Halomonas capable of utilizing multiple carbon sources and application of halomonas in fermentation production of polyhydroxyalkanoate

Abstract

The invention discloses halomonas capable of utilizing multiple carbon sources and application of the halomonas in fermentation production of polyhydroxyalkanoate. The halomonas capable of utilizing the multiple carbon sources disclosed by the invention is halomonas nigrifans X339, and the preservation number of the halomonas nigrifans X339 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 1.37971. The invention further discloses a preparation method of the halomonas capable of utilizing the multiple carbon sources. Experiments prove that the halomonas disclosed by the invention can be used for producing polyhydroxyalkanoate by fermenting various saccharides and organic acids, and the strain has high saline-alkaline adaptability, can be fermented under the condition that a culture medium is not sterilized, and can effectively prevent mixed carbon sources such as straw hydrolysate from going bad in a high-temperature sterilization process; according to the present invention, the method can effectively utilize the sugar and the organic acid therein, can further maintain the high basophilic ability, has the low optimal growth salt concentration, and has the good application prospect.

Description

Technical Field

[0001] The present invention relates to a Halomonas that can utilize multiple carbon sources and its application in fermentative production of polyhydroxyalkanoates in the field of biotechnology. Background Art

[0002] Traditional industrial biological fermentation for producing chemicals usually uses pure carbon sources such as glucose and glycerol. These carbon sources generally originate from grains. Utilizing such carbon sources not only competes with humans for food but also incurs high costs. Moreover, a large amount of biomass remains unutilized in agricultural production, such as straw and sugar residues. How to utilize these substances is an urgent problem to be solved. Patent CN116970538A discloses a Halomonas sp. LY03 that can ferment using carbon sources such as waste soybean oil, C10 saturated fatty acid, C11 saturated fatty acid, C12 saturated fatty acid, C13 saturated fatty acid, C14 saturated fatty acid, C16 saturated fatty acid, C18 saturated fatty acid, oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), glycerol, molasses, fructose, glucose, sucrose, etc. However, it cannot utilize the main components in straw hydrolysate, such as xylose and arabinose. The article (Luo, C.B., Li, H.C., Li, D.Q., Nawaz, H., You, T.T., & Xu, F. (2022). Efficiently unsterile polyhydroxyalkanoate production from lignocellulose by using alkali - halophilic Halomonas alkalicola M2. Bioresource technology, 351, 126919.) introduces a Halomonas for fermentatively producing PHA from lignocellulose. Summary of the Invention

[0003] The problem to be solved by the present invention is how to use microorganisms to ferment with non - grain carbon sources. This includes, but is not limited to, the utilization of complex carbon sources such as straw hydrolysate, as well as the utilization of chemically synthesized carbon sources such as acetic acid and propionic acid. These carbon sources will deteriorate or volatilize when sterilized by conventional high - temperature sterilization methods. Therefore, to utilize these carbon sources and avoid the problem of bacterial contamination during fermentation, more costly sterilization methods are required.

[0004] To solve the above technical problems, the present invention first provides a Halomonas nigrificans X339, whose preservation number in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms is CGMCC No. 1.37971. Halomonas nigrificans X339 has a broad-spectrum carbon source utilization ability and adaptability to high salt and high alkali. Under high saline-alkali conditions, it can perform open fermentation in a culture medium without high-temperature sterilization.

[0005] The present invention also provides a bacterial agent, and the active ingredient of the bacterial agent is the Halomonas nigrificans X339 described in claim 1.

[0006] The above-mentioned bacterial agent may further include a carrier. The carrier can be a solid carrier or a liquid carrier. The solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil or water; the organic solvent can be decane and / or dodecane. In the bacterial agent, the active ingredient can exist in the form of cultured live cells, the fermentation broth of live cells, the filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the composition can be various dosage forms, such as liquid agent, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0007] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the bacterial agent.

[0008] The application of the Halomonas nigrificans X339 or the bacterial agent in the fermentation production of polyhydroxyalkanoates also belongs to the protection scope of the present invention.

[0009] In the above application, the carbon source used for fermentation can be glucose, xylose, arabinose, cellobiose, lactose, maltose, fructose, starch, sucrose, galactose, acetic acid, propionic acid, n-butanol, valeric acid, caproic acid, succinic acid, citric acid, palmitic acid, glycerol, ethanol and / or straw hydrolysis liquor.

