KR0129117B1 - A bacillus sp. which produce polyhydroxyalkanates - Google Patents

Abstract

Bacillus sp. KCTC8552P2 (Bacillus thuringenesis H0079) capable of biosynthesizing polyhydroxybutyrate(PHB)-polyhydroxyvalerate(PHV) copolymer including about 90 mol% of 3-hydroxyvalerate(HV) unit is clamed. PHB-PHV copolymer is synthesizd in the medium containg glucose as main substrate and propionate as cosubstrate by the single step batch culture. When the concentration of propionate in the medium become 1.0%, maximum yield of dry cell weight and polyhydroxyalkanate biomass indicate 7.3 g/l and 43.9% respectively. Copolymer obtained is used for the material of biothermoplastics.

Description

Bacillus microorganism to synthesize polyhydroxyalkynet

1 is a graph of the effect of propionate on the growth of strains of the present invention.

(A) to (d) of Figure 2 is a graph showing the effect of propionate on the synthesis and composition of poly (3HB-co-3HV) in the batch culture of the strain of the present invention, ○ is 0.1% propio Nate, 은 is 0.3% propionate, and ▽ are 0.1% propionate added in logarithmic groups.

3 is a gas chromatography showing fatty acid plasticity present in cells of Bacillus thuringiensis (KCTC1033).

4 is a gas chromatography showing the fatty acid composition present in the cells of the strain according to the present invention.

5 is a component analysis gas chromatography of PHA synthesized by the strain of the present invention.

The present invention relates to a bacterium of the genus Bacillus, which synthesizes polyhydroxyalkanates (hereinafter referred to as PHA), and more particularly, to a step 1 in a medium containing glucose as a main substance and propionate as an auxiliary substrate. The present invention relates to a bacterium of the genus Bacillus, which produces a polyhydroxybutyrate (PHB) / PHV copolymer containing up to 90 mol% of 3HV (hydroxyvalerate) units by batch culture of.

Synthetic plastic and synthetic fiber products, which have been widely used in daily life because of its durability, are contaminated with soil and rivers and inhibit plant growth without being degraded in nature even after decades of use. It is causing serious environmental pollution problems. The cause of environmental pollution by plastic is due to the excellent durability of plastics to withstand the decomposition of microorganisms.

In order to solve the environmental pollution problem caused by such plastics, biodegradable plastics decomposed by microorganisms have been developed. These biodegradable plastics are prepared by adding starch or metal complexes to existing synthetic plastics such as polyethylene or polyester, so that the biodegradable plastics can be easily decomposed by the action of microbial decomposition and photooxidation. However, such biodegradable plastics easily degrade starch in the plastic components, but the synthetic plastic portion still remains undecomposed to contaminate the environment, and the metal complex portion used in the manufacture of these plastics is another source of environmental pollution. As a result, there was a limit to fundamentally solving the problem of environmental pollution caused by plastics.

Recently, in order to solve the environmental pollution problem caused by synthetic plastics more fundamentally, researches on bioplastics generated from living organisms have been actively conducted. These bioplastics, also called biopolymers, are natural polymers obtained directly from microorganisms, animals, and plants and have excellent mechanical durability like conventional synthetic plastics, and can be used as materials for general materials, packaging materials, and agricultural films. It has very good biodegradability, and after a certain time in nature, it is completely decomposed into water and carbon dioxide.It is harmless to human body and has excellent biocompatibility.It is also used as a material for adhesives, bands, sutures, etc. . That is, these bioplastics are biothermoplastics that are completely decomposed by microorganisms in nature and naturally absorbed and decomposed in vivo when used in vivo, and can completely solve environmental pollution problems.

Looking at the related patents related to this, Korea Patent Publication No. 192-19941 filed by Korea Synthetic Island contains a carbon source, a nitrogen source, phosphorus and a trace element that can accumulate PHB / PHV copolymers in cells by microbial fermentation. Disclosed is a method for producing a PHB / PHV copolymer in which an aqueous cell proliferation medium is cultured under a logarithmic supply condition at a cell growth rate to maximize cell concentration, and then a copolymer is recovered.

