CN113862147A - Electromagnetic coupling embedded double-tank device and application thereof - Google Patents
Electromagnetic coupling embedded double-tank device and application thereof Download PDFInfo
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- CN113862147A CN113862147A CN202111353681.6A CN202111353681A CN113862147A CN 113862147 A CN113862147 A CN 113862147A CN 202111353681 A CN202111353681 A CN 202111353681A CN 113862147 A CN113862147 A CN 113862147A
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Abstract
The invention relates to an electromagnetic coupling embedded double-tank device and application thereof.A large fermentation tank is embedded with two single tanks, each single tank is provided with a stirrer, the surface of each single tank is provided with countless regular round uniformly-distributed small holes, the rotation directions of the single tank stirrers are opposite, the rotating speed is adjustable, a magnetic field is distributed around the large tank body, the rotating speeds of the small tank stirrers are different, the whole tank body forms a countless electric field, and industrial microbial strains are efficiently expressed in an electromagnetic coupling field. Because the single tank of the system has different stirring speeds, different fermentation formulas, different material supplementing substances and different electric field strengths (0-100 mV/cm) formed by fluid viscosity, and the magnetic field strength can be adjusted by the distance between N and S poles (0.1-0.6T), the electromagnetic coupling double-tank device has obvious effect of improving the production titer of the biological pesticide industrial strains such as streptomyces aureofaciens, streptomyces aureochromogenes, streptomyces lavendulae varietal and the like through fermentation.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to an electromagnetic coupling embedded double-tank device and application thereof.
Background
Biological fermentation cannot be separated in the industries of biological medicine, food, biological pesticide, sewage treatment and the like, and the current biological fermentation industry has the following defects: (1) the biological fermentation brings a large amount of waste water and waste gas with special smell, which not only is difficult to recover and treat, increases the unit consumption of raw materials and reduces the product conversion rate, but also pollutes the subsequent fermentation process, thus causing further waste of the raw materials; (2) the pollution caused by the production of chemical raw material medicines has the characteristics of multiple types, complex components, high concentration, high toxicity, high volatility, intermittent discharge, unstable pH value, high chemical oxygen demand, difficult biochemical degradation and the like, and great difficulty and cost pressure are brought to the treatment of pollutants; (3) at present, biological fermentation still faces the biggest technical problem, fermentation equipment lacks technical innovation, although the automation degree is high in the aspects of feeding, stirring, acid and alkali adjustment, tank body disinfection, temperature control, air filtration disinfection and the like, online control of the parameters is usually realized by adopting DCS, the industrial fermentation adopts the traditional single-tank fermentation, the target product obtained by the single-tank fermentation has a boundary effect, namely, no matter how to optimize the fermentation parameters, scientifically and reasonably prepare materials, or utilize synthetic biology even gene editing technology to carry out gene modification on industrial strains, the target product obtained by the industrial strains in the single-tank fermentation is improved in a limited way, and the biggest technical bottleneck is faced in the current biological fermentation industry.
Aiming at the bottleneck of conventional biological fermentation, the electromagnetic coupling embedded double-tank device is used for fermentation, and the fermentation mode has the advantages that: (1) two single tanks are embedded in the electromagnetic coupling double-tank fermentation large tank, each single tank is provided with a stirrer, the surface of each single tank is provided with regularly arranged small holes, the rotation directions of the single tank stirrers are opposite, the rotating speed is adjustable, a magnetic field is distributed around the large tank, due to the different rotating speeds of the small tank stirrers, countless biological electric fields are formed in the whole tank, and the gene and protein time sequence efficient expression of industrial microbial strains is carried out in the electromagnetic coupling environment; (2) the fermentation system has adjustable magnetic field intensity, and the electric field intensity is related to the stirring speed of two single tanks, the addition amount of trace elements in ingredients, the viscosity of fluid, feeding and the like; (3) the advantages of industrial strains for producing target products are obviously exerted by the embedded double-tank fermentation, namely the embedded double-tank fermentation contributes to the failure of the side regulation factors and the positive regulation and synergism of target product genes; (4) the embedded double-tank fermentation can shorten the fermentation period and reduce the discharge of by-products (waste water, waste residues and waste gases); finally, energy is saved, consumption is reduced, and a target product is produced in large quantities; (5) by the optimized combination of the electric field and the magnetic field intensity of the system, the fermentation potential of the industrial strain in the system is fully exerted, so that the gene transcription of a target product of the industrial strain is efficiently expressed when the industrial strain is fermented in the system.
