Current Developments in Biotechnology and Bioengineering: Foundations of Biotechnology and Bioengineering

By Ashok Pandey

Current Developments in Biotechnology and Bioengineering: Foundations of Biotechnology and Bioengineering is a package of nine books that compile the latest ideas from across the entire arena of biotechnology and bioengineering. This volume focuses on the underlying principles of biochemistry, microbiology, fermentation technology, and chemical engineering as interdisciplinary themes, constructing the foundation of biotechnology and bioengineering.

• Provides state-of-art information on basics and fundamental principles of biotechnology and bioengineering
• Supports the education and understanding of biotechnology education and R&D
• Contains advanced content for researchers engaged in bioengineering research

• Language: English
• Publisher: Elsevier Science
• Release date: Sep 19, 2016
• ISBN: 9780444636799

Book preview

Current Developments in Biotechnology and Bioengineering

Foundations of Biotechnology and Bioengineering

Editors

Ashok Pandey

José António Couto Teixeira

Table of Contents

Cover image

Title page

Copyright

List of Contributors

About the Editors

Preface

1. Basic Microbiology

1.1. Introduction

1.2. Microbial Diversity and Systematics

1.3. Microbial Physiology and Metabolism

1.4. Different Branches and Scope of Microbiology

1.5. Microbial Reproduction

1.6. Culturable and Nonculturable Microbes

1.7. Microbial Genomics and Metabolomics

1.8. Economic Significance of Microbes

1.9. Conclusions and Perspectives

2. Basic Biochemistry

2.1. Introduction

2.2. Structure and Function of Proteins

2.3. Enzymes

2.4. Physicochemical Properties of Mono-, Oligo-, and Polysaccharides

2.5. Characteristics and Properties of Lipids

2.6. Nucleic Acids

2.7. Energetics

3. Fundamentals of Molecular Biology

3.1. Introduction

3.2. DNA Replication

3.3. Transcription

3.4. Regulation of Transcription: The Operon

3.5. DNA Damage and Repair

3.6. Recombination

3.7. Transposable Elements

3.8. Plasmids

3.9. Horizontal Gene Transfer and Genome Evolution

3.10. Blotting Techniques

3.11. Polymerase Chain Reaction

3.12. DNA Sequencing

3.13. Conclusions and Perspectives

4. Principles of Genetic Engineering

4.1. Introduction

4.2. Basic Techniques

4.3. Recombinant DNA Technology

4.4. Genetic Engineering in Industrial Biotechnology and Bioengineering

4.5. Conclusions and Perspectives

5. Principles of Metabolic Engineering

5.1. Introduction

5.2. Metabolic Pathway Flux Map

5.3. Basis of Metabolic Models and Flux Analysis

5.4. Network Simplifications

5.5. Concept of Reaction Stoichiometry in Flux Analysis

5.6. Flux Estimation in Underdetermined Systems

5.7. Metabolic Control Analysis

5.8. Conclusion and Perspectives

6. Fundamentals of Bio-reaction Engineering

6.1. Introduction

6.2. Enzyme Kinetics

6.3. Cell Growth Kinetics

6.4. Main Bioreactor Types

6.5. Bioreactor Modes of Operation

6.6. Gas–Liquid Mass Transfer

6.7. Bioprocess Monitoring and Control

6.8. Conclusions and Perspectives

7. Fundamentals of Biological Separation Processes

7.1. Introduction

7.2. General Considerations

7.3. Upstream Processing

7.4. Cell Harvest

7.5. Primary Recovery

7.6. Intermediate Purification

7.7. Final Purification

7.8. Conclusions and Perspectives

8. Synthetic Biology: Perspectives in Industrial Biotechnology

8.1. Introduction

8.2. Synthetic Biology Methods and Tools

8.3. Synthetic Biology Applications

8.4. Concerns About Synthetic Biology

8.5. Summary and Conclusion

Index

Copyright

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List of Contributors

T.Q. Aguiar,     University of Minho, Braga, Portugal

M.R. Aires-Barros,     Institute for Bioengineering and Biosciences (IBB), Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisbon, Portugal

A.M. Azevedo,     Institute for Bioengineering and Biosciences (IBB), Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, Lisbon, Portugal

