CN109402013B - High-temperature-resistant bacillus subtilis for producing lipase and application of bacillus subtilis in composting - Google Patents

High-temperature-resistant bacillus subtilis for producing lipase and application of bacillus subtilis in composting Download PDF

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CN109402013B
CN109402013B CN201811381862.8A CN201811381862A CN109402013B CN 109402013 B CN109402013 B CN 109402013B CN 201811381862 A CN201811381862 A CN 201811381862A CN 109402013 B CN109402013 B CN 109402013B
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bacillus subtilis
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microbial agent
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李秀芬
齐希光
任月萍
王新华
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Jiangnan University
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    • C05F17/00Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention discloses high-temperature resistant bacillus subtilis for producing lipase and application thereof in composting, belonging to the technical field of environmental biology. The high-temperature-resistant bacillus subtilis B211 in the microbial inoculum prepared by the invention has good capability of degrading fat in oily kitchen waste, can be well symbiotic with other strains, and has scientific and reasonable compounding proportion.

Description

High-temperature-resistant bacillus subtilis for producing lipase and application of bacillus subtilis in composting
Technical Field
The invention relates to high-temperature resistant bacillus subtilis for producing lipase and application thereof in composting, belonging to the technical field of environmental biology.
Background
China has a large population, is a large country for generating garbage, and has more than two hundred million tons of urban garbage generation amount per year according to urban garbage statistical data issued by the Ministry of construction; still more than 1500 counties produce nearly 0.7 million tons of waste; in the aspect of garbage of villages and towns, accurate statistical data is not available for a while because the quantity of the villages and the towns is too dispersed. In general, the domestic garbage production amount in China is more than four hundred million tons. The most main constituent part of domestic garbage in China is kitchen garbage which is more than 60 percent, and some areas even reach 70 to 80 percent, while the main domestic garbage in Europe and America is paper, and the kitchen garbage only accounts for 25 percent. In addition, the water content of the kitchen waste is high; easy decay and deterioration and environmental pollution. The organic matter content of the kitchen waste is high, and the aerobic composting treatment is one of important ways for realizing the recycling and the harmlessness of the kitchen waste.
However, the kitchen waste sometimes has a crude fat content of 25%, the higher the crude fat content is, the higher the material viscosity is, the poorer the air permeability and the loose degree are, an anaerobic environment is easily generated in the aerobic composting process, and the aerobic composting process is not facilitated. On the other hand, the fat is basically insoluble in water, the microbial degradation effect is poor, and when the temperature is higher, the fat in the kitchen waste is easy to emulsify, so that the contact state of the fat and the microorganisms can be improved, and the degradation efficiency is improved. Therefore, the screening of high-temperature-resistant fat degrading bacteria and the research and development of the compound microbial agent containing different microbial strains and compound proportions have great significance for accelerating the composting process of the oily kitchen waste, improving the composting efficiency and improving the quality of finished products.
Disclosure of Invention
The invention aims to provide a high-temperature resistant bacillus subtilis B211 for producing lipase, which is preserved in China Center for Type Culture Collection (CCTCC) in 7-13.2018, wherein the preservation number is M2018475, and the preservation address is China, Wuhan and Wuhan university. The fermentation culture conditions of the strain are as follows: the temperature is 25-37 ℃, the ventilation volume is 0.8-1.5vvm, the stirring speed is 200-.
The second purpose of the invention is to provide a microbial agent containing the high-temperature-resistant bacillus subtilis B211.
In one embodiment of the invention, the microbial agent comprises bacillus subtilis B211, lysine bacillus fusiformis (Lysinibacillus fusiformis), bacillus licheniformis B105, bacillus brevis B205 and Trichoderma atroviride (Trichoderma atroviride).
In one embodiment of the invention, the Bacillus licheniformis (Bacillus licheniformis misstrain) B105 is preserved in China center for type culture Collection in 2018, 7 and 13 months, with the preservation number of CCTCC No: M2018470, and the preservation address of China, Wuhan university.
In one embodiment of the invention, the Bacillus brevis (Bacillus therobrastine) B205 is preserved in China center for type culture Collection in 7 and 13 months in 2018, with the preservation number of CCTCC No: M2018472, and the preservation address of China, Wuhan university.
In one embodiment of the invention, the lysine bacillus fusiformis (Lysinibacillus fusiformis) and Trichoderma atroviride (Trichoderma atroviride) may be selected from commercially available strains including, but not limited to, Lysinibacillus fusiformis tcc7055, Trichoderma atroviride IMI 206040.
In one embodiment of the present invention, the ratio of viable count of Bacillus subtilis B211, lysine bacillus fusiformis, Bacillus licheniformis B105, Bacillus brevis B205 and Trichoderma atroviride is 18-28:2-3:0.5-1.5:800-1200: 2.8-3.3.
In one embodiment of the present invention, the total viable count of the microbial agent is 1.0 × 109-1.0×1010CFU/mL。
In one embodiment of the present invention, the total viable count of the microbial agent is 1.0 × 109-1.0×1010CFU/g。
The third purpose of the invention is to provide the application of the microbial agent.
In one embodiment of the invention, the application is used for promoting mixed composting of oily kitchen waste, food plant sewage treatment residual sludge and straw.
In one embodiment of the present invention, the preparation method of the microbial agent comprises: respectively inoculating high-temperature resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic short-bud bacillus B205 and trichoderma atroviride into a culture medium to obtain fermentation liquor of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic short-bud bacillus B205 and the trichoderma atroviride, mixing the obtained fermentation liquor in proportion, and adding a carrier auxiliary material to obtain the liquid composite microbial agent containing the high-temperature resistant bacillus subtilis B211.
In one embodiment of the present invention, the carrier auxiliary material includes, but is not limited to, one or more of lignite, zeolite, and stone powder.
In one embodiment of the present invention, the preparation method of the complex microbial agent comprises: mixing high-temperature-resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic Brevibacillus brevis B205 and fermentation liquor of trichoderma atroviride according to a proportion, adding one or more of crushed straws, carbonized straws and bran, and drying by hot air to obtain the solid compound microbial agent containing the high-temperature-resistant bacillus subtilis B211.
In one embodiment of the invention, the fermentation broth for obtaining the high temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105 and the thermophilic brevibacillus brevis B205 is prepared by respectively inoculating the high temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105 and the thermophilic brevibacillus brevis B205 into a nutrient broth culture medium after high temperature sterilization, wherein the inoculation amount is 3-10% (v/v) of the volume of the fermentation culture medium, standing and culturing for 20-40h, the culture temperature of the high temperature resistant bacillus subtilis B211, the fusiform lysine bacillus and the bacillus B105 is 25-37 ℃, the culture temperature of the thermophilic brevibacillus brevis B205 is 30-60 ℃, and when the viable count is more than or equal to 1 × 108And (3) obtaining the fermentation liquor of the high-temperature resistant bacillus subtilis B211, the lysine bacillus fusiformis, the bacillus licheniformis B105 and the bacillus brevis B205 at the CFU/mL.
In one embodiment of the invention, the nutrient broth is a nutrient broth after autoclaving.
In one embodiment of the invention, the ingredients of the nutrient broth medium comprise 3.0g of beef extract, 10.0g of peptone, 5.0g of sodium chloride and 1000mL of water.
In one embodiment of the invention, the fermentation liquor for obtaining trichoderma atroviride is prepared by respectively inoculating trichoderma atroviride into a potato glucose culture medium subjected to high-temperature sterilization, wherein the inoculation amount is 3-10% (v/v) of the volume of the fermentation culture medium, standing and culturing for 20-30h, the culture temperature is 20-30 ℃, and when the viable count is more than or equal to 1 × 108And at CFU/mL, obtaining the fermentation liquor of the trichoderma atroviride.
In one embodiment of the present invention, the potato dextrose culture medium is sterilized at high temperature.
In one embodiment of the invention, the ingredients of the potato dextrose medium comprise 6.0g of potato extract powder, 20.0g of glucose and 1000mL of water.
In one embodiment of the present invention, the method for applying the microbial agent comprises the following steps:
(1) and (3) mixing the compound microbial agent and the compost raw materials according to the ratio of 0.5-1: mixing at a mass ratio of 100;
(2) composting the mixture, measuring the temperature of the pile body, and turning the pile according to the temperature change.
