CN113832070B

CN113832070B - Luminous bacteria capable of producing polyhydroxyalkanoate by using vegetable oil or starch and other various carbon sources and application thereof

Info

Publication number: CN113832070B
Application number: CN202111209909.4A
Authority: CN
Inventors: 李正军; 田林钺
Original assignee: Qingdao Fumei New Energy Technology Co ltd; Beijing University of Chemical Technology
Current assignee: Qingdao Fumei New Energy Technology Co ltd; Beijing University of Chemical Technology
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2023-06-16
Anticipated expiration: 2041-10-18
Also published as: CN113832070A

Abstract

The invention discloses a luminescent bacterium capable of producing polyhydroxyalkanoate by utilizing various carbon sources such as vegetable oil or starch and the like and application thereof. The bacteria is Photobacterium sp.TLY01, and the preservation number of the bacteria in the China general microbiological culture Collection center is CGMCCNo.22976. Experiments prove that the Photobacterium sp.TLY01 is a strain which can utilize various substrates as carbon sources to rapidly grow and produce polyhydroxyalkanoates, and particularly can efficiently utilize vegetable oil or starch. The invention can effectively solve the problems of kitchen waste oil and the like, and is expected to be a low-cost and high-efficiency polyhydroxyalkanoate production mode. The invention has important application value.

Description

Luminous bacteria capable of producing polyhydroxyalkanoate by using vegetable oil or starch and other various carbon sources and application thereof

Technical Field

The invention belongs to the field of microbiology, and particularly relates to a luminescent bacterium capable of producing polyhydroxyalkanoate by utilizing various carbon sources such as vegetable oil or starch and application thereof.

Background

Polyhydroxyalkanoate (PHA) is a generic term for a type of polymeric polyesters polymerized from hydroxy fatty acid monomers that are completely synthesized by microorganisms, and is a substitute for traditional petrochemical-derived non-degradable plastics. Unlike petrochemical plastics, PHAs are produced by microorganisms, generally act as carbon reserves in the event of nutritional imbalances, and accumulate in the form of particles within the cell in amounts up to 90% of the dry weight of the cell. PHA has the characteristics of biodegradability, biocompatibility and the like, is considered as environment-friendly green bioplastic, is favorable for solving the increasingly serious environmental pollution problem, and has good prospect.

Although PHA has many environmental benefits, it is produced at a higher cost than petrochemical plastics due to the use of pure substrates such as glucose, fructose, propionic acid, etc. in the production process, combined with the relatively complex separation and extraction processes. Waste biomass such as waste oil, lignocellulose hydrolysate, agricultural food waste, dairy industry by-product whey and biodiesel industry by-product waste glycerol is used as raw materials for fermentation, so that the production cost of PHA can be reduced.

Waste oils and fats from various sources such as dairy industry, edible oil processing industry, food processing industry, waste edible oil and animal fat waste are waste rich in fat, and are non-edible oils with poor quality, and excessive water content, saponification value, iodine value, acid value, peroxide value, carbonyl value and the like. For example, the long-time frying of the remaining old oil of the food causes a series of chemical reactions such as oxidation, hydrolysis, condensation and the like of the oil and fat, and a plurality of substances such as saturated or unsaturated aldehydes, ketones and the like, which are extremely harmful to the human body, because the long-time heating of Wen Pengzha causes the reaction of the oil and fat with oxygen in the air, the various foods being fried and the like. The waste oil and fat are produced in a large amount in the global scope, and the waste oil and fat are used as microbial fermentation raw materials, and have the advantages of wide sources, low cost and the like compared with the traditional raw materials.

Vegetable oils are generally extracted from oil crops such as soybean, rapeseed, palm, sunflower, corn and the like. Vegetable oils are the main source and important components of waste oils, many of which come from and leave a large amount of common vegetable oils such as soybean oil, palm oil, etc. Some potential oil-based feedstocks, such as soybean oil, corn oil, and various palm oils, have been found to be useful as a source of PHA-producing carbon, with certain advantages over other conventional sugars and volatile fatty acids. Some PHA producing bacteria and PHA producing methods are reported by taking vegetable oil and waste oil as raw materials, but the PHA producing efficiency is still low. In order to further utilize the grease matrix to produce PHA in high yield, further research and development of high-quality vegetable oil utilization strains are required, and fermentation technology for utilizing waste vegetable oil and other vegetable oils is improved.

The luminescent bacteria generally refer to a non-pathogenic gram-negative bacteria which can emit visible fluorescence from cells in the normal growth process, the wavelength of the visible fluorescence emitted by the bacteria is between 450 and 490nm, the fluorescence emitted by a single luminescent bacteria is extremely weak, thousands of luminescent bacteria are gathered together, and the emitted fluorescence is visible to the naked eye in a dark place. The luminous bacteria mainly exist in the ocean, have different forms, are mostly straight bacilli, have blunt ends, have shorter bacilli, have one or a plurality of terminal flagella, can survive and reproduce by simple organic nutrients, and are called as wide-feeding bacteria. The current research on luminescent bacteria has mostly focused on pathogenicity to animals and humans. Several strains of the genus Protobacter have been reported to be capable of PHA synthesis, but the yields are not high and there are significant differences compared to the PHA-producing strains currently in use. For example, completegenome sequence of Photobacterium ganghwense C2.2.2.2 published by microbiology open in 2021: a new polyhydroxyalkanoate production candidate it was reported that after 96 hours of fermentation of a strain of P.luminophore in a medium containing 20g/L glycerol, the dry cell weight and PHA content reached 3.1g/L and 53%, respectively. In 2008, strain Synthesis of reserve polyhydroxyalkanoates by luminescent bacteria published in Microbiology was fermented in glycerol containing 3g/L for 36 hours, and the dry cell weight was less than 1g/L, and the PHA content was 42%.

