h -1 and 2.36h -1. At a dilution rate of 3.73 h - j , the initial A D P conversion dropped to only about 45%. In order to determine the total ATP production within a period of 20 h at these dilution rates, these data were replotted in the format of Fig. 1, The integrated areas under the curves from 0 to 20 h were used to calculates the amounts of A T P produced in 20 h. The results are shown in Table 2. The optimal dilution rate was 2.36 h -~, as indicated by the most A T P produced (63.3 p m o l / m g Chl) in 20 h. For comparison with the total A T P production in the presence of ascorbate, total A T P produced in the CSTR without ascorbate is also shown in Table 2. The fact that the Ts0 of A T P production changed with respect to the dilution rate used suggests that the decaying A T P phosphorylation activity is not due only to the extent of light exposure. Otherwise, the Ts0 should be the same for all dilution rates under the same illumination condition. Examination of the a m o u n t of A T P produced within the Ts0 for each dilution rate (Table 2) suggests the plausibility of the inactivation of the A T P phosphorylation of thylakoid being related to the a m o u n t of A T P produced. That is, the inactivation process is directly related to A T P photophosphorylation. However, it is unclear why this does not seem to be true for dilution rate of 2.36 h -~. It could be due to the fact that the immobilized thylakoid in the reactor were most efficaciously utilized at 2.36 h-~. Below this dilution rate, the flow rate of substrate was not sufficient to allow the utilization of the full capacity of the reactor. Above this dilution rate, the flow rate of substrate was too fast to allow the A T P photophosphorylation reaction to reach its full extent. Long operational stability of the A T P phosphorylation activity of thylakoid is crucial for its application to continuous A T P production on a commercial scale. In this study, it was shown that 50% of the A T P photophospho-
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FIG. 2. Effect of the concentration of ascorbate on the stability of the activity of cyclicphotophosphorylation. Cyclicphotophosphorylation of ATP by agarose-entrapped thylakoid was carried out in a CSTR at the following concentration of ascorbate: 0 mM (O), 5 mM ( • ) , 20 mM (&) and 50 mlvl (•). The insert shows the initial ADP conversion as a function of the concentration of ascorbate, x is the maximum ADP conversion at the beginning of each experiment.
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lb
1;
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FIG. 3. Effectof dilution rate on the stability of the activity of cyclic photophosphorylation. Cyclic photophosphorylation of ATP
by agarose-entrapped thylakoid was carried out in a CSTR at the following dilution rates: 0.73 h -t (e), 1.42h -1 (o), 2.36h -j (,,) and 3.73 h -j (;t). The insert shows the initial ADP conversion as a function of the dilution rate. x is the maximum ADP conversion at the beginning of each experiment.
CONTINUOUS OPERATION OF SPINACH THYLAKOID
VOL. 73, 1992 TABLE 2. Total ATP production by agarose-entrapped thylakoid in the CSTR Dilution rate (h -I) 3.7 2.4 1.4 0.73
ATP produced ~mol/mg Chip within Ts0 within 20 h 20.6+3.2 43.4+7.6 25.6+4.3 20.2+4.9
(5.2h) b (9.6h) b (8.6h) b (12.1 h) b
44.3+ 3.0 63.3+11.7 39.2_+ 6.5 26.2+ 4.8
................................................................................
0,73 c
Not calculated
475
designed and are testing a semi-continuous process for glucose-6-phosphate production based on A T P regeneration by spinach thylakoid in a batch reactor and the downstream recovery of A D P by anion exchange resins. ACKNOWLEDGMENT A. H. C. Yu is a recipient of a Fellowship from the Science& Technology Agency (STA) of Japan.
