The method for molding the polyhydroxyalkanoate of the present invention into a film is not particularly limited; however, examples include T-die extrusion molding, calender molding, roll molding, and inflation molding. The molding temperature employed at the time of film molding is not particularly limited; however, the molding temperature is 130° C. to 190° C.

The method for molding the polyhydroxyalkanoate of the present invention into a nonwoven fabric is not particularly limited; however, the molding method may be an electrospray deposition (ESD) method, a melt blowing method, or another method for producing a nonwoven fabric. An ESD method or a melt blowing method is more preferred.

In the melt blown method, the temperature for melting the polyhydroxyalkanoate of the present invention is preferably from 80° C. to 250° C. The lower limit of the temperature of the nozzle block is more preferably 120° C. or more, further preferably 150° C. or more, and particularly preferably 170° C. or more. The upper limit of the temperature of the nozzle block is more preferably 210° C. or less, further preferably 200° C. or less, and particularly preferably 190° C. or less.

In the melt blown method, the temperature of the nozzle block for fiberizing the polyhydroxyalkanoate of the present invention is preferably from 80° C. to 250° C. The lower limit of the temperature of the nozzle block is more preferably 120° C. or more, further preferably 150° C. or more, and particularly preferably 180° C. or more. The upper limit of the temperature of the nozzle block is more preferably 220° C. or less, further preferably 210° C. or less, and particularly preferably 200° C. or less. Meanwhile, When the temperature of the nozzle block is out of the above range, the polyhydroxyalkanoate of the present invention cannot be fiberized.

In the melt blown method, the temperature of the hot air is preferably 100° C. or more and 250° C. or less. The lower limit of the temperature of the hot air is more preferably 150° C. or more, further preferably 180° C. or more, and particularly preferably 190° C. or more. The upper limit of the temperature of the nozzle block is more preferably 230° C. or less, further preferably 220° C. or less, and particularly preferably 210° C. or less.

By setting the melting temperature, the temperature of the nozzle block, the temperature of the hot air, and the residence time in the melt blown method within the above ranges, a good fiber state is obtained, and the fiber is suitable for producing a nonwoven fabric.

When fiberizing the polyhydroxyalkanoate of the present invention, the content of the 3-hydroxyhexanoic acid unit is preferably from 13.0 mol % to 30.0 mol %. The lower limit is more preferably 15.0 mol % or more. The upper limit is more preferably 25 mol % or less. By setting the content in this range, a good fiber state is obtained, and the fiber is suitable for producing a nonwoven fabric.

[Method for Producing Polyhydroxyalkanoate]

The method for producing a polyhydroxyalkanoate of the present invention may be any production method as long as a polyhydroxyalkanoate having a melt flow rate (MFR) at 160° C. and 2.16 kg/f of 2.5 g/10 minutes or more is obtained.

For example, according to an embodiment of the method for producing a polyhydroxyalkanoate of the present invention, the method includes the following steps.

- Step 1: a step of preparing a microorganism capable of producing a polyhydroxyalkanoate;
- Step 2: a step of growing the microorganism in a medium;
- Step 3: a step of causing an animal to ingest the grown microorganism; and
- Step 4: a step of collecting a polyhydroxyalkanoate from excrements of the animal.

It is preferable that the polyhydroxyalkanoate of the present invention is produced using a microorganism. Examples of the microorganism include microorganisms having a polyhydroxyalkanoate production ability, such as Bacillus megaterium, Cupriavidus necator, and Alcaligenes latus. Cupriavidus necator is particularly preferred.

The microorganism is preferably a microorganism in which a gene participating in the synthesis of a polyhydroxyalkanoate has been deleted or introduced. For example, it is preferable to use a microorganism in which acetoacetyl-CoA reductase gene has been deleted. Furthermore, it is preferable to introduce hydroxyalkanoate synthase gene or enoyl-CoA hydratase gene. Thereby, the content of the 3-hydroxyhexanoate unit included in the polyhydroxyalkanoate can be increased.

An example of hydroxyalkanoate synthase that is expressed by hydroxyalkanoate synthase gene comprises, for example, an amino acid sequence set forth in SEQ ID NO:1 shown in FIG. 1, or an amino acid sequence set forth in SEQ ID NO:1 having one or more amino acids substituted, deleted, or added.

