Preparation method of novel microbial carbon source

Abstract

The invention relates to a preparation method of a microbial carbon source. The method comprises the following steps: firstly, efficiently degrading various plastic wastes including polylactic acid plastic, polyethylene glycol terephthalate plastic and polyurethane plastic into micromolecular substances capable of being utilized by microorganisms in a manner of combining strong acid treatment with a photocatalytic degradation technology; the process not only solves the problem of environmental pollution caused by plastic wastes, but also provides a novel carbon source with low cost and wide source for microbial culture. The microbial carbon source can be used for culturing various microorganisms such as escherichia coli, bacillus subtilis, pseudomonas putida, aspergillus niger, rhizopus oryzae and saccharomyces cerevisiae, and has wide application prospect and economic value. The method has important significance in the fields of environmental protection, resource recycling and biotechnology.

Description

Technical Field

[0001] The invention relates to the technical field of microbial cultivation, and in particular to a method for preparing a novel microbial carbon source. Background Art

[0002] In today's global environmental protection context, the treatment of plastic waste has become an urgent problem to be solved. With the advancement of science and technology and the enhancement of people's environmental awareness, the use of microbial technology to treat plastic waste has gradually become one of the research hotspots. Traditionally, plastics have caused long-term pollution to the environment due to their stable chemical structure and difficulty in natural degradation. However, by degrading plastics into small molecules and allowing microorganisms to use them as carbon sources, this provides a new way to treat plastic waste.

[0003] Although the technology of degrading plastics into small molecules and using them as microbial carbon sources has shown great potential, it still faces some challenges in practical application. There are many types of plastics, and different types of plastics require different microorganisms or enzymes for degradation, which increases the complexity of technical implementation; harmful byproducts may be produced during the degradation process, which need to be strictly controlled to avoid secondary pollution. Therefore, how to improve the degradation efficiency, expand the scope of application, and ensure environmental safety are key issues that must be solved before the technology can be promoted and applied. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preparing a novel microbial carbon source, wherein the method for preparing the carbon source is as follows:

[0005] S1: Weigh the plastic and grind it into powder, pass it through a 50-mesh sieve, add 0.5-0.7 mol / L strong acid, stir at 60°C for 5h, adjust the pH of the solution to between 6.5-7.5 with alkali, filter, and dry to obtain intermediate 1;

[0006] The weight proportions of plastic powder and 0.5-0.7 mol / L strong acid are:

[0007] 1 part plastic powder;

[0008] 0.5-0.7mol / L strong acid 6-7 parts;

[0009] S2: Add photocatalyst and water to intermediate 1 to make a suspension, stir for 12 hours under the conditions of ultrasonic oscillator and ultraviolet light, add non-polar solvent for separation after stirring, retain the organic phase, spin dry, add 3-5 times the weight of semi-polar solvent, reflux at 85°C for 4 hours, and filter to obtain intermediate 2;

[0010] The weight proportions of intermediate 1, photocatalyst and water are as follows:

[0011] Intermediate 1 1 part;

[0012] Photocatalyst 0.05-0.15 parts;

[0013] 8-10 parts water;

[0014] S3: Intermediate 2 is ground into powder and passed through a 75-mesh sieve

[0015] The microbial carbon source can be obtained through the above two steps.

[0016] According to the above technical features, the strong acid is selected from one or more of hydrochloric acid, nitric acid and sulfuric acid;

[0017] Preferred strong acids are one or more of nitric acid and sulfuric acid.

[0018] According to the above technical features, the base is selected from one or more of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, and potassium bicarbonate;

[0019] Preferred alkalis are one or more of sodium carbonate, sodium bicarbonate, and sodium hydroxide.

[0020] According to the above technical features, the photocatalyst is selected from: one or more of titanium dioxide, zinc oxide, and cadmium sulfide;

[0021] The amount of titanium dioxide (TiO2) is: 0.1-0.15 parts;

[0022] The dosage of zinc oxide (ZnO) is: 0.1-0.15 parts;

[0023] The dosage of cadmium sulfide (CdS) is: 0.05-0.1 parts;

[0024] The preferred photocatalyst is one or more of titanium dioxide and zinc oxide.

[0025] According to the above technical features, the semi-polar solvent is selected from: ethanol, methanol.

