Industrial Microbiology E-Book

Michael J. Waites

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 727

Veröffentlichungsjahr: 2013

Beschreibung

Of major economic, environmental and social importance, industrial microbiology involves the utilization of microorganisms in the production of a wide range of products, including enzymes, foods, beverages, chemical feedstocks, fuels and pharmaceuticals, and clean technologies employed for waste treatment and pollution control.

Aimed at undergraduates studying the applied aspects of biology, particularly those on biotechnology and microbiology courses and students of food science and biochemical engineering, this text provides a wide-ranging introduction to the field of industrial microbiology. The content is divided into three sections:

• key aspects of microbial physiology, exploring the versatility of microorganisms, their diverse metabolic activities and products
• industrial microorganisms and the technology required for large-scale cultivation and isolation of fermentation products
• investigation of a wide range of established and novel industrial fermentation processes and products

Written by experienced lecturers with industrial backgrounds, Industrial Microbiology provides the reader with groundwork in both the fundamental principles of microbial biology and the various traditional and novel applications of microorganisms to industrial processes, many of which have been made possible or enhanced by recent developments in genetic engineering technology.

• A wide-ranging introduction to the field of industrial microbiology
• Based on years of teaching experience by experienced lecturers with industrial backgrounds
• Explains the underlying microbiology as well as the industrial application.

Content is divided into three sections:

1. key aspects of microbial physiology, exploring the versatility of microorganisms, their diverse metabolic activities and products

2. industrial microorganisms and the technology required for large-scale cultivation and isolation of fermentation products

3. investigation of a wide range of established and novel industrial fermentation processes and products

Leseprobe

Contents

Preface

Acknowledgements

Introduction to industrial microbiology

Part 1 Microbial physiology

1 Microbial cell structure and function

Prokaryotes

Fungi

Further reading

2 Microbial growth and nutrition

Microbial nutrition

Microbial growth kinetics

Monitoring microbial growth in culture

Effects of environmental conditions on microbial growth

The control (inhibition) of microbial growth

Further reading

3 Microbial metabolism

Catabolism

Anabolism: the synthesis of biomolecules

Autotrophy

Methylotrophic metabolism

Metabolic regulation

Further reading

Part 2 Bioprocessing

4 Industrial microorganisms

Isolation of suitable microorganisms from the environment

Culture collections

Industrial strains and strain improvement

Strain stability

Further reading

5 Fermentation media

Media formulation

6 Fermentation systems

Fermenter design and construction

Control of chemical and physical conditions

Fermenter control and monitoring

Operating modes

Sterilization

Solid-substrate fermentations

Fermentation process development

Further reading

7 Downstream processing

Cell harvesting

Cell disruption

Product recovery

Distillation

Finishing steps

Inclusion bodies and the role of genetic engineering in downstream processing

Further reading

8 Product development, regulation and safety

Product quality and safety

Manufacturing and environmental safety

Further reading

Part 3 Industrial processes and products

9 Microbial enzymes

Commercial microbial enzyme production

Detergent enzymes

Starch processing enzymes and related carbohydrases

Enzymes in cheese production

Enzymes in plant juice production

Enzymes in textile manufacture

Enzymes in leather manufacture

Enzymes used in the treatment of wood pulps

Enzymes as catalysts in organic synthesis

Further reading

10 Fuels and industrial chemicals

Alkanes

Butanol

Industrial ethanol

Hydrogen

Electricity

Amino acids

Organic acids

Polyhydroxyalkanoates

Polyhydric alcohols

Microbial exopolysaccharides

Bioemulsans

Further reading

11 Health care products

Antibiotics

Ergot alkaloids

Steroid biotransformations

Bacterial vaccines

Recombinant therapeutic peptides and proteins

Bacteriophages as therapeutic agents

Further reading

12 Food and beverage fermentations

Alcoholic beverages

Vinegar production

Dairy fermentations

Other traditional fermented foods

Further reading

13 Food additives and supplements

Flavours

Lipids

Natural food preservatives

Nucleosides, nucleotides and related compounds

Vitamins

Further reading

14 Microbial biomass production

Manufacture of baker’s yeast

Single cell protein production

Mushrooms

Further reading

15 Environmental biotechnology

Waste-water and effluent treatment

Composting

Ensiling

Biodegradation of xenobiotics

Bioremediation

Biomining (mineral leaching)

Microbial desulphurization of coal

Bioinsecticides

Further reading

16 Microbial biodeterioration of materials and its control

Biodeterioration of stored plant food materials

Non-food animal products

Stone and related building materials

Cellulosic materials

Fuels and lubricants

Metals

Plastics

Biodeterioration of cosmetics and pharmaceuticals

Biodeterioration testing

Further reading

17 Animal and plant cell culture

Animal cell culture

Plant cell culture

Alternatives to animal cell and plant cell culture

Index

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A catalogue record for this title is available from the British Library

ISBN 0-632-05307-0

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Cataloging-in-Publication Data

Introduction to Industrial Microbiology/Michael J. Waites . . . [et al.].

p. cm.

ISBN 0-632-05307-0 (pbk.)

1. Industrial Microbiology: an Introduction.

I. Waites, Michael J.

QR53 .I522 2001

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Dedication

This book is dedicated to the memory of our great friend and co-author Gary Higton who, at the age of only 40 years, died unexpectedly during the final stages of its preparation. Gary was a knowledgeable microbiologist, a fine teacher, supportive colleague and a loyal friend. He is greatly missed by us all.

Preface

Industrial microbiology is primarily associated with the commercial exploitation of microorganisms, and involves processes and products that are of major economic, environmental and social importance throughout the world. There are two key aspects of industrial microbiology, the first relating to production of valuable microbial products via fermentation processes. These include traditional fermented foods and beverages, such as bread, beer, cheese and wine, which have been produced for thousands of years. In addition, over the last hundred years or so, microorganisms have been further employed in the production of numerous chemical feedstocks, energy sources, enzymes, food ingredients and pharmaceuticals. The second aspect is the role of microorganisms in providing services, particularly for waste treatment and pollution control, which utilizes their abilities to degrade virtually all natural and man-made products. However, such activities must be controlled while these materials are in use, otherwise consequent biodeterioration leads to major economic losses.

This textbook is intended to be an introduction to industrial microbiology. In writing it, the authors have drawn on their experience teaching industrial microbiology and other aspects of applied microbiology to undergraduates and masters students on a range of applied biology, microbiology, biotechnology, food science and chemical engineering courses. It is assumed that the reader will have an elementary knowledge of microbiology and biochemistry. Nevertheless, even those students with only a basic knowledge of chemistry and cell biology, and those who are not specialist micro-biologists, should find the material accessible.

The book is divided into three sections. Part 1 is designed to underpin the main content. Its aim is to provide a sufficient, albeit brief, overview of microbial structure, physiology and biochemistry to enable the student to fully appreciate the diversity of microorganisms and their metabolic capabilities. The reader should soon come to recognize the versatility of microorganisms, their ability to grow on a wide range of substrates and to produce an extensive array of products, many of which are commercially available.

