CN106591178B - Kitchen waste composite microbial degradation microbial inoculum and preparation method and application thereof - Google Patents
Kitchen waste composite microbial degradation microbial inoculum and preparation method and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
Abstract
The invention relates to a kitchen waste composite microbial degradation microbial inoculum and a preparation method and application thereof, wherein the degradation microbial inoculum is formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, methylobacterium radiodurans, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii with a carrier, wherein the mixed strain accounts for 15-25% of the total mass of the degradation microbial inoculum; the mass ratio of the bacillus amyloliquefaciens, the radiation-resistant methylobacterium, the pantoea dispersa, the pseudomonas oryzae, the citrobacter freundii and the staphylococcus cohnii is 1.5-3: 1-1.5; the composite microbial degradation microbial inoculum is used for degrading kitchen garbage. Compared with the prior art, the composite microbial degradation microbial inoculum can effectively degrade common kitchen garbage such as vegetables, grains, fish, poultry and the like, has the degradation rate of over 80 percent, high degradation rate, small generated peculiar smell, no pollutant or toxic substance, low cost and stable performance.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a kitchen waste composite microbial degradation microbial inoculum, and a preparation method and application thereof.
Background
In recent years, China is in a fast stage of urbanization development, and the number of urban population is increasing at a higher speed every year. Along with the gathering of population, the discharge amount of municipal waste is increasing, wherein the proportion of kitchen waste is increasing year by year. Because the kitchen waste contains a large amount of macromolecular organic substances such as starch, cellulose, protein, grease and the like and water, the kitchen waste provides a good living environment for various microorganisms, and further causes mass propagation of germs, mosquitoes and other harmful organisms, so that the kitchen waste has great harm to the health of people and the urban environment. Although the kitchen waste has high utilization value, the water content is high, the heat value is low, the collection, transportation and incineration treatment are inconvenient, secondary pollutants such as leachate and the like are easy to generate and are easy to decay to generate stink if the treatment is not proper.
At present, there are several methods for disposing of such waste around the world:
1. a landfill method: the garbage is collected and transported to a landfill site to be paved and compacted. Its advantages are simple process, easy operation, and less environmental pollution.
2. The burning method comprises the following steps: the comprehensive treatment process of high-temperature decomposition and deep oxidation of solid waste. Its advantages are greatly reduced volume of waste, eliminating harmful bacteria, and supplying heat to generate electricity.
3. A composting method: the garbage becomes harmless humus after fermentation. But the fermentation period is long, the nutrient content is low, the volume is large after treatment, and the method is only suitable for rural areas with inconvenient traffic.
Therefore, the harmless, recycling and reduction treatment of the kitchen waste is significant to the current situation of resource shortage of per capita in China. And the biological technology is adopted to carry out biodegradation or biotransformation, so that the urban garbage can be effectively treated, and the resource can be recycled. Therefore, compared with physical and chemical methods, biological treatment technology is becoming the main development direction of garbage treatment in the future due to its advantages of environmental protection, safety and high efficiency.
The patent with the granted publication number of CN 102010832B discloses a kitchen waste degradation and elimination type microbial agent II, wherein the microbial agent II is 3.0 × 108～1.5×1011The F cu/ml of Actinomucor elegans (Actinomucor elegans) has CGMCC No.3881 and 3.0 × 108～1.3×1011cfu/ml Paenibacillus cineris composite liquid microbial inoculum with CGMCC No. 3883. The preparation method specifically comprises the following steps:
1) and slant culture:
inoculating Actinomucor elegans (Actinomucor elegans) CGMCC No.3881 to a PDA inclined plane, inoculating Paenibacillus cineris CGMCC No.3883 to a beef extract peptone inclined plane, and culturing at 28-30 ℃ for 24-72 h respectively to activate the strains;
2) and first-stage seed liquid culture:
inoculating the activated actinomucor elegans obtained in the step 1) to a PDA liquid culture medium, inoculating the activated Paenibacillus cineris to a beef extract peptone liquid culture medium, and performing shake culture for 48-120 h at 28-30 ℃ and 100-250 r/min respectively; respectively obtaining a first-stage seed liquid of the actinomucor elegans and a first-stage seed liquid of the Paenibacillus cineris;
3) and (3) fermenting and culturing bacterial liquid:
placing a culture solution I in a fermentation tank, inoculating the primary seed liquid of the Paenibacillus cineris obtained in the step 2) into the culture solution I according to the inoculation amount accounting for 5-25% of the volume ratio of the culture solution I, and performing fermentation culture at 30-32 ℃ at 30-100 r/min for 48-72 h; obtaining bacterial liquid;
the culture solution I comprises: diluting the monosodium glutamate waste liquid by 10-200 times, and adding alkali to adjust the pH value to 7.1-7.3;
4) and (3) performing liquid fermentation culture of fungi:
placing a culture solution II in a fermentation tank, inoculating the first-grade seed solution of the actinomucor elegans obtained in the step 2) into the culture solution II according to the inoculation amount accounting for 5-25% of the volume ratio of the culture solution II, and performing fermentation culture at 28-30 ℃ and 30-100 r/min for 48-72 hours; obtaining fungus bacterial liquid;
the culture solution II comprises: diluting the monosodium glutamate waste liquid by 10-200 times, and adding alkali to adjust the pH value to 5.4-5.6;
5) and mixing the bacterial liquid obtained in the step 3) with the fungal liquid obtained in the step 4) to obtain the kitchen garbage degradation and elimination type microbial agent II.