[0026] According to the above technical features, the non-polar solvent is selected from: one or more of tetrahydrofuran, dichloromethane, toluene, and ethyl acetate;

[0027] The preferred non-polar solvent is one or more of tetrahydrofuran, dichloromethane, and ethyl acetate.

[0028] According to the above technical features, the plastic is selected from: one or more of polylactic acid plastic, polyethylene terephthalate plastic, and polyurethane plastic.

[0029] According to the above technical features, it is characterized in that it can be used to replace the carbon source in the microbial culture medium.

[0030] According to the above technical features, a method for preparing a novel microbial culture medium is:

[0031] S1: Prepare the culture medium, the components and mass concentrations of which are:

[0032] 10-20g / L microbial carbon source;

[0033] 1-2g / L(NH4)2SO4;

[0034] 0.3-0.7 g / L KH2PO4;

[0035] 1.5-2.5 g / L K2HPO4·3H2O;

[0036] 0.3-0.7 g / L NaCl;

[0037] 0.1-0.3 g / L MgSO4·7H2O;

[0038] 1.5-2g / L agar powder;

[0039] The solvent was ultrapure water;

[0040] The pH of the culture medium is 6.5-7.5;

[0041] The pH of the culture medium is 6.5-7.5;

[0042] Agar is a solid culture medium, while agar-free culture medium is a liquid culture medium.

[0043] S2: 120℃ high pressure steam sterilization for 25min;

[0044] S3: cooling room room temperature;

[0045] Through the above three steps, a new microbial culture medium can be obtained;

[0046] The preferred components and mass concentrations of the culture medium are:

[0047]

[0048] The pH of the culture medium was 7.0.

[0049] According to the above technical characteristics, a novel microbial culture medium is suitable for culturing Escherichia coli, Bacillus subtilis, Pseudomonas putida, Aspergillus niger, Rhizopus oryzae, Saccharomyces cerevisiae, etc.;

[0050] The preferred culture medium is suitable for Escherichia coli, Aspergillus niger, and Saccharomyces cerevisiae.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1) This method can effectively use plastic waste as raw materials and convert it into microbial carbon sources. This method not only helps solve the problem of plastic pollution, but also finds new uses for plastic waste, which is in line with the global environmental protection trend;

[0053] 2) By recycling waste plastics to produce microbial carbon sources, the cost can be greatly reduced compared to traditional carbon source materials such as glucose. This provides a more economical option for microbial cultivation;

[0054] 3) The use of strong acid treatment combined with photocatalytic degradation can effectively improve the degradation efficiency of plastics and broaden the types of degradable plastics, including polylactic acid plastics, polyethylene terephthalate plastics, polyurethane plastics, etc., demonstrating technological innovation;

[0055] 4) The prepared microbial carbon source can replace the traditional carbon source for use in microbial culture medium, and is suitable for culturing a variety of microorganisms, including Escherichia coli, Bacillus subtilis, Pseudomonas putida, Aspergillus niger, Rhizopus oryzae, Saccharomyces cerevisiae, etc., and has wide applicability and potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is through mass spectrometry using polyethylene terephthalate plastic as a carbon source for microorganisms; DETAILED DESCRIPTION

[0057] In order to further illustrate the method adopted by the present invention and the effect achieved, the technical solution of the present invention will be clearly and completely described in combination with experiments below.

[0058] Preparation of medium 1

[0059] Liquid culture medium with polylactic acid plastic as microbial carbon source:

[0060] S1: Weigh polylactic acid plastic and grind it into powder, pass it through a 50-mesh sieve, weigh 100 g of the powder, add it to 700 mL of 0.5-0.7 mol / L sulfuric acid solution, stir at 60 ° C for 5 h, adjust the pH of the solution to between 6.5-7.5 with alkali, filter, and dry to obtain 185 g of the intermediate;

[0061] S2: Add 8 g of titanium dioxide and 800 mL of water to 85 g of intermediate 1 to prepare a suspension, stir for 12 h under an ultrasonic oscillator and ultraviolet light, add tetrahydrofuran for separation after stirring, retain the organic phase, spin dry, add 3-5 times the weight of ethanol, reflux at 85°C for 4 h, and filter to obtain 261 g of intermediate;

[0062] S3: Intermediate 2 is ground into powder and passed through a 75-mesh sieve

[0063] Through the above two steps, the microbial carbon source of polylactic acid plastic can be obtained;

[0064] S4: Prepare liquid culture medium with polylactic acid plastic as carbon source for microorganisms:

[0065]

[0066] The pH of the culture medium was 7.0.

