JP5555975B2 - Process for producing polyhydroxyalkanoate - Google Patents

Description

  The present invention relates to a method for producing polyhydroxyalkanoate (PHA) and PHA, and in particular, PHA which does not contain 3-hydroxybutyrate (3HB) as a monomer unit and has a glass transition temperature (Tg) of −48 ° C. or less, and The present invention relates to a method for producing PHA using a by-product of biodiesel fuel as a carbon source.

  Biodegradable polyesters are known to be produced in many microorganisms by using renewable bioorganic resources (biomass) such as sugars and vegetable oils as raw materials and accumulated in the bodies of microorganisms. Among them, PHA is a polyester that is attracting attention because it exhibits thermoplasticity like chemical synthetic plastics and has excellent biodegradability and biocompatibility, and more than 90 types of monomer structures have been confirmed in polymers. ing.

  As typical PHA produced by microorganisms in this way, there is one having 3HB as a monomer unit. PHA {P (3HB)} having 3HB as a monomer unit has high crystallinity, is hard and brittle. It is said that it has poor practicality. In order to solve such problems, various attempts have been made to improve the properties of PHA.

  In particular, PHA composed of monomer units other than 3HB is a microorganism that has been transformed using a genetic recombination technique, by changing the type, medium composition, culture conditions, etc. of microorganisms that produce PHA, or by newly separating microorganisms. Manufacture is realized by using, for example.

  For example, JP 4-504205 A discloses a PHA composed of repeating units having 6 to 10 carbon atoms by culturing Pseudomonas fluorescens rRNA branched bacteria in a predetermined nutrient medium. A manufacturing method is disclosed (Patent Document 1).

  In JP-A-7-031490, a newly isolated Pseudomonas sp. 31-1 strain is used to form monomer units 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO). ), 3-hydroxydecanoate (3HD), or 3-hydroxydodecanoate (3HDD). And it is disclosed that the number average molecular weight of the copolymer is 110,000-290,000, and glass transition temperature is -44 degreeC--34 degreeC (patent document 2).

  In JP 2001-178484 A, a newly isolated Pseudomonas chicory eye strain H45 is cultured in a medium containing sodium acetate as a single carbon source, so that the number of carbon atoms is 6 (hereinafter referred to as “C6”). A method for producing a PHA containing at least one monomer unit of C8, C10, C12, and C14 is disclosed (Patent Document 3).

  Japanese Patent Application Laid-Open No. 2004-321167 discloses a method for producing PHA composed of C6 to C10 monomer units by culturing a predetermined transformed microorganism in a medium containing C6 to C10 as a carbon source. (Patent Document 4).

  On the other hand, with regard to biofuels, measures for promoting the use of biofuels are rapidly advancing, and biodiesel fuel (BDF) produced from rapeseed oil and waste cooking oil is attracting attention as an alternative to light oil. Has been. By-product (BDFB) is discharged about 20% during the production of BDF, but most of it is disposed of as industrial waste, and in recent years, an effective use method of BDFB has been studied.

  For example, Japanese Patent Laid-Open No. 2006-180782 reports that hydrogen and ethanol are produced from BDFB by a fermentation technique using microorganisms (Patent Document 5).

  On the other hand, fermentation synthesis of PHA by microorganisms is promoted when the growth of microorganisms is restricted by depleting nitrogen sources, inorganic salts, etc., which are nutrient sources other than carbon sources. A two-stage culture method comprising a stage and a second stage for accumulating PHA is employed.

  The first stage of the two-stage culture method is aimed at culturing and growing microorganisms in a nutrient-rich medium, so that an assimitable carbon source can be freely selected. In the second stage, the microorganisms obtained in the first stage are washed and recovered, and the microorganisms are cultured in a mineral medium to which a carbon source is newly added under the restriction of nutrient sources other than the carbon source, thereby inducing PHA production. To do.

  On the other hand, the one-stage culture method is a method of fermenting and synthesizing PHA by simultaneously performing growth of microorganisms and accumulation of PHA in a nutrient-rich medium containing a nitrogen source and a carbon source. Although this one-stage culture method can also produce PHA, there is a problem that the production amount is very small as compared with the conventional two-stage culture method.

  For example, Patent Document 2 discloses that a copolymer having 3HHx and 3HO as monomer units is produced by one-stage culture given sodium octanoate as a carbon source. Japanese Patent Laid-Open No. 2002-253285 discloses a method for producing PHA in one step by culturing using an inorganic medium obtained by adding a carbon compound and a natural medium component to a predetermined alkanoic acid. (Patent Document 6).

Japanese National Publication No. Hei 4-504205 Japanese Patent Laid-Open No. 7-031490 JP 2001-178484 A JP 2004-321167 A JP 2006-180782 A JP 2002-253285 A

  However, in the inventions described in Patent Documents 1 to 5, the Tg of the produced PHA is high temperature, and the number average molecular weight and weight average molecular weight are also different. Here, even if the types of monomer units constituting PHA are the same, PHA having completely different characteristics such as glass transition temperature, viscosity, adhesiveness, etc. are different due to different constitutional ratios and molecular weights of monomer units. Become. For example, Biopol D400G (manufactured by Monsanto) and Biopol D600G (manufactured by Monsanto) are polyhydroxybutyrate / valerate copolymers and have the same monomer unit, but have a glass transition temperature of 5 each. There is a difference of 15 ° C. Further, Patent Document 2 shows that the composition ratio of the produced PHA monomer units and the glass transition temperature differ by several degrees C., and thus have different properties such as rubber and oil.

