CN114736833B - High-temperature-resistant degradation microbial inoculum for household kitchen waste compost and method thereof - Google Patents
High-temperature-resistant degradation microbial inoculum for household kitchen waste compost and method thereof Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant degradation microbial inoculum for household kitchen waste composting and a method thereof, wherein the high-temperature-resistant degradation microbial inoculum consists of aerobacillus sp, pueribacillus sp, bacillus licheniformis (bacillus licheniformis) and bacillus subtilis (Bacillus subtilis). The high-temperature-resistant degradation microbial inoculum provided by the invention is compounded by four high-temperature-resistant synergistic bacteria with high starch, protein, fat and cellulose degradation capacities respectively obtained by directional separation and screening of a novel gellan gum culture medium, and the effective viable count of the solid composite microbial inoculum obtained by fermentation can reach 2.87 multiplied by 10 11 cfu/g-microbial inoculum, the water content is less than or equal to 30 percent, and the long-time household storage is convenient; the microbial inoculum can improve the quantity and activity of thermophilic microorganisms in a composting system, and effectively solves the bottleneck problems of slow temperature rise, low fermentation temperature, long decomposition period, poor product quality and the like of the household kitchen waste composting.
Description
Technical Field
The invention relates to a high-temperature-resistant degradation microbial agent for household kitchen waste compost and a method thereof, which are used for realizing rapid reduction, recycling and harmlessness of household kitchen waste compost, and belong to the technical fields of kitchen waste treatment and environmental microorganisms.
Background
In the context of garbage classification hot flushes and "two-carbon", home compost is gradually moved from europe into the chinese market. However, in practical application, because self-made equipment lacks a mechanical control system or a user lacks composting experience, household kitchen waste composting generally has the problems of slow starting temperature rise, low fermentation temperature, long decomposition period, poor product quality and the like, and the enthusiasm of the user is eliminated. Therefore, the improvement of technical optimization is important to the popularization of the household composting mode.
The microorganism is the main body of kitchen garbage degradation. The temperature has a direct effect on the microbial community structure and activity and the degradation of organic matters in the heap. In the initial stage of composting, along with the proliferation of microorganisms, organic matters such as easily degradable starch, protein and the like are utilized and release heat energy, and the temperature of the compost is increased. When the temperature rises to above 45 ℃, the compost enters a high temperature stage, thermophilic microorganisms become a dominant population, and the residual soluble organic matters and cellulose complex organic matters in the conversion reactor are mainly decomposed, so that the organic matter degradation capability is higher. The research shows that the temperature can be increased within a certain range to enhance the degradation capability of microorganisms on organic matters by improving the enzyme activity, and meanwhile, a part of indissolvable substances can be converted into soluble substances so as to facilitate the decomposition and utilization of microorganisms, so that the method has important significance for promoting the decomposition and innocuity of materials in a high-temperature stage. However, according to literature reports, during the high temperature stage of composting, the bacterial and actinomycete numbers will be 10 from the initial stage 7 CFU·g -1 And 10 6 CFU·g -1 Down to 10 6 CFU·g -1 And 10 4 CFU·g -1 The copy number of the 16S rRNA gene is also obviously reduced, so that the degradation advantage of high temperature to organic matters is difficult to develop, and the degradation advantage is also one of the important reasons for causing the bottleneck problem of household kitchen waste composting.
The addition of the high temperature resistant microbial inoculum to the heap body can not only improve the quantity of thermophilic microorganisms, improve the physicochemical property of the compost by changing the microbial community structure, accelerate the composting reaction process and improve the quality of the compost, but also has the advantages of simple operation, no secondary pollution, lower cost and the like compared with common natural adsorbents such as peat, zeolite and the like and additives such as calcium magnesium metal salts and the like, and is beneficial to the promotion of household composting technology and the mode popularization.
In conclusion, how to directionally separate, screen and purify the high-temperature resistant bacteria with high starch, protein, fat and cellulose degradation capability and fully utilize the synergistic effect of the high-temperature resistant bacteria to prepare the composite microbial inoculum so as to realize the rapid degradation, decomposition and harmlessness of household kitchen wastes is a problem to be solved by the technicians in the field.
