Gas Exchange of Algae

Transcription

1 APPLIED MICROBIOLOGY, May 1967, p American Society for Microbiology Vol. 15, No. 3 Printed in U.S.A. Gas Exchange of Algae III. Relation Between the Concentration of Carbon Dioxide in the Nutrient Medium and the Oxygen Production of Chlorella pyrenoidosa ELIZABETH C. B. AMMANN AND VICTORIA H. LYNCH1 Research Laboratories, Lockheed Missiles and Space Company, Palo Alto, California 9434 Received for publication 1 October 1966 The oxygen production of a photosynthetic gas exchanger containing Chlorella pyrenoidosa (1% packed cell volume) was measured when various concentrations of carbon dioxide were present within the culture unit. The internal carbon dioxide concentrations were obtained by manipulating the entrance gas concentration and the flow rate. Carbon dioxide percentages were monitored by means of electrodes placed directly in the nutrient medium. The concentration of carbon dioxide in the nutrient medium which produced maximal photosynthesis was in the range of 1.5 to 2.5% by volume. Results were unaffected by either the level of carbon dioxide in the entrance gas or the rate of gas flow. Entrance gases containing 2% carbon dioxide flowing at 32 ml/min, 3% carbon dioxide at 135 ml/min, and 4% carbon dioxide at 55 ml/min yielded optimal carbon dioxide concentrations in the particular unit studied. By using carbon dioxide electrodes implanted directly in the gas exchanger to optimie the carbon dioxide concentration throughout the culture medium, it should be possible to design more efficient large-scale units. An algal photosynthetic gas exchanger is under consideration as a means of supplying oxygen and removing carbon dioxide during man's extraterrestrial and submarine ventures. The feasibility of using such a gas exchanger depends on how well large-scale algal culture units can be designed. Optimal environmental conditions must be supplied to each algal cell so that culture weight does not become an insurmountable obstacle. The availability of carbon dioxide is an important environmental factor which influences the photosynthetic rate of dense algal cultures. Several forms of carbon dioxide are found within a photosynthetic gas exchanger (12). Carbon dioxide gas dissolves in water and forms carbonic acid which dissociates to form hydrogen and bicarbonate ions [CO2 (gas) CO2 (dissolved) H2CO3 H+ + HCOj-]. Bicarbonate can further dissociate to carbonate (HCO3- H+ + COi-2). The present study relates the concentration of carbon dioxide in the nutrient medium to oxygen production. An understanding of the optimal concentration of carbon dioxide in direct contact with the algae cells will allow more efficient photosynthetic gas exchanger designs to be implemented. MATERIALS AND METHODS Culture organism and medium. Of 1.6% packed cell volume (pcv) Chlorella pyrenoidosa (Starr 252), 1 Deceased. 9 ml was maintained in a continuous culture unit which has been described previously (2). Cells from this culture were periodically transferred to a second culture apparatus, described below, for gas exchange analyses. Consequently, the environmental conditions supplied to this inoculum unit were closely controlled. They were: nutrient medium; basal salt medium (1) with 1 g of nitrogen per liter in the form of urea; irradiation, General Electric F4 cool white fluorescent lamp producing 13 X 1's to 14 X 1'5 quanta (4 to 7 nm) per sec per cm; entrance gas, 2. to 2.2% carbon dioxide in air; flow rate, 11 to 16 ml/min; dissolved carbon dioxide (all species), 1.1 to 1.5%;pH 5.8 to 6.1; temperature, 23.5 to25.4 C; growth rate,.4 divisions per hr. Experimental culture apparatus. The previously described culture unit (2) was modified so that two Severinghaus carbon dioxide electrodes (National Welding Equipment Co., Richmond, Calif.) could be inserted directly into the nutrient medium (Fig. 1). One electrode was placed at the bottom of the unit just above the entrance gas (Q), and the other was inserted near the top just below the exit gas (R). A rubber diaphragm stopper port (M), located in the middle of the column, was used to obtain samples for total carbon dioxide analysis. The length of the column was 56 cm. Analysis of carbon dioxide in the nutrient medium. The Severinghaus carbon dioxide electrodes were calibrated by flowing compressed gases containing air enriched with.5, 1, 2, 3, 4, 5, and 6% carbon dioxide through the algae culture unit which was filled with nutrient medium. The carbon dioxide percentage in each gas tank had been previously analyed by a Scholander gas analyer (.5 ml, sample 487

