Method for promoting active carbon to efficiently ferment phas to produce phas by using ethanol by talaromyces stipitatus

Abstract

The application provides a method for promoting Talaromyces cucurbitiradicus to utilize ethanol to produce PHA by activated carbon, and belongs to the technical field of bio-chemical industry.The method comprises the following steps: adding ethanol, a nitrogen source and buffer salt into water to prepare a fermentation substrate, then inoculating Talaromyces cucurbitiradicus into the fermentation substrate, adding activated carbon and uniformly dispersing, fermenting the obtained fermentation liquid, filtering, treating the obtained fermentation liquid with sodium hypochlorite solution, filtering, dispersing the obtained wet filter cake with chloroform, then adding methanol or ethanol to precipitate PHA, filtering, and obtaining PHA solid.The method can significantly strengthen the metabolic ability and PHA accumulation ability of Talaromyces cucurbitiradicus by adjusting the adding amount of activated carbon and setting a nutrition limiting condition, so that the high-efficiency utilization of ethanol carbon source and the high-level accumulation of PHA are realized.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a method for producing PHA by promoting the efficient fermentation of *Talaromyces cucurbitiradicus* using ethanol with activated carbon. Specifically, it relates to a method for enhancing the efficient fermentation of *Talaromyces cucurbitiradicus* using ethanol with activated carbon to produce PHA. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers synthesized by microorganisms. They possess excellent biocompatibility and biodegradability, and can be widely used in packaging materials, biomedical materials, and environmental protection materials. Current PHA production methods primarily use sugars, vegetable oils, or fatty acids as carbon sources, which suffers from high raw material costs, limited substrate sources, and unstable fermentation efficiency, thus restricting the large-scale, low-cost production of PHA.

[0003] *Talaromyces cucurbitiradicus* is a fungus belonging to the genus *Talaromyces*. It was isolated and officially published as a new species in 2018 during a survey of endophytic fungi in Cucurbitaceae plants in China (the orthotype specimen at the Mycological Specimen Museum of the Institute of Microbiology, Chinese Academy of Sciences is HMAS 247175; the MycoBank accession number for this strain is MB 820559). Its carbon sources for survival include ethanol and glucose. Studies have shown that this strain can synthesize PHA using ethanol and glucose as carbon sources, especially when using ethanol as the carbon source, where the PHA content can reach as high as 510 mg PHA / g dry bacteria. However, the PHA synthesis rate is slow, and the fermentation time is relatively long. Summary of the Invention

[0004] Studies have shown that activated carbon can significantly enhance the metabolic process of *Talaromyces cucurbitiradicus* using ethanol as a carbon source, increasing the synthesis rate and accumulation level of PHA, thereby achieving efficient fermentation production of PHA. Therefore, this invention provides a method for using activated carbon to enhance the efficient fermentation of *Talaromyces cucurbitiradicus* to produce PHA using ethanol. By setting nutrient limiting conditions and introducing activated carbon enhancement, efficient utilization of the ethanol carbon source and high-level accumulation of PHA are achieved.

[0005] To address the issue of low yield and accumulation levels in the fermentation of PHA using ethanol as a substrate, the technical solution adopted in this invention is as follows:

[0006] A method for promoting efficient fermentation of talaromyces cucurbitiradicus using ethanol to produce PHA using activated carbon is disclosed. The method involves adding ethanol, a nitrogen source, and a buffer salt to water to prepare a fermentation substrate. Then, talaromyces cucurbitiradicus is inoculated into the fermentation substrate. Activated carbon is then added to the resulting system and dispersed evenly. The resulting fermentation broth is fermented, filtered, and the resulting wet bacterial cells are treated with sodium hypochlorite aqueous solution and filtered. The resulting wet filter cake is dispersed in chloroform, and then methanol or ethanol is added to precipitate PHA. After filtration, solid PHA is obtained.

[0007] Furthermore, the method includes the following specific steps:

[0008] S1. Take ethanol, nitrogen source and buffer salt and add them to water to prepare fermentation substrate;

[0009] S2. Inoculate the fermentation substrate with 30% of the cucurbitiradicus rhizobium, mix well, and obtain the system.

[0010] S3. Add activated carbon to the system to disperse it fully and obtain fermentation broth;

[0011] S4. Ferment the fermentation broth, and filter it after fermentation is terminated to obtain wet bacterial cells;

[0012] S5. Add the wet bacterial cells to a 0.5% sodium hypochlorite aqueous solution, stir, filter, and obtain a wet filter cake;

[0013] S6. Add the wet filter cake to chloroform, heat to 60°C, stir, filter, add methanol or ethanol to the filtrate to precipitate PHA, filter, and obtain solid PHA.

