Method for producing 3-hydroxybutyric acid

Abstract

The present invention aims to provide a method for efficiently purifying 3-hydroxybutyric acid from a fermentation broth containing 3-hydroxybutyric acid, in which lactic acid, one of the by-products, does not remain in the fermentation broth containing 3-hydroxybutyric acid. [Solution] The present invention provides a method for producing 3-hydroxybutyric acid, characterized by culturing 3-hydroxybutyric acid-producing bacteria in a culture medium containing lactic acid to obtain a 3-hydroxybutyric acid fermentation liquid in which no lactic acid remains.

Description

Technical Field

[0001] The present invention relates to a method for obtaining a 3-hydroxybutyric acid-containing fermentation broth free from lactic acid that is produced simultaneously with 3-hydroxybutyric acid during the fermentative production of 3-hydroxybutyric acid by a microorganism. Background Art

[0002] 3-hydroxybutyric acid is a material that serves as a raw material for biodegradable plastics, pharmaceuticals, functional foods and the like, and establishment of an efficient production method therefor is expected. 3-hydroxybutyric acid can be produced by fermentation with microorganisms, and can be produced from renewable resources such as plant raw materials. As fermentative production methods for 3-hydroxybutyric acid using microorganisms, for example, a method of directly producing 3-hydroxybutyric acid from saccharides using a Halomonas microorganism (Patent Document 1), and a method of producing a polymer of 3-hydroxybutyric acid (PHB) from saccharides, then hydrolyzing the polymer to obtain 3-hydroxybutyric acid (Patent Document 2) are known.

[0003] However, it is known that microorganisms that produce 3-hydroxybutyric acid (hereinafter also referred to as "3-hydroxybutyric acid-producing bacteria" or simply "producing bacteria") catabolize 3-hydroxybutyric acid from substrates such as sugars, but also produce other organic acids (lactic acid, formic acid, acetic acid) at the same time. In the fermentative production of 3-hydroxybutyric acid to obtain 3-hydroxybutyric acid, other organic acids produced simultaneously are unnecessary by-products, and thus it is necessary to remove these by-products in the purification step of 3-hydroxybutyric acid. Among these by-products, formic acid and acetic acid are volatile organic acids, and can be easily removed by heating the fermentation broth.

[0004] On the other hand, since lactic acid is non-volatile, it is difficult to remove even by heating the fermentation broth. Furthermore, since the molecular weight and chemical structure of lactic acid are similar to those of 3-hydroxybutyric acid, it is not easy to separate lactic acid from 3-hydroxybutyric acid even when various separation means are used. In other words, if a fermentation production method is chosen that results in lactic acid being mixed into the 3-hydroxybutyric acid-containing fermentation liquid, it becomes impossible to separate 3-hydroxybutyric acid from lactic acid or remove lactic acid from the 3-hydroxybutyric acid-containing fermentation liquid, thus making it difficult to efficiently purify 3-hydroxybutyric acid. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent Publication (Patent No. 6521243) [Patent Document 2] Patent Publication (Patent No. 6281887) [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors investigated methods for controlling the metabolism of lactic acid-producing bacteria by adding various components to the culture medium, and discovered that by adding lactic acid to the culture medium in the early stages of culturing the lactic acid-producing bacteria, the lactic acid in the culture medium disappeared in the later stages of culturing, thus completing the present invention. Therefore, the object of the present invention is to provide a method for producing 3-hydroxybutyric acid from a fermentation broth containing 3-hydroxybutyric acid in such a way that lactic acid, one of the by-products, does not remain in the fermentation broth containing 3-hydroxybutyric acid. [Means for solving the problem]

[0007] The present invention is characterized by a method for efficiently purifying 3-hydroxybutyric acid from a 3-hydroxybutyric acid-containing fermentation broth, which is obtained by activating the metabolic pathway that utilizes lactic acid during the early stages of microbial cell proliferation in the culture phase, thereby obtaining a 3-hydroxybutyric acid-containing fermentation broth in which no lactic acid remains in the later stages of culture. [Effects of the Invention]

