Bioprocess Engineering: Kinetics, Sustainability, and Reactor Design

Bioprocess Engineering

Kinetics, Sustainability, and Reactor Design

Second Edition

Shijie Liu

Suny Esf Department of Paper and Bioprocess Engineering, Syracuse, NY, USA

Table of Contents

Cover image

Title page

Copyright

Preface to the Second Edition

Preface to the First Edition

Acronyms, Abbreviations, and Symbols

Chapter 1: Introduction

Abstract

1.1 Biological Cycle

1.2 Green Chemistry

1.3 Sustainability

1.4 Biorefinery

1.5 Biotechnology and Bioprocess Engineering

1.6 Mathematics, Biology, and Engineering

1.7 The Story of Penicillin: The Dawn of Bioprocess Engineering

1.8 Bioprocesses: Regulatory Constraints

1.9 The Pillars of Bioprocess Kinetics and Systems Engineering

1.10 Summary

A. Green Chemistry

B. Sustainability

C. Biorefinery

D. History of Penicillin

E. Regulatory Issues

Problems

Chapter 2: An Overview of Biological Basics

Abstract

2.1 Cells and Organisms

2.2 Stem Cell

2.3 Cell Chemistry

2.4 Cell Feed

2.5 Summary

Problems

Chapter 3: An Overview of Chemical Reaction Analysis

Abstract

3.1 Chemical Species

3.2 Chemical Reactions

3.3 Reaction Rates

Example 3.1: Is Sodium Hydroxide Reacting?

Example 3.2: Arrhenius Law

Example 3.3: Elemental Balance

Example 3.4: Yield Factors

Example 3.5: Fermented Rice

Example 3.6: Cooling of Fermentation Medium

Example 3.7: Heat and Material Balances in Combustion

Example 3.8: Economic Analysis

Problems

Chapter 4: Batch Reactor

Abstract

4.1 Isothermal Batch Reactors

Example 4.1: Constant Volume Batch Reactor

Example 4.2: Variable Volume Batch Reactor

Example 4.3: Concentration Profile for a Series Reaction

Example 4.4: Batch Reactor Sizing

Example 4.5: Optimum Batch Reactor Size

Example 4.6: H-Factor

Example 4.7: Hot Water Extraction of Woodchips

Example 4.8: Concentration Profile for a Series Reaction With Feedback Regulation

Problems

Chapter 5: Ideal Flow Reactors

Abstract

5.1 Flow Rate, Residence Time, Space Time, Space Velocity, and Dilution Rate

5.2 Plug Flow Reactor

Example 5.1: Constant Density PFR

Example 5.2: Variable Density PFR

Example 5.3: Constant Density CSTR

Example 5.4: CSTR Process Optimization

Example 5.5: Reactor Arrangement for Autocatalytic Reaction

Example 5.6: Autocatalytic Reaction in PFR With Recycle

Example 5.7: Reactor Feeding Pattern

Example 5.8: Optimization for Intermediate Product Production

Example 5.9: Temperature Effect in a CSTR

Example 5.10: Temperature Effect in a PFR

Problems

Chapter 6: Kinetic Theory and Reaction Kinetics

Abstract

6.1 Elementary Kinetic Theory

6.2 Collision Theory of Reaction Rates

6.3 Reaction Rate Analysis/Approximation

6.4 Unimolecular Reactions

6.5 Free Radicals

6.6 Kinetics of Acid Hydrolysis

6.7 Parametric Estimation

6.8 Summary

Problems

Chapter 7: Enzymes

Abstract

7.1 How Enzymes Work

7.2 Simple Enzyme Kinetics

Example 7.1: Specific Activity

Example 7.2: Allosteric Inhibition

Example 7.3: Batch Enzyme Reaction

Example 7.4: Batch Enzymatic Reactor Optimization

Example 7.4

Example 7.5: Enzymatic Reaction in a CSTR

Problems

Chapter 8: Chemical Reactions on Solid Surfaces

Abstract

8.1 Catalysis

8.2 How Does Reaction With Solid Occur?

8.3 Adsorption and Desorption

Example 8.1: Adsorption of Phenol on Activated Carbon From Aqueous Solutions. A Comparison of a Nonideal Isotherm and Ideal Isotherm

Example 8.2: Adsorption of Water Vapor on Silica Gel

Example 8.3: Temkin and Freundlich Isotherms

Example 8.4: The reaction network of isomerization on a solid catalyst follows the following scheme

Problems

Chapter 9: Cell Metabolism

Abstract

9.1 The Central Dogma

9.2 DNA Replication: Preserving and Propagating the Cellular Message

9.3 Transcription: Sending the Message

9.4 Translation: Message to Product

9.5 Metabolic Regulation

Example 9.1: Diauxic Growth

Example 9.2: Sequential Feedback Control of Branched Pathways

Example 9.3: Fluxes in Sequential Feedback Control of Branched Pathways

9.6 How a Cell Senses Its Extracellular Environment

Example 9.4

9.7 Major Metabolic Pathways

9.8 Overview of Biosynthesis

9.9 Overview of Anaerobic Metabolism

9.10 Interrelationships of Metabolic Pathways

9.11 Overview of Autotrophic Metabolism

9.12 The Monod Equation: FES Approximation Through Metabolic Pathways

9.13 Summary

Problems

Chapter 10: Interactive Enzyme and Molecular Regulation

Abstract

10.1 Protein Oligomerization and Interactive Enzyme

10.2 Ligand Binding and Cooperativity

10.3 Competitive Multiligand Binding on an Interactive Enzyme

10.4 Catalytic Reaction Rate on Interactive Enzymes

10.5 Kinetics of Polymorphic Catalysis and Allosteric Modulation

10.6 Influence of a Competitive Effector on Interactive Enzymes

10.7 Summary

Problems

Chapter 11: How Cells Grow

Abstract

11.1 Quantifying Biomass

11.2 Batch Growth Patterns

11.3 Biomass Yield

Example 11.1: Simplistic Growth Kinetics Calculation

Example 11.2: Growth Kinetic Modeling

Example 11.3: Selective Uptake Model Kinetics

Problems

Chapter 12: Cell Cultivation

Abstract

12.1 Batch Culture

Example 12.1: Optimum Batch Reactor Size

Example 12.2: Kinetic Parameter Estimation From Chemostat Data

Example 12.3: Chemostat With Recycle

Example 12.4: Continuous Secondary Metabolite Production Reactor Design Based on Batch Data

Example 12.5: Continuous Wastewater Treatment

Example 12.6: Fermentation in a PFR

Example 12.7: Fed-Batch Reactor

Problems

Chapter 13: Evolution and Genetic Engineering

Abstract

13.1 Mutations

13.2 Selection

13.3 Natural Mechanisms for Gene Transfer and Rearrangement

13.4 Techniques of Genetic Engineering

13.5 Applications of Genetic Engineering

13.6 The Product and Process Decisions

13.7 Host-Vector System Selection

13.8 Regulatory Constraints on Genetic Processes

13.9 Metabolic Engineering

13.10 Protein Engineering

13.11 Summary

Problems

Chapter 14: Sustainability: Humanity Perspective

Abstract

14.1 What is Sustainability?

Example 14.1

14.2 Sustainability of Humanity

14.3 Water

Example 14.2

Example 14.3

14.4 CO2 and Biomass

Example 14.4

14.5 Woody Biomass Use and Desired Sustainable State

Example 14.5

14.6 Solar Energy

14.7 Geothermal Energy

14.8 Summary

Problems

Chapter 15: Sustainability and Stability

Abstract

15.1 Feed Stability of a CSTR

Example 15.1: Multiple Steady States

Example 15.2: Multiple Steady States in a Bioreaction System

Example 15.3: Thermal Hysteresis: Ignition/Extinction Curve

Example 15.4: Changing Steady State Operations

Example 15.5: Segregational Loss

Example 15.6: Genetic Instability of a Continuous Culture

Example 15.7: Stability of a Mixed Culture

Problems

Chapter 16: Mass Transfer Effects: Immobilized and Heterogeneous Reaction Systems

