APPLICATIONS OF CELL IMMOBILISATION BIOTECHNOLOGY

FOCUS ON BIOTECHNOLOGY

Volume 8B

Series Editors

MARCEL HOFMANCentre for Veterinary and Agrochemical Research, Tervuren, Belgium

JOZEF ANNÉRega Institute, University of Leuven, Belgium

Volume Editors

University of Belgrade,Belgrade, Serbia and Montenegro

RONNIE WILLAERTFree University of Brussels,

Brussels, Belgium

VIKTOR NEDOVI

COLOPHON

Focus on Biotechnology is an open-ended series of reference volumes produced bySpringer in co-operation with the Branche Belge de la Société de Chimie Industriellea.s.b.l.

The initiative has been taken in conjunction with the Ninth European Congress onBiotechnology. ECB9 has been supported by the Commission of the EuropeanCommunities, the General Directorate for Technology, Research and Energy of theWallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels CapitalRegion.

Applications of Cell Immobilisation Biotechnology

Edited by

VIKTOR NEDOVI

and

RONNIE WILLAERTFree University of Brussels,

Brussels, Belgium

University of Belgrade,Belgrade, Serbia and Montenegro

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN-10 1-4020-3229-3 (HB) Springer Dordrecht, Berlin, Heidelberg, New YorkISBN-10 1-4020-3363-X (e-book) Springer Dordrecht, Berlin, Heidelberg, New YorkISBN-13 978-1-4020-3229-5 (HB) Springer Dordrecht, Berlin, Heidelberg, New YorkISBN-13 978-1-4020-3363-6 (e-book) Springer Dordrecht, Berlin, Heidelberg, New York

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v

This book is the second of two volumes dedicated to cell immobilisation/encapsulationbiotechnology. The first book (Focus on Biotechnology, vol. 8A) dealt with thefundamentals of cell immobilisation biotechnology, whereas the present book is focused on the applications. Actually it is an outcome of the editors’ intention to gather the vast widespread information on applications of immobilisatiaa on/encapsulation biotechnology into a comprehensive reference book and to give the reader an overview of the most recent results and developments that have been realised in this domain. Extensiveresearch in the field of cell immobilisation/ encapsulation for various processes in biotechnology has been carried out during recent years. This is clearly reflected in the voluminous publications of original research, patents, and symposia, and thedevelopment of successful commercial inventions. In addition, it is obvious that there is a rapidly growing market for such applications in many different areas includingbiomedicine, pharmacology, cosmetology, food technology and beverage production,agriculture, waste treatment, analytical applications and biologics production. The progress in immobilised/encapsulated cell applications has therefore become a real scientific and technological challenge in the last few years.

This book consists of 33 chapters that are arranged into 5 parts: (1) “Tissueengineering”, (2) “Microencapsulation for disease treatment”, (3) “Food and beverageapplications”, (4) “Industrial biochemical production”, and (5) “Environmental and agricultural applications”. In each part various related topics are presented in detail.Specifically, contents of different chapters cover a broad variety of cell types and applications: seeding and cultivation of mammalian cells on different supports for tissueengineering, encapsulation of animal cells for treatment of variety of diseases, immobilisation of microbial cells for utilisation in food industry and fermentationprocesses, encapsulation and stabilisation of probiotics, production of biopharmaceuticals, biologics and biofuel, waste treatment and pollution control, agricultural applications such as artificial plant seeds and artificial insemination of farmanimals and development of biosensors. In addition, particular attention is given toselection, design and characterisation of cell carriers and bioreactors for differentprocesses. 77 carefully selected experts in different fields of biotechnology and bioengineering from 46 research institutes and laboratories all over the world contributed to this volume and collectively provided a unique, rich expertise and knowledge. Today, the book presents the most comprehensive, complete, up-to-datesource of information on various aspects of cell immobilisation/encapsulationapplications.

EDITOR’S PREFACE

vi

This book is intended to cover needs and to be the essential resource for bothacademic and industrial communities interested in cell immobilisation biotechnology. An integrated approach to “biological” and “engineering/technology” aspects is pursued to reach an even wider audience such as specialists in biomedicine, pharmacy, microbiology, biology, food technology, agriculture, environmental protection, chemical, biochemical and tissue engineering who seek a broad view on cellimmobilisation/encapsulation applications. Due to the in depth review of various applications of cell immobilisation, this volume, in combination with the first volume,can be used as a “handbook” of “Cell Immobilisation Biotechnology”.