L. Domingues,     University of Minho, Braga, Portugal

B. Fernandes,     University of Minho, Braga, Portugal

P. Gunasekaran,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

P. Jauregi,     University of Reading, Reading, United Kingdom

A. Mota,     University of Minho, Braga, Portugal

C. Oliveira,     University of Minho, Braga, Portugal

L. Pastrana,     INL – International Iberian Nanotechnology Laboratory, Braga, Portugal

J. Rajendhran,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

K.B. Ramachandran,     Indian Institute of Technology Madras, Chennai, India

S. Ramalingam,     Anna University, Chennai, India

J.L. Rodrigues,     University of Minho, Braga, Portugal

L.R. Rodrigues,     University of Minho, Braga, Portugal

T. Satyanarayana,     University of Delhi South Campus, New Delhi, India

B. Singh,     Maharshi Dayanand University, Rohtak, India

S. Srinivasan,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

A. Vicente,     University of Minho, Braga, Portugal

About the Editors

Ashok Pandey

Professor Ashok Pandey is Eminent Scientist at the Center of Innovative and Applied Bioprocessing, Mohali (a national institute under the Department of Biotechnology, Ministry of Science and Technology, Government of India), and former chief scientist and head of the Biotechnology Division at the CSIR’s National Institute for Interdisciplinary Science and Technology at Trivandrum. He is an adjunct professor at Mar Athanasios College for Advanced Studies Thiruvalla, Kerala, and at Kalasalingam University, Krishnan Koil, Tamil Nadu. His major research interests are in the areas of microbial, enzyme, and bioprocess technology, which span various programs, including biomass to fuels and chemicals, probiotics and nutraceuticals, industrial enzymes, solid-state fermentation, etc. He has more than 1100 publications and communications, which include 16 patents, 50+ books, 125 book chapters, and 425 original and review papers, with an h index of 75 and more than 23,500 citations (Google Scholar). He has transferred several technologies to industries and has been an industrial consultant for about a dozen projects for Indian and international industries.

Professor Pandey is the recipient of many national and international awards and fellowships, which include Elected Member of the European Academy of Sciences and Arts, Germany; Fellow of the International Society for Energy, Environment and Sustainability; Fellow of the National Academy of Science (India); Fellow of the Biotech Research Society, India; Fellow of the International Organization of Biotechnology and Bioengineering; Fellow of the Association of Microbiologists of India; honorary doctorate degree from the Université Blaise Pascal, France; Thomson Scientific India Citation Laureate Award, United States; Lupin Visiting Fellowship; Visiting Professor at the Université Blaise Pascal, France, the Federal University of Parana, Brazil, and the École Polytechnique Fédérale de Lausanne, Switzerland; Best Scientific Work Achievement Award, Government of Cuba; UNESCO Professor; Raman Research Fellowship Award, CSIR; GBF, Germany, and CNRS, France fellowships; Young Scientist Award; and others. He was chairman of the International Society of Food, Agriculture and Environment, Finland (Food & Health) during 2003–04. He is the Founder President of the Biotech Research Society, India (www.brsi.in); International Coordinator of the International Forum on Industrial Bioprocesses, France (www.ifibiop.org); chairman of the International Society for Energy, Environment & Sustainability (www.isees.org); and vice president of the All India Biotech Association (www.aibaonline.com). Professor Pandey is editor-in-chief of Bioresource Technology, Honorary Executive Advisor of the Journal of Water Sustainability and Journal of Energy and Environmental Sustainability, subject editor of the Proceedings of the National Academy of Sciences (India), and editorial board member of several international and Indian journals, and also a member of several national and international committees.

José António Couto Teixeira

José António Couto Teixeira is currently a Professor (Professor Catedrático) at the Biological Engineering Department, University of Minho, Portugal (since 2000). He has a degree in Chemical Engineering from the University of Porto (1980) and a Ph.D. in Chemical Engineering also from the University of Porto (1988). He has been involved in various management activities, being Head of the Department of Biological Engineering, University of Minho, 2000–2012, and Head of the Biological Engineering Research Center, 2012–2015. His main research interests are industrial biotechnology (bioprocess development for the transformation of lignocellulosic materials into second-generation bioethanol and chemicals, valorization of agroindustrial residues, and bioreactor development, including new design bioreactors and continuous processing) and food biotechnology (nonconventional food processing, edible films for packaging, process development for production of prebiotics). He was responsible or coresponsible for the supervision of 31 Ph.D. theses and 20 postdoctoral researchers and has been the coordinator of 32 scientific research projects, 7 of which were international. José Teixeira was awarded the Stimulus to Excellence, 2006, from the Fundação para a Ciência e a Tecnologia; the Seeds of Science in Engineering and Technology, 2011, from Ciência Hoje;, and the Scientific Merit Award, Universidade do Minho, 2015. He is the coeditor of the books Reactores Biológicos—Fundamentos e Aplicações (in Portuguese), Engineering Aspects of Milk and Dairy Products, and Engineering Aspects of Food Biotechnology and the author/coauthor of over 400 peer-reviewed papers (see http://orcid.org/0000-0002-4918-3704).

