PROPOSAL FOR A NEW COST ACTION

Biological and Biochemical Diversity of Hydrogen Metabolism

Short title: Hydrogen Biodiversity

Proposing countries:

The Netherlands and Hungarian Republic

The Netherlands
Proposer: Dr. Wilfred R. Hagen Department of Biochemistry Agricultural University NL-6703 HA Wageningen Dreijenlaan 3 Tel: +31 83 70 83860 Fax: +31 83 70 84 801 E-mail: fred.hagen@epr.bc.wau.nl

COST National Coordinator: Mr. Upton Van der Vliet Senter - EG Liaison Agency of the Ministry of Economic Affairs P.O.Box 30732 (Grote Markstraat, 43) NL-2500 GS The Hague Tel: +31 70 361 02 50 Fax: +31 70 356 28 11

E-mail: u.vd.vliet@egl.nl

Hungarian Republic
Proposer: Dr. Kornl L. Kovcs Department of Biotechnology A. Jzsef University of Szeged H-6726 Temesvri krt. 62 Tel: +36 62 454 351 Fax: +36 62 454 352 E-mail: kornel@szbk.u-szeged.hu

COST National Coordinator: Ms. Mrta Marjay National Committee for Technological Development (OMFB) Department for International Relations P.O.Box 565 (Szervita tr 8, H-1052) H-1374 Budapest Tel: +36 1 117 5782 Fax: +36 1 117 54 36

E-mail: h12779kis@ella.hu

Date of Submission:

July 20, 1998

Draft of a Technical Annex for a Prospective COST Action on

Biological and Biochemical Diversity of Hydrogen Metabolism

A. BACKGROUND A.1. STATE OF KNOWLEDGE Understanding hydrogen metabolism is important for both basic and applied research. The biological diversity of hydrogen metabolism is reflected by the very recent recognition of the role of strictly hydrogen dependent ancient microbes in the evolution of eukaryotes. The key enzyme in hydrogen metabolism is hydrogenase. Hydrogenases catalyse the formation or decomposition of the simplest molecule occurring in biology: hydrogen. H 2H+ + 2 e2

This group of metalloenzymes has gained increasing interest for potential biotechnological exploitation. In spite of the recognized role of hydrogenases in various bioenergetic processes of unicellular organisms, their practical application is hindered by a limited understanding of the structure and mechanisms of function of the key enzymes involved. The simple-looking task is solved by a sophisticated molecular mechanism. A research programme that requires sophisticated biochemical, molecular biology and biophysical infrastructure and knowledge base, which is present in the laboratories forming the COST network is necessary to solve this complex scientific problem. The proposed new COST Action builds on the already established, and very effectively functioning research network formed through the activity of COST Action 8.18. This network combined the efforts of more than 80 leading scientists in 45 laboratories of 13 European nations. COST Action 8.18 focuses on the hydrogenase enzyme itself, the proposed New Action extends this scope to significantly wider aspects of hydrogen metabolism including metalloenzyme model and computational chemistry and various metabolic processes related to gas metabolism in microorganisms. The proposed

research uses the unique combination of the indispensable expertise available only through solid international collaboration. In addition to the scientific thrill of uncovering the various aspects of enzyme structure-function relationship, the integrated international collaboration should contribute to the solution of pressing tasks related to environmental biotechnology. Hydrogen metabolism is involved in biogas production, denitrification, nitrogen fixation, corrosive sulfate reduction and biohydrogen production from water, and thus it is of remarkable commercial importance and environmental applications.

A.2. NEED FOR A COST ACTION The field related to hydrogen metabolism is rather wide and studied in several COSTmember countries and outside the geographic area covered by COST, but not fully covered by existing programmes. Many of the existing activities treat biohydrogen as a strong candidate in the energy carrier structure marginally. The proposed action intends to focus European research efforts leading to the development of such systems operating on large scale as a long term goal. Since there are several potential routes to reach this objective, a thorough and comparative study at the present basic and applied research stage will contribute to the standardization of the methods to be offered for global implementation in the future. Biological hydrogen production has been an active field of research for many years, with significant applied R&D programs currently supported by the governments of Japan and the US, and related basic research is also carried out in these countries in addition to the European Union and Associated States. Most recently, biohydrogen R&D was the subject of a major international conference, held in Kona, Hawaii, in June 1997, attended by over 100 researchers from over a dozen countries. Photobiological hydrogen production is the subject of Subtask B of Annex 10 of the IEA Hydrogen Agreement. Biological Hydrogen production was also identified a technology area for collaborative international R&D under Task Force 7 of the Climate Technology Initiative - CTI - established under the UNFCCC. In Europe COST Action 8.18 has been practically the only vehicle to assure effective contribution to the global problems co-ordinating the various research activities scattered in the participating nations (13 European countries, USA and Russia) during the past few years. The concerted European efforts represent a research strength that confers the leading edge in comparison with the well organized and funded projects launched by major oversees countries. Involvement and training of young scientists and those working in the less-developed European countries are key elements in developing the European biohydrogen network.

B. OBJECTIVES AND BENEFITS B.1. OBJECTIVES Main objective

The main objective of the proposed COST action is to merge interrelated European expertise in order to understand the molecular structural basis of biochemical and physiological processes connected to hydrogen metabolism and its biological and chemical diversity. Secondary objectives understanding the role of various structural units in hydrogen activation by hydrogenases at molecular level exploring biodiversity and chemical diversity of hydrogen producing systems analysing the structural, genetic and physiological basis of the functional hydrogenases establishing the requirements for the development of functionally active stable hydrogen activating catalysts for further practical applications developing methods and technologies for the exploitation of biologically and/or chemically produced hydrogen B.2. BENEFITS The results will have positive environmental effects when hydrogen metabolism will be incorporated into in vivo and in vitro applications. Examples of such processes are: nitrogen fixation (environmental protection), biogas production (alternative energy source), H2 dependent denitrification of industrial waste waters and drinking water (public health), solar energy bioconversion via generation of O2 and H2 from water (sustainable economical development), conversion of methane and/or biogas to easily storable methanol (alternative energy carrier), stereo and site specific hydrogenation of high value compounds (pharmaceutical industry), and cofactor regeneration (biotechnology with redox enzymes).

