Comamonas testosteroni G314 and application thereof

Abstract

The application belongs to the field of environmental microbiology, and particularly relates to a Comamonas testosteroni G314 and application thereof.The technical points are as follows: the Comamonas testosteroni provided by the application is preserved in the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Xibahe Road, Chaoyang District, Beijing, China, and the preservation number is CGMCC No. 32129, and the preservation time is September 30, 2024.The Comamonas testosteroni provided by the application has different abilities to synthesize PHA by using different organic acid carbon sources, and the type of PHA can be regulated by regulating the composition of volatile fatty acids, so that the performance of PHA is improved, new strains for efficient production of PHA are provided, and the production cost of PHA is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology, specifically relating to a type of testosterone-bearing bacterium, Commamonas testosteroni G314, and its applications. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) possess physical properties comparable to traditional plastics, while also exhibiting biodegradability and biocompatibility, which traditional plastics lack, making them an ideal alternative material. However, the limited production volume and high cost of PHA currently significantly restrict its commercial application.

[0003] Currently, over 300 species of microorganisms capable of synthesizing PHA in nature have been discovered. The PHA production capacity and required conditions vary among different species. Most known PHA-synthesizing bacteria are aerobic. During PHA production under aerobic conditions, most of the carbon source enters the tricarboxylic acid cycle and is ultimately converted into CO2. Only the remaining carbon source is used for strain growth and PHA synthesis. This process not only significantly reduces the amount of carbon source converted into PHA but also increases the cost of aeration.

[0004] Under hypoxic or anaerobic conditions, the carbon source for CO2 production through respiratory metabolism is scarce or nonexistent, which can significantly improve carbon source utilization and lead to higher PHA synthesis. However, current research can only modify strains using gene editing technology to enable them to synthesize PHA under hypoxic conditions, thereby increasing PHA yield. However, gene-edited strains can only synthesize specific PHA conformations, and the production efficiency is low. Summary of the Invention

[0005] The purpose of this invention is to provide a method for controlling the configuration of polyhydroxyalkanoates by culturing the strain using different types of carbon sources. The carbon source for synthesizing polyhydroxyalkanoates can be obtained through anaerobic fermentation of organic waste such as kitchen waste or residual sludge, which can simultaneously realize the resource utilization of organic waste and reduce the production cost of polyhydroxyalkanoates.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0007] This invention provides a strain of *Comamonas testosteroni* G314, screened from industrial sludge. This strain can anaerobically synthesize polyhydroxy fatty acid esters without gene editing technology. It is deposited at the China General Biotechnology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China; accession number: CGMCC No. 32129; deposit date: September 30, 2024.

[0008] This invention provides an application of *Comamonas testosteroni* G314, which is used to synthesize polyhydroxy fatty acid esters under anoxic or anaerobic conditions. No oxygenation equipment is required during fermentation, greatly simplifying the fermentation equipment requirements and reducing equipment investment and maintenance costs. Simultaneously, the anaerobic environment helps inhibit the growth of other aerobic contaminating bacteria, improving the purity and reliability of the production.

[0009] Furthermore, the polyhydroxyalkanoate (PHA) is either poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Both poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) are biodegradable materials that can be completely degraded by microorganisms in natural environments (such as soil and oceans), producing harmless byproducts such as carbon dioxide and water. Therefore, they do not cause white pollution and are environmentally friendly green materials that can replace traditional petroleum-based plastics. Moreover, the raw materials for PHA in this invention can be derived from organic waste, making its production process more sustainable, reducing dependence on petrochemical raw materials, and simultaneously providing the dual function of waste treatment.

[0010] Poly(3-hydroxybutyrate) itself has high crystallinity, resulting in high rigidity and strength in mechanical properties, making it suitable as a substitute for certain rigid plastics in products requiring wear resistance or high rigidity. By adding 3-hydroxyvalerate (3HV) units to poly(3-hydroxybutyrate), forming copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), the flexibility and ductility of the material can be significantly improved. The introduction of 3HV reduces the crystallinity of the polymer, making the material softer and more elastic, suitable for applications requiring a certain degree of flexibility, such as packaging materials and medical products.

[0011] Furthermore, the use of *Trichomonas testis* to synthesize polyhydroxy fatty acid esters under anaerobic or hypoxic conditions includes the following steps:

[0012] Testosterone-bearing bacteria were inoculated into LB medium and cultured to obtain seed culture.

