THERMOPHILS: THE PLANET'S PAST, ВIOTECHNOLOGY'S FUTURE

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Опубликовано в библиотеке: 2021-08-29
Источник: Science in Russia, №4, 2010, C.28-34

by Yelizaveta BONCH-OSMOLOVSKAYA, Dr. Sc. (Biol.), deputy research director, head of the laboratory involved with hyperthermophilic communities, Vinogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia

 

Although thermophilic ("heat-loving") microorganisms have been known for more than a hundred years, their active studies were launched but a few decades ago. Growing at high temperatures, these organisms are among the oldest on earth; their communities are viewed as analogs of ecosystems that came into being with the birth of biological life. Enzymes produced by them are remarkable for great heat stability, a factor of strong attraction to biotechnologists.

 

UZON CALDERA AS A MUSEUM OF MICROBES

 

For quite some time thermophilic prokaryotes were thought not to be capable of growing at temperatures above 50 to 60 degrees centigrade. But such views were revised towards the close of the 20th century. Thomas Brock, an American microbiologist studying hot springs of volcanic origin, made a really revolutionary discovery: these springs were found to be inhabited by microorganisms growing at temperatures in the 60 to 80°C range. Subsequently two German scientists, Wolfram Zillig and Karl Stetter, detected thermophils whose growth conditions were optimal at temperatures above 80°C. The upper temperature limit for such microorganisms has been found to be as high as 122°C. This temperature occurs in benthic hot springs in the

 
стр. 28

 

 

Thermal fields of the Uzon caldera with its many hot springs: Eastern Field (a) with its predominantly neutral waters and Orange Field (b) with prevalent acidulous springs.

 

sea where overheated water vapor stays liquid on account of a very high hydrostatic pressure. Our Institute (named in 2003 for Sergei Vinogradsky, one of the fathers of microbiology in this country) began research on thermophilous microorganisms in hot springs of volcanic origin back in the late 1970s, that is almost at the same time as our Western colleagues did. The caldera* of the Uzon volcano (Kronotsky Reserve in Kamchatka) is still the principal object of related studies—it abounds in hundreds of hot springs with most diverse characteristics (mostly in what concerns the temperature and acidity of the medium) inhabited by most different thermophils.

 

Uzon's thermal springs characterized by moderate temperatures in the 50 to 70°C range are inhabited by microbial communities called "mats" and composed of cyanobacteria and associated organotrophic bacteria (organisms consuming photosynthetic substances).

 

Acad. Georgi Zavarzin, heading the Laboratory of Relict Microbial Communities at our Institute, and coworkers came up with a hypothesis in 1980 on the crucial role of cyanobacterial communities in forming the present-day atmosphere of the earth: they proved that it takes "mats" just a few days to convert volcanic gases contained in hot springs into an atmosphere similar to the present one.

 

There was a time when hard ultraviolet radiation made life impossible on the illuminated surface of the earth. That is why present-day thermophilous microbial communities—living on inorganic energy sources of volcanic origin in the absence of oxygen (anaerobes)—are a good object of research as analogs of ancient ecosystems. Thus, Dr. Margarita Miroshnichenko has isolated microorganisms inhabiting the hot waters of the Uzon caldera at temperatures of 75 to 80°C and synthesizing organic matter from CO2 (carbon dioxide); they use the energy of molecular hydrogen. These lithotrophs ("stone-eaters") are obligate anaerobes: they do without oxygen and, what is more, do not grow in its presence. Yet any reaction supplying vital energy needs a highly energized substrate and an oxidant. These are all the various compounds of sulfur also found in great abundance in hot springs.

 

The organic matter of the Uzon caldera formed by photosynthetic and lithotrophic microorganisms is supplemented with plant organics from low-temperature zones. All these components are vigorously decomposed by organotrophic thermophils, bacteria and archaea (a group of the oldest prokaryotes). Among these there are quite a few thermophils flourishing at temperatures above 80°C. For instance, the Desulfurococcus kamchatkens isolated by Dr. Ilya Kub-lanov is capable of decomposing proteins, including such hard substances as alphakeratin (essential for the growth of hair and fur in mammals).  Its optimum

 

 

* Caldera (cauldron)—a kettlehole having steep slopes and flat bottom, formed due to the cave-in of a volcano's top and occasionally of adjacent locality; it can be 10 to 15 km across.-Ed.

 
стр. 29

 

 

New thermophilic microorganisms from the Uzon caldera: lithotrophic bacterium CALDIMICROBIUM RIMAE (a) and organotrophic archaea FERVIDOCOCCUS FONTIS (b).