Abstract

16.1 How Does Transformation Occur in a Heterogeneous System?

16.2 Molecular Diffusion and Mass Transfer Rate

16.3 External Mass Transfer

Example 16.1: External Mass Transfer Effect on Reaction Rate

Example 16.2: Maneuvering a Space Satellite

Example 16.3: Particle Size Effect

Example 16.4: Isothermal Internal Effectiveness Factor for Different Kinetics

Example 16.5: Mass Transfer Effects in a CSTR

Problems

Chapter 17: Bioreactor Design Operation

Abstract

17.1 Bioreactor Selection

17.2 Reactor Operational Mode Selection

17.3 Aeration, Agitation, and Heat Transfer

17.4 Scale-Up

Example 17.1

Example 17.2: Holding Time and Tube Length Requirement for a Continuous Sterilizer

Example 17.3: Continuous Sterilizer

Example 17.4

Problems

Chapter 18: Combustion, Reactive Hazard, and Bioprocess Safety

Abstract

18.1 Biological Hazards

18.2 Identifying Chemical Reactivity Hazards

18.3 Heat, Flames, Fires, and Explosions

18.4 Probabilities, Redundancy, and Worst-Case Scenarios

18.5 Chain Reactions

18.6 Autooxidation and Safety

18.7 Combustion

18.8 Premixed Flames

18.9 Heat Generation

18.10 Combustion of Liquids and Solids

Example 18.1: Burning of Wood

18.11 Solid and Liquid Explosives

18.12 Explosions and Detonations

18.13 Reactor Safety

Example 18.2: Reactor Runaway (This Is a Story Retold Based on Fogler (1999))

Solution

18.14 Summary

Problems

Index

Copyright

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Preface to the Second Edition

Shijie Liu

The success of the first edition as an undergraduate and graduate text on bioprocess engineering has led to the further development in treaties on reactor analysis and enzyme kinetics.

With the goal of making this text as suitable as the first text on bioprocess engineering, there were many aspects of chemical kinetics, microbiology or biochemistry, reaction engineering, and bioreaction engineering that needed to be integrated. The second edition furthers this goal, and we now have an integrated text on bioprocess engineering. We do not make a particular distinction in the treatment of chemical transformations or bio-transformations. An attempt is made to unify the terminologies in the different fields and bring them into bioprocess engineering.

As with the first edition, the straight-line plots for reaction rates, or elaborate ways to render reaction rates to straight lines, are missing from this text. The kinetic parametric estimation (formerly Chapter 7 in the first edition) is now combined into Chapter 6 on chemical kinetics. It is my hope that the future generations of bioprocess engineers will not rely on straight-line interpretations. This text promotes a mechanistic understanding of the bio-transformations and regulations over the artificial, straight-line explanations.

Speaking of straight lines and graphic solutions, the pinch line method is now introduced starting in Chapter 4 on batch reactor analysis. It provides a means for solving a batch reactor problem when the kinetics is not fully understood.

Another merge is seen in Chapter 12 on cell cultivation. This chapter now replaces the chapter on continuous cultivation in the first edition and discusses all of the cultivation methods: batch, continuous, and fed batch. This rearrangement leads to the elimination of the Chapter on fed-batch cultivation from the first edition.

The major changes to this second edition include more thorough discussions on the validation of the Monod equation in Chapters 9 and 11, and enzymes with complex structures or with multiple active centers in Chapters 7 and 10. While the Monod equation is empirical, its theoretical foundations have now been provided in the text. Enzymes with multiple active centers can be interactive, as ligand binding can alter the enzyme structure, affecting further binding and/or reactivity, and therefore, producing desired regulations. Because of the developments on the treatise on these interactive enzymes, Chapter 7 (which was Chapter 8 in the first edition) on enzymes has been rewritten; and a new chapter on interactive enzymes and regulations (Chapter 10) has also been written. Another addition to the second edition is Chapter 18: Combustion, Reactive Hazard, and Bioprocess Safety. Reactive hazard and process safety is becoming a required component in our undergraduate education. This chapter provides a means to include the topic in the undergraduate curriculum.

I hope you will enjoy this second edition.

Preface to the First Edition

Shijie Liu

All I know is just what I read in the papers.

Will Rogers

The quote above is quite intriguing to me, and is reflective of this text. Everything in this text can be found either directly, or through extrapolation or deduction using the books and papers one can find to date. The most influential books at this time include Biochemical Engineering Fundamentals by J.E. Bailey and D.F. Ollis; Elements of Chemical Reaction Engineering by H.S. Fogler; The Engineering of Chemical Reactions by L.D. Schmidt; Chemical Reaction Engineering by O. Levenspiel; Bioprocess Engineering-Basic Concepts by M.L. Shuler and F. Kargi; and many others. All of these texts, as well as others, have helped form a part of this text. It is not intended for this text to replace all these great textbooks. This is a mere rearrangement and/or compilation and is made to give you, the reader, an opportunity to understand some of the basic principles of chemical and biological transformations in bioprocess engineering.

The computer age has truly revolutionized the literature on bioprocess engineering, well beyond the literature revolution, which was brought about by the mass production, or availability, of paper and the distribution of books via libraries. The explosion in the shear amount of literature and the birth of interdisciplinary disciplines, or subject areas, in the past decades have been phenomenal. Bioprocess engineering is born of biotechnology and chemical engineering. With the maturing of bioprocess engineering as a discipline, it evolved from an interdisciplinary subject area of Biology and Chemical Engineering, to a discipline that covers the engineering, and engineering science, aspects of biotechnology, green chemistry, and biomass or renewable resource engineering. As such, textbooks in this area are needed to cover the educational needs of the new generation of fine bioprocess engineers, and not just converting well-versed chemical engineers and engineering-savvy biologists into bioprocess engineers. I hope that this textbook can fill this gap, and bring about the maturity of bioprocess engineering. Yet, some of the materials in this text are deep in analyses that are suited for graduate work and/or for research reference.

The key aspect that makes bioprocess engineering special is that it, as a discipline, is centered on solving problems of transformation stemming from cellular functions, and biological and/or chemical conversions concerning the sustainable use of renewable biomass. The mechanism, rate, dynamic behavior, transformation performance, and manipulations of bioprocess systems are the main topics of this text.

