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THE FIRST INTERNATIONALCONFERENCE ON

CHITIN/CHITOSAN

May 1978

Edited by

R.A.A. Muzzare111 University of Ancona, Italy

E.R. Pariser Sea Grant Program Massachusetts Institute of Technology

The hosts for the conference, which was held in Boston, Massachusettson April 11 through 13, 1977, were the MIT Sea Grant Program and the Massachusetts Science and Technology Foundation.

MIT Sea Grant Report MITSG78-7 Index No. 78-307-Dmb INTRODUCTION

Theobjective of the MITSea Grant Programand the Massachusetts Scienceand Technology Foundation in conveningthe First International Conferenceon /Chitosanwas to bri ng together those experts and researchers from around the world who are working to expandthe uses of this exciting, abundant,renewable natural resource. By providing a forum, we hopedto encouragethe businessand scientific communities to share insights and experiences,to stimulate newideas, and to identify researchthat wouldadvance the applicationof chitin and chitosan in pharmaceuticals,food processing,papermaking, agriculture, adhesive and textile manufacturing, and waste treatment. Weare publishing the proceedings of this conference becausewe believe that continuedinformation sharing is essential if the advances in deve'lopingthis valuable resourceare to continue. Wewish to ex- pressour appreciation to all thosewho participated in andcontributed to the conference, and we sincerely hope that publication of the papers will serve in the future as a comprehensivereference source for them, as well as for those who could not attend.

Mr. Irving Sacks Mr. Dean A. Horn Acting Director Director

Massachusetts Science and MIT Sea Grant Program Technology Foundation

15 May 1978 Wededicate these proceedings to the National Sea

Grant Office, and especially to

Dr. Robert Abel and

Mr. HaroId Goodwin whose vision and wisdom are responsible for making the study of chitin and chitosan a matter uf international

concern.

R. A.A. Muzzarelli E.R, Pariser University of Ancona, Massachusetts Institute Italy of Technology ACKNOWLEDGMENTS

Wewish to acknowledgethe cooperation and support received from the following organizationswhose contributions madethis conference possible, University of Delaware Sea Grant Program University of WashingtonSea Grant Program Nationa 1 Sea Grant Program, NDAA, DOC Massachusetts Institute of Technology The initiation, planning, organizing and managingof a meetingsuch as this conferencealways requires a taxing and pro- tracted team effort involving manyparticipants whoseefforts it is impossibleto acknowledgeadequately. Thenames of at least two peoplemust, however,be recordedhere in appreciation of the great contributionsthey made: Mr. VincentLoCicero, formerly of the MassachusettsScience and Technology Foundation who was largely in chargeof pre-conferencepreparations and conference organization; the other is Ms. Marie Phaneuf, also of the MassachusettsScience andTechnology Foundation, the organizing genius, whokept all major and minor details, correspondenceand papers in mind and in order and who started the enormousand difficult job of retyping the scientific paperspresented at the conference. RichardDeAmicis should also be mentioned here to acknowledgehis important contri- butionssin initiating the editing work on the papers. We owea very specialvote of thanksto Ms,Margaret M. Sevcenkowho undertook the arduousand complicated task of editing, proofreadingand organizing the text of these proceedings,which we fee'I she did superbly. Last, but by nomeans least, wemust thank the wholeMIT Sea Grant staff for its patient and efficient cooperation over the last year, but especially wewould like to expressour thanksto Ms. JoanneSullivan for her meticulous, tireless and alwayscheerful efforts to ready the manuscript for final printing. To co-host the conference and publish the proceedings of the First International Conference on Chitin/Chitosan, the MIT Sea Grant Program received support from the Office of Sea Grant in the National Oceanic and Atmospheric Administration, U,S. Department of Ccmmierce,through grant number 04-6-158-44081 ~ and from the Massachusetts Institute of Technology. The U,S. Government is authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation that may appear hereon.