Part 2 is devoted to bioprocessing. It examines the commercial fermentation operations and requirements for large-scale cultivation of microorganisms. This involves the acquisition and development of suitable production strains that must then be provided with nutrients, especially appropriate carbon and energy sources. The object of any industrial fermentation is then to optimize either growth of the organism or the production of a target microbial product. This is normally achieved by performing fermentations under rigorously controlled conditions in large fermenters with culture capacities often in excess of several thousand litres. The design and operation of these fermentation processes is discussed, along with the downstream processing operations necessary for the recovery and purification of the target products. Aspects of safety and good manufacturing practices are also examined.

Over the last twenty years, many traditional and established industrial fermentation processes have been advanced through the contribution of genetic engineering (in vitro recombinant DNA technology). This technology has also facilitated the development of many novel processes and products. It not only accelerates strain development, leading to improvements in the production microorganisms, but can aid downstream processing and other elements of the process. Initially, it involved the manipulation of bacteria, but has moved to cloning in yeasts, filamentous fungi, and plant and animal cells. Developments in this field continue to grow rapidly.

There are several good texts that cover the fundamental aspects of genetic manipulation of microorganisms. Consequently, we have not attempted to give a detailed account of such techniques here, they are merely introduced and further reading is suggested. Nevertheless, many examples of the application and potential of genetically engineered microorganisms within industrial processes are discussed.

Part 3 explores the wide range of industrial microbial processes and products, including human food and animal feed production, the provision of chemical feedstocks, alternative energy sources, enzymes and products for application in human and animal health. We consider that the economic and scientific importance of traditional and long-established fermentation processes can be somewhat overlooked, as attention is often dominated by more recent developments. Therefore we have aimed to give a balanced and comprehensive coverage of current industrial processes and their products. The production of valuable commodities from animal and plant cell culture is also included, primarily because the culture and handling of such cells involves techniques similar to those used for the propagation of microorganisms.

An additional aspect of industrial microbiology, examined here, is the application of extensive degradative abilities of microorganisms, particularly the harnessing of these properties in waste treatment and pollution control. The necessity to limit these activities in inappropriate situations, i.e. the prevention of biodeterioration of materials while still in use, is also discussed.

Acknowledgements

We would like to thank the following authors and the publishers for allowing us to use figures and tables from their publications:

Figs 1.1 and 3.12 are from Dawes, I. W. & Sutherland, I. W. (1992) Microbial Physiology, 2nd Edition. Blackwell Scientific Publications.

Fig. 1.2 is from Poxton, I. R. (1993) Journal of Applied Bacteriology Supplement 74, 1S–11S.

Figs 4.3, 7.8 and 7.9 are from Brown, C. M., Campbell, I. & Priest, F. G. (1987) Introduction to Biotechnology. Blackwell Scientific Publications.

Fig. 12.7 is from Lewis, M. & Young, T. W. (1995) Brewing. Chapman & Hall.

Tables 12.3 and 12.4 are based on tables in Bamforth, C. W. (1985) The use of enzymes in brewing. Brewers’ Guardian114, 21–26.

In addition, we wish to thank our colleague Dr Tom Coultate for his advice and enthusiastic support.

Introduction to industrial microbiology

Traditional fermentation processes, such as those involved in the production of fermented dairy products and alcoholic beverages, have been performed for thousands of years. However, it is less than 150 years ago that the scientific basis of these processes was first examined. The birth of industrial microbiology largely began with the studies of Pasteur. In 1857 he finally demonstrated beyond doubt that alcoholic fermentation in beer and wine production was the result of microbial activity, rather than being a chemical process. Prior to this, Cagniard-Latour, Schwann and several other notable scientists had connected yeast activities with fermentation processes, but they had largely been ignored. Pasteur also noted that certain organisms could spoil beer and wine, and that some fermentations were aerobic, whereas others were anaerobic. He went on to devise the process of pasteurization, a major contribution to food and beverage preservation, which was originally developed to preserve wine. In fact, many of the early advances of both pure and applied microbiology were through studies on beer brewing and wine making. Pasteur’s publications, Études sur le Vin (1866), Études sur la Bière (1876) and others, were important catalysts for the progress of industrial fermentation processes. Of the further advances that followed, none were more important than the development of pure culture techniques by Hansen at the Carlsberg Brewery in Denmark. Pure strain brewing was carried out here for the first time in 1883, using a yeast isolated by Hansen, referred to as Carlsberg Yeast No. 1 (Saccharomyces carlsbergensis, now classified as a strain of Saccharomyces cerevisiae).

During the early part of the 20th century, further progress in this field was relatively slow. Around the turn of the century there had been major advancements in the large-scale treatment of sewage, enabling significant improvement of public health in urban communities. However, the first novel industrial-scale fermentation process to be introduced was the acetone–butanol fermentation, developed by Weizmann (1913–15) using the bacterium Clostridium acetobutylicum. In the early 1920s an industrial fermentation process was also introduced for the manufacture of citric acid, employing a filamentous fungus (mould), Aspergillus niger. Further innovations in fermentation technology were greatly accelerated in the 1940s through efforts to produce the antibiotic penicillin, stimulated by the vital need for this drug during World War II. Not only did production rapidly move from small-scale surface culture to large-scale submerged fermentations, but it led to great advances in both media and microbial strain development. The knowledge acquired had a great impact on the successful development of many other fermentation industries.

More recent progress includes the ability to produce monoclonal antibodies for analytical, diagnostic, therapeutic and purification purposes, pioneered by Milstein and Kohler in the early 1970s. However, many of the greatest advances have followed the massive developments in genetic engineering (recombinant DNA technology) over the last 20 years. This technology has had, and will continue to have, a tremendous influence on traditional, established and novel fermentation processes and products. It allows genes to be transferred from one organism to another and allows new approaches to strain improvement. The basis of gene transfer is the insertion of a specific gene sequence from a donor organism, via an expression vector, into a suitable host. Hosts for expression vectors can be prokaryotes such as the bacterium Escherichia coli; alternatively, where post-translational processing is required, as with some human proteins, a eukaryotic host is usually required, e.g. a yeast.

A vast range of important products, many of which were formerly manufactured by chemical processes, are now most economically produced by microbial fermentation and biotransformation processes. Microorganisms also provide valuable services. They have proved to be particularly useful because of the ease of their mass cultivation, speed of growth, use of cheap substrates that in many cases are wastes, and the diversity of potential products. In addition, their ability to readily undergo genetic manipulation has opened up almost limitless possibilities for new products and services from the fermentation industries.

Successful development of a fermentation process requires major contributions from a wide range of other disciplines, particularly biochemistry, genetics and molecular biology, chemistry, chemical and process engineering, and mathematics and computer technology. A typical operation involves both upstream processing (USP) and downstream processing (DSP) stages (Fig. i). The USP is associated with all factors and processes leading to and including the fermentation, and consists of three main areas.

1 The producer microorganism. Key factors relating to this aspect are: the strategy for initially obtaining a suitable industrial microorganism, strain improvement to enhance productivity and yield, maintenance of strain purity, preparation of a reliable inoculum and the continuing development of selected strains to improve the economic efficiency of the process. For example, the production of stable mutant strains that vastly overproduce the target compound is often essential.

