Microbial composition for inoculum for promoting anaerobic biodegradation of plastics, accelerant, inoculum and preparation method of inoculum

Abstract

The invention discloses a microbial composition for an inoculum for promoting anaerobic biodegradation of plastics, an accelerant, the inoculum and a preparation method of the inoculum, the microbial composition comprises citronellol pseudomonas, copper chloride pseudomonas and pseudomonas putida, and the colony count ratio of the citronellol pseudomonas to the copper chloride pseudomonas to the pseudomonas putida is 1: (1-3): (1-3). The composition is suspended in normal saline or normal saline containing 10% of glycerin to prepare a degradation accelerant, and after the degradation accelerant is inoculated to a standard inoculum and subjected to adaptive culture, the plastic anaerobic biodegradation period can be shortened. According to the method, the anaerobic biodegradation detection time of the anaerobic biodegradation plastic can be remarkably shortened, the detection cost is reduced, and the method has remarkable significance on a detection mechanism and a detection object inspection method.

Description

Technical Field

[0001] The present invention belongs to the technical field of anaerobic biodegradation, specifically a microbial composition for promoting the anaerobic biodegradation of plastics, a degradation accelerating agent containing the composition, and an additive that can be used as an inoculum for detecting the anaerobic biodegradation performance of plastics and a degradation-promoting additive during centralized garbage degradation treatment. Background Art

[0002] The complete degradation of organic matter is a crucial component of the Earth's ecosystem's carbon cycle. Naturally occurring microorganisms initiate the degradation of organic matter, ultimately breaking it down into CO₂, CH₄, H₂O, and their minerals. These are then transformed through photosynthesis in plants and digestion, absorption, and synthesis by animals or microorganisms, forming organic biomass or new compounds required for modern industrial synthesis. Natural biodegradation of organic matter typically occurs in soil and / or sand, compost, anaerobic digestion, or aqueous culture media, by microorganisms in the soil and water in the presence of light and oxygen, or under anaerobic or light-protected conditions. Therefore, the degradation process is influenced by the type and quantity of microorganisms in the degradation environment, the nutrients they require, temperature, light intensity, humidity, oxygen content, and other conditions.

[0003] Polyolefins such as polyethylene, polypropylene, and polyvinyl chloride biodegrade extremely slowly in the natural environment, and it takes hundreds of years to degrade. Therefore, these polymer compounds are often referred to as non-degradable compounds. With the development of the petrochemical industry and the aggravation of the white pollution problem caused by the large-scale application of plastic products, studying the biodegradation mechanism of plastics, developing biodegradable plastics and centralized plastic degradation treatment processes have become research hotspots and industrial development directions. The anaerobic biodegradability test of plastics plays an important role in the development and production of degradable plastics. In the standards for the degradation performance test of plastics (such as GB / T 33797-2017 and ASTM D 5511), the inoculum used is mostly activated sludge that has been treated with anaerobic digestion. For example, GB / T 33797-2017 specifies that the inoculum should be "household waste that has been anaerobic digested as the sole raw material," "must be digested for at least four months and maintained at appropriate humidity conditions (52±2°C) for a maximum of 30 days," and "short-term fermentation should be conducted for approximately 7 days at the same operating humidity to prevent the inoculum from being fed," and must meet the following requirements: pH: 7.5–8.5, volatile fatty acids (VFA): less than 1 g / kg wet weight, ammonia nitrogen (NH₄⁺-N): 0.5 g / kg–2 g / kg wet weight." ASTM D5511's inoculum requirements are generally consistent with those of GB / T 33797-2017. According to the degradation performance requirements specified in current biodegradable plastic standards, to test for complete degradation or maximum biodegradability, test samples are typically tested continuously for up to 180 days, with a maximum limit of 730 days. However, such long testing cycles not only increase the workload of testing agencies and reduce testing efficiency, but also lead to higher costs and inadequate timeliness of test results for customers. Therefore, if biodegradation performance testing can achieve accelerated biodegradation like accelerated weathering resistance testing, it will be of great significance and an urgent industrial reality need. Summary of the Invention

[0004] In order to achieve accelerated detection of anaerobic biodegradation of plastics, the first object of the present invention is to provide a microbial composition that promotes anaerobic biodegradation, which can be inoculated into the inoculum used for detection - activated sludge, significantly shortening the time and detection cycle of anaerobic biodegradation of plastics.

