EXCRETION OF GLYCOLIC ACID BY ALGAE DURING PHOTOSYNTHESIS*

BY N. E. TOLBERT AND L. I’. ZILL

(From the Biology Division, Oak Ridge National Laboratory, 0a.k Ridge, Tennessee)

(Received for publication, February 10, 1956)

Although pure cultures of algae, in nutrient solution, have been used extensively in research on photosynthesis, the excretion of products into the medium has not been reported, despite their possible importance in the physiology and biochemistry of such cultures. The present investiga- tion with the Cl4 tracer has revealed a significant, and apparently functional, secretion of glycolic acid by Chlorella into the growth medium under aero- bic conditions, concurrent with COS uptake during photosynthesis. In nutrient solutions, below about pH 4.5, Chlorella loses other cell constitu- ents, particularly sucrose.

Procedures

Culturing and Labeling of Chlorellu with Cl-Chlorella pyrenuidosa (Emerson strain) was grown in 1 to 1.3 liters of the Knops nutrient solution in 4 liter, shake culture flasks through which flowed air containing about 4 per cent of COz. The algae received continuous illumination from a bank of red neon tubing supplemented by a small amount of blue fluores- cent light. Since these conditions produced no significant amount of heat, the cultures were shaken without water cooling in a room at about 21-23’. Rapid growth of the algae permitted the harvesting of 80 to 90 per cent of the culture each day, the yield from each flask being about 4 to 6 ml. of packed cells. The harvesting operation consisted in removing the algal suspension aseptically with the aid of a vacuum and replacing it with an equal volume of fresh nutrient solution. The algae were centrifuged at lo”, washed once in water, and again centrifuged in graduated tubes for 10 minutes at full speed in an International clinical centrifuge. The cells were resuspended in water, nutrient solution, or other designated medium, at a density of 1 ml. of packed cells per 100 ml. This culture was aerated and kept at 23” with exposure to at least 1000 foot-candles of light. For most experiments, 3 ml. aliquots of this final suspension were used, and no experiments were run with cultures that had been harvested longer than 5 hours.

* Work performed under contract No. W-7405-Eng-26 for the Atomic Energy Com- mission.

895

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896 EXCRETION OF GLYCOLIC ACID

The 3 ml. aliquot of algal suspension was placed in a flattened test tube, the sides of which were about 0.5 cm. apart so that the suspension was dis- tributed over about a 6 sq. cm. area. These tubes were mounted in a water bath at 23” and illuminated with at least 2000 foot-candles of light from each side by a reflector flood lamp. The cultures were allowed to come to a steady state condition during 30 minutes of exposure to air or to a designated concentration of COZ in air. At zero time, 25 PC. of (25 per cent C14) NaHC1403 in 25 ~1. of solution were injected into the suspen- sion. The tubes were closed and shaken during the Cl4 fixation period.

Separation of Algae from Culture Solution-The cells were separated from the medium on a Super-Cel (Johns-Manville, New York) column 2 to 3 cm. long and slightly less than 1 sq. cm. area. Retardation of flow rate by the formation of an algal layer on the column was prevented by mixing the cells with the upper layer of Super-Cel. Vacuum was applied to the column through two receiving flasks, and effluent could be directed into either receiver by a two-way stopcock. At the end of the Cl4 fixation pe- riod, the cell suspension was poured on the column, and the supernatant fluid and 2 to 3 volumes of wash water were collected in one receiving flask. The total time required for the separation of the cells from their medium was 5 to 10 seconds. Methanol at room temperature was immediately poured on the column in order to kill and extract the cells rapidly. Subse- quent alternate washings, first with methanol and then with water until no more Cl4 was eluted, were collected in the second receiving flask which thus contained the total Cl4 in the extractable constituents of the cell. When the column was essentially free of chlorophyll, extraction of Cl4 was complete. Boiling methanol gave results similar to those with metha- nol at room temperature. Bassham and Benson’ have also found that ChZoreZZu is killed as effectively and rapidly with cold methanol as with hot methanol or ethanol.