Preface

This book is a part of the comprehensive series Current Developments in Biotechnology and Bioengineering (Editor-in-chief: Ashok Pandey), comprising nine volumes, and presents a collection of chapters dealing with the foundations of biotechnology and bioengineering. Biotechnology has been part of human activities for more than 10,000  years and its importance has been increasing with human development. Nowadays, biotechnology has an enormous impact on our everyday life and is a key technology for industry, health, environment, food, and other areas of activity. Biotechnology, according to the Organization of Economic Co-operation and Development, is defined as the application of scientific and engineering principles to the processing of materials by biological agents. In biotechnology, intact organisms, such as yeast, bacteria, or microalgae, or their components, such as enzymes, are used to manufacture useful products and provide services. This makes clear the multidisciplinary character of biotechnology and the importance of professionals from different areas of knowledge interacting and understanding one another so that the full potential of biotechnology can be exploited.

This book comprises eight chapters: the first and second chapters are dedicated to the fundamentals of microbiology and biochemistry; from the third to the fifth, topics addressed are focused on genetics, molecular biology, and genetic and metabolic engineering; Chapters 6 and 7 deal with the most important engineering operations in biotechnology; and, finally, Chapter 8 reviews methods and tools in synthetic biology.

Chapter 1 gives an overview of the basic aspects of microbiology, including microbial metabolism, and its role in various industrial bioprocesses; in Chapter 2 the main biological molecules are introduced, such as proteins, carbohydrates, lipids, and nucleic acids, including their chemical structures, properties, and importance in biotechnological and bioengineering developments such as genetic engineering and bioprocesses. This chapter also points out advances in areas such as proteomics and genetic engineering, and their relevance to advances in the discovery of new drugs and treatment of diseases is discussed.

The basic concepts of the fundamental units of life, encompassing DNA replication, transcription, and gene regulation, are discussed in Chapter 3. Other vital biological phenomena such as transformation, conjugation, transduction, recombination, and horizontal gene transfer are also presented in this chapter. The major techniques in molecular biology, such as blotting, polymerase chain reaction, and sequencing technologies, are also described. Chapter 4 compiles information on the principles of genetic engineering, describing not only the basic techniques used in molecular biology and the basics of recombinant DNA technology, but also presenting more recent developments on these techniques, as well as up-to-date in silico tools. In Chapter 5, metabolic engineering is the topic considered, and the major steps involved in metabolic engineering—analysis and synthesis—are described. Metabolic flux analysis and its importance to bioprocess development are also addressed.

Chapters 6 and 7 deal with the most relevant engineering topics in biotechnology—bio-reaction engineering and downstream processing. Chapter 6 presents the basic concepts of biocatalyst (cell and enzymes) kinetics and the main bioreactor types and operation modes, as well as a comprehensive approach regarding the monitoring of various bio-reactions and control strategies. In Chapter 7, the most commonly used unit operations in the downstream processing of biotechnology products are described, and relevant considerations in designing a purification strategy are discussed.

Finally, in Chapter 8, the relevance of synthetic biology in the improvement of biotechnology is presented together with a review of the tools and methods used.

We hope that this book will be of great value to engineers, microbiologists, geneticists, and others in providing key life science and engineering aspects of the development of biotechnology and bioengineering. We would like to acknowledge the reviewers for their valuable comments to improve the final quality of the chapters included in this volume. We thank Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and the entire production team at Elsevier for their help and support in bringing out this volume. Without their commitment, efficiency, and dedicated work, this volume could not have ever been accomplished.