In one embodiment of the invention, the step (2) is composting, the temperature of the compost is measured at 8:00 am and 6:00 pm, the temperature of the compost is turned once a day when the temperature is higher than 55 ℃, the temperature of the compost starts to decrease along with the increase of the composting time, and the turning is stopped when the temperature is lower than 40 ℃, and the composting is finished.
The invention has the beneficial effects that:
(1) the invention discloses a high-temperature resistant bacillus subtilis B211 for producing lipase. The strain produces lipase, is high temperature resistant, has good fat degradation capability and has extremely wide market application prospect.
(2) The compost composite microbial agent provided by the invention can be used for mixing and composting oily kitchen waste, food plant sewage treatment residual sludge and straws, and can better realize resource utilization of the mixed waste.
(3) After the composite microbial agent provided by the invention is piled for 2 days, the degradation rate of fat in the compost raw material can reach 81.9% -95.7%, and the degradation rate of fat in a control experiment is 56.9%.
(4) The microbial agent provided by the invention has obvious advantages in composting and can effectively shorten the temperature rise time of the compost, so that the time required by the compost to reach the high temperature of 55 ℃ is shortened from 62h to 16-32h in a control experiment.
(5) The microbial agent provided by the invention has obvious advantages in composting and can effectively improve the highest temperature of a compost, so that the highest temperature of the compost is improved to 61-66 ℃ from 59 ℃ of a control experiment.
(6) The microbial agent provided by the invention has obvious advantages in composting and can improve the harmless effect of the composting, the number of faecal coliform and the death rate of roundworm eggs are 71-93/g and 96.0% -98.7% respectively when the composting is finished, and the number of faecal coliform and the death rate of roundworm eggs are 110/g and 91.8% respectively in a control experiment.
Biological material preservation
Bacillus subtilis B211 is preserved in China center for type culture collection (CCTCC No: M2018475) in 2018, 7 and 13 months, and the preservation address is China, Wuhan university.
Bacillus licheniformis (Bacillus licheniformis strain) B105 has been preserved in China center for type culture Collection in 2018, 7-13.7.M, with the preservation number of CCTCC No: M2018470, and the preservation address of China, Wuhan university.
Bacillus brevis (Bacillus theroruber strain) B205, which has been preserved in China center for type culture Collection in 2018, 7.13.M, with the preservation number of CCTCC No. M2018472, and the preservation address of China, Wuhan university.
Detailed Description
The detection method is referred to the quality standard of the biological organic fertilizer in China (NY 884-.
In a specific embodiment, the commercially available strains Lysinibacillus fusiformis ATCC7055 and Trichoderma atroviride IMI 206040 can be used for the L.fusiformis and L.viride (Trichoderma atroviride), but are not limited to the above strains.
EXAMPLE 1 screening of thermostable Bacillus subtilis B211
(1) Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1:1, inoculating 5% (w/w) of summer rotten leaves in a certain area of Wuxi city of Jiangsu province, uniformly mixing, composting, measuring the temperature of a compost at 8:00 am and 6:00 pm every day, turning the compost once every day when the temperature is higher than 55 ℃, beginning to reduce the temperature of the compost along with the extension of composting time, stopping turning the compost when the temperature is lower than 40 ℃, and sampling;
(2) weighing 10g of compost sample, putting the compost sample into a triangular flask filled with 90mL of sterilized water, and shaking the mixture for 1h by a 120r/min shaking table to obtain 10-1Diluting, and sequentially making into 10-4、10-5And 10-6Respectively sucking 100 mu L of the three compost sample diluents, uniformly coating the three compost sample diluents on nutrient agar culture media, Gao's first agar culture media and potato agar culture medium plates, respectively putting the three compost sample diluents into an incubator at 55 ℃ for inverted culture for 1-7 days, and then repeatedly scribing and separating until pure strains are obtained;
(3) and (3) directly dripping a proper amount of 1.6% neutral red water solution on the surface of the bacterial colony, wherein if the bacterial colony is dark blue, the bacterial colony is a lipase producing bacterial strain, can degrade fat, and can judge the lipase producing capability of the bacterial strain according to the color depth, and the deeper the color is, the stronger the lipase producing capability is, and finally, the pure bacterial strain which produces lipase and has the strongest fat degrading capability is obtained.
