by Irina IVSHINA, laboratory head, Laboratory of Alkanotrophic Microorganisms, Institute of Ecology and Genetics of Microorganisms (IEGM), Ural Branch of the Russian Academy of Sciences, Professor of the Microbiology and Immunology Department, State National Research Institute, Perm, Microbes, these infinitesimal living beings, are of infinitely great role in nature. Louis Pasteur

Documentary descriptions alone will never do in studying bacteria, the oldest forms of biological life, and their involvement in human activities. In the absence of investigated bacterial strains a taxonomy of these microorganisms would be like trying to map the starry sky proceeding from random observations of distant luminaries and their scintillations. If we compare a collection of living cultures with a map of an unexplored continent holding many surprises in store-say, sand dunes instead of the expected rocky mountains--such a collection will be like an explored coast, the start-out point of voyages into a terra incognita. One such collection is the subject of the present article.

LABORATORY RESERVATIONS

There is a major turnabout taking place in social mentality: man is coming to realize his critical dependence on the microbial world at large. Ignoring the diversity of this world is fraught with undesirable consequences in medicine, environment and biotechnology. Conversely, a better knowledge in this field stimulates a search for optimal ways of conserving the gene pool of the globe's microbial genetic resources, and opens up prognostication and control prospects with respect to the ecological situation. Such information is also of great help in developing up-to-date technologies with the use of live biological systems. Small wonder that research of microorganisms, above all those implicated in human activities, sparks a wide range of international initiatives.

One effective way is to cultivate tiny microorganisms in laboratory reservations. This business is experiencing a renaissance in many countries against a backdrop of the tempestuous progress of the world bioeconomy. Depositaries are gaining in significance--so much so that microbial collectibles are evaluated in hard money.

The story of obtaining the thermostable DNA polymerase (Taq polymerase) isolated from the extremely thermophilic bacterium Thermus aquatkus is a good example. It inhabits the hot springs (at t≥55 °C) of Yellowstone National Park in the United States famous for its geysers. Dr. Thomas Brock and his student Hudson Freeze were the first ever to isolate this polymerase.

Isolated into a pure culture and identified in 1969, this bacterial strain was handed by its discoverers for deposition in the American collection. Kary Mullis, a biochemist employed at a biotechnological company, who bought the strain for a token sum of $35 in 1993, got a Nobel Prize in 1993 for isolating from it an enzyme capable of resisting high temperatures and for showing its utilitarian prospects. His technology of a polymerase chain reaction made it possible to selectively copy large regions of DNA, which meant a breakthrough in molecular biology, gene engineering and medicine. The Swiss corporation F. Hoffman-la Roche purchased the technology and patent for Taq polymerase by shelling out as much as 300,000,000 USD. An industry based on using this precious enzyme is bringing in billions of dollars in profit.

It would be in place to recall that back in the 1930s Boris Perfilyev, a Soviet microbiologist, isolated a similar thermophilic microorganism (of photosynthetic bacteria) in a hot spring near the settlement of Kurort-noye on the Kerch Peninsula. He named it thiodendron (Thiodendron lateens). Unfortunately this culture was not deposited in a collection. Even though attempts were made to isolate thiodendron again, they did not come off, for the habitat of the thermophilic bacterium and its biocenosis had been irreparably violated and so gone with the culture were unique genes and enzymes.

The tie-in is obvious: microorganisms and their habitats are interconnected, and thus need special protection. This is particularly true of stenobiontic species (local, confined to limited ecological conditions), or else they will vanish, and their unique genes will be gone as well.

That is why in a race for competitive advantages many countries have been setting up and replenishing microbial collections. In line with a conceptual principle initiated by the Organization for Economic Cooperation and Development, pregenomic collections are upgraded into biological resource centers, the depositaries and providers of top-grade biological material and related valuable data. This transition reflects the evolution of microbial depositaries in the wake of scientific and technological progress and under pressure of events unfolding in the socioeconomic, legal and political spheres.

The focus is on specialized collections obeying standard rules and catering to users' needs. Such collections are both microbial storage centers and comprehensive databases on their characteristics and utilitarian prospects; conventional wide-profile collections are essentially unable to supply such information. Broad identification of ever new groups of microorganisms will swell such pools immensely and make a microbiologist's work ever more difficult; accordingly, the formation of giant multiprofile collections has slowed down worldwide, with a network of centralized collections gaining the upper hand.