RELATEDREPORTS

The following reports contain information related to the project described herein:

CHITIN ANDCHITIN DERIVATIVES, MIT/Marine Industry Collegium Opportunity Brief. M!TSG76-5. Cambridge: Massachusetts Institute of Technology, August 1976. 21 pp, $2,5O,

Ashford, Nicholas A., Dale Hattis, and Albert E. Murray. INDUSTRIALPROSPECTS FOR CHITIN AND PROTEINFROM SHELLFISHWASTES. MITSG 77-3. Cambridge: Massachusetts Institute of Technology, 1976. 99 pp. $12.50.

The Sea Grant Information Center maintains an inventory of all technical publications. We invite orders and inquiries to:

Sea Grant Information Center HIT S ea Grant P rogram 77 Mas sachu set t s Avenue Cambridge, Massachusetts 02139 P REFACE ~IIITIN kai'tin!. Zoos, and Chem.Also -ine. [a. F. ch tine, f. Gr. yizDv frock, tunic: see -IN. The etymologicalformation would be chitonin!] The organic substance which forms the elytra and lntegumentsof insects and the carapacesof crustacea. The Oxford English Dictionary, 1961

One of the streets in the old quarter of Nancy, , bears the nameof Henri Braconnot, the discoverer of chitin. Braconnot, who lived from I/80 to 1856, studied chemistry and ln Strasbourgand biology and geology in . Still ln his twenties, he became director of the Botanic Gardens of Nancy. The city honored him, however, apparently not for his scientific contri- butions'~ but for a generous sumof moneyhe left to the town,

In the course of his work with mushrooms in 1 811, Braconnot becameintrigued with a cellulosic substancehe had isolated which did not undergochange ln an alkaline solution. Henamed the substance, "fungine." Tenyears later, in 1821,the name"chitlne" wasgiven to a material seemingly identical to fungine, but found by A. Odier while studying insects. Qdier, whopublished the newname in the M6moiresde la Socibtd d'Histoire Naturelle de Paris and in the Dictionnaire Classique d'Historic Naturelle, noted," It is most remarkable tu find the exoskeleton of insects to be identical with the cuticle of plants." ThoughOdier was incorrect in believing the two substances were identical, he is distinguished for not only namingchitin but for being the first personto observethe remarkablesimilarity betweenthe function of in plants and that of "chitine" in insects. Scientific interest in chitin continued for a time, but eventually muchpublished work fell into disuse and was forgotten until almost 40 years ago whenawareness that this natural polymer hadan impressivenumber of potential applications stimulated new research. The editors of these proceedings believe that this curiosity and subsequentinvestigations into the usesof' chitin contributed to the British war effort by providing a superior adhesive for the woodenfighter planes used by the British during the beginning of World War II. Nowmore than a century and a half after Braconnot madehis discovery, scientists throughoutthe world are fascinated by the potential large-scale, industrial applications for chitin. And they continue to be astonishedby the numberof living organisms in which they are finding chitin, its derivative chitosan, and enzymescapable of interacting with the two substances. Chitin has comeof age.in both basic and applied scienceand technology. At a time when it is accepted, indeed imperative, that the wise utilization of natura'1 resources is a conditio sine gua non f h eeity's se i 1, it legit 1, fe sighted ~i' te e g with the best traditions of international scientific cooperation for the Sea Grant Program of NOAA, U.S. Department of Convaerce, together with listed academic institutions and organizations, to co-sponsor and generously support the first international confer- ence on chitin and chitosan, of which these are the proceedings.

