BIOCHEMICAL ENGINEERING
AND BIOTECHNOLOGY
PRELIMS.qxd 10/27/2006 10:54 AM Page i
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BIOCHEMICAL
ENGINEERING AND
BIOTECHNOLOGY
GHASEM D. NAJAFPOUR
Professor of Chemical Engineering
Noshirvani Institute of Te...
Elsevier
Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands
The Boulevard, Langford Lane, Kidlington, Oxford OX5 ...
Preface
In the new millennium, extensive application of bioprocesses has created an environment
for many engineers to expa...
This book was prepared with the encouragement of distinguished Professor Gaddy, who
made me proud to be his student. I als...
vii
Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
viii TABLE OF CONTENTS
3.3 Effect of agitation on dissolved oxygen . . . . . . . . . . . . . . . . . . . . . . . 23
3.4 Ai...
TABLE OF CONTENTS ix
Chapter 5. Growth Kinetics
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....
x TABLE OF CONTENTS
6.4 Stirred tank bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1...
TABLE OF CONTENTS xi
8.5 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
xii TABLE OF CONTENTS
10.3 Benefits from bioethanol fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253...
TABLE OF CONTENTS xiii
12.2.1 Microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
Chapter 15. Sterilisation
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
16.13.2.1 Preparation of PVA solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
16.13.2.2 Prepar...
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CHAPTER 1
Industrial Microbiology
1.1 INTRODUCTION
Microorganisms have been identified and exploited for more than a centu...
which are formed during the cells’ exponential growth phase. In some bioprocesses, yeast
or fungi are used to produce adva...
INDUSTRIAL MICROBIOLOGY 3
of bread. The degradation of carbohydrates by microorganisms is followed by glycolytic
or Embden...
make use of large-scale advanced technology. However, application of the bioprocess in
large-scale control of microorganis...
INDUSTRIAL MICROBIOLOGY 5
1.4 BIOPROCESS PRODUCTS
Major bioprocess products are in the area of chemicals, pharmaceuticals,...
1.4.2 Cell Products
Products are produced by cells, with the aid of enzymes and metabolites known as cell
products. These ...
INDUSTRIAL MICROBIOLOGY 7
to use whey to make some more useful product. Whey can be converted from being a waste
product t...
The word ‘wine’ is derived from the French term ‘vinaigre’ meaning ‘sour wine’. It is
prepared by allowing a wine to get s...
supplementary food with bread and other foodstuffs. This amino acid is a biological product
which is also used as a food a...
• The major steps in the commercial production of penicillin are:
(1) Preparation of inoculum.
(2) Preparation and sterili...
INDUSTRIALMICROBIOLOGY11
Sampling port
Mixgas(10%CO2,15%Ar,
20%H2,&55%CO
Nitrogen
gas
Fresh
media
Micro-DCU-System
Tempera...
12 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY
syrup from sucrose, which is partly hydrolysed by this enzyme. The proteolyti...
4. Ghose, T.K., “Bioprocess Computation in Biotechnology”, vol. 1. Ellis Horwood Series in Biochemistry and
Biotechnology,...
14
CHAPTER 2
Dissolved Oxygen Measurement and Mixing
2.1 INTRODUCTION
In biochemical engineering processes, measurement of...
DISSOLVED OXYGEN MEASUREMENT AND MIXING 15
electrodes and polarographic electrodes. In both probes, there are membranes th...
16 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY
following sections. If the above experiments are conducted, they may lead us ...
DISSOLVED OXYGEN MEASUREMENT AND MIXING 17
2.3.2 Seed Culture
How do we start the real experiment? A 100ml seed culture is...
18 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY
2.5 OXYGEN TRANSFER RATE (OTR)
Once batch mode studies are completed and the ...
DISSOLVED OXYGEN MEASUREMENT AND MIXING 19
2.6 RESPIRATION QUOTIENT (RQ)
Measurements of inlet and outlet gas compositions...
20 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY
Example 2.1: Calculate Cell Density in an Aerobic Culture
A strain of Azotoba...
DISSOLVED OXYGEN MEASUREMENT AND MIXING 21
Let us assume the solubility of oxygen does not affect on CAL
*
or kLa, the fac...
22
CHAPTER 3
Gas and Liquid System (Aeration and Agitation)
3.1 INTRODUCTION
In the biochemical engineering profession, th...
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Najafpour Biochemical engg and Biotechnology

A textbook for topics of biotechnology.
Published on: Mar 3, 2016
Published in: Engineering      
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  • 1. BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY PRELIMS.qxd 10/27/2006 10:54 AM Page i
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  • 3. BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY GHASEM D. NAJAFPOUR Professor of Chemical Engineering Noshirvani Institute of Technology University of Mazandaran Babol, Iran Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo PRELIMS.qxd 10/27/2006 10:54 AM Page iii
  • 4. Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2007 Copyright © 2007 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier.com. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-52845-2 ISBN-10: 0-444-52845-8 Printed and bound in The Netherlands 07 08 09 10 11 10 9 8 7 6 5 4 3 2 1 For information on all Elsevier publications visit our website at books.elsevier.com PRELIMS.qxd 10/27/2006 10:54 AM Page iv
  • 5. Preface In the new millennium, extensive application of bioprocesses has created an environment for many engineers to expand knowledge of and interest in biotechnology. Microorganisms produce alcohols and acetone, which are used in industrial processes. Knowledge related to industrial microbiology has been revolutionised by the ability of genetically engineered cells to make many new products. Genetic engineering and gene mounting has been devel- oped in the enhancement of industrial fermentation. Finally, application of biochemical engineering in biotechnology has become a new way of making commercial products. This book demonstrates the application of biological sciences in engineering with theo- retical and practical aspects. The seventeen chapters give more understanding of the know- ledge related to the specified field, with more practical approaches and related case studies with original research data. It is a book for students to follow the sequential lectures with detailed explanations, and solves the actual problems in the related chapters. There are many graphs that present actual experimental data, and figures and tables, along with sufficient explanations. It is a good book for those who are interested in more advanced research in the field of biotechnology, and a true guide for beginners to practise and establish advanced research in this field. The book is specifically targeted to serve as a useful text for college and university students; it is mostly recommended for undergraduate courses in one or two semesters. It will also prove very useful for research institutes and postgraduates involved in practical research in biochemical engineering and biotechnology. This book has suitable biological science applications in biochemical engineering and the knowledge related to those biological processes. The book is unique, with practical approaches in the industrial field. I have tried to prepare a suitable textbook by using a direct approach that should be very useful for students in following the many case studies. It is unique in having solved problems, examples and demonstrations of detailed experi- ments, with simple design equations and required calculations. Several authors have con- tributed to enrich the case studies. During the years of my graduate studies in the USA at the University of Oklahoma and the University of Arkansas, the late Professor Mark Townsend gave me much knowledge and assisted me in my academic achievements. I have also had the opportunity to learn many things from different people, including Professor Starling, Professor C.M. Sliepcevich and Professor S. Ellaison at the University of Oklahoma. Also, it is a privilege to acknowledge Professor J.L. Gaddy and Professor Ed Clausen, who assisted me at the University ofArkansas. I am very thankful for their courage and the guidance they have given me. My vision in research and my success are due to these two great scholars at the University of Arkansas: they are always remembered. v Preface.qxd 10/27/2006 10:51 AM Page v
  • 6. This book was prepared with the encouragement of distinguished Professor Gaddy, who made me proud to be his student. I also acknowledge my Ph.D. students at the University of Science Malaysia: Habibouallah Younesi and Aliakbar Zinatizadeh, who have assisted me in drawing most of the figures. I am very thankful to my colleagues who have contributed to some parts of the chapters: Dr M. Jahanshahi, from the University of Mazandaran, Iran, and Dr Nidal Hilal from the University of Nottingham, UK. Also special thanks go to Dr H. Younesi, Dr W.S. Long, Associate Professor A.H. Kamaruddin, Professor S. Bhatia, Professor A.R. Mohamed and Associate Professor A.L. Ahmad for their contribution of case studies. I acknowledge my friends in Malaysia: Dr Long Wei Sing, Associate Professor Azlina Harun Kamaruddin and Professor Omar Kadiar, School of Chemical Engineering and School of Industrial Technology, the Universiti Sains Malaysia, for editing part of this book. I also acknowledge my colleague Dr Mohammad Ali Rupani, who has edited part of the book. Nor should I forget the person who has accelerated this work and given lots of encouragement: Deirdre Clark at Elsevier. G. D. NAJAFPOUR Professor of Chemical Engineering University of Mazandaran, Babol, Iran vi PREFACE Preface.qxd 10/27/2006 10:51 AM Page vi
  • 7. vii Table of Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Chapter 1. Industrial Microbio1ogy 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Process fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Application of fermentation processes . . . . . . . . . . . . . . . . . . . . . . . . 4 1.4 Bioprocess products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.4.1 Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.4.2 Cell products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.4.3 Modified compounds (biotransformation) . . . . . . . . . . . . . . . . . . . . . 6 1.5 Production of lactic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.6 Production of vinegar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.7 Production of amino acids (lysine and glutamic acid) and insulin . . . 8 1.7.1 Stepwise amino acid production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.7.2 Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.8 Antibiotics, production of penicillin . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.9 Production of enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.10 Production of baker’s yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Chapter 2. Dissolved Oxygen Measurement and Mixing 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.2 Measurement of dissolved oxygen concentrations . . . . . . . . . . . . . . . 14 2.3 Batch and continuous fermentation for production of SCP . . . . . . . . 15 2.3.1 Analytical methods for measuring protein content of baker’s yeast (SCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.3.2 Seed culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.4 Batch experiment for production of baker’s yeast . . . . . . . . . . . . . . . 17 2.5 Oxygen transfer rate (OTR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.6 Respiration quotient (RQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.7 Agitation rate studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.8 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Chapter 3. Gas and Liquid System (Aeration and Agitation) 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.2 Aeration and agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 CONTENTS.qxd 10/27/2006 10:52 AM Page vii
