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2 - Microbial Biotechnology: Scope, Techniques, Examples
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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One can be a good biologist without necessarily knowing much about microorganisms, but one cannot be a good microbiologist without a fair basic knowledge of biology!
– Stanier, R. Y., Doudoroff, M., and Adelberg, E. A. (1957). The Microbial World. p. vii, Englewood Cliffs, NJ: Prentice-Hall, Inc.Microorganisms, whether cultured or represented only in environmental DNA samples, constitute the natural resource base of microbial biotechnology. Numerous prokaryotic and fungal genomes have been completely sequenced and the functions of many genes established. For a newly sequenced prokaryotic genome, functions for over 60% of the open reading frames can be provisionally assigned by sequence homology with genes of known function. Knowledge of the ecology, genetics, physiology, and metabolism of thousands of prokaryotes and fungi provides an indispensable complement to the sequence database.
This is an era of explosive growth of analysis and manipulation of microbial genomes as well as of invention of many new, creative ways in which both microorganisms and their genetic endowment are utilized. Microbial biotechnology is riding the crest of the wave of genomics.
The umbrella of microbial biotechnology covers many scientific activities, ranging from production of recombinant human hormones to that of microbial insecticides, from mineral leaching to bioremediation of toxic wastes. In this chapter, we sketch the complex terrain of microbial biotechnology. The purpose of this chapter is to convey the impact, the extraordinary breadth of applications, and the multidisciplinary nature of this technology. The common denominator to the subjects discussed is that in all instances, prokaryotes or fungi provide the indispensable component.
10 - Secondary Metabolites: Antibiotics and More
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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- 01 October 2007, pp 324-397
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The preceding chapter described the industrial production of some primary metabolites, including citric acid and amino acids. In contrast to these compounds, which are present in most living organisms and are produced by the ubiquitous major metabolic pathways, secondary metabolites are produced only by special groups of organisms through specialized pathways. Their chemical structure tends to be complex, and they are often produced only during the special growth phase, most often during the stationary phase. The most important of these secondary metabolites are the antibiotics.
Many science historians argue that among the many scientific discoveries of the twentieth century, that of the first antibiotic, penicillin (Figure 10.1), by Alexander Fleming (reported in 1928) is the discovery that had the largest impact on human life. The story is well known. Fleming is supposed to have kept a rather untidy laboratory and to have discovered, after returning from his vacation, that the bacterial colonies neighboring a contaminating mold colony were lysed on one of the Petri plates left on his bench. This story is often cited as an example of the importance of serendipity in science. This view, however, totally disregards the fact that Fleming dedicated his entire career to the search for natural products that lyse bacterial cells, in an effort to find agents that could be used in the treatment of bacterial infections. He had, in fact, discovered the enzyme lysozyme several years earlier but was disappointed that most human pathogens were intrinsically resistant to its lytic action.
9 - Primary Metabolites: Organic Acids and Amino Acids
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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- 01 October 2007, pp 299-323
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Microorganisms can function as efficient factories for the industrial scale production of the primary metabolites. Among these, ethanol will be discussed in Chapter 13. Other important primary metabolites currently produced by fermentation are listed in Table 9.1. Some organic acids and amino acids are seen to be the most important products in this category (excluding ethanol, of course).
CITRIC ACID
About 1 billion pounds of citric acid are produced worldwide (Table 9.1) by fermentation with the wild-type strains of a fungus, Aspergillus niger. Citric acid is used, for example, as a flavoring agent in food and drinks and to prevent oxidation and rancidity of fats and oils. In the very efficient fermentation process, up to 80% of the source sugar is converted into citric acid. Because this process, in operation since 1916, has been studied “probably more than any other process in mold metabolism,” according to one review, and could serve as a model for primary metabolite fermentation processes including glutamate fermentation (discussed later), we will examine this process in some detail.
Citric acid, unlike ethanol, is not one of the typical waste products of energy metabolism, and is usually degraded completely into CO2 and water through the operation of the citric acid cycle. Thus, the efficient conversion of sugars, such as glucose and sucrose, into citric acid by A. niger is rather unexpected, especially because mitochondria of A. niger contain all the enzymes of the citric acid cycle. The citric acid production occurs predominantly in the stationary phase, and indeed requires several unusual conditions.