We express our gratitude and appreciation to our many colleagues, who as expertsin their fields, have contributed to this volume. We would also like to thank the series editor, Marcel Hofman, and the publisher, Kluwer/Springer, for their excellent support in assuring the high quality of this publication.

Viktor A. Nedovi and Ronnie Willaert

Belgrade/Brussels, September 2004

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TABLE OF CONTENTS

EDITOR’S PREFACE ................................................................................................ v TABLE OF CONTENTS ............................................................................................ 1 PART 1TISSUE ENGINEERINGBio-artificial organs ................................................................................................. 17The example of artificial pancreas ......................................................................... 17Claudio Nastruzzi, Giovanni Luca, Giuseppe Basta and Riccardo Calafiore............17

1. Introduction to bio-artificial organs...................................................................172. Diabetes mellitus ...............................................................................................173. Application of bio-artificial pancreas ................................................................184. General characteristic of bio-artificial pancreas ................................................20f5. Devices for bio-artificial pancreas.....................................................................21

5.1. Macrodevices..............................................................................................215.2. Microcapsule ..............................................................................................22

5.2.1. General considerations.........................................................................225.2.1.1. Uncoated Ca-alginate microcapsules............................................225.2.1.2. Barium alginate microcapsules. ....................................................235.2.1.3. Agarose microcapsules. ................................................................235.2.1.4. Poly-ethylene glycol microcapsules. ............................................245.2.1.5. Other polymers. ............................................................................245.2.1.6. Conformal alginate microcapsules................................................255.2.1.7. Polyaminoacid coated alginate microcapsules..............................25d5.2.1.8. Scaling-up protocols for alginate microcapsules. .........................26

6. Microcapsule immunobarrier capacity ..............................................................287. Site of transplantation........................................................................................288. Intracapsular environmental conditions.............................................................289. Co-encapsulation of drug delivery devices........................................................29

9.1. Effects of anti-oxidants on β-cell function .................................................299.2. Effects of vitamin D3 on insulin secretion ..................................................30

10. Conclusions .....................................................................................................33References .............................................................................................................35

Bioartificial pancreas: an update ...........................................................................39Ales Prokop and Jeffrey M. Davidson.......................................................................39

1. Introduction .......................................................................................................392. Islet immobilisation techniques: towards a perfect capsule...............................40

2.1. Drop generation and capsule size ...............................................................412.2. Process and reactor design, product performance ......................................422.3. Biologically relevant problems...................................................................43

3. Islet sourcing .....................................................................................................443.1. Embryonic stem cells (ESC).......................................................................48m3.2. Adult pluripotent stem cells (APSC) ..........................................................483.3. Embryonic vs. adult SC ..............................................................................50

2

References .............................................................................................................51Bioartificial skin.......................................................................................................55Barbara Zavan, Roberta Cortivo, Paola Brun, Carolin Tonello and Giovanni Abatangelo ..........................................................................................55

1. Introduction .......................................................................................................552. Requirements .....................................................................................................56

2.1. Anatomic ....................................................................................................562.2. Surgical.......................................................................................................57

3. Tissue engineering of human skin .....................................................................583.1. Scaffolds .....................................................................................................583.2. Cells ............................................................................................................59

3.2.1. Epidermis.............................................................................................593.2.2. Dermis .................................................................................................61

4. Conclusions .......................................................................................................65References .............................................................................................................66

Bioartificial liver ......................................................................................................69Clare Selden...............................................................................................................69

1. Introduction .......................................................................................................692. What is the best cell for a bioartificial liver, and how many are required? .......69ff3. Bioreactor design – initial clinical experience...................................................704. Bioreactor designs – experimental models ........................................................735. Cell encapsulation..............................................................................................746. Alginate hydrogels.............................................................................................757. Alternative approach..........................................................................................808. Conclusion.........................................................................................................80References .............................................................................................................80

Tissue-engineered blood vessels and the future of tissue substitutes ..................85Lucie Germain, Karina Laflamme and François A. Auger........................................85

