Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

1. Introduction......................................................................................................	203
2. Gas concentrations in batch-wise cell cultures.................................................	204
2.1. Oxygen Uptake Rate (OUR)......................................................................	204
2.2. Carbon dioxide equilibrium in the gas phase.............................................	205
2.3. Carbon dioxide equilibrium in the liquid phase.........................................	206
3. Software sensor design.....................................................................................	208
3.1. Dynamic model..........................................................................................	208
3.2. The Kalman filter algorithm......................................................................	209
4. Application of the software sensor...................................................................	211
4.1. Validation of the software sensor..............................................................	211
4.2. Application to cell cultivation ...................................................................	213
4.3. Robustness of the software sensor.............................................................	214
5. Concluding remarks.........................................................................................	215
References............................................................................................................	216

Fermentation phase detection using fuzzy clustering techniques and neural networks

for improved control.................................................................................................			217
Takoi K. Hamrita and Shu Wang .............................................................................			217
1. Introduction		......................................................................................................	217
2. Fermentation phase detection ...........................................................................			218
	2.1. Off-line ........................................phase detection using fuzzy clustering		218
	2.1.1. ............................................Variable selection for phase detection		218
	2.1.2. Fuzzy clustering for off-line phase detection of penicillin-G fed-
	batch fermentation....................................................................................		218
	2.1.3.	Fuzzy clustering for off - line phase detection of gluconic acid batch
	fermentation..............................................................................................		221
	2.2. Neural ...................................networks for on-line fuzzy phase detection		222
3.	Conclusion........................................................................................................		225
References............................................................................................................			225
Simulation, design ...........and model based predictive control of photobioreactors			227
J.-F. Cornet, C.G. ............................................................Dussap and J.-J. Leclercq			227
Abstract.................................................................................................................			227
1.	Introduction......................................................................................................		227
2.	Modelling ..............................................................................	photobioreactors	228
	2.1. Radiative ...................................................................transfer formulation		228
	2.2. Computing ...............................................................the optical properties		230
	2.3. Coupling ..........................radiative transfer with rates and stoichiometry		232
3. Results and discussions.....................................................................................			234
	3.1. Simulation ...............................................................................and design		235
	3.2. Model based .................................................................predictive control		236
4.	Conclusions ...........................................................................and perspectives		236
Acknowledgement................................................................................................			238
References............................................................................................................			238
PART	V Reactor ...................................................................................	Engineering	239
Bioreactors for space .......................................: biotechnology of the next century			241

8

Isabelle Walther, Bart Van Der Schoot, Marc Boillat and Augusto Cogoli............		241
Summary..............................................................................................................		241
1. Introduction.....................................................................................................		241
2. Space bioreactors: instrument.........................................................................		242
2.1. Large space bioreactors............................................................................		243
2.2. Miniature space bioreactors......................................................................		244
2.2.1. The DCCS......................................................................................		244
2.1.2. The Swiss space bioreactor: SBR I................................................		245
3. Space bioreactor SBRI: performances in flight..............................................		246
3.1. Liquid handling........................................................................................		246
3.2. Chemical measurement and control..........................................................		247
3.3. System control..........................................................................................		249
3.4. Biological analyses...................................................................................		249
4. Conclusions and perspectives...........................................................................		250
Acknowledgements..............................................................................................		251
References............................................................................................................		251
PART VI Immobilisation and Permeabilisation......................................................		253
State of the art developments in immobilised yeast technology for brewing...........		255
C.A. Masschelein and J. Vandenbussche .................................................................		255
1. Process requirements for high turnover rates in brewery fermentations ..........		255
2. Matrix design for application in the brewing process.......................................		257
3. Reactor design for application in the brewing process.....................................		258
4. Reactor configuration for continuous immobilised yeast fermentation systems
		259
5. Flavour development and control in immobilised yeast systems .....................		261
6. Technological potential of options for immobilised yeast application in the
brewing industry...................................................................................................		262
6.1. Immobilised primary fermentation............................................................		263
6.1.1. Packed bed reactor systems ............................................................		263
6.1.2.	Gas lift draft tube reactor systems ..................................................	264
6.1.3. Loop reactor systems......................................................................		265
6.2. Fast flowing immobilised yeast systems for the production of low and
alcohol-free beer...............................................................................................		267
6.2.1	Packed bed reactors.........................................................................	268
6.2.2	Fluidised bed reactors......................................................................	269
6.2.3 Gas lift loop reactor.........................................................................		270
6.3. Immobilised yeast systems for continuous flavour maturation of beer.....		271
6.3.1. DEAE cellulose carrier (Spezyme®)..............................................		271
6.3.2. Sintered glass bead carrier (Siran ®)...............................................		273
7. Concluding remarks.........................................................................................		274
References ............................................................................................................		274
Immobilized yeast bioreactor systems for brewing – Recent achievements.............		277
Viktor A. Nedovic, Bojana Obradovic, Ida Leskosek-Cukalovic and Gordana
Vunjak-Novakovic...................................................................................................		277

