Method for directly recovering polyhydroxyalkanoate in form of microbeads

Abstract

The present invention relates to a method for directly recovering polyhydroxyalkanoates in the form of microbeads using physical cell disruption. In the present invention, physical cell disruption and powdering processes were optimized to directly recover polyhydroxyalkanoates produced as bead-shaped particles in cells of bacteria producing polyhydroxyalkanoates as they are. By using the optimized polyhydroxyalkanoate preparation method of the present invention, polyhydroxyalkanoates can be prepared with high purity and high yield without an organic solvent, and polyhydroxyalkanoates in the form of microbeads can be directly obtained without a separate molding process. Therefore, the method can be used as a novel polyhydroxyalkanoate preparation platform that can be used for biomedical or cosmetic applications.

Description

Method for direct recovery of polyhydroxyalkanoates in the form of microbeads

[0001] The present invention relates to a method for directly recovering polyhydroxyalkanoate in the form of microbeads using physical cell disruption.

[0002] Plastics are polymer materials that have dramatically improved the convenience of human life. Their lightweight, moldable, processable, economical, and durable properties allow them to be used in a wide range of applications, from industrial materials to disposable consumables. However, most plastics used for general purposes, such as industrial packaging, food packaging, household goods, and agricultural and horticultural use, persist in the natural environment for a semi-permanent period without decomposing. Consequently, the disposal of discarded plastics has become a persistent environmental issue. Research into biodegradable plastics as an alternative is actively underway. Biodegradable plastics are plastics that can be broken down by bacteria or living organisms. Because they decompose without leaving behind pollutants, they are expected to potentially contribute to reducing solid waste, such as ocean microplastics and carbon dioxide emissions from petroleum-based plastics, and are attracting significant attention in the field of biotechnology. Biodegradable bioplastics are bio-based plastics that can be biodegraded, and representative examples include polylactic acid (PLA) and polyhydroxyalkanoates (PHA). Biodegradable bioplastics can decompose into water and carbon dioxide within 6 months to 5 years when landfilled. Currently, bioplastics account for about 1% of the more than 360 million tons of plastics produced annually, or 2 million tons, of which about 1.2 million tons are biodegradable plastics. However, it is expected to increase to 1.8 million tons by 2025, and the market growth rate of polyhydroxyalkanoates in particular is expected to increase by about 10 times. As demand increases and the number of biopolymers and products with improved performance increases, the bioplastics market is expected to continue to grow and diversify.

[0003] Polyhydroxyalkanoates (PHAs) are intracellular energy storage compounds produced by various microorganisms under limited nutrient conditions. They are biodegradable polymers composed of various hydroxycarboxylic acids. While possessing properties similar to those of conventional petroleum-derived synthetic polymers such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), they are completely biodegradable and possess excellent biocompatibility. In addition, because the mechanical properties and melting point can be controlled by adjusting the type and ratio of polyhydroxyalkanoate monomers, it is used as an alternative to petroleum plastics in various industrial fields such as medicine, food, and energy.

[0004] [Prior Art Literature]

[0005] [Non-patent literature]

[0006] Yang, Yung-Hun, et al. “Optimization of growth media components for polyhydroxyalkanoate (PHA) production from organic acids by Ralstonia eutropha.” Applied microbiology and biotechnology 87.6 (2010): 2037-2045.

[0007] An object of the present invention is to provide a method for producing polyhydroxyalkanoate.

[0008] In addition, an object of the present invention is to provide polyhydroxyalkanoate.

[0009] In addition, it is an object of the present invention to provide a use of the polyhydroxyalkanoate for use in the production of bioplastics.

[0010] To solve the above problem, the present invention provides a method for producing polyhydroxyalkanoate, comprising the steps of: culturing bacteria having the ability to produce polyhydroxyalkanoate; physically disrupting bacterial cells to separate polyhydroxyalkanoate; and pulverizing the separated polyhydroxyalkanoate.

[0011] In addition, the present invention provides a polyhydroxyalkanoate produced by the above method.

[0012] In addition, the present invention provides the use of the polyhydroxyalkanoate for use in the production of bioplastics.

