Etextbook 978 0137062706 bioprocess engineering basic concepts 3rd edition

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4.7. Summary 4.8. Appendix: Example Regulation of Complex Pathways Suggestions for Further Reading Problems 5 MAJOR METABOLIC PATHWAYS 5.1. Bioenergetics 5.2. Glucose Metabolism: Glycolysis and the TCA Cycle 5.3. Respiration 5.4. Control Sites in Aerobic Glucose Metabolism 5.5. Metabolism of Nitrogenous Compounds 5.6. Nitrogen Fixation 5.7. Metabolism of Hydrocarbons 5.8. Biodegradation of Xenobiotics 5.9. Overview of Biosynthesis 5.10. Overview of Anaerobic Metabolism 5.11. Overview of Autotrophic Metabolism 5.12. Summary Suggestions for Further Reading Questions 6 HOW CELLS GROW 6.1. Batch Growth 6.1.1. Quantifying Cell Concentration, 6.1.2. Growth Patterns and Kinetics in Batch Culture, 6.1.3. How Environmental Conditions Affect Growth Kinetics, 6.1.4. Heat Generation by Microbial Growth, 6.2. Quantifying Growth Kinetics 6.2.1. Unstructured Nonsegregated Models, 6.2.2. Models for Transient Behavior, 6.2.3. Cybernetic Models, 6.3. Cell Growth in Continuous Culture 6.3.1. Specific Devices for Continuous Culture, 6.3.2. The Ideal Chemostat, 6.3.3. The Chemostat as a Tool, 6.3.4. Deviations from Ideality, 6.4. Summary

Suggestions for Further Reading Problems 7 STOICHIOMETRY OF MICROBIAL GROWTH AND PRODUCT FORMATION 7.1. Coefficients for Atp Consumption and Oxygen 7.2. Stoichiometric Calculations 7.2.1. Elemental Balances, 7.2.2. Degree of Reduction, 7.3. Theoretical Predictions of Yield Coefficients 7.4. Estimation of Elemental Cell Composition 7.5. Stoichiometry by Oxidation-Reduction Half-Reactions 7.6. Thermodynamics of Biological Reactions 7.7. Summary Suggestions for Further Reading Problems 8 HOW CELLULAR INFORMATION IS ALTERED 8.1. Evolving Desirable Biochemical Activities Through Mutation and Selection 8.1.1. How Mutations Occur, 8.1.2. Selecting for Desirable Mutants, 8.2. Natural Mechanisms for Gene Transfer and Rearrangement 8.2.1. Genetic Recombination, 8.2.2. Transformation, 8.2.3. Transduction, 8.2.4. Episomes and Conjugation, 8.2.5. Transposons: Internal Gene Transfer, 8.3. Genetically Engineering Cells 8.3.1. Basic Elements of Genetic Engineering, 8.3.2. Genetic Engineering of Higher Organisms, 8.3.3. Genome Engineering, 8.4. Genomics 8.4.1. Experimental Techniques, 8.4.2. Computational Techniques, 8.5. Summary Suggestions for Further Reading

Problems Part 2 Engineering Principles for Bioprocesses 9 OPERATING CONSIDERATIONS FOR BIOREACTORS FOR SUSPENSION AND IMMOBILIZED CULTURES 9.1. Choosing the Cultivation Method 9.2. Modifying Batch and Continuous Reactors 9.2.1. Chemostat with Recycle, 9.2.2. Multistage Chemostat Systems, 9.2.3. Fed-Batch Operation, 9.2.4. Perfusion Systems, 9.2.5. Membrane Bioreactors, 9.3. Immobilized Cell Systems 9.3.1. Active Immobilization of Cells, 9.3.2. Passive Immobilization: Biological Films, 9.3.3. Diffusional Limitations in Immobilized Cell Systems, 9.3.4. Bioreactor Considerations in Immobilized Cell Systems, 9.4. Hybrid Bioreactors: Attached and Suspended Cells 9.5. Solid-State Fermentations 9.6. Summary Suggestions for Further Reading Problems 10 SELECTION, SCALE-UP, OPERATION, AND CONTROL OF BIOREACTORS 10.1. Scale-Up and its Difficulties 10.1.1. Overview of Traditional Reactor Types, 10.1.2. Reactors with Internal Mechanical Agitation, 10.1.3. Bubble Column and Loop Reactor, 10.1.4. Single-Use Bioreactors, 10.1.5. Considerations in Aeration, Agitation, and Heat Transfer, 10.1.6. Approaches to Scale-Up, 10.1.7. Scale-Down and Microbioreactors, 10.2. Bioreactor Instrumentation and Control 10.2.1. Instrumentation for Measurements of Active

Fermentation, 10.2.2. Using the Information Obtained, 10.3. Sterilization of Process Fluids 10.3.1. The Kinetics of Death, 10.3.2. Sterilization of Liquids, 10.3.3. Sterilization of Gases, 10.4. Summary Suggestions for Further Reading Problems 11 RECOVERY AND PURIFICATION OF PRODUCTS 11.1. Strategies to Recover and Purify Products 11.2. Separation of Insoluble Products 11.2.1. Filtration, 11.2.2. Centrifugation, 11.2.3. Coagulation and Flocculation, 11.3. Cell Disruption 11.3.1. Mechanical Methods, 11.3.2. Nonmechanical Methods, 11.4. Separation of Soluble Products 11.4.1. Liquid–Liquid Extraction, 11.4.2. Aqueous Two-Phase Extraction, 11.4.3. Precipitation, 11.4.4. Dialysis, 11.4.5. Reverse Osmosis, 11.4.6. Ultrafiltration and Microfiltration, 11.4.7. Cross-Flow Ultrafiltration and Microfiltration, 11.4.8. Adsorption, 11.4.9. Chromatography, 11.4.10. Electrophoresis, 11.4.11. Electrodialysis, 11.5. Finishing Steps for Purification 11.5.1. Crystallization, 11.5.2. Drying, 11.6. Integration of Reaction and Separation 11.7. Summary Suggestions for Further Reading

Problems 12 BIOPROCESS CONSIDERATIONS IN USING ANIMAL CELL CULTURES 12.1. Structure and Biochemistry of Animal Cells 12.2. Methods Used for the Cultivation of Animal Cells 12.2.1. Basic Techniques for Animal Cell Culture, 12.2.2. Growth Media, 12.2.3. Growth Dynamics for Animal Cells, 12.3. Bioreactor Considerations for Animal Cell Culture 12.4. Bioreactor Systems for Animal Cell Culture 12.4.1. Nonstirred Reactor Systems, 12.4.2. Systems for Entrapped Cells in Stirred Reactors, 12.4.3. Suspended Cultures, 12.5. Products of Animal Cell Cultures 12.6. Summary Suggestions for Further Reading Problems 13 BIOPROCESS CONSIDERATIONS IN USING PLANT CELL CULTURES 13.1. Why Plant Cell Cultures?

