KR20210068209A - Process for preparing PHB using mixed microbs - Google Patents

Abstract

The present invention provides a composition for producing polyhydroxybutyrate (PHB) comprising the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain, a PHB (polyhydroxybutyrate) production kit comprising the PHB production composition, and a mixed strain of the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain It relates to a method for producing polyhydroxybutyrate (PHB) comprising the step of sequentially and repeatedly culturing in a eutrophic (feast) medium and a famine (famine) medium. By using the composition for producing PHB provided in the present invention, since PHB can be produced more effectively and in high yield, it will be widely used in the production of useful components using microorganisms.

Description

Method for producing PHB using a mixed strain {Process for preparing PHB using mixed microbs}

The present invention relates to a method for producing PHB using a mixed strain, and more specifically, the present invention relates to a composition for producing PHB (polyhydroxybutyrate) comprising Acinetobacter junii BP25 strain and Aeromonas hydrophila strain, PHB comprising the PHB production composition ( A method for producing polyhydroxybutyrate (PHB) comprising the step of culturing a kit for production of polyhydroxybutyrate and a mixed strain of the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain in a eutrophic (feast) medium and a famine (famine) medium. is about

While plastic materials have contributed greatly to the affluent daily life and industrial development of modern people, environmental hormone leakage due to incineration or landfill of various waste vinyl, Styrofoam, and plastic containers that are generated in large quantities, detection of highly toxic dioxins, and incomplete waste It is emerging as a cause of serious environmental pollution such as air pollution caused by combustion. In order to solve this problem, as the pressure to commercialize and make mandatory biodegradable plastics, which are environmentally friendly and harmless plastics that can be used as easily as ordinary plastics and rot by microorganisms in the soil after use, is growing, advanced countries such as Germany, Italy, and the United States Practical use of biodegradable plastics is actively progressing, such as making it mandatory for shopping bags and plastic bottles to use biodegradable resins.

PHB is an energy-storing polymer synthesized by microorganisms that has similar physical properties to chemically synthesized polymers used as plastic raw materials, and has excellent biodegradability, so that it can be completely decomposed into water and carbon dioxide by soil microorganisms within a short time. It is an environmentally friendly plastic raw material. However, compared to the high use value of PHB as an eco-friendly plastic raw material, the production cost is higher than that of general-purpose, non-degradable general plastic raw material, so there is a very limit in its use except for expensive medical supplies.

Most microorganisms in nature use PHB as an energy storage material and it was known that they can only accumulate about 20-40% of the dry cell weight in cells under poor environmental conditions. However, some domestic and foreign companies preparing for commercialization of PHB recently and Research institutes are known to have enabled the accumulation of PHB up to 80% or more of the dry cell weight by using more advanced bioengineering and genetic engineering techniques, such as the method of transferring the PHB biosynthesis gene to E. coli. For the production of such PHB, a method using mainly strains such as Alkaligenes eutrophus and Alkaligenes reuters has been developed, but recently, the PHB synthesis gene of Alkaligenes eutrophus has a fast growth rate and is the most industrially Research to increase the productivity of PHB by transforming the widely used E. coli is being actively conducted. For example, Korean Patent Application Laid-Open No. 10-2004-0046678 discloses a technique using a novel strain capable of improving the productivity of PHB, and Korean Patent No. 10-0454250 discloses transformation with a three-step culture method. A method for improving the productivity of PHB by culturing E. coli is disclosed.

Under this background, the present inventors have made intensive research efforts to develop a method for improving the productivity of PHB. As a result, when using a method of sequentially repeating a mixed strain of Acinetobacter junii BP25 strain and Aeromonas hydrophila strain in two media, PHB It was confirmed that productivity can be significantly improved, and the present invention was completed.

The main object of the present invention is to provide a composition for producing PHB (polyhydroxybutyrate) comprising the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain.

Another object of the present invention is to provide a kit for producing PHB (polyhydroxybutyrate) comprising the composition for producing PHB.

Another object of the present invention is the production of polyhydroxybutyrate (PHB) comprising the step of sequentially and repeatedly culturing a mixed strain of the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain in a nutrient medium and a famine medium. to provide a way

One embodiment of the present invention for achieving the above object provides a composition for producing PHB (polyhydroxybutyrate) comprising the Acinetobacter junii BP25 strain and Aeromonas hydrophila strain.

