Planta (Berl.) 91,220--226 (1970)
Hydrogen Evolution by Several Algae F. P. H~,~m~,Y Scripps Institution of Oceanography, University of California, La Jolla l~eceived October 15, 1969/January 5, 1970
Summary. Out of 33 strains of unicellular algae examined, H a evolution was observed only in species of Chlamydomonas, Chlorella and Scenedcsmus. While the photoevolution of H e by these algae was generally stimulated both by an organic substrate and by the uncoupler CCCP1, response to DCMU1 varied. On the basis of the response to DCM-U, it was concluded that the mechanism of photoevolution of H 2 differed from one alga to another. The reaction in some algae appeared to be dependent on either the photooxidation of water or oxidative carbon metabolism for reductant; that in other algae was supported by reduetant from both these sources. Introduction There is evidence for the occurrence of hydrogenase in a variety of algae, including several green algae (Gaffron, 1940; Frenkel, 1952; Kessler and Maifarth, 1960), some blue-green algae (Frenkel, 1949; Hattori, 1963), the red alga, Porphyridium cruentum, and some seaweeds (Frenkel and Rieger, 1951). However, H~ evolution has been observed only in members of the genera Scenedesmus (Gaffron and Rubin, 1942), Chlamydomonas (Frenkel, 1952), Chlorella (Spruit, 1954) and Ankistrodesmus (Kessler, 1962). There appear to be two mechanisms of photoevolution of H2 among algae. One involves the photooxidation of water and electron transport through both photosystems of photosynthetic electron transport to the release of H 2. I t is characterized b y the simultaneous release of H2 and 02, sensitivity to DCMU, Emerson enhancement, and insensitivity to respiratory inhibitors (Spruit, 1954, 1958; Bishop and Gaffron, 1963). The other involves the movement of reductant from oxidative carbon metabolism through only photosystem I of the photosynthetic electron transport system to the release of H 2. I t is characterized b y the simultaneous release of I t 2 and CO2, insensitivity to DCMU, lack of enhancement, stimulation b y substrates, and inhibition b y respiratory inhibitors 1 CCCP: earbonyleyanide-m-chlorophenylhydrazone; phenyl)-l,l-dimethylurea.
DCMU: 3-(3,4-dichloro-
H 2 Evolution by Algae
221
(Frenkel, 1952 ; Kessler, 1962 ; K a l t w a s s e r a n d Gaffron, 1964; Healey, in press). A s u r v e y of several unicellular algae was u n d e r t a k e n to e x a m i n e more fully the d i s t r i b u t i o n of H 2 evolution a m o n g algae a n d to d e t e r m i n e the c o n t r i b u t i o n of the above two m e c h a n i s m s to p h o t o e v o l u t i o n of H 2 a m o n g algae.
Materials and Methods Stock cultures of the algae were maintained by weekly transfers in liquid medium on a rotary shaker. Experimental cultures, inoculated from the stock cultures, were grown in i-Liter Erlenmeyer flaks gently shaken on a wrist-action shaker and bubbled with air. The temperature was mMntained at 20~ The cultures received light (8000 lux) from cool-white fluorescent tubes placed beneath them. The cultures were harvested in late exponential phase. Except for Chroomonas diplococcus, t~hodosorus marinus, and several of the diatom strains, all cultures were axenie. Stock and experimental cultures of axenie strains were checked for bacterial contamination by inoculating 5 ml of medium containing 0.1% tryptone and 0.1% glucose (w/v) with 1 ml of culture. Freshwater algae were grown in medium FW-2 (Healey, in press), with tile exception of Euglena gracilis, which was grown in the medium of Cramer and Myers (1952). Marine algae were grown in medium LF-3, based on the artificial sea water of Lyman and Fleming (1940). The composition of Medium LF-3 was as follows (in g unless otherwise noted): NaC1, I8.8; MgCI2 9 6H20, 5.7; Na2SO4, 3.1; KC1, 0.56; CaC1292H20, 0.40; NaI-ICOa, 0.15; KBr, 0.077; KNOs, 1.01; K2HPO4, 0.0087; Na2SiO3 99H~O, 0.020; glycylglyeine, 0.50; vitamin B e , 1.0 rag; vitaminBle, 1.0 ~zg; biotin, 1.0 ~zg; trace element solution 1 (Healey, in press), 10.0 ml; made up to 1 Liter with distilled water. The pI-I was adjusted to 7.8--8.0 with 1-N NaOH. The algae were harvested by centrifugation; freshwater species were resuspended in buffer FW-2 (Healey, in press) and marine species in buffer LF-3. The latter was identical with medium LF-3, except for the substitution of C1- for S O t , NO~-, and HCO[ on an equimolar basis, and the omission of KBr, Na2SiOe, and vitamins. A cell concentration of about 0.3 mg dry weight per ml was used in photosynthetic measurements and of 3 mg dry weight per ml for measurements of H e evolution. Dry weight was determined by resuspending an aliquot of cells in distilled water, transferring it to a pre-weighed Muminum-foil cup and drying to constant weight at 80~ The dry weight of the marine Chlorella was not measured directly but was estimated assuming the same relationship between dry weight and packed cell volume as found for Chlamydomonas moewusii (1.0 ~zl packed volume ~ 0.15 mg dry weight). Packed cell