Heterotrophy Compared to Photoautotrophy for Growth Characteristics and Pigment Compositions in Batch Cultures of Four Green Microalgae.

Four strains of green microalgae (Scenedesmus acutus, Scenedesmus vacuolatus, Chlorella sorokiniana, and Chlamydomonas reinhardtii) were compared to determine growth and pigment composition under photoautotrophic or heterotrophic conditions. Batch growth experiments were performed in multicultivators with online monitoring of optical density. For photoautotrophic growth, light-limited (CO2-sufficient) growth was analyzed under different light intensities during the exponential and deceleration growth phases. The specific growth rate, measured during the exponential phase, and the maximal biomass productivity, measured during the deceleration phase, were not related to each other when different light intensities and different species were considered. This indicates species-dependent photoacclimation effects during cultivation time, which was confirmed by light-dependent changes in pigment content and composition when exponential and deceleration phases were compared. Except for C. reinhardtii, which does not grow on glucose, heterotrophic growth was promoted to similar extents by acetate and by glucose; however, these two substrates led to different pigment compositions. Weak light increased the pigment content during heterotrophy in the four species but was efficient in promoting growth only in S. acutus. C. sorokiniana, and S. vacuolatus exhibited the best potential for heterotrophic biomass productivities, both on glucose and acetate, with carotenoid (lutein) content being the highest in the former.

A Fast-Growing Oleaginous Strain of Coelastrella Capable of Astaxanthin and Canthaxanthin Accumulation in Phototrophy and Heterotrophy.

Considering the importance of microalgae as a promising feedstock for the production of both low- and high-value products, such as lipids and pigments, it is desirable to isolate strains which simultaneously accumulate these two types of products and grow in various conditions in order to widen their biotechnological applicability. A novel freshwater strain from the genus Coelastrella was isolated in Belgium. Compared to other Coelastrella species, the isolate presented rapid growth in phototrophy, dividing 3.5 times per day at a light intensity of 400 µmol·m-2·s-1 and 5% CO2. In addition, nitrogen depletion was associated with the accumulation of astaxanthin, canthaxanthin, and fatty acids, which reached ~30% of dry weight, and a majority of SFAs and MUFAs, which are good precursors for biodiesel. This strain also accumulated astaxanthin and canthaxanthin in heterotrophy. Although the content was very low in this latter condition, it is an interesting feature considering the biotechnological potential of the microalgal heterotrophic growth. Thus, due to its rapid growth in the light, its carotenogenesis, and its fatty acids characteristics, the newly identified Coelastrella strain could be considered as a potential candidate for biorefinery purposes of both low- and high-values products.

New Features on the Environmental Regulation of Metabolism Revealed by Modeling the Cellular Proteomic Adaptations Induced by Light, Carbon, and Inorganic Nitrogen in Chlamydomonas reinhardtii.

Microalgae are currently emerging to be very promising organisms for the production of biofuels and high-added value compounds. Understanding the influence of environmental alterations on their metabolism is a crucial issue. Light, carbon and nitrogen availability have been reported to induce important metabolic adaptations. So far, the influence of these variables has essentially been studied while varying only one or two environmental factors at the same time. The goal of the present work was to model the cellular proteomic adaptations of the green microalga Chlamydomonas reinhardtii upon the simultaneous changes of light intensity, carbon concentrations (CO2 and acetate), and inorganic nitrogen concentrations (nitrate and ammonium) in the culture medium. Statistical design of experiments (DOE) enabled to define 32 culture conditions to be tested experimentally. Relative protein abundance was quantified by two dimensional differential in-gel electrophoresis (2D-DIGE). Additional assays for respiration, photosynthesis, and lipid and pigment concentrations were also carried out. A hierarchical clustering survey enabled to partition biological variables (proteins + assays) into eight co-regulated clusters. In most cases, the biological variables partitioned in the same cluster had already been reported to participate to common biological functions (acetate assimilation, bioenergetic processes, light harvesting, Calvin cycle, and protein metabolism). The environmental regulation within each cluster was further characterized by a series of multivariate methods including principal component analysis and multiple linear regressions. This metadata analysis enabled to highlight the existence of a clear regulatory pattern for every cluster and to mathematically simulate the effects of light, carbon, and nitrogen. The influence of these environmental variables on cellular metabolism is described in details and thoroughly discussed. This work provides an overview of the metabolic adaptations contributing to maintain cellular homeostasis upon extensive environmental changes. Some of the results presented here could be used as starting points for more specific fundamental or applied investigations.

