INTRODUCTION

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This centennial view is, of course, a personal one; as students, each of us comes to identify and admire certain scientific heroes/heroines and their experiments as we traverse the microbial terrain. Experiments that we perform today have roots. So, in this brief view from what we now believe to be the lofty vantage point of 1999, I would like to share with you some discoveries that I believe were pivotal to an understanding of anaerobic life.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

	BEGINNING OF UNDERSTANDING

Forty-two years before the founding of the American Society for Microbiology (ASM), Pasteur, Professor of Chemistry at the University of Lille, published a paper which may be viewed as the definitive work that marks the birth of microbiology (9, 21). In this 1857 paper he presented the results of his studies on the conversion of sugar to lactic acid (18). His conclusions were direct challenges to major chemists of the day, Liebig, Wohler, and Berzelius, who opposed the views of biologists that living cells were responsible for fermentation. Pasteur concluded that the cell was the "ferment" and that multiplying cells in the lactic fermentation possessed a distinct morphology. These cells, when removed and added to fresh sugar, produced a rapid fermentation. Pasteur drove home the point that fermentation occurs only in the presence of living cells and is a necessary part of their life. Later he would compose a short sentence that said it all: "Fermentation is the consequence of life without air." He observed that natural fermentations could be mixed, but it became clear to him that each type of fermentation was carried out by a distinct cell type.

So the birth of microbiology involved anaerobic life! Although Leewenhoek's first observations on microbes were published in 1684, for about 175 years after this event the tools for turning his discoveries into science were not adequate to the task, but by the mid-19th century the tools of organic chemistry were equal to the task of determining the molecular structure of a substrate as well as the structures of the products produced by anaerobic life; so with the development of pure-culture techniques, study of the physiology of anaerobes became a science at the forefront of microbiology. Thus, the fermentation balance in which the atoms of the substrate could be accounted for in the products became a standard tool for study of anaerobic physiology and led to controlled industrial processes in food and chemical industries, for example, the butanol-acetone fermentation by clostridia. In the period from 1916 until the mid-1950s the great industrial demand for these solvents was met by the clostridial fermentation of starch. The Fernbach-Weizmann patent on Clostridium acetobutylicum, which involved the breakpoint where butyric and acetic acids are reprocessed by the cell to form solvents, made Weizmann a wealthy man. He was a Zionist and used his wealth to advance this cause; the state of Israel owes much to anaerobic life!

Because fermentation is inefficient, with most of the energy contained in the substrate remaining in the products, anaerobes provided wonderful tools for studying physiology. Fermentation balances of growing cultures were soon extended to washed-cell suspensions under nongrowing conditions. With this technique, the mysteries of growth could be divorced from metabolism, and educated guesses could be made about intermediates in the conversion of substrate to products. Study of the enzymology of microbial metabolism lagged behind biochemical studies with animal tissues such as liver because the technology for rupturing the bacterial cell wall was slow to develop. Study of the propionic fermentation by H. G. Wood, a graduate student, and C. H. Werkman led to the discovery of heterotrophic CO₂ fixation (30). They had added calcium carbonate to the medium to neutralize the acids produced from the substrate, glycerol; they found more carbon in the fermentation products than in the substrate used. Here was something new and different, but at the time photosynthetic organisms were the only organisms known to fix CO₂, and the scientific community reacted unkindly. van Niel, the world authority on propionic bacteria at the time, was most skeptical; he missed this discovery because he was especially careful to include only the substrate to be tested in his media. To prove his results to the scientific community, Wood and fellow graduate students built their own mass spectrometer as well as their own multistory diffusion column to enrich for ¹³CH₄, which they oxidized to ¹³CO₂. They showed that the ¹³C label was found in a carboxyl group of succinate (31). One of the legacies of World War II was the radioactive isotope ¹⁴C, which provided a new tool for the study of microbial physiology and metabolism, one especially valuable for elucidating carbohydrate fermentative pathways in anaerobes. With the development of improved microbial torture chambers such as the ultrasonic probe and the Hughes press at midcentury (and much later the French press), the bacterial cell wall could be broken easily and the era of cell extracts was born. Microbial biochemists were free at last to pursue the enzymology of the microbial cell; the catalysts of anaerobic life were at hand.