Microbial Biotechnology
- Fundamentals of Applied Microbiology
- 2nd edition
- Alexander N. Glazer, Hiroshi Nikaido
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- 01 October 2007
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Knowledge in microbiology is growing exponentially through the determination of genomic sequences of hundreds of microorganisms and the invention of new technologies such as genomics, transcriptomics, and proteomics, to deal with this avalanche of information. These genomic data are now exploited in thousands of applications, ranging from those in medicine, agriculture, organic chemistry, public health, biomass conversion, to biomining. Microbial Biotechnology. Fundamentals of Applied Microbiology focuses on uses of major societal importance, enabling an in-depth analysis of these critically important applications. Some, such as wastewater treatment, have changed only modestly over time, others, such as directed molecular evolution, or 'green' chemistry, are as current as today's headlines. This fully revised second edition provides an exciting interdisciplinary journey through the rapidly changing landscape of discovery in microbial biotechnology. An ideal text for courses in applied microbiology and biotechnology courses, this book will also serve as an invaluable overview of recent advances in this field for professional life scientists and for the diverse community of other professionals with interests in biotechnology.
Preamble
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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Il n'y a pas des sciences appliquées … mais il y'a des applications de la science. (There are no applied sciences … but there are the applications of science.)
– Louis PasteurMicroorganisms are the most versatile and adaptable forms of life on Earth, and they have existed here for some 3.5 billion years. Indeed, for the first 2 billion years of their existence, prokaryotes alone ruled the biosphere, colonizing every accessible ecological niche, from glacial ice to the hydrothermal vents of the deep-sea bottoms. As these early prokaryotes evolved, they developed the major metabolic pathways characteristic of all living organisms today, as well as various other metabolic processes, such as nitrogen fixation, still restricted to prokaryotes alone. Over their long period of global dominance, prokaryotes also changed the earth, transforming its anaerobic atmosphere to one rich in oxygen and generating massive amounts of organic compounds. Eventually, they created an environment suited to the maintenance of more complex forms of life.
Today, the biochemistry and physiology of bacteria and other microorganisms provide a living record of several billion years' worth of genetic responses to an ever-changing world. At the same time, their physiologic and metabolic versatility and their ability to survive in small niches cause them to be much less affected by the changes in the biosphere than are larger, more complex forms of life. Thus, it is likely that representatives of most of the microbial species that existed before humans are still here to be explored.
7 - Bacillus thuringiensis (Bt) Toxins: Microbial Insecticides
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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The concerted effect of the exponentially increasing costs of insecticide development, the dwindling rate of commercialization of new materials, and the demonstration of cross or multiple resistance to new classes of insecticides almost before they are fully commercialized makes pest resistance the greatest single problem facing applied entomology. The only reasonable hope of delaying or avoiding pest resistance lies in integrated pest management programs that decrease the frequency and intensity of genetic selection by reduced reliance upon insecticides and alternatively rely upon multiple interventions in insect population control by natural enemies, insect diseases, cultural manipulations, and host-plant resistance.
–Metcalf, R. L. (1980). Changing role of insecticides in crop protection. Annual Review Entomology, 25, 219–256.The competition for crops between humans and insects is as old as agriculture, but chemical warfare against insects has a much shorter history. Farmers began to use chemical substances to control pests in the mid-1800s. Not surprisingly, the development of insecticides paralleled the development of chemistry: early insecticides were in the main inorganic and organic arsenic compounds, followed by organochlorine compounds, organophosphates, carbamates, pyrethroids, and formamidines, many of which are in use today. In 2001, global sales of chemical insecticides included more than 1.23 million pounds of active ingredients and reached about $9.1 billion a year.
There are disadvantages to relying exclusively on chemical pesticides. Foremost is that widespread use of single-chemical compounds confers a selective evolutionary advantage on the progeny of pests that have acquired resistance to the substances.
