Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures

Unsynchronized Chlamydomonas reinhardtii cells subsequently deprived of sulfur produce H-2 under continuous illumination in the laboratory for 3-4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronize their growth and cell division. While it is clear that only insignificant amounts of H-2 can be produced by sulfur-deprived cells during the night period, little work has been done to examine the effects of the light/dark cycles preceding sulfur deprivation on subsequent H-2 photoproduction. We show that (a) C reinhardtii cells exhibit synchronized growth and cell division in the presence of acetate, (b) cells with the highest specific rates of H-2 photoproduction also have the highest rates of biomass accumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetate accumulation, but not with H-2 photoproduction. This work shows that it is possible to maximize the production of H-2 by sulfur-depriving synchronized cultures at about 4 h after the beginning of the light period. <br /

Continuous hydrogen photoproduction by Chlamydomonas reinhardtii

This study demonstrates, for the first time, that it is possible to couple sulfate-limited Chlamydomonas reinhardtii growth to continuous H-2 photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H-2 production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H2 production occurs under anaerobic conditions. The dependence of the H-2 production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported

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, IN., 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 CO2 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,, production. (c) 2007 Elsevier B.V. All rights reserved

Evaluation of light energy to H2 energy conversion efficiency in thin films of cyanobacteria and green alga under photoautotrophic conditions

Cyanobacteria and green algae harness solar energy to split water and to fix CO2. Under specific conditions, they are capable of photoproduction of molecular hydrogen (H2). This study compares the light-energy-to-hydrogen-energy conversion efficiency (LHCE) in two heterocystous, N2-fixing cyanobacteria (wild-type Calothrix sp. strain 336/3 and the δhupL mutant of Anabaena sp. strain PCC 7120) and in the sulfur-deprived green alga, Chlamydomonas reinhardtii strain CC-124, after entrapment of the cells in thin Ca2+-alginate films. The experiments, performed under photoautotrophic conditions, showed higher LHCEs in the cyanobacteria as compared to the green alga. The highest efficiency of ca. 2.5% was obtained in films of the entrapped δhupL strain under low light condition (2.9Wm-2). Calothrix sp. 336/3 films produced H2with a maximum efficiency of 0.6% under 2.9Wm-2, while C. reinhardtii films produced H2most efficiently under moderate light (0.14% at 12.1Wm-2). Exposure of the films to light above 16Wm-2led to noticeable oxidative stress in all three strains, which increased with light intensity. The presence of oxidative stress was confirmed by increased (i) degradation of chlorophylls and some structural carotenoids (such as β-carotene), (ii) production of hydroxylated carotenoids (such as zeaxanthin), and (iii) carbonylation of proteins. We conclude that the H2photoproduction efficiency in immobilized algae and cyanobacteria can be further improved by entrapping cultures in immobilization matrices with increased permeability for gases, especially oxygen, while matrices with low porosity produced increased amounts of xanthophylls and other antioxidant compounds.</p

A new approach for sustained and efficient H2 photoproduction by Chlamydomonas reinhardtii

Sustained H2 photoproduction is demonstrated in green algae under a train of strong white light pulses interrupted by longer dark phases. The devised protocol relies on the presence of the [FeFe]-hydrogenase in algal chloroplasts, which is activated within a few seconds after the establishment of anaerobiosis. H2 photoproduction proceeds for up to 3 days with the maximum rate occurring in the first 6 hours

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

Photoproduction of H-2 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 O-2-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 H-2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H-2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H-2-production, a shorter duration of H-2-production, lower H-2 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 H-2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch

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 mu m 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 H-2, CO, CO2 or O-2. 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 +/- A 0.2 mmol O-2 m(-2) h(-1), Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47 +/- A 0.04 mmol H-2 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 +/- A 0.25 mmol O-2 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

Analytical approaches to photobiological hydrogen production in unicellular green algae

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of “anaerobic oxygenic photosynthesis”, whereby photosynthetically generated oxygen is consumed by the cell’s own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular “hydrogen metabolism” process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated