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Engineering and Manufacturing for Biotechnology - Marcel Hofman & Philippe Thonart

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ENGINEERING AND MANUFACTURING FOR BIOTECHNOLOGY

VOLUME 4

FOCUS ON BIOTECHNOLOGY

Volume 4

Series Editors

MARCEL HOFMAN

Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

JOZEF ANNÉ

Rega Institute, University ofLeuven, Belgium

Volume Editors

MARCEL HOFMAN

Société de Chimie Industrielle,

Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

PHILIPPE THONART

Faculté Univ. des Sciences Agronomiques Gembloux,

Centre Wallon de Biologie Industrielle, Belgium

Colophon

Focus on Biotechnology is an open-ended series of reference volumes produced by Kluwer Academic Publishers BV in co-operation with the Branche Belge de la Société de Chimie Industrielle a.s.b.l.

The initiative has been taken in conjunction with the Ninth European Congress on Biotechnology. ECB9 has been supported by the Commission of the European Communities, the General Directorate for Technology, Research and Energy of the Wallonia Region, Belgium and J Chabert, Minister for Economy of the Brussels Capital Region.

The series is edited by Marcel Hofman, Centre for Veterinary and Agrochemical Research, Tervuren, and Jozef Anné, Rega Institute, University of Leuven, Belgium.

Engineering and Manufacturing

for Biotechnology

Volume 4

Edited by

MARCEL HOFMAN

Société de Chimie Industrielle,

Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

and

PHILIPPE THONART

Faculté Univ. des Sciences Agronomiques Gembloux,

Centre Wallon de Biologie Industrielle, Belgium

KLUWER ACADEMIC PUBLISHERS

NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

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3ULQW2001 Kluwer Academic Publishers

Dordrecht

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EDITORS PREFACE

Early integration of process engineering and biological research is the key for success in industrial biotechnology. This is true as well when a selected wild-type organism is put to work as when an organism is engineered to purpose.

Focus on Biotechnology reports on biotechnology achievements in the recent past, but also provides a strategic view on the evolution in the next decade. The present volume "Engineering and Manufacturing for Biotechnology" took advantage of the European Congress on Biotechnology (Brussels, Belgium, July 11-15, 1999) : by topics handled and by expertise of the contributors the engineering science symposia of this congress offered just what was needed to cover this important topic.

The editors have solicited the authors of a number of outstanding contributions to illustrate the intimate interaction between productive organism and the numerous processing steps running from the initial inoculation to the packaged product. Upstream processing of the feed streams, selection of medium components, product harvesting, downstream processing and product conditioning are just a few major steps. Each step imposes a number of important choices. Every choice is to be balanced against time to market, profitability, safety and ecology.

It should be readily apparent from this volume that the development of a truly effective biotechnology process requires a broad command of leading-edge engineering science, a spark of genius, and last but not least much hard work. That is why the editors wish to express their gratitude to all the authors of this volume, for finding the time after busy hours at the lab, on the pilot floor or in the production plant to share their experience and enthusiasm.

A final word of esteem is due to all those that through their devoted and outstanding secretarial skills have made the edition of this volume possible.

Marcel Hofman

Philippe Thonart

TABLE OF CONTENTS
Editors Preface................................................................................................................				1
TABLE OF CONTENTS..............................................................................................				3
PART I Upstream processes and fermentation..........................................................				19
Pretreatment processes of molasses for the utilization in fermentation processes......				21
Güzide Çalik, Meliha Berk, Fatma Gül Boyaci, Pinar Çalik, Serpil Takaç, Tunçer H.
Özdamar.....................................................................................................................				21
Abstract...................................................................................................................				21
1.	Introduction........................................................................................................			21
2.	Materials	and	methods.......................................................................................	22
	2.1.PretreatmentProcesses(PP)........................................................................			22
	2.2. Bioprocesses................................................................................................			23
	2.2.1. Glutamic acid fermentation ..............................................................			23
	2.2.2. Alkaline protease fermentation.........................................................			23
3.	Results and discussions......................................................................................			24
	3.1. Effect of PP on metal ion concentrations....................................................			24
	3.2. Effect of PP on glutamic acid fermentation.................................................			25
	3.3. Effect of PP on serine alkaline protease fermentation.................................			26
4.	Conclusions.......................................................................................................			27
Acknowledgements................................................................................................				28
References..............................................................................................................				28
Lactic acid fermentation of hemicellulose liquors and their activated carbon
pretreatments..............................................................................................................				29
Perttunen, J., Myllykoski, L. and Keiski, R.L............................................................				29
Summary.................................................................................................................				29
1. Introduction........................................................................................................				29
2. Materials and methods........................................................................................				30
3. Results and discussion........................................................................................				31
4. Conclusions........................................................................................................				36
5.	References..........................................................................................................			37
Enzymic solubilisation of proteins from tropical tuna using alcalase and some
biological properties of the hydrolysates....................................................................				39
Fabienne Guerard, Rozenn Ravallec-Ple, Denis De La Broise, Adrien Binet and
Laurent Dufosse..........................................................................................................				39
Summary.................................................................................................................				39
1. Introduction........................................................................................................				39
2.	Materials	and	methods........................................................................................	41
	2.1.Materials......................................................................................................			41
	2.2. Preparation of the hydrolysate.....................................................................			41