[0013] After inoculating the fermentation medium with 1%–10% (v / v) of seed culture, the culture is fermented on a constant-temperature shaker to obtain the fermentation broth. This invention allows for adjustment of the inoculum amount according to the fermentation scale and process requirements, thereby optimizing production efficiency. Fermentation on a constant-temperature shaker ensures precise control of temperature and shaker speed, providing a stable environment for cell growth and PHA synthesis. This helps improve the reproducibility and efficiency of the fermentation process and reduces the impact of environmental fluctuations.

[0014] Centrifugation of the fermentation broth yielded *Trichomonas testis* cells, which were then freeze-dried to obtain dried *Trichomonas testis* cells. Centrifugation effectively collected the fermented cells. This centrifugation step is rapid and efficient, suitable for large-scale production. The freeze-dried cells exhibit good stability, maximizing the preservation of the structure and purity of PHA within the cells. This drying method does not damage PHA, ensuring high-quality PHA is obtained in subsequent extraction processes.

[0015] Dry cells of *Trichomonas vaginalis* were mixed with chloroform and acidified methanol containing benzoic acid, and reacted at 105°C for 6 hours. Chloroform, as an organic solvent, effectively dissolves PHA, while acidified methanol helps disrupt the cell wall and release PHA. This extraction process, optimized in terms of reaction time and temperature, enables efficient PHA extraction while avoiding excessive heat that could degrade PHA, thus yielding high-purity PHA.

[0016] Distilled water was added, and the chloroform layer was transferred to anhydrous sodium sulfate for drying to obtain polyhydroxyalkanoates (PHA). The step of adding distilled water followed by drying the chloroform layer in anhydrous sodium sulfate effectively removes moisture from the organic solvent, thus improving the purity of PHA.

[0017] Furthermore, the initial pH of the fermentation culture is 5–9, the fermentation temperature is 25–40°C, the rotation speed of the constant-temperature shaker is 100–200 rpm, and the seed culture time is 10–24 hours. This flexibility allows the fermentation conditions to be adjusted to adapt to different environments and culture objectives, thereby improving fermentation efficiency.

[0018] Furthermore, the initial pH value of the fermentation culture was 8, the fermentation temperature was 258℃, the rotation speed of the constant temperature shaker was 180 rpm, the seed culture time was 22 h, and the inoculum size was 5–10% (v / v), preferably 10% (v / v). Using pH 8 as the initial value provides a suitable alkaline environment for the metabolism of *Trichophyton mentagrophytes*, promoting carbon source utilization. Choosing a higher inoculum size (5–10%, preferably 10%) can accelerate the increase in cell count in the early stages of fermentation, shorten the production cycle, reduce the risk of contamination, and simultaneously ensure the robustness and high yield of the fermentation.

[0019] Furthermore, the carbon source in the fermentation medium can be any one or more of glucose, sucrose, glycerol, or volatile fatty acids. This diversity of carbon sources increases the flexibility and adaptability of fermentation, allowing the selection of the most suitable source based on feedstock availability and cost. For example, glucose and sucrose provide readily available energy, while glycerol and volatile fatty acids are suitable for promoting PHA synthesis under specific conditions. In addition, using a combination of multiple carbon sources can optimize cell growth and PHA accumulation, increasing the final yield.

[0020] Furthermore, the volatile fatty acids are any one or more of sodium acetate, sodium propionate, butyric acid, or valeric acid. The metabolic pathways of these fatty acids directly promote the accumulation of PHA synthetic monomers. Simultaneously, using different types of volatile fatty acids can regulate the composition of PHA, resulting in a final product with better properties such as flexibility and extensibility.

[0021] Furthermore, the fermentation medium composition includes: 3.3 g / L carbon source, 0.08 g / L KH₂PO₄, 0.15 g / L NH₄Cl, 0.1 g / L MgSO₄, 0.01 g / L CaCl₂·2H₂O, 1.5 mg / L FeCl₃·6H₂O, and 1.0 mL / L trace elements. The appropriate concentration of carbon source promotes growth and optimizes PHA accumulation. KH₂PO₄ provides phosphorus to stabilize the pH of the medium; NH₄Cl provides nitrogen to promote protein and cell proliferation; MgSO₄ and CaCl₂ provide essential ions to maintain intracellular and extracellular ion balance; and FeCl₃ acts as a cofactor to promote the activity of various enzymes. This optimized medium composition ensures the nutrient balance required for *Trichophyton mentagrophytes* growth and PHA synthesis, maximizing cell proliferation and PHA synthesis efficiency while minimizing nutrient waste.