Analyses of Total Cl4 Fixed and Cl4 Products-The supernatant fluid from the cells and the soluble extract of the cells were adjusted with HCl t.o an acid pH (red to added methyl orange indicator) and aerated vigorously for 1 hour with C1202 to remove excess C1402. Aliquots of each were then counted for total fixed Cl4 after being adjusted with KOH to a yellow color with the indicator (about pH 6). Aliquots were also analyzed by paper chromatography, water-saturated phenol being used as the first solvent and butanol-propionic acid-water (1) as the second. Compounds were located by autoradiographs made with “no-screen” x-ray film, after which they were counted with a Geiger tube.

Chromatographic Analysis for Glycolic Acid-Although glycolic acid is

1 Bassham, J. A., and Benson, A. A., University of California, Berkeley, personal communication.

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N. E. TOLBERT AND L. I’. ZILL 897

not nearly so volatile as acetic acid, on paper chromatograms the free acid is lost rapidly enough to make its quantitative chromatography and auto- radiography uncertain. In previous chromatographic investigations on t.he function of glycolic acid in plant metabolism (a), autoradiographs were made as quickly as possible for a record, during which time an undeter- mined portion of t,he glycolic acid was lost from the paper. In fact, glycolic acid in the chromatogra,phic system used is unique in that no other acid has been detected at it,s RF value, and that a substantial amount of it dis- appears during a 2 months exposure to x-ray film. Column chromatog- raphy of organic acids on Cclite columns (3) also has been used t,o identify and isolate the glycolic acid.

TABLE I Loss of Glycolic Acid j’ronl Paper chromatograms

Chromatographic solvent j RF

I- ljutanol-propionic acid-water.. 0.58 Water-saturated butanol plus ethyl-

amine atmosphere 0.27

Uutanol saturated with 1.5 M NH,OH. 0.05 100 ml. of ethanol, 2 ml. of concen-

trated NH,OH. 0.24

Glycolate put on PaPer

C.).S.

60

156

123

145

Recovery after develo ment and radioautograp y E

per cent

60 after 2 wks.

76 “ 1 wk. 50 “ 6 nks. 71 “ 14 wks.

77 I‘ 1 wk.

Both glycolic-l-CL4 and glycolic-2-C’4 acids were used to study the ana- lytical procedures. Before use, these preparations were chromatographed on Celite columns to separate them from impurities that had accumulated by radiat.ion decomposition since their synthesis in 1948 (4). The recovery was only 33 per cent, the major cont.aminant,s being formic-V4 and oxalic- Cl4 acids.

In studies with tracer amounts of glycolic-Cl4 acid applied to paper chromatograms as salts, 40 f 10 per cent of the acid was lost during de- velopment of the chromatograms and the 2 week exposure period to the x-ray film (Table I). From 1 to 8 per cent of the acid was lost during development. The rest of the loss could be prevented by spraying the chromatograms with NaOH solution while they were drying.

Alkaline solvents were not satisfactory for paper chromatography for several reasons: (a) in solvents containing KaOH or KOH the organic acids and other constituents of the plant do not separate well, (b) solvents that contain ammonium hydroxide or ethylamine suffer the same disadvantage, and (c) in addition, glycolate-Cl4 is lost from such chromatograms (Table

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898 EXCRETION OF GLYCOLIC ACID

I). At the RF for glycolate, an acid reaction on the paper chromatogram we obtained with bromocresol green spray. The ammonium and ethyl- amine glycolate salts apparently hydrolyze on the paper chromatograms in the moisture from the air to give the volatile-free bases and the less volatile-free acid.

As a result of these difficulties, the paper chromatographic analyses of the whole cells gave only rough estimations of the amount of glycolic acid, based on corrected counts or visual inspection of the autoradiographs. Since the C?* activity in the media at pH values above 5.5 was all present as glycolate, counting of a neutralized aliquot gave accurate values for the total amount of this anion that was excreted; chromatography was used only to check for the presence of other compounds.

