JP7223808B2 - Microbial inoculant for high-temperature biodegradation of garbage and method of decomposition of garbage - Google Patents

Description

CCTCC CCTCC M 2019263 CCTCC CCTCC M 2020014 CCTCC CCTCC M 2020015 CCTCC CCTCC M 2020016

TECHNICAL FIELD The present invention relates to the field of garbage disposal technology, and more particularly to the microbial inoculant and application for high-temperature biodegradation of garbage.

Garbage, also referred to as washing juice, leftover food, etc., is the remaining portion of food (e.g., vegetable leaves, fruit peels and fruit crumbs, etc.) after food processing, processing (including stewing), or leftover after eating. A general term for discarded food. With the transformation of the economic level and social necessities, people attach more and more importance to eating and drinking, resulting in a large amount of wasted food and drink. Studies show that half of the food in the world is wasted.

The main components of food waste include food leftovers such as rice and flour, vegetables, animal/vegetable oils and meat bones. In terms of chemical structure, it contains starch, protein, fibrin, lipids and inorganic salts. These compositions are characterized by high water content and rich nutrition, are prone to spoilage and odor, spread bacteria and viruses, not only affect the cleanliness of cities, but also pollute the environment, and endanger people's health. However, since the rich nutrition contained in food waste is an extremely valuable renewable resource, efficient and rational disposal of food waste can certainly bring high value. In addition, the heterogeneity of garbage itself makes its transportation and disposal very inconvenient and expensive.

With the continued deepening of the concept of environmental protection and sustainable development, the irreversible environmental damage and resource inefficiency of traditional food waste disposal methods, including, for example, incineration and sanitary landfill disposal, As people started looking for new treatment methods, the biological treatment of food waste has become more and more a hotspot of research and attention due to its unique advantages. For example, an invention application with publication number CN105665417A disclosed a composite microbial inoculant for food waste high temperature biodegradation and methods of making and using it. The composite microbial inoculant is composed of a composite fungus and a carrier, and the composite fungus can be a mixture of Candida krusei yeast, Bacillus subtilis, Wicker harmomyces anomalus yeast, Aspergillus niger, and actinomycetes. The carrier is composed of soybean meal, bran, rice husk powder and shavings. All of these microbial inoculants have a good garbage decomposition effect, but due to the difference in eating habits, the microenvironment of garbage and the specificity of microbial enzyme production, high-temperature microorganisms can accelerate the decomposition of garbage and the biological reaction time. It is more advantageous for shortening the time and reducing energy consumption. Therefore, it is necessary to select an advantageous strain that has better garbage decomposition ability and high temperature resistance to speed up the biodegradation of garbage. As a result, garbage is rendered harmless, reduced in weight, and recycled.

Chinese Patent Application Publication No. 105665417

The present invention addresses the deficiencies existing in the existing technology and provides a microbial inoculant and application for food waste high temperature biodegradation.

The present invention provides the following bacteria, namely
(1) Pichia kluyveri yeast with strain number ZJB-091 and accession number CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) Bacillus thuringiensis rod with strain number ZJB19165 and accession number CCTCC NO:M 2020015;
(4) Bacillus paralicheniformis rod with strain number ZJB19166 and accession number CCTCC NO: M 2020016;
It is a microbial inoculant for garbage high-temperature biodegradation, comprising

In the microorganism inoculum for high-temperature biodegradation of garbage, Pichia kluyveri yeast ZJB- 091 , Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165, Bacillus The mass ratio of Bacillus paralicheniformis rod ZJB19166 is 1:1-2:1:1-2.

In the microorganism inoculum for high-temperature biodegradation of garbage, Pichia kluyveri yeast ZJB- 091 , Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165, Bacillus The mass ratio of the dry cell weight of Bacillus paralicheniformis rod ZJB19166 is 1:1:1:1.

The present invention provides the following steps:
(1) activating and culturing each of the four strains;
(2) Obtaining a seed medium by performing seed culture for each of the four strains after activation;
(3) fermenting the culture solution for each of the four strains to obtain a fermentation solution;
(4) A method for preparing the microbial inoculum for high-temperature biodegradation of food waste, comprising mixing the fermentation broth of four strains in proportion to obtain the microbial inoculum for high-temperature biodegradation of food waste. .

The present invention is an application of the microbial inoculant for high-temperature biodegradation of garbage in decomposition of garbage.