12.6_+ 2.8
" ATP produced is expressed as /Jmol ATP per mg chlorophyll used for immobilization. b The time at which 50% of the initial activity was lost. c Ascorbate was not included in the substrate solution. rylation activity was lost within a period of 10 h under optimal conditions in continuous operation. Furthermore, the total A T P production in the CSTR within a period of 20 h is very low (63.3 pmol A T P per mg of input chlorophyll) when it is compared to A T P production with nonimmobilized thylakoid in batch reactors within a period of 1 h (84 ~mol A T P per mg of chlorophyll) (7). Hence, the average rate of A T P production by immobilized thylakoid in the CSTR is about a 28-fold decrease from that by non-immobilized thylakoid in batch reactors. With the agarose-entrapped thylakoid, A T P production in continuous reactors will therefore not be of any commercial interest. We did not investigate A T P regeneration with hexokinase in the CSTR in this study because the analysis of the operational stability of thylakoid would be complicated by the coupled reactions involved. In previously published work on A T P production by chromatophores (5, 11, 12), hexokinase was used as an A T P - c o n s u m i n g enzyme to enhance A T P production. Therefor, the results are not directly comparable with our present results. Nevertheless, immobilized chromatophores were shown to be able to maintain a low level of A T P production ( ~ 2 /zmol A T P / h - m g bacteriochlorophyll (Bchl); calculated by the present authors with the data in reference 12) for more than 10Oh in a CSTR (12). It has also been reported that a productivities of 18/zmol A T P / h . Bchl was obtained when immobilized chromatophores was used in a flat reactor (5). Even though these studies have shown that A T P production can be maintained for a long time, their potential for A T P regeneration is very low ( ~ 1.5 times only) (5, 12). We have previously shown that A T P can be regenerated about 24 times when hexokinase is included with non-immobilized thylakoid in batch reactors (7). Our conclusion was that A T P production by thylakoid would be more efficient when carried out in batch reactors than in continuous reactors; this is due largely to the intrinsically low operational stability of thylakoid. Even though an immobilization method might be found to improve the operational stability of the thylakoid, the rate of photophosphorylation would be low compared to nonimmobilized thylakoid. Furthermore, the cost of immobilization is an additional expense that would negate the benefit gained by improving the operational stability. Because of its low cost and high A T P production rate, nonimmobilized thylakoid can be used in batch reactors as an effective, disposable enzyme source for A T P regeneration. Several batch reactors can be set up with a time offset between each of them such that a semi-continuous production process can be achieved. Presently, we have
REFERENCES 1. Cramer, F.: Preparation of esters, amides, and anhydrides of phosphoric acid, p. 319. In Foerst, W. (ed.), Newer methods of preparative organic chemistry, vol. 8. Academic Press, New York (1964). 2. Gardner, C. R., Colton, C. K., Langer, R. S., Hamilton, B. K., Archer, M. C., and Whitsides, G. M.: Enzymatic regeneration of ATP from AMP and ADP: I. Thermodynamics, kinetics, and process development. Enzyme Eng., 2, 209-216 (1974). 3. Berke, W., Sehuz, H.J., Wandrey, C., Mort, M., Denda, G., and Kula, M.R.: Continuous regeneration of ATP in enzyme membrane reactor for enzymatic syntheses. Biotechnol. Bioeng., 32, 130-139 (1988). 4. Pace, G.W., Yang, H.S., Tannenbaum, S.R., and Archer, M. C.: Photosynthetic regeneration of ATP using bacterial chromatophores. Biotechnol. Bioeng., 18, 1413-1423 (1976). 5. Larreta-Garde, V. and Thomas, D.: Factors controlling initial and long-term ATP regeneration catalyzed by immobilized chromatophores. Biotechnol. Bioeng., 29, 79-84 (1987). 6. Matsuoka, H. and Suzuki, S.: Stabilization of phosphorylating mitochondrial electron transport particles and their use for ATP regeneration. Biotechnol. Bioeng., 23, 1103-1114 (1981). 7. Yu, A.H.C. and Hosono, K.: ATP regeneration by cyclic photophosphorylation using spinach thylakoid. BiotechnoL Lett., 13, 411-416 (1991). 8. Murata, K., Tani, K., Kato, J., and Chibata, I.: Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP. Enzyme Mierob. Technol., 3, 233-241 (1981). 9. Asada, M., Yanamoto, K., Nakanishi, K., Matsuno, R., Kimura, A., and Kamiknbo, T.: Long term continuous ATP regeneration by enzymes of the alcohol fermentation pathway and kinases of yeast. Eur. J. Appl. Microbiol. Biotechnol., 12, 198-204 (1981). 10. Langer, R. S., Hamilton, B. K., Gardner, C. R., Archer, M. C., and Colton, C.K.: Enzymatic regeneration of ATP. AIChE J., 22, 1079-1090 (1976). 11. Larteta-Garde, V., Thomasset, B., Tanaka, A., Gellf, G., and Thomas, D.: Comparative stabilization of biological photosysterns by several immobilization procedures. I. ATP production by immobilized bacterial chromatophores. Eur. J. Appl. Microbiol. Biotechnol., 11, 133-138 (1981). 12. Larreta-Garde, V., Gellf, G., and Thomas, D.: Immobilizedchromatophores-ATP regeneration in batch and open reactors. Eur. J. Appl. Microbiol. Biotechnol., 14, 232-236 (1982). 13. Smeds, A.L. and Enfors, S.O.: Stability of chromatophores during ATP regeneration in an enzyme reactor. Enzyme Microb. Technol., 8, 755-760 (1986). 14. Kyle, D.J. and Ohad, I.: The mechanism of photoinhibition in higher plants and green algae, p. 468-475. In Staehelin, L.A. and Arntzen, C.J. (ed.), Photosynthesis III, Encyclopedia of plant physiol., new series, vol. 19. Springer-Verlag, Berlin (1986). 15. Cornic, G. and Miginiac-Maslow, M.: Photoinhibition of photosynthesis in broken chloroplasts as a function of electron transfer rates during light treatment. Plant Physiol., 78, 72.4-729 0985). 16. Cocquempot, M. F., Thomasset, B., Barbotin, J. N., GeHf, G., and Thomas, D.: Comparative stabilization of biological photosystems by several immobilization procedures. II. Storage and function stability of immobilized thylakoids. Eur. J. Microbiol. Biotechnol., 11, 193-198 (1981).
