JP2021194006A - Microorganism inoculant for high-temperature biodegradation of garbage, and application - Google Patents

Abstract

To provide a microorganism inoculant for high-temperature biodegradation of garbage and an application.SOLUTION: The microorganism inoculant includes the followings: Pichia kluyveri yeast ZJB-091 having the accession number of CCTCC NO:M 2019263, Bacillus licheniformis bacillus ZJB19163 having the accession number of CCTCC NO:M 2020014, Bacillus thuringlensis bacillus ZJB19165 having the accession number of CCTCC NO: M 2020015, Bacillus paralicheniformis bacillus ZJB19166 having the accession number of CCTCC NO:M 2020016. The microorganism inoculant not only accelerates biodegradation of garbage, but also removes offensive odor, and can realize detoxification and volume reduction processing of garbage. Cooperative effect works between microorganism strains, polymeric organic matters in garbage are quickly degraded into small-molecular materials under an aerobic condition to generate a large amount of organic substance and trace element, and conversion to waste matter recycling is realized.SELECTED DRAWING: None

Description

The present invention relates to the field of waste treatment technology, and particularly to a microbial inoculant for high temperature biodegradation of food waste and its application.

Swill, also known as swill juice, leftover food, etc., remains after food processing, processing (including boiling), residual parts of food (eg, vegetable leaves, fruit peels and fruit residue, etc.) or after eating. A general term for abandoned food. With the shift in economic levels and social necessities, people have become more and more concerned about eating and drinking, resulting in a large waste of food and drink. Studies show that half of the food was wasted in the world.

The main components of swill include leftovers of rice and flour foods, vegetables, animal / vegetable oils and meat bones. From the viewpoint of chemical structure, it contains starch, protein, fibrin, lipid and inorganic salt. These compositions have high water content and abundant nutritional characteristics, are prone to rotting and odors, spread bacteria and viruses, affect the cleanliness of the city, pollute the environment, and It harms people's health. However, since the abundant nutrition contained in swill is an extremely valuable renewable resource, effective and rational treatment of swill can always bring high value. In addition, the inhomogeneity of the garbage itself makes its transportation and disposal very inconvenient and costly.

With continued deep penetration into environmental protection and sustainable development principles, due to the irreversible environmental depletion and resource ineffectiveness of traditional garbage disposal methods, including, for example, incineration and sanitary landfill disposal. As people have begun to look for new treatment methods, bio-treatment of garbage has become a hotspot for research and attention due to its unique advantages. For example, the invention application whose publication number is CN105665417A discloses a complex microbial inoculant for high-temperature biodegradation of raw garbage, and its production and usage. The complex microbial inoculum is composed of a complex cell and a carrier, and the complex cell is a mixture of Candida krusei yeast, Bacillus subtilis, Wickerhamomyces anomals yeast, Aspergillus niger and Aspergillus niger. The carrier is composed of soybean meal, bran, rice husk powder, candida and the like. All of these microbial inoculants have a good swill decomposition effect, but due to differences in eating habits, the microenvironment of microbial and the specificity of microbial enzyme production, high temperature microorganisms accelerate the decomposition of swill and the biological reaction time. It will be more advantageous for shortening the cost and reducing energy consumption. Therefore, it is necessary to select a superior strain that has a better ability to decompose swill and can withstand high temperatures to accelerate the biodegradation of swill. This will make garbage harmless, reduce its weight, and recycle it as a resource.

Chinese Patent Application Publication No. 10566541417

The present invention provides a microbial inoculant for high temperature biodegradation of swill and its application, aiming at the deficiencies existing in the existing technology.

The present invention relates to the following bacteria, that is,
(1) Pichia kluyveri yeast whose strain number is ZJB-091 and whose contract number is CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus with strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) Bacillus thuringlensis bacillus with strain number ZJB19165 and accession number CCTCC NO: M 2020015;
(4) Bacillus paralicheniformis bacillus whose strain number is ZJB19166 and whose contract number is CCTCC NO: M 2020016;
It is a microbial inoculator for high temperature biodegradation of swill, including.

In the above-mentioned microbial inoculum for high-temperature biodegradation of garbage, Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165, Bacillus licheniformis bacillus ZJB The mass ratio of Bacillus paralicheniformis bacillus ZJB19166 is 1: 1 to 2: 1: 1 to 2.