The chlorophyll a fluorescence induction curve in the green microalga Haematococcus pluvialis: further insight into the nature of the P-S-M fluctuation and its relationship with the "low-wave" phenomenon at steady-state.

Chlorophyll fluorescence is an information-rich signal which provides an access to the management of light absorbed by PSII. A good example of this is the succession of fast fluorescence fluctuations during light-induced photosynthetic induction after dark-adaptation. During this period, the fluorescence trace exhibits several inflexion points: O-J-I-P-S-M-T. Whereas the OJIP part of this kinetics has been the subject of many studies, the processes that underly the PSMT transient are less understood. Here, we report an analysis of the PSMT phase in the green microalga Haematococcus pluvialis in terms of electron acceptors and light use by photochemistry, fluorescence and non-photochemical quenching (NPQ). We identify additional sub-phases between P and S delimited by an inflexion point, that we name Q, found in the second time scale. The P-Q phase expresses a transient photochemical quenching specifically due to alternative electron transport to oxygen. During the transition from Q to S, the NPQ increases and then relaxes during the S-M phase in about 1 min. It is suggested that this transient NPQ observed during induction is a high energy state quenching (qE) dependent on the alternative electron transport to molecular oxygen. We further show that this NPQ is of the same nature than the NPQ, known as the low-wave phenomenon, which is transiently observed after a saturating light pulse given at steady-state. In both cases, the NPQ is oxygen-dependent. This NPQ is observed at external pH 6.0, but not at pH 7.5, which seems correlated with faster saturation of the PQ pool at pH 6.0.

Photosynthesis of Scenedesmus obliquus in outdoor open thin-layer cascade system in high and low CO2 in Belgium.

Two outdoor open thin-layer cascade systems operated as batch cultures with the alga Scenedesmus obliquus were used to compare the productivity and photosynthetic acclimations in control and CO2 supplemented cultures in relation with the outdoor light irradiance. We found that the culture productivity was limited by CO2 availability. In the CO2 supplemented culture, we obtained a productivity of up to 24gdwm(-2)day(-1) and found a photosynthetic efficiency (value based on the PAR solar radiation energy) of up to 5%. In the CO2 limited culture, we obtained a productivity of up to 10gdwm(-2)day(-1) while the photosynthetic efficiency was up to 3.3% and decreased to 2.1% when the integrated daily PAR increased. Fluorescence and oxygen evolution measurements showed that ETR and oxygen evolution light saturation curves, as well as light-dependent O2 uptake were similar in algal samples from both cultures when the CO2 limitation was removed. In contrast, we found that CO2 limitation conducted to a decreased PSII photochemical efficiency and an increased light-induced heat-dissipation in the control culture compared to the CO2 supplemented culture. These features are in line with a lower light use efficiency and may therefore contribute to the lower productivity observed in absence of CO2 supplementation in outdoor mass cultures of S. obliquus.

Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009.

BACKGROUND: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. RESULTS: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. CONCLUSIONS: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Modeling the dependence of respiration and photosynthesis upon light, acetate, carbon dioxide, nitrate and ammonium in Chlamydomonas reinhardtii using design of experiments and multiple regression.