By now The World Data Center for Microorganisms (WDCM, http:wdcm.nig.ac.jp.) has registered 592 collections from 68 countries. All told, there are as many as 2 mln sustained cultures. It is noteworthy that over 50 percent of these depositaries are getting state support on a regular basis. In the World Directory of Cultures of Microorganisms Russia is represented by 16 collections (for comparison: France has 35, Brazil--52, Sri Lanka-- 4). This statistics is in some way indicative of the level of attention to national genetic resources in a particular country, and her level of readiness to address problems of biodiversity and in the long run, those of biotechnology.

Russian collections of microorganisms are part of state budget institutions within the jurisdiction of different departments. These collections fall into three categories. The first one comprises collections of a wide profile with standard cultures of the already known species kept for the purpose of taxonomy, classification and identification. They also fulfill the functions of patent deposition and expert taxonomic centers. The second category takes in specialized collections (at research and educational institutions) meant for studying and conserving particular taxonomic groups isolated from natural ecosystems and having potentially useful properties. And the third group of collections is designated for purely research purposes (private, monographic for the most part) assembled by individual scientists for specialist research goals.

URAL COLLECTION OF ALKANOTROPHS

The Ural Regional Collection of Alkanotrophic Microorganisms* (official acronym of the IEGM collection, www.iegm.ru/iegmcol) is just one such microbial depositary making rapid progress. It is part of the laboratory of our Institute. It sprung from an individual collection of hydrocarbon-oxidating cultures started in 1975 in an attempt to use them as bioindicators of oil and gas presence and ecological pollution. Our collection specializes in actinobacteria oxidizing natural and anthropogenic hydrocarbons and thus implicated in biochemical processes in the biosphere and in the formation of a hydrocarbon-free atmosphere of the earth.

Conceptualizing the profile of our collection we understood that the Perm territory is one of the nation's oil-and gas-rich regions. The mining of natural hydrocarbons poses ecological problems, oil pollution in the first place. This is a live issue for petroleum-mining territories. But both in the Urals and thousands of miles east, up to the Pacific coast, there are no analogs of our depositary. True, in Siberia the Biophysics Institute (Krasnoyarsk) of the RAS Siberian Branch has gathered a collection of luminous bacteria; and the Institute of Bioor-ganic Chemistry (Vladivostok) of the RAS Far Eastern Branch has a collection of marine microorganisms.

Today the gene pool of our collection has more than 2,000 pure, identified nonpathogenic strains described in much detail and isolated during numerous field expeditions over many years when thousands upon thousands of samples (soil, rhizosphere, surface and stratal waters, snow, air and core samples) were taken in contrast ecogeographical regions, including the Perm Region west of the Urals and eastern Siberia, part of the Volga region (Ulyanovsk Region) and Krasnoyarsk Ter-

* Alkanotrophic microorganisms (alkanotrophs) form organic matter from hydrocarbons. Alkanes, saturated hydrocarbons.--Ed.

ritory. This work is particularly important, for the ecological situation and the home source of microbial cultures determine the diversity of their phenotypes and indicate the presence of such forms of variability that might point to dissimilarity in genotypes. I see the value of this collection in that many of the bacterial species are represented there not by singletons (often by type species), but by numerous natural isolates from different habitation areas taking in the principal geographical zones of Russia and former Soviet republics, which makes it possible to study the ecological flexibility of bacterial species, to purposively select active bioproduc-ers of useful substances and identify biodestructors of organic contaminants.

Widely represented in our collection are cultures capable of living in extreme conditions; such forms, remarkable for high oxygenase* activity, have good commercial prospects; we have also strains capable of producing basic amino acids, vitamins, biosurfactants** as well as strains destroying pollutants, crude oil and petroleum products including.

RHODOCOCCI: WHAT ARE THEY CAPABLE OF?

Bacteria of the Rhodococcus genus take pride of place in our collection. These microorganisms, belonging to the actinomycetic line of prokaryotic evolution, are assigned to the new class of Actinobacteria. Our Institute has assembled the amplest--both in this and in other countries--pool of nonpathogenic strains of rhodococ-ci. Each has been studied in great detail with the use of

* Oxygenases, enzymes catalyzing activation of molecular oxygen and subsequent inclusion of one or two of its atoms into molecules of different substrates.--Ed.

** Biosurfactants, surface-active substances of biogenic origin holding substantial advantages over synthetic detergents and offering good prospects for cleansing biotopes from hydrocarbon congregations.--Auth.

up-to-date methods. Yet not so long ago they were thought to be of little interest.