1. Introduction .......................................................................................................852. TEBV as a conceptual continuum .....................................................................853. Reconstructing small diameter blood vessel by the self-assembly approach ....874. Methodology for TEBV reconstruction by the self-assembly approach ...........885. Histological and phenotypic characteristics of the reconstructed TEBV ..........906. Functional characteristics of the reconstructed TEBV ......................................90f7. Vascularisation of the tissue construct by the addition of capillaries in vitro ...938. Conclusion.........................................................................................................95Acknowledgements ...............................................................................................95References .............................................................................................................95

Tissue engineering of cartilage and myocardium .................................................99Bojana Obradovic, Milica Radisic and Gordana Vunjak-Novakovic ........................99

1. Introduction .......................................................................................................991.1. Tissue engineering requirements ..............................................................1001.2. Tissue engineering model system.............................................................100

2. Cartilage tissue engineering.............................................................................1032.1. Articular cartilage .....................................................................................1032.2. Clinical need .............................................................................................103

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2.3. Tissue engineering....................................................................................1032.4. Cell sources...............................................................................................1042.5. Scaffolds ...................................................................................................1042.6. Bioreactor hydrodynamics........................................................................1052.7. Growth factors ..........................................................................................1062.8. Duration of culture....................................................................................1082.9. Spatial and temporal patterns of chondrogenesis .....................................1092.10. Mathematical model of cartilage development.......................................110f

2.10.1. Mathematical model of GAG accumulation in cultured cartilage explants ..............................................................1102.10.2. Mathematical model of GAG accumulation in engineered cartilage

constructs................................................................................................1133. Cardiac tissue engineering...............................................................................116

3.1. Myocardium (cardiac muscle) ..................................................................1163.2. Clinical need .............................................................................................1163.3. Tissue engineering....................................................................................1173.4. Cells ..........................................................................................................1173.5. Scaffolds ...................................................................................................1183.6. Bioreactor hydrodynamics........................................................................118

3.6.1. Static dishes .......................................................................................1203.6.2. Interstitial flow ..................................................................................121

3.7. Mathematical model of oxygen distribution in a tissue construct ............1233.8. Mechanical stimulation.............................................................................1263.9. Electrical stimulation of construct contractions........................................127f

4. Summary..........................................................................................................129References ...........................................................................................................129

Tissue engineered heart.........................................................................................135Kristyn S. Masters and Brenda K. Mann.................................................................135

1. Introduction .....................................................................................................1352. Heart valves .....................................................................................................135

2.1. Valve biology ...........................................................................................1362.2. Valve substitutes.......................................................................................137

2.2.1. Mechanical valves .............................................................................1372.2.2. Tissue valves......................................................................................138

2.3. Tissue engineered heart valves .................................................................1392.3.1. Cell immobilisation in acellular valves..............................................1392.3.2. Cell immobilisation in porous matrices .............................................1402.3.3. Cell immobilisation in hydrogels.......................................................142

2.4. Critical considerations for tissue engineered heart valves........................1432.4.1. Cell source .........................................................................................1432.4.2. Material properties.............................................................................144

2.5. The ideal tissue engineered valve .............................................................1443. Cardiac muscle ................................................................................................145

3.1. Biological considerations..........................................................................1453.2. Cell source ................................................................................................1453.3. Scaffold materials .....................................................................................146

4

3.4. Cultivation conditions...............................................................................1484. Conclusions .....................................................................................................149References ...........................................................................................................149

Bone tissue engineering .........................................................................................153Pankaj Sharma, Sarah Cartmell and Alicia J. El Haj...............................................153

1. Introduction .....................................................................................................1532. Mesenchymal stem cells ..................................................................................1543. Carrier scaffolds ..............................................................................................1554. Ex vivo conditioning of constructs...................................................................1575. Animal studies .................................................................................................1596. Human studies .................................................................................................161References ...........................................................................................................163

Stem cells – potential for tissue engineering........................................................167M. Minhaj Siddiqui and Anthony Atala ..................................................................167

1. Introduction .....................................................................................................1672. What is a stem cell? .........................................................................................168

2.1. Adult stem cells ........................................................................................1692.2. Foetal stem cells .......................................................................................1702.3. Embryonic stem cell .................................................................................1702.4. Somatic cell nuclear transfer: therapeutic cloning....................................171

