Libmonster ID: JP-952
Author(s) of the publication: Yuri RAMAZANOV, Andrei REPKOV,Ivan KOSYUK

by Yuri RAMAZANOV, Director General;

Andrei REPKOV, Chief Designer;

Ivan KOSYUK, Technical Director "SAYANY" CJSC, Novosibirsk

The Siberian Innovation Company has created a unique gas-vortex biological reactor that has no foreign analogies. Its design is based on new technological principles, which even make it possible to breed stem cells in it. In the judgment of specialists, this design is one of the most promising in the field of Russia's high tech. Patents obtained in the USA and in a number of European countries, medals and diplomas of large international exhibitions bear record to it.

The easiest biotechnical techniques were mastered by people as far back as before Christ. On the land of ancient Sumerians (now Iraq), for example, as early as 6,000 thousand years ago people applied one of them: fermentation. In tubes, wineskins, amphoras, they obtained products of fermentation - beer, wine, koumiss, dough for porous bread. As far as special bio-technological equipment is concerned, its appearance is related to much later periods. The Petri dish has become the first in this row. In 1880s it was especially developed for Robert Koch* by Julius Richard Petri, his fellow-professional. This flat glass jar with a cap is used until now by microbiologists all over the world. It was exactly this device that was used by Koch when he first separated the culture of tubercle bacillus in 1882.

The next push for developing new equipment was the discovery of penicillin in 1928 by British scientist Alexander Fleming (Nobel Prize winner of 1945). The antibiotic has quickly proved the necessity of its large-scale production, however, it took much time to achieve this goal. Only in 1943 in the USA they found its efficient producer - Penicillium chrysogenus fungus, which is capable of reproducing itself not only on the surface of the cultural media, but inside it as well. It was precisely a "hunt" for penicillin that resulted in the creation of biological reactors (fermenters) - devices in

* Koch. Robert (1843 - 1910) - German microbiologist, one of the founders of modern bacteriology and epidemiology, Nobel Prize winner of 1905, foreign corresponding member of the Petersburg Academy of Sciences since 1884. - Ed.

стр. 15

Difference of gas-vortex fermenters (a) from fermenters with agitator (b). A source of undesirable turbulences is removed from liquid medium.

which aerobian or anaerobic breeding of various kinds of biomass was implemented: cells of animals and insects, plants, microorganisms, fungi. This process requires the observance of many conditions. Apart from maintaining temperature and other conditions the achievement of reactor efficient operation requires permanent mixing of cultural fluid contained in it.

Apparently, it would be difficult to mention a field where the process of mixing does not exist. Yet there are some of them in which it is the basis of production. They include biotechnology, chemical synthesis and food industry. Two techniques are well-known and widely used. The first includes the use of a mechanical device immersed into a fluid (the easiest example of such "agitator" is a spoon in a cup of tea). The second consists in gas blowing through a fluid in air-lift apparatus. In reactors with a mechanical agitator more than 70 percent of power intake is spent for overcoming the forces of medium resistance, in this case the fluid is heated excessively, which is harmful. Excessive heat should be removed, and this requires additional expenses.

Drawbacks are present in air-lift bioreactors as well: due to insufficient mixing they are not suitable for viscous media; air bubbles that come to the surface and contact live cells contained in the fluid explode and cause the cells a traumatic cavitation blow. Abundant foam-formation does not make it possible to use the whole apparatus space, and the use of chemical antifoamer decreases the quality of a final product.

By the way, the majority of reactors used over the world are a combination of these types of apparatus, however, a symbiosis like this does not rule out organic defects related to the insufficiency of superficial mass exchange, availability of turbulent and stagnant zones, high energy consumption, poor characteristics, when working with viscous media.