Example 2 identification of high temperature resistant Bacillus subtilis B211
After being cultured on a nutrient agar medium plate at 30 ℃ overnight, the strain forms beige, flaked, wet surface, convex middle and irregular edge colonies, and is gram-positive, rod-shaped and sporulating when observed by an optical microscope.
And (2) performing taxonomic molecular identification by adopting 16SrDNA sequencing, performing Blast comparison on a sequencing result (1496bp) in NCBI (national center of Biotechnology information), and finding that the similarity of a 16SrDNA base sequence and Bacillus subtilis strain is highest.
By combining the morphological characteristics, physiological and biochemical characteristics and hereditary characteristics, the lipase-producing strain can be identified as high-temperature-resistant Bacillus subtilis strain, named as Bacillus subtilis B211 and preserved in China Center for Type Culture Collection (CCTCC) No. M2018475 in 7-13 months in 2018.
The lipase producing ability of the strain is verified. The strain was inoculated on nutrient agar medium plates, cultured at 30 ℃ for 25h, streaked into nutrient broth, and cultured at 30 ℃ for 25 h. And (3) centrifuging the fermentation liquor, and determining the lipase activity by an alkaline titration method, wherein the lipase activity is 9.1U/mL.
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.75: 0.75: 1, mixing the materials in proportion, wherein the water content of the mixed materials is 55 percent, the C/N ratio is 30, uniformly mixing 0.3 percent (by mass) of fermentation liquor of Bacillus subtilis B211 with the materials, composting, measuring the temperature of two heaps at 8:00 am and 6:00 pm every day, turning the heaps once every day when the temperature is higher than 55 ℃, beginning to reduce the temperature of the heaps along with the extension of composting time, stopping turning the heaps when the temperature is lower than 40 ℃, and sampling, wherein the result shows that the fat content in the raw materials is reduced by 78.1 percent after 2 days of composting.
The results of the above procedure using other commercial Bacillus subtilis show that when the temperature of the compost is higher than 35 ℃, the activity of the cells is weakened along with the increase of the temperature, and the fat content in the raw material is not obviously reduced after 2 days of composting.
Example 3 activation and culture of high temperature resistant Bacillus subtilis B211 strains
Inoculating the high-temperature resistant bacillus subtilis B211 to a nutrient agar culture medium plate, and culturing for 25h at 30 ℃. The nutrient agar culture medium comprises 3.0g of beef extract, 10.0g of peptone and 5.0g of sodium chloride.
Inoculating the activated strain into a 250mL shake flask filled with 100mL liquid culture medium, and performing shake culture at 30 ℃ at 450r/min for 25 h. The shake flask culture medium comprises 3.0g of beef extract, 10.0g of peptone and 5.0g of sodium chloride, and the pH value is 7.0.
Inoculating the shake flask culture solution into a 3L fermentation tank according to the volume ratio of 3-10%, stirring at the rotation speed of 200-600r/min, ventilation of 0.8-1.5vvm and dissolved oxygen of more than 15%, and fermenting and culturing at 25-37 ℃ for 20-40 h. The culture medium of the fermentation tank comprises 3.0g of beef extract, 20.0g of peptone, 30g of glucose, 2.0g of potassium dihydrogen phosphate and 5.0g of sodium chloride, and the pH value is 7.0.