[0010] Among them, the straw hydrolysis liquor is a liquid product obtained from straw through a hydrolysis process. During the hydrolysis process, the cellulose, hemicellulose and other organic substances in the straw will be decomposed and converted into hydrolysis liquor. Its main components include sugar substances, organic acid substances, phenolic substances, ketone substances and alcohol substances, etc.

[0011] In one embodiment of the present invention, the straw hydrolysate is a wheat straw hydrolysate.

[0012] The application of the Halomonas nigrificans X339 or the bacterial agent in the preparation of a polyhydroxyalkanoate product by fermentation also falls within the protection scope of the present invention.

[0013] The present invention also provides a method for fermentatively producing polyhydroxyalkanoates, which comprises: fermenting by using the Halomonas nigrificans X339 in a fermentation medium to achieve the fermentative production of polyhydroxyalkanoates;

[0014] The fermentation medium is a medium in which Halomonas can grow, and the fermentation medium contains a carbon source, and the carbon source is glucose, xylose, arabinose, cellobiose, lactose, maltose, fructose, starch, sucrose, galactose, acetic acid, propionic acid, n-butanol, valeric acid, caproic acid, succinic acid, citric acid, palmitic acid, glycerol, ethanol and / or straw hydrolysate.

[0015] In one embodiment of the present invention, the fermentation medium is a medium obtained by adding the carbon source to the MM medium.

[0016] In the above method, the pH of the fermentation medium can be 6.5–11. Further, the pH of the fermentation medium can be 7.5-9.

[0017] In the above method, the fermentation medium may contain NaCl, and the concentration of NaCl can be 1 g / 100 mL to 15 g / 100 mL. Further, the concentration of NaCl in the fermentation medium can be 2 g / 100 mL - 7 g / 100 mL, such as 3 g / 100 mL.

[0018] The present invention also provides a product for producing polyhydroxyalkanoates, and the product contains the Halomonas nigrificans X339 and the fermentation medium.

[0019] The application of the product in the production of polyhydroxyalkanoates also falls within the protection scope of the present invention.

[0020] The Halomonas nigrificans X339 of the present invention has the ability to utilize a broad spectrum of carbon sources and can be used to solve the problem of microbial fermentation utilization of mixed carbon sources for biomass regeneration. The Halomonas nigrificans X339 of the present invention can utilize various sugars and organic acids for fermentative production of polyhydroxyalkanoates, and this strain has high salinity and alkalinity adaptability, can be fermented without sterilizing the culture medium, can effectively prevent the deterioration of mixed carbon sources such as straw hydrolysate during high-temperature sterilization, and can effectively utilize the sugars and organic acids therein. The Halomonas nigrificans X339 of the present invention can also have a lower optimal growth salt concentration (30 g / L NaCl) under the condition of retaining high alkalophilic ability (optimal pH 8-9), which can reduce the addition of NaCl in the culture medium. The Halomonas nigrificans X339 of the present invention has good application prospects.

[0021] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0022] Biomaterial Preservation Instructions

[0023] Abbreviation of the depositary institution: CGMCC

[0024] Name of the depositary institution: China General Microbiological Culture Collection Center

[0025] Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101

[0026] Date of deposit: December 29, 2023

[0027] Accession number registered by the deposit center: CGMCC No. 1.37971

[0028] Taxonomic name: Halomonas nigrificans

[0029] Strain number: X339 Description of the drawings

[0030] Figure 1Optimal salt concentration test for Halomonas nigrificans X339. "%" represents mass-volume concentration (W / V), that is, "g / 100 mL". The concentration labels on the right side of the figure correspond to the curves from top to bottom at the dotted line from top to bottom in sequence.

[0031] Figure 2 Optimal pH test for Halomonas nigrificans X339. The pH labels on the right side of the figure correspond to the curves from top to bottom at the dotted line from top to bottom in sequence.

[0032] Figure 3 Test for the utilization ability of Halomonas nigrificans X339 to different carbohydrate carbon sources.

[0033] Figure 4 Test for the utilization ability of Halomonas nigrificans X339 to different organic acids and alcohols.

[0034] Figure 5 Growth curve of Halomonas nigrificans X339 using different concentrations of acetic acid (g / L) as the sole carbon source.