[0067] S5: 120℃ high pressure steam sterilization for 25min;

[0068] S6: Cooling room temperature;

[0069] Through the above 6 steps, a liquid culture medium with polylactic acid plastic as a carbon source for microorganisms can be obtained.

[0070] Preparation of medium 2

[0071] Liquid culture medium with polyethylene terephthalate plastic as microbial carbon source:

[0072] S1: Weigh polyethylene terephthalate plastic and grind it into powder, pass it through a 50-mesh sieve, weigh 100 g of the powder, add it to 600 mL of 0.5-0.7 mol / L sulfuric acid solution, stir at 60 ° C for 5 h, adjust the pH of the solution to between 6.5-7.5 with alkali, filter, and dry to obtain 172 g of the intermediate;

[0073] S2: Add 7g of zinc oxide and 700mL of water to 72g of intermediate 1 to prepare a suspension, stir for 12h under the conditions of an ultrasonic oscillator and ultraviolet light, add tetrahydrofuran for separation after stirring, retain the organic phase, spin dry, add 3-5 times the weight of ethanol, reflux at 85°C for 4h, and filter to obtain 251g of intermediate;

[0074] S3: Intermediate 2 is ground into powder and passed through a 75-mesh sieve

[0075] Through the above two steps, the microbial carbon source of polyethylene terephthalate plastic can be obtained;

[0076] S4: Preparation of liquid culture medium with polyethylene terephthalate plastic as microbial carbon source:

[0077]

[0078] The pH of the culture medium was 7.0.

[0079] S5: 120℃ high pressure steam sterilization for 25min;

[0080] S6: Cooling room temperature;

[0081] Through the above 6 steps, a liquid culture medium with polyethylene terephthalate plastic as a microbial carbon source can be obtained.

[0082] Preparation of Medium 3:

[0083] Liquid culture medium with polyurethane plastic as microbial carbon source:

[0084] S1: Weigh the polyurethane plastic and grind it into powder, pass it through a 50-mesh sieve, weigh 100g of the powder, add it to 600mL of 0.5-0.7mol / L nitric acid solution, stir at 60℃ for 5h, adjust the pH of the solution to between 6.5-7.5 with alkali, filter, and dry to obtain 169g of the intermediate;

[0085] S2: Add 7 g of cadmium sulfide and 700 mL of water to 69 g of intermediate 1 to prepare a suspension, stir for 12 h under an ultrasonic oscillator and ultraviolet light, add tetrahydrofuran to separate the liquid after stirring, retain the organic phase, spin dry, add 3-5 times the weight of ethanol, reflux at 85°C for 4 h, and filter to obtain 248 g of the intermediate;

[0086] S3: Intermediate 2 is ground into powder and passed through a 75-mesh sieve;

[0087] The polyurethane plastic microbial carbon source can be obtained through the above two steps;

[0088] S4: Prepare liquid culture medium with polyurethane plastic as microbial carbon source:

[0089]

[0090] The pH of the culture medium was 7.0.

[0091] S5: 120℃ high pressure steam sterilization for 25min;

[0092] S6: Cooling room temperature;

[0093] Through the above 6 steps, a liquid culture medium with polyurethane plastic as a microbial carbon source can be obtained.

[0094] Embodiment 1:

[0095] Escherichia coli (CICC 10389) was inoculated into 100 mL LB liquid medium under sterile conditions and cultured at 37°C for 24 hours as a seed fermentation liquid.

[0096] The seed fermentation liquid was inoculated into medium 1, and the inoculation amount was calculated as 1%. After inoculation of the seed fermentation liquid, each treatment group was aerobic cultured in a 37°C constant temperature shaker at 150 r / min for 12 h, 24 h, 36 h, 48 h, and 60 h, and the OD at 600 nm was measured. 600 value.

[0097] Embodiment 2:

[0098] Referring to the method of Example 1, the culture medium 1 in Example 1 was replaced with culture medium 2, and the other conditions were the same as those in Example 1.