Chapter 1 is an introduction to bioprocess engineering profession including green chemistry, sustainability considerations, and regulatory constraints. Chapter 2 is an overview of biological basics or cell chemistry including cells, viruses, stem cell, amino acids, proteins, carbohydrates, and various biomass components, and fermentation media. In Chapter 3, a survey of chemical reaction analysis is introduced. The basic knowledge of reaction rates, conversion, yield, stoichiometry, and energy regularity for bioreactions are reviewed. The concepts of approximate and coupled reactions are introduced, providing the basis of understanding for the metabolic pathway representations later in this book. Mass and energy balances for reactor analyses, as well as the definitions of ideal reactors and commonly known bioreactors are introduced before an introduction to reactor system analyses. Biological and chemical reaction basics are followed by reactor analysis basics in Chapters 4 and 5, including the effect of reaction kinetics, flow contact patterns, and reactor system optimizations. Gasification (of coal and biomass) is also introduced in Chapter 5. How the ideal reactors are selected, what flow reactor to choose, and what feed strategies to use are also covered in Chapter 5.

Chapters 6, 7, 8, 9, 10, and 11 are studies in bioprocess kinetics. In Chapter 6 you will learn the collision theory for reaction kinetics and approximations commonly employed to arrive at simple reaction-rate relations. Kinetics of acid hydrolysis, as an important unit operation in biomass conversion, is introduced as a case study. In Chapter 7, we discuss the techniques for estimating kinetic parameters from experimental data, breaking away from the traditional straight-line approaches developed before the computer age. You can learn how to use modern tools for extracting kinetic parameters reliably and quickly, without complex manipulations of the data. In Chapters 8 and 9, we discuss the application of kinetic theory to catalytic systems. Enzymes, enzymatic reactions, and the application of enzymes are examined in Chapter 8; while adsorption and solid catalysis are discussed in Chapter 9. The derivation of simplified reaction-rate relations, such as the Michaelis-Menten equation for enzymatic reactions and Langmuir-Hinshelwood-Hougen-Watson (LHHW) for solid catalysis, is demonstrated. The applicability of these simple kinetic relations is discussed. In Chapter 9, you will learn both ideal and nonideal adsorption kinetics and adsorption isotherms. Is multilayer adsorption the trademark for physisorption? In 9.5, the heterogeneous kinetic analysis theory is applied to reactions involving woody biomass, where the solid phase is not catalytic. Chapter 10 discusses cellular genetics and metabolism. The replication of genetic information, protein production, substrate uptake, and major metabolic pathways are discussed, hinting at the application of kinetic theory in complicated systems. In Chapter 11 you will learn how cells grow: cellular material quantifications, batch growth patterns, cell maintenance and endogenous needs, medium and environmental conditions, and kinetic models. Reactor analyses are also presented in Chapters 8 and 11.

In Chapters 12 and 13 we discuss controlled cell cultivation. Continuous culture and wastewater treatments are discussed in Chapter 12. Exponential growth is realized in continuous culturing. An emphasis is placed on reactor performance analyses, mostly using the Monod growth model in the examples in both Chapters. Chapter 13 introduces fed-batch operations and their analyses. Fed-batch can mimic exponential growth in a controlled manner, as opposed to batch operations where no control on growth is asserted besides environmental conditions.

Chapter 14 discusses evolution and genetic engineering, with an emphasis on biotechnological applications. You will learn how cells transform, how cells are manipulated, and what some of the applications for cellular transformation and recombinant cells are. Chapter 15 introduces perspectives on sustainability. Bioprocess engineering principles are applied in order to examine the sustainability of biomass economy and atmospheric CO2. Chapter 16 discusses the stability of catalysts including the activity of a chemical catalyst, the genetic stability of cells and mixed cultures, as well as the stability of reactor systems. Sustainability and the stability of bioprocess operations are discussed. A stable process is sustainable. Multiple steady states, the approach toward a steady state, the conditions for stable operations, and predator-prey interactions are also discussed. Continuous culture is challenged by stability of cell biomass. In ecological applications, sustainability of a bioprocess is desirable. For industrial applications, the ability of the bioprocess system to return to the previous set-point after a minor disturbance is an expectation. In Chapter 17, the effect of the mass transfer on the reactor performance, in particular with biocatalysis, is discussed. Both external mass transfers, eg suspended media; and internal mass transfers, eg immobilized systems; as well as temperature effects are discussed. Detailed numerical solutions can be avoided, or greatly simplified, by directly following the examples. Chapter 18 discusses reactor design and operation. Reactor selection, mixing schemes, scale-up, and sterilization and aseptic operations are also discussed.

Acronyms, Abbreviations, and Symbols

a catalyst activity

a specific surface or interfacial area, m²/m³

a thermodynamic activity

a cumulative affinity change factor

ad dimensionless dispersion coefficient

a constant

A chemical species

A adenine

A constant

A heat transfer area, m²

Ac acetyl

ADP adenosine diphosphate

AMP adenosine monophosphate

ATP adenosine triphosphate

B chemical species

B constant

BOD biological oxygen demand

BOD5 biological oxygen demand measured for 5 days

BnS bound species S on n-sited enzyme

c constant

C chemical species

C concentration, mol/L or kg/m³

C constant

C cytosine

CD dimer formed by swapping carboxylic acid termini

CP heat capacity, J/(mol K), or kJ/(kg K)

CoA coenzyme A

CHO Chinese Hamster Ovary cell

COD chemical oxygen demand

CSTR continuously stirred tank reactor

d diameter, m

D diameter, m

D diffusivity, m²/s

D dilution rate, s− 1

D dimer

D- chirality or optical isomers: right-hand rule applies

DNA deoxyribonucleic acid

DO concentration of dissolved oxygen, g/L

DP degree of polymerization

DS domain swapped

e electron

E enzyme

E energy, kJ/mol

En enzyme having n-subunits

jEn polymorph member or the j-th enzyme having n-subunits

EMP Embden-Meyerhof-Parnas

f fractional conversion

f fanning friction factor

F flow rate, kg/s or kmol/s

F Farady constant

FAD flavin adenine dinucleotide in oxidized form

FADH flavin adenine dinucleotide in reduced form

FDA Food and Drug Administration

FES fast equilibrium step (hypothesis)

g gravitational acceleration, 9.80665 m²/s

G Gibbs free energy, kJ/mol

G Guanine

GRAS generally regarded as safe

GMP good manufacture practice

GTP guanosine triphosphate

h height or length

H enthalpy, kJ/mol or kJ/kg

HC harvesting cost

HMP hexose monophosphate (pathway)

Ig immunoglobulin

J total transfer flux, kmol/s or kg/s

J transfer flux, kmol/(m² s) or kg/(m² s)

k kinetic rate constant

k mass transfer coefficient, m/s

K thermodynamic equilibrium constant

K saturation constant, mol/L or kg/L

K enzyme binding saturation constant, mol/L

KL overall mass transfer coefficient (from gas to liquid)

L length

L- chirality or optical isomers: left hand rule applies

m amount of mass, kg

 mass flowrate, kg/s

mS maintenance coefficient

M molecular weight, Darton (D) or kg/kmol

MC molar (or mass) consumption rate

MG mass generation rate

MR mass removal rate

MS molar or mass supply rate

MSS multiple steady state

n total matters in number of moles

N mass transfer rate, kJ/(m² s) or kg/(m² s)

