Bioinspired mechanically stable all-polysaccharide based scaffold for photosynthetic production.

We demonstrate the construction of water-stable, biocompatible and self-standing hydrogels as scaffolds for the photosynthetic production of ethylene using a bioinspired all-polysaccharidic design combining TEMPO-oxidised cellulose nanofibers (TCNF) and a cereal plant hemicellulose called mixed-linkage glucan (MLG). We compared three different molecular weight MLGs from barley to increase the wet strength of TCNF hydrogels, and to reveal the mechanisms defining the favourable interactions between the scaffold components. The interactions between MLGs and TCNF were revealed via adsorption studies and interfacial rheology investigations using quartz crystal microbalance with dissipation monitoring (QCM-D). Our results show that both the MLG solution stability and adsorption behaviour did not exactly follow the well-known polymer adsorption and solubility theories especially in the presence of co-solute ions, in this case nitrates. We prepared hydrogel scaffolds for microalgal immobilisation, and high wet strength hydrogels were achieved with very low dosages of MLG (0.05 wt%) to the TCNF matrix. The all-polysaccharic biocatalytic architectures remained stable and produced ethylene for 120 h with yields comparable to the state-of-the-art scaffolds. Due to its natural origin and biodegradability, MLG offers a clear advantage in comparison to synthetic scaffold components, allowing the mechanical properties and water interactions to be tailored.

Mapping Nanocellulose- and Alginate-Based Photosynthetic Cell Factory Scaffolds: Interlinking Porosity, Wet Strength, and Gas Exchange.

To develop efficient solid-state photosynthetic cell factories for sustainable chemical production, we present an interdisciplinary experimental toolbox to investigate and interlink the structure, operative stability, and gas transfer properties of alginate- and nanocellulose-based hydrogel matrices with entrapped wild-type Synechocystis PCC 6803 cyanobacteria. We created a rheological map based on the mechanical performance of the hydrogel matrices. The results highlighted the importance of Ca2+-cross-linking and showed that nanocellulose matrices possess higher yield properties, and alginate matrices possess higher rest properties. We observed higher porosity for nanocellulose-based matrices in a water-swollen state via calorimetric thermoporosimetry and scanning electron microscopy imaging. Finally, by pioneering a gas flux analysis via membrane-inlet mass spectrometry for entrapped cells, we observed that the porosity and rigidity of the matrices are connected to their gas exchange rates over time. Overall, these findings link the dynamic properties of the life-sustaining matrix to the performance of the immobilized cells in tailored solid-state photosynthetic cell factories.

Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments.

The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.

Photosynthetically produced sucrose by immobilized Synechocystis sp. PCC 6803 drives biotransformation in E. coli.

BACKGROUND: Whole-cell biotransformation is a promising emerging technology for the production of chemicals. When using heterotrophic organisms such as E. coli and yeast as biocatalysts, the dependence on organic carbon source impairs the sustainability and economic viability of the process. As a promising alternative, photosynthetic cyanobacteria with low nutrient requirements and versatile metabolism, could offer a sustainable platform for the heterologous production of organic compounds directly from sunlight and CO2. This strategy has been applied for the photoautotrophic production of sucrose by a genetically engineered cyanobacterium, Synechocystis sp. PCC 6803 strain S02. As the key concept in the current work, this can be further used to generate organic carbon compounds for different heterotrophic applications, including for the whole-cell biotransformation by yeast and bacteria. RESULTS: Entrapment of Synechocystis S02 cells in Ca2+-cross-linked alginate hydrogel beads improves the specific sucrose productivity by 86% compared to suspension cultures during 7 days of cultivation under salt stress. The process was further prolonged by periodically changing the medium in the vials for up to 17 days of efficient production, giving the final sucrose yield slightly above 3000 mg l-1. We successfully demonstrated that the medium enriched with photosynthetically produced sucrose by immobilized Synechocystis S02 cells supports the biotransformation of cyclohexanone to ε-caprolactone by the E. coli WΔcscR Inv:Parvi strain engineered to (i) utilize low concentrations of sucrose and (ii) perform biotransformation of cyclohexanone to ε-caprolactone. CONCLUSION: We conclude that cell entrapment in Ca2+-alginate beads is an effective method to prolong sucrose production by the engineered cyanobacteria, while allowing efficient separation of the cells from the medium. This advantage opens up novel possibilities to create advanced autotroph-heterotroph coupled cultivation systems for solar-driven production of chemicals via biotransformation, as demonstrated in this work by utilizing the photosynthetically produced sucrose to drive the conversion of cyclohexanone to ε-caprolactone by engineered E. coli.

