Interactions between photosynthetic, (chloro)respiratory and hydrogen oxidoreduction pathways in algae and cyanobacteria

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1 Interactions between photosynthetic, (chloro)respiratory and hydrogen oxidoreduction pathways in algae and cyanobacteria Laurent Cournac CEA Cadarache, DSV/DEVM/L3BM, UMR 6191 CNRS CEA Univ Méditerranée, Saint Paul Lez Durance Cedex, France ; Fax : In cyanobacteria and in chloroplasts of higher plants or microalgae, different respiratory or respiratory-like electron transfer pathways have been shown to interact with the photosynthetic electron transfer chain. By analyzing gas exchange of Chlamydomonas reinhardtii and Synechocystis PCC6803 in vivo using membrane-inlet mass spectrometry, we will give some hints on how these pathways can also interact with hydrogen metabolism. Further, we will discuss how their inhibition or stimulation in microalgae or cyanobacteria may be designed in order to derive photosynthetic electron transfer into H 2 production.

2 Perturbing cyanobacterial hydrogen metabolism with simple changes in media components: toward metabolic control of microbial hydrogen producers. Damian J. Carrieri, Gennady Ananyev, G.Charles Dismukes Princeton University, Department of Chemistry and Princeton Environmental Institute Using cyanobacteria as cell factories to generate hydrogen using only water, sunlight and catalytic amounts of inorganic media components is under investigation as a possible route to displacement of fossil fuels. However, practical contributions by this approach will require substantial improvements of hydrogen production rates and yields. We have selected to initially examine the cyanobacterium Arthrospira maxima, which grows in alkaline carbonate lakes. This choice is based in part on its high primary productivity and anticipated capacity to divert water oxidation from photosynthetic carbon fixation to other processes by withholding of inorganic carbon. We are testing whether perturbation of the metabolic pathways (e.g. through disruption of routes that compete with hydrogen production) will lead to cells with optimal solar to hydrogen efficiencies. We demonstrate that hydrogen production from Arthrospira maxima can be improved simply by changing the nickel concentration in the media. Addition of Ni 2+, which is non-essential under photoautotrophic growth conditions, leads to a 2-3 fold increase in hydrogen rates and yields in bulk cultures and up to a 20-fold increase in microvolume samples in which hydrogen is continuously removed. These improvements are found with no observable difference in photoautotrophic growth rates and appear when cells are fermenting their own internal fixed-carbon stores and when exogenous substrates such as glucose or pyruvate are added to cultures. We are currently examining the mechanism of this stimulation. Many mechanistic studies and screening reports of cyanobacteria in the literature have neglected to examine the importance of nickel for photoautotrophic growth or anaerobic hydrogen production. Our results demonstrate the importance of media components for current screenings of cyanobacteria hydrogen producers. A second important implication is that increasing the flux of electrons for hydrogen production (rather than production of competing substrates) can be achieved easily without deleterious effects on cultures, paving the way for further incremental improvements.

3 High-throughput Screening of PSII Efficiency in Phototrophs Using Fluorescence Imaging. Derrick R. J. Kolling #, Brett A Nitchke #, Georgia Tien, Robert R. Bidigare, and G. Charles Dismukes # # Princeton University, NJ 08544, University of Hawaii at Manoa, HI, It is likely that the prime candidate for biosolar hydrogen production will have to be engineered, but there is no reason to start from scratch. Nature, via evolution, has provided us with an amazing palette of biodiversity from which we can choose a robust microbe for biosolar hydrogen production. The cyanobacteria form a diverse subdivision of prokaryotic oxygenic phototrophs with 2,654 unique species classified and 55 draft genomes completed or in progress. Many, but not all strains are capable of H 2 production. Hydrogenase encoding genes are found in all 5 major taxonomic groups and at least 50 genera and a hundred or so strains thus far have been found to metabolize H 2. There is a huge untapped potential for discovery of new hydrogen producers. We are presently acquiring cultures from collections at the University of Hawaii. We are using a video fluorescence imager (FluorCam 900MF, Photon Systems Instruments) to measure the efficiency of charge separation in Photosystem II by measuring the yield of variable fluorescence emission from chlorophyll. High efficiency is a key to fixing large amounts of carbon and generous carbon stores are vital for 2- stage hydrogen production (i.e. carbon fixation in light, followed by proton reduction by fixed carbon in the dark). High throughput is attained using multiwell plates, with each well containing a different phototroph strain. While this screen identifies potentially strong photosynthesizers, a parallel screen is being performed in the Posewitz lab at the Colorado School of Mines in which they use a chemochromic sensor to detect hydrogen production from duplicate plates of the same strains sent to Princeton. Potential candidates should pass both of these screens. Screening of the first shipment of cultures from the University of Hawaii has been completed. The strains Aphanocapsa biformis, Chlorella sp., and Gloeocystis sp. were all found to have Fv/Fm greater than 0.5. In addition, Gloeocystis sp. showed strong fluorescence quenching. Good reproducibility (SD ±0.02, 3 avgs.) and insensitivity to plate location in the imager testify to the robustness of the measurements taken.