N number of species

ND dimer formed by swapping amine termini

NAD nicotinamide adenine dinucleotide

NADP nicotinamide adenine dinucleotide phosphate

OR order of reaction

OTR oxygen transfer rate

OUR oxygen utilization rate

p pressure

pe polymorph enzyme parameter

P probability

P pressure

P product, or product concentration, kg/m³, or g/L, or mol/L

P power (of stirrer input)

PX productivity or production rate of biomass

P0 probability of the vanishing of the entire population

P1 probability of the entire population not vanishing

PCR polymerase chain reaction

PEP phosphoenol pyruvate

PFR plug flow reactor

PP pentose phosphate (pathway)

PSSH pseudosteady state hypothesis

P/O ATP formation per oxygen consumption

q Thermal flux, J/s or W

Q volumetric flow rate, m³/s

 thermal energy transfer rate into the system

r radial direction

r rate of reaction, mol/(m³ s) or kg/(L s)

R correlation coefficient

R ideal gas constant, 8.314 J/(mol K)

R product, or product concentration

R recycle ratio

Re Reynolds number

RNA ribonucleic acid

s selectivity

S entropy

S overall selectivity

S substrate (or reactant)

S substrate concentration, g/L

S surface area, m²

Sh Sherwood number

SMG specific mass generation rate

SMR specific mass removal rate

t time, s

T temperature, K

T thymine

TCA tricarboxylic acid

u superficial velocity, m/s

U average velocity or volumetric flux

U internal energy

U overall heat transfer coefficient, kJ/m²

U uracil

CoQn co-enzyme ubiquinone

v molar volume, m³/kmol

V volume, m³

 rate of work input to the system

 rate of shaft work done by the system

x variable

x axial direction

X cell or biomass

X cell biomass concentration, L− 1, or g/L

XSU biomass storage for managed forest

XSU biomass storage for undisturbed unmanaged forest

y mole fraction

y variable

Y yields

YF yield factor, or ratio of stoichiometric coefficients

z vertical direction

z variable

Z collision frequency

Z valence of ionic species

Greek Symbols

α affinity change factor due to the binding of a ligand

α constant

α chirality or optical isomers: two chiral centers with different hand gestures

α metabolite transfer or equilibrium coefficient

β heat of reaction parameter

β constant

β chirality or optical isomers: two chiral centers with the same hand gestures

β reactivity change factor due to the binding of a ligand(s)

χ fraction

γ thermodynamic activity coefficient

γ activation energy parameter

γDR degree of reduction

δ thickness or distance

Δ difference

ɛ void ratio

ϕ Thiele modulus

η effectiveness factor

θ fractional coverage (on available active sites)

θ binding saturation ratio on enzyme

μ specific rate of formation, or rate of reaction normalized by the catalyst or cell biomass concentration, s− 1 or g/g/s

μ specific biomass growth rate, s− 1 or g/g/s

μf dynamic viscosity of fluid, Pa s

ν stoichiometric coefficient

νf kinematic viscosity of fluid or medium, m²/s

ρ density, kg/m³

σ active site

σ variance

τ space time, s

ω mass fraction

ω rotational speed

ω weighting factor

Subscript

ads adsorption

app apparent

A species A

b reverse reaction

B batch

c combustion

c catalytic

c cyclic

cat catalyst

C species C

C cold stream

C concentration based

C calculation based on model

C carboxylic acid terminus or pertaining to carboxylic acid terminus

d death

d doubling

des desorption

D diffusion coefficient related

e endogenous (growth needs)

e external (mass transfer)

e in effluent stream

eq equilibrium

eff effective

f final or at end

f fluid or medium

f formation

f forward reaction

F in feed stream

G growth

H heat of reaction

H hot stream

i reaction i

i initial

i impeller

in inlet

I inhibition

j species j

m maximum

max maximum

N amine terminus or pertaining to amine terminus

net net

OPT optimum

obs observed

out outlet

p particle

P product

P preparation

R reaction; reactor

R relaxed or active

R reference; reduced

s solid

S sterilization

S saturation

S substrate

S surface

S total species

t tube, or reactor

T tube

T total

T tense or inert

U unloading

0 initial

0 in feed

0 pre-exponential

+ plasmid-containing

− plasmid-free

∞ maximum or at far field

Σ total or sum

Superscript

0 (thermodynamic) standard conditions

* equilibrium

* based on transitional state

′ catalyst mass based

′ variant

Chapter 1

Introduction

Abstract

An overview on the biological cycle, green chemistry, sustainability, biorefinery, the profession of bioprocess engineers, and the text is presented in this introductory chapter. Sustainability and green chemistry are important to the practice of bioprocess engineers. Biorefinery is a desired approach to provide the products of human needs, putting the human needs directly into the biological cycle. Both biotransformation and chemical transformations are important to biorefinery. Genetic manipulations and biotransformation are important to the bioprocess engineering profession. Because of the fundamental impact genetic manipulation and biotransformation can have on the biological cycle, ethics and regulations are critical to the practice of bioprocess engineers. It is the intention that this text can be used as a senior and/or graduate text for bioprocess engineering students. It may also be served as a reference and/or graduate text for chemical kinetics, chemical reactor analysis, and bioprocess systems engineering. In the same time, we have inserted materials, more advanced analysis or complex systems in the text as an extension to the introductory material. It is our aim that this text can serve equally as a reference or resource for further research or development.

Keywords

Biological cycle; Biorefinery; Biotransformation; Green chemistry; Regulatory; Sustainability

Outline

1.1 Biological Cycle

1.2 Green Chemistry

1.3 Sustainability

1.4 Biorefinery

1.5 Biotechnology and Bioprocess Engineering

1.6 Mathematics, Biology, and Engineering

1.7 The Story of Penicillin: The Dawn of Bioprocess Engineering

1.8 Bioprocesses: Regulatory Constraints

1.9 The Pillars of Bioprocess Kinetics and Systems Engineering

1.10 Summary

Bibliography

Problems

1.1 Biological Cycle

Fig. 1.1 illustrates the natural biological processes occurring on Earth. Living systems consist of plants, animals, and microorganisms. Sunlight is used by plants to convert CO2 and H2O into carbohydrates and other organic matter, releasing O2. Animals consume plant matter, converting plant materials into animal cells, and using the chemical energy from oxidizing plant matter into CO2 and H2O (H2O also serves as a key substrate for animals), finishing the cycle. Microorganisms further convert dead animal and/or plant biomass into other form of organic substances fertilizing the growth of plants, releasing CO2 and H2O, and the cycle is repeated. Energy from the Sun is used to form molecules and organisms that we call life. Materials or matter participating in the biological cycle are renewable so long as the cycle is maintained. Bioprocess engineers manipulate and make use of this cycle by designing processes to make desired products, by training microorganisms, plants, and animals, or via direct chemical conversions.

Fig. 1.1 The natural biological processes.

The reactor is the heart of any chemical and/or biochemical processes. With reactors, bioprocesses turn inexpensive sustainably renewable chemicals such as carbohydrates, into valuable ones that humans need. As such, bioprocesses are chemical processes that use biological substrates and/or catalysts. While not limited to such, we tend to refer to bioprocesses as (1) biologically converting inexpensive chemicals or materials into valuable chemicals or materials; and (2) manipulating biological organisms to serve as catalyst for conversion or production of products that human need. Bioprocess engineers are the only people technically trained to understand, design, and efficiently handle bioreactors. Bioprocess engineering ensures that a favorable sustainable state or predictable outcome of a bioprocess is achieved. This is equivalent to saying that bioprocess engineers are engineers with, differentiating from other engineers, training in biological sciences, especially quantitative and analytical biological sciences, and green chemistry.