Some microbial products are primary metabolites, produced during active growth (the trophophase), which include amino acids, organic acids, vitamins and industrial solvents such as alcohols and acetone. However, many of the most important industrial products are secondary metabolites, which are not essential for growth, e.g. alkaloids and antibiotics. These compounds are produced in the stationary phase of a batch culture, after microbial biomass production has peaked (the idiophase).

Fig. i Outline of a fermentation process.

2 The fermentation medium. The selection of suitable cost-effective carbon and energy sources, and other essential nutrients, along with overall media optimization are vital aspects of process development to ensure maximization of yield and profit. In many instances, the basis of industrial media are waste products from other industrial processes, notably sugar processing wastes, lignocellulosic wastes, cheese whey and corn steep liquor.

3 The fermentation. Industrial microorganisms are normally cultivated under rigorously controlled conditions developed to optimize the growth of the organism or production of a target microbial product. The synthesis of microbial metabolites is usually tightly regulated by the microbial cell. Consequently, in order to obtain high yields, the environmental conditions that trigger regulatory mechanisms, particularly repression and feedback inhibition, must be avoided.

Fermentations are performed in large fermenters often with capacities of several thousand litres. These range from simple tanks, which may be stirred or unstirred, to complex integrated systems involving varying levels of computer control. The fermenter and associated pipework, etc., must be constructed of materials, usually stainless steel, that can be repeatedly sterilized and that will not react adversely with the microorganisms or with the target products. The mode of fermenter operation (batch, fed-batch or continuous systems), the method of its aeration and agitation, where necessary, and the approach taken to process scale-up have major influences on fermentation performance.

Conventional DSP includes all unit processes that follow fermentation. They involve cell harvesting, cell disruption, product purification from cell extracts or the growth medium, and finishing steps. However, attempts are now being made to integrate fermentation with DSP operations, which often increases process productivity. Overall, DSP must employ rapid and efficient methods for the purification of the product, while maintaining it in a stable form. This is especially important where products are unstable in the impure form or subject to undesirable modifications if not purified rapidly. For some products, especially enzymes, retention of their biological activity is vital. Finally, there must be safe and inexpensive disposal of all waste products generated during the process.

Fermentation products

The overall economics of fermentation processes are influenced by the costs of raw materials and consumables, utilities, labour and maintenance, along with fixed charges, working capital charges, factory overheads and operating outlay. Fermentation products can be broadly divided into two categories: high volume, low value products or low volume, high value products. Examples of the first category include most food and beverage fermentation products, whereas many fine chemicals and pharmaceuticals are in the latter category.

Food, beverages, food additives and supplements

A wide range of fermented foods and beverages have been produced throughout recorded history. They continue to be major fermentation products worldwide and are of vast economic importance. Fermented dairy products, for example, result from the activities of lactic acid bacteria in milk, which modify flavour and texture, and increase long-term product stability. Yeasts are exploited in the production of alcoholic beverages, notably beer and wine, due to their ability to ferment sugars, derived from various plant sources, to ethanol. Most processes use strains of one species, S. cerevisiae, and other strains of this yeast are used as baker’s yeast for bread dough production.

Several organic acids derived from microbial action are employed in food manufacture and for a wide range of other purposes. The first human use was for acetic acid, as vinegar, produced as a result of the oxidation of alcoholic beverages by acetic acid bacteria. A further aerobic fermentation involves citric acid production by the filamentous fungus, A. niger, which has become a major industrial fermentation product, as it has numerous food and non-food applications. Also, most of the amino acids and vitamins used as supplements in human food and animal feed are produced most economically by microorganisms, particularly if high-yielding overproducing strains are developed. In addition, some microorganisms contain high levels of protein with good nutritional characteristics suitable for both human and animal consumption. This so-called ‘single-cell protein’ (SCP) can be produced from a wide range of microorganisms cultivated on low-cost carbon sources.

Health-care products

In terms of providing human benefit, antibiotics are probably the most important compounds produced by industrial microorganisms. Most are secondary metabolites synthesized by filamentous fungi and bacteria, particularly the actinomycetes. Well over 4000 antibiotics have now been isolated, but only about 50 are used regularly in antimicrobial chemotherapy. The best known and probably the most medically useful antibiotics are the β-lactams, penicillins and cephalosporins, along with aminoglycosides (e.g. streptomycin) and the tetracyclines. New antibiotics are still being sought as resistance to established antibiotics has become a major problem in recent years, through the misuse and overuse of these drugs.

Other important pharmaceutical products derived from microbial fermentation and/or biotransformations are alkaloids, steroids and vaccines. More recently, therapeutic recombinant human proteins such as insulin, interferons and human growth hormone have been produced by a range of microorganisms. This is a rapidly expanding field and many more recombinant therapeutic products are likely to come on to the market over the coming years.

Microbial enzymes

Microbial enzymes, particularly extracellular hydrolytic enzymes, have numerous roles as process aids or in the production of a wide range of specific food and nonfood products. Proteases, for instance, are extensively used as additives to washing powders, in the removal of protein hazes from beer and as microbial rennets for the production of cheese. Several carbohydrases are employed in the production of a diversity of sugar syrups from starch. For example, high-fructose corn syrup is produced by hydrolysing corn starch to glucose using α-amylase and amyloglucosidase, and the resulting glucose is then isomerized to a sweeter molecule, fructose, by a glucose isomerase. All of these examples involve the use of ‘bulk’ enzymes. Smaller quantities of highly purified ‘fine’ enzymes are used for numerous specialized purposes.

Immobilization of enzymes or whole cells, by their attachment to inert polymeric supports, allows easier recovery and reuse of the biocatalyst, and some enzymes are much more stable in this form. Also, the product does not become contaminated with the enzyme. Applications of immobilized biological catalysts include the production of amino acids, organic acids and sugar syrups.

Industrial chemicals and fuels

Industrial feedstock chemicals supplied through fermentation include various alcohols, solvents such as acetone, organic acids, polysaccharides, lipids and raw materials for the production of plastics. Some of these fermentation products also have applications in food manufacture.

Fossil fuels, especially oil, are likely to become exhausted within the next 50–100 years, resulting in the need to develop alternative sources of energy. Biological fuel generation may make an increasing contribution, particularly in the conversion of renewable plant biomass to liquid and gaseous fuels. This plant biomass can be in the form of cultivated energy crops, natural vegetation, and agricultural, industrial and domestic organic wastes. Currently, methane and ethanol are the main products, although other potential fuels can be generated using microorganisms, including hydrogen, ethane, propane and butanol.

Environmental roles of microorganisms

Microorganisms are particularly important in wastewater treatment, which utilizes the metabolic activities of diverse mixed microbial populations capable of degrading any compound that may be presented to them. The two main objectives are to destroy all pathogenic microbes present in the sewage, particularly the causal organisms of the water-borne diseases cholera, dysentery and typhoid. The second objective is to break down the organic matter in waste-water to mostly methane and carbon dioxide, thereby producing a final effluent (outflow) that can be safely discharged into the environment. Microbial activities can also be employed in the degradation of man-made xenobiotic compounds within waste streams and in the bioremediation of environments contaminated by these materials.

