KR101947274B1 - Microbubble generator and method for culturing microbe including microalgae with high-efficiency using the same - Google Patents

Abstract

The present invention relates to a microbubble generator and a method for culturing microorganisms containing high-efficiency microalgae using the microbubble generator, and more particularly, to a method for producing DHA oil by culturing microalgae using a microbubble generator- The present invention relates to a microbubble generator for efficiently producing microalgae oil by increasing the growth of microalgae and increasing lipid content by supplying oxygen at a high concentration in a short time, and a microbial culture method comprising the microalgae.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microbubble generator and a microbial cell culture method using the microbubble generator. 2. Description of the Related Art Microbubble generators and high-

The present invention relates to a microbubble generator and a method for culturing a microorganism containing the microbubble generator, and more particularly, to a method for producing a metabolite by culturing aerobic microorganisms including microalgae, The micro-algae or microorganisms can be grown at a high concentration by supplying air having a minute size by using the culturing apparatus of the present invention. As a result, compared to the conventional culture apparatus, the energy consumption due to excessive consumption of air and excessive stirring A microbubble generator for reducing consumption and reducing the shear stress applied to the microbes, and an application thereof.

In recent years, as an alternative to fossil fuels, interest in biofuel such as biodiesel has increased, and the demand for bio-oil, which is a raw material thereof, is increasing rapidly.

Accordingly, the price of vegetable oil such as rapeseed oil and soybean oil which is a main source of bio oil has been surging, and it has become necessary to secure oil to replace it. In addition, it is less affected by environmental factors such as climate than conventional vegetable oil Studies on the production of bio-oil using microbial oil, such as microalgae, which can be produced in a narrow space, are under way.

Especially, heterotrophic microbial oil can be produced by using disposable organic material as a raw material by utilizing existing microbial cultivation facility. Therefore, it is an alternative oil that can be industrialized in a short time in Korea which has a microbial culture technology with a narrow land area. can do.

Among the heterotrophic microorganisms, Thraustochytrid-based microalgae accumulate 30% or more of oil in the microorganism in relation to the weight of the microorganism. This oil is triacylglycerol, which contains a high concentration of polyunsaturated fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) , Squalene and other high-performance ingredients, it is a very promising source of oil as a next-generation bio-oil supplier.

On the other hand, DHA, a polyunsaturated fatty acid, is partially synthesized from alpha-linolenic acid (C18: 3n3) contained in foods such as soybeans in the human body, but is mainly obtained from deep sea fish such as tuna and salmon. However, due to the reduction of catches due to the strengthening of fishing regulations and the pollution of heavy metals, environmental hormones and radioactive materials including mercury in the fish body due to pollution of the marine environment, the risk of ingestion of deep sea fish increases, As a new means for safely and stably supplying a bio-oil containing a polyunsaturated fatty acid, a micro-algae of a Trout kettre system has been attracting attention.

Conventionally, in order to produce DHA oil at a high concentration through the cultivation of such a microorganism, microorganisms are easily destroyed when the incubation time is long, There is a problem in that the recovery of the microalgae oil (lipid) is problematic and the DHA oil is oxidized and denatured.

Korean Patent Registration No. 1187813 discloses a technique for adding a stabilizer to lower the shearing force in an incubator during the culturing of microtidal strains. In Korean Patent No. 1328090, shearing in the incubator Mutation strains with enhanced resistance to stress have been disclosed.

Thus, in order to minimize oxidative degeneration of DHA oil due to the breakdown of microbial cells, it is effective to produce DHA oil through short-term culture.

However, it is not easy to raise the amount of the bacterium and increase the concentration of DHA through the short-term cultivation of the microorganism of the trout tokit lead system. This is because, in the culture condition suitable for the microbial propagation and the oil production of the microorganism of the trout, This is because the synthesis of DHA is not performed smoothly. For example, in order to supply oxygen at a high concentration using a conventional agitating type incubator, it is necessary to increase the stirring speed and the air supply amount. As a result, the shear stress in the incubator is increased during the cultivation of the microalgae, There are disadvantages, and when using an ordinary airlift incubator to increase the air supply, a large amount of foam is generated and the produced oil is shunted.