[0014] Furthermore, inorganic salts and trace elements are added to the fermentation substrate.

[0015] Furthermore, the inorganic salt includes at least one of phosphate, sulfate and chloride, preferably including KH2PO4, K2HPO4, MgSO4·H2O, NaCl or KCl;

[0016] The phosphate is a phosphate buffer system composed of KH2PO4 and K2HPO4.

[0017] Furthermore, the initial concentration of ethanol in the fermentation substrate is 5~80 g / L, preferably 10~50 g / L.

[0018] Furthermore, the inoculum amount of the pumpkin rhizobacterium Talaromyces cucurbitiradicus in the fermentation substrate is 1-30% of the fermentation substrate volume, preferably 5-20%.

[0019] Furthermore, the activated carbon is coal-based activated carbon made from coal-based raw materials, or biomass activated carbon made from biomass such as wood and fruit shells through activation.

[0020] Furthermore, the amount of activated carbon added in the system is 0.01~10 g / L, preferably 0.1~6 g / L.

[0021] Furthermore, the fermentation temperature is 10~45℃, preferably 20~35℃;

[0022] The fermentation time is 3 to 20 days, preferably 5 to 10 days;

[0023] The initial pH value of the fermentation is 4.0~6.5.

[0024] Furthermore, the carbon-to-nitrogen ratio in the fermentation substrate is 5-18:1;

[0025] The volume of chloroform added is 10-50 mL / g PHA, preferably 20-40 mL / g PHA, based on the theoretical PHA yield.

[0026] The volume of methanol or ethanol added is 1 to 5 times the volume of chloroform added, preferably 2 to 4 times.

[0027] The beneficial effects of the activated carbon method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA according to the present invention are as follows:

[0028] This invention significantly enhances the metabolic and PHA accumulation capabilities of Talaromyces cucurbitiradicus, a rhizobium of pumpkin, by adjusting the amount of activated carbon added and setting nutrient limiting conditions, thereby achieving efficient utilization of ethanol as a carbon source and high-level accumulation of PHA.

[0029] The pumpkin rhizobacterium Talaromyces cucurbitiradicus used in this invention can synthesize and accumulate PHA in the cell using ethanol as a carbon source, thereby realizing the bioconversion and resource utilization of ethanol substrate.

[0030] This invention achieves stable accumulation of PHA under relatively mild fermentation conditions by setting nutrient restrictions and controlling ethanol concentration. The process is clear, easy to operate, and has a wide range of raw material sources, making it suitable for large-scale implementation.

[0031] The present invention, as you just mentioned, can enhance the metabolic process and PHA accumulation capacity of Talaromyces cucurbitiradicus by adding activated carbon, thereby increasing the conversion efficiency of ethanol to PHA and thus facilitating the acquisition of higher PHA yield and accumulation levels; at the same time, the activated carbon raw materials are widely available and easy to obtain, which is convenient for industrial application. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Specific information about the pumpkin rhizobacterium Talaromyces cucurbitiradicus in this invention is published at https: / / nmdc.cn / fungarium / fungi / chinastrain / details?id=5f62fd6f362c262b48ca381d.

[0034] Example 1: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using activated carbon.

[0035] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0036] S1. Add ethanol as a carbon source to 200 mL of water to adjust the initial concentration of ethanol to 5 g / L. Then add ammonium sulfate to adjust the carbon-nitrogen ratio to 5:1. Add a buffer salt consisting of 0.094 g KH2PO4 and 0.694 g K2HPO4 to obtain the fermentation substrate.

[0037] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 30% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation vessel.

[0038] S3. Add coal-derived activated carbon to the system obtained in step S2 at a concentration of 10.0 g / L, stir to ensure full dispersion, and obtain fermentation broth;

[0039] S4. Ferment the fermentation broth obtained in step S3 at 45℃ and with an initial pH of 4.0 for 3 days. Terminate the fermentation when the PHA content in the cells of the fermentation broth reaches 779.6 mg / g dry bacteria. Filter to obtain 5 mL of wet cells (water content 92%).

[0040] S5. Add the wet bacterial cells obtained in step S4 to 100 mL of a 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain a wet filter cake.

[0041] S6. Add the wet filter cake obtained in step S5 to 5 mL of chloroform, heat to 60°C, stir for 1 hour, filter, add 5 mL of methanol to the filtrate to precipitate PHA, filter, and obtain 0.26 g of PHA solid (theoretical PHA yield is 0.31 g), with ethanol utilization rate of 99.6% and ethanol-PHA conversion rate of 31.1%.