Most of the papers in this volume were presented at the conference. Papers by E,R, HAYES, et al., page 103; K. SIMPSON, page 253; O.L. OKE, et al., page 327; lhl. TAKEDA,page 355; B,P. HETTICK, et al,, page 464; M.l . BADE, page 472; S.C. SHRIVASTAVA, et al,, page 492; C.J. BRINE, page 509; K. OKUTANI, page 582; and K.O. PARKER,page 606, however, were received after the conference and are included here because of the contributions they are making to the field. TABLEOF CONTENTS Page Chairman's Address Chitin, an Important Natura'I Polymer Riccardo A. A. Muzzarelli

I . SOURCE OF CHITIN

Distribution and Quantitative Importance of Chitin in Animals Ch. Je uniaux TheDistribution andQuantitative Importanceof Chitin in Fungi Jose Ruiz-Ilerrera The Detection and Estimation of Chitin in Insect Organs 22 A. Glenn Richards

I I, ECONDMICCONSIDERATIONS OF ALTERNATIVESOURCES OF CHITIN 29

Approachesto a PracticalAssessment of Supplyand Demandfor Chitin Products in the United States Albert E. Murray and Dale kattis 3O

The Economicsot' Chitin Recoveryand Production 45 Peter M. Perceval Antartic Krill EuphausiaSuperba! as a Sourceof Chitin and Chitosan C. G. Anderson, N. de Pablo and C. R. Rorno 54 A Criti ca'I Evaluation of the Potent i a1 Sourcesof Chitin and Chitosan G. G. A1Ian, J. R. Fox, and N. Kong 64

III. TECHNOOGV OF CHITIN-CHITOSAN PROCESSING 79

The Production of Chitin and Chitosan Edwin Lee Johnsonand Quintin P. Peniston 80 A Studyof Variables in the ChitosanManufacturing Processin Relation to Molecular-Weight Distribution Chemical Characteristics and Waste-Treatment Effectiveness A. C. M. Wu and W, A. Bough 88 viii Organic Acid Solvent Systems for Chitosan Ernest R. Hayes, Donald H. Devi es, and Vaughan G. Munroe 103

IV. CHARACTERISTICS OF CHITIN-CHITOSAN 107

Determination of the Structures of u- and 8 - by K-Ray Diffraction John Blackwell, Ram Minke, and Kenncorwin Gardner 'I 08

Tensile Mechanical Properties and Transconformational Changes of Chitins H. R, Hepburn and H, D, Chandler 124

Crystal-Defect Mechanisms and the Transconformational Changes of Chitin H. D. Chandler and H. R. Hepburn 144

Bulk and Solution Properties of Chitosan L. J. Filar and M. G. Wirick 169

Marine Chitin Proper ties and Solvents Fran k A. Rutherford, I! I 182

Characterization of Chitosan. I:Thermoreversible Chitosan Gels Ernest R. Hayes and Donald H. Davies 193

Film-Forming Capability of Chitosan B. L. Averbach 199

Chitosan of High Viscosity and Protein as a Valuable By-Product from Squi11a Moorjani, D. Imam Khasim, S. Rajalakshmi, P, Puttaraj appa, and B. L. Amia 210

V. ORGANIC AND MINERAL RECOVERY 217

Treatment of Food-Processing Wastes with Chitosan and Nutritional Evaluation of Coagulated By-Products W. A. Bough 218

Chitosan Applications in Wastewater Sludge Treatment Takashi Asano 231

The Recovery of Protein and Pigments from Shrimp and Crab Meals and their Use in Salmonid Pigmentation Kenneth L. Simpson 253 ix The Chelating Properties of Kytex H Chitosan H. Hauer 263

Chitosan and Chi tosan Derivatives for Removal of Toxic Metallic Ions from Manufacturing Plant Waste Streams M. S. Masri and V. G, Randall 277

The Role of Chitin and Other Organics in the Transport of Metals to the World's Oceans V. Subramanian 288

VI . SPECIAL APPLICATIONS 295

Applications of Chitin and Chitosan in Wound- Healing Acceleration L. L. 6alassa and J. F. Prudden 296

Use of Crosslinked Chitosan in the Finishing Treatment of Wool Fabric for Laundering -Shrinkage Control M. S. Masri, V. G. Randall, and A. G. Pittman 306

