Continuous culture device with mobile vessel, which allows for taking most suitable cell versions

FIELD: chemistry; biochemistry.

SUBSTANCE: device for speeding up reproduction, through faster reproduction and/or increased reproduction output of living cells in a suspension or any cultured organisms through a natural selection process has a flexible sterile pipe 7 with culture medium. A system of movable clamping apparatus 3, 4, 5 divides the pipe 7 into separate zones, containing spent culture (downstream zone), growing culture (growth compartment) and fresh growth medium (upstream zone). In the device there is an apparatus 13 for moving gates and the pipe such that, part of the growth compartment and the culture associated with it can be shut off by clamping apparatus and separated from the growth compartment. That way, part of the pipe which contains unused medium can be linked with part of the culture and medium associated with it, already present in the growth compartment.

EFFECT: realising a method with high reproduction output of living cells or cultured organisms.

36 cl, 10 dwg

 

The technical field to which this invention

The described invention provides a method and device to enable selection of living cells with an increased rate of reproduction, and specific metabolic properties in liquid or semi-solid medium. During the process of selection (adaptive evolution) genetically variant organisms (mutants) occur in the population and compete with other variants of the same origin. Variants with the highest rate of reproduction increases the relative proportions over time, leading to population (and individual organisms with an increased rate of reproduction. This process can improve the performance of the organisms used in industrial processes or for theoretical purposes.

The level of technology

Selection for increased rate of reproduction (i.e. fitness) requires sustained growth, which is achieved by regular thinning of the growing culture. In previous prior art is performed in two ways: serial dilution and continuous cultivation, which differ primarily in the degree of cultivation.

Serial culture includes repeated transfer of a small amount of grown culture in a much larger vessel containing fresh culture medium. When cultured the organisms grow to saturation in this new vessel, this process is repeated. This method is used to achieve the longest demonstrations supported culture in the literature (Lenski and Travisano: Dynamics of adaptation and diversication: a 10,000-generation experiment with bacterial populations. 1994. Proc. Natl. Acad. Sci. USA 15:6808-14), in experiments, which clearly showed persistent improvement of the rate of propagation over a period of several years. This method is usually performed by manual, with a significant investment of labor, and it is vulnerable to contamination due to exposure to outdoor environmental conditions. Serial culture is also inefficient, as described in the next paragraph.

Sample rate or the rate of improvement of the rate of reproduction depends on the size of the population (Fisher: The Genetical Theory of Natural Selection. 1930. Oxford University Press, London, UK). In addition, in a situation similar to the serial transfer, where the population size is subjected to rapid fluctuations, the selection is proportional to the harmonic mean (N) of this population (Wright: Size of population and breeding structure in relation to evolution. 1938. Science 87: 430-431), and, therefore, can be approximated using the lowest population during this cycle.

Population size can be maintained and, therefore, the selection can be made more effective through continuous culture. Continuous culture, as distinguished from serial dilution, includes a lower relative about what they eat, so a small part of the growing culture is regularly replaced with an equal volume of fresh medium for cultivation. This process maximizes the effective population size and increase its minimum size during cyclic cultivation. The device, which makes possible continuous culture, called "chemostate if breeding occur at specific time intervals, and turbidostat"if breeding is done automatically when the culture grows to a specific density.

For simplicity, both types of devices will be further consolidated under the name of "chemostat". Chemostat were invented simultaneously by two groups of researchers in the 1950s (Novick &Szilard: Description of the chemostat. 1950. Science 112: 715-716) and (Monod: La technique de la culture continue - Theorie et applications. 1950. Ann. Inst. Pasteur 79: 390-410). Hemostat used to demonstrate short periods of rapid improvement of the rate of reproduction (Dykhuizen DE. Chemostats used for studying natural selection and adaptive evolution. 1993. Methods Enzymol. 224:613-31).

Traditional chemostat not able to sustain long periods of selection for increased rate of reproduction due to unintentional selection of resistant breeding (static) options. These options are capable of withstanding breeding by attaching to the surface of hemostat and thus win the competition less sticky individual is MOU including those that have a higher rate of reproduction, thus eliminating the intended purpose of this device (Chao & Ramsdell: The effects of wall populations on coexistence of bacteria in the liquid phase of chemostat cultures, 1985. J. Gen. Environ. 131: 1229-36).