Disclosure of Invention
Aiming at the bottleneck problems of low temperature rising, low fermentation temperature, long decomposition period, poor product quality and the like of the domestic kitchen garbage compost, the invention provides the high-temperature degradation resistant microbial inoculum for the domestic kitchen garbage compost and the method thereof, which accelerate the temperature rising of a heap body, prolong the duration of the high-temperature period and promote the decomposition of materials.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a high-temperature-resistant degradation microbial inoculum for household kitchen waste composting, wherein the high-temperature-resistant degradation microbial inoculum is formed by compounding and combining Aspergillus sp, pueribacillus sp, bacillus licheniformis (Bacillus licheniformis) and Bacillus subtilis (Bacillus subtilis); the Aerobacillowsp was deposited at the microorganism strain collection of Guangdong province at 2022, 4/8, under the accession number GDMCC No.62364; the Pueribacillus sp.2022, 4 th and 8 th are deposited with the Guangdong province microorganism strain collection center with the deposit number of GDMCC No.62363; the bacillus licheniformis (Bacillus licheniformis) is deposited with the cantonese province microorganism strain collection at 4 and 8 of 2022 with the deposit number of GDMCC No.62362; the bacillus subtilis (Bacillus subtilis) is deposited with the microorganism strain collection in Guangdong province at 2022, 4 and 8, with the deposit number of GDMCC No.62361.
As a preferable aspect of the first aspect, the aerobacillus sp, the Pueribacillus sp, bacillus licheniformis and Bacillus subtilis contained in the refractory degradation bacteria agent are obtained by directional separation and screening of a refractory gellan gum medium.
As a preference of the first aspect, four microorganisms of the refractory degradation bacteria are aerobacillus sp, pueribacillus sp, bacillus licheniformis and Bacillus subtilis are not antagonistic to each other and have different organic matter degradation advantages.
As a preferable aspect of the above first aspect, the aerobacillus sp, the Pueribacillus sp, bacillus licheniformis and Bacillus subtilis contained in the high temperature degradation resistant microbial agent can all grow at 55 ℃ and maintain the organic matter degradation activity.
As a preferable aspect of the first aspect, the refractory degradation bacteria agent is a solid composite bacteria agent prepared by mixing and doping an aspergillus sp, a Pueribacillus sp, bacillus licheniformis and Bacillus subtilis into a solid substrate according to a mass ratio of bacteria liquid of 1:1:1:1 and performing aerobic fermentation.
As a preferable aspect of the first aspect, the total effective viable count in the high temperature degradation resistant microbial agent is 1×10 11 ～4×10 11 cfu/g-microbial inoculum, wherein the effective viable count of the Aerobacillus sp is not less than 8X10 10 cfu/g-microbial inoculum, the effective viable count of Pueribacillus sp is not less than 3×10 10 The effective viable count of cfu/g-microbial inoculum, bacillus licheniformis is not less than 5×10 10 The effective viable count of cfu/g-microbial inoculum, bacillus subtilis is not less than 4×10 10 cfu/g-microbial inoculum.
In a second aspect, the invention provides a preparation method of the high-temperature-resistant degradation bacteria agent based on any one of the first aspect, which comprises the following steps:
respectively inoculating the Aerobactella sp, pueriactella sp, bacillus licheniformis and Bacillus subtilis into an LB liquid culture medium, and carrying out shake culture for 2-3 d to obtain four bacterial solutions; based on a solid matrix obtained by uniformly mixing sterilized corncobs, bamboo scraps and nutritional ingredients, respectively spraying the four bacterial liquids into the solid matrix, regulating the water content of the solid matrix to 58-62%, uniformly stirring, carrying out aerobic composting fermentation at 44-46 ℃ for 5-7 d, and periodically stirring and ventilating to obtain the high-temperature-resistant degradation bacterial agent.