Pichia kluyveri yeast ZJB-091, Bacillus licheniformis ZJB19163, Bacillus thuringlensis ZJB19165, Bacillus thuringlensis ZJB19165 The mass ratio of the dry cells weight of the Bacillus paralicheniformis ZJB19166 is 1: 1: 1: 1.

The present invention is in the following steps, i.e.
(1) Activate and culture each of the four strains;
(2) Seed culture is obtained for each of the four activated strains;
(3) Fermenting the culture broth for each of the four strains to obtain the fermented broth;
(4) A method for preparing the above-mentioned swill high-temperature biodegradation microbial inoculant, which comprises obtaining the above-mentioned swill high-temperature biodegradation microbial inoculant by mixing fermented liquids of four kinds of strains in proportion. ..

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

The present invention is a method for decomposing swill by putting the above-mentioned microbial inoculant for high-temperature biodegradation of swill into the swill awaiting decomposition and performing biodegradation.

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

In the method of biodegrading the swill, the biodegradation temperature is 55 ° C.

In the method for decomposing swill, the inoculation amount of the microbial inoculant for high temperature biodegradation of swill does not fall below 1% of the mass of swill.

In the method for decomposing swill, the inoculation amount of the microbial inoculant for high temperature biodegradation of swill does not fall below 1.5% of the mass of swill.

It is a morphology diagram of the swill after the processing in Example 7. FIG. It is a schematic diagram of the garbage biodegradation test in Example 8.

Embodiments of the present invention will be described below. The present embodiment is an example of carrying out the present invention, and the present invention is not limited to the present embodiment.

The microbial inoculant for high temperature biodegradation of garbage is the following bacteria, that is,
(1) Pichia kluyveri yeast whose strain number is ZJB-091 and whose contract number is CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus with strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) Bacillus thuringlensis bacillus with strain number ZJB19165 and accession number CCTCC NO: M 2020015,
(4) Includes Bacillus paralicheniformis bacillus 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 thuringlensis bacillus ZJB19165 and Bacillus licheniformis. Bacillus paralicheniformis) The mass ratio of bacillus ZJB19166 is 1: 1 to 2: 1: 1 to 2.

More preferably, in the microbial inoculum, Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165 and Bacillus licheniformis. (Bacillus paralicheniformis) The mass ratio of the dry cells weight of the bacillus ZJB19166 is 1: 1: 1: 1.

Pichia kluyveri yeast ZJB-091 has been deposited at the China Typical Culture Storage Center (CCTCC), the contract number is CCTCC NO: M 2019263, and the contract time is April 17, 2019. be. The 18s rDNA sequence of Pichia kluyveri yeast ZJB-091 is as shown by SEQ ID NO.1. The biological characteristics of the Pichia kluyveri yeast ZJB-091 are as follows. White to cream colony, moist and sticky, easy to wear, the front, back and center of the colony match the color of the edges, with pseudohyphae and fruit during the growth process It has a flavor like that. Shows a spherical or oval shape. It can produce amylase, proteolytic enzymes, lipases and cellulases.

Bacillus licheniformis bacillus ZJB19163 has been deposited at the China Typical Culture Storage Center (CCTCC) with a deposit number of CCTCC NO: M 2020014 and a deposit time of January 6, 2020. The 16S rRNA sequence of Bacillus licheniformis bacillus ZJB19163 is as shown by SEQ ID NO.2. The biological characteristics of the Bacillus licheniformis bacillus are as follows. The colonies are round, pale yellow, slightly white, smooth and sticky on the surface, opaque, well-ordered, wrinkle-free, and produce amylase, proteolytic enzymes and lipases. can.

Bacillus thuringlensis bacillus ZJB19165 has been deposited at the China Typical Culture Storage Center (CCTCC), the deposit number is CCTCC NO: M 2020015, and the commission time is January 6, 2020. The 16S rRNA sequence of Bacillus thuringlensis ZJB19165 is as shown by SEQ ID NO.3. The biological characteristics of the Bacillus thuringlensis bacillus are as follows. The colonies are round, pale yellow, smooth on the surface, opaque, well-ordered, wrinkle-free, rod-shaped, and belong to spore-forming fungi.