[0005] A microbial composition for use as an inoculant to promote anaerobic biodegradation of plastics comprises: Pseudomonas citronellol, Pseudomonas cuprichlorans and Pseudomonas putida, wherein the colony count ratio of Pseudomonas citronellol: Pseudomonas cuprichlorans: Pseudomonas putida is 1:(1-3):(1-3).

[0006] Preferably, the colony count ratio of Pseudomonas citronellol:Pseudomonas cuprichlorans:Pseudomonas putida is 1:2:2.

[0007] Furthermore, the preservation number of the Pseudomonas citronellol is DSM 50332, the preservation number of the Pseudomonas aeruginosa is CGMCC 1.895, and the preservation number of the Pseudomonas putida is ATCC 12633.

[0008] The present invention also provides a degradation promoter for inoculum used in anaerobic biodegradation detection of plastics, which is the aforementioned microbial composition suspended in physiological saline and stored at a low temperature of 3-8°C. The promoter is suitable for short-term storage of no more than 7 days.

[0009] The present invention also provides a second degradation accelerator for inoculum in anaerobic plastic biodegradation testing, which is the aforementioned microbial composition suspended in physiological saline containing 10% glycerol and stored frozen at -80°C. This accelerator is suitable for long-term storage.

[0010] Furthermore, the total colony concentration in the degradation promoter is 106 to 109 CFU / mL, preferably 10 9 CFU / mL.

[0011] The present invention also provides an inoculum for detecting anaerobic biodegradation of plastics, wherein the standard inoculum is inoculated with the aforementioned microbial composition or the aforementioned degradation accelerator.

[0012] Preferably, the volume ratio of the standard inoculum to the degradation promoter is (6-12):1, and most preferably the volume ratio is 9:1.

[0013] The present invention also provides a method for preparing the above-mentioned inoculum, characterized in that it comprises the following steps:

[0014] (1) Prepare standard inoculum according to standard requirements;

[0015] (2) The standard inoculum and the degradation promoter are fully stirred and mixed according to the volume ratio, and allowed to stand to promote bacterial adsorption;

[0016] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

[0017] The standard inoculum in the present invention is an existing inoculum required by the provisions of GB / T 33797-2017, ASTM D5511 or ISO15985 or other relevant international, national and industry standards (including mandatory standards and recommended standards).

[0018] The microbial composition and promoter for promoting anaerobic biodegradation of plastics of the present invention, when inoculated into a standard inoculum, can significantly reduce the anaerobic biodegradation detection time of anaerobic biodegradable plastics and reduce the detection cost, which has significant significance for detection institutions and parties submitting detection objects for inspection. DETAILED DESCRIPTION

[0019] The present invention is described in further detail below with reference to the examples.

[0020] The present invention will be further described below with reference to specific embodiments to facilitate understanding of the present invention.

[0021] In the following examples, the bacterial strains used are deposited in the following locations and their accession numbers:

[0022] Pseudomonas aeruginosa (CGMCC 1.895)

[0023] Pseudomonas putida (ATCC 12633)

[0024] Pseudomonas citronellolis (DSM 50332)

[0025] 1. Culture medium for bacterial strains

[0026] 1. Pseudomonas aeruginosa culture medium

[0027] Culture medium formula (refer to NAC culture medium and fermentation process optimization):

[0028] Sodium nitrate 0.8-1.0%

[0029] Potassium chloride 0.1%

[0030] Phosphoric acid 0.98-1.1%

[0031] Magnesium sulfate heptahydrate 0.1%

[0032] Glucose 0.4-0.5%

[0033] Soybean oil 4-5%

[0034] Agar 1.5%

[0035] pH 7.0-7.2 (before sterilization)

[0036] Preparation method of culture medium: Dissolve the above ingredients in distilled water, sterilize under high pressure at 121℃ for 20 minutes, and pour into plates or make liquid culture medium after cooling.

[0037] Culture conditions: 37°C, aerobic culture for 24-48 hours. Identification can be achieved by the blue-green pigment of the colonies and growth characteristics at 42°C.

