WO2016208542A1

WO2016208542A1 - Method for producing polyhydroxyalkanoic acid using purple photosynthetic bacterium

Info

Publication number: WO2016208542A1
Application number: PCT/JP2016/068282
Authority: WO
Inventors: 圭司沼田; 美栄子樋口; 里奈青木; 哲也藤木
Original assignee: 国立研究開発法人理化学研究所; 株式会社カネカ
Priority date: 2015-06-26
Filing date: 2016-06-20
Publication date: 2016-12-29
Also published as: JP2018133997A

Abstract

The present invention addresses the problem of developing and providing a method for producing a polyhydroxyalkanoic acid (PHA) at reduced cost, with high efficiency and in a large quantity using a microorganism. Provided is a method for producing a PHA, comprising culturing a purple photosynthetic bacterium while stirring a liquid culture medium and while irradiating the liquid culture medium with far infrared light to produce cells of the bacterium in each of which the PHA is accumulated.

Description

Production method of polyhydroxyalkanoic acid using red photosynthetic bacteria

The present invention relates to a method for producing polyhydroxyalkanoic acid, which is a bioplastic, using red photosynthetic bacteria.

Synthetic resins, so-called plastics, are lightweight and strong materials that are easy to mold and color, have high corrosion resistance, and have excellent advantages such as mass production. Although it is highly convenient and used in various ways in daily life and industrial fields, it is an indispensable material in modern life, but it also has major problems to be solved.

For example, many of the plastics currently used are made from petroleum, but there is a concern that oil will be depleted in the future due to the increase in international consumption accompanying the rise of emerging countries. Moreover, since it is a non-renewable resource, carbon dioxide discharged into the atmosphere upon combustion is a major cause of global warming.

Disposal of waste plastics is also a problem. For example, plastics remain in nature without being decomposed by microorganisms or the like due to their high corrosion resistance. Also, when incinerated, not only a large amount of carbon dioxide is generated, but also harmful gases such as dioxins can be generated. Furthermore, since the calorific value at the time of incineration is high, it may cause destruction of the incinerator.

Therefore, research and development of bioplastics (biomass plastics) using biomass as raw materials as new plastic materials to replace the petroleum-based plastics has been promoted, and some of them have already been put into practical use. Biomass is a renewable resource and does not cause the depletion problem of fossil fuels. In addition, carbon dioxide generated by the combustion of biomass is carbon neutral because carbon dioxide in the atmosphere is originally fixed by photosynthesis by photosynthetic organisms, and is therefore carbon neutral, increasing the amount of carbon dioxide in the atmosphere. Nor. Therefore, it can also contribute to prevention of global warming (Non-Patent Document 1).

In addition, biodegradable plastics have been developed to solve the problem of waste plastic treatment. The biodegradable plastic is a plastic having properties and properties as a plastic and having a property of being completely decomposed in nature by an enzymatic action of microorganisms, and examples thereof include polyester synthesized from petroleum. Biodegradable plastic does not hurt the incinerator because it has a low calorific value even when incinerated, and does not release harmful substances.

Furthermore, biodegradable bioplastics that have both the properties of bioplastics and biodegradable plastics have been developed. Known biodegradable bioplastics include polyhydroxyalkanoic acid (PHA), polylactic acid (PLA), starch resin, polybutylene succinate (PBS), and the like.

PHA is a kind of biopolyester in which microorganisms accumulate in the body, and is a carbon and energy storage substance that is synthesized and accumulated in cells using glucose as a carbon source in preparation for poor nutrition. PHA is a hard bioplastic and is expected to be applied in various fields such as pharmaceuticals, agricultural chemicals, medical materials, and industrial materials.

Various methods for producing PHA with microorganisms have been disclosed so far. For example, Patent Document 1 discloses a method for producing polyhydroxybutanoic acid (PHB), which is a kind of PHA, using an alkaligenes bacterium. However, many of the conventional methods including the method disclosed in Patent Document 1 require an organic carbon source as an assimilating carbon source for propagation, and the productivity is low, resulting in an increase in manufacturing cost. there were.

Therefore, various methods for efficiently producing PHA from microorganisms without requiring organic carbon source reducing substances were sought. For example, Non-Patent Document 2 discloses a method for producing PHA using cyanobacteria that perform photosynthesis. Cyanobacteria can acquire assimilable carbon sources by photosynthesis themselves. Although it is excellent as a host bacterium for PHA production in that it does not require an organic carbon source, it can only produce PHB, which is a homopolymer, as PHA. PHB has the same melting point (180 ° C.) and fracture strength as polypropylene, but has a physical defect of being hard and brittle because it has lower fracture elongation and higher crystallinity than polypropylene.

7-14352

The present invention is to develop and provide a method for producing PHA efficiently and in large quantities by using microorganisms while reducing the production cost.

In order to solve the above-mentioned problems, the present inventors paid attention to photosynthetic bacteria as host bacteria for PHA production. As in the case of cyanobacteria, photosynthetic bacteria do not need an organic carbon source because they can acquire an assimilating carbon source by photosynthesis themselves. Furthermore, marine photosynthetic bacteria that inhabit seawater can use a large amount of seawater as a medium, thereby reducing the production cost of PHA. In addition, since the culture is performed in a high salt concentration medium, there is an advantage that there is little contamination of other bacteria. However, the photosynthetic bacterium has a problem that it is difficult to cultivate efficiently for producing PHA because the culturing method is not well established.