[0035] Figure 6 Growth curve of Halomonas nigrificans X339 using different concentrations of propionic acid (g / L) as the sole carbon source. Specific implementation mode

[0036] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. For the quantitative tests in the following examples, at least three repeated experiments are set, and the results are averaged.

[0037] Medium preparation and culture conditions:

[0038] HM medium: 36 g / L sodium chloride, 10 g / L yeast extract, 5 g / L tryptone, 1 g / L magnesium sulfate heptahydrate, 0.27 g / L calcium chloride, 0.23 g / L sodium bromide, 0.06 g / L sodium bicarbonate, 2 g / L potassium chloride, pH is adjusted to 9.0 with NaOH, and the balance is water. (1.5 g / L agar powder is additionally added to the corresponding solid plate medium).

[0039] LB60 Medium: 60 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, with the balance being water. (1.5 g / L agar powder is additionally added to the corresponding solid plate medium).

[0040] LB30 Medium: 30 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, with the balance being water.

[0041] LB Medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone, with the balance being water.

[0042] MM Medium: 60 g / L sodium chloride, 1 g / L yeast powder, 1 g / L ammonium chloride, 0.4 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 9.65 g / L disodium hydrogen phosphate dodecahydrate, 0.05 g / L ammonium ferric citrate, 0.02 g / L calcium chloride dihydrate, 0.03 mg / L sodium molybdate dihydrate, 0.02 mg / L nickel chloride hexahydrate, 0.01 mg / L copper sulfate pentahydrate, 0.3 mg / L boric acid, 0.03 mg / L manganese chloride tetrahydrate, 0.1 mg / L zinc chloride heptahydrate, 0.2 mg / L cobalt chloride hexahydrate. The pH of the medium is adjusted to about 8.5 using 5M NaOH aqueous solution, with the balance being water.

[0043] Unless otherwise specified, the bacteria are cultured at 37°C. The liquid medium is cultured in a shaker at 200 rpm, and the solid plate medium is cultured in an incubator.

[0044] Determination of cell dry weight: Place 10 mL of the fermentation product in a 50 mL centrifuge tube, centrifuge at 10000 rcf for 10 minutes, and discard the supernatant; seal the centrifuge tube with a sealing film and store it in a -80°C refrigerator for 2 h; dry the centrifuge tube containing the bacteria in a vacuum freeze dryer for 24 h; weigh and calculate the cell dry weight (g / L) in the fermentation product.

[0045] Determination of polyhydroxyalkanoate (PHA) content: Add 2 mL of esterification solution (containing methanol, 3% (v / v) concentrated sulfuric acid (98%, w / w) and 1 g / L benzoic acid, the balance is water) and 2 mL of chloroform to about 40 mg of freeze-dried bacteria, and esterify at 100°C for about 4 hours. 20-30 mg of PHB standard was treated in the same way. Then a gas chromatograph GC-6820 (Agilent, USA) was used equipped with a chromatographic column DB-FFAP column (30m×0.32mm×0.25μm film thickness, part number 123-3232, Agilent, USA). The test method is: the initial temperature is maintained at 80°C for 1.5 minutes; in the first stage, the temperature is increased to 140°C at a rate of 30°C / min; in the second stage, it is increased to 220°C at a rate of 40°C / min, and this process takes 2 minutes; the total analysis time is 6 minutes; the injection temperature is 200°C and the detector temperature is 220°C.

[0046] Example 1. Isolation and Characterization of Halomonas nigrificans X339

[0047] 1. Isolation of Halomonas nigrificans X339

[0048] The inventor screened a strain of bacteria X339 from the Hudong Chagan Nao Salt Lake in Inner Mongolia Autonomous Region. The isolation method is as follows:

[0049] The lake water sample was diluted 10 times with 36 g / L NaCl solution, and then 100 μL of the diluted sample was spread on a solid plate of HM medium. After culturing at 28°C for 1 day, a single colony was picked out and cultured with HM medium and 16S rDNA sequencing was performed.

[0050] The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1, and the strain was identified as Halomonas nigrificans, hereinafter referred to as Halomonas nigrificans X339.

[0051] Halomonas nigrificans X339 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 29, 2023, with the deposit number CGMCC No.1.37971.

[0052] 2. Determination of the optimal salt concentration for the growth of Halomonas nigrificans X339

[0053] Prepare LB media with 14 different salt concentrations. The concentrations of other substances are the same as those in normal LB media, and the NaCl concentrations are set to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, and 20 g / 100 mL respectively. Sterilize using high-pressure steam, with the sterilization conditions being 121 °C for 20 minutes.