2 488 AMMANN AND LYNCH APPL. MICROBIOL. FIG. 1. Algae culture apparatus with pco2 electrodes (Q and R) and sampling port (M). Arrows indicate direction ofgas flow. sie). A linear response was obtained when the log of the electrode response was plotted against per cent carbon dioxide. The response was the same whether the unit was filled with nutrient medium, distilled water, or air. The nutrient medium was drained out after calibration, and algae plus nutrient medium were added to obtain the experimental data. The entire setup was torn down after each day's measurements so that the carbon dioxide electrodes could be cleaned and recalibrated. Analysis of total carbon dioxide in the nutrient medium. Total carbon dioxide was measured with a Van Slyke apparatus. A 25 X 13 M sodium bicarbonate standard was used to check instrument accuracy. The reagents used to release and absorb the carbon dioxide were.1 N lactic acid and 1.5 M sodium hydroxide. The experimental samples were obtained by inserting an airtight syringe through the sample port (M) and withdrawing 1 ml of nutrient medium. The liquid was immediately passed through a Swinny hypodermic adaptor containing a.45-p pore sie membrane filter (Millipore Corp., Bedford, Mass.) and into a second airtight syringe to remove the algae prior to analysis. Gas exchange measurements. Beckman gas analyers based on the paramagnetic and infrared properties of oxygen and carbon dioxide were used to determine the oxygen production and carbon dioxide consumption of Chlorella pyrenoidosa under the influence of different CO2 partial pressures. All gas calculations have been previously described (2, 3). RESULTS The experimental data were obtained by first calibrating the CO2 pressure (pco2) electrodes in the experimental culture apparatus and then adding 5 ml of Chlorella pyrenoidosa from the continuous culture unit and 3 ml of fresh nutrient medium to obtain a final cell concentration of.9 to 1.1% pcv. Different concentrations of carbon dioxide in the nutrient medium were obtained by individually bubbling compressed air containing.5, 1, 2, 3, 4, 5, and 6% carbon dioxide through the algal culture at a single flow rate over a period of 1 hr. In this way, different carbon dioxide environments were produced without necessitating a change in gas flow. Oxygen production and carbon dioxide consumption of the alga were measured in each carbon dioxide environment. The effect of similar concentrations of carbon dioxide obtained with different entrance gases was analyed by comparing separate experiments carried out at different flow rates on different days. Three separate experiments were run by use of flow rates of 55, 135, and 32 ml/min. Similar carbon dioxide environments were also obtained by introducing different percentages of carbon dioxide at various flow rates during a single 1-hr experiment for comparison. This procedure decreased the variables due to slight changes in light intensity and cell count, which occurred between experiments, but introduced a second variable due to flow rate within the single experiment. In all experiments, final gas measurements were made only after steady-state conditions had been achieved. The stabiliation of the oxygen and carbon dioxide concentrations in the exit gas and the carbon dioxide concentration within the culture unit indicated when this dynamic equilibrium condition had been reached. The concentration of carbon dioxide at various locations within a photosynthetic gas exchanger under steady-state conditions is shown in Table 1. Data are given for several entrance gas percentages and flow rates. It can be seen that the concentration of carbon dioxide within the culture unit w-as much lower than the entrance gas percentage and matched the concentration found in

3 VOL. 1 5X 1967 GAS EXCHANGE OF ALGAE 489 TABLE 1. Concentration of carboni dioxide at various locations within a photosynthetic gas exchanger under steady-state conditions as a function of entrance gas concentration and flow rate Location CO2 concn (% by vol) 55a Entrance gas Lower pco2 electrode (Q) Upper pco2 electrode (R) Exit gas a Represents flow rate in milliliters per minute. the exit gas more closely under all conditions tried. There appeared to be slightly more carbon dioxide available to the algae at the bottom of the 56-cm column than at the top. Approximately the same carbon dioxide gradient was observed along the column regardless of flow rate. Good mixing is indicated with a 56-cm column length and with flow rates of 55 to 32 ml/min since both lower and upper electrodes recorded fairly similar carbon dioxide values. The slight differences in the CO2 concentration between the exit gas and the upper pco2 electrode are due to instrument accuracy. The standard deviation of the carbon dioxide electrode at the.5% level was -i-.3%, at the 1% level, +.6%, and at the 2% level, i.15%. The standard deviation of the infrared carbon dioxide analyer was.15%. The Van Slyke carbon dioxide determinations matched the carbon dioxide electrode measurements when the ph was 6. or less (Fig. 2). Only at very low concentrations of free carbon dioxide was the concentration of other species of carbon dioxide proportionally larger. The ph at these levels ranged from 6.2 to 6.9; the predominant species of carbon dioxide was, therefore, bicarbonate. It is not possible to determine whether oxygen production favors a particular species of carbon dioxide from these experiments. A good correlation between the partial pressure of carbon dioxide within a photosynthetic exchanger and the amount of oxygen which is produced was observed in the present study (Fig. 3). The optimal carbon dioxide concentration for a 1.