[0013] In the present invention, the physical cell disruption and powdering process is optimized to directly recover polyhydroxyalkanoate produced as bead-shaped particles within the cells of bacteria producing polyhydroxyalkanoate in its original form. By using the optimized polyhydroxyalkanoate production method of the present invention, polyhydroxyalkanoate can be produced with high purity and high yield without an organic solvent, and polyhydroxyalkanoate in the form of microbeads can be directly obtained without a separate molding process. Therefore, it can be utilized as a new polyhydroxyalkanoate production platform that can be used for biomedical or cosmetic purposes.

[0014] Figures 1a and 1b are SEM images showing the morphology of PHA produced by disrupting cells of seven strains exhibiting relatively high PHA productivity using a high-pressure homogenizer.

[0015] Figures 2a and 2b are diagrams showing the particle size of PHA produced by disrupting cells of seven strains exhibiting relatively high PHA productivity using a high-pressure homogenizer, as confirmed by DLS analysis.

[0016] Figure 3 is a TEM image showing the morphology of PHA within Halomonas xianhensisHN-1-3-2 cells before cell disruption.

[0017] Figure 4 is a diagram showing the particle size of Halomonas xianhensisHN-1-3-2 cells before cell disruption confirmed by DLS analysis.

[0018] Figure 5 is a diagram showing the particle size of PHA obtained after disrupting Halomonas xianhensisHN-1-3-2 cells with ultrasound, as confirmed by DLS analysis.

[0019] Figure 6 is a SEM image showing the morphology of PHA obtained after disrupting Halomonas xianhensisHN-1-3-2 cells using ultrasonic waves.

[0020] Figure 7 is a diagram showing the particle size of PHA obtained through physical cell disruption, prepared as a 10% suspension, and powdered by general drying, confirmed by DLS analysis.

[0021] Figure 8 is a diagram showing the particle size of PHA obtained through physical cell disruption, prepared as a 20% suspension, and powdered by general drying, confirmed by DLS analysis.

[0022] Figure 9 is a diagram showing the particle size of PHA obtained through physical cell disruption, prepared as a 10% suspension (w / w), and powdered by spray drying, as confirmed by DLS analysis.

[0023] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are provided as examples of the present invention, and if a detailed description of a technology or configuration well known to those skilled in the art may be judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0024] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0025] Unless otherwise defined, all technical terms used in this invention have the same meanings as those commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0026]

[0027] In one aspect, the present invention relates to a method for producing polyhydroxyalkanoate, comprising the steps of culturing bacteria having the ability to produce polyhydroxyalkanoates (PHA); physically disrupting bacterial cells to isolate polyhydroxyalkanoate; and pulverizing the isolated polyhydroxyalkanoate.

[0028] In one embodiment, the method may not use an organic solvent.

[0029] In one embodiment, the polyhydroxyalkanoate is poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), short-chain or medium-chain length, saturated or unsaturated PHA, or any copolymer thereof, or any combination thereof.

[0030] In one embodiment, the bacteria having polyhydroxyalkanoate producing ability are Cobetia, Ralstonia, Vibrio, Halomonas, Pseudomonas, Rhodopseudomonas, Bacillus, Methylobacterium, Methylosinus, Methylocella, Azotobacter, Alcaligenes, Aeromonas, Nocardia, Methylocapsa, Methylocaldum, Methylocystis, Methyloversatilis, Rubrivivax, It may be a bacterium belonging to the genus Xanthobacter, Cupriavidus or Hydrogenophaga.

[0031] In one embodiment, the bacteria having polyhydroxyalkanoate producing ability may be a halophilic strain, and may be Cobetia marina, Cobetia amphilecti, Halomonas xianhensis, Halomonas alkaliphila, Halomonas hydrothermalis, Halomonas litopenaei, Halomonas ventosae or Halomonas xianhensis, and Cobetia marina HN 1-5-1, Cobetia amphilecti HN 2-9-1, Halomonas alkaliphila HR-72-5, Halomonas hydrothermalis HSS-4, It may be Halomonas lithopenaei M039, Halomonas bentose M434 or Halomonas cyanhensis HN-1-3-2, and more preferably Halomonas lithopenaei YBW-3-4-1 (Accession No. KCTC 19194P), Covetia emphylectii HN 2-9-1 (Accession No. KCTC 8327P) or Halomonas cyanhensis HN-1-3-2 (Accession No. KCTC 19195P).