Example 4 preparation of liquid Complex microbial Agents containing high temperature resistant Bacillus subtilis B211
The compost composite microbial agent is prepared by compounding high-temperature resistant bacillus subtilis B211 fermentation liquor and other strain fermentation liquor according to different viable count ratios, and the preparation method comprises the following steps:
(1) respectively inoculating high-temperature resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105 and thermophilic Brevibacillus brevis B205 into a nutrient broth culture medium subjected to high-temperature sterilization, wherein the inoculation amount is 3-10% (v/v) of the volume of the fermentation culture medium, culturing for 20-40h, the culture temperature of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus and the bacillus licheniformis B105 is 25-37 ℃, the culture temperature of the thermophilic Brevibacillus brevis B205 is 30-60 ℃, and when the viable count is more than or equal to 1 × 108Obtaining fermentation liquor of the high-temperature resistant bacillus subtilis B211, the lysine bacillus fusiformis, the bacillus licheniformis B105 and the bacillus brevis B205 when the concentration is CFU/mL;
(2) the fermentation liquid of Trichoderma atroviride is prepared by inoculating Trichoderma atroviride into potato glucose agar culture medium sterilized at high temperature, wherein the inoculation amount is 3% -10% (v/v) of the fermentation medium volume, culturing at 20-30 deg.C for 20-30 hr, and the viable count is greater than or equal to 1 × 108Obtaining fermentation liquor of the trichoderma atroviride when CFU/mL;
(3) mixing the obtained high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the geobacillus thermophilus B205 and the fermentation liquor of trichoderma atroviride according to a certain proportion, enabling the ratio of the viable count of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the geobacillus thermophilus B205 and the trichoderma atroviride to be 18-28:2-3:0.5-1.5:800-9-1.0×1010CFU/mL liquid compound microbial agent.
Example 5 preparation of solid Complex microbial Agents containing high temperature resistant Bacillus subtilis B211
Mixing the obtained high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic Brevibacillus brevis B205 and the fermentation liquor of the trichoderma atroviride according to a certain proportion, adding one or more of crushed straws, carbonized straws and bran into the mixture, and drying the mixture by hot air to obtain the solid composite microbial agent containing the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic Brevibacillus brevis B205 and the trichoderma atroviride, wherein the ratio of the viable count of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic Brevibacillus brevis B205 and the trichoderma atroviride is 18-28:2-3:0.5-1.5: 800-.
Example 6 application of composite microbial Agents for composting
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1:1, mixing the mixture according to the mass ratio of 0.3: 100, the addition amount of the microbial inoculum is 0.3 percent, composting is carried out after uniform mixing, the temperature of two heaps is measured at 8:00 in the morning and 6:00 in the afternoon every day, when the temperature is higher than 55 ℃, the heaps are turned once every day, the temperature of the heaps begins to drop along with the extension of composting time, when the temperature is lower than 40 ℃, the turning is stopped, sampling is carried out, and various quality indexes of the compost are measured. The result shows that the time required for the pile body to reach the highest temperature is 21h, and the highest temperature is 65 ℃; after being piled for 2 days, the fat degradation rate is 93.2 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 16.9, and the number of faecal coliform bacteria and the death rate of roundworm eggs are respectively 88/g and 97.0 percent.
Example 7 use of compost composite microbial Agents
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1.1: mixing the components according to a ratio of 0.9, wherein the water content of the mixed material is 55%, the C/N ratio is 25, and the ratio of the viable count of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the bacillus brevis B205 and the trichoderma atroviride is 18:2:0.5:800:2.8, and the mixed material are mixed according to a mass ratio of 0.5: 100, the addition amount of the microbial inoculum is 0.5 percent, composting is carried out after uniform mixing, the temperature of two heaps is measured at 8:00 in the morning and 6:00 in the afternoon every day, when the temperature is higher than 55 ℃, the heaps are turned once every day, the temperature of the heaps begins to drop along with the extension of composting time, when the temperature is lower than 40 ℃, the turning is stopped, sampling is carried out, and various quality indexes of the compost are measured. The result shows that the temperature of the pile body rises rapidly, the time required for reaching the highest temperature is 26 hours, and the highest temperature is 63 ℃; after being piled for 2 days, the fat degradation rate is 88.6 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 17.3, and the number of faecal coliform bacteria and the death rate of roundworm eggs are respectively 90/g and 96.5%.