Biotechnological production is constructed in such a way that biomass is increasing gradually: from smaller volumes at the initial stage to bigger ones in the end. Biological cultures may intensively develop in a fermenter, if only they feature a certain initial concentration. To make a "start-up" the quantity of cells in the inoculation dose should be in the order of 300 thou-

стр. 16

sand per ml, and only after that we may expect the 4 to 6 times increase in their number in the apparatus. Then the growth stops, since metabolites produced by the cells prevent their further generation. And the biomass, when diluted to the initial strength, may serve as an inoculation dose for 4 to 6 fermenters similar in volume or for a plant with the volume 4 to 6 times as large. It is easy to calculate how many (and what kind of) fermenters will be required for starting up an apparatus with the working volume of 1 m3. The chain is as follows: 1.6 → 8 → 40 → 200 → 1,000 l.

The question arises: is it acceptable to have 2 apparatus (say, 40 and 1,000 liters) instead of 5, but filling them fractionally at the initial stages of culturing, i. e. adding culture broth into them as the number of cells in them is growing? Unfortunately, it is not possible when using apparatus of traditional types. Fermenters with agitator, as a rule, operate in optimum mode only with a strictly preset level of fluid. Its variations cause the change in the flow dynamics and, accordingly, in culturing conditions, and consequently, to undesirable results.

As a whole the existing types of reactors were suitable for researches until they started to perform experiments with cells easily susceptible to traumas (including primary ones and hybridomas*) and became interested in problems of extra-cellular protein synthesis and in culturing the cells of insects. Standard apparatus turned out to be of little use for these objects.

A certain way out of the situation was found. Reactors were created with rotating bodies, with sail-type agitators made of soft synthetic materials, even with magnetic particles in suspension operated by a rotary magnetic field. However, gimmicks like this are suitable in laboratory conditions and in not in large-scale production. Of course, the new designs of fermenters have been permanently developing: from 1976 to 1996 more than three hundred patents were issued for various kinds of fermenters in different countries. Nevertheless, the engineering solutions included old techniques of mixing that did not go beyond habitual patterns. Although, as it turned out, a brand new version of realizing this process is possible.

Late in the 1980s an employee of S. Kutateladze Thermal Physics Institute, Dr. Sc. (Tech.) Vasily Kislykh, who was engaged in studying vortex processes, after his transfer for the work at the All-Union Research Institute of Molecular Biology, which was established in the settlement of Koltsovo (today - Scientific-Production Association "Vektor") near Novosibirsk, put forward the idea of creating an apparatus that used an organized and controlled air-eddy for mixing a culture medium. Here the proposal was appreciated, since in case of a success there appeared a possibility of obtaining a reactor where injury-susceptible cells

* Hybridoma - a hybrid obtained by merging a normal cell with a transformed one. It features a capability of synthesizing protein, for example, an antisubstance, and the unlimited growth efficiency. Monoclonal antibodies synthesized by hybridomas are used in many fields of biology and medicine. - Ed.

стр. 17

might be cultured. They managed to construct several experimental prototypes but the work was closed down due to insufficient financing. The very idea turned out to be so promising that a team of engineers and designers, in which Kislykh was also incorporated, tried to bring it to embodiment at their own risk. It resulted in the appearance of an absolutely new type of apparatus - gas-vortex non-gradient reactors.

It is important in principle that a mixing device in these apparatus is not immersed into fluid, and this means that negative consequences caused by this factor are absent. The fermenter is essentially a temperature-controlled reservoir with a centrifugal activator installed above the surface of the culture medium; in the course of rotation the activator forms an intensive air swirl. With the aid of a special floating ring plate the swirl is projected into the fluid. Mixing is ensured at the expense of pressure difference above the fluid surface and surface frictional force of the air-eddy.

Three-dimensional motion of a type of "rotating vortex ring" (quasi-stationary flow with an axial counter-flow) is created in the fluid. This ensures gentle, rather efficient mixing without foam-formation, occurrence of hydraulic impacts, cavitation, highly turbulent and stagnant zones. Ninety eight percent of power in a gas-vortex reactor is used directly for mixing, the insertion power is distributed over the entire space uniformly, so no micro-zones of local overheating are formed. Direct energy consumption is reduced 7 to 10 times compared with other installations of similar purpose. The device is capable of managing even more viscous liquids. In our joint work with the Gubkin Institute of Oil and Gas (Moscow) polysaccharides were obtained in a fermenter, and their viscosity was 1,270 times higher than that of water. Gas swirl is also an efficient antifoamer. The flow dynamics of the fermenter just slightly depends on the level of fluid in it.