Disclosure of Invention

The object of the present invention is to produce polyhydroxyalkanoates more economically. Polyhydroxyalkanoates include poly-3-hydroxybutyrate and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

The invention firstly protects the Photobacterium sp.TLY01, the strain is preserved in China general microbiological culture collection center (CGMCC for short, address: north Chen West Lu No. 1, no. 3 in the Beijing area towards the sun) in the 28 th year of 2021, and the preservation number is CGMCC No.22976. Photobacterium sp.TLY01 (CGMCC No. 22976) is abbreviated as Photobacterium TLY01.

The invention also provides a microbial inoculum containing the luminous bacillus TLY01.

The use of the microbial inoculum may be in the production of polyhydroxyalkanoates.

The preparation method of the microbial inoculum comprises the following steps: inoculating the luminous bacillus TLY01 into a bacterial culture medium and culturing to obtain bacterial liquid, namely the bacterial agent.

The bacterial culture medium may be TYS liquid culture medium or LB liquid culture medium.

In the preparation method of the microbial inoculum, the culture conditions can be as follows: culturing at 25-37deg.C (such as 25-30deg.C, 30-37deg.C, 25deg.C, 30deg.C or 37deg.C) at 100-300rpm (such as 100-200rpm, 200-300rpm, 100rpm, 200rpm or 300 rpm) for 12-48 hr (such as 12-24 hr, 24-36 hr, 36-48 hr, 12 hr, 24 hr, 36 hr or 48 hr).

The application of the luminous bacillus TLY01 or any of the bacterial agents in the production of polyhydroxyalkanoates also belongs to the protection scope of the invention.

The application of the luminous bacillus TLY01 and the culture medium rich in carbon sources in the production of polyhydroxyalkanoates also belongs to the protection scope of the invention.

The invention also provides a method for producing polyhydroxyalkanoate, which can comprise the following steps:

(1) Inoculating the above-mentioned light emitting bacillus TLY01 (or light emitting bacillus TLY01 seed solution with inoculation amount of 5%) into culture medium rich in carbon source, and culturing at 25-37deg.C (such as 25-30deg.C, 30-37deg.C, 25deg.C, 30deg.C or 37deg.C), 100-300rpm (such as 100-200rpm, 200-300rpm, 100rpm, 200rpm or 300 rpm) to obtain fermentation broth;

(2) After the step (1) is completed, taking the fermentation liquor, centrifuging and collecting sediment;

(3) After the step (2) is completed, taking the precipitate, and freeze-drying to obtain a freeze-dried product;

(4) After step (3) is completed, polyhydroxyalkanoate is isolated from the freeze-dried product.

In the step (1), the time of the culturing may be 24-48 hours (e.g., 24-36 hours, 36-48 hours, 24 hours, 36 hours or 48 hours).

Any of the above carbon sources may be at least one of glycerol, sugar, volatile fatty acids, and vegetable oils.

The volatile fatty acid may be at least one of acetic acid, propionic acid, and valeric acid.

The sugar may be at least one of glucose, xylose, fructose, sucrose and starch.

The vegetable oil may be at least one of soybean oil, rice oil and palm oil.

Any one of the above-mentioned carbon source-rich media further contains NaCl; the concentration of NaCl in the carbon source-rich medium is 10-40g/L (e.g., 10-20g/L, 20-30g/L, 30-40g/L, 10g/L, 20g/L, 30g/L, or 40 g/L).

Any of the polyhydroxyalkanoates described above may include poly-3-hydroxybutyrate and/or poly (3-hydroxybutyrate-co-3-hydroxyvalerate). The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) may be a copolyester containing two monomer components, 3-hydroxybutyrate and 3-hydroxyvalerate.

Any of the above-mentioned carbon source-rich media may specifically be at least one of the glucose-containing TYS liquid media, xylose-containing TYS liquid media, fructose-containing TYS liquid media, sucrose-containing TYS liquid media, starch-containing TYS liquid media, glycerol-containing TYS liquid media, acetic acid-containing TYS liquid media, propionic acid-containing TYS liquid media, valeric acid-containing TYS liquid media, soybean oil-containing TYS liquid media, rice oil-containing TYS liquid media, and palm oil-containing TYS liquid media mentioned in the examples.

Any of the above-mentioned carbon source-rich media may be specifically a glycerol-and 10g/L NaCl-containing media as mentioned in the examples.

Any of the above-mentioned carbon source-rich media may be specifically exemplified by the media containing soybean oil, 10g/L NaCl and propionic acid at different concentrations mentioned in the examples.

Any of the above-mentioned carbon source-rich media may be specifically exemplified by the media containing soybean oil, 10g/L NaCl and valeric acid at various concentrations mentioned in the examples.

Any of the above-mentioned carbon source-rich media may specifically be the medium containing starch, 10g/L NaCl and propionic acid at different concentrations as mentioned in the examples.

Any of the above-mentioned carbon source-rich media may specifically be the medium containing starch, 10g/L NaCl and different concentrations of valeric acid as mentioned in the examples.

The invention has the following advantages:

1. the luminous bacillus TLY01 provided by the invention can utilize various traditional substrates as carbon sources to rapidly grow and accumulate poly-3-hydroxybutyrate, wherein the raw materials comprise glycerol, glucose, xylose, fructose, sucrose, starch, acetic acid, propionic acid, valeric acid and the like, the proportion of the produced poly-3-hydroxybutyrate to the dry weight of bacteria can be up to about 65%, and the yield in a shake flask can be up to about 5 g/L.

2. The luminous bacillus TLY01 provided by the invention can utilize vegetable oil (comprising soybean oil, rice oil, palm oil and the like) as a carbon source to rapidly grow and accumulate poly-3-hydroxybutyrate, wherein the poly-3-hydroxybutyrate accounts for about 44% of the dry weight of bacteria, and the yield in a shake flask can be about 3.2 g/L.

