BIOHYDROGEN III Renewable Energy System by Biological Solar Energy Conversion Elsevier Internet Homepage http://www.elsevier.nl (Europe) http://www.elsevier.com (America) http://www.elsevier.co.jp (Asia) Consult the Elsevier homepage for full catalogue information on all books, journals and electronic products and services Related Journals Free specimen copy gladly sent on request Elsevier Ltd, The Boulevard, Langford Lane, Kidlington, Oxford, OX5 1GB, UK International Journal of Hydrogen Energy http://www.elsevier.com/locate/ijhydene Renewable Energy http://www.elsevier.com/locate/renene Renewable and Sustainable Energy Reviews http://www.elsevier.com/locate/rser Journal of Power Sources http://www.elsevier.com/locate/jpowsour To Contact the Publisher Elsevier welcomes enquiries concerning publishing proposals: books, journal special issues, conference proceedings, etc All formats and media can be considered Should you have a publishing proposal you wish to discuss, please contact, without obligation, the publisher responsible for Elsevier's renewable energy programme: Tony Roche Senior Publishing Editor, Renewable Energy Elsevier Science Ltd The Boulevard, Langford Lane Kidlington, Oxford OX5 1GB, UK Phone: +44 1865 843887 Fax: +44 1865 843920 E.mail: t.roche@elsevier.com General enquiries, including placing orders, should be directed to Elsevier's Regional Sales Offices - please access the Elsevier homepage for full contact details (homepage details at the top of this page) BIOHYDROGEN III Renewable Energy System by Biological Solar Energy Conversion Edited by Jun Miyake, Tissue Engineering Research Center (TERC), AIST, Amagasaki, Japan Yasuo Igarashi, Department of Biotechnology, University of Tokyo, Tokyo, Japan Matthias Rögner, Plant Biochemistry, Faculty for Biology, Ruhr-University Bochum, Bochum, Germany 2004 ELSEVIER Amsterdam • Boston • Heidelberg • London • New York • Oxford • Paris San Diego • San Francisco • Singapore • Sydney • Tokyo ELSEVIER B.V 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Cataloguing in Publication Data A catalogue record is available from the British Library ISBN: 08 044356 @ The paper used in this publication meets the requirements of ANSI/NISOZ39.48-1992 (Permanence of Paper) Printed in The Netherlands PREFACE Hydrogen is regarded as one of the most promising energy carriers of our future: This is especially due to the fact that it can be regenerated in a cyclic process out of water without emission of CO2, i.e it is environmentally neutral The main problem is that hydrogen gas does not exist as a pure compound in natural resources For this reason it has to be produced by technical processes from fossil energy carriers which in turn usually require high temperatures and high pressure In addition, the production of the unwanted CO2 is inevitably involved in these processes Hydrogen can also be technically produced from water by electrolysis using conventional or regenerative produced electrical energy However, as the efficiency of this process is rather low (about 10%) it is quite expensive An alternative, CO2 neutral method is the photobiological hydrogen production by microalgae which use natural solar energy directly as energy source for these transformation processes These organisms whose growth rates are about 10-times higher than those of higher plants grow with minimal nutrients due to a very efficient photosynthesis Some of them contain hydrogenases with an extreme capacity for the production of hydrogen In contrast to technical processes, photobiological hydrogen production does not require high-tech equipment as all processes occur at room temperature and at atmospheric pressure Moreover, as no electricity has to be generated transiently, the transformation efficiency is rather high - usually more than 10% Biohydrogen is pure hydrogen, so there is no need for further purification processes and conclusively no air pollution occurs The use of such natural hydrogen production machines in combination with the natural process of photosynthesis is the topic of an international NEDO project for the development of a semiartificial device for hydrogen production On the occasion of the second meeting of all groups involved in this project, an international symposium on "Biohydrogen" was organized in Kyoto 2002 The state of the art of biohydrogen production from participants of this symposium is summarized in the chapters of this book October 2002 NEDO International Joint Research Grant "Research team of Molecular Device for Hydrogen Production" Team Leader, Matthias Rögner This page is intentionally left blank CONTENTS I Hydrogen Production New Frontiers of Hydrogen Energy Systems T Ohta Novel Approaches to Exploit Microbial Hydrogen Metabolism 13 K.L Kovacs, Z Bagi, B Balint, B.D Fodor, Gy Csanadi, R Csaki, T Hanczar, A.T Kovdcs, G Maroti, K Perei, A Toth and G Rakhely Application of Hydrogenase for Renewable Energy Model Systems 33 N.A Zorin II Photosynthesis and Photobioreactor Photo-Biological Hydrogen Production by the Uptakehydrogenase and PHB Synthase Deficient Mutant of Rhodobacter Sphaeroides M.S Kim, J.H Ahn and Y.S Yoon 45 Hydrogen Production by Suspension and Immobilized Cultures of Photo trophic Microorganisms Technological Aspects 57 A.A Tsygankov III Hydrogenase The Potential of Using Cyanobacteria as Producers of Molecular Hydrogen 75 P Lindblad Photobiological Hydrogen Production by Cyanobacteria Utilizing Nitrogenase Systems Present Status and Future Development 83 H Sakurai, H Masukawa, S Dawar and F Yoshino Fundamentals and Limiting Processes of Biological Hydrogen Production P.C Hallenbeck 93 viii Contents IV Bio Molecular Device The Isolation of Green Algal Strains with Outstanding H2-Productivity 103 M Winkler, C Maeurer, A Hemschemeier and T Happe Identification of a CIS-Acting Element Controlling Anaerobic Expression of the hydA Gene from Chlamydomonas Reinhardtii 117 M Stirnberg and T Happe Glycolipid Liquid Crystals as Novel Matrices for Membrane Protein Manipulations 129 M Hato and T Baba Artificial Phytanyl-Chained Glycolipid Vesicle Membranes with Low Proton Permeability are Suitable for Proton Pump Reconstitution Matrices 143 T Baba and M Hato Amphipols: Strategies for an Improved PS2 Environment in Detergent-Free Aqueous Solution 151 M Nowaezyk, R Oworah-Nkruma, M Zoonens, M Rogner and J.