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).

Microorganisms play a fundamental role in the global recycling of matter by releasing carbon, nitrogen, phosphorus, and sulfur from an immense variety of complex organic compounds for reuse by living organisms and in generating energy. The ability of the life support systems of the planet to function depends on the activities of these organisms. The spectacular metabolic versatility of prokaryotes and fungi is displayed in the major areas of environmental microbiology examined in this chapter. We examine sewage and wastewater treatment and the degradation of xenobiotics and petrochemicals, and end with a consideration of biomining. Microorganisms, primarily chemolithotrophic prokaryotes, are effective in the bioleaching of low-grade ores and in the removal of toxic heavy metals from the environment.

DEGRADATIVE CAPABILITIES OF MICROORGANISMS AND ORIGINS OF ORGANIC COMPOUNDS

Microorganisms excel at using organic substances, natural or synthetic, as sources of nutrients and energy. These include certain synthetic compounds – detergents, solvents (trichloroethane, toluene, xylenes), and transformer fluids (polychlorobiphenyls) – that seem very different from any natural compounds such an organism would be likely to encounter. The explanation for this remarkable range of degradative abilities is that prior to the advent of humans, microorganisms had already coexisted for billions of years with an immense variety of organic compounds.

What is ivermectin? Chemically, and technically, ivermectin is aligned with a family of “16-membered macrocyclic lactones with a disaccharide attached at the carbon-13 position” (Campbell, 1989). Scientists at Merck, Sharp & Dohme Research Laboratories (MSDRL) in Rahway, New Jersey, initially isolated it. These molecules most closely resemble milbemycins, discovered in Japan and first thought to have toxic effects just for mites. It is now known that these drugs are effective against certain parasitic nematodes as well. Biologically, ivermectin is a broad-spectrum anthelmintic, acaricide, and insecticide.

For me, the story of this drug's discovery is an interesting tale, for a number of reasons. First, one of the folks deeply involved in it is an old friend of mine, Bill Campbell. I have known Bill for more than thirty years. Second, before I began writing this essay, I knew very little about this sort of applied research. So, I had to sit down and do some serious reading. Quite honestly, I found it to be rather intriguing. Third, I was told by a colleague of mine, who should know, that ivermectin is considered by many in the agricultural industry almost as a miracle drug, primarily because of its toxic breadth for both ecto- and endoparasitic organisms.

As I have written elsewhere in this tome, there were two important contributions that allowed those working in the field of parasitology to make breakthrough discoveries in the nineteenth and early twentieth centuries. The first was the microscope. It was the Dutch rug trader, Antonie van Leeuwenhoek, who began to develop and refine this technology in the seventeenth century. Robert Hooke, an early British microscopist and a contemporary of the resourceful Dutchman, created what was ultimately to evolve into one of the most powerful of all biological concepts, in fact, one that still is being cultivated today. While looking through one of his primitive scopes one day, Hooke noted that the structure of a piece of cork he had sliced was divided internally into what he called “cellulae”. With this observation, the cell theory was borne.

The contribution of van Leeuwenhoek provided the way for technology to eventually take us inside cells and build on Hooke's observation. The cell theory, along with Darwin's evolutionary theory, unquestionably did more to alter the biological landscape than any other conceptualization. There was, however, a widely held idea that had to be purged before significant biological research could progress.

Even though they are buried several feet below the desert's scorched sand for most of the year, spadefoot toads, Scaphiopus couchii, definitely feel the rain when it comes. Remarkably, they feel it even though they cannot see it and they are not touched by it.

The beginning of Richard Tinsley's interest in the spadefoot toad, and Pseudodiplorchis americanus, a monogenetic trematode infecting the toad's urinary bladder, began in the late 1960s while he working on his Ph. D. at the University of Leeds in England. It was then, very early in his career, that he came across a publication written in 1940 for an obscure journal (The Wassman Collector) by L. O. Rodgers and Bob Kuntz. In this one and a half paged paper, Rodgers and Kuntz had described P. americanus, but nothing about the biology of the extraordinary interaction between this parasite and its host. Richard said, however, holding a now yellowed Xerox copy of the paper in his Bristol office, “There is enough in here [in the Rodgers/Kuntz paper] to show that there is an exciting story to be told. First of all it's a monogenean. Then, with my interests in this group of vertebrates, I knew that its host was completely terrestrial. But, I knew that the toad returned to water for a very short time each year.

INTRODUCTION

Many xenobiotic chemicals introduced into the environment for agricultural and industrial use are nitro-substituted aromatics. Nitro groups in the aromatic ring are often implicated as the cause of the persistence and toxicity of such compounds. Nitroaromatic compounds enter soil, water, and food by several routes, such as use of pesticides, plastics, pharmaceuticals, landfill dumping of industrial wastes, and the military use of explosives. The nitroaromatic compound, trinitrotoluene (TNT) is introduced into soil and water ecosystems mainly by military activities such as the manufacture, loading, and disposal of explosives and propellants. This contamination problem may increase in future because of the demilitarization and disposal of unwanted weapons systems.

