WO2021015587A1 - Biosurfactant production apparatus and biosurfactant production method using same - Google Patents

Abstract

The present invention relates to a biosurfactant production apparatus comprising: a first and a second air injection pipe for concentrating foam and recovering a culture medium; a foam sensing part; and a foam discharging part. Provided with a foam sensing part and a first and a second air injection pipe, the biosurfactant production apparatus can attain highly concentrated foam by controlling an amount and concentration of foam and can reduce the loss of the culture medium by preventing the overflow of the foam generated during microorganism cultivation or the discharge of the culture medium together with the foam. Furthermore, when cultured using the biosurfactant production apparatus according to the present invention, the microorganism can be protected. Some cells present in the collected foam are removed to prolong the time period of producing a surfactant, thereby improving the production yield of the biosurfactant.

Description

Bio-surfactant production apparatus and bio-surfactant production method using the same

The present invention relates to an apparatus for producing a bio-surfactant and a method for producing a bio-surfactant using the same, and more specifically, to a culture unit for culturing a microorganism; A foam sensing unit positioned at the top of the culture unit and configured to sense a bubble generated from the culture unit; A first air injection tube for injecting air into the culture solution in the culture unit; A foam discharge unit through which the bubbles generated from the culture unit are discharged; A second air inlet tube for injecting air from the upper part of the culture unit toward the culture solution to dispel bubbles generated in the culture process to increase density; And a foam collection unit connected to the foam discharge unit and configured to collect the generated foam. A bio-surfactant production device comprising: a bio-surfactant production device and a bio-surfactant production device comprising controlling the operation of the first and second air injection pipes according to bubble sensing of the bubble sensing unit. It relates to a method for producing a surfactant.

Bio-surfactants are natural surfactants present in animals and plants, such as lecithin, saponin, and bile acids, and surfactants produced by metabolism of microorganisms. Surfactin, a bio-surfactant, is known to be the most powerful material among bio-surfactants.

Bacillus subtilis include Bacillus Belle Zen sheath (B.velezensis), Bacillus amyl kwipe sieon Lowry's (B. amyloliquefaciens) to produce the seopaek tin (surfactin) bio-surfactant (biosurfactants) consisting of a lipid and an amino acid, Bacillus piece nipo miss (B licheniformis ), B. mojavensis , B. pumilus , and B. subtilis are known (Chen et al., 2015).

Surfactin is known to exhibit strong antiviral, antifungal, and antimicroplasma effects, and among them, antibiotic efficacy is the most known. Surfactin is in high demand in the pharmaceutical industry, environmental purification industry, cosmetics industry, etc. because it exhibits various effects as described above, but it has not been largely successful in commercialization because it is difficult to mass-produce due to low production efficiency.

There is a chemical synthesis method as a method for producing bio-surfactants, especially surfactin, but there is a disadvantage that the molecular weight is too large to produce surfactin by this method, or the synthesis cost is high, and it is produced by fermentation in a microbial culture system. The method is economical, but there is a limit to mass production of surfactin.

In addition, in order to overcome the limitations of the surfactin production method as described above, a method has been developed to produce in the field by spraying Bacillus bacteria that produce surfactin directly on crops. Specifically, European Patent No. 03274442 and Korean Patent No. 15557062 use Bacillus Bacillus as agricultural materials to produce surfactin by spraying Bacillus directly onto crops, thereby directly exhibiting effects such as antibacterial and antibiotics on crops. However, naturally occurring bacteria have a low overall amount of surfactant production in their natural state, as well as the amount of production that is greatly affected by temperature, so their efficacy is insignificant, so users avoid using it and rather prefer chemical pesticides.

In addition, surfactin has a strong emulsifying function, but it is used only as an expensive cosmetic and drug additive because its production efficiency is low and its production cost is high when it is used as a kitchen detergent, laundry detergent, and bath detergent, which are mainly used in everyday life. In order to convert synthetic detergents, which are the cause of environmental pollution, into eco-friendly detergents, the technology to lower the production cost by increasing production efficiency is required. Accordingly, various Bacillus bacteria culture devices have been devised to increase the production efficiency of surfactin and lower the production cost. Became. First, Korean Registered Patent No. 1139702 relates to a microbial complex culture device. It is equipped with a sensor that detects temperature, dissolved oxygen, pH and pressure in the culture tank to check each element in real time and attach an automatic control system to automatically or manually However, a configuration for solving a problem such as overflow of the culture medium due to foam generation when culturing microorganisms is not disclosed. Korean Patent Registration No.0737709 uses a silicone foam control agent to prevent bubbles from occurring by causing a malfunction of the incubator or preventing the overflow of the incubator by attaching bubbles generated during cultivation of microorganisms to various sensors. It is difficult to increase the production efficiency of surfactants because foaming is suppressed by using a foam control agent or an antifoaming agent, and it is economically unfavorable because an additional cost occurs because the antifoaming agent must be removed during the purification process.

On the other hand, a device for passively collecting bubbles has been devised as a device for improving the problems caused by the use of a foam control agent during cultivation, as in Korean Patent Registration No. 0737709. Korean Patent Registration No. 1120093 is a culture system that passively collects bubbles generated during microbial cultivation, and uses a passive method of connecting the bubble storage tank to the culture tank so that the bubbles generated in the culture tank pass to the bubble storage tank through a connection pipe. Use. However, the culture system has a problem that the culture solution is moved to the bubble storage tank together with bubbles due to the increased pressure by the narrow connection pipe, and the loss of the culture solution is severe.Even if air is continuously injected, the growth efficiency of microorganisms is increased or the surfactant It is difficult to improve your productivity. In addition, in the culture system, it is difficult to efficiently produce high-density bubbles in which surfactin is concentrated in a high concentration as the low-density foam (non-concentrated foam) generated at the beginning of culture moves to the foam storage tank. It is difficult to produce surfactants in large quantities.

