Chloroplast phosphate transporter CrPHT4-7 regulates phosphate homeostasis and photosynthesis in Chlamydomonas.

In eukaryotic cells, phosphorus is assimilated and utilized primarily as phosphate (Pi). Pi homeostasis is mediated by transporters that have not yet been adequately characterized in green algae. This study reports on PHOSPHATE TRANSPORTER 4-7 (CrPHT4-7) from Chlamydomonas reinhardtii, a member of the PHT4 transporter family, which exhibits remarkable similarity to AtPHT4;4 from Arabidopsis (Arabidopsis thaliana), a chloroplastic ascorbate transporter. Using fluorescent protein tagging, we show that CrPHT4-7 resides in the chloroplast envelope membrane. Crpht4-7 mutants, generated by the CRISPR/Cas12a-mediated single-strand templated repair, show retarded growth, especially in high light, reduced ATP level, strong ascorbate accumulation, and diminished non-photochemical quenching in high light. On the other hand, total cellular phosphorous content was unaffected, and the phenotype of the Crpht4-7 mutants could not be alleviated by ample Pi supply. CrPHT4-7-overexpressing lines exhibit enhanced biomass accumulation under high light conditions in comparison with the wild-type strain. Expressing CrPHT4-7 in a yeast (Saccharomyces cerevisiae) strain lacking Pi transporters substantially recovered its slow growth phenotype, demonstrating that CrPHT4-7 transports Pi. Even though CrPHT4-7 shows a high degree of similarity to AtPHT4;4, it does not display any substantial ascorbate transport activity in yeast or intact algal cells. Thus, the results demonstrate that CrPHT4-7 functions as a chloroplastic Pi transporter essential for maintaining Pi homeostasis and photosynthesis in C. reinhardtii.

Function of iron-stress-induced protein A in cyanobacterial cells with monomeric and trimeric photosystem I.

The acclimation of cyanobacteria to iron deficiency is crucial for their survival in natural environments. In response to iron deficiency, many cyanobacterial species induce the production of a pigment-protein complex called iron-stress-induced protein A (IsiA). IsiA proteins associate with photosystem I (PSI) and can function as light-harvesting antennas or dissipate excess energy. They may also serve as chlorophyll storage during iron limitation. In this study, we examined the functional role of IsiA in cells of Synechocystis sp. PCC 6803 grown under iron limitation conditions by measuring the cellular IsiA content and its capability to transfer energy to PSI. We specifically tested the effect of the oligomeric state of PSI by comparing wild-type (WT) Synechocystis sp. PCC 6803 with mutants lacking specific subunits of PSI, namely PsaL/PsaI (PSI subunits XI/VIII) and PsaF/PsaJ (PSI subunits III/IX). Time-resolved fluorescence spectroscopy revealed that IsiA formed functional PSI3-IsiA18 supercomplexes, wherein IsiA effectively transfers energy to PSI on a timescale of 10 ps at room temperature-measured in isolated complexes and in vivo-confirming the primary role of IsiA as an accessory light-harvesting antenna to PSI. However, a notable fraction (40%) remained unconnected to PSI, supporting the notion of a dual functional role of IsiA. Cells with monomeric PSI under iron deficiency contained, on average, only 3 to 4 IsiA complexes bound to PSI. These results show that IsiA can transfer energy to trimeric and monomeric PSI but to varying degrees and that the acclimatory production of IsiA under iron stress is controlled by its ability to perform its light-harvesting function.

Enhancing biophotovoltaic efficiency: Study on a highly productive green algal strain Parachlorella kessleri MACC-38.

Biophotovoltaic (BPV) devices are a potential decentralized and environmentally friendly energy source that harness solar energy through photosynthesis. BPV devices are self-regenerating, promising long-term usability. A practical strategy for enhancing BPV performance is to systematically screen for highly exoelectrogenic algal strains capable of generating large electric current density. In this study, a previously uncharacterized green algal strain - Parachlorella kessleri MACC-38 was found to generate over 340 µA mg-1 Chl cm-2. This output is approximately ten-fold higher than those of Chlamydomonas reinhardtii and Chlorella species. The current production of MACC-38 primarily originates from photosynthesis, and the strain maintains its physiological integrity throughout the process. MACC-38 exhibits unique traits such as low extracellular O2 and Fe(III) reduction, substantial copper (II) reduction, and significant extracellular acidification during current generation, contributing to its high productivity. The exoelectrogenic and growth characteristics of MACC-38 suggest that it could markedly boost BPV efficiency.