[0032] In one embodiment, the physical crushing may be physical crushing by applying ultrasound or pressure, and ultrasound may be applied at an intensity (amplificaiton) of 25 to 35% for 5 to 20 minutes, and the pressure may be applied at a pressure of 1000 to 1300 bar using a high-pressure homogenizer, and may be applied 2 to 4 times.

[0033] In one embodiment, the polyhydroxyalkanoate separated after physical disruption of bacterial cells may have an average particle size of 400 nm to 1 μm.

[0034] In one embodiment, the method can obtain polyhydroxyalkanoate produced within cells in the form of bead particles without a separate molding process, which is achieved by a cell disruption process using ultrasound under the above-described specific conditions.

[0035] In one embodiment, the cell disruption process using ultrasound under the above conditions of the present invention can directly recover polyhydroxyalkanoate having an average particle size of 400 to 600 nm in the form of beads generated within bacterial cells, while minimizing deformation of the particle size and shape thereof.

[0036] In one embodiment, the method can obtain polyhydroxyalkanoate produced within cells in the form of bead particles without separate molding processing.

[0037] In one embodiment, the step of pulverizing the separated polyhydroxyalkanoate may be performed by mixing the separated polyhydroxyalkanoate with triple distilled water after physical crushing to make a suspension of 5 to 30% (w / w) (water content of 70 to 95% (w / w)), and then pulverizing the mixture.

[0038] In one embodiment, the step of powdering the separated polyhydroxyalkanoate may be performed by normal drying or spray drying, and normal drying may be performed at 30 to 45°C for 36 to 60 hours, and spray drying may be performed under the conditions of an inlet temperature of 90 to 120°C, an outlet temperature of 30 to 60°C, a spray pressure of 140 to 180 kPa, and a hot air flow rate of 40 to 60 m / min.

[0039] In one embodiment, the powdered polyhydroxyalkanoate may have an average particle size of 10 μm or less, and may be from 400 nm to 5 μm.

[0040] In one embodiment, the method comprises the steps of: culturing bacteria having polyhydroxyalkanoate-producing ability and recovering the water content to 50-60% to prepare a cell suspension of 0.1 to 10% (w / w); applying ultrasound for 10 to 30 minutes using 0.05 to 0.2 M NaOH, 1 to 2 mM EDTA or purified water as an ultrasound buffer at an amplification intensity of 25 to 35% and an ultrasound pulse of 3 seconds on / off for 3 seconds to physically disrupt bacterial cells; centrifuging at 2000 to 4000 xg for 15 to 25 minutes to separate polyhydroxyalkanoate; And the separated polyhydroxyalkanoate may be prepared as a sample having a moisture content of 70 to 95% (w / w), and then a step of spray drying and powdering under the conditions of an inlet temperature of 90 to 120°C, an outlet temperature of 30 to 60°C, a spray pressure of 140 to 180 kPa, and a hot air flow rate of 40 to 60 m / min may be included.

[0041] In one embodiment, the cell disruption conditions using ultrasound in the method may be to apply ultrasound to bacteria having a polyhydroxyalkanoate-producing ability at a cell concentration of 0.1-10% (w / w) using an ultrasound buffer of 0.05 to 0.2 M NaOH, 1 to 2 mM EDTA, or purified water as an ultrasound buffer at an intensity of 30% and a pulse of On 3 seconds / Off 3 seconds for 10 minutes, and the polyhydroxyalkanoate produced within the bacterial cells by the optimized ultrasound treatment conditions can be directly recovered in the form of microbeads without deformation.

[0042] In one embodiment, in the step of separating polyhydroxyalkanoate by centrifugation, the separated polyhydroxyalkanoate can be washed using tertiary purified water, 50 to 250 ppm of NaHSO3, or 0.01 to 0.2 M NaOH as a washing buffer, and tertiary purified water or 150 to 250 ppm of NaHSO3 can be used as a washing buffer.

[0043] In one aspect, the present invention relates to polyhydroxyalkanoates prepared by the method of the present invention and their uses.

[0044] In one embodiment, the polyhydroxyalkanoate may be in the form of microbeads having an average particle size of 10 μm or less, and may be in the form of microbeads having an average particle size of 400 nm to 5 μm.

[0045] In one aspect, the present invention relates to the use of polyhydroxyalkanoates for the production of bioplastics.

