A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain

Photoproduction of H(2) gas was examined in the Chlamydomonas reinhardtii tla1 strain, CC-4169, containing a truncated light-harvesting antenna, along with its parental CC-425 strain. Although enhanced photosynthetic performance of truncated antenna algae has been demonstrated previously (Polle etal. Planta 2003; 217:49-59), improved H(2) photoproduction has yet to be reported. Preliminary experiments showed that sulfur-deprived, suspension cultures of the tla1 mutant could not establish anaerobiosis in a photobioreactor, and thus, could not photoproduce H(2) gas under conditions typical for the sulfur-deprived wild-type cells (Kosourov et al. Biotech Bioeng 2002; 78:731-40). However, they did produce H(2) gas when deprived of sulfur and phosphorus after immobilization within thin (similar to 300 mu m) alginate films. These films were monitored for long-term H(2) photoproduction activity under light intensities ranging from 19 to 350 mu E m(-2) s(-1) PAR. Both the tla1 mutant and the CC-425 parental strain produced H(2) gas for over 250 h under all light conditions tested. Relative to the parental strain, the CC-4169 mutant had lower maximum specific rates of H(2) production at low and medium light intensities (19 and 184 mu E m(-2) s(-1)), but it exhibited a 4-times higher maximum specific rate at 285 mu E m(-2) s(-1) and an 8.5-times higher rate at 350 mu E M(-2) s(-1) when immobilized at approximately the same cell density as the parental strain. As a result, the CC-4169 strain accumulated almost 4-times more H(2) than CC-425 at 285 mu E M(-2) s(-1) and over 6-times more at 350 mu E M(-2) s(-1) during 250-h experiments. These results are the first demonstration that truncating light-harvesting antennae in algal cells can increase the efficiency of H(2) photoproduction in mass culture at high light intensity. (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved

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

Hydrogen production by sulfur-deprived Chlamydomonas reinhardtii under photoautotrophic conditions

Thus far, all experiments leading to H-2 production by sulfur-deprived cultures of microalga have been done with photoheterotrophic cultures in the presence of acetate, which increases the cost of the H-2 produced. This study demonstrates that sustained H-2 photoproduction by a sulfur-deprived green alga, Chlamydomonas reinhardtii, is possible under strictly photoautotrophic conditions in the absence of acetate or any other organic substrate in the medium. To accomplish this, we used cultures pre-grown with 2% CO2 under low light conditions (25 mu E m(-2) s(-1)) and also supplemented with CO2 during S-deprivation, along with a special light regime. Maximum H-2 production (56.4 +/- 16.7 ml l(-1) culture, equal to 56.4 x 10(-3) m(3) m(-3) culture) was observed with photoautotrophic cultures: (a) supplied with carbon dioxide for the first 24 h of sulfur deprivation, (b) exposed during the O-2-producing stage to high light (110 - 120 mu E m(-2) s(-1)), and (c) then exposed to low light (20 - 25 mu E m(-2) s(-1)) during the O-2-consumption and H-2-production stages. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved

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 H(2) 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 H(2) production by cycling immobilized cells between minus and plus sulfate conditions. (C) 2008 Elsevier B.V. All rights reserved

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

Maximizing the hydrogen photoproduction yields in Chlamydomonas reinhardtii cultures: The effect of the H-2 partial pressure

Photoproduction of H-2 gas has been examined in sulfur/phosphorus-deprived Chalmydomonas reinhardtii cultures, placed in photobioreactors (PhBRs) with different gas phase to liquid phase ratios (V-g.p/V-l.p). The results demonstrate that an increase in the ratio stimulates H-2 photoproduction activity in both algal suspension cultures and in algae entrapped in thin alginate films. In suspension cultures, a 4x increase (from similar to 0.5 to similar to 2) in V-g.p/V-l.p in a 2x increase (from 10.8 to 23.1 mmol l(-1) or 264-565 ml l(-1)) in the total yield of H-2 gas. Remarkably, 565 ml of H-2 gas per liter of the suspension culture is the highest yield ever reported for a wild-type strain in a time period of less than 190 h. In immobilized algae, where diffusion of H-2 from the medium to the PhBR gas phase is not affected by mixing, the maximum rate and yield of H-2 photoproduction occur in PhBRs with V-g.p/V-l.p above 7 or in a PhBR with smaller headspace, if the H-2 is effectively removed from the medium by continuous flushing of the headspace with argon. These experiments in combination with studies of the direct inhibitory effect of high H-2 concentrations in the PhBR headspace on H-2 photoproduction activity in algal cultures clearly show that H-2 photoproduction in algae depends significantly on the partial pressure of H-2 (not O-2 as previously thought) in the PhBR gas phase. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved

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

Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

Background: Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. Principal Findings: We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L−1 of culture from E. coli with specific activities of 1000 U (U = 1 µmol hydrogen evolved mg−1 min−1). Significance: The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.United States. Dept. of Energy. (contract DE-AC36-08-GO28308

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria