Method for producing polyhydroxyalkanoate

Abstract

A method for producing a polyhydroxyalkanoate, which is a method for culturing a polyhydroxyalkanoate-producing microorganism in a culture medium to obtain a microbial cell in which the polyhydroxyalkanoate is accumulated. The culture medium contains a sulfur source at a sulfur component concentration of 0.0001-13 mM at the time when the culture starts. The production method includes a step of adding a carbon source and a sulfur source to the culture medium during the culture, and the average value of the ratio (C / S ratio) of the carbon weight (C) of the carbon source added per hour to the sulfur weight (S) of the sulfur source added per hour calculated during the period of adding the sulfur source is within the range of 500-10000.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing polyhydroxyalkanoate based on microbial culture.

[0002] With the world's growing attention to sustainable development goals represented by SDGs and increasing concerns about environmental problems such as marine microplastics, the conversion from existing petroleum-derived non-biodegradable plastics to biodegradable materials is being promoted, centered around industries such as packaging and food services, biomedicine, and agriculture. In recent years, biodegradable materials that have become active in industrial production include, for example, polylactic acid (PLA), polyhydroxyalkanoate (PHA), etc. Among them, due to the excellent biodegradability of PHA in a wide range of environments and being a rare biodegradable material that can be biodegraded even in seawater, it is highly anticipated as a solution to the marine microplastic problem and environmental problems.

[0003] PHA is a natural thermoplastic polyester produced and accumulated as an energy storage substance in the cells of most microbial species. Generally, industrial production of PHA is carried out by supplying a nutrient source to PHA-accumulating microorganisms and culturing them.

[0004] As a culture method for microbial-based substance production, generally known methods include: batch culture (a method in which the required nutrients are added to the culture medium in advance at the start of culture), continuous culture (a method in which a specific nutrient is added and the culture solution is discharged to keep the nutrient concentration in the culture solution constant), fed batch culture (a method in which a specific nutrient is added without discharging the culture solution), etc.

[0005] Batch culture is a culture method suitable for small-scale cultivation. As the most commonly used method at the research level, the more the product is desired to be obtained at a high concentration, the more nutrients are required at the start of culture. However, since there are also nutrients that show cytotoxicity when the concentration becomes high, batch culture is rarely used for microbial culture-based substance production at the industrial level.

[0006] From the perspective of suppressing the concentration of the carbon source at the start of culture, a method of adding a carbon source during the culture process, such as fed batch culture and continuous culture, is required. Therefore, multiple methods of adding a carbon source during the culture process have been proposed (for example, refer to Patent Documents 1 and 2).

[0007] In addition, as a characteristic of PHA-accumulating microorganisms, it is generally known that in an environment where a carbon source is abundantly present, depletion of a phosphorus source and / or a nitrogen source stimulates a change in metabolism and accumulation of PHA. Therefore, regarding the cultivation of PHA-accumulating microorganisms, there are also certain limitations on the concentration of the phosphorus source and / or the nitrogen source at the time point of starting cultivation. On the other hand, it has been reported that adding a phosphorus source after depletion of the phosphorus source or adding a nitrogen source after depletion of the nitrogen source is effective in improving PHA productivity (for example, refer to Patent Documents 2 and 3).

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: Japanese Patent Application Laid-Open No. 2015-006181

[0011] Patent Document 2: Japanese Patent Application Laid-Open No. 2005-080529

[0012] Patent Document 3: Japanese Patent Application Laid-Open No. 2013-9628 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] As described above, in the production of PHA based on microbial cultivation, there are limitations on the concentrations of the carbon source, the phosphorus source, and the nitrogen source at the start of cultivation. However, there are few insights regarding the sulfur source.

[0015] A sulfur source is also used in PHA production cultivation. Generally, the sulfur component concentration at the start of cultivation is mostly set to about 10 to 40 mM. However, to the best knowledge of the present inventors, there are no insights regarding the influence of the sulfur component concentration at the start of cultivation and during cultivation on PHA productivity.

[0016] In view of the above, an object of the present invention is to achieve higher PHA productivity when cultivating PHA-producing microorganisms to produce PHA.

[0017] Means for Solving the Problems

[0018] The present inventors have found that during cultivation, in the process of cultivating PHA-producing microorganisms while adding a carbon source, if the sulfur component concentration of the sulfur source contained in the culture medium at the time point of starting cultivation exceeds 13 mM, the production of PHA will be hindered. In addition, when the sulfur component concentration of the sulfur source contained in the culture medium is set to 13 mM or less at the time point of starting cultivation, PHA productivity can be improved by adding a sulfur source in a specific amount during the cultivation process, thereby completing the present invention.

[0019] That is, the present invention relates to a method for producing polyhydroxyalkanoate, which is a method of culturing polyhydroxyalkanoate-producing microorganisms in a culture medium to obtain microbial cells in which polyhydroxyalkanoate is accumulated.

[0020] At the start time of the culture, the above-mentioned culture medium contains a sulfur source in an amount such that the sulfur component concentration is 0.0001 to 13 mM.

[0021] The above-mentioned production method includes: during the culture, a step of adding a carbon source and a sulfur source to the above-mentioned culture medium.

[0022] The average value of the ratio (C / S ratio) of the carbon weight (C) of the above-mentioned carbon source added per hour to the sulfur weight (S) of the above-mentioned sulfur source added per hour calculated during the period of adding the above-mentioned sulfur source is in the range of 500 to 10,000.

[0023] Effects of the Invention

[0024] According to the present invention, when culturing PHA-producing microorganisms to produce PHA, higher PHA productivity can be achieved.

[0025] According to the present invention, by performing fed-batch culture or continuous culture to culture PHA-producing microorganisms, higher PHA productivity can be achieved.

[0026] According to a preferred embodiment of the present invention, since the total usage amount of the sulfur source can be suppressed, a culture tank with a limited capacity can be used to efficiently improve PHA productivity. In addition, the sulfur component contained in the drainage discharged after culturing the microorganisms is reduced, and the cost of sulfur-related drainage treatment can be suppressed. Detailed Embodiments

[0027] Hereinafter, the embodiments of the present invention will be described in detail.

[0028] The embodiment of the present invention relates to a method for producing polyhydroxyalkanoate, which is a method of culturing polyhydroxyalkanoate-producing microorganisms in a culture medium to obtain microbial cells in which polyhydroxyalkanoate is accumulated.

[0029] (Polyhydroxyalkanoate)

[0030] As the type of the above-mentioned polyhydroxyalkanoate (PHA), any PHA that can be produced by microorganisms is acceptable and there is no particular limitation. It can be a homopolymer formed by one type of hydroxyalkanoic acid or a copolymer formed by two or more types of hydroxyalkanoic acids. Specifically, examples include: homopolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, copolymers of one monomer selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms and other hydroxyalkanoic acids (for example, 2-hydroxyalkanoic acids, 4-hydroxyalkanoic acids, 5-hydroxyalkanoic acids, 6-hydroxyalkanoic acids, etc. having 4 to 16 carbon atoms), and copolymers of two or more monomers selected from 3-hydroxyalkanoic acids having 4 to 16 carbon atoms, etc.

[0031] Among them, homopolymers or copolymers containing 3-hydroxybutyric acid as a monomer unit are preferred. Examples of such polymers include: homopolymer P(3HB) of 3-hydroxybutyric acid (abbreviation: 3HB), copolymer P(3HB-co-3HV) of 3HB and 3-hydroxyvaleric acid (abbreviation: 3HV), copolymer P(3HB-co-3HH) (abbreviation: PHBH) of 3HB and 3-hydroxyhexanoic acid (abbreviation: 3HH), copolymer P(3HB-co-4HB) of 3HB and 4-hydroxybutyric acid (abbreviation: 4HB), PHA containing lactic acid (abbreviation: LA) as a constituent, such as copolymer P(LA-co-3HB) of 3HB and LA, etc., but not limited to these. Among them, from the viewpoint of wide application range as a polymer, PHBH is preferred.

[0032] According to the purpose, the type of PHA produced can be appropriately selected according to the type of PHA synthase gene possessed by or newly introduced into the microorganism used, the type of genes related to its synthesis in the metabolic system, culture conditions, etc.

[0033] (PHA-producing microorganism)

[0034] The above-mentioned PHA-producing microorganism only needs to be a microorganism having the ability to produce PHA. This microorganism can be a microorganism having a PHA synthase gene. This microorganism can also be a wild strain originally having a PHA synthase gene, or a mutant strain obtained by subjecting such a wild strain to artificial mutagenesis treatment, or a strain into which an exogenous PHA synthase gene has been introduced by genetic engineering methods.

[0035] The PHA-producing microorganism is not particularly limited as long as it has the ability to produce PHA, and can be a microorganism found in nature, a mutant or a transgenic organism. Specifically, examples include Cupriavidus necator and other Cupriavidus species; Alcaligenes latas and other Alcaligenes species; Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas resinovorans, Pseudomonas oleovorans and other Pseudomonas species; Bacillus megaterium and other Bacillus species; Azotobacter genus; Nocardia genus; Aeromonas caviae, Aeromonas hydrophila and other Aeromonas species; Ralstonia genus; Wautersia genus; Comamonas genus, etc. (Microbiological Reviews, items 450 - 472, 1990). By using genetic engineering methods to introduce PHA synthase genes, etc., it is also possible to use biological cells that are considered to have been modified to produce PHA. It is also possible to use, for example, Gram-negative bacteria such as Escherichia genus, Gram-positive bacteria such as Bacillus genus, yeast such as Saccharomyces genus, Yarrowia genus, Candida genus, and higher biological cells such as plants. Since bacteria can accumulate a large amount of PHA, they are preferred. Particularly preferred are bacteria belonging to the Cupriavidus genus, and particularly preferably Cupriavidus necator.

[0036] (PHA synthase gene)

[0037] The PHA synthase gene introduced by transgenic technology is not particularly limited, and examples include PHA synthase genes derived from Aeromonas caviae, Aeromonas hydrophila, Pseuromonas SP 61-3, Cupriavidus necator, and their variants. The above-mentioned variants refer to base sequences encoding PHA synthases with amino acid sequences in which one or more amino acid residues are deleted, added, inserted, or replaced.

