RU2490323C1 - Microbioreactor and method for operation thereof - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: group of inventions relates to biotechnology, particularly to bioreactors, primarily micro- and mini-bioreactors with an enzyme immobilised on particles and can be used to conduct biotechnological processes in liquid media in pharmaceutical, food and other industries. The microbioreactor has a housing, connecting pipes, pumps for feeding a substrate and auxiliary substances that are connected to said housing by pipes. There is a distribution device and parallel channels in the housing. The channels have a cross-section with a periodically varying length. Each of the channels contains solid particles with an immobilised enzyme. The distribution device consists of a chamber for feeding a substrate and the next one or more chambers for feeding auxiliary substances. There is one or more openings in the side wall of the channels in each chamber for feeding auxiliary substances, wherein channels in the top and bottom parts are equipped with grids, the diameter of openings in which is less than the diameter of the solid particles. Each of the channels can have additional grids which are fitted in sections with the largest area. The method of operating the microbioreactor involves using one or more pumps to feed the substrate and auxiliary substances into the housing with rates that vary periodically over time, wherein the maximum speed of the substrate with auxiliary substances in the wide section of the channels in one period is set higher than the rate of fluidisation of solid particles with the immobilised enzyme, and the minimum speed of the substrate with auxiliary substances in the wide section of the channels in one period is lower than the rate of the onset of fluidisation. Duration of the interval of maximum rate is set such that particles with the immobilised enzyme are able to travel the distance from the bottom to the top grid, and duration of minimum rate is set such that particles with the immobilised enzyme are able to travel the distance from the top to the bottom array. The maximum speed of the substrate with auxiliary substances in the wide section of the channels in one period is set higher than the rate of deposition of solid particles with the immobilised enzyme, and the minimum speed of the substrate with auxiliary substances in the wide section of the channels in one period is set negative.EFFECT: group of inventions increases efficiency of mass-exchange and bioreactor processes and, as a result, increases efficiency of the apparatus.5 cl, 6 dwg

Description

The present invention relates to bioreactors, mainly to micro- and minimally scaled bioreactors (or otherwise microbioreactors) with an enzyme immobilized on particles and can be used to carry out biotechnological processes in liquid media, for example, for carrying out reactions in liquid-liquid and liquid-gas systems pharmaceutical, food and other industries.

Known bioreactor (fermenter) for aerobic cultivation of microorganisms in liquid column type with energy supply to the gas phase (Vinarov A.Yu., Gordeev L.S., Kukharenko A.A., Panfilov V.I. Fermentation apparatus for microbiological synthesis processes / Under the editorship of VA Bykov, Moscow: DeLi Print, 2005, p.165-185) and the supply of energy to the liquid phase (ibid., P.185-193), as well as with a combined supply of energy ( ibid., pp. 193-214). A common feature of these devices is the presence of mixing, aeration and mass transfer units of oxygen and heat transfer. Due to the fact that the known devices (bioreactors) have a large volume, the energy in them is distributed unevenly. When using an enzyme immobilized on solid particles, a significant proportion of these particles is concentrated in the stagnant zones, as a result of which the access of the substrate to their surface is hampered, and the efficiency of the apparatus is reduced.

Closest to the claimed is a biocatalized flow reactor (application 2010122385, 11/18/2008, IPC С12М 1/40 (2006.01), publication date of the application: 12/27/2011) with a layer consisting of a reactor vessel with an internal space connected to the liquid inlet and connected with the exit of the liquid, while in the inner space there is a layer containing the enzyme lovastatin esterase immobilized on a water-insoluble solid carrier, characterized in that the enzyme is covalently linked to a solid carrier activated at least bifunctional th coupling agent, wherein the solid carrier is thus in association with at least bifunctional agent that immobilized lovastatin esterase exhibits at least 5 times higher the hydrolytic activity towards lovastatin, salts of lovastatin in the presence of simvastatin and simvastatin salts than to simvastatin and salts of simvastatin. Known bioreactor allows to obtain and / or purify simvastatin. Its disadvantage is the ineffective mass transfer from the surface of the particles of the solid carrier, due to the presence of stagnant zones at the points of contact of the particles. As a result, only 30–40% of the particle surface fully functions, while convective diffusion is absent in the particle contact zones, and molecular diffusion, which provides transport by 2–3 orders of magnitude lower, significantly limits mass transfer.

