Method for producing biodegradable polyester

Abstract

To solve the problem in which: a remarkable culture outcome was observed under autotrophic conditions with a cell concentration of 91.3 g L-1 and a P(3HB) concentration of 61.9 g L-1, but the oxygen mass transfer coefficient kLa reached an extremely high value of 3000 h-1, rendering it unsuitable for scale-up.SOLUTION: A method for producing biodegradable polyester includes: a primary culture step of culturing, until completion of a logarithmic growth phase, hydrogen-oxidizing, polyhydroxyalkanoate-producing microorganisms in an inorganic salt-based medium supplied with a mixed gas containing H2, O2, and CO2; and a secondary culture step of, after the completion of the logarithmic growth phase of the microorganisms, continuing the culture while maintaining the inorganic salt concentration in the medium at a level to be lower than at the start of the culture, but not to be completely depleted. This method achieves a dry cell weight concentration of 98.3 g L-1 and a PHBH concentration of 81.5 g L-1 with an agitation capacity of about 450 h-1.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing polyhydroxyalkanoates, which are known as biodegradable plastics that accumulate in the bodies of microorganisms, in high yields. In particular, the present invention relates to a method for producing copolymer polyesters, which are biodegradable plastic materials that are composed of two or more monomer units including (R)-3-hydroxybutyric acid and (R)-3-hydroxyhexanoic acid and have excellent flexibility, processability, etc., in high yields per medium volume using genetically modified microorganisms and CO2 as a direct raw material. [Background technology]

[0002] Environmental pollution caused by waste plastic products synthesized from fossil fuels is becoming a serious problem. In particular, there are concerns that "microplastics" not only cause damage to marine life but also pose a risk to human health. As one solution, there is an urgent need to popularize biodegradable plastics that can be decomposed by microorganisms. In particular, polyhydroxyalkanoates (hereinafter abbreviated as "PHA"), which accumulate within the cells of certain bacteria, are expected to be a plastic material that can be decomposed even in the ocean, where there are few microorganisms.

[0003] Polyhydroxybutyric acid (hereinafter abbreviated as "P(3HB)"), a homopolyester of (R)-3-hydroxybutyric acid (hereinafter abbreviated as "3HB"), is a representative PHA. However, it has a major physical drawback in that it is difficult to process due to its "narrow process window," i.e., the small difference between its melting point and decomposition temperature, and the products it produces are hard and brittle.

[0004] For this reason, the development of copolymer PHA with improved physical properties by incorporating other hydroxyalkanoic acids is progressing. In particular, P(3HB-co-3HHx) (hereinafter abbreviated as "PHBH"), a copolymer of 3HB and (R)-3-hydroxyhexanoic acid (hereinafter abbreviated as "3HHx"), shows excellent flexibility and processability at a 3HHx fraction of 10 mol%.

[0005] However, the materials that can be used as raw materials for the production of copolymerized PHA such as PHBH are limited to expensive and highly cytotoxic organic acids, lactones, fatty acids, etc. In order to popularize PHA, it is essential to reduce the price, and considering that PHA is decomposed into CO2 in nature, it is important to use cheap and carbon-neutral resources.

[0006] Kaneka Corporation has developed a technology to efficiently produce PHBH from vegetable oils and fats using a genetically modified strain of the hydrogen bacterium Cupriavidus necator (Patent Document 1), and sells this degradable polymer under the name "Green Planet (registered trademark)." The company's current production volume is 5,000 tons per year, but it has announced plans to increase production.

[0007] The recombinant strain of C. necator created by Fukui et al., a co-inventor of the present invention, accumulates PHBH rich in 3HHx molar fraction from carbohydrates, which are less expensive than vegetable oils (Patent Document 2). In addition, Tanaka et al., the present inventor, discovered that this recombinant strain also synthesizes PHBH under autotrophic conditions using a mixed gas of H2, O2, and CO2 as a substrate (Non-Patent Document 1).

[0008] C. necator is a type of hydrogen bacteria that grows by fixing CO2 using the energy obtained from the oxidation of hydrogen. Hydrogen bacteria grow quickly as autotrophs that fix carbon dioxide, and unlike plants and algae, they do not require a large facility area for lighting and are not affected by weather, so they have been often used in research and development of fermentation technology that uses CO2 as a carbon source.

