Planta (Berl.) 74, 278--285 (1967)

Glycolate Biosynthesis by Scenedesmus and Chlorella in the Presence or Absence of NaHCO~* JOHN L. HESS, N. E. TOLBEIaT and LEE M. PIKE** Department of Biochemistry, Michigan State University, East Lansing, Michigan Received November 23, 1966

Summary. Both Scenedesmus and Chlorella excreted comparable quantities of glycolate. G]ycolate formation was dependent upon light and oxygen, but occured in the absence of added CO2 or NaHC03 for net photosynthesis. In an environment of 3000 ft. c. light and an atmosphere of oxygen, about 35 ~tg glycolate were excreted per hour per milliliter 1% (v/v) algae without NaHCO s or CO2. Upon addition of NaHCO 3 the rate increased to about 55 ~g. Glycolate formation in the light in the absence of COS may result from photometabolism of algal polysaccharides. Glycolate excretion by Scenedesmus occurred at all pH values between 6.5 and 9.5 and was not related to utilization of bicarbonate. Scenedesmus obliquus excreted glycolate when existing in plates of four or eight cells, but not when present as small individual cells. At pH 914C fixation by Scenedesmus was faster than fixation by Chlorella. There was no significant difference in products of 14C fixation formed by Scenedesmus at pH values between 6.5 and 9.5. For unknown reasons ~-hydroxy-2-pyridinemethanesulfonate stimulated CO2 fixation by Scenedesmus by at least 100%. This sulfonate had no effect on glycolate excretion nor upon the distribution of laC among the products of laC02 fixation by Scenedesmus.

Introduction

(~STElCLIND(1952)

observed t h a t the green algae Chlorella pyrenoidosa was u n a b l e to utilize b i c a r b o n a t e directly, b u t t h a t Scenedesmus quadricauda did utilize bicarbonate. S~E~,MAN NIELSEN (1960) a t t r i b u t e d this difference to a " q u a n t i t a t i v e difference i n p e r m e a b i l i t y of the plasma m e m b r a n e s for t t C O s - i o n s " , b u t suggested t h a t the i n a b i l i t y of Chlorella to utilize b i c a r b o n a t e m a y n o t be absolute (STEEMAN NIV,ZSEN, 1966). The capacity of various strains of algae to ntilize CO 2 or b i c a r b o n a t e d u r i n g photosynthesis has n o t been i n v e s t i g a t e d at the biochemical level. I n the initial report on glycolate excretion b y algae, TOLB~,RT a n d ZILL (1956) p o s t u l a t e d t h a t glycolate excretion m i g h t be related to bi* Supported in part by NSF Grant GB-4154 and published with the approval of the Director of the Michigan Agricultural Experiment Station as journal article No. 3946. The research was initiated during the period when N. E. TOLBE~T was supported in part by a National Institutes of Health Senior Fellowship at the Biochemisches Institut, Universiti~t, Freiburg/Br., Germany. ** NSF undergraduate research participant.

Glycolate Biosynthesis by Scenedesmus and Chlorella

279

carbonate u p t a k e . This hypothesis was s u p p o r t e d b y the c o n t i n u e d p r o d u c t i o n of glycolate b y Chlorella a t alkaline p H (ORT~ et al., 1966). I n the present report, 14CO2 fixation rates a n d products a n d glycolate excretion b y Scenedesmus are compared with similar d a t a from Chlorella in order to evaluate a n y differences i n these algae which m i g h t explain their differential abilities to utilize bicarbonate. Since glyeolate product i o n b y Chlorella is greatly e n h a n c e d b y high light i n t e n s i t y a n d high oxygen p a r t i a l pressure (ToLBE~T, a n d ZILL, 1956; TOLS~nT, 1963; BASStIAM a n d K I ~ x , 1962; WI~ITrI~GHA~ a n d PI~ITC~A~D, 1963), these e n v i r o n m e n t a l conditions were used in most of the experiments.