Results

General Observations of Phenomenon and IdentiJication of Glycolic Acid- A large amount of only one Cl*-labeled compound was present on chromato- grams of the supernatant media from Chlorella cells that had fixed Cl*02 near a neutral pH for a short period of time. The Rp values were those of glycolic acid, and part of the radioactivity sublimed off the paper chro- matograms within a few weeks. The radioactive material was cochro- matographed wit.h authentic glycolic acid in several solvents. The compound, without carrier, was extracted with ether from the algal culture fluid in sufficient quant,ities t,o giv‘e an acid reaction when chromat,ograms of it were sprayed with bromocresol green. The unknown gave a purple color test with 2,7-dihydroxynaphthalene in acid solution (5). It was eluted from H&O*-Celite columns, developed with chloroform-butanol, at the effluent volume where only this acid has been reported (3) and t’he eluent gave color tests for glycolic acid.

Chromatograms of the soluble extract from the Chlorella cells revealed the photosynthetic products reported by many other investigators. Glycolic acid is always present in the extracts of Chlorella as well as in extracts from higher plants (6). Under normal conditions, however, the supernatant solution from Chlore&z contains 10 to 100 times as much glycolate as the cells.

Separation and Determination of Glycolate from Mass Cultures of Chlorella -A 10 ml. aliquot of C%loreZZa culture was centrifuged and glycolate was determined in the supernatant fluid with the use of 2,7-dihydroxynaph- thalene (5). Nitrate ion in the culture medium was found to interfere with this test. It was therefore necessary to remove nitrate by passing the supernatant fluid, acidified with HCl to about 0.1 N, through a weak base anion exchanger (Dowex 3) in the hydroxide form and by washing the column with an equal volume of 0.1 N HCl. The amount of glycolate

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N. E. TOLBERT AND L. P. ZILL 899

varied from 3 to 8 mg. per liter of supernatant fluid from algal cultures 24 hours old. Cultures of algae 48 hours old contained only about 1 mg. of glycolate per liter.

Appreciable amounts of other acids were not found in the supernatant medium from actively growing ChZoreZZa cultures 1 to 3 days old. These experiments were run with algae labeled with NaHC140a over a period of several hours. The constituents in the medium were separated on Celite columns to establish the absence of volatile acids, such as formic or acetic acid.

E$ect of pH-The products and their amounts, excreted by Chlorella during the short-time CY402 fixation experiments, were markedly affected by the pH of the media (Table II; Fig. 1). At pH values above about 4.5, the radioactivity in the supernatant solution was present only in glycolate and varied from 5 to 10 per cent of the total soluble Cl4 fixed during a 10 minute photosynthesis test period. Since glycolic acid is a strong organic acid (pK, = 3.8), its secretion by the cells occurred at pH values at which it must exist as a glycolate salt. Although the total glycolate excreted decreased at pH values above 5.5 (Fig. l), the total fixation decreased pro- portionally. The amount of glycolate excreted into a medium of pH 5.5 or above represented a high and rather constant percentage of the total Cl4 fixed (Table II). As the acidity of the medium was increased from pH 4.5 to 2.5, the percentage of the fixed Cl4 excreted as glycolate decreased without an equivalent increase of glycolate inside the cells. The total Cl4 in the supernatant fluid increased markedly around pH 2.5. At the lower pH values, especially below 3.5, the increase in excreted CY4-labeled material was primarily sucrose, but there were also traces of other cell compounds, such as free sugars and amino acids. At these very acid con- ditions, the cells apparently lost the ability to retain their cytoplasmic components. Thus each curve of Fig. 1 represents two phenomena, one for glycolate excretion with a maximum at pH 5.5 and above, and one for loss of sucrose and other cellular compounds at the most acid conditions tested.

The total 04 fixed by the Chlorella increased at the non-physiological acid pH values. Similar results have been reported for Scenedesmus at high light intensities (7). At lower light intensities, the rate of Cl402 uptake by Chlorella also was more nearly constant at pH 4 to 8. The high fixation rates for the added NaHCY403 at the acid pH may be partially explained by the 30 minute preaeration before the test period, which would have removed much of the bound, unlabeled CO2 from the acidic solutions.