Accordingly, the present inventors have made intensive research efforts to overcome the problems of the prior art. In the case of a bio-surfactant production device equipped with a foam sensing unit, a first air injection pipe, a second air injection pipe, and a foam discharge unit, foam By providing a sensing unit, a first air injection pipe and a second air injection pipe, mass production of surfactants is possible and bubbles can be discharged because only high-density bubbles can be moved to the foam collection unit by controlling the amount of bubbles and concentrating the bubbles. By providing a part, it is possible to prevent the phenomenon that the bubble overflows or the culture solution is discharged with the bubble, thereby reducing the loss of the culture solution, and by removing some cells present in the collected bubble, it is possible to form a durable body through quorum sensing. It was confirmed that the production efficiency of surfactin can be improved by not only making it possible to prolong the period for producing surfactants by delaying but also suppressing the autotoxicity of Bacillus bacillus caused by the produced bio-surfactants, especially surfactin. Finished.

Therefore, the main object of the present invention is to control the amount of foam and concentrate the foam to move only high-density foam to the foam collection unit, so that mass production of surfactant is possible, and the foam overflows or the culture solution is discharged with the foam. By preventing the phenomenon, it is possible to reduce the loss of the culture medium, and by removing some cells present in the collected foam, it is possible to prolong the production period of surfactant by delaying the formation of durable bodies through quorum sensing. It is to provide an apparatus for producing a bio-surfactant capable of improving the production efficiency of surfactin by suppressing the autotoxicity of Bacillus bacteria caused by a surfactant.

Another object of the present invention is to provide a method for producing a bio-surfactant using the bio-surfactant production device.

[National R&D project that supported this invention]

[Task identification number] 2019A005

[Department name] Chungcheongbuk-do, Cheongju

[Research Management Professional Institution] Chungbuk Industrial-Academic Convergence Headquarters

[Research Project Name] 2019 Industry-Academic Convergence R&D Support Project

[Research Project Name] Process development for mass acid acid of sulfuric acid, an eco-friendly surfactant

[Contribution rate] 1/1

[Organization] Prox & Rem Co., Ltd.

[Research Period] 2019.02.21 ~ 2019.12.20

[National R&D project that supported this invention]

[Task identification number] 2020A008

[Task number] 2020A008

[Ministry Name] Chungcheongbuk-do, Cheongju

[Name of project management (professional) institution] Chungbuk Industry-Academic Convergence Headquarters

[Research Project Name] 2020 Industry-Academic Convergence R&D Support Project

[Research Title] Pilot Facility Operation Method for Industrialization of Biosurfactant Sulpactin

[Contribution rate] 1/1

[Name of project execution organization] Prox Enrem

[Research Period] 2020.04.01 ~ 2020.12.20

According to an aspect of the present invention, the present invention provides a culture unit for culturing microorganisms, a foam sensing unit positioned at the top of the culture unit and for sensing bubbles generated from the culture unit, and injecting air into the culture solution in the culture unit. A first air inlet pipe for, a foam outlet through which bubbles generated from the cultivation unit are discharged, a second air inlet pipe for high density by blowing air from the upper portion of the cultivation unit toward the culture solution to blow out bubbles generated in the cultivation process, And a foam collection unit connected to the foam discharge unit and configured to collect generated bubbles, wherein the first and second air injection pipes are controlled according to the foam sensing unit. It provides a bio-surfactant production device characterized by.

Conventionally, in order to collect bubbles generated during microbial cultivation, a method of collecting bubbles in a bubble storage tank using a narrow connector has been used. However, when a narrow connector is used, the loss of the culture solution is large because the culture solution moves together with the bubbles due to the increased pressure, and it is difficult to mass-produce the bio-surfactant because the unconcentrated low-density bubbles are first collected in the bubble storage tank. Accordingly, the present inventors reduce the loss of the culture solution by reducing the pressure due to the movement of the bubbles through the bubble discharge unit, and control the operation of the first and second air injection pipes through the bubble sensing unit to control the amount of bubbles and By preventing collection in this storage tank, a bio-surfactant production apparatus was invented that could efficiently produce a bio-surfactant.

In the bio-surfactant production apparatus of the present invention, the first air injection pipe continuously injects air into the foam sensing unit until the foam is sensed, and after the foam is sensed, air is supplied at intervals of 1 to 10 minutes. It is characterized in that air is periodically injected by inflow and blocking.

In the bio-surfactant production apparatus of the present invention, the amount of air introduced into the first air injection pipe is 2.5 to 10 VVM until the foam is sensed, and 0.5 to 2.0 VVM after the foam is sensed.

In the bio-surfactant production apparatus of the present invention, when the foam is sensed in the foam sensing unit, the second air injection pipe is operated or blocked.

In the bio-surfactant production apparatus of the present invention, when the foam sensing unit senses the foam once, the second air injection pipe is operated (on), so that air is introduced in an amount of 0.5 to 1.5 VVM, and the foam is sensed twice. In this case, the amount of air introduced into the second air injection pipe is increased to 2 to 10 VVM, and when the bubbles are sensed three times, the second air injection pipe is blocked (off).

In the bio-surfactant production apparatus of the present invention, the second air injection pipe is in the form of a spray.

In the bio-surfactant production apparatus of the present invention, the bubble discharge part is configured in a bottleneck shape, and the diameter of the upper end of the bubble discharge part is wider than the diameter of the lower end of the bubble discharge part through which bubbles are introduced.

In the bio-surfactant production apparatus of the present invention, the diameter of the upper end of the foam discharge part is 2 to 10 times wider than the diameter of the lower end of the foam discharge part into which the foam is introduced.