  • 8. viii TABLE OF CONTENTS 3.3 Effect of agitation on dissolved oxygen . . . . . . . . . . . . . . . . . . . . . . . 23 3.4 Air sparger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.5 Oxygen transfer rate in a fermenter . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.5.1 Mass transfer in a gas–liquid system . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.6 Mass transfer coefficients for stirred tanks . . . . . . . . . . . . . . . . . . . . . 26 3.7 Gas hold-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 3.8 Agitated system and mixing phenomena . . . . . . . . . . . . . . . . . . . . . . 28 3.9 Characterisation of agitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 3.10 Types of agitator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.11 Gas–liquid phase mass transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 3.11.1 Oxygen transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 3.11.2 Diameter of gas bubble formed D0 . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 3.12 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.13 Case study: oxygen transfer rate model in an aerated tank for pharmaceutical wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 3.13.2 Material and method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 3.13.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.13.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 3.13.5 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 3.14 Case study: fuel and chemical production from the water gas shift reaction by fermentation processes . . . . . . . . . . . . . . . . . . . . 50 3.14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.14.2 Kinetics of growth in a batch bioreactor . . . . . . . . . . . . . . . . . . . . . . . 51 3.14.3 Effect of substrate concentration on microbial growth . . . . . . . . . . . . 55 3.14.4 Mass transfer phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 3.14.5 Kinetic of water gas shift reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.14.6 Growth kinetics of CO substrate on Clostridium ljungdahlii . . . . . . . 65 3.14.7 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 3.14.8 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Chapter 4. Fermentation Process Control 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 4.2 Bioreactor controlling probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 4.3 Characteristics of bioreactor sensors . . . . . . . . . . . . . . . . . . . . . . . . . . 72 4.4 Temperature measurement and control . . . . . . . . . . . . . . . . . . . . . . . . 72 4.5 DO measurement and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 4.6 pH/Redox measurement and control . . . . . . . . . . . . . . . . . . . . . . . . . . 76 4.7 Detection and prevention of the foam . . . . . . . . . . . . . . . . . . . . . . . . . 77 4.8 Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.9 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 CONTENTS.qxd 10/27/2006 10:52 AM Page viii
  • 9. TABLE OF CONTENTS ix Chapter 5. Growth Kinetics 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 5.2 Cell growth in batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 5.3 Growth phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 5.4 Kinetics of batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 5.5 Growth kinetics for continuous culture . . . . . . . . . . . . . . . . . . . . . . . . 84 5.6 Material balance for CSTR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 5.6.1 Rate of product formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 5.6.2 Continuous culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 5.6.3 Disadvantages of batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 5.6.4 Advantages of continuous culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 5.6.5 Growth kinetics, biomass and product yields, YX/S and YP/S . . . . . . . . 91 5.6.6 Biomass balances (cells) in a bioreactor . . . . . . . . . . . . . . . . . . . . . . . 93 5.6.7 Material balance in terms of substrate in a chemostat . . . . . . . . . . . . 94 5.6.8 Modified chemostat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 5.6.9 Fed batch culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 5.7 Enzyme reaction kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 5.7.1 Mechanisms of single enzyme with dual substrates . . . . . . . . . . . . . . 99 5.7.2 Kinetics of reversible reactions with dual substrate reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 5.7.3 Reaction mechanism with competitive inhibition . . . . . . . . . . . . . . . . 106 5.7.4 Non-competitive inhibition rate model . . . . . . . . . . . . . . . . . . . . . . . . 107 5.8 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 5.9 Case study: enzyme kinetic models for resolution of racemic ibuprofen esters in a membrane reactor . . . . . . . . . . . . . . . . . . . . . . . 130 5.9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 5.9.2 Enzyme kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 5.9.2.1 Substrate and product inhibitions analyses . . . . . . . . . . . . . . . . . . . . . 131 5.9.2.2 Substrate inhibition study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 5.9.2.3 Product inhibition study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 5.9.3 Enzyme kinetics for rapid equilibrium system (quasi-equilibrium) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.9.4 Derivation of enzymatic rate equation from rapid Equilibrium assumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 5.9.5 Verification of kinetic mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Chapter 6. Bioreactor Design 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 6.2 Background to bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 6.3 Type of bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 6.3.1 Airlift bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 6.3.2 Airlift pressure cycle bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.3.3 Loop bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 CONTENTS.qxd 10/27/2006 10:52 AM Page ix
  • 10. x TABLE OF CONTENTS 6.4 Stirred tank bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.5 Bubble column fermenter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 6.6 Airlift bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 6.7 Heat transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 6.8 Design equations for CSTR fermenter . . . . . . . . . . . . . . . . . . . . . . . . 154 6.8.1 Monod model for a chemostat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 6.9 Temperature effect on rate constant . . . . . . . . . . . . . . . . . . . . . . . . . . 158 6.10 Scale-up of stirred-tank bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 6.11 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Chapter 7. Downstream Processing 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 7.2 Downstream processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 7.3 Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 7.3.1 Theory of filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 7.4 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 7.4.1 Theory of centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 7.5 Sedimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 7.6 Flotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 7.7 Emerging technology for cell recovery . . . . . . . . . . . . . . . . . . . . . . . . 180 7.8 Cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 7.9 Solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 7.9.1 Product recovery by liquid–liquid extraction . . . . . . . . . . . . . . . . . . . 183 7.9.2 Continuous extraction column process, rotating disk contactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 7.10 Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 7.10.1 Ion-exchange adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 7.10.2 Langmuir isotherm adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 7.10.3 Freundlich isotherm adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 7.10.4 Fixed-bed adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 7.11 Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 7.11.1 Principle of chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 7.12 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 Chapter 8. Immobilization of Microbial Cells for the Production of Organic Acid and Ethanol 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 8.2 Immobilised microbial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 8.2.1 Carrier binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 8.2.2 Entrapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 8.2.3 Cross-linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 8.2.4 Advantages and disadvantages of immobilised cells . . . . . . . . . . . . . 202 8.3 Immobilised cell reactor experiments . . . . . . . . . . . . . . . . . . . . . . . . . 202 8.4 ICR rate model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 CONTENTS.qxd 10/27/2006 10:52 AM Page x