Disclosure of Invention
In order to solve the problems of various defects of single-tank fermentation adopted in the traditional fermentation industry, the invention firstly provides an electromagnetic coupling embedded double-tank device which comprises a large fermentation tank (1), wherein two small tanks with the same shape and volume are embedded in the large fermentation tank, countless small holes (21) which are regularly, circularly and uniformly distributed are distributed around the small tanks, stirrers (22) are arranged inside the two small tanks, a magnetic field (3) is distributed around the large tank, a circulating water heating pipeline (13), a sterilizing air pipeline (14), a pH sensor (11), a dissolved oxygen sensor (12), an automatic acid and alkali adjusting system and an automatic material supplementing system, and the large fermentation tank (1) and the two small tanks adopt a DCS (distributed control system) on line.
In the invention, the industrial microorganism strains carry out high-efficiency expression of target products in an electromagnetic coupling field, and the field intensity formed by different stirring speeds of a single tank body, fermentation formulas, feeding materials and fluid viscosity (0-100 mV/cm) in the device can be adjusted by the distance between N and S poles (0.1-0.6T); the electromagnetic coupling embedded double-tank fermentation system can reduce the emission of fermentation byproducts of industrial strains, increase the production of target products of the industrial strains and shorten the fermentation period; the electromagnetic coupling double-tank device fermentation has obvious effect of improving the production titer of the biological pesticide industrial strains such as streptomyces aureofaciens, streptomyces aureochromogenes, streptomyces lavendulae Hainan variety and the like; the electromagnetic coupling embedded double-tank device is used for fermentation to subvert the traditional industrial fermentation technology, meets the national requirement of advocating green biological intelligent manufacturing, and is a subversive and revolutionary technology for food, enzyme chemical industry, medicine and fermented feed industries.
In the preferred embodiment of the invention, the volume of the large tank is adjustable according to the actual fermentation requirement, and is preferably 0.03-100 m3(ii) a Preferably the small tank volume is 1/3 of the large tank volume.
In a preferred embodiment of the present invention, the diameter of the small hole is 1 to 2mm, and the diameter of the small hole of one small pot is 2 times of the diameter of the small hole of the other small pot.
In a preferred embodiment of the invention, the rotating speed power of the stirrer is 0.75 kW-75 kW, the rotating speeds of the stirrers in the two small tanks are opposite, the magnetic field is distributed around the tank body of the large tank, the rotating speeds of the stirrers in the two small tanks are different, and the whole tank body forms an infinite number of electric fields.
In a preferred embodiment of the invention, the magnetic field is a magnetic field whose magnetic induction is adjusted by the distance between the two N poles and the S pole, and the magnetic induction is 0.1-0.6T, preferably 0.2T; the external electric field of the device passes through a direct current power supply, and the current intensity of the device is adjustable at a distance of 0-100mV/cm, preferably 40 mV/cm.
The invention also protects the application of the device in fermenting the small streptomyces aureofaciens to produce kasugamycin, the streptomyces aureofaciens and the Hainan variety of the streptomyces lavendulae.