As a preferable mode of the second aspect, the screening method of the above-described aspergillus sp., purebacillus sp., bacillus licheniformis, bacillus subtilis is as follows:
s1: extracting the environmental sample with sterile water, and diluting the extractive solution to 10 times by 10 times dilution method -3 To 10 -8 Dilution, suction 10 -6 、10 -7 、10 -8 Adding 200 mu L of each diluent into a high-temperature-resistant beef extract peptone solid culture medium, uniformly coating, and inversely culturing for 2-3 d in a constant-temperature incubator at 55 ℃ until bacterial colonies grow; single colonies were picked, streaked repeatedly and cultured at 55deg.C until a pure culture was obtained.
S2: and (3) respectively carrying out shaking culture on the pure culture strains in an amylase fermentation culture solution, a protease fermentation culture solution, a lipase fermentation culture solution and a cellulase fermentation culture solution at 55 ℃ and at 180rpm for 48 hours, then measuring the activities of amylase, protease, lipase and cellulase, and selecting the strain with high degradation enzyme activity as an alternative strain of the high-temperature degradation resistant microbial inoculum.
S3: and carrying out an antagonism test on the alternative strains, and combining strains which are not antagonized and have different organic matter degradation advantages with the strains of the bacteria sp, the Bacillus licheniformis and the Bacillus subtilis for preparing the high-temperature resistant degradation bacteria.
Further preferably, the high temperature resistant beef extract peptone solid culture medium is prepared by mixing 20.0g/L gellan gum and 4 mL/L20 mM CaCl 2 The solution replaces 20.0g/L agar in the traditional solid culture medium as coagulant, and comprises 5.0g/L NaCl, 10.0g/L tryptone and 3.0g/L beef extract as coagulantThe nutrient substance, pH7.4, is sterilized at 121deg.C for 30min, and then cooled and solidified.
Further preferably, the amylase fermentation broth comprises 10.0g/L soluble starch, 5.0g/L tryptone, 3.06g/L KH 2 PO 4 、2.50g/L(NH 4 ) 2 SO 4 、0.21g/L CaCl 2 Sterilizing at 121deg.C for 30min at pH 7.0; the protease fermentation culture solution comprises 4.0g/L casein and 0.5g/L MgSO 4 ·7H 2 O、0.36g/L KH 2 PO 4 、1.07g/L Na 2 HPO 4 ·12H 2 O、0.16g/L NaCl、0.05g/L Tryptilase、0.014g/L ZnCl 2 、0.002g/L CaCl 2 、0.002g/L FeSO 4 Sterilizing at 121deg.C for 30min at pH 7.0; the lipase fermentation culture solution comprises 12mL/L olive oil polyvinyl alcohol emulsion, 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder and 1.0g/L MgSO 4 ·7H 2 O、0.5g/L KH 2 PO 4 、0.5g/LK 2 HPO 4 Sterilizing at 121 deg.c and pH 7.4-7.6 for 30min; the cellulase fermentation culture solution comprises 7.0g/LCMC-Na, 3.0g/L tryptone and 0.5g/L MgSO 4 ·7H 2 O、2.0g/L(NH 4 ) 2 SO 4 、1.0g/LKH 2 PO 4 、0.5g/L NaCl、0.1g/L CaCl 2 、0.01g/L FeCl 3 Sterilizing at 121 deg.c and pH 7.0-7.2 for 30min.
Further preferably, the antagonism test adopts a filter paper sheet method, one strain of bacteria is uniformly coated on a high-temperature-resistant LB solid culture medium, then the filter paper sheet soaked in the bacterial liquid of the other strain of bacteria is placed on a coated flat plate, the filter paper sheet is inversely cultured for 1-2 d at 55 ℃, and whether the antagonism of the two strains of bacteria is judged according to whether a bacteriostasis ring is generated around the filter paper sheet.
As a preferable aspect of the second aspect, the mass ratio of the four bacterial liquids sprayed into the solid substrate is 1:1:1:1.
As a preferable aspect of the second aspect, the nutrient component contained in the solid matrix includes 5% corn starch, 5% soybean meal, 0.5% (NH) 4 ) 2 SO 4 、0.5％ KH 2 PO 4 、0.1％ NaCl、0.1％ MgSO 4 ·7H 2 O and 0.01% MnSO 4 The percentages are mass percentages of the corresponding components in the total mass of the solid matrix.