volume was measured by suspending 3.0 ml of algal suspension in a hematocrit tube and centrifuging at 1000 • g for 30 rain. After suspension in the appropriate buffer, algae to be examined for the occurfence of H a evolution were placed in anaerobic manometric flasks under N 2 at 20 ~ Alkaline pyrogMlol was included in the centre well. No additions were made to the cell suspensions. The algae were then subjected to the following sequence of dark and light (2151ux) periods: 4 h r darlmess, 2 hr light, 6hr darkness, 2 h r light, 12 hr darkness, 2 hr light. This regime was used in order to detect photoevolution of H e in species requiring either short or long dark-adaptation periods. Algae showing gas release under the above conditions were re-examined in the presence of pMladiummethylene blue to confirm the gas as H 2. Subsequent measurements of H~ evolution 16
Planta (BEN.), Bd. 91
222
F.P. Healey:
were made manomctrieally under N2 in the presence of alkaline pyrogallol. Some of these procedures have been described in more detail (Healey, in press). Results Table 1 shows the algae which were found to evolve H a and also gives the average rates of I t 2 evolution observed. Release of Ha, confirmed by uptake of the gas by palladium-methylene blue, was observed only in members of the genera Chlamydomenas, Chlorella, and Scenedesmus. A 3-hr period of dark anaerobiosis was sufficient for the appearance of the H aevolution rates reported. Under the conditions used, no H a evolution was detected in Dunaliella salina (ICC 1644), Platymonas spec. (author's isolate), Euglena gracilis, strain Z (ICC 753), Porphyridium cruentum (ICC 637), Rhodosorus marinus (West isolate), Chroomonas diplococcus (Butcher isolate), Cryptomonas lis (Provasoli isolate), Rhodomonas D3 (Provasoli isolate) and several strains of pennate diatoms isolated from marine littoral habitats. Those species with an ICC designation are maintained in the Indiana University Culture Collection (Starr, 1964); the others are maintained in the Marine Botany collection at Scripps Institution of Oceanography. Some characteristics of the photoevolution of H a by several green algae are also shown in Table 1. The species can be divided into three groups on the basis of response to sufficient DCMU to completely inhibit photosynthesis. At pH 6, this inhibitor had little or no effect on the release of H a by the two Chlamydomonas species tested. The reaction here was generally stimulated by acetate, both in the presence and in the absence of DCMU. In the second group, Scenedesmus obliquus, Chlorella pyrenoidosa, and Seenedesmus quadrieauda, DCMU sufficient to inhibit photosynthesis completely caused only about 50 % inhibition of tt~ evolution. This inhibition was partially overcome by the addition of glucose. The third and last group included two species of Chlorella in which the photoevolution of H a was largely or completely inhibited by DCMU. In the case of Chlorella vuIgaris, this inhibition was largely reversed by glucose after prolonged incubation, while in the ease of the marine Chlorella, glucose had no effect. In all cases, the uncoupler, CCCP, either stimulated or only slightly inhibited the photoevolution of H a. Because the effect of 105: M DCMU on the photoevolution of H a by S. obliquus following a short adaptation period contrasted with the results obtained by Bishop and Gaffron (1963) using a long adaptation period, the effect of DCMU in this species was examined more thoroughly. Table 2 shows that increasing the length of the dark adaptation period increased inhibition by DCMU. This increased inhibition was largely overcome by adding glucose.
3.3 5.0 1.1 1.6 1.6 1.0
0.2
0.4
0.2
0.3
0.4 -3.5
5.0
0.5
5.5
0.6
Light
2.0
Dark
Average rates of H 2 evolution at p H 6.0 (fzl H 2 h r - i m g dry w t -1)
150 90 100 200 80
40 40 10 10 0
120
120
150
100 100
60
Substrafe c
DCM-Ub
0
20 70
60
70
70
130
150
DCMU -{substrate
80
90 90
110
140
140
--
150
CCCP (10-SM)
400
500 500
--
400
500
400
300
Approximate saturating intensity (lux)
Effect of additions on photoevolution of H e at p H 6.0; r a t e expressed as percent of control, receiving no addition
'~ I C C : I n d i a n a Culture Collection (Starr, 1964); SIO ~ M a r i n e Botany Culture Collecdion, Scripps I n s t i t u t i o n of Oceanography. b DCMU concentration was 10-aM in the case of Chlamydomonas spp. and 10-~M with the others. c Substrate was 10-alV[ acetate in t h e case of Chlamydomonas spp. and 10-2M glucose for the others.
author's collection
Chlorella spec. (marine)
4 hr after addition
Chlamydomonas moewusii ICC 97 Chlamydomonas dysosmos ICC 342 Chlamydomonas debaryana ICC 344 Scenedesmus obliffuus D3 ICC 393 Chtorella pyrenoidosa ICC 251 Scenedesmus guadricauda SIO Chlorella vulgaris SIO
Species a
Table 1. Some characteristics o/hydrogen evolution by several green algae Measurements of dark evolution of H2 were m a d e during t h e 3rd hour of anaerobiosis; those of photoevohltion of I-I~ were m a d e in saturating light after 3 h r of dark anaerobiosis.