Their slow growth, the difficulties involved in their isolation and identification as well as the absence of express pathogenic characteristics left these microorganisms out of the range of fundamental studies. But with the accumulation of new data it became obvious: rhodococci exhibit an extraordinary variety of characters and functions not determined yet even in gross outline.

Rhodococci were found to be capable of accumulating molecular nitrogen in the presence of n-alkanes. This fact points to the probable ancient origins of these nitrogen-fixing bacteria that by consuming hydrocarbons are playing a useful role in sustaining a nitrogen and carbon balance on the globe. By far not all microorganisms are capable of utilizing molecular nitrogen, N₂.

Rhodococci are ubiquitous and dominating components of the biocenosis of harsh habitation areas, among them oil-polluted and oil-mining districts, biotopes with an enhanced concentration of mineral salts, and so forth. Some of the alkanotrophic rhodococci possess unique biochemical properties: they are capable of consuming, along with liquid n-alkanes, also higher gaseous homologs of methane (C₂-C₄). This ability makes them least dependent on the ambient medium--they can live in conditions hostile to other microorganisms.

In contemporary biotechnology rhodococci are among the closely studied bacterial groups with broad commercial prospects. That is why the number of related publications and patents has been on the uptrend lately. Still and all, the collection pools of representatives of this taxon are not so big worldwide: the Russian collection has 38 strains, the Japanese--92, the Pasteur Institute in France--66, the National Collection of cultures in Great Britain--88; the German collection of type cultures contains 77 strains.

All-round studies of this group of actinobacteria have brought forth evidence on the high adaptability of natural rhodococcal isolates. Such bacteria are endowed with exceptional viability in hostile natural media. They can survive thanks to a variety of conducive factors: alkano- and oligotrophic ways of life; diaoxotrophy (ability to change from a hydrocarbon substrate to a carbohydrate one); an ability to synthesize and build up endogenic reserve substances as extra energy resources, the hedge fund in unfavorable, growth-limiting conditions--actinobacteria have good survival chances even when subsistence resources run out. They can grow at low (+4 to +10 °C)--rhodococci are found in soil samples of arctic, subarctic and taiga forestland soils in a wide range of aggressive acidity (at pH between 5-8). And last but not least, a complex morphogenetic growth cycle; a tendency of great cell differentiation, adhesion and colonization of surfaces as well as a capability of aggregation that enables a bacterial population to grow under conditions when even solitary cells are able to multiply.

According to our data, in the presence of n-alkanes, rhodococci synthesize extracellular amino acids, including such basic (irreplaceable) ones as arginine, lysine, histidine and others.* Remarkably, the supersynthesis of amino acids is higher in the presence of propane. Collection strains capable of producing amino acids may be needed as bulk material for selection of forms synthesizing just one amino acid in amounts sufficient for commercial production.

Furthermore, rhodococcal cultures are also used in oil and gas prospecting. We have found individual dominant species of propane- and butane-oxidizing actinobacteria (R.rhodococcus and R.rubber) to be confined to oil pool outlines. A proximate analysis method has been

* Such amino acids cannot be synthesized within the organism and get in with food only.--Ed.

devised for locating indicator rhodococcal species involving the use of specific polyclonal immune serums.

Yet another biotechnologically useful capability: in the presence of liquid n-alkanes, rhodococci can synthesize surfactants decreasing the surface and interfacial water tension (up to 26-28 and 2-5 H/m respectively). Having a high emulsifying and oil removing activity, such surfactants display a wide spectrum of biological effects, for one, an express immunomodulating and anti-inflammatory action. Biosurfactants produced by rhodococci show good remedial effects as cleaners of oil-polluted soils (in bioremedial procedures)*. On this basis an ecologically safe technology of rehabilitating oil-contaminated soil and ground has been worked out and patented; adjusted to temperate and cold climate regions, it removes as much as 90 percent of oil in badly polluted places, and does this job within six weeks**.

We have obtained new evidence on the ability of cultivated rhodococcal species to actively accumulate caesium in the presence of n-alkanes. Our test results and selected strains can be used toward a biotechnological technique of purifying radionuclide- and oil-polluted industrial waters.

* Methods for water, ground and atmosphere purification with the use of the metabolic potential of biological objects like plants, fungi, insects, worms and other organisms.--Ed.

** The research collective headed by Irina Ivshina merited a prize of the federal government (2008) for the rehabilitation technology in polluted or otherwise damaged tracts of this country's northern regions.--Ed.