Example 8 use of compost composite microbial Agents
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1.1: mixing the components according to a ratio of 0.9, wherein the water content of the mixed material is 55%, the C/N ratio is 25, and the ratio of the viable count of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the bacillus brevis B205 and the trichoderma atroviride is 24:2.4:1:1045:2.9, and the mixed material are mixed according to a mass ratio of 0.5: 100, the addition amount of the microbial inoculum is 0.5 percent, composting is carried out after uniform mixing, the temperature of two heaps is measured at 8:00 in the morning and 6:00 in the afternoon every day, when the temperature is higher than 55 ℃, the heaps are turned once every day, the temperature of the heaps begins to drop along with the extension of composting time, when the temperature is lower than 40 ℃, the turning is stopped, sampling is carried out, and various quality indexes of the compost are measured. The result shows that the temperature of the pile body rises rapidly, the time required for reaching the highest temperature is 16h, and the highest temperature is 66 ℃; after stacking for 2 days, the fat degradation rate is 95.7 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 11.2, and the number of faecal coliform bacteria and the death rate of roundworm eggs are 71/g and 98.7 percent respectively.
Example 8 use of compost composite microbial Agents
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1.1: mixing the components according to a ratio of 0.9, wherein the water content of the mixed material is 55%, the C/N ratio is 25, and the solid compound microbial inoculum containing the viable count of the high-temperature resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the bacillus brevis B205 and the trichoderma atroviride with the ratio of 28:3:1.5:1200:3.3 to the mixed material according to a mass ratio of 0.1: 100, the addition amount of the microbial inoculum is 0.1 percent, composting is carried out after uniform mixing, the temperature of two heaps is measured at 8:00 in the morning and 6:00 in the afternoon every day, when the temperature is higher than 55 ℃, the heaps are turned once every day, the temperature of the heaps begins to drop along with the extension of composting time, when the temperature is lower than 40 ℃, the turning is stopped, sampling is carried out, and various quality indexes of the compost are measured. The result shows that the temperature of the pile body rises rapidly, the time required for reaching the highest temperature is 32 hours, and the highest temperature is 61 ℃; after being stacked for 2 days, the fat degradation rate is 81.9 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 18.8, and the number of faecal coliform bacteria and the death rate of roundworm eggs are 93/g and 96.0 percent respectively.
Comparative example 1
Mixing the oily kitchen waste, the food plant sewage treatment residual sludge and the corn straws according to the mass ratio of 0.5: 1:1, mixing the mixture according to the mass ratio of 0.3: 100, and the adding amount of the microbial inoculum is 0.3 percent, wherein the ratio of the viable count of the lysine bacillus fusiformis, the bacillus licheniformis B105, the bacillus brevis B205 and the trichoderma aureoviride is 2.4:1:1045:2.9, composting is carried out after uniform mixing, the temperature of two heaps is measured at 8:00 am and 6:00 pm every day, when the temperature is higher than 55 ℃, the heaps are turned once every day, the temperature of the heaps begins to drop along with the prolonging of the composting time, when the temperature is lower than 40 ℃, the turning is stopped, sampling is carried out, and various quality indexes of the compost are measured.
The result shows that the time required for the pile body to reach the highest temperature is 62h, and the highest temperature is 59 ℃; after being stacked for 2 days, the fat degradation rate is 56.9 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 20.9, and the number of faecal coliform bacteria and the death rate of roundworm eggs are 110/g and 91.8 percent respectively.
Comparative example 2
The specific embodiment is the same as example 6, except that the ratio of the number of viable bacteria of Bacillus subtilis B211, lysine bacillus fusiformis strain, Bacillus licheniformis B105, Bacillus brevis B205 and Trichoderma atroviride is different, as shown in Table 1. The result shows that the time required for the pile body to reach the highest temperature is prolonged to 57-64h, and the highest temperature is 49-58 ℃; after being piled for 2 days, the fat degradation rate is 58.2 to 77.7 percent; when the composting is finished, the carbon-nitrogen ratio of the mixed material is 18.9-24.6, and the number of faecal coliform bacteria and the death rate of ascaris ova are 230-.
TABLE 1
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (12)

1. A high temperature resistant Bacillus subtilis B211 for producing lipase is preserved in China center for type culture Collection in 7 month and 13 month in 2018, with the preservation number of CCTCC No. M2018475, and the preservation address of China, Wuhan university.