Experimental Procedures and Techniques Algae, obtained from the Culture Collection at Indiana University, Bloomington, Indiana, were Scenedesmus obliquus (GAFrRO•) No. 393 and Sc. quadricauda No. 77. Chlorella pyrenoidosa (WA~BURG)was obtained from M. STILLER, Purdue University. The Scenedesmus cultures were grown in sterile inorganic medium V of NORRISet al. (1955); the Chlorella were grown in WAR~V:aG'sK medium as modified by S~ILLE~(1966). Cultures were aerated with 0.2--0.5 % CO2in air at a constant temperature of 200 and a light intensity of 1000 ft.c. from Sylvania Grolux fluorescent lamps. The cultures were shaken in low form 2800 ml Fel~abach flasks at approximately 60 cycles per minute. The algae were harvested each day and about 250 ml were diluted to i liter for continuation of the culture. Harvesting was done at different times in order to avoid any possible endogenous rhythm. These culture procedures were followed in order to maintain random cultures. Cell volume was determined by centrifuging at full speed in a clinical centrifuge for 5 minutes, and a 1% (v/v) resuspension was made in water or designated buffer. At the end of the experiments ceils were removed by centrifugation or by rapid filtration on millipore filters. The supernates were assayed colorimetrically for glycolate (CALKI~S, 1943). l~C02 photosynthetic fixation experiments were run at pH 7, 9 and 11 on 20 ml of 1% (v/v) suspensions of cells in the absence or presence of e-hydroxy-2-pyridinemethanesulfonate as previously described (ToL~ERT and H~ss, 1966). After a 5-min. temperature equilibration with the sulfonate in the light and aeration with air, a solution of NaH14COs (2 ~zmoles) was added and the suspension shaken vigorously. Samples taken at 0, i0, 30, 60 and 120 sec. after the addition of NaH1r were drained into methanol and heated. Excess 14C02was removed by aeration with ~CO 2 in the presence of excess acetic acid. Aliquots were counted in a scintillation counter for total ~4C fixed per ml of algal suspension, and distribution of 14C among products was determined by two dimensional chromatography and radioautography (BE~sON et al., 1950) of the methanol-water soluble fraction.

Results 1. Glycolate Biosynthesis. B o t h Seenedesmus a n d Chlorella excreted comparable q u a n t i t i e s of glycolate in a n e n v i r o n m e n t of high light i n t e n s i t y a n d a n oxygen atmosphere (Fig. 1, a n d Table). Glycolate a c c u m u l a t i o n in a n algal suspension in 0.001 M phosphate w i t h o u t CO~ or N a H C O 3 d e m o n s t r a t e d t h a t glyeolate excretion can occur in the light even i n the absence of a n e x t e r n a l carbon source for n e t photosynthesis. Glycolate excretion in light a n d oxygen was f u r t h e r e n h a n c e d b y NaHCOa. 19"

280

J.L.

HESS,

N. E. TOLBERT and L. M. PIKE:

Glycolate excretion in light a n d air was significant b u t less t h a n in an o x y g e n a t m o s p h e r e (Table). Thus for glyeolate excretion, light was necessary; an o x y g e n a t m o s p h e r e a n d CO 2 s t i m u l a t e d t h e a m o u n t of glycolate p r o d u c t i o n , b u t t h e presence of exogenous N a H C 0 8 or C02 was n o t necessary. These results have p r e v i o u s l y been o b s e r v e d w i t h Chlorella (for review see TOLBEI~T, 1 9 6 3 ) e x c e p t t h a t glyeolate p r o d u c t i o n in t h e absence of a d d e d CO 2 h a d n o t been recognized. F r e e z i n g Chlorella a t - - 180 for 10 to 20 m i n u t e s d e s t r o y e d t h e i r a b i l i t y to form glycolate (Table). I n these e x p e r i m e n t s the frozen algal suspension (1% v/v) was L

SCENEDESMUS

OBLIQUUS

CHLORELLA

PYRENOIDOSA

z 8C o ~c

(,9 ~sc

B[CARBONATE BICARBONATE

c3' 5c -~ 4c

f

LtJ ~c (D >, 2c (.9

15

30

MINUTES

6'0

9'0

[5

3'0

MINUTES

6'0

90

Fig. 1. Glycolate formation and excretion in the presence or absence of 17aIIC03. A 1% suspension of cells were prepared in either 0.01 M bicarbonate or 0.001 ~ phosphate and exposed to 3,000 ft.-c, of light while being aerated with oxygen. Open symbols represent algae in either becarbonate or phosphate, as designated; closed symbols represent experiments containing also 0.001 M u-hydroxy-2pyridinemethanesulfonate

Table. _Formation o/glycolate by Chlorella pyrenoidosa (WAlCBUI~G)during one hour Experimental conditions

Dark; air or oxygen; phosphate buffer or 0.02 M NaHC03 Light; air; 0.02 M NaHCO 3 Light; oxygen; 0.02 M NaHCO s Light; oxygen; No NaHCO 3 Frozen algae; light; oxygen; with or without 0.02 M NaCO 3

~g glyeolate/hr/ml 2 % algae 0 11 75 55 0*

* A value of 1.5 tzg was obtained by the eolorimetric asssay but the absorption spectrum was different from that for authentic glyeolate.