E$ect of Age of Algae-Chlorella cultures were harvested 1, 2, or 3 days after heavy inoculations. The amounts of Cl4 appearing in the super- natant fluid from 1 or 2 day-old cultures are shown in Fig. 1. 3 day-old

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900 EXCRETION OF GLYCOLIC ACID

cultures excreted even less CY4. The 1 day-old cultures were more active photosynthetically, and they also excreted a comparably larger amount of CP-labeled glycolate. The 2 day-old cultures and, particularly, the 3 day-old cultures fixed less CY402 and excreted less glycolate-C14. Thus

TABLE II Eflect of pH on Excretion of Glycolic Acid by Chlorella during Photosynthesis

pH of medium

2.5 3.5 4.4 5.5 6.8 8.0

C” fixed 04 in medium

Total In medium As glycolate As sucrose

6810 6953 5428

1270 987

6.2 30 3.8 65 3.5 85

12.9 98 9.6 95 9.8 106

70 35 15

Trace “

0

* By 3 ml. aliquot from 1 ml. of packed cells suspended in 100 ml. of water; 10 minute photosynthesis period. Cells were from 1 day-old cultures and were pre- aerated for 30 minutes with air before the Cr409 experiment; pH values are the aver- age at the beginning and end of the experiment which may have varied as much as +0.3 of a pH.

f By inspection of autoradiographs of the chromatograms.

6 I 3 5 7 9

PH

FIQ. 1. Effect of age of algal culture on amount of fixed Cl4 excreted into the me- dium. 0, 1 day-old culture; l , 2 day-old culture. Experimental conditions were the same aa described for Table II.

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N. E. TOLBERT AND L. P. ZILL 901

the amount of glycolate excreted may directly reflect the growth and photo- synthetic ability of the cultures.

E$ect of Duration of Photosynthesis-An increase in the amount of gly- colate-Cl4 excretion with longer experiments indicated that the glycolate was labeled after the photosynthetic carbon cycle. For Chlorella, after 2 to 5 minutes of photosynthesis with C40~, the specific activity of the sugar phosphate esters of the photosynthetic carbon cycle approaches constant values (8), but the increase in specific activities of related products is less rapid. From 2 to 5 per cent of the total soluble fixed Cl4 in the medium was excreted as glycolate after 2 minutes of photosynthesis and 3 to 10 per cent after 10 minutes. A great variation among experiments was al- ways encountered. In 30 minute experiments, a further increase in the percentage excreted generally occurred, but the trend was not consistent. Experiments were usually run for 2 to 10 minutes when other variables were being studied.

These data indicate that part of the newly fixed Cl4 moved through com- pounds of the photosynthetic carbon cycle into a glycolate pool that was being excreted. To substantiate this hypothesis, the Chbrellu was treated with C1402 for 2 minutes, which was sufficient time to label heavily the direct intermediates of the photosynthet,ic carbon cycle (Experiment 3, Table IV). The culture was then aerated for 30 minutes with 5 per cent unlabeled COz in air (Experiment 5, Table IV). As a result, the Cl4 was replaced by Cl2 while the Cl4 moved into the products derived from the photosynthetic cycle. Of the Cl4 fuced in the 2 minute period and still in the extractable portion of the culture, 13.6 per cent was in the glycolate of the supernatant fluid. In repetitions of this experiment, 30 to 40 per cent of the total soluble Cl4 fixed has been excreted as glycolate, though 12 to 15 per cent excretion was more common.

Eflect of Nutrient and Phosphate Concentration-Chlorella excreted the same amount of glycolate whether resuspended in glass-distilled water or in the Knops solution. The effect of the concentration of all the various components of the nutrient solution has not been investigated. The data in Table III indicate that, in phosphate at relatively high molarities, the rate of Cl402 fixation was not immediately affected, but the amount of glycolate excretion was diminished. A 200-fold increase in phosphate concentration decreased glycolate excretion by about 5-fold. Thus a very high phosphate concentration did not prevent glycolate excretion.