4 Large Scale Genomic Analysis for Understanding Hydrogen Metabolism in Chlamydomonas reinhardtii Alexandra Dubini 1,3, Florence Mus 2, Michael Seibert 1, Maria L. Ghirardi 1, Matthew C. Posewitz 3 and Arthur R. Grossman 2 1 National Renewable Energy Laboratory, Golden, CO 2 Carnegie Institute of Washington, Stanford, CA 3 Colorado School of Mines, Golden, CO Chlamydomonas reinhardtii, is a unicellular green alga, able to convert the low potential reductant generated from water by photosynthesis into H2. Hydrogenase activity in C. reinhardtii is induced by anaerobiosis. This is achieved either in the dark by using an inert gas (or exogenous reductant) to purge O2 from sealed cultures or in the light by depriving sealed cultures of sulfur. Clearly, a high throughput approach is required to fully understand the influence of metabolism and other environmental factors on the regulation of gene expression and H2-production activity. Our initial studies are focused on determining the suite of genes differentially regulated as a result of shifting cultures of C. reinhardtii strain CC425 from aerobic growth to conditions of anaerobiosis. qpcr was used to demonstrate that the genes coding for the two [FeFe]- hydrogenase structural genes (HydA1 and HydA2 and the two C. reinhardtii [FeFe]- hydrogenase assembly genes (HydEF and HydG) are anaerobically induced during this condition. With the completion of the C. reinhardtii genome sequence it is now possible to thoroughly explore large-scale transcript profiles associated with H2 metabolism in this alga using high-density DNA microarrays. The current array was developed at the Carnegie Institution and is based on specific synthetic 70 mers that represents approximately 10,000 genes [3]. Genome analysis of C. reinhardtii indicates that relative to other microorganisms, this alga has an extremely broad repertoire of enzymes associated with a fermentative metabolism, particularly those involved in metabolizing pyruvate, ethanol and acetate. Our initial microarray data revealed increased levels of transcription for a number of genes including those involved in anaerobic metabolism, cellular electron transport, signal transduction and posttranslational modification. references 1. Wykoff, D. D., Davies, J. P., Melis, A. and Grossman, A. R. (1998). The Regulation of Photosynthetic Electron Transport During Nutrient Deprivation in Chlamydomonas reinhardtii. Plant Physiol. 117, Melis, A., Zhang, L., Forestier, M., Ghirardi, M. L. and Seibert, M. (2000). Sustained Photobiological Hydrogen Gas Production Upon Reversible Inactivation of Oxygen Evolution in the Green Alga Chlamydomonas reinhardtii. Plant Physiol. 122, Eberhard, S., Jain, M., Im, C.S., Pollock, S., Schrager, J., Lin, Y., Peek, A.S., Grossman, A.R., (2006). Generation of an Oligonucliotide Array for Analysis of Gene Expression in Chlamydomonas reinhardtii. Curr. Genet. 49,

5 The Construction of a Synechocystis Recombinant System for Solar Hydrogen Production Jianping Yu, Sharon Smolinski, Gary Vanzin, and Pin-Ching Maness* National Renewable Energy Laboratory 1617 Cole Blvd., Golden, CO 80401, USA Using water as the substrate and driven by solar energy, the cyanobacterium Synechocystis sp. PCC 6803 has immense potential for renewable H 2 production. Yet the H 2 -production reaction catalyzed by the Synechocystis bi-directional [NiFe]- hydrogenase (HoxH) does not sustain in the presence of O 2, which is an inherent byproduct of oxygenic photosynthesis. Research in our laboratory has uncovered an O 2 - tolerant [NiFe]-hydrogenase from the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS. Its hydrogenase exhibits a half life near 21 hours when whole cells were stirred in ambient air. To surmount the O 2 -sensitivity of the Synechocystis H 2 -production system, one strategy is to genetically transfer and express the genes encoding the Rx. gelatinosus O 2 -tolerant hydrogenase in a cyanobacterial host for continuous H 2 production, in light. Via transposon mutagenesis and rescue cloning, we have cloned the genes in Rx. gelatinosus encoding the large (CooH) and small (CooL) subunits of the hydrogenase structural protein along with the hypabcdef genes with putative functions in hydrogenase assembly and maturation. To facilitate the construction of the recombinant system, we have knocked out the native HoxH hydrogenase of the Synechocystis host to eliminate background hydrogenase activity. Furthermore, we constructed an expression system in E. coli to produce the Synechocystis PetF ferredoxin I and demonstrated that when photo-reduced, this ferredoxin could mediate H 2 production catalyzed by the Rx. gelatinosus hydrogenase. Work is underway to express the Rx. gelatinosus hydrogenase structural genes and the putative assembly genes in E. coli as a model host first, later in Synechocystis, for continuous H 2 production.