Photosynthetic hydrogen production: Novel protocols, promising engineering approaches and application of semi-synthetic hydrogenases.

Photosynthetic production of molecular hydrogen (H2 ) by cyanobacteria and green algae is a potential source of renewable energy. These organisms are capable of water biophotolysis by taking advantage of photosynthetic apparatus that links water oxidation at Photosystem II and reduction of protons to H2 downstream of Photosystem I. Although the process has a theoretical potential to displace fossil fuels, photosynthetic H2 production in its current state is not yet efficient enough for industrial applications due to a number of physiological, biochemical, and engineering barriers. This article presents a short overview of the metabolic pathways and enzymes involved in H2 photoproduction in cyanobacteria and green algae and our present understanding of the mechanisms of this process. We also summarize recent advances in engineering photosynthetic cell factories capable of overcoming the major barriers to efficient and sustainable H2 production.

Electrospinning of Electroconductive Water-Resistant Nanofibers of PEDOT-PSS, Cellulose Nanofibrils and PEO: Fabrication, Characterization, and Cytocompatibility.

Electrically conductive composite nanofibers were fabricated using poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT-PSS) and cellulose nanofibrils (CNFs) via the electrospinning technique. Poly(ethylene oxide) (PEO) was used to assist the electrospinning process, and poly(ethylene glycol) diglycidyl ether was used to induce chemical cross-linking, enabling stability of the formed fibrous mats in water. The experimental parameters regarding the electrospinning polymer dispersion and electrospinning process were carefully studied to achieve a reproducible method to obtain bead-free nanofibrous mats with high stability after water contact, with an electrical conductivity of 13 ± 5 S m-1, thus making them suitable for bioelectrochemical applications. The morphology of the electrospun nanofibers was characterized by scanning electron microscopy, and the C/S ratio was determined with energy dispersive X-ray analysis. Cyclic voltammetric studies showed that the PEDOT-PSS/CNF/PEO composite fibers exhibited high electroactivity and high stability in water for at least two months. By infrared spectroscopy, the slightly modified fiber morphology after water contact was demonstrated to be due to dissolution of some part of the PEO in the fiber structure. The biocompatibility of the PEDOT-PSS/CNF/PEO composite fibers when used as an electroconductive substrate to immobilize microalgae and cyanobacteria in a photosynthetic bioelectrochemical cell was also demonstrated.

Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae.

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H2 yield.

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.

Immobilized heterocysts as microbial factories for sustainable nitrogen fixation.

A novel thin-layer biocatalyst for photosynthetic N2 fixation and H2 photoproduction was assembled using a Ca2+-alginate matrix and heterocysts isolated from wild-type Anabaena sp. PCC 7120 filaments. Compared to suspension heterocysts, heterocysts entrapped in Ca2+-alginate films showed improved stability of the nitrogenase system. While suspension heterocysts lost nitrogenase activity within 24 h, immobilized heterocysts supported nitrogenase activity for up to 125 h. The maximum specific rate of acetylene reduction was the same in both cases (∼0.4 µmol C2H2 mg Chl-1 h-1), but the catalyst with entrapped heterocysts required a much longer time to achieve the maximum rate (60 h instead of 3 h in suspension). Simultaneously with acetylene reduction, the immobilized heterocysts were able to photoproduce H2 for 125 h, yielding up to 1.1 mmol H2 mg Chl-1. The absence of acetylene increased the H2 photoproduction rate to a maximum of 25-30 µmol H2 mg Chl-1 h-1, and the catalyst was capable of H2 photoproduction for 190 h, yielding up to 2.5 mmol H2 mg Chl-1. The recovery of the catalyst with entrapped heterocysts was achieved through placing the cells in a N2 atmosphere for 24 h. This engaged a second cycle of H2 photoproduction, which lasted for another 240 h (10 days), thus yielding ∼3 mmol H2 mg Chl-1 in total after 454 h. Together, these findings demonstrate great potential for a heterocyst-based thin-layer platform for the sustainable production of chemicals and biofuels.

Carotenoid Biosynthesis in Calothrix sp. 336/3: Composition of Carotenoids on Full Medium, During Diazotrophic Growth and After Long-Term H2 Photoproduction.