13.2. Plant Cells in Culture Compared to Microbes 13.3. Bioreactor Considerations 13.3.1. Bioreactors for Suspension Cultures, 13.3.2. Reactors Using Cell Immobilization, 13.3.3. Bioreactors for Organized Tissues, 13.4. Economics of Plant Cell Tissue Cultures 13.5. Summary Suggestions for Further Reading Problems 14 UTILIZING GENETICALLY ENGINEERED ORGANISMS 14.1. How the Product Influences Process Decisions 14.2. Guidelines for Choosing Host–Vector Systems 14.2.1. Escherichia Coli, 14.2.2. Gram-Positive Bacteria,

14.2.3. Lower Eucaryotic Cells, 14.2.4. Mammalian Cells, 14.2.5. Insect Cell–Baculovirus System, 14.2.6. Transgenic Animals, 14.2.7. Transgenic Plants and Plant Cell Culture, 14.2.8. Cell-Free Protein Synthesis, 14.2.9. Comparison of Strategies, 14.3. Process Constraints: Genetic Instability 14.3.1. Segregational Loss, 14.3.2. Plasmid Structural Instability, 14.3.3. Host Cell Mutations, 14.3.4. Growth-Rate-Dominated Instability, 14.4. Avoiding Process Problems in Plasmid Design 14.5. Predicting Host–Vector Interactions and Genetic Instability 14.6. Regulatory Constraints on Genetic Processes 14.7. Metabolic Engineering 14.8. Synthetic and Systems Biology 14.9. Protein Engineering 14.10. Summary Suggestions for Further Reading Problems 15 MEDICAL APPLICATIONS OF BIOPROCESS ENGINEERING 15.1. Tissue Engineering 15.1.1. What Is Tissue Engineering?, 15.1.2. Tissue-Engineered Skin Replacements, 15.1.3. Chondrocyte Culture for Cartilage Replacement, 15.2. Gene Therapy Using Viral Vectors 15.2.1. Models of Viral Infection, 15.2.2. Mass Production of Retrovirus, 15.3. Bioreactors 15.3.1. Stem Cells and Hematopoiesis, 15.3.2. Extracorporeal Artificial Liver, 15.3.3. Body-on-a-Chip Systems, 15.4. Summary

Suggestions for Further Reading Problems 16 BIOPROCESSES UTILIZING MIXED CULTURES 16.1. Major Classes of Interactions in Mixed Cultures 16.2. Simple Models Describing Mixed-Culture Interactions 16.3. Mixed Cultures in Nature 16.4. Industrial Utilization of Mixed Cultures 16.5. Biological Waste Treatment 16.5.1. Biological Waste-Treatment Processes, 16.5.2. Advanced Wastewater Treatment Systems, 16.5.3. Conversion of Wastewater to Useful Products, 16.6. Summary Suggestions for Further Reading Problems APPENDIX TRADITIONAL INDUSTRIAL BIOPROCESSES A.1. Anaerobic Bioprocesses A.1.1. Ethanol Production, A.1.2. Lactic Acid Production, A.1.3. Acetone–Butanol Production, A.2. Aerobic Processes A.2.1. Citric Acid Production, A.2.2. Production of Baker’s Yeast, A.2.3. Production of Penicillins, A.2.4. Production of High-Fructose Corn Syrup, A.3. Bioprocess Technologies: Biofuel and Bioenergy Production from Biomass A.3.1. Production of Liquid Fuels, A.3.2. Production of Gaseous Fuels from Biomass, A.3.3. Bioelectricity Generation from Wastes Using Microbial Fuel Cells, Suggestions for Further Reading INDEX

Preface This third edition of Bioprocess Engineering: Basic Concepts updates the prior two editions. Although the principles of bioprocess engineering stated in the first two editions remains sound, the field has made considerable advances in improving the productivity, consistency, and safety of bioprocesses.

Some of the major changes in the book come from biology itself with the development of tools that have greatly increased our ability to both understand and manipulate cell biology more effectively and cheaply. On the bioprocess side, tremendous advances have been made in production of biologicals with greater emphasis on processes incorporating animal cells. In particular, productivity in making many heterologous proteins has increased by several orders of magnitude over the last 16 years. The commercial use of animal cell culture has increased significantly over this period of time, and plant cell culture is starting to see commercial applications. Processes with microbes based on recombinant DNA and related technology have matured greatly. For any cell type, consistent authentic posttranslational processing of proteins remains challenging.

The development of the concepts in systems and synthetic biology is now having an impact on the thinking of bioprocess engineers.

In this edition, we either introduce or expand discussion of the following issues: • The role of small RNAs as regulators • Cell-free processes • Biofuels and energy • Synthetic biology • Role of genomics • Development of single-use technology in bioprocesses • Stem cell technology and utilization • Use of techniques of microfabrication and nanobiotechnology as well as 3D printing • Animal and plant cell biotechnology • Development in environmental biotechnology Teaching a subject as broad as bioprocess engineering in one semester is a difficult challenge, considering the range of backgrounds students may have. Although many engineering students have some formal exposure to specific areas of biology,

they often have not had an opportunity to complete courses from several areas of biology. The biological concepts necessary for an engineer are described in Part 1, “The Basics of Biology: An Engineer’s Perspective.” Although this condensed treatment cannot replace an in-depth knowledge of biochemistry, genetics, microbiology, and cell biology, it serves as a useful starting point for a bioprocess professional. On the process side, an instructor might wish to emphasize Part 2, “Engineering Principles for Bioprocesses,” dealing with biopharmaceuticals, bioenergy, or waste treatment, as they decide which topics would be effective for their students.

We hope this book imparts the excitement of bioprocess engineering as well as the basic concepts and knowledge to become a leader in this field.

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ACKNOWLEDGMENTS The development of this edition has been assisted by comments of many of our colleagues across the world.

Although a full list is too long to include here, we would like to acknowledge some specific contributions: Jeff Chalmers and Brian Pfleger served as technical editors for the book and provided many useful suggestions and corrections. Michael Thurston did yeoman’s work as development editor. Carolyn Lee-Parsons provided detailed input to Chapter 13 as well as extremely helpful suggestions on other chapters. Thomas Mansell and Jason Boock provided detailed input to Chapter 14. Others making significant contributions to various sections of the book include Kent Goklen, Itzcoatl Pla, Raymond Ketchum, Harrison Yoon, Taylor Stevenson, Jeff Varner, and Susan Roberts. Jason Haugh provided a number of homework problems to us. We would like to thank Dr. Serkan Eker of Dokuz Eylul University, Izmir, Turkey, for his expert services in drawing some of the figures in the book. We also acknowledge the excellent technical support provided by Brenda Stevens in the preparation of the manuscript for this book. In addition, many former students and colleagues have provided useful comments and suggestions.

We thank them, as well as many of our other colleagues, for their comments and advice.

About the Authors Dr. Michael L. Shuler is the Eckert Professor of Chemical and Biomolecular Engineering and of Biomedical Engineering at Cornell University. His research has focused on mathematical models of cell, heterologous protein expression systems, bioremediation, plant cell tissue culture processes, and “Bodyon-a-Chip” technology for drug development. Many of those technologies are being used commercially.

Dr. Fikret Kargi is a retired professor of Environmental Engineering from Dokuz Eylul University in Izmir, Turkey. He was the former Chair of Environmental Sciences. His recent research interests include hydrogen gas production from wastes by fermentation and electrohydrolysis, development of novel biofilm, and advanced oxidation processes for wastewater treatment.