[0099] Embodiment 3:

[0100] Referring to the method of Example 1, the culture medium 1 in Example 1 was replaced with culture medium 3, and the other conditions were the same as those in Example 1.

[0101] Embodiment 4:

[0102] Aspergillus niger (CICC 40048) was inoculated into 100 mL of PDA liquid medium under sterile conditions and cultured at 37° C. for 24 hours as a seed fermentation liquid.

[0103] The seed fermentation liquid was inoculated into medium 1, and the inoculation amount was calculated as 1%. After inoculation of the seed fermentation liquid, each treatment group was aerobic cultured in a 37°C constant temperature shaker at 150 r / min for 12 h, 24 h, 36 h, 48 h, and 60 h, and the OD at 600 nm was measured. 600 value.

[0104] Embodiment 5:

[0105] Referring to the method of Example 1, the culture medium 1 in Example 4 was replaced with culture medium 2, and the other conditions were the same as those in Example 4.

[0106] Embodiment 6:

[0107] Referring to the method of Example 1, the culture medium 1 in Example 4 was replaced with culture medium 3, and the other conditions were the same as those in Example 4.

[0108] Embodiment 7:

[0109] Saccharomyces cerevisiae (CICC 32883) was inoculated into 100 mL of wort under sterile conditions and cultured at 37°C for 24 hours as a seed fermentation liquid.

[0110] The seed fermentation liquid was inoculated into medium 1, and the inoculation amount was calculated as 1%. After inoculation of the seed fermentation liquid, each treatment group was aerobic cultured in a 37°C constant temperature shaker at 150 r / min for 12 h, 24 h, 36 h, 48 h, and 60 h, and the OD at 600 nm was measured. 600 value.

[0111] Embodiment 8:

[0112] Referring to the method of Example 1, the culture medium 1 in Example 7 was replaced with culture medium 2, and the other conditions were the same as those in Example 7.

[0113] Embodiment 9:

[0114] Referring to the method of Example 1, the culture medium 1 in Example 7 was replaced with culture medium 3, and the other conditions were the same as those in Example 7.

[0115] Comparative Example 1:

[0116] Escherichia coli (CICC 10389) was inoculated into 100 mL LB liquid medium under sterile conditions and cultured at 37°C for 24 hours as a seed fermentation liquid.

[0117] The seed fermentation liquid was inoculated into the carbon-free medium 1, and the inoculation amount was calculated as 1%. After inoculation of the seed fermentation liquid, the above treatment groups were aerobically cultured in a 37°C constant temperature shaker at 150 r / min for 12 h, 24 h, 36 h, 48 h, and 60 h, and the OD at 600 nm was measured. 600 value.

[0118] The formula of carbon-free medium is:

[0119]

[0120] The pH of the medium was 7.0;

[0121] Comparative Example 2:

[0122] Aspergillus niger (CICC 40048) was inoculated into 100 mL of PDA liquid medium under sterile conditions and cultured at 37° C. for 24 hours as a seed fermentation liquid.

[0123] The remaining conditions are the same as those in Comparative Example 1.

[0124] Comparative Example 3:

[0125] Saccharomyces cerevisiae (CICC 32883) was inoculated into 100 mL of wort under sterile conditions and cultured at 37°C for 24 hours as a seed fermentation liquid.

[0126] The remaining conditions are the same as those in Comparative Example 1.

[0127] Each of Examples 1-9 and Comparative Examples 1-3 was carried out 3 times, and the OD at 12h, 24h, 36h, 48h and 60h was measured. 600 The average value is taken as the final OD of 12h, 24h, 36h, 48h, and 60h 600 value.

[0128] Table 1 shows the OD values ​​of different strains cultured in medium 1-medium 3 at 12h, 24h, 36h, 48h, and 60h 600 value.

[0129] Table 1

[0130]

[0131]

[0132] When cultured in a medium containing specific plastics (polylactic acid, polyethylene terephthalate, or polyurethane), the microorganisms all showed a clear growth trend, that is, as the culture time increased, the OD 600 The values ​​gradually increased, indicating that these microorganisms can use these plastics as carbon sources for growth and reproduction.

