CN110438030B

CN110438030B - A kind of Pseudomonas putida WP07, preparation method and use

Info

Publication number: CN110438030B
Application number: CN201910567313.8A
Authority: CN
Inventors: 唐晓达; 汪印; 余广炜
Original assignee: Institute of Urban Environment of CAS
Current assignee: Institute of Urban Environment of CAS
Priority date: 2019-06-27
Filing date: 2019-06-27
Publication date: 2021-06-15
Anticipated expiration: 2039-06-27
Also published as: CN110438030A

Abstract

The invention discloses a Pseudomonas putida WP07, a preparation method and an application. The Pseudomonas putida WP07 was deposited in the China General Microorganism Culture Collection and Management Center with the deposit number of CGMCC No.17760. The invention converts swill oil into biodegradable plastic PHA with environmental affinity by microbial method, can realize the resource utilization and harmless treatment of swill oil, and reduce the preparation cost of PHA, and has good application prospect.

Description

Pseudomonas putida WP07, preparation method and application

Technical Field

The invention relates to the field of microorganisms, in particular to Pseudomonas putida WP07, a preparation method and application.

Background

Polyhydroxyalkanoate (PHA) is a high molecular polymer that is stored as an intracellular carbon source and energy source in an unbalanced growth state of microorganisms with excess carbon source and insufficient nitrogen source, and is widely present in microbial cells in the form of hydrophobic particles. Because the material properties of the material are similar to those of traditional plastics synthesized by taking petroleum as raw materials, such as polyethylene, polypropylene and the like, the material can be synthesized by renewable energy sources, such as carbohydrate, fatty acid and the like, the structure of the material has diversity, and the material can be completely degraded and enter the ecological cycle of the nature to gain more and more attention. In addition, PHA is synthesized in bacteria, which guarantees its stereospecificity (all chiral carbon atoms in the backbone are in the R-configuration), which is important for the biodegradability and biocompatibility of PHA. At present, PHA is considered as a "green plastic" or an "environment-friendly plastic" which can replace the traditional non-degradable plastic, thus causing more and more attention in the scientific and industrial circles of all countries in the world. It has physical and chemical characteristics similar to those of synthetic plastics and excellent biodegradability, biocompatibility, optical activity, piezoelectricity, gas separation performance, etc. the synthetic plastics have no excellent performance.

Currently in biosafety and environmentFriendly aspects PHA has gained general acceptance in the industry, but the production cost of PHA is still high, thereby restricting its large-scale industrial production and commercial application development. The cost of PHA mainly comprises three aspects of raw material cost, production energy consumption and extraction processing, so that the industrial production of PHA is realized by reducing the cost, and the optimization of raw material, production, extraction process and the like is needed. The high production cost is the main reason for limiting the application of the polyhydroxyalkanoate at present, and the high raw material cost is the key factor. In the PHA production chain, the selection of suitable renewable resources as a source of carbon is a decisive factor in cost, since carbon source costs account for more than 50% of the overall production cost. CO 2₂Is the most desirable carbon source, but is not readily available, and it is preferable that the carbon source is, in the first place, a recyclable carbohydrate, fat, or the like. At present, PHA is mainly produced by a microbial fermentation method, and carbon sources used in industrial large-scale production are mainly sucrose, glucose or small molecular organic matters, and the cost of PHA accounts for 28-50% of the total cost. The selection of alternative, inexpensive carbon sources can reduce the cost of PHA production. The swill oil in the kitchen waste oil is mainly derived from waste grease separated in the restaurant swill treatment process, the main component of the swill oil is animal and vegetable grease, and the chemical component of the swill oil is fatty glyceride. Animal and vegetable oil contacts with oxygen in air in the process of cooking food, and undergoes hydrolysis, oxidation, polymerization and other reactions in a high-temperature environment, so that the content of original unsaturated fatty acid in the oil is reduced, and some polymers with carcinogenic effect such as various odorous substances like aldehyde, ketone, lactone and the like are generated; in recent years, kitchen waste management is more strict, and most kitchen waste generated by catering institutions is collected into kitchen waste treatment plants, so that swill oil is easier to collect and manage, the quantity of the swill oil is large, and how to realize resource utilization and harmless treatment of the swill oil becomes a focus of attention of people. The invention patent 201510203237.4 discloses a process method for synthesizing PHA from kitchen waste oil by using efficient bacteria, which adopts cupriasis cupriavoides dicator (DSM428), takes the kitchen waste oil as a carbon source for culture, and synthesizes the kitchen waste oil into PHAThe cost is high. Moreover, due to the complex components of swill oil, the method of converting and synthesizing PHA by using swill oil as a single carbon source is still insufficient.