3. The luminous bacillus TLY01 provided by the invention can utilize vegetable oil (soybean oil, rice oil, palm oil and the like) and propionic acid (with the concentration of 2g/L-6 g/L) or valeric acid (with the concentration of 2g/L-6 g/L) as mixed carbon sources to rapidly grow and accumulate poly (3-hydroxybutyrate-co-3-hydroxyvalerate), wherein the poly (3-hydroxybutyrate-co-3-hydroxyvalerate) accounts for about 58% of the dry weight of bacteria, the yield in a shake flask can be about 6.28g/L, and the content of 3-hydroxyvalerate monomer is about 64mol%.

4. The luminous bacillus TLY01 provided by the invention can utilize starch and propionic acid (with the concentration of 2g/L-6 g/L) or valeric acid (with the concentration of 2g/L-6 g/L) as mixed carbon sources to rapidly grow and accumulate poly (3-hydroxybutyrate-co-3-hydroxyvalerate), wherein the poly (3-hydroxybutyrate-co-3-hydroxyvalerate) accounts for about 27% of the dry weight of bacteria, the yield in a shake flask can be about 2.64g/L, and the content of 3-hydroxyvalerate monomer is about 41mol%.

5. The invention provides a method for producing poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by utilizing mixed fermentation of vegetable oil or starch and volatile fatty acid, which reduces the fermentation cost of PHBV and has great significance for promoting industrialized production of PHBV.

6. The invention provides a method for synthesizing polyhydroxyalkanoate by using vegetable oil, which not only can solve the problem of environmental pollution caused by improper treatment of waste oil, but also can reduce the PHA production cost due to the advantages of wide source of the waste oil, low price and the like.

In summary, the luminous bacillus TLY01 provided by the invention is a strain capable of utilizing various substrates (such as vegetable oil, starch and the like) as carbon sources to rapidly grow and produce polyhydroxyalkanoates, and particularly can efficiently utilize the vegetable oil or the starch. The invention can effectively solve the problems of kitchen waste oil and the like, and is expected to be a low-cost and high-efficiency polyhydroxyalkanoate production mode. The invention has important application value.

Preservation description

Strain name: photobacterium illus

Latin name: photobacterium sp.

Strain number: TLY01

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3

Preservation date: 2021, 07, 28

Accession numbers of the preservation center: CGMCC No.22976

Drawings

FIG. 1 shows the result of transmission electron microscope observation of the light emitting bacterium TLY01 in the fermentation broth obtained in step 2 of example 2.

FIG. 2 shows the result of transmission electron microscope observation of the light emitting bacterium TLY01 in the fermentation broth obtained in step 3 of example 2.

FIG. 3 shows the result of transmission electron microscope observation of the light emitting bacterium TLY01 in the fermentation broth obtained in step 2 of example 5.

FIG. 4 shows the NaCl concentration optimization of the polyhydroxyalkanoate synthesized by P.photoperiod TLY01 of example 6.

FIG. 5 shows the result of temperature optimization of the synthesis of polyhydroxyalkanoate by P.photoperiod TLY01 of example 7.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Polyhydroxyalkanoate standard was purchased from Sigma-Aldrich under the product name 403121 and was named poly (3-hydroxybutyrate-co-3-hydroxyvalerate), wherein the 3-hydroxybutyrate monomer content was 92mol% and the 3-hydroxyvalerate monomer content was 8mol%.

The freeze drying procedure in the examples below is as follows: fermenting and culturing microorganisms, taking fermentation liquor, centrifuging at 10000rpm for 10min, discarding supernatant, and then re-suspending thalli with deionized water for washing; and centrifuging at 10000rpm for 10min to collect thalli, placing the centrifuge tube with the washed thalli precipitate in a freeze-drying machine for freeze-drying for 10h at-20 ℃ to obtain a freeze-drying product.

The dry cell weight is measured per liter of fermentation broth in the examples described below. The dry weight of the cells is expressed in g/L. Cell dry weight (CDW for short) = (weight of centrifuge tube after freeze-drying-weight of raw empty centrifuge tube)/broth take-up; the weight of the centrifugal tube after freeze drying and the weight of the original empty centrifugal tube are both in g; the unit of the fermentation broth is L.

The following examples are given for the method of detecting the polyhydroxyalkanoate content of a freeze-dried product: the freeze-dried product was subjected to an esterification reaction, and then calculated by measuring the content of the product after the esterification reaction. About 20mg of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) standard was prepared and the esterification reaction was performed in the same manner.

The specific operation steps of the esterification reaction are as follows: adding 30-40mg of freeze-dried product into an esterification pipe, adding 2mL of chloroform and 2mL of esterification liquid (the esterification liquid is obtained by adding 15mL of concentrated sulfuric acid and 0.5g of benzoic acid into 500mL of methanol), uniformly mixing, adding a cover, sealing, and keeping at 100 ℃ for 4h; cooling to room temperature, adding 1mL deionized water, fully oscillating and uniformly mixing by using a vortex oscillator, standing and layering; after the chloroform phase was completely separated from the water, 1. Mu.L of the chloroform phase was taken for gas chromatography.

Gas chromatography analysis parameters: using an HP 6890 type gas chromatograph, wherein the chromatographic column is an HP-5 capillary column, the column length is 30m, the inner diameter is 320 mu m, and the stationary phase is phenyl methyl polysiloxane with the thickness of 25 nm; the detector is a flame ionization detector (Flame ionization detector, FID); high-purity nitrogen is used as carrier gas, hydrogen is used as fuel gas, and air is used as combustion-supporting gas;

the conditions for the gas chromatographic analysis are as follows:

(1) Column temperature: starting at 80deg.C, and standing for 1.5min; heating to 140 ℃ at a speed of 30 ℃/min, and staying for 0min; the temperature is raised to 220 ℃ at a rate of 40 ℃/min, and the mixture stays for 1min. The total time was 6.5min.