Accordingly, the applicant of the present invention has found a method for effectively increasing the amount of dissolved oxygen in the incubator in order to produce a high concentration of DHA oil through the microalgae culture for a short time.

Korean Patent No. 132809 entitled " A strain producing a high content of docosahexaenoic acid and a method for producing the same "(2013.11.05) Korean Patent No. 1187813 "Method for producing polyunsaturated fatty acids from microalgae" (2012.09.26) Korean Patent No. 700486 "Strain Producing Omega-3 Unsaturated Fatty Acids and Method for Producing Omega-3 Unsaturated Fatty Acids Using the Same" (2007.03.21) Korea Patent No. 1541521 "Production method of docosahexaenoic acid using microalgae" (2015.07.28)

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a high concentration of bio-oil and DHA by supplying a high concentration of oxygen through a short-time culture by promoting oxygen delivery efficiency during culturing of a micro- It is an object of the present invention to provide a microbubble generator capable of suppressing the breakdown of microbes and improving the quality of DHA oil, and a microbial culture method including the microbes with high efficiency using the same.

Another object of the present invention is to reduce air consumption and energy cost in culturing heterotrophic microalgae, increase the growth rate of microalgae, and increase the lipid content in microalgae.

According to an aspect of the present invention, there is provided a micro bubble generator comprising: a socket having a cylindrical shape and provided with an air injection nozzle; An air front end portion formed inside the socket portion and formed in the shape of a propeller so that the air is uniformly distributed at the upper end of the socket portion by rotating in a predetermined direction; A micro bubble generator provided at a lower portion of the air front end and having a plurality of blades for mixing water and air at a high flow rate of turbulent flow along with fine bubbles blowing by high speed rotation; And a rotating shaft installed vertically at the center to transmit the rotating force of the motor to the air front end and the micro bubble generating unit, and is installed inside the micro-algae culture apparatus to generate and supply micro bubbles .

A plurality of holes may be formed in the sidewall portion of the socket portion so that air is not stagnated therein.

The micro bubble generator may further include a plurality of holes in the disc to minimize the turning force generated around the rotation axis.

The micro bubble generator may be configured as a cover type blade so as to minimize the generation of swirling flow in the socket portion and to minimize the resistance value of the fluid generated therein.

In addition, the method for culturing a micro-organism containing high-efficiency microalgae using the micro-bubble generator according to the present invention is characterized in that a) culturing the micro-algae of the strain-aquitride system comprises the steps of a) pre-culturing in a culture medium containing a carbon source and a nitrogen source Transplanting the cells into the culture medium and culturing the cells; And b) supplying microbubbles generated by the microbubble generator having the above-described configuration to enhance the efficiency of oxygen transfer to the culture medium.

At this time, in the step b), the amount of microbubbles to be supplied is 2 to 5 L / min, the microbubble size is 250 to 300 탆, and the stirring speed is 200 rpm.

As described above, unlike the conventional method in which micro-algae cells are damaged by excessively injected air and a strong agitation force in the process of culturing the micro-algae of the strain, In the present invention, a propeller shear type micro bubble generator is used to supply oxygen at a high concentration in a short time. In addition, it is possible to improve the oxygen transmission efficiency by more than 30% by supplying the micro bubble of 300 탆 or less, By increasing the amount of dissolved oxygen, increasing the growth rate of microalgae by inhibiting the crushing of microorganisms, and increasing the lipid content of microalgae by more than 60%. It has the advantage of increasing the market competitiveness of the bio-oil industry.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a microbubble generator of the present invention; FIG.
Fig. 2 is a rear perspective view of Fig.
3 is a view showing a socket unit according to the present invention.
4 is a view showing a micro bubble generator according to the present invention;
5 is a view showing another embodiment of the micro bubble generating unit according to the present invention;

Hereinafter, preferred embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the technical idea of the present invention. The present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

The present invention relates to a method for efficiently delivering oxygen during a short time by applying a micro bubble generator in the process of culturing a microorganism of a threustochytrid system in a heterotrophic microorganism, To improve the growth of microalgae and to increase lipid content.