Disclosure of Invention

The invention aims to provide Pseudomonas putida WP 07.

In order to achieve the aim, the invention provides Pseudomonas putida WP07 which is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 17760.

The invention also provides a method for preparing the Pseudomonas putida WP07, which is characterized by comprising the following steps,

domestication of the strain: adding the swill oil into an inorganic salt culture medium according to the proportion of 20g/L, obtaining a soil sample from an environment rich in oil, adding the soil sample into the inorganic culture medium according to the proportion of 5g/L, and culturing and domesticating to obtain a domesticated culture; preferably, the environment rich in grease is the soil at the smoke outlet of a canteen and the soil at the storage place of kitchen waste;

nile blue selective plate screening: 1mL of the culture was taken from the acclimatized culture, and diluted 10 with 0.9% sodium chloride solution⁴～10⁶After doubling, the cells are used for selective plate culture of coated nile blue; then, a strain with high fluorescence brightness and large biomass is selected from strains with fluorescence phenomenon under 360nm ultraviolet light, namely Pseudomonas putida WP 07.

Further, the inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and the microelement mother liquor are mixed; preferably, the inorganic salt culture medium is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L of microelement mother liquor;

optionally, 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂O。

Further, in the acclimation step of the strain, the culture and acclimation conditions are that the temperature is 20-40 ℃, the rotating speed is 200-300 rpm; continuously adding the swill oil into the inorganic salt culture medium at the ratio of 5g/L every 5 days until the addition amount of the swill oil in the inorganic salt culture medium reaches 100g/L, and continuously acclimating for 5 days to finally obtain an acclimated culture.

Optionally, in the step of screening the nile blue selective plate, the plate is cultured in a 30 ℃ incubator for 3-4 days.

The invention also provides a method for converting swill oil into PHA-containing thallus by using the Pseudomonas putida WP07, which is characterized by using the Pseudomonas putida WP 07.

Further, the method comprises the following steps of,

adding swill oil serving as a single carbon source into an inorganic salt culture medium; preferably, the addition amount of the swill oil is 10 g/L-40 g/L; adding tween 80 at the same time, preferably, adding the tween 80 in an amount of 1/10 weight ratio; then inoculating activated Pseudomonas putida WP07 seed culture solution; preferably, the inoculum size is 3 v/v%; culturing at 20-40 deg.C and rotation speed of 200-300rpm to obtain fermentation liquid, sucking the fermentation liquid, centrifuging, and collecting crude thallus as thallus containing PHA;

preferably, the crude cells are mixed with a 0.9% sodium chloride solution to suspend the cells, and the cells are centrifuged again to obtain PHA-containing cells.

The invention also provides a method for synthesizing PHA-containing dry thalli by using the method, which is characterized by further comprising the steps of freezing the PHA-containing thalli and drying the PHA-containing thalli in a freeze dryer to obtain PHA-containing dry thalli;

preferably, the PHA-containing microbial cells are frozen at a temperature of-20 ℃ or lower for 12 to 16 hours, and then dried in a freeze dryer for 12 to 36 hours to obtain PHA-containing dry microbial cells.

The invention also provides a method for synthesizing PHA by using the method, which is characterized by further comprising the steps of putting the obtained PHA-containing dry thalli into a threaded glass tube, adding chloroform and NaClO solution, covering and tightly closing the glass tube, and leaching in an oven; after the leaching is finished, cooling at room temperature, collecting the bottom organic liquid phase, adding cold methanol for precipitation, taking a precipitate, and freeze-drying to obtain PHA;

preferably, the obtained PHA-containing dry thalli are put into a threaded glass tube, 20mL/g chloroform and 20mL/g 12 w/v% NaClO solution are added, the glass tube is covered and closed, and leaching is carried out in an oven at 100 ℃ for 4 hours; after the leaching is finished, cooling at room temperature, collecting the bottom organic liquid phase, adding cold methanol for precipitation, taking a precipitate, and freeze-drying to obtain PHA;

more preferably, the volume ratio of cold methanol to organic liquid phase is 30: 1.

further, the activated Pseudomonas putida WP07 seed culture solution is prepared by inoculating Pseudomonas putida WP07 into a seed culture medium, and culturing for 12-72h at 20-40 ℃, 200-300rpm, preferably at 25-35 ℃, 200-250rpm for 24-48 h; more preferably, culturing for 24h at 30 ℃ and 200 rpm; obtaining activated pseudomonas seed culture solution;

the preparation method of the seed culture medium comprises the steps of adding 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride into deionized water, mixing, and adjusting the pH value to 7.0 by using 0.1N HCl and 0.1N NaOH solution.