  • 11. TABLE OF CONTENTS xi 8.5 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 8.6 Case study: ethanol fermentation in an immobilised cell reactor using Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . 206 8.6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 8.6.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 8.6.2.1 Experimental reactor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 8.6.2.2 Determination of glucose concentration . . . . . . . . . . . . . . . . . . . . . . . 210 8.6.2.3 Detection of ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 8.6.2.4 Yeast cell dry weight and optical density . . . . . . . . . . . . . . . . . . . . . . 211 8.6.2.5 Electronic microscopic scanning of immobilised cells . . . . . . . . . . . . 211 8.6.2.6 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 8.6.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 8.6.3.1 Evaluation of immobilised cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 8.6.3.2 Batch fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 8.6.3.3 Relative activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 8.6.3.4 Reactor set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 8.6.3.5 Effect of high concentration of substrate on immobilised cells . . . . . 219 8.6.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 8.6.5 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 8.6.6 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 8.7 Fundamentals of immobilisation technology, and mathematical model for ICR performance . . . . . . . . . . . . . . . . . . . . . 222 8.7.1 Immobilisation of microorganisms by covalent bonds . . . . . . . . . . . . 222 8.7.2 Oxygen transfer to immobilised microorganisms . . . . . . . . . . . . . . . . 223 8.7.3 Substrate transfer to immobilised microorganisms . . . . . . . . . . . . . . . 223 8.7.4 Growth and colony formation of immobilised microorganisms . . . . . 224 8.7.5 Immobilised systems for ethanol production . . . . . . . . . . . . . . . . . . . 227 Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Chapter 9. Material and Elemental Balance 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 9.2 Growth of stoichiometry and elemental balances . . . . . . . . . . . . . . . . 229 9.3 Energy balance for continuous ethanol fermentation . . . . . . . . . . . . . 230 9.4 Mass balance for production of penicillin . . . . . . . . . . . . . . . . . . . . . . 231 9.5 Conservation of mass principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 9.5.1 Acetic acid fermentation process . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 9.5.2 Xanthan gum production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 9.5.3 Stoichiometric coefficient for cell growth . . . . . . . . . . . . . . . . . . . . . . 243 9.6 Embden–Meyerhoff–Parnas pathway . . . . . . . . . . . . . . . . . . . . . . . . . 244 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Chapter 10. Application of Fermentation Processes 10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252 10.2 Production of ethanol by fermentation . . . . . . . . . . . . . . . . . . . . . . . . 252 CONTENTS.qxd 10/27/2006 10:52 AM Page xi
  • 12. xii TABLE OF CONTENTS 10.3 Benefits from bioethanol fuel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 10.4 Stoichiometry of biochemical reaction . . . . . . . . . . . . . . . . . . . . . . . . 253 10.5 Optical cell density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 10.6 Kinetics of growth and product formation . . . . . . . . . . . . . . . . . . . . . 254 10.7 Preparation of the stock culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254 10.8 Inoculum preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 10.9 Seed culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 10.10 Analytical method for sugar analysis . . . . . . . . . . . . . . . . . . . . . . . . . 257 10.10.1 Quantitative analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 10.11 Analytical method developed for ethanol analysis . . . . . . . . . . . . . . . 257 10.12 Refractive index determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 10.13 Measuring the cell dry weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 10.14 Yield calculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 10.15 Batch fermentation experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 10.16 Continuous fermentation experiment . . . . . . . . . . . . . . . . . . . . . . . . . 258 10.17 Media sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 10.18 Batch experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 10.18.1 Optical cell density, ethanol and carbohydrate concentration . . . . . . . 261 10.18.2 Continuous ethanol fermentation experiment . . . . . . . . . . . . . . . . . . . 261 10.19 Expected results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 Chapter 11. Production of Antibiotics 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 11.2 Herbal medicines and chemical agents . . . . . . . . . . . . . . . . . . . . . . . . 263 11.3 History of penicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 11.4 Production of penicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 11.5 Microorganisms and media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 11.6 Inoculum preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 11.7 Filtration and extraction of penicillin . . . . . . . . . . . . . . . . . . . . . . . . . 268 11.8 Experimental procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 11.9 Fermenter description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 11.10 Analytical method for bioassay and detecting antibiotic . . . . . . . . . . 269 11.11 Antibiogram and biological assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 11.12 Submerged culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 11.12.1 Growth kinetics in submerged culture . . . . . . . . . . . . . . . . . . . . . . . . . 270 11.13 Bioreactor design and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 11.14 Estimation for the dimension of the fermenter . . . . . . . . . . . . . . . . . . 273 11.15 Determination of Reynolds number . . . . . . . . . . . . . . . . . . . . . . . . . . 275 11.16 Determination of power input . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 11.17 Determination of oxygen transfer rate . . . . . . . . . . . . . . . . . . . . . . . . 277 11.18 Design specification sheet for the bioreactor . . . . . . . . . . . . . . . . . . . 278 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 Chapter 12. Production of Citric Acid 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 12.2 Production of citric acid in batch bioreactor . . . . . . . . . . . . . . . . . . . . 280 CONTENTS.qxd 10/27/2006 10:52 AM Page xii
  • 13. TABLE OF CONTENTS xiii 12.2.1 Microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 12.3 Factors affecting the mold growth and fermentation process . . . . . . . 281 12.4 Starter or seeding an inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 12.5 Seed culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 12.6 Citric acid production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 12.7 Analytical method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 12.7.1 Cell dry weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 12.7.2 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 12.7.3 Citric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 12.8 Experimental run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 Chapter 13. Bioprocess Scale-up 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 13.2 Scale-up procedure from laboratory scale to plant scale . . . . . . . . . . 287 13.2.1 Scale-up for constant KLa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 13.2.2 Scale-up based on shear forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 13.2.3 Scale-up for constant mixing time . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 13.3 Bioreactor design criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 13.3.1 General cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 13.3.2 Bubble column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 13.4 CSTR chemostat versus tubular plug flow . . . . . . . . . . . . . . . . . . . . . 298 13.5 Dynamic model and oxygen transfer rate in activated sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 13.6 Aerobic wastewater treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 13.6.1 Substrate balance in a continuous system . . . . . . . . . . . . . . . . . . . . . . 327 13.6.2 Material balance in fed batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 13.7 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 Chapter 14. Single-Cell Protein 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 14.2 Separation of microbial biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 14.3 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 14.4 Production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 14.5 Media preparation for SCP production . . . . . . . . . . . . . . . . . . . . . . . . 335 14.6 Analytical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 14.6.1 Coomassie–protein reaction scheme . . . . . . . . . . . . . . . . . . . . . . . . . . 336 14.6.2 Preparation of diluted BSA standards . . . . . . . . . . . . . . . . . . . . . . . . . 336 14.6.3 Mixing of the coomassie plus protein assay reagent . . . . . . . . . . . . . 337 14.6.4 Standard calibration curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 14.6.5 Standard calibration curve for starch . . . . . . . . . . . . . . . . . . . . . . . . . 337 14.7 SCP processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 14.8 Nutritional value of SCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 14.9 Advantages and disadvantages of SCP . . . . . . . . . . . . . . . . . . . . . . . . 340 14.10 Preparation for experimental run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 CONTENTS.qxd 10/27/2006 10:52 AM Page xiii
  • 14. Chapter 15. Sterilisation 15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 15.2 Batch sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 15.3 Continuous sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 15.4 Hot plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 15.5 High temperature sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 15.6 Sterilised media for microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 15.6.1 Sterilisation of media for stoke cultures . . . . . . . . . . . . . . . . . . . . . . . 347 15.6.2 Sterilisation of bacterial media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 15.6.3 Sterilise petri dishes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 15.7 Dry heat sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 15.8 Sterilisation with filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 15.9 Microwave sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 15.10 Electron beam sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 15.11 Chemical sterilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 Chapter 16. Membrane Separation Processes 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 16.2 Types of membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 16.2.1 Isotropic membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 16.2.1.1 Microporous membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 16.2.1.2 Non-porous, dense membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 16.2.1.3 Electrically charged membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 16.2.2 Anisotropic membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353 16.2.3 Ceramic, metal and liquid membranes . . . . . . . . . . . . . . . . . . . . . . . . 353 16.3 Membrane processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 16.4 Nature of synthetic membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 16.5 General membrane equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 16.6 Cross-flow microfiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 16.7 Ultrafiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 16.8 Reverse osmosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 16.9 Membrane modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 16.9.1 Tubular modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 16.9.2 Flat-sheet modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 16.9.3 Spiral-wound modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 16.9.4 Hollow-fibre modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 16.10 Module selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373 16.11 Membrane fouling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 16.12 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 16.13 Case study: inorganic zirconia ␥-alumina-coated membrane on ceramic support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 16.13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 16.13.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 xiv TABLE OF CONTENTS CONTENTS.qxd 10/27/2006 10:52 AM Page xiv