The formula of the culture medium of the streptomyces aureofaciens can refer to an invention patent entitled "a kasugamycin fermentation culture medium and a fermentation method (CN 201711346595.6)", and the formula of the fermentation culture medium of the streptomyces aureofaciens is as follows: 8.0% of soybean meal, 0.5% of NaCl, 1.0% of corn steep liquor dry powder, 2.5% of maltose, 4.0% of fish oil and KH2PO4 0.05 percent, 0.03 percent of inositol and sterile water, and the pH value is 7.2; the formula of the streptomyces aureochromogenes fermentation medium is as follows: corn flour 10%, low-temperature soybean cake powder 2.5%, beer yeast powder 0.4%, fish meal 0.5%, NaCl 0.3%, CaCO3 0.3%、KH2PO40.05 percent of alpha-amylase, 0.015 percent of sterilized water, and sterilizing at high temperature for later use, wherein the pH value of the fermentation medium is 6.5-6.8, and the fermentation medium is sterilized at high temperature for later use; the formula of the Hainan variant fermentation medium of the Streptomyces lavendulae is as follows: soluble starch (AR) 1.0%, MgSO4·7H2O（AR）0.045%、KH2PO4（AR）0.10%、KCl（AR）0.045%、NaNO3（AR）0.125%、FeSO4·7H2O (AR) 0.001%, and distilled water (pH6.8-7.0), and sterilizing at high temperature.
In a preferred embodiment of the invention, the Hainan varieties of Streptomyces parvus, Streptomyces aureochromogenes and Streptomyces lavendulae are subjected to the same single-tank fermentation conditions and fermentation process as the electromagnetic coupling embedded double-tank fermentation conditions and fermentation process.
In a preferred embodiment of the invention, when the magnetic field intensity is 0.2T and the electric field intensity is 60mV/cm, the average fermentation titer of the streptomyces aureofaciens in a single tank is 14520 +/-125 mu g/mL, the fermentation period is 189h, the average fermentation titer of the streptomyces aureofaciens in an electromagnetic coupling embedded double-tank device is 16252 +/-106 mu g/mL, and the fermentation period is 180 h; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
In a preferred embodiment of the invention, the average titer of the Streptomyces aureochromogenes in the single-tank fermentation is 3125 +/-18 mu g/mL at a magnetic field strength of 0.3T and an electric field strength of 40mV/cm, and the fermentation period is 120 h; the average titer of the golden streptomyces chromogenes fermented in the electromagnetic coupling embedded double-tank device is 3925 +/-13.6 mu g/mL, and the fermentation period is 108 h; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
In a preferred embodiment of the invention, the average titer of the Hainan variant of Streptomyces lavendulae fermented in a single tank is 8795 +/-95 mu g/mL at the magnetic field strength of 0.2T and the electric field strength of 40mV/cm, and the fermentation period is 84 h; the average titer of the Hainan variant of the Streptomyces lavendulae fermented in the electromagnetic coupling embedded double-tank body is 11189 +/-103 mu g/mL, and the fermentation period is 76 h; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
Compared with the prior art, the invention has the following advantages:
1. the electromagnetic coupling embedded double-tank device can shorten the fermentation period of the target industrial strain macroscopically, improve the production yield of enterprises in unit time, save energy and reduce consumption, and further reduce the three-waste discharge of fermentation enterprises in unit time; the three-waste treatment pressure of the enterprise is relieved, the turnover time of the mobile capital of the enterprise is shortened, and the utilization rate of the capital of the enterprise is further increased;
2. the production titer of the target industrial strain can be improved by the electromagnetic coupling embedded double-tank device fermentation, and the market competitiveness and the technical product with high added value are further improved for enterprises; the electromagnetic coupling embedded double-tank device fermentation can fully mobilize the gene time sequence of the target industrial strain to express efficiently in a microscopic way, namely the embedded double-tank fermentation is beneficial to the failure of the side regulation factor and the positive regulation synergistic effect of the target product gene.
Drawings
The following is further described with reference to the accompanying drawings:
fig. 1 is an electromagnetically coupled damascene type dual can device of the present invention.
The device comprises a large fermentation tank (1), a single tank (2), a magnetic field (3), small holes (21), a stirrer (22), a pH sensor (11), a dissolved oxygen sensor (12), a circulating water heating pipeline (13) and a sterilized air pipeline (14).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not to be construed as limiting the invention.
The invention is further illustrated by the following examples.
