The biosphere has been shaped both by physical events and by interactions with the organisms that occupy it. Among living organisms, prokaryotes are much more metabolically diverse than eukaryotes and can also thrive under a variety of extreme conditions where eukaryotes cannot. This is possible because of the wealth of genes, metabolic pathways and molecular processes that are unique to prokaryotic cells. For this reason, prokaryotes are very important in the cycling of elements, including carbon, nitrogen, sulfur and phosphorus, as well as metals and metalloids such as copper, mercury, selenium, arsenic and chromium. A full understanding of the complex biological phenomena that occur in the biosphere therefore requires a deep knowledge of the unique biological processes that occur in this vast prokaryotic world.

After publication in 1995 of the first full DNA sequence of a free-living bacterium, Haemophilus influenzae, whole genome sequences of hundreds of prokaryotes have now been determined and many others are currently being sequenced (www.genomesonline.org/). Our knowledge of the whole genome profoundly influences all aspects of microbiology. Determination of entire genome sequences, however, is only a first step in fully understanding the properties of an organism and the environment in which the organism lives. The functions encoded by these sequences need to be elucidated to give biochemical, physiological and ecological meaning to the information. Furthermore, sequence analysis indicates that the biological functions of substantial portions of complete genomes are so far unknown.

Some prokaryotes grow by using reduced inorganic compounds as their energy source and CO2 as the carbon source. These are called chemolithotrophs. The electron donors used by chemolithotrophs include nitrogen and sulfur compounds, Fe(II), H2, and CO. The Calvin cycle is the most common CO2 fixation mechanism, and the reductive TCA cycle, acetyl-CoA pathway and 3-hydroxypropionate cycle are found in some chemolithotrophic prokaryotes. Some can use organic compounds as their carbon source while metabolizing an inorganic electron donor. This kind of bacterial metabolism is referred to as mixotrophy.

Reverse electron transport

As with chemoorganotrophs, metabolism of chemolithotrophs requires ATP and NAD(P)H for carbon metabolism and biosynthetic processes. Some of the electron donors used by chemolithotrophs have a redox potential higher than that of NAD(P)+/NAD(P)H (Table 10.1). Electrons from these electron donors are transferred to coenzyme Q or to cytochromes. Some of the electrons are used to generate a proton motive force reducing O2 while the remaining electrons reduce NAD(P)+ to NAD(P)H through a reverse of the electron transport chain. The latter is an uphill reaction and coupled with the consumption of the proton motive force (Figure 10.1). This is referred to as reverse electron transport. In most cases, electron donors with a redox potential lower than NAD(P)+/NAD(P)H are oxidized and this is coupled with the reduction of coenzyme Q or cytochromes for the efficient utilization of the electron donors at low concentration.

Photosynthetic organisms use light energy to fuel their biosynthetic processes. Oxygen is generated in oxygenic photosynthesis where water is used as the electron donor. In anoxygenic photosynthesis, organic or sulfur compounds are used as electron donors. Plants, algae and cyanobacteria carry out oxygenic photosynthesis, whereas the photosynthetic bacteria obtain energy from anoxygenic photosynthesis. Aerobic anoxygenic phototrophic bacteria use light energy in a similar way as the purple bacteria, and are a group of photosynthetic bacteria that grow under aerobic conditions.

Phototrophic organisms have a photosynthetic apparatus consisting of a reaction centre intimately associated with antenna molecules (or a light-harvesting complex). The antenna molecules and the reaction centre absorb light energy. The energy is concentrated at the reaction centre that is activated and initiates light-driven electron transport. Halophilic archaea convert light energy through a photophosphorylation process.

Photosynthetic microorganisms

Microorganisms utilizing light energy include eukaryotic algae, and cyanobacteria, photosynthetic bacteria and aerobic anoxygenic phototrophic bacteria among the prokaryotes. The halophilic archaea synthesize ATP through photophosphorylation, but they are not considered to be photosynthetic organisms since they lack photosynthetic pigments.

Algae and cyanobacteria have similar photosynthetic processes, using chlorophyll, as plants. However, cyanobacteria are members of the proteobacteria according to their cell structure and ribosomal RNA sequences. Photosynthetic bacteria are different from other photosynthetic organisms. They have different photosynthetic pigments and do not use water as their electron donor. Some of them can grow chemoorganotrophically in the dark.

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.

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.

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.

Microorganisms 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.