[0009] In the fermentation industry, in order to reduce costs, it is important to increase the amount of production in a reactor with a limited volume, and PHA production is no exception. However, it is not easy to produce a large amount of PHA using the above-mentioned mixed gases as the raw substrate, which are poorly soluble in liquid media and explosive, so it was necessary to create an innovative culture technology that had not been seen before.

[0010] Tanaka et al. demonstrated that it is possible to cultivate this bacterium at high cell density without wasting raw gas by releasing it, by using a "closed circulation gas fermentation system" in which the mixed gas in a gas storage tank is supplied to a reactor containing a culture medium, some of which is consumed by C. necator, and the remainder is returned to the water-containing holder and resupplied (Non-patent Documents 2 and 3; Patent Documents 3 and 4).

[0011] Using an explosion-proof cultivation bench plant created based on this system, a bacterial cell concentration of 91.3 g L -1 , P(3HB) concentration 61.9g·L -1 Under such autotrophic conditions, they have achieved astonishing culture results (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0012] [Patent Document 1] JP 2013-9628 A [Patent Document 2] International Publication No. 2016 / 021604 [Patent Document 3] Japanese Patent Application Publication No. 6-165686 [Patent Document 4] JP 2004-51920 A [Non-patent literature]

[0013] [Non-Patent Document 1] Bioengineering, 8(11), https: / / doi.org / 10.3390 / bioengineering8110179. (2021) [Non-Patent Document 2] J. Ferment. Bioeng., 69(3), 170-174 (1990) [Non-Patent Document 3] J. Ferment. Bioeng., 71(4), 254-257 (1991) [Non-Patent Document 4] Biotechnol. Bioeng, 45(3), 268-275 (1995) Summary of the Invention [Problem to be solved by the invention]

[0014] However, in this bench plant, in order to efficiently dissolve the substrate hydrogen and other poorly soluble inorganic gases in the medium and have them consumed by the bacteria, the oxygen transfer rate volumetric coefficient k L A value is 3000h -1 However, because the reactor used had a volume of 2 L and had extremely powerful stirring capabilities, it was difficult to scale up for commercial production. Moreover, the fact that the product was P(3HB) was a major barrier to practical application. [Means for solving the problem]

[0015] The present invention has been conceived in view of the above problems, and provides a method for maintaining and supplementing inorganic nutrients at a low concentration in the autotrophic culture of a PHBH biosynthetic strain of C. necator, which was developed by Fukui et al. L This enables high-concentration production of PHBH using CO2 as a direct feedstock without using a reactor with an extremely large a value. PHBH is a biodegradable copolyester as defined in the present invention.

[0016] Furthermore, an important point of the present invention is that the yield is dramatically increased by continuing the secondary culture after the end of the logarithmic growth phase, and the produced polyhydroxyalkanoic acid is not limited to PHBH, which is a copolymer, but may be a polyhydroxyalkanoic acid containing P(3HB), which is a homopolyester.

[0017] Specifically, the method for producing biodegradable polyester according to the present invention includes the steps of: a primary culture step in which a hydrogen-utilizing microorganism capable of producing polyhydroxyalkanoic acid is cultured until the end of the logarithmic growth phase in a medium mainly composed of inorganic salts to which a mixed gas containing H2, O2, and CO2 is supplied; The method is characterized in that it includes a secondary culture step in which, after the logarithmic growth period of the culture of the microorganism has been completed, the culture is continued while the concentration of the inorganic salt in the medium is not depleted and is maintained at a lower concentration than at the start of the culture. Effect of the Invention

[0018] There has been little progress in technological development worldwide for the biosynthesis of copolymerized PHA using CO2 as a raw material, and only a few research cases have been reported using mixotrophic cultures of C. necator and algae. In these cases, CO2 is used as a carbon source for bacterial growth and biosynthesis of 3HB monomers, but precursor organic compounds are added during the PHA accumulation phase for the biosynthesis of second monomers such as 3HHx.