[0042] The ethanol utilization rate (%) is calculated as follows: (Initial ethanol amount - Residual ethanol amount) / Initial ethanol amount × 100% = (1g - 0.004g) × 100% = 99.6%.

[0043] Ethanol-PHA conversion rate (%) = Theoretical PHA yield / Ethanol consumption × 100% = 0.31g / 0.996g = 31.1%

[0044] Example 2: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using ethanol with activated carbon

[0045] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0046] S1. Add ethanol as a carbon source to 1000 mL of water to adjust the initial ethanol concentration to 80 g / L. Then add ammonium chloride as a nitrogen source to adjust the carbon-nitrogen ratio to 8:1. Next, add a buffer salt consisting of 0.47 g KH2PO4 and 3.47 g K2HPO4. Then add 0.5 g MgSO4·H2O and 0.24 g NaCl as inorganic salts. Finally, add 1 mL of trace element solution (containing 10.0 g / L FeSO4·7H2O, 2.0 g / L MnSO4·H2O, 2.0 g / L ZnSO4·7H2O, 0.5 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, and 0.5 g / L Na2MoO4·2H2O) to obtain the fermentation substrate.

[0047] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 1% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation container.

[0048] S3. Add 0.01 g / L of woody biomass activated carbon to the system obtained in step S2, stir to disperse it fully, and obtain fermentation broth;

[0049] S4. Ferment the fermentation broth obtained in step S3 at 10℃ and initial pH 6.0 for 20 days. Terminate the fermentation when the PHA content in the cells of the fermentation broth reaches 881.6 mg / g dry bacteria. Filter to obtain 77 mL of wet cells (water content 92%).

[0050] S5. Add the wet bacterial cells obtained in step S4 to 100 mL of a 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain a wet filter cake.

[0051] S6. Add the wet filter cake obtained in step S5 to 154 mL of chloroform, heat to 60°C, stir for 1 hour, filter, add 308 mL of ethanol to the filtrate to precipitate PHA, filter, and obtain 4.62 g of PHA solid (theoretical PHA yield is 5.43 g), with an ethanol utilization rate of 12.35% and an ethanol-PHA conversion rate of 55.0%.

[0052] Example 3: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using ethanol with activated carbon

[0053] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0054] S1. Add ethanol as a carbon source to 500 mL of water to adjust the initial ethanol concentration to 10 g / L. Then add ammonium sulfate as a nitrogen source to adjust the carbon-nitrogen ratio to 10:1. Add a buffer salt consisting of 0.235 g KH2PO4 and 1.735 g K2HPO4, then add 0.01 g KCl as an inorganic salt. Finally, add 0.5 mL of trace element solution (containing 10.0 g / L FeSO4·7H2O, 2.0 g / L MnSO4·H2O, 2.0 g / L ZnSO4·7H2O, 0.5 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, and 0.5 g / L Na2MoO4·2H2O) to obtain the fermentation substrate.

[0055] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 5% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation vessel.

[0056] S3. Add coal-derived activated carbon at a concentration of 0.1 g / L to the system obtained in step S2, stir to disperse it fully, and obtain the fermentation broth;

[0057] S4. Ferment the fermentation broth obtained in step S3 at 20℃ and with an initial pH of 5.5 for 5 days. Terminate the fermentation when the PHA content in the cells of the fermentation broth reaches 782.4 mg / g dry bacteria. Filter to obtain 16 mL of wet cells (92% water content).

[0058] S5. Add the wet bacterial cells obtained in step S4 to 100 mL of a 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain a wet filter cake.

[0059] S6. Add the wet filter cake obtained in step S5 to 48 mL of chloroform, heat to 60 °C, stir for 1 hour, filter, add 144 mL of methanol to the filtrate to precipitate PHA, filter, and obtain 0.85 g of PHA solid (theoretical PHA yield is 1 g), with ethanol utilization rate of 61.2% and ethanol-PHA conversion rate of 32.6%.

[0060] Example 4: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using ethanol with activated carbon

[0061] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0062] S1. Add ethanol as a carbon source to 1000 mL of water to adjust the initial ethanol concentration to 50 g / L. Then add a mixture of 4.1 g ammonium sulfate and 3.32 g ammonium chloride as a nitrogen source to adjust the carbon-nitrogen ratio to 15:1. Add a buffer salt composed of 0.47 g KH2PO4 and 3.47 g K2HPO4, then add 0.5 g MgSO4·H2O and 0.24 g NaCl as inorganic salts. Finally, add 1 mL of trace element solution to obtain the fermentation substrate (the trace element solution contains 10.0 g / L FeSO4·7H2O, 2.0 g / L MnSO4·H2O, 2.0 g / L ZnSO4·7H2O, 0.5 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, and 0.5 g / L Na2MoO4·2H2O).