Therefore, the present inventors have conducted extensive research, and by continuing to stir the medium while irradiating far-red light during the culture period of the red photosynthetic bacteria, the growth of the cells and the efficient production and accumulation of PHA are achieved. An achievable culture method was established. In addition, when red photosynthetic bacteria were used, it was found that PHA was obtained as a copolymer (copolymer) in red non-sulfur bacteria. This invention is based on the said research result and provides the following.
(1) A method for producing PHA using a red photosynthetic bacterium, comprising a culture step of culturing the red photosynthetic bacterium by irradiating far red light while stirring a liquid medium.
(2) The production method according to (1), wherein the far red light is light having a peak wavelength at 720 nm to 860 nm.
(3) The culturing step includes a growth step of cultivating the red photosynthetic bacteria in a growth medium, a cell collection step for recovering the red photosynthetic bacteria in the culture solution after the growth step, and the recovered red photosynthetic bacteria as an organic acid. And / or the production method according to (1) or (2), comprising a PHA accumulation step of culturing in a PHA production medium containing carbon dioxide as a carbon source.
(4) The production method according to (3), wherein in the growth step, the red photosynthetic bacteria are cultured until the logarithmic growth phase.
(5) The production method according to (3) or (4), wherein in the PHA accumulation step, the red photosynthetic bacterium is cultured until the late logarithmic growth phase or the early stationary phase.
(6) The production method according to any one of (3) to (5), wherein the PHA production medium is a medium in which the carbon source of the growth medium is replaced with an organic acid salt and / or carbonate.
(7) The production method according to any one of (3) to (6), wherein the PHA production medium is deficient in any one or more of a nitrogen source, a phosphate source, and a vitamin.
(8) The production method according to any one of (1) to (7), further comprising a recovery step of recovering bacterial cells contained in the culture solution after the culture step.
(9) The production method according to any one of (1) to (8), further comprising an extraction step of extracting PHA from the culture solution after the culturing step or red photosynthetic bacteria.
(10) The method according to any one of (1) to (9), wherein the red photosynthetic bacterium is a marine photosynthetic bacterium.
(11) The red photosynthetic bacteria are selected from Marichromatium bheemlicum, Thiohalocapsa marina, Thiophaeococcus mangrovi, Afifella pfennigii, Afifella marina, Rhodovulum euryhalinum, Rhodovulum imhoffii, Rhodovulum sulfidophilum, Rhodovulum tesquien, Rhodovulum tesquien The production method according to (10).
(12) purple photosynthetic bacteria Marichromatium bheemlicum JCM13911, Thiohalocapsa marina JCM14780, Thiophaeococcus mangrovi JCM14889, Afifella pfennigii ATCC BAA1145, Afifella marina DSM2698, Rhodovulum euryhalinum DSM4868, Rhodovulum imhoffii JCM13589, Rhodovulum sulfidophilum ATCC35886, Rhodovulum tesquicola ATCC BAA1573, Rhodovulum visakhapatnamense JCM13531, Roseospira The production method according to (11), selected from the group consisting of goensis JCM14191 and Roseospira marina ATCC BAA447.

This specification includes the disclosure of Japanese Patent Application No. 2015-128590, which is the basis of the priority of this application.

According to the PHA production method of the present invention, PHA can be efficiently produced by stably cultivating red photosynthetic bacteria. Moreover, PHA can be obtained as a copolymer by using some red photosynthetic bacteria.

It is a figure which shows the dry cell weight after culture | cultivation when the selected 12 kinds of red photosynthetic bacteria are culture | cultivated in each culture medium. The amount of PHA accumulated in the cells after culturing the selected 12 kinds of red photosynthetic bacteria in each medium (weight% per dry cell) (A) and the amount of PHA per liter of medium (mg) (B) FIG.

<Polyhydroxyalkanoic acid production method>
The present invention relates to a method for producing polyhydroxyalkanoic acid (PHA) using a red photosynthetic bacterium.
1. Definition of terms The following terms used in this specification are defined.

The “photosynthetic bacterium” is an eubacteria that performs non-oxygen-generating photosynthesis, and according to the growth mode under a pigment, electron donor, aerobic or anaerobic conditions, a red sulfur bacterium, a red non-sulfur bacterium, a green sulfur bacterium, It is classified into five families: green non-sulfur bacteria and aerobic photosynthetic bacteria.

“Red photosynthetic bacteria” is a general term for red sulfur bacteria and red non-sulfur bacteria in the above photosynthetic bacteria. In the method for producing PHA of the present invention, a red photosynthetic bacterium, that is, a red sulfur bacterium or a red non-sulfur bacterium is used. The red photosynthetic bacterium is more preferable in terms of PHA production than other photosynthetic bacteria because it is relatively easy to cultivate because it does not have a strong anaerobic requirement and has a strong carbon fixation activity.

The red photosynthetic bacteria are roughly classified into freshwater and marine depending on the habitat environment, and any of the red photosynthetic bacteria used in the present invention may be used. Preferably, it is a marine red photosynthetic bacterium. This is because, as described above, contamination of other bacteria can be suppressed by a high salt concentration medium, and a large amount of seawater can be used as a medium, so that the production cost of bioplastic can be suppressed.

