Photosynthetic efficiency of Chlamydomonas reinhardtii in attenuated, flashing light

As a result of mixing and light attenuation, algae in a photobioreactor (PBR) alternate between light and dark zones and, therefore, experience variations in photon flux density (PFD). These variations in PFD are called light/dark (L/D) cycles. The objective of this study was to determine how these L/D cycles affect biomass yield on light energy in microalgae cultivation. For our work, we used controlled, short light path, laboratory, turbidostat‐operated PBRs equipped with a LED light source for square‐wave L/D cycles with frequencies from 1 to 100 Hz. Biomass density was adjusted that the PFD leaving the PBR was equal to the compensation point of photosynthesis. Algae were acclimated to a sub‐saturating incident PFD of 220 µmol m^?2 s^?1 for continuous light. Using a duty cycle of 0.5, we observed that L/D cycles of 1 and 10 Hz resulted on average in a 10% lower biomass yield, but L/D cycles of 100 Hz resulted on average in a 35% higher biomass yield than the yield obtained in continuous light. Our results show that interaction of L/D cycle frequency, culture density and incident PFD play a role in overall PBR productivity. Hence, appropriate L/D cycle setting by mixing strategy appears as a possible way to reduce the effect that dark zone exposure impinges on biomass yield in microalgae cultivation. The results may find application in optimization of outdoor PBR design to maximize biomass yields. Biotechnol. Bioeng. 2012; 109: 2567–2574. © 2012 Wiley Periodicals, Inc.     

Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor

The aim of this study was to establish and validate a model for the photosynthetic growth of Chlamydomonas reinhardtii in photobioreactors (PBRs). The proposed model is based on an energetic analysis of the excitation energy transfer in the photosynthesis apparatus (the Z-scheme for photosynthesis). This approach has already been validated in cyanobacteria (Arthorspira platensis) and is extended here to predict the volumetric biomass productivity for the microalga C. reinhardtii in autotrophic conditions, taking into consideration the two metabolic processes taking place in this eukaryotic microorganism, namely photosynthesis and respiration. The kinetic growth model obtained was then coupled to a radiative transfer model (the two-flux model) to determine the local kinetics, and thereby the volumetric biomass productivity, in a torus PBR. The model was found to predict PBR performances accurately for a broad set of operating conditions, including both light-limited and kinetic growth regimes, with a variance of less than 10% between experimental results and simulations.     

Kinetic modeling of light limitation and sulfur deprivation effects in the induction of hydrogen production with Chlamydomonas reinhardtii: Part I. Model development and parameter identification

Chlamydomonas reinhardtii is a green microalga capable of turning its metabolism towards H2 production under specific conditions. However this H2 production, narrowly linked to the photosynthetic process, results from complex metabolic reactions highly dependent on the environmental conditions of the cells. A kinetic model has been developed to relate culture evolution from standard photosynthetic growth to H2 producing cells. It represents transition in sulfur-deprived conditions, known to lead to H2 production in Chlamydomonas reinhardtii, and the two main processes then induced which are an over-accumulation of intracellular starch and a progressive reduction of PSII activity for anoxia achievement. Because these phenomena are directly linked to the photosynthetic growth, two kinetic models were associated, the first (one) introducing light dependency (Haldane type model associated to a radiative light transfer model), the second (one) making growth a function of available sulfur amount under extracellular and intracellular forms (Droop formulation). The model parameters identification was realized from experimental data obtained with especially designed experiments and a sensitivity analysis of the model to its parameters was also conducted. Model behavior was finally studied showing interdependency between light transfer conditions, photosynthetic growth, sulfate uptake, photosynthetic activity and O2 release, during transition from oxygenic growth to anoxic H2 production conditions.     

Mechanistic modeling of sulfur‐deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii

The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron‐hydrogenase is well known. However, the oxygen‐sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo‐production. Under illumination, sulfur‐deprivation has been shown to accommodate the production of hydrogen gas by partially‐deactivating O₂ evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H₂ production during sulfur‐deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