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17. Weaver, J.C., Williams, B.G., Klibanov, A., and Demain, A.L.: Gel microdroplets: rapid detection and enumeration of individual microorganisms by their metabolic activity. Biotechnology, 6, 1084-1089 (1988). 18. Powell, K.T. and Weaver, J.C.: Gel microdroplets and flow cytometry: rapid determination of antibody secretion by individual ceils within a cell population. Biotechnology, 8, 333-337 (1990). 19. Larreta-Garde, V., Cocquempot, M. F., Barholin, J. N., Thomasset, B., and Thomas, D.: Immobilized thylakoids and chromatophores: hydrogen production and ATP regeneration. Enzyme
J. FERMENT. BIOENG., Eng., 5, 109-118 (1980). 20. Yamashita, T. and Butler, W. L.: Inhibition of chloroplasts by U.V. irradiation and heat treatment. Plant Physiol., 43, 20372040 (1968). 21. Wagner, G. R., Youngman, R. J., and Elstner, E. F.: Inhibition of chloroplast photo-oxidation by flavonoids and mechanisms of the antioxidative action. J. Photochem. Photobiol., B: Biol., 1, 451--460 (1988). 22. Horio, T. and Kameu, M.D.: Optimal oxidation-reduction potentials and endogenous co-factors in bacterial photophosphorylation. Biochem., 1, 144-153 (1962).
Vol. 73, No. 6, 471-476. 1992
Continuous ATP Production by Spinach Thylakoid in a Stirred Tank Reactor ALEX H. C. YU§ A~D KUNIAKI HOSONO* Fermentation Research Institute, Agency of Industrial Science and Technology, 1-1-3 Higashi, Tsukuba, lbaraki 305, Japan Received 7 October 1991/Accepted 14 March 1992 Spinach thylakoid was immobilized by two different methods for the purpose of retention within a continuous-flow stirred tank reactor (CSTR). The glutaraldehyde crosslinked albumin polymer method completely inactivated the cyclic ATP photophosphorylation of thylakoid. In contrast, agarose-entrapped thylakoid retained about 17Yo of the activity of the cyclic photophosphorylation of non-immobilized thylakoid. This activity declined continuously during ATP production in the CSTR. Fifty percent of the initial activity was lost within about 5.5 h. Aseorbate was found to increase the stability of ATP photophosphorylation; about twice as much ATP was produced at the optimal ascorbate concentration of 5 mM. Under the optimal dilution rate of 2.36 h - l , about 60 gmol of ATP per mg chlorophyll were produced in 20 h by agarose-entrapped thylakoid in the CSTR. These results showed that, compared to non-immobilized thylakoid in batch operation, agarose-entrapped thylakoid produced only a low amount of ATP under continuous operation.
