JPH0438763B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to poly-β-hydroxybutyric acid (hereinafter referred to as
(abbreviated as PHB). PHB is accumulated by various microorganisms, mainly bacteria, as a particulate energy storage substance inside microbial cells. PHB extracted from such cells is a thermoplastic polyester with the following repeating units: -O.CH( _CH3 ) _CH2CO- which rapidly crystallizes to relatively high levels, e.g. on the order of 70% or more. do. This crystallization behavior is often a disadvantage when the polymers are used, for example, as molding materials. It was found that the crystallization of this PHB can be modified by incorporating units of dissimilar monomers into the polymer chain. Thus, by culturing microorganisms under specific conditions in the presence of certain organic acids, small proportions of comonomer units can be introduced into the polymer chain. An example of such an acid is propionic acid. Although this invention is not bound by the following theory, it is believed that the metabolic pathway leading to such a polymer is as follows. In the following, CoASH is terminally esterified coenzyme A. Therefore, CH ₃ COSCoA is an acetylthioester of coenzyme A and is generally referred to as acetyl-CoA. NADP is nicotinamide adenine dinucleotide phosphate in its oxidized state.
NADPH ₂ is reduced NADP. The first step in the biosynthesis of PHB by microorganisms is considered to be the synthesis of acetyl-CoA. this is,
For example, it is produced from coenzyme A and acetate, or from the decarboxylation of pyruvate, which is a glycolysis product of carbohydrates, or oxaloacetate, which is a member of the tricarboxylic acid (TCA) cycle or the Krebs cycle. ] is formed by the decarboxylation of Therefore, with acetate ester as a source of acetyl-CoA, PHB is produced through a metabolic pathway that includes the following reactions: (1) CH ₃・CO ・ O ⁻ +CoA ・SH thiokinase――――――――→ CH ₃・CO・S・CoA＋OH ^- (2) 2CH ₃・CO・S・CoAβ−ketothiolase――――――――――→ CH ₃・CO・CH ₂・CO・S・CoA＋CoA・SH ( 3) CH ₃・CO・CH ₂・CO・S・CoA＋NADPH ₂ reductase――――――――→ CH ₃・CHOH・CH ₂・CO・
S・CoA+NADP (3-hydroxybutyryl CoA) (4) CH ₃・CHOH・CH ₂・CO・S・CoA polymerase――――――――→ −O・CH (CH ₃ )・CH ₂・CO− +CoASH (repeat unit in the polymer) Therefore reaction (4) adds one -CoASH to the growing polymer chain.
Add O・CH(CH ₃ )・CH ₂・CO− unit. Since the enzymes involved lack specificity for propionic acid, the corresponding pathway is thought to be as follows. (1a) CH ₃・CH ₂・COO ^- +CoA・SH――→ CH ₃・CH ₂・CO・S・CoA＋OH ^- (2a) CH ₃・CH ₂・CO・S・CoA＋CH ₃・
CO・S・CoA―→ CH ₃・CH ₂・CO・CH ₂・CO・S・CoA＋
CoA・SH (3a) CH ₃・CH ₂・CO・CH ₂・CO・S・CoA
+NADPH ₂ -→ CH ₃・CH ₂・CHOH・CH ₂・CO・S・CoA
+NADP (4a) CH ₃・CH ₂・CHOH・CH ₂・CO・S・
CoA――→ -O・CH(C ₂ H ₅ )・CH ₂・CO ⁻ +CoA・SH In other words, in contrast to PHB having pendant methyl groups, repeating units with pendant ethyl groups form the polymer chain. introduced inside. Certain polymers containing 3-hydroxybutyric acid units, ie the following units -OCH( _CH3 ) _CH2CO- , and other units have already been described in the literature. A polymer that exhibits an infrared band that is said to show ethylenic unsaturation was developed by Davis as an ``Applied
Microbiology” 12 (1964) p.301-304. These polymers, described by Davis as copolymers containing 3-hydroxybutyric acid units and the following 3-hydroxy-2-butenoic acid units -OC( _CH3 )=CHCO-, - Produced by culturing in butane. Outside of Wallen is “Environmentel Science and
Technology 6 (1972) p.161-164 and 8
(1974) p. 576-579, β-hydroxybutyric acid units and the following 3-hydroxyvaleric acid units –OCH(C ₂ H ₅ ) CH ₂ CO - has been published in a ratio of 1:5. Outside of Marchessault, “IUPAC Macro
Florence1980 International Symposium on
Macromoles Preprints” 2 (1980) p.272-275
reported a study of this polymer and confirmed that it mainly contains 3-hydroxyvaleric acid units. USP 3275610 lists certain microorganisms, especially
A method for the microbiological production of polyester by culturing Nocardia salmonicolor with a carboxylic acid containing 4 carbon atoms is presented. In Examples 2 and 3 thereof, 3-butenoic acid and 2-
Using hydroxybutyric acid, the polymer has a melting point of
Since the temperature is on the order of 178-184°C, it appears to be poly-3-hydroxybutyric acid ester. However, in Example 1, 2-methylacrylic acid (ie, methacrylic acid) is used, and the resulting polymer is not identified, but is described as having a melting point of 215-220°C and being soluble in methyl ethyl ketone. In contrast,
The copolymer containing mainly 3-hydroxybutyric acid residues of this invention has a melting point of 180°C or less and is insoluble in cold methyl ethyl ketone. When PHB-accumulating microorganisms are cultured aerobically on suitable substrates, ie, energy and carbon sources, the microorganisms will proliferate until one or more of the essential elements for growth are exhausted. In the following, this growth of microorganisms will be referred to as "breeding". When one of the essential factors for reproduction is exhausted, PHB accumulates in the microorganisms unless the substrate is exhausted, although subsequent reproduction is very limited, if at all. In some microorganisms, PHB-induced inhibitory factors (e.g. restriction of one or more essential reproductive elements)
PHB will accumulate during microbial reproduction even in the absence of
The amount of PHB is generally small, typically less than about 10 wt% of the resulting cells. Therefore, when propagated in batch culture, microorganisms that do not structurally produce PHB produce little or no PHB until one or more of the essential reproductive components are exhausted.