[0019] In the present invention, it is possible to produce a PHA copolymer containing 3HHx units at a high concentration and content comparable to heterotrophic production using vegetable oils and fats as a raw material, using CO2 as the only carbon source. In addition, the present invention makes it possible to produce P(3HB) homopolyester or PHA copolymer containing hydroxyalkanoic acid other than 3HHx as the second or third monomer unit at a high concentration and content, depending on the PHA biosynthesis ability of the hydrogen bacteria used. The spread of biodegradable plastics and the utilization of CO2 as a resource have both become important international issues, and the present invention is expected to contribute to both of these issues. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing a configuration of a manufacturing apparatus for carrying out a manufacturing method according to the present invention. [Diagram 2] 1 is a graph showing the change in concentration of ammonium nitrogen and phosphate versus culture time in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The method for producing a biodegradable copolyester according to the present invention will be described below with reference to the drawings and examples. Note that the following description is merely an example of one embodiment of the present invention and an example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention.

[0022] In addition, the technical scope of the present invention also includes embodiments and examples obtained by appropriately combining the technical means disclosed in each of the different embodiments and examples. In addition, all documents described in this specification are incorporated herein by reference. In addition, when "A to B" is written in this specification, it means A or more (greater than A, including A) and B or less (smaller than B, including B).

[0023] First, the background of the method for producing the biodegradable copolyester according to the present invention will be described. Initially, an autotrophic culture test of the recombinant C. necator strain was carried out using a commercially available aerobic fermentation reactor and a laboratory-scale closed circulation gas fermentation system. The obtained bacterial cell concentration was 25 g L -1 The extent was limited.

[0024] A detailed investigation of the effects of the nutritional environment of the medium revealed that the bacterial cell concentration and PHBH concentration increased beyond the above-mentioned limits when small amounts of inorganic nitrogen compounds (N source) and phosphorus compounds (P source), which are the building blocks of bacterial protein and nucleic acids, and a solution of magnesium salts and trace metal elements (trace elements), which are cofactors, were added after the end of the logarithmic growth phase.

[0025] On the other hand, it was also found that even a small excess of these inorganic salts in the medium inhibits PHBH biosynthesis and prevents the bacterial cell concentration from increasing.

[0026] In the production of PHA through heterotrophic cultivation using organic compounds as a carbon source (C source), a widely used method is to shift the bacterial metabolism from cell growth to PHA accumulation by limiting the amount of nitrogen and phosphorus compounds added. In particular, in high-cell density cultivation, a method is often used to increase PHA production by supplementing these compounds while maintaining a constant supply ratio with respect to the C source, i.e., the C / N ratio and C / P ratio.

[0027] In contrast, in autotrophic cultivation of hydrogen bacteria, the C / N ratio and C / P ratio cannot be set to appropriate values ​​because both the C source and the energy source are inorganic gases that are poorly soluble in water.

[0028] In the method for producing a biodegradable copolyester according to the present invention, autotrophic culture is started using a medium containing a phosphorus compound at a much lower concentration than that in heterotrophic high cell density culture, and the culture is continued until the end of the logarithmic growth phase. This culture period is called the primary culture step.

[0029] After the logarithmic growth phase, a small amount of a solution of phosphorus compounds and trace metal elements is repeatedly added to the medium in the reactor each time the bacterial cell concentration increases by a certain amount, and ammonia is added as both a nitrogen source and a neutralizing agent to maintain the pH within a specified range, maintaining the concentrations of ammonia nitrogen and phosphorus compounds at extremely low values ​​compared to when the culture began, while also preventing a shortage of magnesium salts and trace metal elements. This culture period is called the secondary culture process. This secondary culture process makes it possible to produce PHA at a high concentration that was not previously possible using CO2 as a raw material.

[0030] <Microorganisms> The microorganism used in the present invention is a bacterium belonging to either the genus Cupriavidus or Alcaligenes, and is preferably genetically modified to produce a copolymer polyester. In this specification, the term "microorganism" is synonymous with a bacterium having hydrogen assimilation. The microorganism is also referred to as a bacterial cell.

[0031] <Starting medium composition> The composition of the medium at the start of cultivation consists of N source, P source, Mg source, S source, Na source, K source, and trace elements. These may be called nutrient elements. The inorganic salts (inorganic compounds) that supply these may also be called nutrients. The concentrations of these in the medium change depending on the amount of microorganisms at the time of inoculation.