If one thinks of science as a dream, engineering is making the dream a reality. The maturing of Chemical Engineering to a major discipline and as one of the very few well-defined disciplines in the 1950s has led to the ease in the mass production of commodity chemicals and completely changed the economics or value structure of materials and chemicals, thanks to the vastly available what were then waste and toxic materials: fossil resources. Food and materials can be manufactured from the cheap fossil materials. Our living standards improved significantly. Today, chemical reactors and chemical processes are not built by trial-and-error, but by design. The performance of a chemical reactor can be predicted, not just found to happen that way; the differences between large and small reactors are largely solved. Once a dream for the visional pioneers, it can now be achieved at ease. Fossil chemical and energy sources have provided much of our needs for advancing and maintaining the living standards of today. With the dwindling of fossil resources, we are facing yet another value structure change. The dream has been shifted to realizing a society that is built upon renewable and sustainable resources. Fossil sources will no longer be abundant for human use. Sustainability becomes the primary concern. Who is going to make this dream come true?

On a somewhat different scale, we can now manipulate life at its most basic level: the genetic. For thousands of years people have practiced genetic engineering at the level of selection and breeding, or directed evolution. But now it can be done in a purposeful, predetermined manner with the molecular-level manipulation of DNA, at a quantum leap level (as compared with directed evolution) or by design. We now have tools to probe the mysteries of life in a way unimaginable prior to the 1970s. With this intellectual revolution emerges new visions and new hopes: new medicines, semisynthetic organs, abundant and nutritious foods, computers based on biological molecules rather than silicon chips, organisms to degrade pollutants and clean up decades of unintentional damage to the environment, zero harmful chemical leakage to the environment while producing a wide array of consumer products, and revolutionized industrial processes. Our aim of comfortable living standards is ever higher.

Without hard work, these dreams will remain merely dreams. Engineers will play an essential role in converting these visions into reality. Biosystems are very complex and beautifully constructed, but they must obey the rules of chemistry and physics and they are susceptible to engineering analysis. Living cells are predictable, and processes to use them can be methodically constructed on commercial scales. There lies a great task: analysis, design, and control of biosystems to the greater benefit of a sustainable humanity. This is the job of the bioprocess engineer.

This text is organized such that you can learn bioprocess engineering without requiring a profound background in reaction engineering and biotechnology. To limit the scope of the text, we have left out the product purification technologies, while focusing on the production generation. We attempt to bridge molecular-level understandings to industrial applications. It is our hope that this will help you to strengthen your desire and ability to participate in the intellectual revolution and to make an important contribution to the human society.

1.2 Green Chemistry

Green chemistry, also called sustainable chemistry, is a philosophy of chemical research and engineering that encourages the design of products and processes that minimize the use and generation of hazardous substances while maximizing the efficiency of the desired product generation. Whereas environmental chemistry is the chemistry of the natural environment, and of pollutant chemicals in nature, green chemistry seeks to reduce and prevent pollution at its source. In 1990, the Pollution Prevention Act was passed in the United States. This act helped create a modus operandi for dealing with pollution in an original and innovative way. It aims to avoid problems before they happen.

Examples of green chemistry starts with the choice of solvent for a process: water, carbon dioxide, dry media, and nonvolatile (ionic) liquids, which are some of the excellent choices. These solvents are not harmful to the environment as either emission can easily be avoided or they are ubiquous in nature.

Paul Anastas, then of the United States Environmental Protection Agency, and John C. Warner developed 12 principles of green chemistry, which help to explain what the definition means in practice. The principles cover such concepts as: (a) the design of processes to maximize the amount of (all) raw material that ends up in the product; (b) the use of safe, environment-benign substances, including solvents, whenever possible; (c) the design of energy efficient processes; and (d) the best form of waste disposal: not to create it in the first place. The 12 principles are:

(1) It is better to prevent waste than to treat or clean up waste after it is formed.

(2) Synthetic methods should be designed to maximize atom efficiency.

(3) Wherever practicable, synthetic methodologies should be designed to use and generate substances that possess little or no toxicity to human health and the environment.

(4) Chemical products should be designed to preserve efficacy of function while reducing toxicity.

(5) The use of auxiliary substances (eg, solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.

(6) Energy requirements should be recognized for their environmental and economic impacts and should be minimized. Synthetic methods should be conducted at ambient temperature and pressure.

(7) A raw material or feedstock should be renewable rather than depleting wherever technically and economically practicable.

(8) Reduce derivatives—Unnecessary derivatization (blocking group, protection/deprotection, temporary modification) should be avoided whenever possible.

(9) Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.

(10) Chemical products should be designed so that at the end of their function they do not persist in the environment and break down into innocuous degradation products.

(11) Analytical methodologies need to be further developed to allow for real-time, in-process monitoring and control prior to the formation of hazardous substances.

(12) Substances and the form of a substance used in a chemical process should be chosen to minimize potential for chemical accidents, including releases, explosions, and fires.

One example of green chemistry achievements is the polylactic acid (or PLA). In 2002, Cargill Dow (now NatureWorks) won the Greener Reaction Conditions Award for their improved PLA polymerization process. Lactic acid is produced by fermenting corn and converted to lactide, the cyclic dimmer ester of lactic acid using an efficient, tin-catalyzed cyclization. The l,l-lactide enantiomers is isolated by distillation and polymerized in the melt to make a crystallizable polymer, which has use in many applications including textiles and apparel, cutlery, and food packaging. Wal-Mart has announced that it is using/will use PLA for its produce packaging. The NatureWorks PLA process substitutes renewable materials for petroleum feedstocks, does not require the use of hazardous organic solvents typical in other PLA processes, and results in a high-quality polymer that is recyclable and compostable.

Understanding the concept of green chemistry holds a special position for bioprocess engineers. Processes we design and operate must have minimal potential environmental impacts while optimized for maximum benefit. Basic steps for green chemistry that are what bioprocess engineering do include:

(1) Developing products and processes based on renewable resources, such as plant biomass;

(2) Design processes that bypass dangerous/toxic chemical intermediates;

(3) Design processes that avoid dangerous/toxic solvent use;

(4) Reducing chemical processing steps, but demands high efficiency;

(5) Promoting biochemical processes to reduce chemical processing steps or toxic chemical utilization;

(6) Design milder (closer to atmosphere temperature and pressure) processes and multiple product recovery routes;

(7) Bypassing chemical equilibrium with innovative reactor design, and biocatalysis, rather than adding intermediate steps;

(8) Avoid production of unwanted products other than H2O and CO2.

1.3 Sustainability

Sustainability is the capacity to endure or maintain at the longest timescale permissible. In other words, sustainability is the ability to maintain continuum. In ecology, the word describes how biological systems remain diverse and productive over time. Long-lived and healthy wetlands and forests are examples of sustainable biological systems. For humans, sustainability is the potential for long-term maintenance of well being, which has environmental, economic, and social dimensions.

