Possible toxic effects on Daphnia resulting from the green alga Scenedesmus obliquus

We cultured individuals of two Daphnia species and their hybrid on two different algae, Scenedesmus obliquus and Chlamydomonas globosa, in different concentrations. Our results suggest that culture conditions of S. obliquus can be such that the algal cells become toxic to Daphnia     

High-affinity siderophore-mediated iron-transport system in the green alga Scenedesmus incrassatulus

Under iron-deficient conditions a high-affinity siderophore-mediated iron-transport system is induced in the green alga Scenedesmus incrassatulus R-83. Algal siderophores have a strong avidity for ferric versus ferrous iron, quickly oxidate Fe^II and efficiently solubilize Fe^III hydroxides. The entire ferrated molecule is translocated across the membrane by the specific transport system. The iron-uptake rate in Fe-deficient cells is higher at higher pH adjusted with bicarbonate in the medium, suggesting the presence of an inducible membrane-bound translocator. The iron-reduction step is not involved in uptake of ferrated siderophores. The total absorbed iron from siderophores is high and does not strongly depend on the nutritional status of cells, showing that the critical step for iron uptake is siderophore secretion rather than the membrane-bound iron-transport system.Abbreviations DFOB desferrioxamine B - EDDHA ethylenediamine di (o-hydroxyphenyl) acetic acid - BPDS bathophenanthrolinedisulphonate This work was supported by grant No. B-69 from the National Fund for Scientific Investigations at the Ministery of Education and Science in Bulgaria.     

Superoxide dismutase in Scenedesmus obliquus

In heterotrophically grown Scenedesmus obliquus, the specific activity of superoxide dismutase (SOD; EC 1.15.1.1) declined when glucose was abundant, increased as it was depleated, and remained steady at a high level when it was absent. Transition to autotrophic growth produced only a small (20% over 5 d) increase in specific activity above the values obtained in dark-grown cells after glucose and starch-reserve depletion. This small, but consistent, increase did, however, parallel a similar increase in photosynthetic capacity. Polyacrylamide-gel electrophoresis showed the existence of nine isoenzymes of SOD. The three major and one of the minor isoenzymes were present in all extracts while three minor isoenzymes were found only in autotrophically grown cells and two only in heterotrophically grown cells. Characterization studies indicated that two of the major isoenzymes are dimeric FeSODs the other is a tetrameric MnSOD, and of the minor isoenzymes, two are dimeric FeSODs and four are dimeric MnSODs.Abbreviation SOD superoxide dismutase     

Competition between the green alga Scenedesmus and the cyanobacterium Synechococcus under different modes of inorganic nitrogen supply

In this study, we evaluated growth responses of the green alga Scenedesmus and the cyanobacterium Synechococcus supplied with inorganic nitrogen in different ways. A competitive situation in which nitrogen was limiting was created in mixed cultures as well as in cultures growing in the same vessel but separated by a permeable dialysis membrane. Supplying inorganic nitrogen in small pulses at a high frequency favoured the cyanobacterium Synechococcus, whereas batch additions favoured the green alga Scenedesmus. When using a large-pulse/low-frequency supply mode, the yield of the green alga was higher when ammonium was added as nitrogen source compared to when nitrate was added. By contrast, the yield of the cyanobacterium was higher in the nitrate regime. However, uptake experiments using unialgal cultures showed that both organisms depleted the medium of ammonium more rapidly than they depleted the medium of nitrate; i.e. the higher yield of the cyanobacterium in the nitrate regime than in the ammonium regime can be attributed to the effects of competition with the green alga. Since nitrate assimilation involves the consumption of reductive power, we suggest that the outcome of competition was governed by the fact that green alga was light limited and therefore better able to compete for ammonium than for nitrate. The results from the laboratory studies are discussed in relation to results from an enclosure experiment performed in Lake Erken, Sweden. In that field experiment, in which additions of both phosphate and ammonium were applied every second day to 350-l enclosures, the green algal biomass increased exponentially during an incubation period of 22 days.     

Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO₂-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 M azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.Abbreviations CA carbonic anhydrase - CCM CO₂-concentrating mechanism - IEF isoelectric focusing - Rubisco ribulose-l,5-bisphosphate carboxylase/oxygenase We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation.     

