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BIOCHEMICAL ENGINEERING FUNDAMENTALS

McGraw-Hill Chemical Engineering Series Editorial Advisory Board James J. Carberry, Professor of Chemical Engineering, University of Notre Dame James R. Fair, Professor of Chemical Engineering, University of Texas, Austin Max S. Peters, Professor of Chemical Engineering, University of Colorado William R. Schowalter, Professor of Chemical Engineering, Princeton University James Wei, Professor of Chemical Engineering, Massachusetts Institute of Technology

BUILDING THE LITERATURE OF A PROFESSION Fifteen prominent chemical engineers first met in New York more than 60 years ago to plan a continuing literature for their rapidly growing profession. From industry came such pioneer practitioners as Leo H. Baekeland, Arthur D. Little, Charles L. Reese, John V. N. Dorr, M. C. Whitaker, and R. S. McBride. From the universities came such eminent educators as William H. Walker, Alfred H. White, D. D. Jackson, J. H. James, Warren K. Lewis, and Harry A. Curtis. H. C. Parmelee, then editor of Chemical and Metallurgical Engineering, served as chairman and was joined subsequently by S. D. Kirkpatrick as consulting editor. After several meetings, this committee submitted its report to the McGraw-Hill Book Company in September 1925. In the report were detailed specifications for a correlated series of more than a dozen texts and reference books which have since become the McGraw-Hill Series in Chemical Engineering and which became the cornerstone of the chemical engineering curriculum. From this beginning there has evolved a series of texts surpassing by far the scope and longevity envisioned by the founding Editorial Board. The McGraw-Hill Series in Chemical Engineering stands as a unique historical record of the development of chemical engineering education and practice. In the series one finds the milestones of the subject’s evolution: industrial chemistry, stoichiometry, unit operations and processes, thermodynamics, kinet­ ics, and transfer operations. Chemical engineering is a dynamic profession, and its literature continues to evolve. McGraw-Hill and its consulting editors remain committed to a publishing policy that will serve, and indeed lead, the needs of the chemical engineering profession during the years to come.

THE SERIES Bailey and Ollis: Biochemical Engineering Fundamentals Bennett and Myers: Momentum, Heat, and Mass Transfer Beveridge and Schechter: Optimization: Theory and Practice Carberry: Chemical and Catalytic Reaction Engineering Churehill: The Interpretation and Use of Rate Data— The Rate Concept Clarke and Davidson: Manual for Process Engineering Calculations Coughanowr and Koppel: Process Systems Analysis and Control Daubert: Chemical Engineering Thermodymanics Fahien: Fundamentals o f Transport Phenomena Finlayson: Nonlinear Analysis in Chemical Engineering Gates, Katzer9 and Schuit: Chemistry o f Catalytic Processes Holland: Fundamentals of Multicomponent Distillation Holland and Liapis: Computer Methods for Solving Dynamic Separation Problems Johnson: Automatic Process Control Johnstone and Thring: Pilot Plants, Models, and Scale-Up Methods in Chemical Engineering Katz, Cornell, Kobayashi, Poettmann, Vary, Elenbaas, and Weinaug: Handbook of Natural Gas Engineering King: Separation Processes Klinzing: Gas-Solid Transport Knudsen and Katz: Fluid Dynamics and Heat Transfer Luyben: Process Modeling, Simulation, and Control for Chemical Engineers McCabe, Smith, J. C., and Harriott: Unit Operations of Chemical Engineering Mickley, Sherwood, and Reed: Applied Mathematics in Chemical Engineering Nelson: Petroleum Refinery Engineering Perry and Chilton (Editors): Chemical Engineers' Handbook Peters: Elementary Chemical Engineering Peters and Timmerhaus: Plant Design and Economics for Chemical Engineers Probstein and Hicks: Synthetic Fuels Ray: Advanced Process Control Reid, Prausnitz, and Sherwood: The Properties of Gases and Liquids Resnick: Process Analysis and Design for Chemical Engineers Satterfield: Heterogeneous Catalysis in Practice Sherwood, Pigford, and Wilke: Mass Transfer Smith, B. D.: Design of Equilibrium Stage Processes Smith, J. M.: Chemical Engineering Kinetics Smith, J. M., and Van Ness: Introduction to Chemical Engineering Thermodynamics Thompson and Ceckler: Introduction to Chemical Engineering Treybal: Mass Transfer Operations Valle-Riestra: Project Evolution in the Chemical Process Industries Van Ness and Abbott: Classical Thermodynamics of Nonelectrolyte Solutions: With Applications to Phase Equilibria Van Winkle: Distillation Volk: Applied Statistics for Engineers WaIas: Reaction Kinetics for Chemical Engineers Wei, Russell, and Swartzlander: The Structure of the Chemical Processing Industries Whitwell and Toner: Conservation of Mass and Energy