The microorganisms reproduce without accumulating PHB, and then the microorganisms synthesize PHB. Therefore, to produce a copolymer it is necessary to use an acid or a derivative thereof which provides copolymer units other than 3-hydroxybutyric acid units as at least part of the substrate present during polymer accumulation. The acid or derivative thereof that provides copolymer units other than 3-hydroxybutyric acid units may be referred to herein as the "comonomer component of the substrate" (or simply "comonomer component"). If the culture conditions are such that polyesters (e.g. PHB) do not accumulate significantly, the comonomer components of the substrate are often metabolized by the microorganism in a separate process, e.g. acetyl-CoA or
It becomes a member of the TCA cycle and copolymers are no longer produced. Therefore, as an example, without any reproduction restrictions, propionic acid is metabolized by microorganisms to form propionyl-CoA, then take up carbon dioxide to become methylmalonyl-CoA, and then succinate, a member of the TCA cycle. Metabolism of the "substrate comonomer component" by such a different process also occurs when microorganisms that structurally produce PHB are used. Therefore, structural
Even when using PHB-accumulating microorganisms, cultivation of the microorganisms under conditions that limit the amount of one or more factors essential for reproduction but not essential for PHB accumulation is critical. It is preferable to accumulate coalescence. Even when microorganisms are cultured under conditions that limit the essential elements for growth and thus allow polymers to accumulate by the microorganisms, some of the comonomer components of the substrate are acetyl.
May be metabolized by pathways leading to CoA or members of the TCA cycle. This allows microorganisms to synthesize 3-hydroxybutyric acid units as well as other copolymer units for incorporation into the copolymer even when the comonomer component is the only substrate during the polymer accumulation step. It is. Also, as illustrated by the metabolic pathway suggested above for the production of 3-hydroxyvalerate from propionate, in one step, reaction (2a), propionyl CoA and acetyl
A reaction with CoA takes place. Therefore, when propionate is the only substrate, some of the propionate is metabolized to acetyl-CoA,
3-hydroxyvaleric acid units may be produced. In addition to the metabolic pathways that lead to 3-hydroxyvaleric acid units in the copolymer shown above, other reactions can occur by using various other materials as comonomer components of the substrate. For example, other hydroxy-substituted carboxylic acids can optionally be incorporated into the polymer by the polymerase (if not metabolized by other routes, resulting in e.g. acetyl-CoA or propionyl-CoA). For example, HO・CR ¹ R ²・(CR ³ R ⁴ ) _o・CO・CoA――→ −O・CR ¹ R ² (CR ³ R ⁴ ) _o・CO−+CoASH [here n is zero or an integer, R ¹ , R ² ,
R ³ and R ⁴ may be the same or different;
selected from hydrocarbon groups (eg alkyl, aralkyl, aryl or alkaryl groups); halo- and hydroxy-substituted hydrocarbons; hydroxy; halogen atoms; and hydrogen atoms]. Of course, if n = 1 and R ² = R ³ = R ⁴ = hydrogen,
If R ¹ is a methyl group, the hydroxycarboxylic acid is 3-hydroxybutyric acid which is metabolized directly to give 3-hydroxybutyric acid units. Preferably, each of the groups R ¹ , R ² , R ³ and R ⁴ contains less than 4 carbon atoms. Generally R ¹ ,
At least one of R ² , R ³ and R ⁴ is hydrogen. Preferably, n is zero, 1 or 2. Such hydroxycarboxylic acids may be added neat as part or all of the comonomer component of the substrate, or may be microbially synthesized from other comonomer component materials. Acrylic acid, 3-chloropropionic acid and 3
It has been found that acids such as -hydroxypropionic acid can give polymers containing 3-hydroxybutyric acid units and other units. In some cases, all of these other units are 3-hydroxyvaleric acid units, while in other cases other units (i.e. units A as defined above, which are 3-hydroxyvaleric acid units and (which may arise in connection with)
In particular, protons showing the following (i) and (ii) and ¹³ Cnmr
Identified by spectrum. (i) Proton nmr triplet at 4.3 ppm. (ii) ¹³ Cnmr peaks at 59.89 and 33.83 ppm (tetramethylsilane standard control). From these nmr data, these units are 3-
Hydroxypropionic acid units (n=1, R ¹ = R ²
= R ³ = R ⁴ = H). The copolymer may also contain 4-hydroxyvaleric acid units (n=2, ^R1 = _CH3 , ^R2 = ^R3 = ^R4 =H). 3-Hydroxypropionic acid units and/or 4-hydroxyvaleric acid units are intermediates _CH3 ·OH· _CH2 ·CO·S·CoA and _CH3 ·CHOH· _CH2 · _CH2 ·CO·S·CoA. It may also come from. The former can be produced from 3-hydroxypropionate and CoA.SH. In addition to being supplied by itself, 3-hydroxypropionate can be used in the hydration reaction of acrylates CH ₂ = CH・COO ⁻ +H ₂ O――→CHOH・CH ₂・
Hydrolysis of 3-chloropropionate by COO ^- or CH ₂ Cl・CH ₂・COO ^- +OH ^{- --} →CHOH・
It can be produced by CH ₂・COO ^- +Cl ^- . 4-hydroxyvaleryl
CoA is a condensation of acetyl CoA and acrylic CoA,
The subsequent reduction, i.e. CH ₃ · CO · S · CoA + CH ₂ = CH · CO · S ·
CoA――→ CH ₃・CO・CH＝CH・CO・S・CoA＋
CoA・SH CH ₃・CO・CH=CH・CO・S・CoA+
2NADPH ₂ ---→ CH ₃・CHOH・CH ₂ CH ₂・CO・S・CoA+
Can be produced by 2NADP. Similarly, 4-hydroxyvaleryl CoA is 3-
From hydroxy- or 3-chloro-propionate, condensation with acetyl-CoA and dehydration (3-
In the case of chlorpropionate, it can be produced by an intermediate hydrolysis step). Therefore, by the method of the present invention, the 3-hydroxybutyric acid unit -O.CH(CH ₃ ).CH _2.CO- is converted into the unit of the following formula -O.CR ¹ R ^2. (CR ³ R ⁴ ) _o.CO - It is possible to obtain copolymers containing with. [In the above formula, n is zero or an integer,
Each of R ¹ , R ² , R ³ and R ⁴ represents a hydrocarbon group (e.g. an alkyl, aralkyl, aryl or alkaryl group); a halo- and hydroxy-substituted hydrocarbon group; a hydroxy group; a halogen atom; and a hydrogen atom ; provided that n is 1 and R ² , R ³ and R ⁴ are each a hydrogen atom, then R ¹ is not a methyl group]. Preferably, each of R ¹ , R ² , R ³ and R ⁴ is 4
