Novel Biodegradable Microbial Polymers NATO ASI Series Advanced Science Institutes Series

A Series presenting the results of activities sponsored by the NA TO Science Committee, which aims at the dissemination of advanced scientific and technological knowledge, with a view to strengthening links between scientific communities.

The Series is published by an international board of publishers in conjunction with the NATO Scientific Affairs Division

A Life Sciences Plenum Publishing Corporation B Physics London and New York

C Mathematical Kluwer Academic Publishers and Physical Sciences Dordrecht, Boston and London o Behavioural and Social Sciences E Applied Sciences

F Computer and Systems Sciences Springer-Verlag G Ecological Sciences Berlin, Heidelberg, New York, London, H Cell Biology Paris and Tokyo

Series E: Applied Sciences - Vol. 186 Novel Biodegradable Microbial Polymers edited by Edwin A. Dawes Department of Applied Biology, University of Hull, Hull, U.K.

Kluwer Academic Publishers

Dordrecht / Boston / London

Published in cooperation with NATO Scientific Affairs Division Proceedings of the NATO Advanced Research Workshop on New Biosynthetic Biodegradable Polymers of Industrial Interest from Microorganisms Sitges, Spain May 26-31, 1990

library of Congress Cataloging-in-Publication Data NATO Advanced Research Workshop on New Biosynthetic, Biodegradable Polymers of Industrial Interest from Microorganisms (1990 : Sitges, Spain) Novel biodegradable microbial polymers proceedings of the NATO Advanced Research Workshop on New Biosynthetic, Biodegradable Polymers of Industrial Interest from Microorganisms, Sitges, Spain, May 26-31, 1990 I edited by Edwin A. Dawes. p. cm. -- (NATO ASI series. Series E, Applied sciences; vol. 186)' "PublIshed in cooperation with NATO Scientific Affairs Division." Inc ludes index. lSBN-13: 978-94-010-7458-2 e-1SBN-13:978-94-009-2129-0 DOl: 10.1007/978-94-009-2129-0

1. Microbial polymers--Congresses. I. Dawes, Edwin A. (Edwin Alfred) II. North Atlantic Treaty Organization. SCIentific Affairs Division. III. Title. IV. Series: NATO ASI series. Series E, Appl ied sciences; no. 186. OR92.P59N37 1990 576'.11924--dc20 90-5224 ISBN-13: 978-94-010-7458-2

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No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photo• copying, recording or by any information storage and retrieval system, without written permission from the copyright owner. NATO - ADVANCED RESEARCH WORKSHOP

NEW BIOSYNTHETIC BIODEGRADABLE

POLYMERS OF INDUSTRIAL INTEREST

FROM MICROORGANISMS

Organizing Committee

Co-Directors R.C. Fuller - U.S.A. R.W. Lenz - U.S.A.

Committee E.A. Dawes - U.K., Proceedings Editor R. Guerrero - Spain H.G. Schlegel- FR.G. A.lB. Zehnder - The Netherlands PREFACE

The NATO Advanced Research Workshop from which this book derives was conceived during Biotec-88, the Second Spanish Conference on Biotechnology, held at Barcelona in June 1988. The President of the Conference, Dr. Ricardo Guerrero, had arranged sessions on bacterial polymers which included lectures by five invited participants who, together with Dr. Guerrero, became the Organizing Committee for a projected meeting that would focus attention upon the increasing international importance of novel biodegradable polymers. The proposal found favour with the NATO Science Committee and, with Dr. R. Clinton Fuller and Dr. Robert W. Lenz as the co-Directors, Dr. Edwin A. Dawes as the Proceedings Editor, and Dr. Hans G. Schlegel, Dr. Alexander J.B. Zehnder and Dr. Ricardo Guerrero as members of the Organizing Committee, the meeting quickly took shape. To Dr. Guerrero we owe the happy choice of Sitges for the venue, a pleasant coastal resort 36 kilometres from Barcelona, which proved ideal. The sessions were held at the Palau de Maricel in appropriately impressive surroundings, and invaluable local support was provided by Mr. Jordi Mas-Castella and by Ms. Merce Piqueras. Much of the preparatory work fell upon the broad shoulders of Mr. Edward Knee, whose efforts are deeply appreciated. The Organizing Committee hopes that this Workshop will prove to be the first of a series which will aim to keep abreast of a rapidly expanding and exciting area of research that is highly relevant to environmental and industrial interests.