Compared with the patent, the invention is characterized in that the used bacterial strains are all bacteria, do not contain fungi and are easy to culture. The bacteria acting on the method have high growth speed and short fermentation time which is only 24-48 hours. The composite microbial degradation microbial inoculum prepared by the invention can rapidly degrade most kitchen garbage in a short time, does not generate peculiar smell, and can be put into multiple use at one time.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a kitchen waste composite microbial degrading microbial inoculum which can effectively degrade common kitchen waste such as vegetables, grains, fish, poultry and the like, has high degradation rate, generates little peculiar smell and generates no toxic and harmful substances, and a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
a kitchen waste composite microbial degradation microbial inoculum is formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii with a carrier, wherein the mixed strain accounts for 15-25% of the total mass of the degradation microbial inoculum, the mass percentage content of water in the degradation microbial inoculum is 10-20%, the pH value of the degradation microbial inoculum is 5.5-8.5, and the number of viable bacteria contained in each gram of the degradation microbial inoculum is 106～109And (4) respectively.
The mass ratio of the bacillus amyloliquefaciens, the radiation-resistant methylobacterium, the pantoea dispersa, the pseudomonas oryzae, the citrobacter freundii and the staphylococcus cohnii is 1.5-3: 1-1.5.
The Bacillus amyloliquefaciens is preserved in China Center for Type Culture Collection (CCTCC) in Loa Jia mountain (No. 299 in eight paths) in Wuchang district, Wuhan city, Hubei, the preservation date is 2014, 12 months and 12 days, the preservation number is AB2014337, and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is classified and named.
The bacillus amyloliquefaciens is screened from forest soil accumulated with rotten fallen leaves for a long time, and the specific screening and identifying steps are as follows:
(1) diluting 1g of sieved soil by 104 times with sterile water;
(2) coating 100 mu L of diluent on a sterile Congo red cellulose culture medium, and culturing at 28 ℃ for 36 h;
(3) selecting the colony with a larger transparent ring in the culture medium for purification culture, and storing at-80 ℃;
(4) the purified strain is sent to the Shanghai Mergiz biological medicine science and technology limited company for identification.
The aperture of the standard sieve used in the step (1) is 200 mm.
The Congo red cellulose culture medium used in the step (2) has the solvent of water, the pH of natural pH, and the solutes and the concentrations are as follows: 14g/L of microcrystalline cellulose, 0.25g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 2g/L of gelatin, 0.2g/L of Congo red and 14g/L of agar.
The diameters of transparent rings of the colonies selected in the step (3) are all larger than 6 mm.
The purification culture medium adopted in the purification culture in the step (3) has the following solvent of water, the pH of natural pH, and the solute and the concentration: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agar.
The identification result in the step (4) is bacillus amyloliquefaciens, and the bacillus amyloliquefaciens is preserved in the China center for type culture collection in the Wuchang district Loa Lophanthoides (No. 299 Bayi) in Wuhan city, Hubei province within 12 months and 12 days in 2014.