Contains fewer than 1 carbon atoms. Preferably n is zero, 1 or 2. Copolymers may contain more than one type of unit. Preferably in at least some of the units n=1, R ² =R ³ =R ⁴ =H and R ¹ =ethyl. To be of practical use as plastic materials, the polymers should have a weight average molecular weight M _w (measured, for example, by gel permeation chromatography) of 10,000 or more. The proportion of repeating units in the copolymer is from 0.1 to 50 mol%, in particular from 1 to 40 mol%, of the total repeating units of the copolymer. In some cases, the microbially obtained polymer is a mixture of homopolymers of repeating units and copolymers containing repeating units. In this case, the total proportion of repeating units in the polymer is from 0.1 to 50 mol% of the total repeating units. Most preferably, the proportion of repeating units is between 3 and 30 mole percent. To obtain a significant proportion of comonomer units, the amount of chemically bound carbon in the comonomer component of the substrate must be equal to or less than the total chemical bonds in the substrate present during the culture conditions during which the polymer is being accumulated by the microorganism. at least 2% by weight of carbon, preferably at least 10
It should be % by weight. During such periods, the only comonomer component is preferably the carboxylic acid (or derivative thereof) present in the substrate, although acetate may optionally be present. According to the invention, a microorganism capable of accumulating polyester is cultured in an aqueous medium on a water-soluble assimilable carbon-containing substrate, and the microorganism accumulates polyester during at least a portion of the culture. In a method for producing a thermoplastic polyester by culturing under conditions, at least during the culture period during which polyester accumulates, the assimilable carbon-containing substrate is metabolized by microorganisms under the polyester accumulation conditions. T-O・
Contains an organic acid or organic acid derivative that forms a polyester other than one consisting solely of CH(CH ₃ )/CH ₂ /CO- repeating units, and the amount of chemically bonded carbon in the acid or acid derivative is There is provided a process for the microbiological production of thermoplastic polyesters as described above, characterized in that the polyester comprises at least 2% by weight of the total chemically bound carbon in the substrate present during the polyester accumulation period. The concentration of comonomer components in the aqueous medium is 0.05g/
Should be greater than or equal to 0.1 to 5, preferably 0.1 to 5
The concentration would be g/g/. It will therefore be clear that the water solubility of the comonomer component should be greater than or equal to 0.05 g/m and should be sufficient to provide the desired comonomer component concentration at the culture temperature. The comonomer component may be the acid itself, or it may be a salt, ester (including lactones in the case of hydroxy-substituted acids), anhydride, amide, or halide. As mentioned above, at least part of the culture period
Preferably, the culture is carried out under conditions that limit elements essential for microbial growth but not for polyester accumulation. The most convenient growth factor limitation is nitrogen limitation. For this reason,
When nitrogen limitation is employed, the substrate is preferably nitrogen-free and thus amides are less preferred substrates. The acid capable of forming a copolymer should be one that does not give only repeat units when the culture is in the polymer accumulation stage. Therefore, examples of acids that are undesirable from this point of view include acetic acid, 3-hydroxybutyric acid,
members of the TCA cycle, as well as acetyl-CoA and/or when the culture is in the polymer accumulation phase.
Or there are acids that give only members of the TCA cycle. Thus, unsuitable acids include phosphoglyceric acid, pyruvic acid, citric acid, isocitric acid, alpha-ketoglutaric acid, succinic acid, fumaric acid, maleic acid, malic acid, oxalacetic acid, oxalosuccinic acid, aconitic acid, and There is methylmalonic acid. Amino acids are similarly unsuitable. Other acids found not to give copolymers include formic acid, butyric acid, phenyl acetic acid, benzoic acid, chloroacetic acid, 2-chloropropionic acid, 3-
Hydroxybutyric acid, 4-hydroxybutyric acid, 2-chlorobutyric acid, 2-methylacrylic acid, 2,3-dimethylacrylic acid, 3,3-dimethylacrylic acid, lactic acid, glyoxylic acid and glycolic acid. Acids that have been found to give copolymers include:
Propionic acid, 3-hydroxypropionic acid, 3
-chloropropionic acid, 3-ethoxypropionic acid, 2-hydroxybutyric acid, isobutyric acid, and acrylic acid. Other acids that can be used include:
There are higher saturated carboxylic acids containing an odd number of carbon atoms, such as valeric acid, heptanoic acid, piparic acid and substituted propenoic acids (eg 2- and 3-chloropropenoic acids). As mentioned above, in some cases microorganisms may perform other actions on acids. Isobutyric acid therefore provides repeating units with n=1, R ² =R ³ =R ⁴ =H, R=isopropyl group. n=1, ^R2 = ^R3 =
The repeating unit of R ⁴ =H, R ¹ =ethyl group indicates that the microorganism replaces the methyl group with hydrogen during the metabolic pathway to the copolymer. Preferred acids that can give copolymers are propionic acid, isolactic acid and acrylic acid. Suitable derivatives of such acids include alkali metal salts and lower alkyl esters (where the alkyl group is 1 to 1).
containing 4 carbon atoms). Particularly suitable esters include the methyl, ethyl, isopropyl, propyl, butyl and isobutyl esters of propionic acid; the methyl and ethyl esters of isobutyric acid; and the methyl and ethyl esters of acrylic acid. Mixtures of copolymer-forming acids (or acid derivatives) can be used as the comonomer component of the substrate.
For example, interesting results have been obtained using mixtures of propionic acid and acrylic acid. As mentioned above, even when using microorganisms that constitutively produce PHB, the microbial culture period during which polyesters are accumulated must be kept under conditions of nutrient limitation that is essential for growth but not essential for polyester accumulation. It is preferable to carry out. In addition to substrate and oxygen (which is generally provided by injecting air into the aqueous culture medium), various nutrient salts are necessary for the microorganisms to be able to reproduce. Therefore, sources of the following elements in generally assimilable form (usually water-soluble salts) are required: nitrogen, phosphorus, sulfur, potassium, sodium, magnesium,
Along with calcium and iron, trace elements such as manganese, zinc and copper. Although it is possible to limit the supply of oxygen to the fermenter to induce polyester accumulation, it is preferred to limit the amount of one or more nutrients. The most practical elements to limit are nitrogen, phosphorous, and less preferred are magnesium, sulfur, or potassium. Among these, it is most preferred to limit the amount of nitrogen, which is conveniently supplied as an ammonium salt. The amount of assimilable nitrogen required is approximately 8-15% of the desired weight of cells with low polyester accumulation.