Specific examples of the red photosynthetic bacteria include the red sulfur bacteria shown in Table 1 and the bacteria of the red non-sulfur bacteria genus shown in Table 2. Specifically, Allochromatium genus bacteria, Ectothiorhodospira genus bacteria, Halochromatium genus bacteria, Halorhodospira genus bacteria, Marichromatium genus bacteria, Thiocapsa genus bacteria, Thiohalocapsa genus bacteria, and Thiophaeococcus genus bacteria belonging to red sulfur bacteria, and red non-sulfur bacteria These include Rhodobaca genus bacteria, Rhodobacter genus bacteria, Rhodobium genus bacteria, Afifella (Rhodobium) genus bacteria, Rhodothalassium genus bacteria, Rhodovulum genus bacteria, and Roseospira genus bacteria. Among them, three marine red sulfur bacteria, Marichromatium bheemlicum, Thiohalocapsa marina and Thiophaeococcus mangrovi, and marine red non-sulfur bacteria Afifella pfennigii (Rhodobium pfennigii), Afifella marina rum (Rhodobium hodum Nine species of sulfidophilum, Rhodovulum tesquicola, Rhodovulum visakhapatnamense, Roseospira goensis and Roseospira marina are suitable as the red photosynthetic bacteria of the present invention. These red photosynthetic bacteria are RIKEN BioResource Center (Japan), NITE (Japan), ATCC (USA), DSMZ (Germany), NMLHC (Canada), ECACC (UK), NIBSC (UK), CNCM (France), CBS (Netherlands), BCCM (Belgium), ABC (Italy), NMI (Australia), CCTCC (China), CGMCC (China), etc.

“Polyhydroxyalkanoate” (often referred to herein as “PHA”) is a biopolyester known to accumulate in the cells as a stored energy of certain microorganisms. This is the target product in the production method. PHA can be represented by the following general formula 1.

[In the formula, R may be the same or different, and is a linear or branched alkyl group having 1 to 14 carbon atoms, n is an integer of 2 or more, preferably an integer of 100 or more, preferably Is an integer less than 100,000]
Specific examples of PHA include, but are not limited to, polyhydroxybutanoic acid (PHB: P (3HB)) (Poly-3-hydroxybutyrate) represented by Chemical Formula 1 below, and polyhydroxyvalerin represented by Chemical Formula 2 below. Acid (PHV: P (3HV)) (Poly-3-hydroxyvalerate), polyhydroxyhexanoic acid represented by Chemical Formula 3 (PHH: P (3HH)) (Poly-3-hydroxyhexanoate), Poly represented by Chemical Formula 4 Hydroxybutanoic acid-polyhydroxyvaleric acid (PHBV: P (3HB-co-3HV)) (Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)), polyhydroxybutanoic acid-polyhydroxyhexanoic acid represented by Formula 5 (PHBH: P (3HB-co-3HH)) (Poly (3-hydroxybutyrate-co-3-hydroxyhexanoate)), and poly-3-hydroxybutanoic acid-poly-4hydroxybutanoic acid (PHBB: P ( 3HB-co-4HB)) (Poly (3-hydroxybutyrate-co-4-hydroxybutyrate))) and the like.

[Wherein n is an integer of 2 or more, preferably an integer of 100 or more, preferably an integer of 100,000 or less]

[Wherein, m and n are integers of 2 or more, preferably are integers of 100 or more, and are preferably integers of 100,000 or less]

[Wherein, m and n are integers of 2 or more, preferably are integers of 100 or more, and are preferably integers of 100,000 or less]
The PHA produced by the production method of the present invention is not particularly limited. For example, it may be a homopolymer such as P (3HB) composed of a single monomer of 3HB, or P (3HB-co-3HV) or P (3HB-co-3HH) composed of two or more monomers. Such a copolymer may be used. A copolymer is preferred.
2. Method The PHA production method of the present invention includes a culture step as an essential step, and includes a recovery step and an extraction step as selection steps. Hereinafter, each step will be specifically described.
2-1. Culturing process In the present specification, the "culturing process" is a process for increasing the number of bacterial cells by cultivating red photosynthetic bacteria under specific conditions and accumulating PHA in the bacterial cells.

The above “specific conditions” are stirring of the liquid medium during culturing and irradiation of the cells with far red light.

In this specification, “stirring” refers to flowing a liquid. The stirring method is not particularly limited as long as the liquid can be flowed. For example, a method in which a liquid is swirled by stirring with a stirrer or a stirring bar, a method in which a container containing liquid is shaken using a shaker (including a reversing method), a liquid in a flow path by a liquid feeding device The method of flowing through is mentioned. Stirring aims to homogenize the components and cells in the medium. This makes it possible to uniformly apply nutrients and irradiated light to each microbial cell.

In this specification, “far red light” (Far Red) means light having a peak wavelength range of 700 nm to 860 nm. The “peak wavelength” is a wavelength at which the light emission intensity is maximum. The far-red light irradiated to the red photosynthetic bacteria in the culturing step may be light in the above-mentioned wavelength range, and is not limited, but suitable far-red light is light having a peak wavelength of 720 nm to 860 nm.

In the present invention, “irradiation” means exposure of red photosynthetic bacteria with the far-red light. Irradiation may be continuous or bright and dark light with a bright and dark period. The continuous irradiation here includes not only continuous irradiation but also irradiation of pulsed light that repeats the light and dark period in a very short time. In the case of light / dark cycle light, the light / dark cycle of the day is not particularly limited, but may be, for example, in the range of light period 8 hours: dark period 16 hours to light period 16 hours: dark period 8 hours. Alternatively, it may be bright or dark light in which a dark period (for example, a dark period of 1 second or more, preferably several seconds or more) that can be recognized by the naked eye is inserted periodically or aperiodically during the light period. Preferred irradiation is continuous irradiation. Note that the far-red light to be irradiated does not necessarily have to have a certain peak wavelength, and can be changed within the wavelength range during the irradiation period. For example, there is a case where light having a peak wavelength of 730 nm is irradiated at the start of irradiation, and the light is changed to light having a peak wavelength of 740 nm after the eighth day of irradiation.