	2.3. Determination of the degree of hydrolysis..................................................				41
	2.4. Size Exclusion Chromatography (SEC)......................................................				42
	2.5. Mitogenic			activity........................................................................................	42
	2.6. Gastrin radioimmunoassay (RIA)................................................................				42
	2.7. Microbial cultivations..................................................................................				43
		2.7.1. Microorganisms and cultivation media.............................................			43
		2.7.2. Growth kinetics, modelling the growth curve..................................			43
3.	Results and discussion........................................................................................				44
	3.1. Effect of the enzyme concentration on the degree of hydrolysis.................				44
	3.2. Study of chromatographic profiles ..............................................................				45
	3.3.	Biological		activities of tuna hydrolysates....................................................	47
		3.3.1.	Mitogenic activity.............................................................................		47
		3.3.2. Gastrin radioimmunoassay.............................................................			47
		3.3.3. Nitrogenous substrate for microbial growth.....................................			48
4. Conclusion..........................................................................................................					49
References..............................................................................................................					50
Acknowledgements................................................................................................					50
Influence of the experimental conditions on the hydrolysis process in fish
hydrolysates................................................................................................................					51
Rozenn Ravallec-Ple, Laura Gilmartin, Alain Van Wormhoudt and Yves Le Gal....					51
Summary.................................................................................................................					51
1. Introduction........................................................................................................					51
2. Materials and methods........................................................................................					52
	2.1.	Substrate......................................................................................................			52
	2.2.Enzymes......................................................................................................				52
	2.3.	Hydrolysis....................................................................................................			52
	2.4.	Statistical		analysis........................................................................................	53
	2.5. FPLC		chromatography ................................................................................		54
3.	Results and discussion........................................................................................				54
	3.1. Effect		of the enzyme on the degree of hydrolysis .......................................		54
	3.2.	Optimization of processing conditions using Alcalase®.............................			55
	3.2.	Chromatographic profiles............................................................................			56
4.	Conclusion..........................................................................................................				57
References..............................................................................................................					58
PART	II	Process	Modelling .........................................................................................		59
Mathematical modelling of microbial processes - Motivation and means.................					61
Teit Agger and Jens Nielsen.......................................................................................					61
Abstract...................................................................................................................					61
1. Introduction........................................................................................................					61
2.Motivation..........................................................................................................					62
3. Means - General modelling frameworks ............................................................					64
4.	Selected applications..........................................................................................				70
5.	Future prospects..................................................................................................				72
References..............................................................................................................					73

Nomenclature.........................................................................................................				74
Macroscopic modelling of bioprocesses with			a view to engineering applications	..... 77
Ph. Bogaerts and R. Hanus.........................................................................................				77
Abstract...................................................................................................................				77
1.	Introduction........................................................................................................			77
2. Macroscopic reaction network and associated mass balances............................				80
	2.1. first	sufficient condition of BIBS	stability of (9).........................................	83
	2.2. second sufficient condition of BIBS stability of (9)....................................			83
3.	Kinetic model structure ......................................................................................			84
	3.1. Motivations for a new kinetic model structure............................................			84
	3.2. General kinetic model structure...................................................................			85
4. Parameter identification......................................................................................				87
	4.1. Motivations for a systematic procedure.......................................................			87
	4.2. Systematic procedure for the	parameter identification................................		89
		of the	pseudo-stoichiometric coefficients
		(independently of the kinetic coefficients) .................................................		89
		4.2.2. Second step: first estimation	of the kinetic coefficients ...................	93
		4.2.3. Third step: final estimation	of the kinetic coefficients (and of some
				94
	4.3. Necessary conditions for reaction		scheme validation..................................	97
5. Application on simulated bacteria cultures.........................................................				99
6.	Conclusions and			107
References............................................................................................................				108
A model discrimination approach for data analysis and experimental design..........				111
R. Takors, D. Weuster-Botz, W. Wiechert, C. Wandrey..........................................				111
1.	Introduction......................................................................................................			111
2. Theoretical concept...........................................................................................				113
	2.1. Model discrimination.................................................................................			113
	2.2. Model discriminating design .....................................................................			114
		2.2.1. Extended entropy approach ............................................................		114
		predictive design .................................................................		115
3.	Material			116
	3.1.			116
	3.2.			117
	3.3. Numerical and programming tools............................................................			117
4.	Results and discussion......................................................................................			117
	4.1	Model discrimination of steady - state fermentations ...................................		117
	4.2 Batch and fed-batch fermentation modelling..............................................			120
	4.3 Model discriminating design with Zymomonas mobilis .............................			122
5. Conclusions......................................................................................................				126
References............................................................................................................				127
Model based sequential experimental design for bioprocess optimisation - An
overview				129
Ralph Berkholz and Reinhard Guthke......................................................................				129
Summary...............................................................................................................				129