[0038] 2. Pseudomonas putida culture medium

[0039] Culture medium formula (refer to nutrient broth agar):

[0040] Peptone 5.0g

[0041] Beef extract 3.0g

[0042] NaCl 5.0g

[0043] Agar 15.0g

[0044] 1.0L distilled water

[0045] pH 7.0

[0046] Preparation method of culture medium: After mixing, sterilize at 121℃ for 20 minutes, cool to 50℃ and pour into plates, or directly prepare liquid culture medium.

[0047] Culture conditions: Aerobic culture at 30℃ for 18-24 hours. The colonies are milky white and non-fluorescent.

[0048] 3. Citronellol Pseudomonas culture medium

[0049] Medium formula (adjusted for general LB medium):

[0050] Peptone 1%

[0051] Yeast extract 0.5%

[0052] NaCl 0.5%

[0053] Glycerol 0.5% (carbon source supplement)

[0054] Agar 1.5%

[0055] pH 7.0

[0056] Preparation method: Dissolve peptone, yeast extract and NaCl in distilled water, adjust the pH to 7.2, add glycerol and agar powder, sterilize under high pressure, and pour into plates or make solid culture medium.

[0057] Culture conditions: Aerobic culture at 28℃ for 48 hours, the colonies are light yellow.

[0058] 2. Single strain expansion

[0059] The three bacteria were inoculated into the corresponding culture medium respectively and cultured to the logarithmic growth phase (OD600≈0.8) according to their respective conditions.

[0060] The cells were collected by centrifugation, resuspended in sterile saline, and the concentration was adjusted to 10^9 CFU / mL.

[0061] 3. Preparation of accelerator

[0062] 1. Example 1 (Accelerator Suitable for Short-Term Storage)

[0063] The bacterial suspension was mixed in a volume ratio of 2:2:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL. Store at 4°C and retest the viable count before use.

[0064] 2. Example 2 (Promoter suitable for long-term storage)

[0065] The bacterial suspension was mixed in a volume ratio of 2:2:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL, then add 10% glycerol, divide into aliquots and freeze at -80℃ for long-term storage.

[0066] 3. Example 3 (Accelerator Suitable for Short-Term Storage)

[0067] The bacterial suspension was mixed in a volume ratio of 1:1:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL. Store at 4°C and retest the viable count before use.

[0068] 4. Example 4 (Accelerator Suitable for Short-Term Storage)

[0069] The bacterial suspension was mixed in a volume ratio of 3:3:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL. Store at 4°C and retest the viable count before use.

[0070] Example 5 (Promoter suitable for short-term storage)

[0071] The bacterial suspension was mixed in a volume ratio of 3:2:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL. Store at 4°C and retest the viable count before use.

[0072] Example 6 (Promoter suitable for short-term storage)

[0073] The bacterial suspension was mixed in a volume ratio of 2:3:1 for Pseudomonas aeruginosa, Pseudomonas putida and Pseudomonas citronellol. The total bacterial concentration after mixing was 10 9 CFU / mL. Store at 4°C and retest the viable count before use.

[0074] IV. Preparation of Inoculum

[0075] Example 7

[0076] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0077] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 1 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0078] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria to ensure that they meet the requirements.

[0079] Example 8

[0080] (1) Preparation of standard inoculum: The standard inoculum was prepared using anaerobic sludge from urban kitchen and bathroom organic waste according to ASTM D5511.

[0081] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 2 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0082] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0083] Example 9

[0084] (1) Preparation of standard inoculum: The standard inoculum was prepared according to ISO15985 using anaerobic digestion sludge that simulates the microbial diversity in the natural environment.

[0085] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 3 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0086] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0087] Example 10

[0088] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0089] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 4 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0090] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0091] Example 11

[0092] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0093] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 5 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0094] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0095] Example 12

[0096] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0097] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 6 were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0098] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0099] Example 13

[0100] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0101] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 1 were mixed in a volume ratio of 10:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0102] (3) Culture at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony counts of the three bacteria and the proportion of target bacteria.

[0103] Comparative Example 1

[0104] The standard inoculum of GB / T 33797-2017 was used.

[0105] Comparative Example 2

[0106] The standard inoculum of ASTM D5511 was used.

[0107] Comparative Example 3

[0108] The standard inoculum of ISO15985 was used.

