by Tatyana VOLOVA, Dr. Sc. (Biol.), Deputy Director, Institute of Biophysics, Siberian Branch of the Russian Academy of Sciences, Head of the Department of Biotechnology, Siberian Federal University, Yekaterina SHISHATSKAYA, Cand. Sc. (Med.), senior scientific worker of the same Institute, Oleg SHISHATSKY, economist-engineer, Siberian Federal University (Krasnoyarsk)

The technology for obtaining biocompatible biopolymers, completely resolved in biological media, has been developed using hydrogen bacteria at the Institute of Biophysics, RAS Siberian Branch. The first in the country experimental production of these polymers has been designed and launched in Krasnoyarsk. Use of these polymers is promising for tissue reconstruction in maxillofacial surgery, orthopedics and traumatology, in cell cultivation, and controlled delivery of drugs in diseases of various organs.

AN ADDITIVE TO RATION

By the present time the negative aftereffects of anthropogenic loading of the nature of our planet have reached a borderline, when uncoordinated economic activities can lead to irreversible changes in the biosphere in general*. How can this destructive process be suspended? Among the priority measures are development of closed cycle technologies, directed toward a complex processing of extracted resources, use of ecologically-pure energy carriers and materials involved in the biospheric turnover cycles. The development of these approaches is a task of basic sciences, specifically, of physico-chemical biology. Due to new data on the structure and function of living systems, we can design

* See: Yu. lzrael, "Threat of Climatic Catastrophe?", Science in Russia, No. 4, 2004.-Ed.

Hydrogen bacterial cells, realizing the "protein program" of macromolecular synthesis.

Polyhydroxybutyrate content in hydrogen bacteria varies from low "trace" concentration (1) to 20 percent (2), 40 percent (3), 90 percent (4) of the cell's dry weight.

complexes for the synthesis of a wide spectrum of valuable compounds.

Among the perspective producers are hydrogen oxidizing bacteria (hydrogen serves as a growth substrate for them). These microorganisms first became objects of practical interest in the 1970s in the USA and USSR; they were suggested to be used in space as a regenerative component of closed systems of man's vital activity*. Later on hydrogen bacteria were regarded as a potential source of fodder and nutritive protein. Comprehensive studies were unfolded for this purpose in the USA, Germany, USSR, and Japan. At the end of the 1980s and beginning of the 1990s, these unusual microorganisms again attracted the interest of scientists as one of the most effective producers of polyhydroxyalcanoates (alcanoic acid polyesters)—polypropylene analogs, which, in contrast to polypropylene, are capable of degradation in natural media.

Active studies of these bacteria were unfolded in Russia on the initiative of Academician Georgy Zavarzin. He headed the studies on isolation, classification, and description of representatives of this interesting microbiological group at S. Vinogradsky Institute of Microbiology, the Russian Academy of Sciences (Moscow) in 1970s-1980s. The scientist kindly presented several most productive strains of these bacteria to our Institute.

* See: O. Gazenko, A.  Grigoryev, V. Yegorov.  "Space  Medicine: Yesterday. Today, Tomorrow", Science in Russia, Nos. 3, 4, 2006.—Ed.

3 m³ fermenter is the main unit of experimental production of hydrogen bacteria biomass at the Institute of Biophysics, RAS Siberian Branch.

Due to numerous experimental data on physiology of hydrogen bacteria, accumulated at our Institute, and the knowledge of specific features of their metabolism under effects of environmental factors contributed to the creation of biotechnological systems, synthesizing various products of interest. For example, these microorganisms, cultivated in a complete nutrient medium (medium containing all necessary elements for the cell growth at a maximum rate), produce protein maeromolecules. Modification of these conditions leads to correction of the program and unbalanced cell growth. Thus, under conditions of high content of carbon and nitrogen deficiency they deposit carbon, synthesizing one of the alcanoic acid esters (polyhydroxy-butyrate—a thermoplastic polymer). By its basic characteristics this polymer is similar to polypropylene, but, as other polyhydroxyalcanoates, can degrade in biological media to the end products: carbon dioxide and water under aerobic conditions (in the presence of air oxygen) and to methane and water under anaerobic conditions.

Experimental production of protein on hydrogen was started in the 1980s at the Institute of Biophysics, RAS Siberian Branch. The characteristics of the product were studied in experiments on broiler chickens, hens, piglets, calves, and fur-bearing animals (polar foxes and minks). It was proved that this protein was highly valuable and fit for rations of agricultural animals.

PLASTICS THAT ARE HARMLESS FOR NATURE

The second process, our specialists have developed and realized with hydrogen bacteria, is a synthesis of degraded bioplastics (polyhydroxyalcanoates). These ecological materials should replace synthetic polymers, now widely used in many spheres of industry, medicine, communal sphere, etc. The volume of their production all over the world is tremendous, reaching 180 mln tons annually and increasing by 25 mln tons with every year. Half of this is used for the manufacture of packing materials, and hence, the bulk of this production is then disposed of, thus augmenting the total pollution of the planet. In countries with well-developed economy no more than 16-20 percent of synthetic plastics are repeatedly processed. Hence, the radical solution of the "polymeric waste" problem is creation and introduction of materials capable of degradation (under certain conditions) into components harmless for nature.