[0008] According to the present invention, in the fermentation production of 3-hydroxybutyric acid, 3-hydroxybutyric acid can be produced without leaving lactic acid, which is difficult to separate or remove from the 3-hydroxybutyric acid-containing fermentation liquid, as a byproduct. By ensuring that lactic acid does not remain in the 3-hydroxybutyric acid-containing fermentation liquid, the purification of 3-hydroxybutyric acid becomes easier, and 3-hydroxybutyric acid that can be efficiently used as a raw material for biodegradable plastics and other products can be obtained. [Brief explanation of the drawing]

[0009] [Figure 1] Graph showing the results of the culture test using medium A1 without added lactic acid in Example 3. [Figure 2] This graph shows the results of a culture test using medium A1 in which the lactic acid concentration of Example 3 was adjusted to approximately 0.5 w / v%. [Figure 3] Graph showing the results of the culture test using medium A2 prepared with lactic acid as the sole carbon source in Example 3. [Figure 4] This graph shows the results of a culture test using a culture medium in which the lactic acid concentration of Example 4 was adjusted to approximately 0.5 w / v%. [Figure 5] Graph showing the results of a culture test using a culture medium in which the lactic acid concentration of Example 4 was adjusted to approximately 1 w / v%. [Figure 6] Graph showing the results of a culture test using a culture medium in which the lactic acid concentration of Example 4 was adjusted to approximately 2 w / v%. [Figure 7] Graph showing the results of a culture test using a culture medium in which the lactic acid concentration of Example 4 was adjusted to approximately 4 w / v%. [Figure 8] Graph showing the results of a culture test using a culture medium in which the lactic acid concentration of Example 4 was adjusted to approximately 5 w / v%. [Modes for carrying out the invention]

[0010] In the fermentation production of 3-hydroxybutyric acid according to the present invention, microorganisms belonging to the genus Halomonas (hereinafter also referred to as "Halomonas microorganisms") are used as 3-hydroxybutyric acid-producing bacteria, selected from the group consisting of Halomonas couriensis JCM12237 strain, Halomonas salifodinae JCM14803 strain, Halomonas halophila JCM20791 strain, Halomonas pacifica JCM20633 strain, Halomonas salina JCM21221 strain, Halomonas venusta JCM20634 strain, and Halomonas sp. OITC1261 strain.

[0011] Among microorganisms of the genus Halomonas, Halomonas koriensis, Halomonas salifodinae, Halomonas halophylla, Halomonas pacifica, Halomonas salina, and Halomonas venusta can be used without any particular restrictions, as long as they belong to these species. For example, standard strains such as Halomonas couriensis JCM12237, Halomonas salifodinae JCM14803, Halomonas halophila JCM20791, Halomonas pacifica JCM20633, Halomonas salina JCM21221, and Halomonas venusta JCM20634 are suitable for use because they exhibit excellent 3-hydroxybutyric acid production. Furthermore, Halomonas sp. OITC1261 strain is particularly suitable for use in the present invention because it has a high capacity to produce 3-hydroxybutyric acid. Halomonas sp. strain OITC1261 is deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture) under accession number NITE P-02027.

[0012] In order to culture Halomonas microorganisms and produce a 3-hydroxybutyric acid-containing fermentation liquid that does not contain lactic acid, the nutrient medium must contain a small amount of lactic acid. Lactic acid can be used in the D-isomer, L-isomer, or a mixture thereof. Lactic acid is added to the nutrient medium in the form of free acid or salt. When a free acid is used, after adding the free acid to the nutrient medium, the pH of the nutrient medium is adjusted to a pH suitable for the growth of 3-hydroxybutyric acid-producing bacteria.