One way and hemostatically device (the Genetic Engine) were invented in order to avoid resistance breeding in continuous culture (U.S. Patent 6686194-B1, filed INSTITUT PASTEUR [FR] & RUPERT MUTZEL [DE]). This method uses a controlled valves migration for the periodic movement of culture between the two chemostate that allows you to sterilize and wash each of them between periods of active growth culture. These regular cycles of sterilization to prevent the selection of resistant breeding options by means of their destruction. These method and device to solve the considered problem, but require independent complex manipulation of multiple fluids in a sterile (sealed) environment, including one liquid (NaOH), which is caustic and potentially very reactive, quickly destroying the valves and cause problems of retention and elimination of waste.

The invention

Thus, the subject of this invention is the provision of improved (and completely independent) method and device for the continuous culture of organisms (including bacteria which, archaebacteria, eukaryotes and viruses) without interferences caused resistant breeding options. Like other hemostats this device provides a means for regular cultivation grown fresh culture medium for cultivation and means for gas exchange between the culture and the external environment, sterility and automatic operation or as hemostat, either as a turbidostat.

This invention is designed to achieve this task without transferring any liquid, including sterilization or washing. It is a specific advantage of the present invention relative to the previous prior art because it eliminates the dangers and difficulties associated with sterilization and washing, including the retention and transfer of complex fluids, including corrosive solvents.

Continuous culture is achieved in a flexible sterile tubing filled with growing medium. The environment and the surface of this camera are static relative to each other, and both regularly and simultaneously replaced by a peristaltic movement of the pipeline through the "gate" or the point at which the pipeline sterile split clamping devices (clamps)that prevent PE is Emesene cultured organisms between areas of the pipeline. Can also be added (optional) UV-gate above and downstream from the culture vessel for additional reliability.

These method and apparatus are also improved in comparison with the previous prior art, as they are continuously and not intermittently produce selection against insertion resistant breeding options to the surfaces of hemostat, since the replacement of the affected surfaces are manufactured in tandem with the process of cultivation.

This pipeline is divided temporarily so that there are zones that contain saturated (fully grown) culture, areas that contain a fresh environment, and the area between these two zones is called the growing chamber, in which the grown culture is mixed with fresh medium to obtain cultivation. These gates are periodically opened from one point on the tube and replaced at another point, so that the grown culture with its associated surface of the growth chamber and attached static organisms removed from the growth chamber and replaced with fresh medium and fresh surface of the camera. Using this method static options specifically counter-selected by the destruction of the zone in which the selection (camera height).

Brief description of the graphical material

Neisseria the it and non-limiting way, one possible configuration will include several components, described next. Further, this invention is explained by exemplary image on the basis of the preferred option with reference to the figures, in which:

Figure 1 depicts a General view of the possible configurations of the device, in which:

(1) represents a flexible conduit containing different zones of the device, which are: fresh environment upstream (7), the growing chamber (10), Luggage for sampling (11) and the removal area grown culture (15);

(2) represents a thermostatically controlled box, allowing you to adjust the temperature in accordance with the conditions defined by the user, and in this box can be placed:

a. the growing chamber (10),

b. Luggage sampling (11),

c. are higher in the thread clamping devices (3)defining the beginning of the growth chamber (10),

d. below the thread clamping devices (4)defining the end of the growth chamber (10) and the beginning of the camera for sampling (11),

e. the latter are lower in the thread clamping devices (4)defining the end of the chamber for sampling (11),

f. the turbidimeter (6), allowing the user or the automatic control system to monitor the optical density of the growing culture and to manage the control system on the basis of feedback (13), making possible the movement of the pipeline (1) on the basis of the lotnosti culture (turbidostat),

g. one or more mixers (9).

It should be noted that the components of the device are listed in a-g may also be located outside thermostatic Boxing or may be present in the absence of a thermostatic box.

(7) represents a fresh environment in unused flexible pipe,

(8) represents the tank, loaded full with fresh medium pipeline for distribution of fresh medium and during pipeline operations

(12) represents an optional gate ultraviolet radiation,

(13) represents the control system, which may consist of a computer connected with the means of communication with various interfaces monitoring or operations, such as turbidimetry optical density, devices for measurement and control of temperature, motors, agitators and inclination, etc. that make possible the automation and regulation of operations

(14) represents an optional tank waste removal, on which is wound the pipeline that contains the deleted filled crop pipeline

(15) is deleted grown culture, located downstream from the chamber for sampling.