C. SCIENTIFIC PROGRAMME Hydrogen metabolism in diverse micro-organisms and their potential for various applications will be studied. Data at molecular and/or atomic levels are being collected on the relevant metalloenzymes, in particular on members of the various classes of hydrogenases present in each major group of micro-organisms where hydrogen metabolism plays a physiological role.

The proposed COST Action covers 5 areas as follows: 1. Biological and biochemical diversity of hydrogen metabolism: 1.1. The multiplicity of hydrogenases at the organism level with different functions or activities; 1.2. The diversity and variations of enzymes carrying the same hydrogen activating function at the molecular level 2. Active centres of hydrogenases: 2.1. Structure of the metal centres related to the enzyme mechanism; 2.2. Chemical models and computational chemistry of metal centres 3. Biosynthesis of proteins involved in hydrogen metabolism: 3.1. Expression patterns of bacterial hydrogenases and the role of accessory genes in both hydrogenase synthesis and functional structure; 5

3.2. Regulatory pathways as determined by external factors such as hydrogen, or trace element supply; 4. Towards the realisation of a successful biohydrogen concept: 4.1. Global collaborations for biotechnological exploitation of hydrogenases; 4.2. Utilization of extremophiles in biohydrogen research and development; 5. Events related to hydrogen metabolism: 5.1. Redox and electron transport phenomena involving hydrogenases and biohydrogen production 5.2. Relationship between the metabolism of hydrogen and other gases (N2, NOx, CO, CO2, CH4) All these areas can be approached by a combination of the very powerful tools of molecular biology, biochemistry, biophysics and biotechnology. The work programme will be divided into 5 working groups specially dealing with issues corresponding to the areas listed above. Tasks ranging from structural studies to ecological considerations are to be addressed in the various WGs. The co-ordination of the diverse research approaches in a COST Action is therefore highly desirable.

WORKING GROUP 1. Biological and biochemical diversity of hydrogen metabolism 1.1. The multiplicity of hydrogenases at the organism level with different functions or activities Hydrogenases can serve both for the uptake of hydrogen into the cell and the disposal of electrons via hydrogen production. Since electrons can be used for energy production in electron transport chains, some organisms evidently recover and reoxidize hydrogen which has escaped during nitrogen fixation by the inherent hydrogen producing activity of nitrogenase. It is of fundamental ecological and biotechnological interest to understand the functions of uptake vs. hydrogen producing hydrogenases. In order to approach this question such enzymes have to be characterized and compared. They can then be changed or knocked out by site directed mutagenesis. The physiological effects will give clues as to their differential functions. The same holds true for cases in which multiple, apparently functionally equivalent, hydrogenases are found. In this case the correlation of biochemical characteristics and structural features will guide mutagenesis experiments in order to get insights into the structure-function-relationship of the enzymes and should eventually lead to the definition of minimal enzymes, the construction of which would be an outstanding biotechnological achievement. Other enzymes, e.g., transaminases in cyanobacteria, or products of hydrogenase accessory genes, like HupUV, catalyze in vitro the tritium exchange reaction often considered to be diagnostic for hydrogenases. Related enzymes such as COdehydrogenase, cyanase, and others will give valuable insights in understanding hydrogenase catalysis. 1.2. The diversity and variations of enzymes carrying the same hydrogen activating function at molecular level

Hydrogenases are usually classified on the basis of their metal content: the two main groups being the NiFe and Fe-only enzymes. Although recent comparative spectroscopy indicates an unifying set of basic structural elements to make up the active site of metallohydrogenases, the different enzymes diverge in protein structure and in intramolecular electron transfer. The iron-only hydrogenases have been relatively little studied but are of great potential interest because of their involvement in fermentation by bacteria such as Clostridia and sulfate-reducing bacteria. Moreover they have higher catalytic rates in hydrogen production than the nickel-iron hydrogenases. These enzymes have been shown recently to contain the same infrared signature of diatomic ligands as the NiFe hydrogenases, which may be one of the most important features of the active site. The structure and function of the iron-only hydrogenases represent an important goal of future studies. The organisation of archaeal genes significantly differs from that of eubacterial ones. It is therefore of great importance to study hydrogenases from these organisms. Methanogens have a distinct and complex pathway for hydrogen metabolism including hydrogenases that contain NiFe, NiFeSe or no metal at all. Understanding the structure of these enzymes will certainly complement our knowledge on these enzymes and to develop a functional hydrogenase containing minimal protein components. Thermodynamically, H2 formation becomes more favourable at elevated temperatures. Attention, therefore, should be given to hydrogen formation by thermophilic and hyperthermophilic micro-organisms. Certain hyperthermophilic archaea can be grown on relatively cheap and simple media (e.g., based on seawater and starch) affording very significant yields of biomass. The heat required to sustain large batch cultures at elevated temperatures, typically around 90 C, is readily available as a common industrial waste product.

WORKING GROUP 2. Active centres of hydrogenases 2.1. Structure of the metal centres related to the enzyme mechanism The three-dimensional atomic structure of nickel-iron (Ni-Fe) hydrogenases is now known at the atomic level. Crystallographic studies on the enzyme from several Ni-Fe hydrogenases from sulfate-reducing bacteria have shown consistently that the active site contains one nickel and one iron both co-ordinated to the protein by only four cysteine sulfur ligands, with three non-protein diatomic ligands bound to the iron. Infrared spectroscopy has detected these three diatomic ligands in all the redox states of the enzyme and, more recently, identified their nature (two CN and one CO) by isotopic substitution. Such structure a extraordinary among metalloenzymes and must be linked to the specific reaction catalyzed. Moreover crystallographic and moleculardynamics studies suggest that specific transfer routes are required for the transfer of the chemical reaction partners (H2, H+ and e- ) between the active site, deeply buried inside the protein, and the molecular surface.