(2) Column pressure: 10psi, 1.5min; the pressure was increased to 20psi at a rate of 2.5psi/min and the residence time was 0.5min. (psi is the unit of pressure, i.e., pounds per square inch, 1 psi= 6.89476 kPa)

(3) Sample inlet: the temperature was 200℃and the split ratio was 30 using split mode.

(4) A detector: the temperature was 220℃and the hydrogen flow was 30mL/min and the air flow was 400mL/min.

The polymer was quantitatively analyzed by an internal standard method using a microsyringe from Agilent company with a sample injection amount of 1. Mu.L, and quantified according to the peak area.

For gas chromatography detection, lyophilized cell samples were compared to poly (3-hydroxybutyrate-co-3-hydroxyvalerate) standards. The steps are adopted for esterification reaction and gas chromatography detection, and the freeze-dried cell sample contains signals with the same peak position as the position of the 3-hydroxybutyric acid in the standard, namely the polyhydroxyalkanoate accumulated in the thalli is poly-3-hydroxybutyrate. The freeze-dried cell sample contains signals with the same peak positions as the 3-hydroxybutyric acid and the 3-hydroxyvaleric acid in the standard, namely the polyhydroxyalkanoate accumulated in the thalli is poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

The poly-3-hydroxybutyrate yield algorithm was: PHB yield = (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass×0.905) ]/sample esterification mass×dry cell weight

The poly (3-hydroxybutyrate-co-3-hydroxyvalerate) yield algorithm was: PHBV yield = PHB yield + PHV yield, wherein:

PHB yield = (PHB peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHB peak area in standard) × (standard mass×0.905 ]/sample esterification mass×dry cell weight)

PHV yield = (PHV peak area in sample/internal standard peak area in sample) × [ (internal standard peak area in standard/PHV peak area in standard) × (standard mass×0.095) ]/sample esterification mass×dry cell weight

Polymer content is defined as the ratio of polymer to dry cell weight, polymer content = polymer yield/dry cell weight x 100%.

The following examples relate to the following media:

TYS liquid medium: 5g of peptone, 1g of yeast powder, 20g of sodium chloride, 0.7g of potassium chloride, 2.5g of magnesium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride and 0.2g of sodium bicarbonate are dissolved in a proper amount of deionized water, and then the deionized water is used for fixing the volume to 1L, and the pH value is regulated to 7.2.

TYS solid medium: 15g of agar powder is added into 1L of TYS liquid medium, sterilized for 15min at 121 ℃, and naturally cooled.

TYS liquid medium containing glucose: 20g of glucose, 5g of peptone, 1g of yeast powder, 20g of sodium chloride, 0.7g of potassium chloride, 2.5g of magnesium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride and 0.2g of sodium bicarbonate are dissolved in a proper amount of deionized water, and then the volume is fixed to 1L by using the deionized water, and the pH value is regulated to 7.2.

TYS liquid medium containing xylose: 20g of glucose in the TYS liquid medium containing glucose was replaced with 20g of xylose, and the others were unchanged.

Fructose-containing TYS liquid medium: 20g of glucose in the TYS liquid medium containing glucose was replaced with 20g of fructose, and the other was unchanged.

TYS liquid medium containing sucrose: the glucose 20g in the TYS liquid medium containing glucose was replaced with sucrose 20g, and the others were unchanged.

TYS liquid medium containing starch: the glucose 20g in the TYS liquid medium containing glucose was replaced with the starch 10g, and the others were unchanged.

TYS liquid medium containing glycerol: the glucose 20g in the TYS liquid medium containing glucose was replaced with glycerol 20g, and the others were unchanged.

TYS liquid medium containing acetic acid: the glucose 20g in the TYS liquid medium containing glucose was replaced with acetic acid 10g, and the others were unchanged.

TYS liquid medium containing propionic acid: the glucose 20g in the TYS liquid medium containing glucose was replaced with propionic acid 10g, and the others were unchanged.

TYS liquid medium containing valeric acid: the glucose 20g in the TYS liquid medium containing glucose was replaced with valeric acid 10g, and the others were unchanged.

TYS liquid medium containing soybean oil: 20g of glucose in the TYS liquid medium containing glucose was replaced with 20g of soybean oil, and the other was unchanged.

TYS liquid medium containing rice oil: 20g of glucose in the TYS liquid medium containing glucose was replaced with 20g of rice oil, and the others were unchanged.

Palm oil-containing TYS liquid medium: 20g of glucose in the TYS liquid medium containing glucose was replaced with 20g of palm oil, and the other was unchanged.

Example 1 isolation, identification and preservation of Photobacterium sp.TLY01 (CGMCC No. 22976)

1. Separation

1.1 g of a soil sample (obtained from soil near Tianjin Kong oilfield) was added to a triangular flask containing 100ml of sterile physiological saline and shaken at 200rpm for 1 hour to obtain a water sample.

2.1 mL of the water sample obtained in the step 1 is inoculated into TYS liquid medium containing soybean oil, and the culture is carried out for 24 hours at 30 ℃ and 200rpm, so as to obtain a culture.

3. Streaking the culture obtained in the step 2 on TYS solid medium by using a sterilized inoculating loop, and culturing for 24 hours in an inverted mode.

4. After step 3 is completed, the monoclonal which can grow on TYS solid medium is isolated, cultured and purified.

The isolated and purified strain was designated TLY01.

2. Authentication

1. Morphological identification

TLY01 was inoculated into TYS solid medium, cultured at 30℃for 10 hours, and observed with naked eyes and a microscope.

The result shows that the colony is milky white, round, 1.2-1.8mm in diameter, smooth in edge and raised in surface; TLY01 cells are rod-shaped, oblong and slightly curved.