It is. Fermentation is preferably carried out such that the dry weight of polyester-containing cells is at least 5 g per aqueous medium. Therefore, if, for example, you want to produce 10 g/cell of PHB-containing cells with a PHB content of 40 wt%, the amount of essential nutrients supplied to the culture tank to limit cell proliferation is 6
Therefore, if nitrogen is used as the reproductive limiting nutrient, the nitrogen content of cells without PHB is approximately 8 to
Since it is 15wt%, the amount of assimilable nitrogen required is approximately
0.5-0.9g/, for example, ammonia ion
It is 0.6-1.2g/. Cultivation is carried out under conditions customary for the microorganism, such as pH, temperature, and degree of aeration (when oxygen is not the limiting nutrient source). Similarly, the nutrient salts used (other than the growth-limiting nutrient source, the amount of which is determined in consideration of the above conditions) are the amounts normally used for the growth of the microorganism. The microorganisms are grown in culture to a desired weight on readily metabolizable substrates, such as carbohydrates, in the presence of sufficient amounts of nutrients necessary for growth to be limited during the polymer accumulation stage, and then polymer accumulation is allowed to proceed. Preferably, the culture is carried out under conditions of reproductive element limitation. Optionally, the substrate for at least a portion (and possibly all) of the propagation step may be an acid (or a derivative thereof) that becomes a repeating unit in the polymer accumulation step. Cultures can be carried out in batch cultures, where polymer accumulation occurs when the amount of nutrients necessary for reproduction but not for polymer accumulation is insufficient or exhausted. Alternatively, the culture can be carried out in a continuous culture where the aqueous medium containing the bacterial cells is removed continuously or intermittently from the culture vessel at a rate that corresponds to the rate of addition of fresh aqueous medium and substrate. The amount of limiting nutrient source fed to the culture tank is such that the aqueous medium removed from the tank contains very little of this nutrient, and the aqueous medium removed from the tank is then fed batchwise or preferably continuously. Preferably, the aerated culture is continued with the addition of fresh substrate containing the comonomer component to allow polymer accumulation to occur. During this additional cultivation step, additional amounts of substrate and nutrients may be added, but additional propagation is generally undesirable;
Nutrient sources used to limit reproduction should not be added further. However, the presence and/or addition of some residual amount of the limiting nutrient source to the aqueous medium fed from the first culture tank to one or more other culture tanks is essential for effective operation. It is understood that this is preferable. Alternatively, culturing can be carried out as a one-step continuous process. To achieve polyester accumulation by nutrient limitation, the residence time of the medium in the fermenter must be long enough for the microorganisms to grow and use up the limiting nutrients supplied to the culture tank, and for the microorganisms to then accumulate polyester. As it is, make it longer. In either the batch type or continuous type mentioned above,
The acid (or derivative thereof) used to provide the copolymer repeat units is used as part or all of the substrate during the polymer accumulation phase that occurs when the nutrients necessary for propagation are depleted. The acid may be used in a mixture with a substrate (e.g. a carbohydrate) that provides the repeating units, or it may be the only substrate;
In the latter case, sufficient comonomer content is present in the acetyl
If the alternative route to CoA is metabolized to give the repeat unit, for example if the alternative route involves reaction (2a), then any acetyl-CoA necessary to obtain the repeat unit is used. However, if the comonomer component is the only substrate, polymer yields are often reduced. The comonomer component can also be present in only a portion of the polymer accumulation step; the remainder of the polymer accumulation step, occurring before and/or after the portion of the polymer accumulation step in which the comonomer component is present, contains only repeating units. The substrate provided may be the only substrate. In some cases, it is also possible to prevent the normal metabolism of acid to acetyl-CoA by blocking enzymes required for the normal pathway and/or by using microorganisms that are incapable of synthesizing the necessary enzymes. be. However, to obtain substantial yields of polymer, cultivation for a period of time under conditions that limit and preferably deplete the nutrients required for propagation is generally preferred. Cultivation is preferably carried out such that the amount of accumulated polyester is approximately 50-80% by weight of the bacterial cells. Microorganisms that can be used include any poly(3) that can assimilate the acid or its salt to produce the copolymer.
-It is a hydroxybutyrate ester accumulating microorganism.
Bacteria Alcaligenes entrophus (traditionally
(formerly known as Hydrogenomonas eutropha), such as the H16 strain, which has been widely used in academic studies of this species [ATCC No. 17699, J General
Microbiology (1979) 115, p. 185-192] and mutant strains of the H16 strain, such as 11/7B, S301/C5,
S501/C29 and S501/C41 (respectively the
Natinal Collection of Industrial Bacteria,
Torry Research Station, Aberdeen, Scotland
NCIB No. deposited on August 18, 1980.
11600, 11599, 11597 and 11598) are particularly suitable. The ATCC number is the American Type
Culture Collection, 12301 Park Lawn Drive,
Rockville, Maryland 20852U.SA. As mentioned above, it is preferred to use carbohydrates as a substrate during the breeding stage.
Alcaligenes eutrophus strain H16 (ATCC No.
17699) does not assimilate glucose, but its mutant strains, such as the above-mentioned 11/7B, S301/C5, S501/
C29 and S501/C41 can utilize glucose. Carbohydrates, especially glucose, are preferred substrates for the propagation stage due to their cost and the ability of microorganisms to propagate effectively. Polyesters are produced as granules inside microbial cells. Although the polyester-containing cells can be used as is as a molding material, as shown for example in US Pat. No. 3,107,172, it is generally preferred to separate the polyester from the bacterial cells.
This is accomplished by disrupting the cells and then extracting the polyester with a suitable solvent. An example of a suitable extraction process is described in European Patent Application No. 15123. As mentioned above, in order for the copolymer to be put to practical use,
The copolymer must have a weight average molecular weight ( _Mw ) of 10,000 or more as measured by gel permeation chromatography. Preferably, M _w is 50,000 or more, more preferably 100,000 or more, especially 200,000 or more. The copolymers always have a D-configuration and exhibit lower melting points than 3-hydroxybutyric acid homopolymers. The copolymers are particularly useful in the production of melt-molded articles, where reduced crystallinity comparable to 3-hydroxybutyric acid homopolymers is preferred. Of particular interest is the use of small amounts of copolymers of vinyl chloride-based polymers as high molecular weight processing aids. In this application, the amount of copolymer is 0.5-10 wt% relative to the vinyl chloride polymer. For best results in this application, the copolymer should be random. To obtain a random copolymer, the acid used to obtain the comonomer units is preferably present as the only substrate at least throughout the period of cultivation of the microorganism under propagation factor limiting conditions. After melt extrusion, the copolymer is preferably heated at a temperature between the glass transition temperature (Tg) and the melting point of the polymer.