Dr. Matthew P. DeLisa is the William L. Lewis Professor of Engineering in the Robert Frederick Smith School of Chemical and Biomolecular Engineering at Cornell University. His research focuses on understanding and controlling the molecular mechanisms underlying protein biogenesis—folding and assembly, membrane translocation, and posttranslational modifications—in the complex environment of a living cell. His contributions to science and engineering include the invention of numerous commercially important technologies for facilitating the discovery, design, and manufacturing of human drugs and seminal discoveries in the areas of cellular protein folding and protein translocation.

Part 1: The Basics of Biology: An Engineer’s Perspective

1. What Is a Bioprocess Engineer?

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. But now it can be done in a purposeful, predetermined manner with the molecular-level manipulation of DNA. We now have tools to probe the mysteries of life in a way unimaginable even a few decades ago. We are at the doorstep of being able to chemically synthesize whole genomes and transfer them into a host bacterium to create semisynthetic life. We now have a whole host of new techniques to rationally design novel organisms.

With this intellectual revolution emerged new visions and new hopes: new medicines, semisynthetic organs grown in large vessels, abundant and nutritious foods, computers based on biological molecules rather than silicon chips, superorganisms to degrade pollutants, a wide array of consumer products, and potentially new sources of energy.

These dreams will remain dreams without hard work.

Engineers will play an essential role in converting these visions into reality. Biological systems are very complex and beautifully constructed, but they obey the rules of chemistry and physics and are susceptible to engineering analysis. Living cells are predictable, and the processes to use them can be rationally constructed on commercial scales. Doing this is the job of the bioprocess engineer.

Probably the reason you are reading this book is your desire to participate in this intellectual revolution and to make an important contribution to society. You can do it, but it is not easy to combine the skills of the engineer with those of the biologist. This book’s intent is to help you begin to develop these skills. This book and a one-term course are not enough to make you a complete bioprocess engineer, but it will help you form the necessary foundation.

In this chapter, we discuss the origins of bioprocess engineering, how bioprocess engineering evolved, and the complementary roles of biologists and engineers. We then consider the regulatory constraints on bioprocesses and how they impact the engineer.

1.1. BIOTECHNOLOGY AND BIOPROCESS ENGINEERING When new fields emerge from new ideas, old terminology is

usually not adequate to describe these fields. Biotechnology and what constitutes engineering in this field are best described with examples rather than single words or short phrases. However, it is critical to understand the cell’s inner workings to harness the cellular machinery and engineer the cell to perform a designated task.

Biotechnology usually implies the use or development of methods of direct genetic manipulation for a socially desirable goal. Such goals might be the production of a particular chemical, but they may also involve the production of better plants or seeds, or gene therapy, or the use of specially designed organisms to degrade wastes. The key element for many professionals is the use of sophisticated techniques outside the cell for genetic manipulation. Others interpret biotechnology in a much broader sense and equate it with the technical use of biology for production of pharmaceuticals, chemicals, food, and diagnostics; they may include engineering as a subcomponent of biotechnology.

Many terms are used to describe engineers working with biotechnology. Bioengineering is a broad title and includes work on medical, bioprocess, agricultural, and environmental systems; its practitioners include agricultural, electrical, mechanical, industrial, environmental, and chemical engineers as well as others. Biological engineering is similar but emphasizes applications to plants and animals. Metabolic engineering is the design of cells with genetically altered pathways to make small molecules that are often novel for that cell. This term stresses human design of cells for manufacture of specialty chemicals. 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, particularly for production of biologics, chemicals, and fuels. Biomedical engineering has been considered to be 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. It focuses on the human body with an emphasis on application of engineering principles from a variety of disciplines to design medical devices, synthetic organs, and novel methods for drug delivery and to develop diagnostics and instrumentation. 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.”

There is a difference between bioprocess engineering and biochemical engineering. In addition to chemical engineering, bioprocess engineering includes the work of mechanical, electrical, environmental, and industrial engineers to apply the principles of their disciplines to processes based on using living cells or subcomponents of such cells. The problems of detailed equipment design, sensor development, control algorithms, and manufacturing strategies can utilize principles from these disciplines. Biochemical engineering is more limited in the sense that it draws primarily from chemical engineering principles and broader in the sense that it is not restricted to well-defined, artificially constructed processes but can be applied to natural systems.

This book focuses primarily on the application of chemical engineering principles to systems containing biological catalysts, but with an emphasis on those systems making use of biotechnology. 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.

1.2. DIFFERING APPROACHES TO RESEARCH FOR BIOLOGISTS AND ENGINEERS The fundamental trainings of biologists 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 solid 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 adept at the formation of testable hypotheses, experimental design, and data interpretation from complex systems. Engineers quite often are unfamiliar with the experimental techniques and

strategies used by life scientists.

The skills of the engineer and 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. This book provides sufficient biological background for you to understand how to apply engineering principles to biosystems. However, if you are serious about becoming a bioprocess engineer, you will need to take further courses in microbiology, biochemistry, and cell biology, as well as more advanced work in bioengineering. If you already have these courses, this book can be used for review and to provide a coherent framework for applications to bioprocessing.

1.3. THE STORY OF PENICILLIN: HOW BIOLOGISTS AND ENGINEERS WORK TOGETHER In September 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 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 (see Figure 1.1).

Figure 1.1. Alexander Fleming’s original plate showing the growth of the mold Penicillium notatum and its inhibitory action on bacterial growth. (With permission, from Corbis Corporation.)

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. 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 bobby 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, in addition to microbial physiologists and other life scientists, would be needed.

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 significant 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 (ca. 0.001 g/l). 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. This medium increased productivity about tenfold. 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 liquid 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 tank design and operation. Large volumes of absolutely clean, oil- and dirtfree 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 of about 10,000 gal were built.

Pfizer completed in less than 6 months the first plant for commercial production of penicillin by submerged fermentation (Hobby, 1985). The plant had 14 tanks, each of 7000-gal capacity. Large-scale fermenters (typically 50,000 to 100,000 l) now used in antibiotic fermentations are shown in Figures 1.2 and 1.3.

Figure 1.2. Series of large-scale antibiotic fermenters.

(Courtesy of Pfizer, Inc.)

Figure 1.3. Inside view of a large antibiotic fermenter.

(From Trends in Biotechnology 3(6), 1985. Used with permission of Elsevier Science Publishers.)

By a combination of good luck and hard work, the United States had the capacity by the end of World War II to produce enough penicillin for almost 100,000 patients per year. The impact of penicillin was immediate, and the advent of antibiotics saved many lives. Although the first impact was in war zones, the overall effect was critical globally to fighting many diseases (see Figure 1.4).

Figure 1.4. First impact of penicillin in war zones. (WW II Museum, New Orleans, LA.)

This accomplishment required a high level of multidisciplinary work. For example, Merck realized that men who understood both engineering and biology were not available. Merck assigned a chemical engineer and microbiologist together to each aspect of the problem. They planned, executed, and analyzed the experimental program jointly, “almost as if they were one man” (Silcox in Elder, 1970).

Progress with penicillin fermentation has continued, as has the need for the interaction of biologists and engineers. From 1939 to now, the yield of penicillin has gone from 0.001 g/l to over 50 g/l of fermentation broth. Progress has involved better understanding of mold physiology, metabolic pathways, penicillin structure, methods of mutation and selection of mold genetics, process control, and reactor design (see Figure 1.5).