In the bio-surfactant production apparatus of the present invention, the culture unit is provided in a culture tank for culturing microorganisms, a propeller for agitation of a culture solution for agitating the culture solution, and a low density produced by being provided on the inside of the culture tank It characterized in that it comprises a propeller for removing bubbles for removing bubbles, and a control sensor provided inside the culture tank and for controlling the state of the culture solution.

In the bio-surfactant production apparatus of the present invention, the air injection unit is connected to an air pump equipped with a pressure gauge for injecting air, and is connected to the air pump and is connected to the inside of the culture tank and the bubble discharge unit on the other side connected to the air pump. An air movement pipe for moving the introduced air, a first valve for controlling air inflow to the first air injection pipe, and a second valve for controlling air inflow to the second air injection pipe. To do.

In the bio-surfactant production apparatus of the present invention, the foam collection unit is connected to the upper portion of the foam discharge unit, and is connected to a foam transfer pipe for moving the concentrated foam through the foam discharge unit, and to a foam transfer pipe to collect bubbles. It characterized in that it comprises a container, a foam liquefaction member provided in the interior of the collection container for liquefying bubbles, and an air outlet provided at one end of the collection container and for adjusting the air pressure.

According to another aspect of the present invention, the present invention provides a first step of culturing microorganisms and a culture solution in a culture tank injected with air, a second step of sensing bubbles generated in the culture tank with a foam sensor, and bubbles generated in the culture tank. It provides a method for producing a bio-surfactant comprising a third step of collecting and recovering the culture solution, and a fourth step of obtaining the bio-surfactant from the collected foam.

In the production method of the bio-surfactant of the present invention, the first step is characterized in that during culturing, air in an amount of 2.5 to 10 VVM is continuously injected into the first air inlet tube.

In the production method of the bio-surfactant of the present invention, when the bubble is sensed in the second step, the air introduced into the first air injection pipe is adjusted to an amount of 0.5 to 2.0 VVM and is introduced at intervals of 1 to 10 minutes. do.

In the production method of the bio-surfactant of the present invention, when bubbles are sensed in the second step, the second air injection pipe is operated to inject air, and the air injection is performed in the amount of 0.5 to 1.5 VVM air per bubble sensing first time. Is injected, air is injected in the amount of 2.0 to 10 VVM in the second bubble sensing, and air is not injected in the third bubble sensing.

In the production method of the bio-surfactant of the present invention, the third step of collecting the generated bubbles is step 3-1 of moving the generated bubbles to the foam discharge unit, and the bubbles moved to the foam discharge unit are moved to the foam transfer pipe. It characterized in that it comprises a step 3-2, and a third step of recovering the culture medium moved to the bubble discharge unit to the culture tank.

In the production method of the bio-surfactant of the present invention, the fourth step of obtaining the bio-surfactant is characterized in that the bubble collected in the foam collection container is liquefied to obtain the bio-surfactant.

In the production method of the bio-surfactant of the present invention, it is characterized in that it further comprises the step of additionally supplying a culture solution in the same amount as the culture solution in which the bubbles are liquefied after removing the bubbles in the fourth step.

The bio-surfactant production apparatus according to the present invention includes a foam sensing unit, first and second air injection pipes, and controls the operation of the first air injection pipe according to the sensing of the foam sensing unit, thereby controlling the amount of foam generated during microbial culture. It can be adjusted, and by controlling the operation of the second air inlet pipe according to the sensing of the foam sensing unit, it is possible to control the discharge of unconcentrated bubbles to obtain a high-concentrated foam, and the foam generated during microbial culture by having a foam discharge unit Loss of the culture medium can be reduced by preventing the overflow or the culture medium from being discharged with bubbles.

In addition, in the case of culturing microorganisms using the bio-surfactant production apparatus according to the present invention, the bio-surfactant secreted in the culture solution, especially surfactin, is removed by collecting bubbles generated during cultivation, thereby reducing autotoxicity, thereby preventing the culture bacteria. Can protect.

In addition, by removing some cells present in the collected foam, it is possible to prolong the production period of the surfactant by delaying the formation of durable bodies through quorum sensing, thereby improving the production efficiency of bio-surfactants, especially surfactin.

1 is a view showing an apparatus for producing a bio-surfactant according to an embodiment of the present invention.

2 is a diagram showing an apparatus for producing a bio-surfactant according to another embodiment of the present invention.

FIG. 3 is a diagram showing a bubble discharge part in the bio-surfactant production apparatus according to an embodiment of the present invention, when the diameter of the upper end of the bubble discharge part is a, and the diameter of the lower end of the bubble discharge part into which the bubble flows is b, a Indicates that the diameter of is wider than the diameter of b.

[Explanation of code]

10: Bio-surfactant production device

100: air inlet

101: air pump

102: first air flow meter

103: air filter

104: water tank

105: air transfer pipe

106: second airflow metering

107: first air injection pipe

108: first valve

109: second air injection pipe

110: 2nd valve

111: spray spray

200: culture unit

201: culture tank

202: propeller for stirring the culture medium

203: foam removal propeller

204: bubble sensing unit

205: foam outlet

206: control sensor

300: foam discharge unit

301: inlet of the foam discharge part

400: bubble collection unit

401: foam transfer tube

402: foam collection container

403: member for liquefying foam

403a: propeller for liquefying foam

403b: blower

404: air outlet

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

The bio-surfactant production apparatus 10 of the present invention is a device for producing a bio-surfactant, particularly surfactin, by culturing a microorganism that produces a surfactant, and can produce a bio-surfactant economically and with improved efficiency.

1 is a diagram showing a bio-surfactant production apparatus 10 according to the present invention. As shown in FIG. 1, the bio-surfactant production apparatus 10 according to the present invention includes an air injection unit 100 including a first air injection pipe 107 and a second air injection pipe 109, and a culture unit. (200), consisting of a bubble sensing unit 204, a bubble discharge unit 300 and a bubble collection unit 400, the bubble sensing unit 204 is the first air injection pipe 107 and the 2 Controls the operation of the air injection pipe 109.