[0038] (Cultivation)

[0039] The cultivation of the polyhydroxyalkanoate-producing microorganism in this embodiment refers to the "main cultivation" in the final stage carried out for the purpose of accumulating polyhydroxyalkanoate at a high concentration in the polyhydroxyalkanoate-producing microorganism. The "pre-cultivation" and "seed mother cultivation" carried out before the "main cultivation" are not included in the "cultivation" of this embodiment. Therefore, in the "pre-cultivation" and "seed mother cultivation", the sulfur component concentration is not particularly limited, and whether a sulfur source is added during the cultivation is also not particularly limited.

[0040] (Culture medium)

[0041] As the culture medium used in the "pre-cultivation", "seed mother cultivation" and "main cultivation", any liquid culture medium containing a nutrient source that helps the growth and proliferation of the polyhydroxyalkanoate-producing microorganism to be cultivated is acceptable. It is preferable to mix the PHA-producing microorganism in a liquid containing a carbon source, a nitrogen source, a phosphorus source, a sulfur source, inorganic salts, and other organic nutrient sources, and disperse it by stirring, shaking, etc.

[0042] Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, and nitric acid, nitrates, nitrites, peptone, meat extract, yeast extract, etc. Examples of the phosphorus source include phosphates such as potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium phosphate, ammonium phosphate, and inorganic phosphoric acid, peptone, meat extract, yeast extract, etc. Examples of the inorganic salts include chlorides, phosphates, nitrates, nitrites, sulfates, sulfites, etc. of magnesium, sodium, potassium, and other trace metal elements (iron, cobalt, nickel, copper, etc.). Examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, proline, and vitamins such as vitamin B1, vitamin B12, vitamin C, etc. Regarding the culture medium for the "pre-cultivation" and "seed mother cultivation", it can be freely selected from the nutrient sources that help the growth and proliferation of the selected polyhydroxyalkanoate-producing microorganism. Among them, in the "main cultivation" of this embodiment, for the nutrient source containing sulfur element, it is preferably used by the method of using the sulfur source described below.

[0043] (Carbon source)

[0044] In the "main cultivation" of the polyhydroxyalkanoate-producing microorganism in this embodiment, there is a step of adding a carbon source to the culture medium for cultivation. The addition method of this carbon source is not particularly limited, and continuous addition is preferred. That is, in the "main cultivation", it is preferably carried out while continuously adding a carbon source to the culture medium containing the PHA-producing microorganism. Here, "continuous addition" includes not only the method of continuous addition without interruption over time, but also the method of repeating addition with intermittent temporary rest periods set.

[0045] As the carbon source, oils and fats such as glycerides and fatty acids can be cited; sugars such as glucose and fructose; and organic carbon such as peptone, meat extract, and yeast extract. As the above-mentioned oils and fats, animal fats, vegetable fats, their mixed fats, transesterified oils, fractionated oils, etc. can be used, and there is no particular limitation. Specific examples of vegetable fats can include: rapeseed oil, sunflower oil, soybean oil, olive oil, corn oil, palm oil, palm kernel oil, cottonseed oil, sesame oil, nut oil, jatropha oil, rice bran oil, etc. Specific examples of animal fats can include lard, etc. These can be used alone or in combination of two or more.

[0046] When continuously adding the carbon source, the addition amount is not particularly limited, and it is desirable to pay attention to keeping the carbon source concentration in the medium within a certain range while adding the carbon source.

[0047] (Usage mode of sulfur source)

[0048] In the "main culture" of the polyhydroxyalkanoate-producing microorganism of the present embodiment, at the time point of the start of the culture, the above-mentioned medium contains a sulfur source in an amount such that the sulfur component concentration is 0.0001 mM or more and 13 mM or less. At the time point of the start of the culture, when the sulfur component concentration in the medium exceeds 13 mM, the production of PHA is hindered by the sulfur source, and it is difficult to achieve good PHA productivity. The above sulfur component concentration can be 10 mM or less, can be 8 mM or less, or can also be 6 mM or less.

[0049] At the time point of the start of the culture, the lower limit of the above sulfur component concentration can be 0.0001 mM or more, can be 0.001 mM or more, can be 0.01 mM or more, can be 0.1 mM or more, can be 0.5 mM or more, can be 1 mM or more, can be 3 mM or more, or can also be 5 mM or more.

[0050] The above-mentioned "sulfur component concentration in the medium at the time point of the start of the culture" refers to the molar amount (mol) of sulfur element in the culture solution relative to the volume (L) of the culture solution after inoculating the mother culture solution. When all the sulfur sources contained in the medium are composed of inorganic salts, the "sulfur component concentration in the medium at the time point of the start of the culture" can be calculated based on the medium composition. When sulfur components are introduced by the mother culture solution, the amount of sulfur introduced is calculated based on the medium composition of the mother culture solution and the amount of inoculated bacteria, and it can be added to the amount of sulfur calculated from the medium composition of the main culture solution.

