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1.8Biodiversity and Microorganisms

Although there are three aspects of biodiversity, namely environment (ecosystem), species, and genes, genetic diversity determines biodiversity at the molecular level. Carl Woese schematized phylogenetic relationships by ribosomal RNA sequencing for genetic diversity related to functions common to all organisms (see Fig. 1-4B). Considering the biological classification, although the Monera kingdom is only marginally included in the five-kingdom theory, it occupies the two domains of Bacteria and Archaea classified as per the three-domain theory. The five-kingdom theory focuses on species diversity, while the three-domain theory focuses on genetic diversity. Because microbial cells are less prominent as a result of their microscopic size, it is hard to appreciate their high degree of genetic diversity, that is diversity of the molecular materials that compose cells, compared with that of animals and plants. Even when we perceive diversity in plants and animals, we are not noticing diversity in molecular materials and cells. Diversity in the forms and mechanisms of multicellular organisms is actually determined by diversity in intercellular interactions. From this aspect, diversity of microorganisms is difficult to observe.

All animal cells derive energy from the glycolytic pathway or aerobic respiration. However, microorganisms are not limited to these two methods of deriving energy. With regard to respiration alone, several microorganism species perform respiration using a range of electron acceptors in addition to molecular oxygen, such as nitrate, sulfate, and ferrous ions, or anaerobic respiration. When we consider microorganisms living in environments with temperatures ranging from below the freezing point to those exceeding 100°C, the diversity of microorganisms becomes obvious at the molecular and cellular level (the individual level as well).

Although there are almost one million species of insects, less than 10,000 species of bacteria have been recognized. This is because the criteria for species are not the same. As stated in section 6, the biological species concept for animals and plants is based on the breeding potential and does not include asexual reproduction of prokaryotes. Species of prokaryotes can be examined by similarity in DNA and morphological, physiological, and biochemical traits. A new species is acknowledged when these characteristics clearly separate an organism from other groups. In addition, in case of microorganisms, the common, non-specific search for new species is not performed because they are highly diverse. Isolation of new strains with physiological or pathological properties is attempted, and a new species is formally registered only after it has been isolated. Thus, the number of registered species of microorganisms, in particular, bacteria, is extremely low, despite their diversity. The total number of species of bacteria on the planet is unknown. The phylogenetic diagram of microorganisms is almost completely blank. Another reason is because isolating or culturing all species of bacteria is difficult. Since most bacteria living in the natural environment do not form colonies, bacteria that can be induced to form colonies have only been isolated (see Column at the bottom).

Perspectives on biodiversity are diverse, reflecting diversity of our understanding. While it is easy to overlook the existence of microorganisms when analyzing species diversity, microorganisms contribute significantly to overall genetic diversity of living things. It is important to recognize this difference in perspectives when considering biodiversity.

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Microorganisms from the Human Perspective

In the latter half of the 19th century, the chemist Louis Pasteur indicated that activities of microorganisms, i.e., organisms so small that they could only be observed through a microscope, are responsible for decomposition and fermentation of food, laying the foundation of biochemistry. Furthermore, Pasteur developed sterilization and is renowned for the concept of pasteurization of milk at 65°C for 30 min. Based on traditional food technology including that of alcohol, vinegar, cheese, yogurt, and bread and that of miso and soy sauce in Japan, fermentation processes were developed for producing antibiotics and biologically active substances during the mid-20th century. Amino acid fermentation of glutamic acid and lysine was started in Japan within the last 50 years.

The medical scientist Robert Koch laid the foundation of pure isolation and culture methods for microorganisms. The formulation of research methods for infectious agents led to the discovery of the pathogenic bacteria causing anthrax, tuberculosis, and cholera. By the end of the 19th century, numerous pathogenic bacteria had been discovered, including those by Shibasaburō Kitasato and Kiyoshi Shiga, and research also progressed on serotherapy and vaccine therapy. The development of antibiotics including penicillin from fungi and streptomycin from actinomycetes renewed the battle between humans and infectious diseases, while the new problem of drug-resistant bacteria concurrently emerged.

Thus, microorganisms were recognized for activities beneficial and harmful to humans. This has influenced nomenclature and taxonomy. For example, lactic acid bacteria and nitrogen-fixing bacteria do not have biological classification, but generic names for each species of bacteria that reflect their specific bioactivity. When unicellular microorganisms are studied as cellular models for complex animals and plants because of their universal properties as organisms, the diversity as organisms can be ignored.

As humans, we are naturally interested in ourselves as organisms and other organisms deeply related to human survival. This book is also structured considering this perspective. However, while studying living things, it is important to separate ourselves from human values, and try to be objective. In addition to harming or aiding humans, many microorganisms exist in symbiotic relationships with humans. One hundred trillion bacteria are replenished daily through reproduction in human intestines. However, there are numerous microorganisms in the environment that neither directly harm nor benefit humans. For example, billions of bacteria can be observed in 1 g of soil and millions more can be observed in 1 mL of apparently transparent seawater. There are certainly more varied and diverse microbial resources than those we have currently recognized because only a small proportion of the world of microorganisms has been explored.

In ecology, microorganisms are positioned as decomposers. In terms of practical application, microorganisms are also used in the treatment of marine and soil pollution. From the aspects of animals, plants, and humans, microorganisms certainly degrade dead bodies and pollutants. However, these activities are very different from the perspective of microorganisms. For example, photosynthetic productivity of marine cyanobacteria is equal or greater than the primary productivity of terrestrial plants, and only microorganisms can incorporate atmospheric nitrogen into their body composition. In addition to being decomposers in the ecosystem, microorganisms play roles as primary producers.

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Microorganisms in Nature

Many native microorganisms are difficult to isolate and culture, despite the evidence of their existence. In the pure isolation method for microorganisms proposed by Robert Koch, the sample was diluted, added to a plate with culture medium (initially, potato slices and gelatin medium were used, and later, agar medium was developed), and incubated. Isolated colonies that grew on the medium are selected, cultured, and then can be subjected to further analysis. A single colony is a result of the exponential growth of a single cell and thus is a genetically homogeneous population of hundreds of millions of individuals (clones). On the basis of the number of colonies cultured, the number of cells that can reproduce in the original sample can be estimated. Therefore, the viable cell count is equivalent to the number of colonies formed, and microbiology has gained the quantitative property of modern science. Difficulty in culturing many bacteria existing in the environment has thus become a crucial issue.

Even if a strain cannot be cultured, its presence can be confirmed by PCR (see appendix) using the information of known strains related to it. We can directly clone specific genes from soil and water samples and reconstruct the entire genome in particular cases, without even culturing the organism. However, in the absence of culturing and direct observation of the strain, very little can be known about the properties of the strain as an organism. Some bacteria can be exposed to cold starvation and produced in a viable but non-culturable (VNC or VBNC) state in which they exhibit biological activity without producing colonies. Although some consider non-culturable bacteria in the environment to be in a state similar to the VNC state, if the strain cannot be isolated, it cannot be denied that the culture technique may simply be imperfect or that cultures cannot be produced because the organism does not form colonies. There are also examples of microorganisms that cannot be isolated due to their strong symbiotic relationships with other organisms or microorganisms or their strong dependencies on particular environments.

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