共生真菌Simplicillium lanosoniveum促进衣藻生长和脂类合成

【背景】藻类是生产生物柴油的主要原料,而一些真菌和细菌能够与藻类共生并提高生物柴油产量,因此藻-菌共生培养技术成为国内外研究的热点。【目的】研究共生真菌Simplicilliumlanosoniveum对衣藻Chlamydomonas reinhardtii细胞生长和脂类合成的影响。【方法】将分离的蓝藻共生真菌和衣藻混合(共生)培养。【结果】与衣藻单独培养相比,混合培养衣藻的比生长速率(0.20 d-1)、细胞产率[0.17 g/(L·d)]和生物量(2.85 g/L)分别提高了10.3%、51.3%和55.7%;脂类比合成速率[0.68 mg/(g·d)]、合成速率[1.95 mg/(L·d)]和含量(220.4 mg/g)分别提高了33.3%、107.5%和32.0%,并且脂类中的饱和脂肪酸以及单不饱和脂肪酸C18-1和C18-2的比例上升,有利于生物柴油的加工。【结论】真菌Simplicilliumlanosoniveum能够促进衣藻的生长和脂类合成,因此藻-菌混合培养可用于生物柴油原料的生产。     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).     

Fate of substituted benzoates in the freshwater green alga, Chlamydomonas reinhardtii 11-32b

The removal of chlorinated, nitrated, and sulfonated benzoic acids in cultures of the unicellular green alga, Chlamydomonas reinhardtii 11-32b, was investigated, and the metabolic fate of a model compound, 4-chloro-3,5-dinitrobenzoic acid, was determined. The freshwater alga was able to remove a wide variety of benzoic compounds from the incubation medium. Chlamydomonas discriminated very specifically between the benzoic acids, indicated by the varying degrees of which the test compounds disappeared from the culture medium. Moreover, the alga was capable of transforming 4-chloro-3,5-dinitrobenzoic acid to several metabolites. A release of chloride ions was observed, and 3,5- dinitro-4-hydroxybenzoic acid was identified as a major transient product in the algal metabolism of 4-chloro-3,5-dinitrobenzoic acid.     

Interplay between light intensity,chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur‐deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 µmol photons m^?2 s^?1 in combination with chlorophyll concentrations of 12 and 24 mg L^?1. A high light intensity (140 µmol photons m^?2 s^?1, supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L^?1) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 µmol photons m^?2 s^?1 (on both sides) and 24 mg L^?1 of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L^?1 in the culture mixed with the stir bar, and rose to about 170 mL L^?1 in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50–100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process. Biotechnol. Bioeng. 2009; 104: 76–90 © 2009 Wiley Periodicals, Inc.     

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP‐glucose pyrophosphorylase. This mutant is unable to convert glucose‐1–phosphate to ADP‐glucose, the precursor of starch biosynthesis. During nutrient‐replete culturing, sta6 does not re‐direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC–MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO₂) decrease in sta6 due to attenuated rates of NADPH re‐oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system‐wide consequences of slower NADPH re‐oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high‐light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient‐replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.     

Treatment with NaHSO3 greatly enhances photobiological H2 production in the green alga Chlamydomonas reinhardtii

Treatment with NaHSO₃ induces a 10-fold increase in H₂ photoproduction in the filamentous N₂-fixing cyanobacterium Anabaena sp. strain PCC 7120. However, it is unclear whether this treatment also increases H₂ photoproduction in green alga. In this study, treatment with 13 mM NaHSO₃ resulted in about a 200-fold increase in H₂ production in Chlamydomonas reinhardtii, and this increase was most probably the result of reduced O₂ content and enhanced hydrogenase activity. Compared to the conventional strategy of sulfur deprivation, NaHSO₃ treatment results in a higher maximum rate of H₂ photoproduction, greater efficiency of conversion of light energy into H₂, shorter half-time to produce the maximum accumulated H₂ levels, and reduced costs because no centrifugation is involved. We therefore conclude that NaHSO₃ treatment is an efficient, rapid, and economic strategy for improving photobiological H₂ production in the green alga C. reinhardtii.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Effects of doubled atmospheric CO2 concentration on the growth and photosynthesis of Chlamydomonas reinhardtii (Volvocales, Chlorophyceae)

The freshwater microalga, Chlamydomonas reinhardtii Dangeard, was cultured under 350 and 700 ppmv CO₂ to determine the impact of doubled atmospheric CO₂ concentration on its growth and photosynthesis. No significant difference was observed in the specific growth rate, photosynthetic efficiency, maximal net photo‐synthetic rate and light‐saturating point between the low and high CO₂ cultures. Both the low‐ and high‐CO₂‐grown cells showed reduced light‐dependent O₂ evolution rate and photochemical efficiency (F_v/F_m) owing to photoinhibition when exposed to high photon flux density. However, high‐CO₂‐grown cells were less photoinhibited, and showed better recovery in dim light or darkness during the initial period of the recovery process.     