Contents
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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11 - Biocatalysis in Organic Chemistry
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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In carrying out their metabolic processes, microorganisms interconvert diverse organic compounds. These “biotransformations” are catalyzed with high specificity and efficiency by enzymes. The active site of an enzyme, where substrate binding and catalysis are carried out, is an asymmetric surface whose special geometry frequently guarantees that the enzymecatalyzed reaction will yield a particular stereoisomer as the sole product. Such stereospecific, or enantioselective, reactions may be difficult or impossible to achieve by purely chemical means. The terms used to describe the stereochemistry of organic compounds are defined in Box 11.1, and the determination of enantioselectivity is described in Box 11.2.
Even when an organic compound can be synthesized chemically, the process may require many steps, whereas a single enzyme-catalyzed reaction can often achieve the same end. Also, enzymes can catalyze reactions at ambient temperature, away from extremes of pH, and at atmospheric pressure. Undesired isomerization, racemization, epimerization, and rearrangement reactions that are frequently encountered during chemical processes are generally avoided. The absence of such side reactions is a particular advantage when the desired product is rather labile. Finally, enzymes can accelerate the rates of chemical reactions by factors of 108 to 1012. For all these reasons, biotransformations by microorganisms, or by enzymes purified from microorganisms, are highly useful in preparative organic chemistry.
Certain disadvantages do limit the use of enzymes in organic chemical processes, but these limitations have frequently proved to be surmountable challenges rather than impenetrable barriers.
4 - The World of “Omics”: Genomics, Transcriptomics, Proteomics, and Metabolomics
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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GENOMICS
SEQUENCING OF GENOMES
As mentioned in Chapter 3, we now know the complete nucleotide sequences of genomes of many organisms. The availability of this large amount of data at an unprecedented scale now allows us, and indeed forces us, to think “globally,” that is, on the scale of whole organisms, or even an assemblage of organisms, rather than of individual genes and enzymes. Here we describe very briefly how the genome sequences are determined.
Genome sequencing of viruses began in the late 1970s. The basic technique involved, the random sequencing of fragments by the Sanger dideoxy termination method, was proposed and applied successfully by Fred Sanger and associates to the complete sequencing of bacteriophage DNAs, notably that of phage λ in 1980 (see Figure 4.1 for the principle of the random shotgun method).
Historically, the first attempt to obtain a complete genome sequence of a cellular organism was geared toward Escherichia coli, the best studied organism outside of humans. This project started in 1989 and used a “directed” approach. Because a fairly detailed genetic map of E. coli was available thanks to the efforts of bacterial geneticists, it was possible to first produce a set of λ-based clones, each containing up to 20 kb DNA, with overlapping ends. The sequencing from here on represented the shotgun phase. The inserted segments in the λ vector were then randomly cut into much smaller fragments of a few kilobases, they were cloned into an M13 vector and were sequenced (for sequencing reactions, see Box 4.1), and the sequences were assembled by looking for overlaps.
Index
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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5 - Recombinant and Synthetic Vaccines
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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- 01 October 2007, pp 169-202
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In developing countries, infectious diseases still cause 30% to 50% of all deaths. Effective chemotherapeutic agents simply do not exist for many of the diseases that plague these regions, and many of the agents that do exist are far too costly for much of the population to afford. Vaccines thus have become the most important tool for fighting infectious diseases in those parts of the world.
The situation is very different in developed countries, where infectious diseases account for only 4% to 8% of all deaths. This is not to say, however, that vaccines are not important in those parts of the world. The low rate of infectious diseases in industrialized nations is in fact largely the result of the widespread use of vaccination (Figure 5.1). In addition to the well-known example of the smallpox vaccine, which has succeeded in eradicating the disease completely, other vaccines have brought dramatic decreases in the incidence of numerous grave diseases. For example, at the beginning of the twentieth century, diphtheria (caused by the bacterium Corynebacterium diphtheriae) infected about 3000 children yearly out of every million in developed countries. Because diphtheria targets young children in particular, this incidence corresponds to several percent of children of the susceptible age, and nearly one tenth of the infected children died. Now, thanks to a mass immunization program, diphtheria incidence in the United States is less than 0.2 per million, a decrease of more than a thousandfold.