L~
O
224
F.P. Healey:
Table 2. E/[ect of length of darlc anaerobic 1period on the rate of H 2 evolution by Sccnedesmus obliques and its resl)onse to DCMU at pH 6.5 Light intensity -~ 970 lux (4 • 10-4 W cm-2 between 400 and 720 nm). Reaction
Length of dark adaptation period
Dark evolution of H~ (V1 hr -1 nag dry wt. -1) Photoevolution of H 2 (,al hr -1 mg dry wt. -1) Photoevolution of tt 2 after the addition of 10-~M DCMU (% of control) Photoevolution of H 2 after the addition of 1 0 - ~ DCMU and 10-2 M glucose (% of control)
3 hr
10 hr
0.56 6.6 40
0.40 8.5 19
44
38
Discussion Under the conditions used, t I 2 evolution was detected only in green algae of the genera ChloreUa, Scenedesmus, and Chlamydomonas, where it has been previously observed. Inspire of the presence of hydrogenase in Euglena spee. (Krasna and Rittenberg, 1954), Porphyridium cruentum (Frenkel and Rieger, 1951), and Platymonas spec. (Healey, unpublished observations), no release of I t 2 b y these algae was observed. This m a y have been due to any of a number of reasons. Some aspect of the procedure m a y have been inhibitory to H2 evolution; hydrogenase m a y have been inactivated by 03 release as soon as the cells were put into the light; or H 2 evolution m a y have been dependent on some cellular component not required in other hydrogenase reactions. The variable responses of photoevolution of H 2 to DCMU among the several green algae studied indicate the degree to which the two mechanisms, outlined in the Introduction, are operative. I n the two Chlamydomonas species, DCMU had no effect on H~ release (at p H 6), showing t h a t the DCMU-insensitive mechanism is sufficient to support the m a x i m u m rate of I-I2 evolution by these species. I n the two Scenedesmus species and Chlorella pyrenoidosa, the approximately 50 % inhibition of I-Is release b y DCMU indicates t h a t in these algae the reaction is about equally dependent on both mechanisms. The very strong to complete inhibition of I t 2 release by DCMU shows that H 2 evolution in the remaining two Chlorella species involves only the DCMU-sensitive mechanism. The fact t h a t the rate of photoevolution of H2 in the presence of DCMU and substrate was usually greater than t h a t with DCMU alone shows the ability of the DCMU-insensitive mechanism to increase when the substrafe supply is increased. The general lack of inhibition by CCCP indicates t h a t the photoevolution of H 2 is independent of phosphorylation in all the species examined.
H 2 Evolution by Algae
225
The relationship between the two mechanisms of photoevolution of H 2 in Scenedesmus obliquu8 is illustrated by the effect of DCMU after short and long adaptation periods. The increased inhibition by DCMU following the long adaptation period reflects a decreased ability of the cells to evolve H 2 by the DCMU-insensitive mechanism. This is presumably due to depletion of endogenous reserves by fermentation. Addition of glucose restored the levels of oxidizable substrate upon which DCMUinsensitive H~ evolution is dependent and thus decreased DCMU inhibition. Since the long adaptation period resulted in a greater rather than lower rate of photoevolution of H~, the rate of the DCMU-sensitive mechanism must have increased as that of the DCMU-insensitive one fell. The present study has shown that the photoevolution of H 2 by algae is more complex than previously thought. Furthermore, together with other recent results, it has lessened the apparent contrast between the photoevolution of H 2 by algae and that by photosynthetic bacteria. In place of the two mechanisms outlined by Bishop (1966), recent results suggest the following three mechanisms: 1. Electrons from normal photosynthetic reductant are driven directly by light to a potential level capable of reducing H +, as in H 2 release from water by several algae and from thiosulfate by Chromatium. This reaction is stimulated by uncouplers in both algae and Chromatium and is DCMU-sensitive in algae (Bishop and Gaffron, 1963; Losada et al., 1961; Ivanov and Demina, 1968). 2. Electrons from oxidative carbon metabolism are driven directly by light to a potential level capable of reducing H +, as occurs in several algae. This reaction is sensitive to dark starvation and inhibitors of oxidative carbon metabolism, insensitive to DCMU, and stimulated by uncouplers (Healey, in press). 3. Electrons from oxidative carbon metabolism are driven by a high energy intermediate of photophosphorylation to a potential level capable of reducing H +, as occurs in Rhodospirillum rubrum. This reaction is dependent on an organic substrate and is inhibited both by inhibitors of oxidative carbon metabolism and by uncouplers (Gest et al., 1962). I would like to express my gratitude to Drs. F. T. Haxo, M. D. Kamen, and B. E. Voleani for their guidance and helpful discussions during the present investigation. I am also grateful to Dr. P. G. Heytler of E. I. Dupont de Nemours and Company for his gifts of DCMU and CCCP. This study was supported by the Marine Life Research Group of the University of California. References Bishop, N. I. : Partial reactions of photosynthesis and photoreduetion. Ann. Rev. Plant Physiol. 17, 185--208 (1966). - - Gaffron, It.- On the interrelation of the mechanisms for oxygen and hydrogen evolution in adapted algae. In: Photosynthetic mechanisms of green plants.
