CN101457205B - Siphoning type intermittent spraying bioreactor - Google Patents

Abstract

The invention relates to a siphon type intermittent spray bioreactor in the field of biotechnology, wherein: one port of the liquid storage bottle is connected to a gas-liquid mixing and stirring tank via a first liquid pump, and the inner bottom of the gas-liquid mixing and stirring tank is provided with a gas spray head , the gas nozzle is connected to the gas flow meter outside the gas-liquid mixing tank, the output port of the gas-liquid stirring device is connected to the input port of the spray device, the output port of the spray device is connected to the input port of the reactor body, and the output port of the reactor body The port is connected to the continuous harvesting device, the waste liquid output port of the reactor main body is connected to the waste liquid bottle, the continuous harvesting device is connected to the recovery port of the reactor main body through the second liquid pump, and an oxygen probe and a pH probe are arranged inside the reactor main body. Oxygen probe and pH probe are connected with computer. The invention has the advantages of simple design, convenient operation and low cost.

Description

Siphon intermittent spray bioreactor

technical field

The invention relates to a device in the field of biotechnology, in particular to a siphon type intermittent spray bioreactor.

Background technique

my country is one of the countries with the richest medicinal plant resources in the world. As the earliest natural resources developed, medicinal plant resources are currently facing two crises: one is that the reserves and output of wild plant resources are generally declining; the other is that The demand for medicinal plants continues to increase, resulting in excessive mining of medicinal plant resources. On the one hand, the traditional field cultivation of medicinal plants is affected by "time and place", resulting in large fluctuations in yield and quality; on the other hand, it inevitably brings about problems such as pesticide residue pollution. The use of bioreactors for plant tissue and cell in vitro culture has the characteristics of rapid growth and easy control, which can gradually replace field cultivation to meet the needs of the market.

At present, the reactors used for in vitro culture of medicinal plant tissues, cells and organs mainly include stirring type, air lift type, bubbling type and spray type. The stirred bioreactor has a wide operating range, strong oxygen supply capacity, and good mixing effect, but the shear force generated during the stirring process will cause damage to the isolated plant tissue. Although the shearing force of the bubbling reaction and airlift reactor is small, it is not conducive to the mixing of substances and gas transfer. The spray reactor has the characteristics of simple structure, small shear force, good mass transfer effect and low cost, and is the most commonly used reactor type at present. In addition, when using the first three types of reactors for cultivation, it is necessary to soak the explants in the culture solution, which is not conducive to gas exchange and prone to hydrated seedlings. The spray bioreactor can not only improve the efficiency of mass energy transfer, but also has the advantage of small shear force.

Found through the retrieval of prior art literature, " Controlling Hyper hydration of Carnations (Dianthus caryophyllus L.) Grown in a Mist Reactor " (in spray reaction) published by M.J.Correll etc. on " Biotechnology and bioengineering " (biotechnology and bioengineering) Controlling the hydration of carnations in the device), a spray reactor for cultivating organisms is proposed in this paper. The spray device is designed on the basis of acoustics, and the spraying work is controlled by the acoustic device. The spray device needs to be equipped with sophisticated intelligent control Instruments are expensive and complex to operate.

Contents of the invention

The present invention aims at the deficiencies in the above-mentioned prior art, and proposes a siphon type intermittent spray bioreactor. The present invention adopts the siphon principle to design the spray device, utilizes the potential energy of the culture fluid itself to realize automatic spraying, and adjusts the spray aperture and The volume of the spray tank is used to adjust the spray duration, which makes the design of the whole device simple, easy to operate and cheap in cost.

The present invention is achieved through the following technical solutions, the present invention includes: liquid storage bottle, first liquid pump, spray device, reactor main body, oxygen probe, pH probe, computer, gas-liquid mixing tank, continuous harvesting device, waste liquid bottle, gas flowmeter, and the second liquid pump, wherein: one port of the liquid storage bottle is connected to the gas-liquid mixing tank via the first liquid pump, and the inner bottom of the gas-liquid mixing tank is provided with a gas nozzle, and the gas nozzle is connected to the gas-liquid mixing tank. The gas flow meter outside the mixing tank is connected, the output port of the gas-liquid mixing tank is connected to the input port of the spray device, the output port of the spray device is connected to the input port of the reactor body, and the output port of the reactor body is connected to the continuous harvesting device The waste liquid output port of the reactor body is connected to the waste liquid bottle, and the continuous harvesting device is connected to the recovery port of the reactor body through the second liquid pump. The reactor body is equipped with an oxygen probe and a pH probe, an oxygen probe and a pH probe Connect to computer.