The catalytic mechanism remains in many respects far from understood: e.g., the respective role in the redox events of the nickel and iron atom and the three diatomic ligands at the active site. These studies should provide a more precise picture of the active site during activation and catalysis (e.g., the formal oxidation states of the nickel and iron atoms during the catalytic cycles) and an indirect view of the substrate binding site(s). Moreover genetic studies combined with X-ray and spectroscopic analysis should lead to a better understanding of the hydrogenase function through, for example, appropriate mutations of some amino-acids which seem to play a role in the catalytic mechanism (i.e., those involved in the active site or in the putative transfer routes of the components of the reaction, H2, H+ and e-). 2.2. Chemical models and computational chemistry of metal centres Insights into the function of the different components of the catalytic site of hydrogenase can be gained from chemical models. These fall into two categories: functional, which show aspects of the activity but do not necessarily resemble the active site, and structural, which imitate the structural and spectroscopic properties. Since the determination of the structure and spectroscopic identification of the diatomic ligands, a number of promising models have appeared mimicking the nickel, the bimetallic Ni-Fe, or the Fe(2CN,CO) arrangement. This approach provides a powerful tool to understand the redox properties of nickel, iron and ligands in the active site and hence their respective roles in the catalysis. An added attractive feature of these compounds is the possibility of generating catalysts which have the functionality of hydrogenase but are more robust. The active site of hydrogenases comprises metals which are abundant, iron and nickel, in contrast to the platinum metal catalysts which are used in electrochemistry and fuel cells. Imitating the mixed-metal clusters of hydrogenase, with their higher oxidation states and specific ligand geometry, may provide the basis for the design of a new generation of catalysts for the benefit of mankind. Modern (semi) ab-initio methods, in particular density functional calculations, have now been developed to a level of meaningful application to open-shell systems of a nature and size corresponding to the active site of hydrogenases. These theoretical studies can relate electronic and magnetic properties with structure and structural changes. The results will be complementary to, and supportive of, structural and spectroscopic studies.

WORKING GROUP 3. Biosynthesis of proteins involved in hydrogen metabolism 3.1. Expression patterns of bacterial hydrogenases and the role of accessory genes in both hydrogenase synthesis and function. Hydrogenases are oligomeric enzymes. In Ni-containing enzymes, two ubiquitous catalytic subunits are found, one of which contains a bimetallic Fe-Ni reaction center. The other subunit carries Fe-S clusters involved in electron transport. The enzyme complexes frequently have additional subunits serving as electron acceptor moieties. Their functions are not understood in all cases and deserve further analysis. A second major focal point of the planned research will be the role of the formation of the Fe-Ni8

center. This implies the incorporation of the two metals into the polypeptide backbone during its folding. A particularly intriguing question is the generation and incorporation of diatomic ligands (CO and CN) recently discovered at the Fe atom. At present, nothing is know about their (enzymatic or nonenzymatic?) synthesis which is a major challenge for a molecular biology working group in the proposed Action. 3.2. Regulatory pathways as determined by external factors such as hydrogen, or trace element supply. It is clear that uptake hydrogenase synthesis is of no advantage to the cell if the substrate, hydrogen, is unavailable. Therefore, it was anticipated that hydrogen supply would influence hydrogenase gene expression. Only recently has this expectation been verified. Hydrogen sensors have been detected that are used to transmit the signal availability of hydrogen to the transcription regulatory pathway. The signal transduction remains to be elucidated. Most hydrogenases are metal-containing enzymes. Many of those contain Ni as part of the primary reaction center. The role of the metal in synthesis regulation is a very interesting question. It can apparently occur at the transcriptional level and at the level of posttranslational processing of the protein during the formation of the metal center. Several hydrogenases contain essential selenium atoms in their reactive centres. These enzymes can only be synthesized if this trace element is available. Since hydrogenases are indispensable enzymes in one of these organisms, it has to have alternative hydrogenase genes which are transcribed, when selenium is absent. This is another example, for which the signalling pathway that indicates selenium depletion has to be investigated. Thus a variety of complex patterns governing hydrogenase gene transcription will be amenable to molecular biological analysis.

WORKING GROUP 4. Towards the realisation of a successful biohydrogen concept Realization of practical processes for photobiological hydrogen production from water using solar energy would result in a major, novel source of sustainable and renewable energy, without greenhouse gas emissions. Development of practical processes will require significant scientific and technological advances and relatively long-term (>10 yr.) basic and applied R&D. A five-year collaborative R&D program is proposed under the new Annex IEA Hydrogen Agreement. This work aims to acquire the fundamental and early-stage applied knowledge required to develop biophotolysis processes, including those which involve microalgal hydrogen production from water and sunlight with intermediate CO2 fixation. The main topic areas are of common research interest to the current biohydrogen programs in Japan and the US, as well as of European researchers participating in the proposed new COST Action. However, it only represents part of the overall research effort in this field, as this program aims at mutual research interests, rather than this entire field. The research will be directed and integrated among the various laboratories and countries to aim for rapid advances in both basic and applied R&D areas. 9

4.1. Global collaborations for biotechnological exploitation of hydrogenases It is generally anticipated that hydrogen may become a major energy carrier as environmental problems related to the fossil fuels become more pressing in the near future. Biological systems have great potential to convert solar energy to molecular hydrogen. Production of H2 in these systems is not accompanied by the formation of any deleterious by-products and reactions occur in ambient conditions. Likewise, H2 is a clean source of energy since its combustion is not accompanied by the formation of CO2 or other greenhouse gases. Biohydrogen can be produced according to three distinct strategies. First, photosynthetic microorganisms are able to directly convert solar energy to hydrogen from organic or inorganic substrates or from water. Second, anaerobic heterotrophic microorganisms and acetogens can form hydrogen during the oxidation of organic substrates. Third, a combination of non-biological inorganic systems with hydrogenase in vitro can lead to continuous photoproduction of hydrogen for an extended period of time. The interest in improved or new bioreactor design for biohydrogen production will increase as the need for large scale alternative energy production becomes manifest. Here the collaboration between biologists and engineers will be of great importance. Bioreactors include those equipment specially designed, developed and tested for phototrophic bacteria and cyanobacteria. Efforts should also be focused on finding organisms/mutants suitable to use in such bioreactors for H2 photoproduction. A continuation of work on appropriate photobioreactors is required as well as demonstration of outdoor set-up for generating biohydrogen. 4.2. Utilization of extremophiles in biohydrogen research and development; Hydrogen is presumably produced in hyperthermophilic metabolism as a general dump for excess reducing equivalents. Consequently, biotechnological possibilities can be envisioned in addition to the direct production of molecular hydrogen. Pilot studies indicate that the reducing over-power can be used for the production of (fine) chemicals notably by employing a wide range of aliphatic and aromatic aldehyde reduction-oxidation activities carried by several tungsten-containing aldehyde oxidoreductases, which are coupled to hydrogenase through a redox pool of ferredoxin and nicotinamide nucleotides. Development of these technologies requires fundamental knowledge of the underlying activities and their bioregulation. There is, therefore, a requirement for the development of a novel 'hot' biochemistry. Laboratories must be set up for, e.g., advanced enzymological studies near 100 C. Concepts have to be developed describing the hyperthermophilic nature of structural stability, catalysis, electron transfer, regulation.