The practical employment of alkanotrophic rhodococci in different biotechnological areas calls for reliable methods of conserving their vital activity and original characteristics. Therefore adequate storage methods should be maintained for each particular culture. Our IGEM Institute has developed optimal regimes of cryoconservation of bacterial cultures (in cold). Their viability is estimated as long as 20 to 40 years.

Microbiologists have no definite yardstick in the differentiation of actinobacteria so much complex taxo-nomically. No "gold standard" here! Identification through the available determinants by a great number of characters is not feasible technically. That is why we have come up with an optimized pattern of species differentiation from a limited number of characters. Today it is being used with much success in broad taxonomic studies.

NOT A RESEARCH CENTER ONLY

Our depositary is also doing applied works involving the use of the collection gene pool of alkanotrophs. In particular, we have isolated an agent of immobilized cells from hydrocarbon-oxidating bacteria; we have a novel biopreparation as well as forms good for cleansing oil-polluted ground patches in regions of extreme climatic conditions. And we have many other good things up our sleeve, too.

Making use of the available biotechnological potential calls for cooperative effort. Our Institute is doing joint research with the Technical Chemistry Institute of the Perm Science Center (RAS Ural Branch) on problems implicated in the biotransformation of natural compounds; our partners are using our collection in such joint studies. Among our partners is also the Science Center of the Powder Material Science (Perm National Research Polytechnical University) concerned with biocorrection of the phase composition of high-porous multiphase ceramic materials and upgrading their operational characteristics. Our partners abroad include the University of Edinburgh (Scotland, Great Britain) involved with the synthesis of biosurfactants and land pollution, and Stratclyde University (Glasgow, Scotland, Britain) researching in the biological rehabilitation of oil-polluted soil and ground. On the domestic scene, we might as well name the Perm-based branch of Lukoil-Engineering, a major oil producer.

Our collection is also a science education center catering to budding biologists, for one, students of the Perm Biological Lyceum (junior college) and prospective undergraduates of the Department of Biology at Perm State National Research University; the most gifted among them take a graduate course at our Institute and defend their M.Sc. dissertations.

All bioresources of our depositary are open to users. It is affiliated with the World Fund of Microbial Collections, and is a member of the World Federation for Culture Collections (WFCC), and of an analogous organization in Europe; it has a computer database of its own. Information dealing with sustained strains is published in our regular catalogs. Our collection is actively employed in regional, federal and international programs, including the Seventh European Framework Program fulfilled jointly with colleagues from Belgium, Italy and Switzerland. Among our users are research collectives of this and other countries, experts in the fields of microbiology, biotechnology, ecology and medicine as well as industrial engineers. We are running short-term educational courses on the isolation, cultivation and identification of alkanotrophic organisms.

Unfortunately here in Russia we have no competitions for financial grants to support the collection business. Otherwise we might have been much better off. Its important role is still underrated, as seen from a recent case study on the condition of microbial collections in the Russian Federation; for lack of adequate state support some collections may go out of existence soon, while others have been irretrievably lost. Russia is the world's only country registering a negative growth dynamics in the number of deposited microbiological cultures. To overcome this downturn, as we see it, it is necessary to set up a wide network of microbiological resource centers and upgrade them to a national status; they should be getting regular long-term financial support. Apart from national centers organized on the basis of the well-established All-Russia Collection of Microorganisms and the All-Russia Collection of Industrial Microorganisms, it will be appropriate to form specialized national centers territorially, say, one in the Far East dealing with marine microorganisms, another in Siberia with a collection of luminous bacteria, and a third one in the Urals with a pool of genetic material. And so forth.

To ensure effective work in sustaining collections of a new generation we need a system of their objective identification and evaluation; we should find it out how useful such specialist activities are for the scientific community and users at large. Then we should draw a list of collections of national significance and establish an official coordinating center exercising supervising functions; we also need a Law on Microbial Genetic Resources. We need legal enactments protecting workers who make such collections and store them. Accordingly, law-making should be improved in such areas as access to microbial resources, protection of intellectual property rights and even-handed distribution of profits from resources used in biotechnological projects, their success depending on the correct choice of a particular microorganism.

The collection-making business, we hope, will recover and see its heydey. A warranty of that is the available collections at RAS biology-oriented research centers and the growing research potential of universities. And the long-awaited institution of the BIO-2020 development program for a period of up to 2020 sustains this hope of ours.