The continuous harvesting device includes: a cavity, a pushing device, a filter sieve, a sterilizing filter membrane, a harvesting port, an inlet of the harvesting device, and a culture fluid return pipe. The upper end of the cavity is provided with a harvesting port, and the two sides of the cavity are respectively For the harvesting device inlet and the driving device, a filter screen and a sterilizing filter membrane are arranged inside the cavity, and the filter sieve is located above the sterilizing filter membrane, and a culture solution return pipe is provided at the lower end of the cavity.

The pushing device includes: a spring, a pushing handle, a rubber hose, a piston-type pushing piece, the piston-type pushing piece is close to the cavity inner wall of the continuous harvesting device, the piston-type pushing piece is connected with one end of the pushing handle, A rubber hose is arranged on the outside of the rubber hose, and a spring is arranged between the rubber hose and the push handle.

The spray device includes: a culture solution inlet, a siphon elbow, a siphon, a drain pipe, a spray head, a spray tank, a liquid level gauge, and a spray tube, wherein: the siphon, the siphon elbow, and the spray tube are connected in sequence to form an inverted U-shaped, the siphon tube and the siphon elbow are all placed inside the spray tank, a part of the spray tube is placed inside the spray tank, and the other part is set on the upper end of the reactor body through the spray tank and connected with the spray head. A liquid level gauge is arranged on the side wall, and a culture solution inlet and a liquid discharge pipe are respectively arranged at the upper end and the lower end of the spray tank.

The liquid storage bottle, the first liquid pump, the gas flow meter, the spray device, the reactor main body, the continuous harvesting device, the waste liquid bottle and the second liquid pump are all connected by a silicone tube.

The upper ends of the liquid storage bottle, the spray device, the waste liquid bottle and the gas flow meter are respectively provided with air filters.

When the present invention works, the culture solution flows from the liquid storage bottle into the gas-liquid mixing tank under the action of the first liquid pump, and the mixed culture solution is injected into the spray tank through the culture solution inlet, and starts when the liquid level exceeds the siphon elbow. Spray, when the liquid level is lower than the siphon, the spray ends and enters the next spray cycle. The duration of the spray depends on the size of the spray aperture in the spray head and the volume of the spray tank, and the spray cycle is controlled by the first liquid pump corresponding to the perfusion rate of the culture liquid. During the spraying process, the computer detects the biological growth environment in the main body of the reactor through the oxygen probe and the pH probe for real-time monitoring. The used culture solution in the main body of the reactor flows into the waste liquid bottle, and the growing biomass passes through the continuous harvesting device in real time. reward.

The invention designs the spraying device according to the siphon principle, and the energy required for automatic spraying comes from the potential energy of the culture liquid itself, without consuming additional energy, and the spraying duration can be adjusted by the spray aperture and the volume of the spray tank without expensive precision instruments To control, the automatic intermittent spray just meets the requirements of plant tissue/organ growth, reducing the occurrence of hydration seedlings and other phenomena; the present invention is simple in design, convenient in operation and cheap in cost.

Description of drawings

Fig. 1 is a structural representation of the present invention;

Fig. 2 is the structural representation of continuous harvesting device among the present invention;

Fig. 3 is a structural schematic diagram of the spraying device in the present invention.

Detailed ways

The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings: this embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following the described embodiment.

The spray reactor of this embodiment is used to cultivate the homozygous tetraploid adventitious roots of Radix Pseudostellariae.