Studies on the Preparation of Chitin Fibers Junzo Noguchi, Seiichi Tokura, and Morio Nishi 315

The Possible Use of Chitin and Chitosan as Animal Feed O. L. Oke 327

VII. CHEMICAL ASPECTS OF CHITIN RESEARCH 334

Modified Chitosans and their Chromatographic Performances Riccardo A. A. Muzza rel li 335

Use of Chitin Powder as Adsorbent in Thin-Layer Chromatography M. Takeda 355 Insolubilizing Enzymes with Chitosan and Chitosan- Derived Polymers M. S. Masri, V, G. Randall, and W. L. Stanley 364

Interaction of Wheat-Germ Agglutinin with Chitin Oligomers and Microbial Cell-Wall Polymers Reuben Lotan 375

Chitosan-Metal Complexes and their Function F. Yaku 386 x Characterization of Chitosan. II: The Determination of the Degree of Acetylation of Chitosan and Chitin Ernest R. Hayes 406

Studies on the Acetylation of Chitosan G. K. Moore and G. A. F. Roberts 421

Chitin from Marine Organ i sms and its Use as an Adhesive G. Sundara Rajulu and N. Gowri 430

A Method for Obtaining Pure Chitin of Commercial ValueS. A, Karuppaswamy 437

Metal-Binding Property of Chitosan from Prawn Waste P. Madhaven and K. G. Ramachandran Nair 444

V] II BIOCHEMICAL AND MICROSIOI DGICAL RESEARCH 449

Synthesis of Chitin Microffbrfls fn Vitro by Chitin Synthetase Particles, Chitosomes, Isolated from Mucor Rouxii S. Bartnic~i- arri , . a E. Bracker, and J. Ruiz- ~ errera 450

In Vitro Chitfn Biosynthesis by Cuticular D~sks of Mand ~ ca Sexta Larvae in the V Instar 1t ece P. llettlck a d M 1 t. Bade 464

Enzymatic Breakdown of the Chitfn Component in Insect Cuticle during the Molt Maria L. Sade 472

Ultrastructure of Crab Chitin and Chitosan Revealed with the Scanning Electron Microscope M. S. Masrf and F, T. Jones 483

Chitin Fiber Arrangement in Arachnid and Myrfapod Cuticle S. C. Shrivastava and Archana Dutt 492

Chitin Content and Variation with Molt Stage and Carapace Location fn the Blue Crab, C 1 1l t c ~Sid a Charles J. Brine 509

Chemical Dffferences in the o- and 8-Chitins and their Significance in the IIuality of the By-Products of Chitfn C. K. Kandaswamy 517 Enzymatic Hydrolysis of Chitosan D. Fenton, B. Davis, C. Rotgers, and D. E. Eveleigh 525

Distribution and Activity of Chitinolytic Enzymesin the Digestive Tract of Birds and Mammals Ch. Jeuniaux and C. Cornelius 542 Method of Extracting Chitinase from Gastric Juices and Intestinal Chyme of Chicken A. Smirnoff 550 Chitin Digestion in the Digestive Tract of Fish Koichi 0kutani 554 Chitin Degradation in Estaurine Environments and Implications in Crustacean Biology M. A. Hood, and S. P. Meyers 563 Chitinoly tie and Chitosanolytic Microorganisms and The Potential Biodeterioration Problem in the Commercial Application of Chitin and its Derivatives R.C.W. Berkeley 570 Chitin Decomposition by Microorganisms in Soil Nduka Okafor 578

Chitin- and N-Acetylglucosamine-Decomposing Bacteria in the Sea Koichi Dkutani 5B2 Chitinase Systems in Microorganisms and the Commercial Use of Chitin A. Dhtakara, Y. Uchida nd M. Miisutomi 587

K.M. Rudall: An Appreciation K,D, Parker 606 61 'I Subject Index

Index of Authors 629

Index of Organisms 649