Subcellular location of the tetrapyrrole synthesis enzyme porphobilinogen deaminase in higher plants: an immunological investigation

A recombinant plasmid, pArab8, harbouring the cDNA encoding the mature form of the tetrapyrrole synthesis enzyme porphobilinogen deaminase (EC 4.3.1.8; also known as hydroxymethylbilane synthase) from Arabidopsis thaliana (L.) Heynh. has been constructed, and used to transform Escherichia coli. The porphobilinogen deaminase protein from Arabidopsis was overexpressed in this strain, and purified to homogeneity (3000-fold) with a yield of 20%. Antibodies were raised against the purified plant enzyme, and used in Western blot analysis, immunoprecipitation of enzyme activity and immuno-gold electron microscopy. The results indicate that the enzyme is confined to plastids in both leaves and roots. The implications of this finding for plant tetrapyrrole synthesis are discussed.Abbreviations DEAE diethylaminoethyl - FPLC fast protein liquid chromatography - PBG porphobilinogen This work was supported by Science and Engineering Research Council (SERC) and Agricultural and Food Research Council (AFRC) grants to P.M.J. and an AFRC grant to A.G.S. The protein sequencing was carried out by Mr Lawrence Hunt of the SERC MRI Protein Sequencing Unit (Director Dr M.G. Gore) at Southampton University. We acknowledge the Wellcome Foundation for financial support of the Protein and Nucleic Acid Chemistry Facility at the University of Cambridge, where the oligonucleotide primers were synthesised.     

Action spectra for photosynthetic adaptation in Scenedesmus obliquus

Photosynthetic adaptation of the unicellular green alga Scenedemus obliquus to different light conditions was investigated with respect to chlorophyll synthesis. Cultures were grown under white light (20 W · m^–2) from fluorescent lamps and were then transferred and subjected to the actual adaptation regime which consisted of a 24-h irradiation by different fluence rates and wavelengths. Fluence rate-response curves for chlorophyll synthesis were measured between 4 · 10^–2 and 1 · 10² W · m^–2. In white light from incandescent lamps, in blue and red light the fluence rate-response curves for chlorophyll (Chl) a and also for Chl b were bell-shaped. In red light the threshold was about the same as under blue light. The maximal amounts of Chl a and b were about twofold increased under blue light relative to the values obtained with red light. Action spectra for the stimulation of chlorophyll synthesis (Chl a + Chl b) as well as those for the separate chlorophylls showed two maxima near 450 and 500 nm. However, the action spectrum for Chl b synthesis demonstrated a considerably higher value in the 450-nm peak. Experiments with the photosynthesis inhibitor 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) indicated that photosynthetic energy supply supported the photostimulation of chlorophyll synthesis. The action spectra indicate the cooperation of two photoreceptors. The 460-nm peak is attributed to the typical blue-light receptor, being more active in Chl b formation. The peak at 500 nm may represent carotenoproteins acting as an accessory pigment system.Abbreviations PCV packed cell volume - Chl total amount of chlorophyll - Chl a, b chlorophyll a, b - DCMU 3-(3,4-dichlorphenyl)-1,1-dimethylurea This project was supported by the Deutsche Forschungsgemeinschaft. We thank Ms. K. Bölte for technical assistance.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Autoinduction activity of a conditioned medium obtained from high density cultures of the green alga <Emphasis Type="Italic">Scenedesmus subspicatus</Emphasis>

Culture filtrates (conditioned medium, CM) containing exudates obtained from cells of Scenedesmus subspicatus grown in batch culture were tested for their autoinduction activity. Undiluted CM completely inhibited the proliferation of cells due to depletion of nitrogen in this medium. When undiluted CM was supplemented with fresh bold basal medium (BBM) medium, enhancement of population growth in a dilution-dependent manner was observed. The most effective was 2-fold diluted CM (CM/2), whereas the growth activity of CM/20 decreased considerably. The proliferation of cells at a very low initial density (10 cells mL⁻¹) was induced by the addition of CM/2. Biovolume, dry matter production and oxygen evolution by cells grown in 2-, 5- and 10-fold diluted CM increased markedly in both a concentration- and time-dependent manner. Conditioned medium factor (CMF) was mainly a product of rapidly dividing cells. Preliminary characterisation revealed that CMF is heat-stable, resistant to very low pH, highly hydrophilic (poorly soluble in ethanol or diethyl ether) and diffuses through a 1 kDa dialysis membrane. CM can be stored for long periods due to the time-stable activity of CMF. Depending on the enzyme applied, proteolytic digestion completely (papain, bromelain) or partially (subtilisin) abolished the activity of CM. This effect of CM was enhanced by digestion with trypsin. We suggest that CMF may be a low-molecular-weight peptide(s) or glycopeptide(s).     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