■M i!. .*

BIOCHEMICAL ENGINEERING FUNDAMENTALS Second Edition

James E. Bailey California Institute of Technology

David F. Ollis North Carolina State University

X-

McGraw-Hill, Inc.

New York St. Louis San Francisco Auckland Bogotá Caracas Lisbon London Madrid Mexico City Milan Montreal New Delhi San Juan Singapore Sydney Tokyo Toronto

lis book was set in Times Roman. tie editors were Kiran Verma and Cydney C. Martin. tie production supervisor was Diane Renda; ie cover was designed by John Hite; •oject supervision was done by Albert Harrison, Harley Editorial Services.

IOCHEMICAL ENGINEERING FUNDAM ENTALS opyright © 1986, 1977 by McGraw-Hill, Inc. All rights reserved, rinted in the United States of America. Except as permitted under the United States ,'opyright Act of 1976, no part of this publication may be reproduced or distributed i any form or by any means, or stored in a data base or retrieval system, without ie prior written permission of the publisher. 10 11 12 13 14 BKMBKM 9 9 8 7 6 5 4

;SBN 0 -Q 7 -Q 0 3 2 ia -S library of Congress Cataloging-in-Publication Data lailey, James E. (James Edwin), 1944Biochemical engineering fundamentals. (McGraw-Hill chemical engineering series) Includes bibliographies and index. I. Biochemical engineering. I. Ollis, David F. I. Title. III. Series 85-19744 ΓΡ248.3.Β34 1986 660\63 SBN 0-07-003212-2

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CONTENTS

Preface Chapter I A Little Microbiology LI 1.2

1.3

1.4

Biophysics and the Cell Doctrine The Structure of Cells 1.2.1 Procaryotic Cells 1.2.2 Eucaryotic Cells 1.2.3 CellFractionation Example LI: Analysis of Particle Motion in a Centrifuge Important Cell Types 1.3.1 Bacteria 1.3.2 Yeasts 1.3.3 Molds 1.3.4 Algae and Protozoa 1.3.5 Animal and Plant Cells A Perspective for Further Study Problems References

Chapter 2 Chemicals of Life 2.1

2.2

2.3

2.4

Lipids 2.1.1 Fatty Acids and Related Lipids 2.1.2 Fat-soluble Vitamins, Steroids, and Other Lipids Sugars and Polysaccharides 2.2.1 D-Glucose and Other Monosaccharides 2.2.2 Disaccharides to Polysaccharides 2.2.3 Cellulose From Nucleotides to RNA and DNA 2.3.1 Building Blocks, an Energy Carrier, and Coenzymes 2.3.2 Biological Information Storage: DNA and RNA Amino Acids into Proteins 2.4.1 Amino Acid Building Blocks and Polypeptides

xix i 3 3 3 6 9 9 12 13 16 18 21 22 24 24 26 27 29 29 32 34 34 36 38 42 42 46 53 55 ix

CONTENTS

2.5

2.6

2.4.2 Protein Structure 2.4.3 Primary Structure 2.4.4 Three-Dimensional Conformation: Secondary and Tertiary Structure 2.4.5 Quaternary Structure and Biological Regulation Hybrid Biochemicals 2.5.1 Cell Envelopes: Peptidoglycan and Lipopolysaccharides 2.5.2 Antibodies and Other Glycoproteins The Hierarchy of Cellular Organization Problems References