Figure 1.5. Schematic of penicillin production process.

Before the penicillin process, almost no chemical engineers sought specialized training in the life sciences. With the advent of modern antibiotics, the concept of a bioprocess engineer was born. The penicillin process also established a paradigm for bioprocess development and biochemical engineering. This paradigm still guides much of our profession’s thinking. The mindset of bioprocess engineers was cast with the penicillin experience. It is for this reason that we have focused on the penicillin story rather than on an example for production of a protein from a genetically engineered organism. Although many parallels can be made between the penicillin process and our efforts to use recombinant DNA, no similar paradigm has yet emerged from our experience with genetically engineered cells.

We must continually reexamine the prejudices the field has inherited from the penicillin experience.

It is you, the student, who will best be able to challenge these prejudices.

1.4. BIOPROCESSES: REGULATORY CONSTRAINTS To understand the mindset of a bioprocess engineer, you must understand the regulatory climate in which many bioprocess engineers work. The U.S. FDA (Food and Drug Administration) and its equivalents in other countries must insure the safety and efficacy of medicines. For bioprocess engineers working in the pharmaceutical or biotechnology industry, the primary concern is not reduction of manufacturing cost (although that is still a very desirable goal), but the production of a product of consistently high quality in amounts to satisfy the medical needs of the population.

Consider briefly the process by which a drug obtains FDA approval. A typical drug undergoes 6.5 years of development from the discovery stage through preclinical testing in animals.

Human clinical trials are conducted in three phases. Phase 1 clinical trials (about 1 year) are used to test safety; typically 20 to 80 volunteers are used. Phase II clinical trials (about 2 years) use 100 to 300 patients, and the emphasis is on efficacy (i.e., whether it helps the patient) as well as further determining which side effects exist. Compounds that are still promising enter phase III clinical trials (about 3 years) with 1000 to 3000 patients. Since individuals vary in body chemistry, it is important to test the range of responses in terms of both side effects and efficacy by using a representative cross section of the

population. Data from clinical trials is presented to the FDA for review (about 18 months). If the clinical trials are well designed and demonstrate statistically significant improvements in health with acceptable side effects, the drug is likely to be approved. Even after this point, there is continued monitoring of the drug for adverse effects. The whole drug discoverythrough-approval process takes 12 to 15 years on the average and costs up to about $5 billion (in 2013). Only one in ten drugs that enter human clinical trials receives approval. Recent FDA reforms have decreased the time to obtain approval for lifesaving drugs in treatment of diseases such as cancer and AIDS, but the overall process is still lengthy.

This process greatly affects a bioprocess engineer. FDA approval is for the product and the process together. There are tragic examples in which a small process change has allowed a toxic trace compound to form or become incorporated in the final product, resulting in severe side effects, including death.

For example, heparin contaminated with oversulfated chondroitin sulfate caused significant problems. Thus, process changes may require new clinical trials to test the safety of the resulting product. Since clinical trials are very expensive, process improvements are made under a limited set of circumstances. Even during clinical trials it is difficult to make major process changes.

Drugs sold on the market or used in clinical trials must come from facilities that are certified as good manufacturing practice (GMP). GMP concerns the actual manufacturing facility design and layout, the equipment and procedures, training of production personnel, control of process inputs (e.g., raw materials and cultures), and handling of product. The plant layout and design must prevent contamination of the product and dictates the flow of material, personnel, and air. Equipment and procedures must be validated. Procedures include not only operation of a piece of equipment but also cleaning and sterilization. Computer software used to monitor and control the process must be validated. Off-line assays done in laboratories must satisfy good laboratory practices (GLP).

Procedures are documented by standard operating procedures (SOP).

The GMP guidelines stress the need for documented procedures to validate performance: “Process validation is establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality characteristics” and “There shall be written procedures for

production and process-control to assure that products have the identity, strength, quality, and purity they purport or are † represented to possess.” †

Guidelines on General Principles of Process Validation, Federal Register of May 11, 1987 (52 FR 17638).

The actual process of doing validation is often complex, particularly when a whole facility design is considered. The FDA provides extensive information and guidelines, which are updated regularly. Bioprocess engineers involved in biomanufacturing for pharmaceuticals need to consult these sources. For example, the recent introduction of disposable bioreactors requires considerations of how FDA regulations can be applied. However, certain key concepts do not change. These concepts are written documentation, consistency of procedures, consistency of product, and demonstrable measures of product quality, particularly purity and safety. These tasks are demanding and require careful attention to detail. Bioprocess engineers often find that much of their effort is to satisfy these regulatory requirements.

The key point is that process changes cannot be made without considering their significant regulatory impact.

SUGGESTIONS FOR FURTHER READING Synthetic Biology GIBSON, D. G., ET AL., Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome, Science 329(5987): 52–56, 2010.

History of Penicillin ELDER, A. L., ed., The History of Penicillin Production, Chem.

Eng. Prog. Symp. Ser. 66 (#100), American Institute of Chemical Engineers, New York, 1970.

HOBBY, G. L., Penicillin. Meeting the Challenge, Yale University Press, New Haven, CT, 1985.

LAX, E., The Mold in Dr. Florey’s Coat: The Story of the Penicillin Miracle, Henry Holt & Company, New York, New York, 2004.

MOBERG, C. L., Penicillin’s Forgotten Man: Norman Heatley, Science 253(5021): 734–735, 1991.

Regulatory Issues AGALLOCO, J. P., AND CARLETON, F. J., Validation of

Pharmaceutical Processes, 3d ed., CRC Press, Boca Raton, FL, 2007.

KISHIMOTO, T. K., ET AL., Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System, N. Engl. J. Med. 358(23): 2457–2567, 2008.

MULLARD, A., New Drugs Cost US $2.6 Billion to Develop, Nat. Rev. Drug Discov. 13: 877, 2014.

NAGLAK, T. J., KEITH, M. G., AND OMSTEAD, D. R., Validation of Fermentation Processes, Biopharm. (July/Aug.): 28–36, 1994.

RAO, G., MOREIRA, A., AND BRONSON, K., Disposable Bioprocessing: The Future Has Arrived, Biotechnol. Bioeng.

102: 348–356, 2009.

Education LEE-PARSONS, C. W. T., Biochemical Engineering Taught in the Context of Drug Discovery to Manufacturing. Chem. Eng. Edu. 39(Pt 3): 208–221, 2005.

QUESTIONS 1.1. What is GMP, and how does it relate to the regulatory process for pharmaceuticals?

1.2. When the FDA approves a process, it requires validation of the process. Explain what validation means in the FDA context.

1.3. Why does the FDA approve the process and product together?

1.4. Carefully specify the differences among biotechnologist, bioprocess engineer, and bioengineer.

1.5. What is achieved by submerged fermentation in penicillin production versus surface fermentation?

2. An Overview of Biological Basics A basic understanding of biology is essential to the biochemical engineer. Here we present selected aspects of basic biology at a level that will allow the student to function as a biochemical engineer. Although specific courses in biochemistry, genetics, microbiology, and cell biology provide much more depth, most engineering students do not have an opportunity to take three or four courses in the biological sciences, and some have no opportunity to take any of these courses. Thus, we have selected the parts of microbiology, biochemistry, genetics, and cell biology most directly relevant to bioprocesses to emphasize throughout chapters 2 to 8 and present this material with examples of application to bioprocesses.