Korean Patent Application Publication No. 92-19943, filed with Koryo Synthesis, uses an alcaligenes latus (KFCC-10724) in the process of preparing PHB and PHB / PHV copolymers, which are a kind of PHA. After culturing aerobicly at -49 degrees and pH 6.5-7.2, the nutrient medium with the C / N ratio of carbon source and nitrogen source adjusted to 15-30 was continuously fed into the culture medium, and the cells were grown to 30-50 g / L, P / N ratio is 40-80, sulfur (S), phosphorus (P), calcium (Ca) and potassium (K) are continuously supplied with limited nutrition medium, keeping dissolved oxygen low (10-20%) Disclosed is a method for preparing PHA by oil culture, in which PHB / PHV copolymer containing 5-20% of PHB and 3-HV units in a cell is accumulated at high concentration (60-80%) by inducing accumulation conditions.

Korean Patent Application Publication No. 91-12251, filed by ICI, UK, uses a copolymer of HB and HV monomer units using PHB-accumulating bacteria that cannot proliferate when cultured on a substrate composed of HV components under conditions that do not limit proliferation. In a microbiological production process, a substrate comprising a substrate composed of water-soluble assimilable carbon containing HV and water soluble assimilable carbon containing HB under conditions of growth limitation to aid in the synthesis and accumulation of bacteria copolymers. Incubating the bacteria with dry cells weighed per 11 L of medium in a cell, and then recovering the bacteria containing the copolymer, wherein the strain used is a mutant or variant of Alkarygenes eutrophus NCIMB 40124 Water soluble basically provided by the HV component under conditions that do not limit this proliferation To alkali that can not proliferate when cultured in a lot of substrates, including a desire Ness oil Trojan pyuseu NCIMB40124, or its mutated variants chena.

Japanese Patent Laid-Open No. 90-299590, filed by ICI, UK, is a microbiological method for producing a polymer in which the most frequent number of carbon atoms contained in the polymer monomer is N, which is formed of a magnetizable carbohydrate compound having a carbon number of less than N. A method for microbiological production of a polymer comprising culturing a bacterium on a substrate, wherein the bacterium has at least one bacterium selected from NCIB40135, NCIB9571, NCIB9904, NCIB8626, or NCIB9520 or this microorganism group or at least one characteristic of the bacterial group. It is about.

U.S. Patent No. 4786598 is a process for producing 3-PHB, which is cultured in two stages of Alkali genus ratus or a mutant strain derived from the strain, and is transferred to a two-stage fermenter continuously while incubating at the most suitable conditions for growth in the first stage. While continuing the culture and extracting the polymer from the cells.

In these patents, the content of 3HV in the synthesis of PHB / PHV copolymer using microorganisms depends on the concentration of auxiliary substrate used as precursor of 3HV. However, since propionate or pentanoate, which is used as a precursor of 3HV monomers, is very toxic to cells, increasing the concentration of precursors to increase the content of 3HV greatly inhibits the growth of the cells. Receive. Therefore, in order to produce PHB / PHV containing a high content of 3HV, the cells are grown in a normal fermentation medium (medium without a precursor such as propionate added) in one step, and the cells are grown in two steps. A two-step batch culture method incubated in a medium added as a precursor is used.

The present invention has been made to solve this problem, and a main object of the present invention is to provide a novel bacterium bacterium that has relatively high resistance to propionate and synthesizes PHA.

Another object of the present invention is a method of culturing the microorganism for synthesizing PHB / PHV copolymer containing up to 95 mol% of 3HV monomer in a single stage batch culture in a medium containing glucose as a main substance and propionate as an auxiliary substrate. To provide.

Still another object of the present invention is to provide a method for producing PHA by culturing the microorganism.

The microorganism of the present invention for achieving the above object is a bacterium strain of Bacillus thuringiensis, which was deposited as No. KCTC 8552P to the Genetic Bank of Korea Institute of Science and Technology on February 14, 1994 as a microorganism for synthesizing PHA. (thuringiensis) H0079.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The strain of the present invention frequently appeared as a contaminating strain in the study of the PHA producing strains of the present inventors, a novel strain of the genus Bacillus, which is a gram-positive aerobic bacterium isolated by the present inventors and has endogenous spores (apos). to be.