226
F . P . Healey: H 2 Evolution by Algae
Nat. Acad. Sci. Publ. 5;0. 1145, p. 441---451 (B. Kok and A. T. Jagendorf, eds.). Washington 1963. Cramer, M., Myers, J.: Growth and photosynthetic characteristics of Euglena gracills. Arch. Mikrobiol. 17, 384--402 (1952). Frenkel, A. W. : A study of the hydrogenase systems of green and blue-green algae. Biol. Bull. 97, 261--262 (1949). Hydrogen evolution by the flagellate green alga, Chlamydomonas moeurasii. Arch. Biochem. 38, 219--230 (1952). Rieger, C. : Photoreduction in algae. Nature (Lond.) 167, 1030 (1951). Gaffron, H.: Carbon dioxide reduction with molecular hydrogen in green algae. Amer. J. Bot. 27, 273--283 (1940). Rubin, J. : Fermentative and photochemical production of hydrogen in algae. J. gem Physiol. 26, 219---240 (1942). Gest, H., Ormerod, J. G., Ormerod, K. S. : Photome~abolism of Rhodosplrillum rubrum: Light-dependent dissimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic citric acid cycle. Arch. Biochem. 97, 21--33 (1962). Hattori, A. : Effect of hydrogen on nitrite reduction by Anabaena cylindrica. In: Studies on microalgae and photosynthetic bacteria, p. 485---492 (Japanese Soe. Plant Physiol., eds.). Tokyo: Univ. of Tokyo Press 1963. Healey, F. P. : The mechanism of hydrogen evolution by Chlamydomonas ~noewusil. Plant Physiol., in press. Ivanov, I.D., I)emina, N. S.: The connection between hydrogen evolution and phosphorylation in Chromatium qninutissiraum. Dokl. Acad. Nauk SSSR (Biol. Sei. Sect.) Tr. 180, 312--313 (1968). Kaltwasser, H., Gaffron, H.: Effects of carbon dioxide and glucose on photohydrogen production in Scenedesmus. Plant Physiol. 39, Suppl. xiii (1964). Kessler, E. : Hydrogenase und H2-Stoffwechsel bei Algen. Dtsch. bot. Ges. (N.F.) 1, 92--101 (1962). ]~aifarth, H.: Vorkommen und Leistungsf~higkeit yon Hydrogenase bei einigen Griinalgen. Arch. Mikrobiol. 37, 215--225 (1960). Krasna, A. L, Rittenberg, D. : The mechanism of action of the enzyme hydrogenase. J. Amer. chem. Soc. 76, 3015--3020 (1954). Losada, M., Nozaki, M., Amen, D. I. : Photoproduetion of molecular hydrogen from thiosulfate by Chromatium cells. In: A symposium on light and life, p. 570--575 (W. D. MeElroy and B. Glass, eds.). Baltimore: Johns Hopkins Press 1961. Lyman, J., Fleming, R. H.: Composition of sea water. J. Mar. Res. 3, 134--146 (1940). Spruit, C. J . P . : Photoproduction of hydrogen and oxygen in Chlorella. Proc. First Intern. Photobiological Congr., Amsterdam, p. 323--327 (1954). - - Simultaneous photoproduction of hydrogen and oxygen by Chlorella. Medelel. Landbouwhogesch. Wageningen 58, No 9 (1958). Starr, R. C. : The culture collection of algae at Indiana University. Amer. J. Bot. 51, 1013--1044 (1964). -
-
-
-
-
-
Notc added inproo/: The recent results of Kaltwasser et al. [Kaltwasser, H., Stuart, T. S., Gaffron, H.: Light-dependent hydrogen evolution by Scenedesmus. Planta (Berl.) 89, 309--322 (1969)] add further support to some of the conclusions reached in this paper. Dr. F. P. Healey Department of Zoology University of Texas Austin, Texas 78712, U.S.A.