A PSII photosynthetic control is activated in anoxic cultures of green algae following illumination.

Photosynthetic hydrogen production from microalgae is considered to have potential as a renewable energy source. Yet, the process has two main limitations holding it back from scaling up; (i) electron loss to competing processes, mainly carbon fixation and (ii) sensitivity to O2 which diminishes the expression and the activity of the hydrogenase enzyme catalyzing H2 production. Here we report a third, hitherto unknown challenge: We found that under anoxia, a slow-down switch is activated in photosystem II (PSII), diminishing the maximal photosynthetic productivity by three-fold. Using purified PSII and applying in vivo spectroscopic and mass spectrometric techniques on Chlamydomonas reinhardtii cultures, we show that this switch is activated under anoxia, within 10 s of illumination. Furthermore, we show that the recovery to the initial rate takes place following 15 min of dark anoxia, and propose a mechanism in which, modulation in electron transfer at the acceptor site of PSII diminishes its output. Such insights into the mechanism broaden our understanding of anoxic photosynthesis and its regulation in green algae and inspire new strategies to improve bio-energy yields.

The Functions of Chloroplastic Ascorbate in Vascular Plants and Algae.

Ascorbate (Asc) is a multifunctional metabolite essential for various cellular processes in plants and animals. The best-known property of Asc is to scavenge reactive oxygen species (ROS), in a highly regulated manner. Besides being an effective antioxidant, Asc also acts as a chaperone for 2-oxoglutarate-dependent dioxygenases that are involved in the hormone metabolism of plants and the synthesis of various secondary metabolites. Asc also essential for the epigenetic regulation of gene expression, signaling and iron transport. Thus, Asc affects plant growth, development, and stress resistance via various mechanisms. In this review, the intricate relationship between Asc and photosynthesis in plants and algae is summarized in the following major points: (i) regulation of Asc biosynthesis by light, (ii) interaction between photosynthetic and mitochondrial electron transport in relation to Asc biosynthesis, (iii) Asc acting as an alternative electron donor of photosystem II, (iv) Asc inactivating the oxygen-evolving complex, (v) the role of Asc in non-photochemical quenching, and (vi) the role of Asc in ROS management in the chloroplast. The review also discusses differences in the regulation of Asc biosynthesis and the effects of Asc on photosynthesis in algae and vascular plants.

The lifetime of the oxygen-evolving complex subunit PSBO depends on light intensity and carbon availability in Chlamydomonas.

PSBO is essential for the assembly of the oxygen-evolving complex in plants and green algae. Despite its importance, we lack essential information on its lifetime and how it depends on the environmental conditions. We have generated nitrate-inducible PSBO amiRNA lines in the green alga Chlamydomonas reinhardtii. Transgenic strains grew normally under non-inducing conditions, and their photosynthetic performance was comparable to the control strain. Upon induction of the PSBO amiRNA constructs, cell division halted. In acetate-containing medium, cellular PSBO protein levels decreased by 60% within 24 h in the dark, by 75% in moderate light, and in high light, the protein completely degraded. Consequently, the photosynthetic apparatus became strongly damaged, probably due to 'donor-side-induced photoinhibition', and cellular ultrastructure was also severely affected. However, in the absence of acetate during induction, PSBO was remarkably stable at all light intensities and less substantial changes occurred in photosynthesis. Our results demonstrate that the lifetime of PSBO strongly depends on the light intensity and carbon availability, and thus, on the metabolic status of the cells. We also confirm that PSBO is required for photosystem II stability in C. reinhardtii and demonstrate that its specific loss also entails substantial changes in cell morphology and cell cycle.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level.

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the "Tulip" device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this "Tulip" platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the "Pot" device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant "Pot" device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

Thin cell layer cultures of Chlamydomonas reinhardtii L159I-N230Y, pgrl1 and pgr5 mutants perform enhanced hydrogen production at sunlight intensity.