A preparation method of a kitchen waste composite microbial degradation microbial inoculum specifically comprises the following steps:
(1) respectively carrying out single strain fermentation culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii to obtain bacillus amyloliquefaciens zymocyte liquid, radiation-resistant methylobacterium zymocyte liquid, disperse pantoea zymocyte liquid, pseudomonas oryzae zymocyte liquid, citrobacter freundii zymocyte liquid and staphylococcus cohnii zymocyte liquid;
(2) mixing the following bacterial strain fermentation bacteria liquids in parts by weight to obtain a composite bacterial liquid: 15-30 parts of bacillus amyloliquefaciens zymocyte liquid, 10-15 parts of radiation-resistant methylobacterium zymocyte liquid, 10-15 parts of pantoea dispersa zymocyte liquid, 10-15 parts of pseudomonas oryzae zymocyte liquid, 10-15 parts of citrobacter freundii zymocyte liquid and 10-15 parts of staphylococcus cohnii zymocyte liquid;
(3) mixing the dried sawdust and bran according to a volume ratio of 1: 3-5 to prepare a carrier;
(4) and (3) uniformly mixing the compound bacterial liquid obtained in the step (2) with the carrier obtained in the step (3) according to a mass ratio of 1: 25-30 to obtain the kitchen waste compound microbial degradation microbial inoculum.
The culture medium used for carrying out strain culture on bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa in the step (1) comprises water as a solvent, and solutes and concentrations of: 3-10 g/L beef extract, 3-10 g/L peptone, 2-7 g/L sodium chloride, 2-7 g/L dipotassium hydrogen phosphate, 2-5 g/L potassium dihydrogen phosphate, 0.1-1 g/L magnesium sulfate heptahydrate and 1-1.5 g/L ammonium sulfate.
The culture medium used for carrying out strain culture on the pseudomonas solanacearum in the step (1) has the following solvent of water, natural pH and solutes and concentrations: 200-300 g/L of potato cooking juice and 15-20 g/L of glucose;
the preparation method of the potato cooking juice comprises the following steps: taking 200-300 g of peeled potatoes, cutting into slices, weighing, heating and boiling for 30min, then filtering by using gauze and reserving filtrate.
The culture medium for the bacterial strain culture of the staphylococcus cohnii in the step (1) comprises water as a solvent, pH of 7.0-7.6, solutes and concentrations: 5-15 g/L of casein, 5-10 g/L of soybean meal, 15-20 g/L of glucose and 5-10 g/L of sodium chloride.
The culture conditions for culturing the strains of the bacillus amyloliquefaciens, the citrobacter freundii, the methylobacter radiodurans and the pantoea dispersa in the step (1) are as follows: the temperature is 25-35 ℃, the rotating speed is 180-220 r/min, and the culture time is 24-48 h.
The culture conditions for strain culture of the pseudomonas solanacearum in the step (1) are as follows: carrying out shake flask shake bed culture at the temperature of 30-35 ℃ at the rotating speed of 200-220 r/min for 24-36 h;
the culture conditions for strain culture of the staphylococcus cohnii are as follows: the temperature is 28-32 ℃, shaking and shaking culture is carried out, the rotating speed is 180-220 r/min, and the culture time is 24-36 h.
In the step (3), the moisture content in the bran and the sawdust is not more than 10% by mass.
An application of a kitchen waste composite microbial degradation microbial inoculum is used for degrading kitchen waste.
In practical application, 0.5-2 kg of kitchen waste and the prepared composite microbial degradation microbial inoculum are put into a garbage disposer together and are uniformly stirred, the temperature is controlled to be 50-60 ℃, and the degradation time is 24-36 hours.
In the present invention, the radiation-resistant methylobacterium, Pantoea dispersa, Pseudomonas oryzae, Citrobacter freundii and Staphylococcus cohnii are selected from commercially available commercial strains.
The composite microbial degradation microbial inoculum prepared by the invention can be repeatedly used, namely, after the kitchen garbage which is put in last time is fully degraded, new kitchen garbage to be treated is added again for continuous degradation.
The bacillus amyloliquefaciens used by the invention has the main functions of degrading protein and starch, the radiation-resistant methylobacterium has the main functions of degrading fat and multi-carbon compounds, the pantoea dispersa has the main functions of degrading macromolecular carbohydrate substances, and the pseudomonas solani, citrobacter freundii and staphylococcus cohnii have the main functions of degrading cellulose. The bacterial strains adopted by the invention have a synergistic interaction effect when degrading kitchen garbage, namely, under the condition of the same bacterial concentration, the degradation effect of the mixed bacterial agent of the six bacterial strains is better than that of a single bacterial agent or other mixed bacterial agents.