In a third aspect, the invention provides a method for using the high-temperature-resistant degradation microbial inoculum in household kitchen waste composting based on any one of the first aspect, which fully utilizes the linkage effect of mechanical stirring, aeration oxygen supply, material conversion and water removal, enables the growth and metabolism of microorganisms to be in an optimal temperature and humidity oxygen environment, maximally exerts the organic matter degradation capability of the high-temperature-resistant degradation microbial inoculum, and realizes the effects of rapid heat production and decomposition promotion; the linkage effect of mechanical stirring, aeration oxygen supply, material conversion and water removal is concretely realized as follows:
the high-temperature-resistant degradation microbial inoculum is added into household kitchen waste composting equipment, and the porosity of the material is maintained at 35% -50% by periodically stirring and turning; through regular ventilation and aeration, sufficient oxygen is provided for the pile body, and the activities of high amylase, protease, lipase and cellulase are ensured to be maintained by microorganisms; the metabolism of microorganisms is utilized to convert macromolecular organic matters into micromolecular compounds including carbon dioxide and water, and simultaneously, biological heat is released, so that the temperature of a stack body is quickly raised, and high temperature resistant bacteria are further promoted to take a dominant position, and the degradation effect of each strain is exerted; and a large amount of water generated by organic matter degradation can be converted into water vapor and removed from a composting system under the high-temperature condition generated by heating up a pile body, so that the water content of the material is ensured to be maintained within the optimal humidity range of microbial metabolism of 50% -65%.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts gellan gum with high thermal stability, good water holding capacity, high gel speed, high strength and wide acid resistance range to replace traditional solid culture medium coagulant agar, carrageenan and the like with lower gel temperature (40 ℃) by adding 20mM CaCl 2 The solution is used as a thickening agent (curing agent) to modify gellan gum so as to overcome the defect that the gellan gum is easy to crack in a large amount of high-temperature water-splitting evaporation environment, increase the gel strength and transparency of a solid culture medium at high temperature (55-60 ℃), and improve directional separationThe high temperature resistant single strain is cultured with high temperature resistant single strain efficiency, and the high temperature resistant single strain is successfully separated at 55 ℃ by taking the high temperature resistant single strain as a carrier.
(2) The invention obtains a plurality of high temperature resistant microorganisms through early-stage environmental genome analysis, directional separation and screening, and screens four strains which are complementary in function and are not antagonistic to each other through high temperature (55 ℃) organic matter degrading enzyme activity test and antagonistic test, and is used for preparing high temperature resistant degrading bacteria. The four strains of bacteria can promote the degradation of four macromolecular organic matters with the highest proportion in kitchen waste respectively, and the high temperature resistant microbial agent compounded by the four strains is measured to have the amylase activity of 38.65U/g-microbial agent, the protease activity of 92.48U/g-microbial agent, the lipase activity of 52.15U/g-microbial agent and the cellulase activity of 77.89U/g-microbial agent, which is higher than the product enzyme activity technical index in agricultural microbial agent (GB 20287-2006).
(3) The high-temperature resistant degradation microbial inoculum has high total effective viable bacteria content, and the maximum effective viable bacteria number of the product can reach 1-4 multiplied by 10 11 cfu/g-microbial inoculum, wherein the effective viable count of the Aerobacillus sp is not less than 8X10% 10 cfu/g-microbial inoculum, pueribacillus sp with effective viable count not less than 3×10 10 cfu/g-microbial inoculum, bacillus licheniformis effective viable count is not less than 5×10 10 cfu/g-microbial inoculum, bacillus subtilis effective viable count is not less than 4×10 10 The cfu/g-microbial inoculum is higher than the technical indexes of the effective viable count of the products in organic material decomposing inoculant (NY 609-2002) and agricultural microbial inoculum (GB 20287-2006).
(4) The invention fully utilizes the linkage effect of mechanical stirring, aeration oxygen supply, material conversion and water removal, so that the microbial growth metabolism is in the most appropriate temperature and humidity environment, the quantity and activity of thermophilic microorganisms in a composting system are improved, the degradation capability of the high-temperature degradation resistant microbial agent organic matters is exerted to the greatest extent, the temperature rise speed of a pile body is obviously accelerated, the duration of a high-temperature period is prolonged, the degradation and decomposition period of kitchen waste is shortened, and the toxicity of products is reduced.