The effect of the Pseudomonas putida WP07 on PHA synthesis by conversion of swill oil can reach 0.49 g/L.

The invention has the following beneficial effects:

(1) the pseudomonas strain which can convert and synthesize PHA by using the swill oil as a single carbon source is obtained by swill oil domestication and nile blue selective plate screening, the screening process is relatively simple, and the cost is lower.

(2) The pseudomonas is used for converting the swill oil into the PHA, so that the resource utilization and harmless treatment of the swill oil are realized, the synthetic raw materials of the PHA are expanded and reduced, and the production cost is reduced.

(3) The technological process is simple, PHA yield is high, swill oil conversion rate is high, and the method has a good application prospect.

The method converts the swill oil into the biodegradable plastic PHA with environmental affinity by a microbiological method, can realize resource utilization and harmless treatment of the swill oil, can reduce the preparation cost of the PHA, and has good application prospect.

Drawings

FIG. 1 is a graph showing the effect of fermentation temperature on the efficiency of PHA synthesis by conversion of Pseudomonas putida WP 07;

FIG. 2 is a graph showing the effect of pH of an inorganic salt medium on the efficiency of PHA synthesis by conversion of Pseudomonas putida WP 07;

FIG. 3 is a graph showing the effect of swill oil addition on PHA synthesis efficiency of Pseudomonas putida WP 07;

FIG. 4 effect of cultivation time on PHA synthesis efficiency by Pseudomonas putida WP 07.

FIG. 5 phylogenetic tree of strain Pseudomonas putida WP 07.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: acclimatization and screening of Pseudomonas putida WP07 strain

Selecting canteen smoke outlet soil, and obtaining the pseudomonas strain which takes the swill oil as a single carbon source to be converted and synthesized into PHA through high-concentration swill oil domestication and nile blue selective screening. The domestication and screening steps of the strain comprise:

(1) domestication of the strain: firstly, adding the swill oil into an inorganic salt culture medium according to the proportion of 20g/L, then obtaining a soil sample from the soil at the smoke outlet of a canteen, and adding the soil sample into the inorganic culture medium according to the proportion of 5g/L for culture and domestication; the domestication condition of the strain is that the temperature is 20-30 ℃, the rotating speed is 200-300 rpm; continuously adding the swill oil into the inorganic salt culture medium at the ratio of 5g/L every 5 days until the addition amount of the swill oil in the inorganic salt culture medium reaches 100g/L, and continuously acclimating for 5 days to finally obtain an acclimated culture. Wherein the inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and microelement mother liquor, and the addition amount is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L of microelement mother liquor. 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂O。

(2) Nile blue selective plate screening: 1mL of the culture was taken from the acclimatized culture, and diluted 10 with 0.9% sodium chloride solution⁴～10⁶After doubling, the cells are used for selective plate culture of coated nile blue; culturing the coated nile blue selective plate in a constant-temperature incubator at 30 ℃ for 3-4 days; then, a strain with high fluorescence brightness and large biomass is screened from strains with fluorescence phenomenon under 360nm ultraviolet light, and the strain is identified by Meiji organism in Shanghai, and is pseudomonas. The bacterium is gram-negative bacterium, and has a short rod shape, and one end of the bacterium has 1-2 long flagella. The colony grown on LB culture medium is ivory white, round and smooth. The thalli obtained by culturing in inorganic salt liquid culture medium is slightly pink. Simultaneously comparing the gene with 31 housekeeping genes of other strains in the same genusThe phylogenetic tree was constructed by selecting NJ (Neighbor-join) using MEGA software (see FIG. 5). The strain of the invention is numbered Pseudomonas putida WP 07. Pseudomonas putida WP07 is deposited in China general microbiological culture collection center with the preservation number of CGMCC No. 17760.