  • 15. 16.13.2.1 Preparation of PVA solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 16.13.2.2 Preparation of zirconia-coated alumina membrane . . . . . . . . . . . . . . . 385 16.13.2.3 Preparation of porous ceramic support . . . . . . . . . . . . . . . . . . . . . . . . 386 16.13.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 16.13.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 16.13.5 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 Chapter 17. Advanced Downstream Processing in Biotechnology 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 17.2 Protein products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 17.3 Cell disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 17.4 Protein purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 17.4.1 Overview of the strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 17.4.2 Dye-ligand pseudo-affinity adsorption . . . . . . . . . . . . . . . . . . . . . . . . 394 17.5 General problems associated with conventional techniques . . . . . . . . 394 17.6 Fluidised bed adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 17.6.1 Mixing behaviour in fluidised/expanded beds . . . . . . . . . . . . . . . . . . 396 17.7 Design and operation of liquid fluidised beds . . . . . . . . . . . . . . . . . . 397 17.7.1 Hydrodynamic characterisation of flow in fluidised/expanded beds and bed voidage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 17.7.2 Minimum fluidisation velocity of particles . . . . . . . . . . . . . . . . . . . . . 398 17.7.3 Terminal settling velocity of particles . . . . . . . . . . . . . . . . . . . . . . . . . 399 17.7.4 Degree of bed expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 17.7.5 Matrices for fluidised bed adsorption . . . . . . . . . . . . . . . . . . . . . . . . . 402 17.7.6 Column design for fluidised bed adsorption . . . . . . . . . . . . . . . . . . . . 403 17.8 Experimental procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 17.9 Process integration in protein recovery . . . . . . . . . . . . . . . . . . . . . . . . 404 17.9.1 Interfaced and integrated fluidised bed/expanded bed system . . . . . . 405 17.10 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 17.11 Case study: process integration of cell disruption and fluidised bed adsorption for the recovery of labile intracellular enzymes . . . . . 409 17.11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409 17.11.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 17.11.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 17.11.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 17.11.5 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 TABLE OF CONTENTS xv CONTENTS.qxd 10/27/2006 10:52 AM Page xv
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  • 17. CHAPTER 1 Industrial Microbiology 1.1 INTRODUCTION Microorganisms have been identified and exploited for more than a century. The Babylonians and Sumerians used yeast to prepare alcohol. There is a great history beyond fermenta- tion processes, which explains the applications of microbial processes that resulted in the production of food and beverages. In the mid-nineteenth century, Louis Pasteur understood the role of microorganisms in fermented food, wine, alcohols, beverages, cheese, milk, yoghurt and other dairy products, fuels, and fine chemical industries. He identified many microbial processes and discovered the first principal role of fermentation, which was that microbes required substrate to produce primary and secondary metabolites, and end products. In the new millennium, extensive application of bioprocesses has created an environ- ment for many engineers to expand the field of biotechnology. One of the useful applica- tions of biotechnology is the use of microorganisms to produce alcohols and acetone, which are used in the industrial processes. The knowledge related to industrial microbiology has been revolutionised by the ability of genetically engineered cells to make many new prod- ucts. Genetic engineering and gene mounting have been developed in the enhancement of industrial fermentation. Consequently, biotechnology is a new approach to making com- mercial products by using living organisms. Furthermore, knowledge of bioprocesses has been developed to deliver fine-quality products. Application of biological sciences in industrial processes is known as bioprocessing. Nowadays most biological and pharmaceutical products are produced in well-defined industrial bioprocesses. For instance, bacteria are able to produce most amino acids that can be used in food and medicine. There are hundreds of microbial and fungal products purely available in the biotechnology market. Microbial production of amino acids can be used to produce L-isomers; chemical production results in both D- and L-isomers. Lysine and glu- tamic acid are produced by Corynebacterium glutamicum. Another food additive is citric acid, which is produced by Aspergillus niger. Table 1.1 summarises several widespread applications of industrial microbiology to deliver a variety of products in applied industries. The growth of cells on a large scale is called industrial fermentation. Industrial fermen- tation is normally performed in a bioreactor, which controls aeration, pH and temperature. Microorganisms utilise an organic source and produce primary metabolites such as ethanol, 1 Ch001.qxd 10/27/2006 10:49 AM Page 1
  • 18. which are formed during the cells’ exponential growth phase. In some bioprocesses, yeast or fungi are used to produce advanced valuable products. Those products are considered as secondary metabolites, such as penicillin, which is produced during the stationary phase. Yeasts are grown for wine- and bread-making. There are other microbes, such as Rhizobium, Bradyrhizobium and Bacillus thuringiensis, which are able to grow and utilise carbohydrates and organic sources originating from agricultural wastes. Vaccines, anti- biotics and steroids are also products of microbial growth. 1.2 PROCESS FERMENTATION The term ‘fermentation’ was obtained from the Latin verb ‘fervere’, which describes the action of yeast or malt on sugar or fruit extracts and grain. The ‘boiling’ is due to the pro- duction of carbon dioxide bubbles from the aqueous phase under the anaerobic catabolism of carbohydrates in the fermentation media. The art of fermentation is defined as the chem- ical transformation of organic compounds with the aid of enzymes. The ability of yeast to make alcohol was known to the Babylonians and Sumerians before 6000 BC. The Egyptians discovered the generation of carbon dioxide by brewer’s yeast in the preparation 2 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY TABLE 1.1. Industrial products produced by biological processes12 Fermentation product Microorganism Application Ethanol (non-beverage) Saccharomyces cerevisiae Fine chemicals 2-Ketogluconic acid Pseudomonas sp. Intermediate for D-araboascorbic acid Pectinase, protease Aspergillus niger, A. aureus Clarifying agents in fruit juice Bacterial amylase Bacillus subtilis Modified starch, sizing paper Bacterial protease B. subtilis Desizing fibres, spot remover Dextran Leuconostoc mesenteroides Food stabilizer Sorbose Gluconobacter suboxydans Manufacturing of ascorbic acid Cobalamin (vitamin B12) Streptomyces olivaceus Food supplements Glutamic acid Brevibacterium sp. Food additive Gluconic acid Aspergillus niger Pharmaceutical products Lactic acid Rhizopus oryzae Foods and pharmaceuticals Citric acid Aspergillus niger or A. wentii Food products, medicine Acetone-butanol Clostridium acetobutylicum Solvents, chemical intermediate Insulin, interferon Recombinant E. coli Human therapy Baker’s yeast Yeast and culture starter Lactobacillus bulgaricus Cheese and yoghurt production Lactic acid bacteria Microbial protein (SCP) Candida utilis Food supplements Pseudomonas methylotroph Penicillin Penicillium chrysogenum Antibiotics Cephalosporins Cephalosparium acremonium Antibiotics Erythromycin Streptomyces erythreus Antibiotics Ch001.qxd 10/27/2006 10:49 AM Page 2
  • 19. INDUSTRIAL MICROBIOLOGY 3 of bread. The degradation of carbohydrates by microorganisms is followed by glycolytic or Embden–Myerhof–Parnas pathways.1,2 Therefore the overall biochemical reaction mechanisms to extract energy and form products under anaerobic conditions are called fer- mentation processes. In the process of ethanol production, carbohydrates are reduced to pyruvate with the aid of nicotinamide adenine dinucleotide (NADH); ethanol is the end product. Other fermentation processes include the cultivation of acetic acid bacteria for the production of vinegar. Lactic acid bacteria preserve milk; the products are yoghurt and cheese. Various bacteria and mold are involved in the production of cheese. Louis Pasteur, who is known as the father of the fermentation process, in early nineteenth century defined fermentation as life without air. He proved that existing microbial life came from pre- existing life. There was a strong belief that fermentation was strictly a biochemical reac- tion. Pasteur disproved the chemical hypothesis. In 1876, he had been called by distillers of Lille in France to investigate why the content of their fermentation product turned sour.3 Pasteur found under his microscope the microbial contamination of yeast broth. He discovered organic acid formation such as lactic acid before ethanol fermentation. His greatest contribution was to establish different types of fermentation by specific microor- ganisms, enabling work on pure cultures to obtain pure product. In other words, fermenta- tion is known as a process with the existence of strictly anaerobic life: that is, life in the absence of oxygen. The process is summarised in the following steps: • Action of yeast on extracts of fruit juice or, malted grain. The biochemical reactions are related to generation of energy by catabolism of organic compounds. • Biomass or mass of living matter, living cells in a liquid solution with essential nutrients at suitable temperature and pH leads to cell growth. As a result, the content of biomass increases with time. In World War I, Germany was desperate to manufacture explosives, and glycerol was needed for this. They had identified glycerol in alcohol fermentation. Neuberg discovered that the addition of sodium bisulphate in the fermentation broth favored glycerol production with the utilization of ethanol. Germany quickly developed industrial-scale fermentation, with production capacity of about 35 tons per day.3 In Great Britain, acetone was in great demand; it was obtained by anaerobic fermentation of acetone–butanol using Clostridium acetobutylicum. In large-scale fermentation production, contamination was major problem. Microorganisms are capable of a wide range of metabolic reactions, using various sources of nutrients. That makes fermentation processes suitable for industrial applications with inexpensive nutri- ents. Molasses, corn syrup, waste products from crystallisation of sugar industries and the wet milling of corn are valuable broth for production of antibiotics and fine chemicals. We will discuss many industrial fermentation processes in the coming chapters. It is best to focus first on the fundamental concepts of biochemical engineering rather than the applications. There are various industries using biological processes to produce new products, such as antibiotics, chemicals, alcohols, lipid, fatty acids and proteins. Deep understanding of bio- processing may require actual knowledge of biology and microbiology in the applications of the above processes. It is very interesting to demonstrate bench-scale experiments and Ch001.qxd 10/27/2006 10:49 AM Page 3