% pcv C. pyrenoidosa suspension was in the range of 1.5 to 2.5% carbon dioxide. Air containing 2% CO2 flowing at 32 ml/min, 3% CO2 at 135 or 32 ml/min, or 4% CO2 at 55 ml/min yielded carbon dioxide concentrations within this range and caused the maximal oxygen production in the particular unit studied. Neither the concentration of carbon dioxide in the entrance gas nor the rate of flow affected the results. C. pyrenoidosa produced the same amount of oxygen when equivalent carbon dioxide concentrations.l.4 L 2 x 1. pc2 ELECTRODE Al I - /VAN SLYKE I /, CARBON DIOXIDE IN THE NUTRIENT MEDIUM (% BY VOLUME ) FIG. 2. Oxygen production by Chlorella pyrenoidosa as a function ofdifferent species ofcarbon dioxide (free and total C2). Measurements were made for a period of 1 hr. 1.6 ~~~~~~~~~~~ 1l4 2Ō12 Om ML/MIN 6' W_ 1.2 ~~~55ML/MN 1.' 32 ML/MIN CARBON DIOXIDE IN THE NUTRIENT MEDIUM (% BY VOLUME) FIG. 3. Oxygen production by Chlorella pyrenioidosa as a function of carbon dioxide in the nutrient medium. The numbers within the symbols indicate the carboni dioxide percentage of the entrance gas. Three separate experiments were run, each at a single flow rate. Each experiment lasted for a period of 1 hr. were obtained within the culture unit by using different entrance gases and different flow rates during a single experiment (Table 2). The photosynthetic quotient (CO2 consumed/2 produced) remained steady during all measurements. An average of.9 i.3 was obtained. A long-term experiment was run on a single continuous culture containing 1 % pcv C. pyrenoidosa to verify the short-term results. All environmental conditions were similar to those described previously except that a lamp of higher

4 49 AMMANN AND LYNCH APPL. MICROBIOL. TABLE 2. Oxygen production by Chlorella pyrenoidosa as a function of carbon dioxide in the nutrienit mediuma Carbon Carbon dioxide Oye dioxide in Flow rate in nutrient Oxygen entrance gas (ml/min ) medium production (% (% by vol) (m/) a Similar concentrations were obtained by varying both the carbon dioxide percentage in the entrance gas and the flow rate in a single 1-hr experiment. TABLE 3. Long-term oxygen productioi of Chlorella pyrenoidosa as a funiction of carboni dioxide in the nutrient medium Dissolved CO2 (% 2 (ml/min) production duration Exp(days) light intensity was used [Sylvania F48 warm white fluorescent lamp producing 27 x 115 quanta (4 to 7 nm) per sec per cm]. The culture was maintained at a single dissolved carbon dioxide concentration for a 2- to 6-day time interval during which time daily gas exchange measurements were made. Maximal oxygen production occurred when the culture contained above 1 and less than 3%,X carbon dioxide in solution (Table 3). DISCUSSION The parameter employed to define the optimal carbon dioxide environment for algal photsynthesis should be one that can be applied to any algal system. A measure of carbon dioxide in the entrance or exit gas is the standard means for determining the amount of carbon dioxide present in a photosynthetic gas exchanger. Results of the present investigation suggest that this method is not the best means of comparison among units. The amount of external carbon dioxide that actually becomes available to each algal cell is influenced by many factors. In the present study, maximal oxygen production was obtained, regardless of the per cent carbon dioxide in the entrance gas, by manipulating the flow rate to obtain carbon dioxide values in the nutrient medium from 1.5 to 2.5%. Differences in culture dimensions, cell density, and method of gas introduction undoubtedly also influence the amount of carbon dioxide available within an algal culture. Thus, carbon dioxide analyses should be made directly in the nutrient medium surrounding the algal cells so that comparisons among units can be made. It should be possible to design more efficient, large-scale photosynthetic gas exchangers by using carbon dioxide electrodes to optimie the carbon dioxide concentration throughout a culture unit. Once a design has been established, the electrodes would be removed. Hannan and Patouillet (6) reported data on a photosynthetic gas exchanger which tends to support the data obtained in the present experiment pertaining to the optimal carbon dioxide concentration within a culture unit. The two workers found that an increase in the carbon dioxide percentage in the exit gas from.25 to.69% caused a continuing increase in oxygen production. It is most probable that the concentration of carbon dioxide within their unit was approximately equal to that of the exit gas as was observed in the present study. The length of their culture was 25.4 cm, the cell density was approximately 1.