In order to improve the productivity of PHB, the present inventors confirmed that the growth rate of the strain was remarkably improved when a combination of a strain known to produce PHB was conventionally cultured while conducting various studies. That is, among the PHB-producing strains known so far, the Acinetobacter junii BP25 strain and Aeromonas hydrophila strain significantly improved the growth rate of the strains when cultured after mixing 1:1 based on the number of cells than when culturing each of them individually. was confirmed. When the growth rate of the cells is improved and the biomass of the cells is increased, the productivity of PHB produced from such cells is increased, so the composition comprising the two strains can be used as a composition for producing PHB with improved productivity of PHB.

The term "Acinetobacter junii BP25 strain" of the present invention is known as a subspecies of Acinetobacter junii strain, which is a type of pathogen known to cause sepsis or respiratory disease, and is known to have the ability to produce PHB.

The term " Aeromonas hydrophila strain " as used in the present invention is a kind of Gram-negative globular bacterium belonging to the genus Aeromonas, in the form of aquatic bacteria with flagella, and produces various organic gases by fermenting glucose, and in humans, It is known to be a kind, and is known to have the ability to produce PHB.

Another embodiment of the present invention provides a kit for producing polyhydroxybutyrate (PHB) comprising the composition for producing PHB.

A kit for producing PHB comprising the composition for producing PHB of the present invention may include various components necessary to increase PHB productivity using the composition. These components are not particularly limited thereto, but may include one or more other component compositions, solutions or devices suitable for culturing the strains included in the composition. As an example, it may be a culture vessel, a culture medium, a shaking incubator for culture, a culture timer, and the like.

Another embodiment of the present invention provides a method for producing PHB using a mixed strain of the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain.

Specifically, the PHB production method of the present invention comprises the steps of (a) obtaining a mixed strain containing the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain at the same level; (b) inoculating and culturing the obtained mixed strain in a eutrophic medium, and then sequentially repeating the process of inoculating and culturing in a famine medium; and. (c) after the end of the culture, recovering the PHB from the culture.

The term "culture" of the present invention means to grow microorganisms in an appropriately artificially controlled environmental condition, and includes flask culture, batch culture, etc., but is not particularly limited thereto.

In the present invention, the eutrophic (feast) medium of step (b) is a medium capable of growing the strain, and may be interpreted as a medium containing a carbon source and various nutrients.

The carbon source contained in the eutrophic medium is not particularly limited thereto, but as an example, it may be a carbon source that can support the growth of the two PHB-producing strains provided in the present invention, and as another example, sodium acetate, Sodium butyrate and the like may be used alone or in combination.

Nutrient components included in the feeding medium are not particularly limited thereto, but as an example, may be various mineral components that can support the growth of the two PHB-producing strains provided in the present invention, and as another example, NaCl, Na ₂ HPO ₄ , KH ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , MgSO ₄ 7H ₂ O, peptone, yeast extract, etc. may be used alone or in combination. have.

The oligotrophic medium in contrast to the eutrophic medium has no significant effect on the growth of the strain, and can be interpreted as a medium that improves the productivity of PHB produced in the strain.

The oligotrophic medium contains only a carbon source, and the carbon source included therein is the same as described above.

The culturing in step (b) can be performed by culturing in a nutrient medium, then culturing in a nutrient medium, and then culturing in a nutrient medium again, sequentially repeating culture using two mediums. .

At this time, the time of culturing in the nutrient medium and the time of culturing in the nutrient medium should be set to be the same. These incubation times are not particularly limited thereto, but as an example, may be 36 to 60 hours, and as another example , may be 72 to 54 hours, and as another example, may be 48 hours.

In addition, the temperature conditions for performing the culture are not particularly limited thereto, but as an example, may be 20 to 45 ℃, as another example, may be 25 to 40 ℃, as another example, 30 to 35 It can be °C.

In addition, precursors suitable for the culture medium may be used. The above-mentioned raw materials may be added in a batch, fed-batch or continuous manner by an appropriate method to the culture during the culturing process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture.

In addition, an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles. Oxygen or oxygen-containing gas (eg air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. Cultivation is continued until the maximum amount of the peptide is obtained. This is usually achieved in 10 to 100 hours for this purpose.

The PHB recovery of step (c) may be performed by methods known in the art, such as dialysis, centrifugation, filtration, solvent extraction, chromatography, and crystallization. For example, a method of recovering the desired PHB by centrifuging the culture to obtain a supernatant from which the transformant is removed, and applying the obtained supernatant to a solvent extraction method can be used, in addition to the characteristics of the desired PHB Any method capable of recovering the PHB by combining known experimental methods in accordance with the present invention is not particularly limited and may be used.