Compared with the prior art, the invention carries out liquid culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii, mixes bacterial liquids of all strains to prepare composite microbial bacterial liquid, and then mixes the composite microbial bacterial liquid with bran and sawdust carrier materials to obtain the kitchen waste degrading microbial inoculum, which has the following characteristics:
1) the degrading bacteria agent contains various strains with different degrading functions, and can effectively degrade kitchen garbage containing various substances such as vegetables, grains, meat and the like;
2) the microbial activity in the degrading microbial inoculum is high, the degrading rate reaches more than 80%, the degrading speed is high, the using amount is small when kitchen garbage is treated, the generated peculiar smell is small, the cost is low, the use is convenient, toxic and harmful substances are not generated, and the method is safe, reliable and environment-friendly.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1
The preparation method of the kitchen waste composite microbial degradation microbial inoculum specifically comprises the following steps:
1. preparation of composite microbial liquid
(1) The culture medium for strain culture of bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa adopts water as a solvent, and comprises the following solutes in concentration: 8g/L beef extract, 5g/L peptone, 5g/L sodium chloride, 5g/L dipotassium hydrogen phosphate, 5g/L potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate, 1g/L ammonium sulfate and pH of 7.2.
The culture conditions were: the temperature is 28 ℃, the rotating speed is 200r/min, and the culture time is 20 h.
The culture medium for strain culture of the pseudomonas solanacearum adopts water as a solvent, and comprises the following solutes in concentration: 200g/L of potato, 15g/L of glucose and 7.5 of pH. The potato juice cooking method comprises the following steps: peeling potato 200g, cutting into slices, weighing, heating to boil for 25min, filtering with gauze and retaining the filtrate.
The culture conditions were: the temperature is 30 ℃, the rotating speed is 220r/min, and the culture time is 24 h.
(3) The culture medium for strain culture of staphylococcus cohnii has water as solvent, the following solutes and concentrations: 10g/L of casein, 10g/L of soybean meal, 15g/L of glucose, 5g/L of sodium chloride and 7.0 of pH.
The culture conditions were: the temperature is 28 ℃, the rotating speed is 220r/min, and the culture time is 28 h.
(4) And uniformly mixing 90mL of bacillus amyloliquefaciens bacterial liquid, 45mL of radiation-resistant methylobacterium bacterial liquid, 45mL of disperse pantoea bacterial liquid, 45mL of pseudomonas oryzae bacterial liquid, 45mL of citrobacter freundii bacterial liquid and 30mL of staphylococcus cohnii bacterial liquid to obtain the composite microbial liquid.
2. Preparation of the support Material
Mixing 3L of testa Tritici and 15L of sawdust to obtain carrier material.
Preparation of degradation microbial inoculum
And (3) uniformly mixing 300mL of the compound microorganism bacterium solution in the step (1) with 12L of the carrier material in the step (2) to obtain the kitchen waste degrading microbial inoculum.
Pouring 12L of kitchen waste degrading microbial inoculum into a waste stirring processor, adding 0.6-1.2 kg of kitchen waste above the degrading microbial inoculum, covering a cover of the processor to form a closed environment, and controlling the heating and stirring temperature to be 50 ℃. And weighing the total weight of the garbage disposer every 24 hours, and calculating the degradation efficiency.
The degradation efficiency is reflected in the weight reduction efficiency:
the degradation efficiency is (B-C)/A multiplied by 100%
Weight (kg) of kitchen garbage input B: total weight before treatment (kg) C: total weight (kg) after treatment.
The results are shown in Table 1.
TABLE 1 degradation efficiency of degrading bacteria to kitchen waste as a function of time
As can be seen from the analysis in Table 1, the degrading microbial inoculum of the invention can rapidly reduce the weight of kitchen waste in a short time, has good effect, and provides theoretical basis and experimental basis for treating the kitchen waste by applying biotechnology.
Example 2
The kitchen waste composite microbial degradation microbial inoculum is formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, radiation-resistant methylobacterium, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii and a carrier, wherein the mixed strain accounts for 15% of the total mass of the degradation microbial inoculum, the mass percentage content of water in the degradation microbial inoculum is 20%, the pH value of the degradation microbial inoculum is 5.5, and the number of viable bacteria contained in each gram of the degradation microbial inoculum is 106And (4) respectively.