Figure 2 depicts two possible States of this device, which is an example of the fact that a thermostatically controlled box (2) other net assets the ti device associated with the chamber for culture, can be tilted to various degrees for the purposes of mixing, for the purpose of circulation and removal of gas and for the purposes of ensuring the removal of granular (aggregated) cells, which can avoid breeding from settling to the bottom.

Figure 3-9 represent Flex (1), placed in a thermostatically controlled box (2) and inserted through the clamping device (3), (4) and (5), through which the pipeline will stop during all stages of the process and through which the pipeline will move in accordance with its peristaltically movement.

Figure 3 depicts symbolically the state t0 of the device in which all zones Flex full with fresh medium before the introduction of organisms intended for continuous cultivation.

Figure 4 depicts symbolically the condition T1 Flex directly after the introduction of the strain of the organism.

Figure 5 depicts symbolically the state T2 of the device, which is the cultivation period, during which the culture is growing in the area defined by the growing chamber (10), a limited clamping devices (3) and (4).

Figure 6 depicts symbolically the status of the T3 device, directly after the first peristaltic movement of the pipeline is associated environment, which determines the beginning of the second cycle of cultivation, the introduction of fresh pipeline and environment through movement of clamping devices (3) simultaneously with the transfer of the equivalent volume of the pipeline, the environment and cultures of the growth area of the culture (10) and into the chamber area for sampling (11) by moving the clamping devices (4).

It is important to understand that the pipeline environment in the pipeline, and any culture that has grown up in this environment, all move together. The movement of fluid occurs because of fresh medium and grown culture mixed together by stirring in the chamber area of growth.

Figure 7 depicts symbolically the state T4 of the device that is the second cultivation cycle; during this cycle of organisms that remain in the cell growth after the peristaltic movement of the pipeline can now grow with the use of nutrients provided in fresh medium, which is mixed with the remaining culture during this stage.

Figure 8 depicts symbolically the state T5 device, directly after the second peristaltic movement of the pipeline and its environment, which determines the beginning of the third cycle of cultivation, the introduction of fresh pipeline and environment through movement in the mouth 3 simultaneously with the transfer of the equivalent volume of the pipeline, environment and cultures of the area of culture growth (10) and into the chamber area for sampling (11) by moving the clamping devices (4).

Figure 9 depicts symbolically the T6 condition of the device, which is the third cycle of cultivation; this stage is equivalent to the condition T4 and points to the recurring nature of subsequent operations. Samples selected organisms can be removed at any time from the zone camera for sampling (11) using a syringe or other device to retrieve.

Figure 10 depicts a possible profile of the teeth defining the clamping devices in a configuration that consists of two abutting chain teeth, clamps Flex. Clamping devices could also be determined by pressing a single chain teeth to floating strap, removable clamps or other mechanisms that prevent the movement of organisms through these clamping devices and which can alternate way to fit and be disposed in different positions along the pipeline.

Information confirming the possibility of carrying out the invention

The basic operation of the device depicted in figures 3-9.

One possible configuration of this device is shown in figure 1, in the form in which it is after downloading a fresh pipeline sterilin the th environment /shown as environment, divided in zones A-H clamping devices (3), (4) and (5)/.

Inoculation of this device the selected organism can be achieved by the introduction of the organism into the growing chamber (3) via injection (figure 4, area B). Then this culture give rise to the desired density and begin continuous cultivation (figure 5).

Continuous cultivation is repeated movements split clamping devices zones of the pipeline. This includes simultaneous movement of the gate, pipeline, environment and any culture in the pipeline. The pipeline will always move in the same direction; unused pipeline containing fresh medium (hereinafter referred to as being "upstream" from the growth chamber (7)), will move into the growing chamber and mixed with the remaining culture here, providing a substrate for further growth of the contained organisms. Before introduction into the chamber area of growth in this environment and associated piping will be maintained in sterile condition by separation from the growth chamber located upstream clamping devices (3). Used pipe containing grown culture, will simultaneously move "downstream" and separated from the growth chamber located "downstream" clamping lighting is tions (4).

The configuration of clamping devices is not a specific moment of this patent application. For example, in a specific configuration, clamping devices can be constructed through the same chain of multiple teeth, moving at the same time, or, in another configuration, separated by individually synchronized circuits, as shown in figure 1. Clamping devices can consist of a system made of two chains of teeth, clamping the pipe coupling as described in figure 10, in order to avoid contamination between zones G and H of the pipeline, through the accuracy of the inner surface between the teeth. In another configuration sterile clamping devices can be obtained by pressing one of the chain teeth to the side of the pipe and thereby a strong pressure pipeline to a fixed chassis (chassis), along which the pipeline slides during its peristaltic movement, as shown in figures 3-9, grades 3, 4 and 5.