9

1. Immobilised cell systems in biotechnology......................................................			277
2. Applications of immobilised yeast systems in brewing....................................			278
2.1. Cell carriers and immobilization methods.................................................			278
	2.1.1. Adsorption to a pre-formed carrier.................................................		279
	2.1.2.	Cell entrapment...............................................................................	280
	2.1.3.	Self-aggregation..............................................................................	280
	2.1.4.	Containment of cells behind a barrier............................................	280
2.2. Bioreactor design.......................................................................................			281
	2.2.1. Packed bed reactor..........................................................................		281
	2.2.2.	Fluidised bed reactor.......................................................................	282
	2.2.3.	Silicon carbide cartridge loop Reactor...........................................	282
	2.2.4.	Internal loop gas-lift reactor.........................................................	283
3. Alginate-gas-lift bioreactor system...................................................................			283
3.1. Alginate microbeads loaded with yeast cells.............................................			283
3.2. Internal loop gas-lift bioreactor.................................................................			285
3.3. Beer fermentation in alginate-gas-lift bioreactor system...........................			285
4. Conclusion........................................................................................................			289
References ............................................................................................................			289
New matrices and bioencapsulation processes.........................................................			293
Ulrich Jahnz, Peter Wittlich, Ulf Prüsse and Klaus-Dieter Vorlop...........................			293
Summary...............................................................................................................			293
1. Introduction......................................................................................................			293
1.1. Techniques for the immobilisation process...............................................			293
1.2. Short overview of suitable materials for encapsulation.............................			294
1.3. Shapes of particles with immobilised biocatalysts ....................................			294
2. Techniques for bead production.......................................................................			295
2.1. Blow-off-devices.....................................................................................			297
2.2.	Vibration....................................................................................................		297
2.3.	Atomizers................................................................................................		297
2.4.	JetCutting................................................................................................		298
3. Materials for encapsulation...............................................................................			301
3.1. Natural polysaccharides for ionotropic gelation........................................			301
3.2. Synthetic hydrogels by chemical reaction.................................................			301
3.3. Hydrogels from polyvinyl alcohol.............................................................			302
4. LentiKats®.........................................................................................................			302
4.1. Description of properties...........................................................................			302
4.2. Production devices for laband technical scale.........................................			303
4.3. Examples for applications of LentiKats®...................................................			305
5. Conclusions......................................................................................................			306
References............................................................................................................			306
PART VII Downstream Processing...........................................................................			309
Industrial downstream processing............................................................................			311
MadsLaustsen..........................................................................................................			311
1. Introduction......................................................................................................			311