WORKING GROUP 5. Events related to hydrogen metabolism

10

5.1. Redox and electron transport phenomena involving hydrogenases and biohydrogen production A very considerable divergence is reached at the level of natural redox partners of hydrogenases. Flavodoxins, ferredoxins, rubredoxins, monoheme cytochromes, multiheme cytochromes, NADH, NADPH have all been implicated as putative partners of hydrogenase. This multitude defines electron transfer to and from hydrogenase as a research area in itself. The redox partner can change in response to nutrient variation (the flavodoxin/ferredoxin switch depending on available Fe levels) or to a changing function (high- potential/low-potential partner depending on whether H2 should be consumed or produced). Different hydrogenases in the same cell can use different redox partners, and one particular hydrogenase can be redox-connected to different streams of metabolism. All this switching and branching of electron transfer requires research for enzymological, physiological, and technological implications. The majority of the putative redox partners consists of small, highly charged proteins for which the direct electrochemistry on solid electrodes has been well established. Consequently, several aspects of hydrogenase are amenable to studies employing cyclic voltammetry and related techniques. For example catalytic-wave analysis in coupled systems (reconstituted chains) defines the rate-limiting step in electron transfer. Furthermore, the relative ease with which these electrode-protein chain constructs can be set up would seem to suggest a spin-off of several prospective applications for hydrogenase systems in biosensors and in bioelectrochemical production of fine chemicals. Photosynthesis can achieve relatively high light energy conversion efficiencies, but only at low light intensity. At full sunlight, efficiencies drastically decline. The reason is that the large amounts of the light-harvesting pigments capture more photons at full sunlight than the photosynthetic apparatus can actually handle. These excess photons are thus wasted, with their energy released as heat or fluorescence, even causing damage to the photosynthetic apparatus. Reducing antenna sizes is a method for increasing photosynthetic efficiencies, and this is a central need in photobiological hydrogen production. Use of photosynthetic bacteria as model systems to demonstrate increased photosynthetic efficiencies in pigment-reduced mutants of single photosystem microbes requires concerted research efforts. In an additional step green algae and cyanobacteria strains should be developed with greatly reduced lightgathering pigment contents in both photosystems using molecular genetic techniques. Among the in vitro systems, heterogeneous photocatalysis involving hydrogen is very promising. The coupling of TiO2 with hydrogenases may link hydrogen production to waste photodegradation. From the point of view of stability and longevity, a good TiO2-hydrogenase system will probably be based on hydrogenases from thermophilic or hyperthermophilic microorganisms.

5.2. Relationship between the metabolism of hydrogen and other gases (N2, NOx, CO, CO2, CH4) In addition to offering an alternative for the global environmental and energy crisis, biologically produced hydrogen may also serve as reductant for numerous microbiological activities of environmental significance. Reductants are needed to 11

convert CO2, atmospheric nitrogen, nitrate, sulfate, and municipal, agricultural, or industrial waste into useful products. In nitrogen fixing phototrophic bacteria and cyanobacteria, H2 production is mainly catalyzed by a nitrogenase, but its partial consumption is quickly performed by an unidirectional uptake hydrogenase. In addition, a bi-directional enzyme may also oxidize or produce some of the hydrogen, depending on the physiological conditions. In the future, it may become technically possible to express foreign hydrogenases in photosynthetic microorganisms, which are the best potential candidates for biological solar energy conversion to hydrogen. The rationale is to couple an oxygen stable hydrogenase to photosynthetically produced reducing power and exploit the maximal H2 production capacity of these microorganisms. Nitrogen fixation by plants is only possible through symbiosis of the plant with bacteria. These bacteria also occur in free living forms. They contain hydrogenases. The physiological conditions of the cells change drastically upon symbiotic interaction with the plant. Oxygen supply in the nitrogen fixing organelles, the plant root nodule is drastically reduced. This allows the activity of nitrogenase (a hydrogenase in itself) and induces additional hydrogenase activities. Nitrogenase catalyze the ATP-dependent reduction of protons in the absence of any other substrate, and N2 is reduced to ammonia in a process accompanied with H2 production. Alternative nitrogenases (V-, Fe only-enzymes) produce more H2 than the conventional, Mo-containing enzyme complex. It is of great interest to understand the parameters and mechanisms leading to the change of hydrogenase expression patterns. In particular, the molecular details of the link between hydrogenase expression and the regulation of nitrogen fixation will be a major issue in the complex approach of the next few years. Nitrate, a hazardous material for human health, is most commonly taken up through drinking water contaminated as a result of human activity. Nitrate is the most oxidized form of nitrogen and can be returned into the natural nitrogen cycle after reduction to the completely benign nitrogen gas. Biological denitrifying processes comprise of mixed bacterial populations or enzymes, which are capable of reducing nitrate to nitrogen in an effective and economic way using the in situ produced hydrogen for reduction. H2 metabolism is important in acetogenic and methanogenic environments and in the anaerobic bioconversion of chlorinated pollutants. Special attention is to be given to acetogens, which can be co-cultivated with other bacteria on organic wastes and effluents. Beside hydrogen evolution, these microbes produce large amounts of acetic acid, which can be used efficiently, for example as a biodegradable road deicer after addition of Ca2+ and Mg2+. Many sulfate reducing bacteria are able to use hydrogen as an electron donor for sulfate reduction and metal corrosion. In addition, it has been shown that sulfate reducers are also able to grow as acetogens in the absence of sulfate and under these environmental conditions they produce H2 from waste.

