1. IEA-HIA Annex21 BioHydrogen Production Annex 21 Athen, Nov.05-07, 2008 OA: Jun Miyake, AIST/U. Tokyo

2. BioHydrogen: Concept

3. BioHydrogen In the World Japan Russia India China Korea Thailand Turkey U.S.A. Canada U.K. Netherlands Sweden Italy Singapore Taiwan France Israel Bulgaria Portugal Switzerland Poland Denmark Norway Hungary Germany Malaysia Cuba

4. H 2 Price Evaluation

5. Annex21 Members Potential HIA Members Singapore Turkey Russia Taiwan Hungary Latvia Thailand New Zealand India China Portugal Annex21 Members Canada Finland France Germany Japan Korea Netherlands Norway Sweden UK USA

6. Annex21 4 Subtasks A. Biohydrogen Systems. Goal: increase achievable H2 production from substrates above currently achievable yields (e.g. 3 to 4 moles H2/ mole of glucose). Activity Leader: Netherlands B. Basic Studies for Photobiological Hydrogen Production. Goal: demonstrate potentially practical processes for conversion of water or organic substrates to H2 with solar energy. Activity Leader: United States of America C. Bio-inspired Systems. Goal: identify promising applications of enzymes and biologically-inspired processes for hydrogen production and fuel cells Activity Leader: Sweden/France D. Overall Analysis. Goal: to provide independent evaluations and analyses for the various technologies considered by the Annex. Activity Leader: Japan

7. Dark fermentation Continuous H 2 Production from soybean-curd (TOFU) processing waste Using anaerobic mixed microflora at 60 ℃ Maximal H 2 production rate 7 L H 2 /L/day Maximal yield 1.6 mol H /mol glucose Hollow-fiber microfiltration membrane module improved H 2 production rate showing the ranges of 0.9-1.3 m 3 /day in a 100 L scaled system Subtask A

8. Genetic research for photosynthetic bacteria Genetic study of Rhodobacter sphaeroides KD131, isolated from the bay area in Korea Mainly consists of an uptake-hydrogenase and two nitrogenases gene clusters that are identified by genome analysis of a bio- hydrogen producer, R. sphaeroides KD131. Circle view of Rhodobactor sphaeroides KD 131 Genetic approach to improve H 2 production Subtask A

9. Cellulolytic, anaerobic bacterium Clostridium thermocellum Clostridium termitidis C. thermocellum possesses the highest rate of cellulose-degradation of all known microorganisms Dr. Levin Subtask A

10. Microbial electrolysis cell (MEC) Membrane-less continuous flow microbial electrolysis cell (MEC) with a gas- phase cathode. This MEC had the highest volumetric rate of hydrogen reported or this type of device, 6.3 L/L/day. Subtask A

11. Dr. Lalman’s group (University of Windsor) Supplement with linoleic acid improved hydrogen production from glucose using a mixed anaerobic culture toward 2.4 mol H 2 / mol glucose, while yields were lower at pH 7.6 (1.4 mol H 2 / mol glucose). Subtask A

12. Dr. Nakhla’s group (University of Western Ontario) The maximum yields of 2.3 and 1.6 mol H 2 /mol glucose by resulting microflora were achieved for the 65 degrees C pretreated anaerobically digested and activated sludges, respectively. DNA analysis of the microbial community showed that the elevated pretreatment temperatures reduced the species diversity. Subtask A

13. Dr. Beland’s group (Environment Canada) examined hydrogen production from food waste (FW), primary sludge (PS) and waste activated sludge (WAS). FW was found to have low pH buffering capacity while the values for PS and WAS were relatively higher. All combinations of the feedstocks (FW+PS, FW+WAS and FW+PS+WAS) showed enhanced hydrogen production potential as compared with the individual wastes. A mixing ratio of 1:1 was found to be the best among the ratios tested for all three co-digestion groups. A hydrogen yield of 112 mL/g volatile solid (VS) added was obtained from a combination of FW, PS and WAS. Subtask A

14. the Maness group at NREL has been studying the fermentation of dilute-acid, pretreated corn stover lignocellulose by the thermophilic cellulose-degrader Clostridium thermocellum 27405. Yields and rates of H 2 production depend on carbon loadings, with higher carbon substrate loading yielding faster rates of H 2 production but with lower molar yields of H 2. Nevertheless, this is the first demonstration that corn stover lignocellulose can serve as a feedstock in support of H 2 production. Using cellobiose as the substrate, the group also observed an increase in total amount of H 2 when various metabolic pathways are blocked using chemical inhibitors. These findings provide the proof of concept that metabolic pathway engineering is a promising approach in directing more cellular flux toward H 2 production provided a genetic system can be developed for this microbe. Subtask A