[0032] As far as we can currently confirm, the amount of inoculated microorganisms is the OD of the culture medium. 600 When evaluated by the OD value 600 For example, if the nitrogen source is (NH4)2SO4, the nitrogen source is 0.5 g L -1 ~5.0g·L -1 For example, if the P source is KH2PO4, the amount is 0.5 to 1.0 g L -1 For example, if the magnesium source is MgSO4·7H2O, the amount is 0.1 g·L -1 ~1.0g L -1 For example, NaHCO3 is 0.5 to 1.0 g L -1 If the amount is set at 100 mg / kg, the amount will be sufficient to allow the microorganisms to grow until the logarithmic growth phase. In addition, the amounts of K and S sources contained in these inorganic salts are sufficient, so there is no need to add them separately.

[0033] Specific examples of nutritional element sources include inorganic nitrogen compounds such as ammonium salts, such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, as N sources. Examples of P sources include phosphorus compounds such as potassium monohydrogen phosphate, potassium dihydrogen phosphate, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, and phosphoric acid. Other inorganic salts that can be used include, for example, magnesium sulfate.

[0034] Trace elements can be composed of FeSO4·7H2O, CoCl2, CaCl2·2H2O, CuSO4, NiCl·6H2O, CrCl2·6H2O, citric acid, HCl, etc. Trace elements are not limited to these, and other compositions can be used. Trace elements can also be called nutritional elements.

[0035] <Addition of pH regulator and N source during primary culture> During the culture period, it is desirable to maintain the pH at 6.7 to 7.3. Since the medium tends to be acidic due to the activity of microorganisms and the dissolution of CO2, an aqueous solution of an alkaline substance is appropriately added as a pH adjuster. Potassium hydroxide, sodium hydroxide, ammonia, etc. can be suitably used as a pH adjuster during primary culture. These can also be used as a K source, a Na source, or a N source, and the substances used may be changed as the culture progresses. In addition, during primary culture, since the medium contains nutrient elements at the start of culture, it is not necessary to add nutrient elements other than the above pH adjuster.

[0036] However, potassium hydroxide and sodium hydroxide are toxic to the bacteria, so care must be taken not to add excessive amounts during cultivation. Ammonia is used as a nitrogen source to synthesize bacterial components such as proteins and nucleic acids, so it is best to supply it in the form of ammonia water or ammonia gas.

[0037] <Mixed substrate gas> In the present invention, a mixed gas of hydrogen, oxygen, and carbon dioxide is supplied to the culture medium as an energy source and a carbon source for the microorganism. The mixing ratio of these gases is preferably in the range of H2: 80% to 65%, O2: 20% to 10%, and CO2: 15% to 10%. The mixing ratio of the gases may be changed during the entire process of the culture (primary culture and secondary culture) as the culture progresses.

[0038] Gases such as NOx, CO (carbon monoxide), and N2 (nitrogen) can also be added to make a mixed gas. It is known that these gases are contained in the exhaust gas of an internal combustion engine, so a mixed gas in which the gas components are adjusted with the exhaust gas as the main component may be used. A mixed gas that is mainly made of exhaust gas has a low risk of explosion.

[0039] <pH regulator during secondary culture> In the secondary culture step, it is also desirable to add a pH adjuster appropriately so as to maintain the pH at a value of 6.7 to 7.3. As the pH adjuster, in addition to potassium hydroxide or sodium hydroxide, ammonia water or ammonia gas, which also serves as a N source, is preferably used, but since the accumulation of ammonia nitrogen in the medium has the effect of suppressing the biosynthesis of PHA, it is necessary to add it so as to maintain the ammonia nitrogen concentration at a predetermined concentration or lower, as described below in <Substances to be added during secondary culture>.

[0040] <Additives during secondary culture> As the culture progresses, the amount of nutrients in the medium decreases. The inorganic compounds added as nutrients are used to maintain physiological functions within the bacteria and as cofactors for enzymes, in addition to being components of the bacterial body. Although it depends on the concentration added at the start of the culture, most of these will be depleted by the end of the primary culture process (logarithmic growth phase). Therefore, nutrients are added as the bacterial body grows. The nutrients to be added include a nitrogen source, a phosphorus source, a magnesium source, and trace elements. Therefore, potassium dihydrogen phosphate and magnesium sulfate are suitable for use.