Fast and reversible response of thylakoid-associated polyamines during and after UV-B stress: a comparative study of the wild type and a mutant lacking chlorophyll b of unicellular green alga Scenedesmus obliquus

The functional and biochemical aspects of the photosynthetic apparatus in response to UV-B radiation were examined in unicellular oxygenic algae Scenedesmus obliquus. The wild type (Wt) and a chlorophyll b-less mutant (Wt-lhc) were used as a specific tool for the understanding of antenna role. Photosynthesis was monitored during and after UV-B stress by time resolved fluorescence spectroscopy and polarography. Carotenoids, such as neoxanthin, loroxanthin, lutein, violaxanthin, antheraxanthin, zeaxanthin, alpha- and beta-carotene, cellular and thylakoid-associated putrescine, spermidine, spermine and subcomplexes of light-harvesting complex (LHCII) of photosystem II (PSII) were investigated to assess their possible involvement in response to UV-B. Oxygen evolution depression by UV-B was higher in the Wt-lhc mutant than in the Wt. Photosynthesis recovery occurred in the Wt, but not in the mutant. The dissipation of excess excitation energy during UV-B stress was accompanied by changes in the thylakoid-associated polyamines which were much higher than changes in xanthophylls. We conclude that, at least in the unicellular green alga S. obliquus, mutants lacking chlorophyll b have significant lower capacity for recovery after UV-B stress. In addition, the comparison of xanthophylls and thylakoid-associated polyamines reveals that the latter are more responsive to UV-B stress and in a reversible manner.     

Photoisomerization of lycopene during carotenogenesis in mutant C-6D of Scenedesmus obliquus

Mutant C-6D of the unicellular green alga Scenedesmus obliquus has lost the ability to form cyclic carotenoids during heterotrophic growth in the dark. In the dark it accumulates acyclic intermediates, i.e. lycopene, neurosporene and ζ-carotene. The lycopene and two neurosporene forms were identified to be cis-isomers. Upon transfer to light, intermediates decrease and a normal set of carotenoids is synthesized. Inhibition of the cyclization reaction by nicotine reveals a lightinduced isomerization of cis-lycopene to trans-lycopene. Since the spectral characteristics of these two isomers differ drastically the isomerization can be followed in vivo by measuring a light-induced absorbance change. This absorbance change has its maximum at 520 nm and shows fast kinetics under high light intensities reaching a saturation level after about 2 min. Fluence-response curves for this absorbance change were performed for different wavelengths of actinic light. From the linear parts of these curves an action spectrum was caculated showing maxima at 670, 630 and 440 nm originating from chlorophyll and a maximum at shorter wavelengths (400–510 nm) which is interpreted to derive from ζ-carotene. A model for the light regulation of carotenogenesis in mutant C-6D is presented and the relation to the so-called 520-change observed in many plants is discussed.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.Abbreviations CWH complete cell walls isolated from the homogenates - CWM maternal cell walls accumulated in the medium - KC ketocarotenoid - SC secondary carotenoids - SP sporopollenin     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of areporter gene, LacZ, in the unicellular green alga Haematococcuspluvialis. By employing the micro-particle bombardment method,motilecells in the exponential phase showed transient expression oflacZ. This was detected in bombarded motile cells undertherupture-disc pressures of 3103 KPa and 4137 KPa.Transient expression of LacZ gene could not be observed in non-motile cells ofthis alga under the same transformation condition. No LacZ background was foundin either the motile cells or the non-motile cells. The study suggests apromising potential of the SV40 promoter and the lacZreporter gene in genetic engineering of unicellular green algae.     

Adenosine deaminase from Azotobacter vinelandii. Purification and properties

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65°C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.     