Bacillus paralicheniformis bacillus ZJB19166 has been deposited at the China Typical Culture Storage Center (CCTCC), the deposit number is CCTCC NO: M 2020016, and the commission time is January 6, 2020. be. The 16S rRNA sequence of Bacillus paralicheniformis bacillus ZJB19166 is as shown by SEQ ID NO.4. The biological characteristics of the Bacillus paralicheniformis bacillus ZJB19166 are as follows. The colonies are round, pale yellow, smooth on the surface, opaque, well-ordered, wrinkle-free, rod-shaped, and belong to spore-forming fungi. It can produce amylase, proteolytic enzymes, lipases and cellulases.

The present invention also provides a method for preparing the microbial inoculant. The following steps, i.e.
(1) Activate and culture each of the four strains;
(2) Seed culture is obtained for each of the four activated strains;
(3) Fermenting the culture broth for each of the four strains to obtain the fermented broth;
(4) The above-mentioned microbial inoculant is obtained by mixing fermented liquids of four strains in proportion.

Specifically, the method for preparing the microbiological inoculant fine is described in the following steps, that is,
(1) Inoculate Pichia kluyveri yeast ZJB-091 into PDA slope medium and incubate at a constant temperature to activate the strain.
(2) Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 To perform activation culture of the strain by culturing in
(3) After inoculating the activated Pichia kluyveri yeast ZJB-091 into the PDA slope medium and culturing the seeds, the seed medium of the Pichia kluyveri yeast is obtained. What to do; Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165 and Bacillus paralicheniformis pebbles 166 Then, after seed culture, Bacillus licheniformis (Bacillus licheniformis) bacillus ZJB19163, Bacillus thuringlensis (Bacillus thuringlensis) bacillus ZJB19165 and Bacillus paralicheniformis (Bacillus paralicheniformis) 166 seed bacterium Z ;
(4) Pichia kluyveri yeast ZJB-091 seed medium and Bacillus licheniformis Bacillus licheniformis ZJB19163, Bacillus thuringlensis Bacillus thuringlensis ZJB19165 and Bacillus licheniformis para After inoculating the seed medium of the bacillus ZJB19166 into a fermented can containing the fermentation medium and culturing the fermentation, Pichia kluyveri yeast ZJB-091 fermented liquid and Bacillus licheniformis bacillus ZJB19163, respectively. , Obtaining fermented liquids of Bacillus thuringlensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166;
(5) Pichia kluyveri yeast ZJB-091 fermented liquor and Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165 and Bacillus licheniformis para After proportionally mixing the fermented liquor of ZJB19166, immobilizing it in a ratio of 1:20 using 5 to 150th wheat stalk powder, and then performing low-temperature drying, a microbial inoculant for raw garbage biodegradation. Is to get.

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

In step (2), the composition of the peptone slope medium of beef pesto 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 for 12 to 24 hours at 35 to 55 ° C.

In step (3), the composition of the PDA liquid medium is as follows. 200 g of potato, 20 g of glucose, 1 L of water; seed culture conditions: under the conditions of 25 to 40 ° C. and 150 to 200 r / min, the cells were cultured in a shaking bed for 24 to 48 hours. 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: under the conditions of 35 to 55 ° C. and 150 to 200 r / min, the cells were shake-cultured in a shaker for 24-48 hours.

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

The present invention also provides the application of the microbial inoculant in the decomposition of swill.

The present invention also provides a method for decomposing swill. That is, the microbial inoculant is put into the swill that is waiting for decomposition and biodegraded. The biodegradation temperature is preferably 55 ° C.

Compared with the existing technique, the present invention has the following beneficial effects.
(1) The microbial inoculant provided by the present invention not only accelerates the biodegradation of swill, but also removes offensive odors, and can realize detoxification and weight reduction treatment of swill.
(2) According to the present invention, by operating the complex microbial inoculant for the treatment of raw garbage, a cooperative effect is effective between each microbial strain, and high molecular weight organic matter in raw garbage is produced under aerobic conditions. To quickly decompose into small molecule substances to generate a large amount of organic substances and trace elements, and to realize the conversion of waste resources.
(3) The microbial inoculant provided by the present invention can effectively suppress the growth of harmful microorganisms at 55 ° C. and realize a detoxification treatment of swill.
(4) After the microbial inoculant provided by the present invention acts on swill, no offensive odor is generated, and the prospect of application is good.