15. The fermentative consortium at NREL (Maness, Ghirardi, and Seibert) was also shown to metabolize algal starch, lipid and protein at a non-optimal yield of 0.45 mg H 2 /mg biomass; is able to utilize algal biomass isolated from a variety of growth conditions, both fresh and frozen (damaged), without the need for pre-treatment; and in collaboration with the Tsygankov laboratory (Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Russia), the integration of fermentative with photosynthetic bacterial H 2 production was demonstrated using potato wastes as the initial substrate for fermentation. Subtask A

16. In Netherlands, the model hydrogen-producing extreme thermophilic Caldicellulosiruptor saccharolyticus was studied in detail. By using 13 C-NMR the occurrence of the Embden-Meyerhof (EM) pathway in the extreme thermophile C. saccharolyticus was shown. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mol of glucose. The measured yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H 2 per mol glucose) were obtained at low growth rates. whole genome of C. saccharolyticus became available. Subtask A

17. The formate metabolism of a Moorella strain and a Desulfovibrio strain was studied. It was found that these strains were able to grow by the hydrolysis of formate to hydrogen and bicarbonate. However, these strains were only able to grow when the hydrogen was efficiently removed by methanogens. This led to syntrophic growth. Subtask A

18. In the UK, the global flour industry produces 96 million ton/year of wheatfeed, which is mainly used for animal feed. This co-product is high in carbohydrates and potentially a significant substrate for biohydrogen production. A 10 l bioreactor, inoculated with sewage sludge, was operated on wheatfeed (10 g l −1 ) at pH 5.5 and 35 °C in batch and semi-continuous mode (15 h hydraulic retention time (HRT)). Wheatfeed hydrolysate was also investigated in continuous mode (15 h HRT). NaOH–H 2 O 2 treatment of 25 g l −1 wheatfeed resulted in hydrolysate containing on average 8.1 g l −1 total sugar. Hydrogen yields of 64 and 56 m 3 H 2 per ton dry weight were produced from wheatfeed in batch and 56 m 3 H 2 per ton dry weight of wheatfeed in semi-continuous mode. Hydrogen yields from hydrolysate were only 22 and 31 m 3 H 2 per ton dry weight, (or 0.9 mol H 2 per mol hexose degraded, assuming all sugar is hexose). Fermentation of unhydrolysed wheatfeed is therefore recommended. It is calculated that approximately 264 m 3 /ton of CH 4 can be produced from a subsequent anaerobic digestion stage. The biohydrogen produced (diesel equivalents) from the 1.2 million ton/year of wheatfeed in the UK would be more than twice that required for transportation by the UK flour industry. (Hawkes et al. (2008) Bioresource Technol. 99 5020–5029. ) Subtask A

19. Biohydrogen from olive mill waste water: Hydrogen production from olive mill waste water by Rhodobacter sphaeroides was achieved in a two-step process involving clay pretreatment and photofermentation. Compared to the photofermentation using raw olive mill waste water (14 lH2/lOMW) (Eroglu et al., 2008a), the amount of photobiological hydrogen production was doubled by using the effluent of a clay pretreatment process (31.5 lH2/lOMW). Subtask A

20. Biohydrogen from waste ground wheat: The objective of this study was the optimization of biohydrogen production from ground waste wheat solution by dark and photofermentation processes using mesophilic dark fermentative bacteria (heat-treated, anaerobic sludge) and photosynthetic bacteria (Rhodobacter species). In dark fermentations, maximum hydrogen yield (1.53 molH2/mol glucose) and the specific rate (98 mlH2/ (g biomass.h)) were obtained when the C/N and C/P ratios were 200 and 1000, respectively. (Argun et al., 2008a; Argun et al., 2008b; Oztekin et al., 2008). Subtask A

21. Biohydrogen from dark fermenter effluents: Biohydrogen production by Rhodobacter capsulatus on thermophilic dark fermenter effluent of miscantus hydrolysate in indoor batch- operation was investigated (Uyar et al., 2008). The pretreatment of dark fermenter effluent with iron and buffer addition resulted in a drastic increase (from 0.3 l H2 /l c ) to 1.0 l H2 /l c ) in H 2 production. Photobiological hydrogen production on dark fermenter effluents of molasses and potato steam peels were investigated with Rhodobacter capsulatus wild type and hup deleted mutant strains. The maximum productivity achieved was 2.73 g H2 /(m 3.h) on molasses effluent and 1.1 gH 2 /(m 3.h) on potato steam peels effluent adjusted with Fe, Mo and buffer in indoor batch-operations. Subtask A

22. Genetics and Strain Improvement: Genetically improved strains of Rhodobacter capsulatus and Rhodobacter sphaeroides were carried out by deletion of the uptake hydrogenase gene. A 70% increase with hup- strain of R. capsulatus (Ozturk et al., 2006) and 20% increase with hup- strain of R. sphaeroides in hydrogen production were observed (Kars et al., 2008). Subtask A