The present invention is a method for biodegrading garbage, wherein the microbial inoculant for high-temperature biodegradation of garbage is put into garbage waiting to be decomposed and biodegraded.

In the method for decomposing garbage, the biodegradation temperature is 50°C to 55°C.

In the method for decomposing garbage, the biodegradation temperature is 55°C.

In the method for decomposing garbage, the inoculum amount of the microbial inoculant for high-temperature biodegradation of garbage is not less than 1% of the mass of garbage.

In the method for decomposing food waste, the inoculum amount of the microbial inoculant for high-temperature biodegradation of food waste does not fall below 1.5% of the mass of food waste.

FIG. 11 is a morphological diagram of food waste after completion of treatment in Example 7; FIG. 10 is a schematic diagram of a garbage biodegradation test in Example 8. FIG.

An embodiment of the present invention will be described below. This embodiment is an example of implementing the present invention, and the present invention is not limited to this embodiment.

The microbial inoculant for garbage high-temperature biodegradation is the following bacteria, namely
(1) Pichia kluyveri yeast with strain number ZJB-091 and accession number CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) a Bacillus thuringiensis rod having a strain number of ZJB19165 and an accession number of CCTCC NO: M 2020015;
(4) Bacillus paralicheniformis with strain number ZJB19166 and accession number CCTCC NO:M 2020016;

Preferably, in the microbial inoculum, Pichia kluyveri yeast ZJB- 091 , Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis The mass ratio of Bacillus paralicheniformis bacillus ZJB19166 is 1:1-2:1:1-2.

More preferably, the microbial inoculant comprises Pichia kluyveri yeast ZJB- 091 , Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheni The mass ratio of dry cells of Bacillus paralicheniformis ZJB19166 is 1:1:1:1.

Pichia kluyveri yeast ZJB-091 has been deposited in the Chinese Typical Culture Conservation Center (CCTCC) with the accession number CCTCC NO: M 2019263 and the deposit time on April 17, 2019. be. The 18s rDNA sequence of Pichia kluyveri yeast ZJB-091 is shown in SEQ ID NO.1. The biological characteristics of Pichia kluyveri yeast ZJB-091 are as follows. White to cream-coloured colonies, moist, sticky, easy to dig, front, back and center of the colonies matching the color of the margins, pseudohyphae, fruity during the growth process. have a flavor like Shows a spherical or oval shape. Amylase, proteolytic enzyme, lipase and cellulase can be produced.

Bacillus licheniformis bacillus ZJB19163 has been deposited in the Chinese Typical Culture Conservation Center (CCTCC) with the accession number CCTCC NO: M 2020014 and the accession time on January 6, 2020. The 16S rRNA sequence of Bacillus licheniformis bacillus ZJB19163 is shown in SEQ ID NO.2. The biological characteristics of the Bacillus licheniformis rod are as follows. Colonies are circular, pale yellow, slightly white, smooth, sticky, opaque, with regular edges, no wrinkles, and produce amylase, proteolytic enzymes and lipase. can.

The Bacillus thuringiensis bacillus ZJB19165 has been deposited in the Chinese Typical Culture Conservation Center (CCTCC), the accession number is CCTCC NO: M 2020015, and the deposit time is January 6, 2020. be. The 16S rRNA sequence of Bacillus thuringiensis bacillus ZJB19165 is shown in SEQ ID NO.3. The biological characteristics of the Bacillus thuringiensis rod are as follows. The colonies are circular, pale yellow, smooth, opaque, with regular edges, no wrinkles, rod-like, and belong to Spore-forming fungi.

Bacillus paralicheniformis bacillus ZJB19166 has been deposited in the Chinese Typical Culture Conservation Center (CCTCC), the accession number is CCTCC NO: M 2020016, and the accession time is January 6, 2020. be. The 16S rRNA sequence of Bacillus paralicheniformis bacillus ZJB19166 is shown in SEQ ID NO.4. The biological characteristics of Bacillus paralicheniformis rod ZJB19166 are as follows. The colonies are circular, pale yellow, smooth, opaque, with regular edges, no wrinkles, rod-like, and belong to Spore-forming fungi. Amylase, proteolytic enzyme, lipase and cellulase can be produced.