The irradiance of far-red light may be in the range of 0.1 / m ² to 15 W / m ² . It is preferably 6 W / m ² to 10 W / m ² , more preferably 8 W / m ² ± 1 W / m ² .

The irradiation method is not particularly limited as long as the method can sufficiently expose the red photosynthetic bacteria in the culture solution. For example, a method of irradiating light to the entire culture tank by installing a light source around the transparent culture tank, irradiating the culture tank with light from one or more directions, and stirring the culture solution in the culture tank And a method of passing light over the whole culture solution. The light irradiation is preferably direct irradiation from a light source, but may be indirect irradiation via a reflector or the like for the purpose of reaching the entire culture solution. In the case of indirect irradiation, since the irradiated light may be attenuated, it is preferable to increase the light intensity of the light source as necessary so that the light intensity is in the above-described range.

Light irradiation is a period of growth culture of red photosynthetic bacteria (a “growth step” period described later) and / or a period of promoting biosynthesis and intracellular accumulation of PHA by the red photosynthetic bacteria (a “PHA accumulation step” period described later) You can go to Irradiation can be performed throughout the entire period of the PHA production method of the present invention. Preferred irradiation periods are a growth step period and a PHA accumulation step period.

The light source used in this step is not particularly limited as long as it is a light source capable of emitting light in the wavelength range. For example, a white light emitting diode (Light Emitting Diode: hereinafter referred to as “LED”), a high intensity discharge (HID: High Intensity Discharge) lamp, a halogen, which can emit white light including far-red light Light bulbs, incandescent light bulbs, sunlight, and the like can be mentioned. In consideration of continuous irradiation and power costs, it is preferable and effective to use an LED having a peak wavelength in the wavelength range of 700 nm to 860 nm, preferably in the wavelength range of 720 nm to 860 nm.

In this step, the culture conditions other than the above-mentioned “specific conditions” are not limited, and may be performed according to a method known in the bacterial culture field.

For example, the medium used in this step is not particularly limited as long as it is a growth medium that can culture red photosynthetic bacteria. For example, in the case of a red sulfur bacterium, 0.05% KH ₂ PO ₄ , 1.5 × 10 ^-3 % CaCl ₂ · 2H ₂ O, 0.2% MgSO ₄ · 7H ₂ O, 6.4 × 10 ^-3 % NH _{4 is} used as a growth medium. Cl, 2% NaCl, 0.3% Sodium pyruvate, 0.04% yeast extract, 2 × 10 ^-4 % Vitamin B12, 2.4 × 10 ^-3 % Na ₂ S ・ 9H ₂ O, 0.15% Na ₂ S ₂ O ₃・ 5H ₂ O, 0.15% FeCl ₂・ 6H ₂ O, 7 × 10 ^-3 % ZnCl ₂・ 5H ₂ O, 0.01% MnCl ₂・ 4H ₂ O, 6.2 × 10 ^-3 % H ₃ BO ₃ , 1.9 × 10 ^-3 % CoCl ₂ · 6H ₂ O, 1.7 × 10 ^-3 % CuCl ₂ · 2H ₂ O, 2.4 × 10 ^-3 % NiCl ₂ · 6H ₂ O, 3.6 × 10 ^-3 % Na ₂ MoO ₄ · H ₂ O (medium In addition to growth medium A having a composition of pH 7.5), a commercially available medium such as Marine Agar 2216 from Difco can be used. For red non-sulfur bacterial growth, 0.05% KH ₂ PO ₄ , 2.5 × 10 ^-3 % CaCl ₂ · 2H ₂ O, 0.3% MgSO ₄ · 7H ₂ O, 6.8 × 10 ^-3 % NH ₄ Cl , 2% NaCl, 0.3% Sodium malate, 0.3% Sodium pyruvate, 0.04% East extract, 5 × 10 ^-4 % Iron citrate, 2 × 10 ^-4 % Vitamin B12, 7 × 10 ^-3 % ZnCl ₂・ 5H ₂ O, 0.01% MnCl ₂・ 4H ₂ O, 6 × 10 ^-3 % H ₃ BO ₃ , 0.02% CoCl ₂・ 6H ₂ O, 2 × 10 ^-3 % CuCl ₂・ 2H ₂ O, 2 Growth medium B having a composition of × 10 ⁻³ % NiCl ₂ .6H ₂ O, 4 × 10 ⁻³ % Na ₂ MoO ₄ .H ₂ O (medium pH 6.8) can be used.

The culture temperature may be in the temperature range where the red photosynthetic bacteria can grow and / or proliferate. For example, the culture temperature disclosed by each depository that has obtained red photosynthetic bacteria can be used. The temperature may be in the range of 23 ° C to 32 ° C, and usually in the range of 28 ° C to 30 ° C.

When inoculating red photosynthetic bacteria in the medium at the start of this step, the number of cells is not particularly limited, but the cells have an OD ₆₆₀ value in the range of 0.05 to 0.3, preferably 0.05 to 0.15 after inoculation. Any number is acceptable.