First, the culture unit 200 is for culturing microorganisms, and accommodates and cultures a culture solution containing a microorganism for producing a bio-surfactant, particularly Bacillus Bacillus for producing surfactin. Microorganisms for the production of the bio-surfactants Bacillus Belle Zen sheath (B. velezensis), Bacillus amyl kwipe sieon Lowry's (B.amyloliquefaciens), Bacillus piece nipo miss (B. licheniformis), Bacillus cap Ben sheath (B. mojavensis), Bacillus pumi Ruth (B. pumilus may be), and Bacillus subtilis at least one microorganism selected from Bacillus subtilis consisting of (B.subtilis).

The culture unit 200 includes a culture tank 201 for culturing microorganisms, a propeller 202 for agitation of a culture solution provided at the bottom of the inside of the culture tank and for agitation of the culture solution, a low density foam provided at the top of the inside of the culture tank It is configured to include a propeller 203 for removing bubbles for extinguishing (non-concentrated bubbles), and a control sensor 206 provided inside the culture tank and for controlling the state of the culture solution. Inside the culture tank 201, a propeller 202 for stirring the culture medium, a propeller 203 for removing bubbles, and a control sensor 206 are provided. The culture solution supplied to the culture tank 201 is preferably supplied at 30 to 70% of the capacity of the culture tank 201, and when cultured by supplying 70% or more of the culture solution, the culture solution overflows and the effect of collecting bubbles decreases. . By operating the propeller 202 for stirring the culture solution to agitate the culture solution, the cells are prevented from sinking in the culture solution, and the air injected by the first air injection tube 107 of the air injection unit 100 described later is transferred to the culture solution. By dissolving it increases the efficiency of the culture. In addition, by activating the foam removal propeller 203 and blowing out the unconcentrated foam, that is, the low-density foam, together with the air injection by the second air injection pipe 109 to be described later, the foam discharge unit 300 ) To control the movement. In addition, the temperature sensor 206a or the pH sensor 206b is operated as the control sensor 206 to measure the temperature and pH of the culture solution. In addition, by further including a dissolved oxygen amount measuring sensor or a foam measuring sensor in addition to the control sensor, the amount of dissolved oxygen in the culture medium may be measured and the amount of foam generated may be adjusted.

In the bio-surfactant production apparatus 10 according to the present invention, it was exemplified that a temperature sensor 206a or a pH sensor 206b is provided as the control sensor 206 provided in the culture tank 201, but Any control sensor installed to control the culture medium in the microbial culture apparatus in the industry may be provided, preferably a pressure sensor, a water level sensor, or a foam sensor, but is not limited thereto.

A foam sensing unit 204 for sensing bubbles generated from the culture unit is provided at an upper end of the culture unit 200. The foam sensing unit 204 controls the operation of the first air injection pipe 107 and the second air injection pipe 109 to be described later through foam sensing, and continuously according to microbial culture in the culture tank 201 The generated bubbles are sensed by the bubble sensing unit 204 to operate or block the first and second air injection pipes, or to control the amount of incoming air. The foam sensing unit 204 is a contact sensor that senses foam by contacting the foam generated from the surface of the culture medium of the culture tank 201 and moved to the top of the culture tank, or a laser sensor that senses the foam by recognizing the generated foam. However, it is not limited thereto. When the sensor is a contact sensor, it is preferable to be located at the top of the inside of the culture tank because the foam must directly contact the sensor, and only one contact sensor may be provided, but a plurality of contact sensors may be provided for accurate foam sensing. If the sensor is a laser sensor, it should be recognized that the bubbles generated on the surface of the culture medium move to the top of the culture tank, so it is preferable to be located on the upper side of the outside of the culture tank, and only one laser sensor is required, but accurate foam sensing is performed. For this purpose, two or more laser sensors may be provided to be located on both sides of the upper end of the culture tank.

In the bio-surfactant production apparatus 10 according to the present invention, it is exemplified that a contact sensor is provided as the foam sensing unit 204 provided in the culture tank 201, but in addition to sensing materials such as foam in the art Any sensor for this can be provided.

Next, the air injection unit 100 including the first air injection pipe 107 and the second air injection pipe 109 is for injecting air into the culture unit 200 or the bubble discharge unit 300, It is connected to the culture unit 200 or the discharge unit 300 to continuously or periodically inject air.

The first air injection pipe 107 of the air injection unit 100 is for injecting the air introduced from the air injection unit 100 into the culture solution in the culture unit 200 to the culture unit, and the bubble sensing unit 204 Until the bubble is sensed, air of 2.5 to 10 VVM is continuously introduced through the first air injection pipe 107, and after the bubble is sensed once in the bubble sensing unit 204, air of 0.5 to 2.0 VVM is Inflow and blocking are repeated at intervals of 1 to 10 minutes through the first air injection pipe 107. When the inflow of air flowing into the culture unit through the first air injection pipe 107 is periodically controlled, the production amount of bio-surfactant may be increased compared to the case of continuously injecting air.