The carotenoid composition of the filamentous heterocystous N2-fixing cyanobacterium Calothrix sp. 336/3 was investigated under three conditions: in full medium (non-diazotrophic growth); in the absence of combined nitrogen (diazotrophic growth); and after long-term H2 photoproduction (diazotrophic medium and absence of nitrogen in the atmosphere). Anabaena sp. PCC 7120 and its ΔhupL mutant with disrupted uptake hydrogenase were used as reference strains. Analysis of identified carotenoids and enzymes involved in carotenogenesis showed the presence of three distinct biosynthetic pathways in Calothrix sp. 336/3. The first one is directed towards biosynthesis of myxoxanthophylls, such as myxol 2'-methylpentoside and 2-hydroxymyxol 2'-methylpentoside. The second pathway results in production of hydroxylated carotenoids, such as zeaxanthin, caloxanthin and nostoxanthin, and the last pathway is responsible for biosynthesis of echinenone and hydroxylated forms of ketocarotenoids, such as 3'-hydroxyechinenone and adonixanthin. We found that carotenogenesis in filamentous heterocystous cyanobacteria varies depending on the nitrogen status of the cultures, with significant accumulation of echinenone during diazotrophic growth at the expense of ß-carotene. Under the severe N deficiency and high CO2 supply, which leads to efficient H2 photoproduction, cyanobacteria degrade echinenone and ß-carotene, and accumulate glycosylated and hydroxylated carotenoids, such as myxol (or ketomyxol) 2'-methylpentosides, 3'-hydroxyechinenone and zeaxanthin. We suggest that the stability of the photosynthetic apparatus in Calothrix sp. 336/3 cells under N deficiency and high carbon conditions, which also appeared as the partial recovery of the pigment composition by the end of the long-term (∼1 month) H2 photoproduction process, might be mediated by a high content of hydroxycarotenoids.

Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation.

The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O(2) in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O(2) downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO(2) levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H(2) photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport.

Hydrogen photoproduction by immobilized n2-fixing cyanobacteria: understanding the role of the uptake hydrogenase in the long-term process.

We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.

Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120.

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

A versatile method for preparation of hydrated microbial-latex biocatalytic coatings for gas absorption and gas evolution.

We describe a latex wet coalescence method for gas-phase immobilization of microorganisms on paper which does not require drying for adhesion. This method reduces drying stresses to the microbes. It is applicable for microorganisms that do not tolerate desiccation stress during latex drying even in the presence of carbohydrates. Small surface area, 10-65 µm thick coatings were generated on chromatography paper strips and placed in the head-space of vertical sealed tubes containing liquid to hydrate the paper. These gas-phase microbial coatings hydrated by liquid in the paper pore space demonstrated absorption or evolution of H2, CO, CO2 or O2. The microbial products produced, ethanol and acetate, diffuse into the hydrated paper pores and accumulate in the liquid at the bottom of the tube. The paper provides hydration to the back side of the coating and also separates the biocatalyst from the products. Coating reactivity was demonstrated for Chlamydomonas reinhardtii CC124, which consumed CO2 and produced 10.2 ± 0.2 mmol O2 m⁻² h⁻¹, Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47 ± 0.04 mmol H2 m⁻² h⁻¹, Clostridium ljungdahlii OTA1, which consumed 6 mmol CO m⁻² h⁻¹, and Synechococcus sp. PCC7002, which consumed CO2 and produced 5.00 ± 0.25 mmol O2 m⁻² h⁻¹. Coating thickness and microstructure were related to microbe size as determined by digital micrometry, profilometry, and confocal microscopy. The immobilization of different microorganisms in thin adhesive films in the gas phase demonstrates the utility of this method for evaluating genetically optimized microorganisms for gas absorption and gas evolution.

Phenotypic diversity of hydrogen production in chlorophycean algae reflects distinct anaerobic metabolisms.

Several species of green algae use [FeFe]-hydrogenases to oxidize and/or produce H(2) during anoxia. To further define unique aspects of algal hydrogenase activity, the well-studied anaerobic metabolisms of Chlamydomonas reinhardtii were compared with four strains of Chlamydomonas moewusii and a Lobochlamys culleus strain. In vivo and in vitro hydrogenase activity, starch accumulation/degradation, and anaerobic end product secretion were analyzed. The C. moewusii strains showed the most rapid induction of hydrogenase activity, congruent with high rates of starch catabolism, and anoxic metabolite accumulation. Intriguingly, we observed significant differences in morphology and hydrogenase activity in the C. moewusii strains examined, likely the result of long-term adaptation and/or genetic drift during culture maintenance. Of the C. moewusii strains examined, SAG 24.91 showed the highest in vitro hydrogenase activity. However, SAG 24.91 produced little H(2) under conditions of sulfur limitation, which is likely a consequence of its inability to utilize exogenous acetate. In L. culleus, hydrogenase activity was minimal unless pulsed light was used to induce significant H(2) photoproduction. Overall, our results demonstrate that unique anaerobic acclimation strategies have evolved in distinct green algae, resulting in differential levels of hydrogenase activity and species-specific patterns of NADH reoxidation during anoxia.