The culture process can include a growth step, a cell recovery step, and a PHA accumulation step as necessary. The culture process including this process is characterized in that different media are used in the growth step and the PHA accumulation step. Other conditions relating to the culture may in principle be the same as those already described. Therefore, the description of the described culture conditions is omitted here, and the characteristic points of each step are specifically described below.
(1) Proliferation step The “proliferation step” is a step of cultivating red photosynthetic bacteria in a growth medium, and mainly aims at increasing the number of bacteria in the culture solution.

The medium used in this step is a growth medium. As the growth medium, any medium can be used as long as the red photosynthetic bacteria can be cultured, and the type and composition of the medium are not particularly limited. In this field, a medium known for red photosynthetic bacteria can be used. For example, the above-mentioned growth medium A for red sulfur bacteria and growth medium B for red non-sulfur bacteria can be mentioned. Commercially available media can also be used.

The culture period of this step is preferably until the inoculated red photosynthetic bacterium reaches the log-phase, preferably the mid-log phase to the late-log phase. Specifically, the OD ₆₆₀ value of the culture solution is in the range of 0.5 to 4.0, 0.5 to 2.0, 0.5 to 1.5, or 0.6 to 1.2, preferably 0.8 to 1.1, more preferably 0.9 to 1.0. It is sufficient to culture until the range of
(2) Cell recovery step The “cell recovery step” is a step of recovering the red photosynthetic bacteria in the culture solution after the growth step, and the red photosynthetic bacteria cultured in the growth step are used as a PHA production medium in the PHA accumulation step. The purpose is to remove as much growth medium and coarse substances as possible from the culture medium.

However, this step is a selection step that may be performed as needed, unlike the other two steps. That is, a part of the culture solution after the growth step can be directly transplanted to the PHA production medium of the PHA accumulation step without collecting the red photosynthetic bacteria after the growth step.

As a method for recovering the bacterial cells from the culture solution, a conventional method known in the art may be used. For example, a method of removing the culture medium by centrifuging or filtering the culture solution can be mentioned. Specifically, after centrifuging the culture solution with an appropriate gravitational acceleration (g) for an appropriate period of time using a centrifuge, the supernatant is removed and the cells are collected, or the pore size is smaller than the cell size of red photosynthetic bacteria. There is a method of removing the culture solution by filtering through a filter having the above and recovering the cells remaining on the filter. The collected cells may be washed once or more with the medium used in the culturing step, or with a buffer or physiological saline having the same osmotic pressure as the medium as necessary.

The cells collected in this step are used in the next PHA accumulation step.
(3) PHA accumulation step The “PHA accumulation step” is a step in which the red photosynthetic bacteria cultured in the growth step are transferred to the PHA production medium and cultured, and it promotes the biosynthesis and intracellular accumulation of PHA by the bacteria. Objective.

The medium used in this step is a PHA production medium. This medium is a medium in which the carbonate source of the growth medium is replaced with an organic acid salt and / or carbonate. A medium in which the carbonic acid source of the growth medium is replaced with an organic acid salt, or an organic acid salt and a carbonate is preferable. Hydrate may be sufficient as organic acid salt and carbonate.

Organic acid salts include, for example, alkanoates (including formate, acetate, propionate, butyrate, valerate, and caprate), citrate, malate, sulfonate , Tartrate, and lactate. Acetate is particularly preferred. Examples of acetates include sodium acetate and calcium acetate. Examples of carbonates include sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate and the like.

As an example of a PHA production medium, in the growth medium A for red sulfur bacteria, a PHA production medium for red sulfur bacteria in which sodium pyruvate as a carbon source is replaced with 0.1% sodium bicarbonate and 0.5% sodium acetate, Examples of the growth medium B for non-sulfur bacteria include a PHA production medium for red non-sulfur bacteria in which sodium malate and sodium pyruvate, which are carbon sources, are substituted with 0.1% sodium bicarbonate and 5 g / L sodium acetate.

The PHA production medium may be a deficient medium in which any one or more of a nitrogen source, a phosphate source, and vitamins are further removed from the growth medium in addition to the carbon source replacement. Specific examples include a nitrogen-deficient medium, a phosphate-deficient medium, a vitamin-deficient medium, a nitrogen / phosphate-deficient medium, a nitrogen / vitamin-deficient medium, a phosphoric acid / vitamin-deficient medium, and a nitrogen / phosphate / vitamin-deficient medium. The type of vitamin is not particularly limited, but vitamin B1 (thiamine), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, and pyridoxamine), vitamin B7 (biotin), and vitamin B12 (cobalamin) are preferable. Multiple vitamins may be depleted.

This is because PHA production in photosynthetic bacteria is known to be promoted by restriction of nitrogen, phosphate, and / or vitamins (eg, H. Brandl, et al., 1989, Int J Biol Macromol). . 11, 49-55.).

In this step, the cells recovered after the growth step are appropriately diluted as necessary, inoculated into a PHA production medium, and cultured. The number of cells in the medium at the start of this step is not limited, but the OD ₆₆₀ value of the culture solution is in the range of 0.03-2.0, in the range of 0.03-1.5, in the range of 0.03-1.0, or in the range of 0.05-0.8, preferably Adjustment may be made so as to be in the range of 0.1 to 0.5, more preferably in the range of 0.1 to 0.3.