10

2. General aspects connected to				downstream processing.....................................	311
	2.1. Intellectual property rights			.........................................................................	311
	2.2. Public research in downstream processing................................................				312
	2.3. Quality.......................................................................................................				312
	2.4. Upstream process.......................................................................................				313
3.	Pharmaceutical production...............................................................................				314
	3.1. General downstream issues........................................................................				314
	3.2.	Recovery....................................................................................................			315
		3.2.1. Primary separation..........................................................................			315
		3.2.2.	Intracellular products......................................................................		315
		3.2.3.	Concentration..................................................................................		316
		3.2.4.	Precipitation/crystallisation...........................................................		316
	3.3 Purification.................................................................................................				316
		3.3.1.	Chromatographic	principles............................................................	316
		3.3.2. Matrix quality.................................................................................			317
	3.4. Research and development of particular interest for pharmaceutical
	downstream		processing.....................................................................................		318
4. Enzyme production...........................................................................................					318
	4.1. General downstream issues related to enzyme production........................				318
	4.2. Harvest.......................................................................................................				320
	4.3. Concentration.............................................................................................				320
	4.4. Purification................................................................................................				321
	4.5. Future challenges connected to downstream processing of bulk enzymes.
					322
5. Summary...........................................................................................................					323
References............................................................................................................					323
Separation		of	lactalbumin and	lactoglobulin by preparative chromatography
using simulated moving beds...................................................................................					325
S.L. Lucena, P.T.V. Rosa, L.T. Furlan and C.C. Santana ........................................					325
Abstract.................................................................................................................					325
1.	Introduction....................................................................................................				325
2.	Basic concepts of processes of separation with simulated moving beds..........				327
3.	Mathematical formulation................................................................................				328
	3.1. Application for a column of adsorption.....................................................				329
	3.2. Application to the simulated moving bed..................................................				329
4.	Adsorption isotherms........................................................................................				330
5.	Results and discussion......................................................................................				330
	5.1. Individual column of adsorption................................................................				330
	5.2. Simulated moving bed...............................................................................				332
6. Conclusions......................................................................................................					335
References............................................................................................................					336
Appendix..............................................................................................................					337
High-speed pectic enzyme fractionation by immobilised metal ion affinity
membranes...............................................................................................................					339
Silvia Andrea Camperi, Mariano Grasselli and Osvaldo Cascone ...........................					339

11

Abstract.................................................................................................................				339
1.	Introduction......................................................................................................			339
2. Materials and methods......................................................................................				341
	2.1. Enzymes and reagents ...............................................................................			341
	2.2. Histidine, lysozyme, myoglobin and haemoglobin concentration
	measurements...................................................................................................			341
	2.3. Pectic enzyme assay ..................................................................................			341
	2.4. Chelating hollow fibre synthesis................................................................			341
	2.5. pure water flux determination for a single chelating hollow fibre.............			342
	2.6. Measurement of the amount of ida introduced..........................................			342
	2.7. Adsorption isotherms measurement...........................................................			343
	2.8. Assembing a hollow-fibre membrane module...........................................			343
	2.9. Breakthrough curves for pe and pl adsorption...........................................			343
	2.10. Utilisation of the Cu(II)IDA-cartridge for pectic enzyme fractionation.. 343
3. Results and discussion......................................................................................				344
	3.1. Chromatographic characterisation of the derivatised membranes.............			344
	3.2. Properties of the hollow-fibre membrane module.....................................			346
	3.3. Breakthrough curves for pe and pl adsorption...........................................			346
	3.4. Utilisation of the ida-cartridge for pectic enzyme fractionation................			346
4.	Conclusions......................................................................................................			348
Acknowledgements..............................................................................................				348
References............................................................................................................				348
PART VIII Economic finalities..................................................................................				351
Economic benefits of the application of biotechnology - Examples........................				353
Marlene Etschmann, Peter Gebhart and Dieter Sell.................................................				353
Summary...............................................................................................................				353
Overview..............................................................................................................				353
1.	Production of 7-aminocephalosporanic		acid ...................................................	354
2.	Stonewashing of jeans......................................................................................			354
3.	Production of riboflavin ...................................................................................			355
4. Biopulping........................................................................................................				356
5.	Bleach cleanup..................................................................................................			356
	5.1. Materials and	methods...............................................................................		357
	5.1.1. Selection	of the production	plant....................................................	357
	5.1.2. The process.....................................................................................			357
	5.1.3. Economic analysis..........................................................................			358
	5.2. Results.......................................................................................................			358
6.	Conclusions......................................................................................................			360
References............................................................................................................				360
Enzyme stability and stabilisation : applications and case Studies...........................				361
Dr. Guido A. Drago and Dr. Tim D. Gibson............................................................				361
Summary..............................................................................................................				361
1.	Introduction...............................................................................................................			361
2.	Materials and methods.....................................................................................			363