[0054] Pick the Halomonas nigrificans X339 stored in the -80 °C refrigerator and inoculate and activate it in a test tube containing 4 mL of LB30 medium. Incubate overnight at 37 °C with 200 rpm to obtain the X339 bacterial solution. Add 200 μL of the medium with different salt concentrations to each well of a 10×10 100-well microplate, and inoculate 2 μL of the X339 bacterial solution into each well. Set 4 parallels for each salt concentration. Place the 10×10 100-well microplate in a growth curve analyzer (Bioscreen C, Finland) and incubate it with shaking at 37 °C for 24 h. Measure the OD value at 600 nm every 1 h during this period to plot its growth curve.

[0055] The results are as Figure 1 shown. Halomonas nigrificans X339 can grow at NaCl concentrations ranging from 1 g / 100 mL to 15 g / 100 mL. The optimal NaCl concentration is 2 g / 100 mL - 7 g / 100 mL, and the optimal salt concentration range of Halomonas nigrificans X339 is relatively broad.

[0056] 3. Determination of the optimal growth pH of Halomonas nigrificans X339

[0057] Prepare 12 portions of LB30 medium, and then add Tris base with a final concentration of 50 mmol / L. Use 5 mol / L HCl or 5 mol / L NaOH aqueous solution to adjust the pH to pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, and 11.0 respectively to obtain LB30 media with different pH values. Sterilize using an autoclave, with the sterilization conditions being 121 °C for 20 minutes.

[0058] Pick the Halomonas nigrificans X339 strain stored in an -80°C refrigerator, inoculate it into a test tube containing 4 mL of LB30 medium, and culture it overnight at 37°C with 200 rpm to activate the strain, obtaining the X339 bacterial solution. Add 200 μL of LB30 medium with different pH values to each well of a 10×10 100-well microplate, inoculate 2 μL of the X339 bacterial solution into each well, and set 4 parallels for each pH value. Place the 10×10 100-well microplate in a growth curve analyzer (Bioscreen C, Finland), and culture it with shaking at 37°C for 24 h. During this period, measure the OD value at 600 nm every 1 h to plot its growth curve.

[0059] The results are as Figure 2 shown. Halomonas nigrificans X339 can grow in the range of pH 6.5 - pH 11, and its optimal pH is between 7.5 and 9.0. Halomonas nigrificans X339 has a broad pH adaptability and good alkali tolerance.

[0060] 4. Carbon source utilization ability of Halomonas nigrificans X339

[0061] The carbon source utilization ability test was carried out using MM medium. After autoclaving, different sterile carbon sources were added to it, and the pH was adjusted to about 8.5 with 5 mol / L aqueous NaOH solution to obtain MM media with different carbon sources. The added carbon sources and their concentrations in the medium are shown in Table 1.

[0062] Pick the Halomonas nigrificans X339 strain stored in an -80°C refrigerator, inoculate it into a test tube containing 4 mL of LB60 medium, and culture it overnight at 37°C with 200 rpm to activate the strain, obtaining the X339 bacterial solution. Add 200 μL of MM medium with different carbon sources to each well of a 10×10 100-well microplate, inoculate 2 μL of the X339 bacterial solution into each well, and set 4 parallels for each carbon source. Place the 10×10 100-well microplate in a growth curve analyzer (Bioscreen C, Finland), and culture it with shaking at 37°C for 72 h. During this period, measure the OD value at 600 nm every 1 h to plot its growth curve. Use the MM medium without added carbon source as the carbon-free control, and use the MM medium without added carbon source and without inoculation as the blank control.

[0063] Table 1. Addition amounts of different carbon sources

[0064]

[0065]

[0066] The carbon source utilization ability of Halomonas nigrificans X339 is as follows Figure 3 , Figure 4 shown. In the test, the carbon sources that Halomonas nigrificans X339 can utilize include glucose, xylose, arabinose, cellobiose, lactose, maltose, fructose, starch, sucrose, galactose, n-butanol, valeric acid, caproic acid, succinic acid, citric acid, palmitic acid, glycerol, and ethanol. The carbon sources that cannot be utilized are methanol, isopropanol, capric acid, and lauric acid.

