Application of pseudomonas strain y2 in synthesis of polyhydroxyalkanoate

Abstract

The invention relates to the field of microorganisms, in particular to application of a pseudomonas strain y2 in synthesis of polyhydroxyalkanoate, a microbial agent prepared from the pseudomonas strain y2 is added into wastewater to be treated, and the pseudomonas strain y2 is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M 20231228. The strain realizes microbial efficient synthesis of PHA, has good synthesis capability and environmental adaptability when being used for PHA synthesis, and can realize efficient synthesis of PHA in a pure culture state. The chemical wastewater contains abundant organic matters and can be used as a carbon source for synthesizing the PHA by the pseudomonas strain y2, so that the production cost of the PHA is further reduced, a higher PHA synthesis yield is obtained, and a feasible way is provided for reducing the industrial production cost of the PHA.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to application of Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates. Background Art

[0002] The COD content of high-salinity organic sewage is more than several thousand milligrams per liter, and some are even as high as tens of thousands to hundreds of thousands of milligrams per liter. However, from the perspective of sustainable development, the large amount of organic matter contained in high-salinity organic sewage is a resource. Studies have found that volatile fatty acids produced by anaerobic fermentation of high-salinity organic sewage can be used as an ideal carbon source for the synthesis of polyhydroxyalkanoates (PHA). PHA is a biodegradable polymer produced by microorganisms under conditions of sufficient carbon source and lack of nutrients such as nitrogen and phosphorus. PHA has many excellent properties such as biodegradability, biocompatibility, optical activity, piezoelectricity, gas barrier properties, etc. It has broad application prospects in biodegradable packaging materials, tissue engineering materials, sustained-release materials, electrical materials and medical materials.

[0003] In recent decades, the production of biopolymers has received great attention. However, their widespread application is limited by factors such as high production costs and insufficient production, among which the limitation of bacterial species synthesis is a prominent problem. The PHA synthesis ability of different bacterial species determines whether they have the potential for large-scale production. According to relevant reports, a variety of bacteria with PHA synthesis ability have been isolated, including Pseudomonas , Bacillus and Paracococcus etc., Liang et al. reported Bacillus cereus L17 can synthesize poly-β-hydroxybutyrate (PHB) using sodium acetate as the sole carbon source, with a PHB yield of 28.1%. Li et al. first isolated a strain named Brevundimonas vesicularis The PHA-producing bacteria of UJN1 were optimized with sucrose as the carbon source, producing 34.1% PHA of the cell dry weight. However, the above strains generally have problems such as low synthesis efficiency. Therefore, screening out efficient, stable, and environmentally adaptable PHA-synthesizing strains will become the key to achieving large-scale and efficient production of PHA. Due to the complexity of the marine environment, the ocean is rich in microbial resources, which is a treasure trove that needs to be developed. How to organically combine the two has become the focus of the inventor's research. Summary of the invention

[0004] In order to solve the problems of poor environmental adaptability and low synthesis efficiency in the prior art of using microorganisms to synthesize PHA, the present invention provides an application of Pseudomonas strain y2 in the synthesis of polyhydroxyalkanoates. The present invention utilizes Pseudomonas strain y2 ( Pseudomonas Khazarica y2) realizes efficient microbial synthesis of PHA. It has good synthesis ability and environmental adaptability when used for PHA synthesis, and can realize efficient synthesis of PHA in pure culture. Chemical wastewater contains rich organic matter, which can be used as a carbon source for Pseudomonas strain y2 to synthesize PHA, further reducing the production cost of PHA, and providing a feasible way to reduce the industrial production cost of PHA.

[0005] In the present invention, the influence of environmental factors such as pH, temperature, carbon source, carbon-nitrogen ratio, etc. on the synthesis process of polyhydroxyalkanoates by Pseudomonas strain y2 is firstly deeply studied and discussed, and the optimal conditions and optimal yield of PHA synthesis by Pseudomonas strain y2 are obtained, which can provide a theoretical basis and technical guidance for the resource utilization of industrial wastewater. The Pseudomonas strain y2 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC M 20231228.