  In addition, effective use of BDFB has just begun to be developed and is currently used to produce hydrogen, ethanol, and 1,3-propanediol, and can be used effectively to produce other substances. There have been no reports.

  Furthermore, the production of PHA by the two-stage culture method requires more facilities than the one-stage culture method, and accordingly, the work process becomes complicated and requires extra labor and time. Not suitable for production. In addition, the conventional method for producing PHA by the one-stage culture method such as the invention described in Patent Document 2 has a problem that the production amount is less than that of the two-stage culture and the production amount is about half. was there. Furthermore, in the invention described in Patent Document 6, although PHA is produced by a one-stage culture method, there is a problem that it is necessary to add a carbon source compound and an expensive yeast extract.

  The present invention has been made to solve such a problem, and is a PHA having a Tg of −48 ° C. or less without containing 3HB, and it is not necessary to add an expensive yeast extract or the like. Can be used effectively as a carbon source, and PHA can be produced by microorganisms, and the produced PHA can be easily separated and recovered. It is an object of the present invention to provide a PHA and a method for producing PHA that can efficiently produce PHA in one-stage culture.

  The polyhydroxyalkanoate according to the present invention is characterized by at least one selected from the group consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate. It is a polyhydroxyalkanoate as a monomer unit and has a glass transition temperature of −48 ° C. or lower.

  Moreover, in this invention, it is preferable that the sum total of the composition rate of the said 3-hydroxy octanoate and the composition rate of the said 3-hydroxy decanoate is 70% or more.

And in this invention, the viscosity in -5 degreeC or more and 45 degrees C or less is 1.0 * 10 < ⁴ > mPa / s or more and 1.2 * 10 < ⁵ > mPa / s or less, Comprising: The said viscosity falls with a raise in temperature. It is preferable to do.

  Furthermore, in the present invention, the molecular weight distribution is preferably 1 to 2.

  On the other hand, the production method of the polyhydroxyalkanoate according to the present invention is characterized in that it is produced by a microbial fermentation method using a by-product of biodiesel fuel obtained by transesterification of fats and oils as a carbon source.

  Another feature of the method for producing a polyhydroxyalkanoate according to the present invention is that a microbial fermentation method uses a neutralized byproduct obtained by neutralizing a byproduct of biodiesel fuel obtained by transesterification of fats and oils as a carbon source. It is in the point to manufacture.

  Moreover, in this invention, it is preferable that the culture | cultivation in the said microbial fermentation method is a one-stage culture | cultivation which used the by-product of the said biodiesel fuel, or the said neutralization by-product for the culture medium.

  In the present invention, a polyhydroxyalkanoate in which the monomer unit of the first composition is 3-hydroxyoctanoate and the monomer unit of the second composition is 3-hydroxydecanoate, or the monomer unit of the first composition It is possible to selectively produce polyhydroxyalkanoates in which is 3-hydroxydecanoate and the monomer unit of the second composition is 3-hydroxyoctanoate by controlling the culture time.

  In the present invention, the microorganism in the microorganism fermentation method is preferably Ralstonia eutropha or polyhydroxyalkanoate-producing recombinant Escherichia coli.

  Furthermore, in this invention, it is preferable to manufacture polyhydroxyalkanoate which does not contain 3-hydroxybutyrate as a monomer unit by using Pseudomonas putida (Pseudomonas putida) as a microorganism in the said microorganism fermentation method.

  Moreover, in this invention, it is preferable that culture | cultivation in the said microbial fermentation method is performed by a fed-batch culture method or a continuous culture method.

  According to the present invention, PHA can be easily and effectively obtained by a production method adapted to industrial production by effectively using BDFB that has been treated as industrial waste. And since obtained PHA does not contain 3HB and Tg is -48 degrees C or less, it has adhesiveness without losing fluidity also in a cold region, and it utilizes for adhesives, binder resin, etc. be able to. In addition, since the composition ratio of PHA can be adjusted by appropriately selecting the culture time, PHA having desired properties can be produced according to the purpose of use.

  Hereinafter, the PHA and the method for producing PHA according to the present invention will be described based on the method for producing PHA and PHA in the present embodiment.

  First, the PHA according to the present invention will be described. The PHA according to the present invention has at least one selected from the group consisting of 3HHx, 3HO, 3HD, and 3HDD as a monomer unit. That is, the monomer unit in the present invention is one to four selected from four types of monomer units of 3HHx, 3HO, 3HD, and 3HDD, and does not include monomer units other than the four types of monomer units. However, as a whole PHA, “at least one selected from the group consisting of 3HHx, 3HO, 3HD, and 3HDD is used as a monomer unit” and at least one selected from the group consisting of “3HHx, 3HO, 3HD, and 3HHD” Also included is a PHA having a PHA as a part of “one monomer unit”. The PHA in this embodiment is composed of 3HHx, 3HO, 3HD, and 3HDD as shown in FIG. 3, FIG. 4, FIG. 5, or FIG.