[0051] On the other hand, when the sulfur source contained in the medium contains an organic nutrient source, the "sulfur component concentration in the medium at the time point of the start of the culture" can be calculated by taking out a part of the medium after preparing the medium and measuring the amount of sulfur element by an elemental analyzer.

[0052] From the viewpoint of facilitating the adjustment of the sulfur component concentration at the start of culture, it is preferred to use an inorganic salt.

[0053] The sulfur source used at the start of the culture is not particularly limited and can be selected from magnesium sulfate, potassium sulfate, sodium sulfate, ammonium sulfate, other trace metal elements (iron, cobalt, nickel, copper, etc.) sulfate, sulfite and other inorganic salts, sulfuric acid, and organic nutrient sources such as peptone, meat extract, yeast extract, etc. These sulfur sources can be used alone or in combination. Among them, sulfuric acid or inorganic salts are preferred, and sulfuric acid or sulfates are more preferred.

[0054] The "main culture" of the polyhydroxyalkanoate-producing microorganism of the present embodiment includes a step of adding a sulfur source to the culture medium in the culture. The sulfur source added in the culture is not particularly limited, and can be selected from magnesium sulfate, potassium sulfate, sodium sulfate, ammonium sulfate, other trace metal elements (iron, cobalt, nickel, copper, etc.) sulfates, sulfites and other inorganic salts, sulfuric acid, and organic nutrient sources such as peptone, meat extract, and yeast extract. These sulfur sources can be used alone, or multiple sulfur sources can be used in combination. From the viewpoint of easy adjustment of the amount of sulfur source added, sulfuric acid or inorganic salts are preferred, and sulfuric acid or sulfates are more preferred. The sulfur source can be added alone, or it can be dissolved or dispersed in water and added.

[0055] The method of adding the sulfur source to the culture medium during the culture is not particularly limited, but continuous addition is preferred. That is, the "main culture" is preferably performed while the sulfur source is continuously added to the culture medium containing the PHA-producing microorganism. Here, "continuous addition" includes not only a method of continuous addition without interruption over time, but also a method of repeated addition while intermittently setting a temporary rest period.

[0056] When the sulfur source is continuously added to the culture medium during the culture, the sulfur source may be added at the same time as the culture is started, but it is preferred that the sulfur source be added after a certain period of time has passed since the start of the culture and the concentration of sulfur components in the culture medium has decreased. For example, the sulfur source may be added after 1 hour or more, 3 hours or more, or 5 hours or more from the start of the culture.

[0057] When adding a carbon source and a sulfur source to the culture medium in the culture, it is desired to control the addition amounts of the carbon source and the sulfur source as described below. That is, setting 1 hour as the unit time, calculating the ratio (C / S ratio) of the carbon weight (C) of the carbon source added per hour to the sulfur weight (S) of the sulfur source added per hour, and obtaining its average value. At this time, control the addition amounts of the carbon source and the sulfur source so that the average value of the C / S ratio falls within the range of 500 to 10,000. That is, the amount of the sulfur source added in the culture is on average more than 1 / 10,000 and less than 1 / 500 of the amount of the carbon source added in the culture, which is an extremely small amount compared to the carbon source.

[0058] The above C / S ratio is calculated during the "period of adding the sulfur source".

[0059] The above "period of adding the sulfur source" refers to the period from the start of adding the sulfur source until the end. The starting point of the above "period of adding the sulfur source" is the time point when the sulfur source starts to be added. That is, the period from the start of the culture until the start of adding the sulfur source is excluded from the above "period of adding the sulfur source".

[0060] In principle, the end point of the above "period of adding the sulfur source" is the time point when the addition of the sulfur source finally ends before the end of the above culture. However, dividing the above "period of adding the sulfur source" into units of 1 hour starting from the starting point of the "period of adding the sulfur source", taking this 1 hour as the unit time. In the case where the last unit time is less than 1 hour and the culture continues after the addition of the sulfur source finally ends, the time point when the last unit time reaches 1 hour is taken as the end point of the "period of adding the sulfur source". In the case where the last unit time is less than 1 hour and the culture ends, the culture end time point is taken as the end point of the "period of adding the sulfur source".

[0061] The above "ratio (C / S ratio) of the carbon weight (C) of the above carbon source added per hour to the sulfur weight (S) of the above sulfur source added per hour" means dividing the above "period of adding the sulfur source" into units of 1 hour starting from the starting point of the "period of adding the sulfur source", taking this 1 hour as the unit time, calculating the C / S ratio for each unit time, and calculating the average value of the C / S ratio from the values of the C / S ratio for each obtained unit time. In the case where the last unit time is less than 1 hour, this time less than 1 hour is also considered as a unit time, and the value of the C / S ratio for this unit time is also used to calculate the average value of the C / S ratio.

[0062] However, when no carbon source is added within a certain unit time, the C / S ratio for that unit time is zero, and this C / S ratio value (zero) is not considered when calculating the average value of the C / S ratio. Additionally, within a certain unit time, when the amount of sulfur source added is extremely small or zero compared to the amount of carbon source added, the effect brought about by the addition of the sulfur source cannot be achieved. Therefore, when calculating the average value of the C / S ratio, C / S ratio values exceeding 20,000 for each unit time are not considered.