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 high-temperature-degrading swill bacteria>
5 g of soil sample near Zhejiang Institute of Technology's Koshu Shokudo was taken, placed in a sterilized small glass ball and an Erlenmeyer flask with 50 mL of sterile water, and cultured at 55 ° C. with 180 r / min shaking, and enriched for one week. Later, gradient dilution of the bacterial suspension was performed on a super clean bench. Set the concentration gradient to 10 ^-1 , ^10-2 , 10 ^-3 , 10 ^-4 , ^10-5 , ^10-6 , ^10-7 , ^10-8 , ^10-9 , and prepare 100 μL of bacterial suspension at each concentration. After sucking and inoculating the PPA slope and the Peptone slope medium of beef pesto, respectively, the panel was sealed with a sealing film, then turned upside down and placed in a constant temperature incubator at 28 to 40 ° C. to incubate for 24 to 48 hours. Then, a panel with an appropriate number of colonies was selected, a typical colony was selected, and after separation and purification, the protein, starch, fat and fibrin identification medium panels were inoculated, respectively. After that, it was uniformly applied with a sterilized application rod. Then, the petri dish was sealed with a sealing film, turned upside down, and cultured in a constant temperature incubator at 28 to 40 ° C. for 48 to 37 hours, and then the diameter of the lysis zone and the colony diameter ratio were observed. Strains with relatively large lysis zones are inoculated into PDA liquid medium and Peptone liquid medium of beef pesto, respectively, and after culturing for 24 hours, an appropriate amount of bacterial solution is taken and 30% glycerin is used in a 1: 1 ratio. Stored in a refrigerator at 80 ° C. A part of the bacterial solution was taken and morphologically, physiologically biochemically and molecularly biologically evaluated.

Strain genomic DNA was extracted using the FastDNA ™ Spin Kit for Soil Reagent Kit, and then PCR amplification was performed using this as a template. The amplification primers are as follows.

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

The bacterial PCR system is as shown in Table 1.

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

The PCR system for fungi is as shown in Table 2.

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

As a result of PCR product sequencing by Hangzhou Tsingke Biotechnology Co., Ltd., the sequences obtained are SEQ ID NO.1, SEQ ID NO.2, SEQ ID. It is as shown by NO.3 and SEQ ID NO.4.

After inputting the obtained DNA sequence into GenBank, it was collated with all the sequences in the database according to the Blast procedure. After that, in combination with the morphological and physiological and biochemical identification results, the strains obtained by selection were Pichia kluyveri yeast, Bacillus licheniformis 杆, and Bacillus, respectively. After confirming that they are thuringlensis and Bacillus paralicheniformis, they are Pichia kluyveri yeast ZJB-091, Bacillus licheniformis, Bacillus licheniformis, and Bacillus licheniformis, respectively. It was named Bacillus thuringlensis ZJB19165 and Bacillus paralicheniformis ZJB19166.

Pichia kluyveri yeast ZJB-091, Bacillus licheniformis Bacillus licheniformis ZJB19163, Bacillus thuringlensis Bacillus ZJB19165 and Bacillus paralicheniformis It has been deposited at the Culture Storage Center (CCTCC). Specific information is as follows.

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

Address of China Typical Culture Storage Center (CCTCC): Wuhan University China Typical Culture Storage Center (CCTCC), Yaichi Road, Hongshan District, Wuhan City, Hubei Province.

The medium components related to the method are as follows.
PDA solid medium: potato 200 g, glucose 20 g, agar 20 g, water 1 L, pH natural.
PDA liquid medium: potato 200 g, glucose 20 g, water 1 L, pH natural.
The composition of the beef pesto peptone solid medium is as follows. Beef pesto 5g, peptone 10g, sodium chloride 5g, agar 20g, water 1L, pH natural.
The composition of the Peptone liquid medium of beef pesto is as follows. Beef pesto 5g, peptone 10g, sodium chloride 5g, water 1L, pH natural.
Protein identification medium: defatted milk powder 50 g, soluble starch 10 g, yeast extract _{5g, KH 2 PO 4 1g,} MgSO 4 · 7H 2 O 0.2g, agar 20g, water 1L, pH 7.0.
Starch identification medium: 10 g of peptone, 2 g of soluble starch, 5 g of beef pesto, 5 g of NaCl, 20 g of agar, 1 L of water, pH 7.0-7.2.
Fat Identification medium: peptone _{10g, K 2 HPO 4 1g,} MgSO 4 · 7H 2 O 0.5g, polyvinyl alcohol 1.2g, Victoria blue B 0.04 g, olive oil 10 g, agar 20g, water 1L, pH 8.0 ..
Cellulose Identification _{medium: K 2 HPO 4 0.5g, MgSO} 4 · 7H 2 O 0.2g, CMC-Na 2g, Congo Red 0.2 g, agar 16g, gelatin 2g, water 1L, pH 7.0.