Editors

Ashok Pandey

José António Couto Teixeira

1

Basic Microbiology

B. Singh¹,  and T. Satyanarayana²,∗     ¹Maharshi Dayanand University, Rohtak, India     ²University of Delhi South Campus, New Delhi, India

Abstract

Microbiology deals with the study of microorganisms and their interactions with biotic and abiotic components of the environment. Microbes are so small that they are not seen by the naked eye, and include bacteria and archaea, fungi, algae, protozoa, and viruses. Microbes are the main decomposers of organic matter and, therefore, influence all living beings and contribute to chemical and physical processes. Microbiology is a multidisciplinary science that depends on the skills and knowledge of individuals specializing in many different fields of life sciences, physical sciences, and engineering. Microbiology continues to benefit from independent scientific and medical disciplines like bacteriology, virology, public health science, clinical microbiology, immunology, parasitology, vaccinology, and others. Microbes are used in basic and applied research, manufacturing of food and other products, public health, environmental protection, and other areas. This chapter gives an overview of the basic aspects of microbiology, including microbial metabolism and its roles in various industrial bioprocesses.

Keywords

Culturable and nonculturable microbes; Genomics; Microbial metabolism; Microbiology; Microorganisms; Proteomics; Transcriptomics

1.1. Introduction

Microbiology was initially focused on the causes of infectious diseases but now includes practical applications of the science. A large number of scientists have made significant contributions to the development of microbiology. There is no evidence for the exact discovery of microorganisms, but the microscope was available in the mid-1600s. Robert Hooke, an English scientist, initially observed microorganisms. Antonie van Leeuwenhoek made several observations of microscopic organisms during the 1670s and he called them animalcules. He was the first to provide accurate descriptions of protozoa, fungi, and bacteria. During this period, the theory of spontaneous generation was disputed by Francesco Redi by proving that fly maggots do not arise from decaying meat when the meat is covered to prevent the entry of flies.

Louis Pasteur experimentally proved that bacterial growth is the main reason for sour taste in wine and dairy products. Pasteur also disproved the theory of spontaneous generation using swan-necked flasks filled with broth. His work also provided support for the belief that microorganisms are in the air and can cause diseases. Pasteur postulated the germ theory of disease, but he did not prove this theory unequivocally. Later this theory was proved by the German scientist Robert Koch by cultivating anthrax bacilli from infected animals. He injected pure cultures of the bacilli into mice, which resulted in anthrax. All these findings are collectively called Koch's postulates (Fig. 1.1). During his work with staphylococci, Alexander Fleming (1929) observed the inhibition of bacterial growth by a mold that was identified as Penicillium notatum [21]. Selman Abraham Waksman discovered over 20 antibiotics along with the well-known streptomycin.

During the era of discovery of microbes (1625–1850) many agents of various infectious diseases were identified. The treatment of infected people was not, however, known well, except for some precautionary measures. Antibiotics were introduced after World War II, which resulted in a decline in cases of pneumonia, tuberculosis, meningitis, syphilis, and other diseases. With the development of the electron microscope in the 1940s, the knowledge and understanding of viruses were possible. The development of vaccines during the decade 1950–60 led to the control of viral diseases such as polio, measles, mumps, and rubella.

Figure 1.1  The steps of Koch's postulates used to relate a specific microorganism to a specific disease. (A) Microorganisms are observed in a sick animal and (B) cultivated in the lab. (C) The organisms are injected into a healthy animal, and (D) the animal develops the disease. (E) The organisms are observed in the sick animal and (F) reisolated in the lab. Adapted from http://www.cliffsnotes.com/sciences/biology/microbiology/.

Modern microbiology has expanded into many fields of human endeavor such as the development of pharmaceutical products, the use of quality control methods in food and dairy products, the control of pathogens in drinking water, and the industrial applications of microorganisms. Microbes have been employed in the production of vitamins, amino acids, enzymes, growth supplements, and several others. They are also useful in the manufacture of fermented foods such as dairy products (sour cream, yogurt, buttermilk) as well as other foods/beverages (pickles, sauerkraut, bread, wines, beer, and other alcoholic beverages).

Biotechnology, an area related to applied microbiology, deals with the use of microbes in the production of important pharmaceuticals (human insulin, interferon, blood-clotting factors, clot-dissolving enzymes, vaccines). It is extremely difficult to synthesize these products by other means. Microbes can be engineered to increase plant resistance to microbial pathogens, insects, frost, and more.

1.2. Microbial Diversity and Systematics

Like all other living beings, microbes are placed into a system of classification. Classification highlights characteristics that are common among certain groups while providing an order to the variety of living organisms. Taxonomy displays the unity and diversity among microorganisms. Carolus Linnaeus (1750–60) classified all known plants and animals according to set rules of nomenclature.