  Here, in the present invention, “being as a monomer unit” means not only the case where all of the selected monomer units constitute the PHA, but also, for example, entangled with the chain of the PHA without constituting the PHA. This also includes the case where it remains as a monomer.

  The PHA according to the present invention has a glass transition temperature (Tg) of −48 ° C. or lower. Here, Tg refers to a temperature at which a substance such as glass, which is liquid at a high temperature, suddenly increases its viscosity in a certain temperature range due to a drop in temperature, almost loses fluidity and becomes a non-solid solid. At a temperature higher than the glass transition temperature, the thermal motion of each part of the chain polymer forming the network structure is intense and exhibits rubber-like elasticity, but below the glass transition temperature, the thermal motion is suppressed and hardened by a decrease in free volume. .

  Note that the Tg of PHA in this embodiment varies depending on the carbon source of the microorganism that produces PHA. As shown in FIG. 7, when purified glycerin is used as the carbon source, the Tg of −48.6 ° C. to −57.3 ° C. When the by-product (BDFB) of biodiesel fuel obtained by transesterification of fats and oils is used as a carbon source, the neutralized by-product (neutralized) is neutralized by -5FB to -60.9 ° C. When BDFB) is used as a carbon source, the temperature is -49.0 ° C to -57.7 ° C.

  Further, the composition ratio of the PHA according to the present invention is not particularly limited as long as at least one selected from the group consisting of 3HHx, 3HO, 3HD, and 3HDD is used as a monomer unit. 3, FIG. 4, FIG. 5, or FIG. 6, among the monomer units, the sum of the composition ratio of 3HO and the composition ratio of 3HD is 70% or more. In addition, as shown in FIG. 3 or FIG. 7, the molecular weight distribution of PHA in this embodiment is preferably 1 to 2, more preferably 1.5 to 2, even more preferably from the viewpoint of physical properties. Is 1.7 to 2.

Moreover, the viscosity of the PHA according to the present invention preferably has a property that the viscosity changes in a temperature-dependent manner, and in particular, the viscosity decreases with increasing temperature. As shown in FIG. 9 or FIG. 10, the PHA in this embodiment has a viscosity at −5 ° C. or more and 45 ° C. or less of about 1.0 × 10 ⁴ mPa / s or more and about 1.2 × 10 ⁵ mPa / s or less. Thus, the viscosity decreases with increasing temperature. That is, since the viscosity of the PHA according to the present invention changes depending on temperature, it can be easily processed, and can be used for, for example, an adhesive or a binder resin.

  Next, a method for producing PHA according to the present invention will be described. The method for producing PHA according to the present invention uses purified glycerin, biodiesel fuel by-product (BDFB) and neutralized by-product (neutralized BDFB) neutralized by BDFB obtained by transesterification of oil and fat. It is a production method using a microbial fermentation method as a source. In particular, from the viewpoint of environmental protection, cost, adaptability to industrial production, and the like, a production method by microbial fermentation using BDFB or neutralized BDFB as a carbon source is preferable.

  Here, BDFB will be described in detail. BDFB is produced as a by-product when producing biodiesel fuel (BDF), and BDFB used in the present embodiment contains glycerin and fatty acids as main components as shown in FIG. BDFB used in the method for producing PHA according to the present invention is not limited as long as it is produced as a by-product when producing BDF from fats and oils, for example, organic impurities derived from other foods, ash, minerals, etc. It may have other compositions.

  BDFB is generally obtained by adding alcohol and an alkali catalyst such as potassium hydroxide to fats and oils such as rapeseed oil and waste edible oil, and causing alcoholysis to cause a transesterification reaction. It can be obtained at the same time. Therefore, BDFB has alkalinity. Therefore, when producing PHA, BDFB can be produced by a microbial fermentation method using BDFB as a carbon source, and neutralized BDFB can also be produced using a carbon source.

  In the present embodiment, as shown in FIG. 7 or FIG. 8, when purified glycerin is used as a carbon source, solid PHA can be produced at room temperature (up to about 40 ° C.). When Japanese BDFB is used as a carbon source, PHA having fluidity at room temperature can be produced.

  In the method for producing PHA according to the present invention, purified glycerin, BDFB, or neutralized BDFB may be used as it is as the only carbon source, and fatty acid, sugar, etc. are used as the carbon source for purified glycerin, BDFB, neutralized BDFB. May be used.