[0063] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 20% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation vessel.

[0064] S3. Add coconut shell activated carbon to the system obtained in step S2 at a ratio of 6.0 g / L, stir to disperse it fully, and obtain the fermentation broth;

[0065] S4. Ferment the fermentation broth obtained in step S3 at 35℃ and with an initial pH of 4.5 for 10 days. Terminate the fermentation when the PHA content in the cells of the fermentation broth reaches 864.2 mg / g dry bacteria. Filter to obtain 120 mL of wet cells (92% water content).

[0066] S5. Add the wet bacterial cells obtained in step S4 to 200 mL of 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain wet filter cake.

[0067] S6: Add the wet filter cake obtained in step S5 to 300 mL of chloroform, heat to 60 °C, stir for 1 hour, filter, add 600 mL of ethanol to the filtrate to precipitate PHA, filter, and obtain 7.03 g of PHA solid (theoretical PHA yield is 8.3 g), with an ethanol utilization rate of 36.4% and an ethanol-PHA conversion rate of 45.4%.

[0068] Example 5: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using ethanol with activated carbon.

[0069] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0070] S1. Add ethanol as a carbon source to 500 mL of water to adjust the initial ethanol concentration to 30 g / L. Then add ammonium sulfate as a nitrogen source to adjust the carbon-nitrogen ratio to 12:1. Add a buffer salt consisting of 0.235 g KH2PO4 and 1.735 g K2HPO4, then add 0.25 g MgSO4·H2O and 0.1 g KCl as inorganic salts. Finally, add 1 mL of trace element solution (containing 10.0 g / L FeSO4·7H2O, 2.0 g / L MnSO4·H2O, 2.0 g / L ZnSO4·7H2O, 0.5 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, and 0.5 g / L Na2MoO4·2H2O) to obtain the fermentation substrate.

[0071] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 15% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation vessel.

[0072] S3. Add coal-derived activated carbon at a concentration of 1.0 g / L to the system obtained in step S2, stir to fully disperse it, and obtain the fermentation broth;

[0073] S4. Ferment the fermentation broth obtained in step S3 at 28℃ and with an initial pH of 5.0 for 7 days. Fermentation is terminated when the PHA content in the cells of the fermentation broth reaches 824.2 mg / g dry bacteria. The broth is then filtered to obtain 43 mL of wet cells (92% water content).

[0074] S5. Add the wet bacterial cells obtained in step S4 to 100 mL of a 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain a wet filter cake.

[0075] S6. Add the wet filter cake obtained in step S5 to 86 mL of chloroform, heat to 60 °C, stir for 1 hour, filter, add 430 mL of methanol to the filtrate to precipitate PHA, filter, and obtain 2.41 g of solid PHA (theoretical PHA yield is 2.84 g), with an ethanol utilization rate of 48.5% and an ethanol-PHA conversion rate of 39.0%.

[0076] Example 6: A method for promoting efficient PHA production by rhizobia fermentation of pumpkin using ethanol with activated carbon

[0077] This embodiment describes a method for using activated carbon to promote efficient fermentation of pumpkin rhizobia using ethanol to produce PHA. The specific preparation process includes the following steps:

[0078] S1. Add ethanol as a carbon source to 1000 mL of aqueous solution and adjust the initial ethanol concentration to 2 g / L. Then add ammonium sulfate as a nitrogen source and adjust the carbon-nitrogen ratio to 18:1. Add a buffer salt consisting of 0.47 g KH2PO4 and 3.47 g K2HPO4, then add 0.5 g MgSO4·H2O, 0.12 g NaCl, and 0.05 g KCl as inorganic salts. Finally, add 1 mL of trace element solution (containing 10.0 g / L FeSO4·7H2O, 2.0 g / L MnSO4·H2O, 2.0 g / L ZnSO4·7H2O, 0.5 g / L CuSO4·5H2O, 0.5 g / L CoCl2·6H2O, and 0.5 g / L Na2MoO4·2H2O) to obtain the fermentation substrate.

[0079] S2. Inoculate the fermentation substrate obtained in step S1 with Talaromyces cucurbitiradicus at an inoculation rate of 8% of the fermentation substrate volume, mix well, and then transfer the resulting system into a fermentation container.