Chapter 3 The Kinetics of Enzyme-Catalyzed Reactions 3.1 The Enzyme-Substrate Complex and Enzyme Action 3.2 Simple Enzyme Kinetics with One and Two Substrates 3.2.1 Michaelis-Menten Kinetics 3.2.2 Evaluation of Parameters in the Michaelis-Menten Equation 3.2.3 Kinetics for Reversible Reactions, Two-Substrate Reactions, and Cofactor Activation 3.3 Determination of Elementary-Step Rate Constants 3.3.1 RelaxationKinetics 3.3.2 SomeResultsofTransient-KineticsInvestigation 3.4 Other Patterns of Substrate Concentration Dependence 3.4.1 SubstrateA ctivationandInhibition 3.4.2 Multiple Substrates Reacting on a Single Enzyme 3.5 Modulation and Regulation of EnzymaticActivity 3.5.1 TheM echanism sofReversibleEnzym eM odulation 3.5.2 Analysis of Reversible M odulator Effects on Enzyme Kinetics 3.6 Other Influences on Enzyme Activity 3.6.1 The Effect of pH on Enzyme Kinetics in Solution 3.6.2 Enzyme Reaction Rates and Temperature 3.7 Enzyme Deactivation 3.7.1 M echanism sandM anifestationsofProteinD enaturation 3.7.2 Deactivation Models and Kinetics 3.7.3 Mechanical Forces Acting on Enzymes 3.7.4 Strategies for Enzyme Stabilization 3.8 Enzyme Reactions in Heterogeneous Systems Problems References

Chapter 4 Applied Enzyme Catalysis 4.1

ApplicationsofHydrolyticEnzymes 4.1.1 Hydrolysis of Starch and Cellulose Example 4.1: Influence of Crystallinity on Enzymatic Hydrolysis o f Cellulose 4.1.2 Proteolytic Enzymes 4.1.3 Esterase Applications

60 61 63 68 70 71 74 76 79 85

86 92 95 101 105 108 111 111 114 114 115 116 120 122 124 129 130 132 135 136 136 144 146 148 152 156 157 161 163 169 172 175

CONTENTS Xl

4.1.4

4.2

4.3

4.4

4.5

Enzyme Mixtures, Pectic Enzymes, and Additional Applications Other Applications of Enzymes in Solution 4.2.1 MedicalApplicationsofEnzymes 4.2.2 Nonhydrolytic Enzymes in Current and Developing Industrial Technology Immobilized-Enzyme Technology 4.3.1 Enzyme Immobilization 4.3.2 Industrial Processes 4.3.3 Medical and Analytical Applications of Immobilized Enzymes 4.3.4 Utilization and Regeneration of Cofactors Immobilized Enzyme Kinetics 4.4.1 EffectsofExternalM ass-TransferResistance 4.4.2 Analysis of Intraparticle Diffusion and Reaction Example 4.2: Estimation of Diffusion and Intrinsic Kinetic Parameters for an Immobilized Enzyme Catalyst 4.4.3 Simultaneous Film and Intraparticle Mass-Transfer Resistances 4.4.4 Effects of Inhibitors, Temperature, and pH on Immobilized Enzyme Catalytic Activity and Deactivation Concluding Remarks Problems References

Chapter 5 Metabolic Stoichiometry and Energetics 5.1 5.2

5.3

5.4

5.5

5.6

5.7

5.8

Thermodynamic Principles Metabolic Reaction Coupling: ATP and NAD 5.2.1 ATP and Other Phosphate Compounds 5.2.2 Oxidation and Reduction: Coupling via NAD Carbon Catabolism 5.3.1 Embden-Meyerhof-Parnas Pathway 5.3.2 Other Carbohydrate Catabolic Pathways Respiration 5.4.1 TheTCA Cycle 5.4.2 The Respiratory Chain Photosynthesis: Tapping the Ultimate Source 5.5.1 Light-Harvesting 5.5.2 Electron Transport and Photophosphorylation Biosynthesis 5.6.1 Synthesis of Small Molecules 5.6.2 Macromolecule Synthesis Transport across Cell Membranes 5.7.1 Passive and Facilitated Diffusion 5.7.2 Active Transport Metabolic Organization and Regulation 5.8.1 Key Crossroads and Branch Points in Metabolism 5.8.2 Enzyme Level Regulation of Metabolism