[0046] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0047]

[0048] Example 1. Cultivation of PHA-producing microorganisms

[0049] 1-1. Selection of PHA producing strains

[0050] Among the eight strains producing PHA, the final strain to be used for PHA production was selected using cell density (OD 600 nm), dry cell weight (g / L), PHA content (wt %), and PHA concentration (g / L) as indicators. Specifically, the cell density of the culture solution diluted with 0.85% NaCl aqueous solution was measured at a wavelength of 600 nm using a spectrophotometer (TECAN). After uniformly suspending the cells in the culture solution using a vortex mixer, 1 ml of the sample was placed in a 1.5 ml microtube, and the cells were sedimented using a mini-centrifuge at 5000 × g for 20 min at 4℃. The supernatant was removed as much as possible, dried at 65℃ for 24 hours, and the dry cell mass was determined by subtracting the weight of the empty tube from the total weight to measure the weight of the dried cells. For the analysis of PHA content (wt %) and PHA concentration (g / L), 10 ml of sulfuric acid (Deajung, Sulfuric acid 98%, GR) was added to 20 g / L of Crotonic acid (Sigma-Aldrich, 98%, 107-93) and reacted at 100 °C for 10 minutes to prepare a standard solution. This was diluted to 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40, 50, 60, and 80 ug / L with tertiary purified water, and the absorbance was measured at 230 nm using a spectrophotometer (TECAN). Then, samples with concentrations showing absorbance values ​​within the range of 0.1000-0.9999 were used to prepare R 2 A standard curve with a value of 0.99 or higher was derived. Afterwards, 10 ml of sulfuric acid was added to the biomass samples crushed under each condition, reacted at 100°C for 10 minutes, and diluted with tertiary purified water to within the range of the standard curve. The absorbance was measured at the same wavelength and the crotonic acid content in the sample was calculated by substituting it into the standard curve, thereby analyzing the PHA content and concentration.

[0051] №Isolate numberEzBioCloudDry Cell Weight (g / L)PHA concentration (g / L)PHA content (%)Closest match1HR-72-5Halomonas alkaliphila0.9420.4649.22YBW-3-4-1Halomonas litopenaei1.0730.57±0.0153.33±1.073M434Halomonas ventosae1.891.36±0.0472±2.314HSS-4Halomonas hydrothermalis1.0520.3±0.0128.53±1.465HN-1-9-2Halomonas xianhensis1.090.73±0.2367.06±3.546HN-1-3-2Halomonas xianhensis2.6441.76±0.1671.5±2.157HN-2-9-1Cobetia amphilecti0.5810.2339.88HN-1-5-1Cobetia marina0.5340.2547

[0052] Among the analyzed strains, the halophilic strains Cobetia marinaHN 1-5-1, Cobetia amphilectiHN 2-9-1 (accession no. KCTC 8327P), Halomonas hydrothermalisHSS-4, Halomonas alkaliphilaHR-72-5, Halomonas ventosaeM434, Halomonas litopenaeiYBW-3-4-1 (accession no. KCTC 19194P), and Halomonas xianhensisHN 1-3-2 (accession no. KCTC 19195P) were selected as strains showing relatively high dry cell mass and PHA concentration.

[0053]

[0054] 1-2. PHA production

[0055] Each of the seven strains selected in the above Example 1-1 was cultured in a 1 L flask in an ASS medium containing ASS (Artificial sea salt) 30 g / L, Glucose 20 g / L, Soy peptone 5 g / L, and Yeast extract 1 g / L, which is the optimal growth and PHA production condition of Halomonas xianhensis HN 1-3-2, at 40°C and 180 rpm for 48 hours, to produce PHA. Specifically, each strain was cultured overnight at 37°C and 180 rpm in a 3% salt concentration medium (Soy peptone 5 g / L, Yeast extract 1 g / L, Artificial sea salt 30 g / L) (Pre-culture). The culture solution, whose cell density measured using a spectrophotometer (TECAN) was 1.5-1.8 in optical density (600 nm), was inoculated into ASS medium supplemented with 20 g / L glucose at 2% (v / v) and cultured at 40°C and 180 rpm (Transfer-culture). The culture solution, whose optical density (600 nm) was 4.5-5.0, was inoculated into ASS medium supplemented with 20 g / L glucose at 2% (v / v) and cultured at 40°C and 180 rpm (Production-culture). At this time, when using a flask, the culture was performed in a volume of 1 / 5 of the flask, and when using a 5 L fermenter, the culture was performed at a maximum working volume of 3 L, air 0.5 L / min (DO sat point 15%), 200-800 RPM, pH 7.6 (20-25% Ammonia solution), and 40 ℃.After the culture was completed, the culture solution was centrifuged at 5000 ×g at 4℃ for 20 minutes to remove the supernatant, and the moisture content of the recovered cells was measured using a moisture content analyzer (Kern, DAB 100-3 Moisture Analyzer). To directly check the biomass before disruption, the cells in the culture solution were uniformly suspended using a shaking mixer, and 1 ml of the cell culture solution was centrifuged at 5000 ×g at 4℃ for 20 minutes to obtain the precipitated cells.