[0022] Furthermore, the trace elements included were: CuSO4·5H2O 0.01 g / L, ZnSO4·H2O 0.1 g / L, MnCl2·4H2O 0.03 g / L, H3BO3 0.3 g / L, CoCl2·6H2O 0.2 g / L, NiCl2·6H2O 0.02 g / L, and NaMoO4·2H2O 0.03 g / L. Supplementation with trace elements effectively improved bacterial growth and metabolic activity. Cu and Zn play important roles in redox reactions and enzyme activity; Mn contributes to protein synthesis and enzyme stability; Co is a component of vitamin B12 and is essential for cellular metabolism; Ni and Mo are related to specific enzyme activities, particularly crucial for the synthesis pathway of PHA. The precise addition of these trace elements ensured the metabolic efficiency of *Trichophyton mentagrophytes* and the rate of PHA synthesis, significantly improving the stability of fermentation production and the yield of PHA.

[0023] In summary, the present invention has the following beneficial effects:

[0024] This invention provides a *Comamonas testosteroni* G314 strain. Cultured under anoxic and anaerobic conditions using sodium acetate, the *Comamonas testosteroni* G314 strain achieved PHB accumulation of 69.0% and 63.7% of cell dry weight, respectively, which is higher than the PHB accumulation under aerobic conditions. This indicates that the cell's carbon utilization efficiency during metabolism is higher, suggesting that carbon sources are preferentially used for PHA synthesis rather than other metabolic pathways under anoxic or anaerobic conditions. Since anoxic and anaerobic conditions do not require forced oxygen supply, energy consumption can be significantly reduced. Especially in large-scale industrial fermentation, this reduction not only saves energy costs but also reduces the investment and maintenance of process equipment (such as oxygenation systems), significantly improving economic efficiency. Simultaneously, the lower oxygen demand also reduces environmental impact, aligning with green and environmentally friendly production principles.

[0025] Furthermore, when the strain was cultured using valeric acid as a carbon source, the highest PHBV accumulation was achieved under hypoxic conditions, reaching 74.3% of the cell dry weight, with HV accounting for as high as 91.1%. This high HV proportion indicates that the obtained PHA is mainly poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and its flexibility is significantly improved. The increase in HV content can significantly improve the mechanical properties of the polymer, such as reducing brittleness and increasing ductility and elasticity, making this material more suitable for applications requiring high flexibility and plasticity, such as biomedical materials and packaging films.

[0026] This invention allows for the regulation of the composition and properties of PHA by altering the type of carbon source. For example, it enables the synthesis of more PHB or PHBV under different environments and the adjustment of the HV ratio. This method of regulating PHA configuration through carbon source control can provide diversified material choices for industrial applications, allowing the physical and chemical properties of PHA to be flexibly varied according to different needs, thereby expanding its application range and enabling better adaptation in fields such as biodegradable plastics, food packaging, and biomedical devices.

[0027] Preservation of biological materials

[0028] Commonas testosteroni G314 was deposited on September 30, 2024, at the China General Biotechnology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 32129, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] Figure 1 This is a phylogenetic tree constructed from the 16S rRNA sequence of Commonas testosteroni G314.

[0031] Figure 2 This study describes the growth and PHA synthesis of *Comamonas testosteroni* G314 under different oxygen conditions using sodium acetate as a carbon source.

[0032] Figure 3 This describes the growth and PHA synthesis of *Comamonas testosteroni* G314 using sodium propionate as a carbon source.

[0033] Figure 4 This describes the growth and PHA synthesis of *Comamonas testosteroni* G314 using butyric acid as a carbon source.

[0034] Figure 5 This describes the growth and PHA synthesis of *Comamonas testosteroni* G314 using valeric acid as a carbon source. Detailed Implementation

[0035] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific implementation methods, features and effects of a testosterone-producing bacterium (Comamonas testosteroni) G314 and its application according to the present invention are described in detail below.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] Currently, based on the monomer configuration of the polymer, PHA can be classified into homopolymers or copolymers. Common homopolymers include poly(3-hydroxybutyrate) (P(3HB), abbreviated as PHB in this article) and poly(4-hydroxybutyrate) (P(4HB)), while copolymers include poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV), abbreviated as PHBV in this article). The physical and chemical properties of PHA depend on the chemical and structural composition of the monomer units. Compared with homopolymers, copolymers have significantly improved mechanical properties. With the increase of monomer content in copolymers, the material has a lower Young's modulus, higher elongation at break, and better mechanical properties. The carbon source, as the most important culture parameter in the PHA synthesis process, greatly affects the yield and type of PHA synthesized by microorganisms.