EDWIN A. DAWES

vii ACKNOWLEDGEMENTS

The Organizing Committee wishes to thank the Scientific Affairs Division of the North Atlantic Treaty Organization and also the U.S. Department of the Navy's Office of Naval Research for the financial support which made this Workshop possible. They are also indebted to the following organizations which contributed to the support of the Workshop as corporate sponsors:

btF biotechnologische Forschungsgesellschaft m.b.H. CIBA - Geigy Ltd DSM Research EJ. du Pont de Nemours & Company, Inc. Gesellschaft ftir biotechnologische Forschung m.b.H. Johnson & Johnson Company Marlborough Biopolymers Ltd. (ICI pIc) Pharmacia LEO Therapeutics AB Proctor & Gamble Company Rohm and Haas Company Shell Research Ltd. U.S. Army - Natick Research, Development & Engineering Center

viii PARTICIPANTS

A.l ANDERSON, Department of Applied Biology, University of Hull, Hull HU6 7RX, U.K. E. ATKINS, H.H. Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL, U.K. A. BALLISTRERI, Department of Chemical Sciences, University of Catania and Institute for the Chemistry and Technology of Polymeric Materials, 95125 Catania, Italy. PJ. BARHAM, H.H. Wills Physics Laboratory, University of Bristol, Bristol BS8 1TL, U.K. H. BRANDL, Mikrobiologie Institut fur Pflanzenbiologie, Zollikerstrasse 107, CH-8008 Zurich, Switzerland. M.W. BREEDVELD, Department of Microbiology, Agricultural University, 6703 CT Wageningen, The Netherlands. H.l BRIGHT, Office of Naval Research, 800 North Quincy Street, Arlington, VA 22217, U.S.A. D. BYROM, ICI Biological Products, ICI PLC, Billingham, Cleveland, TS23 lLB, U.K. D.C. CAMERON, Department of Chemical Engineering, University of Wisconsin, Madison, WI 53706, U.S.A. E. CHIELLlNI, Instituto di Chimical Organico e Industriale, University of Pisa, 56100 Pisa, Italy. lE. CLARK, Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284, U.S.A. V. CRESCENZI, Departimento di Chimica, Universita di Roma "La Sapienza", 00185 Roma, Italy. S. DABI, Johnson & Johnson Co., Personal Products, Research and Development Division, Miltown, NJ 08850, U.S.A. E.A. DAWES, Department of Applied Biology, University of Hull, Hull HU6 7RX, U.K. D.E. DENNIS, Department of Biology, James Madison University, Harrisonburg, VA 22087, U.S.A. Y. DOl, Department of Chemical Engineering, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan. G. EGGINK, Ato Agrotechnologie, Agrotechnological Research Institute, Haagsteeg 6, Postbus 17,6700 AA Wageningen, The Netherlands. R. FERRER, Department of Microbiology, Faculty of Pharmacy, Universidad de Granada, Granada, Spain. R.C. FULLER, Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, U.S.A. K. GAGNON, Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, U.S.A. H. van GEMERDEN, Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands. D. GILMORE, Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, U.S.A.

ix x

R GROSS, Department of Chemistry, University of Lowell, One University Avenue, Lowell, MA 01854, U.S.A. P. GUERIN, Laboratoire de Chimie Biologique et Macromoleculaire, Ecole Nationale Superieure de Chimie de Rennes, 35700 Rennes, France. R GUERRERO, Department of Microbiology, University of Barcelona, Diagonal, 645, 08028 Barcelona, Spain. 1. GUEZENNEC, IFREMER, Centre de Brest, BP 70, 29280 Plouzane, France. H. HA~VORSON, Marine Biological Laboratory, Woods Hole, MA 02543, U.S.A. UJ. HANGGI, btF Biotechnologische, St. Peter Strasse, A-4020 Linz, Austria. F. HERMES, Department of Academic & Scientific Contacts, DSM Research, P.O. Box 18, 6160 MD Geleen, The Netherlands. G.W. HUISMAN, Department of Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands. D. JENDROSSEK, Institut ftir Mikrobiologie, UniversitiH G6ttingen, D-3400 G6ttingen, F.R.G. B.-W. KIM, Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, U.S.A. EJ. KNEE, Jr., Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, U.S.A. I.S. KULAEV, Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, 142292 Pushchino, Moscow Region, USSR. R.M. LAFFERTY, Technische Universitat, Institute for Biotechnology, Sch10egalgasse 9, A-8010 Graz, Austria. RW. LENZ, Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, U.S.A. T. LINDELL, Pharmacia Straffanstrop, Pharmacia LEO Therapeutics AB, Box 161, S- 245 00 Straffanstrop, Sweden. 1.D. LINTON, Shell Research Ltd., Sittingbourne, Kent ME9 8AG, U.K. D. LOHMANN, Central Research Laboratories, Ciba-Geigy AG, CH-4002, Basel, Switzerland. M.T. MADIGAN, Department of Microbiology, Southern Illinois University, Carbondale, IL 62901, U.S.A. RH. MARCHESSAULT, Chemistry Department, McGill University, 3420 University Street, Montreal, Quebec H3A 2A 7, Canada. 1. MAS-CASTELLA, Department of Microbiology, University of Barcelona, Diagonal, 645, 08028 Barcelona, Spain. 1. MAS-GORDI, Department of Genetics and Microbiology, Autonomous University of Barcelona, E-08193 Bellaterra, Spain. 1. MAYER, Biotechnology Branch, Biological Sciences Division, Department of the Army, Natick Research Development & Engineering Center, Natick, MA 01760, U.S.A. M. MILAS, C.E.R.M.A.V.-C.N.R.S., Domaine Universitaire de Grenoble, 38402 Saint Martin D'Heres Cedex, B.P. 68 Grenoble, France. G. MONTAUDO, Department of Chemical Sciences, University of Catania, and Institute for the Chemistry and Technology of Polymeric Materials, 95125 Catania, Italy. M.P. NUTI, Departamento di Biotecnologie Agrarie, Universita di Padova, Via Gradenigo 6, 35131 Padova, Italy. W.J. PAGE, Department of Microbiology, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. xi