  In addition, in the method for producing PHA according to the present invention, any method can be used as long as it can cultivate microorganisms and produce PHA by using purified glycerin, BDFB, or neutralized BDFB as a carbon source in the medium. You may employ | adopt the culture | cultivation which passes through not only a culture | cultivation and a two-stage culture but more steps (step). The two-stage culture can be performed in a single culture apparatus by using a medium prepared such that nitrogen sources and inorganic salts to be restricted in the medium are consumed and deficient by the growth of microorganisms. is there. Therefore, the one-stage culture according to the present invention is a culture in which PHA is fermented and synthesized in one culture stage, and the growth of microorganisms and the accumulation of PHA in a nutrient-rich medium in which a nitrogen source and a carbon source are added. By using not only the method of fermenting and synthesizing PHA by carrying out at the same time, but also using a medium prepared as described above so that nitrogen sources and inorganic salts to be restricted in the medium are consumed and deficient by the growth of microorganisms. The culture performed in the culture apparatus is also included.

  For example, Pseudomonas putida (Pseudomonas putida) is a microorganism that normally requires two-stage culture to produce PHA. However, by using BDFB or neutralized BDFB as a carbon source, there is no decrease in production. The PHA containing 3HB as a monomer unit can be produced by one-stage culture. As described above, since the one-stage culture can be performed, the work process and necessary equipment can be reduced. Therefore, it can be said that the method for producing PHA according to the present invention is applied to industrial production.

  In the method for producing PHA according to the present invention, PHA having two kinds of composition ratios can be selected and produced by controlling the culture time. That is, by setting the culturing time to be shorter than a certain time, it is possible to produce a PHA in which 3HO is a monomer unit having the first composition and 3HD is a monomer unit having the second composition. If it is set longer, a PHA in which 3HD is a monomer unit of the first composition and 3HO is a monomer unit of the second composition can be manufactured. Since the two types of PHA have different composition ratios, they become PHA having different properties such as viscosity, adhesiveness, and shearing characteristics, and it is possible to select and manufacture a PHA having a desired property according to the usage method. It is.

  Furthermore, in the method for producing PHA according to the present invention, there is no particular limitation as long as it is a microorganism capable of producing PHA using purified glycerin, BDFB, or neutralized BDFB as a carbon source, but in this embodiment, Ralstonia eutropha (Ralstonia eutropha) ), Pseudomonas putida, Bacillus megaterium, Aeromonas caviae, Allochromatium vinosum, Zoogloea ramigera, Parococcus denitrificans), Rhisobium meliloti, Acinetobacter sp., Alcaligenes latus, Thiocystis violacea, Thiocapsa phennigii, Synechocystis sp. PCC6803, Pseudomonas aerug, etc. Other HA natural producing microorganism, a recombinant microorganism is used which is artificially imparted an ability to produce PHA by a genetic engineering technique.

  For example, when it is desired to improve the production amount of PHA, a microorganism in which PHA production is induced by culturing under nitrogen-restricted conditions is preferable. Ralstonia eutropha and Pseudomonas used in this embodiment are preferred. -It is preferable to use Pseudomonas aeruginosa etc. other than Pseudomonas putida. On the other hand, when it is desired to produce PHA that does not contain 3HB as a monomer unit, microorganisms belonging to the genus Pseudomonas are preferable. For example, Pseudomonas putida used in the present embodiment or desired transformed PHA production Preferably recombinant microorganisms are used. In the present embodiment, Pseudomonas putida is used as a preferred embodiment in terms of PHA content and production amount.

  Moreover, in the method for producing PHA according to the present invention, the enzyme gene to be introduced, the origin of the gene, and the host are not particularly limited as long as they are recombinant microorganisms capable of producing PHA. For example, an enzyme gene that can be introduced includes a PHA-producing bacterium-derived PHA-producing enzyme gene, an artificially prepared DNA encoding a protein having a hydroxyalkanoyl CoA polymerization ability, and a monomer supply-related enzyme gene (acetoacetyl CoA). Reductase gene, β-ketothiolase, transferase, hydratase and the like, and as hosts, E. coli, Bacillus subtilis, Bacillus megateriumu, Ralstonia eutropha, Pseudomonas putida, Corynebacterium, etc. can be mentioned.

  In the PHA production method of this embodiment, NADPH-acetoacetyl CoA reductase gene, β-ketothiolase, and PHA polymerization, which are genes encoding enzymes that generate PHA derived from Ralstonia / eutropha, which are generally used in general, are used. PHA-producing recombinant Escherichia coli in which an enzyme and E. coli are introduced is used.

  Moreover, as a culture method of the microbial fermentation method in the method for producing PHA according to the present invention, the timing of adding a medium is not particularly limited. That is, it is possible to produce PHA not only in batch culture but also in fed-batch culture method and continuous culture method advantageous for industrial production. In this embodiment, a fed-batch culture method or a continuous culture method is used.

  The culture temperature of the microorganism may be appropriately selected according to the genus species of the microorganism, the culture method, the substrate specificity, the solubility of the substrate, and the productivity. However, the microorganism used can grow and proliferate to generate PHA. The culture temperature may be within a range, preferably 30 to 45 ° C.

  Furthermore, the PHA extraction / purification step can be performed by a conventional method. For example, the culture solution is collected by a centrifuge, washed with distilled water and methanol, and dried cells are obtained by decompression or freeze-drying. The dried cells are suspended in chloroform, and PHA is extracted by warming. There is a method in which PHA is precipitated and recovered with methanol after the body is removed.