176 177 177 179 180 181 189 194 199 202 204 208

216 218 220 222 222 226 228 233 235 235 237 239 239 241 245 246 246 251 251 252 253 254 261 262 263 265 269 270 271

KU CONTENTS

5.9

5.10

5.11

Chapter 6 6.1

6.2

6.3

6.4

End Products of Metabolism 5.9.1 Anaerobic Metabolism (Fermentation) Products 5.9.2 Partial Oxidation and Its End Products 5.9.3 Secondary Metabolite Synthesis Stoichiometry of Cell Growth and Product Formation 5.10.1 Overall Growth Stoichiometry: Medium Formulation and Yield Factors 5.10.2 Elemental Material Balances for Growth 5.10.3 Product Formation Stoichiometry 5.10.4 Metabolic Energy Stoichiometry: Heat Generation and Yield Factor Estimates 5.10.5 Photosynthesis Stoichiometry Concluding Remarks Problems References

273 274 275 277 277 280 285 289

Molecular Genetics and Control Systems

307

Molecular Genetics 6.1.1 The Processes of Gene Expression 6.1.2 Split Genes and mRNA Modification in Eucaryotes 6.1.3 Posttranslational Modifications of Proteins 6.1.4 Induction and Repression: Control of Protein Synthesis 6.1.5 DNA Replication and M utation 6.1.6 O verview oflnform ationF low intheC ell Alteration of Cellular DNA 6.2.1 Virus and Phages: Lysogeny and Transduction 6.2.2 Bacterial Transformation and Conjugation 6.2.3 Cell Fusion Commercial Applications of Microbial Genetics and Mutant Populations 6.3.1 Cellular Control Systems: Implications for Medium Formulation 6.3.2 Utilization of Auxotrophic Mutants 6.3.3 Mutants with Altered Regulatory Systems Recombinant DNA Technology 6.4.1 Enzymes for Manipulating DNA 6.4.2 Vectors for Escherichia coli 6.4.3 Characterization of Cloned DNAs 6.4.4 Expression of Eucaryotic Proteins in E. coli 6.4.5 Genetic Engineering Using Other Host Organisms 6.4.6 Concluding Remarks Growth and Reproduction of a Single Cell 6.5.1 Experimental Methods: Flow Cytometry and Synchronous Cultures 6.5.2 The Cell Cycle of E. coli 6.5.3 The Eucaryotic Cell Cycle Problems References

307 308 314 316 317 321 326 327 327 330 332

292 297 300 300 305

335 335 336 339 340 341 345 346 349^ 353 356 357 358 360 361 364 370

CONTENTS Χϋί

Chapter 7

7.3

7.4

7.5

7.6 7.7 7.8

Kinetics of Substrate Utilization, Product Formation, and Biomass Production in Cell Cultures Ideal Reactors for Kinetics Measurements 7.1.1 The Ideal Batch Reactor 7.1.2 The Ideal Continuous-Flow Stirred-Tank Reactor (CSTR) Kinetics of Balanced Growth 7.2.1 Monod Growth Kinetics 7.2.2 Kinetic Implications of Endogenous and Maintenance Metabolism 7.2.3 Other Forms of Growth Kinetics 7.2.4 Other Environmental Effects on Growth Kinetics Transient Growth Kinetics 7.3.1 Growth-Cycle Phases for Batch Cultivation 7.3.2 Unstructured Batch Growth Models 7.3.3 Growth of Filamentous Organisms Structured Kinetic Models 7.4.1 Compartmental Models 7.4.2 Metabolic Models 7.4.3 Modeling Cell Growth as an Optimum Process Product Formation Kinetics 7.5.1 Unstructured Models Example 7.1: Sequential Parameter Estimation for a Simple Batch Fermentation 7.5.2 Chemically Stuctured Product Formation Kinetics Models 7.5.3 Product Formation Kinetics Based on Molecular Mechanisms: Genetically Structured Models 7.5.4 Product Formation Kinetics by Filamentous Organisms Example 7.2: A Morphologically Structured Kinetic Model for Cephalosporin C Production Segregated Kinetic Models of Growth and Product Formation Thermal-Death Kinetics of Cells and Spores Concluding Remarks Problems References