[0056]

[0057] Example 2. Direct recovery analysis of bead-type PHA using high-pressure homogenization.

[0058] To confirm the extraction of PHA by physical cell disruption, the cells (water content: 50-60%) of each of the strains Cobetia marinaHN 1-5-1, Cobetia amphilectiHN 2-9-1, Halomonas hydrothermalisHSS-4, Halomonas alkaliphilaHR-72-5, Halomonas ventosaeM434, Halomonas litopenaeiYBW-3-4-1, and Halomonas xianhensisHN 1-3-2 recovered in Example 1-2 were each made into a 10% (w / w) uniform suspension using purified water. 5 ml of each suspension was placed in a 15 ml conical container, placed on ice, and passaged twice using a high pressure homogenizer (HPH) at a range of 1000-1300 bar to disrupt the cells. The disrupted cells were centrifuged at 3000 xg at 4℃ for 20 minutes to remove the supernatant, and the washing process of refilling the original volume with purified water and centrifuging under the same conditions to remove the supernatant was repeated twice. In order to confirm the characteristics of the PHA thus obtained, it was coated with platinum (Pt) for 120 to 180 seconds, and the microstructure and morphology of the PHA were analyzed by SEM, and the particle size thereof was analyzed by DLS (Dynamic Light Scattering) using purified water as a buffer.

[0059] SEM analysis results showed that all PHAs obtained from the seven strains were spherical nanoparticles (Fig. 1a and Fig. 1b), and the average particle sizes analyzed by DLS were Cobetia marina HN-1-5-1 766.43 ± 27.74 nm, Cobetia amphilecti HN-2-9-1 720.18 ± 43.21 nm, Halomonas hydrothermalis HSS-4 612.69 ± 4.95 nm, Halomonas alkaliphile HR-72-5 815.00 ± 23.96 nm, Halomonas ventosae M434 535.31 ± 7.64 nm, Halomonas litopenaei YBW-3-4-1 686.13 ± 29.43 nm, and Halomonas xianhensis HN 1-3-2 715.24 ± 27.74 nm. It was found to be 26.39 nm (Figs. 2a and 2b). Through this, it was confirmed that PHA biosynthesized within microorganisms can be directly recovered with an average particle size of 400 nm to 1 μm using a physical cell disruption method.

[0060]

[0061] Example 3. Direct recovery analysis of bead-type PHA using ultrasonic disruption

[0062] 3-1. Analysis of PHA yield and purity according to ultrasonic treatment conditions

[0063] The physical / mechanical disruption conditions for high-purity and high-efficiency extraction of PHA from strains in the form of microbeads were set as the basic conditions in the cell concentration experiment: sonication buffer 3 times purified water, sonication time 10 minutes, sonication intensity (Amplification) 30%, and post-sonication washing buffer 3 times purified water. Afterwards, for each condition, the experiment was conducted by applying the experimental results after the experiment and establishing the optimal conditions in the order of cell concentration, buffer used during sonication, sonication time (Reaction time), sonication intensity (Amplification), and post-sonication washing buffer (Washing buffer). Specifically, the cells of the Halomonas xianhensis HN-1-3-2 strain recovered in Example 1-2 (water content: 50-60%) were made into a uniform suspension (Table 1) of 0.1 to 20% (w / w) using tertiary purified water, and then 5 ml of the suspension was placed in a 15 ml conical container and placed on ice. The suspension was then subjected to ultrasonic disruption using an UltrasonicatorHD 4100 (BANDELIN) equipped with a TS103 probe under various ultrasonic buffer (Table 2), treatment time (Table 3), and treatment intensity (Table 4) conditions. After the cells were disrupted, they were centrifuged at 3000 ×g at 4℃ for 20 minutes to separate PHA and cells. Then, the washing buffer (Table 6) of 3rd purified water, 110 or 20% EtOH, 100 or 200 ppm NaHSO3, 0.05 or 0.1 M NaOH, or 0.5 or 1% NaClO) was filled to the original volume, centrifuged under the same conditions, and the washing process of removing the supernatant was repeated 3 times. The content (wt %) of PHA in the biomass recovered after ultrasonic disruption was analyzed using the Crotonic acid content as in Example 1-1. The recovery rate (Recovery) and purity (Purity) of PHA were calculated using the following mathematical equations 1 and 2 using the analyzed PHA content.