For almost two decades, the development of a cofactor regeneration system has been recognized as a prerequisite to the industrial development of economically viable biosynthetic processes. Since many cofactors (e.g. ATP and NADH) are consumed as co-reactants in biosynthesis reactions, they must be supplied at stoichiometric amounts. The obvious way to lower the high cost of these cofactors in biosynthetic processes is to regenerate and recycle them. The regeneration of ATP by various means (chemical synthesis, cell-free enzymes, isolated organelles, and whole ceils) has been attempted in the past (1-9), and has been reviewed and discussed by Langer et al. (10). The use of photosynthetic organelles to convert light energy into ATP by phosphorylation of ADP is an attractive system, since no expensive substrate is required as compared to ATP regeneration by cell-free enzymes. Chromatophores, isolated membrane vesicles from photosynthetic bacteria, have been extensively studied for this purpose (4, 5, 11-13). Recently, we have carried out studies on the alternative photosynthetic organelle, namely, thylakoid membranes from spinach leaves (7). We have demonstrated that spinach thylakoid is an inexpensive ATP regeneration source which can be coupled to ATPconsuming kinases to produce phosphorylated products at an order of magnitude reduction of the requirement of ATP (7). However, it is known that the photochemical activities of the isolated photosynthetic organelles are very unstable. Under the typical reaction conditions, light and oxygen both play a role in inactivating the photosynthetic activities (14, 15). In the past, immobilization was attempted in order to stabilize the photochemical activity of these organelles (11, 16). The commercial usefulness of the photosynthetic organelles in continuous ATP regeneration in bioreactors will be largely dependent on their operational stability. The present paper reports on one part of our continuing
effort to develop an economical ATP regeneration system; namely, the continuous operation of spinach thylakoid in a continuous stirred tank reactor (CSTR) was studied. We are interested in comparing the capabilities of ATP production by thylakoid carried out under batch and continuous operations. Previously, we have shown that a production rate of 84/~mol A T P / h . m g Chlorophyll (Chl) can be achieved by thylakoid in batch reactors (7). However, the photophosphorylation activity of thylakoid typically ceased after about 2 h (7). In this study, we have investigated the ATP productivities under continuous operation to determine if more ATP can be produced than in batch reactors within the operational life-time of the thylakoid. Isolated spinach thylakoid was immobilized in order to allow for the retention of the organelle within the reactor under continuous operation. The thylakoid membranes were immobilized by two different methods: agarose gel entrapment and glutaraldehyde crosslinked albumin polymer. Attempts were made to improve the operational stability of immobilized thylakoid by using rutin, a scavenger of active oxygen species, and by adjusting the redox potential with ascorbate. Total ATP production was optimized by adjusting the dilution rate of the reactor. MATERIALS AND METHODS Isolation of spinach thylakoid membranes The isolation of thylakoid membranes from spinach leaves was described previously (7). Immobilization of isolated thylakoid
Crosslinked albumin polymer method This immobilization procedure is based on the method described by Coequempot et al. (16) with modifications as follows. Thylakoid suspension (0.32 ml) corresponding to approximately 0.9 mg of chlorophyll was added to 1.68 ml of a solution containing 8.7 mM potassium phosphate, pH 6.8, 6.5% bovine serum albumin (BSA) and 0.360/~ glutaraldehyde. The resulting mixture was deposited as semispherical microdroplets of about 15-20 ~I volume by a micropipette onto a piece of Parafilm®. The Parafilm®
* Corresponding author. § Present address: Pulp and Paper Research Institute of Canada, 3800 Wesbrook Mall, Vancouver, Canada. 471
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YU AND H O S O N O
with the thylakoid/albumin microdroplets was put into a light-tight box and was kept at - 2 0 ° C for 2.5 h. The crosslinked albumin/thylakoid microbeads were thawed and then washed with distilled water. Agarose gel entrapment method This immobilization procedure is based on the method described by Weaver et al. (17) with modification as follows. Thylakoid suspension (0.2 ml) corresponding to 0.85-0.94 mg of chlorophyll was mixed with 1.8 ml of 4% agarose solution at 300C. The agarose solution was prepared by dissolving ultra-low gelling temperature agarose (type IX; Sigma) in a pH 7.6 solution containing 50raM Hepes, 5 mM NaC1, 5 mM MgCI2. The agarose-thylakoid mixture was added dropwise by a 1-ml pipette into a 100-ml beaker containing 50 ml of mineral oil at 25-30°C under constant stirring by a magnetic bar. The speed of stirring was adjusted so as to disperse the agarose-thylakoid droplets into spherical microdroplets of 100 pro-l,000 pm in diameter. The beaker was then transferred to an ice-water bath. After 20 min, the solidified agarose-thylakoid beads were washed with a pH 7.6 