Healthy ecosystems and environments provide vital goods and services to humans and other organisms. Utilization and release of substances at a rate that is harmonious with a steady state nature is the key for sustainability. This naturally leads to a carrying capacity for each substance or species with which humans interact. The sustainable state can be influenced by (process conditions or) how we interact with nature. There are two major ways of reducing negative human impact and enhancing ecosystem services. The first is environmental management; this approach is based largely on information gained from earth science, environmental science, and conservation biology. The second approach is management of human consumption of resources, which is based largely on information gained from economics. Practice of both of these major steps can ensure a more favorable sustainable state to be evolved into.

Sustainability interfaces with economics through the social and ecological consequences of economic activity. Sustainable economics involves ecological economics where social, cultural, health-related, and monetary/financial aspects are integrated. Moving towards sustainability is also a social challenge that entails international and national law, urban planning and transport, local and individual lifestyles, and ethical consumerism. Ways of living more sustainably can take many forms from reorganizing living conditions (eg, ecovillages, eco-municipalities, and sustainable cities), reappraising economic sectors (permaculture, green building, sustainable agriculture), or work practices (sustainable architecture), using science to develop new technologies (green technologies, renewable energy), to adjustments in individual lifestyles that conserve natural resources. These exercises reduce our reliance or demand on disturbing the environment. To make all these concepts come to light, bioprocess engineers will be at the forefront of developing and implementing the technologies needed. On a grand scale, maintaining renewability or looking for a favorable predictable steady state on everything we touch or interact is the key to sustainability (Fig. 1.2). This falls right in the arena of bioprocess engineering.

Fig. 1.2 Change of sustainable state owing to human interruption.

Are you ready for the challenge of designing processes that meets sustainability demands?

1.4 Biorefinery

On a grand scale, sustainability is the basis of nature. Enforcing sustainability at the time scale of humanity is an insurance of our way of life to continue. Prior to the 1900s, agriculture and forestry were the predominant sources of raw materials for energy, food, and a wide range of everyday commodities, and the human civilization depended almost entirely on renewable materials. Humanity was restricted by the sustainable supply inefficiently harvested from the biomass, which drew energy from the sun. The industrial revolution has brought a leap in the human civilization. Mass production of goods by machines dominates our daily life. The industrial revolution was brought to mature by the development of combustion engines and subsequent development of fossil energy and chemical industry. Besides the more than doubling of useful biomass production/harvest, mankind has increasingly tapped into the large fossil energy reserves. At first the fossil chemicals were regarded as waste and thus any use was welcomed. It soon became the cheapest chemical and energy sources for the industrial revolution. As a result, our living standards have seen a leap. There is no turning back to the primitive way of life in the past. However, fossil energy and chemical sources are depleting despite the cyclic price change of energy and commodity materials. There is a critical need to change the current industry and human civilization to a sustainable manner, assuring that our way of life today continues on the path of improvement after the depletion of fossil sources. Our way of life exists only if sustainability is maintained on a time scale no longer than our life span.

Biorefinery is concept in analogous to a petroleum refinery, whereby a raw material feed (in this case plant ligocellulosic biomass instead of petroleum) is refined to a potpourri of products (on demand). In a biorefinery, lignocellulosic biomass is converted to chemicals, materials, and energy that runs on the human civilization, replacing the needs of petroleum, coal, natural gas, and other nonrenewable energy and chemical sources. Lignocellulosic biomass is renewable as shown in Fig. 1.1, in that plant synthesizes chemicals by drawing energy from the sun and, carbon dioxide and water from the environment, while releasing oxygen. Combustion of biomass releases energy, carbon dioxide, and water. Therefore, biorefinery plays a key role in ensuring the cycle of biomass production and consumption included satisfying human needs for energy and chemicals.

A biorefinery integrates a variety of conversion processes to produce multiple product streams such as transportation liquid fuels, steam/heat, electricity, and chemicals from lignocellulosic biomass. Biorefinery has been identified as the most promising route to the creation of a sustainable biobased economy. Biorefinery is a collection of the essential technologies to transform biological raw materials into a range of industrially useful intermediates. By producing multiple products, a biorefinery maximizes the value derived from a lignocellulosic biomass feedstock. A biorefinery could produce one or more low-volume, high-value chemical products together with a low-value, high-volume liquid transportation fuel, while generating electricity and process heat for its own use and/or export.

Fig. 1.3 shows a schematic of various biorefinery processes. There are at least three steps in converting woody biomass: pretreatment, cracking or rendering biomass to intermediate molecules, and conversion (of the intermediate molecules to desired products). There are three major categories or approaches in pretreatment: systematical disassembling processes, pyrolysis, and gasification processes. In systematical disassembling processes, the lignocellulosic biomass is commonly disassembled to individual components systematically for optimal conversions that followed. The basic approach is based on a systematical disassembling and conversion to desired chemicals. This pretreatment method and the rout of conversion followed depend heavily on separation and/or physical fractionation of the intermediates as well as the final desired products. Pyrolysis applies heat and some Oxygen (or none) to render the biomass to liquid, while gasification renders the biomass to syngas. Biological conversions are preferred over chemical conversions due to their selectivity or green chemistry concepts. However, owing to the complexity of the lignocellulosic biomass, a multitude of biological processes is required for optimal operations. The biological reactions are also very slow and thus require larger facility foot prints. Especially following the gasification, bioconversion is in the primitive stage of development.

Fig. 1.3 A schematic of various biorefinery processes. (With permission: Liu., S., 2015. A synergetic pretreatment technology for woody biomass conversion, Appl. Energy 144, 114–128.)

Pyrolysis resembles more closely to the refinery, whereby the products can be controlled in a more systematical manner. It may be classified as systematical disassembling as well. However, there are restrictions on the type of products the process can produce. Gasification as shown in Fig. 1.3 is at the extreme side of conversion technology, whereby the lignocellulosic biomass is disassembled to the basic building block for hydrocarbons: H2 and CO and then reassembled to desired products as desired. The final products can be more easily tailored from syngas or CO + H2. For example, Fisher-Tropsch process can turn CO + H2 into higher alcohol, alkenes, and many other products. Syn gas (together with air: mixture of N2 and O2) is also the starting point for ammonia synthesis, from which nitrogen fertilizers and many other products are produced. However, all thermochemical processes suffer from selectivity. During the disassembling process, coke or carbon is produced especially at high temperatures and thus reduces the conversion efficiency if H2 and CO are the desired intermediates. Thermal-chemical processes are generally considered less green than biological and other sequential disassembling processes due to their severe operating conditions, poor selectivity or byproducts generation, and thermodynamic restrictions.

The promise of a biorefinery to supply the products human needs is shown in Fig. 1.4 for the various examples of building blocks or platform chemicals that sugar (a specific example of glucose) can produce, besides the very basic building blocks of CO and H2. For example, glucose can be fermented to ethanol by yeast and bacteria anaerobically, and lactic acid can be produced by lacto bacteria. As shown in Fig. 1.4, each arrow radiates from the glucose in the center represents a route of biotransformation (or fermentation) by default, whereas chemical transformations are shown with labeled arrows. For example, glucose can be dehydrated to 5-hydroxymethylfurfural catalyzed with an acid, which can be further decomposed to levulinic acid by hydration. All these chemicals shown in Fig. 1.4 are examples of important platform (or intermediate) chemicals, as well as commodity chemicals. For example, ethanol is well known for its use as a liquid transportation fuel. Ethanol can be dehydrated to ethylene, which is the monomer for polyethylene, or dehydrognated and dehydrated to make 1,3-butadiene, monomer for the synthetic rubber. Ethanol can also be employed to produce higher alcohols and alkenes.