Photobleaching in the unicellular green alga Dunaliella parva 19/9

The change in the pigment composition of the unicellular alga Dunaliella parva 19/9 during exposure to high light (4000 mol m^-2 s^-1) has been investigated. During photobleaching the carotenoids were lost at a greater rate than the chlorophylls. In these photoinhibitory conditions, -carotene and especially the minor carotenes, - and -carotene, were more susceptible to oxidative destriction than the xanthophylls. Lutein, the major carotenoid present, was the most stable of the carotenoids in these conditions. In addition to the direct photobleaching of carotenoids and chlorophylls, high light treatment induced the de-epoxidation of violaxanthin to antheraxantin and zeaxanthin. Small amounts of zeaxanthin were present in cells prior to illumination but the amount increased 2.4 fold following high light treatment. The effects of extremes of temperature during exposure to high light intensities were also investigated. The destruction of chlorophylls was found to be more temperature sensitive than that of the carotenoids. The general pattern of loss for the individual carotenoids was similar to that found at 25°C, i.e., the carotenes were more readily degraded than the xanthophylls.     

Effects of 3,4-benzopyrene on the course of cell cycle events in the chlorococcal alga Scenedesmus quadricauda

Synchronous cultures of the chlorococcal alga Scenedesmus quadricauda were grown under optimal growth conditions. The mean length of their cell cycle was approximately 20 h. The cultures were treated at the start, at the 4th, and 8th hour of the cell cycle with 3,4-benzo(a)pyrene (BP) in the range of 0.1–0.5 g ml^-1 of final concentration. A period about 4 h was found within which no inhibitory effects could be detected even at the highest BP concentrations used. In presence of BP the rates of RNA and protein syntheses gradually decreased until complete inhibition of net syntheses occurred. In a similar way chlorophyll synthesis was inhibited, and this was followed by gradual degradation of the chlorophyll. The higher the concentration of BP the more rapid the decrease of the rates of syntheses and the earlier their complete inhibition. At low BP concentrations while DNA replications were initiated, the number of replications was lowered. At higher concentrations the initiations of DNA replications were delayed or completely suppressed. Syntheses of saccharides were the least inhibited processes in presence of BP. Starch synthesis was slowed down at the end of the cell cycle and fructose synthesis (free and sucrose bound) was even stimulated later in the cell cycle. The release of daughter coenobia, and protoplast fissions were most susceptible to BP treatment, being affected at concentrations which produced no measureble disturbances of macromolecular syntheses. At BP concentrations at which the inhibition of macromolecular syntheses occurred, the delay or suppression of mitoses was observed.Abbreviations BP 3,4-benzo(a)pyrene - PhAR photosynthetically active radiation     

Observations on the photohydrogen producing activity during the synchronous cell cycle of Scenedesmus obliquus

In anaerobically adapted samples of synchronized cultures of the unicellular green alga Scenedesmus obliquus it was observed that both the rate and the maximum volume of hydrogen produced in the light changed in a parallel fashion over the life cycle. These two parameters of cells of the 16th h were 3 times greater than the comparable values for cells of the 8th h. Although both photosystems are involved in photohydrogen production the patterns seen over a complete life cycle (24 h) for hydrogen metabolism was inverse to that noted for changes in the photosynthetic capacity. The provision of either glucose, ethanol or acetate to 8th and 16th h cultures enhanced photohydrogen production of the 8th to the same level as the 16th h. From these findings, and also from the observation that the starch content is low at the 8th but 4 fold at the 16th h, it is apparent that in autotrophic cultures an endogenous organic compound, and not water, serves as the electron donor for photohydrogen production. Since free glucose was not detected the natural substrate is most likely starch. From experiments with monochromatic light and observations on the inhibitory action of DCMU and DBMIB on photohydrogen production we conclude that the major portion of the machinery for photohydrogen production in Scenedesmus requires both PS I and PS II participation and the input of electrons from the natural substrate proceeds through PS II.The alternate possibility that glucose, acetate and ethanol also act as inhibitors of reactions, most probably photophosphorylation, which compete with photohydrogen production was suggested by some experiments. The subsequent modulation of hydrogenase activity was discussed as a possible reason for the enhancement of photohydrogen production.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethyl-urea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - NAD nicotinamide adenine dinucleotide - PSI photosystem I - PSII photosystem II - PCV packed cell volume