12

3.	Results..............................................................................................................				364
	3.1. Alkaline phosphatase solution stability: enzyme source and buffer
	parameters.........................................................................................................				364
	3.2. Horseradish peroxidase stability in solution..............................................				366
	3.3. Alcohol oxidase dry stability : alcohol biosensors ....................................				367
	3.4. Acetylcholineesterase stability and biosensors..........................................				368
	3.5. Recombinant luciferase stability in solution..............................................				370
	3.6. Immobilised glucose oxidase : pre-stabilised complexes..........................				371
	3.7. Detection			of protein-polyelectrolyte complexes by isoelectric focusing...	373
4.	Discussion and conclusions.............................................................................				374
Acknowledgement................................................................................................					375
References............................................................................................................					375
Improvements of enzyme stability and specificity by genetic engineering..............					377
M. Pohl and M.-R. Kula...........................................................................................					377
1.	Introduction......................................................................................................				377
2.	Results.............................................................................................................				377
	2.1.	Formate		dehydrogenase.............................................................................	377
	2.2. Pyruvate decarboxylase.............................................................................				379
3.	Conclusion........................................................................................................				382
Acknowledgement................................................................................................					382
References............................................................................................................					382
An approach to desiccation-tolerant bacteria in starter culture production..............					383
Weekers F., Jacques P., Mergeay M. and Thonart P..............................................					383
1. Introduction......................................................................................................					383
2.	Selection of desiccation-tolerant bacteria.........................................................				384
3. Targets of desiccation damages and the proposed mechanisms responsible for
dessication			tolerance.............................................................................................		385
	3.1. Membranes ................................................................................................				385
		3.1.1. Membrane desiccation-damage mechanisms..................................			386
		3.1.2. Role of disaccharides in membrane tolerance to desiccation .........			387
	3.2.	Proteins......................................................................................................			388
		3.2.1. The anhydrobiotic cell and a water replacement hypothesis..........			388
		3.2.2. Vitrification of the cytoplasm as mechanism of tolerance to
		desiccation................................................................................................			389
	3.3.	Nucleic		acids..............................................................................................	391
		3.3.1. Mechanisms of tolerance to DNA damages during desiccation .....			392
		3.3.2. UV irradiation as a tool for the selection of drought-tolerant bacteria
					392
4. Factors influencing survival.............................................................................					393
	4.1. Bacterial species........................................................................................				393
	4.2. Growth conditions.....................................................................................				393
	4.3. Protective			additives....................................................................................	394
	4.4. Cell		concentration......................................................................................		394
	4.5. Drying gas, rate and extend.......................................................................				394
	4.6. Rehydration ..............................................................................................				395

13

4.7. Stability during storage..............................................................................			395
5. Conclusions	......................................................................................................		395
Acknowledgements..............................................................................................			396
References............................................................................................................			396
Biotechnological research and the dairy industry:....................................................			399
Heike Neubauer and Beat Mollet.............................................................................			399
Abstract.................................................................................................................			399
1. Introduction......................................................................................................			399
1 . 1 . The history of lactic acid bacteria..............................................................			399
2. Classification of lactic acid bacteria.................................................................			400
2.1. The group of lactic acid bacteria...............................................................			400
2.2. Classical bacterial taxonomy combined with molecular biology ..............			401
2.3. Isolation of new strains of lactic acid bacteria...........................................			401
3. Lactic acid bacteria as starter cultures..............................................................			402
3.1. The role of lactic acid bacteria in the fermentation of milk.......................			402
3.2. The new age of strain and product development.......................................			404
4. Improved starter strains – case studies.............................................................			405
4.1. Selection of naturally improved strains.....................................................			405
4.1.1. Mild, shelf-stable yoghurt...............................................................			405
4.1.2.	Probiotics, bacteria with health	beneficial properties....................	406
4.2. The genetic engineering approach.............................................................			408
4.2.1. Texture producing strains...............................................................			408
4.2.2.	Novel flavour producing strains....................................................		408
5. Outlook and Conclusions..................................................................................			409
Acknowledgements..............................................................................................			410
References............................................................................................................			410
Immobilised cell technology in winery and fruit wine production...........................			413
Remy Cachon and Charles Divies...........................................................................			413
Summary..............................................................................................................			413
1. Introduction......................................................................................................			413
2. Immobilised cell concept.................................................................................			414
3. Possible applications in winery and fruit wine production..............................			415
3.1. Alcoholic fermentation.............................................................................			415
3.1.1. Alcoholic fermentation without		pressure...............................	415
3.1.2.	Alcoholic fermentation with	pressure : elaboration of sparkling
wines.........................................................................................................			416
3.2. Malolactic fermentation of wine................................................................			418
4. Conclusion........................................................................................................			419
References............................................................................................................			419
A new polysaccharide derived from plant rhizosphere : production, purification and
physico-chemical properties.....................................................................................			423
Crompin J.M., Gamier T., Payot T., De Baynast R..................................................			423
Summary..............................................................................................................			423
1. Introduction......................................................................................................			423
2. Materials and methods.....................................................................................			424