Seasonal and diel changes in photosynthetic activity of the snow alga Chlamydomonas nivalis (Chlorophyceae) from Svalbard determined by pulse amplitude modulation fluorometry

The seasonal and diel dynamics of the physiological state and photosynthetic activity of the snow alga Chlamydomonas nivalis were investigated in a snowfield in Svalbard. The snow surface represents an environment with very high irradiation intensities along with stable low temperatures close to freezing point. Photosynthetic activity was measured using pulse amplitude modulation fluorometry. Three types of cell (green biflagellate vegetative cells, orange spores clustered by means of mucilaginous sheaths, and purple spores with thick cell walls) were found, all of them photosynthetically active. The pH of snow ranged between 5.0 and 7.5, and the conductivity ranged between 5 and 75 microS cm(-1). The temperature of snow was stable (-0.1 to +0.1 degrees C), and the incident radiation values ranged from 11 to 1500 micromol photons m(-2) s(-1). The photosynthetic activity had seasonal and diel dynamics. The Fv/Fm values ranged between 0.4 and 0.7, and generally declined over the course of the season. A dynamic response of Fv/Fm to the irradiance was recorded. According to the saturating photon fluence values Ek, the algae may have obtained saturating light as deep as 3 cm in the snow when there were higher-light conditions, whereas they were undersaturated at prevalent low light even if on the surface.     

Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii

Eukaryotic microalgae serve as indicators of environmental change when exposed to severe seasonal fluctuations. Several environmental stress conditions are known to produce reactive oxygen species in cellular compartments, resulting in oxidative damage and apoptosis. The study of cell death in higher plants and animals has revealed the existence of an active ‘programmed cell death’ (PCD) process and similarities between such processes suggest an evolutionary origin. A study was undertaken to examine the morphological, biochemical and molecular responses of the unicellular green alga Chlamydomonas reinhardtii after exposure to oxidative (10 mM H₂O₂) and osmotic (200 mM NaCl and 360 mM sorbitol) stress. Concentrations of H₂O₂ (2–50 mM), NaCl and sorbitol (100–800 mM) were negatively correlated with growth. Biochemical analyses showed an increase in intracellular H₂O₂ production (2.2-fold with H₂O₂ and ~1.2–1.4-fold with NaCl and sorbitol) and activities of some antioxidant enzymes [super oxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)]. Alteration of mitochondrial membrane potential (MMP) was observed upon treatment with H₂O₂ and NaCl, but not with sorbitol, indicating that the ionic stress component of NaCl altered the MMP. In addition, H₂O₂ led to the activation of a caspase-3-like protein, increase in the cleavage of a poly(ADP) ribose polymerase-1 (PARP-1)-like enzyme and formation of DNA nicks and laddering. With NaCl and sorbitol, no caspase activation, nor oligonucleosomal DNA laddering was observed, indicating non-apoptotic death. However, genomic DNA of NaCl (800 mM)-stressed cells, but not those of sorbitol-treated cells showed complete shearing. We conclude that the ionic rather than the osmotic component of NaCl leads to necrosis. These results unequivocally suggest that the vegetative cells of C. reinhardtii respond differentially to various stress agents, leading to different death types in the same organism. Moreover, unlike most other organisms, when exposed to NaCl this alga does not undergo PCD.     

Nitric oxide production mediated by nitrate reductase in the green alga Chlamydomonas reinhardtii: an alternative NO production pathway in photosynthetic organisms

Biological activity of nitric oxide (NO) production was investigated in the unicellular green alga Chlamydomonas reinhardtii. An NO specific electrode detected a rapid increase in signal when nitrite (NO(2)(-)) was added into a suspension of C. reinhardtii intact cells in the dark. The addition of KCN or the NO quencher bovine hemoglobin completely abolished the signal, verifying that the nitrite-dependent increase in signal is due to enzymatic NO production. L-arginine, the substrate for NO synthase, did not induce detectable NO production and the NOS inhibitor N(omega)-nitro-L-arginine showed no inhibitory effect on the nitrite-dependent production of NO. Illuminating cells showed a significant suppressive effect on NO production. When the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea was present in the suspension, C. reinhardtii cells produced NO after the addition of nitrite even under illumination. Kinetic and microscopic observations, using the intracellular fluorescent NO probe 4,5-diaminofluorescein-2 diacetate, both demonstrated that NO was produced within the cells in response to the addition of nitrite. The Chlamydomonas mutant cc-2929, which lacks nitrate reductase (NR) activity, did not display any of the responses observed in the wild-type cells. The results presented here provide direct in vivo evidence to confirm that NR is involved in the nitrite-dependent NO production in the green alga.     

Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light‐driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light‐harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI‐bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.     

The influence of zinc and light conditions on the cadmium-repressed growth of the green alga Coelastrum proboscideum

The growth response of Coelastrum proboscideum Bohlin to cadmiun (3 × 10^?9M to 2.4 × 10^?7M) was studied. The inorganic media used varied in zinc concentration (1.3 × 10^?7M to 3.6 × 10^?6M). The data were evaluated by factorial analyses. The influence of zine on the growth depression by cadmium depended on the light conditions (16:8 h light:dark cycles or 24 h continuous illumination periods). Intermittent illumination caused a negative interaction of zinc and cadmium in contrast to a positive interaction or additive effects of these elements during continuous illumination.     

The CO2-concentrating mechanism is absent in the green alga Coccomyxa: a comparative study of photosynthetic CO2 and light responses of Coccomyxa, Chlamydomonas reinhardtii and barley protoplasts

Photosynthesis was characterized for the unicellular green alga Coccomyxa sp., grown at low inorganic carbon (C_i) concentrations, and compared with Chlamydomonas reinhardtii, which had been grown so that the CO₂ concentrating mechanism (CCM) was expressed, and with protoplasts isolated from the C₃ plant barley (Hordeum vulgare). Chlamydomonas had a significantly higher C_i-use efficiency of photosynthesis, with an initial slope of the C_i-response curve of 0.7 mol(gChl)⁻¹ h⁻¹ mmol C_im⁻³)⁻¹, as compared to 0.3 and 0.23 mol(gChl)⁻¹ h⁻¹ (mmol C_im⁻³)⁻¹ for Coccomyxa and barley, respectively. The affinity for C_i was also higher in Chlamydomonas, as the half maximum rate of photosynthesis [K_0.5 (C_i)] was reached at 0.18 mol m⁻³, as compared to 0.30 and 0.45 mol m⁻³ for Coccomyxa and barley, respectively. Ethoxyzolamide (EZ), an inhibitor of the enzyme carbonic anhydrase (CA) and the CCM, caused a 17-fold decrease in the initial slope of the photosynthetic Cj-response curve in Chlamydomonas, but only a 1.5- to two-fold decrease in Coccomyxa and barley. The photosynthetic light-response curve showed further similarities between barley and Coccomyxa. The rate of bending of the curve, described by the convexity parameter, was 0.99 (sharp bending) and 0.81–0.83 (gradual bending) for cells grown under low and high light, respectively. In contrast, the maximum convexity of Chlamydomonas was 0.85. The intrinsically lower convexity of Chlamydomonas is suggested to result from the diversion of electron transport from carbon fixation to the CCM. Taken together, these results suggest that Coccomyxa does not possess a CCM and due to this apparent lack of a CCM, we propose that Coccomyxa is a better cell model system for studying C₃ plant photosynthesis than many algae currently used.     

Kinetic modeling of light limitation and sulfur deprivation effects in the induction of hydrogen production with Chlamydomonas reinhardtii. Part II: Definition of model‐based protocols and experimental validation

Photosynthetic hydrogen production under light by the green microalga Chlamydomonas reinhardtii was investigated in a torus‐shaped PBR in sulfur‐deprived conditions. Culture conditions, represented by the dry biomass concentration of the inoculum, sulfate concentration, and incident photon flux density (PFD), were optimized based on a previously published model (Fouchard et al., 2009. Biotechnol Bioeng 102:232–245). This allowed a strictly autotrophic production, whereas the sulfur‐deprived protocol is usually applied in photoheterotrophic conditions. Experimental results combined with additional information from kinetic simulations emphasize effects of sulfur deprivation and light attenuation in the PBR in inducing anoxia and hydrogen production. A broad range of PFD was tested (up to 500 µmol photons m⁻² s⁻¹). Maximum hydrogen productivities were 1.0 ± 0.2 mL H₂/h/L (or 25 ± 5 mL H₂/m² h) and 3.1 mL ± 0.4 H₂/h L (or 77.5 ± 10 mL H₂/m² h), at 110 and 500 µmol photons m⁻² s⁻¹, respectively. These values approached a maximum specific productivity of approximately 1.9 mL ± 0.4 H₂/h/g of biomass dry weight, clearly indicative of a limitation in cell capacity to produce hydrogen. The efficiency of the process and further optimizations are discussed. Biotechnol. Bioeng. 2011;108: 2288–2299. © 2011 Wiley Periodicals, Inc.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.