C PEDROS-ALIO, Instituto de Ciencias del Mar, Passeig Nacional sin, 08003 Barcelona, Spain. E. PISKIN, Chemical Technology Division, Hacettepe University, Beytepe, P.K. 716, Kizilay - Ankara, Turkey. A PRIES, Institiit fUr Mikrobiologie, Universitat G6ttingen, 3400 G6ttingen, F.R.G. H. PREUSTING, Department of Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands. M. RINAUDO, CE.R.M.AV.-C.N.R.S., Domaine Universitaire de Grenoble, 38402 Saint Martin D'Heres Cedex, B.P. 68 Grenoble, France. F. RODRIGUEZ-VALERA, Department of Molecular Genetics and Microbiology, Universidad de Alicante, 03080 Alicante, Spain. J. ROMESSER, Experimental Station, E.I duPont de Nemours & Co., Inc., P.O. 80228, Wilmington, DE 19880-0228, U.S.A. H.G. SCHLEGEL, Institut fUr Mikrobiologie, Universitat G6ttingen, D-3400 G6ttingen, F.R.G. P. SCHUBERT, Institut fiir Mikrobiologie, Universitat G6ttingen, D-3400 G6ttingen, F.R.G. AJ. SINSKEY, Departments of Chemistry and Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A J.F. STAGEMAN, ICI PLC, Biological Products, Billingham, Cleveland TS23 lLB, U.K. L. STAL, Laboratorium voor Microbiologie, Universiteit van Amsterdam, Nieuwe Achte~gracht 127, 1018 WS Amsterdam, The Netherlands. A STEINBUCHEL, Institut fUr Mikrobiologie, Universitat G6ttingen, D-3400 G6ttingen, F.R.G. G. SWIFT, Rohm and Haas Co., Spring House, PA 19477, U.S.A J. SWINGS, Laboratory for Microbiology & Microbial Genetics, Rijksuniversitat Gent, KL Ledegankstraat 35, B-9000 Gent, Belgium. S. TANENBAUM, Department of Chemistry, College of Environmental Science & Forestry, Baker Laboratories, Syracuse, New York 13210, U.S.A A TIMM, Institut fUr Mikrobiologie, Universitat G6ttingen, D-3400 G6ttingen, F.R.G. B. TINLAND, C.E.R.M.AV.-CN.R.S., Domaine Universitaire de Grenoble, 38402 Saint Martin D'Heres Cedex B.P. 68 Grenoble, France. H. ULMER, Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, U.S.A. M. VINCENZINI, Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita, e Centro di Studio dei Microrganismi Au totrofi , Universita di Firenze, 50144 Firenze, Italy. G.C WALKER, Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A D.R. WILLIAMS, Department of Applied Biology, University of Hull, Hull HU6 7RX, U.K. B. WITHOLT, Department of Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands. AJ.B. ZEHNDER, Department of Microbiology, Agricultural University, 6703 CT Wageningen, The Netherlands. L.P.T.M. ZEVENHUIZEN, Department of Microbiology, Agricultural University, 6703 CT Wageningen, The Netherlands. CONTENTS