It is also used to make films by passing them through one or more rolls to reduce the film thickness and introduce some molecular orientation. The invention is illustrated in the following examples. Example 1 The normal metabolism of propionate converts it to succinate, which is oxidized to oxaloacetate in the TCA cycle and then decarboxylated to acetyl-CoA. In decarboxylation of oxaloacetate, both terminal acid groups are removed as carbon dioxide gas. Therefore, if a propionate with a radioactively labeled carbon atom in the carboxy group, i.e. 1- ¹⁴ C-propionate,
If fed into cells for conversion to acetyl-CoA, ¹⁴ CO ₂
Radioactivity is lost as some kind of polymer
Incorporation of ¹⁴ C results from the conversion of propionyl CoA to 3-hydroxyvaleryl CoA, followed by polymerization. Alcaligenes eutrophus mutant strain NCIB11599,
It was propagated aerobically in a batch fermenter using an aqueous medium A containing sufficient assimilable nitrogen and glucose as substrate to support 3.5 g/accumulated polyester. Aqueous medium A had the following composition per portion of deionized water. (NH ₄ ) ₂ SO ₄ 2g MgSO ₄・7H ₂ O 0.8g K ₂ SO ₄ 0.45g H ₃ PO ₄ (1.1M) 12ml FeSO ₄・7H ₂ O 15mg Trace element solution 24ml Trace element solution is deionized water Each sample had the following composition: CuSO ₄・5H ₂ O 0.02g ZnSO ₄・6H ₂ O 0.1g MnSO ₄・4H ₂ O 0.1g CaCl ₂・2H ₂ O 2.6g When the biomass concentration reaches 4.5g/, that is, the assimilable nitrogen of the system After depletion, 1 g of sodium propionate containing ^1-14C -propionate was added to the fermenter together with glucose, and fermentation was continued for 5 minutes. Cells were then collected by filtration and the polymer was extracted with chloroform. The labeled carbon was almost completely in the chloroform solution, indicating that no labeled terminal carbon atoms were lost as carbon dioxide gas. Therefore, at least some propionate was incorporated into the polymer other than as acetyl CoA. Example 2 (comparative example) AlCaligenes eutrophus mutant strain NCIB11599,
An aqueous medium having the following composition per 1 part deionized water:
In a 5-batch fermenter containing 4000ml of B, PH6.8,
Propagated by aerobic culture at 34°C. (NH ₄ ) ₂ SO ₄ 4g MgSO ₄・7H ₂ O 0.8g K ₂ SO ₄ 0.45g H ₃ PO ₄ (1.1M) 12ml FeSO ₄・7H ₂ O 15mg 24ml trace element solution used in Example 1 Glucose , was supplied to the fermenter at a rate of 8 g/hr. The amount of assimilable nitrogen in medium B is 26 g of PHB.
This was sufficient to support cells containing no. After 40 hours, cells were collected by centrifugation. Cells were lyophilized and polymers were extracted with chloroform. Example 3 Example 2 was repeated, but when the cell weight reached 34 g, 2.8 g of propionic acid was added instead of glucose.
It was fed to the fermenter at a rate of g/hr. Example 4 Example 3 was repeated, but the propionic acid feed was started when a cell weight of 39 g was reached. Example 5 Example 3 was repeated, but the propionic acid feed was started when a cell weight of 56 g was reached. Example 6 Example 3 was repeated, but when a cell weight of 48 g was reached, 12 g of propionic acid was added at once. Example 7 Example 2 was repeated, but using medium A and propionic acid at a rate of 4 g/hr instead of glucose.
It was fed throughout the fermentation. Example 8 Example 2 was repeated, but when the cell weight reached 38 g, glucose 5.2 was added instead of glucose.
A mixture of glucose and propionic acid was fed to the fermenter at a rate of 2.8 g/hr and 2.8 g/hr of propionic acid. Example 9 Example 8 was repeated but when a cell weight of 28 g was reached, glucose 6.8 g/hr and propionic acid
Feed of the mixture was started at a rate of 1.2 g/hr. In Examples 2 to 9, propionic acid was 400g/
It was added as a solution containing. Example 10 Example 2 was repeated, but when the cell weight reached 28 g, isobutyric acid was fed to the fermenter at a rate of 2 g/hr instead of glucose. Isobutyric acid is 150
It was added as a solution containing g/g. In Examples 3-6 and 8-10, the weight of acid fed to the fermenter versus the weight of glucose fed to the fermenter after the cell weight reached 26 g (i.e., when the system was depleted of nitrogen) Fermentation was continued until the ratio of "total weight of acid supplied to the fermenter" reached the value shown in Table 1. Example 11 Example 2 was repeated, but when the cell weight reached 26.4 g, 3-chloropropionic acid was fed to the fermenter instead of glucose at a rate of 4 g/hr for 5 hours. Example 12 Example 11 was repeated, but the 3-chloropropionic acid feed was started when a cell weight of 34.4 g was reached. Example 13 Example 12 was repeated, but when a cell weight of 30 g was reached, 4 g of 3-chloropropionic acid was added at once, and then glucose was fed at a rate of 6.8 g/hr for 7 hours. In Examples 11-13, 3-chloropropionic acid was added in a solution containing 50 g/ml. Example 14 Example 2 was repeated, but when the cell weight reached 31 g, 4 g/hr of acrylic acid was added instead of glucose.
was fed to the fermenter for 5 hours at a ratio of Acrylic acid was added in the form of a solution containing 100 g/ml.

[Table] This is the quotient divided by the sum of the weight of lucose and the weight of acid supplied to the fermenter.
The amount of each comonomer unit in the polymers of Examples 2-14 was determined by (a) hydrolysis and gas chromatography and (b) ¹³ C nuclear magnetic resonance spectroscopy. The molecular weight of the polymer was determined by gel permeation chromatography. Chlorine analysis was also performed on the polymers of Examples 2, 11, 12 and 13. The results are shown in Table 2. In Table 2, 3-HV represents a 3-hydroxyvaleric acid unit, and the current unit A is the unit described above and is considered to be a 3-hydroxypropionic acid unit. Almost no chlorine from 3-chloropropionic acid was found in the polymer. Therefore, chlorine is lost during the metabolism of 3-chloropropionic acid,
The resulting product is metabolized to units A and 3-
It appears to give hydroxyvaleric acid units. The chlorine content of the polymers of Examples 11-13 shows that some chlorine is present in the unit as a chlorine-containing group R, possibly R ¹ =chloroethyl,
Probably, R ² = R ³ = R ⁴ = H, and n = 1.