One or more second air injection pipes 109 of the air injection unit 100 are connected to one end of the upper end of the culture tank 201 or connected to one end of the foam discharge unit 300 to provide a foam discharge unit 300 The second air injection pipe 109 is formed inside, and the second air injection pipe 109 is generated from the culture unit 200 by spraying the air introduced from the air injection unit 100 from the top to the bottom of the culture tank (toward the culture solution). This is for high density by removing low-density bubbles (non-concentrated bubbles), and when the bubbles are sensed by the bubble sensing unit 204, the second air injection pipe is operated or blocked. In addition, low-density bubbles are first selectively blown out by the air injected from the second air injection pipe. Specifically, when the bubble sensing unit 204 senses the bubble once, the second air injection pipe is activated (on) and air is introduced in an amount of 0.5 to 1.5 VVM, and when the bubble is sensed the second time, the second air is injected. The amount of air introduced into the tube is increased to 2 to 10 VVM, and when the bubbles are sensed 3 times, the second air injection tube is cut off. The operation control of the second air injection pipe 109 by the foam sensing unit 204 is for collecting only the concentrated foam (high-density foam) by the foam collection unit 400, which is sensed at the first and second times. Low-density bubbles are first selectively extinguished by spraying air flowing into the second air injection pipe 109 by blowing unconcentrated bubbles (low-density bubbles), and the concentrated bubbles (high-density bubbles) sensed in the third time are second air. By blocking (off) the injection pipe 109, it is moved to the foam discharge unit 300 and collected by the foam collection unit 400. Since the second air injection pipe 109 needs to blow out the generated bubbles, it is preferable that air is sprayed over the largest area, and for this purpose, the second air injection pipe 109 may be in the form of a spray 111.

In the bio-surfactant production apparatus 10 according to the present invention, the second air injection pipe 109 has been exemplified that one second air injection pipe 109 is provided at one end of the foam discharge unit 300. , The second air injection pipe 109 may be provided at any position capable of extinguishing low-density bubbles generated from the culture unit 200 using a spray-type second air nozzle, and at least one second air injection A tube 109 may be provided.

The air injection unit 100 includes an air pump 101, an air movement pipe 105, a first valve 108 and a second valve 110. The air pump 101 is equipped with a pressure gauge, is connected to the air pump 101, the air fastened to be connected to the inside of the culture tank 201 and the bubble discharge unit 300 on the other side connected to the air pump 101 Air is moved and injected into the culture tank 201 and the bubble discharge unit 300 through the moving pipe 105, in particular, the first air injection pipe 107 and the second air injection pipe 109. In addition, the first valve 108 and the second valve 110 connected to the other side of the air moving pipe connected to the air pump 101 move to the first air injection pipe 107 and the second air injection pipe 109, respectively. And controlling incoming air.

In addition, the air injection unit 100, a first air flow meter 102 for adjusting the flow of air injected from the air pump 101, an air filter 103 for filtering the supplied air, A water tank 104 for supplying moisture to the air, and a second air flow meter 106 for measuring the injected air flow may be additionally provided in the air injection unit 100 as necessary, and the above member By providing it, it is possible to control the flow of air.

Next, the foam discharge unit 300 is for collecting the concentrated foam discharged to the foam discharge unit inlet 301 and recovering the culture solution to the culture unit 200, the foam discharge port 205 of the culture tank 201 ) And the generated bubbles are continuously or intermittently collected, and the culture solution that has moved to the bubble discharge unit 300 along with the bubbles is recovered to the culture tank 201. Specifically, the foam discharge unit 300 is connected to the upper portion of the culture tank 201 and is concentrated by the foam sensing unit 204 and the first and second air injection pipes 107 and 109 to the culture tank 201 The concentrated foam (high-density foam) that has moved from the lower part of the to the upper part is collected, and some of the culture liquid that has moved together with the foam is recovered to the culture tank 201 again. The high-density foam (concentrated foam) collected in the foam discharge unit 300 is collected into the foam collection container 402 through a foam transfer pipe 401 connected to the upper portion of the foam discharge unit 300 to be described later. The foam discharge unit 300 and the foam transfer pipe 401 are vertically connected to the top of the culture tank 201 to prevent the phenomenon that some of the culture liquid moved along the foam moves to the foam collection container 402 due to gravity. I can. The bubble discharge part 300 is connected to the bubble discharge part inlet 301 of the bubble discharge part and the bubble discharge port 205 of the culture tank 201. In addition, as shown in FIGS. 2 and 3, the bubble discharge unit 300 may be configured in a bottleneck shape, and the diameter of the upper end of the bubble discharge unit 300 is greater than the diameter of the lower end of the bubble discharge unit 300 into which the bubbles are introduced. It may be formed to be wide. Specifically, when the top diameter of the bubble discharge part 300 is a, and the diameter of the lower end of the bubble discharge part 300 into which the bubble flows is b, the diameter of a may be larger than the diameter of b, and the The diameter of a may be 2 to 10 times wider than the diameter of b. When the diameter of the lower end of the bubble discharge unit 300 into which bubbles are introduced is narrower than the diameter of the upper end of the bubble discharge unit 300, a large foam of low density is formed by the lower end of the bubble discharge unit 300. It can be prevented from being discharged through, and since the diameter of the upper end of the bubble discharge port 300 is wider than the lower end of the bubble discharge unit 300 through which bubbles are introduced, the pressure caused by the lower end of the bubble discharge unit 300 through which bubbles are introduced By suppressing the increase, it is possible to prevent the culture medium from moving to the foam collection unit 400 together with the foam.

Next, the bubble collection unit 400 is for collecting and collecting the bubbles generated in the culture tank 201 by the bubble discharge unit 300, and continuously or intermittently collects the generated bubbles.

The foam collection unit 400 includes a foam moving pipe 401, a foam collection container 402, a foam liquefaction member 403, and an air outlet 404. The bubble collection container 402 is provided with a bubble liquefaction member 403 for liquefying bubbles, and a propeller 403a for liquefying bubbles and/or a blower 403b to liquefy the collected bubbles to thereby liquefy the collected bubbles and biointerface An active agent, in particular surfactin, is obtained.

In the bio-surfactant production apparatus 10 according to the present invention, a foam liquefaction propeller 403a and/or a blowing device 403b as a foam liquefaction member 403 provided in the foam collection container 402 However, if the foam liquefaction member 403 is not provided, the collected foam may be transferred to another container and allowed to liquefy by leaving it in a refrigerator for a certain period of time. In addition, an air outlet 404 for adjusting air pressure is provided at one end of the collection container 402.