As shown in Figure 1, this embodiment includes: a liquid storage bottle 1, a first liquid pump 3, a spray device 7, a reactor body 8, an oxygen probe 9, a pH probe 10, a computer 11, a gas-liquid mixing tank 12, a continuous Harvesting device 13, waste liquid bottle 14, gas flow meter 15, second liquid pump 17, wherein:

One port of the liquid storage bottle 1 is connected to the gas-liquid mixing tank 12 via the first liquid pump 3. The inner bottom of the gas-liquid mixing tank 12 is provided with a gas nozzle 5, and the gas nozzle 5 is connected to the gas outside the gas-liquid mixing tank 12. The flow meter 15 is connected, the output port of the gas-liquid mixing tank 12 is connected with the input port of the spray device 7, the output port of the spray device 7 is connected with the input port of the reactor main body 8, and the output port of the reactor main body 8 is connected with the continuous harvesting device 13 connected, the waste liquid output port of the reactor main body 8 is connected with the waste liquid bottle 14, the continuous harvesting device 13 is connected with the recovery port of the reactor main body 8 through the second liquid pump 17, and the inside of the reactor main body 8 is provided with an oxygen probe 9 and The pH probe 10 , the oxygen probe 9 and the pH probe 10 are connected to a computer 11 .

In this embodiment, the reactor main body 8 has a volume of 4.8 liters.

In this embodiment, the liquid storage bottle 1, the first liquid pump 3, the gas flow meter 15, the spray device 7, the reactor main body 8, the continuous harvesting device 13, the waste liquid bottle 14, and the second liquid pump 17 are all connected by silicone tubing.

As shown in Figure 2, the described continuous harvesting device 13 includes: a cavity (not marked in the figure), a pushing device, a filter screen 22, a sterilizing filter membrane 23, a harvesting port 18, a harvesting device inlet 19, a reflux of the culture solution Pipe 21, the upper end of the cavity is provided with a harvesting port 18, the two sides of the cavity are the harvesting device inlet 19 and the pushing device respectively, and the inside of the cavity is provided with a filter screen 22 and a sterilizing filter membrane 23, and the filter sieve 22 is located at the sterilizing filter. Above the membrane 23, the lower end of the cavity is provided with a culture solution return pipe 21.

The pushing device includes: a spring 20, a push handle 24, a rubber hose 25, a piston push piece 26, the piston push piece 26 is closely attached to the inner wall of the cavity, and the piston push piece 26 is connected to one end of the push handle 24 , the outside of the push handle 24 is provided with a rubber hose 25, and a spring 20 is arranged between the rubber hose 25 and the push handle 24. During continuous harvesting, the plants/organs flow into the continuous harvesting device 13 along with the culture solution, and then the Achieve separation. The culture solution returns to the reactor main body 8 under the action of the liquid pump 3 after passing through the sterilizing filter membrane 23, and the medicinal plant tissues and organs are pushed into the inlet 19 of the harvesting device.

In this embodiment, the volume of the spray device 7 is 20ml.

As shown in Figure 3, the spray device 7 includes: a culture solution inlet 27, a siphon elbow 29, a siphon 30, a drain pipe 31, a spray head 16, a spray tank 33, a liquid level gauge 34, and a spray pipe 35, wherein : siphon pipe 30, siphon elbow 29, and spray pipe 35 are connected successively to form an inverted U-shaped shape. Siphon pipe 30 and siphon elbow 29 are all placed inside the spray tank 33, and a part of the spray tube 35 is placed inside the spray tank, and the other part Pass through the spray tank 33 and be arranged on the upper end of the reactor main body 8, and be connected with the spray head 16. The side wall of the spray tank 33 is provided with a liquid level gauge 34, and the upper and lower ends of the spray tank 33 are respectively provided with a culture solution inlet 27. And drain pipe 31.

In this embodiment, the upper ends of the liquid storage bottle 1 , the spray device 7 , the waste liquid bottle 14 , and the gas flow meter 15 are respectively provided with an air filter head 2 .

In this embodiment, the air filter head 2 is a glass filter element with a pore size of 100 m.

In this embodiment, the spray head 16 is a glass filter element with a pore size of 180 μm.

In this embodiment, the spray duration depends on the spray aperture and the volume of the spray tank 33, and the spray cycle is determined by the filling rate.