[0067] 5. Acetic acid and propionic acid utilization ability of Halomonas nigrificans X339

[0068] The acetic acid utilization ability test was carried out using MM medium. After autoclaving, different concentrations of sterile acetic acid were added to it. The concentrations of acetic acid were set to 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 g / L. The pH was adjusted to about 8.5 with 5 mol / L NaOH aqueous solution to obtain MM media with different acetic acid concentrations.

[0069] Pick the strain of Halomonas nigrificans X339 stored in the -80°C refrigerator and inoculate it into a test tube containing 4 mL of LB60 medium. Incubate overnight at 37°C and 200 rpm to activate the strain to obtain X339 bacterial liquid. Add 200 μL of MM medium with different acetic acid concentrations to each well of a 10×10 100-well microplate, and inoculate 2 μL of X339 bacterial liquid into each well. Four parallels were set for each acetic acid concentration. Place the 10×10 100-well microplate in a growth curve analyzer (Bioscreen C, Finland) and shake-culture at 37°C for 72 h. During this period, measure the OD value at 600 nm every 1 h to plot its growth curve. Use MM medium without adding acetic acid as the carbon source-free control.

[0070] According to the above method, replace acetic acid with propionic acid. The concentrations of propionic acid are set to 5, 10, 20, 30, 40, 50, and 60 g / L, and other steps remain unchanged to detect the propionic acid utilization ability of Halomonas nigrificans X339.

[0071] The results are as follows Figure 5 , Figure 6 shown. The results show that the carbon sources that Halomonas nigrificans X339 can utilize also include acetic acid and propionic acid, and its utilization and tolerance abilities for acetic acid and propionic acid are very strong, and it can grow well under the condition of 60 g / L.

[0072] Example 2. Fermentation of PHA by Halomonas nigrificans X339 using different carbon sources

[0073] The carbon source utilization ability test was carried out using MM medium. After autoclaving, different sterile carbon sources and sterile MOPS solution (500 g / L) were added to it. The pH was adjusted to about 8.5 with 5 mol / L aqueous NaOH solution to obtain MM media with different carbon sources. The carbon sources and their concentrations in the medium are shown in Table 2, and the final concentration of MOPS in the medium is 40 g / L.

[0074] The strain of Halomonas nigrificans X339 stored in a -80 °C refrigerator was picked and inoculated into a test tube containing 4 mL of LB60 medium, and cultured overnight at 37 °C and 200 rpm to activate the strain, obtaining the X339 seed solution. Then the X339 seed solution was inoculated into 100 mL of LB60 medium at a volume ratio of 1% and activated for 12 h to obtain the secondary seed solution. The secondary seed solution was inoculated into a 500 mL shake flask fermentation medium containing 50 mL of MM medium with different carbon sources at a volume ratio of 5%. The shake flask was placed in a shaker and cultured at 37 °C and 200 rpm. The fermentation duration was controlled at 48 h.

[0075] After the fermentation was completed, the dry cell weight, PHA% (wt%), and PHA yield in the fermentation product were measured. Among them, PHA% (wt%) represents the mass percentage of PHA to the dry cell weight in the fermentation product per unit volume.

[0076] The results of fermentation of PHA by Halomonas nigrificans X339 using different carbon sources are shown in Table 2. Halomonas nigrificans X339 can effectively utilize glucose, xylose, starch, acetic acid, fructose, citric acid, and maltose to produce PHA. Among them, the fermentation PHA contents of acetic acid and maltose are the highest, 61% and 58% respectively. The yield using maltose as the carbon source is also the highest, reaching 8.88 g / L.

[0077] Table 2. Yields of PHA fermented by Halomonas nigrificans X339 using different carbon sources

[0078] Carbon source Cell dry weight (g / L) PHA% (wt%) PHA yield (g / L) Fermentation duration Glucose 30 g / L 14.45±0.16 51.34±1.07 7.37±0.17 48h Xylose 30 g / L 8.97±0.21 21.01±1.80 1.88±0.18 48h Starch 30 g / L 14.71±0.43 42.95±1.88 6.32±0.29 48h Acetic acid 40 g / L 10.29±0.27 61.32±7.16 6.31±0.70 48h Fructose 30 g / L 12.94±0.32 39.31±4.75 5.08±0.49 48h Citric acid 30 g / L 11.28±0.70 37.41±2.58 4.21±0.25 48h Maltose 30 g / L 15.40±0.01 58.25±1.09 8.88±0.06 48h

[0079] Example 3. Fermentation of PHA by Halomonas nigrificans X339 using straw hydrolysate

[0080] The wheat straw hydrolysate is a product of Anhui Fengyuan Biotech Co., Ltd. The main components are as follows, in units of (m / m): glucose 23.2%, xylose 14.07%, arabinose 1.22%, lactic acid 1.57%, formic acid 0.83%, acetic acid 2.06%. The density of the hydrolysate is 1.35 g / mL.