2.1. MICROBIAL DIVERSITY Life is very tenacious and can exist in extreme environments.

Living cells can be found almost anywhere that water is in the liquid state. The right temperature, pH, and moisture levels vary from one organism to another.

Organisms from extreme environments (extremophiles) often provide the human race with important tools for processes to make useful chemicals and medicinals. They are also key to the maintenance of natural cycles and can be used in the recovery of metals from low-grade ores or in the desulfurization of coal or other fuels. The fact that organisms can develop the capacity to exist and multiply in almost any environment on earth is extremely useful.

Some cells can grow at –20°C (in a brine to prevent freezing), while others can grow at 120°C (where water is under high enough pressure to prevent boiling). Cells that grow best at low temperatures (below 20°C) are usually called psychrophiles, and those with temperature optima in the range of 20° to 50°C are mesophiles. Organisms that grow best at temperatures greater than 50°C are thermophiles.

Most organisms grow around neutral pH (6 to 8) and are called neutrophiles. There are some, however, that have pH optima far from neutrality, preferring pH values down to 1 or 2 (acidophiles) or well above pH 9 (alkaliphiles). Some organisms can grow at both low pH values and high temperatures.

Although most organisms can grow only where water activity is high, others can grow on barely moist solid surfaces or in solutions with high salt concentrations (halophiles).

Some cells require oxygen for growth and metabolism. Such organisms can be termed aerobic. Other organisms are inhibited by the presence of oxygen and grow only anaerobically. Some organisms can switch the metabolic pathways to allow them to grow under either circumstance.

Such organisms are facultative.

Often, organisms can grow in environments with almost no obvious source of nutrients. Some cyanobacteria (formerly called blue-green algae) can grow in an environment with only a little moisture and a few dissolved minerals. These bacteria are photosynthetic and can convert CO2 from the atmosphere into the organic compounds necessary for life. They can also convert N2 into NH3 for use in making the essential building blocks of life. Such organisms are important in colonizing nutrientdeficient environments.

Not only do organisms occupy a wide variety of habitats, but they also come in a wide range of sizes and shapes. Spherical, cylindrical, ellipsoidal, spiral, and pleomorphic cells exist.

Special names are used to describe the shape of bacteria. A cell with a spherical or elliptical shape is often called a coccus (plural, cocci); a cylindrical cell is a rod or bacillus (plural, bacilli); a spiral-shaped cell is a spirillum (plural, spirilla).

Some cells may change shape in response to changes in their local environment.

Thus, organisms can be found in the most extreme environments and have a wondrous array of different shapes, sizes, and metabolic capabilities. This great diversity provides the engineer with an immense variety of potential tools, many of which have been exploited. Take, for example, the heatresistant enzyme Taq DNA polymerase, an enzyme which is derived from a thermophilic bacterium named Thermus aquaticus. Taq has become one of the most important enzymes in molecular biology because of its use in a DNA amplification technique known as the polymerase chain reaction (PCR; discussed in Chapter 8, “How Cellular Information Is Altered”).

2.1.1. Naming Cells How cells are named is complicated by the bewildering variety of organisms present. A systematic approach to classifying these organisms is an essential aid to their intelligent use. Taxonomy is the development of approaches to organize and summarize our knowledge about the variety of organisms that exist. Although knowledge of taxonomy may seem remote from the needs of the engineer, it is necessary for efficient communication among engineers and

scientists working with living cells. Taxonomy can also play a critical role in patent litigation involving bioprocesses.

While taxonomy is concerned with approaches to classification, nomenclature refers to the actual naming of organisms. For microorganisms, we use a dual name (binary nomenclature). The names are given in Latin, or are Latinized.

A genus is a group of related species, and a species includes organisms that are substantially alike. A common gut organism that has been well studied is Escherichia coli. Escherichia is the genus and coli the species. When writing a report or paper, it is common practice to give the full name when the organism is first mentioned but in subsequent discussion to abbreviate the genus to the first letter. In this case, we would use E. coli.

Although organisms that belong to the same species all share the same major characteristics, there are subtle and often technologically important variations within species. An E. coli used in one laboratory may differ from that used in another.

Thus, various strains and substrains are designated by the addition of letters and numbers. For example, E. coli B/r A will differ in growth and physiological properties from E. coli K12.

Now that we know how to name organisms, we could consider broader classification up to the level of kingdoms.

There is no universal agreement on how to classify microorganisms at this level. Such classification is rather arbitrary and need not concern us. However, we must be aware that there are two primary cell types: eucaryotic and procaryotic. The primary difference between them is the presence or absence of a membrane around the cell’s genetic information.

Procaryotes have a simple structure with a single chromosome. Procaryotic cells have no nuclear membrane and no organelles, such as the mitochondria and endoplasmic reticulum. Eucaryotes have a more complex internal structure, with more than one chromosome (DNA molecule) in the nucleus. Eucaryotic cells have a true nuclear membrane and contain mitochondria, endoplasmic reticulum, Golgi apparatus, and a variety of specialized organelles. We describe each of these components in Section 2.1.4. A detailed comparison of procaryotes and eucaryotes is presented in Table 2.1. Structural differences between procaryotes and eucaryotes are discussed later.

TABLE 2.1. A Comparison of Procaryotes with Eucaryotes

There is further distinction of subgroups within procaryotes.

Evidence suggests that a common or universal ancestor gave rise to three distinctive branches of life: eucaryotes, eubacteria (or “true” bacteria), and archaebacteria. Table 2.2 summarizes some of the distinctive features of these groups. All of the genes of whole organisms, known as the genome, can now be readily sequenced and this information is having a great impact on our understanding of how these families evolved and are related.

TABLE 2.2. Primary Subdivisions of Cellular Organisms That Are Now Recognized

Viruses cannot be classified under any of these categories, as they are not free-living organisms. Let’s consider first some of the characteristics of these rather simple “organisms.”

2.1.2. Viruses Viruses are very small and are obligate parasites of other cells, such as bacterial, yeast, plant, and animal cells. Viruses cannot capture or store free energy and are not functionally active except when inside their host cells. The sizes of viruses vary from 30 to 200 nanometers (nm). Viruses contain either DNA (DNA viruses) or RNA (RNA viruses) as genetic material. DNA stands for deoxyribonucleic acid, and RNA is ribonucleic acid; we will soon discuss these molecules in more detail. In free-living cells, all genetic information is contained in the DNA, whereas viruses can use either RNA or

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DANCE ON STILTS AT THE GIRLS’ UNYAGO, NIUCHI

Newala, too, suffers from the distance of its water-supply—at least the Newala of to-day does; there was once another Newala in a lovely valley at the foot of the plateau. I visited it and found scarcely a trace of houses, only a Christian cemetery, with the graves of several missionaries and their converts, remaining as a monument of its former glories. But the surroundings are wonderfully beautiful. A thick grove of splendid mango-trees closes in the weather-worn crosses and headstones; behind them, combining the useful and the agreeable, is a whole plantation of lemon-trees covered with ripe fruit; not the small African kind, but a much larger and also juicier imported variety, which drops into the hands of the passing traveller, without calling for any exertion on his part. Old Newala is now under the jurisdiction of the native pastor, Daudi, at Chingulungulu, who, as I am on very friendly terms with him, allows me, as a matter of course, the use of this lemon-grove during my stay at Newala.