First, the micro bubble generator 10 according to the present invention will be described.

The micro bubble generator G is installed in a known culture apparatus for generating and supplying micro bubbles when microalgae are cultured, and includes a socket unit 12 as shown in Figs. 1 and 2; An air front end portion 13; A micro bubble generator 14; And a rotary shaft (15).

As shown in FIG. 3, the socket portion 12 is formed in a substantially cylindrical shape, and an air injection nozzle 121 is provided on the inner side to allow the outside air to flow into an air front end portion described later.

A plurality of holes 12a are formed in the side surface of the socket portion 12. This is because when the size of the socket portion 12 is small, air is injected into the air through the air injection nozzle, So that the inside of the socket is stagnated without being able to escape to the outside.

That is, although the micro bubble generator 14 to be described later must rotate in a state where water and air are in contact with each other in the water, rotation may occur only in the air layer, so microbubbles may not be generated properly. So that the air flows out to the outside through the socket portion without stagnation.

The plurality of holes 12a may be shielded by a means such as a bolt if necessary.

The air front end portion 13 is formed in the shape of a propeller so that the air is uniformly distributed at the upper end of the socket portion 12 by rotating in a certain direction.

The micro bubble generator 14 is formed in a substantially disc shape as shown in FIG. 4 and has a plurality of blades 14a at the upper portion thereof. The micro bubble generator 14 is installed at a lower portion of the air front end portion 13, The microbubbles are produced by mixing the water and the air by the fast flow rate of the turbulent flow as well as the bubbling of the large size bubble several times.

That is, the propeller 13a of the air front end portion 13 pushes the upper fluid (water and air) downward, and the blade 14a of the micro bubble generating portion 14 rotates at high speed The micro bubble is generated by cutting, and the size of the micro bubble is preferably 250 to 300 mu m.

At this time, the blade 14a generates a swirling flow of the fluid in the culture apparatus and greatly influences agitation. The blade 14a has a plurality of slits 12b formed in the longitudinal direction at the lower end of the socket portion 12 And the air flows out at the same time.

The micro bubble generator 14 is made of stainless steel (SUS) so that it can be sterilized and maintained in a cultured state without being interfered with by other harmful microorganisms for culturing microorganisms. And the swing motion is maintained in a state in which it is possible.

In addition, the micro bubble generator 14 may further include a hole 14b formed in the disc portion to minimize the turning force generated around a rotation axis, which will be described later.

5, the micro bubble generator 14 'may be configured to form a cover-shaped blade 14a' to seal the lower end portion of the socket portion, The generation of swirling flow of the fluid can be minimized and the resistance value of the fluid generated therein can be minimized.

The rotary shaft 15 is vertically provided at the center to transmit the rotational force of a motor (not shown) to the air front end portion 13 and the micro bubble generating portion 14, so that damage does not occur even in long- And a separate part and a cover may be fabricated to prevent frictional damage and heat due to rotation.

Since the size of the microbubbles obtained by the microbubble generator (G) having the above structure is small compared with the conventional bubbles, the residence time in the water is relatively long, and the continuous operation efficiency and the bubble generation rate are high, .

In addition, the microbubble generation by the microbubble generator (G) of the present invention is not based on a conventional high pressure tank or passing through a high-speed pump but a high-speed shear type, It is possible to reduce the factors as much as possible.

Next, a method for culturing microalgae by applying the microbubble generator (G) according to the present invention will be described.

As the microalgae cultured in the present invention, Schizochytrium sp. Strain SH103, which is a microalgae of Thraustochytrid type, is used.

A basic medium consisting of a carbon source, a nitrogen source, a sea salt, a man and a vitamin mixture is used for culturing the microalgae. The carbon source is 60 g / L of glucose, the nitrogen source is 10 g / L of yeast extract, L of a mixture of biotin (0.2 g / L) and cyanocobalamin B12 (1.0 g / L) was used as a phosphate buffer (20 g / L of seawater salt and 9 g / L of potassium phosphate (KH ₂ PO ₄ ) / L is used.