During the culture period, the inoculated red photosynthetic bacterium is in the late logarithmic growth phase or early stationary phase, specifically, the OD ₆₆₀ value of the culture solution is in the range of 0.8 to 2.0, or in the range of 0.8 to 1.2, The culture is preferably performed until the range is 0.8 to 1.0.
2-2. Recovery step The “recovery step” is a step of recovering red photosynthetic bacteria from the culture solution in the culture step. The purpose of this step is to recover the red photosynthetic bacteria in which PHA is accumulated in the cells by removing the medium and coarse substances from the culture solution in the selection step.

The method for recovering the bacterial cells from the culture solution is in accordance with the method described in the aforementioned bacterial cell recovery step.

This step makes it possible to obtain red photosynthetic bacteria that accumulate PHA in the cells as a slurry. If necessary, the collected red photosynthetic bacteria may be dried by a known drying method.

The drying method is not particularly limited as long as moisture can be reduced. For example, a ventilation drying method in which hot or cold air is applied using a blower, a windless drying method in which moisture is evaporated by heating, a slurry is suspended in an appropriate buffer, and then the suspension is sprayed into a gas to rapidly Spray drying to dry, freeze drying (freeze drying) method, vacuum drying method to deaerate in a sealed container using a vacuum pump etc., natural drying method to leave exposed to the outside air (including sun drying), or a combination thereof Can be mentioned. When the cells are actually dried, various drying apparatuses to which the above method is applied may be used. For example, a drum dryer, a far-red band dryer, a continuous vacuum dryer, a spray dryer, a freeze dryer, and the like can be given.

The dried algal body may be in a solid state, a granule state, or a powder state.
2-3. Extraction Step The “extraction step” is a step of extracting PHA from the culture solution after the culturing step, or the red photosynthetic bacteria (including cell debris) in the culture solution or after the recovery step. This step is a selection step and aims to collect PHA accumulated in cells of red photosynthetic bacteria.

Any method known in the art can be used as the method for extracting PHA from the culture solution or red photosynthetic bacteria cells in this step.

For example, when extracting PHA accumulated in cells of red photosynthetic bacteria, there is a method of disrupting cells by a physical method and / or chemical method and isolating PHA from the obtained cell extract. It is done. The cell extract here includes a solid extract and a liquid extract (including a cell extract mixed in a culture solution).

Examples of the physical method for crushing bacterial cells include a crushing / squeezing method, an ultrasonic method, an osmotic shock method, or a combination thereof.

Examples of the chemical method for crushing microbial cells include an alkali method and an enzyme method. Specific methods for lysing bacterial cells are described in, for example, Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Please refer to the method.

The obtained cell extract may be subjected to treatment for denaturing and removing proteins such as cell disruption as necessary.

The method for separating and purifying PHA from the cell extract is not particularly limited. For example, an organic solvent in which PHA can be dissolved is added to the cell extract as an extraction solvent and mixed, and then another organic solvent having a lower solubility than that of the extraction solvent is added as a precipitation solvent. There is a method to precipitate and purify PHA. Specifically, for example, chloroform may be added to the cell extract as an extraction solvent to dissolve PHA, and then hexane may be added as a precipitation solvent to recover the precipitated PHA. As the extraction solvent or the organic solvent as the precipitation solvent, alcohols such as methanol and ethanol, and halogenated hydrocarbons such as hexane, acetone, chloroform, and 1,2-dichloroethane can be used. In addition, separation and purification may be performed using a known purification method described in JP-A-2005-348640.

If the PHA is leaked or discharged out of the cells of the red photosynthetic bacteria and contained in the culture solution, the extraction solvent is directly added to the culture solution, mixed, centrifuged, and the organic solvent layer is recovered. Thereafter, a precipitation solvent is added, and the precipitated PHA may be recovered in the same manner as described above.

(the purpose)
Using the PHA production method of the present invention, it was confirmed that the target PHA was accumulated in the cells by culturing red photosynthetic bacteria.
(Method)
1. Red photosynthetic bacteria The red photosynthetic bacteria in this example were obtained from the depository institution, including 16 marine red sulfur bacteria shown in Table 1 below and 17 marine red non-sulfur bacteria shown in Table 2 in total. Used. Strains with “JCM” code in the “Resource number” in the table are from RIKEN BioResource Center (Japan), strains with “DSM” code are from DSMZ (Germany), and strains with “ATCC” code are Obtained from ATCC (USA). The “growth temperature” in the table is the culture temperature disclosed by each depository organization.