Microbial-based ‘clean technology’ is also being increasingly used in the desulphurization of fuels and the leaching of metals (e.g. copper, iron, uranium and zinc) from low-grade mineral ores and wastes using species of Thiobacillus and Sulfolobus. Environmental biological control is a further area where microorganisms are employed in an effort to reduce our reliance on synthetic chemical pesticides. Bacteria, fungi, protozoa and viruses are cultivated to produce biomass or cell products for the control of fungal, insect and nematode pests of agricultural crops, along with some vectors of human and animal diseases.

Conclusion

As can be seen from this brief introduction, microorganisms have a major role in providing food, raw materials and essential services. This role is likely to expand due to our increasing requirements for resources and the ability to manipulate microorganisms to improve their yields and the range of their products and activities.

Part 1

Microbial physiology

1

Microbial cell structure and function

The cell is the basic unit of all living organisms, many of which are unicellular, whereas others are multicellular forms, enabling cell specialization. All cells are filled with a fluid matrix and surrounded by a cytoplasmic membrane, primarily composed of lipids and proteins. They also contain nucleic acids, the physical carriers of genetic information, along with ribosomes that take part in protein synthesis. Cells are divided into two categories: those of archaeans and eubacteria are prokaryotic, whereas the cells of fungi, protozoa, algae and other plants, and animals are eukaryotic.

Prokaryotic cells are normally less than 5μm in diameter, with a very few notable exceptions such as the marine bacterium, Thiomargarita namibiensis, at over 100 times larger. Prokaryotes rarely possess membrane-bound organelles and have little recognizable internal ultrastructure, apart from inclusion bodies (granules of organic or inorganic compounds), various vacuoles and some specialized invaginations of the cell membrane, e.g. the lamellae of photosynthetic cells (Fig. 1.1a). Most prokaryotic cells contain a single chromosome composed of deoxyribonucleic acid (DNA), which is located in a region of the cell referred to as the nucleoid. The chromosome is usually circular, although in some prokaryotes it is linear. Prokaryotic ribosomes are 70S (Svedberg units), which refers to their rate of sedimentation on centrifugation and is a measure of their size, although density and shape of the particle can also influence this value. Almost all prokaryotes have cell walls or cell envelopes located outside the cytoplasmic membrane, which usually contain some peptidoglycan. Outside this wall they may have capsules or slime coats and propelling flagellae that are less complex than those of eukaryotic cells. Cell division in prokaryotes is normally by simple binary fission.

Eukaryotic cells are generally larger than those of prokaryotes and contain a range of membrane-bound organelles, including mitochondria, lysosomes, Golgi bodies and an extensive endoplasmic reticulum (Fig. 1.1b). Photosynthetic cells also contain chloroplasts.

The DNA of eukaryotic cells, in the form of several linear chromosomes, is characteristically complexed with histone proteins and is housed in a double membrane-bound nucleus. Eukaryotic ribosomes, apart from those located within certain organelles, are 80 S, somewhat larger than those of prokaryotes. If cell walls are present, they are composed of materials other than peptidoglycan, such as cellulose and related β-glucans, chitin or silica. Eukaryotic cells divide by a complex process of mitosis and usually have a sexual life-cycle, involving meiosis (reduction division). This process halves the number of chromosomes from diploid (2n) chromosome pairs to produce haploid (n) cells containing a single set of chromosomes, facilitates genetic recombination and results in the formation of gametes.

Prokaryotes

Prokaryotes have been separated into two distinct groups on the basis of the study of phylogenetic (evolutionary) relationships. They are the archaebacteria or archaea (‘ancient’ bacteria) and the eubacteria (‘true’ bacteria), the group that contains almost all established industrial prokaryotes (Table 1.1).

Archaea

These prokaryotes are quite different from eubacteria and have some features, especially aspects of the transcription and translation machinery associated with protein synthesis, that are similar to eukaryotic cells. Most archaeans live in extreme environments similar to those that early life forms are thought to have endured. Three basic physiological types are found, namely halophiles (adapted to high salt concentrations), methanogens (methane producers) and thermophiles (adapted to high temperatures), and some of these are also barophiles (adapted to high pressure) (see Chapter 2). Archaeans have relatively small genomes containing less than half the DNA of eubacteria. For example, the genome of Methanococcus jannaschii has been sequenced and found to contain 1760 genes composed of 1700 kilobase pairs (kbp). Although few archaeans are currently used for industrial purposes, they possess many interesting properties that could be exploited for biotechnological uses in the future. Their enzymes are of particular interest.

Table 1.1 Microbial genera with established industrial roles (for specific examples of their roles see Table 4.1)

Fig. 1.1 Diagrammatic representation of the main structures of (a) prokaryotic and (b) eukaryotic microbial cells. Not all structures are always present, including capsules, chloroplasts, flagellae, pili, storage granules and vacuoles (from Dawes & Sutherland (1992)).

All prokaryotes may be initially subdivided according to their characteristics in the Gram staining procedure, which is determined by cell wall/envelope structure. Archaeal cell wall composition varies considerably, some appearing Gram-positive whereas others are Gram-negative. Their cell wall constituents are quite different from those of the eubacteria, in that they contain the unique polymers, methanochondroitin and pseudomurein. They also possess distinctive membrane lipids. The archaea may be divided into three kingdoms.

1 Euryarchaeota are primarily methanogens, such as Methanobacterium and Methanosarcina, and the extreme halophiles Halobacterium and Halococcus.

2 Crenarchaeota are mostly extreme barophiles and thermophiles that include Pyrodictium, Pyrolobus, Sulfolobus and Thermoproteus.

3 Korarchaeota are hyperthermophiles which, as yet, have not been isolated as pure cultures.

Eubacteria

The eubacteria are a very diverse group that may be divided into 12 subgroups. However, almost all industrial bacteria are contained within just two of them, the proteobacteria and the Gram-positive eubacteria.

1 The Proteobacteria is a major kingdom of Gram-negative bacteria that is divided into five groups, α, β, γ, δ, and ε. They include purple photosynthetic bacteria and non-photosynthetic relatives, notably the Enterobacteriaceae (e.g. Escherichia coli), along with Hyphomicrobium, Nitrobacter, Pseudomonas, Thiobacillus and Vibrio.

2 The Gram-positive eubacteria are composed of two major subdivisions:

(a) the low guanine (G) + cytosine (C) group, which refers to the proportion of this base pair within the organism’s DNA and includes Bacillus, Clostridium, Lactobacillus, Leuconostoc, Staphylococcus, Streptococcus and Mycoplasma; and

(b) the high G + C group, which contains the actinomycetes (filamentous bacteria, e.g. Streptomyces), Corynebacterium, Mycobacterium and Micrococcus.

The other subgroups, which contain few examples of industrial bacteria, are as follows.

3Cyanobacteria and relatives, which are oxygenic phototrophs, e.g. Anabaena, Nostoc and Spirulina.

4Chlamydia, a group of obligate intracellular parasites.

5Planctomyces and Pirella, bacteria lacking peptidoglycan; some with membrane-bound nucleoid.

6Bacteroides and Flavobacteria, a subgroup that contains a mixture of physiological types.

7 Green sulphur bacteria, such as Chlorobium, an anaerobic phototroph.

8 Spirochetes and relatives which are Gram-negative coiled bacteria.

9 Deinococci, radioresistant micrococci and relatives, e.g. Deinococcus radiodurans and Thermus aquaticus.

10 Green non-sulphur bacteria and anaerobic phototrophs.

11Thermotoga and Thermosulfobacteria, thermophiles from hot springs and marine sediments.