FEET MUTILATED BY THE RAVAGES OF THE “JIGGER” (Sarcopsylla penetrans)

The water-supply of New Newala is in the bottom of the valley, some 1,600 feet lower down. The way is not only long and fatiguing, but the water, when we get it, is thoroughly bad. We are suffering not only from this, but from the fact that the arrangements at Newala are nothing short of luxurious. We have a separate kitchen—a hut built against the boma palisade on the right of the baraza, the interior of which is not visible from our usual position. Our two cooks were not long in finding this out, and they consequently do—or rather neglect to do—what they please. In any case they do not seem to be very particular about the boiling of our drinking-water—at least I can attribute to no other cause certain attacks of a dysenteric nature, from which both Knudsen and I have suffered for some time. If a man like Omari has to be left unwatched for a moment, he is capable of anything. Besides this complaint, we are inconvenienced by the state of our nails, which have become as hard as glass, and crack on the slightest provocation, and I have the additional infliction of pimples all over me. As if all this were not enough, we have also, for the last week been waging war against the jigger, who has found his Eldorado in the hot sand of the Makonde plateau. Our men are seen all day long—whenever their chronic colds and the dysentery likewise raging among them permit—occupied in removing this scourge of Africa from their feet and trying to prevent the disastrous consequences of its presence. It is quite common to see natives of this place with one or two toes missing; many have lost all their toes,

or even the whole front part of the foot, so that a well-formed leg ends in a shapeless stump. These ravages are caused by the female of Sarcopsylla penetrans, which bores its way under the skin and there develops an egg-sac the size of a pea. In all books on the subject, it is stated that one’s attention is called to the presence of this parasite by an intolerable itching. This agrees very well with my experience, so far as the softer parts of the sole, the spaces between and under the toes, and the side of the foot are concerned, but if the creature penetrates through the harder parts of the heel or ball of the foot, it may escape even the most careful search till it has reached maturity.

Then there is no time to be lost, if the horrible ulceration, of which we see cases by the dozen every day, is to be prevented. It is much easier, by the way, to discover the insect on the white skin of a European than on that of a native, on which the dark speck scarcely shows. The four or five jiggers which, in spite of the fact that I constantly wore high laced boots, chose my feet to settle in, were taken out for me by the all-accomplished Knudsen, after which I thought it advisable to wash out the cavities with corrosive sublimate. The natives have a different sort of disinfectant—they fill the hole with scraped roots. In a tiny Makua village on the slope of the plateau south of Newala, we saw an old woman who had filled all the spaces under her toe-nails with powdered roots by way of prophylactic treatment. What will be the result, if any, who can say?

The rest of the many trifling ills which trouble our existence are really more comic than serious. In the absence of anything else to smoke, Knudsen and I at last opened a box of cigars procured from the Indian store-keeper at Lindi, and tried them, with the most distressing results. Whether they contain opium or some other narcotic, neither of us can say, but after the tenth puff we were both “off,” three-quarters stupefied and unspeakably wretched. Slowly we recovered—and what happened next? Half-an-hour later we were once more smoking these poisonous concoctions—so insatiable is the craving for tobacco in the tropics.

Even my present attacks of fever scarcely deserve to be taken seriously. I have had no less than three here at Newala, all of which have run their course in an incredibly short time. In the early afternoon, I am busy with my old natives, asking questions and making notes. The strong midday coffee has stimulated my spirits to

an extraordinary degree, the brain is active and vigorous, and work progresses rapidly, while a pleasant warmth pervades the whole body. Suddenly this gives place to a violent chill, forcing me to put on my overcoat, though it is only half-past three and the afternoon sun is at its hottest. Now the brain no longer works with such acuteness and logical precision; more especially does it fail me in trying to establish the syntax of the difficult Makua language on which I have ventured, as if I had not enough to do without it. Under the circumstances it seems advisable to take my temperature, and I do so, to save trouble, without leaving my seat, and while going on with my work. On examination, I find it to be 101·48°. My tutors are abruptly dismissed and my bed set up in the baraza; a few minutes later I am in it and treating myself internally with hot water and lemon-juice.

Three hours later, the thermometer marks nearly 104°, and I make them carry me back into the tent, bed and all, as I am now perspiring heavily, and exposure to the cold wind just beginning to blow might mean a fatal chill. I lie still for a little while, and then find, to my great relief, that the temperature is not rising, but rather falling. This is about 7.30 p.m. At 8 p.m. I find, to my unbounded astonishment, that it has fallen below 98·6°, and I feel perfectly well. I read for an hour or two, and could very well enjoy a smoke, if I had the wherewithal—Indian cigars being out of the question.

Having no medical training, I am at a loss to account for this state of things. It is impossible that these transitory attacks of high fever should be malarial; it seems more probable that they are due to a kind of sunstroke. On consulting my note-book, I become more and more inclined to think this is the case, for these attacks regularly follow extreme fatigue and long exposure to strong sunshine. They at least have the advantage of being only short interruptions to my work, as on the following morning I am always quite fresh and fit.

My treasure of a cook is suffering from an enormous hydrocele which makes it difficult for him to get up, and Moritz is obliged to keep in the dark on account of his inflamed eyes. Knudsen’s cook, a raw boy from somewhere in the bush, knows still less of cooking than Omari; consequently Nils Knudsen himself has been promoted to the vacant post. Finding that we had come to the end of our supplies, he began by sending to Chingulungulu for the four sucking-pigs which we had

bought from Matola and temporarily left in his charge; and when they came up, neatly packed in a large crate, he callously slaughtered the biggest of them. The first joint we were thoughtless enough to entrust for roasting to Knudsen’s mshenzi cook, and it was consequently uneatable; but we made the rest of the animal into a jelly which we ate with great relish after weeks of underfeeding, consuming incredible helpings of it at both midday and evening meals. The only drawback is a certain want of variety in the tinned vegetables. Dr. Jäger, to whom the Geographical Commission entrusted the provisioning of the expeditions—mine as well as his own—because he had more time on his hands than the rest of us, seems to have laid in a huge stock of Teltow turnips,[46] an article of food which is all very well for occasional use, but which quickly palls when set before one every day; and we seem to have no other tins left. There is no help for it—we must put up with the turnips; but I am certain that, once I am home again, I shall not touch them for ten years to come.

Amid all these minor evils, which, after all, go to make up the genuine flavour of Africa, there is at least one cheering touch: Knudsen has, with the dexterity of a skilled mechanic, repaired my 9 × 12 cm. camera, at least so far that I can use it with a little care.

How, in the absence of finger-nails, he was able to accomplish such a ticklish piece of work, having no tool but a clumsy screw-driver for taking to pieces and putting together again the complicated mechanism of the instantaneous shutter, is still a mystery to me; but he did it successfully. The loss of his finger-nails shows him in a light contrasting curiously enough with the intelligence evinced by the above operation; though, after all, it is scarcely surprising after his ten years’ residence in the bush. One day, at Lindi, he had occasion to wash a dog, which must have been in need of very thorough cleansing, for the bottle handed to our friend for the purpose had an extremely strong smell. Having performed his task in the most conscientious manner, he perceived with some surprise that the dog did not appear much the better for it, and was further surprised by finding his own nails ulcerating away in the course of the next few days. “How was I to know that carbolic acid has to be diluted?” he mutters indignantly, from time to time, with a troubled gaze at his mutilated finger-tips.