D. ORGANISATION AND TIMETABLE 12

D.1. ORGANISATION The Action will be divided into the following 5 working groups (short titles are indicated in parenthesis) 1. Biological and biochemical diversity of hydrogen metabolism. (Biodiversity) 2. Active centres of hydrogenases. (Structure and Function) 3. Biosynthesis of proteins involved in hydrogen metabolism. (Biosynthesis) 4. Towards the realisation of a successful biohydrogen concept. (Biohydrogen) 5. Events related to hydrogen metabolism. (Related events) Two co-ordinators will be elected to lead these working groups (WG) by the Management Committee. The work in the WG will include annual workshops, smaller meetings of selected groups of experts within and between WGs. Several laboratories will be involved in more than one WG as the subjects of the WGs are broad and encompass a range of interrelated disciplines. Interactions between WGs are important as several issues should be studied using a variety of approaches.

Interactions between WGs

WG1 Biodiversity

WG3 Biosynthesis

WG4 Biohydrogen

WG2 Structure

WG5 Related events

D.2. TIME-TABLE 2-3 WG meetings are planned for each year so that each WG meets at least once a year. The WG meeting will include a scientific workshop to present and discuss the latest developments related to that WGs activity. The WGs preferably assemble in one of the member laboratories. In years 2000 and 2003 all participants of the Action will gather at the International Conferences on Hydrogenases, these will be the fora for demonstration of COST results to the hydrogenase research community. The Conferences will therefore be combined with WG workshops and MC meetings.

13

The Management Committee will meet twice a year. As a general rule MC meetings will be held in conjunction with a WG meeting. The COST National Co-ordinator (CSO member), the national representative in the Technical Committee for Agriculture and Biotechnology of the host country and eventually non-COST Experts will be invited. Other management details will be decided upon at the first MC meeting. The time required to pursue the scientific programme will be five years. The evaluation of the progress of the Action will be carried out at the last MC meeting of each year. The MC will review the developments and will adjust the programme and specific tasks accordingly. International Conferences on Hydrogenases have been organized in every three years since 1985. Two of these large meetings will be held during the time frame of the proposed COST Action. The conference in Berlin (D) in 2000 will be a suitable forum to prepare the Action for the midterm evaluation of achievements and the next conference coincides with the final assessment of the COST Action. International experts from outside Europe and members of the Technical Committee for Agriculture and Biotechnology will be invited to provide a critical analysis of the outcome and to establish further goals for biohydrogen research and development in Europe.

TIME TABLE

Start

1999 2000 WG WG 2,3&4 12345 WG 1&5

2001 WG 2,4,&5 WG 3&4

2002

2003

WG WG 1,3&4 12345 WG 2&5

International Conference on Hydrogenases

E. ECONOMIC DIMENSION Fourteen COST countries (A, B, CH, CZ, D, E, F, GR, H, I, NL, P, S, UK) have indicated interest in the planned COST Action. On the basis of national estimates and taking into account the co-ordination costs to be covered, the overall budget is estimated at roughly MECU 16 in 1998 prices.

14

Other way of providing and idea of the national effort to be devoted to the project is the number of person-years expected to be involved in the Action. This figure is estimated at about 60 person-years per year, 300 person-years in total. These estimates are valid under the assumption that all the laboratories, but no other parties and no other countries will participate in the Action. A departure from this is expected and will change the financial figures accordingly.

F. OUTPUTS , DISSEMINATION AND EXPLOITATION F.1. OUTPUTS AND DISSEMINATION The COST Action will result in outputs that will not otherwise be realised. Communication within the Action and WGs will be maintained mainly through electronic means after revision and extension of the E-mail connections already in place and an Internet site established by COST Action 8.18. Annual Reports will be also produced in hard copies yearly, which will contain relevant data of participants, short accounts of each WG summarizing the main achievements of the year, and abstracts of WG workshops. The results will be made available for the scientific community through communications in international journals and through an interactive Web site on the Internet. Exchange of scientists and joint experimentation will be facilitated by Short Term Scientific Missions, at least 8 missions are planned each year. Special attention will be paid to award these fellowships to young scientists, particularly from less-developed regions. As many of the participants are affiliated with higher education, other existing European mobility schemes, e.g., SOCRATES, will be used to increase exchange and improve training of young researchers. Increasing public awareness to accept the biohydrogen concept, teaching the younger generation about these technologies and dissemination of the relevant information within and outside of the scientific community are especially important tasks for the proposed COST Action. F.2. EXPLOITATION Clearly, within the time frame of this Action, implementation of large scale biohydrogen facilities cannot be expected. This period is to be devoted to the understanding of the basic phenomena at research level. The results of these efforts will be published in refereed international scientific journals. Joint publications by the Action members will be encouraged. Direct applications of hydrogen producing microbes in the fields of biodegradation of organic waste, denitrification, biocorrosion and nitrogen fixation are technically feasible. Such results will be jointly patented and submitted for further technological development through separate EUREKA projects in collaboration with industrial partners. These agricultural and environmental issues address global challenges, an internationally concerted venture will yield benefits faster and at better cost/benefit ratio than similar technical solutions developed in each country separately. 15