Example 1:
(1) single jar of tertiary fermentation equipment of body purchases Nanjing Tianhui biotechnology limited company, zero level fermentation cylinder total volume is 5L, one-level fermentation cylinder volume is 30L, second grade fermentation cylinder volume is 50L, sterile air pipe is installed to every fermentation cylinder, the sterile air is produced by small-size air compressor and is inputed to the fermentation cylinder (being equipped with the air flow meter) by the pipe, the vapor of the disinfection jar body is produced by steam generator and is inputed to the fermentation cylinder by the pipe (the centre is equipped with the solenoid valve, the steam gauge), every fermentation cylinder is equipped with real time monitoring dissolved oxygen, pH, the temperature, the rotational speed, the feed supplement, defoaming and the acid and alkali system of making up. The electromagnetic coupling embedded double-tank body is designed and processed, the peripheral tank of the zero-level electromagnetic coupling double-tank body is 5L, two 1.5L single tanks are arranged in the embedded double-tank body, a magnetic field and an electric field are distributed around the embedded double-tank body, the diameters of the built-in single tanks are 1mm and 2mm, the volumes of the embedded double-tank body and the embedded double-tank body are 30L, two 10L single tanks are arranged in the embedded double-tank body, a magnetic field and an electric field are distributed around the embedded double-tank body, the diameters of the built-in single tanks are 1mm and 2mm, the volumes of the secondary fermentation tanks are 50L, two 20L single tanks are arranged in the embedded double-tank body, the magnetic field and the electric field are distributed around the embedded single tanks, the diameters of the built-in single tanks are 1mm and 2mm, the rest fermentation conditions, the inoculation quantity and other parameters are the same with the single-tank body fermentation medium formula of each stage fermentation tank of the small streptomyces aureofaciens: corn flour 0.3%, glucose 0.35%, yeast extract powder 0.35%, KH2PO4 0.15%，CaCO3 0.45%, soybean meal 0.45%, fish meal 0.13%, NaCl0.15%, soybean oil 0.015%, (NH)4)2SO40.1 percent, 0.006 percent of GPE defoaming agent, 0.0005 percent of amylase and the balance of sterilized water. The batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 29h, and the first-level fermentation time is 34h and the second-level fermentation time is 189 h. The fermentation conditions were: the temperature is 28-30 ℃, the ventilation rate is 1:1, the stirring speed is 150-200 rpm, the tank pressure is 0.02MPa, and the fermentation clear liquid is collected and purified for 189 hours to obtain the kasugamycin. The supernatant of 120h fermentation liquor after the secondary tank fermentation is ultracentrifuged for 30min at 12000rmp is sequentially filtered by a ceramic membrane (the average membrane pore diameter is 0.1 mu m, and the transmembrane pressure is 0.2-0.3mPa) to determine the kasugamycin content, and specific results are shown in Table 1 below.
TABLE 1 comparison of electromagnetic coupling embedded double-tank fermentation effect of Streptomyces aureofaciens and conventional single-tank fermentation effect
Example 2:
(2) single jar of tertiary fermentation equipment of body purchases Nanjing Tianhui biotechnology limited company, zero level fermentation cylinder total volume is 5L, one-level fermentation cylinder volume is 30L, second grade fermentation cylinder volume is 50L, sterile air pipe is installed to every fermentation cylinder, the sterile air is produced by small-size air compressor and is inputed to the fermentation cylinder (being equipped with the air flow meter) by the pipe, the vapor of the disinfection jar body is produced by steam generator and is inputed to the fermentation cylinder by the pipe (the centre is equipped with the solenoid valve, the steam gauge), every fermentation cylinder is equipped with real time monitoring dissolved oxygen, pH, the temperature, the rotational speed, the feed supplement, defoaming and the acid and alkali system of making up. The electromagnetic coupling embedded double-tank body is designed and processed, the peripheral tank of the zero-level electromagnetic coupling double-tank body is 5L, two 1.5L single tanks are arranged in the embedded double-tank body, a magnetic field and an electric field are distributed around the single tanks, the diameters of the single tanks are 1mm and 2mm, the volume of the embedded double-tank body is 30L, two 10L single tanks