Since we came to Newala we have been making excursions in all directions through the surrounding country, in accordance with old habit, and also because the akida Sefu did not get together the tribal elders from whom I wanted information so speedily as he had promised. There is, however, no harm done, as, even if seen only from the outside, the country and people are interesting enough.

The Makonde plateau is like a large rectangular table rounded off at the corners. Measured from the Indian Ocean to Newala, it is about seventy-five miles long, and between the Rovuma and the Lukuledi it averages fifty miles in breadth, so that its superficial area is about two-thirds of that of the kingdom of Saxony. The surface, however, is not level, but uniformly inclined from its south-western edge to the ocean. From the upper edge, on which Newala lies, the eye ranges for many miles east and north-east, without encountering any obstacle, over the Makonde bush. It is a green sea, from which here and there thick clouds of smoke rise, to show that it, too, is inhabited by men who carry on their tillage like so many other primitive peoples, by cutting down and burning the bush, and manuring with the ashes. Even in the radiant light of a tropical day such a fire is a grand sight.

Much less effective is the impression produced just now by the great western plain as seen from the edge of the plateau. As often as time permits, I stroll along this edge, sometimes in one direction, sometimes in another, in the hope of finding the air clear enough to let me enjoy the view; but I have always been disappointed.

Wherever one looks, clouds of smoke rise from the burning bush, and the air is full of smoke and vapour. It is a pity, for under more favourable circumstances the panorama of the whole country up to the distant Majeje hills must be truly magnificent. It is of little use taking photographs now, and an outline sketch gives a very poor idea of the scenery. In one of these excursions I went out of my way to make a personal attempt on the Makonde bush. The present edge of the plateau is the result of a far-reaching process of destruction through erosion and denudation. The Makonde strata are everywhere cut into by ravines, which, though short, are hundreds of yards in depth. In consequence of the loose stratification of these beds, not only are the walls of these ravines nearly vertical, but their upper end is closed by an equally steep escarpment, so that the

western edge of the Makonde plateau is hemmed in by a series of deep, basin-like valleys. In order to get from one side of such a ravine to the other, I cut my way through the bush with a dozen of my men.

It was a very open part, with more grass than scrub, but even so the short stretch of less than two hundred yards was very hard work; at the end of it the men’s calicoes were in rags and they themselves bleeding from hundreds of scratches, while even our strong khaki suits had not escaped scatheless.

NATIVE PATH THROUGH THE MAKONDE BUSH, NEAR MAHUTA

I see increasing reason to believe that the view formed some time back as to the origin of the Makonde bush is the correct one. I have no doubt that it is not a natural product, but the result of human occupation. Those parts of the high country where man—as a very slight amount of practice enables the eye to perceive at once—has not yet penetrated with axe and hoe, are still occupied by a splendid timber forest quite able to sustain a comparison with our mixed forests in Germany. But wherever man has once built his hut or tilled his field, this horrible bush springs up. Every phase of this process

may be seen in the course of a couple of hours’ walk along the main road. From the bush to right or left, one hears the sound of the axe— not from one spot only, but from several directions at once. A few steps further on, we can see what is taking place. The brush has been cut down and piled up in heaps to the height of a yard or more, between which the trunks of the large trees stand up like the last pillars of a magnificent ruined building. These, too, present a melancholy spectacle: the destructive Makonde have ringed them— cut a broad strip of bark all round to ensure their dying off—and also piled up pyramids of brush round them. Father and son, mother and son-in-law, are chopping away perseveringly in the background—too busy, almost, to look round at the white stranger, who usually excites so much interest. If you pass by the same place a week later, the piles of brushwood have disappeared and a thick layer of ashes has taken the place of the green forest. The large trees stretch their smouldering trunks and branches in dumb accusation to heaven—if they have not already fallen and been more or less reduced to ashes, perhaps only showing as a white stripe on the dark ground.

This work of destruction is carried out by the Makonde alike on the virgin forest and on the bush which has sprung up on sites already cultivated and deserted. In the second case they are saved the trouble of burning the large trees, these being entirely absent in the secondary bush.

After burning this piece of forest ground and loosening it with the hoe, the native sows his corn and plants his vegetables. All over the country, he goes in for bed-culture, which requires, and, in fact, receives, the most careful attention. Weeds are nowhere tolerated in the south of German East Africa. The crops may fail on the plains, where droughts are frequent, but never on the plateau with its abundant rains and heavy dews. Its fortunate inhabitants even have the satisfaction of seeing the proud Wayao and Wamakua working for them as labourers, driven by hunger to serve where they were accustomed to rule.

But the light, sandy soil is soon exhausted, and would yield no harvest the second year if cultivated twice running. This fact has been familiar to the native for ages; consequently he provides in time, and, while his crop is growing, prepares the next plot with axe and firebrand. Next year he plants this with his various crops and

lets the first piece lie fallow. For a short time it remains waste and desolate; then nature steps in to repair the destruction wrought by man; a thousand new growths spring out of the exhausted soil, and even the old stumps put forth fresh shoots. Next year the new growth is up to one’s knees, and in a few years more it is that terrible, impenetrable bush, which maintains its position till the black occupier of the land has made the round of all the available sites and come back to his starting point.

The Makonde are, body and soul, so to speak, one with this bush.

According to my Yao informants, indeed, their name means nothing else but “bush people.” Their own tradition says that they have been settled up here for a very long time, but to my surprise they laid great stress on an original immigration. Their old homes were in the south-east, near Mikindani and the mouth of the Rovuma, whence their peaceful forefathers were driven by the continual raids of the Sakalavas from Madagascar and the warlike Shirazis[47] of the coast, to take refuge on the almost inaccessible plateau. I have studied African ethnology for twenty years, but the fact that changes of population in this apparently quiet and peaceable corner of the earth could have been occasioned by outside enterprises taking place on the high seas, was completely new to me. It is, no doubt, however, correct.

The charming tribal legend of the Makonde—besides informing us of other interesting matters—explains why they have to live in the thickest of the bush and a long way from the edge of the plateau, instead of making their permanent homes beside the purling brooks and springs of the low country.

“The place where the tribe originated is Mahuta, on the southern side of the plateau towards the Rovuma, where of old time there was nothing but thick bush. Out of this bush came a man who never washed himself or shaved his head, and who ate and drank but little.

He went out and made a human figure from the wood of a tree growing in the open country, which he took home to his abode in the bush and there set it upright. In the night this image came to life and was a woman. The man and woman went down together to the Rovuma to wash themselves. Here the woman gave birth to a stillborn child. They left that place and passed over the high land into the valley of the Mbemkuru, where the woman had another child, which

was also born dead. Then they returned to the high bush country of Mahuta, where the third child was born, which lived and grew up. In course of time, the couple had many more children, and called themselves Wamatanda. These were the ancestral stock of the Makonde, also called Wamakonde,[48] i.e., aborigines. Their forefather, the man from the bush, gave his children the command to bury their dead upright, in memory of the mother of their race who was cut out of wood and awoke to life when standing upright. He also warned them against settling in the valleys and near large streams, for sickness and death dwelt there. They were to make it a rule to have their huts at least an hour’s walk from the nearest wateringplace; then their children would thrive and escape illness.”