[0064] Cell concentration (%, w / w)Recovery (wt%)Purity (%)Non-Treatment2.29 ± 1.050.54 ± 0.070.150.56 ± 1.4974.47 ± 1.25149.50 ± 1.5765.30 ± 1.20548.52 ± 1.5669.39 ± 0.371046.32 ± 1.1069.65 ± 1.281523.80 ± 1.0341.35 ± 0.852015.99 ± 1.7832.01 ± 0.01

[0065] TestDry cellweight (g / L)ExtractedPHA biomass (g / L)QuantifiedPHA biomass (g / L)Recovery (wt%)Purity (%)Non-Treatment67.6043.002.393.535.553차 정제수67.6036.0027.2840.3575.77DisruptionBuffer2 mMEDTA67.6036.7029.6043.7980.651mMEDTA67.6039.6031.1946.1378.750.2 M NaOH67.6039.6032.7648.4682.720.05 M NaOH67.6039.0032.0247.3782.110.2M SDS67.6039.8031.3246.3478.700.05M SDS67.6040.0031.1446.0777.86

[0066] Reaction time (min)Recovery (wt%)Purity (%)02.22 ± 0.269.88 ± 1.19548.85 ± 0.2155.64 ± 4.801050.12 ± 0.2675.55 ± 3.591550.75 ± 0.5472.55 ± 2.322052.47 ± 0.1085.78 ± 2.992553.84 ± 0.8773.87 ± 4.613061.06 ± 1.5193.22 ± 0.16

[0067] Amplification (%)Recovery (wt%)Purity (%)02.90 ± 0.232.50 ± 0.372031.02 ± 2.4970.89 ± 6.912546.89 ± 0.4867.69 ± 1.393052.46 ± 2.6574.91 ± 4.343567.42 ± 0.3976.74 ± 4.18

[0068] BufferDry cell weight (g / L)Extracted PHA (g / L)Quantified PHA (g / L)Recovery (%)Purity (%)Non-washed21.1920.6014.5468.6570.613차 정제수21.1919.3014.7969.8176.6420% EtOH21.1920.6018.3386.5188.9710% EtOH21.1920.6018.3286.4788.94200 ppm NaHSO321.1920.6018.4086.8689.33100 ppm NaHSO321.1919.4016.4077.4284.550.1 M NaOH21.1920.8017.4782.4683.990.05 M NaOH21.1921.7017.5882.9981.021% NaClO21.192.802.2610.6680.620.5% NaClO21.195.404.8923.0690.46

[0069]

[0070]

[0071] As a result, the cell concentration conditions that significantly increased the recovery rate and purity of PHA were 0.1 to 10% (w / w), the ultrasonic buffer conditions were 0.05 to 0.2 M NaOH, the ultrasonic treatment time was 10 min (Pulse On 3 sec / Off 3 sec), the ultrasonic treatment intensity was 25 to 35%, and the washing buffer was 200 ppm NaHSO3 (Tables 2 to 6). However, in selecting the ultrasonic buffer and washing buffer, in addition to Recovery (wt%) and Purity (%), considerations such as process applicability and PHA suspension manufacturing possibility may be included, so the use of tertiary purified water as the ultrasonic buffer and washing buffer was also judged to be meaningful in the process, and the crushing conditions were established with tertiary purified water.