A thermostatically controlled box (2) is obtained using the already known means, such as a thermometer associated with heating or cooling unit.

Aeration (gas exchange), when it is necessary for the growth of cultured organism or in accordance with the plan of the experiment, before thetsa directly and without mechanical assistance through the use of gas permeable tubing. For example, without limitation, a flexible gas-permeable tubing may be made of silicone. Aeration can be achieved through gas exchange with the surrounding atmosphere or by gas exchange with artificially certain flair (fluid or gas), which is in contact with the growing chamber or with the whole hemostats. If you want to use in the experiment of anaerobiosis this Flex can be impermeable to gas. For example, without limitation, a flexible impervious to gas piping may be made of silicone or may be treated with silicone.

For anaerobic conditions, the evolution of the area of the pipeline can also be made in a specific and controlled atmospheric zone to control the dynamics of gas exchange. This can be achieved either by performing sealing thermostatically controlled box that he was impervious to gas, and then the introduction of a neutral gas, or by placing the device in a location with controlled atmosphere.

Counter-selection static options perform the replacement of the surface of the growth chamber together with the growing medium.

This device is additionally designed to operate in different orientations relative to gravity, i.e. in inclined positions, as shown in figure 2, in the range up to 360°

Resistant breeding options can avoid breeding adhesion to each other, and not to the wall of the chamber, if aggregated cells can be deposited above the stream and thereby to avoid removal from the camera. Thus, it is desirable that the pipeline was usually tilted downwards, so that the aggregated cells will fall in the direction of the zone that is to be removed from the growth chamber during the cycle of the pipeline. This configuration includes tilt the device so that the gate located downstream, are below the clamping devices located upstream, relative to gravity.

Luggage for cultivation may be a reduced pressure or excess pressure in accordance with the terms chosen by the experimenter. Can be used in different ways and the pressure correction. For example, the application of vacuum or compressed air to fresh medium and the pipeline through his lying upstream end and through the growing chamber; another way of reducing the pressure or increasing pressure line can be made alternating compression or locking the pipeline upstream from the growth chamber.

When the environment contained in the gas-permeable tubing, in this environment, it may cause air bubbles. They will rise to the top of the hermetically closed zone of the pipeline and become trapped there, while moving in this zone (and determine its gate) will not release this zone or chamber growth chamber for sampling or end point of hemostat (figures 6, zone D-C, b or a, respectively). If the device is tilted downward, such bubbles will accumulate in the cell growth chamber for sampling and push culture. This device is designed for periodic tilt up during the displacement cycle of the pipeline, which allows you to remove the accumulated gas from these cameras.

Sloping move of this device and/or shaking the camera growth through external device (9) can be used to reduce aggregation of cells in the cell growth. Alternatively, one or more stirring rods can be included in the pipeline filled with fresh medium before sterilization and mixed under the action of magnetic fields during operations with the culture.

Proportional to the length of the zones of fresh medium, as defined above flow gates, in comparison with the length of the culture of the camera will determine the level of dilution that can be achieved during the cycle.

The frequency of breeding can be defined either by timing (function hemostat)or regulation by the type of feedback, whereby the density of the culture in the cell growth was measured by turbidimetry (Figo is a 1 - mark 6), and the breeding cycle occurs when the turbidity reaches a threshold (function of thermostat).

Luggage for sampling makes possible the extraction of cultures for analysis of the experiment, collection of organisms with improved growth rate for further cultivation, storage or functional use or for other purposes, such as the expense of the population, checking the chemical composition of the environment or the pH of the grown culture. To achieve permanent monitoring of the pH inside the cell growth pipeline may include by construction line indicator pH, sealed/inlaid in the wall of the pipeline.

Any form of liquid or semi-solid material can be used as a medium for the cultivation of this device. The ability to use semi-solid substrates for cultivation is a significant progress in comparison with the previous prior art. Growing medium that will determine metabolic processes, improved method of selection, can be selected and set by the user.