3. Potential and how cells are differentiated........................................................1713.1. Non-specific differentiation and selection................................................1723.2. Gene transduction induced differentiation................................................1723.3. Growth factor and media formulation induced dimm fferentiation .................173

4. Advantages in tissue engineering ....................................................................1734.1. Self-renewal..............................................................................................1744.2. Multipotency.............................................................................................174

5. Research directions..........................................................................................1745.1. Skin...........................................................................................................1755.2. Bone..........................................................................................................1755.3. Cartilage....................................................................................................1755.4. Renal.........................................................................................................1765.5. Skeletal and cardiac muscle......................................................................177

6. Ethical and political considerations in stem cell biology.................................178References ...........................................................................................................178

PART 2MICROENCAPSULATION FOR DISEASE TREATMENTChallenges in cell encapsulation ...........................................................................185Gorka Orive, Rosa Mª Hernández, Alicia R. Gascón and José Luis Pedraz ...........185

1. Introduction .....................................................................................................1852. Immunoisolation approaches ...........................................................................1863. Potential advantages of cell encapsulation technology....................................1874. Materials used in cell encapsulation ................................................................1875. Cell lines ..........................................................................................................1906. Therapeutic applications..................................................................................1917. Perspectives and concluding remarks..............................................................193

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References ...........................................................................................................193Protein therapeutic delivery using encapsulated cell platform .........................197Marcelle Machluf ....................................................................................................197

1. Introduction .....................................................................................................1972. Anti-angiogenic protein therapy......................................................................1983. Anti-angiogenic gene delivery.........................................................................1984. Genetically engineered cells delivering therapeutics.......................................1995. Cell encapsulation – a platform for delivering therapeutics ............................200m6. Cell encapsulation – delivering anti-angiogenic therapeutics .........................2017. Future perspective............................................................................................205References ...........................................................................................................205

Cell encapsulation therapy for malignant gliomas .............................................211Anne Mari Rokstad, Rolf Bjerkvig, Terje Espevik and Morten Lund-Johansen.....211

1. Introduction .....................................................................................................2112. Glioma growth and invasiveness .....................................................................2123. Glioma treatment .............................................................................................213

3.1. Surgery .....................................................................................................2133.2. Irradiation .................................................................................................2133.3. Chemotherapy...........................................................................................213

4. Glioma treatment with alginate bioreactors.....................................................2134.1. Optimising the alginate bioreactor............................................................2144.2. Improvement of the alginate microcapsules for proliferating cells ..........2174.3. Choosing cell-lines suited for encapsulation ............................................2184.4. The biocompatibility of foreign materials in the brain .............................2194.5. The biocompatibility of the alginate bioreactors ......................................220

5. Conclusion.......................................................................................................222References ...........................................................................................................222

Gene therapy using encapsulated cells ................................................................229Gonzalo Hortelano...................................................................................................229

1. Introduction .....................................................................................................2292. Gene therapy....................................................................................................2303. Genetic engineering of cells ............................................................................231

3.1. Viral vectors .............................................................................................2323.2. Retrovirus .................................................................................................2323.3. Lentivirus..................................................................................................2333.4. Adenovirus ...............................................................................................2333.5. Adeno-associated virus.............................................................................2343.6. Herpes.......................................................................................................2343.7. Non-viral vectors ......................................................................................234

4. Selection of cells for encapsulation .................................................................2354.1. Established cell lines/primary cells...........................................................2354.2. Proliferative/quiescent cells......................................................................2364.3. Allogeneic/xenogeneic cells .....................................................................237

5. Applications of encapsulated cells in gene therapy .........................................2375.1. Cancer.......................................................................................................2375.2. Neurological conditions............................................................................238

6

5.3. Erythropoietin...........................................................................................2385.4. Encapsulated cells to treat metabolic diseases..........................................2395.5. Gene therapy of haemophilia B ................................................................2405.6. Gene therapy of haemophilia A................................................................242

6. Concluding remarks.........................................................................................242References ...........................................................................................................243

Artificial cells for blood substitutes, enzyme therapy, cell therapy and drug delivery ..............................................................................................................249