12Aquiflex, a group of obligate chemolithotrophic and autotrophic hyperthermophiles.

There is probably no such thing as a typical prokaryotic cell. There is a vast amount of diversity, including: morphological diversity (size and shape; rods, cocci, spirals, filaments, etc.), structural diversity (Gram-positive or Gram-negative cell walls/envelopes, external structures such as flagellae and pili, and the ability to form spores), along with metabolic, ecological and behavioural diversity. However, it is worthwhile considering, in some detail, the key cellular features of examples of industrially valuable Gram-negative and Gram-positive bacteria.

ESCHERICHIA COLI, A GRAM-NEGATIVE BACTERIUM

E. coli was discovered in 1885 by the German bacteriologist Theodor Escherich. It is a major inhabitant of the colon of humans and the lower gut of other warmblooded animals. Some strains can cause food- and water-borne diseases that produce diarrhoea and can be especially problematical for human infants and young animals. A particularly virulent strain is E. coli 0157:H7, the cause of haemorrhagic colitis, which has recently emerged and has been associated with the ingestion of undercooked beef and raw milk.

Extensive information has been accumulated about the biochemistry, physiology and genetics of E. coli. This knowledge and the organism’s rapid growth rate, with a doubling time as low as 20 min (see Chapter 2), has led to it becoming an important industrial microorganism. E. coli has been used extensively as a model for the study of molecular biology and is often considered to be the ideal host in gene-cloning experiments. Consequently, it has proved to be extremely useful for the production of heterologous proteins, derived from other organisms. However, protein secretion by E. coli is rather more problematical, due to the nature of its cell envelope (see below), whereas secretion from Gram-positive bacteria is often more readily achieved.

E. coli is a Gram-negative facultative anaerobe, belonging to the family Enterobacteriaceae, whose members are often referred to as enterobacteria or enteric bacteria. The cells are short straight rods, approximately 0.3–1.0 μm wide and 1.0–3.0 μm long, that divide by binary fission after elongating to approximately twice their normal length. Members of the genus Escherichia are oxidase-negative (lacking cytochrome c oxidase) and carry out mixed acid fermentation, producing mainly lactate, acetate, succinate and formate. The formate may be further degraded to form H2 and CO2.

Outer membrane

The outer coverings of Gram-negative bacterial cells are often referred to as envelopes, rather than walls, as they are more complex than the cell walls of Gram-positive bacteria (Fig. 1.2a). They are essentially composed of two layers that protect the cell and provide rigidity. The outermost layer is called the outer membrane, which is approximately 7–8 nm thick, containing lipopolysaccharide and mucopeptide. This structure does not impede the movement of small molecules, charged or uncharged, and is more permeable than the cell/cytoplasmic membrane (see below), but is a barrier to hydrophobic molecules and proteins. It contains porin proteins, composed of three subunits, that form narrow channels of about 1–2 nm diameter through which small molecules can pass. Non-specific porins allow the passage of molecules up to 600–700 Da, whereas specific porins have binding sites for one or more substances of up to 5000 Da. Lipopolysaccharide components of the envelope are effective in protecting the cell from detergents and other antimicrobial agents. The most common outer membrane protein is Braun’s lipoprotein, which extends through the outer membrane and links to the underlying peptidoglycan.

Flagellae of motile strains propel the cell through aqueous media. In E. coli, flagellae are arranged around the entire cell, referred to as a peritrichous arrangement, whereas other bacteria have polar flagellae or alternative specific arrangements. Each flagellum is several micrometres long, composed of the protein flagellin, and is attached to the outer membrane layer through a basal body, composed of four rings. In addition, fibrils (fimbriae or pili), may be attached to the outer membrane, which are short hair-like projections, 5–7 nm in diameter and 400 nm long. They enable E. coli to attach to surfaces, such as intestinal epithelium.

Some strains also possess capsules located outside the outer membrane, which are composed of polysaccharides. Their production is influenced by the chemical and physical conditions within the local environment. These exopolysaccharides may provide a barrier to certain molecules, help protect against desiccation, or aid attachment of pathogenic strains to host cell surfaces.

Peptidoglycan and the periplasmic space

Within the outer membrane of Gram-negative bacteria, and covalently attached to it through lipoprotein, is a thin layer of peptidoglycan some 2–3 nm thick. It constitutes only 5–10% of the cell envelope and is composed of one to three layers, compared with the 20–25 layers of peptidoglycan in the walls of many Gram-positive bacteria. Nevertheless, it is a very important structural component. When the peptiglycan layer is incomplete, bacterial cells may swell and ultimately burst (see Chapter 3, Peptidoglycan biosynthesis).

The peptidoglycan extends down into the underlying periplasmic space, which is approximately 12–15 nm wide. This region is not empty, it contains a range of proteins, binding proteins, chemoreceptors and various enzymes. Binding proteins initiate transport of specific substances into the cell by taking them to their membrane-bound carriers. The chemoreceptors are involved in chemotaxis, which is the movement of a cell towards attractant and away from repellant chemicals. Hydrolytic enzymes, notably alkaline phosphatase, nucleases and proteases, are secreted into the periplasm from the cytoplasm and are retained close to the cell as they cannot normally pass through the outer membrane. They are associated with the breakdown of large impermeable nutrients into smaller molecules that can be transported across the cell membrane into the cell (see Chapter 2, Nutrient uptake). Some detoxifying and defence enzymes are also located here, e.g. penicillinase.

Cell (cytoplasmic) membrane

Below the periplasmic space lies the inner cell (cytoplasmic) membrane that encloses the cytoplasmic matrix (Fig. 1.2a). This structure is highly selective, controlling the entry of nutrients and the secretion of ions and larger compounds. The membrane is in the form of a lipid bilayer, primarily composed of phosphatidyl ethanolamine. It is interspersed with both transport proteins, such as lactose permease, and pores made up of porins that selectively control the entry of molecules and charged ions into cells. Respiratory proteins, including cytochromes and other electron transfer proteins, are also located within this membrane (see Chapter 3).

Cytoplasmic matrix and cell contents

The cytoplasmic matrix is maintained at pH 7.6–7.8, with differences between the intracellular and extracellular pH being controlled by the primary proton pumps associated with electron transport and respiration. It contains metabolic intermediates, and the enzymes and coenzymes required for catabolic and anabolic metabolism (see Chapter 3). Machinery for protein synthesis, both transcription and translation, is also located here. This includes RNA polymerases for transcribing the genetic code of DNA into messenger RNA (mRNA), and around 18000 ribosomes and transfer RNAs for translating the mRNA message sequences into proteins.

The chromosome resides in the nucleoid region that occupies approximately 10% of the cell’s volume. It is a single circular molecule of double-stranded DNA made up of about 4600 kbp, constituting over 4000 genes, and is attached to the inside of the cell membrane at, or close to, the origin of replication. This DNA molecule is approximately 1 mm long and 1 nm thick, but is extensively folded and coiled, and stabilized by associated proteins.

Plasmids, relatively small circular extrachromosomal DNA molecules, may also be present. They include the fertility (F) factor, resistance (R) plasmids and Col plasmids that code for colicins, specific antibacterial bacteriocins. In enteropathogenic strains of E. coli at least two toxins are known to be plasmid encoded.