[0006] The Pseudomonas strain y2 was inoculated into a basal salt medium (MSM) medium with glucose as the sole carbon source, and its PHA synthesis was investigated by changing the inoculation amount, carbon source, dissolved oxygen, temperature, carbon-nitrogen ratio, and pH. It was found that the strain y2 had the ability to synthesize PHA when the inoculation amount was 5-25%, the carbon source was glucose and sodium acetate, the dissolved oxygen was 140-220 rpm, the temperature was 20-40°C, the carbon-nitrogen ratio was (5-25):1, and the pH was 4-10, indicating that the Pseudomonas strain y2 has excellent environmental adaptability and good application potential.

[0007] Preferably, the inventors found that the Pseudomonas strain y2 was inoculated into MSM medium with glucose as the carbon source at an optimal inoculation amount of 15% (bacterial solution OD 600 =3.0), temperature 30 ℃, carbon-nitrogen ratio of 15:1, pH 7, and culture at 200 rpm. Samples were taken regularly to detect the synthesis of PHA. The PHA yield of Pseudomonas strain y2 could reach 42.91% of the cell dry weight.

[0008] The formula of MSM basic fermentation medium is: Na2HPO4 9 g, KH2PO4 1.5 g, NH4Cl 1 g, trace elements 1 mL, distilled water is added to make up to 1 L, and the pH is adjusted to 7.0 with 5 M NaOH and 1 M HCl solution.

[0009] The composition of the trace elements is: MnSO4·H2O 1.5 g, H3BO3 1.0 g, EDTA·2Na 1.0 g, CuSO4·5H2O 0.2 g, CoCl2·6H2O 0.2 g, ZnSO4·7H2O 0.2 g, NaSiO3·9H2O 0.2 g, Na2MoO4·2H2O 0.05 g, NiSO4·6H2O 0.05 g, dilute to 1 L with distilled water and mix well.

[0010] Based on this, the present invention further provides the use of Pseudomonas strain y2 in the synthesis of polyhydroxyalkanoates, and the bacterial agent prepared by the strain is added to the wastewater to be treated. The biological deposit information of the Pseudomonas strain y2 is: Preservation time: July 7, 2023; Name of depository: China Center for Type Culture Collection; Deposit number: CCTCC M 20231228; Address of depository: Wuhan University, Wuhan, China; Taxonomic nomenclature: Pseudomonas Khazarica y2.

[0011] Preferably, the wastewater to be treated is simulated propylene oxide saponification wastewater, which contains sodium acetate 4000-4600 mg / L, glucose 500-600 mg / L, CaCl2 2500-3500 mg / L, NH4Cl 20-300 mg / L, NaNO3 10-350 mg / L, KH2PO4 3-10 mg / L, and NaCl 10-50 g / L.

[0012] The composition of this simulated wastewater is similar to that of propylene oxide saponification wastewater in industry and can be used to verify the treatment effect of the bacterial agent.

[0013] Preferably, the method for preparing the bacterial agent from the Pseudomonas strain y2 is: inoculating the Pseudomonas strain y2 into LB liquid culture medium, placing the LB liquid culture medium in a constant temperature shaking incubator, and culturing for 12 h under the conditions of 30° C. and 200 rpm.

[0014] As a further preferred embodiment, the OD of the bacterial agent prepared by the Pseudomonas strain Y2 is 600 =3.0.

[0015] Preferably, the conditions for adding the bacterial agent prepared by Pseudomonas strain y2 to the wastewater to be treated are: inoculation amount is 5-25% (v / v), temperature is 20-40°C, and pH is 4-10.

[0016] As a further preference, the conditions for adding the bacterial agent prepared by the Pseudomonas strain y2 to the wastewater to be treated are: an inoculation amount of 10-20%, a temperature of 25-30° C., and a pH of 6-8.