Drawings
FIG. 1 is a temperature change line diagram of a simulated degradation test of household kitchen waste by using a high-temperature-resistant degradation microbial inoculum in an embodiment of the invention;
FIG. 2 is a line graph of pH change in a simulated degradation test of household kitchen waste by using a high-temperature-resistant degradation microbial inoculum in an embodiment of the invention;
FIG. 3 is a graph showing the variation line of Germination Index (GI) of seeds in a simulated degradation test of household kitchen waste by using the high-temperature-resistant degradation microbial inoculum in the embodiment of the invention.
Preservation of organisms
The four microorganism strain preservation information of the invention is as follows:
the American sp.4.8 days 2022 was deposited with the microbiological bacterial strain collection center, guangdong province, the academy of sciences of Guangdong province, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with the accession number GDMCC No.62364.
Pueribacillus sp., 4.8.2022, deposited at the Cantonese national institute of microbiology, cantonese university, microbiological strain collection center, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with the accession number GDMCC No.62363.
Bacillus licheniformis Bacillus licheniformis, deposited at 4 and 8 days 2022 at the Cantonese national institute of microbiology, cantonese university, microbiol, inc., address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with the accession number GDMCC No.62362.
Bacillus subtilis Bacillus subtilis, deposited at 4.8 of 2022 at the Cantonese national institute of microbiology, cantonese university, microorganism strain collection, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with the accession number GDMCC No.62361.
Detailed Description
In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to the appended drawings. The technical features of the embodiments of the invention can be combined correspondingly on the premise of no mutual conflict. The test materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
The invention provides a high-temperature-resistant degradation microbial inoculum for household kitchen waste composting, which is prepared by compounding and combining Aspergillus sp, pueribacillus sp, bacillus licheniformis (Bacillus licheniformis) and bacillus subtilis (Bacillus subtilis). Wherein, the Aerobacillus sp.4.8 of 2022 is deposited with the microorganism strain collection of Guangdong province under the accession number GDMCC No.62364, and the homology with Aeribacillus composti (NR 159152.1) is 99.93%; pueribacillus sp.2022, 4/8 was deposited with the Cantonese microorganism strain collection under accession number GDMCC No.62363, 98.53% homologous to Pueribacillus theae (MG 725951.1); bacillus licheniformis (Bacillus licheniformis) was deposited with the Cantonese microorganism strain collection at 4 and 8 of 2022 under the accession number GDMCC No.62362; bacillus subtilis (Bacillus subtilis) was deposited at 4.8 of 2022 with the collection of microorganisms and cell cultures, guangdong province under the accession number GDMCC No.62361.
In this example, the aerobacillus sp, pueribacillus sp, bacillus licheniformis and Bacillus subtilis contained in the refractory degradation bacteria agent are obtained by directional separation and screening of a novel refractory gellan gum medium; the aerobacillus sp, pueribacillus sp, bacillus licheniformis and Bacillus subtilis all grew at 55 ℃ and maintained high organic matter degrading activity. Four strains of bacteria are inoculated into a culture medium to be cultured to form bacterial liquid, and then the bacterial liquid is mixed and sprayed into a solid culture medium according to the mass ratio of the bacterial liquid of 1:1:1, and the solid composite bacterial agent is prepared through aerobic fermentation. The total effective viable count of the microbial inoculum can reach 1 to 4 multiplied by 10 11 cfu/g-microbial inoculum, wherein the effective viable count of the Aerobacillus sp is not less than 8X10% 10 cfu/g-microbial inoculum, pueribacillus sp with effective viable count not less than 3×10 10 cfu/g-microbial inoculum, bacillus licheniformis effective viable count is not less than 5×10 10 cfu/g-microbial inoculum, bacillus subtilis effective viable count is not less than 4×10 10 cfu/g-microbial agent.