 

growth is at 85°C with the upper limit of 92°C. Another representative of such archaean organisms is the Acidilobus saccharovorans discovered by Dr. Maria Prokofyeva; it grows at 98°C, and even under acidic conditions (growth optimum at pH 3.8). It decomposes a large number of sugars and polysaccharides like starch.

 

Modern microbiology is able to evaluate microbial diversity via direct analysis of DNA isolated from a natural sample. This is certainly a great achievement making it possible to identify both the known microorganisms and the new ones not yet obtained in laboratory cultures. For example, Dr. Anna Perevalova has detected a group of archaean thermophils at Uzon, formerly considered uncultivable, and then she obtained their first cultivated representative, the Fervidococcus fontis.

 

The presence of microorganisms responsible for definite biogeochemical processes can be determined by studying corresponding functional genes in native DNA samples. This is how Dr. Nikolai Chernykh has detected groups of thermophilic bacteria producing organic substance from CO2 in the hottest springs of Uzon. Alexander Merckel, our laboratory's graduate student, has discovered new thermophils forming methane. These data, valuable per se, are a guide for a further search for microorganisms by cultivation methods.

 

ON THE OCEANIC FLOOR

 

Hot springs of volcanic origin are found both on the ground surface and underwater, also at great depths. In the early 1980s Kholger Jannash of the United States and colleagues discovered unique ecosystems associated with such springs. Microorganisms populating deep-water thermal springs and their environs, both thermophils and mesophils*, subsist on volcanic inorganic substrates and synthesize organic substance from carbon dioxide; this organic matter is consumed by numerous invertebrates. Abyssal hydrothermae are a source of a great number of new thermophilic and hyperthermophilic microorganisms isolated at our institute, too. Many of the isolates thus obtained are lithotrophs making use of inorganic energy sources-like Caldithrix abyssi, for instance, isolated by Dr. Miroshnichenko. It utilizes molecular hydrogen with a nitrate oxidant being reduced to ammonium. Caldithrix is remarkable in that it has no related forms among the bacteria known to date: it forms a separate branch of the phylogenetic tree. Further studies must provide more information on this group of microbes.

 

Organisms of the Thermaceae family were among the first thermophils once discovered by Thomas Brock in Yellowstone National Park (Wyoming, USA). It came

 

 

* Mesophils-organisms growing and multiplying at to between 20 and 40°C.-Ed.

 
стр. 30

 

out that microorganisms of the Thermus genus are of wide occurrence in ground surface hydrothermae throughout the world; they grow owing to the oxidation of diverse organic substrates by atmospheric oxygen. We have discovered related organisms in deep-water thermal springs of the Pacific and Atlantic Oceans, peculiar in many ways. Although aerobes, they grow only at low concentrations of oxygen. Unlike Therma-ceae, the abyssal "mavericks" are capable of litho-trophic growth in the presence of molecular oxygen— and under microaerophilic conditions, with nitrate as oxidizer; in this case nitrate is reduced to nitrite.

 

Thus, found both in ground surface and in marine thermal springs are thermophilic bacteria and archaea subsisting on molecular hydrogen as a substrate and on various inorganic substances as oxidants in anaerobic respiration. The range of the known reactions used by thermophils for energy supply keeps expanding. Two groups of suchlike microorganisms discovered by Georgi Zavarzin's team are being studied in our laboratory, and they are a subject in its own right.

 

THIS TASTY CARBON MONOXIDE

 

Carbon monoxide (CO) and hydrogen (H) enter into the composition of volcanic gases getting into hydrothermae. The ability to consume compounds that are poisonous to most of the living beings has been found to be quite common among thermophils. They ingest CO in an anaerobic fashion, that is oxidize it to CO2 in the absence of oxygen. Hydrogen, the other product of their vital activity, comes from water: CO + H2O = CO2 + H2.

 

One thermophil responsible for this process, now known as hydrogenogenic carboxidotrophia, was isolated by Dr. Vitaly Svetlichny in 1990. In these two decades, thanks to the work done by Dr. Tatyana Sokolova and her pupils, many more new data have been collected on these microorganisms. Detected both in round surface and in marine deep-water hydrothermae, they include many bacterial genera as well as hypothermophilous archean organisms. The first hydrogenogenic carboxidotrophs isolated by us grow in an atmosphere of 100 percent carbon monoxide. However, as Dr. Tatyana Kochetkova brought down the initial concentration of CO to 45 percent and then to 15 percent, their diversity went up considerably. Since natural concentrations of CO in hot springs make up mere percent fractions (percentagewise), this diversity may be much wider, since the ability of these organisms to utilize carbon monoxide as the energy substrate could be coupled with sensitivity to its high concentrations.