2. Molecular characterization

The nucleotide sequence of the 16S rRNA of TLY01 is shown as SEQ ID NO. 1. The 16S rRNA sequence was subjected to EZBioCloud database (https:// www.ezbiocloud.net /) analysis. The results showed that TLY01 has high similarity to the gene sequence of the strain in the genus Photobacterium (Photobacterium), and 98.71% similarity to Photobacterium galatheaeS 2753.

Thus, TLY01 was assigned to the genus Photobacterium, designated as Photobacterium TLY01, by combining the above morphology and molecular identification results.

3. Preserving

The TLY01 separated in the first step is preserved in China general microbiological culture collection center (CGMCC) for 28 days in 2021, wherein the preservation number is CGMCC No.22976.

EXAMPLE 2 production of polyhydroxyalkanoate by Photobacterium TLY01 Using Glycerol as carbon

1. The luminous bacillus TLY01 monoclonal is inoculated in 20mLTYS liquid culture medium, and is cultured for 12 hours at 30 ℃ under shaking at 200rpm, so as to obtain luminous bacillus TLY01 seed liquid.

2. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL in size) containing 30mL of TYS liquid medium (5% of the inoculum size) containing glycerol, and cultured at 30℃and 200rpm for 24 hours to obtain a fermentation broth 1.

The results of observation of the fermentation broth 1 by a transmission electron microscope are shown in FIG. 1 (scale: 500 nm). The results showed that the P.luminophore TLY01 in fermentation broth 1 accumulated a large amount of white polyhydroxyalkanoate particles in the cells.

3. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL in size) containing 30mLTYS liquid medium (5% inoculum size) and cultured at 30℃and 200rpm for 24 hours to obtain a fermentation broth 2.

The results of observation of the fermentation broth 2 by a transmission electron microscope are shown in FIG. 2 (scale: 500 nm). The results showed that the white polyhydroxyalkanoate particles were not observed in the cells of the light emitting bacilli TLY01 in fermentation broth 2.

4. Placing the fermentation broth (fermentation broth 1 or fermentation broth 2) into a centrifuge tube (50 mL) and centrifuging at 10000rpm for 10min, and collecting precipitate 1; the precipitate was washed with deionized water 1, centrifuged at 10000rpm for 10min, and the precipitate was collected 2.

5. And (3) placing the centrifuge tube containing the precipitate 2 collected in the step (4) at the temperature of minus 20 ℃ for 2 hours, and then placing the centrifuge tube into a freeze vacuum dryer for freeze-drying for 10 hours to obtain a freeze-dried product.

Before adding the fermentation liquor and after freeze-drying, accurately weighing the weight of the centrifuge tube, and calculating the dry weight of the cells. Dry cell weight = weight of centrifuge tube after lyophilization-weight of centrifuge tube

6. Detecting the content of polyhydroxyalkanoate in the freeze-dried product.

The result shows that the dry cell weight of the fermentation liquid 1 is 8.58g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 4.02g/L, and the polyhydroxyalkanoate accounts for 46.85% of the dry cell weight. The dry cell weight of fermentation broth 2 was 1.90g/L, and no polyhydroxyalkanoate was produced.

EXAMPLE 3 production of polyhydroxyalkanoate by Protobacter sphaeroides TLY01 Using sugar as carbon

2. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL) containing 30mL of a medium (glucose-containing TYS liquid medium, xylose-containing TYS liquid medium, fructose-containing TYS liquid medium, sucrose-containing TYS liquid medium or starch-containing TYS liquid medium) (5% of the inoculum size), and cultured at 30℃and 200rpm for 24 hours to obtain a fermentation broth.

3. Placing the fermentation broth into a centrifuge tube (50 mL) for centrifugation at 10000rpm for 10min, and collecting precipitate 1; the precipitate was washed with deionized water 1, centrifuged at 10000rpm for 10min, and the precipitate was collected 2.

4. And (3) placing the centrifuge tube containing the precipitate 2 collected in the step (3) at the temperature of minus 20 ℃ for 2 hours, and then placing the centrifuge tube into a freeze vacuum dryer for freeze-drying for 10 hours to obtain a freeze-dried product.

5. Detecting the content of polyhydroxyalkanoate in the freeze-dried product.

The result shows that when the culture medium is a TYS liquid culture medium containing glucose, the dry weight of the cells is 7.71g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 5.05g/L, and the polyhydroxyalkanoate accounts for 65.49% of the dry weight of the cells; when the culture medium is a TYS liquid culture medium containing xylose, the dry weight of the cells is 3.90g/L, polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 1.78g/L, and the mass fraction of the polyhydroxyalkanoate in the cells is 45.11% of the dry weight of the cells; when the culture medium is a TYS liquid culture medium containing fructose, the dry weight of the cells is 6.23g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 3.60g/L, and the polyhydroxyalkanoate accounts for 57.83% of the dry weight of the cells; when the culture medium is a TYS liquid culture medium containing sucrose, the dry weight of the cells is 7.37g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 4.47g/L, and the polyhydroxyalkanoate accounts for 60.69% of the dry weight of the cells; when the culture medium is a TYS liquid culture medium containing starch, the dry weight of the cells is 7.08g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 2.10g/L, and the polyhydroxyalkanoate accounts for 29.66% of the dry weight of the cells.

EXAMPLE 4 production of polyhydroxyalkanoate by Photobacterium TLY01 Using volatile fatty acid as carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL in size) containing 30mL of a medium (TYS liquid medium containing acetic acid, TYS liquid medium containing propionic acid or TYS liquid medium containing valeric acid) (5% of the inoculum size), and cultured at 30℃and 200rpm for 48 hours to obtain a fermentation broth.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The result shows that when the culture medium is TYS liquid culture medium containing acetic acid, the dry weight of the cells is 4.49g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.80g/L, and the mass fraction of the polyhydroxyalkanoate is 17.82% of the dry weight of the cells; when the culture medium is TYS liquid culture medium containing propionic acid, the dry weight of the cells is 5.76g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 0.40g/L, and the mass fraction of the polyhydroxyalkanoate in the cells is 6.93%; when the culture medium is a TYS liquid culture medium containing valeric acid, the dry weight of the cells is 6.97g/L, the polyhydroxyalkanoate accumulated in the cells is poly (3-hydroxybutyrate-co-3-hydroxyvalerate), the yield is 4.16g/L, the mass fraction of the polyhydroxyalkanoate is 59.78%, and the content of the 3-hydroxyvalerate monomer in the copolyester is 91.16mol%.