[0063] When the average value of the C / S ratio is less than 500, the total usage amount of the sulfur source increases, and the effect brought about by the addition of the sulfur source reaches its limit. Instead, the sulfur source will hinder the production of PHA, and it is sometimes difficult to achieve good PHA productivity. Additionally, due to the increase in the total usage amount of the sulfur source, it places a burden on the water supply and drainage treatment, and sometimes the cost increases. On the other hand, when the average value of the C / S ratio exceeds 10,000, the amount of sulfur source added is small, and it is sometimes difficult to achieve good PHA productivity. From the perspective of suppressing the total usage amount of the sulfur source and simultaneously achieving good PHA productivity, the above-mentioned average value of the C / S ratio is preferably 1000 - 6000, more preferably 1000 - 4000.

[0064] The cultivation method can be continuous cultivation or fed-batch cultivation.

[0065] In addition to adding the above-mentioned carbon source and sulfur source, the cultivation conditions can be carried out according to the usual microbial cultivation method, and there are no particular limitations on the cultivation scale, aeration and agitation conditions, pH during cultivation, etc. The cultivation temperature can be appropriately selected as a temperature suitable for the growth of the bacteria and the production of PHA. For example, it is preferably about 20 - 40 °C. Additionally, the cultivation time can also be appropriately set, but it is preferably about 1 - 7 days.

[0066] During cultivation, in addition to the carbon source and sulfur source, a phosphorus source and / or a nitrogen source can be appropriately added in batches or continuously.

[0067] The PHA accumulation amount in the PHA-producing microorganism at the end of cultivation is not particularly limited and can be appropriately determined. It is preferably 80% by weight or more, more preferably 90% by weight or more.

[0068] (PHA Recovery)

[0069] After culturing for an appropriate period to accumulate PHA in the cells, PHA can be recovered from the cells using known methods. The recovery method is not particularly limited and can be carried out, for example, by the method described below. As an example, the cells are separated from the culture broth using a centrifuge or the like, and the cells are washed with distilled water, methanol, or the like and dried. PHA is extracted from the dried cells using an organic solvent such as chloroform. The cell components are removed from the solution containing PHA by filtration or the like, and a poor solvent such as methanol or hexane is added to the filtrate to precipitate PHA. In addition, PHA can be recovered by filtering, centrifuging to remove the supernatant, and drying.

[0070] As another example, the cells are separated from the culture broth using a centrifuge or the like and washed with distilled water, ethanol, or the like. Then, the washed sample is mixed with a sodium dodecyl sulfate (SDS) solution, the cell membrane is disrupted by sonication, and PHA is separated from the cell components using a centrifuge or the like and dried, whereby PHA can be recovered.

[0071] (PHA productivity)

[0072] PHA productivity can be evaluated by the content (g / L) of PHA contained in each 1 L of the culture broth after the culture is completed. Specifically, PHA productivity can be calculated by the following method: PHA is recovered from an arbitrary amount of the culture broth by the above-described PHA recovery method and its weight is measured, and the obtained PHA weight is divided by the volume of the culture broth. Any method can be selected as the method for recovering PHA, but the same PHA recovery method is selected when comparing PHA productivity.

[0073] According to the present embodiment, compared with the culture conditions in which no sulfur source is added during the culture, PHA can be produced with high PHA productivity by adding a sulfur source in a given amount during the culture.

[0074] In the following items, preferred embodiments in the present disclosure are listed, but the present invention is not limited to the following items.

[0075] [Item 1]

[0076] A method for producing a polyhydroxyalkanoate, which is a method for culturing a polyhydroxyalkanoate-producing microorganism in a medium to obtain microbial cells in which polyhydroxyalkanoate is accumulated, wherein,

[0077] at the start time of the culture, the medium contains a sulfur source in an amount such that the sulfur component concentration is 0.0001 to 13 mM,

[0078] the production method includes a step of adding a carbon source and a sulfur source to the medium during the culture,

[0079] The average value of the ratio (C / S ratio) of the carbon weight (C) of the carbon source added per hour to the sulfur weight (S) of the sulfur source added per hour, calculated during the addition of the sulfur source, is in the range of 500 to 10,000.

[0080] [Item 2]

[0081] The method for producing a polyhydroxyalkanoate according to Item 1, wherein

[0082] the average value of the C / S ratio is 1,000 to 6,000.

[0083] [Item 3]

[0084] The method for producing a polyhydroxyalkanoate according to Item 1 or 2, wherein

[0085] the sulfur source contains at least one selected from sulfuric acid and sulfates.

[0086] [Item 4]

[0087] The method for producing a polyhydroxyalkanoate according to any one of Items 1 to 3, wherein

[0088] the polyhydroxyalkanoate-producing microorganism belongs to the genus Cupriavidus.

[0089] [Item 5]

[0090] The method for producing a polyhydroxyalkanoate according to any one of Items 1 to 4, wherein

[0091] the polyhydroxyalkanoate-producing microorganism is Cupriavidus necator.