 

As a matter of fact, thermohilic hydrogenogenic carboxidotrophs do not constitute a single philogenetic group but belong to different genera, branches and even domains (archaea and bacteria) in the prokaryot-ic tree. Dr. Alexander Lebedinsky, who has studied the genetic apparatus of hydrogenogenic carboxidotrophia, has demonstrated that the CO-dehydroge-nase and hydrogenase complexes responsible for this process are of the same type in different microorganisms. Possibly the wide occurrence of this characteristic in thermophils may be due to the transfer of a corresponding gene cluster from one group to another enabling them to live in thermal springs deficient in organic matter.

 

THEY REDUCE NOT ONLY METALS ALONE

 

In the course of anaerobic respiration thermophilic microorganisms can reduce metals of fluctuating valence—like iron, for instance. Thus, one thermo-philous archae, Geoglobus acetivorans, whose growth is sustained through the oxidation of molecular hydrogen and the reduction of iron oxide proceeding simultaneously has been isolated by Dr. Galina Slobodkina of our laboratory from abyssal hydrothermae (as deep as 4,000 m from the ocean surface) of the Mid-Atlantic Ridge, namely in the Ashadze field.

 

This capability of thermophilic microorganisms can be put to good use, specifically, for purification of

 
стр. 31

 

 

New thermophilic microorganisms in abyssal hydrothermae- Caldithrix abyssi (a) and Oceanithermus profundus (b).

 

industrial effluents and elimination of toxic metals or radionuclides, since the solubility of reduced forms of many metals is much lower than that of oxidized ones; therefore the former (reduced metals) can be reliably removed through precipitation. Dr. Alexander Slobod-kin and colleagues have shown thermophilic prokary-otes to be effective in eliminating compounds of chromium, uranium and technetium.

 

Microorganisms can interact with insoluble substances, too, resorting to different strategies thereby. According to Dr. Sergei Gavrilov's data, iron oxide (electron acceptor), if enclosed within porous envelopes, attracts microorganisms that come in touch with it by using special superficial structures, the pili, penetrating the pores. This mechanism is being closely studied, for pili are natural conductors of electrons, i.e. of electric current.

 

Yet another side of the thermophil-mediated reduction of insoluble acceptors: in an experimental power network thermophils transfer electrons to one of the two electrodes, the positively charged anode. A "microbial fuel element" can thus be created, in which the liberated energy of chemical bonds is converted to electricity.

 

DENIZENS OF THE PLUTONIC KINGDOM

 

The microorganisms we are dealing with populate hot springs, or hydrothermae—on the ground surface or out in the sea. There is yet another habitat: though hard of access, it dwarfs thermal springs in scope. At a depth of 1,500 to 3,000 meters from the ground surface the temperature can rise to 60-80°C and more; this is a comfortable medium for the hyperthermophilic underground biosphere. Thus, Dr. Margarita Miroshnichen-ko has isolated hyperthermophilic archean and bacterial organisms in stratal waters of the Samotlore oil deposit (Western Siberia) which, strange as it might seem, are closely related to similar microorganisms detectable in marine hydrothermae. There can be no doubt that these are aboriginal isolates: the oil well, where they were found, had never been water-injected for oil pressure increase. We named one of these archaean thermophils of the Thermococcus order Thermococcus sibiricus, for it was found in Siberia, thousands of kilometers away from seas and volcanic hot springs. Jurassic deposits the stratal waters of which are inhabited by the Thermococcus sibiricus are sediments of an ancient sea, while the stratal water is like sea water in its composition. The temperature as high as 84°C at a depth of 1,800 m is optimal for microorganisms of this genus. According to subsequent studies carried out by other scientists, archaean organisms related to the species we have discovered are also found in oil well samples recovered in China and Japan. Consequently, they inhabit the high-temperature hydrosphere over a vast area.

 

THERMOSTABLE ENZYMES

 

There is yet another, utilitarian side to thermophils, and this is their enzymes that are being widely used in most different fields of human activity, say, in the production of detergents, food products, fodder, in the paper-and-pulp and textile industries, and in the reprocessing of various wastes. If an enzymatic reaction proceeds at high temperature, its rate increases, and

 
стр. 32

 

the danger of contamination by foreign microorganisms is reduced dramatically. Their stability at high temperatures is combined with resistance to other agents, e.g. detergents, which is important when thermostable hydrolytic enzymes are used there. Industrial needs in such enzymes have stimulated research into the diversity of thermophilic microorganisms.