[0013] If an excessively large amount of lactic acid is added to the nutrient medium, lactic acid that is not assimilated by the 3-hydroxybutyric acid-producing bacteria will remain in the nutrient medium, making it impossible to obtain a 3-hydroxybutyric acid-containing fermentation broth that is free of residual lactic acid. Therefore, the maximum amount (maximum concentration) of lactic acid added to the nutrient medium must be an amount (concentration) such that lactic acid is consumed (assimilated) or eliminated by the end of culturing. For example, assuming that the lactic acid consumption rate of a *Halomonas* microorganism is 0.388 g / L / h and the culture time is 100 hours, the maximum concentration of lactic acid in the nutrient medium is 3.9 w / v%. Accordingly, the concentration of lactic acid in the nutrient medium after addition must be at most 4.0 w / v%, and more preferably 0.5 to 2.0 w / v%. It has been confirmed that even if the amount of lactic acid added to the nutrient medium is very small, the metabolic pathway of 3-hydroxybutyric acid-producing bacteria that assimilate lactic acid is activated, and the production of lactic acid is suppressed.

[0014] Nutrient medium components other than lactic acid are not particularly limited, but those containing an organic carbon source are preferable, and those further containing inorganic salts and a nitrogen source are more preferable.

[0015] Examples of the organic carbon source include glucose, sucrose, maltose, fructose, gluconic acid, glyceric acid, ethanol, citric acid, acetic acid, butyric acid, malic acid, succinic acid, α-ketoglutaric acid, formic acid, pyruvic acid, molasses, sugarcane juice, sugar beet juice, starch syrup, shochu distillation stillage, and sake lees, and one or more of these may be used.

[0016] The inorganic salts are not particularly limited, and examples thereof include metal salts of metals such as sodium, magnesium, potassium, calcium, iron, manganese, cobalt, zinc, and copper, and sulfates, and one or more of these may be used. Examples of nitrogen sources include nitrates, nitrites, ammonium salts, and organic nitrogen, and one or more of these can be added.

[0017] It is preferable to further add a phosphorus source to the nutrient medium. Examples of phosphorus sources include phosphates, polymerized phosphoric acid, and organic phosphorus, and one or more of these can be added.

[0018] The pH of the nutrient medium is usually 5-10, and preferably 8.5-9.5. Halomonas microorganisms are cultured aerobically in a nutrient medium under the above conditions. Specifically, the culture medium is incubated while being stirred in an environment where the surface of the culture medium is in contact with an oxygen-containing gas.

[0019] To culture Halomonas microorganisms, inoculate the Halomonas microorganisms into a nutrient medium, pre-culture the cells under aerobic conditions at 30-37°C, dilute them 20-50 times, and then perform the main culture under aerobic conditions. The aerobic conditions in this culture are a culture temperature of approximately 30-37°C, preferably 33-37°C, and a culture time of 20-100 hours, preferably 40-70 hours.

[0020] The culture method is not particularly limited, and any culture method such as batch culture, fed-batch culture, or continuous culture can be used. However, fed-batch culture is preferred because it avoids growth inhibition due to high concentrations of substrate and can improve yield.

[0021] Halomonas microorganisms are known to consume (assimilate) organic acids in culture media that contain only organic acids as an organic carbon source. Furthermore, Halomonas microorganisms are known to produce organic acids by utilizing sugars when sugars are the only organic carbon source in the culture medium. Furthermore, Halomonas microorganisms are known to assimilate organic acids when the only organic carbon source in the culture medium is organic acid. Therefore, when Halomonas microorganisms are grown in a culture medium using sugars as an organic carbon source, they begin to utilize the organic acids they have produced up to that point in the later stages of culture, after the sugars have disappeared.