Photobiological hydrogen (H2) production is a promising renewable energy source. HydA hydrogenases of green algae are efficient but O2-sensitive and compete for electrons with CO2-fixation. Recently, we established a photoautotrophic H2 production system based on anaerobic induction, where the Calvin-Benson cycle is inactive and O2 scavenged by an absorbent. Here, we employed thin layer cultures, resulting in a three-fold increase in H2 production relative to bulk CC-124 cultures (50 µg chlorophyll/ml, 350 µmol photons m-2 s-1). Productivity was maintained when increasing the light intensity to 1000 µmol photons m-2s-1 and the cell density to 150 µg chlorophyll/ml. Remarkably, the L159I-N230Y photosystem II mutant and the pgrl1 photosystem I cyclic electron transport mutant produced 50% more H2 than CC-124, while the pgr5 mutant generated 250% more (1.2 ml H2/ml culture in six days). The photosynthetic apparatus of the pgr5 mutant and its in vitro HydA activity remained remarkably stable.

Ascorbate inactivates the oxygen-evolving complex in prolonged darkness.

Ascorbate (Asc, vitamin C) is an essential metabolite participating in multiple physiological processes of plants, including environmental stress management and development. In this study, we acquired knowledge on the role of Asc in dark-induced leaf senescence using Arabidopsis thaliana as a model organism. One of the earliest effects of prolonged darkness is the inactivation of oxygen-evolving complexes (OEC) as demonstrated here by fast chlorophyll a fluorescence and thermoluminescence measurements. We found that inactivation of OEC due to prolonged darkness was attenuated in the Asc-deficient vtc2-4 mutant. On the other hand, the severe photosynthetic phenotype of a psbo1 knockout mutant, lacking the major extrinsic OEC subunit PSBO1, was further aggravated upon a 24-h dark treatment. The psbr mutant, devoid of the PSBR subunit of OEC, performed only slightly disturbed photosynthetic activity under normal growth conditions, whereas it showed a strongly diminished B thermoluminescence band upon dark treatment. We have also generated a double psbo1 vtc2 mutant, and it showed a slightly milder photosynthetic phenotype than the single psbo1 mutant. Our results, therefore, suggest that Asc leads to the inactivation of OEC in prolonged darkness by over-reducing the Mn-complex that is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits. Our study is an example that Asc may negatively affect certain cellular processes and thus its concentration and localization need to be highly controlled.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

Ascorbate Deficiency Does Not Limit Nonphotochemical Quenching in Chlamydomonas reinhardtii.

Ascorbate (Asc; vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance, and photoprotection. In vascular plants, violaxanthin de-epoxidase requires Asc as a reductant; thereby, Asc is required for the energy-dependent component of nonphotochemical quenching (NPQ). To assess the role of Asc in NPQ in green algae, which are known to contain low amounts of Asc, we searched for an insertional Chlamydomonas reinhardtii mutant affected in theVTC2 gene encoding GDP-l-Gal phosphorylase, which catalyzes the first committed step in the biosynthesis of Asc. The Crvtc2-1 knockout mutant was viable and, depending on the growth conditions, contained 10% to 20% Asc relative to its wild type. When C. reinhardtii was grown photomixotrophically at moderate light, the zeaxanthin-dependent component of NPQ emerged upon strong red illumination both in the Crvtc2-1 mutant and in its wild type. Deepoxidation was unaffected by Asc deficiency, demonstrating that the Chlorophycean violaxanthin de-epoxidase found in C. reinhardtii does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component characteristic of photoautotrophic C. reinhardtii cultures grown at high light was not limited by Asc deficiency either. On the other hand, a reactive oxygen species-induced photoinhibitory NPQ component was greatly enhanced upon Asc deficiency, both under photomixotrophic and photoautotrophic conditions. These results demonstrate that Asc has distinct roles in NPQ formation in C. reinhardtii as compared to vascular plants.

Elimination of the flavodiiron electron sink facilitates long-term H2 photoproduction in green algae.

BACKGROUND: The development of renewable and sustainable biofuels to cover the future energy demand is one of the most challenging issues of our time. Biohydrogen, produced by photosynthetic microorganisms, has the potential to become a green biofuel and energy carrier for the future sustainable world, since it provides energy without CO2 emission. The recent development of two alternative protocols to induce hydrogen photoproduction in green algae enables the function of the O2-sensitive [FeFe]-hydrogenases, located at the acceptor side of photosystem I, to produce H2 for several days. These protocols prevent carbon fixation and redirect electrons toward H2 production. In the present work, we employed these protocols to a knockout Chlamydomonas reinhardtii mutant lacking flavodiiron proteins (FDPs), thus removing another possible electron competitor with H2 production. RESULTS: The deletion of the FDP electron sink resulted in the enhancement of H2 photoproduction relative to wild-type C. reinhardtii. Additionally, the lack of FDPs leads to a more effective obstruction of carbon fixation even under elongated light pulses. CONCLUSIONS: We demonstrated that the rather simple adjustment of cultivation conditions together with genetic manipulation of alternative electron pathways of photosynthesis results in efficient re-routing of electrons toward H2 photoproduction. Furthermore, the introduction of a short recovery phase by regular switching from H2 photoproduction to biomass accumulation phase allows to maintain cell fitness and use photosynthetic cells as long-term H2-producing biocatalysts.