Biotransformation of TNT and other nitroaromatics by aerobic bacteria in the laboratory has been reported frequently (Boopathy et al., 1994a; 1994b; Dickel and Knackmuss, 1991; Duque et al., 1993; Funk et al., 1993; McCormick et al., 1976; 1981; Nishino and Spain, 1993; Spain and Gibson, 1991; Zeyer and Kearney, 1984). Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp. has been reported to occur via 4-methyl-5-nitrocatechol in a dioxygenase-mediated reaction (Spanggord et al., 1991). Duque et al. (1993) successfully constructed a Pseudomonas hybrid strain that mineralized TNT. White rot fungus has been shown to mineralize radiolabelled TNT (Fernando et al., 1990). The work of Spiker et al. (1992) showed that Phanerochaete chrysosporium is not a good candidate for bioremediation of TNT contaminated sites containing high concentration of explosives because of its high sensitivity to contaminants.

Obviously, the ‘unholy trinity’ mentioned in the title of this essay is a play on words. However, when I make the connection between the so-called ‘unholy trinity’ and the geohelminths, any parasitologist would know the three parasites to which I refer. These would include Ascaris lumbricoides, Trichuris trichiura, and the hookworms. Generally, the two species of human hookworms, Ancylostoma duodenale and Necator americanus, are lumped together and considered as one, mainly because their biology is so similar, and because the disease they cause is so nearly the same. While their geographic distributions are essentially sympatric in today's world, A. duodenale probably had an Asian origin. Charles Wardell Stiles of the U.S. Bureau of Animal Industry in Beltsville, Maryland, first described Necator americanus in 1906, but its origins are not of the New World. It most likely evolved in Africa and was imported into the western hemisphere, along with malaria, yellow fever, schistosomiasis, and several other diseases, during the slave trade.

Estimates regarding the numbers of people infected with the ‘unholy trinity’ vary, but all would agree that these parasites, collectively, have perhaps the greatest impact on DALYs (Disability-Adjusted Life Years) on a worldwide basis when it comes to the helminth parasites.

INTRODUCTION

Sulphate-reducing bacteria (SRB), or more generally speaking sulphate-reducing prokaryotes (SRP), are terminal oxidizers in the natural recycling of bio-organic compounds to CO2 in anoxic environments, in particular in marine sediments. SRP play this geochemically important role because they make use of a globally abundant electron acceptor, sulphate (in seawater up to 28 mM), and possess numerous degradative (oxidative) capacities with respect to electron donors. The study of the degradative potentials of SRP via de novo enrichment (including direct counting) and isolation from natural samples has been of interest over some decades and formed the basis for our knowledge of the phylogenetic diversity of SRP. Common electron donors and carbon sources of SRP are the low-molecular mass products from the primary anaerobic (fermentative) breakdown of polysaccharides, proteins, lipids and other substances of dead biomass. Several of the involved degradative capacities, for instance complete oxidation or the channelling of branched-chain fatty acids or aromatic compounds into the central metabolism, require special enzymatic reactions (for overview see Rabus et al., 2000) which are not encountered in fermentative bacteria. The study of such and other metabolic capacities in SRP has led to the recognition of principles of general importance or heuristic value in our understanding of the biochemistry and energetics of anaerobes.

A chemical class of organic substrates which have become of interest relatively recently in the study of SRP (and other anaerobes) are hydrocarbons, in particular those from crude oil (petroleum).

INTRODUCTION

The unique ability of the anaerobic sulphate-reducing bacteria (SRB) to respire sulphate provides access to niches that may be restricted from other bacteria. However, environmental niches are by definition constantly in flux. Thus, for scientists to reach a level of understanding that will allow prediction and/or control of the activities of the SRB, it is necessary to learn how the bacteria respond to changes in environmental parameters; such as, nutrient availability, presence of toxic substances, altered salt concentrations, temperature fluctuations, and a myriad of other variables. With the recent sequencing of a number of SRB (Klenk et al., 1997; Heidelberg et al., 2004; Rabus et al., 2004), the available proteins and regulatory sites of the bacteria have been revealed. Nevertheless, a significant percentage of the predicted open reading frames (ORFs) encode hypothetical or conserved hypothetical proteins for which functions remain obscure. Much work is yet to be done to elucidate the interplay of functions that allow the SRB to survive or even flourish in the changing conditions prevailing in their environment. Here we discuss preliminary transcriptional analyses of the responses of Desulfovibrio vulgaris Hildenborough to a number of environmental stressors.

A description of optimal growth conditions for D. vulgaris Hildenborough is derived from its early characterization. This strain is a mesophilic Gram-negative anaerobe which was isolated in 1946 from Wealden Clay near Hildenborough, Kent, in the United Kingdom (Postgate, 1984).