[0022] By utilizing these properties, if Halomonas microorganisms are grown in a culture medium that uses only sugars as an organic carbon source, in the later stages of cultivation, after the sugars have been assimilated and disappeared, the microorganisms will begin to assimilate the organic acids they have produced up to that point, thereby suppressing the lactic acid remaining in the culture medium after cultivation. However, in this case, Halomonas microorganisms utilize 3-hydroxybutyric acid, an organic acid, at the same time as lactic acid, resulting in a decrease in the yield of 3-hydroxybutyric acid.

[0023] Therefore, in this invention, by adding a certain concentration of lactic acid to the culture medium in which Halomonas microorganisms are grown, the 3-hydroxybutyric acid produced up to that point is not utilized, and only the lactic acid is utilized, thereby obtaining a 3-hydroxybutyric acid-containing fermentation liquid in which no lactic acid remains. Furthermore, there is currently no known method for producing (metabolizing) Halomonas microorganisms in a culture medium containing both sugars and organic acids.

[0024] Methods for producing 3-hydroxybutyric acid using Halomonas microorganisms include a method of directly producing 3-hydroxybutyric acid by aerobic culture, and a method of producing it via a polymer (PHB) of 3-hydroxybutyric acid produced by aerobic culture. These methods can be used individually or in combination. In the method of production via PHB, 3-hydroxybutyric acid is obtained by hydrolyzing the PHB produced and accumulated within the cells of Halomonas microorganisms and releasing the converted PHB into a solution outside the cells.

[0025] The following describes a method for producing 3-hydroxybutyric acid based on examples, but the present invention is not limited to these examples.

[0026] (Example 1) Productivity of 3-hydroxybutyrate by Halomonas microorganisms and the effect of lactate-supplemented medium Culture tests were conducted using multiple reference strains classified under the genus Halomonas. The reference strains are shown in Table 1, and were obtained from the RIKEN BioResource Center (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture).

[0027] In the culture test, we used MB9 medium, which was prepared by adding 5 w / v% sucrose as a substrate to commercially available Marine Broth medium (Marine Broth 2216, manufactured by Becton Dickinson & Co.) and adjusting the pH to 9 with 10N sodium hydroxide. For the lactic acid supplemented medium, 5 w / v% sucrose and DL-sodium lactate (final lactic acid concentration of 0.1 w / v% or 0.2 w / v%) were added to marine broth medium, and the pH was adjusted to 9 with 10N sodium hydroxide to create MBL medium. 50 mL each of filter-sterilized MB9 medium and MBL medium were dispensed into Erlenmeyer flasks, and 2.5 mL of pre-cultured Halomonas microorganisms were inoculated onto each flask. The flasks were then cultured aerobically for 48 hours (35°C, 250 rpm). The concentrations of lactic acid and 3-hydroxybutyric acid in the culture medium were quantified by high-performance liquid chromatography. The results are shown in Table 1.

[0028] [Table 1]

[0029] As shown in Table 1, the four strains of Halomonas salifodinae (JCM14803), Halomonas pacifica (JCM20633), Halomonas halophila (JCM20791), and Halomonas corliensis (JCM12237) produced 3-hydroxybutyrate and lactic acid simultaneously in aerobic culture (48 hours) using MB9 medium without lactic acid supplementation. However, in cultures using MBL medium supplemented with lactic acid, when compared over the same culture time, no lactic acid remained in the medium, and the production of 3-hydroxybutyrate was equivalent to or greater than that of MB9 medium.

[0030] In culture media without added lactic acid, the amount of lactic acid produced by the metabolism of Halomonas microorganisms was approximately 3 to 8% of the total amount of 3-hydroxybutyric acid produced. On the other hand, lactic acid was not detected in the culture medium to which lactic acid was added. This is thought to be because the addition of lactic acid to the culture medium activated the metabolic pathway by which Halomonas microorganisms utilize lactic acid, resulting in a state where lactic acid production into the extracellular environment (culture medium) was prevented.