Glycolate Excretion in Presence and Absence of Oxygen and Bicarbonate -Excretion of glycolate by Chlorella depends on aerobic conditions, since it does not occur in a nitrogen atmosphere. The first two experiments in Table IV were identical, except in aeration conditions. In Experiment 1, the Chlorella culture was aerated for 10 minutes with a 99:l mixture of

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902 EXCRETION OF GLYCOLIC ACID

nitrogen and oxygen, and, in Experiment 2, only nitrogen (Linde-pure) was used. The total radioactivity fixed in the subsequent 5 minutes of photosynthesis in the presence of added h’aHC?QOJ was the same for both experiments, but glycolate excretion was markedly inhibited in the absence of added oxygen. Since the experiments were conducted in the light, some oxygen must have been present from the photosynthesis; therefore, com- plete inhibition of glycolate excretion could not be expected.

TABLE III Effect of Increasing Phosphate Concentration

Experimental condition’ Total 13’ fixed 0’ in medium

c.g.r. )LI cent

Knops nutrient solution (0.0015 M in KzHPO,). 10,326 5.4 0.01 M KtHPO, in water. 12,700 2.0 0.15 “ “ “ “ 13,910 1.9 0.3 “ “ (‘ “ _, 13,835 1.1

* 3 ml. aliquot of Chlorella suspension from 1 ml. of packed cells in 100 ml. of solu- tion; pH adjusted to 6.8; 10 minute photosynthesis period.

TABLE IV Efect of Oxygen and Carbon Dioxide

Experi- ment No. Conditions

1 10 min. aeration, NY02; 5 min. PS* 2 10 I‘ “ NI; 5 min. PS 3 30 “ “ air; 2 “ “ 4 (3) + 30 min. in light in N2-02 5 (3) + 30 “ ‘I “ “ 5% CO2 and air

Cl4 in medium Cl’-Glycolatc in cells

per cen1

4.0 Trace 0.5 Large 4.8 Trace 0.1 Very large

13.6 Trace

* PS represents photosynthetic fixation of NaIICr’03 under conditions described in the procedures.

The inhibition of glycolate excretion by partial anaerobiosis resulted in about the same amount of glycolate accumulation in the cell as would normally have been excreted under aerobic conditions (Experiment 2, Table IV). This was in marked contrast to the small quantity of glycolate normally found inside the cell during similar aerobic experiments.

Active bicarbonate uptake was an additional requirement for glycolate excretion. In Experiments 3 and 4 (Table IV), the Chlorella was permitted to fix NaHCr403 for 2 minutes in air, with the result that 4.3 per cent of the soluble Cl4 was found in the medium as glycolate. When similarly treated cells were kept in a COz-free atmosphere of nitrogen and oxygen

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N. E. TOLBERT AND L. P. ZILL 903

(99: l), with shaking to prevent clumping, the glycolate was reabsorbed from the medium and accumulated inside the cells in large amounts. After 30 minutes of aeration wit,h nitrogen-oxygen in the light, the major soluble products inside the cells were glycolate and sucrose. If the cells were again allowed to fix bicarbonate, the glycolate was quickly excreted. This accumulation of glycolate inside the cells, in the absence of bicarbonate but under aerobic conditions, indicates that bicarbonate must exert the main regulatory effect on the glycolate pool. Conversion of glycolate to glyoxylate and then to glycine, under these conditions, would not be a limiting factor. These experiments indicate a rapid and reversible move- ment of glycolate between the algal cells and their nutrient solution.

The rapid excretion of glycolate by Chlorella is dependent on (a) the presence of bicarbonate, (b) aerobic conditions, (c) light for active photo- synthesis, since there must be a net bicarbonate uptake, and (d) the age of the cells, excretion being greatest in the youngest cultures. The glyco- late did not accumulate in the culture medium to more than 3 to 8 mg. per liter. It is reabsorbed by the Chlorella when one or more of the stated conditions for its excretion are not met.

The presence of glycolic acid in media that support growth of several species of Chlamydomonas has also been observed (9).2 Lewin’s data indi- cate that certain Chlamydomonas species have approximately twice as much glycolate in their culture medium as ChZoreZZu. In preliminary ex- periments with some aquatic plants and roots, excretion of glycolate has not been demonstrated.