[0017] As a further preferred embodiment, the conditions for adding the bacterial agent prepared by the Pseudomonas strain y2 to the wastewater to be treated are: an inoculation amount of 15%, a temperature of 30°C, and a pH of 7, at which time the synthesis amount of PHA of the strain is 30.8%.

[0018] In summary, the present invention utilizes Pseudomonas strain y2 ( Pseudomonas Khazarica y2) realizes efficient microbial synthesis of PHA. It has good synthesis ability and environmental adaptability when used for PHA synthesis, and can realize efficient synthesis of PHA in pure culture. Chemical wastewater contains rich organic matter, which can be used as a carbon source for Pseudomonas strain y2 to synthesize PHA, further reducing the production cost of PHA and achieving a higher PHA synthesis yield, providing a feasible way to reduce the industrial production cost of PHA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the PHA synthesis capacity of Pseudomonas strain y2 under different inoculation conditions, Figure 2 is the PHA synthesis capacity of Pseudomonas strain y2 under different carbon source conditions, Figure 3 is the PHA synthesis capacity of Pseudomonas strain y2 under different dissolved oxygen conditions, Figure 4 is the PHA synthesis capacity of Pseudomonas strain y2 under different temperature conditions, Figure 5 is the PHA synthesis capacity of Pseudomonas strain y2 under different carbon-nitrogen ratios, Figure 6 is the PHA synthesis capacity of Pseudomonas strain y2 under different pH conditions, Figure 7 is the PHA synthesis capacity of Pseudomonas strain y2 under optimal conditions, Figure 8 PHA synthesis ability of Pseudomonas strain y2 in simulated propylene oxide saponification wastewater. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the technical solution of the present invention in more detail and should not be construed as limiting the protection scope of the present invention.

[0021] The experimental reagents and consumables used, unless otherwise specified, are all commercially available products; if the conditions are not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer.

[0022] The Pseudomonas strain y2 used is disclosed in patent CN117801986A, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC M 20231228, and the inventor is not described in detail.

[0023] The MSM basic fermentation medium formula used in the following examples is: Na2HPO49 g, KH2PO41.5 g, NH4Cl 1 g, 1 mL of trace elements, distilled water to 1 L, and pH adjusted to 7.0 with 5 M NaOH and 1 M HCl solution.

[0024] The composition of trace elements is: MnSO4·H2O 1.5 g, H3BO3 1.0 g, EDTA·2Na 1.0 g, CuSO4·5H2O 0.2 g, CoCl2·6H2O 0.2 g, ZnSO4·7H2O 0.2 g, NaSiO3·9H2O 0.2 g, Na2MoO4·2H2O 0.05 g, NiSO4·6H2O 0.05 g. Add distilled water to make up to 1 L and mix well.

[0025] Example 1 The fermentation method of Pseudomonas strain Y2 is carried out according to the following steps: 1) A single colony of Pseudomonas strain y2 was inoculated into a 500 mL conical flask containing 300 mL LB liquid medium (yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L), and the medium was placed in a constant temperature shaking incubator and cultured at 30 °C and 200 rpm for 12 h to obtain a seed solution. At this time, the OD of the seed solution was 600 =3.0.

[0026] 2) After the seed solution is cultivated, centrifuge it at 4°C and 9000 rpm for 10 min, wash it with sterile water to remove the remaining LB liquid medium, and centrifuge it again at the same conditions for 10 min. Mix the centrifuged bacteria with sterile water to obtain the bacterial solution OD 600 =3.0, and the bacterial liquid was inoculated into a 500 mL conical flask containing 300 mL MSM fermentation medium, and cultured at a constant temperature of 200 rpm and 30 °C for 3 days.

[0027] 3) Samples were taken every 12 hours, centrifuged at 9000 rpm for 10 min at 4 °C, the supernatant was discarded, the precipitate was frozen at -80 °C overnight and then dried using a vacuum freeze dryer. The dried samples were used to determine the PHA yield by gas chromatography.