  Examples of PHA and a method for producing PHA according to the present invention will be described below. Note that the scope of the present invention is not limited to the features shown by these examples.

  In order to examine the composition of the biodiesel fuel by-product (BDFB) obtained by transesterification of fats and oils, the Japan Food Analysis Center was requested to analyze the composition. The result is shown in FIG. Among the compositions, glycerin is subjected to gas chromatography, fatty acid-containing ether-solubilized components are subjected to Soxhlet extraction, and ignition residue is directly ashed (measurement conditions are temperature: 550 ° C., constant weight). ), The fatty acid composition of ether-solubilized components is analyzed by gas chromatography, and methanol is analyzed by gas chromatography. Although heavy metals were analyzed by the sodium sulfide colorimetric method, they were not detected with a detection limit of 5 ppm. Moreover, pH was measured by the glass electrode method about BDFB10% aqueous solution, and the result was pH 10.3.

  Next, using a known PHA-producing bacterium, the productivity of PHA in media using BDFB and glycerin as carbon sources was compared.

  First, a PHA production test was performed in a flask. Specifically, first, three known PHA-producing microorganisms such as JM109 / pTI305, Ralstonia eutropha ATCC17699 (Ralstonia eutropha ATCC17699) and Pseudomonas putida KT2440 (Pseudomonas putida KT2440), which are freeze-stocked, are inoculated in 6 mL of NR medium. And cultured with shaking at 30 ° C. overnight.

  JM109 / pTI305 is a PHA-producing recombinant Escherichia coli (Polyhydroxyalkanoate synthase from Bacillus sp. INT005 is) obtained by introducing plasmid pTI305 incorporating phaCAB derived from Ralstonia eutropha into E. coli JM109 (E. coli. JM109). composed of PhaC and PhaR. Journal of Bioscience and Bioengineering v94, p. 343-350 (2002)).

  Next, 5 mL of the culture solution obtained above was added to a medium obtained by adding 1% (wt / vol) of neutralized BDFB or glycerol to 100 mL of MS medium prepared in a 500 mL erlenmeyer flask (manufactured by Iwaki). The cells were cultured at 30 ° C. and 160 rpm. After 24 hours, 1% (wt / vol) of BDFB or glycerol was further added and cultured for 48 hours. After culturing, the cells were collected by centrifugation, washed with distilled water and 5% methanol, and then dried under reduced pressure or lyophilized to obtain dry cells.

  The composition of the NR medium and MS medium used in this example is as follows (J. Bacteriol. V179, p4821-4830 (1997), Appl. Microbiol. Biotechnol. V45, p363-370 (1996). )).

(Composition of NR medium) 3 g of Bacto yeast extract in 1 L of medium
Bacto peptone 10g
Erlich Skipjack Extract 10g

(MS medium) Disodium hydrogen phosphate in 1 L of medium 3.6 g
Potassium dihydrogen phosphate 1.5g
Ammonium chloride 0.5g
Magnesium sulfate heptahydrate 0.0104g
Trace element aqueous solution 0.05mL

<PHA production>
The dried cells thus obtained were suspended in a mixed solution of 2 mL of 15 wt% methanol sulfate solution and 2 mL of chloroform and treated at 100 ° C. for 140 minutes for alcoholysis. After cooling, 1 mL of ultrapure water was added and mixed well, allowed to stand and separated into two layers. This lower layer was recovered, and insolubles were removed by filtration through a filter. Of these, 0.5 mL and 0.5 mL of a chloroform solution containing 0.1 vol% methyl caprylate were mixed and subjected to temperature rise analysis with a capillary gas chromatograph GC-2010 (manufactured by Shimadzu Corporation) equipped with FID, and a monomer unit. The methyl esterified product was identified. The column used was a fused silica capillary column DB-5 (column inner diameter 0.25 mm, liquid layer film thickness 0.25 μm, column length 30 m; manufactured by Shimadzu Corporation). The analysis conditions were as follows: initial temperature 90 ° C., 5 minutes, temperature rising rate 5 ° C./minute, final temperature 250 ° C., 2 minutes. The amount of each PHA produced is shown in FIG.

  As shown in FIG. 2, when glycerin and neutralized BDFB were used as the carbon source, JM109 / pTI305 showed no significant difference in PHA content and PHA production. On the other hand, when Ralstonia eutropha ATCC17699 and Pseudomonas putida KT2440 were used, when neutralized BDFB was used as a carbon source, the PHA content and the amount of PHA produced increased significantly.

  Recombinant microorganisms producing PHA such as JM109 / pTI305 are capable of producing PHA at the same time as growth. Ralstonia eutropha ATCC 17699 and Pseudomonas putida KT2440 are induced to produce PHA by culturing under conditions where nutrients such as nitrogen are restricted. Therefore, neutralized BDFB was shown to be a very useful carbon source for PHA-producing natural bacteria.

  As described above, according to the present Example 1, the known PHA-producing bacteria can use BDFB as a carbon source and can obtain a high PHA production amount. It has been shown to be useful.