EXAMPLE 5 production of polyhydroxyalkanoate by Photobacterium TLY01 Using vegetable oil as carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL in size) containing 30mL of a medium (TYS liquid medium containing soybean oil, TYS liquid medium containing rice oil or TYS liquid medium containing palm oil) (5% of the inoculum size), and cultured at 30℃and 200rpm for 24 hours to obtain a fermentation broth.

The fermentation broth was observed by transmission electron microscopy and the results are shown in FIG. 3 (scale 500 nm). As a result, when soybean oil was used as a carbon source, the fermentation broth of the P.luminophorum TLY01 accumulated a large amount of white polyhydroxyalkanoate particles in the cells.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The result shows that when the culture medium is TYS liquid culture medium containing soybean oil, the dry weight of the cells is 7.34g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 3.27g/L, and the polyhydroxyalkanoate accounts for 44.42% of the dry weight of the cells; when the culture medium is TYS liquid culture medium containing rice oil, the dry weight of the cells is 5.75g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 1.82g/L, and the mass fraction of the polyhydroxyalkanoate is 31.80% of the dry weight of the cells; when the medium was palm oil-containing TYS liquid medium, the dry weight of the cells was 5.09g/L, the polyhydroxyalkanoate accumulated in the cells was poly-3-hydroxybutyrate, and the yield was 1.69g/L, accounting for 33.18% by mass of the dry weight of the cells.

EXAMPLE 6 preparation of polyhydroxyalkanoate by Photobacterium TLY01 NaCl concentration optimization

To allow for a more efficient synthesis of polyhydroxyalkanoates by the light emitting bacilli TLY01, the fermentation medium was optimized for NaCl concentration.

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed liquid of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a shake flask (250 mL in size) containing 30mL of a medium (5% inoculum size) containing glycerol and NaCl of different concentrations, and cultured at 30℃and 200rpm for 0h or 24h to obtain a fermentation broth. The glycerol content of the fermentation broth was detected and the carbon source consumption was calculated. Carbon source consumption = glycerol content of 24h of incubation-glycerol content of 0h of incubation

Medium containing glycerol and different concentrations of NaCl: dissolving 20g of glycerin, 5g of peptone, 1g of yeast powder, 0.7g of sodium chloride, 0.5g of potassium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride and 0.2g of sodium bicarbonate in a proper amount of deionized water, then using deionized water to fix the volume to 1L, and regulating the pH value to 7.2; the concentration of sodium chloride in the culture medium is 0g/L, 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L or 80g/L.

The glycerol content was quantitatively determined by high performance liquid chromatography. The specific conditions are as follows: instrument: shimadzu corporation EssenA ria LC series HPLC apparatus is equipped with DGU-20A degasser, LC-16 liquid feed pump, SIL-16 autosampler, RID-20A detector. Chromatographic conditions: bio-Rad

HPX-87H (7.8X100 mm); the flow rate is 0.50mL/min; column temperature 55 ℃; the mobile phase was 7mM aqueous sulfuric acid. The detection method comprises the following steps: taking 5, 8, 10, 20 and 40g/L glycerol standard aqueous solutions, filtering with a 0.22 mu m microporous filter membrane, injecting 10 mu L of sample, performing HPLC detection, using chromatographic peak areas of glycerol standard solutions with different concentrations as ordinate and different concentrations as abscissa, and drawing a standard curve. Taking 1mL of fermentation liquor, centrifuging at 12000rpm for 10min, collecting supernatant, transferring to a new centrifuge tube, filtering with a 0.22 mu m microporous filter membrane, injecting 10 mu L of sample, and performing HPLC detection to obtain the glycerol chromatographic peak area of the fermentation liquor. And substituting the glycerol chromatographic peak area of the fermentation broth into the standard curve to obtain the glycerol content.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

And drawing a bar graph by taking NaCl concentration in the culture medium as an abscissa and carbon source consumption, cell dry weight and polyhydroxyalkanoate yield as an ordinate. The results are shown in FIG. 4.

The results show that when the NaCl concentration in the culture medium is 10g/L, the contents of the cell dry weight and the polyhydroxyalkanoate reach the maximum value, the cell dry weight is 9.18g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 5.60g/L, and the polyhydroxyalkanoate accounts for 60.98% of the mass fraction of the cell dry weight.

Therefore, when the medium is a medium containing glycerol and 10g/L NaCl, the P.photoperiod TLY01 is able to synthesize polyhydroxyalkanoates more efficiently. Culture medium containing glycerol and 10g/L NaCl: 20g of glycerin, 5g of peptone, 1g of yeast powder, 10g of sodium chloride, 0.7g of potassium chloride, 2.5g of magnesium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride and 0.2g of sodium bicarbonate are dissolved in a proper amount of deionized water, and then the deionized water is used for fixing the volume to 1L, and the pH value is regulated to 7.2.

EXAMPLE 7 temperature optimization of the Synthesis of polyhydroxyalkanoates by Photobacterium TLY01

In order to allow for a more efficient synthesis of polyhydroxyalkanoates by the light emitting bacilli TLY01, the fermentation temperature is optimized.