Chapter 8 Transport Phenomena in Bioprocess Systems 8.1

8.2 8.3

8.4

Gas-Liquid Mass Transfer in Cellular Systems 8.1.1 Basic Mass-Transfer Concepts 8.1.2 Rates of Metabolic Oxygen Utilization Determination of Oxygen Transfer Rates 8.2.1 Measurement of Itla' Using Gas-Liquid Reactions Mass Transfer for Freely Rising or Falling Bodies 8.3.1 Mass-Transfer Coefficients for Bubbles and Bubble Swarms 8.3.2 Estimation of Dispersed Phase Interfacial Area and Holdup Example 8.1: HoldupCorrelations Forced Convection Mass Transfer 8.4.1 General Concepts and Key Dimensionless Groups

373 378 378 380 382 383 388 391 392 394 394 403 405 408 409 413 418 421 421 424 426 429 432 434 438 441 445 446 454 457 459 460 467 470 470 473 473 476 482 484 484

XÍV CONTENTS

8.4.2

8.5 8.6 8.7

8.8

8.9 8.10

8.11

Correlations for Mass-Transfer Coefficients and Interfacial Area Example 8.2: Correlations for Maximum (Dc) or Sauter Mean (Dsm) Bubble or Droplet Diameters Overall kxd Estimates and Power Requirements for Sparged and Agitated Vessels Mass Transfer Across Free Surfaces Other Factors Affecting kta' 8.7.1 EstimationofDiffusivities 8.7.2 Ionic Strength 8.7.3 Surface Active Agents Non-Newtonian Fluids 8.8.1 Models and Parameters for Non-Newtonian Fluids 8.8.2 Suspensions 8.8.3 Macromolecular Solutions 8.8.4 Power Consumption and Mass Transfer in Non-Newtonian Fluids Scaling of Mass-Transfer Equipment HeatTransfer 8.10.1 Heat-Transfer Correlations Example 8.3: Heat Transfer Correlations SterilizationofG asesandL iquidsbyFiltration Problems References

Chapter 9 Design and Analysis of Biological Reactors 9.1

9.2

9.3

9.4

9.5

Ideal Bioreactors 9.1.1 Fed-Batch Reactors 9.1.2 Enzyme-Catalyzed Reactions in CSTRs 9.1.3 CSTR Reactors with Recycle and Wall Growth 9.1.4 The Ideal Plug-Flow Tubular Reactor Reactor Dynamics 9.2.1 Dynamic Models 9.2.2 Stability Reactors with Nonideal Mixing 9.3.1 Mixing Times in Agitated Tanks 9.3.2 Residence Time Distributions 9.3.3 Models for Nonideal Reactors 9.3.4 Mixing-Bioreaction Interactions Example 9.1: Reactor Modeling and Optimization for Production of a-Galactosidase by a Monascus sp. Mold Sterilization Reactors 9.4.1 Batch Sterilization 9.4.2 Continuous Sterilization Immobilized Biocatalysts 9.5.1 Formulation and Characterization of Immobilized Cell Biocatalysts 9.5.2 Applications of Immobilized Cell Biocatalysts

486 487 488 495 498 498 499 500 501 501 502 504 505 508 512 517 521 522 523 529

533 535 536 537 539 541 544 545 547 551 551 553 560 573 S 578 586 587 592 595 598 601

CONTENTS XV

9.6

9.7

9.8

9.9

C h a p te r 10 10.1

10.2 10.3

10.4

10.5

10.6

10.7

10.8

Multiphase Bioreactors 9.6.1 ConversionofHeterogeneousSubstrates Example 9.2: Agitated-CSTR Design for a Liquid-Hydrocarbon Fermentation 9.6.2 Packed-Bed Reactors 9.6.3 Bubble-ColumnBioreactors 9.6.4 Fluidized-Bed Bioreactors 9.6.5 Trickle-Bed Reactors Fermentation Technology 9.7.1 Medium Formulation 9.7.2 Design and Operation of a Typical Aseptic, Aerobic Fermentation Process 9.7.3 Alternate Bioreactor Configurations Animal and Plant Cell Reactor Technology 9.8.1 Environmental Requirements for Animal Cell Cultivation 9.8.2 Reactors for Large-Scale Production Using Animal Cells 9.8.3 Plant Cell Cultivation Concluding Remarks Problems References