The glycolate excretion and absorption may represent a glycolate-bi- carbonate anionic exchange across the Chlorella cell wall without the neces- sity of a similar cationic shift. Since bicarbonate uptake during photosyn- thesis and excretion during respiration represent a major ionic movement, a diffusion of some other anion in the opposite direction would lessen an un- necessary loss or absorption of cations. If a Donnan equilibrium exists across the cell membrane, a change in bicarbonate ion concentration should result in an opposite movement of glycolate ion to help restore the equal- ity.

The rapid excretion of glycolate-Cl4 during active bicarbonate uptake of photosynthesis indicates that this anion is uniquely able to respond quickly to the upset of equilibrium when HCOa- in the cell suddenly diminishes in

2 Lewin, R. A., personal communication.

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904 EXCRETION OF GLYCOLIC ACID

concentration and moves in from the medium. However, glycolate does not accumulate in the medium beyond a few mg. per liter. If bicarbonate fixation ceases, the glycolat,e is quickly reabsorbed by the cells. The reab- sorption during steady state photosynthesis, rather than cont,inued a?- cumulation in the medium, would be favored by a high concentration gradient of glycolate outside the cell. However, it may be necessary t.hat energy be expended by the cell in order to reabsorb this anion. In the dark, reabsorption of glycolate represents a shift in the opposit,e direction for the Domlan equilibrium, and the bicarbonate arises from respiration in the cell and moves into the medium.

Several properties of glycolate indicate its unique properties for partici- pation in anionic exchange with bicarbonate. It is a small organic acid that conserves carbon, yet is one of the strongest acids associated with the cell. In plants, it is readily available from the photosynthetic carbon cycle. In this respect, it is believed to be formed on a side oxidation pathway from the photosynthetic carbon cycle, starting from a Cp complex of the oxida- tion level of glycolaldehyde or phosphoglycolaldehyde from sugar phos- phates. This has been indicated by the same distribut,ion of its Cl4 label during short-time photosynthesis experiments as in the or and fl carbons of phosphoglyceric acid (lo), by the activation of the pathway for glycolic acid metabolism to glycine in greening plants only after the establishment of the photosynthebic carbon cycle (11, 12), by the enzymatic studies i?z V&O now in progress (13, 14), and by the ea,se of air oxidation of ribulose diphosphate to phosphoglycolic acid (15).

The consequences of the excretion of glycolate during phot.osynthesis and its reabsorption and accumulation inside Chlorella during dark respira- tion have not yet been evaluated. This bicarbonate-glycolate shift may be involved in acid or COZ bursts, pH shifts, or quant,um efficiency as calcu- lated from short-time or flashing light photosynthesis experiments.

Metabolism studies indicate that glycolate-Cl4 in Chlorella and higher plants is utilized readily, particularly as a precursor for glycine and serine (16, 2). Thus glycolate reabsorption and accumulation in large amounts inside the cell after a period of active photosynthesis should not persist. This reasoning might partially explain extended induction effects in photo- synthesis after periods of darkness longer than 1 hour. Little glycolate would be left in the cell to exchange for the bicarbonate ion. When the algae are placed in the light, a photosynthetic induction period would ensue while the glycolate pool was replenished by a slow rate of photosynthesis, or by the reduction of glyoxylic acid arising from glycine (17) or from iso- citrate (18, 19).

Studies on glycolic-Cl4 acid metabolism have also shown that this acid was readily absorbed from solutions at a pH near 2.5, or below its pK,,

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N. E. TOLBERT AND L. P. ZILL 905

but t,hat it was not absorbed as a sodium or potassium salt. This was observed for Chlorella by Schou et al. (16) and by Myers (20) ; it has been observed also for etiolated or green barley and wheat leaves (11). How- ever, in the present experiments under anaerobic conditions or the absence of COs, glycolate was absorbed easily from solutions of pH 5 to 9, but was not readily metabolized. In the previous experiments (11, 16, ZO), since the authors could not determine whether the glycolate was inside or out- side the cells, it has been assumed that glycolate was not absorbed from neu- tral or alkaline solutions. The present data show that, at pH 5 to 9, gly- colate accumulates inside the Chlorella cell at a predominant rate over its metabolism.