The polysaccharide glycogen is a main store of carbon and energy, and may be seen as inclusion bodies within the cytoplasmic matrix. Under certain circumstances osmoprotective betaines (N,N,N-trimethyl glycine) are also accumulated.

BACILLUS SUBTILIS, A GRAM-POSITIVE BACTERIUM

The genus Bacillus consists of a large number of diverse, rod-shaped, chemoheterotrophic, Grampositive bacteria. These cells are usually somewhat larger than E. coli, at 0.5–2.5 μm wide and 1.2–10 μm long. Some species are strictly aerobic, others are facultative anaerobes or microaerophilic, but all are catalase positive (see Chapter 2). Bacillus species also produce oval or cylindrical endospores that are resistant to adverse environmental conditions and provide a selective advantage for survival and dissemination (Fig. 1.3). Several members of the genus have important industrial roles, particularly as sources of enzymes, antibiotics (bacitracin, gramicidin and polymyxin) and insecticides.

B. subtilis is a common soil microorganism that is often recovered from water, air and decomposing plant residues. This bacterium is considered to be benign as it does not possess any disease traits, unlike some of its relatives, notably B. anthracis, the cause of anthrax. The range of extracellular enzymes produced by this microorganism enables it to degrade a variety of natural substrates and contributes to nutrient cycling. Many of these enzymes are exploited commercially and B. subtilis has become widely used for the production of industrial enzymes, particularly amylases and proteases. Some of these amylases are used extensively in starch modification processes and the proteases are employed as cleaning aids in biological detergents. B. subtilis has also proved very useful for the manufacture of fine chemicals, especially nucleosides, vitamins and amino acids, and some strains are used in crop protection against fungal pathogens. This bacterium is also a valuable cloning host for the production of heterologous proteins.

Fig. 1.3 The structure of a typical bacterial endospore.

The main features of B. subtilis that distinguish it from E. coli are the cell wall structure and the ability to produce spores. B. subtilis cell walls are typical of Gram-positive bacteria, being much less complex than those of Gram-negative bacteria such as E. coli. They are 20–50 nm thick and simply composed of 20–25 layers of peptidoglycan, associated with some lipid, protein and teichoic acid (Fig. 1.2b). Teichoic acid is a distinctive anionic polymer of glycerol phosphate, ribitol phosphate and other sugar phosphates. It is covalently linked to the N-acetylmuramic acid units of the peptidoglycan or to lipids of the underlying cell membrane. This component is not found in Gram-negative bacteria. Outside the cell wall, B. subtilis produces a polypeptide capsule that contains both D- and L-glutamic acid units. Flagellae may be present and flagellated forms are capable of chemotaxis.

The B. subtilis chromosome is a little smaller than that of E. coli at 4188 kbp, but other intracellular features are similar. However, as mentioned above, a major difference is the ability of members of this genus to form spores (Fig. 1.3). Vegetative cells continue to grow and divide by binary fission until nutrients become limiting. Deprivation of nutrients initiates sporulation through an as yet unknown chemical signal. Unequal cell division produces a smaller forespore cell and a larger mother cell by the formation of an asymmetric septum near one pole of the cell. The mother cell goes on to engulf the forespore. A primordial wall of peptidoglycan is then formed around the forespore, which later becomes the cell wall of the vegetative cell that emerges when the spore germinates. This primordial wall is then overlain by a much thicker layer of complex peptidoglycan, known as the spore cortex. This cortex has a unique spore-specific composition, where over 50% of the muramic acid residues are present as muramic acid δ-lactam. Following cortex deposition, the structure is finally enclosed within a proteinaceous coat, and the surrounding mother cell dies and lyses to release the spore. These spores are extremely dormant, exhibiting a lack of metabolism, and are highly resistant to desiccation, heat, radiation and harsh chemical treatments. They can remain viable for long periods. Under favourable growth conditions the spore germinates to form a vegetative cell.

Fungi

Fungi are a diverse group of eukaryotic microorganisms that occupy a variety of habitats. The majority of fungal species are composed of filamentous hyphae and are often referred to as moulds, whereas the yeasts, which will be described later, are unicellular fungi. Of the thousands of species known, relatively few filamentous fungi are used for industrial purposes (Table 1.1). Filamentous fungi are non-photosynthetic and have strict chemoheterotrophic absorptive nutrition (see Chapter 2). Many secrete a range of hydrolytic enzymes to degrade the complex polymeric molecules encountered into smaller units that can be absorbed. Most are saprophytic, utilizing dead animal or plant remains. Some are facultative parasites of plants or animals, and several form symbiotic and mutualistic relationships with other organisms, e.g. with an alga to form a lichen (a fungus, the mycobiont, in symbiotic association with an alga, the phycobiont).

Filamentous fungi originate from either fragments of hyphae or dispersed spores that germinate under suitable environmental conditions. Hyphae can grow rapidly in length, at rates of up to several micrometres per minute. However, there is generally little increase in girth, which maximizes the surface area for absorption of nutrients. They branch to form intertwining mats that are usually established around and within their food source, and are structurally organized into macroscopic vegetative mycelium. In higher fungi, hyphae can be aggregated together to produce large complex solid structures, such as the fruiting bodies of mushrooms, rhizomorphs, sclerotia and stromata.

Individual hyphae are 1–15 μm in diameter depending upon the species. Their cell walls are composed of 80–90% polysaccharide, along with some lipid and protein constituents. Except in certain lower fungi, the polysaccharide is primarily microfibrils of chitin, a linear polymer of β-1,4-linked N-acetylglucosamine units, which is a strong and flexible material, similar to that found in arthropod exoskeletons. Unlike yeast cell walls (see p. 16), glucan and mannan are relatively minor components. Glucans are mostly laid down at the growing hyphal tips. Here the cell walls are only 50 nm thick, but behind the tip wall thickness increases up to 125nm through the further deposition of chitin.

Fungal hyphae may be aseptate or septate. Hyphae of aseptate fungi lack cross-walls and such cells are referred to as coenocytic. This is a multinucleate state resulting from repeated nuclear division without cytokinesis (cell division). Hyphae of septate fungi are divided into cells by cross-walls called septa, containing pores that allow organelles and other cellular materials to move from cell to cell (Fig. 1.4). Cellular organelles of fungi are typically eukaryotic and are particularly associated with the growing hyphal tips, which are often packed with mitochondria, ribosomes and small vesicles. In addition, the hyphae tend to have numerous vacuoles containing storage products, normally glycogen, lipid and volutin (a polymer of metaphosphate).

Fungal chromosomes and nuclei are relatively small, and nuclear division is somewhat different from that in most other eukaryotes. During mitosis, the nuclear envelope remains intact with the spindle located within. After separation of replicated chromosomes, the nuclear envelope constricts to form two new nuclei, during which the spindle disappears.

Although fungal hyphae and spores are normally haploid, except for transient diploid stages in the sexual life-cycles, some mycelia may be genetically heterogeneous, resulting from the fusion of hyphae of separate origins. In such cases, each hypha contributes nuclei that may remain segregated into different parts of the same mycelium, or may mingle and even exchange genes in a process similar to crossing-over events during meiosis.