Furthermore, an example of enoyl-CoA hydratase that is expressed by enoyl-CoA hydratase gene comprises, for example, an amino acid sequence set forth in SEQ ID NO:2 shown in FIG. 2, or an amino acid sequence set forth in SEQ ID NO:2 having one or more amino acids substituted, deleted, or added.

By using a microorganism in which a gene expressing the hydroxyalkanoate synthase set forth SEQ ID NO:1 and/or the enoyl-CoA hydratase set forth in SEQ ID NO:2 has been introduced, a polyhydroxyalkanoate P(3HB-co-3HHx) comprising 3-hydroxybutanoate and 3-hydroxyhexanoate, which has high melt fluidity and excellent processability, can be produced.

The medium used for the culture of the microorganism is not particularly limited as long as a microorganism grows therein. For example, the medium is a medium including, as a carbon source, an alcohol such as methanol, ethanol, or butanol; a fatty acid, such as a saturated or unsaturated fatty acid such as acetic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, or myristic acid; a sugar such as glucose or fructose; an organic acid such as lactic acid; or oils and fats including a large quantity of saturated/unsaturated fatty acids having 10 or more carbon atoms. Examples of the oils and fats include plant oils and fats such as coconut oil, palm kernel oil, palm oil, palm olein, jatropha oil, rapeseed oil, soybean oil, rice oil, and sesame oil; animal oils and fats such as lard and beef tallow; and fish oil. Incidentally, for the oils and fats, products before purification, waste edible oil, and the like can also be used. Regarding the oils and fats to be added as carbon sources into a medium, palm kernel oil or coconut oil, both of which contain lauric acid, is preferred. When palm kernel oil or coconut oil is incorporated, the 3HHx content of the polyhydroxyalkanoate can be increased.

It is preferable that the production conditions for the polyhydroxyalkanoate of the present invention are aerobic conditions. If necessary, a nitrogen source or an inorganic substance may also be added. Examples of the nitrogen source include ammonia, and ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate. Examples of the inorganic substance include potassium monobasic phosphate, potassium dibasic phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.

The culturing temperature is preferably 20° C. to 40° C., and more preferably 25° C. to 35° C. The culturing time is not particularly limited; however, the culturing time is preferably 48 to 72 hours.

In regard to the method for producing a polyhydroxyalkanoate of the present invention, the content of 3HHx can be controlled by controlling the amounts of expression of the acetoacetyl-CoA reductase gene and enoyl-CoA hydratase gene.

The content of 3HHx can also be controlled by controlling the residual amount of the carbon source, adjusting the inorganic component concentration in the culture fluid, and adjusting the airflow quantity of oxygen and the culturing time.

The purification method for the polyhydroxyalkanoate of the present invention is not particularly limited; however, a method of collecting the polyhydroxyalkanoate from the medium by centrifugation and performing extraction using a solvent or the like; or a method of causing an animal to digest and absorb the microorganism and collecting the polyhydroxyalkanoate as excrements, may be employed. From the viewpoint that the concentration of the polyhydroxyalkanoate can be easily concentrated, a method of causing an animal to digest and absorb a microorganism, and collecting the polyhydroxyalkanoate as granular polyhydroxyalkanoate included in the excrements, is preferred.

Examples of the animal include animals such as a rodent, goat, sheep, cow, and a bird; aquatic organisms; beetles; and insects. A beetle larva such as mealworm is preferred, and a 35-day old larva of Tenebrio molitor is more preferred.

After the microorganism is fed to larvae such as mealworms, fecal pellets are collected and sieved using a screen. Subsequently, the fecal pellets are washed with water and a base such as sodium hydroxide, and are dried. Thereby, the polyhydroxyalkanoate of the present invention can be collected.

EXAMPLES

Hereinafter, the present invention will be specifically described by way of Examples; however, the technical scope of the present invention is not intended to be limited by these Examples.

Example 1
Production of Polyhydroxyalkanoate Comprising 3-Hydroxybutanoate Unit (3HB) and 3-Hydroxyhexanoate Unit (3HHx)
Preparation of Mineral Medium for Production of P(3HB-co-3HHx)

A mineral medium for the production of P(3HB-co-3HHx) was formed from 4.0 g/L of NaH₂PO₄, 4.6 g/L of Na₂HPO₄, 0.45 g/L of K₂SO₄, 0.39 g/L of MgSO₄, 62 mg/L of CaCl₂), and 1 mL/L of a trace element solution (a trace element solution includes 15 g/L of FeSO₄·7H₂O, 2.4 g/L of MnSO₄H₂O, 2.4 g/L of ZnSO₄·7H₂O, and 0.48 g/L of CuSO₄·5H₂O dissolved in 0.1 M HCl), and the medium was adjusted to pH 7.0 before being sterilized by an autoclave.

