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Author(s) of the publication: Nikolai RAVIN

by Nikolai RAVIN, Dr. Sc. (Biol.), Vice-Director for Research, Laboratory Head, Molecular Cloning Systems, Bioengineering Center, Russian Academy of Sciences, Moscow

The natural communities of microorganisms may include thousands of species, most of which cannot be cultured under laboratory conditions and thus cannot be properly studied. However, this problem has been solved with the development of new methods of genome analysis. Using these methods, we have studied the composition of bacterial communities of hot springs in Kamchatka and within methane hydrate deposits at the bottom of Lake Baikal.


One of the most important tasks of microbiology (as in other classical biological sciences) is identification and classification of research objects. Zoology and botany make use of the entire variety of morphological signs of animals and plants for this purpose, but this approach is not productive for microorganisms, as the number of differentiating external characteristics of unicellular organisms is scanty. That is why in addition to cell size and shape, the presence or absence of flagella, etc., the classification of bacteria is based on the variety of their functional characteristics, such as the capacity to grow on different substrates, to form certain metabolites, their oxygen dependence, etc. It is impossible to study these characteristics at the level of one

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cell--a whole organism per se--because of its very small size. This problem was solved with the development of culturing methods--reproduction of pure cultures of identical cells under laboratory conditions. Analysis of cell cultures reveals the diversity of metabolic and biochemical processes realized by microorganisms, and their ecological role in the biosphere; it enables scientists to develop methods for identification and classification of microorganisms.

New trends, based on concepts of the functional characteristics of any living organism are determined by its genome (nucleotide sequence in DNA), have been gaining ground in microbiology since the middle of the 20th century. An important result of the use of molecular methods in this branch of science was the creation of a system for identification and classification, based on sequencing* and comparison of nucleotide sequences of genes of 16S ribosomal RNA**, present in all microorganisms. The homology of genes of this RNA reflects their evolutionary relatedness. Proceeding from these facts, Karl Woese of the USA suggested at the end of the 1970s a universal phylogenetic system of prokaryotes (organisms with no distinct cell nucleus, in contrast to eukaryotes which have it). He also found that archaei*** constituted the third domain of organisms, along with bacteria and eukaryotes.

Databases of 16S rRNA nucleotide sequences have been expanding with the development of genome sequencing techniques. These techniques were used on metagenomic DNA**** isolated from natural sources (soil, sea water, human intestine, etc.). It was in those studies that limitations of the culturing methods used in microorganism analysis became obvious. It was found that the majority of these organisms, identified in natural communities by 16S rRNA gene sequences, had no cultured relatives and represented individual phylogenetic branches, the metabolism and ecological role of their microorganisms being quite unknown. Moreover, no more than 0.1-1 percent of microorganisms from natural communities could be cultured under laboratory conditions. The existence of others became known only in the course of molecular analysis.

In the USSR studies of nucleic acid sequences started in the 1960s--in fact, simultaneously with the first analogous studies of foreign scientists. In 1967 Alexander Baev, a biochemist, determined the sequence of the yeast transport valine (carrying valine amino acid) RNA. Deciphering of the genome sequences proper (that is, DNA) necessitated a search for essentially new approaches. Methods for sequencing this

* Sequencing-determination of nucleotide sequences in the DNA chain.--Auth.

** Ribosomal RNA (rRNA)--several molecules of ribonucleic acid, forming the base of a ribosome on which protein synthesis is realized.--Ed.

*** Archaea are unicellular prokaryotes, differing at the molecular level both from bacteria and from eukaryotes. Their specific characteristics are manifest in protein synthesis components, cell wall structure, biochemistry, and resistance to environmental factors, e.g., high temperature.--Ed.

**** Metagenomic DNA, total DNA of microorganisms in a particular community.--Auth.

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Composition of bacterial community of ZAVARZIN SPRING.

The graph (a) shows relationship between the number of identified bacterial species and the number of analyzed 16S rRNA gene sequences. The diagram (b) shows shares of the main microorganism groups in the community.

complex biomolecule have been developed at laboratories of Walter Gilbert, American biophysicist (Nobel Prize, 1980) and Frederick Sanger, English biochemist (Nobel Prize, 1958 and 1980). At the end of the 1970s sequencing methods were used for a complete decoding of the operon* of yeast ribosomal RNA genes by Konstantin Skryabin, Acad. Baev's pupil.