Thomas Ming Swi Chang ........................................................................................249Abstract................................................................................................................2491. Introduction .....................................................................................................2492. Artificial cells containing enzymes for inborn errors of metabolism and other

conditions .......................................................................................................2503. Artificial cells for cell therapy.........................................................................2504. Red blood cell substitutes ................................................................................251

4.1. Polyhemoglobin as blood substitutes........................................................2514.2. Polyhemoglobin containing catalase and superoxide disumutase ............2514.3. Recombinant human hemoglobin .............................................................2524.4. Other new generations of modified hemoglobin blood substitutes ..........252

5. General ............................................................................................................252Acknowledgements .............................................................................................252References ...........................................................................................................253

PART 3FOOD AND BEVERAGE APPLICATIONSBeer production using immobilised cells .............................................................259Viktor Nedovi , Ronnie Willaert, Ida Leskošek- ukalovi , Bojana Obradovi and

Branko Bugarski.................................................................................................2591. Introduction .....................................................................................................2592. Carrier selection and design ............................................................................2613. Reactor design .................................................................................................2634. ICT applications for the brewing industry.......................................................264

4.1. Flavour maturation of green beer .............................................................2644.2. Production of alcohol-free or low-alcohol beer........................................2654.3. Production of acidified wort using immobilisedd lactic acid bacteria........2664.4. Continuous main fermentation .................................................................266

5. Summary..........................................................................................................2686. References .......................................................................................................269

Application of immobilisation technology to cider production: a review.........275Alain Durieux, Xavier Nicolay and Jean-Paul Simon .............................................275

Abstract................................................................................................................2751. Introduction .....................................................................................................2752. Cell immobilisation in cider production ..........................................................277

2.1. Immobilisation of yeast for apple juice fermentation ...............................2782.2. Immobilisation of O. oeni for malolactic fermentation in cider ...............280

3. Conclusions .....................................................................................................282References ...........................................................................................................282

7

Wine production by immobilised cell systems ....................................................285Charles Divies and Remy Cachon ...........................................................................285

1. Introduction .....................................................................................................2852. Immobilised cell technology and heterogeneous bioreactors ..........................2853. Potential of immobilised cell systems for applications in oenology................287

3.1. Alcoholic fermentation .............................................................................2873.2. Bottle-fermented sparkling wines ("méthode champenoise")...................2883.3. Production of sparkling wines in closed reactors .....................................2893.4. The malolactic fermentation .....................................................................290

4. Conclusion.......................................................................................................290References ...........................................................................................................291

Immobilised cell technologies for the dairy industry .........................................295Christophe Lacroix, Franck Grattepanche, Yann Doleyres and Dirk Bergmaier ....295

1. Introduction .....................................................................................................2952. Immobilisation techniques...............................................................................296

2.1. Entrapment within polymeric networks....................................................2962.2. Adsorption to a preformed carrier ............................................................2962.3. Membrane entrapment ..............................................................................2972.4. Microencapsulation ..................................................................................297

3. Microbial growth in gel beads .........................................................................2993.1. Biomass distribution in gel beads .............................................................2993.2. Cell release from beads.............................................................................3003.3. Cross-contamination phenomenon ...........................................................300

4. Changes of culture characteristics during immobilised-cell fermentations .....3015. Biological stability...........................................................................................303

5.1. Psychrotroph contaminants.......................................................................3035.2. Bacteriophage contaminants .....................................................................3035.3. Plasmid stability........................................................................................305

6. Applications of immobilised cell technology ..................................................3056.1. Biomass production ..................................................................................305

6.1.1. Starter production ..............................................................................3066.1.1.1. Lactic acid bacteria starter ..........................................................3066.1.1.2. Probiotic cultures ........................................................................307

6.1.2. Prefermentation of milk .....................................................................3086.2. Metabolite production...............................................................................310

6.2.1. Lactic acid production .......................................................................3106.2.2. Exopolysaccharide production...........................................................3116.2.3. Bacteriocin production.......................................................................312

6.3. Cell protection ..........................................................................................3137. Conclusions .....................................................................................................314References ...........................................................................................................314

Food bioconversions and metabolite production ................................................321P. Heather Pilkington...............................................................................................321

1. Introduction .....................................................................................................3212. Food bioconversions........................................................................................322