6 An Investigation into the Diversity of Chlorophycean H 2 Metabolism. Jonathan E. Meuser 1, Lauren Nagy 1, Maria Ghirardi 2, Mike Seibert 1, Matthew Posewitz 1 1 Environmental Science and Engineering Division, Colorado School of Mines, Golden, CO, 80401, USA. 2 National Renewable Energy Laboratory, Golden, CO, 80401, USA The photobiological hydrogen (H 2 ) metabolism of the model chlorophycean alga, Chlamydomonas reinhardtii has been extensively examined as a potential source of renewable fuel. H 2 evolution in Chlamydomonas is catalyzed by the [FeFe]-H 2 ase, one of the most efficient H 2 catalysts known, yet the diversity of chlorophycean light-driven H 2 evolution is poorly resolved. Moreover, the phylogenetic root(s) of the cholorophycean [Fe-Fe]-H 2 ase, ancient heritage or lateral gene transfer, is unknown. To better understand the variation of H 2 production in the green algae and the physiological basis of this diversity, Chlamydomonas strains with reportedly diverse phenotypes were selected for inter-strain comparison. Both H 2 photoproduction and H 2 ase enzyme activity varied greatly between strains at various times following anaerobic induction. To control for overall metabolic activity, concomitant O 2 evolution and respiration data were also collected. To better understand the phylogenetic root of chlorophycean hydrogen metabolism, taxa with interesting phylogenetic and/or environmental relationship to Chlamydomonas were also screened for H 2 ase activity. This research was supported by the United States Air Force Office of Science Research and the NASA Graduate Student Research Program.

7 Fe-only hydrogenase structure, function, and biosynthesis John W. Peters*, Shawn McGlynn, Anatoli Naumov, Shane Ruebush, Lauren E. Nagy, Trevor Douglas, Robert Szilagyi, Paul W. King, Matthew Posewitz, Fe-only hydrogenases possess a uniquely organometallic active site, termed the H cluster, where the electronic properties of an iron-sulfur cluster are tuned with distinctly non-biological ligands, carbon monoxide and cyanide. There is considerable interest in the biosynthesis of this metal cluster due to the novelty of this metal site and the potential technological importance of these enzymes. Recently, a significant advance was made concerning the biosynthesis of the H cluster when it was discovered that radical SAM enzymes were involved in active hydrogenase expression. The occurrence of these enzymes within the genomes of organisms that harbor Fe-only hydrogenases and the nature of radical SAM enzyme biochemistry are highly suggestive of a mechanism for the cluster biosynthesis and active hydrogenase expression. H cluster biosynthesis is proposed to occur through a sequence of two reactions that are related to chemistry previously established for members of the radical SAM enzyme class. In the current work, we present a mechanistic scheme for hydrogenase H-cluster biosynthesis in which both carbon monoxide and cyanide ligands can be derived from the decomposition of an amino acid radical at the cluster site. Computational models of the proposed intermediates for this reaction sequence have been energetically validated using density functional calculations. We have recently shifted our focus on this project to examining the above hypotheses experimentally and have made a key advancement showing that an apo-hydrogenase presumably devoid of its intact H-cluster can be activated in vitro. The in vitro reconstitution of active [FeFe]-hydrogenases is accomplished by combining heterologously expressed accessory or maturation protein extracts with apo-hydrogenase. Interestingly, the activation process occurs in a manner suggesting that when the hydrogenase accessory proteins are coexpressed, an H-cluster precursor is synthesized that can be rapidly transferred to the hydrogenase enzyme to affect activation.

8 Genetic Engineering of Fermentation Pathways in Cyanobacteria for Improving H 2 Production Kelsey McNeely 1 (kmcneely@princeton.edu), G. C. Dismukes 1, E. Steifel 1, D. Bryant 2 Princeton University 1 and The Pennsylvania State University 2 Many cyanobacteria produce variable amounts of hydrogen as a byproduct of fermentation during dark periods of anaerobisis at night. Fermentation produces ATP and NAD(P)H for cell homeostasis during dark anaerobic periods. The carbon reaction products and enzymatic pathways of fermentation in most species of cyanobacteria have not been elucidated with a few exceptions. Synechocystis 6803 and Synechococcus 7002 are unicellular cyanobacteria with evidently divergent fermentative pathways based on whole genome sequences. Synechocystis 6803 has the genes encoding enzymes needed for ethanol, acetate, and D-lactate production during fermentation while Synechococcus 7002 appears to only contain the D-lactate genes. The presumed pathways are depicted in the schematic below. 6803: Glycogen G6P G1P Pyruvate * Acetyl-CoA + CO 2 * * D-lactate H 2 acetate ethanol 7002: Glycogen G6P G1P Pyruvate * Acetyl-CoA + CO 2 * D-lactate H 2 Once the pathways of fermentation have been elucidated, they can be manipulated in order to increase hydrogen yield during anaerobic glycolysis of either internally produced glycogen or added reductant. Several pathways in 6803 are sinks for NAD(P)H that could otherwise be re-directed to proton reduction by hydrogenase. NADH and possibly NADPH provide the reducing electrons for hydrogen production and enzyme activation. In fermentation, NAD(P) + is reduced from ferredoxin by ferredoxin:nad(p) reductase, while ferredoxin is the co-substrate for pyruvate:ferredoxin oxidoreductase (PFOR). PFOR is catalyzes the conversion of pyruvate to acetyl-coa. Most fermentative processes in 6803 involve the conversion of acetyl-coa into acetate, ethanol, and perhaps other organic acids. In 7002, however, there may be no further fermentative metabolism of acetyl-coa. Our current work includes examining hydrogen production in knock-outs of PFOR, D-lactate dehydrogenase, and aldehyde dehydrogenase in 6803 and PFOR and D-lactate dehydrogenase in 7002, indicated by asterisks in the schematic. These and other mutants will help to elucidate the fermentative pathways of 7002 and 6803 and likely give insight into stimulating H 2 production by hydrogenase.