6 - Plant–Microbe Interactions
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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Humans need food in order to survive, and most of the food in the modern world is the product of agriculture. In 1798, Thomas Robert Malthus published the famous essay in which he argued that the human population increases geometrically yet food production can increase only arithmetically. What he could not predict at that time was the contribution of science to the increased production of food. As Malthus foretold, the world population has increased at an almost alarming rate. It took slightly more than 100 years to double from the 1.25 billion in Malthus's day to 2.5 billion in 1950, but the next doubling, to 5 billion, was achieved in less than 40 years, as seen in Figure 6.1. However, the yield of major food crops per unit area (represented by wheat in Figure 6.1) has increased at an even steeper rate, tripling in slightly more than 40 years. One of the major contributing factors to this increase has been the development of high-yielding varieties of crops, for example, semi-dwarf varieties of wheat and rice, which direct a larger portion of their energy to the production of seeds (grains) than to plant growth; this development, which occurred in the 1960s and 1970s, is often called the “Green Revolution.” Thanks to this increase in yield, the world production of food (represented by cereals in Figure 6.1) could more than keep pace with the increase in population, in spite of the steadily decreasing total land area devoted to agricultural production.
13 - Ethanol
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- 01 October 2007, pp 458-486
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The potential quantity of ethanol that could be produced from cellulose is over an order of magnitude larger than that producible from corn. In contrast to the corn-to-ethanol conversion, the cellulose-to ethanol route involves little or no contribution to the greenhouse effect and has a clearly positive net energy balance (five times better). As a result of such considerations, microorganisms that metabolize cellulose have gained prominence in recent years.
– Demain, A. L., Newcomb, M., and Wu, J. H. D. (2005). Cellulase, clostridia, and ethanol. Microbiology and Molecular Biology Reviews, 69, 124–154.The preceding chapter described the major components of plant biomass – cellulose, hemicelluloses, and lignin – and their natural pathways of biodegradation. Many view the sugars locked up in cellulose and the hemicelluloses as an immense storehouse of renewable feedstocks for the fermentative production of fuel alcohol. This chapter begins with a discussion of the conversion of such sugars to ethanol and ends with an assessment of the future impact of fermentation alcohol as a fuel.
In microbiology, fermentation is defined as a metabolic process leading to the generation of ATP and in which degradation products of organic compounds serve as hydrogen donors as well as hydrogen acceptors. Oxygen is not a reactant in fermentation processes. In the words of Louis Pasteur, “[l]a fermentation est la vie sans l'air” – fermentation is life without air. The long history of brewing and wine making has produced highly refined technologies for large-scale fermentation and for the recovery of ethanol.
3 - Production of Proteins in Bacteria and Yeast
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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The human body functions properly only when thousands of bioactive peptides and proteins – hormones, lymphokines, interferons, various enzymes – are produced in precisely regulated amounts, and serious diseases result whenever any of these macromolecules are in short supply. Until 1982, however, the only available pharmaceutical preparations of these peptides and proteins for the treatment of such diseases were obtained from animal sources, and they were sometimes prohibitively expensive. Bioactive proteins and peptides typically occur at low concentrations in animal tissues, so it was difficult to purify significant amounts for medical use. Some important proteins, such as pituitary growth hormone, differ in animals and humans to the extent that a preparation of animal origin is useless for treating humans. Finally, it was extremely difficult to isolate labile macromolecules from human and animal tissues without running some risk that the products might be contaminated by viral particles and viral nucleic acids.
The introduction of recombinant DNA techniques brought about a revolution in the production of these compounds (Chapter 2). It is now possible to clone a DNA segment coding for a protein and introduce the cloned fragment into a suitable microorganism, such as Escherichia coli or the yeast Saccharomyces cerevisiae. The “engineered” microorganism then works as a living factory, producing very large amounts of rare peptides and proteins from the inexpensive ingredients of the culture medium. And with such products obtained in this way from pure cultures of microorganisms, there is no chance of contamination by viruses harmful to humans.
12 - Biomass
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- 01 October 2007, pp 430-457
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We all have dreamed of producing an abundance of useful food, fuel, and chemical products from the cellulose in urban trash and the residues remaining from forestry, agricultural, and food-processing operations. Such processes potentially could: 1) help solve modern waste-disposal problems; 2) diminish pollution of the environment; 3) help alleviate shortages of food and animal feeds; 4) diminish man's dependence on fossil fuels by providing a convenient and renewable source of energy in the form of ethanol; 5) help improve the management of forests and range lands by providing a market for low-quality hardwoods and the other “green junk” that develops on poorly managed lands; 6) aid in the development of life-support systems for deep space and submarine vehicles; and 7) increase the standard of living – especially of those who develop the technology to do the job! At present, all of these aspirations are frustrated by two major features of natural cellulosic materials, crystallinity and lignification.