Wherein the mass ratio of the bacillus amyloliquefaciens, the radiation-resistant methylobacterium, the pantoea dispersa, the pseudomonas oryzae, the citrobacter freundii and the staphylococcus cohnii is 1.5:1:1:1: 1.
The bacillus amyloliquefaciens used in this example was preserved in the China Center for Type Culture Collection (CCTCC) in Lophania terrae Hainanensis (No. 299, Bayi), Wuhan, Hubei, with a preservation date of 2014, 12 months and 12 days. The preservation number is CCTCC: AB2014337, and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is classified and named.
In the embodiment, the bacillus amyloliquefaciens is screened from forest soil accumulated with rotten fallen leaves for a long time, and the specific screening and identifying steps are as follows:
(1) diluting 1g of sieved soil by 104 times with sterile water;
(2) coating 100 mu L of diluent on a sterile Congo red cellulose culture medium, and culturing at 28 ℃ for 36 h;
(3) selecting the colony with a larger transparent ring in the culture medium for purification culture, and storing at-80 ℃;
(4) the purified strain is sent to the Shanghai Mergiz biological medicine science and technology limited company for identification.
The aperture of the standard sieve used in the step (1) is 200 mm.
The Congo red cellulose culture medium used in the step (2) has the solvent of water, the pH of natural pH, and the solutes and the concentrations are as follows: 14g/L of microcrystalline cellulose, 0.25g/L of magnesium sulfate, 0.5g/L of dipotassium phosphate, 2g/L of gelatin, 0.2g/L of Congo red and 14g/L of agar.
The diameters of transparent rings of the colonies selected in the step (3) are all larger than 6 mm.
The purification culture medium adopted in the purification culture in the step (3) has the following solvent of water, the pH of natural pH, and the solute and the concentration: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and 15g/L of agar.
The identification result in the step (4) is bacillus amyloliquefaciens, and the bacillus amyloliquefaciens is preserved in the China center for type culture collection in the Wuchang district Loa Lophanthoides (No. 299 Bayi) in Wuhan city, Hubei province within 12 months and 12 days in 2014.
The preparation method of the kitchen waste composite microbial degradation microbial inoculum specifically comprises the following steps:
(1) respectively carrying out single strain fermentation culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii to obtain bacillus amyloliquefaciens zymocyte liquid, radiation-resistant methylobacterium zymocyte liquid, disperse pantoea zymocyte liquid, pseudomonas oryzae zymocyte liquid, citrobacter freundii zymocyte liquid and staphylococcus cohnii zymocyte liquid;
(2) mixing the following bacterial strain fermentation bacteria liquids in parts by weight to obtain a composite bacterial liquid: 15 parts of bacillus amyloliquefaciens zymocyte liquid, 10 parts of radiation-resistant methylobacterium zymocyte liquid, 10 parts of pantoea dispersa zymocyte liquid, 10 parts of pseudomonas oryzae zymocyte liquid, 10 parts of citrobacter freundii zymocyte liquid and 10 parts of staphylococcus cohnii zymocyte liquid;
(3) mixing dried sawdust and bran according to a volume ratio of 1:3 to prepare a carrier;
(4) and (3) uniformly mixing the compound bacterial liquid obtained in the step (2) and the carrier obtained in the step (3) according to the volume ratio of 1:25 to obtain the kitchen waste compound microbial degradation microbial inoculum.
The culture medium used for carrying out strain culture on bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa in the step (1) comprises water as a solvent, and solutes and concentrations of: 3g/L of beef extract, 3g/L of peptone, 2g/L of sodium chloride, 2g/L of dipotassium phosphate, 2g/L of monopotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate and 1g/L of ammonium sulfate.
The culture medium used for carrying out strain culture on the pseudomonas solanacearum in the step (1) has the following solvent of water, natural pH and solutes and concentrations: 200g/L of potato cooking juice and 15g/L of glucose;
the preparation method of the potato cooking juice comprises the following steps: taking peeled potato 200g, cutting into slices, weighing, heating and boiling for 30min, then filtering with gauze and retaining the filtrate.
The culture medium for the bacterial strain culture of the staphylococcus cohnii in the step (1) comprises water as a solvent, pH 7.0, solutes and concentrations: 5g/L of casein, 5g/L of soybean meal, 15g/L of glucose and 5g/L of sodium chloride.