Determination of DNA nucleotide sequences opened up new vistas for identification of proteins coded for by respective genes. The first research of this kind in our country (we mean here fragments of the bacteriophage lambda genome) was reported in 1979 by a team of scientists: Academicians Yuri Ovchinnikov, Konstantin Skryabin, Yevgeny Sverdlov, and Alexander Baev. The sequencing of human, animal, plant, and microorganism genes and later, of total viral genomes was continued in the 1980s at several institutes of the Academy of Sciences of the USSR. Yet actually no molecular identification of microorganisms had been carried out in Russia before the middle of the 1990s, when a special research division was set up at the Bioengineering Center of the Russian Academy of Sciences. More than a thousand bacteria and archaea were identified by 16S rRNA sequences.

Genome sequencing methods have been constantly upgraded over these last three decades, their productivity has improved and their price cut down. For example, the parallel pyrosequencing* method, suggested by the American 454 Life Sciences Company (now a member of the Roche firm) in 2005, became a great qualitative

* Operon, the site of genetic material composed of two or more genes transcribed in one RNA and functionally related, e.g., in coding for proteins (enzymes) realizing successive stages of a metabolite's biosynthesis.--Ed.

* Pyrosequencing-an analytical method called so because pyrophosphates (pyrophosphoric acid anions, salts, and esters) play an important role in it.--Ed.

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Presumable scheme of methane hydrate formation in bottom precipitate of Lake Baikal.

leap forward. Using this method, it is possible to determine simultaneously hundreds of thousands of DNA nucleotide sequences in individual reactions, while the traditional capillary electrophoresis allows a simultaneous analysis of no more than several tens of specimens.

I should like to emphasize that many bacterial communities have a complex composition including bacteria and archaea of thousands of species. Analysis of 16S rRNA gene sequences by the pyrosequencing method identifies not only predominating microorganisms, but also minor ones present in very low quantities which, however, can play an important functional role. As a result, we can reliably identify individual groups of bacteria, which is essential for the reconstruction of the metabolic pathways of the community in general.

In Russia studies of microbial communities by high-efficiency sequencing methods were initiated by the Bioengineering Center of the Russian Academy of Sciences in 2008--in fact, simultaneously with the first works of foreign colleagues in this sphere. Now, what are our results?


It was assumed for a long time that microorganisms, like other living forms, cannot live on at temperatures above 50-60 ºC. Thomas Broque, an American scientist, made this important discovery in the 1970s. He found bacteria growing at temperatures of up to 80 "C in the hot springs of Yellowstone Park in the USA. Later thermophils (mainly archaea) were detected in volcanic hot springs, deep-sea thermal springs, and high-temperature oil wells. The upper temperature threshold at which microorganisms grow (122 "C) has been found for archaea inhabiting the deep springs at the bottom of the ocean under high pressure* conditions.

The interest in thermophils is explained by their significance for basic studies and practical uses. Thermophils belong to the evolutionally ancient groups of microorganisms, and their communities can be considered to be analogs of ecological systems which had existed billions of years ago on the still hot Earth in an oxygen-free atmosphere. One of the examples are archaea which have the smallest genome among all living organisms. As for practical uses, thermostable enzymes produced by thermophils can be used in various spheres of biotechnology, for example, as cleavage lipids and bioadditive proteins in detergents.

The main object of our research realized in collaboration with the team headed by Yelizaveta Bonch-Osmolovskaya**, Dr. Sc. (Biol.) from Vinogradsky RAS Institute of Microbiology, are the thermal springs of Kamchatka. There are hundreds of such springs out there, in the Geyser Valley, in the Uzon volcano crater, and elsewhere. These springs are noted for a great variety of physicochemical characteristics--temperature, acidity, chemical composition of water, and presence of volcanic gases.

See: A. Lisitsyn, A. Sagalevich, "Breakthrough Discovery of the Century", Science in Russia, No. 1, 2001.--Ed.

** See: Ye. Bonch-Osmolovskaya. "Thermophils: The Planet's Past, Biotechnology's Future", Science in Russia, No. 4, 2010.--Ed.

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We have analyzed bacterial communities of five thermal sources from the Uzon volcano caldera, differing in two parameters most significant for growth--water temperature and acidity. One of them is the Zavarzin spring. It is a shallow basin 4.5 x 2.3 m in size. The bottom of its colder sites at the periphery is covered with a layer of cyanobacteria several millimeters thick. The water temperature is moderately high (58 ºC), its acidity is neutral (pH 6.3), and the level of suspended sulfur is high. Another spring, Burlyashchy ("boiling") is Uzon's hottest, its water temperature reaching the boiling point (90-94 ºC), taking into account the height above the sea level; its pH is also neutral (6.7). Three others are "1884", "1805", and "1810"; they are acid (pH 3.7-4.1) but differ in water temperature (50, 60, and 90 ºC).