Glycolate Biosynthesis by Scenedesmus and ChloreUa

281

thawed and used similarly to unfrozen cells. In the cells which had been frozen, cell carbohydrate reserves for glycolate biosynthesis ought to have been present, but addition of excess sugars or sugar phosphates also did not result in glycolate formation. The absence of glycolate formation by whole cells which had been briefly frozen illustrates the lability of the process for glyeo]ate biosynthesis. Rates as high as 35 ~g glyeolate excretion per hour per ml of 1% algal suspension have been observed with Scenedesmus and Chlorella pyrenoidosa (WARBU•G) without added COs or NaIICOa. The dry weight of the algae was determined experimentally to be 20% of their wet weight. Since 35 ~zg of glycolate is equal to 0.5 ~moles of glyeolate or 1 ~mole of carbon, the algae were excreting 0.5 ~mole of carbon per hour per mg dry weight. This value is the same as rates of CO s production during dark respiration. THOMAS (1965) states that 7 to 18 txmoles of CO2 are photosynthetically fixed per hour per mg dry weight of Chlorella and that respiration rates are about 1/25 of photosynthetic rates of 0.3 to 0.7 ~moles COs per hr per mg dry weight. Glycolate production in the absence of added CO s or NaHCOa may be attributed either to its direct production as the end product of a photometabo]ism process, or to complete photosynthetic fixation of CO s formed internally during respiration. In another investigation we have shown that in a]l of these algae, a typical glycolate oxidase cannot be detected (H~ss and TOLBERT, 1967), and thus glyeolate excretion seems to occur as an obligate end product of "photometabolism." We have not been able to quantitate glyeolate excretion in the light in the absence of CO s with carbohydrate utilization. In fact, in initial experiments, total methanol-soluble reducing sugars of the algal cells,, as determined colorimetrically, also increased in the cells when glycolate was being excreted in the light without CO s. Thus photometabolism in light and oxygen may originate at the level of polysaceharide hydrolysis which leads to sugars and ultimately to glycolate formation. High levels of photometabolism or photorespiration as measured by oxygen uptake would be explained in higher plants (Fon~STER et al., 1966; GOLDSWOrThY, 1966; ZELITC~, 1959) by the presence of ample glycolate oxidase to metabolize the glycolate. In algae the absence of this enzyme would result in low or unchanged levels of respiration even though light may catalyze a similar photometabolism for polysaccharide breakdown. Thus in algae photometabolism may result in glycolate production rather than oxygen uptake and COs release as reported in higher plants. Nearly equal glyeolate excretion occurred by algae which could utilize bicarbonate, e.g., Scenedesmus, as well as by algae which did not rapidly use bicarbonate, e.g. Chlorella. No direct correlation between glycolate excretion and bicarbonate utilization was apparent.

282

J.L. HEss, N. E. TOLBERTand L. M. PIKE:

WARBU~G and KRIPPAHL (1960) have shown that in their experimental conditions, glyeolate production could account for 90% of the total CO s fixed photosynthetically. However, the formation of glycolate in the light in the absence of NaHCO a indicate that it m a y be misleading to equate moles of CO s fixation to moles of glycolate formation. Synchronized cultures of Scenedesmus obliquus excreted glycolate only if plates of four or eight cells were present. Little glycolate appeared in the supernatant if small individual cells from the log phase of growth represented 85--90 % of the population. Sc. quadricauda, on the other hand, never produced large populations of individual cells, since they separate singly and in a random manner from their parent plates of four or eight cells. Thus with the latter algae we could not determine which phase of cell development was related to glycolate excretion. In most experiments randomized cultures of both algae were used in order to obtain good glycolate excretion. Certainly further investigations of glycolate excretion by synchronized algae are required. 2. COs Fixation Rates and Products. The p H value of Seenedesmus suspensions in bicarbonate increased at least one p H unit during a 90 minute experiment. When phosphate buffer was present an increase was not detected. The increasing p H in bicarbonate can be attributed to the uptake of CO s during photosynthesis and the accumulation of N a O H in the medium. The p H increase occured independently of the amount of glycolate which was being excreted. Total 14C02 fixed in 2 minutes by either strain of Scenedesmus decreased with increasing p H (Fig. 2). The fixation at p H 9 was approximately half that at p H 7. However with Chlamydomonas (O~T~ et al., 1966) and Chlorella (JIMI~EZ, 1962; TOLBERT and HEss, 1966), the total laC02 fixed at p H 8 to 9 was only 10 to 12% of the fixation rate at p H 6 to 7. This greater drop at higher p t I in total fixation rate for Chlamydomonas and Chlorella has been attributed to poor utilization of NaHCO a. The utilization of NaHCO 8 by Scenedesmus at p i t 9 to 10 has previously been reported (0STERLIND, 1950). The low fixation at p H l I has been attributed to the inability of algae to use CO a ions (STE~MA~ NIeLSEn, 1960). The products of 14CO2 fixation b y both strains of Scenedesmus were not significanthy altered at the different p H values and neither strain produced more glycolate at the higher pH. About 3 % of the total 14C fixed in 1 minute was incorporated into glycolate, 1% in glycine and 2 % into serine and in 2 minutes 14C incorporating into glycine and serine had doubled. These results, showing no change in glycolate-14C production between p H 7 and 11, are markedly different from those with Chlamydomonas and Chlorella (ORTH et al., 1966). With the latter algae at p H 8 or above glycolate-14C labeling dominated as percent of the 1~C fixed even