  • 20. make use of large-scale advanced technology. However, application of the bioprocess in large-scale control of microorganisms in 100,000 litres of media may not be quite so simple to manage. Therefore trained engineers are essential and highly in demand; this can be achieved by knowledge enhancement in the sheathe bioprocesses. To achieve such objec- tives we may need to explain the whole process to the skilled labour and trained staff to implement bioprocess knowhow in biotechnology. 1.3 APPLICATION OF FERMENTATION PROCESSES Man has been using the fermentative abilities of microorganisms in various forms for many centuries. Yeasts were first used to make bread; later, use expanded to the fermentation of dairy products to make cheese and yoghurt. Nowadays more than 200 types of fermented food product are available in the market. There are several biological processes actively used in the industry, with high-quality products such as various antibiotics, organic acids, glutamic acid, citric acid, acetic acid, butyric and propionic acids. Synthesis of proteins and amino acids, lipids and fatty acids, simple sugar and polysaccharides such as xanthan gum, glycerol, many more fine chemicals and alcohols are produced by bioprocesses with suit- able industrial applications. The knowledge of bioprocessing is an integration of biochem- istry, microbiology and engineering science applied in industrial technology. Application of viable microorganisms and cultured tissue cells in an industrial process to produce specific products is known as bioprocessing. Thus fermentation products and the ability to cultivate large amounts of organisms are the focus of bioprocessing, and such achievements may be obtained by using vessels known as fermenters or bioreactors. The cultivation of large amounts of organisms in vessels such as fermenters and bioreactors with related fermenta- tion products is the major focus of bioprocess. A bioreactor is a vessel in which an organism is cultivated and grown in a controlled manner to form the by-product. In some cases specialised organisms are cultivated to pro- duce very specific products such as antibiotics. The laboratory scale of a bioreactor is in the range 2–100 litres, but in commercial processes or in large-scale operation this may be up to 100 m3 .4,5 Initially the term ‘fermenter’ was used to describe these vessels, but in strict terms fermentation is an anaerobic process whereas the major proportion of fermenter uses aerobic conditions. The term ‘bioreactor’ has been introduced to describe fermentation vessels for growing the microorganisms under aerobic or anaerobic conditions. Bioprocess plants are an essential part of food, fine chemical and pharmaceutical indus- tries. Use of microorganisms to transform biological materials for production of fermented foods, cheese and chemicals has its antiquity. Bioprocesses have been developed for an enormous range of commercial products, as listed in Table 1.1. Most of the products orig- inate from relatively cheap raw materials. Production of industrial alcohols and organic solvents is mostly originated from cheap feed stocks. The more expensive and special bio- processes are in the production of antibiotics, monoclonal antibodies and vaccines. Industrial enzymes and living cells such as baker’s yeast and brewer’s yeast are also com- mercial products obtained from bioprocess plants. 4 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY Ch001.qxd 10/27/2006 10:49 AM Page 4
  • 21. INDUSTRIAL MICROBIOLOGY 5 1.4 BIOPROCESS PRODUCTS Major bioprocess products are in the area of chemicals, pharmaceuticals, energy, food and agriculture, as depicted in Table 1.2. The table shows the general aspects, benefits and application of biological processes in these fields. Most fermented products are formed into three types. The main categories are now discussed. 1.4.1 Biomass The aim is to produce biomass or a mass of cells such as microbes, yeast and fungi. The commercial production of biomass has been seen in the production of baker’s yeast, which is used in the baking industry. Production of single cell protein (SCP) is used as biomass enriched in protein.6 An algae called Spirulina has been used for animal food in some coun- tries. SCP is used as a food source from renewable sources such as whey, cellulose, starch, molasses and a wide range of plant waste. TABLE 1.2. Products and services by biological processes Sector Product and service Remark Chemicals Ethanol, acetone, butanol Bulk Organic acids (acetic, butyric, propionic and citric acids) Enzymes Fine Perfumeries Polymers Pharmaceuticals Antibiotics Enzymes Enzyme inhibitors Monoclonal antibodies Steroids Vaccines Energy Ethanol (gasohol) Non-sterile Methane (biogas) Food Diary products (cheese, yoghurts, etc.) Non-sterile Baker’s yeast Beverages (beer, wine) Food additives Amino acids Vitamin B Proteins (SCP) Agriculture Animal feeds (SCP) Non-sterile Waste treatment Vaccines Microbial pesticides Mycorrhizal inoculants Ch001.qxd 10/27/2006 10:49 AM Page 5
  • 22. 1.4.2 Cell Products Products are produced by cells, with the aid of enzymes and metabolites known as cell products. These products are categorised as either extracellular or intracellular. Enzymes are one of the major cell products used in industry. Enzymes are extracted from plants and animals. Microbial enzymes, on the other hand, can be produced in large quantities by con- ventional techniques. Enzyme productivity can be improved by mutation, selection and per- haps by genetic manipulation. The use of enzymes in industry is very extensive in baking, cereal making, coffee, candy, chocolate, corn syrup, dairy product, fruit juice and bever- ages. The most common enzymes used in the food industries are amylase in baking, pro- tease and amylase in beef product, pectinase and hemicellulase in coffee, catalase, lactase and protease in dairy products, and glucose oxidase in fruit juice. 1.4.3 Modified Compounds (Biotransformation) Almost all types of cell can be used to convert an added compound into another compound, involving many forms of enzymatic reaction including dehydration, oxidation, hydroxyla- tion, amination, isomerisation, etc. These types of conversion have advantages over chem- ical processes in that the reaction can be very specific, and produced at moderate temperatures. Examples of transformations using enzymes include the production of steroids, conversion of antibiotics and prostaglandins. Industrial transformation requires the production of large quantities of enzyme, but the half-life of enzymes can be improved by immobilisation and extraction simplified by the use of whole cells. In any bioprocess, the bioreactor is not an isolated unit, but is as part of an integrated process with upstream and downstream components. The upstream consists of storage tanks, growth and media preparation, followed by sterilisation. Also, seed culture for inoc- ulation is required upstream, with sterilised raw material, mainly sugar and nutrients, required for the bioreactor to operate. The sterilisation of the bioreactor can be done by steam at 15 pounds per square inch guage (psig), 121 °C or any disinfectant chemical reagent such as ethylene oxide. The downstream processing involves extraction of the product and purification as normal chemical units of operation.7 The solids are separated from the liquid, and the solution and supernatant from separation unit may go further for purification after the product has been concentrated. 1.5 PRODUCTION OF LACTIC ACID Several carbohydrates such as corn and potato starch, molasses and whey can be used to produce lactic acid. Starch must first be hydrolysed to glucose by enzymatic hydrolysis; then fermentation is performed in the second stage. The choice of carbohydrate material depends upon its availability, and pretreatment is required before fermentation. We shall describe the bioprocess for the production of lactic acid from whey. Large quantities of whey constitute a waste product in the manufacture of dairy products such as cheese. From the standpoint of environmental pollution it is considered a major problem, and disposal of untreated wastes may create environmental disasters. It is desirable 6 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY Ch001.qxd 10/27/2006 10:49 AM Page 6
  • 23. INDUSTRIAL MICROBIOLOGY 7 to use whey to make some more useful product. Whey can be converted from being a waste product to something more desirable that can be used for the growth of certain bacteria, because it contains lactose, nitrogenous substances, vitamins and salts. Organisms can utilise lactose and grow on cheese wastes; the most suitable of them are Lactobacillus species such as Lactobacillus bulgaricus, which is the most suitable species for whey. This organism grows rapidly, is homofermentative and thus capable of converting lactose to the single end-product of lactic acid. Stock cultures of the organism are maintained in skimmed milk medium. The 3–5% of inoculum is prepared and transferred to the main bioreactor, and the culture is stored in pasteurised, skimmed milk at an incubation temperature of 43 °C. During fermentation, pH is controlled by the addition of slurry of lime to neutralise the product to prevent any product inhibition. The accumulation of lactic acid would retard the fermentation process because of the formation of calcium lactate. After 2 days of com- plete incubation, the material is boiled to coagulate the protein, and then filtered. The solid filter cake is a useful, enriched protein product, which may be used as an animal feed sup- plement. The filtrate containing calcium lactate is then concentrated by removing water under vacuum, followed by purification of the final product. The flow diagram for this process is shown in Figure 1.1. 1.6 PRODUCTION OF VINEGAR The sugars in fruits such as grapes are fermented by yeasts to produce wines. In wine- making, lactic acid bacteria convert malic acid into lactic acid in malolactic fermentation in fruits with high acidity. Acetobacter and Gluconobacter oxidise ethanol in wine to acetic acid (vinegar). 5000 gallon bioreactor Lactic acid recovery Whey Fermentation of lactose using Lactobacillus bulgaricus Seed culture inoculum 150 gallons Preparation of inocula Stock culture FIG. 1.1. Production of lactic acid from whey. Ch001.qxd 10/27/2006 10:49 AM Page 7