Cells cultured 30 hours before in the medium having the above-described structure were transplanted into 3L of the microalgae culture medium in a 5L incubator, and microbubbles were injected using an incubator equipped with the microbubble generator (G) (batch culture).

At this time, as the air injection amount is more than 5 L / min or the stirring speed is increased, the carbon source consumption rate is also increased. Therefore, the culture of the SH103 strain of the Schizochytrium is optimized and the air injection amount is preferably 2 to 5 L / min in consideration of the dry cell weight and the lipid content Is 3 L / min, and the stirring speed is 200 rpm.

Accordingly, conventional apparatuses such as aerator, which has been applied to a conventional culture apparatus for culturing microorganisms, have a disadvantage in that the oxygen transmission efficiency is uneven and the culture efficiency is not good, In the case of a culture apparatus equipped with a generator, the efficiency of oxygen transfer into the culture tank is increased, and the growth rate of the microorganism is influenced, thereby improving the operation efficiency of the culture apparatus.

Test Example 1

The microbial culture samples cultured by the microbubble generator according to the present invention were analyzed for dry cell mass and lipid content. For this purpose, microbial culture samples cultured according to the present invention were analyzed by time Collect and prepare.

(A) Measurement of dry cell mass

Dry cell mass was measured by the atmospheric pressure heating drying method. 10 mL of the sample collected at each incubation time was placed in a 50 mL conical tube (SPL Life Sciences) which had been weighed in advance, and the supernatant was removed by centrifugation at 4 ° C and 3500 rpm for 20 minutes. To remove the media components contained in the above samples, the cells were washed three times with 10 mL of distilled water, dried in a constant temperature drier at 105 ° C for 3 hours, and then the lid was closed. After cooling to room temperature, the weight was measured. The lid was opened again and dried in a constant temperature drier at 105 ° C for 30 minutes. The lid was then closed. The lid was allowed to cool at room temperature. When the temperature reached room temperature, the weight was measured and repeatedly dried until there was no change in weight. The dry cell mass (g / L) contained in the culture solution was calculated by the following formula, and the results are shown in Table 2 below.

⒝ Measurement of lipid content

Lipid content was analyzed using the modified Bligh-dyer method (Burja et al., 2007). 10 mL of microalgae culture collected at the time of incubation was placed in a 50 mL conical tube and centrifuged at 3500 rpm for 20 minutes at 4 ° C to remove supernatant. To remove the medium components contained in the above samples, the cells were washed 3 times with 10 mL of distilled water and lyophilized for 12 hours. 5 mL of chloroform, 10 mL of methanol and 5 mL of 50 mM potassium phosphate (K ₂ HPO ₄ ) buffer (pH 7.4) were added to the lyophilized sample, and the mixture was reacted at 28 ° C. and 200 rpm for 1 hour. Then, 5 mL of chloroform, 5 ml of potassium (K ₂ HPO ₄ ) buffer was added and mixed for about 30 times. Then, the mixture was allowed to stand for 1 hour to separate the aqueous layer and the organic solvent containing lipid. 5 mL of the chloroform layer was carefully transferred to an aluminum plate which had been previously weighed and then dried in a constant temperature drier at 90 ° C. for 30 minutes and then weighed. Lipid content (g / L) was calculated by the following equation and the results are shown in Table 3 below.

Test Example 2: Concentration of oxygen transfer efficiency by incubator

First, as a culture apparatus for culturing microalgae, an incubator to which the microbubble generator according to the present invention is applied, and a conventional stirring type incubator and a bubble column type incubator were used.

Prior to preparing the oxygen transfer coefficient for each incubator in the present test, a conventional culture apparatus will be briefly described. The agitation type (standard type) incubator is configured such that stirring and air supply are independently configured, The air-lift incubator is equipped with a stirrer and a stirrer in the range of 0 to 1,200 RPM using an impeller. The air-lift incubator is equipped with a stirrer Stirring is performed through circulation.