【table】

[Table] The monomer sequences of the copolymers of Examples 3 to 10 were investigated using high-resolution ¹³ C NMR. It was found that the signals obtained from the carbon atoms of carbonyl groups occur at different chemical shifts, depending on their environment. Therefore, the unit and (n=1, R ²
=C ₂ H ₅ , R ² =R ³ =H), the possible sequences are as follows. A Butyrate-Butyrate B Valerate - Valerate C Butyrate-Valerate NMR testing of the polymers of Examples 2-10 showed three resonances occurring at 169.07, 169.25 and 169.44 ppm, respectively. Outside M.Iida〔Macromoles 11
(1978) p490], the resonance at 169.07ppm is
Butyrate-butyrate sequence A,
169.44 ppm is the valerate-valerate arrangement B. By inference, the signal at 169.25 ppm arises from sequence C of butyrate-valerate. Quantitative analysis of the NMR results of the polymer of Example 10 gave the following results. Sequence A (butyrate-butyrate) 55% Sequence B (valerate-valerate) 14% Sequence C (butyrate-valerate) 31% These results demonstrate that the polymer of Example 10 has ^a ₂ H ₅ , R ² = R ³ = R ⁴ = H)
It clearly shows that it contains a substantial amount of the copolymer. However, some homopolymers of repeating units may also be present. The polymers of Examples 2-14 all had the D(-) configuration. The melting behavior of the as-extracted copolymer was first determined by differential scanning calorimetry (DSC) using a DuPont 1090 system with computer data analysis. The DSC method was also carried out on the samples after compression molding at 190° C. and cooling during pressing to obtain a fully crystallized product. In each case, the swatches were heated in air at 20° C./min and the temperatures of the start (Ts) and peak (Tp) of endothermic melting were recorded along with their area. Heating of the annealed sample was continued to 200°C and after isothermal for 1 minute to ensure complete melting, the sample was quenched in liquid nitrogen.
DSC tests were again performed to determine the glass transition temperature (Tg) of the amorphous region. Finally, the density of the annealed copolymer was measured by density gradient flotation method. The results are shown in Table 3.

TABLE The wide melting point range of each copolymer indicates that the copolymers are rather heterogeneous compositions. However, the melting endotherm becomes sharper and the area slightly decreases, so when annealing,
Significant randomization occurs due to transesterification. This is an indication that the polymer is a true copolymer and not a physical mixture of homopolymers. Multiple DSC peaks are shown in Examples 3, 5, 8 and
10 of the as-extracted polymers were observed. Melting endothermic area is an indicator of crystallinity. After annealing, the polymers of Examples 3-14 were all significantly less crystalline than the control homopolymer of Example 2. Example 15 Alcaligenes eutrophus mutant strain NCIB11599
, aqueous medium C (which is the same as medium B, but
The cells were propagated aerobically at 34° C. and pH 6.8 in a 5-batch fermenter containing 4000 ml of ammonia sulfate (5.2 g/m) sufficient to support 8.5 g/m of PHB-free cells. The substrate was glucose provided at a rate of 5.5 g/hr. When the cell concentration reached 7 g/g, in addition to glucose, propionic acid was added at 1.58 g/g/g.
/hr. Cell dry weight is 15g/
Cells were harvested when . The cell suspension was spray dried, lipids were extracted by refluxing the dry cells with methanol, and polymers were extracted by refluxing chloroform.
The polymer was recovered by a precipitation method in which a chloroform solution was added to a methanol/water mixture. The copolymer has repeating units (R=C ₂ H ₅ , R ² =R ³
=R ⁴ =H, n=1) contained 20 mol%. The copolymer had a molecular weight of 350,000 and was insoluble in cold methyl ethyl ketone. When 2 g of the copolymer was refluxed with 100 ml of methyl ethyl ketone for 1 hour, the entire amount was dissolved. Upon cooling the solution a gelatinous mass formed. On the other hand, when 2 g of 3-hydroxybutyrate homopolymer was refluxed with 100 ml of methyl ethyl ketone, less than 0.1 g of the homopolymer was dissolved. If the solubility test is repeated with ethanol instead of methyl ethyl ketone, after 1 hour of reflux, the copolymer weighs about 0.7 g and the homopolymer weighs 0.04 g.
The following dissolved. In contrast, Wallen et al.
tal Science and Technology 8 (1974) p.576~
The polymer described in No. 579 is said to be soluble in hot ethanol. Example 16 Aqueous media D, E and F were made with the following composition per part deionized water. Medium D (NH ₄ ) ₂ SO ₄ 12g MgSO ₄・7H ₂ O 1.2g K ₂ SO ₄ 1.5g CaCl ₂ 0.12g FeSO ₄・7H ₂ O 0.1g ZnSO ₄・7H ₂ O 0.006g MnSO ₄・4H ₂ O 0.006g CuSO ₄・5H ₂ O 0.0015g H ₂ SO ₄ (concentrated) 1ml Medium E H ₃ PO ₄ (1.1M) 2.4ml Glucose 40g Medium F H ₃ PO ₄ (1.1M) 2.4ml Propionic acid 40g Sterilized nominal A 250 capacity batch fermenter was filled to the 130 mark with an approximately equal volume mixture of media D and E. A small sample of the medium in the fermenter was analyzed for nitrogen content. Then, in an incubator
The Alcaligenes eutrophus mutant strain NCIB 11599 was inoculated and the fermentation was carried out at 34°C and the pH was adjusted by adding caustic soda solution.
was automatically controlled at 6.8 and carried out aerobically. The amount of assimilable nitrogen present in the fermenter was sufficient to allow microbial growth of only about 1.2 kg of PHB-free cells. When the cell weight reached approximately 1.05 Kg, medium E feeding was started at a rate of 6.5/hr. When the cell weight reached approximately 1700 g, the supply of medium E was stopped and the supply of medium F was started at a rate of 6.5/hr, and fermentation was continued until approximately 2.6 Kg of cells were produced. The cell suspension is then centrifuged to a concentration of ca.
It was concentrated to 60 g/ml and the polymer was extracted by contacting 1 volume of the suspension with 2 volumes of 1,2-dichloroethane (DCE) in a Silverson mixer at 20°C for 15 minutes. The DCE phase was separated from the aqueous phase containing cell debris and filtered. One volume of the filtered DCE phase was added to four volumes of a methanol/water (4/1, by volume) mixture to precipitate the polymer. Separate the precipitated polymer,
After washing with methanol, it was dried in the open at 100°C for 4 hours. The polymer had a melting range of about 100-180°C with a peak melting endotherm at 168°C as determined by DSC method. Example 17 The fermentation process of Example 16 was repeated, but switching from supplying medium E to supplying medium F was performed until the cell weight was approx.
I went when I reached 3.5Kg. Medium F is 11.4
/hr for 4 hours and then reduced to 3.2/hr and maintained at this level for a further 9 hours, at which point the cell weight was approximately 3.9Kg. In this example, the amount of assimilable nitrogen present in the fermenter was sufficient to propagate the microorganisms to only about 1.5 Kg of polymer-free cells. The cell suspension was concentrated by centrifugation and then
Polymers were extracted from concentrated cell suspensions using a method of 15. Example 18 Charge a 250 fermenter as in Example 16,
Inoculation was carried out. The amount of assimilable nitrogen was sufficient to propagate microorganisms in only about 1.9 kg of polymer-free cells. Fermentation was carried out as in Example 16 for 34 hours.