By using the bio-surfactant production apparatus 10 as described above, the production efficiency of a bio-surfactant, particularly surfactin, can be improved, and a specific method is as follows.

The method for producing a bio-surfactant includes a first step of culturing microorganisms and a culture medium in a culture tank injected with air, a second step of sensing bubbles generated in the culture tank with a foam sensor, and collecting bubbles generated in the culture tank. And, it can be divided into a third step of recovering the culture solution, and a fourth step of obtaining a bio-surfactant from the collected foam.

The first step is a step of culturing by supplying a microorganism producing a bio-surfactant and a culture solution to the inside of the culture tank 201 and injecting air. The culture solution supplied to the culture tank 201 is 30 compared to the capacity of the culture tank 201 It is preferable to supply in an amount of 70% to 70%, and when the culture solution is cultured by supplying 70% or more of the culture solution, the culture solution overflows and the effect of collecting bubbles decreases. In addition, microorganisms that produce bio-surfactants, especially surfactin, are Bacillus velezensis , Bacillus amyloliquefaciens, Bacillus licheniformis , and Bacillus mobbenscis . (B. mojavensis), Bacillus pumi loose may be a (B. pumilus), and Bacillus subtilis Bacillus subtilis one or more kinds selected from the group consisting of (B.subtilis), not limited to this. In addition, by operating the air pump 101 and injecting air into the culture medium of the culture tank, bubbles may be generated by supplying air, and a specific method of generating bubbles will be described in detail below.

The method of generating bubbles by culturing the culture solution injected with air is to generate bubbles by injecting air according to the operation of the air pump 101 and activating the propeller 202 for stirring the culture solution provided in the culture tank 201. I can. In the air injection according to the operation of the air pump 101, air in an amount of 2.5 to 10 VVM is continuously introduced into the culture tank through the first air injection pipe 107. The time for generating the foam is not particularly limited, but foam may be repeatedly generated for a short time, such as 20 to 30 minutes, or may be continuously generated for 6 hours or more.

Next, the second step is a step of sensing the foam generated in the culture tank with a foam sensor, which is the most essential step for improving the production efficiency of a bio-surfactant, particularly surfactin, in the present invention. The initial foam generated during cultivation is an unconcentrated foam (low density foam), and since the amount of the bio-surfactant contained in the initial foam is very small, it is difficult to collect the low-density foam to obtain a bio-surfactant in high yield. Accordingly, as described later, a method for collecting only the concentrated foam was devised.

Specifically, as the bubbles generated through the first step move from the bottom of the culture tank to the top, the bubbles come into contact with the bubble sensor of the bubble sensing unit 204 and are sensed. At this time, when the bubble is sensed, the operation of the first and second air injection pipes 107 and 109 is controlled. Specifically, when the bubble is sensed, the amount of air continuously flowing into the first air injection pipe 107 is adjusted to 0.5 to 2.0 VVM in the first step and periodically flows in, preferably 0.5 to 2.0 VVM amount of air. Is injected and blocked at intervals of 1 to 10 minutes. Such control of the first air injection pipe 107 may increase the production amount of bio-surfactant. In addition, when the bubbles are sensed, the second air injection pipe 109 is operated to inject or block air into the culture tank 201. In the second air injection pipe 109, air in an amount of 0.5 to 1.5 VVM is injected in the first time of bubble sensing, and the second time of bubble sensing is injected by increasing the amount of air in an amount of 2.0 to 10 VVM, and the third time of bubble sensing From then on, air injection is stopped. Control of the second air inlet pipe 109 as described above prevents unconcentrated bubbles (low-density bubbles) from being discharged to the bubble discharge unit 300 and collected by the bubble collection unit 400 and concentrates the bubbles. It can increase the production of surfactants.

Next, the third step is a step of collecting the generated bubbles in the bubble collection container 402, which includes the step of collecting the concentrated bubbles (high density bubbles) by moving the generated bubbles to the bubble discharge unit 300, , This step is also essential in order to obtain a bio-surfactant in high yield.

For example, when fermentation is carried out using Bacillus bacillus, as fermentation proceeds, surfactin, a bio-surfactant having an antibiotic effect, accumulates in the culture medium. At this time, even if there are enough nutrients in the culture medium, Bacillus bacteria do not grow further and die because of the surfactin produced by themselves, so there is a difficulty in increasing the production of surfactin. On the other hand, cells are essential for the production of surfactin, and the number of cells must be increased to increase the production of surfactin, but if the number of cells is too large, it results in disturbing the production of surfactin through quorum sensing. Therefore, when the above problems are summarized, the concentration of surfactin is lowered during culturing so that host cells are not killed by surfactin, or the number of cells is appropriately reduced or the cells do not grow further during culturing. It was determined that the production of cotton surfactin could be increased, and a method of collecting foam was devised as described later.

Specifically, the third step of collecting the generated bubbles is a step 3-1 of moving the generated bubbles to the bubble discharge unit 300 through the bubble discharge port 205, to the bubble discharge unit 300 Bubbles generated through the 3-2 step of moving the moved bubbles to the bubble transfer pipe 401 and the 3-3 step of recovering the culture solution moved to the bubble discharge unit 300 to the culture tank 201 It is collected in the collection container 402. In general, when bubbles are collected without the foam discharge unit 300, some of the bubbles overflow, and when the bubbles are recovered, some culture solutions are also recovered, so that the culture solution and the produced bio-surfactant are lost. The production of bio-surfactant is low, and bio-surfactant-producing bacteria do not survive due to the bio-surfactant produced at high concentration in the culture medium, making it difficult to continuously produce bio-surfactant. On the other hand, when bubbles are collected through the bubble discharge unit 300 as in the present invention, some cells present in the collected bubbles are removed to delay the formation of durable bodies through quorum sensing, thereby prolonging the period for producing surfactants. As a result, it is possible to continuously produce bio-surfactants, and because a high-concentration bio-surfactant is present in the collected foam, the concentration of the bio-surfactant produced in the culture medium can be lowered, thereby forming conditions in which the bio-surfactant can continue to grow. , It can improve the production efficiency of bio-surfactants.