[0041] It is best to add more depending on the amount of bacterial cell growth. However, this does not mean that adding more every time the culture is started is excluded. The amount of bacterial cell growth that is the standard for adding more depends on how the bacterial cell concentration is measured. For example, the OD 600 If you use the OD value as a guide, 600 For every 60-80 increase in pH, add 0.2 g L of potassium dihydrogen phosphate as a P source. -1 , O.D. 600 For every 100 increase in -1 and 60 μL L of a trace element solution with the composition shown in the Examples below. -1 By repeatedly adding the amount of fertilizer, optimal growth results were obtained.

[0042] Among the nutrient elements, N sources are added as pH adjusters to maintain the pH at 6.7 to 7.3. As described below, N and P sources also have an inhibitory effect on PHA biosynthesis, so they must be added so that their concentrations in the medium are kept low.

[0043] It is important to control the N (ammonia nitrogen) and P (phosphate) concentrations during secondary culture. They should be maintained at low levels without being depleted or oversupplied. As mentioned above, N and P sources have an inhibitory effect on PHA biosynthesis. The upper limit of these concentrations should be lower than those in the initial medium, preferably 300 mg L−1. -1 Less than or equal to 150 mg L -1 Less than or equal to 30 mg L, most preferably -1 The lower limit to avoid depletion is 5 mg L. -1 ~10 mg L -1 More preferably, it is equal to or greater than this.

[0044] Therefore, the N concentration (ammonia nitrogen concentration) to be maintained during secondary culture is 10 mg L -1 ~ Initial culture concentration, preferably 10 mg L -1 ~300 mg L -1 , more preferably 10 mg L -1 ~100 mg L -1 , most preferably 10 mg L -1 ~30 mg L -1 In addition, the P concentration (phosphate concentration) is 5 mg L -1 ~ Initial culture concentration, preferably 5 mg L -1 ~60 mg L -1 , more preferably 5 mg L -1 ~40 mg L -1 , most preferably 5 mg L -1 ~20 mg L -1 It can be said.

[0045] If the concentration is outside this range, the continued growth of the cells and the accumulation of PHA after the logarithmic growth phase are suppressed. Therefore, it is preferable to prepare the culture supernatant when measuring the cell concentration and to quantitatively analyze the N and P concentrations in the obtained sample to adjust the timing and amount of addition.

[0046] <The reactor's stirring capacity required for optimal dissolution of the substrate gas into the medium> In the method for producing a biodegradable copolyester according to the present invention, the stirring capacity of the reactor, which is a factor that greatly affects the efficiency of dissolving the substrate gas in the medium and absorbing it by the bacterial cells, can be within a range that is feasible even when scaled up to a commercial production scale. L a value of 300h -1 ~700h -1 Reactors with a range of k are available, which is within the mixing power achievable with commercially available baffled stirred tanks. L The value a varies depending not only on the inherent characteristics of the reactor but also on the substrate gas aeration rate and stirring speed during cultivation and other conditions, and there is no problem with having a stirring force greater than this value.

[0047] <Culture temperature> The culture temperature is preferably the optimum temperature for the growth of the microorganism used. The hydrogen-utilizing bacteria that can be used in the present invention grow optimally within the range of about 28° C. to 36° C. Therefore, it is necessary to maintain the culture medium in the reactor at that temperature.

[0048] <Decision on starting the secondary culture process> In the present invention, it is effective to confirm whether the growth of the microorganisms in the reactor is in the middle of the logarithmic growth phase or has ended in order to determine whether to start the secondary culture step, which involves the addition of nutrients and other operations. For example, this can be done by checking the culture OD, which is an index of the bacterial cell concentration. 600 The specific growth rate is calculated from the change over time of the value, and the time when this value starts to clearly decrease can be determined as the end of the logarithmic growth phase.

[0049] <End of culture period> As the secondary culture progresses, the amount of PHA accumulated in the microorganisms increases. The end of the secondary culture period can be determined by a significant decrease in the bacterial cell concentration or the specific growth rate described above. In the case of bacterial cell concentration, the OD of the culture medium is 600 The target value is 350 or more. After the cultivation is completed, the cells are removed from the reactor and PHA is extracted from the cells. This can be done by a known method.