14

	2.1. Bacterial strain...........................................................................................				424
	2.2. Inoculum preparation and cultural conditions...........................................				424
	2.3. Recovery and			purification of the exopolysaccharide.................................	424
	2.4. Rheological analysis..................................................................................				424
3. Results and discussion......................................................................................					425
	3.1. Fermentation data......................................................................................				425
	3.2. Downstream			processing.............................................................................	425
	3.3. Gelling properties......................................................................................				426
4.	Conclusion........................................................................................................				427
Acknowledgements..............................................................................................					428
References............................................................................................................					428
Initiation, growth and immobilisation of cell cultures of Taxus spp. for paclitaxel
production.................................................................................................................					429
Chi Wai Tang, Eman Zalat and Ferda Mavituna......................................................					429
Summary...............................................................................................................					429
1.	Introduction.....................................................................................................				429
	1.1. Pharmaceuticals from plants......................................................................				429
	1.2.	Plant	Biotechnology.................................................................................		431
	1.3.	Antitumor compounds from Taxus spp....................................................			431
2.	Materials		and methods.....................................................................................		432
	2.1. Plant material and chemicals .....................................................................				432
	2.2. Culture initiation and maintenance............................................................				433
		2.2.1. Callus initiation..............................................................................			433
		2.2.2. Suspension culture..........................................................................			433
	2.3.	Cell	immobilisation ..................................................................................		434
	2.4.	Bioreactors................................................................................................			435
	2.5.	Analytical measurements..........................................................................			435
		2.5.1. Growth............................................................................................			435
		2.5.2. Viability..........................................................................................			435
		2.5.3. Sugar		analysis.................................................................................	436
		2.5.4. Taxaneanalysis...............................................................................			436
3.	Results and discussion.....................................................................................				436
	3.1. Callus initiation..........................................................................................				436
		3.1.1 Effect of media and plant growth regulators ..................................			436
		3.1.2	Effect of light on callus initiation...................................................		437
		3.1.3	Effect of plant species and explant type on callus initiation...........		437
		3.1.4	Effect of coconut water on callus initiation....................................		438
	3.2. Callus growth and maintenance.................................................................				438
		3.2.1	Effect of explant type .....................................................................		438
		3.2.2	Effect of light on callus growth ......................................................		439
	3.3. Suspension cultures...................................................................................				440
	3.4.Immobilisation			...........................................................................................	442
	3.5. Growth in bioreactors................................................................................				443
	3.6.	Paclitaxel production................................................................................			443
4.	Conclusions.....................................................................................................				444