[0092] [Item 6]

[0093] The method for producing a polyhydroxyalkanoate according to any one of Items 1 to 5, wherein

[0094] the polyhydroxyalkanoate is a copolymer containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid as monomer units.

[0095] Examples

[0096] Hereinafter, the present invention will be further specifically described by way of examples. However, the present invention is not limited to these examples.

[0097] In all of the following reference examples, comparative examples, and examples, the KNK-005 strain was used as the PHA-producing microorganism.

[0098] The KNK-005 strain is a transgenic organism produced by the method described in the specification of US Patent No. 7,384,766, in which the PHA synthase gene derived from Aeromonas caviae has been introduced into the chromosome of the Cupriavidus necator H16 strain.

[0099] (Reference Example 1)

[0100] Using the KNK-005 strain, (1) preculture, (2) seed culture, and (3) main culture were sequentially performed by the methods shown below.

[0101] (1) Preculture

[0102] First, 20 μL of the glycerol stock of the KNK-005 strain was inoculated into 20 mL of the preculture medium and cultured at 30 °C for 18 hours.

[0103] The composition of the preculture medium was 1 w / v% Meat-extract, 1 w / v% Bacto-Tryptone, 0.2 w / v% Yeast-extract, 0.9 w / v% Na2HPO4·12H2O, 0.15 w / v% KH2PO4, (pH 6.8).

[0104] (2) Seed culture

[0105] The obtained preculture broth was inoculated at 1.0 v / v% into a 3 L fermenter (MDL-8C manufactured by Marubishi Bioengineering) containing 1.8 L of the seed culture medium. The operating conditions were set as a culture temperature of 30 °C, a stirring speed of 500 rpm, an aeration rate of 1.8 L / min, and the pH was controlled between 6.5 and 6.6 while culturing for 24 hours to perform the seed culture. A 14% aqueous ammonium hydroxide solution was used for pH control.

[0106] The composition of the seed culture medium was 0.385 w / v% Na2HPO4·12H2O, 0.067 w / v% KH2PO4, 0.15 w / v% (NH4)2SO4, 0.1 w / v% MgSO4·7H2O, 0.155 w / v% NH4Cl, 2.5 w / v% palm oil, 0.5 v / v% trace metal salt solution (1.6 w / v% FeCl3·6H2O, 1 w / v% CaCl2·2H2O, 0.02 w / v% CoCl2·6H2O, 0.016 w / v% CuSO4·5H2O, 0.012 w / v% NiCl2·6H2O dissolved in 0.1 N hydrochloric acid).

[0107] (3) Main culture

[0108] The obtained mother seed culture solution was inoculated at 5.0 v / v% into a 5 L fermenter (Bioneer-Neo manufactured by Marubishi Bioengineering) containing 1.8 L of the main culture medium. The operating conditions were set as a culture temperature of 30 °C, a stirring speed of 500 rpm, an aeration rate of 3.0 L / min, and the pH was controlled between 6.3 and 6.7. A 25% aqueous ammonia solution was used for pH control.

[0109] During the culture period, palm oil was intermittently added as a carbon source so that the oil concentration in the culture supernatant was 0.3 - 2%, and at the same time, the main culture was carried out.

[0110] In addition, during the culture period, a phosphoric acid solution was intermittently added as a phosphorus source, and at the same time, the main culture was carried out.

[0111] Medium A shown in Table 1 was used as the main culture medium. The sulfur component concentration in Medium A containing the mother seed culture solution at the start of the main culture was 16.0 mM.

[0112] The main culture was carried out for 72 hours.

[0113] [Table 1]

[0114] Additive Molecular weight Medium A Medium B Medium C Medium D Medium E <![CDATA[Na2HPO4·12H2O (w / v%)]]> 163.94 0.385 0.385 0.385 0.385 0.385 <![CDATA[KH2PO4 (w / v%)]]> 136.09 0.067 0.067 0.067 0.067 0.067 <![CDATA[MgSO4·7H2O (w / v%)]]> 246.47 0.100 0.100 0.100 0.100 0.100 <![CDATA[(NH4)2SO4 (w / v%)]]> 132.14 0.150 0.125 0.100 0.060 0.015 <![CDATA[NH4Cl (w / v%)]]> 53.49 0.114 0.134 0.155 0.187 0.223 Trace metal salt solution (w / v%) (refer to Table 2) - 0.500 0.500 0.500 0.500 0.500 Sulfur component concentration (mM) (before addition of the mother culture solution) - 15.4 13.5 11.6 8.6 5.2 Sulfur component concentration (mM) (including the sulfur source of the mother culture solution) 16.0 14.1 12.2 9.2 5.8

[0115] [Table 2]

[0116] Trace metal salt solution (0.1N hydrochloric acid) Molecular weight Concentration <![CDATA[FeCl3·6H2O (w / v%)]]> 270.29 1.600 <![CDATA[CaCl2·2H2O (w / v%)]]> 147.01 1.000 <![CDATA[CoCl2·6H2O (w / v%)]]> 237.93 0.020 <![CDATA[CuSO4·5H2O (w / v%)]]> 249.69 0.016 <![CDATA[NiCl2·6H2O (w / v%)]]> 129.60 0.012 Sulfur component concentration (mM) - 0.6

[0117] After the culture was completed, a certain amount of the culture solution was recovered, washed with distilled water and ethanol, and then dried under vacuum. The dry cell weight containing PHA was measured. After washing the cells in the same manner as above, they were suspended in an SDS aqueous solution and disrupted by ultrasonic waves to disrupt / dissolve the cell components, and at the same time, PHA and cell components were separated. Only PHA was recovered by centrifugation, and thus the PHA accumulation amount was measured. Based on this, the PHA productivity was calculated. The results of the PHA productivity are shown in Table 3.