[0028] 4) Determination of PHA content After detection by gas chromatography, the peak area is compared with the standard curve to obtain the PHA content. The measured content is converted with the cell dry weight to obtain the strain PHA yield. The calculation formula is: PHA yield (g / L) · Dry weight of bacteria (g / L); Where x = (peak area + 9558.1) / 273680; PHA Percentage = 100%.

[0029] Example 2 The PHA synthesis ability of Pseudomonas strain y2 at different inoculation amounts was compared by the following method.

[0030] 1) Prepare the target MSM fermentation medium, using sodium acetate as the carbon source and ammonium chloride as the nitrogen source. Weigh the corresponding sodium acetate and add it to the MSM basic fermentation medium, control the carbon-nitrogen ratio to 10:1, adjust the pH to 7 with 5 M NaOH, and dispense 300 mL into a 500 mL conical flask.

[0031] 2) Obtain seed solution according to the method described in Example 1 (at this time, the seed solution OD 600 =3.0) and the seed liquid was processed to obtain a bacterial liquid, and the MSM fermentation medium prepared in step 1) of this embodiment was cultured at a constant temperature with shaking at different inoculation amounts, the culture temperature was 30 ° C, the rotation speed was set to 200 rpm, and the inoculation amount was set to 5%, 10%, 15%, 20%, and 25%, respectively. After the fermentation culture was completed, samples were taken according to the method described in Example 1 to obtain dried samples, and the dried samples were used for the determination of PHA.

[0032] 3) Multiple experimental groups were set up, and three parallel experiments were set up for all experimental groups. Each experimental group used the inoculation amount of 5%, 10%, 15%, 20%, and 25% (v / v) described in step 2), respectively, and samples were taken according to the method described in Example 1, and the optimal inoculation amount was determined based on the cell dry weight and PHA yield.

[0033] The results are as follows Figure 1As shown in the figure, at the beginning of fermentation, the PHA content was high because the strain was enriched and cultured in LB liquid medium. The LB medium was rich in carbon sources and met the conditions for the synthesis of PHA, so the strain synthesized more PHA in the LB medium. The PHA synthesizing bacteria had a low bacterial content under low inoculation conditions and were greatly affected by environmental stress. It took a long time to adapt to the culture environment and synthesized less PHA. Under the conditions of 15%, 20% and 25% inoculations, more PHA was synthesized, but with the increase of inoculation, the PHA content did not increase significantly. Therefore, the inoculation of 15% was selected as the optimal inoculation. When the inoculation was 15%, the maximum PHA synthesis amount and the PHA yield were 31.26%.

[0034] Example 3 The PHA synthesis ability of Pseudomonas strain y2 under different carbon sources was compared by the following method.

[0035] 1) Prepare the target MSM fermentation medium, use sodium acetate, sodium propionate, sodium butyrate, glucose, and sucrose as carbon sources, and ammonium chloride as nitrogen source, weigh the corresponding sodium acetate, sodium propionate, sodium butyrate, glucose, and sucrose and add them to the MSM basic fermentation medium, control the carbon-nitrogen ratio to 10:1, adjust the pH to 7 with 5 M NaOH, and dispense 300 mL into a 500 mL conical flask.

[0036] 2) Prepare seed solution (OD 600 = 3.0) and the seed liquid was treated to obtain a bacterial liquid, which was cultured at a constant temperature and shaken in the MSM fermentation medium with different carbon sources prepared in step 1) of this embodiment (inoculation amount 15%), and samples were taken according to the method described in Example 1 to obtain freeze-dried samples for PHA content determination.

[0037] 3) Multiple experimental groups were set up, and three parallel experiments were set up for all experimental groups. Each experimental group used the MSM fermentation medium with sodium acetate, sodium propionate, sodium butyrate, glucose, and sucrose as carbon sources described in step 1) for fermentation and sampling according to the method described in step 2), and the optimal culture carbon source was determined based on the cell dry weight and PHA yield.