9 Poster abstract for L. Nagy The use of hydrogen (H 2 ) as a non-polluting energy carrier is an attractive alternative to fossil fuel. Presently, H 2 gas production requires the use of finite precious metals, as well as substantial amounts of fossil fuel. The hydrogenase enzyme family is a promising biological alternative to the conventional methods of H 2 production. These enzymes, which include [NiFe] and [FeFe]-hydrogenases, efficiently catalyze the reversible oxidation of H 2 according to the following equation: H 2 2H + + 2e -. Presently, several strategies are being investigated to leverage the catalytic activity of hydrogenases for H 2 production. However, additional optimization of the presently characterized native [FeFe]-hydrogenases is required for many biotechnological applications. Gene-shuffling approaches were therefore investigated to identify a suitable technique for the rapid generation of large and diverse [FeFe]-hydrogenase libraries to screen for novel enzymes. Six native [FeFe]-hydrogenase genes from the genus Clostridium were first cloned and separately transformed into Escherichia coli along with the requisite [FeFe]-hydrogenase assembly genes hyde, hydf and hydg from Clostridium acetobutylicum. With the exception of Clostridium thermocellum, all wildtype enzymes co-expressed with the C. acetobutylicum assembly genes exhibited significant activity. The Family Shuffling with Single-Stranded DNA technique, was then utilized to increase hydrogenase diversity by shuffling two parental [FeFe]- hydrogenase sequences. Activity assays demonstrate that several shuffled products have enzymatic activity and that a single set of [FeFe]-hydrogenase assembly proteins is suitable for the maturation of shuffled enzymes in E. coli. This represents the first report regarding the successful expression of shuffled [FeFe]-hydrogenases with activity. The rapid generation of large libraries containing shuffled [FeFe]-hydrogenases will provide the framework for future screening and recombinant strategies aimed at identifying novel [FeFe]-hydrogenases for biotechnological applications.

10 Density functional theory and QM/MM calculations of intermediates in the catalytic mechanism of [FeFe] hydrogenases S. Trohalaki, G. Hong, R. Pachter Air Force Research Laboratory, Materials & Manufacturing Directorate Wright-Patterson Air Force Base, Ohio Although X-ray structures of the Fe-hydrogenases from Desulfovibrio desulfuricans (DdH) and Clostridium pasteurianum (CpI) were determined, an understanding of the catalytic activity by theoretical density functional theory (DFT) has been limited so far to small model compounds of the active site ([Fe 2 ] H, so-called H-cluster ). In this work, we studied intermediates in the catalytic mechanism of CpI, addressing structure and energetics, by inclusion of the proximate Fe 4 S 4 cubane cluster in the DFT calculations, and compared our results to previous suppositions. In addition, because of our interest in a validated QM/MM method to model the protein environment, also for understanding the characteristics of the oxygen pathway to address oxygen intolerance, we initially applied a DFT/frozen DFT for the cubane clusters for which force-field parameters are not available/mm molecular dynamics method, also including solvent, to assess the effects of the protein environment upon the proton transfer to [Fe 2 ] H for the alternate proton pathway in DdH.