Fig. 1.4 The platform chemicals derived from glucose (the molecule in the center of the diagram). Most of these chemicals are produced via fermentation (or biotransformation), while a few of them are produced via chemical reaction (or chemical transformation) as indicated.

Are you prepared to be at the forefront of developing, designing, and operating these processes for the sustainability and comfortability in humanity?

1.5 Biotechnology and Bioprocess Engineering

Biotechnology is the use or development of methods of direct genetic manipulation for a socially desirable goal. Such a goal might be the production of a particular chemical, but it may also involve the production of better plants or seeds, gene therapy, or the use of specially designed organisms to degrade wastes. The key element is the use of sophisticated techniques outside the cell for genetic manipulation. Biotechnology is applied biology; it bridges biology to bioprocess engineering, just like applied chemistry or chemical technology bridges chemistry to chemical engineering.

Many terms have been used to describe engineers working with biotechnology. The two terms that are considered general: Biological Engineering and Bioengineering come from the two major fields of applications that require deep understanding of biology: agriculture and medicine. Bioengineering is a broad title including work on industrial, medical, and agricultural systems; its practitioners include agricultural, electrical, mechanical, industrial, environmental, and chemical engineers, and others. As such, Bioengineering is not a well-defined term and usually it refers to biotechnology applications that are not easily categorized or in medical fields. Biological Engineering stems from engineering of biology which is also a general term. However, the term Biological Engineering is initially used by agricultural engineers for the engineering applications to or manipulation of plants and animals and is thus specific. For example, the Institute of Biological Engineering has a base in, although not limited to, agricultural engineering. Accreditation Board for Engineering and Technology, Inc. brands Biological Engineering together with Agricultural Engineering when accrediting Bachelor of Science and Master of Science in engineering and technology educational programs. On the other hand, the Society for Biological Engineers was created within American Institute of Chemical Engineers. Therefore, biological engineering is also regarded as a not well-defined term and can at times refer to biotechnology applications in agricultural fields. Biochemical engineering has usually meant the extension of chemical engineering principles to systems using a biological catalyst to bring about desired chemical transformations. It is often subdivided into bioreaction engineering and bioseparations. Biomedical engineering has traditionally been considered totally separate from biochemical engineering, although the boundary between the two is increasingly vague, particularly in the areas of cell surface receptors and animal cell culture. Another relevant term is biomolecular engineering, which has been defined by the National Institutes of Health as …research at the interface of biology and chemical engineering and is focused at the molecular level. In all, a strong background in quantitative analysis, kinetic/dynamic behaviors, and equilibrium behaviors are strongly desirable. Increasingly, these biorelated engineering fields have become interrelated, although the names are restricting.

Bioprocess engineering is a broader and at the same time a narrower field than the commonly used terms referred above: biological engineering, biochemical engineering, biomedical engineering, and biomolecular engineering. Bioprocess engineering is a profession that spans all the biorelated engineering fields as mentioned earlier. It is a profession that has emerged to stand alone, as compared to the interdisciplinary profession once it was. Unlike the term bioengineering, the term bioprocess engineering is specific and well defined. Bioprocess engineering emphasizes the engineering and sciences of industrial processes that are biobased: (1) biomass feedstock conversion for a sustainable society or biorefinery; (2) biocatalysis based processing; and (3) manipulation of microorganisms for a sustainable and socially desirable goal. Bioprocess engineering neither is product based nor is substrate based. Therefore, bioprocess engineering deals with biological and chemical processes involved in all areas, not just for a particular substrate or species (of feedstock or intermediate), outcome or product. Thus, bioprocess engineering intercepts chemical, mechanical, electrical, environmental, medical, and industrial engineering fields, applying the principles to designing and analysis of processes based on using living cells or subcomponents of such cells, as well as nonliving matters. Bioprocess engineering deals with both microscale (cellular/molecular) and large-scale (systemwide/industrial) designs and analyses. Science and engineering of processes converting biomass materials to chemicals, materials, and energy are therefore part of bioprocess engineering by extension. Predicting and modeling system behaviors, detailed equipment and process design, sensor development, control algorithms, and manufacturing or operating strategies are just some of the challenges facing bioprocess engineers. At the heart of bioprocess engineering lay the process kinetics, reactor design, and analysis for biosystems, which forms the basis for this text.

We will focus primarily on the kinetics, dynamics, and reaction engineering involved in the bioprocess engineering. A key component is the application of engineering principles to systems containing biological catalysts and/or biomass as feedstock, but with an emphasis on those systems making use of biotechnology and green chemistry. The rapidly increasing ability to determine the complete sequence of genes in an organism offers new opportunities for bioprocess engineers in the design and monitoring of bioprocesses. The cell, itself, is now a designable component of the overall process.

For practitioners working in the bioprocess engineering, some of journals and periodicals provide the latest developments in the field. Here we name a few:

Biochemical Engineering Journal

Journal of Biological Engineering

Journal of Biomass Conversion and Biorefinery

Journal of Bioprocess Engineering and Biorefinery

Journal of Bioprocess and Biosystems Engineering

Journal of Biotechnology

Journal of Biotechnology Advances

Journal of Biotechnology and Bioprocess Engineering

1.6 Mathematics, Biology, and Engineering

Mathematical modeling holds the key for engineers. Physics at its fundamental level examines forces and motion; one can view it as applied mathematics. In turn, chemistry examines molecules and their interactions. Since physics examines the motions of atoms, nuclei, and electrons, at the basis of molecules, one can view chemistry as applied physics. This directly connects chemistry to mathematics at a fundamental level. Indeed, most physicists and chemists rely extensively on mathematical modeling. While one can view biology as applied chemistry through the connection of chemicals and molecules, the fundamental trainings of biologists today and engineers are distinctly different. In the development of knowledge in the life sciences, unlike chemistry and physics, mathematical theories and quantitative methods (except statistics) have played a secondary role. Most progress has been due to improvements in experimental tools. Results are qualitative and descriptive models are formulated and tested. Consequently, biologists often have incomplete backgrounds in mathematics but are very strong with respect to laboratory tools and, more importantly, with respect to the interpretation of laboratory data from complex systems.

Engineers usually possess a very good background in the physical and mathematical sciences. Often a theory leads to mathematical formulations, and the validity of the theory is tested by comparing predicted responses to those in experiments. Quantitative models and approaches, even to complex systems, are strengths. Biologists are usually better at the formation of testable hypotheses, experimental design, and data interpretation from complex systems. At the dawn of the biotechnology era, engineers were typically unfamiliar with the experimental techniques and strategies used by life scientists. However, today bioprocess engineers have entered even more sophisticated experimental techniques and strategies in life sciences (than biologists) due to the understanding and progress in the prediction and modeling of living cells.