[0031] (Example 2) Productivity of 3-hydroxybutyrate via PHB and the effect of lactate-supplemented medium It is known that PHB, which accumulates in the cells of Halomonas microorganisms during aerobic culture, is converted to 3-hydroxybutyrate and excreted outside the cell under anaerobic conditions. Therefore, using this property, the following culture tests were conducted using several reference strains classified under the genus Halomonas. The reference strains used were the same distributed strains as in Example 1.

[0032] In the culture test, we used SG9 medium, which was prepared by adding 2 w / v% sucrose and 2 w / v% glucose as substrates to commercially available Marine Broth medium (Marine Broth 2216, manufactured by Becton Dickinson & Co.) and adjusting the pH to 9 with 10N sodium hydroxide.

[0033] For the lactate-supplemented medium, DL-sodium lactate (final lactate concentration 0.2 w / v%) was added to SG9 medium to create SGL medium. 20 mL each of filter-sterilized SG9 medium and SGL medium were dispensed into Erlenmeyer flasks, and 1 mL of pre-cultured Halomonas microorganisms was inoculated onto each flask. The flasks were then cultured under aerobic conditions at 30°C (200 rpm). After 24 hours of cultivation, the culture medium was separated and its components were analyzed in the same manner as in Example 1.

[0034] Furthermore, a mixture of 4 mL of the culture medium and 1 mL of buffer (0.4 M carbonic acid-bicarbonate, pH 9.4) was dispensed into a sealed plastic tube, sealed, and left to stand at room temperature. After 5 hours, a portion of the culture medium was taken and its components were analyzed. The amount of PHB was determined by sulfuric acid decomposition of the dried bacterial cells and then calculating the amount of crotonic acid produced by the PHB decomposition. Specifically, after drying the collected bacterial cells, concentrated sulfuric acid was added and the mixture was reacted at 100°C for 1 hour. The resulting crotonic acid was then analyzed using high-performance liquid chromatography and converted to a certain value. These results are shown in Table 2.

[0035] [Table 2]

[0036] Halomonas microorganisms produce and accumulate PHB within their cells under aerobic culture. This PHB is a polymer of 3-hydroxybutyric acid, and under anaerobic conditions, it is converted to 3-hydroxybutyric acid by hydrolytic enzymes within the bacterial cells. As shown in Table 2, Halomonas coriansis (JCM12237), Halomonas salina (JCM21221), and Halomonas venusta (JCM20634) do not yet produce 3-hydroxybutyrate after 24 hours of aerobic culture, but PHB has already accumulated within the bacterial cells. By allowing the PHB inside the bacterial cells to stand in the culture medium and creating anaerobic conditions, the PHB is broken down to produce 3-hydroxybutyric acid.

[0037] As shown in Table 2, the production of PHB was 1.3 to 1.4 times higher when cultured in SGL medium than in SG9 medium, and similarly, the production of 3-hydroxybutyric acid obtained by hydrolyzing PHB was 1.2 to 1.5 times higher in SGL medium. Furthermore, lactic acid was detected in the culture medium using SG9 medium, but not in the culture medium using SGL medium.

[0038] Based on these results, even in the method of producing 3-hydroxybutyric acid by decomposing PHB, it was possible to obtain a 3-hydroxybutyric acid fermentation liquid without residual lactic acid by culturing in a lactic acid-supplemented medium. Furthermore, in the lactic acid-supplemented medium, PHB production increased compared to the medium without lactic acid, and the production of 3-hydroxybutyric acid obtained by decomposing this PHB also increased.

[0039] (Example 3) Comparison of metabolic changes in lactate-added medium and lactate-free medium We investigated the changes in the metabolic rates of lactic acid, 3-hydroxybutyrate, and PHB when Halomonas sp. OITC1261 (NITE P-02027) was aerobically cultured using a benchtop culture system.