  PHA was produced using Pseudomonas putida KT2440 (Pseudomonas putida KT2440) in a medium supplemented with glycerin as a carbon source, and the composition of the obtained PHA was analyzed. Moreover, the productivity of PHA was compared by manufacturing with flasks and fermenters. The production in the flask is the result of analyzing the dried cells of Example 1 by a conventional method. The fermenter was produced with reference to Biotechnology and Bioengineering v68, pp. 446-470 (2002).

  For production with fermenta, specifically, Pseudomonas putida KT2440 (Pseudomonas putida KT2440), which is freeze-stocked, was inoculated in NR medium and cultured overnight at 30 ° C. with shaking. 1 mL of the culture solution was transferred to a 250 mL Erlenmeyer flask (manufactured by Iwaki) containing 50 mL of NR medium and cultured at 30 ° C. for 10 hours.

  Next, 20 mL of the obtained culture solution was transferred to a 3 L Erlenmeyer flask containing 980 mL of MR medium and cultured at 30 ° C. overnight. Thereafter, the culture broth was transplanted to MR medium excluding potassium dihydrogen phosphate. At the time of transplantation, a jar fermenter containing 2.7 L of MR medium was added with 300 mL of culture solution, Jar-1, and a jar fermenter containing 2.5 L of MR medium was added with 500 mL of culture solution, Jar. -2. The agitation speed was 200 rpm, the culture temperature was 30 ° C., and fed-batch culture (fed-batch culture) was performed. The feeding solution was added when the pH of the medium exceeded 7.0, and 120 mL was added until 24 hours and 180 mL from 24 hours until the end of the culture. The aeration rate was 1 vvm until 24 hours and 2 vvm from 24 hours until the end of the culture. Moreover, the dissolved oxygen concentration (DO) which falls with the proliferation of a microbial cell was controlled to 30% by controlling the stirring speed.

The composition of the NR medium, MS medium and fed solution used in this example is as follows.
(Composition of NR medium) 3 g of Bacto yeast extract in 1 L of medium
Bacto peptone 10g
Erlich Skipjack Extract 10g
(Composition of MR medium) About pH 7.0 in 1 L of medium
3g diammonium hydrogen phosphate
Citric acid 0.8g
Glycerol 20g
Magnesium sulfate 1.4g
Trace metal solution 10mL
(Composition of fed solution) 500 g of glycerin in 1 liter
Magnesium sulfate heptahydrate 1g

  After culturing for 48 hours, the cells were collected from each culture solution with a centrifuge, washed twice with distilled water and 5% aqueous methanol solution, and then dried under reduced pressure or freeze-dried to obtain dried cells.

<Composition analysis>
Each dry cell thus obtained was suspended in chloroform and extracted at 100 ° C. for 3 hours. Thereafter, the cells were removed by filtration, and 10 times the amount of methanol was added to the chloroform extract to precipitate and recover PHA. Each obtained PHA was subjected to methanolysis at 100 ° C. for 140 minutes in the same manner as in Example 1, and the temperature of the extract was analyzed by capillary gas chromatograph GC-2010 (manufactured by Shimadzu Corporation) equipped with FID. The methyl esterified product of the monomer unit was identified. As the column, a fused silica capillary column DB-5 (column inner diameter 0.25 mm, liquid layer film thickness 0.25 μm, column length 30 m) (manufactured by Shimadzu Corporation) was used. The analysis conditions were an initial temperature of 90 ° C., 5 minutes, a heating rate of 5 ° C./minute, and a final temperature of 250 ° C. for 2 minutes. The result is shown in FIG.

<Molecular weight measurement>
Moreover, the molecular weight of the obtained compound was measured by gel permeation chromatography (GPC). For GPC measurement, two TSKgel SuperHZM-H columns (6.0 nm ID × 150 mm, manufactured by Tosoh) were connected in series, and the mobile phase was measured using chloroform at 40 ° C. and a flow rate of 0.3 mL / min. did. A calibration curve was prepared using pure polystyrene, and the number average molecular weight (Mn) and the weight average molecular weight (Mw) were calculated. The result is shown in FIG.

  As shown in FIG. 3, PHA containing 3HB and 3HO as main components without 3HB as a monomer unit was produced in both the flask and fermenter production methods.

  According to the present Example 2 as described above, it was shown that it is possible to produce an equivalent PHA using not only a flask but also a fermenter, which is useful for industrial production.

  Next, the composition, molecular weight and physical properties of PHA produced in a medium supplemented with BDFB or neutralized BDFB as a carbon source and PHA produced in a medium supplemented with glycerin were compared.

  Specifically, Pseudomonas putida KT2440 (Pseudomonas putida KT2440) was used to manufacture with a fermenter. Fermenter production was performed by fed-batch culture (fed-batch culture) in the same manner as in Example 2.

  However, when BDFB or neutralized BDFB is used as a carbon source, the feed solution is a liquid (BDFB) obtained by autoclaving BDFB of pH 12 at 121 ° C. for 15 minutes, or BDFB is adjusted to pH 8.0 and then 15 minutes at 121 ° C. Autoclaved liquid (neutralized BDFB) was used. When this BDFB or neutralized BDFB is used as a substrate, the substrate requirement of the bacterial cells is high, and the amount of the fed solution added exceeds the amount of fermenter charged. Therefore, 5.7 L or 6.7 L of the culture solution is added during the cultivation. After each withdrawal, the addition of the feed solution was continued. Extraction of the culture solution was performed when the pH shifted to alkali and the solution became acidic, and 10% (vol / vol) or more of the cells were left in order to add the fed solution again to obtain the same culture solution. .