By using the composition for producing PHB provided in the present invention, since PHB can be produced more effectively and in high yield, it will be widely used in the production of useful components using microorganisms.

1 is a graph showing the change in the level of biomass with the passage of culture time when two strains ( Acinetobacter junii BP25 strain, Aeromonas hydrophila strain) are individually or co-cultured.
Figure 2 is a graph showing the change in the carbon source absorption rate with the lapse of incubation time while culturing the Acinetobacter junii BP25 strain under Cycle-2 conditions twice.
3 is a graph showing the change in carbon source absorption rate with the lapse of incubation time while culturing Aeromonas hydrophila strain under Cycle-2 conditions twice.
4 is a graph showing the change in the carbon source absorption rate with the lapse of incubation time while culturing the co-cultured strain under Cycle-2 conditions twice.
5a is a graph showing the results of analyzing the pH change of the medium when Acinetobacter junii BP25 strain and Aeromonas hydrophila strain are individually or co-cultured, respectively.
Figure 5b is a graph showing the results of analyzing the change in dissolved oxygen in the medium when Acinetobacter junii BP25 strain and Aeromonas hydrophila strain are individually or co-cultured, respectively.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Culture of PHB-producing strains

Two strains (Acinetobacter junii BP25 strain, Aeromonas hydrophila strain) known to exhibit PHB production characteristics were each individually or co-cultured, and after the culture was terminated, the level of biomass contained in the culture was analyzed (Fig. One). The level of biomass was calculated by the following Gompertz equation application formula (1).

f = a x Exp (-Exp (b*2.71828 x (c-x)/a+1))+d ......(1)

In the above formula,

f represents the biomass concentration (g/L);

a represents maximum biomass growth (g/L);

b represents the maximum growth rate (g/L-h);

c represents the lag phase (h);

x represents time (h);

d represents the initial biomass concentration (g/L).

1 is a graph showing the change in the level of biomass with the passage of culture time when two strains ( Acinetobacter junii BP25 strain, Aeromonas hydrophila strain) are individually or co-cultured.

As shown in FIG. 1, the maximum growth rate of the Acinetobacter junii BP25 strain was 0.25/h, the maximum growth rate of the Aeromonas hydrophila strain was 0.17/h, and the maximum growth rate of the co-cultured strain was 0.30/h.

From the above results, it can be seen that the co-cultured strains exhibit a relatively good production of biomass because the growth rate is superior to that of each strain individually cultured.

Example 2: Acinetobacter junii Culture of BP25 strain

Example 2-1: Strain culture

First, a carbon source (sodium acetate 6g/L and sodium butyrate 4g/L) and other nutrients (NaCl 1 g/L, Na ₂ HPO ₄ 0.37 g/L, KH ₂ PO ₄ 0.1 g/L, (NH ₄ ) ₂ A _{eutrophic medium (pH 7.0±0.2) and a carbon source (sodium acetate) containing HPO 4} 0.05 g/L, MgSO ₄ 7H ₂ O 0.02 g/L, peptone 0.5 g/L, yeast extract 0.05 g/L) A famine medium (pH 7.0±0.2) containing only 6 g/L and sodium butyrate 4 g/L) was prepared, respectively.

The prepared eutrophic medium was inoculated with 10% (v/v) of Acinetobacter junii BP25 strain, and cultured for 48 hours under aerobic conditions, then the medium was replaced with a nutrient-dense medium and cultured under aerobic conditions for 48 hours (Cycle). -One). Then, the medium was again replaced with a eutrophic medium and cultured under aerobic conditions for 48 hours, then the medium was replaced with a oligotrophic medium and cultured under aerobic conditions for 48 hours (Cycle-2).

Example 2-2: Carbon Source Analysis

While culturing in the method of Example 2-1, changes in the concentration and utilization rate of the carbon source in the medium according to the lapse of culture time were measured (FIG. 2).

Figure 2 is a graph showing the change in the carbon source absorption rate with the lapse of incubation time while culturing the Acinetobacter junii BP25 strain under Cycle-2 conditions twice.

As shown in Figure 2, in the eutrophic stage, it was confirmed that the maximum utilization of sodium acetate was about 75%, and the maximum utilization of sodium butyrate was about 68%. On the other hand, in the oligotrophic stage, it was confirmed that the maximum utilization of sodium acetate was about 69%, and the maximum utilization of sodium butyrate was about 61%.

Example 2-3: PHB Productivity Analysis

The productivity of PHB produced in the culture obtained from the method of Example 2-1 was analyzed (Table 1). The productivity analysis of the PHB was performed by separating and purifying PHB from the culture, and quantitatively analyzing the level of the separated and purified PHB using HPLC.