 

Caldoanaerobacter 1004 keratinase identified by Dr. Ilya Kublanov is just one example of such studies. The microorganism producing this enzyme was isolated from a hot spring in the Barguzin river valley next to Lake Baikal; it can grow on alpha- and beta-keratins, that is on proteins contained in the fur of animals and feathers of birds. The extracellular keratinase it secretes hydrolyzes keratins resistant to ordinary proteinases. This enzyme can be used in the reprocessing of waste in animal husbandry and poultry farming in particular, for feathers decompose very slowly, and their disposal poses a great problem.

 

THERMOPHIL GENOMES AS BANKS OF PRECIOUS GENES

 

Thermophilic microorganisms grow but slowly under laboratory conditions. Although the time of one cell division may take a mere 30 minutes, the overall cellular mass is not dense enough due to the low output of related processes (anaerobic by and large). So to obtain a required amount of this or that enzyme, we should clone the encoding gene and then express it; only then will the recombinant strain produce a significant amount of the desired protein. To achieve this we should find the gene's structure, which is a laborious process that involves all-out purification and amino acid sequencing of the protein. Here again we have to deal with the slow growth of the initial microorganism in laboratory cultivation.

 

It will be easier to come on top of these problems once we get a complete genome sequences of the organism and then, using bioinformation methods, identify genes coding for enzymes valuable to biotechnology. While the search for complete genomic sequences of various microorganisms, thermophilous among them, was proceeding apace for quite some time in other countries, we in Russia were lagging behind.

 

The situation has changed for the better over the last two years as the Bioengineering Center of the Russian Academy of Sciences—with our participation and with the financial support of the Ministry of Education and Science of the Russian Federation—has obtained 12 complete sequences of the genomes of the thermophilic microorganisms (11 archaean organisms and one bacterial) isolated by us. Three sequences have already been analyzed in full, while the others are still under study. The three studied organisms—the thermophilic archaea Desulfurococcus kamchatkensis, Thermococcus sibiricus and Acidilobus saccharovorans—grow at 85 to 88°C. New hydrolytic enzymes were detected in their genomes. In the Desulfurococcus kamchatkensis these are proteinases of different molecular weight, and amylases. The Thermococcus sibiricus has sprung many surprises on us: an organism known as using peptides only in its growth, it proved to be a carrier of genes causing

 
стр. 33

 

 

Thermincola carboxidiphilum-a hydrogenogenic carboxidotrophic thermophilic bacterium.

 

 

Magnetite formed by a thermophilic iron reducer: left, a test tube in which the iron-reducing bacterium was cultivated; right, control; in between, magnet.

 

hydrolysis of various polysaccharides. They form a single genomic cluster, the "polysaccharide island", that apparently enables this microorganism to live on in Jurassic deposits via decomposition of the buried organic matter of an erstwhile sea. In the Acidilobus saccharovorans, in addition to genes coding for enzymes that hydrolyze polysaccharides, we detected a set of other enzymes responsible for hydrolysis of fats and their oxidation. This archaean species oxidizes organic matter completely, with sulfur compounds as electron acceptors.

 

Thus, the information on the set of genes enables access to encoding thermostable enzymes essential to biotechnology. Their range proves to be much wider than what one could expect proceeding from laboratory cultivation data alone. At the same time new details on metabolism come to light, including those elucidating the evolution of a particular group of microorganisms, and the place of microbial species in contemporary thermophilic communities.

 

Over the past ten years we have done much to catch up in the field of molecular microbiology. Unfortunately we had no part in the breakthrough achieved elsewhere in the world in the 1990s with the help of molecular biology methods. In fact, it is these approaches that have brought research in ecology and microbiology to an absolutely new level. But thereafter things took a different turn—in many respects, thanks to action on the program of the Presidium of the Russian Academy of Sciences "Molecular and Cellular Biology" under the guidance of Acad. Georgi Georgiev. Launched in 2002 in an open and transparent competition, this program enabled the nation's best laboratories to carry on work by up-to-date methods. Our collective is among them, too. In these past seven years we have made good headway in applying molecular biology methods in studying microbial communities within their volcanic habitats. The combination of new approaches with the conventional cultivation techniques, still alive in Russia, of microorganisms remarkable for unique types of metabolism has fructified: we are able both to detect new groups of thermophilic microbes and obtain them under laboratory conditions, too. Furthermore, we are able to evaluate their characteristics and role in natural ecosystems. Genomic data are a good complement to orthodox cultivation methods: they have considerably expanded and revolutionized our knowledge of microorganisms.

 

Works of the laboratory involved with thermophilic microbial communities are supported by RFFI grants: 06-04-49045, 09-04-00251, and 09-04-49045.


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© Yelizaveta BONCH-OSMOLOVSKAYA () Источник: Science in Russia, №4, 2010, C.28-34

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