PREFACE vii

ACKNOWLEDGEMENTS viii

PARTICIPANTS ix

OPENING LECTURE 1

Novel Microbial Polymers: an Introductory Overview 3 Edwin A. Dawes

POLYESTERS I: POLYMER CHARACTERISTICS 17 AND PROPERTIES

Bacterial Polyesters: Past, Present and Future 19 R. Clinton Fuller

Functionalized Poly-~-hydroxyalkanoates Produced 23 by Bacteria R.W. Lenz, B.-W. Kim, H.W. Ulmer, K. Fritzsche, E. Knee and R.C. Fuller

Production of Biodegradable Copolyesters by Alcaligenes eutrophus 37 Y. Doi, A. Segawa, S. Nakamura and M. Kunioka

Microstructure of Bacterial Poly(~-hydroxybutyrate-co-~ 49 hydroxyvalerate) by Fast Atom Bombardment Mass Spectrometry Analysis of their Partial Degradation Products A. Ballistreri, D. Garozzo, M. Giuffrida and G. Montaudo

Pilot Scale Production of PHB with Alcaligenes latus 65 U.J. Hiinggi

POLYESTERS II: POLYMER PRODUCTION - PROPERTIES 71

Culture Substrate Effect in the Production of Poly(~-hydroxy 73 butyrate) by Rhizobium "hedysari" S. Casella, C. Leporini, A. Corti, G. Picci, E. Chiellini, E. Casini and R. Solaro xiv

Physical Properties ofPoly(hydroxybutyrate) and Poly(hydroxy- 81 butyrate-co-hydroxyvalerate) PJ. Barham Properties of Poly-f3-hydroxyalkanoate Latex: Nascent Morphology, 97 Film Formation and Surface Chemistry R.H. Marchessault, C.J. Monasterios and P. Lepoutre

Industrial Production of Copolymer from Alcaligenes eutrophus 113 D. Byrom The Production of Polyhydroxyalkanoates from Unrelated 119 Carbon Sources A.J. Anderson, G.W. Haywood, D.R. Williams and E.A. Dawes

POLYESTERS III: MOLECULAR BIOLOGY AND ENZYMOLOGY 131

Alcaligenes eutrophus and its Scientific and Industrial Career 133 H.G. Schlegel

Genetic and Molecular Analysis of the Alcaligenes eutrophus 143 Polyhydroxyalkanoate - Biosynthetic Genes and Accumulation of PHA in Recombinant Bacteria Alexander Steinbiichel, Peter Schubert, Arnulf Timm and Andreas Pries Bacterial Poly(3-hydroxyalkanoates) 161 B. WithoIt, G.W. Huisman and H. Preusting

Molecular Characterization of the Poly-f3-hydroxybutyrate 175 Biosynthetic Pathway of Alcaligenes eutrophus H16 Brian Janes, Jeff Hollar and Douglas Dennis PHB: A Model System for Biopolymer Engineering II 191 O.P. Peoples and A.J. Sinskey

POLYPHOSPHATES 203

Some Possible Roles of Polyphosphate in Microorganisms 205 H.O. Halvorson Purification of Polyphosphate Glucokinase from Propionibacterium 213 shermanii J.E. Clark The Physiological Role of Inorganic Polyphosphates in 223 Microorganisms: Some Evolutionary Aspects I.S. Kulaev xv

Accumulation of Polyphosphate by Acinetobacter sp.: 235 Physiology, Ecology, and Application Alexander J.B. Zehnder and Johan W. van Groenestijn

NATURAL DISTRIBUTION OF MICROBIAL POLYMERS 245 AND SULFUR

Environmental Control of Sulfur Deposition in Anoxygenic 247 Purple and Green Sulfur Bacteria Hans van Gemerden, Pieter T. Visscher and Jordi Mas

Polyhydroxyalkanoate Accumulation in Planktonic and 263 Anaerobic Environments Carlos Pedr6s-Ali6, Jordi Mas-Castella, Jordi Mas and Ricardo Guerrero

POLYSACCHARIDES I 275

Solution and Gelling Properties of Microbial Polysaccharides 277 of Industrial Interest: the Case of Gellan V. Crescenzi, M. Dentini and T. Coviello

Genetic Analyses and Manipulations of Rhizobium meliloti 285 Exopolysaccharides T.L. Reuber, A. Urzainqui, J. Glazebrook, J.W. Reed and G.C. Walker