Another pH effect, which may be fortuitous, is that, above pH -5.5, the percentage of the total Cl402 fixed and subsequently excreted as glyco- late remains constant. This is the pH range above which bicarbonate ion exists in solution.

SUMMARY

During 2 to 30 minute, steady state photosynthesis experiments with Chlorella, 3 to 10 per cent of the total Cl402 fixed was excreted into the algal medium as glycolate. -4bove pH w-4.5 in the medium, glycolate was the only product excreted, though at lower pH values many other cell constituents were lost. At pH above 5.5, the percentage of the Ci402 fixed and subsequently excreted as glycolate remained about constant. Alternating exposure to Cl402 and Ci202 showed that glycolate was pro- duced from the photosynthetic carbon cycle.

Glycolate excretion into the supernatant fluid was dependent on (a) light for photosynthesis, (b) aerobic condit,ions, and (c) the presence of the bicarbonate anion. When any one of these conditions was not met, the glycolate moved from the medium into the cells, where it accumulated in relatively large amounts. Even in continuous photosynthesis, however, accumulation of glycolate in the medium did not exceed a few mg. per liter.

This phenomenon suggest’s a Donnan equilibrium for a bicarbonate- glycolate shift to facilitate rapid bicarbonate movement. The shift is characteristic of glycolate for Chlorella, since no other organic acid showed comparable movements. The possible effect of this equilibrium on induc- tion periods and quantum efficiency calculations has been discussed.

The authors acknowledge the competent assistance of Miss Pat Kerr during these investigations.

BIBLIOGRAPHY

1. Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. C., Haas, V. A., and Stepka, W., J. Am. Chem. Sot., 73, 1710 (1950).

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2. Tolbert, N. E., and Cohan, M. S., J. Biol. Chem., 204, 649 (1953). 3. Phares, E. F., Mosbach, E. H., Denison, F. W., Jr., and Carson, S. F., Anal.

Chem., 24, 660 (1952). 4. Hughes, D. M., Ostwald, R., and Tolbert, B. hf., J. Am. Chem. Sot., 74, 2434

(1952). 5. Calkin, V. I’., Ind. and Eng. Chem., Anal. Ed., 16,762 (1943). 6. Benson, A. A., and Calvin, M., J. Exp. Bot., 1,63 (1950). 7. Ouellet, C., and Benson, A. A., J. Exp. Bot., 3, 237 (1952). 8. Benson, A. A., Bassham, J. A., Calvin, M., Hall, A. G., Hirsch, H. E., Bawaguchi,

S., Lynch, V., and Tolbert, N. E., J. Biol. Chem., 196, 703 (1952). 9. Allen, M. B., Plant Physiol., 30, suppl. 38 (1955).

10. Calvin, M., Bassham, J. A., Benson, A. A., Lynch, V. H., Ouellet, C., Schou, L., Stepka, W., and Tolbert, S. E., Symposia Sot. Exp. Biol., 6, 284 (1951).

11. Tolbert, 5. E., and Cohan, M. S., J. Biol. Chem., 204, 639 (1953). 12. Tolbert, N. E., and Gailey, F. B., Plant Physiol., 30, 491 (1955). 13. Weissbach, A., and Horecker, B. L., in McElroy, W. D., and Glass, B., Amino

acid metabolism, Baltimore (1955). 14. Gibbs, M., Plant Physiol., 39, suppl. 19 (1955). 16. Goodman, M., Benson, A. A., and Calvin, M., J. Am. Chem. Sot., 77,4257 (1955). 16. Schou, L., Benson, A. A., Bassham, J. A., and Calvin, M., Physiol. Plantarum, 3.

487 (1950). 17. Zelitch, I., J. Biol. Chem., 216, 553 (1955). 18. Smith, R. A., and Gunsalus, I. C., J. Am. Chem. Sot., 76, 5002 (1954). 19. Olson, J. A., Nature, 174, 695 (1954). 20. Myers, J., J. Gen. Physiol., 30, 217 (1946).

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N. E. Tolbert and L. P. ZillALGAE DURING PHOTOSYNTHESISEXCRETION OF GLYCOLIC ACID BY

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