Glycolate Biosynthesis by Scenedesmus and Chlorella I

I

283

I

25

.=Z 9~ ,

2o

0_

~x E_

u5

b io x

u I---

C

I

I

I

7

9

II

pH

Fig. 2. Effect of p I I on rate of photosynthesis b y Scenede.~mus. Tote1 '~CO~ fixed in 2 m i n u t e s d a z i n g photosynthesis in 3,500 ft.-c, light b y Scenedesmus quadricaude (squares) or Scenedesmus obliquus (circles) in 0,001 M phosphate IS

25

I I I I CENEDESMUS OBLIOUUS/e /

I I I I CENEDESMUS OUADRICAUD

"~ 2O ,.=, a. ,.9 x

15

u

p-

20 SECONDS

60

120

Fig. 3. Effect of ~-hydroxy-2-pyridinemethanesulfonate on rate of photosynthesis b y Scenedesmus. Total ~4CO~ fixed during photosynthesis a t p t I 7 in 3,500 ft.-c, light in 0.001 1~ phosphate w i t h 0.01 IV[ sulfonate (squares), w i t h 0.001 M sulfonate (closed circles), or w i t h no sulfonate (open circles)

t h o u g h total fixation was so m u c h less t h a t total glycolate- 14 C production did not increase.

284

J.L. H~ss, N. E. TOLBEI~Tand L. M. PIKe:

3. E]/ects o/~-Hydroxy-2-pyridinemethane-Sul/onate. In higher plants this sulfonate inhibits glycolate oxidase and causes the accumulation of glycolate during photosynthesis (Z~LITCH, 1959). However, this sulfonate did not effect glycolate production by Chlorella or Chlamydomonas (ToL]~ERT and HESS, 1966), presumably because the algae do not contain glycolate oxidase (It~ss and TOLBV,~% 1967). TOLBERT and HEss (1966) also reported that ~-hydroxy-2-pyridinemethanesulfonate stimulated the rate o f 14C fixation by Chlorella and Chlamydomonas at p H 8.3 about sixfold; the sulfonate-stimulated rate at pH 8.3 approximated tile normal rate at pH 6. Similar experiments on the addition of the sulfonate to Scenedesmus showed that the total 14C fixation was also markedly stimulated even at pH 7 (Fig. 3). The reason for this stimulation for both types of algae is unknown. The stimulation of total 1~CO2 fixation by the sulfonate was not observed with suspensions of Sc. quadricauda which excreted glycolate at rates of less than 5 ~g per ml of 1% (v/v) algae in 90 minutes. However, if the Scenedesmus were able to excrete glycolate, the sulfonate only slightly inhibited this glyeolate excretion (Fig. 1) while stimulating total 1~CO2fixation (Fig. 3). For Chlorella and Chlamydomonas the sulfonate also resulted in a marked inhibition of 14C incorporation into cellular amino acids and a great increase in cellular laC-labeled sugar diphosphates (ToLBEnT and HEss, 1966). These latter effects of the sulfonate on Chlamydomonas and Chlorella metabolism were not observed with Scenedesmus. For Scenedesmus the stimulation of 14CO2 fixation by the sulfonate occurred without any detectable alteration in the distribution of laC-]abeled products. Professor H~LMUT HOLZER (University, Freiburg/Br., Germany) facilitated the initiation of the research in Germany. Competent technical assistance of A. OESER expedited the research.