Among the above 33 strains, as a result of culturing by the following culture method using the following growth medium, good growth was observed within a relatively short period of 3 to 7 days on at least one of the liquid medium and the agar medium. 12 strains (3 strains of red sulfur bacteria, 9 strains of red non-sulfur bacteria: strains with a star in the species name in Tables 1 and 2) were selected and used for analysis on PHA production ability.
2. Bacterial culture The red photosynthetic bacteria in this example were cultured by the following medium and culture method.
(1) Medium / growth medium A (for red sulfur bacteria growth)
(composition)
KH ₂ PO ₄ : 0.5g / L, CaCl ₂・ 2H ₂ O: 0.15g / L, MgSO ₄・ 7H ₂ O: 2.0g / L, NH ₄ Cl: 0.64g / L, NaCl: 20g / L, pyruvin sodium acid: 3.0g / L, yeast extract: 0.4g / L, vitamin _{B12: 2mg / L, Na 2} S · 9H 2 O: 240mg / L, Na 2 S 2 O 3 · 5H 2 O: 1.5g / L , FeCl ₂・ 6H ₂ O: 1.5g / L, ZnCl ₂・ 5H ₂ O: 70mg / L, MnCl ₂・ 4H ₂ O: 100mg / L, H ₃ BO ₃ : 62mg / L, CoCl ₂・ 6H ₂ O : 190mg / L, CuCl ₂・ 2H ₂ O: 17mg / L, NiCl ₂・ 6H ₂ O: 24mg / L, Na ₂ MoO ₄・ H ₂ O: 36mg / L
The pH of the medium was adjusted to 7.5.
・ Growth medium B (for red non-sulfur bacterial growth)
(composition)
KH ₂ PO ₄ : 0.5g / L, CaCl ₂・ 2H ₂ O: 0.25g / L, MgSO ₄・ 7H ₂ O: 3.0g / L, NH ₄ Cl: 0.68g / L, NaCl: 20g / L, Apple sodium acid: 3.0 g / L, sodium pyruvate: 3.0 g / L, yeast-extract: 0.4 g / L, ferric citrate: 5 mg / L, vitamin _{B12: 2mg / L, ZnCl 2} · 5H 2 O: 70 mg / L, MnCl ₂・ 4H ₂ O: 100 mg / L, H ₃ BO ₃ : 60 mg / L, CoCl ₂・ 6H ₂ O: 200 mg / L, CuCl ₂・ 2H ₂ O: 20 mg / L, NiCl ₂・ 6H ₂ O: 20mg / L, Na ₂ MoO ₄・ H ₂ O: 40mg / L
The pH of the medium was adjusted to 6.8.
・ PHA production medium A (for red sulfur bacteria PHA production)
(composition)
NH ₄ Cl and sodium pyruvate were removed from the growth medium A, and 1 g of sodium bicarbonate (NaHCO ₃ ) and / or 5 g of sodium acetate (CH ₃ COONa) were added as a carbon source per liter. The pH of the medium was adjusted to 7.5 similarly to the growth medium A.
・ PHA production medium B (for production of red non-sulfur bacteria PHA)
(composition)
NH ₄ Cl, sodium malate and sodium pyruvate were removed from the growth medium B, and 1 g of sodium bicarbonate (NaHCO ₃ ) and / or 5 g of sodium acetate (CH ₃ COONa) was added per liter as a carbon source. The pH of the medium was adjusted to 6.8 similarly to the growth medium B.
(2) Cultivation method / Proliferation culture Each red photosynthetic bacterium is a multi-position stirrer (remote-light) at 30 ° C under far-red LED light conditions (peak wavelength 730nm, irradiance 8W / m ² ). Incubation was performed in a screw cap bin (IWAKI) using Multi 60 type Isis) with continuous shaking.
PHA production culture Red sulfur bacteria were cultured in growth medium A and red non-sulfur bacteria were grown in growth medium B under growth culture conditions, and bacterial cells were collected at the time of logarithmic phase (OD ₆₆₀ = about 1.0). The collected cells are washed with PHA production medium, diluted with PHA production medium so that the starting OD ₆₆₀ is 0.1, and then continuously shaken in a screw cap bottle at 30 ° C using a multi position stirrer. The culture was continued for 1 week. Cells were collected after 1 week, washed with 20 mM Tris-HCl (pH 7.0), and lyophilized.
3. Analysis of Polymer Amount and Composition Approximately 0.5-2 mg of lyophilized cells was incubated for 1 hour at 70 ° C. in 100% ethanol to remove the dye. Using these cells, ethanolysis (ester exchange reaction using ethanol) was performed at 100 ° C. for 4 hours in the presence of 250 μL of chloroform, 100 μL of hydrochloric acid, and 850 μL of ethanol. After cooling, phosphate buffer (pH 8.1) was added to the reaction mixture and neutralized with 0.65N NaOH. After centrifugation at 1,500 rpm for 5 minutes, the chloroform layer was filtered through anhydrous sodium sulfate and incubated on a molecular sieve for 30 minutes. The amount and composition of PHA was determined using a GCMS-QP2010 Ultra (Shimadzu) equipped with a 30 m × 0.25 mm DB-1 capillary-gas chromatography column (Agilent Technologies).

Helium as a carrier gas was injected into 1 μL of the sample solution at 3.30 mL / min. The ethyl ester was separated using a temperature program that increased to 117 ° C. with a temperature ramp of 7 ° C./min at 45 ° C. for 1 minute. The interface temperature and ion source temperature were 250 ° C. and 230 ° C., respectively. The amount of 3HB was determined from a calibration curve. The relative amount of 3HV was estimated from the constituent polarity of 3HB. The amount of PHA was calculated as% dry cell weight.
4). Extraction of PHA PHA was extracted from lyophilized cells in chloroform. The extract was filtered using filter paper (5C ADVANTEC) and concentrated using a rotary vacuum evaporator (As One). Thereafter, PHA purification was performed by precipitating chloroform-extracted PHA with hexane. The PHA precipitate was filtered and then air dried overnight. PHA dissolved in chloroform was concentrated again using a rotary vacuum evaporator (As One) and purified by precipitation with chilled methanol. The PHA precipitate was filtered and then air dried overnight. Purified PHA was dissolved in chloroform and used for further analysis.
5. Characterization of PHA Purified PHA was analyzed by proton nuclear magnetic resonance ( ¹ H NMR) (JNM-Excalibur 270; JEOL, Ltd.) to determine the detailed chemical structure and composition. The NMR sample was dissolved in CDCl ₃ at a concentration of 4 mg / mL using 0.05 v / v% tetramethylsilane (TMS) (Wako Pure Chemical Industries, Ltd.). The molecular weight of PHA was determined by gel permeation chromatography (GPC) (RI-2031, PU-2086, AS-2055, CO-2056; JASCO) equipped with Shodex K-806M, K802 and KG columns at 40 ° C. . Chloroform was used as the mobile phase with a flow rate of 0.8 mL / min. The concentration of purified PHA was about 1 mg / mL. Its molecular weight is 1.32 × 10 ³ , 3.25 × 10 ³ , 1.01 × 10 ⁴ , 2.85 × 10 ⁴ , 6.60 × 10 ⁴ , 1.56 × 10 ⁵ , 4.60 × 10 ⁵ , 1.07 × 10 ⁶ , and 3.15 × 10 Estimated by comparison with ⁶ polystyrene standards.
(result)
The results are shown in Tables 3 and 4 and FIGS.