[0038] Therefore, this specific embodiment screened a microorganism capable of synthesizing PHA under anoxic / anaerobic conditions. The ability to synthesize PHA varies depending on the organic acid carbon source. By regulating the composition of volatile fatty acids, the type of PHA can be controlled, thereby improving the performance of PHA. This not only provides a new strain for efficient PHA production but also significantly reduces the production cost of PHA, especially the high cost of aeration during large-scale production.

[0039] Example 1: Screening and identification of Commonas testosteroni G314

[0040] The *Comamonas testosteroni* G314 provided in this embodiment is deposited at the China General Biotechnology Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China; the accession number is CGMCC No. 32129, and the deposit date is September 30, 2024.

[0041] The culture medium used includes:

[0042] Luria-Bertani (LB) medium: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L NaCl. Take 20 g and add ultrapure water to 1 L. Autoclave at 121°C for 20 min before use. Alternatively, 2% agar powder can be added to prepare LB solid medium.

[0043] Minimal medium (MM): KH2PO4 (1.5 g / L); NH4Cl (0.2 g / L); MgSO4 (0.1 g / L); Na2HPO4 (3.5 g / L); CaCl2·2H2O (0.01 g / L); ferric ammonium citrate (0.06 g / L); 1.0 mL / L trace elements (MnCl2·4H2O (0.03 g / L); ZnSO4·H2O (0.1 g / L); H3BO3 (0.3 g / L); CoCl2·6H2O (0.2 g / L); CuSO4·5H2O (0.01 g / L); NaMoO4·2H2O (0.03 g / L); NiCl2·6H2O (0.02 g / L)), and 4 g / L sodium acetate.

[0044] The pH of the culture medium was adjusted to 7.0 with 2M NaOH. Calcium salts and trace elements were added after being filtered with a syringe (0.22μm) before inoculation. The remaining components were sterilized in an autoclave at 121℃ for 20 min before use.

[0045] The separation and screening process specifically includes the following steps:

[0046] (1) This strain was screened from sludge from a wastewater treatment plant in Shenzhen. 10g of sludge sample from the wastewater treatment plant in Shenzhen was placed in a 250mL Erlenmeyer flask, 90mL of sterile water was added, and the mixture was shaken for 30min at 30℃ and 150rpm to obtain a suspension. Then, a serial dilution was performed, selecting 10... -2 ~10 -6 Dilutes were spread onto LB agar plates (with 50 μg / mL Nile Blue added), with three plates for each dilution. Each plate contained 100 μL of diluted sample suspension. The plates were sealed, labeled, and incubated at 30°C for 48 h.

[0047] Single fluorescent colonies were picked from different plates for isolation and purification. After repeated streaking, pure bacteria were obtained.

[0048] The obtained single colonies were inoculated into MM medium for rescreening and cultured in a shaker at 30℃ and 150rpm for 72h. Finally, the strain with the highest PHA accumulation was screened to obtain Commonas testosteroni G314, which was stored in 25% glycerol and placed in a refrigerator at -20℃.

[0049] Identification of 16S rDNA in the strain: The isolated strain G314 was classified and identified using the 16S rRNA gene sequencing method. The obtained 16S rRNA gene sequence was compared with sequences in GenBank. The results showed that strain G314 had 100% homology with *Comamonas testosteroni* GCF_014076415.1 and 99.8% similarity with *Comamonas phosphati*. Therefore, this strain was identified as a species of *Comamonas*, specifically *Comamonas testosteroni*. The phylogenetic tree of *Comamonas testosteroni* G314 is shown below. Figure 1 .

[0050] Example 2: Determination of the ability of *Comamonas testosteroni* G314 to produce PHA under different oxygen conditions

[0051] Production of PHA: The strain Commonas testosteroni G314 obtained in Example 1 was streaked again onto LB solid medium and cultured in a 30°C incubator for 24 hours to obtain the activated strain;

[0052] A single colony was hooked from an LB plate and placed into LB liquid medium that had been sterilized at high temperature. The culture was incubated at 25°C and 180 rpm for 18 hours to obtain a seed culture. The seed culture was then inoculated into a 500 mL Erlenmeyer flask containing 200 mL of sterilized PHA-producing liquid medium at a 10% inoculation rate. The flask was then incubated at 25°C and 180 rpm for 72 hours.