11 The influence of metabolic state on photosynthesis derived H 2 production in cyanobacteria G. Charles Dismukes, Gennady Ananyev, Damian Carrieri, Stephanie Kriston Princeton University, dismukes@princeton.edu The main goals of the first year of the BioSolarH 2 team have been selecting and screening strains of cyanobacteria (prokaryotes) and algae(eukaryotes) with naturally high endogenous capacity for hydrogenasedependent H 2 production (see: Kolling et al.), building new tools that can measure intracellular electron/proton fluxes and product gases (see Ananyev, et al.), and evaluating modern LC-MS facilities for biohydrogen metabolomics(see Bennette et al.). We have selected for initial studies the alkalophillic carbonate-requiring cyanobacterium Arthrospira maxima. Arthrospira species are cultivated commercially on a large scale. The maxima strain exhibits several attributes expected to contribute to a high basal level of hydrogenase-dependent H 2 production and control, namely: apparent absence of the uptake hydrogenase (hup genes), absence of N 2 fixation, high primary photosynthetic productivity (5.8 days doubling, max 1.5 mg dry cell wt/ml), fastest PSII water oxidation/o 2 evolution rate in vivo (5x algal rate), vigorous O 2 consumption by a highly efficient respiratory chain, and reversible control of water oxidation and CO 2 fixation rates via (bi)carbonate removal. We observe fermentative H 2 production by A. maxima under dark anaerobic conditions at a rate equal to or greater than 2.5 ml H 2 /mg dry wt cells/h for sustained periods up to 6 days at 23 C and at zero H 2 back pressure. This rate increases by 2x at 30 C and is expected to double again at the natural growth temperature of 35 C. An optimal H 2 rate was attained by adjusting the Ni 2+ concentration used for photoautotrophic growth (20x range observed). Micromolar Ni 2+ causes chlorosis of pigments and a lag in the growth phase, but no effect on the rate or yield of cell growth thereafter (see Carreiri et al.). Cell lysis begins on the third day of fermentation and reaches 90% by day 6, owing to elevated osmotic pressure which limits the H 2 production period. The H 2 rate is stimulated by fermentable substrates: pyruvate, glucose and lysed cells each substantially increases the H 2 production rate and yield. The maximum in vitro rate using the methyl violygen assay is 17 ml H 2 /mg dry wt cells/h. Elucidation of the metabolic pathways that provide electrons and protons to the bidirectional hydrogenase in A. maxima has been undertaken. This work is being supported by gene knock-out experiments produced by genetic engineering of Synechococcus 7002 and Synechocystis 6803 (see McNeely et al.). H 2 production in Arthrospira species is catalyzed by a bidirectional NiFe-hydrogenase (hox genes) that uses NADH as its cognate reductant. PSI photogenerated NADPH is either not an effective or directly available reductant for H 2 production. Light which excites both PSI and PSII produces a transient burst of H 2 via rapid PSII chemistry (H 2 O PQH 2 NADH) and a delayed consumption of NADH via PSI chemistry (NADH PQH 2 NADPH). Selective far red illumination of PSI suppresses dark H 2 production, via oxidation of NADH mediated by PQ-NADH dehydrogenase (Ndh1). Subsequently in the dark, H 2 production resumes at a greater rate than prior to PSI illumination, attributed to slow enzymatically controlled reduction of NAD + by photogenerated NADPH. Sustained H 2 evolution occurs over 2-3 days involving two kinetic phases. In phase- 1, the onset of H 2 production occurs within the first hour after anaerobic induction of photoautotrophically grown cultures that have attained stationary growth (>12 days). Phase-1 H 2 is unaffected by agents that consume ATP (FCCP, quinidine) and can be rapidly induced by addition of exogenous glucose or pyruvate. We postulate that this phase represents pyruvate-ferredoxin oxidoreducase PFOR/FNR mediated NAD + reduction. Phase-2 H 2 appears between hours after anaerobic induction in cells of any age, but is strongest in older cultures and cultures grown at lower light intensity (70 μe m2s). Phase-2 H 2 is inhibited by dissipation of ATP (FCCP and quinidine). We postulate that phase-2 represents NAD + reduction via the ATPdependent initial steps of anaerobic glycolysis of glycogen. These studies set the stage for engineering of pathways for optimal H 2 yield.

12 Electrochemical Tools for Ultra-sensitive Rapid H 2 Detection: Optimization of H 2 Production by Cyanobacteria Arthrospira. Gennady Ananyev, Damian Carrieri and G. Charles Dismukes Department of Chemistry, Princeton University, and Princeton Environmental Institute Princeton, NJ Phone: (609) , ananyev@princeton.edu Arthrospira species are the most robust oxygenic phototrophs known. These filamentous cyanobacteria thrive under extreme environmental conditions in alkaline soda lakes (ph ) at concentrations of carbonate that approach saturation ( M). Our fluorescence measurements show that it possesses high quantum efficiency of PSII and the fastest water-oxidizing complex of all oxygenic phototrophs we have examined to date, including several cyanobacteria and green algae. Arthrospira filaments have no heterocysts and do not fix N 2. However, a NiFe-hydrogenase is present. An ultra-sensitive Clark type electrode for measuring dissolved H 2 concentration has been fabricated and applied to detection of microbial H 2 production. The Princeton H 2 electrode can measure dissolved H 2 concentration as low as 1x10-8 M and with time resolution 0.1 s in a sample of 5 μl volume; light pulse duration from 10 ms to 99 s. We found that H 2 production from Arthrospira maxima in batch culture depends on age: in Stage 1 (6-12 days old culture) very little H 2 is produced; in Stage 2 (12 up to 60 days) culture at 23 O C produces up to 4 % of H 2 in a bioreactor headspace. This corresponds to μmol H 2 /mg dry wt/h (33 % of the maximum from Oscillatoria, strain Miami BG7, the cyanobacterium with the highest reported production rate). Light-induced H 2 evolution (at 640 nm) occurs only in the presence of fermentative H 2 production, but increasing light intensity significantly inhibits fermentative H 2 evolution at alkaline ph. Low intensity IR-735 nm activates photo H 2 (1s light/99 dark or integral optical power 50 μw cm -2 ) while high intensity IR-735 nm completely inhibits fermentative and photo H 2 (2s light/98 dark or integral optical power >250 μw cm -2 ). This effect isn t related to inhibition of H 2 -ase by photosynthetic oxygen as IR-735 is unable to drive PSII water oxidation, but instead suggests a decrease in the reduced to oxidized ratio of the electron donor for hydrogen production. Restoration of fermentative H 2 after IR-735 irradiation occurs in min of darkness. Experiments with inhibitors and external substrates suggest a strong link between cellular energetic requirements and fermentative hydrogen production. Quinidine (a Na + -channel blocker) causes a strong increase in photo-h 2 at alkaline ph, while acidifying cultures in the absence of quinidine gives a similar hydrogen evolution profile. Uncoupler FCCP inhibits both photoand dark-h 2 production at high ph but stimulates H 2 production at low ph. Taken together these observations suggest a stimulation of fermentation in response to osmotic and ph stresses. Succinate (direct substrate for respiration) doesn t increase H 2 evolution, yet glucose and especially pyruvate significantly increases H 2 evolution. Elevation of Ni 2+ concentration in growth media from 0.0 to 3.0 μm raises hydrogenase activity and increases hydrogen yields by as much as 360 %. Conditions for activation of hydrogen production by Arthrospira included: (a) extra Ni 2+ in Zarrouk medium (up to 3 μm); (b) light intensity 260 μe m -2 s -1 for biomass accumulation (Stage 1) and 70 μe m -2 s -1 for hydrogen conditioning and extension of lifetime (Stage 2); (c) bicarbonate depletion. Ideal temporal separation of H 2 and O 2 production in Arthrospira solves gas separation problem. Supported by Air Force Office of Scientific Research (grant FA ).