23. Physiology and Biochemistry of PNS Bacteria The effect of light intensity, wavelength and different dark-light cycle protocols and different initial dark periods on growth and hydrogen production of Rhodobacter sphaeroides were examined in indoor batch operations (Uyar et al., 2007). The rate of hydrogen production increased with increased light intensity and reached saturation at around 270 W/m 2. The effect of fluctuating temperature in outdoor conditions on photobiological hydrogen production was investigated by Rhodobacter capsulatus (Özgür et al., 2008). Hydrogen production decreased by 50% if the outdoor temperature fluctuates between 15°C and 40°C. Subtask A

24. Process Development Investigation of performance of the flat plate solar bioreactor (8 l) operating in outdoor conditions with different carbon sources and olive mill waste water was studied by Rhodobacter sphaeroides O.U.001 (Eroglu et al., 2008b). Hydrogen production rate was the highest (0.01 l H2 /(l c.h)) when malate was the carbon source. Large scale panel (40 L) and tubular (80 L) photobioreactors have been constructed and continuous hydrogen production have been achieved by different strains of R.capsulatus on acetate. Subtask A

25. The unicellular green alga Chlamydomonas reinhardtii possesses a [FeFe]- hydrogenase HydA1 (EC 1.12.7.2), which is coupled to the photosynthetic electron transport chain. Large amounts of H 2 are produced in a light- dependent reaction for several days when C. reinhardtii cells are deprived of sulfur. The analysis of the H 2 metabolism of S-depleted C. reinhardtii cultures showed that electrons for H 2 -production are provided both by PSII activity and by a non-photochemical plastoquinone reduction pathway, which is dependent on previous PSII activity. A kinetic model has been developed to relate culture evolution from standard photosynthetic growth to H 2 producing cells. Subtask B

26. Chlamydomonas reinhardtii H 2 production, narrowly linked to the photosynthetic process, results from complex metabolic reactions highly dependent on the environmental conditions of the cells. A kinetic model has been developed to relate culture evolution from standard photosynthetic growth to H 2 producing cells (Fouchard et al (2009) Biotechnol. Bioeng. 102: 232-245). It represents transition in sulfur-deprived conditions and the two main processes then induced which are an over-accumulation of intracellular starch and a progressive reduction of PSII activity for anoxia achievement. Because these phenomena are directly linked to the photosynthetic growth, two kinetic models were associated, the first (one) introducing light dependency (Haldane type model associated to a radiative light transfer model), the second (one) making growth a function of available sulfur amount under extracellular and intracellular forms (Droop formulation). The model parameters identification was realized from experimental data obtained with especially designed experiments and a sensitivity analysis of the model to its parameters was also conducted. Model behavior was finally studied showing interdependency between light transfer conditions, photosynthetic growth, sulfate uptake, photosynthetic activity and O 2 release, during transition from oxygenic growth to anoxic H 2 production conditions. Subtask B

27. In the USA, the S-systems analysis formalism was used to model the metabolic pathways involved in H 2 production by sulfur-deprived algae, and a sensitivity analysis was performed (Jorquera et al., 2008, Int. J. Hydrogen Energy 33:2167-2177). The mathematical model successfully described the literature data with respect to O 2 and H 2 evolution rates in response to sulfur deprivation and to the re-addition of sulfate to the system. The sensitivity analyses identified targets for future protein engineering aimed at increasing the rates of H 2 production by sulfur-deprived algae. Subtask B

28. Green Algea, seven strains found positive for H 2 production under anaerobic conditions were also screened for the ability to produce H 2 under S deprivation. Subtask B

29. Cyanobactera, in the unicellular cyanobacterium Synechocystis PCC 6803 the pentameric bidirectional NiFe-hydrogenase (HoxEFUYH, encoded by the hox genes) is the sole enzyme involved in the H 2 metabolism. Novel and unique finding already published. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme. The level of analysis provided by this investigation presents various tools and knowledge that are important for future development of cyanobacterial biohydrogen production. Subtask B

30. Bio-inspired Systems: The synthesis and characterisation of the pro-ligand complexes are interesting model of the active site of [NiFe] hydrogenase, indicating the imidazole functions has a strong effect on the electronic structure of the complexes. Dissymetrically disubstituted di-iron azadithiolate complexes protonate exclusively at the N atom of the bridge, like the hexacarbonyl precursor but in contrast to symmetrically disubstituted analogues; substitution of dppe for two CO groups noticeably increases the kinetics of the electrocatalytic proton reduction process. Electrochemical investigations on a structural analogue of the [2Fe](H) subsite of [FeFe]H(2)ases were conducted in MeCN/NBu(4)PF(6). The most efficient molecular photocatalytic systems reported so far for hydrogen production could compete with some platinum-based systems. Quantum yield values up to 16% under visible irradiation. Subtask C