The explanation of the name Makonde given by my informants is somewhat different from that contained in the above legend, which I extract from a little book (small, but packed with information), by Pater Adams, entitled Lindi und sein Hinterland. Otherwise, my results agree exactly with the statements of the legend. Washing?

Hapana—there is no such thing. Why should they do so? As it is, the supply of water scarcely suffices for cooking and drinking; other people do not wash, so why should the Makonde distinguish himself by such needless eccentricity? As for shaving the head, the short, woolly crop scarcely needs it,[49] so the second ancestral precept is likewise easy enough to follow. Beyond this, however, there is nothing ridiculous in the ancestor’s advice. I have obtained from various local artists a fairly large number of figures carved in wood, ranging from fifteen to twenty-three inches in height, and representing women belonging to the great group of the Mavia, Makonde, and Matambwe tribes. The carving is remarkably well done and renders the female type with great accuracy, especially the keloid ornamentation, to be described later on. As to the object and meaning of their works the sculptors either could or (more probably) would tell me nothing, and I was forced to content myself with the scanty information vouchsafed by one man, who said that the figures were merely intended to represent the nembo—the artificial deformations of pelele, ear-discs, and keloids. The legend recorded

by Pater Adams places these figures in a new light. They must surely be more than mere dolls; and we may even venture to assume that they are—though the majority of present-day Makonde are probably unaware of the fact—representations of the tribal ancestress.

The references in the legend to the descent from Mahuta to the Rovuma, and to a journey across the highlands into the Mbekuru valley, undoubtedly indicate the previous history of the tribe, the travels of the ancestral pair typifying the migrations of their descendants. The descent to the neighbouring Rovuma valley, with its extraordinary fertility and great abundance of game, is intelligible at a glance—but the crossing of the Lukuledi depression, the ascent to the Rondo Plateau and the descent to the Mbemkuru, also lie within the bounds of probability, for all these districts have exactly the same character as the extreme south. Now, however, comes a point of especial interest for our bacteriological age. The primitive Makonde did not enjoy their lives in the marshy river-valleys.

Disease raged among them, and many died. It was only after they had returned to their original home near Mahuta, that the health conditions of these people improved. We are very apt to think of the African as a stupid person whose ignorance of nature is only equalled by his fear of it, and who looks on all mishaps as caused by evil spirits and malignant natural powers. It is much more correct to assume in this case that the people very early learnt to distinguish districts infested with malaria from those where it is absent.

This knowledge is crystallized in the ancestral warning against settling in the valleys and near the great waters, the dwelling-places of disease and death. At the same time, for security against the hostile Mavia south of the Rovuma, it was enacted that every settlement must be not less than a certain distance from the southern edge of the plateau. Such in fact is their mode of life at the present day. It is not such a bad one, and certainly they are both safer and more comfortable than the Makua, the recent intruders from the south, who have made USUAL METHOD OF good their footing on the western edge of the CLOSING HUT-DOOR plateau, extending over a fairly wide belt of country. Neither Makua nor Makonde show in their dwellings anything of the size and comeliness of the Yao houses in the plain, especially at Masasi, Chingulungulu and Zuza’s. Jumbe Chauro, a Makonde hamlet not far from Newala, on the road to Mahuta, is the

most important settlement of the tribe I have yet seen, and has fairly spacious huts. But how slovenly is their construction compared with the palatial residences of the elephant-hunters living in the plain.

The roofs are still more untidy than in the general run of huts during the dry season, the walls show here and there the scanty beginnings or the lamentable remains of the mud plastering, and the interior is a veritable dog-kennel; dirt, dust and disorder everywhere. A few huts only show any attempt at division into rooms, and this consists merely of very roughly-made bamboo partitions. In one point alone have I noticed any indication of progress—in the method of fastening the door. Houses all over the south are secured in a simple but ingenious manner. The door consists of a set of stout pieces of wood or bamboo, tied with bark-string to two cross-pieces, and moving in two grooves round one of the door-posts, so as to open inwards. If the owner wishes to leave home, he takes two logs as thick as a man’s upper arm and about a yard long. One of these is placed obliquely against the middle of the door from the inside, so as to form an angle of from 60° to 75° with the ground. He then places the second piece horizontally across the first, pressing it downward with all his might.

It is kept in place by two strong posts planted in the ground a few inches inside the door. This fastening is absolutely safe, but of course cannot be applied to both doors at once, otherwise how could the owner leave or enter his house? I have not yet succeeded in finding out how the back door is fastened.

MAKONDE LOCK AND KEY AT JUMBE CHAURO

This is the general way of closing a house. The Makonde at Jumbe Chauro, however, have a much more complicated, solid and original one. Here, too, the door is as already described, except that there is only one post on the inside, standing by itself about six inches from one side of the doorway. Opposite this post is a hole in the wall just large enough to admit a man’s arm. The door is closed inside by a large wooden bolt passing through a hole in this post and pressing with its free end against the door. The other end has three holes into which fit three pegs running in vertical grooves inside the post. The door is opened with a wooden key about a foot long, somewhat curved and sloped off at the butt; the other end has three pegs corresponding to the holes, in the bolt, so that, when it is thrust through the hole in the wall and inserted into the rectangular opening in the post, the pegs can be lifted and the bolt drawn out.[50]

MODE OF INSERTING THE KEY

With no small pride first one householder and then a second showed me on the spot the action of this greatest invention of the Makonde Highlands. To both with an admiring exclamation of “Vizuri sana!” (“Very fine!”). I expressed the wish to take back these marvels with me to Ulaya, to show the Wazungu what clever fellows the Makonde are. Scarcely five minutes after my return to camp at Newala, the two men came up sweating under the weight of two heavy logs which they laid down at my feet, handing over at the same time the keys of the fallen fortress. Arguing, logically enough, that if the key was wanted, the lock would be wanted with it, they had taken their axes and chopped down the posts—as it never occurred to them to dig them out of the ground and so bring them intact. Thus I have

two badly damaged specimens, and the owners, instead of praise, come in for a blowing-up.

The Makua huts in the environs of Newala are especially miserable; their more than slovenly construction reminds one of the temporary erections of the Makua at Hatia’s, though the people here have not been concerned in a war. It must therefore be due to congenital idleness, or else to the absence of a powerful chief. Even the baraza at Mlipa’s, a short hour’s walk south-east of Newala, shares in this general neglect. While public buildings in this country are usually looked after more or less carefully, this is in evident danger of being blown over by the first strong easterly gale. The only attractive object in this whole district is the grave of the late chief Mlipa. I visited it in the morning, while the sun was still trying with partial success to break through the rolling mists, and the circular grove of tall euphorbias, which, with a broken pot, is all that marks the old king’s resting-place, impressed one with a touch of pathos.

Even my very materially-minded carriers seemed to feel something of the sort, for instead of their usual ribald songs, they chanted solemnly, as we marched on through the dense green of the Makonde bush:—