2.1. Sake production ........................................................................................322

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2.2. Soya sauce production ..............................................................................3232.3. Mead production.......................................................................................3242.4. Removal of malic acid from coffee beans ................................................3242.5. De-bittering of citrus juice........................................................................3242.6. “Ugba” food snack production .................................................................3252.7. Removal of simple sugars.........................................................................325

2.7.1. Purification of food-grade oligosaccharides ......................................3252.7.2. Glucose removal from egg.................................................................326

2.8. Sugar conversions.....................................................................................3262.9. Hydrolysis of triglycerides and proteins in milk ......................................3272.10. Vitamins..................................................................................................328

3. Metabolite production......................................................................................3283.1. Amino acids..............................................................................................3283.2. Organic acids ............................................................................................3293.3. Alcohols....................................................................................................3303.4. Enzymes....................................................................................................3313.5. Bacteriocins ..............................................................................................331

References ...........................................................................................................331Immobilised-cell technology and meat processing..............................................337Linda Saucier and Claude P. Champagne................................................................337aa

Introduction .........................................................................................................3371. Historical use of meat fermentation.................................................................3382. New approach to meat preservation ................................................................3393. Probiotic cultures and health ...........................................................................3414. Immobilisation/encapsulation and starter production......................................341

4.1. Technological cultures for meat fermentation ..........................................3424.2. Probiotic cultures for meats ......................................................................345

5. Applications of ICT cultures in meat...............................................................3455.1. Meat fermentation using immobilised cells ..............................................3455.2. Improving the use of protective cultures via ICT .....................................3475.3. Other applications of ICT in meat ............................................................348

6. Conclusion.......................................................................................................349References ...........................................................................................................350

Bioflavouring of foods and beverages ..................................................................355Ronnie Willaert, Hubert Verachtert, Karen Van Den Bremt, Freddy Delvaux and Guy

Derdelinckx ........................................................................................................3551. Introduction .....................................................................................................3552. Definition of bioflavouring..............................................................................3563. Processes for flavour production .....................................................................3564. Bioproduction of natural flavours....................................................................358

4.1. Microorganisms ........................................................................................3584.1.1. De novo biosynthesis.........................................................................359

4.1.1.1. Biosynthesis of lactones. ............................................................3594.1.1.2. In situ bioflavouring. ..................................................................3594.1.1.3. Production of flavour metabolite organic acids. .........................360

4.1.2. Bioconversion by microorganisms ....................................................362

9

4.1.2.1. Biosynthesis of vanillin. .............................................................3624.1.2.2. Biosynthesis of flavours starting from fatty acids. .....................3634.1.2.3. Biosynthesis of aldehydes...........................................................3654.1.2.4. Bioflavouring of beer..................................................................365

References ...........................................................................................................368PART 4INDUSTRIAL BIOCHEMICAL PRODUCTIONProduction of ethanol using immobilised cell bioreactor systems.....................375Argyrios Margaritis and Peter M. Kilonzo ..............................................................375

1. Introduction .....................................................................................................3752. Immobilised cell systems.................................................................................376

2.1. Basic principles of cell immobf ilisation.....................................................3782.2. Cell immobilisation by adsorption............................................................3792.3. Cell immobilisation by covalent bonding .................................................3802.4. Cell immobilisation by physical entrapment within porous matrices .......381

2.4.1. Calcium alginate matrix.....................................................................3812.4.2. Carrageenan matrix............................................................................3842.4.3. Polyacrylamide gel matrix .................................................................3862.4.4. Epoxy resin matrix.............................................................................3862.4.5. Gelatin polymer matrix......................................................................387

2.5. Cell immobilisation by containment behind a membrane barrier .............3882.6. Cell immobilisation by self-aggregation...................................................389

3. Immobilised cell bioreactor types....................................................................3914. Ethanol production from non-conventional feedstock using immobilised cell

systems ...........................................................................................................3954.1. The production of ethanol from cheese whey...........................................3964.2. The production of ethanol from jerusalem artichokes ..............................3964.3. The production of ethanol from cellulose and cellobiose.........................3974.4. Production of ethanol from xylose ...........................................................399

Acknowledgement ...............................................................................................400References ...........................................................................................................400