Such agitated and bubble column incubators can be made by various types of spargers, such as spinning, ring, ladder, single orifice, multi orifice type, .

Tests were conducted to compare oxygen delivery efficiency using incubators having the above structure.

)를 측정한바, 각각 5L 배양기에 0.1~0.5M의 3L 아황산나트륨용액을 넣고 온도를 28℃로 유지한 후 공기주입량과 교반속도를 다양한 조건으로 조절하였다.For this purpose, the sodium absorption rate per unit volume

) Was measured, and 0.1 L to 0.5 M 3 L sodium sulfite solution was added to each 5 L incubator, and the temperature of the solution was maintained at 28 ° C. Then, the amount of air injected and the stirring speed were adjusted under various conditions.

The aqueous solution of copper sulfate was added to the solution of the above conditions, and the final concentration of 3 mM was added. The air was supplied and the time was measured. The sample was sampled every 5 minutes. The sample was diluted to an appropriate concentration, Solution (0.16M iodine, 0.3M potassium iodide) was added to measure the amount of sodium sulfite consumed per unit time at an absorbance of 600 nm.

)를 산출한 후 그 결과를 표 4에 나타내었다.The oxygen uptake rate per unit volume was calculated from the consumption of the measured sodium sulfite and the oxygen transfer coefficient

), And the results are shown in Table 4. < tb >< TABLE >

According to Table 4, when applying a conventional stirred with a bubble column-type incubator, whereas the oxygen transfer efficiency of approximately 88.5h ^-1, 285h ^-1, the case of applying the microbubbles incubator according to the present invention, 2130h ^- the ¹ High efficiency was obtained.

That is, in the conventional incubator, since the efficiency of transferring the carbon dioxide or air by the sparger is as low as about 13%, more than 80% of the amount of air to be injected is not used for growth of microalgae, , It is possible to cultivate microalgae at a high concentration by inducing high-speed growth of microalgae and improving oxygen transmission rate by efficient air supply.

Test Example 3: Production of DHA-containing oil by culture apparatus

Lipid productivity and DHA (microalgae) were obtained through culture of Schizochytrium sp. Microalgae using the incubator to which the microbubble generator of the present invention was applied and the conventional stirrer type and air-lift type incubator. The content (DHA content) was measured.

First, a mixture of glucose 60 g / L as a carbon source and 10 g / L yeast extract as a nitrogen source, 20 g / L artificial seawater salt, 9 g / L potassium diphosphate (KH ₂ PO ₄ ), 9.5 g / L thiamine, / L, cyanocobalamin B12 1.0 g / L), and the cells were cultured in a microalgae culture medium for 30 hours before transplantation into 3 L microalgae culture medium , A microbubble-type and a stirring-type incubator were batch-cultured under the conditions of a culture temperature of 28 ° C, a stirring speed of 200 rpm and an air injection amount of 5 L / min. The bubble column culture incubator had a culture temperature of 28 ° C, a stirring speed of 700 rpm, min. In addition, the bubble column type incubator was batch-wise cultivated at a temperature of 28 ° C and an air flow rate of 6 L / min.

The culture broth was recovered by centrifugation, and the cells were washed three times with PBS buffer (phosphate buffered saline, pH 7.2) and lyophilized for 12 hours to measure dry cell mass. The lipid content of DHA was determined by the modified Bligh-Dyer method al., 2007).

The dried cells of the culture 10mL chloroform 3mL, methanol 6mL, 50mM dipotassium hydrogen phosphate (K ₂ HPO ₄₎ buffer (pH 7.4) and the reaction mixture at 28 ℃ to 200rpm after the reaction for 1 hour, chloroform 3mL, phosphate dibasic 3mL 3 ml of potassium (K ₂ HPO ₄ ) buffer was added thereto, and the mixture was mixed for about 30 times. The mixture was centrifuged at 4 ° C and 3500 rpm for 10 minutes to separate the aqueous layer and the lipid-containing organic solvent layer. The chloroform layer was carefully transferred with an aluminum dish preliminarily weighed, dried at a temperature of 90 ° C for 30 minutes, and then the lipid content was measured. The yield of the obtained oil was shown in Table 5 below.