1.	Bioprocess modelling for experimental design procedures..............................	130
	1.1. Bioprocess modelling................................................................................	130
	1.2. Transparency.............................................................................................	131
	1.3. Restricted validity of bioprocess models...................................................	132
	1.4. Identifiability.............................................................................................	132
	1.5. Knowledge and data based hybrid bioprocess modelling..........................	132
2. Direct experimental design method..................................................................		133
3. Indirect experimental design method................................................................		134
4. optimal experimental design method............................................................		137
5.	Experimental Example.....................................................................................	138
References............................................................................................................		140

Metabolic flux modelling as a tool to analyse the behavior of a genetically modified

strain of Saccharomyces cerevisiae..........................................................................			143
Urrieta-Saltijeral J.M., Dussap C.G., Pons A., Creuly C. and Gros J.B...................			143
Abstract.................................................................................................................			143
1.	Introduction......................................................................................................		143
2.	Materials and methods......................................................................................		145
	2.1. Microorganisms and growth conditions ....................................................		145
	2.2.	Analysis of metabolites..............................................................................	145
	2.3. Flux estimation and statistical analysis......................................................		145
3. Results and discussion ......................................................................................			148
	3.1. Growth yields determination .....................................................................		148
	3.2. Selection of a reliable metabolic network..................................................		149
	3.3.	Discussion..................................................................................................	150
	3.4. Thermodynamic analysis...........................................................................		151
4.	Conclusion and perspectives.............................................................................		154
References............................................................................................................			155
Metabolic investigation of an anaerobic cellulolytic bacterium : Fibrobacter
succinogenes.............................................................................................................			157
C. Creuly, A. Pons, and C.G. Dussap.......................................................................			157
Abstract.................................................................................................................			157
1. Introduction......................................................................................................			157
2.	Material and method.........................................................................................		158
	2.1. Strain and cultivation.................................................................................		158
	2.2. Experimental design..................................................................................		158
	2.3.	Metabolites assays.....................................................................................	158
	2.4. Flux estimation and statistical analysis......................................................		159
3.	Results and discussion......................................................................................		161
	3.1. Metabolic network.....................................................................................		161
	3.2.Fluxcalculation.........................................................................................		164
	3.3. Validation..................................................................................................		164
4. Conclusions and perspectives...........................................................................			165
Acknowledgement................................................................................................			166
References............................................................................................................			166

PART III Integrated Processes..................................................................................			169
Crossflow ultrafiltration of Bacillus licheniformis fermentation medium to separate
protease enzymes......................................................................................................			171
Serpil Takaç, Sema Elmas, Pinar Çalik, Tunçer H. Özdamar..................................			171
Abstract.................................................................................................................			171
1.	Introduction......................................................................................................		171
2.	Materials and methods......................................................................................		173
	2.1. Experimental runs......................................................................................		173
	2.2. Analyses.....................................................................................................		173
	2.3. Cake resistance model...............................................................................		174
3. Results and Discussion .....................................................................................			174
	3.1. Effect of initial enzyme concentration.......................................................		174
	3.2. Effects of recirculation velocity and transmembrane pressure..................		176
	3.3. The recovered activity of SAP enzyme after separation............................		177
4.	Conclusions......................................................................................................		177
Acknowledgements..............................................................................................			178
References............................................................................................................			178
Nomenclature .......................................................................................................			179
PART IV Monitoring and Control............................................................................			181
Evaluating		a during fermentation using many methods simultaneously..............	183
K. Pouliot, J. Thibault, A. Garnier, G. Acuna Leiva ................................................			183
Abstract.................................................................................................................			183
1. Introduction......................................................................................................			183
2. Materials and methods......................................................................................			185
	2.1. Organism and medium...............................................................................		185
	2.2. Experimental system..................................................................................		185
	2.3. Review of the methods for measuring a during the course of fermentation
			186
	2.3.1. Dynamic method.............................................................................		187
	2.3.2. Steady-state methods.......................................................................		188
	2.4. Data reconciliation method........................................................................		189
	2.4.1. Weighting factors............................................................................		192
3.	Results and discussion......................................................................................		193
4. Conclusion........................................................................................................			199
Acknowledgements..............................................................................................			199
References............................................................................................................			200
Nomenclature .......................................................................................................			200
	Greek	letters......................................................................................................	201
	Subscripts..........................................................................................................		201
	Symbols............................................................................................................		201
Respiration quotient: estimation during batch cultivation in bicarbonate buffered
media........................................................................................................................			203
Ronald Neeleman .....................................................................................................			203
Abstract.................................................................................................................			203

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