[0109] Comparative Example 4

[0110] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0111] (2) Preparation of composite inoculum: The standard inoculum in (1) and the expanded Pseudomonas aeruginosa were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0112] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

[0113] Comparative Example 5

[0114] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0115] (2) Preparation of composite inoculum: The standard inoculum in (1) and the expanded Pseudomonas putida were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0116] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

[0117] Comparative Example 6

[0118] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0119] (2) Preparation of composite inoculum: The standard inoculum in (1) and the expanded Pseudomonas citronellol were mixed in a volume ratio of 9:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0120] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

[0121] Comparative Example 7

[0122] (1) Preparation of standard inoculum: According to GB / T 33797-2017, the standard inoculum was prepared using anaerobic sludge from a municipal sewage treatment plant.

[0123] (2) Preparation of composite inoculum: The standard inoculum in (1) and the promoter in Example 1 were mixed in a volume ratio of 15:1, stirred thoroughly, and allowed to stand for 2 hours to promote bacterial adsorption.

[0124] (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

[0125] 5. Anaerobic degradation test

[0126] Anaerobic degradable plastic film sample: brand PE7042, film sample preparation should be carried out in accordance with GB / T38787, the sample is a film with a thickness of 0.25 mm.

[0127] Experimental Methods: Using the inoculum prepared in Examples 7-13 and Comparative Examples 1-7, the degradation time required for anaerobic degradable plastic film samples to reach 60% absolute biodegradation was determined according to the methods of GB / T 33797-2017, ASTM D5511, and ISO 15985, based on the standards for standard inoculum. The results are shown in Table 1.

[0128] Table 1 Degradation timetable of different inocula

[0129]

[0130]

[0131] As shown in Table 1, the degradation time of PE7042 plastic film samples using the inoculum containing the promoters of Examples 7-13 of the present invention was significantly shortened compared to the standard inoculum of Comparative Examples 1-3, demonstrating that the compositions of the present invention can significantly shorten the anaerobic biodegradation time of plastics. Examples 7-13 also significantly shortened the degradation time compared to Comparative Examples 4-6 when a single bacterial species was added, demonstrating the significant synergistic effect of the three microorganisms. Comparison of Examples 7 and 13 with Comparative Example 7 demonstrates that the relationship between promoter dosage and degradation time is not linear.

[0132] Those skilled in the art will understand that the foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A microbial composition for inoculum to promote anaerobic biodegradation of plastics, characterized in that It contains Pseudomonas citronellol, Pseudomonas copper-chlorinated and Pseudomonas putida, and the colony number ratio of Pseudomonas citronellol: Pseudomonas copper-chlorinated: Pseudomonas putida is 1:(1-3):(1-3).

2. The composition according to claim 1, wherein: The colony count ratio of Pseudomonas citronellol:Pseudomonas cuprichlorans:Pseudomonas putida is 1:2:

2.

3. The composition according to claim 1, wherein: The Pseudomonas citronellol deposit number is DSM 50332, the Pseudomonas aeruginosa deposit number is CGMCC 1.895, and the Pseudomonas putida deposit number is ATCC 12633.

4. A degradation accelerator for inoculum used in anaerobic biodegradation detection of plastics, characterized in that The total colonies of the microbial composition according to claim 1 are suspended in physiological saline and stored at 3-8°C.

5. A degradation accelerator for inoculum used in anaerobic biodegradation detection of plastics, characterized in that The total colonies of the microbial composition according to claim 1 are suspended in physiological saline containing 10% glycerol and stored at -80°C.

6. The degradation accelerator according to claim 4 or 5, characterized in that The total colony concentration is 106~109 CFU / mL.

7. An inoculum for detecting anaerobic biodegradation of plastics, characterized in that The standard inoculum is inoculated with the microbial composition according to claim 1 or the degradation promoter according to claim 2 or 3.

8. The inoculum of claim 7, wherein the volume ratio of the standard inoculum to the degradation promoter is (6-12):

1.

9. A method for preparing the inoculum according to claim 7, characterized in that The steps include: (1) Prepare standard inoculum according to standard requirements; (2) The standard inoculum and the degradation promoter are fully stirred and mixed according to the volume ratio, and allowed to stand to promote bacterial adsorption; (3) Cultivate at 30-37°C under anaerobic conditions for 24-48 hours, and monitor the total colony count and the proportion of target bacteria.

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