When the present embodiment works, the fresh culture solution flows from the liquid storage bottle 1 into the gas-liquid mixing tank 12 under the action of the first liquid pump 3, and the mixed culture solution is injected into the spray tank 33 through the culture solution inlet 27, when When the liquid level exceeds the siphon elbow 29, it begins to spray, and when the liquid level is lower than the siphon 30, the spray ends and enters the next spray cycle. The duration of the spray depends on the size of the spray aperture in the spray head and the volume of the spray tank, and the spray cycle is controlled by the first liquid pump 3 corresponding to the perfusion rate of the culture liquid. The biological growth environment in the reactor main body 8 is detected by the computer 11 through the oxygen probe 9 and the pH probe 10, so as to monitor in real time. The used culture solution in the reactor main body 8 flows into the waste liquid bottle 14, and the increased biomass passes through the continuous harvesting device 13 instant harvest.

In this example, the homozygous tetraploid adventitious roots of Radix Pseudostellariae were inoculated into a spray reactor with a volume of 4.8 liters and cultivated. The initial inoculation density was 5 g (dry weight)/L, and the culture solution was: 1/2MS+1.5 mg/L IBA+0.05mg/L 6-BA+30g/L sucrose, the ventilation rate is 0.3vvm, the perfusion rate is 50% (volume percentage) per day, the spray time is once 5 minutes, and the spray cycle is 4 times per hour, The cultivation temperature was 25°C, and the culture was carried out in the dark. The results showed that under this culture condition, the maximum average growth rate reached 0.25±0.08 per day, which was higher than 0.22±0.12 per day when cultured in an air-lift reactor.

This embodiment is a multifunctional bioreactor, which can be used for the continuous culture of medicinal plant tissues, cells and organs, and is suitable for the in vitro continuous culture of most medicinal plant tissues and organs.

Claims (1)

1. A siphon type intermittent spray bioreactor, comprising: liquid storage bottle, the first liquid pump, reactor main body, oxygen probe, pH probe, computer, it is characterized in that also comprising: spray device, gas-liquid mixing tank, Continuous harvesting device, waste liquid bottle, gas flow meter, and second liquid pump, wherein: one port of the liquid storage bottle is connected to the gas-liquid mixing tank through the first liquid pump, and the inner bottom of the gas-liquid mixing tank is provided with a gas spray head , the gas spray head is connected to the gas flow meter outside the gas-liquid mixing tank, the output port of the gas-liquid mixing tank is connected to the input port of the spray device, the output port of the spray device is connected to the input port of the reactor body, and the reactor body The output port is connected to the continuous harvesting device, the waste liquid output port of the reactor main body is connected to the waste liquid bottle, the continuous harvesting device is connected to the recovery port of the reactor main body through the second liquid pump, and the oxygen probe and the pH probe are arranged inside the reactor main body , the oxygen probe and the pH probe are connected to the computer; The continuous harvesting device includes: a cavity, a pushing device, a filter sieve, a sterilizing filter membrane, a harvesting port, an inlet of the harvesting device, and a culture fluid return pipe. The upper end of the cavity is provided with a harvesting port, and the two sides of the cavity are respectively For the harvesting device inlet and the driving device, the cavity is equipped with a filter screen and a sterilizing filter membrane, the filter sieve is located above the sterilizing filter membrane, and the lower end of the cavity is provided with a culture solution return pipe; The pushing device includes: a spring, a pushing handle, a rubber hose, and a piston-type pushing piece. The piston-type pushing piece is closely attached to the inner wall of the cavity. A rubber hose, a spring is arranged between the rubber hose and the push handle; The spray device includes: a culture solution inlet, a siphon elbow, a siphon, a drain pipe, a spray head, a spray tank, a liquid level gauge, and a spray tube, wherein: the siphon, the siphon elbow, and the spray tube are connected in sequence to form an inverted U-shaped, the siphon tube and the siphon elbow are all placed inside the spray tank, a part of the spray tube is placed inside the spray tank, and the other part is set on the upper end of the reactor body through the spray tank and connected with the spray head. The side wall is provided with a liquid level gauge, and the upper end and the lower end of the spray tank are respectively provided with a culture solution inlet and a liquid discharge pipe; The liquid storage bottle, the first liquid pump, the gas flow meter, the spray device, the reactor main body, the continuous harvesting device, the waste liquid bottle, and the second liquid pump are all connected by silicone tubes; The upper ends of the liquid storage bottle, the spray device, the waste liquid bottle and the gas flow meter are respectively provided with air filters.

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