At this time, the content of DHA contained in the lipids was measured by gas chromatography, and 6 mL of a 5% sulfuric acid-methanol solution was added to an appropriate amount of dried cells, followed by reaction at 90 ° C for 1 hour to produce a fatty acid methyl ester. 1 mL and analyzed by gas chromatography.

According to the References 1 to 3, the maximum dry cell mass and lipid content of the stirring type incubator were confirmed to be 26.1 g / L and 13.8 g / L at 48 hours, respectively, and the maximum dry cell mass and lipid content of the bubble column- The maximum amount of dry cell mass and lipid content of the microbubble-type incubator were confirmed to be 29.4 g / L and 14.6 g / L at 18 hours of culture, respectively, The incubation time could be shortened by 2 times as compared with the conventional stirring type incubator.

In addition, as shown in Table 5, the microbubble-type incubator showed the highest lipid productivity of 19.5 g / L-d when cultured for 18 hours by each incubator.

Test Example 4: Optimization of culture according to air injection amount

The microalgae were cultured under the same culture conditions as in Test Example 3 except that the amounts of air were varied at 2 L / min, 3 L / min, 4 L / min and 5 L / min, respectively, And the results are shown in Table 6 below.

According to the above Table 6, when the amount of air injected was 3 L / min, the dry bacterial weight, lipid content, and DHA yield were the highest at 33.1 g / L, 19.0 g / L and 7.2 g / L, respectively.

Test Example 5: Optimization of culture according to stirring speed

The microalgae were cultured under the same culture conditions as in Test Example 3 except that the batch culture was performed for 30 hours at a stirring speed of 100 rpm, 200 rpm, 300 rpm, and 400 rpm, respectively, and the culture performance was measured and shown in Table 7 below .

According to Table 7, it was confirmed that when the stirring speed was 200 rpm, the dry cell mass, lipid content and DHA production amount were the highest, 31.9 g / L, 18.6 g / L, and 7.1 g / L, respectively.

12: Socket section 121: Air injection nozzle
13: air front end portion 14: micro bubble generating portion
15: rotation axis G: micro bubble generator

Claims (8)

A socket portion formed in a cylindrical shape and provided with an air injection nozzle and having a plurality of holes formed in a side periphery thereof so as to prevent air from stagnating therein;
An air front end portion formed inside the socket portion and formed in the shape of a propeller so that the air is uniformly distributed at the upper end of the socket portion by rotating in a predetermined direction;
A micro bubble generator provided at a lower portion of the air front end and having a plurality of blades for mixing water and air at a high flow rate of turbulent flow along with fine bubbles blowing by high speed rotation; And
And a rotary shaft installed vertically at the center for transmitting the rotational force of the motor to the air front end and the micro bubble generating unit,
The micro bubble generator may further include a plurality of holes formed in the disc to minimize the turning force generated about the rotation axis,
Wherein the microbubble generator is installed inside the micro-algae culture apparatus to generate and supply microbubbles. delete delete The method according to claim 1,
Wherein the micro bubble generator includes a cover-shaped blade for minimizing the generation of swirling flow in the socket portion and minimizing a resistance value of fluid generated inside the micro bubble generator. A method for cultivating a microalgae of a Troutukitrid system,
a) transplanting and culturing the cells pre-cultured in a culture medium containing a carbon source and a nitrogen source into the culture medium; And
b) supplying microbubbles produced by the microbubble generator of claim 1 or 4 to the culture medium to increase the efficiency of oxygen delivery, and setting the feed rate to 2 to 5 L / min Wherein the microbubble generator is a microbubble generator. delete 6. The method of claim 5,
Wherein the size of the microbubbles in the step b) ranges from 250 to 300 탆. 8. The method of claim 7,
Wherein the microbubble generator is cultivated at a stirring speed of 200 rpm in the step b).

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