At this time. Separation and purification of PHB was performed as follows: First, the culture was centrifuged (4° C., 3000 x g, 10 minutes) to obtain a precipitated biomass, which was washed with 70% ethanol and then centrifuged again. A biomass from which impurities were removed was obtained. Equal volumes of chloroform and 10% sodium hypochlorite were added to the obtained biomass, followed by a shaking reaction (120 rpm, 35±0.2° C., 3 hours) to pulverize the biomass, and then the chloroform lower layer fraction containing PHB was obtained. collected. Cold methanol was added to the collected chloroform lower layer fraction to form a precipitate, which was centrifuged to recover the precipitate, and then dried to separate and purify PHB.

Quantitative analysis of the separated and purified PHB was performed using HPLC. Roughly, a solution obtained by dissolving the separated and purified PHB sample in sulfuric acid was heated at 90° C. for 30 minutes to obtain a reactant, and the obtained reactant HPLC analysis (Aminex HPX-87H column (Bio-Rad Laboratories, USA), 210 nm UV detector (Waters 2487, MA, USA) and 5 mM H2SO4 mobile phase) was performed on the .

PHB productivity of Acinetobacter junii BP25 strain carbon source cycle Culture method PHB (g/L) PHB (g/g carbon source _added ) Cycle-1 eutrophication 0.38 0.054 oligotroph 1.78 0.272 Cycle-2 eutrophication 0.41 0.056 oligotroph 1.82 0.270

As shown in Table 1, biomass was rapidly increased in the eutrophic stage, but the maximum yield of PHB was 0.41 g/L, and the growth of biomass was not confirmed in the oligotrophic stage, but the maximum yield of PHB was It was confirmed that 1.82 g/L was shown.

From the results of FIG. 2 and Table 1, it was analyzed that the growth of the cells was performed in the eutrophic stage and the production of PHB was performed in the oligotrophic stage when the Acinetobacter junii BP25 strain was cultured.

Example 3: Aeromonas hydrophila culture of strains

Example 3-1: Strain culture

The strain was cultured in the same manner as in Example 2-1, except that the Aeromonas hydrophila strain was used instead of the Acinetobacter junii BP25 strain.

Example 3-2: Carbon Source Analysis

While culturing in the method of Example 3-1, changes in the concentration and utilization rate of the carbon source in the medium were measured according to the lapse of culture time (FIG. 3).

3 is a graph showing the change in carbon source absorption rate with the lapse of incubation time while culturing Aeromonas hydrophila strain under Cycle-2 conditions twice.

As shown in Figure 3, in the eutrophic stage, it was confirmed that the maximum utilization of sodium acetate was about 67%, and the maximum utilization of sodium butyrate was about 52%. On the other hand, in the oligotrophic stage, it was confirmed that the maximum utilization of sodium acetate was about 61%, and the maximum utilization of sodium butyrate was about 44%.

Example 3-3: PHB Productivity Analysis

PHB productivity in the same manner as in Example 2-3, except that the culture obtained from the method of Example 3-1 was used instead of the culture obtained from the method of Example 2-1. were analyzed (Table 2).

PHB productivity of Aeromonas hydrophila strains carbon source cycle Culture method PHB (g/L) PHB (g/g carbon source _added ) Cycle-1 eutrophication 0.15 0.026 oligotroph 0.96 0.168 Cycle-2 eutrophication 0.17 0.027 oligotroph 1.12 0.175

As shown in Table 2, in the eutrophic stage, the biomass was rapidly proliferated, but the yield of PHB was 0.17 g/L, and in the oligotrophic stage, the growth of biomass was not confirmed, but the yield of PHB was 1.12 g It was confirmed that /L was represented.

From the results of FIG. 3 and Table 2, it was analyzed that the growth of cells was performed in the eutrophic stage, and the production of PHB was performed in the oligotrophic stage even when the Aeromonas hydrophila strain was cultured.

Example 4: Co-culture

Example 4-1: Strain culture

Acinetobacter junii BP25 instead strain, Acinetobacter junii the strain to strain BP25 and Aeromonas same manner as in Example 2-1 except that the hydrophila strain using a mixed strain mixed in equal volume and incubated.

Example 4-2: Carbon Source Analysis

While culturing in the method of Example 4-1, changes in the concentration and utilization rate of the carbon source in the medium were measured according to the lapse of culture time (FIG. 4).