	NITROGEN

Although observations had been made previously which suggested that microbes could use nitrogen in the atmosphere, Winogradsky performed a definitive experiment in 1895 that conclusively changed concepts about the abilities of anaerobes (29). To water that contained only sugar and no fixed nitrogen, he added a crumb of soil. After a few days a scum of microbes formed on the surface, and later bubbles arose from the soil; soon the odor of butyric acid could be detected in the flask. The anaerobe was isolated in pure culture and named Clostridium pastorianum. Careful studies on the amount of sugar fermented and the amount cell nitrogen formed clearly established the fixation of nitrogen by anaerobes. Subsequent decades of research would show that only microbes possess the unique ability to activate and fix dinitrogen from the vast resources of the atmosphere through the fascinating biochemistry of nitrogenase. In this experiment, Winogradsky also discovered one of nature's strategies: in a habitat where nutrients are available, anaerobic microbes can grow because aerobes deplete the available oxygen. The surface growth contained an aerobic nitrogen fixer, Azotobacter.

Although the anaerobic metabolism of sugars took front stage early, observations that pure cultures of organisms could also grow anaerobically on single-amino-acid nitrogenous compounds such as glutamate, which was converted to butyrate, formate, and ammonia, were made in the early 1900s. But it was Stickland who opened the modern era of study of anaerobic metabolism of nitrogenous compounds in 1934 (24). Anyone who has ever grown Clostridium sporogenes cannot escape the impact of its dynamic biochemistry! By use of cell suspensions Stickland made sense out of one of nature's strategies of anaerobic amino acid metabolism by showing that whereas single amino acids could not be metabolized, certain amino acids when added in pairs were rapidly metabolized, one amino acid being oxidized (the electron donor) and the second being reduced (the electron acceptor). Thus, for example, alanine was oxidized to carbon dioxide and ammonia, and glycine (2 mol) was reduced to acetic acid and ammonia. In the next decade these experiments were extended by others who showed that at least 15 species of clostridia could grow by Stickland reactions. The gateway to knowledge of the anaerobic metabolism of nitrogenous compounds was greatly widened by H. A. Barker's laboratory in the 1940s and 1950s (4, 5) with the isolation of different species and strains of anaerobes that could metabolize amino acids, purines, and pyrimidines. By use of ¹⁴C-labelled substrates, the fate of each carbon atom in the substrate could be determined by chemical degradation of each product of fermentation. Similarly, specific nitrogen atoms in the substrate could be labelled with ¹⁵N and the origin of nitrogen atoms in products could be ascertained.

	ANOXIC PHOTOSYNTHESIS

The paper that made a science out of the study of anoxic photosynthesis was the 1931 publication by van Niel on the purple and the green sulfur bacteria (25). After Englemann's intriguing 1883 study (10), where pigmented, motile bacterial cells responded to a spectrum of light projected on a microscope slide by collecting in discrete bands according to the absorption bands of their pigments, studies on the phototrophic bacteria fell into confusion for decades. Were these organisms really photosynthetic? Was sulfide oxidized as an energy source? What was the role of light? Why was oxygen so difficult to detect as a product of photosynthesis? van Niel performed careful stoichiometric studies on selected strains of purple and of green sulfur bacteria that he had isolated from nature. For the purple bacteria he showed that the light-dependent conversion of 1 mol of sulfide to 1 mol of sulfate was coupled to the fixation of 2 mol of carbon dioxide; for the green sulfur bacteria the conversion of 2 mol of sulfide to 2 mol of elemental sulfur was coupled to the fixation of 1 mol of carbon dioxide. Anoxic photosynthesis by the purple and the green sulfur bacteria was real; these organisms could be cultured in a defined inorganic medium, and sulfide was the light-dependent source of reducing power for the reduction of carbon dioxide; so oxygen was not a product of photosynthesis by these organisms. Although a vast effort was invested in study of the mechanism of photosynthesis in plants, algae, cyanobacteria, and bacteria, it was a member of the purple nonsulfur phototrophs, Rhodopseudomonas viridis, isolated in Pfennig's laboratory that in the hands of Michel's group provided the crystalline photosynthetic reaction center for X-ray analysis (16). Recently, Widdel and coworkers made the discovery that ferrous ions may serve as the electron donors for certain purple nonsulfur phototrophs (27)! At last there is an explanation for the banded iron oxide geological formations which were deposited when the earth's atmosphere was anoxic: anoxic phototrophs probably did it.