A Novel Exopolysaccharide from a Filamentous Cyanobacterium: 295 Production, Chemical Characterization and Rheological Properties M. Vincenzini, R. De Philippis, C. Sili and R. Materassi

Physiology of Exopolysaccharide Production 311 J.D. Linton

POLYSACCHARIDES II 331

Structural Diversity and Functional Versatility of 333 Polysaccharides D. Lohmann

Relation Between Molecular Structure and Physicochemical 349 Properties for Some Microbial Polysaccharides M. Rinaudo, M. Milas and B. Tinland

Structure of Microbial Polysaccharides using X-Ray Diffraction 371 E. Atkins xvi

Recent Developments in Rhizobium Polysaccharides 387 L.P.T.M. Zevenhuizen

PANEL DISCUSSION 403

UNIVERSITY, INDUSTRY AND GOVERNMENT: FUTURE 405 DIRECTIONS AND PRIORITIES IN RESEARCH AND DEVELOPMENT - PANEL DISCUSSION S.W. Tanenbaum (Chairman)

POSTERS 417

POLYHYDROXYALKANOATES 417

Poly(malic acid), a Functional Poly(l3-hydroxy acid)-type 419 Polyester Available from Chemical and Biological Synthesis Ph. Guerin, C. Braud, J.P. Girault, M. Vert, E. Holler, H. Fischer and C. Windisch

The Degradation of Shampoo Bottles in a Lake 421 H. Brandl and Petra Piichner

Production of Poly-l3-hydroxybutyrate by Azotobacter vinelandii 423 UWD in Beet Molasses Culture at High Aeration W.J. Page

Halobacteria as Producers of Poly-l3-hydroxyalkanoates 425 F. Rodriguez-Valera and J.A. Garcia Lillo

Acetone as a Substrate for Poly-l3-hydroxybutyrate 427 Production by Phototrophic Purple Bacteria Vasiliki G. Karayiannis and Michael T. Madigan

Poly-l3-hydroxyalkanoate (PHA) Accumulation in Rhizobium 431 meliloti Affected in the Dicarboxylate Transport (DCT) Genes R. Tombolini, B. Boesten, F. O'Gara and M.P. Nuti

Occurrence and Role of Poly-hydroxy-alkanoate in the 435 Cyanobacterium Oscillatoria limosa Lucas J. Stal, Heike Heyer and Gabi Jacobs

Influence of Growth Conditions on Production and Composition 439 of PHA by Pseudomonas oleovorans E.1. Knee, Jr., M. Wolf, R.W. Lenz and R.C. Fuller xvii

Production of Poly-3-hydroxyalkanoates by P. putida during 441 Growth on Long-chain Fatty Acids G. Eggink, H. van der Wal and G. Huyberts

Fonnation ofpoly-3-hydroxyalkanoates) by wild type and 445 recombinant strains of Pseudomonas aeruginosa and other fluorescent pseudomonads Arnulf Timm and Alexander Steinbtichel

Molecular analysis of the Alcaligenes eutrophus PHB-biosynthetic 447 genes: identification of the NH2-tenninus of PHB synthase and identification of the transcription start site of phbC. Peter Schubert, Andreas Pries, Niels Kriiger and Alexander Steinbtichel

Yield Study of the Poly-beta-hydroxyalkanoate) Produced by 449 Pseudomonas oleovorans grown on Sodium Octanoate K.D. Gagnon, D.B. Bain, R.W. Lenz and R.C. Fuller

Genetic Analysis of Polyester Synthesis in Pseudomonas 451 oleovorans GjaIt W. Huisman, Rob Meima, Eric Won ink and Bernard WithoIt

Production and Characterization of Poly(3-hydroxyalkanoates) 453 Hans Preusting, Atze Nijenhuis and Bernard WithoIt

POLY(GLUTAMIC ACID) 455

Poly(glutamic acid) Produced by Bacterial Fermentation 457 Steven A. Giannos, Devang Shah, Richard A. Gross, David L. Kaplan and Jean M. Mayer

POLYSACCHARIDES 461

Role of Counterions on Gellan Properties 463 M. Milas, M. Rinaudo and X. Shi

Polysaccharides, Modified Polysaccharides and Polysaccharide 465 Blends for Biodegradable Materials 1.M. Mayer

Degradation Kinetics of Polymer Films in Marine and Soil 469 Systems under Accelerated Conditions Jean M. Mayer

Bioconversion of Pentosans to Heteropolysaccharides 473 S.W. Tanenbaum, B. Scott, P.J. Fisher, A. Henwood, 1.S. Novak, D. Slomczynski and J.P. Nakas INDEX 479