4 is a graph showing the change in the carbon source absorption rate with the lapse of incubation time while culturing the co-cultured strain under Cycle-2 conditions twice.

As shown in FIG. 4 , it was confirmed that the maximum utilization of sodium acetate was about 78% in the eutrophic stage, and the maximum utilization of sodium butyrate was about 74%. On the other hand, in the oligotrophic stage, it was confirmed that the maximum utilization of sodium acetate was about 73%, and the maximum utilization of sodium butyrate was about 70%.

Example 4-3: PHB Productivity Analysis

PHB productivity in the same manner as in Example 2-3, except that the culture obtained from the method of Example 4-1 was used instead of the culture obtained from the method of Example 2-1. were analyzed (Table 3).

PHB productivity of co-cultured strains carbon source cycle Culture method PHB (g/L) PHB (g/g carbon source _added ) Cycle-1 eutrophication 0.42 0.056 oligotrophic 2.12 0.280 Cycle-2 eutrophication 0.44 0.069 oligotrophic 2.46 0.376

As shown in Table 3, biomass was rapidly increased in the eutrophic stage, but the yield of PHB was 0.44 g/L, and the growth of biomass was not confirmed in the oligotrophic stage, but the yield of PHB was 2.46 g It was confirmed that /L was represented.

From the results of Figures 4 and 3, it was found that when co-culturing was performed, the utilization rate of the carbon source was increased, and the production yield of PHB was also increased, compared to when each strain was individually cultured.

Example 5: Medium analysis

Changes in pH and dissolved oxygen in the medium contained in the cultures cultured in Examples 2 to 4 were analyzed.

Example 5-1: Medium pH Analysis

The pH change of the medium contained in the three cultures cultured in Examples 2 to 4 was analyzed (FIG. 5a).

5a is a graph showing the results of analyzing the pH change of the medium when Acinetobacter junii BP25 strain and Aeromonas hydrophila strain are individually or co-cultured, respectively.

As shown in Figure 5a, in the eutrophic stage, when co-culturing each strain individually, the pH of the overall medium shows a higher level, whereas in the oligotrophic stage, the Acinetobacter junii BP25 strain is individually or co-cultured. It was confirmed that the pH of the entire medium exhibited a higher level when culturing Aeromonas hydrophila strains individually than when culturing.

Example 5-2: Analysis of dissolved oxygen in medium

Changes in dissolved oxygen in the medium contained in the three cultures cultured in Examples 2 to 4 were analyzed (FIG. 5b).

Figure 5b is a graph showing the results of analyzing the change in dissolved oxygen in the medium when Acinetobacter junii BP25 strain and Aeromonas hydrophila strain are individually or co-cultured, respectively.

As shown in Figure 5b, when the medium is replaced according to the performance of the eutrophic phase and the oligotrophic phase, the level of dissolved oxygen in the medium increases sharply, and then, as the incubation time elapses, the level of dissolved oxygen in the medium increases It was confirmed that the decreasing trend was repeated.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (10)

A composition for producing polyhydroxybutyrate (PHB) comprising the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain.
A kit for producing PHB (polyhydroxybutyrate) comprising the composition for producing PHB of claim 1.
(a) obtaining a mixed strain comprising the Acinetobacter junii BP25 strain and the Aeromonas hydrophila strain at the same level;
(b) inoculating and culturing the obtained mixed strain in a eutrophic medium, and then sequentially repeating the process of inoculating and culturing in a famine medium; and.
(c) after the culture is terminated, a method for producing PHB (polyhydroxybutyrate) comprising the step of recovering PHB from the culture.
4. The method of claim 3,
The eutrophic (feast) medium is a medium containing a carbon source and nutrients, the production method of PHB.
5. The method of claim 4,
The carbon source is sodium acetate and sodium butyrate, the method for producing PHB.
5. The method of claim 4,
The nutritional component is selected from the group consisting of NaCl, Na ₂ HPO ₄ , KH ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , MgSO ₄ 7H ₂ O, peptone, yeast extract, and combinations thereof A method for producing PHB, which is a component that becomes
4. The method of claim 3,
Culturing in the eutrophic medium will be performed for 36 to 60 hours under aerobic conditions, the production method of PHB.
4. The method of claim 3,
The method for producing PHB, wherein the famine medium is a medium containing only a carbon source.
4. The method of claim 3,
The method for producing PHB, wherein the culture in the nutrient medium is performed for 36 to 60 hours under aerobic conditions.
4. The method of claim 3,
The method for producing PHB, wherein the culture in the nutrient medium and the culture in the nutrient medium are performed for the same time.

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