[0038] like Figure 2As shown, at the start of fermentation, the strain was enriched and cultured in LB liquid medium, and the LB medium was rich in carbon sources, so the strain synthesized more PHA in the LB medium. In the medium with sodium butyrate as the carbon source, the PHA synthesizing bacteria cannot use sodium butyrate as a carbon source to supply its own life activities, and can only decompose and consume PHA to supply life activities, and cannot synthesize PHA. Therefore, the PHA content is greatly reduced, indicating that sodium butyrate cannot be used as a fermentation carbon source, and cannot be used by the strain, indicating that the strain may lack enzymes related to sodium butyrate metabolism. Under conditions where the carbon source is more suitable, such as glucose, sucrose and sodium acetate as carbon sources, there is more PHA synthesis, among which the PHA synthesis amount is higher when the carbon source is glucose, so glucose is considered to be a carbon source suitable for fermentation. Under this condition, the maximum PHA yield is 33.43%. Glucose, sucrose and sodium acetate can all be used as carbon sources suitable for fermentation.

[0039] Example 4 The PHA synthesis ability of Pseudomonas strain y2 under different dissolved oxygen conditions was compared by the following method.

[0040] 1) Prepare the target MSM fermentation medium, using glucose as the carbon source and ammonium chloride as the nitrogen source. Weigh the corresponding glucose and add it to the MSM basic fermentation medium. Control the carbon-nitrogen ratio to 10:1. Adjust the pH to 7 with 5 M NaOH and dispense 300 mL into a 500 mL conical flask.

[0041] 2) Obtain seed solution (OD 600 =3.0) and the seed liquid was processed to obtain a bacterial liquid, and the MSM fermentation medium prepared in step 1) of this embodiment was cultured at different speeds under constant temperature shaking (inoculation amount 15%), the culture temperature was 30 ° C, and the speeds were set to 140 rpm, 160 rpm, 180 rpm, 200 rpm, and 220 rpm, respectively, for a total of five speeds. After the fermentation culture was completed, sampling was performed according to the method described in Example 1 to obtain dried samples, and the dried samples were used for the determination of PHA.

[0042] 3) Setting up multiple experimental groups, all of which were set up with three parallel experiments, and each experimental group used the culture speed of 140 rpm, 160 rpm, 180 rpm, 200 rpm, and 220 rpm described in step 2) respectively, and sampling was performed according to the method described in Example 1, and the optimal culture speed (dissolved oxygen) was determined based on the cell dry weight and PHA yield.

[0043] The results are as follows Figure 3As shown in the figure, at the beginning of fermentation, the PHA content was high because the strain was enriched and cultured in LB liquid medium. The LB medium was rich in carbon sources and met the conditions for PHA synthesis, so the strain synthesized more PHA in the LB medium. The PHA synthesizing bacteria were greatly affected by environmental stress in a low dissolved oxygen environment, and the synthesis of PHA was less. At 180 rpm and 200 rpm, where the conditions were more suitable and the dissolved oxygen content was higher, more PHA was synthesized, indicating that the bacteria were aerobic bacteria. The PHA synthesis was the highest at 200 rpm, so 200 rpm was considered to be the appropriate fermentation speed (dissolved oxygen content). The maximum PHA yield at this speed was 36.71%.

[0044] Example 5 The PHA synthesis ability of Pseudomonas strain y2 under different temperature conditions was compared by the following method.

[0045] 1) Prepare MSM fermentation medium, using glucose as the carbon source and ammonium chloride as the nitrogen source. Weigh the corresponding amount of glucose and add it to the MSM basic fermentation medium, control the carbon-nitrogen ratio to 10:1, adjust the pH to 7 with 5 M NaOH, and dispense 300 mL into a 500 mL conical flask.