The Zavarzin spring is characterized by the greatest biological variety. More than a hundred bacterial species have been detected here. Bacteria account for 95 percent of all microorganisms inhabiting the spring, and only 5 percent are archaea. One of the phylogenetic branches of bacteria is a heretofore unknown evolutionally ancient group; its representatives have been found only in this spring. Complete characterization of the bacterial community became possible only thanks to the pyrosequencing technology (about 35,000 sequences of 16S rRNA analyzed). Traditional methods would not have detected even one-tenth of this great variety. Proceeding from these new data, we put forward a hypothesis on the nature of ecological relationships among the main groups of microorganisms in this spring.

It was found that the initial production of organic substances is realized by photosynthetic cyanobacteria and by various groups of chemolithoautotrophs oxidizing reduced volcanic substances (hydrogen, sulfur, etc.), brought by a geothermal stream. All these organic substances and those brought with surface water from adjacent regions are consumed by various organotrophic microorganisms, including bacteria fermenting the organic substrates and completely oxidizing them by oxygen, sulfur, or nitrate electrons as acceptors. The presence of a great number of phylogenetically different groups, none of them predominating, indicates that this complex community is well-balanced and each group occupies its own ecological niche. A community of this kind can be regarded as a model of the initial ecosystems of the ancient Earth.

The "population" of the Burlyashchy thermal spring is quite different. It comprises just two groups of microorganisms: Aquificae type bacteria and Thermo-proteales order archaea. Both are chemolithoautotrophs synthesizing organic substances from C02, by utilizing the energy of molecular hydrogen and other reduced volcanic substances. Presumably, this simple structure of the community is determined by a limited spectrum of available substrates and bacteria growing at extremely high temperatures.

In contrast to thermal sources with the neutral pH, acid sources have been but little studied all over the world, and so, no wonder that we found many "surprises" when analyzing the abovementioned "1884", "1805", and "1810" springs. Let us note that the majority of microorganisms in those springs were not bacteria, but archaea, better adapted to survival under high temperature and acidity conditions. The greatest bacterial

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variety was found in the "cool" (50 ºC) "1884" spring. One of the most numerous groups of bacteria were the Acidithiobacillus order, aerobically oxidizing iron and sulfur. Sulfur oxidation gives rise to sulfuric acid, and thus the Acidithiobacillus activity could be responsible for high acidity of the medium. Bacteria of another group, Verrucomicrobia, oxidize methane (gas) brought with the geothermal stream. They grow at moderately high temperatures in an acid medium, created by Acidithiobacillus.

The most unexpected results were obtained by analysis of the "archaeic" component of the community. More than 90 percent of detected archaea were phylo-genetic strains among which there were no microorganisms cultured by the known classical microbiological methods. Their metabolism and ecological role call for further studies.

About half of archaea in the "1805" and "1810" thermal springs were representatives of the Sulfolobales order, found in sulfatares* and acid hot springs all over the world. These archaea oxidize sulfur, hydrogen, and organic compounds; their activity can be responsible for high acidity of the springs. Nanoarchaea were the most interesting and unexpected finding: their percentage was the highest under the extreme conditions of the "1810" spring (90 ºC, pH 4.1). As we have said, they represent an evolutionally ancient branch. The only one cultivable of them, the Nanoarchaeum equitans, previously found in a deep sea thermal spring, is a symbiont of another archaeum, Ignicoccus hospitalis. The absence of its "relatives" in this spring and a large share of nanoarchaea (24 percent of all archaea) suggest that these organisms are free living and relicts of ancient ecosystems.


One more object of our studies are bacterial communities found in methane hydrate deposits. But first, some digression. Methane hydrates are a prospective fuel and energy resource. Their mining will become possible in the near future. These are solid crystal water and gas compounds formed at high pressure and low temperatures. These deposits are at least several times larger than traditional gas resources. Their main deposits were found in sea and ocean sediments, and in permafrost areas; significant deposits were also found in Russia's Arctic shelf.

The interest in these deposits is explained by not only prospects of their use as energy carriers, but also by their potential effects on global changes in the climate. In fact, temperature rises destabilize hydrates and cause emission of gaseous methane. Increase of the atmospheric concentration of methane (an active "hothouse" gas) intensifies the hothouse effect and causes further temperature rises.

Various hypotheses on the origin of methane hydrates are discussed now. Some scientists think their origin is biogenic, that is, they are produced by microorganisms, while others suppose they are abiogenic products of thermal degradation of carbohydrates. Studies of sea methane hydrates have shown their relationship to communities of different microorganisms, methane oxidizing and methane forming. However, it is well known that sea and fresh water bacterial communities and the biochemical processes they realize differ significantly depending on the mineral composition of water.