The present invention also provides a method for preparing said microbial inoculum. the following steps, i.e.
(1) activating and culturing each of the four strains;
(2) Obtaining a seed medium by performing seed culture for each of the four strains after activation;
(3) fermenting the culture solution for each of the four strains to obtain a fermentation solution;
(4) including obtaining the microbial inoculum by mixing the fermentation broth of the four strains in proportion;

Specifically, the method for preparing the microbial inoculum includes the following steps:
(1) Pichia kluyveri yeast ZJB-091 is inoculated into a PDA slant medium and cultured at a constant temperature to activate the strain;
(2) Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 were each inoculated into peptone slants of beef pesto. , culturing at constant temperature to perform activation culture of the strain;
(3) The activated Pichia kluyveri yeast ZJB-091 was inoculated into a PDA slant medium for seed culture, and then a Pichia kluyveri yeast seed medium was obtained. After activation, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 were placed on beef pesto peptone slants, respectively. After inoculating and seed culture, Bacillus licheniformis Bacillus ZJB19163, Bacillus thuringi ensis Bacillus ZJB19165 and Bacillus paralicheniformis Bacillus paralicheniformis Bacillus ZJB19166 seed medium to obtain;
(4) the Pichia kluyveri yeast ZJB-091 seed medium and Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis After inoculating the seed medium of Bacillus paralicheniformis ZJB19166 into the fermentation cans containing the fermentation medium and fermenting culture, respectively, Pichia kluyveri yeast ZJB-091 fermentation liquid and Bacillus licheniformis (Bacillus licheniformis) obtaining a fermentation broth of bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166;
(5) Pichia kluyveri yeast ZJB-091 fermentation liquid and Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis ) The fermented liquid of Bacillus ZJB19166 is proportionally mixed, and then fixed at a ratio of 1:20 using wheat stalk powder of 5 to 150 meshes, and then dried at low temperature to produce biodegradable microorganisms for garbage. to obtain the inoculum.

In step (1), the composition of the PDA slants is as follows. 200 g of potato, 20 g of glucose, 20 g of agar, 1 L of water; activation culture conditions: constant temperature culture at 25-40° C. for 24-48 hours.

In step (2), the composition of the beef pesto peptone slants is as follows. 5 g of beef pesto, 10 g of peptone, 5 g of sodium chloride, 20 g of agar, 1 L of water; activation culture conditions: constant temperature culture at 35-55° C. for 12-24 hours.

In step (3), the composition of PDA liquid medium is as follows. 200 g of potato, 20 g of glucose, 1 L of water; Seed culture conditions: 25-40° C., 150-200 r/min, cultured on a shaking bed for 24-48 h. The composition of the peptone liquid medium of beef pesto is as follows. 5 g of beef pesto, 10 g of peptone, 5 g of sodium chloride, 1 L of water; Seed culture conditions: 35-55° C., 150-200 rpm, shake culture for 24-48 hours in a shaker.

200 g of potato, 20 g of glucose, 1 L of water; Fermentation culture conditions: 25° C. to 40° C., 200 to 450 r/min for 36 to 80 hours. The composition of the fermentation medium for Bacillus licheniformis, Bacillus thuringi ensis and Bacillus paralicheniformis is as follows. 5 g of beef pesto, 10 g of peptone, 5 g of sodium chloride, 1 L of water; Fermentation culture conditions: 35° C. to 55° C., 200 to 450 rpm, fermentation was performed for 36 to 80 hours.

The present invention also provides the application of said microbial inoculants in garbage decomposition.

The present invention also provides a method of garbage decomposition. That is, the microbial inoculant is put into garbage waiting to be decomposed for biodegradation. Preferably, the biodegradation temperature is 55°C.

Compared with existing technology, the present invention has the following beneficial effects.
(1) The microbial inoculant provided by the present invention not only speeds up the biodegradation of food waste, but also removes offensive odors and realizes detoxification and weight reduction treatment of food waste.
(2) By applying the compound microbial inoculant to the treatment of food waste in the present invention, there is a cooperative effect between each microbial strain, and under aerobic conditions, macromolecular organic matter in food waste is produced. , to quickly decompose into small molecule substances, produce a large amount of organic substances and trace elements, and realize the conversion of waste to resources.
(3) The microbial inoculant provided by the present invention can effectively suppress the growth of harmful microorganisms at 55° C. and realize the detoxification of food waste.
(4) The microbial inoculant provided by the present invention does not produce offensive odor after acting on garbage, and has good prospects for application.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to the following examples.