A known method of operating bioreactors (Vinarov A.Yu., Gordeev L.S., Kukharenko A.A., Panfilov V.I. Fermentation apparatus for microbiological synthesis processes / Edited by V.A. Bykov. M .: "DeLi print ", 2005, p.125), which consists in supplying the apparatus with the necessary components of a nutrient medium (salt, substrate, water), a titrating agent, aeration gas and the selection of a suspension from the fermenter. Mixing is carried out using a mixer, jets of liquid or by sparging gas.

The disadvantages of this method include problems with the uniform distribution of the introduced mixing energy over the volume of the apparatus, the uneven distribution of concentration and temperature over the volume of the apparatus. All this also leads to a decrease in the efficiency of the fermenter.

The objective of the invention is to increase the efficiency of mass transfer and bioreaction processes, and ultimately to increase the productivity of the apparatus due to:

- increase the uniformity of the distribution of input energy over the volume of the apparatus;

- ensuring equal accessibility of the surface of solid particles with immobilized enzyme;

- Improving the process of dispersing drops, bubbles;

- a significant increase in the quality of mixing inside bubbles, drops and liquid shells.

The problem is solved in that in a microbioreactor containing a housing, connecting pipes connected to them by means of tubes, pumps for feeding the substrate and auxiliary substances, according to the invention, a switchgear is arranged in the housing, parallel channels are arranged having a cross-section periodically varying in length, solid particles with an immobilized enzyme are loaded into each channel; the distribution device consists of a substrate input chamber and one or several there are one or several holes in the side wall of the channels in each of the channels for the introduction of auxiliary substances, the channels in the upper and lower parts provided with gratings, the diameter of the holes in which is smaller than the diameter of the solid particles, while in each of the channels placed additional gratings installed in sections with a maximum area.

The problem is also solved by the fact that in the method of operation of the microbioreactor, which consists in supplying one or more pumps of the substrate and auxiliary substances to the housing, according to the invention, the substrate with auxiliary substances is supplied at a time-varying flow rate, while the maximum substrate speed with auxiliary substances in a wide cross section of the channels are set higher than the rate of fluidization of solid particles with an immobilized enzyme, and the minimum substrate velocity over a period of excipients in a wide cross section of the channels are set below the velocity of the beginning of fluidization, and the duration of the maximum speed interval is set such that the particles with the immobilized enzyme have time to pass the distance from the lower to the upper lattice, and the duration of the minimum speed interval is set such that the particles with the immobilized enzyme have time to pass the distance from the upper to the lower lattice, the maximum for a period the speed of the substrate with auxiliary substances in a wide section of channels is set higher than the deposition rate of solid particles with an immobilized enzyme, and the minimum substrate rate with auxiliary substances over a period in a wide section of channels is set to negative.

The inventive microbioreactor and the method of its operation can improve the efficiency of mass transfer and bioreaction processes, and ultimately increase the productivity of the apparatus.

As auxiliary substances can act substances in a gaseous or liquid state. The minimum fluid velocity can be either positive or negative.

The claimed technical solution is new, has an inventive step and is industrially applicable.

Figure 1 presents a diagram of a microbioreactor, figure 2 is a General schematic diagram of the strapping of a microbioreactor that implements the proposed method. Figure 3 shows an embodiment of the tubular part of the housing of the microbioreactor. Figure 4 shows the movement of elements of a dispersed medium (droplets), a continuous medium (liquid shells) and solid particles with an immobilized enzyme. Figure 5 shows the time diagram of the instantaneous flow rate of liquid media in the apparatus (for example, a two-phase system - a substrate with one auxiliary substance), Fig.6 - time dependence of the instantaneous volume of liquid (two-phase system) passing through the cross section of the microbioreactor.