Hydrogen Evolution by Several Algae F. P. H~,~m~,Y Scripps Institution of Oceanography, University of California, La Jolla l~eceived October 15, 1969/January 5, 1970
Summary. Out of 33 strains of unicellular algae examined, H a evolution was observed only in species of Chlamydomonas, Chlorella and Scenedcsmus. While the photoevolution of H e by these algae was generally stimulated both by an organic substrate and by the uncoupler CCCP1, response to DCMU1 varied. On the basis of the response to DCM-U, it was concluded that the mechanism of photoevolution of H 2 differed from one alga to another. The reaction in some algae appeared to be dependent on either the photooxidation of water or oxidative carbon metabolism for reductant; that in other algae was supported by reduetant from both these sources. Introduction There is evidence for the occurrence of hydrogenase in a variety of algae, including several green algae (Gaffron, 1940; Frenkel, 1952; Kessler and Maifarth, 1960), some blue-green algae (Frenkel, 1949; Hattori, 1963), the red alga, Porphyridium cruentum, and some seaweeds (Frenkel and Rieger, 1951). However, H~ evolution has been observed only in members of the genera Scenedesmus (Gaffron and Rubin, 1942), Chlamydomonas (Frenkel, 1952), Chlorella (Spruit, 1954) and Ankistrodesmus (Kessler, 1962). There appear to be two mechanisms of photoevolution of H2 among algae. One involves the photooxidation of water and electron transport through both photosystems of photosynthetic electron transport to the release of H 2. I t is characterized b y the simultaneous release of H2 and 02, sensitivity to DCMU, Emerson enhancement, and insensitivity to respiratory inhibitors (Spruit, 1954, 1958; Bishop and Gaffron, 1963). The other involves the movement of reductant from oxidative carbon metabolism through only photosystem I of the photosynthetic electron transport system to the release of H 2. I t is characterized b y the simultaneous release of I t 2 and CO2, insensitivity to DCMU, lack of enhancement, stimulation b y substrates, and inhibition b y respiratory inhibitors 1 CCCP: earbonyleyanide-m-chlorophenylhydrazone; phenyl)-l,l-dimethylurea.
DCMU: 3-(3,4-dichloro-
H 2 Evolution by Algae
221
(Frenkel, 1952 ; Kessler, 1962 ; K a l t w a s s e r a n d Gaffron, 1964; Healey, in press). A s u r v e y of several unicellular algae was u n d e r t a k e n to e x a m i n e more fully the d i s t r i b u t i o n of H 2 evolution a m o n g algae a n d to d e t e r m i n e the c o n t r i b u t i o n of the above two m e c h a n i s m s to p h o t o e v o l u t i o n of H 2 a m o n g algae.
Materials and Methods Stock cultures of the algae were maintained by weekly transfers in liquid medium on a rotary shaker. Experimental cultures, inoculated from the stock cultures, were grown in i-Liter Erlenmeyer flaks gently shaken on a wrist-action shaker and bubbled with air. The temperature was mMntained at 20~ The cultures received light (8000 lux) from cool-white fluorescent tubes placed beneath them. The cultures were harvested in late exponential phase. Except for Chroomonas diplococcus, t~hodosorus marinus, and several of the diatom strains, all cultures were axenie. Stock and experimental cultures of axenie strains were checked for bacterial contamination by inoculating 5 ml of medium containing 0.1% tryptone and 0.1% glucose (w/v) with 1 ml of culture. Freshwater algae were grown in medium FW-2 (Healey, in press), with tile exception of Euglena gracilis, which was grown in the medium of Cramer and Myers (1952). Marine algae were grown in medium LF-3, based on the artificial sea water of Lyman and Fleming (1940). The composition of Medium LF-3 was as follows (in g unless otherwise noted): NaC1, I8.8; MgCI2 9 6H20, 5.7; Na2SO4, 3.1; KC1, 0.56; CaC1292H20, 0.40; NaI-ICOa, 0.15; KBr, 0.077; KNOs, 1.01; K2HPO4, 0.0087; Na2SiO3 99H~O, 0.020; glycylglyeine, 0.50; vitamin B e , 1.0 rag; vitaminBle, 1.0 ~zg; biotin, 1.0 ~zg; trace element solution 1 (Healey, in press), 10.0 ml; made up to 1 Liter with distilled water. The pI-I was adjusted to 7.8--8.0 with 1-N NaOH. The algae were harvested by centrifugation; freshwater species were resuspended in buffer FW-2 (Healey, in press) and marine species in buffer LF-3. The latter was identical with medium LF-3, except for the substitution of C1- for S O t , NO~-, and HCO[ on an equimolar basis, and the omission of KBr, Na2SiOe, and vitamins. A cell concentration of about 0.3 mg dry weight per ml was used in photosynthetic measurements and of 3 mg dry weight per ml for measurements of H e evolution. Dry weight was determined by resuspending an aliquot of cells in distilled water, transferring it to a pre-weighed Muminum-foil cup and drying to constant weight at 