% pcv (about 5, cells per mm3), and they obtained the same general values for entrance and exit carbon dioxide gas concentrations. If this actually is the case, Hannan and Patouillet's data indicate that carbon dioxide values of approximately.7% are suboptimal for maximal photosynthesis. This value is below the 1.5 to 2.5% demonstrated to be in the optimal range in the present study. Rosenberg (9), working with 4-ml cultures containing 1% pcv C. pyrenoidosa, also indicated that carbon dioxide concentrations below 1 % are suboptimal for maximal gas exchange. When very dilute concentrations of Chlorella (1 cells per mm3) are employed, both photosynthesis and growth have been reported to be independent of the concentration of carbon dioxide down to.1 to.3% (4, 1). It is not possible to resolve the different results obtained with dilute versus dense cell populations at this time. Cells of dense cultures must necessarily compete more strongly among themselves for the available inorganic salts and light energy. The supply of light energy to individual cells fluctuates widely from limiting to saturating levels owing to the fact that the cells are being agitated in close proximity. Perhaps, such conditions affect the concentration of carbon dioxide which is optimal for photosynthesis. Experiments in which low cell concentrations are used have little application to the design of an optimal photosynthetic gas

5 VOL. 15, 1967 GAS EXCHANGE OF ALGAE 491 exchanger in which dense algae suspensions are required to obtain maximal gas exchange per unit of volume. Inhibition of photosynthesis was observed in the present experiments when carbon dioxide concentrations in the dense algal culture exceeded approximately 3%. Similar narcotic effects have been observed by other workers, using dilute algal suspensions and lower carbon dioxide concentrations (7, 1), with one exception (5). The biochemistry of the inhibition by carbon dioxide is not completely understood at the present. Chlorophyll synthesis has been found to be reduced by a 1% concentration of the gas in illuminated etiolated leaves (11). High levels of carbon dioxide are also known to inhibit several reactions associated with the Krebs cycle (8, 13). Perhaps, similar reactions are associated with the reduced oxygen production in Chlorella. ACKNOWLEDGMENT We thank John E. Durichek for the mechanical assembly and maintenance of the culture apparatus, Ruth F. Zeits for the Van Slyke carbon dioxide analyses, and Eugene. Ammann for critical reading of the manuscript. LrTERATURE CIED 1. AMMANN, E. C. B., AND V. H. LYNCH Purine metabolism by unicellular algae. II. Adenine, hypoxanthine, and xanthine degradation by Chlorella pyrenoidosa. Biochim. Biophys Acta 87: AMMANN, E. C. B., AND V. H. LYNCH Gas exchange of algae. I. Effects of time, light intensity, and spectral-energy distribution on the photosynthetic quotient of Chlorella pyrenoidosa. Appl. Microbiol. 13: AMMANN, E. C. B., AND V. H. LYNCH Gas exchange of algae. II. Effects of oxygen, helium, and argon on the photosynthesis of Chlorella pyrenoidosa. Appl. Microbiol. 14: BRIGGS, G. E., AND C. P. WHrrrINGHAM Factors affecting the rate of photosynthesis of Chlorella at low concentrations of carbon dioxide and in high illumination. New Phytologist 51: DAVIS, E. A., J. DEDRICK, C. S. FRENCH, H. W. MILNER, J. MYERS, J. H. C. SMITH, AND H. A. SPOEHR Laboratory experiments on Chlorella culture at the Carnegie Institution of Washington, Department of Plant Biology. Carnegie Inst. Wash. Publ. 6, p HANNAN, P. J., AND C. PATOUILLET Gas exchange with mass cultures of algae. I. Effects of light intensity and rate of carbon dioxide input on oxygen production. Appl. Microbiol. 11: OSTERLIND, S The retarding effect of high concentrations of carbon dioxide and carbonate ions on the growth of a green alga. Physiol. Plantarum 1: RANSON, S. L., D. A. WALKER, AND I. D. CLARKE Effects of carbon dioxide on mitochondrial enymes from Ricinus. Biochem. J. 76: ROSENBERG, J. L Use of a glass electrode for measuring rapid changes in photosynthetic rates. J. Gen. Physiol. 37: STEEMANN NIELSEN, E Carbon dioxide as carbon source and narcotic in photosynthesis and growth of Chlorella pyrenoidosa. Physiol. Plantarum 8: STEER, B. T., AND D. A. WALKER Inhibition of chlorophyll synthesis by high concentrations of carbon dioxide. Plant Physiol. 4: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER Manometric techniques. Burgess Publishing Co., Minneapolis. 13. WALKER, D. A., AND J. M. A. BROWN Effects of carbon dioxide concentration on phosphoenolpyruvic carboxylase activity. Biochem. J. 67:79-83.