Biosynthesis of P(3HB-co-3HHx) Using 13-L Fermentation Tank

Biosynthesis of P(3HB-co-3HHx) was carried out using Cupriavidus necator having polyhydroxyalkanoate synthase gene introduced therein.

First, Cupriavidus necator in which a gene encoding polyhydroxyalkanoate synthase as set forth in SEQ ID NO:1 had been introduced was streaked on an agar plate and was cultured for 24 hours at 30° C. Next, as pre-culture, the Cupriavidus necator was inoculated two times into 50 mL of culture fluid using a platinum loop, and the culture system was shaken in an incubator shaker at 30° C. for 8 hours until the OD600 nm of the culture fluid reached 4. To 100 mL of the mineral medium in which 0.54 g/L of urea, 0.39 g/L of MgSO₄, 62 mg/L of CaCl₂, 1 mL/L of the trace element solution, and 1% by mass of crude palm kernel oil had been added, about 3 mL of the culture fluid was inoculated. The crude palm kernel oil was subjected to an autoclave treatment before being added to the mineral medium. Furthermore, this mineral medium was cultured for 18 hours and then was inoculated into a 6-L fermentation tank. The form of the Cupriavidus necator thus inoculated was checked before being transferred into the fermentation tank (10% v/v). While the temperature of the culture medium was maintained at 30° C., the pH of the medium was set to 7.0±0.1 by addition of 3 M NaOH and 3 M H₃PO₄. Stirring was carried out at a stirring rate of 200 to 900 rpm using a Rushton turbine. Air was supplied at a volume of 1 vvm (air volume/working volume of fermenter/minute) through a filter cartridge (Sartorius Stedim, Germany), and the dissolved oxygen concentration was maintained at or above 40%. MgSO₄·7H₂O was added on the 18th hour after culture, and urea was added every 6 hours. Trace elements were added in an amount of 1 mL between planting and on the 18th hour of culture. Crude palm kernel was supplied at a concentration of 10 g/L to 20 g/L every 6 hours according to the consumption of the oil by the microorganism. In order to determine the residual oil content, wetted cell weight, and optical density of the bacterial culture, sampling was performed every 6 hours. The culturing time was in the range of from 48 hours to 72 hours according to the growth of the bacterial cells.

Biological Collection of P(3HB-co-3HHx)

35-day old mealworms (larvae of Tenebrio molitor) were reared in a plastic container at ambient temperature (about 25° C.). 100 g of the reared mealworms were fed with the dried microorganisms including P(3HB-co-3HHx) (FIG. 3). The amount of the microorganisms thus fed was supplied based on the body weight of the mealworms (5% per day of the body weight). Before a new batch of microorganisms was supplied, fecal pellets (FIG. 4) of the mealworms were collected and sieved using screens having a mesh size of 0.50 mm and 0.25 mm. By performing double sieving, other impurities were eliminated, and the subsequent washing process could be made easier.

Purification of P(3HB-co-3HHx) Using Distilled Water

About 10% (w/v) of the fecal pellets were added to tap water to a concentration of 100 g/L. The fecal pellet suspension was rinsed several times and was settled before the supernatant was discarded. The supernatant was removed, and the P(3HB-co-3HHx) thus collected was dried in an oven at 50° C. until a constant mass was obtained.

Furthermore, the P(3HB-co-3HHx) thus dried was rinsed for one hour in 0.25 M NaOH, the mixture was settled, and the supernatant was removed. The pellets thus collected were stirred for another one hour in tap water until the pH decreased to a value less than 9.5. Subsequently, granules of P(3HB-co-3HHx) thus collected were dried in an oven at 50° C. until a constant mass was obtained, and thus, the intended P(3HB-co-3HHx) was collected.

Examples 2 to 5

In Examples 2 to 5, P(3HB-co-3HHx) was produced using the microorganisms and media described in the following Table 1, and P(3HB-co-3HHx) was collected by the purification method described in Table 1.

Measurement of Average Molecular Weight and Molecular Weight Distribution

Measurement of the average molecular weight (number average molecular weight (Mn) and weight average molecular weight (Mw)) was carried out for the polyhydroxyalkanoate obtained as described above, and the molecular weight distribution D (Mw/Mn) was calculated.