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions.

A new technique for immobilizing H2-photoproducing green algae within a thin (<400 microm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus-deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol mg(-1) Chl h(-1)) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2-photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2-producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale-up and possible future applications.

Hydrogen production by photoautotrophic sulfur-deprived Chlamydomonas reinhardtii pre-grown and incubated under high light.

We have previously demonstrated that Chlamydomonas reinhardtii can produce hydrogen under strictly photoautotrophic conditions during sulfur deprivation [Tsygankov et al. (2006); Int J Hydrogen Energy 3:1574-1584]. The maximum hydrogen photoproduction was achieved by photoautotrophic cultures pre-grown under a low light regime (25 microE m(-2) s(-1)). We failed to establish sustained hydrogen production from cultures pre-grown under high light (100 microE m(-2) s(-1)). A new approach for sustained hydrogen production by these cultures is presented here. Assuming that stable and reproducible transition to anerobiosis as well as high starch accumulation are important for hydrogen production, the influence of light intensity and dissolved oxygen concentration during the oxygen evolving stage of sulfur deprivation were investigated in cultures pre-grown under high light. Results showed that light higher than 175 microE m(-2) s(-1) during sulfur deprivation induced reproducible transition to anerobiosis, although the total amount of starch accumulation and hydrogen production were insignificant. The potential PSII activity measured in the presence of an artificial electron acceptor (DCBQ) and an inhibitor of electron transport (DBMIB) did not change in cultures pre-grown under 20 microE m(-2) s(-1) and incubated under 150 microE m(-2) s(-1) during sulfur deprivation. In contrast, the potential PSII activity decreased in cultures pre-grown under 100 microE m(-2) s(-1) and incubated under 420 microE m(-2) s(-1). This indicates that cultures grown under higher light experience irreversible inhibition of PSII in addition to reversible down regulation. High dissolved O(2) content during the oxygen evolving stage of sulfur deprivation has a negative regulatory role on PSII activity. To increase hydrogen production by C. reinhardtii pre-grown under 100 microE m(-2) s(-1), cultures were incubated under elevated PFD and decreased oxygen pressure during the oxygen evolving stage. These cultures reproducibly reached anaerobic stage, accumulated significant quantities of starch and produced significant quantities of H(2). It was found that elevation of pH from 7.4 to 7.7 during the oxygen producing stage of sulfur deprivation led to a significant increase of accumulated starch. Thus, control of pH during sulfur deprivation is a possible way to further optimize hydrogen production by photoautotrophic cultures.

Prolongation of H2 photoproduction by immobilized, sulfur-limited Chlamydomonas reinhardtii cultures.

Two approaches to prolong the duration of hydrogen production by immobilized, sulfur-limited Chlamydomonas reinhardtii cells are examined. The results demonstrate that continuous H2 photoproduction can occur for at least 90 days under constant flow of TAP medium containing micromolar sulfate concentrations. Furthermore, it is also possible to prolong the duration of H2 production by cycling immobilized cells between minus and plus sulfate conditions.

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

A comparison of hydrogen photoproduction by sulfur-deprived Chlamydomonas reinhardtii under different growth conditions.

Continuous photoproduction of H(2) by the green alga, Chlamydomonas reinhardtii, is observed after incubating the cultures for about a day in the absence of sulfate and in the presence of acetate. Sulfur deprivation causes the partial and reversible inactivation of photosynthetic O(2) evolution in algae, resulting in the light-induced establishment of anaerobic conditions in sealed photobioreactors, expression of two [FeFe]-hydrogenases in the cells, and H(2) photoproduction for several days. We have previously demonstrated that sulfur-deprived algal cultures can produce H(2) gas in the absence of acetate, when appropriate experimental protocols were used (Tsygankov, A.A., Kosourov, S.N., Tolstygina, I.V., Ghirardi, M.L., Seibert, M., 2006. Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions. Int. J. Hydrogen Energy 31, 1574-1584). We now report the use of an automated photobioreactor system to compare the effects of photoautotrophic, photoheterotrophic and photomixotrophic growth conditions on the kinetic parameters associated with the adaptation of the algal cells to sulfur deprivation and H(2) photoproduction. This was done under the experimental conditions outlined in the above reference, including controlled pH. From this comparison we show that both acetate and CO(2) are required for the most rapid inactivation of photosystem II and the highest yield of H(2) gas production. Although, the presence of acetate in the system is not critical for the process, H(2) photoproduction under photoautotrophic conditions can be increased by optimizing the conditions for high starch accumulation. These results suggest ways of engineering algae to improve H(2) production, which in turn may have a positive impact on the economics of applied systems for H(2) production.