solution containing 0.1 M sorbitol, 5 mM MgCI2, 5 mM K2HPO4, 50 mM Hepes to remove the mineral oil. The washed agarose-entrapped beads were then size-fractionated with a steel mesh (360/zm x 400/~m). Determination of initial rate of cyclic photophosphorylation Cyclic photophosphorylation mediated by phenazinc methosulfate (PMS) was carried out as described previously (7). In the case of immobilized thylakoid, the reaction solution was directly used for ATP determination without the trichloroacetic acid treatment that was done in the case of non-immobilized thylakoid. For reaction with agarose-entrapped thylakoid, the amount of thylakoid used in the reaction was determined by recovering all the thylakoid beads after the reaction. The agarose-cntrapped thylakoid beads were melted at 60°C and the chlorophyll content was determined as previously described (7). For reaction with crosslinked albumin-thylakoid beads, the amount of thylakoid used in the reaction was determined by the weight of the beads since the recovery of thylakoid in this immobilized method was almost quantitative. Continuous ATP production in CSTR The cyclic ATP photophosphorylation was carried out in a temperature-controlled stirred tank reactor of 7 ml at 15°C. A simplified diagram of the experimental set-up is shown in the insert of Fig. 1. Saturating light (60 m W / c m 2) was provided by a 250-W light bulb placed 17 cm from the reactor. To absorb the heat from the light source, water at 15°C was circulated through a bottle placed in front of the reactor. Immobilized thylakoid, placed inside the reactor, was agitated by a magnetic stir bar. Substrate solution (0.1 M sorbitol, 5 mM K2HPO4, 0.4raM ADP, 5 m M MgC12, 5 mM NaCI, 0.1 mM PMS and 50ram Hepes, pH 7.6) in a light-tight container was continuously fed into the reactor by a peristaltic pump at a fixed speed. Another channel of the same pump was used to remove the product from the reactor. The pore size of the outlet tube was small relative to the bead size of the immobilized thylakoid, so that immobilized thylakoid was not removed from the reactor. Fractions of the outlet stream were collected. Selected fractions were used for the determination of ATP by Luciferin/Luciferase assay, as previously described (7). The continuous operation of this process was carried out in a cold room at 4°C.
J. FERMENT. BIOENO.,
RESULTS AND DISCUSSION Immobilization of spinach thylakoid In order to retain the thylakoid membranes within a CSTR, the soluble thylakoid membranes needed to be immobilized. Immobilization often inadvertently affects the activity of the catalyst; different procedures cause different degrees of inactivation. Therefore, two defferent methods were used to immobilize the thylakoid in this study. The first method, utilizing glutaraldehyde to crosslink albumin and thylakoid into a foam structure, was described by Larreta-Garde et al. in the immobilization of bacterial chromatophores (11) and lettuce thylakoid (16). The second method, utilizing low-temperature gelling agarose to entrap thylakoid into spherical microdroplets, was a modification of the method described by Weaver et al. in the immobilization of microorganisms and mammalian cells (17, 18). As shown in Table 1, the initial rate of cyclic ATP photophosphorylation of thylakoid after immobilization were greatly affected. In the case of the albuminthylakoid foam method, essentially complete inactivation of the cyclic photophosphorylation of the immobilized thylakoid was observed. The inactivation observed was different from the previous reports (19) where 9.5% of cyclic ATP photophosphorylation was recovered after immobilization. It was also reported that 85-90% of oxygen production activity (i.e. electron transport chain activity) of non-immobilized thylakoid were obtained for thylakoid immobilized by this albumin foam method (16). Thus, this suggests the likelihood that the inactivation observed is due to the uncoupling of ATP phosphorylation from electron transport and/or damage to the components of the ATPase. For agarose-entrapped thylakoid, only 17% of the cyclic ATP phosphorylation activity was recovered after immobilization. The physical recovery of thylakoid membranes in this immobilization procedure was found to be about 65%. Therefore, the total activity of ATP photophosphorylation recovered in terms of initial thylakoid used for immobilization was only about 11%. In this immobilization method, the thylakoid was briefly exposed ( ~ 2 m i n ) to a temperature of 30°C when liquid agarose was mixed with the thylakoid suspension. We have observed in other experiments that 80% of the cyclic photophosphorylation activity were lost after pre-incubation of the thylakoid at 25°C in the dark for 1 h. It has also been reported that heat treatment at 50°C for 3 min is sufficient to almost completely inactivate the electron transfer between water and photosystem II (20). Thus, the observed inactivation with this immobilization method is likely to be due to the heat inactivation of some components of the electron transfer chain. As expected, immobilization of the thylakoid by either TABLE 1. Recovery of the activity of cyclic ATP photophosphorylation after thylakoid immobilization Thylakoid Free Agarose entrapped BSA foam structure