Table 3 shows that when 12 selected red photosynthetic bacteria were cultured in the growth medium only (Growth condition), and after culturing in the growth medium, NH ₄ Cl as a nitrogen source was removed from the growth medium, and the carbon source was 0.1%. Amount of PHA per dry cell weight when cultured in PHA production medium (w / o NH ₄ Cl; 0.1% NaHCO ₃ + 0.5% accetate) substituted with 5% sodium bicarbonate and 0.5% sodium acetate, and The composition of the polymer in PHA is shown.

Table 4 shows PHA production medium (w) in which 12 selected red photosynthetic bacteria were cultured in a growth medium, NH ₄ Cl as a nitrogen source was removed from the growth medium, and the carbon source was replaced with only 0.1% sodium bicarbonate. / o NH ₄ Cl; 0.1% NaHCO ₃ ), and PHA production medium (w / o NH ₄ Cl; 0.5% accetate) substituted only with 0.5% sodium acetate. The amount of PHA and the composition of the polymer in PHA are shown.

It turned out that all the red photosynthetic bacteria examined as shown in the table can accumulate PHA.

Three kinds of red sulfur bacteria and three kinds of red non-sulfur bacteria Afifella marina, Rhodovulum tesquicola and Roseospira visakhapatnamensis were able to confirm significant PHA accumulation under nitrogen restriction compared to growth conditions. For the other 6 species, no change in PHA accumulation due to nitrogen limitation was observed.

Moreover, the red sulfur photosynthetic bacterium verified in this example produced a monopolymer (monopolyester) composed only of 3-hydroxybutanoic acid (3HB) as PHA. On the other hand, it became clear that all the red non-sulfur photosynthetic bacteria verified in this example produced a copolyester composed of 3-hydroxybutanoic acid (3HB) and 3-hydroxyvaleric acid (3HV).

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

A method for producing polyhydroxyalkanoic acid using red photosynthetic bacteria,
The said production method including the culture | cultivation process of irradiating far-red light, stirring a liquid culture medium, and culture | cultivating a red photosynthetic bacterium.
The production method according to claim 1, wherein the far-red light is light having a peak wavelength at 720 nm to 860 nm.
The culturing step comprises:
A growth step of cultivating the red photosynthetic bacteria in a growth medium;
A cell recovery step for recovering red photosynthetic bacteria in the culture solution after the growth step, and a polyhydroxyalkane in which the recovered red photosynthetic bacteria are cultured in a polyhydroxyalkanoic acid production medium using an organic acid and / or carbonic acid as a carbon source. The production method according to claim 1, comprising an acid accumulation step.
The production method according to claim 3, wherein in the growth step, the red photosynthetic bacteria are cultured until the logarithmic growth phase.
The production method according to claim 3 or 4, wherein in the polyhydroxyalkanoic acid accumulation step, the red photosynthetic bacteria are cultured until the late logarithmic growth phase or the early stationary phase.
The production method according to any one of claims 3 to 5, wherein the polyhydroxyalkanoic acid production medium is a medium in which the carbon source of the growth medium is replaced with an organic acid salt and / or carbonate.
The production method according to any one of claims 3 to 6, wherein the polyhydroxyalkanoic acid production medium is deficient in any one or more of a nitrogen source, a phosphate source, and a vitamin.
The production method according to any one of claims 1 to 7, further comprising a recovery step of recovering bacterial cells contained in the culture solution after the culture step.
The production method according to any one of claims 1 to 8, further comprising an extraction step of extracting polyhydroxyalkanoic acid from the culture solution after the culturing step or the red photosynthetic bacterium.
The production method according to any one of claims 1 to 9, wherein the red photosynthetic bacterium is a marine photosynthetic bacterium.
Red photosynthetic bacteria are selected from Marichromatium he bheemlicum, Thiohalocapsa marina, Thiophaeococcus mangrovi, Afifella pfennigii, Afifella marina, Rhodovulum euryhalinum, Rhodovulum imhoffii, Rhodovulum sulfidophilum, Rhodovum 項The production method described in.
Purple photosynthetic bacteria Marichromatium bheemlicum JCM13911, Thiohalocapsa marina JCM14780, Thiophaeococcus mangrovi JCM14889, Afifella pfennigii ATCC BAA1145, Afifella marina DSM2698, Rhodovulum euryhalinum DSM4868, Rhodovulum imhoffii JCM13589, Rhodovulum sulfidophilum ATCC35886, Rhodovulum tesquicola ATCC BAA1573, Rhodovulum visakhapatnamense JCM13531, Roseospira goensis JCM14191 and The production method according to claim 11, which is selected from the group consisting of Roseospiraomarina ATCC BAA447.