13 Evaluation and Procurement of an LC-MS System for Metabolomic Studies of H 2 -Evolving Phototrophs Nicholas Bennette, John Eng, and G. Charles Dismukes 2 nd BioSolarH 2 Mini-Symposium, July 28 th & 29 th, 2006 The main goal of the multi-disciplinary AFSOR MURI project is to identify new and engineer existing lines of phototropic microbial species capable of high yields of hydrogenase-derived H 2 via photosynthesis, socalled bio-solar hydrogen. One objective spanning the six institutions involved in the project is the successful elucidation of the biochemical pathways and metabolic networks that govern H 2 production and competing reactions in eukaryotic algae and prokaryotic cyanobacteria. The current incomplete knowledge of the operational components of metabolomic systems in microbial oxygenic phototrophs prevents the use of flux balance analysis, through which the optimization of key products H 2 and its precursors may be controlled. This approach has been applied to a few well-studied chemotropic bacteria to optimize the yield of specific cellular components, principally through information obtained via metabolome/proteome extraction and quantitation. Of the various combinations of separation and analysis techniques, High-Performance Liquid Chromatography (HPLC) coupled directly to tandem Mass Spectroscopy (MS/MS) is generally regarded as perhaps the best approach in terms of reproducibility, stability, robustness, accuracy, and breadth of analysis of the cellular metabolome and proteome. Following the awarding of a DOD-DURIP instrument grant this June, we are now in a position to purchase an (HP)LC-MS system for metabolomics studies of microbial hydrogen production. Though there are numerous published examples of applications of LC-MS to metabolomic as well as proteomic analysis of oxygenic phototrophs including cyanobacteria none to date have been performed correlating proteolic/metabolic flux to hydrogen evolution. Currently we are in the process of evaluating and selecting instrumentation that will permit such an analysis and become the foundation of metabolomics studies for the BioSolarH 2 project. In our initial proposal, we cited the Thermo-Finnegan triple-quadrupole (QQQ) Quantum Ultra MS as optimal for our budget and purposes. A competing system, the Agilent 6410 QQQ MS, has since been released and promises comparable through-put and performance at a significantly lower cost, enabling us to consider its purchase along with a second MS instrument designed for proteomics. We are evaluating the performance of each instrument using samples that test the limit-of-detection, linearity, and robustness in a head-to-head manner. The selected system will be supported by campus resources provided by the Departments of Chemistry, Molecular Biology, and the Lewis-Sigler Institute for Integrative Genomics: This includes siting in the newly founded joint mass spectrometry facility located in the Department of Molecular Biology which is serviced by 2.5 full-time research staff. Initial experiments will focus on establishing and validating the protocol for the extraction, chromatographic separation, and mass spectrometric detection of the algal/cyanobacterial metabolome. Following MS/MS optimization of all targeted metabolites, chromatography conditions will be adjusted for optimal elution and detection of the desired portion of the metabolome. For this purpose, we are currently evaluating LC systems most suitable for rapid, reproducible separations of whole cell lysates. Once column and QQQ parameters are established, extraction methods will be compared and likely modified for maximal metabolome recovery. With these methods in place, cellular levels of metabolites key to hydrogen evolution may be reliably, efficiently, and simultaneously measured.