<Example 2: Strain enzyme activity measurement>
The isolated and obtained Pichia kluyveri yeast ZJB-091 was inoculated into a PDA liquid medium, and Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165. Bacillus paralicheniformis bacillus ZJB19166 was cultured in a shaker at 55 ° C. with shaking for 48 hours. The fermented liquor was centrifuged at 4 ° C. and 8000 r / min for 10 min, and then the supernatant was taken to measure the enzymatic activity of amylases, proteolytic enzymes, lipase and cellulase. The DNS method was used to measure the amylase activity. Definition of enzyme activity: The amount of enzyme required to produce 1 μg maltose per minute with 1 mL enzyme solution is one enzyme activity unit and is indicated by "U / mL". The Folin-Ciocalt method was used to measure the activity of proteolytic enzymes. Definition of enzyme activity: The amount of enzyme required to hydrolyze casein with 1 mL of enzyme solution every minute to produce 1 μg tyrosine is one enzyme activity unit and is indicated by "U / mL". .. The national standard GB / T 23535-2009 is used to measure lipase activity. Definition of enzyme activity: 1 mL of enzyme solution hydrolyzes the substrate to 1 min at constant temperature and pH (55 ° C., pH 7.0 was used in this example) (olive in this example). The amount of enzyme required to produce 1 μmol of titable fatty acid (using oil as a substrate) is one enzyme activity unit and is indicated by “U / mL”. The 3,5-dinitrosalicylic acid method was used to measure cellulase activity. The amount of enzyme required to produce 1 μg glucose per minute with 1 mL enzyme solution is one enzyme activity unit and is indicated by “U / mL”. The results are as shown in Table 3.

<Example 3: Preparation of microbiological inoculant fine>
[1] Slope culture Pichia kluyveri yeast ZJB-091 was inoculated on the PDA slope, and Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19 Bacillus paralicheniformis bacillus ZJB19166 was inoculated into the peptone slope medium of beef pesto, and then cultured at a constant temperature of 24 to 48 hours at 25 to 40 ° C. for 24 to 48 hours, respectively, to activate the strain. Activated PDA slope medium composition used: potato 200 g, glucose 20 g, agar 20 g, water 1 L, pH natural. It was sterilized for 30 minutes at 115 ° C. Peptone slope medium composition of beef pesto used: beef pesto 5 g, peptone 10 g, sodium chloride 5 g, agar 20 g, water 1 L, pH natural. It was sterilized for 20 minutes at 121 ° C.

[2] Seed medium The activated Pichia kluyveri yeast ZJB-091 obtained in step [1] was inoculated into a potato / glucose liquid medium. Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 infused into beef pe. After culturing with shaking at 25 to 40 ° C. and 150 to 200 r / min shaker for 24 to 48 hours, Pichia kluyveri yeast ZJB-091, Bacillus licheniformis bacillus ZJB19163, and Bacillus licheniformis, respectively. thuringlensis) bacillus ZJB19165 and Bacillus paralicheniformis bacillus ZJB19166 seed medium were obtained. The potato / glucose liquid medium used is as follows. Potato 200g, glucose 20g, water 1L, pH natural. It was sterilized for 30 minutes at 115 ° C. The composition of the Peptone liquid medium of beef pesto is as follows. Beef pesto 5g, peptone 10g, sodium chloride 5g, water 1L, pH natural. It was sterilized for 20 minutes at 121 ° C.

[3] Expansion culture:
Put a medium with a volume ratio of 50 to 67% in a fermentation can, and then obtain Pichia kluyveri yeast ZJB-091, Bacillus licheniformis 杆 bacterium ZJB19163, Bacillus licheniformis obtained in step [2]. Bacillus thuringlensis ZJB19165 and Bacillus paralicheniformis seed medium were inoculated into fermentation cans containing a medium having a volume ratio of 50 to 67%, and then 30 ° C to 45. Fermentation culture was carried out for 36 to 80 hours under the conditions of ° C. and 200 to 450 r / min. Pichia kluyveri yeast ZJB-091, Bacillus licheniformis ZJB19163, Bacillus thuringlensis Bacillus ZJB19165 and Bacillus paralicheniformis Obtained.