80~ The dry weight of the marine Chlorella was not measured directly but was estimated assuming the same relationship between dry weight and packed cell volume as found for Chlamydomonas moewusii (1.0 ~zl packed volume ~ 0.15 mg dry weight). Packed cell volume was measured by suspending 3.0 ml of algal suspension in a hematocrit tube and centrifuging at 1000 • g for 30 rain. After suspension in the appropriate buffer, algae to be examined for the occurfence of H a evolution were placed in anaerobic manometric flasks under N 2 at 20 ~ Alkaline pyrogMlol was included in the centre well. No additions were made to the cell suspensions. The algae were then subjected to the following sequence of dark and light (2151ux) periods: 4 h r darlmess, 2 hr light, 6hr darkness, 2 h r light, 12 hr darkness, 2 hr light. This regime was used in order to detect photoevolution of H e in species requiring either short or long dark-adaptation periods. Algae showing gas release under the above conditions were re-examined in the presence of pMladiummethylene blue to confirm the gas as H 2. Subsequent measurements of H~ evolution 16
Planta (BEN.), Bd. 91
222
F.P. Healey:
were made manomctrieally under N2 in the presence of alkaline pyrogallol. Some of these procedures have been described in more detail (Healey, in press). Results Table 1 shows the algae which were found to evolve H a and also gives the average rates of I t 2 evolution observed. Release of Ha, confirmed by uptake of the gas by palladium-methylene blue, was observed only in members of the genera Chlamydomenas, Chlorella, and Scenedesmus. A 3-hr period of dark anaerobiosis was sufficient for the appearance of the H aevolution rates reported. Under the conditions used, no H a evolution was detected in Dunaliella salina (ICC 1644), Platymonas spec. (author's isolate), Euglena gracilis, strain Z (ICC 753), Porphyridium cruentum (ICC 637), Rhodosorus marinus (West isolate), Chroomonas diplococcus (Butcher isolate), Cryptomonas lis (Provasoli isolate), Rhodomonas D3 (Provasoli isolate) and several strains of pennate diatoms isolated from marine littoral habitats. Those species with an ICC designation are maintained in the Indiana University Culture Collection (Starr, 1964); the others are maintained in the Marine Botany collection at Scripps Institution of Oceanography. Some characteristics of the photoevolution of H a by several green algae are also shown in Table 1. The species can be divided into three groups on the basis of response to sufficient DCMU to completely inhibit photosynthesis. At pH 6, this inhibitor had little or no effect on the release of H a by the two Chlamydomonas species tested. The reaction here was generally stimulated by acetate, both in the presence and in the absence of DCMU. In the second group, Scenedesmus obliquus, Chlorella pyrenoidosa, and Seenedesmus quadrieauda, DCMU sufficient to inhibit photosynthesis completely caused only about 50 % inhibition of tt~ evolution. This inhibition was partially overcome by the addition of glucose. The third and last group included two species of Chlorella in which the photoevolution of H a was largely or completely inhibited by DCMU. In the case of Chlorella vuIgaris, this inhibition was largely reversed by glucose after prolonged incubation, while in the ease of the marine Chlorella, glucose had no effect. In all cases, the uncoupler, CCCP, either stimulated or only slightly inhibited the photoevolution of H a. Because the effect of 105: M DCMU on the photoevolution of H a by S. obliquus following a short adaptation period contrasted with the results obtained by Bishop and Gaffron (1963) using a long adaptation period, the effect of DCMU in this species was examined more thoroughly. Table 2 shows that increasing the length of the dark adaptation period increased inhibition by DCMU. This increased inhibition was largely overcome by adding glucose.
3.3 5.0 1.1 1.6 1.6 1.0
0.2
0.4
0.2
0.3
0.4 -3.5
5.0
0.5
5.5
0.6
Light
2.0
Dark
Average rates of H 2 evolution at p H 6.0 (fzl H 2 h r - i m g dry w t -1)
150 90 100 200 80
40 40 10 10 0
120
120
150
100 100
60
Substrafe c
DCM-Ub
0
20 70
60
70
70
130
150
DCMU -{substrate
80
90 90
110
140
140
--
150
CCCP (10-SM)
400
500 500
--
400
500
400
300
Approximate saturating intensity (lux)
Effect of additions on photoevolution of H e at p H 6.0; r a t e expressed as percent of control, receiving no addition
'~ I C C : I n d i a n a Culture Collection (Starr, 1964); SIO ~ M a r i n e Botany Culture Collecdion, Scripps I n s t i t u t i o n of Oceanography. b DCMU concentration was 10-aM in the case of Chlamydomonas spp. and 10-~M with the others. c Substrate was 10-alV[ acetate in t h e case of Chlamydomonas spp. and 10-2M glucose for the others.