  • 24. The word ‘wine’ is derived from the French term ‘vinaigre’ meaning ‘sour wine’. It is prepared by allowing a wine to get sour under controlled conditions. The production of vinegar involves two steps of biochemical changes: (1) Alcoholic fermentation in fermentation of a carbohydrate. (2) Oxidation of the alcohol to acetic acid. There are several kinds of vinegar. The differences between them are primarily associated with the kind of material used in the alcoholic fermentation, e.g. fruit juices, sugar and hydrolysed starchy materials. Based on US Department of Agriculture (USDA) definitions, there are a few types of vinegar: vinegar, cider vinegar, apple vinegar. The products are made by the alcoholic and subsequent acetous fermentations of the apple juice. The acetic acid content is about 5%.Yeast fermentation is used for the production of alcohol. The alco- hol is adjusted to 10–13%, then it is exposed to acetic acid bacteria (Acetobacter species), whereby oxygen is required for the oxidation of alcohol to acetic acid. The desired tem- perature for Acetobacter is 15–34 °C. The reaction is: (1.6.1) (1.6.2) 1.7 PRODUCTION OF AMINO ACIDS (LYSINE AND GLUTAMIC ACID) AND INSULIN Many microorganisms can synthesise amino acids from inorganic nitrogen compounds. The rate and amount of some amino acids may exceed the cells’ need for protein synthesis, where the excess amino acids are excreted into the media. Some microorganisms are capa- ble of producing certain amino acids such as lysine, glutamic acid and tryptophan. 1.7.1 Stepwise Amino Acid Production One of the commercial methods for production of lysine consists of a two-stage process using two species of bacteria. The carbon sources for production of amino acids are corn, potato starch, molasses, and whey. If starch is used, it must be hydrolysed to glucose to achieve higher yield. Escherichia coli is grown in a medium consisting of glycerol, corn- steep liquor and di-ammonium phosphate under aerobic conditions, with temperature and pH controlled. • Step 1: Formation of diaminopimelic acid (DAP) by E. coli. • Step 2: Decarboxylation of DAP by Enterobacter aerogenes. E. coli can easily grow on corn steep liquor with phosphate buffer for an incubation period of 3 days. Lysine is an essential amino acid for the nutrition of humans, which is used as a 2CH CH OH O 2CH COOH 2H O3 2 2 sp. 3 2ϩ ϩ2 Acetobacter æ Ææææææ C H O 2CH CH OH 2CO6 12 6 3 2 2 Zymomonas mobilis æ Æææææææ ϩ 8 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY Ch001.qxd 10/27/2006 10:49 AM Page 8
  • 25. supplementary food with bread and other foodstuffs. This amino acid is a biological product which is also used as a food additive and cereal protein. Many species of microorganisms, especially bacteria and fungi, are capable of produc- ing large amounts of glutamic acid. Glutamic acid is produced by microbial metabolites of Micrococcus, Arthrobacter, and Brevibacterium by the Krebs cycle. Monosodium gluta- mate is known as a flavour-enhancing amino acid in food industries. The medium gener- ally used consists of carbohydrate, peptone, inorganic salts and biotin. The concentration of biotin has a significant influence on the yield of glutamic acid. The ␣-ketoglutaric acid is an intermediate in the Krebs cycle and is the precursor of glutamic acid. The conversion of ␣-ketoglutaric acid to glutamic acid is accomplished in the presence of glutamic acid dehydrogenase, ammonia and nicotinamide adenine dinucleotide dehydrogenase (NADH2). The living cells assimilate nitrogen by incorporating it into ketoglutaric acid, then to glu- tamic acid and glutamine. Therefore glutamic acid is formed by the reaction between ammonia and ␣-ketoglutaric acid in one of the tricarboxylic acid (TCA) cycle or Krebs cycle intermediates.2,9 1.7.2 Insulin Insulin is one of the important pharmaceutical products produced commercially by geneti- cally engineered bactera. Before this development, commercial insulin was isolated from animal pancreatic tissue. Microbial insulin has been available since 1982. The human insulin gene is introduced into a bacterium like E. coli. Two of the major advantages of insulin production by microorganisms are that the resultant insulin is chemically identical to human insulin, and it can be produced in unlimited quantities. 1.8 ANTIBIOTICS, PRODUCTION OF PENICILLIN The commercial production of penicillin and other antibiotics are the most dramatic in industrial microbiology. The annual production of bulk penicillin is about 33 thousand met- ric tonnes with annual sales market of more than US$400 million.8 The worldwide bulk sales of the four most important groups of antibiotics, penicillins, cephalosporins, tetracy- clines and erythromycin, are US$4.2 billion per annum.10 The mold isolated by Alexander Fleming in early 1940s was Penicillium notatum, who noted that this species killed his culture of Staphylococcus aureus. The production of peni- cillin is now done by a better penicillin-producing mould species, Penicillium chryso- genum. Development of submerged culture techniques enhanced the cultivation of the mould in large-scale operation by using a sterile air supply. • Streptomycin produced by Actinomycetes • Molasses, corn steep liquor, waste product from sugar industry, and wet milling corn are used for the production of penicillin • Penicillium chrysogenum can produce 1000 times more penicillin than Fleming’s original culture8 INDUSTRIAL MICROBIOLOGY 9 Ch001.qxd 10/27/2006 10:49 AM Page 9
  • 26. • The major steps in the commercial production of penicillin are: (1) Preparation of inoculum. (2) Preparation and sterilisation of the medium. (3) Inoculation of the medium in the fermenter. (4) Forced aeration with sterile air during incubation. (5) Removal of mould mycelium after fermentation. (6) Extraction and purification of the penicillin. 1.9 PRODUCTION OF ENZYMES Many moulds synthesise and excrete large quantities of enzymes into the surrounding medium. Enzymes are proteins; they are denatured by heat and extracted or precipitated by chemical solvents like ethanol and by inorganic salts like ammonium sulphate.11 Coenzymes are also proteins combined with low molecular mass organics like vitamin B. It is industri- ally applicable and economically feasible to produce, concentrate, extract and purify enzymes from cultures of moulds such as Aspergillus, Penicillium, Mucor and Rhizopus. Mould enzymes such as amylase, invertase, protease, and pectinase are useful in the processing or refining of a variety of materials. Amylases hydrolyse starch to dextrin and sugars. They are used in preparing sizes and adhesives, desizing textile, clarifying fruit juices, manufacturing pharmaceuticals and other purposes. Invertase hydrolyses sucrose to form glucose and fruc- tose (invert sugar). It is widely used in candy making and the production of non-crystallizable 10 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY Cane molasses Beet molasses Nutrients Mixing and cooking Finished mash storage Stock inoculum Large scale fermenter Filter Filter press CentrifugePackaging and cool storage FIG. 1.2. Commercial production of baker’s yeast. Ch001.qxd 10/27/2006 10:49 AM Page 10
  • 27. INDUSTRIALMICROBIOLOGY11 Sampling port Mixgas(10%CO2,15%Ar, 20%H2,&55%CO Nitrogen gas Fresh media Micro-DCU-System Temperature controller TEMP : 30 OC STIRR : 500 rpm pH : 6.5 pH pO2 : 00.0 % Pressure regulator Pump Mass flow controller Base Lamp Fermenter AFoam Pump Pump Pump Acid Liquid flow breaker Motor Gas sampling vent Pump Liquid sampling port Effluent Nitrogen gas Vent FIG. 1.3. One complete set of fermenters with all accessory controlling units. Ch001.qxd10/27/200610:49AMPage11
  • 28. 12 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY syrup from sucrose, which is partly hydrolysed by this enzyme. The proteolytic enzymes such as protease are used for bating in leather processing to obtain fine texture. Protease is also used in the manufacture of liquid glue, degumming of silks and clarification of beer protein. It is used in laundry detergents and as an adjunct with soaps. Pectinase is used in the clarifi- cation of fruit juice and to hydrolyse pectins in the retting of flax for the manufacture of linen. Apoenzyme is the protein portion of the enzyme, which is inactive. The reaction between low molecular mass coenzymes and apoenzyme gives active holoenzyme: (1.9.1) 1.10 PRODUCTION OF BAKER’S YEAST The use of yeast as a leavening agent in baking dates back to the early histories of the Egyptians, Greeks and Romans. In those days, leavened bread was made by mixing some leftover dough from the previous batch of bread with fresh dough. In modern baking, pure cultures of selected strains of Saccharomyces cerevisiae are mixed with the bread dough to bring about desired changes in the texture and flavour of the bread. Characteristics of S. cerevisiae strains are selected for commercial production of baker’s yeast. It has the abil- ity to ferment sugar in the dough vigorously and rapidly. The selected strains must be stable and produce carbon dioxide, which results from the fermentation process for leavening or ris- ing the dough. The quality of the bread depends on the selected strain of yeast, the incubation period and the choice of raw materials. Sugars in the bread dough are fermented by yeast to ethanol and CO2; whereby the CO2 causes the bread to rise. In the manufacture of baker’s yeast, the stock strain is inoculated into a medium that containing molasses and corn steep liquor. The pH of the medium is adjusted to be slightly acidic at pH 4–5. The acidic pH may retard the bacterial growth. The inoculated medium is aerated during the incubation period. At the end, the cells are harvested by centrifuging out the fermentation broth, and they are recovered by filter press. A small amount of veg- etable oil is added to act as plasticiser, and then the cell mass is moulded into blocks. The process is shown in Figure 1.2. A full set of bioreactors with pH and temperature controllers are shown in Figure 1.3. The complete set of a 25 litre fermenter with all the accessory controlling units creates a good opportunity to control suitable production of biochemical products with variation of process parameters. Pumping fresh nutrients and operating in batch, fed batch and continuous mode are easy and suitable for producing fine chemicals, amino acids, and even antibiotics. REFERENCES 1. Aiba, S., Humphrey, A.E. and Millis, N.F., “Biochemical Engineering”, 2nd edn. Academic Press, New York, 1973. 2. Baily, J.E. and Ollis, D.F., “Biochemical Engineering Fundamentals”, 2nd edn. McGraw-Hill, NewYork, 1986. 3. Demain, A.L. and Solomon, A.N. Sci. Am. 245, 67 (1981). Apoenzyme coenzyme holoenzyme (active)ϩ Ch001.qxd 10/27/2006 10:49 AM Page 12