Liquid medium used in step [3]: Potato / glucose liquid medium was taken as 1 L, 1 L of water was added to 200 g of potato, boiled for 20 minutes, and then filtered to remove the filtrate. Then, after adding 20 g of glucose, water was added and replenished to 1 L. It was sterilized for 30 minutes at 115 ° C. Using 1 L of beef pesto's peptone liquid medium, beef pesto was 5 g, peptone was 10 g, and sodium chloride was 5 g, and the mixture was sterilized at 121 ° C. for 20 min.

(2) Preparation of complex microbial inoculant Bacillus licheniformis cultivated Pichia kluyveri yeast ZJB-091, Bacillus licheniformis 杆 bacterium ZJB19163, Bacillus thuringlensis 杆 bacterium ZJB19165 Bacillus paralicheniformis (Bacillus paralicheniformis) Bacillus ZJB19166 bacterial solution was centrifuged at 8000 rpm for 10 minutes in a centrifuge (desktop) high-speed freezer centrifuge, respectively, and then obtained Pichia kluyveri yeast ZJB-091, Bacillus licheniformis Bacillus licheniformis (Bacillus licheniformis) Bacillus ZJB19163, Bacillus thuringlensis (Bacillus thuringlensis) Bacillus paralicheniformis (Bacillus paralicheniformis) Bacillus ZJB19166 By water mixing in the proportion of .5: 1) or (1: 1: 1: 1), different bacterial cell-proportional microbial cell solutions were obtained. Then, the wheat stalk powder of the 5th to 150th order was used to immobilize in a proportion of 1:20, and then low-temperature drying was performed to obtain a complex microbial inoculant. The drying temperature was 30 to 50 ° C., and the viable cell count was ≧ 3.0 × 10 ⁹ CFU / mL.

<Example 4: Evaluation of microbial inoculant stability>
The complex microbial inoculum was placed in a corner of the laboratory and stored at room temperature. Samples were taken every 15 days and the change in viable cell count was analyzed. The viable cell count was measured by the dilution panel application method.

During the microbial inoculum storage period, different environmental temperatures, water content of the microbial inoculant, category of microbial strain, etc. all affect the viable microbial count. Among them, the temperature of the storage environment is one of the important factors that affect the viable cell count in the microbial inoculant. Under low temperature conditions, the activity of the microorganism is universally low and its growth and metabolism rate is correspondingly slowed, so that the effect of prolonging the storage time of the microbial strain is effectively achieved. However, for complex microbial inoculants, long-term storage under constant temperature conditions increases production costs and thus limits their further industrialization and application. Therefore, we choose to store under room temperature conditions. From the table, it can be found that the viable cell count of the microbial inoculant is basically maintained stable within 45 days.

<Example 5: Selection of inoculation amount>
Inoculation amounts of 0.5%, 1%, 1.5%, 2%, 3%, and 5% (mass ratio of the mass of the microbial inoculant to the garbage waiting to be processed) are used for the microorganisms in the garbage processing facility. After inoculating the inoculant and running for 48 hours, the decomposition proportions of raw garbage starch, protein and fat in different inoculation volume experiments were measured. It is as shown in Table 5.

From the results of the implementation, it is preferable that the inoculation amount of 1.5% is proportional to the input of the complex microbial inoculant, and a better effect can be obtained under the condition of the lowest possible cost.

<Example 6: Temperature selection>
The decomposition treatment result (dust decomposition rate) of the microbial inoculant is embodied in the weight reduction efficiency.
Weight loss rate (%) = (AB) ÷ A × 100%
Among them, A: total weight of added garbage and microbial inoculant (kg); B: total weight of post-treatment residue (kg).