[0053] PHA extraction: The fermentation broth cultured above was transferred to a 50 mL centrifuge tube and centrifuged at 7800 rpm for 15 min. The supernatant was removed. The cell pellet was pre-frozen at -80℃ for 12 h and then freeze-dried to obtain the freeze-dried sample of the strain. 20 mg of the freeze-dried sample of the strain was weighed and placed in a 10 mL esterification tube. In a fume hood, 2 mL of chloroform and 2 mL of acidified methanol solution containing 0.2% benzoic acid were added to the esterification tube. After mixing well, the mixture was placed in an oven at 105℃ for 6 h for digestion.

[0054] After the reaction was completed, the mixture was cooled to room temperature. Then, 1 mL of deionized water was added to the esterification tube, and the mixture was thoroughly mixed for 30 seconds. After standing and separating the layers, the lower organic phase was transferred to 0.75 g of anhydrous sodium sulfate for drying and then transferred to a gas chromatograph analysis bottle.

[0055] PHA determination: The above samples were analyzed using an Agilent 7890AGC-FID system (Agilent Technologies) employing a DB-5 column (30 m long, 0.25 mm inner diameter, 0.25 μm film thickness). The injection port temperature was set to 200 °C, the split ratio to 25:1, and the injection volume to 1 μL. The temperature program was set to 40 °C for 3 min, then increased to 140 °C at a rate of 10 °C / min, held for 2 min, and then increased to 200 °C at a rate of 20 °C / min. Hydrogen was used as the carrier gas at a flow rate of 40 mL / min, and air was supplied at a flow rate of 450 mL / min. Purchased PHB and PHBV (Sigma-Aldrich) were used as external standards.

[0056] PHA content (%) = PHA concentration (g / L) / CDW (g / L) × 100%,

[0057] CDW represents the dry weight of the strain's cells.

[0058] See results Figure 2 Although strain G314 grew slowly under anaerobic conditions for the first 24 hours, it maintained good growth regardless of the oxygen content after 24 hours. After 48 hours of culture, the PHB accumulation of the strain under hypoxic and anaerobic conditions was consistently higher than that under aerobic conditions. This indicates that anaerobic conditions not only do not inhibit the growth and reproduction of strain G314, but also promote PHB synthesis.

[0059] Example 3: Determination of the ability of *Comamonas testosteroni* G314 to produce PHA using volatile fatty acids

[0060] After inoculating strain Commonas testosteroni G314 into LB medium and culturing for 18 h, a seed culture was obtained. This seed culture was then inoculated at a rate of 10% into 500 mL Erlenmeyer flasks containing 200 mL of fermentation medium and cultured anaerobically on a shaker at 25 °C and 180 rpm for 72 h. The fermentation medium consisted of volatile fatty acids (3.3 g / L), KH₂PO₄ (0.08 g / L), NH₄Cl (0.15 g / L), MgSO₄ (0.1 g / L), CaCl₂·2H₂O (0.01 g / L), FeCl₃·6H₂O (1.5 mg / L), and 1.0 mL / L of trace elements.

[0061] After fermentation began, the carbon source was replenished every 24 hours. The extraction and determination methods for PHA were the same as in Example 2, and the results of PHA accumulation are shown below. Figure 3 , 4 5.

[0062] The results showed that when strain G314 used sodium propionate as the carbon source, i.e., when the volatile fatty acid selection was sodium propionate, such as Figure 3 Cell dry weight and PHBV production gradually increased over time, reaching their maximum values ​​at 48 hours, at 1.14 g / L and 0.55 g / L, respectively. At this point, PHBV accumulation reached 48.3% of cell dry weight, with HV monomers accounting for 14.3%.

[0063] When strain G314 uses butyric acid as its carbon source, i.e., selects butyric acid as its volatile fatty acid, such as Figure 4 Cell dry weight and PHB production initially increased and then gradually decreased over time, reaching their maximum values ​​of 1.23 g / L and 0.81 g / L, respectively, at 24 hours. At this point, the accumulated PHB reached 58.2% of the cell dry weight.

[0064] When strain G314 uses valeric acid as its carbon source, i.e., selects valeric acid as its volatile fatty acid, such as Figure 5 Cell dry weight and PHBV production initially increased and then decreased slightly over time, reaching their maximum values ​​at 48 hours, at 1.60 g / L and 1.20 g / L, respectively. At this point, PHBV accumulation also reached its maximum, accounting for 74.3% of cell dry weight, with HV monomers accounting for as high as 91.1%.