If necessary, this device may contain multiple growth chamber, so that the clamping devices located below in the course of the stream from the camera growth, become clamping devices, are placed the above in the course of the flow to the other chamber growth. This could, for example, to allow the cultivation of one organism separately in the first chamber, and then use it as a power source for the second body (or virus) in the second chamber.

This device and method allows researchers and developers of the product to perform the evolution of any strain of cultured living cells in suspension through sustained growth (continuous culture); the resulting improved body can be a new strain or species. These new organisms can be identified by mutations acquired in the course of cultivation, and these mutations can provide the possibility of differences between these new organisms on the characteristics of the genotype of their predecessors. These apparatus and method allows the researcher to select new strains of any living organism by splitting individuals with superior rate of reproduction, through a process of natural selection.

1. Device for increasing the rate of reproduction (through increased speed reproduction and/or increased reproductive output) of living cells in suspension or any cultured organisms through a process of natural selection that contains:
a) a flexible sterile tubing containing culture medium,
b) the roaming system of clamping devices, each of which can be in open or closed positions, moreover, these clamping devices located so that they are able to divide the pipeline into separate zones containing spent culture (zone downstream), growing culture (growth chamber) and fresh culture medium (zone upstream),
(c) means to move the clamping fixtures and piping in such a way that a part of the cell growth and the associated culture can be disabled with clamping devices and separated from the growth chamber, and thus that of the fresh pipeline containing unused medium can be connected with a part of this culture and its associated environment, already present in the growing chamber, and each of these clamping devices is not moved relative to the pipe when the clamping device is in the closed position.

2. The device according to claim 1, in which the pipeline is flexible to allow tightening and separating cells.

3. The device according to claim 1, in which the pipeline is a gas-permeable, for example, consisting primarily of silicone, to allow gas exchange between the cultured organism and the external environment, in accordance with the type of experiment.

4. The device according to claim 1, in which the pipeline is gas-tight to prevent gas is BMENA between the pipe and the outside environment, if this experiment requires anaerobiosis.

5. The device according to claim 1, in which the pipeline is transparent or translucent to allow turbidity measurements.

6. The device according to claim 1, in which the pipeline camera growth and the associated environment and culture can have a reduced pressure or excess pressure relative to the atmospheric environment, if it is necessary for the experimental requirements.

7. The device according to claim 1, in which the pipeline allows you to measure the pH of the medium through the inclusion of the pH indicator in the composition of the pipeline or its sheath.

8. The device according to claim 1, in which the pipeline camera growth and the associated environment and culture can be heated or cooled depending on the conditions of the experiment.

9. The device according to claim 1, in which the pipeline camera growth and the associated environment and the culture can be maintained stationary or mixed by any known method.

10. The device according to claim 9, in which the pipeline may include one or more stirring rods for the purpose of mixing.

11. The device according to claim 1, in which zone of the pipeline can be made in a specific and adjustable atmospheric zone to control the dynamics of gas exchange.

12. The device according to claim 1, in which the pipeline camera growth and associated with h is m environment and culture can be tilted up or down to remove aggregated cells, either upwards to remove the air by means of the features described in claim 1 (C).

13. Method of increasing the rate of reproduction (through increased speed reproduction and/or increased reproductive output) of living cells in suspension or any cultured organisms through a process of natural selection, including: (a) provide a source of culture described in the cell growth by sterile injection starter culture (fermentation) in sterile tubing containing sterile culture medium; (b) maintaining growth conditions according to the experimental requirements; (c) after a certain period of time and the associated growth of culture correcting the position of clamping devices thus to move equal parts of fresh medium and grown culture (respectively in the zone and the zone defined as the growing chamber, allowing the remaining part of the grown culture mixed with the introduced part of the fresh environment and continue growth; (d) the reproduction stages b) and c) until the end of the experiment with continuous culture and selection of variants with increased rate of reproduction; (e) removing the necessary samples grown culture from the chamber for sampling.

14. The method according to item 13, in which the simultaneous application of peristaltic movement of sainyabuli pipeline environment and culture in the pipeline can deliver a certain amount of fresh medium into the growing chamber, while equal amount of culture stands out and is removed through the other end of the growth chamber, to complete the growth cycle and start a new growth cycle.

15. The method according to item 13, in which the experiment may include multiple cycles of growth depending on the requirements of the experimenter without possible contamination of isolated growth and without the possible proliferation of resistant breeding population.

16. The method according to item 13, in which during operation the experimenter can support the growth conditions according to the experimental requirements, which may include temperature, pressure, optical, chemical, pH, agitation and aeration of various gases.