1.2.1. Classification Schemes

The fundamental rank of the classification set by Linnaeus is the species, which is defined as a population of individuals that breed among themselves [4,11,19]. Various species are grouped together to form a genus. Among bacteria, Bacillus subtilis and Bacillus licheniformis are in the genus Bacillus because the microbes are at least 70% similar. Various genera are then grouped as a family because of similarities, and various families are placed together in an order. Continuing the classification scheme, a number of orders are grouped as a class, and several classes are categorized in a single phylum or division. Various phyla or divisions are placed in the broadest classification entry, the domain/kingdom (Fig. 1.2). Morphological, structural, biochemical, and molecular characteristics are considered in classifying the organisms. Microorganisms are grouped into two major groups, prokaryotes and eukaryotes. Bacteria and archaea are prokaryotes because of their cellular structure lacking nucleus and organelles, while other microorganisms such as fungi, protozoa, and algae are eukaryotes, possessing a nucleus and cell organelles. Viruses and prions are neither prokaryotes nor eukaryotes owing to their simple and unique characteristic features.

The five-kingdom system of classification of living organisms was proposed by Robert Whittaker of Cornell University in 1969. The first kingdom is Monera, including prokaryotes such as bacteria and cyanobacteria; the second kingdom, Protista, includes single-celled eukaryotes like protozoa, unicellular algae, and slime molds; the third kingdom Fungi, comprises the molds, mushrooms, and yeasts. These organisms are eukaryotes, which absorb simple nutrients from the soil. The remaining two kingdoms are Plantae and Animalia, which include plants and animals, respectively (Fig. 1.3). Based on ribosomal RNA sequence analysis, in 1977 Carl Woese classified all living organisms into three domains: Archaea, Bacteria, and Eukarya. Archaea and Bacteria are included in the Prokaryotes.

Figure 1.2  A generalized classification scheme.

Figure 1.3  Five-kingdom classification proposed by Whittaker.

1.2.2. Brief Descriptions of Various Groups of Microorganisms

Bacteria and archaea are simple prokaryotic organisms lacking a nucleus and other cell organelles [4,11,19]. They may appear as rods (bacilli), spheres (cocci), commas (vibrio), or spirals (spirilla or spirochetes). They are ubiquitous in occurrence, reproduce by binary fission, and possess unique components in their cell walls. They thrive at temperatures ranging from 0 to 100°C and in the absence or presence of oxygen.

Fungi are eukaryotic microorganisms that are unicellular (yeasts) and multicellular (molds). The yeasts are larger than bacteria, whereas molds are filamentous and branched fungi. The fungi prefer acidic environments, mostly living at room temperature under oxygen-rich conditions. Saccharomyces cerevisiae is a yeast and Aspergillus niger is a mold.

Protozoa are unicellular eukaryotic organisms. Motion is a characteristic feature associated with many species; some protozoa use flagella, some use cilia, and others use pseudopodia. Some nonmotile species are also found. They exist in an infinite variety of shapes owing to the absence of cell walls. Many species cause human diseases such as malaria, sleeping sickness, dysentery, and toxoplasmosis.

Algae are plantlike organisms ranging from single celled to multicellular in nature. Diatoms and dinoflagellates are found in oceans and at the base of marine food chains. They are able to carry out the process of photosynthesis.

Viruses are ultramicroscopic organisms comprising genetic material, either DNA or RNA, enclosed in a protein envelope called a capsid. They do not have a metabolism. Viruses multiply in living cells and use the host machinery for their growth and development. They are nonliving in the absence of host cells. Prions are made of protein and cause several human and animal diseases.

1.2.3. Nomenclature of Microorganisms

The binomial nomenclature system, established by Linnaeus for naming all living organisms, is also applicable to microorganisms [4,11,19]. The binomial name consists of two parts; the first is the genus to which the organism belongs and the second is the species. In binomial names, the first letter of the genus name is capitalized and the remaining part of the genus and the species are written in lowercase. The entire binomial name is italicized. It can be abbreviated by using the first letter of the genus and the full species. For example, Escherichia coli, a rod-shaped bacterium found in the human intestine, is abbreviated as E. coli.