The well-groundedness in mathematical modeling gives bioprocess engineers an edge and responsibility in enforcing sustainability demands. In practice, the sustainable (or steady) state can be different from what we know today or when no human interruption is imposed. The sustainable state could even be fluctuating with a noticeable degree. Engineers hold great responsibility to convincing environmentalists and the public what to expect by accurately predicting the dynamic outcomes without speculating on the potential dramatic changes ahead.

The skills of the engineer and of the life scientist are complementary. To convert the promises of molecular biology into new processes to make new products requires the integration of these skills. To function at this level, the engineer needs a solid understanding of biology and its experimental tools. In this book, we provide sufficient biological background for you to understand the chapters on applying engineering principles to biosystems. However, if you are serious about becoming a bioprocess engineer, it is desirable if you had taken courses in microbiology, biochemistry, and cell biology, as you would appreciate more of the engineering principles in this text.

1.7 The Story of Penicillin: The Dawn of Bioprocess Engineering

Penicillin is an antibiotic of significant importance. The familiar story of penicillin was well presented by Kargi and Shuler in their text "Bioprocess Engineering—Basic Concepts." As you would expect, the discovery of the chemical was accidental and the production of the chemical is elaborate. In Sep. 1928, Alexander Fleming at St. Mary’s Hospital in London was trying to isolate the bacterium, Staphylococcus aureus, which causes boils. The technique in use was to grow the bacterium on the surface of a nutrient solution. One Friday, the basement window was accidentally left open overnight. The experiments were carried out in the basement and one of the dishes had been contaminated inadvertently with a foreign particle. Normally, such a contaminated plate would be tossed out. However, Fleming noticed that no bacteria grew near the invading substance.

Fleming’s genius was to realize that this observation was meaningful and not a failed experiment. Fleming recognized that the cell killing must be due to an antibacterial agent. He recovered the foreign particle and found that it was a common mold of the Penicillium genus (later identified as Penicillium notatum). Fleming nurtured the mold to grow and, using the crude extraction methods then available, managed to obtain a tiny quantity of secreted material. He then demonstrated that this material had powerful antimicrobial properties and named the product penicillin. Fleming carefully preserved the culture, but the discovery lay essentially dormant for over a decade.

World War II provided the impetus to resurrect the discovery. Sulfa drugs have a rather restricted range of activity, and an antibiotic with minimal side effects and broader applicability was desperately needed. In 1939, Howard Florey and Ernst Chain of Oxford decided to build on Fleming’s observations. Norman Heatley played the key role in producing sufficient material for Chain and Florey to test the effectiveness of penicillin. Heatley, trained as a biochemist, performed as a bioprocess engineer. He developed an assay to monitor the amount of penicillin made so as to determine the kinetics of the fermentation, developed a culture technique that could be implemented easily, and devised a novel back-extraction process to recover the very delicate product. After months of immense effort, they produced enough penicillin to treat some laboratory animals.

Eighteen months after starting on the project, they began to treat a London policeman for a blood infection. The penicillin worked wonders initially and brought the patient to the point of recovery. Most unfortunately, the supply of penicillin was exhausted and the man relapsed and died. Nonetheless, Florey and Chain had demonstrated the great potential for penicillin if it could be made in sufficient amount. To make large amounts of penicillin would require a process, and for such a process development, engineers would be needed, in addition to microbial physiologists and other life scientists.

The war further complicated the situation. Great Britain’s industrial facilities were already totally devoted to the war. Florey and his associates approached pharmaceutical firms in the United States to persuade them to develop the capacity to produce penicillin, since the United States was not at war at that time. Many companies and government laboratories, assisted by many universities, took up the challenge. Particularly prominent were Merck, Pfizer, Squibb, and the USDA Northern Regional Research Laboratory in Peoria, Illinois.

The first efforts with fermentation were modest. A large effort went into attempts to chemically synthesize penicillin. This effort involved hundreds of chemists. Consequently, many companies were at first reluctant to commit to the fermentation process, beyond the pilot-plant stage. It was thought that the pilot-plant fermentation system could produce sufficient penicillin to meet the needs of clinical testing, but large-scale production would soon be done by chemical synthesis. At that time, U.S. companies had achieved a great deal of success with chemical synthesis of other drugs, which gave the companies a great deal of control over the drug’s production. The chemical synthesis of penicillin proved to be exceedingly difficult. (It was accomplished in the 1950s, and the synthesis route is still not competitive with fermentation.) However, in 1940 fermentation for the production of a pharmaceutical was an unproved approach, and most companies were betting on chemical synthesis to ultimately dominate.

The early clinical successes were so dramatic that in 1943 the War Production Board appointed A. L. Elder to coordinate the activities of producers to greatly increase the supply of penicillin. The fermentation route was chosen. As Elder recalls, I was ridiculed by some of my closest scientific friends for allowing myself to become associated with what obviously was to be a flop-namely, the commercial production of penicillin by a fermentation process (from Elder, 1970). The problems facing the fermentation process were indeed very formidable.

The problem was typical of most new fermentation processes: a valuable product made at very low levels. The low rate of production per unit volume would necessitate very large and inefficient reactors, and the low concentration (titer) made product recovery and purification very difficult. In 1939 the final concentration in a typical penicillin fermentation broth was one part per million (c.0.001 g/L); gold is more plentiful in sea water. Furthermore, penicillin is a fragile and unstable product, which places significant constraints on the approaches used for recovery and purification.

Life scientists at the Northern Regional Research Laboratory made many major contributions to the penicillin program. One was the development of a corn steep liquor-lactose based medium. Andrew J. Moyer succeeded to increase productivity about 10-fold with this medium in Nov. 26, 1941. A worldwide search by the laboratory for better producer strains of Penicillium led to the isolation of a Penicillium chrysogenum strain. This strain, isolated from a moldy cantaloupe at a Peoria fruit market, proved superior to hundreds of other isolates tested. Its progeny have been used in almost all commercial penicillin fermentations.

The other hurdle was to decide on a manufacturing process. One method involved the growth of the mold on the surface of moist bran. This bran method was discarded because of difficulties in temperature control, sterilization, and equipment size. The surface method involved growth of the mold on top of a quiescent medium. The surface method used a variety of containers, including milk bottles, and the term bottle plant indicated such a manufacturing technique. The surface method gave relatively high yields, but had a long growing cycle and was very labor intensive. The first manufacturing plants were bottle plants because the method worked and could be implemented quickly. However, it was clear that the surface method would not meet the full need for penicillin. If the goal of the War Production Board was met by bottle plants, it was estimated that the necessary bottles would fill a row stretching from New York City to San Francisco. Engineers generally favored a submerged tank process. The submerged process presented challenges in terms of both mold physiology and in tank design and operation. Large volumes of absolutely clean, oil- and dirt-free sterile air were required. What were then very large agitators were required, and the mechanical seal for the agitator shaft had to be designed to prevent the entry of organisms. Even today, problems of oxygen supply and heat removal are important constraints on antibiotic fermenter design. Contamination by foreign organisms could degrade the product as fast as it was formed, consume nutrients before they were converted to penicillin, or produce toxins.

In addition to these challenges in reactor design, there were similar hurdles in product recovery and purification. The very fragile nature of penicillin required the development of special techniques. A combination of pH shifts and rapid liquid-liquid extraction proved useful.

Soon processes using tanks