[0133] By comparing the effects of different plastics as carbon sources, it can be found that for Escherichia coli, its growth is best when polylactic acid plastic is used as a carbon source; while for Aspergillus niger and Saccharomyces cerevisiae, although all three can support their growth, there are differences in growth rate and final density. This may be because different microorganisms have different preferences and utilization efficiencies for carbon sources, which also suggests the importance of choosing suitable combinations of microorganisms and specific plastics in the field of biodegradable plastics.

[0134] Finally, compared with Comparative Examples 1-3, when the culture medium did not contain any carbon source, all tested microorganisms could hardly grow, and the OD 600 The value remained at a very low level, which further proves that the microbial growth observed in the above experiments is the result of adding specific plastics as carbon sources, and also emphasizes the key role of carbon sources in the growth of microorganisms. These results are of great significance for studying how to use microorganisms to degrade difficult-to-treat plastic waste, and provide a theoretical basis for the development of new biodegradation technologies and methods.

Claims

1. A method for preparing a microbial carbon source, characterized in that: The following steps are involved: S1: Weigh the plastic and grind it into powder, pass it through a 50-mesh sieve, add 0.5-0.7 mol / L strong acid, stir at 60°C for 5h, adjust the pH of the solution to between 6.5-7.5 with alkali, filter, and dry to obtain intermediate 1; The weight proportions of plastic powder and 0.5-0.7 mol / L strong acid are: 1 part plastic powder; 0.5-0.7mol / L strong acid 6-7 parts; S2: Add photocatalyst and water to intermediate 1 to make a suspension, stir for 12 hours under the conditions of ultrasonic oscillator and ultraviolet light, add non-polar solvent for separation after stirring, retain the organic phase, spin dry, add 3-5 times the weight of semi-polar solvent, reflux at 85°C for 4 hours, and filter to obtain intermediate 2; The weight proportions of intermediate 1, photocatalyst and water are as follows: Intermediate 1 1 part; Photocatalyst 0.05-0.15 parts; 8-10 parts water; S3: Intermediate 2 is ground into powder and passed through a 75-mesh sieve; The microbial carbon source is obtained through the above three steps.

2. The method for preparing a microbial carbon source according to claim 1, characterized in that: The strong acid is selected from one or more of hydrochloric acid, nitric acid and sulfuric acid.

3. The method for preparing a microbial carbon source according to claim 1, characterized in that: The alkali is selected from one or more of sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, potassium carbonate and potassium bicarbonate.

4. The method for preparing a microbial carbon source according to claim 1, characterized in that: The photocatalyst is selected from one or more of titanium dioxide, zinc oxide and cadmium sulfide.

5. The method for preparing a microbial carbon source according to claim 1, characterized in that: The semi-polar solvent is selected from: one of ethanol and methanol.

6. The method for preparing a microbial carbon source according to claim 1, characterized in that: The non-polar solvent is selected from one or more of tetrahydrofuran, dichloromethane, toluene and ethyl acetate.

7. The method for preparing a microbial carbon source according to claim 1, characterized in that: The plastic is selected from: one or more of polylactic acid plastic, polyethylene terephthalate plastic and polyurethane plastic.

8. A method for preparing a microbial carbon source according to any one of claims 1 to 7, characterized in that: The microbial carbon source obtained by the above preparation method is used as a carbon source in a microbial culture medium.

9. A novel microbial culture medium, comprising the microbial carbon source prepared according to claim 8, characterized in that: A method for preparing a novel microbial culture medium is as follows: S1: Prepare the culture medium, the components and mass concentrations of which are: 10-20g / L microbial carbon source; 1-2g / L(NH4)2SO4; 0.3-0.4g / L KH2PO4; 1.5-2.5 g / L K2HPO4·3H2O; 0.3-0.7 g / L NaCl; 0.1-0.3 g / L MgSO4·7H2O; 1.5-2g / L agar powder; The solvent was ultrapure water; The pH of the culture medium is 6.5-7.5; Agar is a solid culture medium, while agar-free culture medium is a liquid culture medium. S2: 120℃ high pressure steam sterilization for 25min; S3: cooling room room temperature; The novel microbial culture medium is obtained through the above three steps.

10. A novel microbial culture medium according to claim 9, characterized in that: A novel microbial culture medium is suitable for culturing Escherichia coli, Bacillus subtilis, Pseudomonas putida, Aspergillus niger, Rhizopus oryzae and Saccharomyces cerevisiae.

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