31. Bio-inspired Systems: The extreme thermophile Pyrococcus furiosus hydrogenase was proposed to be a new type of H 2 evolution hydrogenase, solubule and membrane- bound ones. Subtask C

32. In Canada, Dr. Hallenbeck’s group has been looking at the heterologous expression of Desulfovibrio vulgaris Hildenborough. The highest yields (at, or somewhat higher than 2 mol H 2 /mol glucose) were obtained with cultures limited for glucose.H 2 /mol Bio-hybrid/ biofuel cell efforts are investigating hydrogenases as biocatalysts for creating hybrid charge-transfer complexes with nanomaterials, and as catalysts in photoelectrochemical devices. Subtask C

33. In the USA, bio-hybrid/ biofuel cell efforts are investigating hydrogenases as biocatalysts for creating hybrid charge-transfer complexes with nanomaterials, and as catalysts in photoelectrochemical devices. Subtask C

34. Biofuel cells: In France, modification of gold and graphite electrodes with commercially available carbon nanotubes is a useful technique for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption (Lojou et al. (2008) J Biol Inorg Chem. 13:1157-67). Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl- functionalized SWCNs were used and compared. Subtask C

35. In korea, Bioelectrocatalytic hydrogen (H 2 ) production was studied using Thiocapsa roseopersicina hydrogenase in a two-compartment proton-exchange-membrane (PEM) fuel-cell system equipped with carbon-paper electrodes. Sodium dithionite(SD), as an electron donor, and hydrogenase, as a catalyst, were used in the anodic oxidation reaction and in the cathodic reduction reaction, respectively. Subtask C

36. In Netherlands, the interaction between bacteria and electricity is being studied. Bacterial processes can be used to produce electricity in biofuel cells. However, microbial processes can also be stimulated by electricity, e.g. for hydrogen production from acetate in the dark. Subtask C

37. In the USA, the Maness group at NREL has collaborated with Dr. Bruce Logan (Penn State Univ.) in testing a novel concept, combining dark fermentation with a microbial electrolysis cell (MEC) to improve H 2 molar yield. In their work, the organic acids and ethanol present in the fermentation effluent from either cellobiose or corn stover lignocellulose fermentors were successfully converted to H 2 via the MEC. The integrated process resulted in a combined H 2 molar yield that substantially exceeded the biological maximum, which is 4 mol H 2 per mol hexose. Subtask C

38. In the UK, a hydrogen-producing Bio-Fuel Cell was developed. Ammonia losses during swine wastewater treatment were examined using single- and two-chambered microbial fuel cells. Ammonia removal was 60% over 5 days for a single-chamber MFC with the cathode exposed to air (air.cathode), versus 69% over 13 days from the anode chamber in a two-chamber MFC with a ferricyanide catholyte. In both types of systems, ammonia losses were accelerated with electricity generation. The possibility of biological ammonia oxidation with current generation was investigated by intermittent dosing of reactors with ammonia and by conducting cyclic voltametry. Subtask C

39. Enzyme engineering: Hydrogenases are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Two quantitative methods for probing the rates of diffusion within hydrogenases were recently described (Leroux et al. 2008 Proc Natl Acad Sci U S A. 2008 105:11188-93. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. The study of structurally characterized mutants of a NiFe hydrogenase revealed that two mutations, which significantly narrow the tunnel near the entrance of the catalytic centre, decrease the rates of diffusion of CO and H 2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. It was therefore demonstrated that diffusion rates cannot be predicted and should be measured and that the role of side-chains are essential in the dynamics of this mechanism. Subtask C

40. A theoretical QM/MM study of the [NiFe] hydrogenase from Desulfovibrio fructosovorans has been performed to investigate possible routes of proton transfer between the active site and the protein surface (Galvàn et al. 2008 Proteins. 73: 195- 203). The minimum energy paths, were obtained using a modified version of the nudged elastic band method, for a set of proposed pathways. The calculations were carried out for the crystallographic structure and for several structures of the protein obtained from a molecular dynamics simulation. The results show one of the studied pathways to be preferred for transport from the active site to the surface, but the preference is not so strong when transport occurs in the opposite direction. Subtask C

41. Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. The structure at high resolution of the recombinant, reconstituted S- adenosine-L-methionine- dependent HydE from Thermotoga maritima was solved recently (Nicolet et al. 2008 J. Biol. Chem. 283:18861-72). Besides the conserved [Fe(4)S(4)] cluster involved in the radical- based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] centre, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes. Subtask C