[0050] <Culture equipment> An overview of the culture apparatus 1 used in the examples described below is shown in Figure 1. The culture apparatus 1 is composed of a reactor 10, a mixed gas supply section A1, an agitator 14, a pH adjuster supply section 16, and a nutrient salt supply section 18. The reactor 10 is a sealable container. Although not shown, it is preferable that the reactor 10 is provided with a discharge means for discharging the medium and microorganisms (hereinafter referred to as the "cultured product") in the reactor 10 after culture.

[0051] The gas supply section A1 includes a gas storage tank 12, a gas mixing ratio regulator 12a that adjusts the flow rate of each gas from multiple gas supply sources (not shown) to the gas storage tank 12, an air supply pipe 12s that supplies the mixed gas from the gas storage tank 12 to the reactor 10, an air supply pump 12p arranged on the air supply pipe 12s, and a return air pipe 12r that returns the mixed gas from within the reactor 10 to the gas storage tank 12.

[0052] The gas mixture ratio regulator 12a supplies the substrate gas (H2, O2, CO2) to the gas storage tank 12 at a constant mixture ratio.

[0053] The agitator 14 agitates the culture in the reactor 10 to which the culture has been added. The pH adjuster feed section 16 feeds a pH adjuster into the reactor 10. The nutrient salt feed section 18 feeds nutrients into the reactor 10 mainly during secondary culture. Note that multiple types of pH adjusters may be used depending on the time of culture, but the type of adjuster should be selected and the timing and amount of addition should be adjusted so that the ammonia nitrogen concentration in the reactor medium falls within the range described above. Also, multiple types of nutrients may be added at different times, but the type of adjuster should be selected and the timing and amount of addition should be adjusted so that the phosphate concentration in the reactor medium falls within the range described above.

[0054] The state of the culture may be detected online using various sensors, or offline by sampling the nutrients in the reactor 10. When detecting the state of the culture offline, the pH regulator supply unit 16 and the nutrient salt supply unit 18 are operated manually. In addition, the pH, temperature, nutrient salt concentration, and bacterial cell count (bacterial cell concentration) of the culture may be measured either online or offline at regular intervals.

[0055] In the following, an example is shown for producing a copolymer polyester of (R)-3-hydroxybutyric acid and (R)-3-hydroxyhexanoic acid, but the monomer composition of the polyester product can be changed depending on the type of microorganism or recombinant gene used, etc. EXAMPLES

[0056] A 1 L glass jar fermenter was used as the reactor, containing 4.0 g L of (NH4)2SO4. -1 , KH2PO4 0.5g L -1 , MgSO4 7H2O 0.2g L -1 , NaHCO3 0.5g L -1 , trace elements solution 100μl·L -1 The recombinant strain MF01 / pBPP-ccr was then grown in a 600 ml inorganic salt synthetic medium. Me JAc -emd (for the MF01 strain, see Patent Document 2) 600 The bacteria were inoculated to a bacterial concentration of 0.3 on the basis of the value, and cultured at 30°C by supplying mixed gas from a water-containing holder (50 L) as a gas storage tank at a flow rate of 0.7 vvm and circulating it within the system.

[0057] The trace elements solution was prepared by dissolving 16.2 g of FeSO4·7H2O, 119.2 mg of CoCl2, 10.3 g of CaCl2·2H2O, 100 mg of CuSO4, 118.0 mg of NiCl·6H2O, 33.0 mg of CrCl2·6H2O, and 15.6 g of citric acid in 100 ml of 1 M HCl.

[0058] From the start of culture until the middle of the logarithmic growth phase, a mixed gas with a composition of H2:O2:CO2 = 80 / 10 / 10% (v / v) was supplied, and thereafter, gas with an O2 concentration up to a maximum of 18% (v / v) and a CO2 concentration up to a maximum of 12% (v / v) was supplied.

[0059] The k of the fermenter used in this test L a value of 450 h at an aeration volume of 0.7 vvm and an agitation speed of 630 rpm -1 The mixing performance is not high. In order to prevent the O2 concentration in the culture system from decreasing drastically due to consumption by the bacteria, the composition ratio of the supply gas was changed as described above during the culture.