<Example 1: Selection and appraisal of food waste high-temperature decomposition bacteria>
5 g of a soil sample near the Luxiu cafeteria of Zhejiang Institute of Technology was taken, placed in a sterilized small glass ball and an Erlenmeyer flask with 50 mL of sterile water, and cultured with shaking at 55 ° C. and 180 r/min for one week of enrichment culture. Afterwards, a gradient dilution of the bacterial suspension was performed on a super clean bench. The concentration gradient was set to 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , and 100 μL of each concentration of bacterial suspension was After blotting and inoculating PDA slants and beef pesto peptone slants respectively, the panels were sealed with a sealing film, turned upside down and placed in a 28-40° C. incubator for 24-48 h of incubation. After that, a panel with an appropriate number of colonies was selected, and typical colonies were selected, separated and purified, and then inoculated into protein, starch, fat and fibrin identification medium panels, respectively. After that, it was applied evenly with a sterilized applicator stick. Thereafter, the petri dish was sealed with a sealing film, turned upside down, and cultured in a constant temperature incubator at 28-40° C. for 48-37 hours, after which the lysis zone diameter and colony diameter ratio were observed. A strain with a relatively large lysis zone was inoculated into PDA liquid medium and beef pesto peptone liquid medium, respectively. Stored in a refrigerator at 80°C. A portion of the fungal fluid was taken for morphological, physiological biochemical and molecular biological examination.

After extracting strain genomic DNA using FastDNA (trademark) Spin Kit for Soil reagent kit, PCR amplification was performed using this as a template. Amplification primers are as follows.

Bacteria adopt the following 16S rRNA consensus primers.
27F: 5'-AGAGTTTGATCCTGGCTCAG-3';
1492R: 5'-GGTTACCTTGTTACGACTT-3'.

The bacterial PCR system is shown in Table 1.

Fungi adopt the following rDNA-ITS consensus primers.
ITS1: 5'-TCCGTAGGTGAACCTGCGG-3';
ITS4: 5'-TCCTCCGCTTATTGATATGC-3'.

The fungal PCR system is shown in Table 2.

PCR procedure: Initial heat denaturation was performed at 95°C for 5 min. 30 cycles of drawing: 95°C 40s, 55°C 30s, 72°C 2min; 72°C 10min.

Hangzhou Tsingke Biotechnology Co., Ltd. carried out PCR product sequencing, and the sequences obtained were SEQ ID NO.1, SEQ ID NO.2, SEQ ID As shown in NO.3 and SEQ ID NO.4.

After entering the obtained DNA sequences into GenBank, they were matched with all sequences in the database based on the Blast procedure. After that, combined with the morphological and physiochemical identification results, the strains obtained by screening were identified as Pichia kluyveri yeast, Bacillus licheniformis bacilli, and Bacillus thuringiensis, respectively. thuring i ensis) rod and Bacillus paralicheniformis rod, then Pichia kluyveri yeast ZJB-091, Bacillus licheniformis rod ZJB19163, Bacillus paralicheniformis, respectively. They were named Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166.

Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 are It has been deposited in the Chinese Typical Culture Conservation Center (CCTCC). Specific information is as follows.

(1) Pichia kluyveri yeast ZJB-091:
Accession number: CCTCC NO: M 2019263;
Acceptance time: April 17, 2019.
(2) Bacillus licheniformis bacillus ZJB19163:
Accession number: CCTCC NO: M 2020014;
Acceptance time: January 6, 2020.
(3) Bacillus thuringiensis rod ZJB19165:
Accession number: CCTCC NO: M 2020015;
Acceptance time: January 6, 2020.
(4) Bacillus paralicheniformis rod ZJB19166:
Accession number: CCTCC NO: M 2020016;
Acceptance time: January 6, 2020.

Chinese Typical Culture Conservation Center (CCTCC) Address: China Typical Culture Conservation Center (CCTCC), Wuhan University, Bayi Road, Jiashan Road, Hongshan District, Wuhan City, Hubei Province.