-L Popot Monolayers and Longmuir-Blodgett Films of Photosystem I on Various Subphase Surfaces 161 D J Qian, T Wakayama, C Nakamura, S.O Wenk and J Miyake Modular Device for Hydrogen Production: Optimization of (Individual) Components 171 A Prodohl, M Ambill, E El-Mohsnawy, J Lax, M Nowaezyk, R Oworah-Nkruma, T Volkmer, S.O Wenk and M Rogner V Appendices List of Participants 183 Author Index 187 I Hydrogen Production 174 A Prodöhl etal manner, His-tags have been fused to the genes of appropriate subunits of these complexes Care was taken to select sites which guarantee that - according to the model shown in Figure - the acceptor side of PS2 and the donor side of PSl are equipped with the His-tag in order that this site is oriented towards the electrode surface upon binding In order to improve the probability for binding of the His-tag to the column matrix - despite various loops of these membrane complexes exposed to their hydrophilic surfaces -a 10-His-tag instead of a 6-His-tag has been chosen Due to the structural information available for both photosystems [Schubert et al 1997; Zouni et al 2001] the genes encoding the following subunits have been selected for His-tag attachment: A The C-terminus of PsbB (CP47) was chosen according to[Bricker et at 1998]; this report showed that the isolation of a highly active PS2 from Synechocystis sp PCC 6803 using a His-tagged mutant of CP47 was possible B Structural data of PsaF led to the conclusion that the N-terminus of the protein is located on the donor side of PSl Another reason for choosing the N-terminus of PsaF is the fact that psaF and psaJ gene are organized in one operon and therefore insertions between the two genes might lead to a functional disorder [Xu et at.2001] Figure Constructs of His-tagged PSl + PS2 a) Vector map of pHispsaF Vector elements: /wwFJ-DS: /waFJ-operon + downstream region (DS); His-tag: 10-Histidine-tag; US: upstream-region b) Vector map of pCP47 Vector elements: psbB; His-tag: 10-Histidine-tag; downstream region (DS) Purification of the His-tagged Photosystems His-tagged PSl and PS2 were purified in a first step on an IMAC-column (chelating sepharose fast flow column, Pharmacia, see M&M) as shown for PS2 in Figure 3; especially free pigments and unwanted proteins were separated by this step Purification of PSl was done accordingly and resulted in a similar elution profile (data not shown) Routinely, after this column PS2 was further purified on an anion exchange column, while PSl was further purified by a hydrophobic interaction chromatography step ace to [Wenk and Kruip 2000] (data not shown) Both procedures resulted in preparations of increased purity and homogeneity as shown by HPLC size exclusion chromatography and SDS-PAGE analysis: Figure (upper part) shows the elution profile of PSl from a size exclusion column; comparison with marker proteins indicates that PSl is nearly exclusively isolated as stable Modular Device for Hydrogen Production 175 trimeric complex SEC also shows (Fig 4, lower part) that PS2 is mainly isolated as a dimer, with a smaller peak of PS2-aggregates and a shoulder indicating some PS2 monomers The purity of both complexes was analysed by SDS-PAGE (Fig 5) The subunit pattern of PS1 indicates the presence of many impurities after the HVIAC column (track 1), which are removed by the second column step i.e the HIC-HPLC (track 2) Figure Purification of PS2-His-tagged on a chelating In case of PS2, the purification by the second column step, i.e the IEC, is not so obvious in comparison to the second column step of PS1 (compare track and of PS2) However, for PS2 this step is necessary to separate monomeric from dimeric PS2 with the latter being prefered for our hydrogen device due to higher activity and longer stability [see also Kuhl et al 2000] For both PS1 and PS2, the known subunits of each complex (as indicated on the right side of the gel) have been identified by homology with bands from WT preparations, by specific antibodies or by mass spectroscopy (data not shown) In general, all major bands of both complexes and - as far as identified - all small subunits could be preserved in our Histagged preparations in comparison with gels of WT-PSl [Wenk and Kruip 2000] and WTPS2[Kuhl et al 2000] In conclusion, SDS-gels after the second column not display major unknown proteins nor the loss of major subunits of the PS1- and PS2-complex, respectively A Prodöhl etal 176 Figure Size-exclusion chromatography of purified PS1 and PS2 Figure SDS-gel of purified PSl-His-tag (left) and PS2-His-tag (right) after IMAC-column (1) and after HIC-column (PS1) or IEC- Table compares the specific activities of PS1 and PS2, respectively, in comparison with PS1- and PS2 complexes isolated from WT These data show, that the activities of His-tagged complexes are only slightly lower than the respective WT complexes, i.e the His-tag in these complexes apparently does not interfer with the activity under these conditions 111 Modular Device for Hydrogen Production Table Activity of FS1 and PS2-His-tag in comparison with untagged PS1 and PS2, respectively Activity / umol O2 • mg CM"1 h"1 WT-PS2 His-tagged PS2 < 2,300 < 2,200 WTPS1 His-tagged PS1 < 1,000