  • 29. 4. Ghose, T.K., “Bioprocess Computation in Biotechnology”, vol. 1. Ellis Horwood Series in Biochemistry and Biotechnology, New York, 1990. 5. Scragg, A.H., “Bioreactors in Biotechnology, A Practical Approach”. Ellis Horwood Series in Biochemistry and Biotechnology, New York, 1991. 6. Bradford, M.M., J. Analyt. Biochem. 72, 248 (1976). 7. Doran, P.M., “Bioprocess Engineering Principles”. Academic Press, New York, 1995. 8. Pelczar, M.J., Chan, E.C.S. and Krieg, N.R., “Microbiology”. McGraw-Hill, New York, 1986. 9. Shuler, M.L. and Kargi, F., “Bioprocess Engineering, Basic Concepts”. Prentice-Hall, New Jersey, 1992. 10. Aharonowitz, Y. and Cohen, G., Sci. Am. 245, 141 (1981). 11. Thomas, L.C. and Chamberlin, G.J., “Colorimetric Chemical Analytical Methods”. Tintometer Ltd, Salisbury, United Kingdom, 1980. 12. Phaff, H.J., Sci. Am. 245, 77 (1981). INDUSTRIAL MICROBIOLOGY 13 Ch001.qxd 10/27/2006 10:49 AM Page 13
  • 30. 14 CHAPTER 2 Dissolved Oxygen Measurement and Mixing 2.1 INTRODUCTION In biochemical engineering processes, measurement of dissolved oxygen (DO) is essential. The production of SCP may reach a steady-state condition by keeping the DO level con- stant, while the viable protein is continuously harvested. The concentration of protein is proportional to oxygen uptake rate. Control of DO would lead us to achieve steady SCP production. Variation of DO may affect retention time and other process variables such as substrate and product concentrations, retention time, dilution rate and aeration rate. Microbial activities are monitored by the oxygen uptake rate from the supplied air or oxygen. Microbial cells in the aerobic condition take up oxygen from the gas and then liquid phases. The rate of oxygen transfer from the gas phase to liquid phase is important. At high cell densities, the cell growth is limited by the availability of oxygen in the medium. The growth of aerobic bacteria in the fermenter is then controlled by the availability of oxygen, substrate, energy sources and enzymes. Air has to be supplied for aerobic process in order to enhance the cell growth. Oxygen limitation may cause a reduction in the growth rate. The supplied oxygen from the gas phase has to penetrate into the microorganism. Several steps are required in order to let such a phenomenon take place. The oxygen first must travel through the gas–liquid interface, then the bulk of liquid and finally into the microbial cell. The solubility of air in water at 10 °C and under atmospheric conditions is 11.5ppm; as the temperature is increased to 30 °C, the solubility of air drops to 8ppm. The solubility of air decreases to 7ppm at 40 °C. Availability of oxygen in the fermentation broth is higher than the air, if pure oxygen is used. The solubility of pure oxygen in water at 10 °C and 1atm pressure is 55ppm. As the temperature increases to 30 °C, the solubility of pure oxy- gen drops to 38.5ppm. The solubility of pure oxygen decreased to 33.7ppm at 40 °C. The above data show that in case of high oxygen demand for SCP production, oxygen drasti- cally depletes in 12–24 hours of incubation. Therefore pure oxygen is commonly used to enhance oxygen availability in the fermentation media. 2.2 MEASUREMENT OF DISSOLVED OXYGEN CONCENTRATIONS The concentration of dissolved oxygen in a fermenter is normally measured with a dissolved oxygen electrode, known as a DO probe. There are two types in common use: galvanic Ch002.qxd 10/27/2006 10:49 AM Page 14
  • 31. DISSOLVED OXYGEN MEASUREMENT AND MIXING 15 electrodes and polarographic electrodes. In both probes, there are membranes that are per- meable to oxygen. Oxygen diffuses through the membrane and reaches to cathode, where it reacts to produce a current between anode and cathode proportional to the oxygen partial pressure in the fermentation broth. The electrolyte solutions in the electrode take part in the reactions and must be located in the bulk of liquid medium. There several DO probes available. Some well-known branded fermenters, like New Brunswick, Bioflo series and the B. Braun Biotstat B fermenters are equipped with a DO meter. This unit has a 2 litre fermentation vessel equipped with DO meter and pH probe, antifoam sensor and level controllers for harvesting culture. The concentration of DO in the media is a function of temperature. The higher operating temperature would decrease the level of DO. A micro-sparger is used to provide sufficient small air bubbles. The air bub- bles are stabilized in the media and the liquid phase is saturated with air. The availability of oxygen is major parameter to be considered in effective microbial cell growth rate. 2.3 BATCH AND CONTINUOUS FERMENTATION FOR PRODUCTION OF SCP The fermentation vessel is a jacketed vessel with a defined working volume. The media are made of phosphate buffer at neutral pH with 3.3g KH2PO4 and 0.3g Na2HPO4, 1g yeast extract and 30g glucose in 1 litre of distilled water. The media should be sterilised in a 20 litres carboy. The fermentation vessel with a working volume of 2 litres may have 500ml media initially sterilised by stream under 15psig and 121 °C. The seed culture is transferred to the fermentation vessel with filtered and pressurized air; the production of SCP is mon- itored by pumping fresh nutrients and supplying air. Continuous culture with constant vol- ume and controlled dilution rate is conducted in SCP production, as fresh and sterilised media are pumped into the culture vessel. It is desirable to control pH, temperature and aera- tion with a constant air flow rate. The most common continuous culture system is the chemostat. The word chemostat refers to the constant chemical environment at steady-state condition.1 Another continuous culture vessel is the turbidostat, where the cell concentra- tion in the culture vessel is kept constant by monitoring cell optical density. The chemostat experiment is carried out for 24 hours at a constant temperature of 32 °C, and by control- ling pH and monitoring DO concentration. The medium consists of an excess amount of nutrients which is required to synthesise the desired concentration of SCP. The growth- limiting nutrient controls the steady-state SCP production rate. The data for optical density, DO level, cell dry weight and measurements of protein and carbohydrates are carried out at 8, 12, 16 and 24 hours in batch mode. The continuous operation is extended for another 24 hours to monitor all parameters and measure SCP. The results should be compared with batch-wise production. The expected results for reduction of sugar in real experiments are similar, as shown in Figure 2.1. The data plotted in Figure 2.1 were obtained by aeration of pharmaceutical wastewater. A well-known reagent for determination of carbohydrates dini- trosalicylic acid (DNS), was used to reduce the organic chemicals in the above wastewater for the course of 3 days incubation.2,3 The method of measurement will be discussed in the Ch002.qxd 10/27/2006 10:49 AM Page 15
  • 32. 16 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY following sections. If the above experiments are conducted, they may lead us to a new set of data that are totally different from Figure 2.1, and only the reduction trend would be about the same. SCP production has to be determined by experimentation, and research is needed to obtain the data. Maximum carbohydrate reduction took place after 24 hours of aeration. Since the carbon source was initially quite low, the rate of biomass production was not appreciable. Figure 2.2 shows the cell density and DO level in a pilot-scale aeration vessel. The role of dissolved oxygen in the treatment system is absolutely vital. Therefore the DO level must be maintained at not less than 3–4ppm in the wastewater for effective aeration. SCP production is very oxygen-dependent. The results would be very satisfactory if pure oxygen is used. 2.3.1 Analytical Methods for Measuring Protein Content of Baker’s Yeast (SCP) Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combina- tion with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595nm when the reagent binds to the protein. A 20mg/l bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for deter- mination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. Time, day 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Carbohydrateconcentration,mg/l 0 50 100 150 200 250 300 0.22 l/min 0.83 l/min 1.14 l/min 1.30 l/min FIG. 2.1. Reduction of carbohydrate in an aeration tank at various air flow rates. Ch002.qxd 10/27/2006 10:49 AM Page 16
  • 33. DISSOLVED OXYGEN MEASUREMENT AND MIXING 17 2.3.2 Seed Culture How do we start the real experiment? A 100ml seed culture is prepared in advance. A 100ml media consists of 0.1g yeast extract and 1g glucose with 0.33g KH2PO4 and 0.03g Na2HPO4. It is sterilised, then the microorganism, Saccharomyces cerevisiae (ATCC 24860), on aYM slant tube used as stock culture is transferred to the sterile cooled media.5 The inoc- ulated media is incubated and harvested after 24 hours. The first stage of the work is the batch experiment, which is then changed to a continuous experimental run with glucose as carbon source and the microorganism, S. cerevisiae, as an organism sensitive to aeration. The last stage demonstrates how agitation plays an important role in the mass transfer process. 2.4 BATCH EXPERIMENT FOR PRODUCTION OF BAKER’S YEAST The fermentation vessel is a jacketed vessel with working volume of 2 litres. The media is made of phosphate buffer at neutral pH with 3.3g KH2PO4 and 0.3g Na2HPO4, 1g yeast extract and 50g glucose in 1 litre of distilled water. The media should be sterilised in a 20 litre carboy. The fermentation vessel with 500ml media is initially sterilised under 15psig steam at 121 °C for 20min.6 The seed culture is transferred to the fermentation ves- sel and feed is gradually pumped in at a flow rate of 350mlhϪ1 . The filtered pure oxygen or pressurised air is continuously supplied. The production of baker’s yeast should be mon- itored for 48 hours by batch experiment at a constant temperature of 32 °C, controlling pH and monitoring DO level. Sufficient air is blown at a flow rate of 2000mlminϪ1 (1vvm).7,8 Data collection for optical density, DO level, cell dry weight, protein and carbohydrates is done at 6, 12, 18, 24, 36 and 48 hours in batch mode as projected in Table 2.1. Time, hours 0 20 40 60 80 CDWandCODconcentration,mg/l 200 300 400 500 600 700 800 900 Carbohydrateconcentration,mg/l 80 100 120 140 160 180 200 220 Disolvedoxygenconcentration,mg/l 0 2 4 6 8 CDW, mg/l COD, mg/l Carbohydrate, mg/l DO, mg/l FIG. 2.2. COD, cell dry weight (CDW), carbohydrate and dissolved oxygen concentrations in a 15 litres aeration tank at an air flow rate of 5 litres/min. Ch002.qxd 10/27/2006 10:49 AM Page 17