The medium components involved in the method are as follows.
PDA solid medium: 200 g potato, 20 g glucose, 20 g agar, 1 L water, pH natural.
PDA liquid medium: 200 g potato, 20 g glucose, 1 L water, pH natural.
The composition of the beef pesto peptone solid medium is as follows. 5 g beef pesto, 10 g peptone, 5 g sodium chloride, 20 g agar, 1 L water, pH natural.
The composition of the peptone liquid medium of beef pesto is as follows. 5 g beef pesto, 10 g peptone, 5 g sodium chloride, 1 L water, pH natural.
Protein identification medium: 50 g skim milk powder, 10 g soluble starch, 5 g yeast extract, 1 _{g KH2PO4} , _0.2 g _MgSO4.7H2O , 20 g agar, 1 L water, pH 7.0.
Starch identification medium: 10 g peptone, 2 g soluble starch, 5 g beef pesto, 5 g NaCl, 20 g agar, 1 L water, pH 7.0-7.2.
Fat identification medium: 10 g of peptone, 1 _{g of K2HPO4} , 0.5 g of _MgSO4.7H2O , _1.2 g of polyvinyl alcohol, 0.04 g of Victoria Blue B, 10 g of olive oil, 20 g of agar, 1 L of water, pH 8.0 .
Cellulose identification medium: _{0.5 g} K2HPO4, 0.2 g _MgSO4.7H2O , ₂ g CMC-Na, 0.2 g Congo red, 16 g agar, 2 g gelatin, 1 L water, pH 7.0.

<Example 2: Strain enzymatic activity measurement>
The isolated Pichia kluyveri yeast ZJB-091 was inoculated into a PDA liquid medium to produce Bacillus licheniformis bacillus ZJB19163, Bacillus thuringiensis bacillus ZJB19165, and Bacillus paralicheniformis bacillus ZJB19166 was shake-cultured on a shaker at 55° C. for 48 h. After centrifuging the fermented liquid for 10 minutes under the conditions of 4° C. and 8000 r/min, the supernatant was taken and the enzymatic activities of amylase, proteolytic enzyme, lipase and cellulase were measured. DNS method was used for amylase activity measurement. Definition of enzyme activity: The amount of enzyme required to produce 1 µg of maltose per minute with 1 mL of enzyme solution is one unit of enzyme activity, and is expressed as "U/mL". The Folin-Ciocalteu method was used to measure proteolytic enzyme activity. Definition of enzyme activity: One enzyme activity unit is the amount of enzyme required to hydrolyze casein with 1 mL of enzyme solution every minute to produce 1 μg of tyrosine, and is expressed as "U/mL". . National standard GB/T 23535-2009 is used for lipase activity determination. Definition of enzymatic activity: 1 mL of enzyme solution hydrolyzes the substrate (in this example, olive oil as substrate), the amount of enzyme required to produce 1 μmol of titratable fatty acid is one enzymatic activity unit and is expressed as “U/mL”. The 3,5-dinitrosalicylic acid method was used to measure cellulase activity. One enzymatic activity unit is the amount of enzyme required to produce 1 μg of glucose per minute with 1 mL of the enzyme solution, and is expressed as “U/mL”. The results are shown in Table 3.

<Example 3: Preparation of microbe inoculum>
[1] Slant culture Pichia kluyveri yeast ZJB-091 was inoculated onto a PDA slant , and Bacillus licheniformis bacilli ZJB19163, Bacillus thuringiensis bacillus ZJB19165 and Bacillus thuringiensis ZJB19165 were grown. Bacillus paralicheniformis rod ZJB19166 was inoculated into a peptone slant medium of beef pesto, and then cultured at a constant temperature of 25 to 40° C. for 24 to 48 hours to activate the strain. Activated PDA slant composition used: 200 g potato, 20 g glucose, 20 g agar, 1 L water, pH natural. Sterilized at 115°C for 30 minutes. Beef pesto peptone slant composition used: 5 g beef pesto, 10 g peptone, 5 g sodium chloride, 20 g agar, 1 L water, pH natural. Sterilized at 121°C for 20 minutes.

[2] Seed medium The activated Pichia kluyveri yeast ZJB-091 obtained in step [1] was inoculated into potato-dextrose liquid medium. Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 were inoculated into peptone broth of beef pesto. After shaking culture at 25 to 40 ° C. and 150 to 200 r/min shaker for 24 to 48 hours, Pichia kluyveri yeast ZJB-091, Bacillus licheniformis rod ZJB19163, Bacillus thuringiensis thuring i ensis) bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 seed media were obtained. The potato/glucose liquid medium used is as follows. 200 g potatoes, 20 g glucose, 1 L water, pH natural. Sterilized at 115°C for 30 minutes. The composition of the peptone liquid medium of beef pesto is as follows. 5 g beef pesto, 10 g peptone, 5 g sodium chloride, 1 L water, pH natural. Sterilized at 121°C for 20 minutes.