Figure 1 shows a microbioreactor containing a housing 1, connecting pipes 2-8 for supplying a substrate (pipe 2) and auxiliary substances (pipes 3, 4), product removal (pipes 5, 6), supply and removal of coolant (pipes 7, 8). Pumps are connected to the nozzles using tubes (not shown conditionally in Fig. 1). In the housing 1 there is a distribution device consisting of a chamber 9 for introducing a substrate and subsequent one or more chambers 10 (two of them in FIG. 1) for introducing auxiliary substances. In the housing 1 are also placed parallel channels (tubes) 11 having a periodically varying cross-sectional length, while one or more openings 12 are made in the side wall of the channels in each of the input channels of the auxiliary substances 12. To ensure a high heat transfer coefficient during the transverse flow around the channels (tubes) 11 in the annular space of the housing 1 is installed transverse partitions 13. The channels in the upper and lower parts are provided with gratings 14, the diameter of the holes in which is smaller than the diameter of the solid particles 15 with immobilized enzyme loaded into each of the channels 11 of the microbioreactor (in figure 1, particles are conventionally shown in one of the channels).

In each of the channels 11 can be placed additional lattice 14, installed in sections with a maximum area of the channels. This allows, if necessary, to limit the progress of the solid particles 15 with the immobilized enzyme and to ensure their more uniform distribution along the length of the channels 11, and hence the working volume of the apparatus.

Figure 2 shows the General schematic diagram of the strapping of the microbioreactor, including the housing 1 with channels 11, pumps 16, connected by tubes to three-way controlled valves 17 and 18, time relay 19, and also (if necessary cooling or additional phase separation) intermediate tank 20. Pumps 16 are installed on each of the phases — the substrate and additional components (one pump is conventionally shown in FIG. 2), followed by their mixing in distribution chambers when dispersing auxiliary substances in the substrate. The time relay allows you to automatically switch the direction of the flows from the bottom up (direct flow - stream A in figure 2) and from top to bottom (reverse flow - stream B in figure 2). Hereinafter, reverse flow refers to a flow with a minimum speed. In any direction of flow, the valve 17 delivers fluid flows to the apparatus, and the valve 18 removes products from it. The microbioreactor can operate both in recycling (as a batch apparatus) and on the duct (as a continuous apparatus). The primary phase separation occurs in the upper part of the microbioreactor: the light phase is discharged through the nozzle 5, the heavy phase is discharged through the nozzle 6. Further separation is carried out in a separate separator (not shown in Fig. 2).

Figure 3 shows an embodiment of the housing 1 with channels 11, when the channels at the point of transition to the distribution chamber have a maximum cross section (pipes and partitions are not conventionally shown). In this case, the supply tubes in the distribution chambers may have a diameter corresponding to the maximum diameter of the channel.

Figure 4 shows the movement of the phases: drops 21 of liquid Zh2 (dispersed medium), liquid shells 22 of liquid Zh1 (continuous medium) and solid particles 15 with an immobilized enzyme in one of the channels 11.

The proposed device operates as follows. Figure 5 shows the timing diagrams of the pulsed supply to the apparatus for the case when the apparatus has three phases - a continuous medium (substrate), a dispersed medium (excipients) and solid particles with an immobilized enzyme. The substrate with auxiliary substances is supplied with flow rates periodically varying in time, using a time relay 19 and valves 17 and 18 controlled by it. Moreover, the maximum speed of the substrate with auxiliary substances (corresponding to flows A) over a period in a wide section of channels is set higher than the velocity of fluidization of solid particles with immobilized enzyme, due to which the particles are transferred to a suspended state, when the particles are separated from each other, and each of the particles is washed from all sides by a fluid flow (with bstratom with excipients). Due to this, the presence of any stagnant zones in the apparatus is completely eliminated, and the surface of the immobilized enzyme is used 100%. The speed in a narrow section is even higher than in a wide one, which guarantees the movement of particles upward along the entire length of the apparatus without the occurrence of return flows along the entire length of the channels. The entrainment of particles from the working zone — from channels 11 — is prevented due to the presence of gratings in the upper part of the channels 11. The duration τ _{1 of} the maximum speed interval is set such that particles with immobilized enzyme have time to travel from the lower grate 14 to the upper grate 14.

The minimum over the period, the speed of the substrate with auxiliary substances in a wide section of channels, i.e. the velocity corresponding to the motion of flows B is set lower than the velocity of the beginning of fluidization, which allows particles to settle on the lower lattice 14. Moreover, the velocity can have a negative sign, i.e. directed downward, contributing to a more rapid deposition of particles. At the same time, in the channels 11, the vibrational velocity of the relative motion of solid particles with an immobilized enzyme with a large amplitude is created, providing a high coefficient of mass transfer from their surface. The duration t3 of the minimum speed interval is set such that particles with the immobilized enzyme have time to travel the distance from the upper lattice 14 to the lower lattice 14.