[0118] (Reference Example 2)

[0119] Except that Medium B shown in Table 1 was used as the main culture medium, (1) pre-culture, (2) mother seed culture, and (3) main culture were carried out in sequence under the same conditions as in Reference Example 1. The sulfur component concentration in Medium B containing the mother seed culture solution at the start of the main culture was 14.1 mM.

[0120] After the culture was completed, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0121] (Comparative Example 1)

[0122] Except that medium C shown in Table 1 was used as the main culture medium, (1) pre-culture, (2) seed mother culture, and (3) main culture were successively carried out under the same conditions as in Reference Example 1. The sulfur component concentration in medium C containing the seed mother culture broth at the time point when the main culture started was 12.2 mM.

[0123] After the culture was completed, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0124] (Comparative Example 2)

[0125] Except that medium D shown in Table 1 was used as the main culture medium, (1) pre-culture, (2) seed mother culture, and (3) main culture were successively carried out under the same conditions as in Reference Example 1. The sulfur component concentration in medium D containing the seed mother culture broth at the time point when the main culture started was 9.2 mM.

[0126] After the culture was completed, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0127] (Comparative Example 3)

[0128] Except that medium E shown in Table 1 was used as the main culture medium, (1) pre-culture, (2) seed mother culture, and (3) main culture were successively carried out under the same conditions as in Reference Example 1. The sulfur component concentration in medium E containing the seed mother culture broth at the time point when the main culture started was 5.8 mM.

[0129] After the culture was completed, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0130] (Example 1, Example 3, Example 5, Example 7, Example 9, Example 11)

[0131] In (3) the main culture, starting from the 20th hour after the start of the culture until the end of the culture, an aqueous sodium sulfate solution of 43 g / L was intermittently added. Except for this, (1) pre-culture, (2) seed mother culture, and (3) main culture were successively carried out under the same conditions as in Comparative Example 1. During the period from the start of adding the aqueous sodium sulfate solution until the end of the culture, the average value of the C / S ratio was calculated by calculating the ratio of the carbon weight (C) in the added carbon source to the sulfur weight (S) in the added sulfur source (C / S ratio). The results are shown in Table 3.

[0132] During the period until the addition ended (i.e., the 20th hour to the 72nd hour), the ratio of the carbon weight (C) of the carbon source added per hour to the sulfur weight (S) of the sulfur source added per hour (C / S ratio) was calculated. The minimum value, maximum value, and average value of the C / S ratio are shown in Table 3.

[0133] The carbon weight (C) of the carbon source added per hour was calculated by the following formula.

[0134] (Weight of palm oil added per hour (g)) / (Molecular weight of palm oil triglyceride) × (Number of carbon atoms in 1 molecule of palm oil triglyceride) × (Molecular weight of carbon)

[0135] The molecular weight and number of carbon atoms of palm oil triglyceride were calculated by analyzing the fatty acid composition of palm oil and assuming that palm oil consists of 100% triglyceride.

[0136] The sulfur weight (S) of the sulfur source added per hour was calculated by the following formula.

[0137] (Concentration of sodium sulfate aqueous solution added (g / L)) / (Specific gravity of the sodium sulfate aqueous solution at this concentration) × (Weight of sodium sulfate aqueous solution added per hour) / (Molecular weight of sodium sulfate) × (Molecular weight of sulfur)

[0138] After the cultivation ended, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0139] (Example 2, Example 4, Example 6, Example 8, Example 10, Example 12)

[0140] In the (3) main cultivation, starting from the 40th hour at the start of cultivation until the end of cultivation, a 43 g / L sodium sulfate aqueous solution was intermittently added. Except for this, (1) pre-cultivation, (2) seed culture, and (3) main cultivation were carried out in sequence under the same conditions as in Comparative Example 1. In addition, the minimum value and maximum value of the C / S ratio were determined under the same conditions as in Example 1, Example 3, Example 5, Example 7, Example 9, Example 11, and the average value was further calculated. The minimum value, maximum value, and average value of the C / S ratio are shown in Table 3.

[0141] After the cultivation ended, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3.

[0142] [Table 3]

[0143]

[0144] As can be seen from Table 3, the following situations are obtained. From Comparative Examples 1 to 3, it can be seen that within the range where the sulfur component concentration in the culture medium at the start of cultivation is 13 mM or less, the PHA productivity increases as the sulfur component concentration increases. However, in Reference Examples 1 and 2 where the sulfur component concentration exceeds 13 mM, the PHA productivity is lower than that of Comparative Example 1 where the sulfur component concentration is 12.2 mM, indicating that the production of PHA is hindered when the sulfur component concentration at the start of cultivation exceeds 13 mM.