[3] Expansion culture:
Into the fermenter, 50-67% volume ratio of medium was added, and then Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus licheniformis obtained in step [2]. Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 seed medium were inoculated into a fermentation canister containing a medium having a volume ratio of 50 to 67%, respectively, and then heated at 30°C. Fermentation culture was performed for 36 to 80 hours under conditions of ~45°C and 200 to 450 r/min. Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163 , Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166, respectively. liquid was obtained.

Liquid medium used in step [3]: 1 L of potato/glucose liquid medium was added to 200 g of potato and 1 L of water was boiled for 20 minutes, followed by filtration to obtain a filtrate. After that, 20 g of glucose was added, and then water was added to fill up to 1 L. Sterilized at 115°C for 30 minutes. 1 L of beef pesto peptone liquid medium was made into 5 g of beef pesto, 10 g of peptone and 5 g of sodium chloride, and sterilized at 121° C. for 20 minutes.

(2) Preparation of complex microbial inoculum Cultured Pichia kluyveri yeast ZJB- 091 , Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus parali Bacillus paralicheniformis Bacillus paralicheniformis ZJB19166 bacterial solution was centrifuged at 8000 rpm for 10 minutes in a centrifuge (desktop type) high-speed refrigerating centrifuge, and then obtained Pichia kluyveri yeast ZJB-091, Bacillus Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 bacterial solution, the weight ratio (dry cell weight) is (0.5) : 1:0.5:1) or (1:1:1:1) to obtain microbial liquids with different cell proportions. Then, 5-150th wheat stalk powder was used to immobilize at a ratio of 1:20, followed by low-temperature drying to obtain a composite microbial inoculum. The drying temperature was 30-50° C. and the viable cell count was ≧3.0×10 ⁹ CFU/mL.

<Example 4: Microbial inoculant stability evaluation>
The combined microbial inoculum was placed in a corner of the laboratory and stored under room temperature. Samples were taken every 15 days and analyzed for changes in the viable count. Viable cell counts were measured by the dilution panel coating method.

During the microbial inoculant storage period, the temperature of different environments, the moisture content of the microbial inoculum and the category of the microbial strain all affect the viable microbial count. Among them, the temperature of the storage environment is one of the important factors affecting the viable cell count in the microbial inoculant. Under the condition of low temperature, the activity of microorganisms is generally low and its growth metabolic rate is correspondingly slowed down, thus effectively achieving the effect of prolonging the storage time of microbial strains. However, for multiple microbial inoculants, long-term storage under constant temperature conditions will increase production costs, thus limiting its further industrialization and application. Therefore, we prefer to store under room temperature conditions. From the table, it can be found that the viable cell count of the microbial inoculum remains basically stable within 45 days.

<Example 5: Selection of inoculation amount>
The amount of inoculum (mass ratio of the microbial inoculant to the amount of food waste awaiting treatment) of 0.5%, 1%, 1.5%, 2%, 3%, and 5%, respectively, was applied to the food waste treatment facility. After 48 hours of operation after inoculation, the decomposition proportions of garbage starch, protein and oil were determined in different inoculum experiments. It is as shown in Table 5.

From the practical results, it is preferable that the inoculum amount of 1.5% is suitable for the multi-microbial inoculant input proportion, so that better effects can be obtained under the condition of the lowest possible cost.

<Example 6: Selection of temperature>
The decomposition treatment result (garbage decomposition rate) of the microbial inoculant is embodied in the weight reduction efficiency.
Weight loss rate (%) = (A - B) / A x 100%
Among them, A: total weight (kg) of raw garbage and microbial inoculant put in; B: total weight (kg) of residue after treatment.

A test was carried out on food waste collected from Luxiu Canteen of Zhejiang University of Technology. Test process: Excluding large lumps and hard parts such as bones, plastic pipettes, etc. in the food waste, the water content of the food waste was measured to be 70-75%. After adjusting the moisture content to 55 to 65% with wood chips, at 35 ° C., 50 ° C., and 55 ° C., respectively, 1: 1: 1: 1 ratio (mass ratio of dry fungus weight) prepared in Example 3 ) was put into the microbial inoculant of ), and a 48-hour garbage biodegradation test was performed. , the weight reduction rate of food waste is 79.38%, and when the temperature is 55° C., the weight reduction rate of food waste is 82.35%.