[0080] S3. Add biomass activated carbon to the system obtained in step S2 at a rate of 0.5 g / L, stir to fully disperse it, and obtain fermentation broth;

[0081] S4. Ferment the fermentation broth obtained in step S3 at 15℃ and with an initial pH of 6.5 for 3 days. Terminate the fermentation when the PHA content in the cells of the fermentation broth reaches 797.5 mg / g dry bacteria. Filter to obtain 10 mL of wet cells (92% water content).

[0082] S5. Add the wet bacterial cells obtained in step S4 to 100 mL of 0.5% sodium hypochlorite aqueous solution, stir for 30 minutes, filter, and obtain wet filter cake.

[0083] S6. Add the wet filter cake obtained in step S5 to 25 mL of chloroform, heat to 60 °C, stir for 1 hour, filter, add 60 mL of ethanol to the filtrate to precipitate PHA, filter, and obtain 0.52 g of solid PHA (theoretical PHA yield is 0.64 g), with an ethanol utilization rate of 99.9% and an ethanol-PHA conversion rate of 31.9%.

[0084] In summary, the method for producing PHA by efficient fermentation of *Talaromyces cucurbitiradicus* using ethanol with activated carbon enhanced by this invention has the following advantages:

[0085] (1) Talaromyces cucurbitiradicus, a fungus that grows in pumpkins, can synthesize and accumulate PHA in the fungus using ethanol as a carbon source, thereby realizing the biotransformation and resource utilization of ethanol substrates.

[0086] (2) By setting nutritional restrictions and controlling ethanol concentration, this invention can achieve stable accumulation of PHA under relatively mild fermentation conditions. The process is clear, easy to operate, and has a wide range of raw material sources, making it suitable for large-scale implementation.

[0087] (3) The addition of activated carbon can enhance the metabolic process and PHA accumulation capacity of Talaromyces cucurbitiradicus, improve the conversion efficiency of ethanol to PHA, and thus help to obtain higher PHA yield and accumulation level; at the same time, the source of activated carbon raw materials is wide and easy to obtain, which is convenient for industrial application.

[0088] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for promoting efficient fermentation of PHA by *Rhizoctonia solani* using ethanol with activated carbon, characterized in that, The method involves adding ethanol, a nitrogen source, and a buffer salt to water to prepare a fermentation substrate. Then, *Talaromyces cucurbitiradicus* is inoculated into the fermentation substrate. Activated carbon is added to the resulting system and dispersed evenly. The resulting fermentation broth is fermented, filtered, and the resulting wet bacterial cells are treated with sodium hypochlorite aqueous solution and filtered. The resulting wet filter cake is dispersed in chloroform, and then methanol or ethanol is added to precipitate PHA. After filtration, solid PHA is obtained.

2. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, characterized in that, The method includes the following specific steps: S1. Take ethanol, nitrogen source and buffer salt and add them to water to prepare fermentation substrate; S2. Inoculate the fermentation substrate with 30% of the cucurbitiradicus rhizobium, mix well, and obtain the system. S3. Add activated carbon to the system to disperse it fully and obtain fermentation broth; S4. Ferment the fermentation broth, and filter it after fermentation is terminated to obtain wet bacterial cells; S5. Add the wet bacterial cells to a sodium hypochlorite aqueous solution, stir, filter, and obtain a wet filter cake; S6. Add the wet filter cake to chloroform, heat, stir, filter, add methanol or ethanol to the filtrate to precipitate PHA, filter, and obtain solid PHA.

3. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1 or 2, characterized in that, Inorganic salts and trace elements are also added to the fermentation substrate.

4. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 3, characterized in that, The inorganic salt includes at least one of phosphate, sulfate and chloride; The phosphate is a phosphate buffer system composed of KH2PO4 and K2HPO4.

5. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The initial concentration of ethanol in the fermentation substrate is 5~80 g / L.

6. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The inoculum size of *Talaromyces cucurbitiradicus* in the fermentation substrate is 1-30% of the fermentation substrate volume.

7. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The activated carbon is coal-based activated carbon made from coal-based raw materials, or biomass activated carbon made from biomass such as wood and fruit shells through activation.

8. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The amount of activated carbon added in the system is 0.01~10 g / L.

9. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The fermentation temperature is 10~45℃, the time is 3~20 days, and the initial pH value is 4.0~6.

5.

10. The method for promoting efficient fermentation of pumpkin rhizobia using ethanol to produce PHA using activated carbon according to claim 1, 2 or 4, characterized in that, The carbon-to-nitrogen ratio in the fermentation substrate is 5-18:1; The volume of chloroform added is 10-50 mL / g PHA, which is the theoretical PHA yield. The volume of methanol or ethanol added is 1 to 5 times the volume of chloroform added.