[0072]

[0073] 3-2. Extraction analysis of PHA in bead form

[0074] In order to confirm the ultrasonic extraction conditions that can extract intracellular biosynthesized bead-shaped PHA while maintaining its shape, the shape of intracellular biosynthesized PHA particles before cell disruption using ultrasonic waves was analyzed using TEM images, and the shape of PHA recovered after disruption was analyzed using SEM images. In order to confirm the shape of intracellular biosynthesized PHA particles before cell disruption, 1 ml of the cell culture solution of the Halomonas xianhensisHN-1-3-2 strain recovered in Example 1-2 was placed in a 1.5 ml e-tube and centrifuged at 5000 ×g for 3 minutes to recover the precipitated cells, which were then washed three times using PBS (Phosphate buffer saline with pH 7.2) under the same centrifugation conditions. The washed cells were reacted with 2% Glutaraldehyde (pH 7.2) at room temperature for 2 hours, and washed three times under the same washing conditions as above. Afterwards, secondary fixation and primary staining using Osmium Tetroxide were performed, followed by TEM analysis. In addition, SEM analysis was performed to confirm the morphology of PHA recovered after ultrasonic disruption under various conditions of Example 2-1. In addition, the PHA particle size before and after disruption was confirmed by DLS analysis.

[0075] After observing the cells with TEM before ultrasonic cell disruption and analyzing them with ImageJ, the cell length was found to be 1.474 ± ​​0.466 nm, and the size of intracellular PHA was found to be 584 ± 175 nm (Fig. 3), which was similar to the results of the cell size analysis using DLS (Fig. 4) and the size of the extracted PHA (Fig. 5). In addition, as a result of SEM and DLS analysis after crushing under the conditions of high recovery rate (yield) and purity of PHA derived from Example 3-1, the PHA particles crushed by sonication under the conditions of cell concentration of 0.1-10% (w / w), ultrasonic buffer with purified water for 3 times, ultrasonic treatment time of 10 minutes, ultrasonic treatment intensity of 30%, and washing buffer with purified water for 3 times were found to be spherical (Fig. 6), and their size was approximately 522.95 ± 2.59 nm (Fig. 5), confirming that PHA in the form of microbeads generated within cells was directly recovered without deformation through physical cell disruption. Therefore, it was confirmed that the above conditions are the most optimized conditions for extracting PHA in the form of microbeads within cells while being able to extract PHA in a high yield and purity (recovery rate: 69.81% and purity: 76.64%, see Table 6).

[0076]

[0077] Example 4. Powdering of PHA

[0078] 4-1. Powdering of physical cell disruption samples using general drying

[0079] In Examples 2 and 3, the PHA (400 nm to 1 ㎛) samples recovered by physical cell disruption using an ultrasonic or high-pressure homogenizer were mixed with triple-distilled water to prepare a 10% (condition with relatively high particle aggregation) or 20% (condition with relatively low particle aggregation) (w / w) PHA suspension. The suspension was dried at 35 or 40°C for 48 hours using an incubator (LabTech / LIB-1002M / Dual Chamber Incubator), pulverized using a mortar and pestle, redispersed in triple-distilled water, and the size of the redispersed particles after drying was analyzed by DLS.

[0080] As a result, when a 10% (w / w) PHA suspension was dried, PHA having a particle size of 0.9 to 2 μm was obtained (Fig. 7), and when a 20% (w / w) PHA suspension was dried, PHA having a particle size of approximately 700 nm was obtained (Fig. 8).

[0081]

[0082] 4-2. Powdering of physical cell disruption samples using spray drying

[0083] Using the spray dryer SD-1001 (Huco), the inlet temperature is maintained at 110℃, the outlet temperature is maintained at 50℃, the spray pressure is set at 160 kPa, the pump is set at 2% operating rate, and the blower is set at 48-50 m 3 After adjusting the flow rate to / min, the PHA (400 nm to 1 ㎛) sample recovered by physical cell disruption using ultrasonic or high-pressure homogenizer in Examples 2 and 3 was mixed with triple-distilled water to prepare a 10% (w / w) PHA suspension (water content 90%), which was sprayed and dried. The particle size of the dried sample was analyzed by DLS.

[0084] As a result, PHA with an average particle size of 4 to 5 μm was obtained outside the cyclone after spray drying, and PHA with an average particle size of 2 to 3 μm was obtained inside (Fig. 9). This confirmed that PHA maintained a bead shape with a particle size of 10 μm or less even after drying.