Strain name: pseudomonas putida WP 07;

date of deposit: year 2019, month 5, day 13;

the deposit unit: west road No.1 hospital No. 3, china committee for culture collection of microorganisms, china general microbiological culture collection center (CGMCC);

deposit number: CGMCC No. 17760.

Example 2 Effect of fermentation temperature on the efficiency of conversion of Pseudomonas putida Putida WP07 to PHA

Pseudomonas putida WP07 obtained in example 1 was inoculated into a seed medium in an amount of 1% (V/V), and cultured at 30 ℃ and 200rpm for 24 hours for activation to obtain an activated Pseudomonas seed culture solution.

Adding swill oil serving as a single carbon source into an inorganic salt culture medium according to the proportion of 20g/L, and adding tween 80 serving as an emulsifier at the same time in a ratio of 1:10 (w/w); then inoculating the activated pseudomonas seed culture solution into an inorganic salt culture medium added with swill oil in an inoculation amount of 3% (v/v), and culturing for 72h under the conditions that the culture temperature is respectively controlled to be 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ and the rotation speed is 300rpm (the result is shown in figure 1 for comparison). Wherein the preparation method of the seed culture medium comprises the steps of adding 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride into deionized water, mixing, and adjusting the pH value to 7.0 by using 0.1N HCl and 0.1N NaOH solution. The inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and microelement mother liquor, and the addition amount is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L traceAnd (4) element mother liquor. 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂And O. And sterilizing the inorganic salt culture medium at 121 deg.C for 20min before fermentation culture.

After the culture is finished, sucking the fermentation liquor, centrifuging for 6min at 6000rpm, mixing the collected crude thalli with a proper amount of 0.9% sodium chloride solution to suspend the thalli, and centrifuging again to collect the thalli containing PHA; freezing the PHA-containing thallus at-20 deg.C for 12h, and drying in a freeze dryer for 24h to obtain PHA-containing dry thallus. Putting the obtained PHA-containing dry bacteria into a threaded glass tube, adding 20mL/g chloroform and 20mL/g 12% NaClO solution, covering and tightly closing the glass tube, and leaching the PHA-containing dry bacteria in an oven at 100 ℃ for 4 hours; and after the leaching is finished, cooling at room temperature, collecting the bottom organic liquid phase, adding cold methanol for precipitation (the volume ratio of the cold methanol to the organic liquid phase is 30: 1), and freeze-drying the precipitate to obtain the PHA. The PHA yield was calculated as follows:

PHA yield (dry cell yield) multiplied by PHA content

Wherein the dry thallus yield is obtained in each liter of fermentation liquor, and the unit is g/L; the PHA content is the percentage of PHA in dry cells, and the unit is%.

FIG. 1 is a graph showing the conversion efficiency of Pseudomonas to synthesize PHA from hogwash oil at different culture temperatures. As can be seen from FIG. 1, the PHA yield was found to be 0.49g/L, which was large at 30 ℃.

Example 3 Effect of pH of mineral salts Medium on the efficiency of PHA Synthesis by Pseudomonas putida WP07 transformation

Pseudomonas putida WP07 obtained in example 1 was inoculated into a seed culture medium, and cultured at 30 ℃ and 200rpm for 24 hours for activation to obtain an activated Pseudomonas seed culture solution.

Adding swill oil serving as a single carbon source into an inorganic salt culture medium according to the proportion of 20g/L, and adding tween 80 serving as an emulsifier at the same time in a ratio of 1:10 (w/w); then inoculating the seed culture solution into an inorganic salt culture medium added with swill oil in an inoculation amount of 3% (v/v), and respectively culturing for 72h under the conditions that the culture temperature is controlled at 30 ℃ and the rotation speed is 300 rpm. Wherein the preparation method of the seed culture medium comprises the steps of adding 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride into deionized water, mixing, and adjusting the pH value to 7.0 by using 0.1N HCl and 0.1N NaOH solution.

The inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and microelement mother liquor, and the addition amount is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L of microelement mother liquor. With 1N H₂SO₄Or 1N NaOH solution to adjust the initial pH of the fermentation medium to 6.5, 7.0, 7.5, 8.0, 8.5, respectively (see FIG. 2 for comparison of results). 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂And O. And sterilizing the inorganic salt culture medium at 121 deg.C for 20min before fermentation culture.