In a word, it is quite justified that studies of polymers of biological origin are becoming a priority task for the recent decade or so. The main purpose is a search for and study of new materials of this kind and creation of the base for construction of biological systems synthesizing polymers with preset characteristics.

Among the biodegraded substances, including those of medical use, which are actively developed and already applied, are aliphatic polyesters, polyamides, segmented polyester urethanes, lactic and glycolic acid

polymers, and, from recent time, polyhydroxyal-canoates. The latter compounds belong to a class of natural macromolecules (fatty acid hydroxyderivative polymers), synthesized by bacteria on various substrates (sugars, organic acids, alcohols).

It had been found that under certain conditions hydrogen bacteria were capable of "producing" polymers of a different chemical structure. For example, we developed the synthesis of polyhydroxybutyrate (mentioned above) with a high output (up to 80-90 percent of the cell substance weight) on various substrates: sugars, plant hydrolysates, acetate, hydrogen mixtures with carbon dioxide. Using bacterial strains capable of growing in the presence of carbon monoxide, we realized for the first time in biotechnological practice the synthesis of polymers on brown carbon gasification products. New composition substances were obtained: hydroxy-butyric, hydroxyvaleric, and hydroxyhexanoic acid copolymers.

Our scientists studied the problems of appropriate engineering realization and wide-scale production technologies. The first in Russia experimental production of biopolymers, satisfying the needs of all research and pilot institutions of the country, was created within the framework of the project of International Research and Technological Center in collaboration with Bio-khimMash Research Institute in Moscow.

However, to obtain a new material is just one part of the problem. The next stage, which we have also passed, is reprocessing of the polymer to obtain specialized articles—monothreads, elastic films, microparticles, bulky constructions—by various methods (casting from solutions, pressing at ambient and high temperatures, extrusion from melted mass). It was found that these plastics were fit for paper lamination, fertilizer and grain enveloping, deposition of chemical toxins, construction of ultrahigh frequency transmitters in radio-electronics, etc.

"BIOPLASTOTANE" AND ITS POTENTIALITIES

It is noteworthy that polymers for biomedical use, for example, for creation of systems capable of reproducing biological functions of human body, are required most of all. Hence, biological man-made organs and tissues on the base of these polymers are now actively developed. However, despite the progress in biotechnology, there are still no materials completely compatible with a living organism. Scanty choice of biopolymers precludes their wide use. The problem of regulation of biopolymer functioning and destruction processes in a living organism also inhibits their application.

We should like to emphasize that special attention in Krasnoyarsk is paid to studies of the biomedical characteristics of polyhydroxyalcanoates. Specialized medical articles made with the use of highly purified specimens of these materials are offered for medical use. Polymeric constructions of different form and weight

have been introduced in the muscle, bone, bloodflow, and viscera of experimental animals. For the first time the reaction of tissues of different structure to implantation of polyhydroxyalcanoates have been studied. Their high biocompatibility and hence, the possibility of implantation for long periods without development of negative reactions of the blood system, cells, tissues, and the organism as a whole have been demonstrated. It was established that these materials could be effectively used as endoprostheses, bone-replacing implants, and matrices for drug deposition and delivery. Certificates at the Rospatent were received and the Bioplastotane trademark for polyhydroxyalcanoates and biomedical articles from this material was registered in 2006.

Using this polymer in solutions, emulsions, and as a powder, we studied the structure and characteristics of two- and three-dimensional matrices in the form of elastic transparent films, membranes, microparticles, sponges, bulky compact and porous constructions. It was found that the generally accepted methods for matrix sterilization could be used without changes in its structure, loss of strength, and deterioration of the surface adhesive characteristics. The absence of cytotoxicity of these matrices and their fitness for in vitro cell cultivation were demonstrated in cultures of fibroblasts, liver, bone, and skin cells.

Durable monofilaments were obtained by gel-technology and extrusions from the melt, and their fitness for reconstructive operations on the intestine and bile ducts was demonstrated. We constructed volumetric compact and porous bone-replacing implants from hybrid composite polyhydroxybutyrate/hydroxyapatite

with different proportions of the components. Studies of the primary osteoblast (bone cell precursors) culture derived from embryonal bone marrow connective tissue cells have shown that the composite is biocompatible and promotes bone tissue formation.

A series of experimental samples of Bioplastotane were offered to medical institutions of the country for studies of the probable application of the material for treatment of tissue and organ defects in patients (in reconstructive technologies of orthopedics and traumatology, maxillofacial surgery). Cooperation with the Department of New Biomaterials of V. Shumakov Institute of Transplantology and Artificial Organs, Ministry of Public Health of the Russian Federation (Moscow), headed by Viktor Sevastyanov, Dr. Sc. (Biol.), has become particularly fruitful. It has resulted in the issue of the first Russian monograph on biotechnology of polymers of this class.

Thus, the technological and economic analysis, carried out by the RAS Siberian Branch Institute of Biophysics in cooperation with the Siberian Federal University, has revealed an acute need in biopolymers, particularly in materials for medical use. The need in them is growing constantly and today amounts to more than 400,000 tons annually. The suggested business plan has demonstrated the efficiency of their manufacture using our technology.