Production of biopharmaceuticals through microbial cell immobilisation ......407Tajalli Keshavarz.....................................................................................................407

1. Introduction .....................................................................................................4072. The use of immobilised microorganisms in production of antibiotics.............408

2.1. Penicillin production.................................................................................4082.2. Cephalosporin-c production .....................................................................4132.3. Other antibiotics .......................................................................................414

3. The use of immobilised microorganisms in biotransformation in two liquid phasesystems ...........................................................................................................417

4. Concluding remarks.........................................................................................419References ...........................................................................................................420

Production of biologics from animal cell cultures ..............................................423James Warnock and Mohamed Al-Rubeai ..............................................................423

1. Introduction .....................................................................................................4232. Cell retention ...................................................................................................424

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3. Cell encapsulation............................................................................................4264. Cell entrapment................................................................................................427

4.1. Stirred tank bioreactors.............................................................................4294.2. Fluidised-bed bioreactors .........................................................................4314.3. Packed-bed bioreactors.............................................................................433

5. Conclusions .....................................................................................................435References ...........................................................................................................436

Stabilisation of probiotic microorganisms...........................................................439Helmut Viernstein, Josef Raffalt and Diether Polheim ...........................................439

1. Introduction .....................................................................................................4392. Standard pathways for production of microorganism and related stabilisation

techniques.......................................................................................................4423. Stabilisation methods.......................................................................................444

3.1. Capsule forming processes .......................................................................444LBC ME10 ..................................................................................................445Bifina® ........................................................................................................445BiActon®.....................................................................................................445

3.2. Specific solutions......................................................................................446LifeTop™ Cap.............................................................................................446LifeTop™ Straw..........................................................................................446

3.3. Extrusion...................................................................................................4463.4. Hard (gelatine) capsules ...........................................................................447

Bioflorin® capsules .....................................................................................447Probio-Tec®QUATRO-cap-4 .....................................................................447Omniflora® N..............................................................................................447BioTura®.....................................................................................................447

3.5. Liquid formulations ..................................................................................4473.6. Techniques for solid particle coating and aggregation .............................4483.7. Pastes and ointments.................................................................................448

Lactiferm® Ironpaste...................................................................................4493.8. Powder formulations.................................................................................449

Antibiophilus sachets and capsules .............................................................449Effidigest® sachets ......................................................................................449Lactiferm® ..................................................................................................449Effervescent formulation .............................................................................450

3.9. Tablet processing......................................................................................450Bion® 3 and Multibionta®..........................................................................450Gynoflor® ...................................................................................................451Paidoflor® ...................................................................................................451Lacto ............................................................................................................451

4. Summary..........................................................................................................451References ...........................................................................................................452

Growth of insect and plant cells immobilised using electrified liquid jets .......455Mattheus F. A. Goosen ............................................................................................455

1. Introduction .....................................................................................................4552. Droplet generation using an electrified liquid jet ............................................456

11

2.1. Production of alginate beads using electrified liquid jets .........................456d2.2. Effect of electrostatic field on cell viability..............................................4582.3. Forces acting on droplet in an electric field..............................................458t

3. Culture of encapsulated insect cells.................................................................462t3.1. Growth of cells and virus in microcapsules..............................................4633.2. Biocompatibility of encapsulation solutions.............................................464

4. Encapsulation and growth of plant tissue inf alginate ......................................4665. References .......................................................................................................467

Plant cell immobilisation applications .................................................................469Ryan Soderquist and James M. Lee.........................................................................469

1. Introduction .....................................................................................................4692. Significance of plant cell immobilisation ........................................................4703. Methods of plant cell immobilisation ..............................................................4714. Important considerations for plant cell immobilisation ...................................4735. Potential benefits of plant cell immobilisation ................................................4746. Conclusions .....................................................................................................477References ...........................................................................................................477

PART 5ENVIRONMENTAL AND AGRICULTURAL APPLICATIONSWastewater treatment by immobilised cell systems ...........................................481Hiroaki Uemoto .......................................................................................................481

1. Introduction .....................................................................................................4812. Immobilisation techniques...............................................................................481

2.1. Adsorption ................................................................................................4822.2. Entrapment................................................................................................4822.3. Carrierless immobilisation........................................................................483