Due to the periodically changing cross-sectional length of the channels 11, the fluid velocity (substrate with auxiliary substances) along the lengths of the channels varies in accordance with the continuity equation. This leads to the fact that solid particles 15 with an immobilized enzyme in a narrow section of channels 11 are accelerated, carried away by the flow, and in a wide section of channels 11 are inhibited. With this movement and the presence of a difference in the densities of the solid and liquid phases, a relative vibrational movement of particles and liquid occurs, which helps to reduce diffusion resistance, which leads to a significant acceleration of mass transfer from the surface of the particles.

In necessary cases (for example, if solid particles with an immobilized enzyme have an increased tendency to stick together with each other and stick to the walls of the apparatus), the maximum velocity of the substrate with auxiliary substances in a wide section of channels over a period of time is set higher than the deposition rate of solid particles with an immobilized enzyme, which is higher fluidization initiation rates.

If necessary, to realize the minimum substrate velocity with auxiliary substances in a wide cross section of the channels, which is superimposed on the translational reverse flow movement in the reverse current phase, for the period, is set to negative. In this case, on average, over a period, the flow rate (supply) should be positive (see formula (2)) to ensure the directional movement of fluid in the apparatus.

The period (cycle duration) is the same for continuous and dispersed media (see figure 5) and is equal to

$$T={\tau }_{\mathrm{one}}+{\tau }_2,(\mathrm{one})$$

where τ ₁ is the duration of the stage of the maximum speed interval (direct flow), s;

τ ₂ - the duration of the stage interval of the minimum speed (reverse flow), C.

The average flow rate of the fluid (substrate and excipients) is equal to

$$〈Q〉=\frac{\mathrm{one}}{T}{\displaystyle \underset{0}{\overset{T}{\int }}qdt}.(2)$$

The volume of fluid passing through the cross section of the microbioreactor in direct flow

$$V_{\text{ïð}}={\displaystyle \underset{0}{\overset{{\tau }_{\mathrm{one}}}{\int }}qdt}.(3)$$

the volume of fluid passing through the cross section of the microbioreactor in the reverse flow

$$V_{î\text{áð}}={\displaystyle \underset{{\tau }_{\mathrm{one}}}{\overset{T}{\int }}qdt}.(\mathrm{four})$$

From formulas (2) - (4) it follows

$$〈Q〉=\frac{V_{\text{ïð}}+{\text{V}}_{î\text{áð}}}{\text{T}}.(5)$$

Figure 5 shows a graph of the instantaneous flow rate q of a two-phase system versus time: q ₁ - instantaneous flow rate of direct flow (stream A), q ₂ - instantaneous flow rate of reverse flow (stream B). The average flow rate, as can be seen from figure 5, has a positive sign, i.e. on average, a heterogeneous system is displaced from the apparatus during a period, and it can be considered as a displacement type apparatus (average for a period). Figure 6 shows the time dependence of the instantaneous volume of liquid (two-phase mixture) passing through the cross section of the microbioreactor, obtained by integrating the dependence in figure 5.

An example of a specific implementation. The microbioreactor for transesterification of triglycerides (vegetable or animal fats, acting here as a substrate) into biodiesel with the formation of glycerol, the scheme of which is shown in figure 1, contains a housing 1 with six channels 11 1 m long. The channels have a cross-section periodically varying in length the smaller diameter of which is 3 mm and the larger diameter of 6 mm. Solid polymer particles 15 are loaded into each channel 11 with a zymase enzyme immobilized on their surface and having a diameter of 0.3 mm. The distribution device consists of a triglyceride input chamber 9 and a single auxiliary chamber 10, next to it, of methyl alcohol. One or two holes 12 with a diameter of 0.6-0.8 mm are made in the side wall of channels 11 in the chamber 10 for introducing auxiliary substances, through which methyl alcohol is introduced and dispersed in a stream of continuous medium — triglycerides. The channels 11 in the upper and lower parts are provided with gratings 14, the pore diameter of which is 0.2 mm. Optionally, in each of the channels 11, additional gratings 14 can be placed, installed in sections with a maximum area, as shown in FIG.