are arranged in the embedded double-tank body, a magnetic field and an electric field are distributed around the embedded double-tank body, the diameters of the single tanks are 1mm and 2mm, the volume of the secondary fermentation tank is 50L, two 20L single tanks are arranged in the embedded double-tank body, the magnetic field and the electric field are distributed around the embedded single tanks, the diameters of the single tanks are 1mm and 2mm, and other fermentation conditions, inoculation amount and other parameters are the same as those of the single tank bodyThe fermentation is the same, and the formula of the streptomyces aureochromogenes fermentation medium is as follows: corn flour 10%, low-temperature soybean cake powder 2.5%, beer yeast powder 0.4%, fish meal 0.5%, NaCl 0.3%, CaCO3 0.3%、KH2PO40.05 percent, 0.015 percent of alpha-amylase and sterile water, and sterilizing at high temperature for standby. The batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 24 hours, the first-level fermentation time is 48 hours, and the second-level fermentation time is 120 hours. The fermentation conditions were: the temperature is 28-30 ℃, the ventilation volume is 1.5:1, the stirring speed is 150-200 rpm, the tank pressure is 0.02MPa, the fermentation clear liquid is collected after secondary fermentation is carried out for 120h, the polyoxin is obtained by purification, the titer of the polyoxin is measured by adopting a bioassay method, and the specific measurement results are shown in Table 2 below.
TABLE 2 comparison of the electromagnetic coupling embedded type double-tank fermentation Streptomyces aureochromogenes with the conventional single-tank fermentation effect
Example 3:
(3) single jar of tertiary fermentation equipment of body purchases Nanjing Tianhui biotechnology limited company, zero level fermentation cylinder total volume is 5L, one-level fermentation cylinder volume is 30L, second grade fermentation cylinder volume is 50L, sterile air pipe is installed to every fermentation cylinder, the sterile air is produced by small-size air compressor and is inputed to the fermentation cylinder (being equipped with the air flow meter) by the pipe, the vapor of the disinfection jar body is produced by steam generator and is inputed to the fermentation cylinder by the pipe (the centre is equipped with the solenoid valve, the steam gauge), every fermentation cylinder is equipped with real time monitoring dissolved oxygen, pH, the temperature, the rotational speed, the feed supplement, defoaming and the acid and alkali system of making up. The electromagnetic coupling embedded double-tank body is designed and processed, the peripheral tank of the zero-level electromagnetic coupling double-tank body is 5L, two 1.5L single tanks are arranged in the embedded double-tank body, a magnetic field and an electric field are distributed around the single tanks, the diameters of the single tanks are 1mm and 2mm, the volume of the embedded double-tank body primary fermentation tank is 30L, two 10L single tanks are arranged in the embedded double-tank body, the magnetic field and the electric field are distributed around the embedded double-tank body, the diameters of the single tanks are 1mm and 2mm, the volume of the secondary fermentation tank is 50L, two 20L single tanks are arranged in the embedded double-tank body, the magnetic field and the electric field are distributed around the embedded single tanks, and the diameter of the single tanks is 1mm and 2mmThe aperture of 1mm and 2mm, the other fermentation conditions, the inoculation amount and other parameters are the same as those of single-tank fermentation, and the formula of the fermentation medium of the streptomyces lavendulae is as follows: soluble starch (AR) 1.0% MgSO4·7H2O、（AR）0.045%、KH2PO4（AR）0.10%、KCl（AR）0.045%、NaNO3（AR）0.125%、FeSO4·7H2O (AR) 0.001%, distilled water in balance, pH6.8-7.0, and high-temperature sterilizing. The batch inoculation amount is 10-15% of the fermentation volume, the zero-level fermentation time is 24 hours, the first-level fermentation time is 48 hours, and the second-level fermentation time is 84 hours. The fermentation conditions were: the temperature is 28-30 ℃, the ventilation rate is 1:1, the stirring speed is 150-200 rpm, the tank pressure is 0.02MPa, the fermentation clear liquid is collected after secondary fermentation is carried out for 84h, the zhongshengmycin is obtained by purification, the titer of the zhongshengmycin is measured by adopting a bioassay method, and the specific measurement result is shown in the following table 3.