In general, the precursor (auxiliary substrate) of the 3HV monomers used for the biosynthesis of Poly (3HB-co-3HV) copolymers is propionate or valenate. However, these precursors have a significant effect on the cell structure of the cell, so even at very low concentrations, they significantly affect cell growth and PHA biosynthesis (Byrom, 1987, Ramsay et al., 1990). Therefore, in the present invention, the effect of dust propionate on the growth of the strain according to the present invention was investigated. As shown in FIG. 1, more cell production was obtained when 0.1% propionate was added than without supplementary propionate, which was found after the depletion of glucose, the main carbon source. Seems to be used as a carbon source. However, when the concentration of propionate was 0.3%, it was greatly influenced by cell growth, and the higher the concentration of propionate, the longer the lagphase was observed. Thus, the strains of the present invention synthesize high concentrations of PHB / PHV copolymers containing homopolymers or 3HV monomers of PHB in fermentation broths based on glucose and 0-1.0%, preferably 0.1-0.3% propionate, as a carbon source. can do.

As described above, the strain is resistant to propionate and PHB / PHV air containing 70-90 mol% of 3HV by one-step batch culture with 0-1.0% propionate added. The coalescence can be synthesized. In addition, by synthesizing PHA up to 43.9% of the total amount in batch culture with 0.1% propionate, PHA accumulation rate is very high among Gram-positive microorganisms known to date.

On the other hand, the results of the copolymerization of the copolymer production of the strain of the present invention by batch culture is as shown in FIG. As can be seen in FIG. 2, as in the case of the clasque culture, the addition of 0.1% of the auxiliary substrate resulted in the highest cell mass and copolymer formation, and the content of 3HV was 50-80mol% during the incubation time. . However, when the same amount (0.1%) of auxiliary substrate was added in the logarithmic phase of microbial growth, propionate showed greater inhibition in cell growth and copolymer production. Showed done.

The identification results of the strains of the present invention are shown in Table 1 below with reference to Bacillus thuringiensis (KCTC1033).

TABLE 1

NT: Not measured.

From the above measurement results, the strain of the present invention is judged to be a kind of Bacillus thuringiensis, but the typical Bacillus thuringiensis synthesizes a small amount of PHA and is known to mainly synthesize δ-toxin (Bergy's manual of determinative bacteriology , 8th edition). Therefore, the strain of the present invention is considered to be a novel microorganism belonging to the subgenus of Bacillus thuringiensis. In order to explain this in more detail, the PHA production capacity of the strain of the present invention and Bacillus thuringiensis (KCTC 1033) cultured in the medium composition shown in Table 6 was measured and described in Table 2 below. The fatty acid composition present in the cells was described based on the gas chromatography of FIGS. 3 and 4.

TABLE 2

TABLE 3

* In the above table, for example, 12: 0 is 12 has 12 carbon atoms, 0 means no double bond number.

In Tables 2 and 3 it can be easily seen that both microorganisms are separate microorganisms. On the other hand, since the strain of the present invention can obtain a biopolymer of various physical properties through the control of the 3HV content in the PHA copolymer, it is possible to widen the production range of the copolymer having a high content of 3HV and its use development. The industrial value is very large.

Although the configuration and effects of the present invention will be described in more detail with reference to the following examples, the scope of the present invention is not limited to the following examples.

Example 1

1) Medium and culture conditions for strain preservation

YPB (yeast extract, 2g / L; peptone, 5g / L; beef extract 1g / L, NaCl, 5g / L; agar, 20g / L) solids for the preservation of this strain identified as a kind of Bacillus thuringiensis Incubated at 30 ° C. in medium and passaged every 3-4 days. In addition, for long-term preservation by using the above YPB liquid medium except agar (Agar) shaking culture for 12 hours at 30 ℃ and put in a cryopreservation vial (vial) and stored at -70 ℃.

2) Medium and culture conditions for PHA production

In a 300 ml Erlenmeyer flask containing 50 ml of seed medium and shaken (160 rpm) for 12 hours at 30 ° C, 5 ml of this seed was inoculated into a 300 ml Erlenmeyer flask containing 45 ml of fermentation medium. Shake culture was carried out for 30 hours under the same conditions. In order to investigate the effect of sodium propionate addition, 0, 0.1, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2.0% propionate was added to the fermentation medium, respectively. Seed medium and fermentation medium composition is shown in Table 4 below.

Composition of spawn and fermentation broth

TABLE 4

* Trace element

For batch culture using a fermenter, 100 ml of the spawn culture medium incubated for 12 hours in the spawn medium was inoculated into a 5L self-permator containing 2 L of sterilized fermentation broth, and the aeration rate was 1 vvm and the stirring speed was 400 rpm. It was. Culture temperature and pH were automatically adjusted to 30 ℃ and 7.0 respectively. The results are shown in Table 5 below.