BACKGROUND: In photosynthetic organisms, the influence of light, carbon and inorganic nitrogen sources on the cellular bioenergetics has extensively been studied independently, but little information is available on the cumulative effects of these factors. Here, sequential statistical analyses based on design of experiments (DOE) coupled to standard least squares multiple regression have been undertaken to model the dependence of respiratory and photosynthetic responses (assessed by oxymetric and chlorophyll fluorescence measurements) upon the concomitant modulation of light intensity as well as acetate, CO2, nitrate and ammonium concentrations in the culture medium of Chlamydomonas reinhardtii. The main goals of these analyses were to explain response variability (i.e. bioenergetic plasticity) and to characterize quantitatively the influence of the major explanatory factor(s). RESULTS: For each response, 2 successive rounds of multiple regression coupled to one-way ANOVA F-tests have been undertaken to select the major explanatory factor(s) (1st-round) and mathematically simulate their influence (2nd-round). These analyses reveal that a maximal number of 3 environmental factors over 5 is sufficient to explain most of the response variability, and interestingly highlight quadratic effects and second-order interactions in some cases. In parallel, the predictive ability of the 2nd-round models has also been investigated by k-fold cross-validation and experimental validation tests on new random combinations of factors. These validation procedures tend to indicate that the 2nd-round models can also be used to predict the responses with an inherent deviation quantified by the analytical error of the models. CONCLUSIONS: Altogether, the results of the 2 rounds of modeling provide an overview of the bioenergetic adaptations of C. reinhardtii to changing environmental conditions and point out promising tracks for future in-depth investigations of the molecular mechanisms underlying the present observations.

Liver proteomic response to hypertriglyceridemia in human-apolipoprotein C-III transgenic mice at cellular and mitochondrial compartment levels.

BACKGROUND: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the mitochondrial, sub-mitochondrial and cellular proteomic impact of hypertriglyceridemia in the hepatocytes of hypertriglyceridemic transgenic mice (overexpressing the human apolipoproteinC-III). METHODS: Quantitative proteomics (2D-DIGE) analysis was carried out on both "low-expressor" (LE) and "high-expressor" (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. RESULTS: The mitoproteome analysis has revealed a large-scale phenomenon in transgenic mice, i.e. a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. These data also demonstrate that the magnitude of proteomic changes strongly depends on the TG plasma level. Our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for TG de novo biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes has also been observed, suggesting that the cytoplasm of HTG mice is subjected to oxidative stress. Moreover, our results suggest that iron over-accumulation takes place in the cytosol of HE mice hepatocytes and may contribute to enhance oxidative stress and to promote cellular proliferation. CONCLUSIONS: These results indicate that the metabolic response to HTG in human apolipoprotein C-III overexpressing mice may support a high TG production rate and that the cytosol of hepatocytes is subjected to an important oxidative stress, probably as a result of FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes.

PSI Mehler reaction is the main alternative photosynthetic electron pathway in Symbiodinium sp., symbiotic dinoflagellates of cnidarians.

Photosynthetic organisms have developed various photoprotective mechanisms to cope with exposure to high light intensities. In photosynthetic dinoflagellates that live in symbiosis with cnidarians, the nature and relative amplitude of these regulatory mechanisms are a matter of debate. In our study, the amplitude of photosynthetic alternative electron flows (AEF) to oxygen (chlororespiration, Mehler reaction), the mitochondrial respiration and the Photosystem I (PSI) cyclic electron flow were investigated in strains belonging to three clades (A1, B1 and F1) of Symbiodinium. Cultured Symbiodinium strains were maintained under identical environmental conditions, and measurements of oxygen evolution, fluorescence emission and absorption changes at specific wavelengths were used to evaluate PSI and PSII electron transfer rates (ETR). A light- and O2 -dependent ETR was observed in all strains. This electron transfer chain involves PSII and PSI and is insensitive to inhibitors of mitochondrial activity and carbon fixation. We demonstrate that in all strains, the Mehler reaction responsible for photoreduction of oxygen by the PSI under high light, is the main AEF at the onset and at the steady state of photosynthesis. This sustained photosynthetic AEF under high light intensities acts as a photoprotective mechanism and leads to an increase of the ATP/NADPH ratio.

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth.

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO2 is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹4N/¹5N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in ß-oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.

Analysis of PSII antenna size heterogeneity of Chlamydomonas reinhardtii during state transitions.