[0040] For the pre-culture of Halomonas sp. strain OITC1261, 100 mL of nutrient medium prepared by adding 2 w / v% sucrose to production medium A1 (2% sodium nitrate, 0.6% potassium sulfate, 0.15% potassium hydrogen phosphate, 0.7% sodium chloride, and other trace inorganic components), adjusting the pH to 9 with sodium hydroxide solution, was dispensed into an Erlenmeyer flask, the strain was inoculated, and the culture was carried out overnight at 35°C and 200 rpm. In this culture, a benchtop culture device (manufactured by Marubishi Bioengin Co., Ltd., MDL 5L) was used to inoculate 2 L of the main culture medium with the entire amount of the pre-culture medium before starting the main culture. The culture media were compared using three different media: Production Medium A1 with sucrose added (no lactic acid added), Production Medium A1 with sucrose and 0.5 w / v% lactic acid added (lactic acid added), and Production Medium A2 prepared with lactic acid as the sole carbon source (5% lactic acid, 1% sodium nitrate, 0.1% potassium sulfate, 0.2% potassium hydrogen phosphate, 0.5% sodium chloride, and other trace inorganic components).

[0041] During the main culture period using production medium A1, sucrose was added in three separate additions (totaling 30 w / v%). Furthermore, during the main culture period using production medium A2, lactic acid was added in two separate doses (totaling 20 w / v%). For lactic acid, an aqueous solution of DL-sodium lactate was used. While maintaining constant culture conditions (temperature 35°C, stirring speed 700 rpm, pH 8.5, aeration rate 5 L / min), a portion of the culture medium was taken during cultivation and its components were analyzed in the same manner as in Example 1. These results are shown in Figures 1 and 2.

[0042] As shown in Figure 1, when using medium A1 (lactic acid-free medium) without added lactic acid, lactic acid, which was not detected at the start of the culture, reached 16.3 g / L 17 hours after the start of the culture and was maintained at a high concentration until 65 hours after the start of the culture (16.1 g / L). 65 hours after the start of this culture, the production of 3-hydroxybutyric acid and PHB was 74 g / L and 27 g / L, respectively.

[0043] On the other hand, when using medium A1 (lactic acid-supplemented medium) with added lactic acid, as shown in Figure 2, the lactic acid, which was 4.8 g / L at the start of the culture, was almost completely assimilated 24 hours after the start of the culture, and thereafter it was not detected at all until 65 hours after the start of the culture. Based on these results, the rate of lactic acid consumption up to 24 hours after the start of the culture is 0.2 g / L / h. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB 65 hours after the start of this culture were 74 g / L and 37 g / L, respectively.

[0044] When using culture medium A2, which uses lactic acid as the sole carbon source, lactic acid was assimilated to produce 3-hydroxybutyric acid and PHB, as shown in Figure 3. The lactic acid consumption rate from the start of this culture up to 24 hours later was 3.1 g / L / h, which was higher than the rate observed in medium A1 containing sucrose.

[0045] These results suggest that while Halomonas microorganisms normally produce lactic acid along with 3-hydroxybutyrate through metabolism, adding lactic acid to the culture medium during the growth phase (early stage of culture) activated the metabolic pathway for lactic acid utilization in Halomonas microorganisms, resulting in a state where lactic acid production to the extracellular environment (culture medium) was suppressed. Furthermore, it was found that this state of no residual lactic acid continued until the later stages of culture, ultimately resulting in a 3-hydroxybutyrate culture medium free of residual lactic acid.

[0046] Compared to a lactate-free medium, the lactate-added medium did not affect the productivity of 3-hydroxybutyric acid, only that lactic acid was not present, and the productivity of PHB improved by 1.4 times. PHB accumulates within the cells of microorganisms, but after cultivation is complete, it is converted to 3-hydroxybutyrate (hydrolyzed) by standing in the culture medium, and 3-hydroxybutyrate is released into the culture medium. Therefore, the method for producing 3-hydroxybutyric acid using PHB as a precursor was shown to be effective in producing high-purity 3-hydroxybutyric acid, and at the same time, it was shown to be an effective method for improving the yield of 3-hydroxybutyric acid because no lactic acid, a by-product, remains.