  Each dried cell was obtained from each culture solution using glycerin, BDFB, and neutralized BDFB as carbon sources in the same manner as in Example 2. In addition, composition analysis and molecular weight measurement were also performed in the same manner as in Example 2.

<Change in PHA composition over time>
PHA was separated and collected from each culture solution over time up to a culture time of 48 hours, and PHA composition analysis was performed for each culture time to calculate the content. The results are shown in FIG. 4 and FIG.

  As shown in FIG. 4 and FIG. 5, PHA containing 3HB as a monomer unit and containing 3HD and 3HO as main components was produced regardless of whether BDFB or neutralized BDFB was used as a carbon source. In addition, when the culture time is more than 10 hours, the PHA having the first composition of 3HO and the second composition of 3HD is changed to the PHA having the first composition of 3HD and the second composition of 3HO. Produced PHA with different compositions.

  In addition, the PHA having the first composition of 3HO and the second composition of 3HD and the PHA having the first composition of 3HD and the second composition of 3HO are clearly different in viscosity, and the first composition is The PHA having 3 HO and the second composition of 3 HD has low viscosity and fluidity, and the PHA having the first composition of 3 HD and the second composition of 3 HO has high viscosity.

<Composition analysis>
In the same manner as described above, PHA was separated and collected from each culture solution after 24 hours and 48 hours of culture time, and the composition analysis of each PHA was performed with a capillary gas chromatograph GC-2010. The culture time was 24 hours and 48 hours. The composition of each PHA was the same. The results after 48 hours of culture are shown in FIG.

  As shown in FIG. 6, even when BDFB and neutralized BDFB were used as a carbon source, 3HB was not included as a monomer unit, and PHA containing 3HD and 3HO as main components was produced. The PHA containing glycerin as a carbon source showed a difference in the content of 3HO and 3HD, and the content of 3HD was higher than that of 3HO. On the other hand, PHA using BDFB and neutralized BDFB as a carbon source showed no significant difference in the content of 3HO and 3HD.

  Moreover, since there was no change in the composition of PHA after 24 hours and 48 hours of culture, it was suggested that continuous culture by fed-batch culture (fed-batch culture) is possible when BDFB and neutralized BDFB are used as a carbon source. .

<Molecular weight and Tg measurement>
Further, as described above, PHA was separated and collected from each culture solution after 48 hours of culture, the molecular weight was measured by GPC, and Tg was determined by a calorimetric analyzer (TGA) and a differential scanning calorimeter (DSC). . TGA was measured using BRUKER AXS TG-DTA 2010 (BRUKER) from nitrogen to 500 ° C. at a rate of temperature increase of 10 ° C./min in nitrogen. DSC measurement was performed from −5 ° C. to 210 ° C. using a BRUKER AXS DSC 3100 at a heating rate of 10 ° C./min in nitrogen. The result is shown in FIG.

  As shown in FIG. 7, the molecular weight distribution (Mw / Mn) was 1.5 to 2.0 and Tg was −48 ° C. or lower when any of them was used as the carbon source. On the other hand, PHA using glycerin as a carbon source is solid at room temperature (up to about 40 ° C.), whereas PHA using BDFB or neutralized BDFB as a carbon source has fluidity at room temperature.

<Thermal characteristics and viscosity measurement>
Next, as described above, PHA was separated and recovered from each culture solution after 48 hours of culture, and the thermal characteristics and viscosity of the obtained PHA were examined.

  First, thermal characteristics were examined as a preliminary experiment for measuring the viscosity of PHA using glycerin as a carbon source. Specifically, PHA using glycerin as a carbon source was left at 60 ° C. for 10 minutes, and the state was observed by naked eye and palpation with a spatula. The experimental process and results are shown in FIGS.

  As shown in FIG. 8A and as described above, PHA using glycerin as a carbon source at room temperature was solid at room temperature, and changed to a fluid liquid at 60 ° C. (FIG. 8B). When this was scrubbed with a spatula and the state was observed, it was viscous (FIG. 8C).

  Subsequently, the viscosity of PHA using glycerin, BDFB and neutralized BDFB as a carbon source was measured with a rheometer. Viscosity was measured by temperature dependent measurement. The results are shown in FIG. 9 and FIG.

  As shown in FIG. 9 and FIG. 10, PHA using BDFB and neutralized BDFB as a carbon source has a lower viscosity than PHA using glycerin as a carbon source, and a decrease in viscosity with an increase in temperature was observed. . In PHA using glycerin as a carbon source, it melted at about 40 ° C. and completely melted at 45 ° C. or more. The melted PHA solidified again as the temperature decreased, and an increase in viscosity was observed compared to before melting.