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed liquid of the P.luminoi TLY01 obtained in step 1 was inoculated into a shake flask (250 mL in size) containing 30mL of a medium (5% inoculum size) containing glycerol and 10g/L NaCl, and cultured at 25 ℃,30 ℃, 37 ℃ or 42 ℃ for 0h or 24h at 200rpm to obtain a fermentation broth. The glycerol content of the fermentation broth was detected and the carbon source consumption was calculated. Carbon source consumption = glycerol content of 24h of incubation-glycerol content of 0h of incubation

The method for detecting the glycerol content is the same as that of the step 2 in the example 6.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

And (3) taking fermentation temperature as an abscissa, carbon source consumption, cell dry weight and polyhydroxyalkanoate yield as an ordinate, and drawing a bar graph. The results are shown in FIG. 5. The results show that when the fermentation temperature is 30 ℃, the dry cell weight and the polyhydroxyalkanoate content reach the maximum value, the dry cell weight is 9.18g/L, the polyhydroxyalkanoate accumulated in the cells is poly-3-hydroxybutyrate, the yield is 5.60g/L, and the polyhydroxyalkanoate accounts for 60.98% of the dry cell weight in mass percent.

Therefore, when the medium is a medium containing glycerol and 10g/L NaCl and the fermentation temperature is 30 ℃, the P.luminophore TLY01 is capable of synthesizing polyhydroxyalkanoate most effectively.

EXAMPLE 8 production of polyhydroxyalkanoate by Photobacterium TLY01 Using Soybean oil and propionic acid as Mixed carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed solution of the luminous bacillus TLY01 obtained in the step 1 is inoculated into a culture medium containing 30mL of soybean oil, 10g/L NaCl and propionic acid with different concentrations, and the culture is carried out at 30 ℃ for 24 hours at 200rpm, so as to obtain a fermentation broth.

Culture medium containing soybean oil, 10g/L NaCl and propionic acid with different concentrations: dissolving 20g of soybean oil, 5g of peptone, 1g of yeast powder, 10g of sodium chloride, 0.7g of potassium chloride, 2.5g of magnesium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride, 0.2g of sodium bicarbonate and propionic acid in a proper amount of deionized water, then using deionized water to fix the volume to 1L, and regulating the pH value to 7.2; the concentration of propionic acid in the culture medium was 2g/L, 4g/L or 6g/L.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The test results are shown in Table 1.

TABLE 1 production of polyhydroxyalkanoates with Soybean oil and propionic acid as Mixed carbon

The results show that poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture with soybean oil and propionic acid as mixed carbon sources, and the addition of 6g/L propionic acid has little effect of inhibiting cell growth, and the monomer content of 3-hydroxyvalerate increases with the increase of propionic acid concentration, and is up to more than 18mol%. PHBV yield was highest when 2g/L propionic acid was added, 4.08g/L, with 8.69mol% 3-hydroxyvaleric acid monomer.

EXAMPLE 9 production of polyhydroxyalkanoate Using Soybean oil and valeric acid as Mixed carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed solution of the luminous bacillus TLY01 obtained in the step 1 is inoculated into a culture medium containing 30mL of soybean oil, 10g/L NaCl and different concentrations of valeric acid, and the culture is carried out at 30 ℃ for 48 hours at 200rpm, so as to obtain a fermentation broth.

Culture medium containing soybean oil, 10g/L NaCl and valeric acid with different concentrations: the propionic acid in the culture medium containing soybean oil, 10g/L NaCl and propionic acid with different concentrations is replaced by valeric acid, and the rest is unchanged.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The test results are shown in Table 2.

TABLE 2 production of polyhydroxyalkanoates with Soybean oil and valeric acid as Mixed carbon

The results show that poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture when soybean oil and valeric acid are used as mixed carbon sources, the dry cell weight decreases with increasing concentration of valeric acid, the PHBV yield and the proportion of dry cell weight also decrease with increasing concentration of valeric acid, and the 3-hydroxyvaleric acid monomer content increases with increasing concentration of valeric acid, and is up to more than 64mol%. PHBV yield was highest when 2g/L valeric acid was added, 6.28g/L, with 25.00mol% 3-hydroxyvaleric acid monomer.

EXAMPLE 10 production of polyhydroxyalkanoate by Photobacterium TLY01 Using starch and propionic acid as Mixed carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed solution of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a medium containing 30mL of starch, 10g/LNaCl and propionic acid of different concentrations, and cultured at 30℃for 24 hours at 200rpm to obtain a fermentation broth.

Culture medium containing starch, 10g/L NaCl and propionic acid with different concentrations: dissolving 10g of starch, 5g of peptone, 1g of yeast powder, 10g of sodium chloride, 0.7g of potassium chloride, 2.5g of magnesium chloride, 3.3g of magnesium sulfate, 1g of calcium chloride, 0.2g of sodium bicarbonate and propionic acid in a proper amount of deionized water, then using deionized water to fix the volume to 1L, and regulating the pH value to 7.2; the concentration of propionic acid in the culture medium was 2g/L, 4g/L or 6g/L.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The test results are shown in Table 3.

TABLE 3 production of polyhydroxyalkanoates with starch and propionic acid as Mixed carbon

The results show that poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained by fermentation culture with starch and propionic acid as mixed carbon sources, and the addition of 6g/L propionic acid has little effect of inhibiting cell growth, and the monomer content of 3-hydroxyvalerate increases with the increase of propionic acid concentration, and is up to more than 10mol%. PHBV yield was highest when 6g/L propionic acid was added, 2.64g/L, with a 3-hydroxyvaleric acid monomer content of 10.56mol%.

EXAMPLE 11 production of polyhydroxyalkanoate by Photobacterium TLY01 Using starch and valeric acid as Mixed carbon

1. Step 1 was performed as in example 3.

2. 1.5mL of the seed solution of the light emitting bacterium TLY01 obtained in the step 1 was inoculated into a medium containing 30mL of starch, 10g/LNaCl and different concentrations of valeric acid, and cultured at 30℃for 48 hours at 200rpm to obtain a fermentation broth.