Paradigm Shift in Algal H2 Production: Bypassing Competitive Processes.

Hydrogen is a promising energy carrier, but producing it sustainably remains a challenge. Green algae can produce hydrogen photosynthetically using their efficient but oxygen-sensitive hydrogenases. Recent strategies aiming to bypass competing processes provide a promising route for scaling up algal hydrogen production.

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in Arabidopsis.

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE 1 (P5CS1) and downregulation of PROLINE DEHYDROGENASE 1 (PDH1) genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of P5CS1 and a C-box motif of PDH1. Salt-induced proline accumulation and P5CS1 expression were reduced in the hy5hyh double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.

Water-splitting-based, sustainable and efficient H2 production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassham cycle.

BACKGROUND: Photobiological H2 production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H2, only water is produced. The [Fe-Fe]-type hydrogenases of green algae are highly active, although extremely O2-sensitive. Sulphur deprivation is a common way to induce H2 production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus. RESULTS: We report on the establishment of an alternative H2 production method by green algae that is based on a short anaerobic induction, keeping the Calvin-Benson-Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O2. Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H2 produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic. CONCLUSION: Our protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H2 production, which enables industrial application of algal biohydrogen production.

The mechanism of photosystem-II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii.

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation.

Concentration Does Matter: The Beneficial and Potentially Harmful Effects of Ascorbate in Humans and Plants.

SIGNIFICANCE: Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction. CRITICAL ISSUES: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. FUTURE DIRECTIONS: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels.

On the pathways feeding the H2 production process in nutrient-replete, hypoxic conditions. Commentary on the article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures", by Jurado-Oller et al., Biotechnology for Biofuels, published September 7, 2015; 8:149.

BACKGROUND: Under low O2 concentration (hypoxia) and low light, Chlamydomonas cells can produce H2 gas in nutrient-replete conditions. This process is hindered by the presence of O2, which inactivates the [FeFe]-hydrogenase enzyme responsible for H2 gas production shifting algal cultures back to normal growth. The main pathways accounting for H2 production in hypoxia are not entirely understood, as much as culture conditions setting the optimal redox state in the chloroplast supporting long-lasting H2 production. The reducing power for H2 production can be provided by photosystem II (PSII) and photofermentative processes during which proteins are degraded via yet unknown pathways. In hetero- or mixotrophic conditions, acetate respiration was proposed to indirectly contribute to H2 evolution, although this pathway has not been described in detail. MAIN BODY: Recently, Jurado-Oller et al. (Biotechnol Biofuels 8: 149, 7) proposed that acetate respiration may substantially support H2 production in nutrient-replete hypoxic conditions. Addition of low amounts of O2 enhanced acetate respiration rate, particularly in the light, resulting in improved H2 production. The authors surmised that acetate oxidation through the glyoxylate pathway generates intermediates such as succinate and malate, which would be in turn oxidized in the chloroplast generating FADH2 and NADH. The latter would enter a PSII-independent pathway at the level of the plastoquinone pool, consistent with the light dependence of H2 production. The authors concluded that the water-splitting activity of PSII has a minor role in H2 evolution in nutrient-replete, mixotrophic cultures under hypoxia. However, their results with the PSII inhibitor DCMU also reveal that O2 or acetate additions promoted acetate respiration over the usually dominant PSII-dependent pathway. The more oxidized state experienced by these cultures in combination with the relatively short experimental time prevented acclimation to hypoxia, thus precluding the PSII-dependent pathway from contributing to H2 production. CONCLUSIONS: In Chlamydomonas, continuous H2 gas evolution is expected once low O2 partial pressure and optimal reducing conditions are set. Under nutrient-replete conditions, the electrogenic processes involved in H2 photoproduction may rely on various electron transport pathways. Understanding how physiological conditions select for specific metabolic routes is key to achieve economic viability of this renewable energy source.