After the culture is finished, sucking the fermentation liquor, centrifuging for 6min at 6000rpm, mixing the collected crude thalli with a proper amount of 0.9% sodium chloride solution to suspend the thalli, and centrifuging again to collect the thalli containing PHA; freezing the PHA-containing thallus at-20 deg.C for 12h, and drying in a freeze dryer for 24h to obtain PHA-containing dry thallus. Putting the obtained PHA-containing dry bacteria into a threaded glass tube, adding 20mL/g chloroform and 20mL/g 12% NaClO solution, covering and tightly closing the glass tube, and leaching the PHA-containing dry bacteria in an oven at 100 ℃ for 4 hours; and after the leaching is finished, cooling at room temperature, collecting the bottom organic liquid phase, adding cold methanol for precipitation (the volume ratio of the cold methanol to the organic liquid phase is 30: 1), and freeze-drying the precipitate to obtain the PHA.

FIG. 2 is a graph showing the effect of pH value of inorganic salt medium on PHA conversion and synthesis efficiency of Pseudomonas. As can be seen from FIG. 2, the PHA yield was maximal at pH7.0, which was 0.49 g/L. Therefore, the optimal pH value of the inorganic salt culture medium is recommended to be 7.0.

Example 4 Effect of swill oil addition on PHA synthesis efficiency of Pseudomonas putida Putida WP07 transformation

Adding swill oil serving as a single carbon source into an inorganic salt culture medium according to the proportion of 10g/L, 15g/L, 20g/L, 25g/L, 30g/L and 40g/L (see a result comparison in figure 3), and simultaneously adding 1:10(w/w) of tween 80 serving as an emulsifier; then inoculating the seed culture solution into an inorganic salt culture medium added with swill oil in an inoculation amount of 3% (v/v), and respectively culturing for 72h under the conditions that the culture temperature is controlled at 30 ℃ and the rotation speed is 300 rpm. Wherein the preparation method of the seed culture medium comprises the steps of adding 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride into deionized water, mixing, and adjusting the pH value to 7.0 by using 0.1N HCl and 0.1N NaOH solution.

The inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and microelement mother liquor, and the addition amount is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L of microelement mother liquor. With 1N H₂SO₄Or 1N NaOH solution to adjust the initial pH of the fermentation medium to 7.0. 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂And O. And sterilizing the inorganic salt culture medium at 121 deg.C for 20min before fermentation culture.

FIG. 3 is a graph showing the effect of swill oil addition on PHA conversion and synthesis efficiency of Pseudomonas. As can be seen from FIG. 3, the PHA yield increases with the increase of the addition amount of the kitchen waste oil (i.e., swill oil), reaches a peak value of 0.49g/L when the addition amount of the kitchen waste oil is 20g/L, and then sharply decreases. Therefore, the optimal addition amount of the kitchen waste oil is 20 g/L.

Example 5 Effect of cultivation time on the efficiency of PHA Synthesis by conversion of Pseudomonas putida Putida WP07

Adding swill oil serving as a single carbon source into an inorganic salt culture medium according to the proportion of 20g/L, and adding tween 80 serving as an emulsifier at the same time in a ratio of 1:10 (w/w); then inoculating the seed culture solution into an inorganic salt culture medium added with swill oil in an inoculation amount of 3% (v/v), respectively culturing for 120h under the conditions of controlling the culture temperature to be 30 ℃ and the rotating speed to be 300rpm, and sampling every 12h for detection (the result is shown in figure 3 for comparison). Wherein the preparation method of the seed culture medium comprises the steps of adding 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride into deionized water, mixing, and adjusting the pH value to 7.0 by using 0.1N HCl and 0.1N NaOH solution.

The inorganic salt culture medium is prepared by adding KH into deionized water₂PO₄、Na₂HPO₄·12H₂O、NH₄Cl、MgSO₄·7H₂O and trace amountThe element mother liquor is mixed, and the addition amount is 1.1g/L KH₂PO₄、12g/L Na₂HPO₄·12H₂O、2g/L NH₄Cl、1.4g/L MgSO₄·7H₂O and 1mL/L of microelement mother liquor. With 1N H₂SO₄Or 1N NaOH solution to adjust the initial pH of the fermentation medium to 7.0. 0.22mg/mL CoCl was added to 0.1N HCl of the mother solution of trace elements₂·H₂O、9.7mg/mL FeCl₃、0.12mg/mL NiCl₂·6H₂O、0.11mg/mL CrCl₃·6H₂O and 0.16mg/mL CuSO₄·5H₂And O. And sterilizing the inorganic salt culture medium at 121 deg.C for 20min before fermentation culture.