Claims

1. 1) A step of culturing bacteria having the ability to produce polyhydroxyalkanoates (PHA); 2) a step of physically disrupting bacterial cells to separate polyhydroxyalkanoates; and 3) A method for producing polyhydroxyalkanoate, comprising a step of powdering the separated polyhydroxyalkanoate.

2. In the first paragraph, the polyhydroxyalkanoate is poly(3-hydroxypropionate) (PHP or P3HP), poly(3-hydroxybutyrate) (PHB or P3HB), poly(4-hydroxybutyrate) (P4HB), poly(3-hydroxyvalerate) (PHV or P3HV), poly(4-hydroxyvalerate) (P4HV), poly(5-hydroxyvalerate) (P5HV), poly(3-hydroxyhexanoate) (PHHx or P3HHx), poly(3-hydroxyoctanoate) (PHO or P3HO), poly(3-hydroxydecanoate) (PHD or P3HD), poly(3-hydroxyundecanoate) (PHU, P3HU), short-chain or medium-chain length, A method for producing a polyhydroxyalkanoate comprising a saturated or unsaturated PHA, or any copolymer thereof, or any combination thereof.

3. In the first paragraph, the bacteria having the ability to produce polyhydroxyalkanoate are Cobetia, Ralstonia, Vibrio, Halomonas, Pseudomonas, Rhodopseudomonas, Bacillus, Methylobacterium, Methylosinus, Methylocella, Azotobacter, Alcaligenes, Aeromonas, Nocardia, Methylocapsa, Methylocaldum, Methylocystis, Methyloversatilis, Rubrivivax, A method for producing polyhydroxyalkanoate belonging to the genus Xanthobacter, Cupriavidus or Hydrogenophaga.

4. A method for producing polyhydroxyalkanoate according to claim 1, wherein the bacteria having the ability to produce polyhydroxyalkanoate are Cobetia marina, Cobetia amphilecti, Halomonas xianhensis, Halomonas alkaliphila, Halomonas hydrothermalis, Halomonas litopenaei, Halomonas ventosae or Halomonas xianhensis.

5. A method for producing polyhydroxyalkanoate, wherein the bacterium having polyhydroxyalkanoate production ability in paragraph 1 is Halomonas lithopenaeiYBW-3-4-1 (Accession No. KCTC 19194P), Covetia emphylectii HN 2-9-1 (Accession No. KCTC 8327P), or Halomonas cyanhensis HN-1-3-2 (Accession No. KCTC 19195P).

6. A method for producing polyhydroxyalkanoate, wherein the physical crushing in paragraph 1 is performed by physically crushing using ultrasound or pressure.

7. A method for producing polyhydroxyalkanoate, wherein, in claim 6, ultrasonic waves are applied for 5 to 20 minutes under conditions of an ultrasonic treatment intensity (amplification) of 25 to 35% to physically disrupt cells.

8. A method for producing polyhydroxyalkanoate, wherein the method comprises physically crushing the polyhydroxyalkanoate by applying a pressure of 1000 to 1300 bar using a high-pressure homogenizer in paragraph 6.

9. A method for producing polyhydroxyalkanoate, wherein the polyhydroxyalkanoate separated by physical crushing in step 2) in paragraph 1 has an average particle size of 400 nm to 1 ㎛.

10. A method for producing polyhydroxyalkanoate, wherein the separated polyhydroxyalkanoate is powdered by general drying or spray drying in the first paragraph.

11. A method for producing polyhydroxyalkanoate, wherein spray drying is performed under conditions of an inlet temperature of 90 to 120°C and an outlet temperature of 30 to 60°C in the 10th paragraph.

12. A method for producing polyhydroxyalkanoate, wherein the polyhydroxyalkanoate powdered in step 3) has an average particle size of 10 ㎛ or less in the first paragraph.

13. Polyhydroxyalkanoate manufactured by the method of paragraph 1.

14. Polyhydroxyalkanoate according to claim 13, having an average particle size of 10 ㎛ or less.

15. Polyhydroxyalkanoate in the form of microbeads according to claim 13.

16. Use of polyhydroxyalkanoate of claim 13 for the manufacture of bioplastics.

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