ANNEX1 Recent publications of the proposed participants

Andrews, S. C., B. C. Berks, J. McClay, A. Ambler, M. A. Quail, P. Golby, and J. R. Guest. 1997. A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology 143: 3633-3647. Appel, J., S. Phunpruch, and R. Schulz. Hydrogenase(s) in Synechocystis: Tools for photohydrogen production? In: BioHydrogen 97 - Proceedings of the International Conference on Biological Hydrogen Production, Kona, Hawaii, USA, 23.-26. June 1997, Plenum Publishing Corporation, New York.(submitted). Bachofen, R., L. Birch, U. Buchs, P. Ferloni, I. Flynn, G. Jud, H. Tahedl and T. G. Chasteen. 1995. Volatilization of arsenic compounds by microorganisms. In: Bioremediation of inorganics (R.E. Hinchee, J.L. Means and D.R. Burris, eds.), Battelle Press, Columbus, pp. 103-108. Barta, F., G. Rkhely, K. L. Kovcs, Molecular biology of hydrogenase(s) from Chromatium vinosum. 1997. Proc. 5th Int. Conf. on the Molecular Biology of Hydrogenases. Albertville, France. p. 110. Bernhard, M., B. Benelli, A. Hochkoeppler, D. Zannoni, and B. Friedrich. 1997. Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16. Eur. J. Biochem. 248:179-186. Biagini, G. A., M. van der Giezen, B. Hill, C. Winters, and D. Lloyd. 1997. Ca2+ accumulation in the hydrogenosomes of Neocallimastix frontalis L2: A mitochondrial-like physiological role. FEMS Microbiol.Lett. 149:227-232. Bodrossy, L., E. M. Holmes, A. J. Holmes, K. L. Kovcs, J. C. Murrell, 1997. Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov. Arch. Microbiol. 168, 493-503. Bodrossy, L., J. C. Murrell, G. Rkhely, K. L. Kovcs, 1997. Hydrogenases in methanotrophs and their biotechnological potential. Proc. 5th Int. Conf. on the Molecular Biology of Hydrogenases. Albertville, France. p. 111. Boison, G., Schmitz, O., Mikheeva, L., Shestakov, S., Bothe, H. 1996 Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans. FEBS Lett. 394:153-158 Boison, G., Schmitz, O., Schmitz, B., Bothe, H. 1997 Unusual gene arrangement of the bidirectional hydrogenase and functional analysis of its diaphorase subunit HoxU in respiration of the unicellular cyanobacterium Anacystis nidulans. Current Microbiol. accepted. Bothe, H., Boison, G. Schmitz, O. 1997 Hydrogenases in Cyanobacteria. In Proceedings of the IX. International Congress on Photosynthetic Prokaryotes, Wien, September 1997, G. Peschek, W. Loeffelhardt, G. Schmetterer, (eds.), Plenum Press, in press Brito, B., M. Martnez, D. Fernndez, L. Rey, E. Cabrera, J. M. Palacios, J. Imperial, and T. Ruiz-Argeso. 1997. Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein NifA. Proc. Natl. Acad. Sci. USA 94:60196024. Brunner, U., T. G. Chasteen, P. Ferloni and R. Bachofen. 1995. Chromatographic determination of phosphine (PH3) and hydrogen sulfide (H2S) in the headspace of anaerobic bacterial enrichments using flame photometric detection. Chromatographia 40: 399-403. Colbeau, A., S. Elsen, M. Tomiyama, N.A. Zorin, B. Dimon, P.M. Vignais. 1998. Rhodobacter capsulatus HypF is involved in regulation of hydrogenase synthesis through the HupUV proteins. Eur. J. Biochem. 251:65-71. de Lacey A., Hatchikian E.C., Volbeda A., Frey M., Fontecilla-Camps J.C., Fernandez V.M. InfraredSpectrochemical Characterization of the Ni-Fe Hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. 119: 7181-7189 (1997) de Sury d'Aspremont, R., B. Toussaint, P.M. Vignais. 1996. Isolation of Rhodobacter capsulatus transketolase. Cloning and sequencing of its structural cbbT gene. Gene 169:81-84.

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Dole F., Fournel A, Magro V., Hatchikian E.C., Bertrand P., Guigliarelli B. Nature and electronic structure of the Ni-X dinuclear center of Desulfovibrio gigas hydrogenase. Implications for the enzymatic mechanism. Biochemistry 36: 7847-7854 (1997) Drapal, N., and A. Boeck. 1998. Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation. Biochemistry (in press). Eitinger, T., and B. Friedrich. 1997. Microbial nickel transport and incorporation into hydrogenases, p. 235-256. In G. Winkelmann and C. Carrano (eds.), Transition Metals in Microbial Metabolism, Harwod Academic Publishers, London. Eitinger, T., L. Wolfram, J. Dernedde, I. Wolf, N. Patenge, and B. Friedrich.1997. Nickel metabolism in Alcaligenes eutrophus: Uptake by a Ni2+-specific permease and metal insertion into the hydrogenases, p. 37-50. In A. X. Trautwein (ed.), Bioinorganic Chemistry: Transition Metals in Biology and their Coordination Chemistry, VCH Verlagsgesellschaft, Weinheim. Elsen, S., A. Colbeau, J. Chabert, P.M. Vignais. 1996. The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. J. Bacteriol. 178:5174-5181. Elsen, S., A. Colbeau, P.M. Vignais. 1997. Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus. J. Bacteriol. 179:968-971. Gutirrez, D. Y., Y. Hernando, J. M. Palacios, J. Imperial, and T. Ruiz-Argeso. 1997. FnrN controls symbiotic nitrogen fixation and hydrogenase activities in Rhizobium leguminosarum biovar viciae UPM791. J. Bacteriol. 179:5264-5270. Happe R.P., Roseboom W., Pierik A.J., Albracht S.P.J., Bagley K.A. Biological Activation of Hydrogen. Nature 385: 126 (1997) Huyett J.E., Carepo M., Pamplona A., Franco R., Moura I., Moura J.J.G. and Hoffman B.M. 57Fe QBand Pulsed ENDOR of the Hetero-Dinuclear Site of Nickel Hydrogenases : Comparisons of the Ni-A, NiB, and NiC states J. Amer. Chem. Soc. 119: 9291-9292 (1997) Ksling, S., J. Appel, R. Schulz, and K. Steinmller. The complex I-homologous NAD(P)Hplastoquinone-oxidoreductase of Synechocystis sp. PCC 6803. In: ISPP 97 - Proceedings of the IXth International Symposium on Phototrophic Prokaryotes, Vienna, Austria, 6.-12. September 1997, Plenum Publishing Company, London. (submitted). Kovcs K. L., G. Rkhely, A. Colbeau, P. M. Vignais. 1997. Molecular biology of hydrogenases from Thiocapsa roseopersicina. Proc. 9th Int. Symp. on Photosynthtic Prokaryotes. G. A. Peschek, W. Lffelhardt, G. Schmetterer (eds.) Vienna, Austria. p. 163. Kovcs, K. L., Cs. Bagyinka, F. Barta, L. Bodrossy, A. Colbeau, K. Gyrfi, J. C. Murrell, G. Rkhely, P. M. Vignais, 1997. Studies on [NiFe] hydrogenases from phototrophic, methanotrophic, and hyperthermophilic microorganisms. Proc. 5th Int. Conf. on the Molecular Biology of Hydrogenases. Albertville, France. p. 60. Kovcs, K. L., Cs. Bagyinka, G. Rkhely. 1996. Molecular biology, physiology, and redox centres of hydrogenases from photosynthetic bacteria. Proc. 11th Internatl. Conf. on Photochemical Conversion and Storage of Solar Energy, Bangalore, India (ed V. Krishnan) pp. 231-232. Kovcs, K. L., G. Rkhely, A. Colbeau, P. M. Vignais, 1997. Molecular biology of hydrogenase(s) from Thiocapsa roseopersicina. Proc. 5th Int. Conf. on the Molecular Biology of Hydrogenases. Albertville, France. p. 74. Kovcs, K. L., L. Bodrossy, Cs. Bagyinka, K. Perei, B. Polyk, On the microbial contribution to practical solutions in bioremediation. OECD Documents on Wider Application and Diffusion of Bioremediation Technologies. Amsterdam, The Netherlands. pp. 381-390. 1996. Lenz, O., A. Strack, A. Tran-Betcke, and B. Friedrich. 1997. A hydrogen-sensing system in transcriptional regulation of hydrogenase gene expression in Alcaligenes species. J. Bacteriol. 179:1655-1663. Lichtl, R. R., M. J. Bazin and D. O. Hall. 1997. The biotechnology of hydrogen production by Nostoc flagelliforme grown under chemostat conditions. Appl. Microbiol. Biotechnol. 47: 701-707. Lindblad, P., "Cyanobacterial hydrogen metabolism - Genetics and enzymology". 97-04-17. Workshop IEA Hydrogen Program Annex 10 - Photoproduction of Hydrogen; Lindblad, P., "Cyanobacterial hydrogen metabolism/hydrogenases - Recent advances using molecular techniques". 97-11-13. Biological Research Center, Szeged, Hungary. Lindblad, P., "Nostoc PCC 73102 and H2: Knowledge, Research and Biotechnological Challenges". 97-06-23. BioHydrogen 97 - International Conference; Kona (HI), USA.