[0145] In Examples 1 to 12, the sulfur component concentration in the culture medium at the start of cultivation was set to 13 mM or less, and a sulfur source was intermittently added so that the average value of the C / S ratio fell within a specific range, and cultivation was carried out simultaneously. Based on this, compared with Comparative Example 1 where no sulfur source was added during cultivation, the PHA productivity increased. Among them, in Examples 3 to 12 where the average value of the C / S ratio was 6000 or less, the productivity increased by more than 5% compared with Comparative Example 1.

[0146] (Example 13)

[0147] In the (3) main cultivation, starting from the 30th hour from the start of cultivation until the end of cultivation, a 31 g / L potassium sulfate aqueous solution was intermittently added. Except for this, (1) pre-cultivation, (2) seed culture, and (3) main cultivation were carried out in the same conditions as Comparative Example 1 in sequence. In addition, the minimum value, maximum value, and average value of the C / S ratio were determined under the same conditions as in Example 1, Example 3, Example 5, Example 7, Example 9, and Example 11, and the average value was further calculated. The minimum value, maximum value, and average value of the C / S ratio are shown in Table 3.

[0148] The sulfur weight (S) of the sulfur source added per hour was calculated by the following formula.

[0149] (Concentration of the added potassium sulfate aqueous solution (g / L)) / (Specific gravity of the potassium sulfate aqueous solution at this concentration) × (Weight of the potassium sulfate aqueous solution added per hour) / (Molecular weight of potassium sulfate) × (Molecular weight of sulfur)

[0150] After the cultivation was completed, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3. Compared with Comparative Example 1, the PHA productivity in Example 13 increased by more than 5%.

[0151] (Example 14)

[0152] In the (3) main culture, starting from the 10th hour from the start of the culture until the end of the culture, an aqueous sodium sulfate solution of 43 g / L was intermittently added. Other than this, the (1) pre-culture, (2) seed mother culture, and (3) main culture were carried out in the same conditions as in Comparative Example 2. In addition, the minimum value and the maximum value of the C / S ratio were determined under the same conditions as in Example 1, Example 3, Example 5, Example 7, Example 9, and Example 11, and the average value was further calculated. The minimum value, the maximum value, and the average value of the C / S ratio are shown in Table 3.

[0153] After the culture ended, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3. Compared with Comparative Example 2 having the same sulfur component concentration at the start time point of the main culture, the PHA productivity in Example 14 was increased by more than 20%. Compared with Comparative Example 1, it was increased by more than 5%.

[0154] (Example 15)

[0155] In the (3) main culture, starting from the 10th hour from the start of the culture until the end of the culture, an aqueous sodium sulfate solution of 43 g / L was intermittently added. Other than this, the (1) pre-culture, (2) seed mother culture, and (3) main culture were carried out in the same conditions as in Comparative Example 3. In addition, the minimum value and the maximum value of the C / S ratio were determined under the same conditions as in Example 1, Example 3, Example 5, Example 7, Example 9, and Example 11, and the average value was further calculated. The minimum value, the maximum value, and the average value of the C / S ratio are shown in Table 3.

[0156] After the culture ended, the PHA productivity was calculated under the same conditions as in Reference Example 1. The results of the PHA productivity are shown in Table 3. Compared with Comparative Example 3 having the same sulfur component concentration at the start time point of the main culture, the PHA productivity in Example 15 was increased by more than 60%. Compared with Comparative Example 1, it was increased by more than 5%.

Claims

1. A method for manufacturing polyhydroxyalkanoate, the method comprising: Polyhydroxyalkanoate-producing microorganisms are cultured in a medium to obtain microbial cells accumulating polyhydroxyalkanoate, wherein, at the start time point of the culture, the medium contains a sulfur source in an amount such that the sulfur component concentration is 0.0001 to 13 mM, the production method includes a step of adding a carbon source and a sulfur source to the medium during the culture, the average value of the ratio (C / S ratio) of the carbon weight (C) of the carbon source added per hour to the sulfur weight (S) of the sulfur source added per hour calculated during the period of adding the sulfur source is in the range of 500 to 10,000.

2. The method for producing polyhydroxyalkanoate according to claim 1, wherein, the average value of the C / S ratio is 1000 to 6000.

3. The method for producing polyhydroxyalkanoate according to claim 1 or 2, wherein, the sulfur source contains at least one selected from sulfuric acid and sulfates.

4. The method for producing polyhydroxyalkanoate according to claim 1 or 2, wherein, the polyhydroxyalkanoate-producing microorganisms belong to the genus Cupriavidus.

5. The method for producing polyhydroxyalkanoate according to claim 1 or 2, wherein, the polyhydroxyalkanoate-producing microorganism is Cupriavidus necator.

6. The method for producing polyhydroxyalkanoate according to claim 1 or 2, wherein, the polyhydroxyalkanoate is a copolymer containing at least 3-hydroxybutyric acid and 3-hydroxyhexanoic acid as monomer units.

Images