TABLE 3 comparison of electromagnetic coupling embedded type double-tank fermentation of Hainan variety of Streptomyces lavendulae with conventional single-tank fermentation
Although the invention has been described in detail in the foregoing with reference to general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto based on the invention. Therefore, it is intended that the present invention cover the modifications and improvements made without departing from the spirit and scope of the present invention.
Claims (10)
1. The utility model provides an electromagnetic coupling inserted double-tank body device, a serial communication port, the device includes large-scale fermentation cylinder (1), two shape and the same little jars of volume of inlaying in, lay countless circular evenly distributed's of rule aperture (21) around the little jar, two little jar internally mounted agitators (22), lay magnetic field (3) around the big jar, settle circulating water heating pipeline (13) in the big jar, disinfection air pipeline (14), pH sensor (11), dissolved oxygen sensor (12), automatic acid-base system of transferring, automatic feed supplement system, large-scale fermentation cylinder (1) and two little jars adopt DCS on-line control system.
2. The device according to claim 1, characterized in that the large tank volume is adjustable according to actual fermentation requirements, preferably 0.03-100 m3(ii) a Preferably the small tank volume is 1/3 of the large tank volume.
3. The device of claim 1, wherein the diameter of the small holes is 1-2 mm, and the diameter of one small pot of small holes is 2 times of the diameter of the other small hole.
4. The device of claim 1, wherein the rotating speed of the stirrer is 0.75 kW-75 kW, the rotating speeds of the stirrers in the two small tanks are opposite, the rotating speed is adjustable, a magnetic field is distributed around the tank body of the large tank, the rotating speeds of the stirrers in the two small tanks are different, and an infinite number of electric fields are formed in the whole tank body.
5. The device of claim 1, wherein the magnetic field is a magnetic induction intensity adjusted by the distance between the two N poles and the S pole, and the magnetic induction intensity is 0.1-0.6T, preferably 0.2T; the external electric field of the device passes through a direct current power supply, and the current intensity of the device is adjustable at a distance of 0-100mV/cm, preferably 40 mV/cm.
6. Use of the device according to any one of claims 1-5 for fermenting Streptomyces parvus kasugamycin, Streptomyces aureochromogenes and Streptomyces lavendulae Hainan variety.
7. The use of claim 6, wherein the Streptomyces parvus, Streptomyces aureochromogenes and Streptomyces lavendulae Hainan variant are fermented in a single tank under the same conditions as the fermentation process and in an electromagnetic coupling embedded double-tank under the same conditions as the fermentation process.
8. The use of claim 6 or 7, wherein the average titer of the Streptomyces aureofaciens fermented in a single tank is 14520 +/-125 μ g/mL, the fermentation period is 189h, the average titer of the Streptomyces aureofaciens fermented in an electromagnetic coupling embedded double-tank device is 16252 +/-106 μ g/mL, and the fermentation period is 180h, when the magnetic field strength is 0.2T and the electric field strength is 60 mV/cm; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
9. The use according to claim 6 or 7, characterized in that the average titer of Streptomyces aureochromogenes in a single-pot fermentation is 3125 ± 18 μ g/mL at a magnetic field strength of 0.3T and an electric field strength of 40mV/cm, the fermentation period being 120 h; the average titer of the golden streptomyces chromogenes fermented in the electromagnetic coupling embedded double-tank device is 3925 +/-13.6 mu g/mL, and the fermentation period is 108 h; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
10. The use according to claim 6 or 7, wherein the average titer of S.lavendulae Hainan variant in a single-pot fermentation is 8795 ± 95 μ g/mL at a magnetic field strength of 0.2T and an electric field strength of 40mV/cm, and the fermentation period is 84 h; the average titer of the Hainan variant of the Streptomyces lavendulae fermented in the electromagnetic coupling embedded double-tank body is 11189 +/-103 mu g/mL, and the fermentation period is 76 h; the fermentation titer is higher than any other combination of magnetic field and electric field strength.
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