Depending on process requirements, the degree of apparatus filling varies from 10 to 90 percent of its actual volume, thus the number of installations of intermediate dimensions may be reduced. In practice it means that to start into operation a 1,000-liter fermenter equipped with gas-vortex mixing only one 20-liter bioreactor of the same type is required.

A serious problem at introducing many laboratory designs into production consists in the complexity of process reproduction in conditions of increased volumes of culturing. This is particularly noticeable during the work with sensitive cells (hybrid, embryonic ones, etc.), and this is related to the hydrodynamics of the mixing process in existing bioreactors - it changes considerably subject to apparatus volume. When gas-vortex method of mixing is used, such problems do not exist. Nearly ideal cell culture conditions result in the reduction of production process.

When studying the possibilities of a new fermenter, experiments on cultivating embryonic cells, hybridomas, cells of insects, plants, fungi, yeast, bacteria were carried out in various research establishments. And all of them were successful.

стр. 18

For the first time embryonic corpuscles* were cultivated from the culture of embryonal stem cells of man** in a gas-vortex fermenter on the basis of the RAS SB Institute of Cytology and Genetics by Dr. Sc. (Biol.) Sergei Kiselev and Cand. Sc. (Biol.) Maria Lagarkova. In structure the said corpuscles are similar to a bilaminar egg-shaped cylinder of the 6-day embryo of a mouse. This similarity in the evolution of a biological process makes it possible to use them as a model system for studying early stages of embryonal evolution. Today they are necessary for obtaining specialized cells - neuronal, hemafacient, insulin-producing and a number of other cells-by way of oriented embryonization, since they contain stem cells of all three germ layers. On the basis of gas-vortex reactors it is planned to create a program-apparatus complex for reproducing/maintaining founder early colony-formings and embryonic corpuscles. This will solve the problem of obtaining them in necessary quantities for medical and research purposes.

One of the promising directions is the use of gas-vortex reactors in food industry. At present Russia imports 62 percent of sugar consumed in the country being its largest importer in the world; however, the products of this line might be obtained from domestic feed (including low-grade grain of wheat and rye of Siberia) by way of fermentative biological conversion of starch. Introduction of grain complex processing is largely slowed down by extremely low efficiency of equipment used in starch hydrolysis. As was demonstrated by the results of pilot-plant equipment with the volume of 2,500 liters, gas-vortex reactors are quite suitable for settling this problem as well.

Probable field of application of the new-type apparatus is rather extensive. Design simplicity and low cost of manufacture of gas-vortex fermenters with the volume below 1 liter make it possible to fabricate them in a disposable version, and this, in turn, permits to develop individual biological forms and medicines adapted to a concrete organism under the usual hospital conditions. For example, it is possible to recover the bacterial population of intestinal tract which that perishes from antibiotics action, by way of cultivating own microorganisms of a person undergoing treatment.

In the end we would like to note the following: since in the course of operation the activator directly contacts gaseous medium only, gas-vortex apparatus can be efficient for the process of mixing high-corrosive liquid media. Experiments carried out show the perspective nature of such bioreactors in chemical synthesis.

Illustrations supplied by the authors

* Embryonic corpuscles present a transition phase from early colony-formings capable of giving birth to the multiplicity of cell lines that are ready to form fully adult cells, for example, cells of nerve tissue or muscular tissue. - Auth.

** The lines of embryonal stem cells of man are obtained from the inner cell mass of pre-implantation blastocysts, which represent a phase of germ development in the process of oosperm fission and remain unclaimed after extracorporal fertilization. - Ed.


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