A test was conducted using the swill collected at the Zhejiang Institute of Technology's Huishu Shokudo. Test process: As a result of measurement excluding large lumps and hard parts such as bones and plastic pipettes in the swill, the water content of the swill was 70 to 75%. After adjusting the water content with wood chips to 55-65%, 1: 1: 1: 1 proportional (mass ratio of dry cell weight) prepared in Example 3 at 35 ° C, 50 ° C, and 55 ° C, respectively. ), And the 48h garbage biodegradation test was performed. As a result, when the temperature was 35 ° C, the weight loss rate of the garbage was 58.12%, and the temperature was 50 ° C. It was discovered that the weight loss rate of swill is 79.38% and the weight reduction rate of swill is 82.35% when the temperature is 55 ° C.

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

A test was conducted using the swill collected at the Zhejiang Institute of Technology's Huishu Shokudo. The test process is as follows.

(1) The composition of the collected sample was analyzed. The steps are as follows. After manually selecting vegetables, staple foods (rice, face meal), meat and other impurities in the sample, mass weighing was performed on each of the selected compositions with an electronic balance. Based on the weighing results, it was converted into the ratio of each composition to the total mass of the food waste sample (Table 6). Moisture content was measured by drying the sample at 105 ° C. and manipulating until the quality loss for 24 hours fell below 0.5%. Moisture content of garbage 70-75%.

(2) Add 1.5% of the 0.5: 1: 0.5: 1 proportional (mass ratio of dry cell weight) microbial inoculant prepared in Example 3 into the waste decomposition treatment machine. placed. The temperature was 55 ° C., 5 kg of swill was added, and then the system moisture was adjusted to 55-65% with wood chips. Then, after operating for 24 hours, 2 kg of material was replenished. Then, the material was replenished once (2 kg) every 48 hours. After operating for 240 hours, the residue was taken out and weighed. After removing the amount of wood chips added, 2.15 kg was obtained, so that the weight loss rate was about 83.46%. The fat content in the residue was measured by acid hydrolysis. The protein content in the residue was measured by the Kjeldahl method. The fat decomposition rate was 9.36%, and the proteolysis rate was 63.12%. The residue is sepia-colored particles or powdery, as shown in FIG.

<Example 8: Application of microbial inoculant>
The decomposition treatment result (dust decomposition rate) of the microbial inoculant is embodied in the weight reduction efficiency.
Weight loss rate (%) = (AB) ÷ A × 100%
Among them, A: the total weight of the added garbage and the microbial inoculant (kg); B: the total weight of the residuals after the treatment (kg).

A test was conducted using the swill collected at the Zhejiang Institute of Technology's Huishu Shokudo. The test process is as follows.

(1) As a result of measurement excluding large lumps such as bones, plastic straws and hard parts in the swill, the water content of the swill was 70 to 75%.
(2) A 1: 1: 1: 1 proportional (mass ratio of dry cell weight) microbial inoculant prepared in Example 3 of 1.5% was added and placed in a waste decomposition treatment machine. The temperature was 55 ° C. After adding 15 kg of swill, the system moisture was adjusted to 55-65% with wood chips. Then, after operating for 48 hours, the contents of ammonia gas and hydrogen sulfide were measured with a "five-in-one" gas inspection device. From now on, after replenishing the material once (15 kg), and after continuing the operation for 48 hours, after removing the amount of wood chips added, 5.6 kg was obtained, so that the weight loss rate was about 81.3%. The taupe paste was formed for the first time after the mixed action of the swill and the microbial inoculant, but as the decomposition time was continuously extended, the swill was continuously decomposed and utilized, and finally sepia-colored particles. Or it will be in the form of powder.

Smell detection: First, the sense of smell was used to experience the gas generated during the swill decomposition process and determine whether or not there was an odor. After that, the contents of ammonia gas and hydrogen sulfide were measured with a "five-in-one" gas inspection device. As shown in Figure 2, the conversion of ppm and mg / ^{m 3} Official:
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 limit value intensity grading of odors is as shown in Table 3.

After 48 hours of biodegradation, garbage has a weak odor of residue and is similar to dried plums. Then, a result of measuring a gas testing equipment, eventually a NH ₃ content of 8.32mg / ^{m 3,} since _{H 2} S content is 0.53 mg / ^{m 3,} the microorganism inoculant is, raw It was explained that no obvious offensive odor was produced during the waste decomposition process.

<Example 9: Application of microbial inoculant>
A test was conducted using the swill collected at the Zhejiang Institute of Technology's Huishu Shokudo. The test process is as follows.