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Lindblad, P., "Nostoc PCC 73102 and H2: Knowledge, Research and Biotechnological Challenges". 97-07-16. Hydrogenases 97 - Vth International Conference on the Molecular Biology of Hydrogenases; Albertville, France. Markov, S. A., A. D. Thomas, M. J. Bazin and D. O. Hall. 1997. Photoproduction of hydrogen by cyanobacteria under partial vacuum in batch culture or in a photobioreactor. Int. J. Hydrogen Energy 22: 521-524. Massanz, C., V. M. Fernandez, and B. Friedrich. 1997. C-terminal extension of the H2-activating subunit, HoxH, directs maturation of the NAD-reducing hydro-genase in Alcaligenes eutrophus. Eur. J. Biochem. 245:441-448. McCarty, S. L., T. G. Chasteen, V. Stalder and R. Bachofen. 1995. Bacterial bioremediation of selenium oxoanions using a dynamic flow bioreactor and headspace analysis. In: Bioremediation of inorganics (R.E. Hinchee, J.L. Means and D.R. Burris, eds.), Battelle Press, Columbus, pp. 95-102. McCarty, S., T. Chasteen, M. Marshall, R. Fall and R. Bachofen. 1993. Emission of volatile methylated compounds of sulfur and selenium by phototrophic bacteria. FEMS Letters 112: 9398. Michotey, V., B. Toussaint, P. Richaud, P.M. Vignais. 1996. Characterization of the mcpA and mcpB genes capable of encoding methyl-accepting type chemoreceptors in Rhodobacter capsulatus. Gene 170:73-76. Montet, Y., Amara, P., Volbeda, A., Vernde, X., Hatchikian, E.C., Field, M.J., Frey, M., Fontecilla-Camps, J.C. Gas access to the active site of Ni-Fe Hydrogenase probed by X-ray crystallography and molecular dynamics Nature Structural Biology 4: 523-526 (1997) Monza, J., P. Diaz, O. Borsani, T. Ruiz-Argeso, and J. M. Palacios. 1997. Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti indiginous to Uruguay. World Journal of Microbiology and Biotechnology 13:565-571. Nepple B. and R. Bachofen. 1997. Induction of stress proteins in the phototrophic bacterium Rhodobacter sphaeroides. FEMS Lett. 153: 173-180. Oude Elferink, S.J.W.H., Rinia, H.A., Bruins, M.E., de Vos, W.M., and Stams, A.J.M. 1997 Detection and quantifization of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification. J. Appl. Bacteriol. 83: 102-110 Polyk B., K. L. Kovcs, I. Somogyi, Zs. Rcz, P. M. Agcs, 1997 Chemical removal and biological elimination of nitrate from drinking water. Proc. 8th European Congress on Biotechnology. Budapest, Hungary. p. 317. Rkhely G., K. L. Kovcs, Genes encoding for the hyperthermophilic hydrogenase from Thermococcus litoralis. Proc. 5th Int. Conf. on the Molecular Biology of Hydrogenases. Albertville, France. p. 122. 1997 Rkhely, G., A. Colbeau, J. Garin, P.M. Vignais, K.L. Kovacs. 1997. Unusual organization of genes coding for HydSL, the stable [NiFe]hydrogenase in the photosynthetic bacterium Thiocapsa roseopersicina. J. Bacteriol. in press). Rkhely, G., K. L. Kovcs. 1996. Plating hyperthermophilic archaea on solid surface. Anal. Biochem. 243:181-183 Rkhely, G., Z. Zhou, M. W. W. Adams, K. L. Kovcs, 1996. Cloning, sequencing, and biochemical analysis of the [NiFe] hydrogenase and putative sulfite reductase genes from Thermococcus litoralis. Proc. Int. Conf. Thermophiles 96 Athens, GA. USA, p. 168. Schmitz, O., Bothe, H. 1996 A possible role for the diaphorase subunit HoxU of the bidirectional hydrogenase in cyanobacteria as electron transferring protein connecting the oxidations of H2 and NADH with the respiratory chain in the cytoplasmic membrane. Naturwissenschaften 84: 14 Schmitz, O., Bothe, H. 1996 NAD(P)+-dependent hydrogenase activity in extracts from the cyanobacterium Anacystis nidulans. FEMS Microbiol. Lett. 135: 97 - 101 Schulz, R., J. Schnackenberg, K. Stangier, R. Wnschiers, T. Zinn, and H. Senger. Lightdependent hydrogen production of the green alga Scenedesmus obliquus. In: BioHydrogen 97 Proceedings of the International Conference on Biological Hydrogen Production, Kona, Hawaii, USA, 23.-26. June 1997, Plenum Publishing Corporation, New York. (submitted).