1.2.4. Introduction to Prokaryotes and Eukaryotes

All living organisms, including microorganisms, are classified into prokaryotes and eukaryotes. Prokaryotes and eukaryotes are distinguished on the basis of their cellular structures. Prokaryotic cells lack a nucleus and other membrane-bound structures called organelles, whereas eukaryotic cells have both a nucleus and cell organelles (Fig. 1.4A and B). Both types of cells are enclosed in cell membranes possessing DNA as the genetic material. Prokaryotes include bacteria, cyanobacteria, and archaea, and eukaryotes include fungi, protozoa, and algae. Viruses and prions are considered as neither prokaryotes nor eukaryotes because they lack several characteristics of living beings.

1.3. Microbial Physiology and Metabolism

1.3.1. Cellular Respiration

Some microorganisms, like cyanobacteria, utilize solar energy for photosynthesis. Glucose is the principal carbohydrate formed during photosynthesis [4,11,19]. Other microorganisms, like nonphotosynthetic bacteria, archaea, fungi, and protozoa, are unable to carry out photosynthesis. Microorganisms obtain their energy from carbohydrates by cellular respiration. Carbohydrates are broken down in the metabolic pathways and energy is generated in the form of adenosine triphosphate (ATP) molecules, and CO2 is released as the waste product. Carbon dioxide can be used by photosynthetic microbes to synthesize carbohydrates. Oxygen serves as an electron acceptor in the process of cellular respiration. The overall mechanism of cellular respiration involves four subdivisions: glycolysis, in which glucose molecules are broken down into pyruvic acid; the Krebs cycle, in which pyruvic acid is broken down and high-energy compounds such as NADH and NADPH are formed; the electron transport system, in which electrons are transported along a series of coenzymes and cytochromes to release energy in the form of electrons; and chemiosmosis, in which the electrons are used to pump protons across the mitochondrial membrane for ATP synthesis.

Figure 1.4  Morphology and structure of (A) a prokaryotic and (B) a eukaryotic cell. Adapted from http://www.cliffsnotes.com/sciences/biology/microbiology/.

Glycolysis is a metabolic pathway that occurs in the cytoplasm of all cells though the activities of enzymes. During the first and third steps, ATP is consumed and the glucose molecule is converted into two C3 compounds through a series of intermediates. Pyruvic acid is the end product of glycolysis. Four ATP molecules are synthesized in the latter phase of the pathway. As a result, four ATP molecules are synthesized and two ATP molecules are utilized, with a net gain of two ATP molecules in glycolysis. During glycolysis, two NADH molecules are produced, which will be used in the electron transport system for releasing energy. Glycolysis is an anaerobic process and is the sole source of energy for anaerobic microorganisms.

Glycolysis is followed by another multistep process called the Krebs cycle. This is also called the citric acid cycle or the tricarboxylic acid cycle. It utilizes two molecules of pyruvic acid formed in glycolysis and results in the formation of high-energy molecules of NADH and FADH and some ATP and carbon dioxide (Fig. 1.5). The Krebs cycle occurs at the cell membrane of prokaryotes (bacteria) and in the mitochondria of eukaryotes. Mitochondria, the sausage-shaped organelles, possess inner and outer membranes; the inner membrane is folded over itself many times, and these folds are called cristae. Cristae contain important enzymes necessary for the proton pump and ATP formation. Before entering the Krebs cycle, pyruvic acid is converted into acetyl-coenzyme A (acetyl-CoA) and CO2 with concomitant formation of high-energy NADH. Acetyl-CoA enters the Krebs cycle after combining with a four-carbon oxaloacetic acid, resulting in the formation of six-carbon citric acid, which undergoes a series of enzyme-catalyzed conversions. The Krebs cycle forms (per two molecules of pyruvic acid) two ATP molecules, NADH molecules, and some FADH2 molecules. Both NADH and FADH2 will be utilized in the electron transport system.

Figure 1.5  An overview of the processes of cellular respiration showing the major pathways and the places where ATP is synthesized. Adapted from http://www.cliffsnotes.com/sciences/biology/microbiology/.

In the electron transport system, a series of cytochromes (cell pigments) and coenzymes act as carriers and transfer molecules by accepting high-energy electrons and passing them to the next molecule in the system. The energy of the electrons is used to transport protons across the cell membrane or into the outer compartment of the mitochondria. Each NADH molecule transfers six protons across the membrane, whereas FADH2 transfers only four protons. Electrons passing from NAD to FAD and to other cytochromes and coenzymes are finally accepted by oxygen atoms, resulting in the formation of water.