author's collection
Chlorella spec. (marine)
4 hr after addition
Chlamydomonas moewusii ICC 97 Chlamydomonas dysosmos ICC 342 Chlamydomonas debaryana ICC 344 Scenedesmus obliffuus D3 ICC 393 Chtorella pyrenoidosa ICC 251 Scenedesmus guadricauda SIO Chlorella vulgaris SIO
Species a
Table 1. Some characteristics o/hydrogen evolution by several green algae Measurements of dark evolution of H2 were m a d e during t h e 3rd hour of anaerobiosis; those of photoevohltion of I-I~ were m a d e in saturating light after 3 h r of dark anaerobiosis.
L~
O
224
F.P. Healey:
Table 2. E/[ect of length of darlc anaerobic 1period on the rate of H 2 evolution by Sccnedesmus obliques and its resl)onse to DCMU at pH 6.5 Light intensity -~ 970 lux (4 • 10-4 W cm-2 between 400 and 720 nm). Reaction
Length of dark adaptation period
Dark evolution of H~ (V1 hr -1 nag dry wt. -1) Photoevolution of H 2 (,al hr -1 mg dry wt. -1) Photoevolution of tt 2 after the addition of 10-~M DCMU (% of control) Photoevolution of H 2 after the addition of 1 0 - ~ DCMU and 10-2 M glucose (% of control)
3 hr
10 hr
0.56 6.6 40
0.40 8.5 19
44
38
Discussion Under the conditions used, t I 2 evolution was detected only in green algae of the genera ChloreUa, Scenedesmus, and Chlamydomonas, where it has been previously observed. Inspire of the presence of hydrogenase in Euglena spee. (Krasna and Rittenberg, 1954), Porphyridium cruentum (Frenkel and Rieger, 1951), and Platymonas spec. (Healey, unpublished observations), no release of I t 2 b y these algae was observed. This m a y have been due to any of a number of reasons. Some aspect of the procedure m a y have been inhibitory to H2 evolution; hydrogenase m a y have been inactivated by 03 release as soon as the cells were put into the light; or H 2 evolution m a y have been dependent on some cellular component not required in other hydrogenase reactions. The variable responses of photoevolution of H 2 to DCMU among the several green algae studied indicate the degree to which the two mechanisms, outlined in the Introduction, are operative. I n the two Chlamydomonas species, DCMU had no effect on H~ release (at p H 6), showing t h a t the DCMU-insensitive mechanism is sufficient to support the m a x i m u m rate of I-I2 evolution by these species. I n the two Scenedesmus species and Chlorella pyrenoidosa, the approximately 50 % inhibition of I-Is release b y DCMU indicates t h a t in these algae the reaction is about equally dependent on both mechanisms. The very strong to complete inhibition of I t 2 release by DCMU shows that H 2 evolution in the remaining two Chlorella species involves only the DCMU-sensitive mechanism. The fact t h a t the rate of photoevolution of H2 in the presence of DCMU and substrate was usually greater than t h a t with DCMU alone shows the ability of the DCMU-insensitive mechanism to increase when the substrafe supply is increased. The general lack of inhibition by CCCP indicates t h a t the photoevolution of H 2 is independent of phosphorylation in all the species examined.
H 2 Evolution by Algae
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The relationship between the two mechanisms of photoevolution of H 2 in Scenedesmus obliquu8 is illustrated by the effect of DCMU after short and long adaptation periods. The increased inhibition by DCMU following the long adaptation period reflects a decreased ability of the cells to evolve H 2 by the DCMU-insensitive mechanism. This is presumably due to depletion of endogenous reserves by fermentation. Addition of glucose restored the levels of oxidizable substrate upon which DCMUinsensitive H~ evolution is dependent and thus decreased DCMU inhibition. Since the long adaptation period resulted in a greater rather than lower rate of photoevolution of H~, the rate of the DCMU-sensitive mechanism must have increased as that of the DCMU-insensitive one fell. The present study has shown that the photoevolution of H 2 by algae is more complex than previously thought. Furthermore, together with other recent results, it has lessened the apparent contrast between the photoevolution of H 2 by algae and that by photosynthetic bacteria. In place of the two mechanisms outlined by Bishop (1966), recent results suggest the following three mechanisms: 1. Electrons from normal photosynthetic reductant are driven directly by light to a potential level capable of reducing H +, as in H 2 release from water by several algae and from thiosulfate by Chromatium. This reaction is stimulated by uncouplers in both algae and Chromatium and is DCMU-sensitive in algae (Bishop and Gaffron, 1963; Losada et al., 1961; Ivanov and Demina, 1968). 2. Electrons from oxidative carbon metabolism are driven directly by light to a potential level capable of reducing H +, as occurs in several algae. This reaction is sensitive to dark starvation and inhibitors of oxidative carbon metabolism, insensitive to DCMU, and stimulated by uncouplers (Healey, in press). 3. Electrons from oxidative carbon metabolism are driven by a high energy intermediate of photophosphorylation to a potential level capable of reducing H +, as occurs in Rhodospirillum rubrum. This reaction is dependent on an organic substrate and is inhibited both by inhibitors of oxidative carbon metabolism and by uncouplers (Gest et al., 1962). I would like to express my gratitude to Drs. F. T. Haxo, M. D. Kamen, and B. E. Voleani for their guidance and helpful discussions during the present investigation. I am also grateful to Dr. P. G. Heytler of E. I. Dupont de Nemours and Company for his gifts of DCMU and CCCP. This study was supported by the Marine Life Research Group of the University of California. References Bishop, N. I. : Partial reactions of photosynthesis and photoreduetion. Ann. Rev. Plant Physiol. 17, 185--208 (1966). - - Gaffron, It.- On the interrelation of the mechanisms for oxygen and hydrogen evolution in adapted algae. In: Photosynthetic mechanisms of green plants.