PSII antenna size heterogeneity has been intensively studied in the past. Based on DCMU fluorescence rise kinetics, multiple types of photosystems with different properties were described. However, due to the complexity of fluorescence signal analysis, multiple questions remain unanswered. The number of different types of PSII is still debated as well as their degree of connectivity. In Chlamydomonas reinhardtii we found that PSIIα possesses a high degree of connectivity and an antenna 2-3 times larger than PSIIß, as described previously. We also found some connectivity for PSIIß in contrast with the majority of previous studies. This is in agreement with biochemical studies which describe PSII mega-, super- and core-complexes in Chlamydomonas. In these studies, the smallest unit of PSII in vivo would be a dimer of two core complexes hence allowing connectivity. We discuss the possible relationships between PSIIα and PSIIß and the PSII mega-, super- and core-complexes. We also showed that strain and medium dependent variations in the half-time of the fluorescence rise can be explained by variations in the proportions of PSIIα and PSIIß. When analyzing the state transition process in vivo, we found that this process induces an inter-conversion of PSIIα and PSIIß. During a transition from state 2 to state 1, DCMU fluorescence rise kinetics are satisfactorily fitted by considering two PSII populations with constant kinetic parameters. We discuss our findings about PSII heterogeneity during state transitions in relation with recent results on the remodeling of the pigment-protein PSII architecture during this process.

Function of the chloroplast hydrogenase in the microalga Chlamydomonas: the role of hydrogenase and state transitions during photosynthetic activation in anaerobiosis.

Like a majority of photosynthetic microorganisms, the green unicellular alga Chlamydomonas reinhardtii may encounter O2 deprived conditions on a regular basis. In response to anaerobiosis or in a respiration defective context, the photosynthetic electron transport chain of Chlamydomonas is remodeled by a state transition process to a conformation that favours the photoproduction of ATP at the expense of reductant synthesis. In some unicellular green algae including Chlamydomonas, anoxia also triggers the induction of a chloroplast-located, oxygen sensitive hydrogenase, which accepts electrons from reduced ferredoxin to convert protons into molecular hydrogen. Although microalgal hydrogen evolution has received much interest for its biotechnological potential, its physiological role remains unclear. By using specific Chlamydomonas mutants, we demonstrate that the state transition ability and the hydrogenase function are both critical for induction of photosynthesis in anoxia. These two processes are thus important for survival of the cells when they are transiently placed in an anaerobic environment.

A dual strategy to cope with high light in Chlamydomonas reinhardtii.

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition-deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.

Function of the chloroplastic NAD(P)H dehydrogenase Nda2 for H2 photoproduction in sulphur-deprived Chlamydomonas reinhardtii.

The relative contributions of the PSII-dependent and Nda2-dependent pathways for H2 photoproduction were investigated in the green microalga Chlamydomonas reinhardtii after suphur-deprivation. For this purpose, H2 gas production was compared for wild-type and Nda2-deficient cells with or without DCMU (a PSII-inhibitor) in the same experimental conditions. Nda2-deficiency caused a 30% decrease of the maximal H2 photoevolution rate observed shortly after the establishment of anoxia, and an acceleration of the decline of H2 photoevolution rate with time. DCMU addition to Nda2-deficient cells completely inhibited H2 photoproduction, showing that the PSII-independent H2 photoproduction relies on the presence of Nda2, which feeds the photosynthetic electron transport chain with electrons derived from oxidative catabolism. Nda2-protein abundance increased as a result of sulphur deprivation and further during the H2 photoproduction process, resulting in high rates of non-photochemical plastoquinone reduction in control cells. Nda2-deficiency had no significant effect on photosynthetic and respiratory capacities in sulphur-deprived cells, but caused changes in the cell energetic status (ATP and NADPH/NADP+ ratio). The rapid decline of H2 photoevolution rate with time in Nda2-deficient cells revealed a more pronounced inhibition of H2 photoproduction by accumulated H2 in the absence of non-photochemical plastoquinone reduction. Nda2 is therefore important for linking H2 photoproduction with catabolism of storage carbon compounds, and seems also involved in regulating the redox poise of the photosynthetic electron transport chain during H2 photoproduction.

Isolation and partial characterization of mutants with elevated lipid content in Chlorella sorokiniana and Scenedesmus obliquus.