<Adhesion measurement>
Next, as described above, PHA was separated and recovered from a culture solution containing glycerin as a carbon source after 48 hours of culture, and the adhesiveness of the obtained PHA was examined.

  Specifically, the amount of load deflection was measured. That is, as shown in FIG. 11, first, 0.1 mm each of PHA dissolved at 45 ° C. was attached to one side surface of two plastic containers (FIG. 11 (a)). Adhesiveness was confirmed by joining the side surfaces attached with PHA (FIG. 11B). Thereafter, the container was allowed to stand at −80 ° C. for 30 minutes, a weight was applied to the container adhesion site, and the amount of deflection was measured (FIG. 11C). Weighing was performed at 500 g, 800 g, and 1000 g. Moreover, each observation figure about the adhesion state during and after weighting in the added weights 587.5 g and 678.5 g is shown in FIGS.

  As shown in FIG. 12 and FIG. 13, even when a weight of 587.5 g or 678.5 g is applied to the container bonding site, the containers remain in an adhesive state and are bent in the load direction (FIGS. 12A and 13). (A)). After that, it was observed that when the load was released, the bending disappeared and the adhesive surface returned to the state before the load (FIGS. 12B and 13B). And in addition weight 500g, 800g, 1000g, the state similar to FIG. 12 and FIG. 13 was observed, and each deflection amount showed 2.0 mm, 5.0 mm, and 6.0 mm, respectively.

  According to the present Example 3 as described above, it is shown that PHA can be produced in a fermenter even when BDFB and neutralized BDFB are used as a carbon source, as well as glycerin, which is useful for industrial production. It was. In addition, PHA using glycerin as a carbon source and PHA using BDFB or neutralized BDFB as a carbon source have obvious differences in the composition content, room temperature properties, polymer solubility, and viscosity of 3HO and 3HD, and BDFB or It has been clarified that PHA using neutralized BDFB as a carbon source has characteristics useful for the use of products requiring tackiness.

  The PHA and the method for producing PHA according to the present invention are not limited to the above-described embodiments, and can be appropriately changed.

It is a figure showing the result of the composition analysis test by the Japan Food Research Center foundation of BDFB used for manufacture of PHA concerning the present invention. It is a figure which shows the production amount of PHA in the present Example 1. It is a figure which shows the composition of PHA, the production | generation amount, and the molecular weight in the present Example 2. In Example 3, it is a figure which shows a time-dependent composition change of PHA at the time of culture | cultivating with the culture medium which used BDFB as a carbon source. In Example 3, it is a figure which shows a time-dependent composition change of PHA at the time of culture | cultivating on the culture medium which used neutralized BDFB as the carbon source. It is a figure which shows the composition and production amount of PHA in the present Example 3. It is a figure which shows the molecular weight of PHA in this Example 3, Tg, room temperature property, and solubility. It is a figure which shows the thermal characteristic of PHA in the present Example 3. It is a figure which shows the viscosity of PHA in the present Example 3. It is a figure which shows the correlation of the viscosity of PHA in this Example 3, and temperature. It is a reference figure which shows the measurement process of the adhesiveness of PHA in the present Example 3. In this Example 3, it is an observation figure which shows the adhesiveness of PHA when a weight of 587.5g is applied. In Example 3, it is an observation figure which shows the adhesiveness of PHA at the time of applying a weight of 687.5g.

Claims (5)

A method for producing a polyhydroxyalkanoate characterized in that a biodiesel fuel byproduct obtained by transesterification of fats and oils or a neutralized byproduct obtained by neutralizing the byproduct is produced by a microbial fermentation method using a carbon source. Then, using Pseudomonas putida as a microorganism in the microbial fermentation method, the monomer unit of the first composition is 3-hydroxyoctanoate and the monomer unit of the second composition is 3-hydroxydecanoate. Controlling the incubation time of a certain polyhydroxyalkanoate or a polyhydroxyalkanoate in which the monomer unit of the first composition is 3-hydroxydecanoate and the monomer unit of the second composition is 3-hydroxyoctanoate Select by Manufacturing method characterized by manufacturing. 2. The production method according to claim 1 , wherein polyhydroxyalkanoate not containing 3-hydroxybutyrate as a monomer unit is produced by using Pseudomonas putida as a microorganism in the microorganism fermentation method.   A method for producing a polyhydroxyalkanoate characterized in that a biodiesel fuel byproduct obtained by transesterification of fats and oils or a neutralized byproduct obtained by neutralizing the byproduct is produced by a microbial fermentation method using a carbon source. And the microorganism in the microorganism fermentation method is Ralstonia eutropha.   A method for producing a polyhydroxyalkanoate characterized in that a biodiesel fuel byproduct obtained by transesterification of fats and oils or a neutralized byproduct obtained by neutralizing the byproduct is produced by a microbial fermentation method using a carbon source. Then, the production method of producing a polyhydroxyalkanoate not containing 3-hydroxybutyrate as a monomer unit by using Pseudomonas putida as a microorganism in the microorganism fermentation method. In any one of claims 1 to 4, wherein the manufacturing method of cultivation in the microbial fermentation method and performing by fed-batch culture or continuous culture.