17. The method according to item 13, in which the combination of inclination and agitators leads to a suitable stirring to mix the growing culture to prevent or suppress aggregation of living organisms.

18. Device for increasing the rate of reproduction (through increased speed reproduction and/or increased reproductive output) of living cells in suspension or any cultured organisms through a process of natural selection that contains
flexible, sterile tubing continuous length;
the clamping system located at points along the segment of the Tr is aprovada, each of these clamping devices are located and placed with the opportunity adjustable to compress the pipeline location clamping device in a closed position in which the pipeline is divided into separate zones on the respective sides of the clamping device, combining again in a single zone in the return of the clamping device in the open position;
in which clamping devices and piping are arranged so that the pipe is compressed by the first four points along the pipeline, determining the first or third zone downstream from the first to third pixels, respectively; and
in which the volume of the second zone, the boundaries of which are defined by points 2 and 3, is greater than the volume of the first and third zones;
in which the clamping system is designed in such a way that duplicate, the tubing is compressed upstream from the first point, the tubing is compressed at a point between the second and third points, and the second point is returned to the open position, whereby the second zone is divided into a portion above the stream and part of the lower thread, the first zone and the part above the stream together, whereby to set new first-fourth point and the first-third zones.

19. The device according to p in which the pipeline is g is nepronitsaemym.

20. The device according to p in which the pipeline is gas-tight.

21. The device according to p, in which the tubing is translucent.

22. The device according to p in which the pipeline is transparent.

23. The device according to p, in which the contents of the pipeline in the second zone may be adjustable image subjected to reduced pressure or excess pressure relative to the atmospheric environment.

24. The device according to p, optionally containing a pH indicator in the pipeline.

25. The device according to p, optionally containing a heating and cooling device that can regulate the temperature of the contents of the pipeline.

26. The device according to p, optionally containing a stir bar.

27. The device according to p in which the agitator comprises at least one stirring rod.

28. The device according to p, in which zone of the pipeline can be made in a specific and controlled atmospheric zone to control the dynamics of gas exchange.

29. The device according to p, optionally containing a device for regulating the inclination of the second part of the pipeline.

30. Method of increasing the rate of reproduction (through increased speed reproduction and/or increased reproductive output) of living cells in suspension or any cultured organisms% by the SSA, natural selection, providing stage: flexible, sterile tubing continuous length; ensure a system of clamping devices located at points along the segment of the pipeline, each of these clamping devices are located and placed in such a way that it is able orderly manner to compress the pipeline location clamping device in a closed position in which the pipeline is divided into separate zones on the respective sides of the clamping device, combining again in a single area when you return the specified clamping device in the open position;
premises culture medium in the pipeline;
closing of the clamping devices with the first four points along the pipeline to determine the first-the third zone downstream from the first to third pixels, respectively, and the volume of the second zone, the boundaries of which are defined by points 2 and 3, is greater than the volume of the first and third zones;
the introduction of cultured organism to the second area between the second and third points and the abandonment of this culture to grow in a culture medium; and
the repetition of the stage, which involves the compression of the pipeline upstream from the first point, the compression of the tubing at a point between the second and third points and return the second point to QCD is atomu position, whereby the second zone is divided into a portion above the stream and part of the lower thread, the first zone and the part above the stream together, whereby to set new first-fourth point and the first-third zones.

31. The method according to item 30, in which the simultaneous application of peristaltic movement of clamping devices, pipeline and environment and culture in the pipeline can deliver a certain amount of fresh medium to the second area of the pipeline, while an equal amount of culture stands out and is removed through the opposite end of the second zone, to complete one cycle of growth and begin a new growth cycle.

32. The method according to item 30, further providing for stage regulation pressure of the contents of the pipeline in the second zone.

33. The method according to item 30, additionally providing for the phase regulation of the temperature of the contents of the pipeline.

34. The method according to item 30, additionally providing for the stage of mixing the contents of the pipeline.

35. The method according to item 30, additionally providing stage to ensure that specific and controlled atmospheric zone around the pipeline to control the dynamics of gas exchange.

36. The method according to item 30, further providing for stage adjustable inclination of the second part of the pipeline.



 

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FIELD: chemistry.