Next, the fourth step is a step of collecting the bio-surfactant, and through the third step, the bubble collected in the bubble collection container 402 may be liquefied to obtain a bio-surfactant. The liquefaction of the foam is generally any method capable of liquefying the foam, but preferably, a foam liquefaction propeller (403a) or a blower (403b) installed inside the foam collection container 402 of the present invention is used. To liquefy it, or if the bubble liquefaction member is not installed in the bubble collection container 402, the bubble may be collected and allowed to stand in a refrigerating chamber to liquefy.

Through a specific experiment, the effect of increasing the production efficiency of surfactin was confirmed through the bio-surfactant production apparatus 10 according to the present invention and a method of improving the production efficiency of the bio-surfactant using the same, and the specific experimental method and results are It is as follows.

Experimental Example 1: Confirmation of surfactin generation according to air inflow conditions using the first air inlet tube

The effect of air inflow conditions using the first air inlet tube on the generation of surfactin In order to confirm the effect, the generation of surfactin under various air inlet conditions was confirmed using the first air inlet tube. The air inflow conditions and the surfactin production amount of the first air injection pipe are shown in Table 1 below.

	Injection method	Volume of air	Surfactin production
Main invention	On/off every 5 minutes	0.5 VVM	1.9 ± 0.17
Comparative Example 1	Continuous injection	0.25 VVM	1.6 ± 0.09
Comparative Example 2	Continuous injection	0.5 VVM	1.4 ± 0.11

As a result, as can be seen in Table 1 above, when a certain amount of air (0.5VVM) is periodically introduced into the first air injection pipe at intervals of 5 minutes (the original invention), the production of surfactin increased, but 0.25 VVM and 0 5. When air in the amount of VVM was continuously introduced (Comparative Example 1 and Comparative Example 2), unconcentrated foam (low density foam) overflowed and the final production amount of surfactin decreased.

Experimental Example 2: Confirmation of surfactin generation according to the presence or absence of a bubble sensing unit and a second air injection tube

In order to check the effect of the presence or absence of the foam sensing unit and the second air injection pipe on the generation of surfactin, the bio-surfactant generating device of the present invention and the foam sensing unit and the second air-injection pipe are both provided. It was confirmed that surfactin was generated by a bio-surfactant generating device without an air injection tube. The results are shown in Table 2 below. Indicated.

	Bubble sensing unit	2nd air inlet pipe	Surfactin production
Main invention	○	○	2.2g
Comparative Example 3	×	×	0.9~1.6g

As a result, as can be seen in Table 2 above, the bio-surfactant generating device of the present invention equipped with a foam sensing unit and a second air inlet tube generates concentrated foam (high-density foam) to increase the production of surfactin, whereas the foam sensing unit And in the apparatus of Comparative Example not provided with the second air inlet tube, the foam was not concentrated, so the production amount of surfactin decreased. In the absence of the second air injection pipe, the amount of air injected through the first air injection pipe and the amount of surfactin produced are inversely proportional.

Experimental Example 3: Confirmation of surfactin generation according to air inflow conditions using a second air inlet tube

In order to confirm the effect of the air inlet condition using the second air inlet pipe on the generation of surfactin, the second air inlet pipe was used to confirm the surfactin generation under various air inlet conditions. The air inflow conditions and the surfactin production amount of the second air injection pipe are shown in Table 3 below.

	Bubble sensing unit	2nd air inlet pipe	First round air volume	Secondary air volume	Surfactin production
Main invention	○	○	1.0 VVM	5.0 VVM	2.2g
Comparative Example 4	○	○	1.0 VVM	1.0 VVM	0.9g

As a result, as can be seen in Table 3 above, when the bubble sensing unit senses the bubble twice, when the amount of air introduced into the second air injection pipe is increased (the original invention), a concentrated bubble (high density bubble) is generated. When the amount of air was not increased while the amount of surfactin was increased (Comparative Example 4), the amount of surfactin was decreased because the foam was not well concentrated.

Experimental Example 4: Confirmation of the concentration effect of surfactin

It was intended to confirm the effect of concentrating surfactin using the surfactin production apparatus according to the invention. The concentration of the foam was calculated by comparing the amount of the foam produced once after culturing for 60 hours and the amount of cells and surfactin in the remaining culture solution, and the results are shown in Table 4 below.

	Culture medium (L)	Wet cell weight (g)	Dry cell weight (g)	Surfactin (g)	Thickening effect
bubble	450	30	6	2	33
Remaining medium	1500	61	12	0.2	One
synthesis	1950	91	18	2.22

As a result, as can be seen in Table 4, about 6 g of cells and about 2 g of surfactin were included in the 450 mL liquefied foam, and about 12 g of cells and about 0.2 g of surfactin were included in the remaining 1500 mL of culture solution. , It can be seen that the generation of bubbles according to the surfactin production apparatus of the present invention exhibits about 33 times the surfactin concentration effect.

Experimental Example 5: Confirmation of the yield of surfactin according to continuous culture

Using the surfactin production apparatus according to the present invention, the foams were collected and the production efficiency of surfactin was confirmed when the foam was continuously cultured with or without supplementation, and the results are shown in Table 5 below.