15

Acknowledgement................................................................................................				444
References............................................................................................................				445
Effective biofuel production by an intelligent bioreactor.........................................				449
Hideki Fukuda, Akihiko Kondo, and Hideo Noda...................................................				449
Abstract.................................................................................................................				449
1. Introduction......................................................................................................				449
2. Key technologies for biofuel production..........................................................				450
2.1	Intelligent bioreactor using immobilized yeast cells ..................................			450
2.2	Immobilizing proteins on the surface of yeast cells....................................			451
3. Outline of ongoing research .............................................................................				452
3.1	Development of highly functional yeast cells.............................................			452
3.2	Development of an intelligent bioreactor system ......................................			453
3.3	Development of an optimal control system		i n conjunction with efficient
monitoring........................................................................................................				453
4. Conclusion........................................................................................................				454
References............................................................................................................				454
PART IX Patents and Licenses..................................................................................				457
Translating European biotech into US patents do’s,			don’ts, & costs.......................	459
Thomas M. Saunders................................................................................................				459
Introduction..........................................................................................................				459
1. Five important patent differences between Europe and the US........................				459
1.1. One-year us grace period from first use or sale.........................................				459
1.2.	Grace period (continued): tempus fugit.....................................................			459
1.3.	Duty	of disclosure.....................................................................................		460
1.4. Computer algorithms now patentable.......................................................				460
1.5.	First to invent versus first to file...............................................................			460
2. Basic patent game theory..................................................................................				460
3. Invention germination ......................................................................................				461
3.1. Invention disclosure forms ........................................................................				461
	3.1.1. Short forms only.............................................................................			461
	3.1.2. Who gets the forms?.......................................................................			461
3.2.	No forms....................................................................................................			462
4. Invention selection............................................................................................				462
4.1. IP focus		......................................................................................................		462
4.2. The learning curve.....................................................................................				463
4.3.The star wars test.....................................................................................				463
4.4. Is there a market?.......................................................................................				463
	4.4.1. Money.............................................................................................			463
	4.4.2. Perceived need................................................................................			463
5. Points of decision..............................................................................................				464
5.1.	Patent	committee.......................................................................................		464
5.2.	Ratings.......................................................................................................			464
	5.5.1.	A = File immediately.....................................................................		464
	5.5.2.	B = Review in six months..............................................................		464

16

	5.5.3. C = Indefinite hold .......................................................................	465
	5.2.3. Hard financial facts.......................................................................	465
6. Its just business................................................................................................		466
6.1. What is it really worth to develop and maintain ...your patent portfolio? 466
6.2. The one true answer..................................................................................		466
6.3.	Nice package.............................................................................................	468
	6.3.1. Human pharmaceuticals.................................................................	468
	6.3.2. Windage.........................................................................................	468
	6.3.3. Exclusivity.....................................................................................	468
	6.3.4. Morepatentstrategy, packages II.................................................	469
7. Filing a patent application ...............................................................................		469
7.1. Input from the inventor..............................................................................		469
7.2. More input from the inventor....................................................................		470
7.3. Compile all relevant art.............................................................................		471
	7.3.1. Why................................................................................................	471
	7.3.2. Yes, everything...............................................................................	471
	7.3.3. When...............................................................................................	472
	7.3.4. Searches..........................................................................................	472
8. The prior of prior art.........................................................................................		472
8.1. In the US...................................................................................................		472
8.2. Non-US......................................................................................................		473
9. Making U.S. filings/incurring...........................................................................		473
9.1. Application preparation.............................................................................		473
	9.1.1. New applications............................................................................	473
	9.1.2. Provisional applications..................................................................	474
	9.1.3. Later applications............................................................................	474
9.2.	Patent prosecution......................................................................................	475
	9.2.1. First matters....................................................................................	475
	9.2.2. Information disclosure statement (cont.)........................................	475
	9.2.3. First office action............................................................................	475
	9.2.4. First response..................................................................................	475
	9.2.3. Further office actions and responses...............................................	477
9.4. Maintaining pending applications..............................................................		477
	9.4.1. Re-examination...............................................................................	477
	9.4.2. Reissue............................................................................................	478
	9.4.3. Pendency.........................................................................................	478
10.	Timing............................................................................................................	479
10.1. U.S. application filing..............................................................................		479
10.2. On-sale bar to patentability......................................................................		479
	10.2.1. Out source disaster........................................................................	479
	10.2.2. Concept offered for sale................................................................	480
10.3. International application filing ................................................................		481
	10.3.1. Priority dates.................................................................................	481
	10.3.2. Internationally file the CIP ...........................................................	481
	10.3.3. Filing costs....................................................................................	481

17