606 607

Instrumentation and Control

658

Physical and Chemical Sensors for the Medium and Gases 10.1.1 Sensors of the Physical Environment 10.1.2 Medium Chemical Sensors Example 10.1: Electrochemical Determination of k¡a 10.1.3 Gas Analysis On-Line Sensors for Cell Properties Off-Line Analytical Methods 10.3.1 Measurements of Medium Properties 10.3.2 Analysis of Cell Population Composition Computers and Interfaces 10.4.1 ElementsofDigitalCom puters 10.4.2 Computer Interfaces and Peripheral Devices 10.4.3 Software Systems D ata Analysis 10.5.1 Data Smoothing and Interpolation 10.5.2 State and Parameter Estimation Process Control 10.6.1 Direct Regulatory Control 10.6.2 Cascade Control of Metabolism Advanced Control Strategies 10.7.1 Programmed Batch Bioreaction 10.7.2 Design and Operating Strategies for Batch Plants 10.7.3 Continuous Process Control Concluding Remarks Problems References

658 659 661 664 669 670 674 674 676 684 685 687 691 693 693 695 698 698 700 703 704 711 713 717 718 722

607 609 610 614 617 620 620 622 626 630 631 633 641 643 644 653

ivi CONTENTS

Chapter 11 11.1

11.2

11.3

11.4 11.5

11.6 11.7

11.8

11.9

Product Recovery Operations

726

Recovery of Particulates: Cells and Solid Particles 11.1.1 Filtration 11.1.2 Centrifugation 11.1.3 Sedimentation 11.1.4 EmergingTechnologiesforCellRecovery 11.1.5 Summary ProductIsolation 11.2.1 Extraction 11.2.1.1 SolventExtraction 11.2.1.2 ExtractionusingAqueousTwo-PhaseSystems 11.2.2 Sorption Precipitation Example 11.1: Procedures for Isolation of Enzymes from Isolated Cells 11.3.1 K ineticsofPrecipitateForm ation Chromatography and Fixed-BedAdsorption: Batch Processing with Selective Adsorbates M embraneSeparations 11.5.1 ReverseOsmosis 11.5.2 Ultrafiltration Electrophoresis CombinedOperations 11.7.1 Immobilization 11.7.2 W holeBrothProcessing 11.7.3 MassRecycle ProductRecoveryTrains 11.8.1 Commercial Enzymes 11.8.2 Intracellular Foreign Proteins from Recombinant E. coli 11.8.3 Polysaccharide and BiogumRecovery 11.8.4 Antibiotics 11.8.5 OrganicAcids 11.8.6 Ethanol 11.8.7 Single-CellProtein Summary Problems References

728 730 733 734 736 738 738 738 739 741 741 745

Chapter 12 Bioprocess Economics 12.1 12.2 12.3 12.4 12.5

Process Economics Bioproduct Regulation General Fermentation ProcessEconomics A Complete Example FineChemicals 12.5.1 Enzymes 12.5.2 ProteinsviaRecom binantD N A 12.5.3 Antibiotics 12.5.4 Vitamins, Alkaloids, Nucleosides, Steroids 12.5.5 MonoclonalAntibodies(MAb)

749 749 753 764 764 767 770 770 772 772 774 775 776 778 782 782 785 786 786 788 789 796 798 799 801 802 804 815 816 816 818 822 826

< CONTENTS XVÜ

12.6

12.7 12.8 12.9

Bulk Oxygenates 12.6.1 Brewing and Wine Making 12.6.2 Fuel Alcohol Production 12.6.3 Organic and Amino Acid Manufacture Single-Cell Protein (SCP) Anaerobic Methane Production Overview Problems References