14 Metapopulation dynamics and directed evolution of single cell organisms on a microfabricated chip Peter Galajda, Juan Keymer, Cecilia Muldoon and Robert H. Austin Department of Physics, Princeton University, Princeton NJ, USA It is known in ecology that the landscape structure of the environment results in the formation of metapopulations, groups of spatially fragmented interacting populations of organisms. Metapopulations play a role in the survival, extinction and evolution of species. Micro- and nafobrication technologies offer a way to create precisely engineered spatially structured microenvironments for microorganisms. We produced microfluidic chips in which channels and chambers make up a system of interacting 'micro environmental patches' and resource supply network. Escherichia coli bacteria and the unicellular algae Chlamydomonas reinhardtii both showed the formation of metapopulations in these chips. The basic features of the spatial and temporal behavior of these populations can be explained and modeled. In some experiments we created 'quality landscapes' or 'fitness landscapes' with which the populations interacted and adapted and/or evolved accordingly. Our results suggest that a combination of ecology, microfabrication technology and molecular biology may be an interesting way to study the social behavior and the adaptation and evolution of microorganisms.

15 BioHydrogen with emphasis on cyanobacteria as the model system Peter Lindblad Dept Physiological Botany, EBC, Uppsala Univ, Villavägen 6, SE-75236, Uppsala, Sweden Photobiological production of H 2 by microorganisms is of great public interest since it promises a renewable energy carrier from nature s most plentiful resources, solar energy and water. Cyanobacteria and green algae are the only organisms known so far capable of both oxygenic photosynthesis and hydrogen production. In cyanobacteria up to three enzymes can be directly involved in hydrogen (H 2 ) metabolism: nitrogenase(s) which produces H 2 concomitantly with nitrogen fixation, a membrane-bound uptake hydrogenase which reoxidizes the H 2 evolved by nitrogenase, and a bidirectional hydrogenase that catalyses both oxidation of molecular hydrogen and reduction of protons. Two different NiFe-hydrogenases have been found in cyanobacteria. A membrane-bound uptake hydrogenase (encoded by hupsl) transfers/donates electrons from H 2 to the photosynthetic and the respiratory electron transport chains, thereby regaining energy and reducing equivalents lost as H 2 during nitrogen fixation. A reversible, bidirectional hydrogenase (encoded by the hox genes) catalyzes both uptake and evolution of H 2 and shows high similarities to the NAD + -reducing hydrogenase of e.g. Ralstonia eutropha. The heteropentameric enzyme consists of a H 2 -oxidizing dimer and a diaphorase part transferring electrons to NAD +. A number of reports suggest a function of bidirectional hydrogenase in disposing excess of reducing equivalents during dark anaerobic conditions (e.g. fermentation) or during periods of adaptation to higher light intensities (Schütz et al 2004, Tamagnini et al 2002). The present overview will focus on the presence and diversity of hydrogenases in cyanobacteria ranging from unicellular to filamentous strains. Possibilities and capacities for photobiological hydrogen production based on cyanobacteria will be examined. Additional focus will be on recent results on the biochemical and molecular regulation of specific hydrogenases in selected strains (Antal et al 2006, Oliveira & Lindblad 2005). Antal, T.K., Oliveira, P. & Lindblad, P The bidirectional hydrogenase in the cyanobacterium Synechocystis sp. strain PCC International Journal of Hydrogen Energy (in press) Oliveira, P. & LIndblad, P LexA, a transcription regulator binding in the promoter region of the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC FEMS Microbiology Letters 251: Schütz, K., Happe, T., Troshina, O., Lindblad, P., Leitao, E., Oliveira, P. & Tamagnini, P Cyanobacterial H 2 production a comparative analysis. Planta 218: Tamagnini, P., Axelsson, R., Lindberg, P., Oxelfelt, F., Wünschiers, R. & Lindblad, P Hydrogenases and Hydrogen Metabolism of Cyanobacteria. Microbiology and Molecular Biology Reviews 66: 1-20