FIG. 4 is a graph showing the effect of culture time on the efficiency of PHA synthesis by Pseudomonas transformation. FIG. 4 shows the overall energy consumption and efficiency problems, with 72h being the optimal cultivation time.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A Pseudomonas putida WP07, which is preserved in the China General Microorganism Culture Collection and Management Center with the preservation number of CGMCC No.17760.

2. a kind of method utilizing the described Pseudomonas putida WP07 of claim 1 to transform swill oil into a method for synthesizing PHA-containing thalline.

3. method as claimed in claim 2, is characterized in that, comprises the steps,

Add swill oil as a single carbon source to the inorganic salt medium; at the same time, add tween 80 and inoculate the activated Pseudomonas putida WP07 seed culture solution; at a temperature of 20°C-40°C and a rotating speed of 200-300rpm Cultivated under the conditions, the fermentation liquid was obtained after the cultivation, and the crude thalline was collected by centrifugation of the fermented liquid, which was the thalline containing PHA.

4. method as claimed in claim 3, is characterized in that, the addition of described swill oil is 10g/L～40g/L.

5. The method of claim 3, wherein the added amount of the tween 80 is a total weight ratio of 1/10.

6. method as claimed in claim 3, is characterized in that, the inoculum of described inoculated Pseudomonas putida WP07 seed culture liquid after activation is 3 v/v %.

7. method as claimed in claim 3, is characterized in that, after thick thalline is mixed with 0.9% sodium chloride solution to make thalline suspended, centrifugation again collects to obtain the thalline containing PHA.

8. The method of claim 3, further comprising: freezing the PHA-containing thalline, and then placing it in a freeze dryer to dry to obtain the PHA-containing dried thalline.

9. The method according to claim 8, wherein the PHA-containing thalline is frozen for 12-16h at a temperature of -20°C and below, and then placed in a freeze dryer for drying for 12-36h to obtain Dried cells with PHA.

10. method as claimed in claim 8 is characterized in that, it is characterized in that, also comprises, the dry thalline that obtains containing PHA is put into the glass tube with thread, adds chloroform and NaClO solution, affixes the lid and closes tightly, in the oven Carry out leaching; after the leaching is completed, cool at room temperature, collect the organic liquid phase at the bottom, add cold methanol for precipitation, and take the precipitated material for freeze-drying to obtain PHA.

11. method as claimed in claim 10, it is characterised in that the obtained dry thalline containing PHA is put into the glass tube with thread, adds the 12 w/v %NaClO solution of 20mL/g chloroform and 20mL/g , sealed tightly, and leached in an oven at 100°C for 4 hours; after leaching, cool at room temperature, collect the organic liquid phase at the bottom, add cold methanol for precipitation, and take the precipitated material for freeze-drying to obtain PHA.

12. The method of claim 11, wherein the volume ratio of the cold methanol to the organic liquid phase is 30:1.

13. The method of claim 9, wherein the activated Pseudomonas putida WP07 seed culture solution is obtained by inserting Pseudomonas putida WP07 into the seed culture medium at 20°C- 40 ℃, under the condition of rotating speed 200-300rpm, cultivate for 12-72h; obtain the activated Pseudomonas seed culture solution;

The preparation method of the seed medium is to add 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride in deionized water, and adjust the pH value with 0.1N HCl and 0.1N NaOH solution after mixing. is 7.0.

14. The method of claim 13, wherein the activated Pseudomonas putida WP07 seed culture solution is to insert Pseudomonas putida WP07 into the seed culture medium at 25- Cultivate for 24-48 hours at 35° C. under the condition of 200-250 rpm rotation speed; obtain the activated Pseudomonas seed culture solution.

15. The method of claim 14, wherein the activated Pseudomonas putida WP07 seed culture solution is to insert Pseudomonas putida WP07 into the seed culture medium at 30° C. , cultivated for 24h under the condition of 200rpm rotating speed; obtained the activated Pseudomonas seed culture solution.