– Cowling, E. B., and Kirk, T. K. (1976). Properties of cellulose and lignocellulosic materials as substrates for enzymatic conversion processes. Biotechnology and Bioengineering Symposium, 6, 95–123.Biomass can have broader definitions, but in the context of biotechnology, it is generally taken to mean “all organic matter that grows by the photosynthetic conversion of solar energy.” The sun, either directly or indirectly, is the principal source of energy on earth, its power converted to a usable organic form – biomass – by green plants, algae, and photosynthetic bacteria.
8 - Microbial Polysaccharides and Polyesters
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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The use of green plants as industrial factories will potentially become an important component of “green chemistry” efforts. Realization of this technology will likely require metabolic engineering of multi-step pathways and significant use of plant primary metabolites.
– Slater, S., et al. (1999). Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production. Nature Biotechnology, 17, 1011–1016.This chapter deals with two classes of biopolymers: polysaccharides and polyesters. Polysaccharides include some of the most abundant carbon compounds in the biosphere, the plant polysaccharides, cellulose, and hemicelluloses (discussed in Chapter 12), as well as the much less abundant but useful algal polymers, such as agar and carrageenan. Bacteria and fungi also produce many different types of polysaccharides, some in amounts well in excess of 50% of cell dry weight. High molecular weight polyesters are produced exclusively by prokaryotes and for a long time were of interest only to students of microbial physiology.
Polysaccharides are used to modify the flow characteristics of fluids, to stabilize suspensions, to flocculate particles, to encapsulate materials, and to produce emulsions. Among many other examples is the use of polysaccharides as ion-exchange agents, as molecular sieves, and, in aqueous solution, as hosts for hydrophobic molecules. Polysaccharides are used in enhanced oil recovery and as drag-reducing agents for ships.
The discovery that many bacteria synthesize large amounts of biodegradable polyester polymers of high molecular weight, which can be used to manufacture plastics, has aroused considerable interest. There are hundreds of varieties of synthetic plastics; their uses are too many to enumerate. Current annual production of these materials in the United States alone exceeds 30 billion pounds.
Frontmatter
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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1 - Microbial Diversity
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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Molecular phylogenies divide all living organisms into three domains – Bacteria (“true bacteria”), Archaea, and Eukarya (eukaryotes: protists, fungi, plants, animals). The place of viruses (Box 1.1) in the phylogenetic tree of life is uncertain. In this book, we focus on the contributions of Bacteria, Archaea, and Fungi to microbial biotechnology. In so doing, we include organisms from all three domains. We also devote some attention to the uses of viruses as well as to the problems they pose in certain technological contexts.
The domains of Bacteria and Archaea encompass a huge diversity of organisms that differ in their sources of energy, their sources of cell carbon or nitrogen, their metabolic pathways, the end products of their metabolism, and their ability to attack various naturally occurring organic compounds. Different bacteria and archaea have adapted to every available climate and microenvironment on Earth. Halophilic microorganisms grow in brine ponds encrusted with salt, thermophilic microorganisms grow on smoldering coal piles or in volcanic hot springs, and barophilic microorganisms live under enormous pressure in the depths of the seas. Some bacteria are symbionts of plants; other bacteria live as intracellular parasites inside mammalian cells or form stable consortia with other microorganisms. The seemingly limitless diversity of the microorganisms provides an immense pool of raw material for applied microbiology.
The morphological variety of organisms classified as fungi rivals that of the bacteria and archaea. Fungi are particularly effective in colonizing dry wood and are responsible for most of the decomposition of plant materials by secreting powerful extracellular enzymes to degrade biopolymers (proteins, polysaccharides, and lignin).
Plate section
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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Acknowledgments
- Alexander N. Glazer, University of California, Berkeley, Hiroshi Nikaido, University of California, Berkeley
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- Microbial Biotechnology
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