Most fungi reproduce by releasing vast quantities of unicellular spores. These propagules are dispersed over a wide area by wind or water. They are stable for long periods, but will germinate if the environmental conditions and substratum are suitable. There are various ways by which spores may be formed asexually, depending upon the fungus (Fig. 1.5). Their mode of formation determines whether they are blastospores, sporangiospores, conidiospores, and so on. Variations in shape and arrangement of spore-bearing structures are often used as a basis for fungal identification.

Fig. 1.4 Diagrammatic representation of a septate fungal hypha.

The sexual cycle in fungi differs from that in other eukaryotic organisms in that syngamy, sexual union of haploid cells of opposite mating strains, + and −, occurs in two stages separated in time: first, plasmogamy, the fusion of cytoplasm, followed some time later by karyogamy, the fusion of nuclei. Following plasmogamy, a dikaryon is formed through the pairing of haploid nuclei from each parental strain, but the pairs of nuclei do not fuse immediately. These nuclear pairs within a dikaryon may remain separate and go on dividing synchronously for long periods. Eventually they fuse to form a diploid cell that directly undergoes meiosis, ultimately producing genetically diverse haploid spores.

Traditionally, the 100 000 known species of fungi have been divided into four main classes, primarily based upon how and where they specifically generate their sexual spores.

Phycomycetes

Phycomycetes are the lower fungi, which are subdivided into Mastigomycotina (zoosporic, motile spores) and Zygomycotina (zygosporic). They are the simplest fungi and their vegetative hyphae are aseptate. The Mastigomycotina contains the water moulds, Saprolegnia, and the important plant pathogens Pythium and Phytophthora. Unusually, their cell walls are composed of cellulose, along with other glucans or chitin. Industrially important members of the Zygomycotina include Mucor, Rhizomucor and Rhizopus species, which are used in some traditional food fermentations, and whole cell and enzyme bioconversions.

Asexual reproduction results in the formation of saclike sporangia at the tips of upright hyphae or sporangiophores (Fig. 1.5a). Hundreds of haploid spores are produced by mitosis within the sporangium, which are then wind dispersed. In sexual reproduction mycelia of opposite mating types each form gametangia containing several haploid nuclei. Plasmogamy of + and − gametangia, and pairing of haploid nuclei, results in the formation of a dikaryotic zygosporangium. This structure is metabolically inactive and resistant to desiccation. When conditions become favourable, karyogamy occurs between paired nuclei and the resulting diploid nuclei undergo meiosis to produce haploid spores.

Ascomycetes or Ascomycotina (sac fungi)

This is the largest class of fungi and includes the yeasts that are utilized in many industrial fermentation processes. Other filamentous members that have industrial and commercial roles are Neurospora species, Claviceps species, and important edible fungi, including Morchella species (morels) and Tuber species (truffles). Their hyphae are septate, and in asexual reproduction, the tips of specialized hyphae (conidiophores) form chains of haploid conidiospores that are usually wind dispersed. Sexual spores or ascospores are contained within a sac-like ascus, which may be enclosed within a fruiting body or ascocarp. The yeasts, such as Saccharomyces cerevisiae, produce the equivalent of an ascus during sexual reproduction and most bud during asexual reproduction in a manner similar to the formation of conidiospores (see below). However, certain yeasts, notably Schizosaccharomyces species, do not bud, but undergo binary fission in a similar manner to bacteria.

Fig. 1.5 Asexual spore formation (a) in the sporangia of Mucor, a coenocytic filamentous fungus and (b) conidia of Penicillium, a septate filamentous fungus.

Basidiomycetes or Basidiomycotina (club fungi)

Members of this division produce septate hyphae and their sexual haploid basidiospores are borne on club-shaped structures called basidia. In some cases, these are contained within large sexual fruiting bodies, the basidiocarps, as in the mushrooms, e.g. Agaricus bisporus (button mushroom) and Lycoperdon species (puff balls). Other industrially important basidiomycetes are certain wood-rotting fungi involved in biodeterioration and biodegradation processes, e.g. Phanerochaete chrysosporium (white rot). Also included in this division are the rusts and smuts, which are important plant pathogens.

Deuteromycetes or Deuteromycotina (‘imperfect’ fungi)

This division contains a diverse group of around 25000 species, which have been grouped together simply because they lack a defined sexual (perfect) stage, or one has not been observed. It is assumed that this group may represent the conidial stages of ascomycetes whose ascus stage has not yet been discovered or has been lost in the course of evolution. Parasexuality has been demonstrated in some species, which has proved important for genetic study and strain development. Many industrially important fungi are classified as deuteromycetes, including species of Aspergillus, Cephalosporium, Fusarium, Penicillium (Fig. 1.5b) and Trichoderma.

Yeasts

Yeasts, notably S. cerevisiae, are worthy of special attention, due to their major contribution as industrial microorganisms. The term ‘yeast’ refers to a unicellular form rather than to a phylogenetic classification. It can be used to describe a unicellular phase of the life-cycles of fungi that may be predominantly filamentous, but exhibit yeast–mould dimorphism. Changes from one form to the other are often influenced by the prevailing nutritional conditions. However, this term is used more commonly as a generic name for fungi that have only a unicellular phase, such as baking or brewing yeasts. These true yeasts and many others of industrial importance are strains of S. cerevisiae, a member of the ascomycetes.

Yeasts are heterotrophic and are found in a wide range of natural habitats, being particularly common on aerial plant organs, especially flowers, and plant debris in the surface layers of soil. Unlike most fungi, which are obligate aerobes, many yeasts are facultative anaerobes, able to grow in the absence of oxygen. They generally have relatively simple nutritional needs that include a reduced carbon source, which can be compounds as simple as acetate, and a variety of minerals. Inorganic nitrogen sources, such as ammonium salts, are readily assimilated, although a variety of organic nitrogen compounds can also be used, including urea and various amino acids. Often the only other complex compounds or growth factors required are vitamins, e.g. biotin, pantothenic acid and thiamine.

Several yeasts have industrial and food uses, and some, notably S. cerevisiae, have GRAS (generally regarded as safe) status. Strains of S. cerevisiae are the most well-known and commercially important yeasts, and have long been employed to produce alcoholic beverages and leaven bread. They are now also used in the production of several other fermentation products, particularly fuel ethanol, single cell protein, enzymes and heterologous proteins, e.g. human insulin. In addition, S. cerevisiae has been the model system in molecular genetics research because its basic mechanisms of replication, recombination, cell division and metabolism are very similar to those of higher eukaryotes, including mammals.

Very few yeasts are animal pathogens. Cryptococcus neoformans causes a relatively rare form of meningitis, but the best known is Candida albicans. This organism is carried by most people in a benign form, but it may commonly infect the skin, and mucosal membranes of the mouth, vagina and alimentary tract. C. albicans can become a serious opportunistic pathogen, particularly in individuals whose immune response is weakened. Such infections are difficult to treat, due to the similarity between the host’s and the pathogen’s metabolism. This yeast exhibits dimorphism, growing predominantly as a yeast form, but develops into branching hyphae, with accompanying changes in cell wall composition, under certain cultural conditions.