3. Applications of immobilised cells ...................................................................483d3.1. Nitrification ..............................................................................................4833.2. Simultaneous nitrification and denitrification ..........................................4863.3. Denitirification..........................................................................................4893.4. Metal accumulation ..................................................................................490

4. Conclusions .....................................................................................................491References ...........................................................................................................491

Immobilised cell strategies for the treatment of soil and groundwaters..........495Ludo Diels ...............................................................................................................495

1. Introduction .....................................................................................................4952. In situ treatment ...............................................................................................4963. Soil and aquifer biofilms .................................................................................4984. Viruses in soil and groundwater: do they play a role?.....................................4995. Crossing the bridge between groundwater and surface water .........................4996. Pump and treat .................................................................................................5007. In situ bioprecipitation.....................................................................................5038. Bacterial contamination of aquifers.................................................................5039. Conclusions .....................................................................................................504References ...........................................................................................................504

12

Application of immobilised cells for air pollution control .................................507Marc A. Deshusses ..................................................................................................507

1. Introduction .....................................................................................................5072. Microbiology of gas phase bioreactors............................................................511

2.1. Microflora.................................................................................................5112.2. Biofilm architecture ..................................................................................5132.3. Secondary degraders and predators ..........................................................5142.4. Biodegradation and growth kinetics .........................................................515

3. Biofilter and biotrickling filter applications ....................................................5173.1. Definitions and performance reporting.....................................................5173.2. Examples of applications..........................................................................519

4. Current research, emerging topics ...................................................................521References ...........................................................................................................524

Artificial seeds........................................................................................................527Eugene Khor and Chiang Shiong Loh.....................................................................527

1. Introduction .....................................................................................................5272. Definitions of artificial seed ............................................................................5273. Plant materials used for encapsulation.............................................................528d

3.1. Somatic embryos and microspores-derived embryos ...............................5283.2. Shoot buds and shoot-tips.........................................................................5293.3. Seeds.........................................................................................................5293.4. Orchid protocorms and protocorm-like bodies.........................................5303.5. Mycorrhizal fungi .....................................................................................5303.6. Hairy roots-derived materials ...................................................................531

4. Matrix material selection for artificial seeds ...................................................5324.1. Fluid drilling .............................................................................................5324.2. Polymeric coating .....................................................................................5324.3. Hydrogels .................................................................................................5334.4. Two-coat system.......................................................................................5334.5. Taking stock .............................................................................................535

5. Outlook ............................................................................................................536References ...........................................................................................................536

Spermatozoal microencapsulation for use in artificial insemination of farm animals...............................................................................................................539

Raymond L. Nebel and Richard G. Saacke .............................................................5391. Introduction .....................................................................................................5392. Procedures for microencapsulation of sperm...................................................5403. Capsule size and maintenance of sperm viability in vitro ...............................5414. Microcapsule behaviour in the female tract.....................................................542r5. Evaluation of encapsulation technology in cattle artificial insemination – field

trials ................................................................................................................5456. Conclusions .....................................................................................................547References ...........................................................................................................547

Biosensors with immobilised microbial cells using amperometric and thermal detection principles...........................................................................................549

Ján Tká , Vladimír Štefuca and Peter Gemeiner .....................................................549

13

1. Biosensors........................................................................................................5492. Microbial biosensors........................................................................................5493. Immobilisation strategies.................................................................................5504. Improvement of microbial biosensor characteristics .......................................5515. Amperometric and thermal microbial biosensors ............................................551

5.1. Amperometric biosensors with the use of Gluconobacter oxydans..........5525.1.1. Gluconobacter oxydans as a prospective biocatalyst.........................5525.1.2. Whole cell Gluconobacter oxydans biosensors .................................553

5.1.2.1. Oxygen-based G. oxydans biosensors ........................................5535.1.2.2. Mediated Gluconobacter sp. biosensors .....................................555

5.1.3. Conclusion and future perspectives of G. oxydans biosensors..........5565.2. Thermal biosensors...................................................................................557

5.2.1. Analytical applications ......................................................................5585.2.2. Investigation of properties of immobilised cells................................5595.2.3. Conclusions and perspectives ............................................................563

References ........................................................................................................... 563Index .......................................................................................................................... 567