Chapter 13 Analysis of Multiple Interacting Microbial Populations 13.1 13.2 13.3

13.4

13.5

13.6

Neutralism, Mutualism, Commensalism, and Amensalism ClassificationoflnteractionsBetweenTwoSpecies Example 13.1: Two-Species Dynamics near a Steady State Competition: Survival of the Fittest 13.3.1 Volterra’s Analysis of Competition 13.3.2 Competition and Selection in a Chemostat Example 13.2: Competitive Growth in Unstable Recombinant Cultures Predation and Parasitism 13.4.1 The Lotka-Volterra Model of Predator-Prey Oscillations 13.4.2 A Multispecies Extension of the Lotka-Volterra Model 13.4.3 Other One-Predator-One-Prey Models Example 13.3: Model Discrimination and Development via Stability Analysis Effects of the Number of Species and Their Web of Interactions 13.5.1 Trophic Levels, Food Chains, and Food Webs: Definitions and an Example 13.5.2 Population Dynamics in Models of Mass-Action Form Example 13.4: An Application of the Mass-Action Theory 13.5.3 Qualitative Stability Example 13.5: Qualitative Stability of a Simple Food Web 13.5.4 Stability of Large, Randomly Constructed Food Webs 13.5.5 Bifurcation and Complicated Dynamics Spatial Patterns Problems References

Chapter 14 Mixed Microbial Populations in Applications and Natural Systems 14.1

14.2

Uses of Well-Defined Mixed Populations Example 14.1: Enhanced Growth of Methane-Utilizing Pseudomonas sp. due to Mutualistic Interactions in a Chemostat Spoilage and Product Manufacture by Spontaneous Mixed Cultures

827 830 831 835 839 847 849 849 852

854 854 860 862 864 865 867 870 871 872 876 876 879 883 883 885 888 888 889 890 892 892 896 900

903 903

907 911

CONTENTS

14.3

14.4

Microbial Participation in the Natural Cycles of Matter 14.3.1 Overall Cycles of the Elements of Life 14.3.2 Interrelationships of Microorganisms in the Soil and Other Natural Ecosystems Biological Wastewater Treatment 14.4.1 Wastewater Characteristics 14.4.2 The Activated-Sludge Process 14.4.3 Design and Modeling of Activated-Sludge Processes 14.4.4 Aerobic Digestion 14.4.5 Nitrification Example 14.2: Nitrification Design 14.4.6 Secondary Treatment Using a Trickling Biological Filter 14.4.7 Anaerobic Digestion 14.4.8 Mathematical Modeling of Anaerobic-Digester Dynamics Example 14.3: Simulation Studies of Control Strategies for Anaerobic Digesters 14.4.9 Anaerobic Denitrification 14.4.10 Phosphate Removal Problems References

Index

913 914 916 919 923 926 929 938 938 939 940 943 946 954 957 957 958 963

965

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PREFACE

Processing of biological materials and processing using biological agents such as cells, enzymes, or antibodies are the central domains of biochemical engineering. Success in biochemical engineering requires integrated knowledge of governing biological properties and principles and of chemical engineering methodology and strategy. Work at the forefront captures the latest, best information and technology from both areas and accomplishes new syntheses for bioprocess design, operation, analysis, and optimization. Reaching this objective clearly requires years of careful study and practice. This textbook is intended to start its readers on this challenging and exciting path. Central concepts are defined and explained in the context of process applications. Principles of current bioprocesses for reaction and separation are presented. Special attention is devoted throughout to the central roles of biological properties in facilitating and enabling desired process objectives. Also, process constraints and limitations imposed by sensitivities and instabilities of biological components are highlighted. By focusing on pertinent fundamental principles in the biological and engineering sciences and by repeatedly emphasizing the impor­ tance of their syntheses, the text seeks to endow its readers with a strong foundation for future study and practice. Learning fundamental properties and mechanisms on an ongoing basis is absolutely essential for long-term professional viability in a technically vibrant area such as biotechnology. The book has been written for the first course in biochemical engineering for senior or graduate students in chemical engineering. However, selected portions of the text can provide bases for other courses in chemical, environmental, civil, or food engineering. As in the first edition, the book is presented in a systematic, logical sequence building from the most fundamental biological concepts. It is therefore well suited for self study by industrial practitioners. To facilitate the book’s accessibility for independent reading and to provide required background in a one- or two-term course taken as an elective or introduction, the text includes a self-contained presentation of key concepts from xix