Chemiosmosis is the process of generation of ATP in cellular respiration due to the pumping of protons through special channels of the mitochondrial membrane from the inner to the outer compartment. This pumping establishes a proton gradient. Once the gradient is established, protons pass down the gradient through molecular particles in the membrane resulting in the formation of ATP. In prokaryotic microorganisms, a total of 36 molecules of ATP can be produced during aerobic cellular respiration. In eukaryotic cells, the number is 34 molecules of ATP. Two molecules of ATP are produced as the net gain of glycolysis, so the grand total is 36 molecules of ATP in eukaryotes and 38 prokaryotes. The ATP molecules are used in the cellular functions.

Fermentation is an anaerobic process in which energy can be released from glucose in the absence of oxygen and the electron donors and electron acceptors are organic molecules. Fermentation occurs in yeast cells and in some bacteria. In yeast cells, glucose can be metabolized through cellular respiration, as in other cells. Under anaerobic conditions, glucose is converted via glycolysis to pyruvic acid, which is further converted to acetaldehyde and then to ethanol. The net gain of ATP to the yeast cell is only two ATPs. Yeasts have the necessary enzymes to convert pyruvic acid to ethanol. Yeasts are, therefore, used in making bread as well as alcohol.

1.3.2. Photosynthesis

Photosynthetic microbes synthesize their food from simple molecules such as carbon dioxide and water in the presence of solar energy [4,11,19]. Among microorganisms, photosynthesis occurs in unicellular algae and bacteria (cyanobacteria, green and purple sulfur bacteria). Photosynthesis takes place in two phases: in the first phase, energy-rich electrons flow through a series of coenzymes and other molecules, and this electron energy is trapped in ATP and NADPH molecules. These molecules are utilized in the second phase, in which carbon dioxide is converted into glucose. Photosynthesis occurs along the thylakoid membranes of plastids of eukaryotic organisms. The thylakoids are similar to the cristae of mitochondria in morphology and structure. Sunlight is captured by pigment molecules of photosystems present in the thylakoid. The photosystem includes the pigment molecules, coenzymes, proton pumps, and molecules of the electron transport systems. In prokaryotes, the chlorophyll molecules are present in the cytoplasm and are called bacteriochlorophylls. Photosynthesis is divided into light (energy-fixing) and dark (light-independent) reactions.

1.3.3. Light Reaction

The light reaction begins with the absorption of light by the photosystem. The energy activates electrons to jump out of chlorophyll molecules in the reaction center. These electrons pass through a series of cytochromes in the electron transport system and the energy is used to pump protons across the membrane, setting up the potential for chemiosmosis. The energy-rich electrons now enter another photosystem and get activated by sunlight. The electrons pass through a second electron transport system and reduce NADP to NADPH. Two oxygen atoms combine with one another to form molecular oxygen, which is released by cyanobacteria and green algae as the by-product of photosynthesis. This entire process is called a noncyclic reaction. Certain microorganisms involve the use of a cyclic energy-fixing reaction. Electrons excited by sunlight pass through molecules of the electron transport system and then follow a special pathway back to the chlorophyll molecules. Each electron powers the proton pump and transports a proton across the membrane. This results in the generation of a proton gradient leading to the production of ATP. Both ATP and NADPH provide the energy necessary for the synthesis of carbohydrates during the dark reaction.

1.3.4. Dark Reaction

The dark reaction occurs in the cytoplasm of the microbial cell and involves the synthesis of glucose and other carbohydrates. This is also called the Calvin cycle after the scientist Melvin Calvin, who performed extensive research on the cycle. The carbon dioxide obtained from the atmosphere is attached to a five-carbon compound called ribulose bisphosphate (RuBP) to form a C6 product. This product immediately breaks into two C3 molecules, i.e., phosphoglycerate (PGA) (Fig. 1.6). Each PGA molecule is converted to phosphoglyceraldehyde (PGAL) using the ATP and NADPH synthesized during the light reaction. Two PGAL molecules interact with each other and form a single six-carbon glucose. The process also generates RuBP molecules to enter again into the cycle. This reaction results in the production of glucose that is utilized as a source of energy by photosynthetic/nonphotosynthetic microorganisms.

1.3.5. Chemical Reactions and Energy

Energy is needed by all microorganisms to maintain their cellular and molecular organization. All cellular activities are also dependent on energy. According to the second law of thermodynamics, "energy can