(1) As a result of measurement excluding large lumps such as bones and plastic straws and hard parts in the swill, the water content of the swill was 65 to 75%.
(2) A 1: 1: 1: 1 proportional (mass ratio of dry cell weight) microbial inoculant prepared in Example 3 of 1.5% was added and placed in a waste decomposition treatment machine. After adding 10 kg of swill, 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 continuing the operation for 12 hours, the surplus amount of the decomposition residue was about 0.75 kg, and the weight loss rate was about 92.5%. The starch decomposition rate was 87.51%, the protein decomposition rate was 91.35%, and the fat decomposition rate was 29.12%. After the mixed action of the swill and the microbial inoculant for 24 hours, no obvious offensive odor was generated, the NH ₃ ^{content was 6.42 mg / m 3} , and the H ₂ S content was 0.46 mg / m ³ .

<Example 10: Application of microbial inoculant>
A test was conducted using the swill collected at the Zhejiang Institute of Technology's Huishu Shokudo. The test process is as follows.

(1) As a result of measurement excluding large lumps such as bones and plastic straws and hard parts in the swill, the water content of the swill was 65 to 75%.
(2) A 1: 1: 1: 1 proportional (mass ratio of dry cell weight) microbial inoculant prepared in Example 3 of 1.5% was added and placed in a waste decomposition treatment machine. After adding 10 kg of swill, the system moisture was adjusted to 55% with wood chips. Then, after operating for 12 hours under 45 ° C, adjusting the temperature to 55 ° C, and then operating for 36 hours, the surplus amount of decomposition residue is about 0.53 kg, and the weight loss rate is about. It was 94.7%. The starch decomposition rate was 90.37%, the protein decomposition rate was 91.35%, and the fat decomposition rate was 27.58%. After the mixed action of the swill and the microbial inoculant for 24 hours, no obvious offensive odor was generated, the NH ₃ ^{content was 6.35 mg / m 3} , and the H ₂ S content was 0.33 mg / m ³ .

Claims (10)

The following bacteria, ie
(1) Pichia kluyveri yeast whose strain number is ZJB-091 and whose contract number is CCTCC NO: M 2019263;
(2) Bacillus licheniformis bacillus with strain number ZJB19163 and accession number CCTCC NO: M 2020014;
(3) Bacillus thuringlensis bacillus with strain number ZJB19165 and accession number CCTCC NO: M 2020015;
(4) Bacillus paralicheniformis bacillus whose strain number is ZJB19166 and whose contract number is CCTCC NO: M 2020016;
A microbial inoculant for high temperature biodegradation of swill, characterized by containing. Pichia kluyveri bacillus ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165, Bacillus paralicheniformis 166 , 1: 1 to 2: 1: 1 to 2, the microbial inoculum for high-temperature biodegradation of raw garbage according to claim 1. Bacillus licheniformis bacillus ZJB-091, Bacillus licheniformis bacillus ZJB19163, Bacillus thuringlensis bacillus ZJB19165, Bacillus paralicheniformis 166 The microbial inoculum for high-temperature biodegradation of raw garbage according to claim 2, wherein the mass ratio of weight is 1: 1: 1: 1. The following steps, i.e.
(1) Activate and culture each of the four strains;
(2) Seed culture is obtained for each of the four activated strains;
(3) Fermenting the culture broth for each of the four strains to obtain the fermented broth;
(4) Any one of claims 1 to 3, which comprises obtaining the above-mentioned microbial inoculant for high-temperature biodegradation of swill by mixing fermented liquids of four kinds of strains in proportion. The method for preparing a microbial inoculant for high-temperature biodegradation of swill described in 1. Application of the microbial inoculant for high temperature biodegradation of swill according to any one of claims 1 to 3 in swill decomposition. A method for decomposing swill, which comprises putting the microbial inoculant for high-temperature biodegradation of swill according to any one of claims 1 to 3 into swill awaiting decomposition and performing biodegradation. The method for decomposing swill according to claim 6, wherein the biodegradation temperature is 50 ° C to 55 ° C. The method for decomposing swill according to claim 7, wherein the biodegradation temperature is 55 ° C. The method for decomposing swill according to claim 6, wherein the inoculation amount of the microbial inoculator for high-temperature biodegradation of swill is not less than 1% of the mass of swill. The method for decomposing swill according to claim 9, wherein the inoculation amount of the microbial inoculator for high-temperature biodegradation of swill is not less than 1.5% of the mass of swill.

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