	ISOLATION TECHNIQUES

In the first half of this century, the Delft school, founded by Beijerinck, played an important role in widening concepts about the diversity of anaerobes. An enrichment technique for anaerobes involved the use of a small bottle with a ground-glass stopper. By use of a selective substrate and nutrients in a completely filled bottle, the population of an anaerobe that could outgrow other microbes became enriched under defined conditions. Subculture transfers in the same medium produced an overwhelming population that, when used for isolation procedures, usually by serial dilution in agar deeps, produced isolate colonies and subsequent isolation of pure cultures. The enrichment technique became widely used, and in the hands of H. A. Barker opened new vistas (4).

When Hungate began to study anaerobes in the termite hindgut and rumen, he realized that the anaerobes therein already represented a natural, stable enrichment; a way to quantitatively cultivate them was needed. He developed procedures for preparing prereduced media at very low reducing potential and for maintaining these conditions during aseptic culture and transfer of an organism (12). It is difficult to overestimate the impact that the Hungate technique as well as Hungate's students had on our knowledge of fastidious anaerobes. The work of Holdeman et al. (11) convinced reluctant medical microbiologists of its importance in isolating anaerobes from "sterile abscesses." Later, the Balch extension (2) of the Hungate technique, which used a pressurized atmosphere in vials or tubes, was widely accepted as a more user friendly procedure and in the hands of Stetter and Zillig opened up a new world of extreme anaerobic thermophiles for study (23). However, preparation of the Hungate agar roll tube demanded considerable expertise and had severe limitations as a tool for genetic analysis of anaerobes; for this purpose the petri plate is unsurpassed. So anaerobes played no role in the molecular and genetic revolution of post-mid-century microbiology. Even in this year of ASM's centennial, knowledge of the genetics of strict anaerobes lags far behind that of other organisms, and the reason is not hard to find: as one of my colleagues told me years ago"When you can plate them on the lab bench, let me know." Aranki and Freter (1) made a major contribution to the technology of cultivating strict anaerobes by developing an anaerobic chamber equipped with catalytic oxygen scrubbers and an air lock. The chamber could be filled with any desired gas mixture, and standard microbiological techniques could be carried out therein. Many typical anaerobes could be grown on petri plates incubated within the chamber. However, for serious anaerobes such as the methanoarchaea, which require a reducing potential below 330 mV, the need for more-stringent anaerobic conditions may be met by use of anaerobic jars or an intrachamber incubator of special design (15).