INTRODUCTION

Early in 1886, it was demonstrated that the addition of gypsum (CaSO4ċ2H2O) to anaerobic mud enrichments containing cellulose led to the production of the malodorous gas, hydrogen sulphide (Hoppe-Seyler, 1886). Soon after, Beijerinck first provided evidence of a microorganism reducing sulphate into sulphide, named as Spirillum desulphuricans (Beijerinck, 1895), which was the first sulphate-reducing bacterium (SRB) isolated in the world. As pointed out by Voordouw (1995), Beijerinck had already addressed, at the end of the nineteenth century, questions to the scientific community with regard to the metabolism and ecological distribution of the SRB, which are still nowadays themes of debate. SRB were first believed to use a limited range of substrates as energy sources (e.g. hydrogen, lactate, ethanol, etc. …), but recent biochemical and microbiological studies have greatly extended the range of electron donors and electron acceptors known to be used by SRB (Fauque et al., 1991; Widdel, 1988). Indeed the latter may have an autotrophic, lithoautotrophic, heterotrophic, or respiration type of life under anaerobiosis and their possible microaerophilic nature has been discussed in the literature (Fauque and Ollivier, 2004). Besides their common ability to use sulphate as terminal electron acceptor, many of them were shown to utilize other mineral sulphur compounds, including elemental sulphur, thiosulphate, sulphite, polythionates and polysulphide (Le Faou et al., 1990). In addition, SRB have been demonstrated to reduce a wide range of heavy metals and radionuclides including Fe(III), and U(VI).

Ross, Bailey, Hairston, Bradley, Michel, McDonald, are all names that will be recognized immediately by any epidemiologist/modeler working on eukaryotic parasites. Almost all of these people studied and published prior to 1971, beginning with Sir Ronald Ross who, I am sorry to say, rather feebly attempted to predict the prevalence of malaria in mathematical terms. Most investigators in this area would agree that the seminal publications for the modern epidemiology of helminth parasites were those of Crofton (1971a, b). His efforts were truly the ‘seeds’ for what followed. Harry Crofton, most unfortunately, died very young, not long in fact after publishing his two papers in 1971. I have often wondered what would have followed had he lived a longer life.

Early in my career, I had the pleasure of spending almost an entire year (1971–72) at Imperial College in London with Desmond Smyth, who was trying to teach me the intricacies of in vitro culture. He succeeded, but in doing so, I also learned that this sort of research is tedious, very expensive, and, frankly (for me, at least), terribly boring. It was about that time that I turned my complete attention to ecological pursuits.

Jim Oliver began work in 1988 on Borrelia burgdoferi, the etiological agent of Lyme disease, and has been a leading figure in its study since that time. According to Jim, “The primary symptom of Lyme disease is a bulls-eye lesion on the skin that continues to expand.” This lesion is not just a localized hypersensitivity “reaction like you would see with the bite of a mosquito or a chigger. Clinically, this is the single-most diagnostic feature of infection with the spirochete that causes the disease.” I then asked if this is due to inflammation, or an indication of bacteria within the skin? Jim responded that he was not certain, but probably both are involved. “I say that because if I want to isolate spirochetes, a biopsy at the margin of the skin lesion would give me the best chance for success. The spreading of the lesion is referred to as erythema migrans.” This characteristic of the disease was initially described in Europe in the late nineteenth century. At the time, it was not associated with any other symptoms of the disease, or with an etiological agent. The disease in North America was first noted in Wisconsin in 1970. Subsequently, there was an outbreak of the disease in Old Lyme (hence Lyme disease) and surrounding counties in Connecticut in the mid 1970s. It seems that a group of children presented juvenile arthritic symptoms.

INTRODUCTION

Sulphate-reducing bacteria (SRB) are those prokaryotic microorganisms, both bacteria and archaea, that can use sulphate as the terminal electron acceptor in their energy metabolism, i.e. that are capable of dissimilatory sulphate reduction. Most of the SRB described to date belong to one of the four following phylogenetic lineages (with some examples of genera): (i) the mesophilic δ-proteobacteria with the genera Desulfovibrio, Desulfobacterium, Desulfobacter, and Desulfobulbus; (ii) the thermophilic Gram-negative bacteria with the genus Thermodesulfovibrio; (iii) the Gram-positive bacteria with the genus Desulfotomaculum; and (iv) the Euryarchaeota with the genus Archaeoglobus (Castro et al., 2000). A fifth lineage, the Thermodesulfobiaceae, has been described recently (Mori et al., 2003).

Many SRB are versatile in that they can use electron acceptors other than sulphate for anaerobic respiration. These include elemental sulphur (Bottcher et al., 2005; Finster et al., 1998), fumarate (Tomei et al., 1995), nitrate (Krekeler and Cypionka, 1995), dimethylsulfoxide (Jonkers et al., 1996), Mn(IV) (Myers and Nealson, 1988) and Fe(III) (Lovley et al., 1993; 2004). Some SRB are even capable of aerobic respiration (Dannenberg et al., 1992; Lemos et al., 2001) although this process appears not to sustain growth, and probably provides these organisms only with energy for maintenance. Since dissimilatory sulphate reduction is inhibited under oxic conditions, SRB can grow at the expense of sulphate reduction only in the complete absence of molecular oxygen. SRB are thus considered to be strictly anaerobic microorganisms and are mainly found in sulphate-rich anoxic habitats (Cypionka, 2000; Fareleira et al., 2003; Sass et al., 1992).