The test was carried out aerobically at ℃ and pH 6.8. When the cell weight reached approximately 1.0 Kg, feeding of medium E and medium G at a rate of 8.7/hr and 4.6/hr, respectively, was started and continued until the cell weight reached 3.9 Kg. Medium G had the following composition per 1 ml of deionized water: 1.2 ml H ₃ PO ₄ (1.1 M) 20 g propionic acid The cell suspension was concentrated by centrifugation and weighed using the method of Example 15. Combines were extracted from the concentrated cell suspension. Example 19 The process of Example 17 was repeated on a larger scale and the nominal volume
A 1000 fermenter was used and filled to the 500 mark with approximately equal volumes of medium D and E. In this example, feeding of medium E was started at a rate of 25/hr when the cell weight was about 4 kg, and feeding of medium F was started at a rate of 37.5/hr when the cell weight was about 8 kg. The supply of media E and F is such that the cell weight is approximately 10
This continued until Kg was reached. The amount of assimilable nitrogen present was sufficient to propagate the microorganisms to approximately 4.1 kg of polymer-free cells. Example 20 Example 19 was repeated, but the feeding rate of medium F was 25
/hr, fermentation continued until the cell weight was about 11 Kg. In this case, the amount of assimilable nitrogen was sufficient for microorganisms to propagate up to about 4 kg of polymer-free cells. The polymers of Examples 16 to 20 were copolymers containing 3-hydroxybutyric acid (HB) units and β-hydroxyvaleric acid (HV) units, and had weight average molecular weights of 300,000 or more. Each copolymer had a D(-) configuration. 100 parts by weight of each of the copolymers of Examples 16 to 20 and the 3-hydroxybutyric acid homopolymer were added to about chloroform.
Slurry with 10 parts by weight and 1 part by weight of talc,
Granulated at room temperature in a domestic meat mincer. The composition was then dried to remove chloroform, extracted at 190°C, and granulated again. The resulting granules were injection molded into test bars at 185°C and mold temperature 65°C.
°C and a cooling time of 20 seconds were used. tensile properties,
The impact strength was measured by ASTM D638-77a at a speed of 50 mm/min and the impact strength was evaluated by the Izot impact test according to ASTM D256-78. The results are shown in Table 4.

[Table] Example 21 The following ingredients were dry mixed at room temperature to make a PVC formulation: Part by weight (i) Vinyl chloride homopolymer (K62) 100 (ii) Thiooctyl based di-N-dithioglycolic acid ester Tin complex stabilizer 1.5 (iii) Methyl methacrylate/butadiene/styrene PVC impact modifier 8 (iv) Wax (external lubricant) 0.8 (v) Glyceryl monoester (internal lubricant) 1 (vi) HB polymer ( Processing aids) 2 The HB polymer processing aids were as follows: (a) 3-hydroxybutyrate homopolymer obtained in Example 2 (b) Copolymer of Example 7 (copolymer Copolymer A) (c) Copolymer of Example 16 (Copolymer B) The processing aid was slurried with approximately 10 wt% chloroform, granulated in a domestic meat grinder at room temperature, dried, and processed at 190°C. Melt extruded, re-granulated, PVC
It was ground to a particle size of less than 150 μm before being incorporated into the dry mixture. The dry mixture was tested as follows: 1. 50 g of the mixture was rotated at 18 rpm under a pressure unevenness loaded with a 5 Kg weight and maintained at 180°C.
Loaded into the mixing head of a Brabender Plastograph. The time required for gelation to occur was recorded. 2 Make the mixture into a cold-pressed candle and pour it into
Maintained at 170℃, diameter 1mm and land length 20mm
The sample was placed in an extrusion rheometer equipped with a die having a circular orifice. After the charge was heated to 170°C, it was extruded at increasing speed. The appearance of the extrudate was recorded and the extrudate was pulled through the die to assess melt extensibility. The results are shown in Table 5.

TABLE This example shows that the copolymer is superior to 3-hydroxybutyric acid homopolymer as a vinyl chloride polymer processing aid. More random copolymer A is clearly superior to copolymer B. Example 22 Medium H was prepared with the following composition: (NH ₄ ) ₂ SO ₄ 1 g KH ₂ PO ₄ 2 g (Na) ₂ HPO ₄ 3 g MgSO _{4.7H 2} O 0.2 g CaCl ₂ 0.01 g FeSO _{4.7H 2} O 0.005g MnSO ₄・4H ₂ O 0.002g Na ₂ CO ₃・10H ₂ O 0.1g (NH ₂ ) ₂ CO 1.5g Deionized water Set the total to 1. The pH of the medium was 7. Medium H500 with 0.5g of methacrylic acid dissolved in advance
into 8 shake flasks each containing ml.
5 ml of a seed culture of Nocardia salmonicolor strain ATCC 19149 was inoculated and cultured at 32°C on an orbital shaker. at intervals of 24, 48 and 72 hours after inoculation.
Add 0.5g of methacrylic acid to each flask,
A final addition of 0.25 g of methacrylic acid was made after 96 hours. Each flask was inspected 108 hours after inoculation.
There was almost no growth of microorganisms in any of the flasks. The flask contents were combined, centrifuged to pellet cells, dried in an oven, and weighed. The pellet weight was 2.81 g. The cell content of the inoculum was also determined and was 69.75 g/.
Therefore, the total weight of cells added to the flask as inoculum was 2.79 g. At the methacrylic acid concentration used, this strain did not assimilate methacrylic acid. Examples 23-45 In these runs, a range of acids and acid derivatives were screened for their ability to give copolymers. The method used was as follows. Alcaligenes eutrophus mutant strain NCIB11599,
Aqueous medium with the following composition per 1 deionized water: 3500 ~
PH6.8 in a 5 batch fermenter containing 4000ml
and cultured aerobically at 34°C. Glucose 17g (NH ₄ ) ₂ SO ₄ 4g MgSO ₄・7H ₂ O 0.8g H ₃ PO ₄ (1.1M) 12ml FeSO ₄・7H ₂ O 15mg Trace element solution 36ml (from Example 1) PH is 4M hydroxide Controlled at 6.8 by automatic addition of a 9:1 (vol/vol) mixture of potassium solution and 4M sodium hydroxide. For such PH control
The addition of KOH/NaOH mixture also served to supply potassium and sodium to the medium. The amount of assimilable nitrogen (provided by the 4 g/ammonium sulfate above) is only about 6.5
The amount was sufficient to support g/g of HB polymer-free cells. Approximately 16g/glucose is 6.5g/
The amount of glucose present was sufficient to provide a small carbon excess as required to produce HB polymer-free cells. Monitoring the residual glucose concentration and also tracking the dissolved oxygen tension provided a clear indication of when the system was starved of assimilable nitrogen. At this stage, the feed of the comonomer component (ie, test acid or acid derivative) was started and continued until a total of about 0.1-5 g/g of comonomer component had been added. After the addition of the comonomer components, the cells were collected by centrifugation. Centrifuged cell samples were lyophilized and the polymers were extracted with chloroform. The polymers were analyzed by gas chromatography/mass spectrometry (GCMS) (which included a preliminary transesterification step) or nmr spectroscopy (NMR). The results are shown in Table 6. In this table, NMR results are shown where possible since the NMR method does not involve a transesterification step and is considered to be more definitive.