[0046] 2) Obtain seed solution (OD 600 =3.0) and the seed liquid was treated to obtain a bacterial liquid, and the MSM fermentation medium prepared in step 1) of this embodiment was cultured at different temperatures with constant temperature shaking (inoculation amount 15%, rotation speed 200 rpm), the culture temperature was 5 ° C as a gradient, the temperature range was 20 ° C-40 ° C, a total of five temperatures, the rotation speed was set to 200 rpm, and after the fermentation culture was completed, the sample was taken according to the method described in Example 1 to obtain a dried sample, and the dried sample can be used for the determination of PHA.

[0047] 3) Setting up multiple experimental groups, all of which were set up with three parallel experiments, and each experimental group used 20°C, 25°C, 30°C, 35°C and 40°C as the culture temperature described in step 2), and sampled according to the method described in Example 1, and determined the optimal culture temperature based on the cell dry weight and PHA yield.

[0048] The results are as follows Figure 4As shown in the figure, at the beginning of fermentation, the PHA content was high because the strain was enriched and cultured in LB liquid medium. The LB medium was rich in carbon sources and met the conditions for the synthesis of PHA, so the strain synthesized more PHA in the LB medium. The PHA synthesizing bacteria were greatly affected by environmental stress under low and high temperature environments, and the synthesis of PHA was less. In order to ensure the normal progress of cell life activities, PHA was decomposed to supply cell life activities. At the more suitable conditions of 30℃ and 25℃, more PHA was synthesized, among which the PHA synthesis amount was the highest at 30℃, so 30℃ was considered to be the suitable fermentation temperature, and the maximum PHA yield at this temperature was 37.23%.

[0049] Example 6 The PHA synthesis ability of Pseudomonas strain y2 under different carbon-nitrogen ratios was compared by the following method.

[0050] 1) Prepare MSM fermentation medium, using glucose as the carbon source and ammonium chloride as the nitrogen source. Weigh the corresponding glucose and add it to the MSM basic fermentation medium. Control the carbon-nitrogen ratio to 5:1, 10:1, 15:1, 20:1, and 25:1. Adjust the pH value with 5 M NaOH and set the pH value to 7. Dispense 300 mL in a 500 mL conical flask.

[0051] 2) Obtain seed solution (OD 600 =3.0) and treating the seed liquid to obtain a bacterial liquid, and culturing the MSM fermentation medium with different carbon-nitrogen ratios prepared in step 1) of this embodiment under different carbon-nitrogen ratios at constant temperature and shaking (inoculation amount 15%, temperature 30°C, rotation speed 200 rpm), and sampling according to the method described in Example 1 to obtain dried samples, which can be used for the determination of PHA.

[0052] 3) Setting up multiple experimental groups, all of which were set up with three parallel experiments, using the MSM fermentation medium with a carbon-nitrogen ratio of 5:1, 10:1, 15:1, 20:1, and 25:1 as described in step 1), respectively, and fermenting and sampling were performed according to the method described in step 2), and the optimal culture carbon-nitrogen ratio was determined based on the cell dry weight and PHA yield.

[0053] according to Figure 5Analysis shows that at the start of fermentation, the strain was enriched and cultured in LB liquid medium, which was rich in carbon sources, and the strain synthesized more PHA in LB medium. The carbon source concentration of the PHA synthesizing bacteria was low in the medium with a low carbon-nitrogen ratio. Due to the limitation of the carbon source concentration, the strain's PHA synthesis was limited. Along with the consumption of external carbon sources, the normal life activities of the strain were limited, and it could only decompose and consume PHA to supply life activities. Under relatively suitable carbon and nitrogen conditions, such as high carbon-nitrogen ratios of 15:1, 20:1, and 25:1, more PHA was synthesized. Among them, the PHA synthesis amount was higher when the carbon-nitrogen ratio was 15:1, so it is believed that a carbon-nitrogen ratio of 15:1 is a carbon-nitrogen ratio suitable for fermentation. Under this condition, the maximum PHA yield is 39.47%.

[0054] Example 7 The PHA synthesis ability of Pseudomonas strain y2 at different pH was compared by the following method.