[0065] As can be seen, when acetic acid and butyric acid are used as carbon sources, the strain mainly synthesizes HB monomers, and the content of synthesized HV monomers is very small and almost negligible. However, when propionic acid or valeric acid is used as carbon sources, the content of HV monomers synthesized by the strain increases significantly. In particular, when valeric acid is used as a carbon source, it can significantly promote the synthesis of HV monomers. This indicates that valeric acid is more suitable than propionic acid for regulating the proportion of HV monomers in PHBV.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been shown above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A type of *Trichomonas testis* ( Comamonas testosteroni G314, characterized in that, The Comamonas testosteroni is preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanmingyuan Road, Beichen West Road, Chaoyang District, Beijing, China, and the preservation number is CGMCC No. 32129. The preservation date is September 30, 2024.

2. Use of a P. testudinis (P. testudini) strain G314 according to claim 1, characterized in that, Comamonas testosteroni ) G314, characterized in that, The Comamonas testosteroni of claim 1 for use in the synthesis of polyhydroxyalkanoates under anoxic or anaerobic conditions, said polyhydroxyalkanoates being poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate- co -3-hydroxyvalerate).

3. Use of a P. testudinis (P. testudini) strain G314 according to claim 2, characterized in that, Comamonas testosteroni ) G314, characterized in that, The Comamonas testosteroni is used for synthesizing polyhydroxyalkanoate under anoxic or anaerobic condition, comprising the following steps: The Comamonas testosteroni is inoculated into LB medium for culture to obtain a seed liquid; After inoculating 1%~10% (v / v) of the seed liquid into a fermentation medium, fermentation culture is carried out on a constant-temperature shaker to obtain a fermentation broth; After centrifugation of the fermentation broth, the Comamonas testosteroni is obtained, and after freeze-drying, the dry Comamonas testosteroni is obtained; The dry Comamonas testosteroni is mixed with chloroform and acidified methanol containing 0.2% benzoic acid, and reacted at 100~105℃ for 4~6 hours; Distilled water is added, and the chloroform layer is transferred to anhydrous sodium sulfate for drying to obtain polyhydroxyalkanoate.

4. Use of a P. testudinis (P. testudini) strain according to claim 3, characterized in that, Comamonas testosteroni ) G314, characterized in that, The initial pH value of the fermentation culture is 5~9, the fermentation culture temperature is 25~40℃, the rotation speed of the constant-temperature shaker is 100~200 rpm, and the seed liquid culture time is 10~24 h.

5. A Comamonas testosteroni (C. testosteroni) according to claim 3, wherein the C. testosteroni is a C. testosteroni strain of the group G314, characterized in that, Comamonas testosteroni ) G314 application, characterized in that, When the Comamonas testosteroni is used for synthesizing poly(3-hydroxybutyrate) under anoxic or anaerobic condition, the carbon source in the fermentation medium is any one or more of sodium acetate, sodium propionate, butyric acid or valeric acid.

6. A Comamonas testosteroni (C. testosteroni) according to claim 3, wherein the C. testosteroni is a C. testosteroni strain of the group G314. Comamonas testosteroni ) G314 application characterized by, When the M. testaceum is used to synthesize poly(3-hydroxybutyrate- co -3-hydroxyvalerate) under anoxic or anaerobic conditions, the carbon source in the fermentation medium is any one or more of sodium propionate or valeric acid.

7. Use of a Comamonas testosteroni (C. testosteroni) strain G314 according to claim 5 or 6, characterized in that, Comamonas testosteroni ) G314, characterized in that, The composition of the fermentation medium includes: carbon source 2~7g / L, KH2PO40.08~0.17g / L, NH4Cl 0.15~0.56g / L, MgSO40.08~0.12g / L, 0.008~0.012g / L, 1.2~1.8mg / L and trace elements 0.8~1.2mL / L.

8. Use of a P. testudinis (P. testudini) strain G314 according to claim 7, characterized by, Comamonas testosteroni Comamonas testosteroni ) G314, characterized by, The microelements include: 0.008~0.012g / L, 0.08~0.12 g / L, 0.025~0.035g / L, H3BO30.25~0.35g / L, 0.16~0.24g / L, 0.02~0.024g / L and 0.025~0.035g / L.

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