SUBSTANCE: when culturing phototrophs, the culture fluid is stirred and aerated through agitation by moving cultivators back and forth in the horizontal plane at given temperature and pH values. The cultivators are illuminated with a pulsed light source with pulse duration of 0.00001-0.001 s and pulse spacing of 0.01-0.1 s. In the apparatus used, the culture fluid is illuminated with diodes located under transparent bottoms of vessels of the same geometric shape and powered by a pulse generator with controlled frequency and light pulse duration.

EFFECT: group of inventions enables to reduce power consumption when culturing phototroph biomass.

2 cl, 2 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: enzyme preparations are obtained using a fermenter with a heating jacket for submerged culturing of microorganisms of the enzyme preparations with continuous aeration with compressed air and mechanical agitation at culturing temperature of 30…32°C on the entire volume of the fermenter; the culture liquid obtained in the fermenter is cultured to remove the residue and the filtrate of the culture liquid with moisture content of 92…95% is fed into a vacuum-sublimation drier, in which the desublimator used is a two-section evaporator of a vapour compression refrigerating machine, the working and spare section of which alternately operate in condensation and regeneration modes, respectively; wherein "hot" water is obtained by heating thereof in the condenser of the refrigeration machine using condensation heat of the coolant to temperature of 68…73°C, one part of which is fed into the heating jacket of the fermenter and the other is first fed for thawing the evaporator section operating in regeneration mode, and then the water cooled to temperature of 5…7°C is then removed from the evaporator section into a storage tank together with the liquid formed from the ice cover thawed on the surface of the cooling element in an amount of moisture evaporated from the enzyme preparation, and further, in closed cycle mode, fed in two streams, one of which is mixed with waste water after the fermenter before the condenser, and the second with "hot" water before the fermenter, wherein excess water from the recirculation loop is removed through the storage tank, followed by measurement of the culturing temperature in the fermenter, the flow rate and temperature of the water at the inlet of the heating jacket of the fermenter, the flow rate and moisture content of the filtrate of the culture liquid, residual pressure in the working volume of the vacuum-sublimation drier, the moisture content of the dried enzyme preparation, the flow rate and temperature of water vapour removed from the vacuum-sublimation drier into the working section of the evaporator, the boiling point of the coolant in the working section of the evaporator, the temperature of uncondensed vapour at the outlet of the working section of the evaporator and the level of water in the storage container; mass and heat flow of the mixture of cooled and "hot" water into the heating jacket of the fermenter is established by changing the ratio of flow rates thereof with adjustment of the culturing temperature in the fermenter; the measured values of moisture content and flow rate of the filtrate of the culture liquid are used to determine the power of the drive of the compressor of the refrigeration machine and the required residual pressure in the working volume of the sublimation drier by action on the power of the controlled drive of the vacuum pump with adjustment of the residual pressure on the final moisture content of the enzyme preparation; the flow rate and temperature of water vapour removed from the vacuum-sublimation drier into the working section of the evaporator, the temperature of uncondensed vapour at the outlet of the working section of the evaporator and the boiling point of the coolant in the working section of the evaporator are used to determine the current value of the coefficient of heat transfer from the water vapour to the coolant on the cooled surface of the working section of the evaporator, and upon achieving minimum permissible value of the coefficient of heat transfer, power of the drive of the compressor of the refrigeration machine is increased first, and the working section of the evaporator is then switched from condensation mode to regeneration mode while simultaneously switching to condensation mode the section operating in regeneration mode.

EFFECT: improved quality of enzyme preparations by increasing accuracy and reliability of controlling the process parameters, high energy efficiency and environmental safety of production processes and vacuum-sublimation drying.

1 dwg

FIELD: biotechnologies.

SUBSTANCE: method includes inspection of an inoculator with process equipment for tightness, sterilisation of the inoculator with steam via an aeration device at the pressure of 0.20 - 0.25 MPa for 30…40 min., its filling with nutrient medium heated by steam to the temperature of 100°C. Then the temperature of the nutrient medium is increased to 121 - 123°C at steam pressure of 0.10 - 0.15 MPa, and the nutrient medium is maintained at these parameters for 15-60 min., afterwards the pressure in the inoculator is reduced down to 0.03…0.05 MPa. The nutrient medium is cooled down to cultivation temperature of 31…32°C with cold water with temperature of 7 - 10°C. After cooling of the nutrient medium, it is seeded with a seeding material with simultaneous mixing and aeration with sterile air. Cultivation of the produced liquid seeding culture is carried out at pH 4.2 - 4.5 and temperature of 31 - 32°C to achieve the phase of exponential growth for 12 - 14 hours. Then it is sent by means of displacement with sterile air from the inoculator into the prepared fermenter in the amount of 3…10% of the nutrient medium amount with its filling by 7/10 of its volume, and the microorganism culture is grown at fermentation temperature of 28 - 40°C for 96 - 120 hours with continuous aeration with sterile air, mechanical mixing and supply of warm water with temperature of 27 - 47°C into a heating jacket of the fermenter. After fermentation the cultural fluid with accumulated biomass is supplied into previously sterilised collectors of finished culture.