Chlorophyll recovery
Initial rate of cyclic Total recovery of photophosp.horylation activity after
(°A)
(%)
immobilization (0A)
100 65
100 17
100 11
~95
~0.05
~0.05
VOL.73, 1992
CONTINUOUS OPERATION OF SPINACH THYLAKOID 473 0.4 •
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•
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FIG. 1. ContinuousATP production by agarose-entrapped thylakoid in a CSTR. CyclicATP photophosphorylationwas carried out in the absence of ascorbate (o) or in the presence of 5 mM ascorbate (•). Other reaction conditions were as described in Materials and Methods. The insert shows a simplified diagram of the experimental set-up. of these two methods resulted in the inactivation of the cyclic ATP photophosphorylation, albeit to a different extent in each case. It is not the objective of this study to try to find the best immobilization method which produces the least inactivation, or extends the life-time of the photophosphorylation activity. Rather, immobilization was used here to allow for the retention of thylakoid in a CSTR for the study of continuous ATP production. Hence, agarose-entrapped thylakoid was used in the following studies of continuous ATP production. Continuous production of ATP by immobilized thylakoid Continuous ATP production was carried out in a CSTR containing agarose-entrapped thylakoid. Throughout the operation, the CSTR was exposed to light, and substrate solution was continuously fed into the reactor. The reactor temperature of 15°C, the pH of the reaction solution and the inlet ADP concentration of 0.4 mM were chosen according to the results of our previous batch experiments (7), so as to give the maximum rate of cyclic photophosphorylation. A representative result of the continuous ATP production is shown in Fig. 1 (line with open circles). The activity of the cyclic ATP photophosphorylation decreased continuously during operation. Inactivation of this activity was very rapid for the first 8 h of continuous operation under saturating light; only about 30% of the initial activity remained. This was followed by a slower decline in the next 15 h. The residual activity (about 10% of initial activity) was fairly constant for at least another 48 h. Since the inactivation did not follow first order decay, as often observed for cell-free enzymes, no attempt was made to model this observed decay. For the purpose of comparing the stability of the photophosphorylation activity under different operation conditions, the time taken for the reactor to lose 50% of its initial activity (T~0) was used as a crude parameter of decay. The maximum conversion of input ADP, usually achieved after I-2 h from the start of operation, was between 75-I00% in the CSTR. The variations of the percent ADP conversion in repetitive experiments might be a reflection of the variations in the recovery of thylakoid activity
after the immobilization procedure. Since immobilized thylakoid was prepared on the same day as the continuous ATP production experiment, the total amount of photophosphorylation activity recovered and subsequently used in each experiment would be slightly different. To test the possibility that the decay in the photophosphorylation activity might be due to the instability of some components of the substrate solution (e.g. PMS), fresh substrate solution was fed to the CSTR after 18 h of continuous operation. It was found that the photophosphorylation activity was not resurrected to the initial level. The ATP production remained unchanged upon the feeding of fresh substrate solution. This suggests that the decay in ATP production was due to the inactivation of the thylakoid. The declining activity of ATP production by immobilized thylakoid during continuous operation is most likely due to the light-induced, chlorophyll-sensitized oxidation of electron-rich components of the thylakoid membranes (14, 15). The photooxidation of the double bonds in membranes could have a detrimental effect on the structure and function of the thylakoid. The protective mechanism, which involves the carotenoid pigments and operated normally in intact chloroplast, might not be functioning in the isolated thylakoid. Carotenoids have the ability to quench excited chlorophyll molecules and to scavenger potentially toxic singlet oxygen. Rutin, a flavonoid which has a chemical structure that could act as a scavenger of active oxygen species, has been shown to retard pigment loss and inhibit lipid peroxidation that are associated with photooxidation of thylakoid membranes (21). Therefore, we had included 0.2 mM rutin in some of the continuous ATP production experiments. But rutin was found not to have any effect on the operational stability of the immobilized thylakoid in the CSTR. It has been established that there is an optimum redox potential for photophosphorylation in bacterial ehromatophores (22). Previously, Pace et aL had found that an ascorbate concentration of 17 mM under anaerobic condition was optimum for photophosphorylation by chromatophores in a batch reactor (4). In order to test whether
474
YU AND HOSONO
J. FERMENT.BmEr~G.,