2. A microbial agent comprising the high-temperature-resistant Bacillus subtilis B211 of claim 1.
3. The microbial agent according to claim 2, comprising Bacillus subtilis B211, lysine Bacillus fusiformis (lysnibacillus fusiformis), Bacillus licheniformis B105, Bacillus brevis thermophilus (Bacillus thermophilus) B205, and Trichoderma atroviride (Trichoderma atroviride); the bacillus licheniformis B105 is preserved in China Center for Type Culture Collection (CCTCC) No. M2018470 in 2018, 7 months and 13 days, and the preservation address is China, Wuhan and Wuhan university; the Bacillus brevis (Bacillus terroruber) B205 is preserved in China center for type culture collection (CCTCC No: M2018472) in 2018, 7 months and 13 days, and the preservation address is China, Wuhan and Wuhan university.
4. The microbial inoculant according to claim 3, wherein the ratio of the viable count of the bacillus subtilis B211, the lysinibacillus fusiformis, the bacillus licheniformis B105, the bacillus brevis B205 and the trichoderma atroviride is 18-28:2-3:0.5-1.5:800-1200: 2.8-3.3.
5. The microbial inoculant according to claim 3 or 4, wherein the total number of viable bacteria is 1.0 × 109-1.0×1010CFU/mL or CFU/g.
6. The microbial agent according to claim 3 or 4, which is prepared by: respectively inoculating high-temperature-resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic short-bud bacillus B205 and trichoderma atroviride into a culture medium to obtain fermentation liquor of the high-temperature-resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic short-bud bacillus B205 and the trichoderma atroviride, mixing the obtained fermentation liquor in proportion, and adding a carrier auxiliary material to obtain a liquid composite microbial agent containing the high-temperature-resistant bacillus subtilis B211; the carrier auxiliary materials include but are not limited to one or more of lignite, zeolite and stone powder.
7. The microbial agent according to claim 5, which is prepared by the following steps: respectively inoculating high-temperature-resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic short-bud bacillus B205 and trichoderma atroviride into a culture medium to obtain fermentation liquor of the high-temperature-resistant bacillus subtilis B211, the fusiform lysine bacillus, the bacillus licheniformis B105, the thermophilic short-bud bacillus B205 and the trichoderma atroviride, mixing the obtained fermentation liquor in proportion, and adding a carrier auxiliary material to obtain a liquid composite microbial agent containing the high-temperature-resistant bacillus subtilis B211; the carrier auxiliary materials include but are not limited to one or more of lignite, zeolite and stone powder.
8. The microbial agent according to claim 3 or 4, which is prepared by: mixing high-temperature-resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic Brevibacillus brevis B205 and fermentation liquor of trichoderma atroviride according to a proportion, adding one or more of crushed straws, carbonized straws and bran, and drying by hot air to obtain the solid compound microbial agent containing the high-temperature-resistant bacillus subtilis B211.
9. The microbial agent according to claim 5, which is prepared by the following steps: mixing high-temperature-resistant bacillus subtilis B211, fusiform lysine bacillus, bacillus licheniformis B105, thermophilic Brevibacillus brevis B205 and fermentation liquor of trichoderma atroviride according to a proportion, adding one or more of crushed straws, carbonized straws and bran, and drying by hot air to obtain the solid compound microbial agent containing the high-temperature-resistant bacillus subtilis B211.
10. A method for promoting mixed composting of oily kitchen waste, food plant sewage treatment residual sludge and straws, which is characterized in that the composting is carried out by using a microbial agent containing the Bacillus subtilis B211 of claim 1.
11. The method of claim 10, comprising the steps of:
(1) mixing the microbial agent as claimed in any one of claims 2 to 9 with compost raw materials according to the ratio of 0.5-1: mixing at a mass ratio of 100;
(2) composting the mixture, measuring the temperature of the pile body, and turning the pile according to the temperature change.
12. The method of claim 11, wherein step (2) comprises composting the mixture, measuring the temperature of the compost at 8:00 a day in the morning and at 6:00 a.m., turning the compost once a day when the temperature is higher than 55 ℃, wherein the temperature of the compost starts to decrease with the increase of the composting time, and stopping the turning when the temperature is lower than 40 ℃ to finish the composting.
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