Whether to replenish the culture medium after removing the foam	Culture medium (L)	Wet cell weight (g)	Dry cell weight (g)	Crude surfactant (g)	Production increase effect
×	6	140 ± 15	28 ± 0.11	2.0 ± 0.04	1 times
○	6+3	1193 ± 25	NA	2.7 ± 0.30	1.4 times

Claims (18)

1. A culture unit for culturing microorganisms;

   A foam sensing unit positioned at the top of the culture unit and configured to sense a bubble generated from the culture unit;

   A first air injection tube for injecting air into the culture solution in the culture unit;

   A foam discharge unit through which the bubbles generated from the culture unit are discharged;

   A second air inlet tube for injecting air from the upper part of the culture unit toward the culture solution to dispel bubbles generated in the culture process to increase density; And

   A foam collection unit connected to the foam discharge unit and configured to collect the generated foam; As a bio-surfactant production device comprising a,

   Bio-surfactant production apparatus, characterized in that for controlling the operation of the first and second air injection pipes according to the foam sensing of the foam sensing unit.
2. The method of claim 1,

   The first air injection pipe continuously injects air into the bubble sensing unit until the bubbles are sensed, and after the bubbles are sensed, air is introduced and blocked at intervals of 1 to 10 minutes to periodically inject air. Bio-surfactant production device, characterized in that.
3. The method of claim 1,

   The amount of air introduced into the first air injection pipe is 2.5 to 10 VVM until the bubble is sensed, and 0.5 to 2.0 VVM after the bubble is sensed.
4. The method of claim 1,

   Bio-surfactant production apparatus, characterized in that the second air injection pipe is operated or blocked when the foam is sensed in the foam sensing unit.
5. The method of claim 1,

   When the bubble sensing unit senses the bubble once, the second air injection tube is turned on and air is introduced in an amount of 0.5 to 1.5 VVM. When the bubble is sensed twice, the air flowing into the second air injection tube is Bio-surfactant production apparatus, characterized in that the amount is increased to 2.0 to 10 VVM, and the second air injection pipe is blocked when the foam is sensed 3 times.
6. The method of claim 1,

   The second air injection pipe is a bio-surfactant production apparatus, characterized in that in the form of a spray.
7. The method of claim 1,

   The bubble discharge unit is configured in a bottleneck shape, and the diameter of the upper end of the bubble discharge unit is larger than the diameter of the lower end of the bubble discharge unit into which the bubbles are introduced.
8. The method of claim 7,

   Bio-surfactant production apparatus, characterized in that the diameter of the upper end of the foam discharge unit is 2 to 10 times wider than the diameter of the lower end of the foam discharge unit into which bubbles are introduced.
9. The method of claim 1,

   The culture unit,

   A culture tank for culturing microorganisms;

   A propeller for stirring the culture solution provided at the lower end of the inside of the culture tank and for stirring the culture solution;

   A foam removal propeller provided on the inner top of the culture tank and for removing the generated low-density foam; And

   A control sensor provided inside the culture tank and configured to control a state of the culture medium; Bio-surfactant production apparatus comprising a.
10. The method of claim 1,

    The air injection unit,

    An air pump attached with a pressure gauge for injecting air;

    An air moving pipe for moving the introduced air, which is connected to the air pump and connected to the inside of the culture tank and the bubble discharge unit on the other side connected to the air pump;

    A first valve for controlling air inflow into the first air injection pipe; And

    A second valve for controlling air inflow into the second air injection pipe; Bio-surfactant production apparatus comprising a.
11. The method of claim 1,

    The foam collection unit,

    A foam moving pipe connected to the upper part of the foam discharge part and for moving the concentrated foam through the foam discharge part;

    A foam collection container connected to the foam transfer pipe and for collecting foam;

    A member for liquefying bubbles provided inside the collection container and liquefying bubbles; And

    An air outlet provided at one end of the collection container and configured to adjust air pressure; Bio-surfactant production apparatus comprising a.
12. A first step of culturing the microorganisms and the culture medium in the culture tank injected with air;

    A second step of sensing the bubbles generated in the culture tank with a foam sensor;

    A third step of collecting the bubbles generated in the culture tank and recovering the culture solution; And

    A fourth step of obtaining a bio-surfactant from the collected foam; Method for producing a bio-surfactant comprising a.
13. The method of claim 12,

    The first step is a method of producing a bio-surfactant, characterized in that during culturing, air in an amount of 2.5 to 10 VVM is continuously injected into the first air injection tube.
14. The method of claim 12,

    When the bubbles are sensed in the second step, the air flowing into the first air injection pipe is adjusted to an amount of 0.5 to 2.0 VVM and introduced at intervals of 5 to 10 minutes.
15. The method of claim 12,

    When the bubbles are sensed in the second step, the second air injection pipe is operated to spray or block air, and in the air injection, 0.5 to 1.5 VVM amount of air is injected in the first bubble sensing, and 2.0 is injected in the second bubble sensing. A method of producing a bio-surfactant, characterized in that air in an amount of to 10 VVM is injected, and air is not injected in the third bubble sensing cycle.
16. The method of claim 12,

    The third step of collecting the generated bubbles,

    3-1 step of moving the generated foam to the foam discharge unit;

    A 3-2 step of moving the bubbles moved to the bubble discharge unit to a bubble moving pipe; And

    3-3 step of recovering the culture solution moved to the foam discharge unit to the culture tank; Bio-surfactant production method comprising a.
17. The method of claim 12,

    The fourth step of obtaining a bio-surfactant,

    Bio-surfactant production method, characterized in that to obtain a bio-surfactant by liquefying the bubbles collected in the foam collection container.
18. The method of claim 12,

    After removing the bubbles in the fourth step, the method of producing a bio-surfactant, further comprising the step of additionally supplying the culture solution in the same amount as the culture solution in which the collected bubbles are liquefied to the culture tank.

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