[Table] Similar results were obtained when the culture of Examples 23-44 was repeated in which the mixture of glucose and comonomer components was continuously added after the nitrogen was exhausted. It was done. Examples 46-51 At a dilution rate (reciprocal of residence time) of 0.085/hr, approximately 4
in 5 fermenters using an effective medium volume of
Alcaligenes eutrophus mutant strain NCIB11599 was continuously cultured aerobically at PH6.8 and 34°C. The aqueous medium used was 1 part deionized water.
The composition was as follows. MgSO ₄・7H ₂ O 0.8g K ₂ SO ₄ 0.45g Na ₂ SO ₄ 0.05g H ₃ PO ₄ (1.1M) solution 12ml Trace element solution (Example 1) 36ml Also each component shown in Table 7 Continuously fed to the fermenter.

[Table] The amount of nitrogen was sufficient to support only 6.5 g/cell without HB polymer. The pH was controlled by automatic addition of a 9:1 (vol/vol) mixture of 4M potassium hydroxide solution and 4M sodium hydroxide solution. Fermentation was started using propionic acid as the only substrate. Three days after reaching steady state, samples of the aqueous cell suspension product were taken and acrylic acid was then added as a co-substrate in increasing amounts. Steady-state fermentation was carried out at each acrylic acid concentration for at least 3 days before each sample was taken. Each sample of aqueous cell suspension product was centrifuged to recover the cells and then lyophilized. The polymer was recovered from the cells with chloroform and then analyzed by NMR methods. Table 8 shows the results.
Shown below.

[Table] Example 52 In this example, under the limitation of phosphorus instead of nitrogen,
Alcaligenes eutrophus mutant strain NCIB11599 at 34°C.
Cultured aerobically. 5 fermenters, 1 part deionized water
An aqueous medium having the following composition was prepared. (NH ₄ ) ₂ SO ₄ 6.2g H ₃ PO ₄ (1.1M) 1.5ml MgSO ₄・7H ₂ O 0.8g FeSO ₄・7H ₂ O 15mg Trace element solution (from Example 1) 36ml PH during fermentation is , 4M KOH and 4M NaOH
automatically by adding a 9:1 (vol/vol) mixture of
Controlled to 6.8. The amount of phosphorus was sufficient to support 8 g/h of HB polymer-free cells. The fermentor was inoculated with a 48 hour shake flask culture and then 5 g/g glucose was added. When all the glucose was used up (at this point the cell concentration was about 2.5 g/hr), propionic acid (in the form of a solution of 300 g/hr) was added at a rate of 0.8 g/hr for 54 hours. Cells were then collected, and the polymer was extracted with chloroform and analyzed by GCMS method. The results were as follows. Final cell concentration 20g/Polymer content 60% Mol% of 3-HV 40mol% Examples 53-57 These examples use Nocardia Salmoni-
Two strains of color species were cultured on glucose substrate,
Polymer accumulation was then induced using various acids. In each example, in a 250 ml shake flask,
Add an aqueous medium containing the following ingredients per 1 ounce of deionized water.
I prepared 50ml. Glucose 10g K ₂ HPO ₄ 1.9g NaHPO ₄ 1.56g (NH ₄ ) ₂ SO ₄ 1.8g MgSO ₄・7H ₂ O 0.2g FeCl ₃・6H ₂ O 0.001g Trace element solution (from Example 1) 1ml of this aqueous The pH of the medium was 7.0. A flask was inoculated with microorganisms (Table 9) and cultured at 30° C. for 24 hours using a rotary shaking method. The resulting suspension was then centrifuged and the supernatant aqueous medium was discarded. The remaining centrifuged pellet was transferred to the above aqueous medium (but
1 g/acid instead of 10 g/glucose and 1.8 g/ammonium sulfate omitted). The resuspended cells were shaken for an additional 24 hours at 30°C, and then the cell suspension was centrifuged. The resulting centrifuged pelleted cells were washed twice with methanol and analyzed for polymer content. The polymer was analyzed by GCMS method. The results are shown in Table 9.

【table】

Claims (1)

[Scope of Claims] 1. A microorganism capable of accumulating polyβ-hydroxybutyric acid ester is cultured in an aqueous medium on a water-soluble assimilable carbon-containing substrate, and at least part of the period of the culture is maintained by the microorganism. In a method for producing a thermoplastic polyester by culturing under conditions in which polyester is accumulated, at least during the portion of the culture period in which polyester is accumulated, the assimilable carbon-containing substrate is is metabolized by microorganisms to produce -O.
Propionic acid, 3-hydroxypropionic acid, 3-chloropropionic acid, 3-ethoxypropionic acid, which is a polyester other than one consisting exclusively of CH(CH ₃ )/CH ₂ /CO- repeating units;
comprising an organic acid or organic acid derivative selected from 2-hydroxybutyric acid, isobutyric acid, acrylic acid and higher saturated carboxylic acids containing an odd number of carbon atoms, and the amount of chemically bonded carbon in the acid or acid derivative is The method for producing thermoplastic polyesters as described above, characterized in that at least 2% by weight of the total chemically bound carbon is present in the matrix present during the polyester accumulation period. 2. Claim 1 provides for the accumulation of polyester by microorganisms under conditions that limit the supply of one or more elements essential for the growth of the microorganisms but not essential for the accumulation of polyesters. The method described. 3 Weight average molecular weight is 10,000 or more, repeating unit of -O・CH( _CH3 )・_CH2・CO− and propionic acid, 3-hydroxypropionic acid, 3-chloropropionic acid, 3-ethoxypropionic acid, 2 -Hydroxybutyric acid, isobutyric acid,
repeating units of ester residues based on organic acids or acid derivatives selected from acrylic acid and higher saturated carboxylic acids containing an odd number of carbon atoms, and said repeating units account for the total number of repeating units.
A poly-β-hydroxybutyric acid copolymer comprising 0.1 to 50 mol%.