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Schumacher, W., Holliger, C., Zehnder, A.J.B., Hagen, W.R. 1997 Redox chemistry of cobalamin and iron-sulfur cofactors in the tetrachoroethene reductase of Dehalobacter restrictus. FEBS Lett. 109:421-425 Schwitzgubel, J. P., M. Talabardon, T. Etter and P. Pringer. 1997. Interspecies hydrogen transfer and production of acetate in methane suppressed fermentation. Proc. 8th International Conference on Anaerobic Digestion, Vol. 3, Sendai, Japan, 393-396. Smidt, H., Song, D., van der Oost, J., and de Vos, W.M. 1997 Molecular characterization of halorespiration. in: International Symposium Environmental Biotechnology (Ostende, Belgium) (eds. Verachtert H., Verstraete W.), pp. 19-22 Sorgenfrei, O., Duin, E. C., Klein, A., and Albracht, S. P. J. Changes in the electronic structure around Ni in oxidized and reduced selenium-containing hydrogenases from Methanococcus voltae. Eur. J. Biochem. 247: 681-687 (1997) Sorgenfrei, O., S. Mller, M. Pfeiffer, I. Sniezko, and A. Klein. 1997. The [NiFe] hydrogenases of Methanococcus voltae: genes, enzymes and regulation. Arch. Microbiol. 167:189-195. Stalder, V., N. Bernard, K. W. Hanselmann, R. Bachofen and T. G. Chasteen. 1995. A method of repeated sampling of static headspace above anaerobic bacterial cultures. Anal. Chim. Acta 303: 91-97. Sveshnikov, D. A., N. V. Sveshnikova, K. K. Rao and D. O. Hall. 1997. Hydrogen metabolism of mutant forms of Anabaena variabilis in continuous cultures and under nutritional stress. FEMS Microbiology Letters 147: 297-301. Tamagnini, P., O. Troshina, F. Oxelfelt, R. Salema, and P. Lindblad. 1997. Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme. Appl. Environ. Microbiol. 63: 1801-1807. Toussaint, B., R. de Sury d'Aspremont, I. Delic-Attre, V. Berchet, S. Elsen, A. Colbeau, W. Dischert, Y. Lazzaroni, and P. M. Vignais. 1997. The Rhodobacter capsulatus hupSLC promoter: identification of cis-acting regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription. Mol. Microbiol. 26:927-937. Tsygankov, A. A., T. V. Laurinavichene, V. E. Bukatin, I. N. Gogotov and D. O. Hall. 1997. Biomass production by continuous cultures of Rhodobacter capsulatus grown in various bioreactors. Applied Biochem. and Microbiol. 33: 485-490. Tsygankov, A. S., L. T. Serebryakova, D. A. Sveshnikov, K. K. Rao, I. N. Gogotov and D. O. Hall. 1997. Hydrogen photoproduction by three different nitrogenases in whole cells of Anabaena variabilis and the dependence on pH. Int. J. Hydrogen Energy 22: 859-867. van Dam, P.J., Reijerse, E. J., Hagen, W. R. Identification of a putative histidine base and of a non-protein nitrogen ligand in the active site of Fe-hydrogenases by one-dimensional and two-dimensional electron spin-echo envelope-modulation spectroscopy. Eur. J. Biochem. 248: 355-61 (1997) van der Giezen, M., K. A. Sjollema, R. R. Artz, W. Alkema, and R. A. Prins. 1997. Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome. FEBS Letters 408:147-150. van der Giezen, M., K. B. Rechinger, I. Svendsen, R. Durand, R. P. Hirt, M. Fvre, T. M. Embley, and R. A. Prins. 1997. A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria. Mol. Microbiol. 23:11-21. Van Fleet-Stalder, V., H. Grleyk, R. Bachofen and T. G. Chasteen. 1997. Effects of growth conditions on methyl selenide production by Rhodobacter sphaeroides 2.4.1. J. Industr. Microbiol. 19: 98-103. Van Soom, C., and Vanderleyden, J. 1997. A quest for symbiosis-specific genes urges itself upon Rhizobium scientists. Mol. Microbiol. 24: 669-670. Van Soom, C., de Wilde, P., and Vanderleyden, J. 1997. HoxA is a transcriptional regulator for expression of the hup structural genes in free- living Bradyrhizobium japonicum. Mol. Microbiol. 23:967-977. Villigen, Switzerland. Venien-Bryan, C., F. Balavoine, B. Toussaint, C. Mioskowski, E. A. Hewat, B. Helme, and P. M. Vignais. 1997. Structural study of the response regulator HupR from Rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid. J. Mol. Biol. 274:687-692.

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Vignais, P. M., B. Dimon, N. A. Zorin, A. Colbeau, and S. Elsen. 1997. The HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction. J. Bacteriol. 179:290-292. Wnschiers, R., and R. Schulz. Photosynthese und Wasserstoff: Grundlagen und Nutzung. BIUZ (in press). Wnschiers, R., J. Appel, K. Stangier, S. Phunpruch, and R. Schulz. 1996. Investigations to apply biologically produced photohydrogen as a future energy source, p. 1668-1673. In: P. Chartier, G.L. Ferrero, U.M. Henius, S. Hultberg, J. Sachau, and M. Wiinblad (eds.), Biomass for Energy and the Environment, Proceedings of the 9th European Bioenergy Conference, vol. III, Pergamon, Elsevier Science, Oxford, New York, Tokyo. Wnschiers, R., T. Zinn, D. Linder, and R. Schulz. 1997. Purification and characterization of cytochrome c6 from the unicellular green alga Scenedesmus obliquus. Z. Naturforsch. 52c:740746. Zorin, N. A., B. Dimon, J. Gagnon, J. Gaillard, P. Carrier, P.M. Vignais. 1996. The H-D exchange reaction catalysed by Thiocapsa roseopersicina hydrogenase: inhibition by iodoacetamide and by acetylene. Eur. J. Biochem. 241:675-681. Zorin, N.A., T. Lissolo, A. Colbeau, P.M. Vignais. 1996. Increased hydrogen photoproduction by Rhodobacter capsulatus strains deficient in uptake hydrogenase. J. Marine Biotechnol. 4:28-33.

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ANNEX2

List of experts who already expressed interest in the COST network

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