ascorbate will increase the stability of photophosphorylation by thylakoid, continuous A T P production was carried out with 5 mM ascorbate. The result is shown in Fig. 1 (line with full circles). It can be seen that the decay of the cyclic photophosphorylation activity was slowed down significantly in the first 14 h when 5 mM ascorbate was included in the substrate solution. The Ts0 for the continuous reaction with ascorbate was about 10 h compared to a Tso of about 5.5 h in the absence of ascorbate. To optimize this effect of ascorbate on the Ts0 of A T P production, different concentrations of ascorbate were included in the substrate solution for the continuous A T P production. As shown in Fig. 2, continuous operation of the immobilized thylakoid reactor with 5 mM ascorbate had the longest Ts0 ( ~ 11 h). At 0, 20 and 50 mM ascorbate, the T50 was between 4.5-5.6 h. Furthermore, the use of 20 and 50raM ascorbate in the experiments had a deleterious effect on the rate of A T P photophosphorylation. This was indicated by the drop in the maximum ADP conversion at the beginning of the experiments, as shown in the insert of Fig. 2. These results suggested that an increase of A T P production by a factor of 2 can be obtained in the immobilized thylakoid reactor by including 5 mM ascorbate in the substrate solution (see later and Table 2). The optimal concentration of ascorbate for the continuous A T P production in immobilized thylakoid CSTR is lower than the optimal concentration (17 mM) of ascorbate for chromatophores found previously by Pace et aL in a batch reactor (4). The mechanism by which ascorbate affects the stability of cyclic photophosphorylation of thylakoid is u n k n o w n at present. It could be due to the reduction of some components of the photosystems. To optimize the production of A T P , continuous operation of the immobilized thylakoid reactor was carried out at different dilution rates. The Ts0 of A T P production was affected by the dilution rate used as shown in Fig. 3. The Ts0 values for dilution rates of 0.73 h -~, 1.42 h -~, 2.36 h -~ and 3.73 h -~ were 12.1___1.7 h, 8.6+--0.0 h, 9.6+-0.5 h and 5.17___0.6 h, respectively. The maximum A D P conversions
oo \\ 01\
at the beginning of the experiments with respect to dilution rates are shown in the insert of Fig. 3. The initial A D P conversions were about the same (between 75-100%) for dilution rates of 0.73h -t, 1.42h -1 and 2.36h -1. At a dilution rate of 3.73 h - j , the initial A D P conversion dropped to only about 45%. In order to determine the total ATP production within a period of 20 h at these dilution rates, these data were replotted in the format of Fig. 1, The integrated areas under the curves from 0 to 20 h were used to calculates the amounts of A T P produced in 20 h. The results are shown in Table 2. The optimal dilution rate was 2.36 h -~, as indicated by the most A T P produced (63.3 p m o l / m g Chl) in 20 h. For comparison with the total A T P production in the presence of ascorbate, total A T P produced in the CSTR without ascorbate is also shown in Table 2. The fact that the Ts0 of A T P production changed with respect to the dilution rate used suggests that the decaying A T P phosphorylation activity is not due only to the extent of light exposure. Otherwise, the Ts0 should be the same for all dilution rates under the same illumination condition. Examination of the a m o u n t of A T P produced within the Ts0 for each dilution rate (Table 2) suggests the plausibility of the inactivation of the A T P phosphorylation of thylakoid being related to the a m o u n t of A T P produced. That is, the inactivation process is directly related to A T P photophosphorylation. However, it is unclear why this does not seem to be true for dilution rate of 2.36 h -~. It could be due to the fact that the immobilized thylakoid in the reactor were most efficaciously utilized at 2.36 h-~. Below this dilution rate, the flow rate of substrate was not sufficient to allow the utilization of the full capacity of the reactor. Above this dilution rate, the flow rate of substrate was too fast to allow the A T P photophosphorylation reaction to reach its full extent. Long operational stability of the A T P phosphorylation activity of thylakoid is crucial for its application to continuous A T P production on a commercial scale. In this study, it was shown that 50% of the A T P photophospho-
.-> 75
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FIG. 2. Effect of the concentration of ascorbate on the stability of the activity of cyclicphotophosphorylation. Cyclicphotophosphorylation of ATP by agarose-entrapped thylakoid was carried out in a CSTR at the following concentration of ascorbate: 0 mM (O), 5 mM ( • ) , 20 mM (&) and 50 mlvl (•). The insert shows the initial ADP conversion as a function of the concentration of ascorbate, x is the maximum ADP conversion at the beginning of each experiment.
o
g
lb
1;
2o
2s
Time (h)
FIG. 3. Effectof dilution rate on the stability of the activity of cyclic photophosphorylation. Cyclic photophosphorylation of ATP
by agarose-entrapped thylakoid was carried out in a CSTR at the following dilution rates: 0.73 h -t (e), 1.42h -1 (o), 2.36h -j (,,) and 3.73 h -j (;t). The insert shows the initial ADP conversion as a function of the dilution rate. x is the maximum ADP conversion at the beginning of each experiment.