[0047] (Example 4) Metabolic changes when using culture media with different lactic acid concentrations We investigated the effects of varying the lactic acid concentration added to the culture medium using Halomonas sp. strain OITC1261.

[0048] For the pre-culture of Halomonas sp. strain OITC1261, 100 mL of nutrient medium prepared by adding 2 w / v% sucrose to production medium B (2% sodium nitrate, 0.43% potassium sulfate, 0.2% potassium hydrogen phosphate, 0.15% sodium chloride, and other trace inorganic components), adjusting the pH to 9 with sodium hydroxide solution, was dispensed into an Erlenmeyer flask, the strain was inoculated, and the flask was cultured overnight (35°C, 200 rpm). In this culture, a tabletop culture apparatus was used, and the entire volume of the pre-culture solution was inoculated into 2 L of the main culture medium to perform the main culture. The culture media were compared using production medium B with 30 w / v% sucrose added, and different amounts of lactic acid added to each medium.

[0049] Sucrose was added in two separate doses during the culture period. Lactic acid was added by adding an aqueous solution of DL-sodium lactate to the culture medium so that the lactic acid concentration in the culture medium was 0.5, 1, 2, 4, and 5 w / v%. While maintaining constant culture conditions (temperature 35°C, stirring speed 700 rpm, pH 8.5, aeration rate 5 L / min), a portion of the culture medium was taken during cultivation and its components were analyzed in the same manner as in Example 1. These results are shown in Figures 3 to 7.

[0050] When cultured in a lactic acid-supplemented culture medium prepared by adding 4.6 g / L of DL-sodium lactate aqueous solution to achieve a lactic acid concentration of 0.5 w / v%, all the lactic acid was consumed within 48 hours after the start of the culture, as shown in Figure 3 (consumption rate 0.096 g / L / h). After 48 hours, the production of 3-hydroxybutyric acid and PHB was 60 g / L and 33 g / L, respectively. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB after 65 hours were 68 g / L and 34 g / L, respectively.

[0051] When cultured in a lactic acid-supplemented culture medium prepared by adding 9.2 g / L of DL-sodium lactate aqueous solution to achieve a lactic acid concentration of 1 w / v%, all the lactic acid was consumed within 48 hours after the start of the culture, as shown in Figure 4 (consumption rate 0.192 g / L / h). After 48 hours, the production of 3-hydroxybutyric acid and PHB was 58 g / L and 32 g / L, respectively. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB after 65 hours were 70 g / L and 30 g / L, respectively.

[0052] When cultured in a lactic acid-supplemented culture medium prepared by adding 18.6 g / L of DL-sodium lactate aqueous solution to achieve a lactic acid concentration of 2 w / v%, all the lactic acid was consumed within 48 hours after the start of the culture, as shown in Figure 5 (consumption rate 0.388 g / L / h). After 48 hours, the production of 3-hydroxybutyric acid and PHB was 61 g / L and 33 g / L, respectively. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB after 65 hours were 73 g / L and 31 g / L, respectively.

[0053] When cultured in a lactic acid-supplemented culture medium to which 38.2 g / L of DL-sodium lactate aqueous solution was added so that the lactic acid concentration in the culture medium was 4 w / v%, all the lactic acid was consumed within 48 hours after the start of the culture, as shown in Figure 6 (consumption rate 0.796 g / L / h). After 48 hours, the production of 3-hydroxybutyric acid and PHB was 55 g / L and 43 g / L, respectively. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB after 65 hours were 73 g / L and 44 g / L, respectively.

[0054] When cultured in a lactic acid-supplemented culture medium to which 49.2 g / L of DL-sodium lactate aqueous solution was added so that the lactic acid concentration in the culture medium was 5 w / v%, 0.5 g / L of lactic acid remained, as shown in Figure 7. After 48 hours, the production of 3-hydroxybutyric acid and PHB was 41 g / L and 36 g / L, respectively. Furthermore, the production volumes of 3-hydroxybutyric acid and PHB after 65 hours were 60 g / L and 35 g / L, respectively.