16 Identification of Genes and Pathway Facilitating Hydrogen Production in Algae Matthew C. Posewitz 1,3, Lauren E. Nagy 2, Alexandra Dubini 1,2, Jonathan Meuser 2, Rodney Smith 1,2 Arthur R. Grossman 3, Florence Mus 3, Matthew Wecker 1,2, Georgia Tien 4, Robert Bidigare 4, Susan Brown 4 Paul W. King 2, Maria L. Ghirardi 1, and Michael Seibert 2 1 National Renewable Energy Laboratory, Golden, CO; 2 Colorado School of Mines, Golden, CO; 3 Carnegie Institution of Washington, Stanford, CA; and 4 Universtiy of Hawaii, Honolulu, HI While many taxonomically diverse microbes have the ability to produce H 2, only certain photosynthetic organisms, including the green alga, Chlamydomonas reinhardtii, are able to directly couple water oxidation to the photoproduction of H 2. A more fundamental understanding of this prototype alga might enable the future development of a sustainable system for biological H 2 production. In order to identify genes that influence H 2 production in C. reinhardtii, a library of random mutants (provided by Prof. A. Melis, University of California, Berkeley), was screened using sensitive chemochromic H 2 - sensor films for clones defective in H 2 production. Two mutants of particular interest were fully characterized. One mutant, hydef-1, is unable to assemble an active [FeFe]-hydrogenase and the second mutant, sta7-10, is unable to accumulate insoluble starch. The hydef-1 mutant is the only reported C. reinhardtii mutant that is unable to produce H 2 and the sta7 mutant is adversely effected in fermentative metabolism and has drastically reduced H 2 -photoproduction rates in comparison to the wild-type cells. These two unique mutants are being studied further using omic -based strategies to better understand H 2 metabolism in C. reinhardtii. Moreover, additional mutants are being characterized and a new library of random mutants has been generated to screen for mutants with increased levels of hydrogenase activity. Characterization of the hydef-1 mutant led to the discovery of the maturation genes required for assembly of an active [FeFe]-hydrogenase. The coexpression of these proteins in combination with an [FeFe]-hydrogenase structural gene led to the first reported heterologous expression of an active [FeFe]- hydrogenase in E. coli. We have leveraged this new expression system to identify a suitable geneshuffling technique to generate large and diverse [FeFe]-hydrogenase libraries for screening purposes. In a demonstration of the viability of the shuffling approach, single-stranded DNAs from [FeFe]- hydrogenase genes were used to generate novel enzyme libraries. Activity assays demonstrated that several shuffled products encode active hydrogenases. Moreover, we show that a single set of [FeFe]- hydrogenase maturation proteins is sufficient for assembly of the unique catalytic H-cluster in a diversity of native and shuffled [FeFe]-hydrogenases. The rapid generation of large libraries containing shuffled [FeFe]-hydrogenases, which can be expressed as active enzymes, will provide the framework for future screening and recombinant strategies aimed at identifying novel [FeFe]-hydrogenases. We are also examining the possibility of expressing [FeFe]-hydrogenases in hosts such as cyanobacteria, which could subsequently be used to express shuffled enzymes. Effective screening and/or selection strategies must then be refined to isolate enzymes of interest. The chemochromic H 2 sensors are currently being adapted for higher throughput screening and will be used to assay organisms expressing shuffled enzymes, as well as diverse native phototrophs for hydrogenases more amenable to the photoproduction of H 2. This work is supported by the U.S. AFOSR and the U.S. DOE, Office of Science.

17 An Update on Algal Hydrogen Production at NREL Michael Seibert National Renewable Energy Laboratory, Golden, CO Chlamydomonas reinhardtii is able to directly couple water oxidation to the photoproduction of H 2. We have been examining the volumetric production of H 2, using the sulfur-deprivation technique, first reported by NREL and UC Berkeley in Many experiments were done to understand the mechanism of H 2 production (which involves the co-occurrence of aerobic photosynthesis, anaerobic fermentation and respiration) and to optimize the process in batch culture. With our colleagues in Russia and at the Univ. of Minn., we have (a) eliminated the need for acetate in the batch process by replacing it with CO 2 and controlling the light regime; (b) converted the batch system to a continuous process in which H 2 photoproduction could be maintained for 6 months (but at low production rates); (c) immobilized the algae on glass fiber, increasing the light conversion efficiency up to about 0.35%, while maintaining continuous production of H 2 for up to 3 months; and (d) developed a new immobilization technique, which uses low cost materials and is suitable for mass production. While helpful in learning how to produce H 2 in different types of photobioreactor systems, all of these techniques use a fraction of the photosynthetic potential of the alga. This is due to the nature of sulfur-deprivation, which greatly decreases the activity of photosynthesis. In order to utilize the maximum photosynthetic potential of the organism, we must address the inherent O 2 -sensitivity of [FeFe]-hydrogenases. To this end we have collaborated extensively with the Beckman Institute (U. of Ill.) and the NREL Computer Sciences Center, to develop Molecular Dynamics methods for simulating H 2 and O 2 gas diffusion in [FeFe]-hydrogenases. These studies have identified two well-defined pathways by which O 2 can access the catalytic site, and many more pathways for H 2 to escape the protein. A strategy to protect the catalytic site from O 2 is to employ site-directed mutagenesis to restrict access of O 2 along the two pathways. To identify candidate amino acid residues for mutagenesis, we generated Potential Mean Force maps, which plot the free energy for O 2 placed at positions along the two pathways. These show regions of low (cavities) and high (barriers) energy, and provide us with the identity of potential specific residues to mutate. Site-directed mutants have been generated, the mutated proteins expressed in E. coli using the [FeFe]-hydrogenase expression system developed at NREL/CSM, and the initial results are encouraging. Finally, we have begun to develop a global understanding of the factors that promote H 2 production by microarray transcript profiling and qpcr methods. The work is elucidating fermentative pathways used by WT C. reinhardtii during anaerobiosis and will provide insights into how mutants, altered in normal H 2 metabolism, acclimate to H 2 -production conditions. More detailed knowledge of the metabolic and regulatory context that facilitates H 2 production will be necessary to understand and ultimately eliminate current limitations in H 2 -production yields. PW King, K Kim, S Kosourov, S. Smolinski, P-C Maness, and ML Ghirardi (NREL); A. Dubini, L Nagy, J Meuser, and MC Posewitz (CSM); J Cohen and K Schulten (U. Ill); AS Federov and AA Tsygankov (RAS, Russia); J Gosse and M. Flickinger (U. Minn.); and F Mus and A Grossman (Carnegie Inst.) have all been intimately involved in this research. Sponsorship by the U.S. DOE Office of Science; the HFC&IT Program; and the AFOSR is greatly appreciated.

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