The culture conditions for culturing the strains of the bacillus amyloliquefaciens, the citrobacter freundii, the methylobacter radiodurans and the pantoea dispersa in the step (1) are as follows: the temperature is 25 ℃, the rotating speed is 180r/min, and the culture time is 48 h.
The culture conditions for strain culture of the pseudomonas solanacearum in the step (1) are as follows: shaking and culturing at 30 deg.C for 36h at a rotation speed of 200 r/min;
the culture conditions for strain culture of the staphylococcus cohnii are as follows: the temperature is 28 ℃, shaking culture is carried out, the rotating speed is 180r/min, and the culture time is 36 h.
The kitchen waste composite microbial degradation microbial inoculum prepared by the embodiment is used for degrading kitchen waste.
Example 3
The kitchen waste composite microbial degradation microbial inoculum is formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, radiation-resistant methylobacterium, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii and a carrier, wherein the mixed strain accounts for 25% of the total mass of the degradation microbial inoculum, the mass percentage content of water in the degradation microbial inoculum is 10%, the pH value of the degradation microbial inoculum is 8.5, and the number of viable bacteria contained in each gram of the degradation microbial inoculum is 109And (4) respectively.
Wherein the mass ratio of the bacillus amyloliquefaciens, the radiation-resistant methylobacterium, the pantoea dispersa, the pseudomonas oryzae, the citrobacter freundii and the staphylococcus cohnii is 3:1.5:1.5:1.5:1.
The Bacillus amyloliquefaciens used in this example was collected in the China Center for Type Culture Collection (CCTCC) in Lojia mountain in Wuchang district (No. 299, Bayi, Hubei, Wuhan City, the collection date was 2014, 12 months and 12 days, the collection number was AB2014337, and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) was named after classification.
The preparation method of the kitchen waste composite microbial degradation microbial inoculum specifically comprises the following steps:
(1) respectively carrying out single strain fermentation culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii to obtain bacillus amyloliquefaciens zymocyte liquid, radiation-resistant methylobacterium zymocyte liquid, disperse pantoea zymocyte liquid, pseudomonas oryzae zymocyte liquid, citrobacter freundii zymocyte liquid and staphylococcus cohnii zymocyte liquid;
(2) mixing the following bacterial strain fermentation bacteria liquids in parts by weight to obtain a composite bacterial liquid: 30 parts of bacillus amyloliquefaciens zymocyte liquid, 15 parts of radiation-resistant methylobacterium zymocyte liquid, 15 parts of pantoea dispersa zymocyte liquid, 15 parts of pseudomonas oryzae zymocyte liquid, 15 parts of citrobacter freundii zymocyte liquid and 10 parts of staphylococcus cohnii zymocyte liquid;
(3) mixing the dried sawdust and bran according to a volume ratio of 1:5 to prepare a carrier;
(4) and (3) uniformly mixing the compound bacterial liquid obtained in the step (2) and the carrier obtained in the step (3) according to the volume ratio of 1:30 to obtain the kitchen waste compound microbial degradation microbial inoculum.
The culture medium used for carrying out strain culture on bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa in the step (1) comprises water as a solvent, and solutes and concentrations of: 10g/L of beef extract, 10g/L of peptone, 7g/L of sodium chloride, 7g/L of dipotassium phosphate, 5g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate and 1.5g/L of ammonium sulfate.
The culture medium used for carrying out strain culture on the pseudomonas solanacearum in the step (1) has the following solvent of water, natural pH and solutes and concentrations: 300g/L of potato cooking juice and 20g/L of glucose;
the preparation method of the potato cooking juice comprises the following steps: taking 300g of peeled potatoes, cutting into slices, weighing, heating and boiling for 30min, then filtering by gauze and reserving filtrate.
The culture medium for the bacterial strain culture of the staphylococcus cohnii in the step (1) comprises water as a solvent, pH 7.6 and solutes and concentrations as follows: 15g/L of casein, 10g/L of soybean meal, 20g/L of glucose and 10g/L of sodium chloride.
The culture conditions for culturing the strains of the bacillus amyloliquefaciens, the citrobacter freundii, the methylobacter radiodurans and the pantoea dispersa in the step (1) are as follows: the temperature is 35 ℃, the rotating speed is 220r/min, and the culture time is 24 h.
The culture conditions for strain culture of the pseudomonas solanacearum in the step (1) are as follows: the temperature is 35 ℃, shaking table cultivation is carried out, the rotating speed is 220r/min, and the cultivation time is 24 hours;
the culture conditions for strain culture of the staphylococcus cohnii are as follows: the temperature is 32 ℃, shaking culture is carried out, the rotating speed is 220r/min, and the culture time is 24 h.
The kitchen waste composite microbial degradation microbial inoculum prepared by the embodiment is used for degrading kitchen waste.
Example 4
The kitchen waste composite microbial degradation microbial inoculum is formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, radiation-resistant methylobacterium, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii and a carrier, wherein the mixed strain accounts for 20% of the total mass of the degradation microbial inoculum, the mass percentage content of water in the degradation microbial inoculum is 16%, the pH value of the degradation microbial inoculum is 7.5, and the number of viable bacteria contained in each gram of the degradation microbial inoculum is 108And (4) respectively.
Wherein the mass ratio of the bacillus amyloliquefaciens, the radiation-resistant methylobacterium, the pantoea dispersa, the pseudomonas oryzae, the citrobacter freundii and the staphylococcus cohnii is 2:1.2:1.2: 1.5.
The Bacillus amyloliquefaciens used in this example was collected in the China Center for Type Culture Collection (CCTCC) in Lojia mountain in Wuchang district (No. 299, Bayi, Hubei, Wuhan City, the collection date was 2014, 12 months and 12 days, the collection number was AB2014337, and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) was named after classification.
The preparation method of the kitchen waste composite microbial degradation microbial inoculum specifically comprises the following steps:
(1) respectively carrying out single strain fermentation culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii to obtain bacillus amyloliquefaciens zymocyte liquid, radiation-resistant methylobacterium zymocyte liquid, disperse pantoea zymocyte liquid, pseudomonas oryzae zymocyte liquid, citrobacter freundii zymocyte liquid and staphylococcus cohnii zymocyte liquid;
(2) mixing the following bacterial strain fermentation bacteria liquids in parts by weight to obtain a composite bacterial liquid: 20 parts of bacillus amyloliquefaciens zymocyte liquid, 12 parts of radiation-resistant methylobacterium zymocyte liquid, 12 parts of pantoea dispersa zymocyte liquid, 10 parts of pseudomonas oryzae zymocyte liquid, 12 parts of citrobacter freundii zymocyte liquid and 15 parts of staphylococcus cohnii zymocyte liquid;
(3) mixing dried sawdust and bran according to a volume ratio of 1:4 to prepare a carrier;
(4) and (3) uniformly mixing the compound bacterial liquid obtained in the step (2) and the carrier obtained in the step (3) according to the volume ratio of 1:28 to obtain the kitchen waste compound microbial degradation microbial inoculum.
The culture medium used for carrying out strain culture on bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa in the step (1) comprises water as a solvent, and solutes and concentrations of: 7g/L beef extract, 7g/L peptone, 3g/L sodium chloride, 4g/L dipotassium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 0.8g/L magnesium sulfate heptahydrate and 1.2g/L ammonium sulfate.
The culture medium used for carrying out strain culture on the pseudomonas solanacearum in the step (1) has the following solvent of water, natural pH and solutes and concentrations: 240g/L of potato cooking juice and 18g/L of glucose;
the preparation method of the potato cooking juice comprises the following steps: cutting 240g of peeled potatoes into slices, weighing, heating and boiling for 30min, then filtering by gauze and reserving filtrate.
The culture medium for the bacterial strain culture of the staphylococcus cohnii in the step (1) comprises water as a solvent, pH 7.3 and solutes and concentrations as follows: 12g/L of casein, 7g/L of soybean meal, 16g/L of glucose and 8g/L of sodium chloride.
The culture conditions for culturing the strains of the bacillus amyloliquefaciens, the citrobacter freundii, the methylobacter radiodurans and the pantoea dispersa in the step (1) are as follows: the temperature is 30 ℃, the rotating speed is 200r/min, and the culture time is 36 h.
The culture conditions for strain culture of the pseudomonas solanacearum in the step (1) are as follows: the temperature is 35 ℃, shaking table cultivation is carried out, the rotating speed is 200r/min, and the cultivation time is 28 h;
the culture conditions for strain culture of the staphylococcus cohnii are as follows: the temperature is 30 ℃, shaking culture is carried out, the rotating speed is 200r/min, and the culture time is 28 h.