[0081] The wheat straw hydrolysate was added to the MM medium as a carbon source at a volume ratio of 5% to obtain the straw-MM medium.

[0082] The Halomonas nigrificans X339 strain preserved in a -80°C refrigerator was picked and inoculated into a test tube containing 4 mL of LB60 medium, and cultured overnight at 37°C and 200 rpm to activate the strain, obtaining the X339 seed solution. Then, the X339 seed solution was inoculated into 100 mL of LB60 medium at a volume ratio of 1% and activated for 12 h to obtain the secondary seed solution. The secondary seed solution was inoculated into a 500 mL shake flask fermentation medium containing 50 mL of straw-MM medium at a volume ratio of 5%. The shake flask was placed in a shaker and cultured at 37°C and 200 rpm. The fermentation duration was controlled at 72 h.

[0083] After fermentation, the cell dry weight, PHA% (wt%), and PHA yield in the fermentation product were measured. Among them, PHA% (wt%) represents the mass percentage of PHA to cell dry weight in the fermentation product per unit volume.

[0084] The results are shown in Table 3. The results show that Halomonas nigrificans X339 can effectively utilize the wheat straw hydrolysate to produce PHA, and the PHA content after 72 h can reach 34% of the cell dry weight.

[0085] Table 3. Yield of PHA fermented by Halomonas nigrificans X339 using straw hydrolysate

[0086] Cell dry weight (g / L) PHA content (wt%) PHA yield (g / L) Fermentation duration 13.51±0.68 31.40±2.6 4.25±0.56 48h 15.21±0.98 34.37±1.63 5.23±0.47 72h

[0087] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. Halomonas nigrificans X339, with the preservation number of CGMCC No. 1.37971 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms.

2. A bacterial agent, the active ingredient of which is Halomonas nigrificans X339 described in claim 1.

3. The application of Halomonas nigrificans X339 described in claim 1 or the bacterial agent described in claim 2 in the fermentation production of polyhydroxyalkanoates.

4. The application according to claim 3, wherein: The carbon source used for the fermentation is glucose, xylose, arabinose, cellobiose, lactose, maltose, fructose, starch, sucrose, galactose, acetic acid, propionic acid, n-butanol, valeric acid, caproic acid, succinic acid, citric acid, palmitic acid, glycerol, ethanol and / or straw hydrolysate.

5. The application of Halomonas nigrificans X339 described in claim 1 or the bacterial agent described in claim 2 in the preparation of products for the fermentation production of polyhydroxyalkanoates.

6. A method for fermentatively producing polyhydroxyalkanoates, comprising: Ferment using Halomonas nigrificans X339 described in claim 1 in a fermentation medium to achieve the fermentation production of polyhydroxyalkanoates; The fermentation medium is a growth medium for Halomonas, and the fermentation medium contains a carbon source, and the carbon source is glucose, xylose, arabinose, cellobiose, lactose, maltose, fructose, starch, sucrose, galactose, acetic acid, propionic acid, n-butanol, valeric acid, caproic acid, succinic acid, citric acid, palmitic acid, glycerol, ethanol and / or straw hydrolysate.

7. The method according to claim 6, wherein: The pH of the fermentation medium is 6.5–11; Furthermore, the pH of the fermentation medium is 7.5-9.

8. The method according to claim 6 or 7, characterized in that: The fermentation medium contains NaCl, and the concentration of NaCl is 1 g / 100 mL to 15 g / 100 mL; furthermore, the concentration of NaCl is 2 g / 100 mL - 7 g / 100 mL.

9. A product for the production of polyhydroxyalkanoates, containing Halomonas nigrificans X339 described in claim 1 and any one of the fermentation media described in claims 6-8.

10. The application of the product described in claim 9 in the production of polyhydroxyalkanoates.

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