<Example 7: Application of microbial inoculant>
The decomposition treatment result (garbage decomposition rate) of the microbial inoculant is embodied in the weight reduction efficiency.
Weight loss rate (%) = (AB) ÷ A × 100%
Among them, A: total weight (kg) of garbage and microbial inoculant put in; B: total weight (kg) of residue after treatment.

A test was carried out on food waste collected from Luxiu Canteen of Zhejiang University of Technology. The test process is as follows.

(1) A composition analysis was performed on the collected samples. The steps are as follows. Vegetables, staple foods (rice, side dishes), meat and other impurities in the samples were manually screened, and then mass weighing was performed on each screened composition with an electronic balance. The results of the weighing were converted into percentages of each composition in the total mass of the food waste sample (Table 6). Moisture content was measured by drying the samples at 105° C. and running until the 24 hour quality loss was less than 0.5%. Moisture content of garbage is 70-75%.

(2) Put 1.5% of the microbial inoculant prepared in Example 3 above at a ratio of 0.5:1:0.5:1 (mass ratio of dry cell weight), and put it in the garbage decomposition machine. placed. The temperature was 55° C., and after adding 5 kg of food waste, the system moisture was adjusted to 55-65% with wood chips. Then, after 24 hours of ventilation, 2 kg of material was added. Thereafter, the material was replenished once (2 kg) every 48 h. After running for 240 hours, the leftovers were taken out and weighed. Since 2.15 kg was obtained after removing the added amount of wood chips, the weight reduction rate was about 83.46%. The acid hydrolysis method determined the fat content in the retentate. The protein content in the retentate was determined by the Kjeldahl method. The fat and oil decomposition rate was 9.36%, and the protein decomposition rate was 63.12%. The residue is sepia grainy or powdery, as shown in FIG.

<Example 8: Application of microbial inoculant>
The decomposition treatment result (garbage decomposition rate) of the microbial inoculant is embodied in the weight reduction efficiency.
Weight loss rate (%) = (A - B) / A x 100%
Among them, A: total weight (kg) of garbage and microbial inoculant put in; B: total weight (kg) of residue after treatment.

A test was carried out on food waste collected from Luxiu Canteen of Zhejiang University of Technology. The test process is as follows.

(1) As a result of excluding large lumps and hard parts such as bones and plastic straws in the garbage, the moisture content of the garbage was 70-75%.
(2) 1.5% of the microbial inoculant prepared in Example 3 was added at a ratio of 1:1:1:1 (mass ratio of dry cell weight), and placed in a garbage decomposition machine. The temperature was 55°C. After 15 kg of food waste was added, the moisture content of the system was adjusted to 55-65% with wood chips. Then, after 48 h of draft operation, the contents of ammonia gas and hydrogen sulfide were measured with a "five-in-one" gas inspection instrument. After replenishing the material once (15 kg), after running continuously for 48 hours, after removing the added amount of wood chips, 5.6 kg was obtained, and the weight reduction rate was about 81.3%. After the food waste and the microbial inoculant were mixed together, it became a grayish brown paste for the first time. Or, it turned out to be powdery.

Odor detection: First, with the sense of smell, the gas produced during the garbage decomposition process was experienced to determine whether there was an odor. Ammonia gas and hydrogen sulfide content were then measured with a "five-in-one" gas inspection instrument. As shown in Figure 2, the conversion formula for ppm and mg/ ^m3 :
ppm=(22.4×mg/m ³ )/molecular weight of gas to be measured The molecular weight of ammonia gas is 17.031 and the molecular weight of hydrogen sulfide is 34.08. The odor threshold intensity grading is shown in Table 3.

After 48 hours of biodegradation, the food waste has a weak residual odor and resembles pickled pickled vegetables. After that, as a result of measuring with a gas inspection instrument, the final NH ₃ content was 8.32 mg/m ³ and the H ₂ S content was 0.53 mg/m ³ , so the microbial inoculant was viable. It was explained that during the garbage decomposition process, no obvious off-flavours were produced.

<Example 9: Application of microbial inoculant>
A test was carried out on food waste collected from Luxiu Canteen of Zhejiang University of Technology. The test process is as follows.