  • 34. 18 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY 2.5 OXYGEN TRANSFER RATE (OTR) Once batch mode studies are completed and the required data are collected, without dis- mantling the bioreactor, liquid media is prepared with 33g KH2PO4 and 3g Na2HPO4, 10g yeast extract and 500g glucose in 10 litres of distilled water. The liquid media can be steri- lised in an autoclave at 121 °C, 15psig for 20min. The liquid media is cooled down to room temperature with air flow rate of 100mlminϪ1 . The fluid residence time of 10 hours is expected to give maximum cell optical density. Otherwise, the effect of media flow rate has to be carried out separately. This is the basic assumption made in this experiment. The aim of this set of experiments is to determine a suitable air flow rate with variation from 0.025 to 1vvm. Table 2.2 shows the data collected in a continuous mode of operation for 3.5 days using isolated strains from the waste stream of a food processing plant. The time intervals for sampling are 12 hours. The steady-state condition of the system may be reached at about 10 hours. If any samples are taken at shorter time intervals, steady-state condition did not reach then overlapping in the experimental condition may occurs. TABLE 2.1. Batch production of baker’s yeast with air flow rate of 1vvm and an agitation speed of 350rpm Time, DO, Optical density, Cell dry Protein Sugar Yield, YX/S hours mg/l absorbance, weight, concentration, concentration, l520,nm mg/ml g/l g/l 0 7.9 0.00 0.00 0.00 32.0 – 6 5.6 0.28 0.14 0.83 30.0 0.42 12 4.1 0.50 0.29 4.75 21.0 0.43 18 2.4 1.55 1.10 8.90 13.0 0.47 24 1.5 2.00 1.45 11.15 5.0 0.41 36 1.1 4.20 2.00 11.95 1.7 0.40 48 0.2 4.45 2.65 12.35 0.5 0.39 TABLE 2.2. Effect of aeration rate on baker’s yeast production Air flow DO, Optical density, Cell dry Protein Sugar rate, mg/l absorbance, l520,nm weight, concentration, concentration, ml/min mg/ml mg/l g/l 50 2 0.2 0.26 1300 16.8 100 4 0.45 0.59 1450 14.5 200 6 0.69 1.24 2500 13.9 500 7.7 1/10 diluted 0.72 3.95 3000 12.5 1000 8 1/10 diluted 0.74 4.63 3100 9.8 1500 8 1/10 diluted 0.77 4.68 3350 8.6 2000 8 1/10 diluted 0.79 4.75 4800 6.7 Ch002.qxd 10/27/2006 10:49 AM Page 18
  • 35. DISSOLVED OXYGEN MEASUREMENT AND MIXING 19 2.6 RESPIRATION QUOTIENT (RQ) Measurements of inlet and outlet gas compositions of a culture vessel have been considered as an indicator for cell activities in the fermentation broth. The continuous monitoring of gas analysis would lead us to understand the oxygen consumption rate and carbon dioxide production, which originate from catabolism of carbon sources. Respiration is a sequence of biochemical reactions resulting in electrons from substances that are then transferred to an exogenous electron accepting terminal. Respiration in a cell is an energy-delivery process in which electrons are generated from oxidation of substrate and transferred through a series of oxidation–reduction reactions to electron acceptor terminals. In biosynthesis, the end products result from a respiration process. Since oxidation of carbonaceous substrate ends with carbon dioxide and water molecules, the molar ratio of carbon dioxide generated from oxidation–reduction to oxygen supplied is known as the respiration quotient: (2.6.1) There are several methods to monitor the off-gas analysis. Online gas chromatography is commonly used. The daily operation for inlet and outlet gases is balanced to project growth in the bioprocess. High operating cost is the disadvantage of the online system. For an online bioreactor a few important process variables should be monitored contin- uously. The off-gas analysis provides the most reliable information for growth activities. Measurement of oxygen and carbon dioxide in the off-gas is a fairly standard procedure used for a pilot-scale bioreactor. Knowing air flow rate and exit gas compositions or having a simple material balance can quantify oxygen uptake rate (OUR) and carbon diox- ide production rate (CPR), which would lead us to a value for RQ. The three indicators for growth can be correlated and give cell growth rate. From RQ the metabolic activity of the bioprocess and the success of a healthy operation can be predicted. The off-gas analysis will show the specific CO2 production rate, which is used to calculate oxygen consumption rate. 2.7 AGITATION RATE STUDIES In the following experiment we shall assume that the optimum air flow rate of 0.5vvm is desired. This means for an aeration vessel with a 2 litre working volume, the experiment requires 1000ml air per minute. The rest of process parameters and media conditions remain unchanged. Another 10 litres of fresh aseptic media must be prepared. The operation is continued for 3.5 days at an agitation speed from 100 to 700rpm; samples are drawn at intervals of 12 hours. Table 2.3 shows the effect of agitation rate on cell dry weight and protein production using a starchy wastewater stream. The active strain was isolated from a food-processing plant. RQ dC /d dC /d dC dC CO O CO O 2 2 2 2 ϭ ϭ t t Ch002.qxd 10/27/2006 10:49 AM Page 19
  • 36. 20 BIOCHEMICAL ENGINEERING AND BIOTECHNOLOGY Example 2.1: Calculate Cell Density in an Aerobic Culture A strain of Azotobacter vinelandii was cultured in a 15m3 stirred fermenter for the produc- tion of alginate. Under current conditions the mass transfer coefficient, kLa, is 0.18sϪ1 . Oxygen solubility in the fermentation broth is approximately 8 ϫ10Ϫ3 kgmϪ3 .9 The spe- cific oxygen uptake rate is 12.5mmolgϪ1 hϪ1 . What is the maximum cell density in the broth? If copper sulphate is accidentally added to the fermentation broth, which may reduce the oxygen uptake rate to 3mmolgϪ1 hϪ1 and inhibit the microbial cell growth, what would be the maximum cell density in this condition? The oxygen uptake rate (OUR) is defined as:10 (E.2.1) Solution We make an assumption based on the fact that all of the dissolved oxygen in the fermenta- tion broth is used or taken by microorganisms. In this case the DO goes to zero. The value for CAL can be zero since it is not given in the problem statement. Also the cell density has to be maximised. Therefore the above assumption is valid. In the above equation x represented the cell density, that is: (E.2.2) Substituting values into (E.2.2), the maximum biomass production is calculated as follows: ϭ Ϫ 12960 g/m or 2.96 g l3 1 x g h max ϭ ϫ Ϫ Ϫ Ϫ (0.18 s )(8 10 kg/m ) (12.5 mmol/ )(1 h/3600 s)(1 mol/ 1 3 3 11000 mmol)(32 g/1 mol)(1 kg/1000 g) x k aC q max * ϭ L AL O2 OUR )O L AL AL2 ϭ ϭ Ϫ( ( ) ( )* q x k a C C TABLE 2.3. Effect of agitation rate on baker’s yeast production Agitation DO, Optical density, Cell dry Protein Sugar rate, rpm mg/l absorbance, l520,nm weight, concentration, concentration, mg/ml mg/l g/l 100 2 1/10 diluted 0.22 0.29 1250 15.5 200 3 1/10 diluted 0.36 0.46 1360 14.7 300 5 1/10 diluted 0.45 0.59 1450 13.6 400 6 1/10 diluted 0.65 0.85 1850 12.1 500 8 1/10 diluted 0.79 1.12 2150 11.5 600 8 1/10 diluted 0.82 1.16 2250 10.8 700 8 1/10 diluted 0.83 1.19 2300 8.4 Ch002.qxd 10/27/2006 10:49 AM Page 20
  • 37. DISSOLVED OXYGEN MEASUREMENT AND MIXING 21 Let us assume the solubility of oxygen does not affect on CAL * or kLa, the factor affected on the oxygen uptake rate that is 12.5/3 ϭ4.167, then xmax is: xmax ϭ(12.96)(4.167) ϭ54glϪ1 To achieve the calculated cell densities, other conditions must be favourable, such as substrate concentration and sufficient time. 2.8 NOMENCLATURE CAL * Equilibrium concentration of A at the liquid phase, mmol/g CAL Concentration of A at liquid phase, mmol/g CPR Carbon dioxide production rate, mmol/gиs kLa Mass transfer coefficient at liquid phase, sϪ1 OUR Oxygen uptake rate, mmol/gиs RQ Respiration quotient, mmol CO2/mmol O2 x Biomass concentration, mg/l xmax The maximum biomass production, sϪ1 Specific oxygen uptake rate, sϪ1 REFERENCES 1. Wang, D.I.C. Cooney, C.L. Deman, A.L. Dunnill, P. Humphrey, A.E. and Lilly, M.D., “Fermentation and Enzyme Technology”. John Wiley & Sons, New York, 1979. 2. Miller, G.L., Analyt. Chem. 31, 426 (1959). 3. Thomas, L.C. and Chamberlin, G.J., “Colorimetric Chemical Analytical Methods”. Tintometer Ltd, Salisbury, United Kingdom, 1980. 4. Bradford, M.M., J. Analyt. Biochem. 72, 248 (1976). 5. Pelczar, M.J. Chan, E.C.S. and Krieg, N.R., “Microbiology”. McGraw Hill, New York, 1986. 6. Scragg, A.H., “Bioreactors in Biotechnology, A Practical Approach”. Ellis Horwood Series in Biochemistry and Biotechnology, New York, 1991. 7. Ghose, T.K., “Bioprocess Computation in Biotechnology”, vol. 1. Ellis Horwood Series in Biochemistry and Biotechnology, New York, 1990. 8. Doran, P.M., “Bioprocess Engineering Principles”. Academic Press, New York, 1995. 9. Shuler, M.L. and Kargi, F., “Bioprocess Engineering, Basic Concepts”. Prentice Hall, New Jersey, 1992. 10. Baily, J.E. and Ollis, D.F., “Biochemical Engineering Fundamentals”, 2nd edn. McGraw-Hill, NewYork, 1986. qO2 Ch002.qxd 10/27/2006 10:49 AM Page 21
  • 38. 22 CHAPTER 3 Gas and Liquid System (Aeration and Agitation) 3.1 INTRODUCTION In the biochemical engineering profession, there are various bioprocesses actively involved in the synthesis and production of biological products. Understanding of all the processes may require basic knowledge of biology, biochemistry, biotechnology, and real knowledge of engineering processes. Transfer of oxygen is a major concern in many bioprocesses that require air for microbial growth such as single cell protein and production of antibiotics. Agitation in a fermentation unit is directly related to oxygen transported from the gas phase to liquid phase followed by oxygen uptake by the individual microbial cell. The activities of microorganisms are monitored by the utilisation of oxygen from the supplied air and the respiration quotient. The primary and secondary metabolites in a bioprocess can be estimated based on projected pathways for production of intracellular and extracellular by-products. In the previous chapter, dissolved oxygen was discussed; in this chapter, mechanisms of oxygen transport are focused on. The details of process operation are also discussed in this chapter. 3.2 AERATION AND AGITATION Aeration and agitation are implemented in most fermentation processes. The word ‘aerobe’ refers to the kind of microorganism that needs molecular oxygen for growth and metabo- lism. ‘Aerobic’is the condition of living organisms surviving only in the presence of molec- ular oxygen. Aerobic bacteria require oxygen for growth and can be incubated to be grown in atmospheric air. Oxygen is a strong oxidising agent which has the ability to accept elec- trons for yielding energy, a process known as respiration. A bioreactor is a reaction vessel in which an organism is cultivated in a controlled manner to produce cell bodies and/or product. Initially the term ‘fermenter’ was used to describe these vessels, but in strict terms, fermentation is an anaerobic process whereas the major proportion of fermenters use aero- bic processes. Thus, in general terms, ‘bioreactor’ means a vessel in which organisms are grown under aerobic or anaerobic conditions. If a bioreactor or a reaction vessel operates under aerating conditions, the system is called an aerobic bioreactor. Sterile air is supplied Ch003.qxd 10/27/2006 10:47 AM Page 22

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