XX PREFACE

biochemistry, cell biology, enzyme kinetics, and molecular genetics. Clearly, this treatment is intended as an introductory exposure to these topics and not as complete coverage of the life science fundamentals needed by those who will study biochemical engineering in depth or who practice in the field. Further formal or self study in biological fundamentals and practical properties is essential in these cases. Throughout, we have tried to interweave descriptive material on the life sciences with engineering processes and analytical techniques. The implications of bioscience fundamentals for bioprocess engineering are frequently indicated in sections dealing with biological principles. Treatment of engineering analysis is presented after required descriptive, background material has been covered. Thus, enzyme kinetics and reaction engineering are introduced immediately following description of proteins and other biochemicals, and cell kinetics follows description of metabolic pathway structure, stoichiometry, and regulation. Text examples and end-of-chapter problems provide the student with oppor­ tunities to apply the concepts presented and to broaden understanding of the subject. More than 150 problems, spanning a range of difficulty, require discus­ sions, derivations, and/or calculations by the student. Compelling motivations for this second edition have come from explosive developments in the biological sciences which provide revolutionary new organ­ isms and materials with tremendous promise for new products and processes. Recombinant DNA and hybridoma technology have stimulated a new biotech­ nology industry. The text has been expanded and updated to present the mate­ rials and methods of gene cloning and expression and cell fusion. New process challenges and strategies for large-scale manufacture of new, ultra-pure protein products are summarized. Several engineering topics have received greater emphasis in the second edition. This is immediately apparent from the new chapters on separation processes, bioprocess instrumentation and control, and bioprocess economics. Important new topics such as metabolic stoichiometry, multiphase reactor engi­ neering, and animal and plant cell reactor technology have also been integrated into the earlier text. In addition, the opportunity of preparing a second edition has enabled numerous improvements in organization and presentation of material included in the first edition. This contributes, for example, to more concise yet more informa­ tive description of background material, and to a more systematic approach to stoichiometry, kinetics, and bioreactor design. The importance of coalescence and dispersion processes in multiphase reactor contacting exemplifies another area of^ enhanced presentation. Cogent and critical comments on the second edition from Michael Shuler, Douglas LauITenbergcr, Peter Reilly, Frances Arnold, Donald Kirwan, and Elmer Gaden provided many improvements. Numerous colleagues and current and former students including Dinesh Arora, Ruben Carbonell, Douglas Clark, Kathy Dennis, Jorge Galazzo, L. Gary Leal, Sun Bok Lee, Harold Monbouquette, Mustafa Ozilgcn, Steven Peretti, Alcx Seressiotis, Robert Siegel, Friedrich Srienc, and Gregory Stcphanopoulos contributed ideas, background research, and/or new

PRKhACK Xxl

homework exercises to the second edition. To those who contributed in numerous ways to the first edition, including Peter Reilly, Elmer Gaden, Harold Bungay, Murray Moo-Young, and George Tsao, we again offer our thanks. Of course t lie authors take full responsibility for any errors, and welcome comments and suggestions from readers. This book would not exist without the patient, steadfast efforts of April Olson, Kathy Lewis, Heidi Youngkin, Sandra Cantrell, Bessie See, and Kathy Cannady who typed the several drafts. Hundreds of hours of proofreading assistance were generously donated by Doug Axe, Nancy da Silva, Jorge Galazzo, Chris Guskc, Justin Ip, Anne McQueen, Kim O’Connor, Steve Peretti, Mike Prairie, Todd Przybycien, Ken Reardon, Jin-Ho Seo, Alex Seressiotis, Jackie Shanks, Friedrich Srienc, and Dane Wittrup. Finally, we would like to extend our heartfelt gratitude to many friends, colleagues, students, and sponsors who have stimulated our development as biochemical engineers in the years since the first edition. They are in many ways the true authors of this book. James E. Bailey David F. Ollis

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