(1) As a result of excluding large lumps and hard parts such as bones and plastic straws in the garbage, the moisture content of the garbage was 65 to 75%.
(2) 1.5% of the microbial inoculant prepared in Example 3 was added at a ratio of 1:1:1:1 (mass ratio of dry cell weight), and placed in a garbage decomposition machine. After adding 10 kg of garbage, the system moisture was adjusted to 55% with wood chips. After that, it was ventilated for 12 hours at 45°C. After adjusting the temperature to 55° C. and continuously running for 12 hours, the remaining amount of decomposition residue was about 0.75 kg, and the weight reduction rate was about 92.5%. The starch decomposition rate was 87.51%, the protein decomposition rate was 91.35%, and the oil and fat decomposition rate was 29.12%. After the garbage and the microbial inoculant were mixed for 24 hours, no obvious offensive odor was generated, the _NH3 content was 6.42 mg/ ^m3 , and the _H2S content was 0.46 mg/ ^m3 .

<Example 10: Application of microbial inoculant>
A test was carried out on food waste collected from Luxiu Canteen of Zhejiang University of Technology. The test process is as follows.

(1) As a result of excluding large lumps and hard parts such as bones and plastic straws in the garbage, the moisture content of the garbage was 65 to 75%.
(2) 1.5% of the microbial inoculant prepared in Example 3 was added at a ratio of 1:1:1:1 (mass ratio of dry cell weight), and placed in a garbage decomposition machine. After adding 10 kg of garbage, the system moisture was adjusted to 55% with wood chips. Then, after 12 hours of ventilation at 45°C, the temperature was adjusted to 55°C, and after 36 hours of operation, the residual amount of decomposition residue was about 0.53 kg, and the weight reduction rate was about 0.53 kg. It was 94.7%. The starch decomposition rate was 90.37%, the protein decomposition rate was 91.35%, and the oil and fat decomposition rate was 27.58%. After the garbage and the microbial inoculant were mixed for 24 hours, no obvious offensive odor was produced, the _NH3 content was 6.35 mg/ ^m3 , and the _H2S content was 0.33 mg/ ^m3 .

Claims (10)

the following bacteria, i.e.
(1) Pichia kluyveri yeast with strain number ZJB-091 and accession number CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) Bacillus thuringiensis bacilli with strain number ZJB19165 and accession number CCTCC NO:M 2020015; and (4) strain number ZJB19166 with accession number CCTCC NO:M. Bacillus paralicheniformis bacilli which is 2020016;
A microbial inoculant for high-temperature biodegradation of garbage, characterized in that it contains all of Mass of Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165, Bacillus paralicheniformis bacillus ZJB19166 The microbial inoculant for high temperature biodegradation of garbage according to claim 1, characterized in that the ratio is 1:1-2:1:1-2. Drying of Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringi ensis bacillus ZJB19165, Bacillus paralicheniformis bacillus ZJB19166 3. The microbial inoculant for high-temperature biodegradation of garbage according to claim 2, wherein the mass ratio of the bacterial mass is 1:1:1:1. the following steps, i.e.
(1) activating and culturing each of the four strains;
(2) Obtaining a seed medium by performing seed culture for each of the four strains after activation;
(3) fermenting the culture solution for each of the four strains to obtain a fermentation solution;
(4) The microbial inoculant for high-temperature biodegradation of garbage is obtained by mixing the fermentation liquids of four strains in proportion. 2. A method for preparing a microbial inoculant for high-temperature biodegradation of garbage according to 1. A method of decomposing garbage, which comprises using the microbial inoculant for high-temperature biodegradation of garbage according to any one of claims 1 to 3. A method of biodegrading garbage, which comprises adding the microbial inoculant for high-temperature biodegradation of garbage according to any one of claims 1 to 3 to garbage waiting to be decomposed. The method for decomposing garbage according to claim 6, characterized in that the biodegradation temperature is 50°C-55°C. The method of garbage decomposition according to claim 7, characterized in that the biodegradation temperature is 55°C. The method of decomposing garbage according to claim 6, characterized in that the inoculum amount of the microbial inoculant for high-temperature biodegradation of garbage is not less than 1% of the mass of the garbage. 10. The method of decomposing garbage according to claim 9, wherein the inoculum amount of the microbial inoculant for high-temperature biodegradation of garbage is not less than 1.5% of the weight of the garbage.

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