CELL GROWTH AND THE YEAST LIFE-CYCLE

Members of the genus Saccharomyces produce cells that are spherical to ellipsoidal and vary in size from approximately 1–7 μm wide and 5–10 μm long. Polyploid industrial strains, containing multiple sets of chromosomes, tend to be at the larger end of this range. The yeast cell is surrounded by a thick wall, within which is the cell membrane, enclosing the cytoplasmic matrix that contains enzymes, storage granules, several different types of organelle and membrane systems.

Their asexual cell division involves budding of a daughter cell from a mother cell and cell growth is largely associated with bud development (Fig. 1.6).

A mature cell initiates a bud at a site where the cell wall has been weakened by lytic enzymes and the bud grows to approximately the same size as the mother cell. Wall growth is polarized, mainly by deposition of new cell wall material at its tip, which is associated with microvesicles in the underlying cytoplasmic matrix. During this period mitosis occurs. The chromosomes replicate, and the mitotic spindle forms and aligns across the junction between the two cells. It then elongates and facilitates the formation of two separated sets of chromosomes that become enclosed within separate nuclei, one in each cell. The cell wall between the mother cell and bud becomes completed, and the two cells may then separate. This cycle can then begin again for both cells, each may bud 10–20 times.

Sexual reproduction involves haploid cells of two mating types (a and α) and mating is mediated by the secretion of small peptide pheromones. Each mating type responds by halting bud formation, then each cell elongates and differentiates to become a specialized pear-shaped gamete. Cells of opposite mating types that are in contact or close proximity undergo plasmogamy and karyogamy to form a diploid cell. These diploid forms are stable and can perform repeated cell division. However, if the physiological conditions are suitable meiosis occurs. This results in sporulation, normally producing four haploid nuclei, which are incorporated into four stress-resistant ascospores, encapsulated within an ascus.

CELL WALL STRUCTURE

Some yeasts develop slimy polysaccharide capsules outside the cell wall that may have a protective function or aid in attachment to surfaces. The cell wall itself is approximately 100–200 nm thick and comprises 15–25% of the dry weight of a cell. Its major components, some 80–90% of wall material, are the polysaccharides glucan, phosphomannan and chitin, along with some proteins (Fig. 1.7). Glucan is a highly branched polymer of β-linked glucose units, predominantly β-1,3 linkages, in the form of microfibrils that provide strength in the inner wall adjacent to the cell membrane. Phosphomannan, a branched polymer of the hexose sugar mannose, is located towards the outside of the cell wall. Chitin may form less than 1% of the yeast wall and is primarily found around bud scars. The number of bud scars on the wall indicates the number of times that a cell has budded and as they never occur twice in the same place, they can be used to estimate the ‘age’ of a cell. Protein wall components are both structural and enzymic; the former are largely associated with mannan. Enzymes that have been found here and in the periplasm include those associated with wall biosynthesis and the hydrolysis of substrates unable to cross the cell membrane. They include glucanase, mannanase, lipase, alkaline phosphatase and invertase, a mannoprotein involved in sucrose utilization.

Fig. 1.6 Diagrammatic representation of a budding yeast cell.

Flocculation of cells is a common feature in some yeasts and is particularly important for certain industrial strains. Flocs are aggregates of cells, not chains of unseparated buds. There are two main theories of floc formation. The first concerns calcium bridging, where a calcium ion links two cells via negatively charged cell wall components. Support for this theory comes from the observation that flocs are dispersed by the chelating agent ethylenediamine tetraacetic acid (EDTA). Secondly, the lectin hypothesis, involving protein–carbohydrate binding, proposes that a surface protein of one cell links to a mannose residue on an adjacent cell. This hypothesis is supported by the fact that sugars, particularly mannose, inhibit floc formation. In addition, proteinaceous cell wall protrusions of 0.1–10 μm, termed fimbriae, are present in many strains and may be associated with cell–cell interactions, including flocculation.

CELL MEMBRANE STRUCTURE

Between the cell wall and underlying membrane, there is a periplasmic space of 3.5–4.5 nm containing secreted proteins that cannot escape through the wall, including the enzymes mentioned above. The cell membrane controls all materials entering and exiting the cell, and is thought to manage cell wall biosynthesis. Yeast cell membranes, like those of other cells, are primarily composed of a lipid bilayer some 7.5 nm thick that arises from the tail-to-tail alignment of lipid molecules (Fig. 1.7). This produces inner and outer hydrophilic regions that sandwich the hydrophobic lipid ‘tails’. The lipid constituents are composed of mono-, di- and triglycerides, glycerophosphatides and sterols. Unsaturated sterol components include ergosterol and zymosterol, which are not found in prokaryotic cell membranes. They are vital for membrane stabilization and membrane rigidity, and molecular oxygen is required for their biosynthesis. Membrane lipid composition varies depending upon growth conditions and also affects the cell’s tolerance to ethanol. For example, brewing strains have been found to contain higher levels of phosphatidyl choline than baking strains.

Fig. 1.7 Diagrammatic representation of the structure of a yeast cell wall and cell membrane.

Located on, within and across the lipid bilayer are both structural and functional protein molecules, together with a small portion of carbohydrate. Protein components may be involved with solute transport, or are signal transduction components that respond to external stimuli and ultimately initiate an internal response. Membrane-associated enzymes include a range of permeases for the transportation of compounds, such as sugars and amino acids; along with adenosine triphosphatases (ATPases), in association with systems responsible for the generation of proton-motive force across the membrane, which provide energy for solute transport. There are also wall synthesizing enzymes. For example, chitin synthetase is present in an inactive form, which is activated by proteolytic cleavage when required.

THE NUCLEUS

The nucleus is surrounded by a double membrane that has regularly spaced pores. This organelle contains the majority (80–85%) of the cellular DNA; the remainder is present as circular molecules in the mitochondria or cytoplasm (see below). Haploid cells of S. cerevisiae contain approximately 12000kbp of DNA, which is 3–10 times more than a typical bacterium and about 100–150 times less than mammalian cells. Nuclear DNA is associated with basic histone proteins and is in the form of 16 linear chromosomes made up of 150–2500kbp. Over 6000 genes have been identified, but around 50% have unknown function. Associated with the nucleus is a structure referred to as a plaque, which has microtubules that pass into both the nucleus and cytoplasmic matrix. It functions in a similar way to the centrioles of animal cells, forming the spindle upon which the replicated chromosomes separate during mitosis.

THE MITOCHONDRIA

Fully developed mitochondria are present only in yeasts that are growing aerobically. Under anaerobic conditions they are simple structures, referred to as promitochondria, as anaerobic growth induces their dedifferentiation. Fully developed organelles are spherical to rod-shaped, enclosed by a double membrane. The inner membrane possesses proteins involved in electron transport and oxidative phosphorylation, and is folded into the lumen of the organelle to form finger-like structures called cristae. Located within the lumen is a fluid matrix containing most of the enzymes of the tricarboxylic acid (TCA) cycle, ribosomes and 75kbp circular DNA molecules that code for some 10% of mitochondrial proteins. ‘Petite’ yeast mutants lack mitochondria and are therefore incapable of respiration. They can grow only fermentatively and on solid media their colonies are very small, compared with wild-type colonies, hence their name.

OTHER ORGANELLES AND CYTOPLASMIC STRUCTURES