[0055] 1) Prepare MSM fermentation medium, using glucose as the carbon source and ammonium chloride as the nitrogen source. Weigh the corresponding amount of glucose and add it to the MSM basic fermentation medium. Control the carbon-nitrogen ratio to be 15:1. Adjust the pH value with 5 M NaOH and 1 M HCl solution. Set the pH values ​​to 5, 6, 7, 8, and 9, respectively. Dispense 300 mL in 500 mL conical flasks.

[0056] 2) Obtain seed solution (OD 600 =3.0) and treating the seed liquid to obtain a bacterial liquid, and culturing the MSM fermentation medium with different pH prepared in step 1) of this embodiment at a constant temperature and shaking conditions of 15% inoculation amount, 30°C and 200 rpm, and sampling according to the method described in Example 1 to obtain dried samples, which can be used for the determination of PHA.

[0057] 3) Setting up multiple experimental groups, all experimental groups were set up with three parallel experiments, each experimental group used the MSM fermentation medium with pH of 5, 6, 7, 8, and 9 described in step 1), respectively, and fermented and sampled according to the method described in step 2), and determined the optimal culture pH according to the cell dry weight and PHA yield.

[0058] The results are as follows Figure 6As shown in the figure, at the start of fermentation, the strain was enriched and cultured in LB liquid medium, which was rich in carbon sources, and the strain synthesized more PHA in LB medium. The PHA synthesizing bacteria were greatly affected by environmental stress under high and low pH environments, and were not suitable for growth and PHA synthesis. The synthesis of PHA was less, and PHA was decomposed to supply cell life activities. At more suitable pH values, pH=6, pH=7 and pH=8, more PHA was synthesized, which shows that the strain is more adapted to a neutral environment. Among them, the amount of PHA synthesis was higher at pH=7, indicating that pH=7 is a suitable fermentation pH, and the maximum PHA yield at this pH value is 40.17%.

[0059] Example 8 The PHA synthesis ability of Pseudomonas strain y2 under optimal conditions was detected and analyzed by the following method.

[0060] 1) Prepare MSM fermentation medium with glucose as carbon source and ammonium chloride as nitrogen source. Weigh the corresponding glucose and add it to MSM basic fermentation medium. Control the carbon-nitrogen ratio to 15:1. Adjust the pH value with 1 M hydrochloric acid solution and set the pH value to 7. Dispense 300 mL in 500 mL conical flasks.

[0061] 2) Obtain seed solution (OD 600 =3.0) and treating the seed liquid to obtain a bacterial liquid, and culturing the MSM fermentation medium prepared in step 1) of this embodiment at an inoculation amount of 15%, a temperature of 30°C and a constant temperature shaking of 200 rpm, and sampling according to the method described in Example 1 to obtain a dried sample, which can be used for the determination of PHA.

[0062] 3) The experimental group set up three parallel experiments, and carried out fermentation and sampling according to the method described in step 2), and determined the PHA yield under the optimal culture conditions based on the cell dry weight and PHA yield.

[0063] according to Figure 7 Analysis showed that under the optimal fermentation conditions, suitable temperature, pH and sufficient carbon source provided suitable conditions for the growth of the strain and the synthesis of PHA, and removed the restrictions of environmental factors on the synthesis of PHA by the strain. The PHA yield was greatly improved compared with that before optimization, reaching the highest level at 48 h, with the maximum PHA yield reaching 42.91%. During the fermentation process, thanks to the suitable fermentation conditions, the production speed of PHA by the strain was significantly improved, and the yield was also greatly increased. However, after 48 h, the strain may be limited by the consumption of nutrients and the decline of the strain itself, and the PHA content began to decline, that is, PHA began to be consumed by the strain.

[0064] Example 9 The PHA synthesis ability of Pseudomonas strain y2 in simulated propylene oxide saponification wastewater was detected and analyzed by the following method.