[0055] These results indicate that, in the cultivation of Halomonas microorganisms, adding lactic acid to the culture medium yields a culture medium containing 3-hydroxybutyric acid and PHB, without any residual lactic acid. At lactic acid addition concentrations of 0.5 to 4 w / v%, there was no effect on the amount of 3-hydroxybutyric acid produced. On the other hand, when the lactic acid concentration in the culture medium was 5 w / v%, lactic acid remained, and the production of 3-hydroxybutyric acid also decreased. These results indicate that a lactic acid concentration of less than 5 w / v% is appropriate, as high concentrations in the culture medium result in residual lactic acid at the end of the culture.

[0056] (Summary) Production rate of 3-hydroxybutyrate and optimal culture time Table 3 shows the hourly production rate of 3-hydroxybutyrate (production rate at each time point) calculated from the results of each culture test.

[0057] [Table 3]

[0058] In all culture tests, the production rate at 24 hours was the lowest, more than 30% lower than the highest value for each stage. Furthermore, in all culture tests, the production rate reached its peak after 41 hours. Therefore, a culture period of approximately 40 hours or more is required to efficiently produce 3-hydroxybutyric acid. After reaching its peak production rate, it gradually decreased, but the value at 65 hours was only 3-20% lower than the peak, maintaining a higher production efficiency than in the initial stages of cultivation (24 hours). Based on these findings, the optimal culture period for efficiently producing 3-hydroxybutyric acid is 40 to 70 hours.

Claims

1. 3-hydroxybutyrate-producing bacteria, By culturing using a culture medium containing lactic acid, Obtain a 3-hydroxybutyric acid fermentation liquid that does not contain residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

2. 3-hydroxybutyrate-producing bacteria, By culturing using a culture medium containing lactic acid at a concentration of less than 5 w / v%, Obtain a 3-hydroxybutyric acid fermentation liquid that does not contain residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

3. 3-hydroxybutyrate-producing bacteria, By culturing for 40 to 70 hours in a medium containing lactic acid at a concentration of less than 5 w / v%, Obtain a 3-hydroxybutyric acid fermentation liquid that does not contain residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

4. 3-hydroxybutyrate-producing bacteria, By performing aerobic culture using a culture medium containing lactic acid, After obtaining a polymer of 3-hydroxybutyric acid, The polymer of 3-hydroxybutyric acid is decomposed, Obtain 3-hydroxybutyric acid without residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

5. 3-hydroxybutyrate-producing bacteria, By performing aerobic culture using a medium containing lactic acid at a concentration of less than 5 w / v%, After obtaining a polymer of 3-hydroxybutyric acid, The polymer of 3-hydroxybutyric acid is decomposed, Obtain 3-hydroxybutyric acid without residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

6. 3-hydroxybutyrate-producing bacteria, By culturing aerobically for 40 to 70 hours using a medium containing lactic acid at a concentration of less than 5 w / v%, After obtaining a polymer of 3-hydroxybutyric acid, The polymer of 3-hydroxybutyric acid is decomposed, Obtain 3-hydroxybutyric acid without residual lactic acid. A method for producing 3-hydroxybutyric acid, characterized by the features described above.

7. The aforementioned 3-hydroxybutyrate-producing bacteria are Halomonas coriensis JCM12237, Halomonas salifodinae JCM14803, Halomonas halophylla JCM20791, Halomonas pacifica JCM20633, Halomonas salina JCM21221, Halomonas venusta JCM20634, and Halomonas sp. OITC1261 strain, all belonging to the genus Halomonas. Use any of these, or a combination thereof. A method for producing 3-hydroxybutyric acid according to any one of features 1 to 6.

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