This paper describes the isolation and partial biomass characterization of high triacylglycerol (TAG) mutants of Chlorella sorokiniana and Scenedesmus obliquus, two algal species considered as potential source of biodiesel. Following UV mutagenesis, 2000 Chlorella and 2800 Scenedesmus colonies were screened with a method based on Nile Red fluorescence. Several mutants with high Nile Red fluorescence were selected by this high-throughput method in both species. Growth and biomass parameters of the strongest mutants were analyzed in detail. All of the four Chlorella mutants showed no significant changes in growth rate, cell weight, cell size, protein and chlorophyll contents on a per cell basis. Whereas all contained elevated total lipid and TAG content per unit of dry weight, two of them were also affected for starch metabolism, suggesting a change in biomass/storage carbohydrate composition. Two Scenedesmus mutants showed a 1.5 and 2-fold increased cell weight and larger cells compared to the wild type, which led to a general increase of biomass including total lipid and TAG content on a per cell basis. Such mutants could subsequently be used as commercial oleaginous algae and serve as an alternative to conventional petrol.

Regulation of the pigment optical density of an algal cell: filling the gap between photosynthetic productivity in the laboratory and in mass culture.

An increasing number of investors is looking at algae as a viable source of biofuels, beside cultivation for human/animal feeding or to extract high-value chemicals and pharmaceuticals. However, present biomass productivities are far below theoretical estimations implying that a large part of the available photosynthetically active radiation is not used in photosynthesis. Light utilisation inefficiency and rapid light attenuation within a mass culture due to high pigment optical density of wild type strains have been proposed as major limiting factors reducing solar-to-biomass conversion efficiency. Analysis of growth yields of mutants with reduced light-harvesting antennae and/or reduced overall pigment concentration per cell, generated by either mutagenesis or genetic engineering, could help understanding limiting factors for biomass accumulation in photobioreactor. Meanwhile, studies on photo-acclimation can provide additional information on the average status of algal cells in a photobioreactor to be used in modelling-based predictions. Identifying limiting factors in solar-to-biomass conversion efficiency is the first step for planning strategies of genetic improvement and domestication of algae to finally fill the gap between theoretical and industrial photosynthetic productivity.

Interplay between non-photochemical plastoquinone reduction and re-oxidation in pre-illuminated Chlamydomonas reinhardtii: a chlorophyll fluorescence study.

In photosynthetic eukaryotes, the redox state of the plastoquinone (PQ) pool is an important sensor for mechanisms that regulate the photosynthetic electron transport. In higher plants, a multimeric nicotinamide adenine dinucleotide (phosphate) (NAD(P))H dehydrogenase (NDH) complex and a plastid terminal oxidase (PTOX) are involved in PQ redox homeostasis in the dark. We recently demonstrated that in the microalgae Chlamydomonas reinhardtii, which lacks the multimeric NDH complex of higher plants, non-photochemical PQ reduction is mediated by a monomeric type-II NDH (Nda2). In this study, we further explore the nature and the importance of non-photochemical PQ reduction and oxidation in relation to redox homeostasis in this alga by recording the 'dark' chlorophyll fluorescence transients of pre-illuminated algal samples. From the observation that this fluorescence transient is modified by addition of propyl gallate, a known inhibitor of PTOX, and in a Nda2-deficient strain we conclude that it reflects post-illumination changes in the redox state of PQ resulting from simultaneous PTOX and Nda2 activity. We show that the post-illumination fluorescence transient can be used to monitor changes in the relative rates of the non-photochemical PQ reduction and reoxidation in response to different physiological situations. We study this fluorescence transient in algae acclimated to high light and in a mutant deficient in mitochondrial respiration. Some of our observations indicate that the chlororespiratory pathway participates in redox homeostasis in C. reinhardtii.

Hydrogen photo-evolution upon S deprivation stepwise: an illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H(2) production.

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidize reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago, Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. in Plant Physiol 122:127-136, 2000). This observation boosted research in algal H(2) evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H(2) generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H(2) production procedures. Ten years later most of the critical steps and limitations of H(2) production by this protocol have been studied from different angles particularly with the model organism Chlamydomonas reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review.

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.