Bibliography BASSJ~AM, J.A., and M. KXaK: The effect of oxygen on the reduction of CO2 to glycolic acid and other products during photosynthesis by ChlorelIa. Biochem. biophys, t~es. Commun, 9, 376--380 (1962). BENsosr A.A., J. A. BAss]~?a, M. CALVIN,T. C. GOO])ALE,V. A. ttASS, and W. STE~:A: The path of carbon in photosynthesis. V. Paper chromatography and radioautography of the products. J. Amer. chem. Soc. 72, 1710--1718 (1950). CALKI~S, V.P.: Micro-determination of glycolic and oxalic acids. Industr. eng. Chem., Anal. Ed. 15, 762--763 (1943). FOR~STWI~ M. L., G. K~OT~:OV, and C. D. NELSON: Effect of oxygen on photosynthesis, photorespiration and respiration in detached leaves. I. Soybean. Plant Physiol. 41, 422-427 (1966). GOLDSWO~Tn-g,A. : Experiments on the origin of CO~ released by tobacco leaf segments in the light. Phytochemistry g, 1013--1019 (1966). H~ss, J. L., and N. E. TOL~ERT:Glycolate pathway in algae. Plant Physiol. 42, (in press) (1967).

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JIMI~EZ, E.: Studies on Photosynthesis. 11. Bicarbonate utilization by washed algae. Doct. Diss., Michigan State University, East Lansing, Michigan (1962). NORRIS, L. R., R. E. Nom~IS, and M. CALvin: A survey of the rates and products of short-term photosynthesis in plants of nine phyla. J. exp. Bot. 6, 67--74 (1955). 0STE~LI]~]), S.: Inorganic carbon sources of green algae. 1. Growth experiments with Scenedesmus guadricauda and CHorella pyrenoidosa. Physiol. Plantarum (Kbh.) 3, 353--360 (1950). Inorganic carbon sources of green algae. VI. Further experiments concerning photoactivation of bicarbonate assimilation. Physiol. Plantarum (Kbh.) 5, 4 0 3 ~ 0 8 (1952). ORTU, G. M., N. E. TOLBERW,and E. JI~INEZ: Rate of glycolate formation during photosynthesis at high pH. Plant Physiol. 41, 143--147 (1966). STEE~• NIELSEN, E. : Uptake of CO2 by the plant. Encyclopedia of plant physiology (W.R~mA~D, ed.), vol. I, p. 70--84. Berlin-GSttingen-Heidelberg: Springer 1960. - - The uptake of free CO2 and HC03 during photosynthesis of plankton algae with special reference to the Coccolihophorid coccolithus huxbyl. Physiol. Plantarum (Kbh.) 19, 232--240 (1966). STILES, M. : Hydrogenase mediated nitrite reduction in Chlorella. Plant Physiol. 41, 348--352 (1966). Tgo~As, M. D. : Photosynthesis (carbon assimilation) : Environmentaland metabolic relationships. In: Plant physiology, a treatise (F. C. STEWARD,ed.), vol. IV/A, p. 9--183. New York: Academic Press 1965. TOLBE~% N. E. : Glyeolate pathway. In: Photosynthetic mechanism in green plants, p. 648--662. Nat. Sci. Foundation - - Nat. Res. Council Publ. No. 1145 (1963). - - , and J. L. HESS: The effect of e-hydroxymethanesulfonates on ~4C0~ photo synthesis by algae. J. biol. Chem. ~41, 5707--5711 (1966). --, and L. P. ZILL: Excretion of glyco]ic acid by algae during photosynthesis, g. biol. Chem. 22~, 895--906 (1956). WA~U~G, 0., and G. KRIPPAttL: Glykolsgurebildung in Chlorella. Z. Naturforsch. 15b, 197--199 (1960). WI:tlTTINGHAM,C. P., and G. G. PRITCttARD: Production of glycolate during photosynthesis in Chlorella. Proe. roy. Soe. B 157, 366--382 (1963). Z~LI~c~, I. : The relationship of glycolic acid to respiration and photosynthesis in tobacco leaves. J. biol. Chem. 234, 3077--3081 (1959). -

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N. E. TOLBERT Department of Biochemistry Michigan State University East Lansing, Mich. 48823, USA