EFFECT: increased yield of cultural liquid with accumulated biomass of aerobic microorganisms, reduced specific power inputs and provision of environmental safety at all stages of production.

1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Claimed is device for obtaining nanoparticles by reduction of metals from initial salts in presence of cultivated cells of microorganisms. Device includes control computer (1), connected with it electronic block of regulation and control (2) of all functional units and blocks of fermenter (3), pH-stabilising block (4) with pH sensor (5) and hoses for supply of titering solutions by pumps (6, 7), block (8) for regulation of redox-potential of culture mixture, provided with redox sensor (9), independently controlled pumps (10, 11) for introduction of initial solutions of metal salts, reducing agents and growth factors into fermenter (3), block (12) for regulation of dissolved oxygen level with sensor pO2 (13), pump (14) for supply of growth substrate, block (15) for measurement of optic culture density with application of optic fibre sensor (16), block (17) for measurement of spectral characteristics of culture mixture with application of optic fibre sensor (18), isolated with impermeable for cells membrane with pore size 100-250 nm, block (19) for thermoregulation of fermenter (3), equipped with temperature sensor (20), block (21) for regulation of culture mixture mixing, which brings into motion blade mixer (22), block (23) for regulation of culture mixture illumination in case of cultivating phototrophic microorganisms and control of spectral parameters of submersible diode lamp (24), block (25) for ultrafiltration of sampled culture mixture with sterilising membrane with pore size 100-250 nm with possibility of output of only nanoparticle suspension from fermenter, condenser of output moisture (26), preventing loss of culture mixture.

EFFECT: invention contributes to extension of arsenal of technological methods of obtaining nanoparticles of metals and makes it possible to achieve controllability of modes of nanoparticle formation.

2 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is a method of growing colonies of microbial cells on a surface of a porous plate. The method includes supply of a nutrient solution from bottom to top through the porous plate into zones of growth of colonies of the microbial cells on its upper surface, supply of a suspension of the microbial cells onto the upper surface of the porous plate, creation of controlled conditions for the colony growth, performing observation of the colony growth, separation of the grown colonies of the microbial cells from the zones of growth and their transfer into external means of identification. The nutrient solution is supplied into the zones of growth of the colonies of the microbial cells by creation of a pressure difference between the hole input and output. Holes are made in the plate from an anode aluminium oxide orthogonally to its large plane and are topologically coded. The said zones of growth are formed in them in the form of porous membranes. The porous membranes are located at the same level as the upper surface of the plate or with formation of a hollow and do not pass the microbial cells. After supply of the nutritional solution, the suspension of the microbial cells of a specified concentration is supplied onto the upper surface of the plate until their homogenous distribution is achieved. Between the zones of growth on the surface of the plate a film, preventing attachment of the microbial cells, is formed. Separation of the grown microcolonies from the zones of growth is performed by hydroblow. A hydroblow is directed from the side of the input of cylindrical holes of the plate and spreads along them and farther through the pores of the porous membranes with force, which does not destroy the microcolonies but is sufficient for their separation from the growth zones. Also claimed is a device for growing the colonies of the microbial cells by the claimed method.

EFFECT: providing conditions of automation of processes of the nutrient solution supply and processes of separation and transfer of the grown colonies, possibility of integration into miniature portable devices, and application in laboratories on a chip and provision of the device portability.

6 cl, 14 dwg, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: what is presented is a medical waste collection device. The device comprises a load device and a former configured to be mounted in a tabletop hole. The load device comprises a branch pipe and a receiving unit configured to take the shape of a rectangle with rounded short sides in the former. The load device is thin-walled and single-space. The receiving unit of the load device belongs within the former, whereas the branch pipe is configured to be mounted in a waste vessel.

EFFECT: laboratory assistant's safety when handling and recycling extremely hazardous biological materials, and reagents.

4 dwg

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