The kitchen waste composite microbial degradation microbial inoculum prepared by the embodiment is used for degrading kitchen waste.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (3)
1. The kitchen waste composite microbial degradation microbial inoculum is characterized by being formed by mixing a mixed strain consisting of bacillus amyloliquefaciens, radiation-resistant methylobacterium, pantoea dispersa, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii with a carrier, wherein the mixed strain accounts for 15-25% of the total mass of the degradation microbial inoculum, the mass percentage of water in the degradation microbial inoculum is 10-20%, and the pH value of the degradation microbial inoculum is 5.5-58.5, each gram of degrading bacteria agent contains 10 viable bacteria6~109A plurality of;
the Bacillus amyloliquefaciens is preserved in the China center for type culture collection, has the address of eight-path No. 299 in Lojia mountain in Wuchang district, Wuhan city, Hubei, the preservation date of 2014, 12 months and 12 days, the preservation number of CCTCC (China center for culture collection) AB2014337, and is named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) in classification;
the degrading microbial inoculum is prepared by the following method:
the culture medium for strain culture of bacillus amyloliquefaciens, citrobacter freundii, methylobacter radiodurans and pantoea dispersa adopts water as a solvent, and comprises the following solutes in concentration: 8g/L beef extract, 5g/L peptone, 5g/L sodium chloride, 5g/L dipotassium hydrogen phosphate, 5g/L potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate, 1g/L ammonium sulfate, pH 7.2, and culture conditions are as follows: the temperature is 28 ℃, the rotating speed is 200r/min, and the culture time is 20 h;
the culture medium for strain culture of the pseudomonas solanacearum adopts water as a solvent, and comprises the following solutes in concentration: 200g/L of potatoes, 15g/L of glucose and 7.5 of pH, wherein the potato cooking method comprises the following steps: taking 200g of peeled potatoes, cutting into slices, weighing, heating and boiling for 25min, then filtering by gauze and reserving filtrate, wherein the culture conditions are as follows: the temperature is 30 ℃, the rotating speed is 220r/min, and the culture time is 24 h;
the culture medium for strain culture of staphylococcus cohnii has water as solvent, the following solutes and concentrations: 10g/L of casein, 10g/L of soybean meal, 15g/L of glucose, 5g/L of sodium chloride and 7.0 of pH, wherein the culture conditions are as follows: the temperature is 28 ℃, the rotating speed is 220r/min, and the culture time is 28 h;
according to the volume ratio of 3:1.5:1.5:1.5:1.5:1, respectively taking bacillus amyloliquefaciens bacterial liquid, radiation-resistant methylobacterium bacterial liquid, disperse pantoea bacterial liquid, pseudomonas oryzae bacterial liquid, citrobacter freundii bacterial liquid and staphylococcus cohnii bacterial liquid, and uniformly mixing to obtain composite microbial liquid;
taking bran and sawdust according to the volume ratio of 1:5, and uniformly mixing the bran and the sawdust to obtain a carrier material;
and uniformly mixing the compound microbial strain liquid and a carrier material according to the volume ratio of 1:40 to obtain the kitchen waste degrading microbial inoculum.
2. The preparation method of the kitchen waste composite microbial degradation microbial inoculum according to claim 1, which is characterized by comprising the following steps:
(1) respectively carrying out single strain fermentation culture on bacillus amyloliquefaciens, radiation-resistant methylobacterium, disperse pantoea, pseudomonas oryzae, citrobacter freundii and staphylococcus cohnii to obtain bacillus amyloliquefaciens zymocyte liquid, radiation-resistant methylobacterium zymocyte liquid, disperse pantoea zymocyte liquid, pseudomonas oryzae zymocyte liquid, citrobacter freundii zymocyte liquid and staphylococcus cohnii zymocyte liquid;
(2) mixing the fermentation bacteria liquid of each strain according to a ratio to obtain a composite bacteria liquid;
(3) mixing the dried sawdust and bran according to a volume ratio of 1:5 to prepare a carrier;
(4) and (3) mixing the composite bacterial liquid obtained in the step (2) with the carrier obtained in the step (3) according to a volume ratio of 1:40, and uniformly mixing to obtain the kitchen waste composite microbial degradation microbial inoculum.
3. The use of the kitchen waste composite microbial degradation bacterial agent as claimed in claim 1, wherein the composite microbial degradation bacterial agent is used for degrading kitchen waste.
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