PHA Accumulation Rate According to Propionate Concentration

TABLE 5

Note) D.C.W (Dry Cell Weight) is a value measured after culturing cells in this medium for 30 hours and drying for 24 hours in a dry oven (105 ℃).

P / X: (PHA / Biomass)

3HB: 3-Hydroxybutyric acid, 3HV: 3-Hydroxyvaleriic acid

PHA Accumulation Rate by Different Bacillus Strains When Auxiliary Substrates Added

TABLE 6

Table 5 shows the PHA accumulation rate of the strain of the present invention according to the concentration change of propionate. The maximum cell mass and maximum accumulation rate were found when 0.1% propionate was added, and the measurements were 7.3 g / L and 43.9%, respectively. On the other hand, as the concentration of auxiliary substrates increased, cell mass as well as accumulation rate tended to decrease significantly. In addition, PHA synthesized in the absence of an auxiliary substrate was a PHB homopolymer, whereas when the auxiliary substrate was added, all of them were poly (3HB-co-3HV) having a high content of 3HV, and 3HV even at a propionate concentration of 0.3%. The content of was over 80 mol%. The PHA accumulation rate and the content of 3HV according to the strain of the present invention can be said to be very high when compared to the results reported by other researchers of Bacillus genus microorganisms (Table 6 below).

Example 2. Quantitative and Composition Analysis of PHA

The cultures collected in Example 1 were centrifuged twice (7,000 rpm / 8 minutes) to harvest the cells, and the speed vac. The cells were dried using a concentrator. The composition and quantitative analysis of PHA in cells was carried out by the method of Braunegg et al. (1978). That is, after crushing the dried cells finely, 20mg cells were added to a Tefron cap tube and 2ml of chloroform and 2ml of methanol containing 15% sulfuric acid were added. After reacting for a minute, 1 ml of distilled water was added to the cap tube after the reaction was mixed vigorously, and a sample was taken from the organic solvent layer formed below and subjected to gas chromatography analysis. ICI Biopol containing 13 mol of 3HV was used as the PHA standard sample. The results are shown in FIG.

Taken together, these strains of the present invention not only have a higher PHA accumulation rate than the Gram-positive bacteria known so far, but also have a very high poly (3HB-co- 3HV content by low concentration of propionate. 3HV) copolymer has the characteristic of synthesizing. In particular, in the case of other strains of the genus Alcaligenes, it is known that the content of 3HV can be increased only through the two-step batch culture in which a high concentration of auxiliary substrate is added. It is thought that the utility is very large in that the content can be improved.

Claims (12)

A bacterium of the genus Bacillus deposited as KCTC8552P at the Genetic Bank of Korea Institute of Science and Technology, which synthesizes polyhydroxyalkanate. A bacterium of genus Bacillus deposited as KCTC8552P in the Genetic Engineering Center Genetic Bank of Korea Institute of Science and Technology for synthesizing polyhydroxyalkanate is cultured in a culture medium containing glucose as a main substance and propionate as an auxiliary substrate. Cultivation method. The method according to claim 2, wherein the polyhydroxyalkanate is polyhydroxybutyrate (PHB). The method according to claim 2, wherein the polyhydroxyalkanate is a PHB / PHV copolymer containing 3HV units. The culture method according to claim 2, wherein the concentration of propionate is 0 to 1.0% with respect to the whole medium. The culture method according to claim 2, wherein the culture is batch culture in one step. It is characterized by synthesizing and purifying polyhydroxyalkanates by culturing Bacillus microorganisms deposited with KCTC8552P at the Genetic Engineering Center Gene Bank of Korea Advanced Institute of Science and Technology in a medium containing glucose as a main substance and propionate as an auxiliary substrate. Process for the preparation of polyhydroxyalkanate. The method of claim 7, wherein the polyhydroxyalkanate is polyhydroxybutyrate (PHB). 8. The method of claim 7, wherein the polyhydroxyalkanate is a PHB / PHV copolymer containing 3HV units. The method of claim 7, wherein the concentration of propionate is 0-1.0% with respect to the charter medium. 10. The method of claim 9, wherein the maximum content of the 3HV unit is 90 mol%. The method according to claim 7 or 10, wherein the polyhydroxyalkanate is up to 43.9% of the total amount of the microorganisms.