[0060] The medium pH was maintained at 7.0 by intermittently adding 3MKOH using a pH controller until the mid-logarithmic phase, and then by intermittently adding 14 wt% ammonia water, which also served as a N source.

[0061] Culture solution OD 600 For every 60 increase in the value, 0.2 g of KH2PO4, 30 mg of MgSO4·7H2O, and 40 μl of trace elements solution were added per liter of medium.

[0062] Figure 2 shows the relationship between the elapsed culture time and the concentration of nutrients. Referring to Figure 2, the horizontal axis is the culture time (h) and the vertical axis is the concentration (mg / L). The open squares indicate the ammonium nitrogen concentration and the closed squares indicate the phosphate concentration. As the bacteria grew from the start of culture, the ammonium nitrogen and phosphate concentrations decreased.

[0063] When the culture time reached 50 hours, it was determined that the logarithmic growth phase had ended, and the secondary culture additives were added. As a result, the ammonium nitrogen and phosphate concentrations increased slightly. After that, when the ammonium nitrogen concentration reached 30 mg L -1 ~10 mg L -1 , phosphate concentration is 20 mg L -1 ~5 mg L -1 The secondary culture additives were added so that the secondary culture was in the range of 0.1 to 0.5%. The culture was continued.

[0064] With this method, the ammonia nitrogen in the fermentation tank was reduced to 30 mg L -1 Below, phosphate is 15 mg L -1 The OD was almost completely removed even though the concentration was suppressed to the following range (see Figure 2). 600 The value was 370.3 (dry cell weight concentration 98.3 g L -1 ), and the PHBH concentration was 81.5 g L -1 The production volume of PHA using CO2 directly as a raw material reached 100,000 m3, exceeding that of P(3HB).

[0065] At this time, the intracellular PHBH content was 82.9 wt %, and the 3HHx molar fraction was 13.8 mol %. [Industrial Applicability]

[0066] The present invention can be suitably used for producing copolymerized polyesters of (R)-3-hydroxybutyric acid and (R)-3-hydroxyhexanoic acid, which are highly processable and degradable even in the ocean, where there are few microorganisms, under processing conditions that can be realized even when scaled up. [Explanation of symbols]

[0067] 1 Culture device 10. Reactor 12 Gas storage tank 12a Gas mixture ratio regulator 12p Air Pump 12s Air supply piping 12r Return air piping 14 Stirrer 16 pH regulator input section 18 Nutrient salt input section 34 Liquid delivery pump

Claims

1. H ２ , O ２ , CO ２ a primary culture step of culturing a hydrogen-utilizing microorganism capable of producing polyhydroxyalkanoic acid in a medium mainly composed of inorganic salts to which a mixed gas containing A method for producing biodegradable polyester, comprising a secondary culture step of continuing the culture after the logarithmic growth period of the culture of the microorganism has ended without depleting the concentration of the inorganic salt in the medium and while maintaining the concentration lower than at the start of the culture.

2. The method for producing biodegradable polyester according to claim 1, wherein the microorganism is a microorganism capable of utilizing hydrogen and producing a copolymer polyester composed of two or more monomer units including (R)-3-hydroxybutyric acid and (R)-3-hydroxyhexanoic acid.

3. The concentration of the inorganic salt is at least 10 mg / L of ammonia nitrogen. －１ ~ Initial culture concentration, phosphorus concentration is 5 mg L －１ The method for producing a biodegradable polyester according to claim 1 or 2, wherein the initial culture concentration is from 1 to 20%.

4. 3. The method for producing a biodegradable polyester according to claim 1, wherein the pH of the medium is maintained in the range of 6.7 to 7.3 throughout the primary culture step and the secondary culture step.

5. 3. The method for producing biodegradable polyester according to claim 1, wherein the mixed gas contains exhaust gas from an internal combustion engine.

6. The mixed gas contains N ２ The method for producing biodegradable polyester according to claim 1 or 2, wherein the method further comprises the step of:

7. 3. The method for producing biodegradable polyester according to claim 1 or 2, wherein the microorganism is a bacterium belonging to either the genus Cupriavidus or the genus Alcaligenes, and is genetically modified to produce a copolymer polyester.

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