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Nat. Acad. Sci. Publ. 5;0. 1145, p. 441---451 (B. Kok and A. T. Jagendorf, eds.). Washington 1963. Cramer, M., Myers, J.: Growth and photosynthetic characteristics of Euglena gracills. Arch. Mikrobiol. 17, 384--402 (1952). Frenkel, A. W. : A study of the hydrogenase systems of green and blue-green algae. Biol. Bull. 97, 261--262 (1949). Hydrogen evolution by the flagellate green alga, Chlamydomonas moeurasii. Arch. Biochem. 38, 219--230 (1952). Rieger, C. : Photoreduction in algae. Nature (Lond.) 167, 1030 (1951). Gaffron, H.: Carbon dioxide reduction with molecular hydrogen in green algae. Amer. J. Bot. 27, 273--283 (1940). Rubin, J. : Fermentative and photochemical production of hydrogen in algae. J. gem Physiol. 26, 219---240 (1942). Gest, H., Ormerod, J. G., Ormerod, K. S. : Photome~abolism of Rhodosplrillum rubrum: Light-dependent dissimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic citric acid cycle. Arch. Biochem. 97, 21--33 (1962). Hattori, A. : Effect of hydrogen on nitrite reduction by Anabaena cylindrica. In: Studies on microalgae and photosynthetic bacteria, p. 485---492 (Japanese Soe. Plant Physiol., eds.). Tokyo: Univ. of Tokyo Press 1963. Healey, F. P. : The mechanism of hydrogen evolution by Chlamydomonas ~noewusil. Plant Physiol., in press. Ivanov, I.D., I)emina, N. S.: The connection between hydrogen evolution and phosphorylation in Chromatium qninutissiraum. Dokl. Acad. Nauk SSSR (Biol. Sei. Sect.) Tr. 180, 312--313 (1968). Kaltwasser, H., Gaffron, H.: Effects of carbon dioxide and glucose on photohydrogen production in Scenedesmus. Plant Physiol. 39, Suppl. xiii (1964). Kessler, E. : Hydrogenase und H2-Stoffwechsel bei Algen. Dtsch. bot. Ges. (N.F.) 1, 92--101 (1962). ]~aifarth, H.: Vorkommen und Leistungsf~higkeit yon Hydrogenase bei einigen Griinalgen. Arch. Mikrobiol. 37, 215--225 (1960). Krasna, A. L, Rittenberg, D. : The mechanism of action of the enzyme hydrogenase. J. Amer. chem. Soc. 76, 3015--3020 (1954). Losada, M., Nozaki, M., Amen, D. I. : Photoproduetion of molecular hydrogen from thiosulfate by Chromatium cells. In: A symposium on light and life, p. 570--575 (W. D. MeElroy and B. Glass, eds.). Baltimore: Johns Hopkins Press 1961. Lyman, J., Fleming, R. H.: Composition of sea water. J. Mar. Res. 3, 134--146 (1940). Spruit, C. J . P . : Photoproduction of hydrogen and oxygen in Chlorella. Proc. First Intern. Photobiological Congr., Amsterdam, p. 323--327 (1954). - - Simultaneous photoproduction of hydrogen and oxygen by Chlorella. Medelel. Landbouwhogesch. Wageningen 58, No 9 (1958). Starr, R. C. : The culture collection of algae at Indiana University. Amer. J. Bot. 51, 1013--1044 (1964). -
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Notc added inproo/: The recent results of Kaltwasser et al. [Kaltwasser, H., Stuart, T. S., Gaffron, H.: Light-dependent hydrogen evolution by Scenedesmus. Planta (Berl.) 89, 309--322 (1969)] add further support to some of the conclusions reached in this paper. Dr. F. P. Healey Department of Zoology University of Texas Austin, Texas 78712, U.S.A.