Culture medium containing starch, 10g/L NaCl and different concentrations of valeric acid: the propionic acid in the culture medium containing starch, 10g/L NaCl and propionic acid with different concentrations was replaced with valeric acid, and the others were unchanged.

3. Step 3 was the same as in example 3.

4. Step 4 in example 3.

5. Step 5 in example 3.

The test results are shown in Table 4.

TABLE 4 production of polyhydroxyalkanoates with starch and valeric acid as Mixed carbon

The results show that when starch and valeric acid are used as mixed carbon sources, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) can be obtained through fermentation culture, PHBV accounts for the proportion of dry weight of cells and decreases along with the increase of the concentration of the valeric acid, and the monomer content of the 3-hydroxyvalerate increases along with the increase of the concentration of the valeric acid, and is up to more than 41mol%. PHBV yield was highest when 6g/L valeric acid was added, 2.54g/L, with 41.81mol% 3-hydroxyvaleric acid monomer content.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> Qingdao Fumei New technology Co., ltd, beijing chemical university

<120> A luminescent bacterium capable of producing polyhydroxyalkanoate by using various carbon sources such as vegetable oil or starch and the like, and use thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1609

<212> DNA

<213> Artificial sequence

<400> 1

tagtaacggc cgccagtgtg ctggaattgc ccttagagtt tgatcatggc tcagattgaa 60

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tgcgttgatg ggcggcgagc ggcggacggg tgagtaatgc ctgggaacat gccttagtgt 180

gggggataac cattggaaac gatggctaat accgcataat gtcttcggac caaagcgggg 240

gaccttcggg cctcgcgcgc taagattggc ccaggtggga ttagctagta ggtggggtaa 300

tagctcacct aggcgacgat ccctagctgg tctgagagga tgatcagcca cactggaact 360

gagacacggt ccagactcct acgggaggca gcagtgggga atattgcaca atgggggaaa 420

ccctgatgca gccatgccgc gtgtgtgaag aaggccttcg ggttgtaaag cactttcagc 480

agtgaggaag aggtggtgtt taatagatgc catctttgac gttagctgca gaagaagcac 540

cggctaactc cgtgccagca gccgcggtaa tacggagggt gcgagcgtta atcggaatta 600

ctgggcgtaa agcgcatgca ggcggcgtgt taagccagat gtgaaagccc ggggctcaac 660

ctcggaatcg catttggaac tggcatgcta gagtcttgta gaggggggta gaatttcagg 720

tgtagcggtg aaatgcgtag agatctgaag gaataccggt ggcgaaggcg gccccctgga 780

caaagactga cgctcagatg cgaaagcgtg gggagcaaac aggattagat accctggtag 840

tccacgccgt aaacgatgtc tacttggagg ttggtgtctt gaacactggc tttcggagct 900

aacgcgttaa gtagaccgcc tggggagtac ggtcgcaaga ttaaaactca aatgaattga 960

cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta 1020

cctactcttg acatccagag aactttccag agatggattg gtgccttcgg gaactctgag 1080

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cacaaagtac gtcgtagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg 1380

ctagtaatcg tggatcagaa tgccacggtg aatacgttcc cgggccttgt acacaccgcc 1440

cgtcacacca tgggagtggg ctgcaccaga agtagatagc ttaaccttcg ggagggcgtt 1500

taccacggtg tggttcatga ctggggtgaa gtcgtaacaa ggtaaccgaa gggcaattct 1560

gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggcc 1609

Claims

1. Photobacterium sp.TLY01 has a preservation number of CGMCC No.22976 in the China general microbiological culture Collection center.

2. A microbial inoculum, characterized in that: the microbial inoculum comprises Photobacterium sp.TLY01 of claim 1.

3. Use of a bacterium photorhabdus sp.tly01 according to claim 1 or a microbial agent according to claim 2 for the production of poly-3-hydroxybutyrate or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

4. Use of Photobacterium sp.TLY01 and a carbon source rich medium according to claim 1 for the production of poly-3-hydroxybutyrate or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

5. The use according to claim 4, characterized in that: the carbon source is at least one of glycerol, sugar, volatile fatty acid and vegetable oil.

6. The use according to claim 5, characterized in that:

the sugar is at least one of glucose, xylose, fructose, sucrose and starch;

the volatile fatty acid is at least one of acetic acid, propionic acid and valeric acid;

the vegetable oil is at least one of soybean oil, rice oil and palm oil.

7. Use according to any one of claims 4 to 6, characterized in that: the culture medium rich in carbon source also contains NaCl; the concentration of NaCl in the carbon source-rich medium is 10-40g/L.

8. Use according to any one of claims 4 to 6, characterized in that: the culture medium rich in carbon source also contains NaCl; the concentration of NaCl in the carbon source-rich medium was 10g/L.

9. A method for producing polyhydroxyalkanoates comprising the steps of:

(1) Inoculating the Photobacterium sp.TLY01 of claim 1 into a medium rich in carbon source, and culturing at 25-37deg.C and 100-300rpm to obtain fermentation broth;

(4) Separating polyhydroxyalkanoate from the freeze-dried product after step (3) is completed;

the polyhydroxyalkanoate is poly-3-hydroxybutyrate or poly (3-hydroxybutyrate-co-3-hydroxyvalerate).

10. The method according to claim 9, wherein: the carbon source is at least one of glycerol, sugar, volatile fatty acid and vegetable oil.

11. The method according to claim 10, wherein:

the sugar is at least one of glucose, xylose, fructose, sucrose and starch;

the vegetable oil is at least one of soybean oil, rice oil and palm oil.

12. The method according to any one of claims 9 to 11, wherein: the culture medium rich in carbon source also contains NaCl; the concentration of NaCl in the carbon source-rich medium is 10-40g/L.

13. The method according to any one of claims 9 to 11, wherein: the culture medium rich in carbon source also contains NaCl; the concentration of NaCl in the carbon source-rich medium was 10g/L.