Pump 16 in the housing 1 (see figure 2) serves triglycerides and methyl alcohol with a periodically variable flow rate, as shown in figure 5. In this case, the maximum velocity of the substrate with auxiliary substances over a period in a wide section of channels is set to 0.03 m / s, i.e. higher than the rate of fluidization of solid particles with an immobilized enzyme (which is 0.01 m / s), and the minimum over the period, the speed of the substrate with auxiliary substances in a wide section of channels is set equal to - 0.01 m / s, i.e. lower than the onset of fluidization (in this case, negative). Moreover, the duration (τ ₁ = 30 s) of the maximum speed interval is set such that the particles with the immobilized enzyme have time to pass the distance from the lower to the upper lattice, and the duration (τ ₂ = 45 s) of the minimum speed interval is set such that the particles with the immobilized enzyme have time walk the distance from the upper to the lower grill. In a narrow section, the velocity is higher than in a wide one, which ensures unidirectional movement of particles in each of the periods.

Due to the cross-section of the channels periodically varying in length, the radial transfer of momentum and energy is intensified, as a result of which the uniformity of the distribution of the introduced energy over the apparatus volume is improved, and the surface of solid particles with immobilized enzyme is equally accessible. These effects are also enhanced by the pulsed supply of liquid phases according to the invention.

The process of dispersing drops, bubbles (for liquid-gas systems) is improved due to the implementation of the dispersant in the form of holes in the side wall of the channels in each of the input chambers of auxiliary substances.

To increase the efficiency of mass transfer from the surface of particles with an immobilized enzyme, the speed of a two-phase mixture (substrate with auxiliary substances) can be set higher than the deposition rate of solid particles with an immobilized enzyme, which in this case is 7 m / s.

Due to the high mass transfer coefficients from the surface of particles with an immobilized enzyme, uniform heating of channels with a two-phase liquid system in them, intensive mixing (due to the so-called Taylor vortices) in drops of methyl alcohol and in liquid shells of triglycerides, the process duration is reduced from 48 hours for a prototype bioreactor (apparatus with a mixing device) up to 4 hours, i.e. 12 times. Therefore, with equal volumes of apparatus due to the reduction in the duration of the process, there is an increase in the productivity of the microbioreactor compared to existing analogues by 12 times.

Thus, the proposed microbioreactor and the method of its operation can improve the efficiency of mass transfer and bioreaction processes, and ultimately increase the productivity of the apparatus.

Claims (5)

1. A microbioreactor comprising a housing, connecting nozzles, pumps for supplying a substrate and auxiliary substances attached to them by means of tubes, characterized in that a distributing device is placed in the housing and parallel channels are installed having a cross-section periodically varying in length and placed in each from channels solid particles with an immobilized enzyme, the distribution device consists of a substrate input chamber and one or more input chambers following it mogatelnyh substances, while in the sidewall of the channel in each of the input chambers adjuvants one or more holes, wherein the channels in the upper and lower parts are provided with gratings, in which the hole diameter less than the diameter of solid particles. 2. The microbioreactor according to claim 1, characterized in that in each of the channels there are additional gratings installed in sections with a maximum area. 3. The method of operation of the microbioreactor according to claims 1 and 2, comprising supplying one or more pumps with a substrate and auxiliary substances into the housing, outputting the finished product, characterized in that the substrate with auxiliary substances is supplied with flow rates periodically varying, with the maximum over a period the speed of the substrate with auxiliary substances in a wide section of the channels is set higher than the velocity of fluidization of solid particles with the immobilized enzyme, and the minimum substrate speed with auxiliary and substances in a wide cross section of the channels are set below the velocity of the beginning of fluidization, and the duration of the maximum speed interval is set such that the particles with the immobilized enzyme have time to pass the distance from the lower to the upper lattice, and the duration of the minimum speed interval is set such that the particles with the immobilized enzyme have time to pass the distance from top to bottom grill. 4. The method according to claim 3, characterized in that the maximum over a period the speed of the substrate with auxiliary substances in a wide section of the channels is set higher than the deposition rate of solid particles with an immobilized enzyme. 5. The method according to claim 3, characterized in that the minimum over the period the speed of the substrate with auxiliary substances in a wide section of the channels is set to negative.

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