[0065] 1) Simulated propylene oxide saponification wastewater was used as the fermentation medium for PHA fermentation production. The simulated wastewater formula was sodium acetate 4487 mg / L, glucose 560 mg / L, CaCl2 2967 mg / L, NH4Cl 214 mg / L, NaNO3 337 mg / L, KH2PO4 5.9 mg / L, and NaCl 30 g / L. 300 mL was dispensed into 500 mL conical flasks.

[0066] 2) Obtain seed solution (OD 600 =3.0) and the bacterial solution obtained by treating the seed liquid is inoculated into the simulated wastewater prepared in step 1) of this embodiment at an inoculum amount of 15% (v / v) for constant temperature shaking culture, and sampling is performed according to the method described in Example 1 to obtain a dried sample, which can be used for the determination of PHA.

[0067] 3) The experimental group set up three parallel experiments, and carried out fermentation and sampling according to the method described in step 2), and determined the PHA yield under the optimal culture conditions based on the cell dry weight and PHA yield.

[0068] according to Figure 8 The analysis showed that under the conditions of simulated propylene oxide saponification wastewater, PHA was decomposed in large quantities in the initial stage of strain fermentation and used to combat environmental stress. During the fermentation process, environmental factors had a strong restriction on the synthesis of PHA by the strain, and the PHA production was lower than that in the fermentation medium to a certain extent. The PHA synthesis time and the peak time were prolonged. However, after adapting to the environmental pressure, the strain still had a good PHA production, and the production reached the highest at 60 h, with the maximum PHA production reaching 30.8%. However, after 60 h, the strain may be limited by the consumption of nutrients, the decline of the strain itself, and other reasons, and the PHA content began to decline, that is, PHA began to be consumed by the strain.

[0069] It can be seen that the present invention uses Pseudomonas strain y2 to achieve efficient microbial synthesis of PHA. It has good synthesis ability and environmental adaptability when used for PHA synthesis, and can achieve efficient synthesis of PHA in a pure culture state. Chemical wastewater, especially propylene oxide saponification wastewater, contains rich organic matter, which can be used as a carbon source for Pseudomonas strain y2 to synthesize PHA, further reducing the production cost of PHA, achieving a higher PHA synthesis yield, and providing a feasible way to reduce the industrial production cost of PHA.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions, modifications, etc. made by technicians in this field without any creative work within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates, characterized in that: The bacterial agent prepared by the strain is added to the wastewater to be treated. The Pseudomonas strain y2 is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC M 20231228. The wastewater to be treated is simulated propylene oxide saponification wastewater.

2. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 1, characterized in that: The simulated propylene oxide saponification wastewater contains 4000-4600 mg / L of sodium acetate, 500-600 mg / L of glucose, 2500-3500 mg / L of CaCl2, 20-300 mg / L of NH4Cl, 10-350 mg / L of NaNO3, 3-10 mg / L of KH2PO4, and 10-50 g / L of NaCl.

3. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 1, characterized in that: The method for preparing the bacterial agent from the Pseudomonas strain y2 is: inoculating the Pseudomonas strain y2 into an LB liquid culture medium, placing the LB liquid culture medium in a constant temperature shaking incubator, and culturing for 12 hours under the conditions of 30°C and 200 rpm.

4. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 3, characterized in that: The bacterial agent OD prepared by Pseudomonas strain Y2 600 =3.

0.

5. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 1, characterized in that: The conditions for adding the bacterial agent prepared by the Pseudomonas strain y2 to the wastewater to be treated are: inoculation amount of 5-25%, temperature of 20-40°C, and pH of 4-10.

6. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 5, characterized in that: The conditions for adding the bacterial agent prepared by the Pseudomonas strain y2 to the wastewater to be treated are: an inoculation amount of 10-20%, a temperature of 25-30°C, and a pH of 6-8.

7. The use of the Pseudomonas strain Y2 in the synthesis of polyhydroxyalkanoates according to claim 6, characterized in that: The conditions for adding the bacterial agent prepared by Pseudomonas strain y2 to the wastewater to be treated are: inoculation amount of 15%, temperature of 30° C., and pH of 7.

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