CONTINUOUS OPERATION OF SPINACH THYLAKOID
VOL. 73, 1992 TABLE 2. Total ATP production by agarose-entrapped thylakoid in the CSTR Dilution rate (h -I) 3.7 2.4 1.4 0.73
ATP produced ~mol/mg Chip within Ts0 within 20 h 20.6+3.2 43.4+7.6 25.6+4.3 20.2+4.9
(5.2h) b (9.6h) b (8.6h) b (12.1 h) b
44.3+ 3.0 63.3+11.7 39.2_+ 6.5 26.2+ 4.8
................................................................................
0,73 c
Not calculated
475
designed and are testing a semi-continuous process for glucose-6-phosphate production based on A T P regeneration by spinach thylakoid in a batch reactor and the downstream recovery of A D P by anion exchange resins. ACKNOWLEDGMENT A. H. C. Yu is a recipient of a Fellowship from the Science& Technology Agency (STA) of Japan.
12.6_+ 2.8
" ATP produced is expressed as /Jmol ATP per mg chlorophyll used for immobilization. b The time at which 50% of the initial activity was lost. c Ascorbate was not included in the substrate solution. rylation activity was lost within a period of 10 h under optimal conditions in continuous operation. Furthermore, the total A T P production in the CSTR within a period of 20 h is very low (63.3 pmol A T P per mg of input chlorophyll) when it is compared to A T P production with nonimmobilized thylakoid in batch reactors within a period of 1 h (84 ~mol A T P per mg of chlorophyll) (7). Hence, the average rate of A T P production by immobilized thylakoid in the CSTR is about a 28-fold decrease from that by non-immobilized thylakoid in batch reactors. With the agarose-entrapped thylakoid, A T P production in continuous reactors will therefore not be of any commercial interest. We did not investigate A T P regeneration with hexokinase in the CSTR in this study because the analysis of the operational stability of thylakoid would be complicated by the coupled reactions involved. In previously published work on A T P production by chromatophores (5, 11, 12), hexokinase was used as an A T P - c o n s u m i n g enzyme to enhance A T P production. Therefor, the results are not directly comparable with our present results. Nevertheless, immobilized chromatophores were shown to be able to maintain a low level of A T P production ( ~ 2 /zmol A T P / h - m g bacteriochlorophyll (Bchl); calculated by the present authors with the data in reference 12) for more than 10Oh in a CSTR (12). It has also been reported that a productivities of 18/zmol A T P / h . Bchl was obtained when immobilized chromatophores was used in a flat reactor (5). Even though these studies have shown that A T P production can be maintained for a long time, their potential for A T P regeneration is very low ( ~ 1.5 times only) (5, 12). We have previously shown that A T P can be regenerated about 24 times when hexokinase is included with non-immobilized thylakoid in batch reactors (7). Our conclusion was that A T P production by thylakoid would be more efficient when carried out in batch reactors than in continuous reactors; this is due largely to the intrinsically low operational stability of thylakoid. Even though an immobilization method might be found to improve the operational stability of the thylakoid, the rate of photophosphorylation would be low compared to nonimmobilized thylakoid. Furthermore, the cost of immobilization is an additional expense that would negate the benefit gained by improving the operational stability. Because of its low cost and high A T P production rate, nonimmobilized thylakoid can be used in batch reactors as an effective, disposable enzyme source for A T P regeneration. Several batch reactors can be set up with a time offset between each of them such that a semi-continuous production process can be achieved. Presently, we have
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17. Weaver, J.C., Williams, B.G., Klibanov, A., and Demain, A.L.: Gel microdroplets: rapid detection and enumeration of individual microorganisms by their metabolic activity. Biotechnology, 6, 1084-1089 (1988). 18. Powell, K.T. and Weaver, J.C.: Gel microdroplets and flow cytometry: rapid determination of antibody secretion by individual ceils within a cell population. Biotechnology, 8, 333-337 (1990). 19. Larreta-Garde, V., Cocquempot, M. F., Barholin, J. N., Thomasset, B., and Thomas, D.: Immobilized thylakoids and chromatophores: hydrogen production and ATP regeneration. Enzyme
J. FERMENT. BIOENG., Eng., 5, 109-118 (1980). 20. Yamashita, T. and Butler, W. L.: Inhibition of chloroplasts by U.V. irradiation and heat treatment. Plant Physiol., 43, 20372040 (1968). 21. Wagner, G. R., Youngman, R. J., and Elstner, E. F.: Inhibition of chloroplast photo-oxidation by flavonoids and mechanisms of the antioxidative action. J. Photochem. Photobiol., B: Biol., 1, 451--460 (1988). 22. Horio, T. and Kameu, M.D.: Optimal oxidation-reduction potentials and endogenous co-factors in bacterial photophosphorylation. Biochem., 1, 144-153 (1962).