Lake Baikal is the only fresh water body, at the bottom of which gaseous hydrate deposits were found. Their formation in bottom sediments is possible due to the great depth of the lake (up to 1,642 m), providing high pressure, and to low temperature of water (3-4 ºC).

* Sulfatares-craters filled with water warmed by hot steam with admixtures of hydrogen sulfide, carbon dioxide, and other volcanic gases.--Auth.

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The aim of our work, carried out in collaboration with a team headed by Tamara Zemskaya, Dr. Sc. (Biol.), from the Limnological Institute of the Siberian Branch of the Russian Academy of Sciences in Irkutsk was an analysis of the composition of bacterial communities associated with gaseous hydrates in Lake Baikal as well as identification of various groups of microorganisms responsible for methane formation and oxidation. Samples of benthic water and two sediment samples--in the near surface (up to 1 cm) and deep layers (85-90 cm), directly adjacent to the hydrate, were collected at a depth of about 1,400 m in the methane hydrate deposition region for analysis. Samples of benthic water were collected from the Mir deepsea apparatus during the expedition of 2009.

The composition of bacterial communities was analyzed by 16S rRNA genes pyrosequencing. About 500 species were found in benthic water. Of these, 99 percent were bacteria and just 1 percent, archaea. About half of the bacteria were common "water" species, characteristic of fresh water bodies, while the other half were methanotrophic bacteria of the Methylococcacea family, growing by oxidizing methane with oxygen dissolved in water. Importantly, none of the 29,000 bacteria identified in Lake Baikal by 16S rRNA sequences, were referred to the known pathogenic species; this fact indicates the purity of water and its safety for humans.

In contrast to water, samples of bottom sediments contained a significant portion of archaea: about two-thirds in the near-surface layer and about 30 percent in the deep layer. Various organotrophic bacteria and archaea decomposing the organic substances accumulated in the form of bottom sediments (dead algae, cyanobacteria, etc.) were found in the upper layer. The sediments serve as an anaerobic (oxygen-free) ecological niche, in which the organic substances are fermented by organotrophs with the formation of acetate and hydrogen as the main products. These substances can serve as substrates for methanogenes*--the most numerous group of archaea, found down there. Since the Baikal water is poor in minerals, methanogenesis is the final stage of the decomposition of organic sediments. The resultant gas is either deposited in the form of hydrates, or goes up and is effectively oxidized in benthic water by aerobic methanotrophic bacteria. Thus, the ecosystem of the lake provides for the transformation of methane hydrates and prevents emission of this hothouse gas into the atmosphere.

As for the bacterial community of the sedimental layer, it includes various groups of bacteria and archaea.

More than 90 percent of them belong to different phy-logenetic strains, which have no cultivated representatives. The microorganisms of the deep-water biosphere and their metabolism are yet obscure.

Concluding, I should like to say that the potential of Russian microbiology has increased significantly in recent years due to the use of modern molecular methods in combination with classical microbiological and biochemical approaches. In many respects this became possible thanks to projects of the Ministry of Education and Science of the Russian Federation, Russian Foundation for Basic Research, and basic research programs of the Russian Academy of Sciences. For example, while not a single complete genome of a microorganism had been deciphered in Russia before 2007, as many as twenty have been decoded in recent years; it is remarkable that about 10 percent of all the known complete archaeic genomes have been determined at the Bioengineering Center.

It stands to reason that the potentialities of high-performance sequencing are not exhausted by the taxonomic status of communities. Phylogenetic affinity not always correlates with the similarity of metabolitic pathways of microorganisms. Therefore, the most reliable reconstruction of metabolism should be based not only on data on a community's composition, but also on the fullest possible genetic information--that is, nucleotide sequence of the entire "metagenome" has to be determinated, its genes identified and their probable functions analyzed. Until recently, these problems could be solved at just few research centers, and this work was quite expensive. The development of new technologies of genome sequencing, leading to higher productivity and lower costs, expands the possibilities of such studies.

These studies have been supported by the Ministry of Education and Science of the Russian Federation (contracts P1049 and 02,740. 11.0765) and grants from the Russian Foundation for Basic Research 08-04-01273 and 11-04-00671.

* Methanogenes--methane-forming microorganisms; belong to archaei.--Auth.


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Nikolai RAVIN, GENOME ANALYSIS AND THE ECOLOGY OF MICROORGANISMS // Minsk: Belarusian Electronic Library (BIBLIOTEKA.BY). Updated: 22.09.2021. URL: (date of access: 02.10.2022).

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