	ANAEROBIC RESPIRATION

The first clue that respiration could be an anaerobic way of life was obtained in 1895 by Beijerinck, who showed that sulfate could be reduced to sulfide in sediments (6). In 1931, Stephenson and Stickland isolated a sulfate reducer from river mud and discovered that it possessed an unusual microbial enzyme, hydrogenase, which catalyzed the reversible oxidation-reduction of hydrogen (22). The importance of this enzyme in anaerobic life is now legend, for it allows fermentative anaerobes to use protons in water as electron acceptors, producing molecular hydrogen, which is an ideal substrate for anaerobic respiration by methanogens, acetogens, and sulfate reducers. Bryant and coworkers (7) used the hydrogenase of Desulfovibrio to extend our knowledge of anaerobe interactions by showing that this enzyme allowed the organism to grow in the absence of sulfate in coculture with a methanogen, which used the hydrogen produced to reduce carbon dioxide to methane. Pfennig and Biebl (19) discovered the importance of S⁰ as an electron acceptor in anaerobic respiration by isolation of an organism, Desulfuromonas acetoxidans that could anaerobically oxidize acetate to carbon dioxide and reduce S⁰ to H₂S; determining how this could be done became a challenge to biochemists. S⁰ is rarely found in sediments because it is readily reduced by a great variety of organisms. The narrow field of study of sulfate and sulfur reduction was expanded enormously to encompass extreme microbial diversity by the discoveries of Widdel (26).

The "S" organism isolated by Bryant et al. from the culture known as Methanobacillus omelianskii was the first obligate proton reducer to be described (8). Resolution of the culture into a methanogen that grew on hydrogen and carbon dioxide and the S organism, which oxidized ethanol to acetate and hydrogen, opened up a new concept, interspecies hydrogen transfer. What set this process apart from interspecies transfers such as lactate or formate was the fact that the removal of hydrogen to very low concentrations (10⁵ atm) allowed the anaerobic oxidation of products such as propionate, butyrate, and higher fatty acids to become thermodynamically feasible. Thus, nature's way out of the fermentation dead end is to employ anaerobic respiration to pull biodegradation. Anaerobic protozoa that lack mitochondria maintain a high population of intracellular methanogens which remove the hydrogen produced by fermentation (17).

A major breakthrough in the study of anaerobic respiration was made by Söhngen in 1910, who though working with enrichment cultures, defined some of the reactions of methanogenesis and showed that when hydrogen and carbon dioxide were provided, 4 mol of hydrogen was required to reduce 1 mol of carbon dioxide to methane (20). It would be many decades before conclusive proof of this equation was obtained. H. A. Barker's laboratory (4) made a science out of the physiology of methanogenesis with pure cultures. Among their many findings was the elegant proof that a methyl group can be reduced intact to methane and essentially serves as the terminal electron acceptor for reduction to methane. Isolation of the first thermophilic methanogen, Methanobacterium thermoautotrophicum, by Zeikus (32) was an achievement that provided an ideal tool for biochemical analysis of methanogenesis in several laboratories; the organism was easily mass cultured, and its enzymes could be fractionated at room temperature! John Leigh isolated the first extreme thermophilic methanogen, Methanococcus jannaschii, from a deep marine hydrothermal vent (13).

Acetogenesis by hydrogen oxidation and carbon dioxide reduction was not considered to be an important component of anaerobic microbial ecology until the last third of this century. Although Wieringa in 1936 had isolated the first acetogen, Clostridium aceticum, that could grow on hydrogen and acetic acid (28), it was the isolation of Acetobacterium woodii by Balch et al. in 1977 (3) that pointed to the importance of this form of anaerobic respiration. The recent finding by Leadbetter et al. that spirochetes are important acetogens in the termite hindgut extends our knowledge of the importance of acetogenesis (14). In anaerobic freshwater environments methanogenesis and acetogenesis are major hydrogen sinks, whereas in marine environments, sulfate reduction predominates.

	COMMENTS

On this centennial of ASM, microbiology is in the midst of the great age of molecular geography; paddling upstream and downstream explorers are busily describing the locations of genes on chromosomes. We are at the beginning of the age of genomics. Complete genome sequences of microbes are being reported with increasing frequency; there has never been a more exciting time for the study of phylogeny and evolution. Recently, the genomes of several thermophilic anaerobic archaea have been sequenced, and only 40% of each genome represents known genes. For microbes most of these genes code for catalysts, and there is much to be learned about new catalysts, how they work, and how each anaerobe regulates them and responds to its environment, including that poison oxygen. Study of obligate anaerobes continually provides that extra challenge for all procedures, but for some of us, challenge is what it is all about.