The high-temperature-resistant degradation microbial inoculum is prepared by the following method:
s1: extracting the environmental sample with sterile water, and diluting the extractive solution to 10 times by 10 times dilution method -3 To 10 -8 Dilution, suction 10 -6 、10 -7 、10 -8 200 mu L of each dilution was added with high temperature resistant beef extract peptone solid medium (pH 7.4, including 20.0g/L gellan gum, 4 mL/L20 mM CaCl) 2 Uniformly coating the solution, 5.0g/L NaCl, 10.0g/L tryptone and 3.0g/L beef extract, and inversely culturing for 2-3 d (preferably 2 d) in a constant temperature incubator at 55 ℃ until colonies grow out; single colonies were picked, streaked repeatedly and cultured at 55deg.C until a pure culture was obtained.
S2: the pure culture strains were cultured in an amylase fermentation broth (pH 7.0, including 10.0g/L soluble starch, 5.0g/L tryptone, 3.06g/L KH) 2 PO 4 、2.50g/L(NH 4 ) 2 SO 4 、0.21g/L CaCl 2 ) Protease fermentation broth (pH 7.0, including 4.0g/L casein, 0.5g/L MgSO) 4 ·7H 2 O、0.36g/LKH 2 PO 4 、1.07g/L Na 2 HPO 4 ·12H 2 O、0.16g/L NaCl、0.05g/L Tryptilase、0.014g/LZnCl 2 、0.002g/L CaCl 2 、0.002g/L FeSO 4 ) Lipase fermentation broth (pH 7.4-7.6, including 12mL/L olive oil polyvinyl alcohol emulsion, 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder, 1.0g/L MgSO) 4 ·7H 2 O、0.5g/L KH 2 PO 4 、0.5g/L K 2 HPO 4 ) And cellulase fermentation broth (pH 7.0-7.2, including 7.0g/L CMC-Na, 3.0g/L tryptone, 0.5 g/LMgSO) 4 ·7H 2 O、2.0g/L(NH 4 ) 2 SO 4 、1.0g/L KH 2 PO 4 、0.5g/L NaCl、0.1g/L CaCl 2 、0.01g/L FeCl 3 ) After 48h of shaking culture at 55℃and 180rpm, the protease activity in appendix D of the 3, 5-dinitrosalicylic acid colorimetric method and the Standard for agricultural microbial agents (GB 20287-2006) mentioned in the methods for soil enzymes and research thereof is preferably referred toThe method comprises the steps of (1) measuring lipase activity in general test methods of industrial enzyme preparations (QB/T1803-1993), measuring cellulase activity in annex D of agricultural microbial agent standard (GB 20287-2006), measuring amylase, protease, lipase and cellulase activities of strains respectively, and selecting strains with high degradation enzyme activity as candidate strains of high-temperature degradation resistant bacteria.
S3: and (3) carrying out an antagonism test on the alternative strains by adopting a filter paper sheet method, uniformly coating one strain on a high-temperature-resistant LB solid culture medium, placing a filter paper sheet soaked in a bacterial liquid of the other strain on a coated flat plate, culturing for 2 days in an inverted mode at 55 ℃, and judging whether the two strains antagonize or not according to whether a bacteriostasis ring is generated around the filter paper sheet. Strains which are not antagonistic to each other and have different organic matter degradation advantages are combined with the bacteria sp, the Pueribacillus sp, bacillus licheniformis and Bacillus subtilis for preparing the high-temperature resistant degradation bacteria agent.
S4: inoculating the above-mentioned Aspergillus sp, puecibacillus sp, bacillus licheniformis and Bacillus subtilis into LB liquid culture medium (pH 7.4, including 10.0g/L NaCl, 10.0g/L tryptone and 5.0g/L yeast extract), and shake culturing for 2-3 d (preferably 2d, and shake culturing condition is preferably 55deg.C and 180 rpm) to obtain four bacterial solutions; spraying the four bacterial liquids according to the mass ratio of 1:1:1 to uniformly mix the four bacterial liquids with the mass ratio of 2:3 after sterilizing corncob (particle size of 1-2 cm) and bamboo chips (passing through a 4-mesh sieve) by high-pressure steam at 121 ℃ for 30min, and adding 5% corn starch, 5% soybean meal and 0.5% (NH) 4 ) 2 SO 4 、0.5％ KH 2 PO 4 、0.1％ NaCl、0.1％ MgSO 4 ·7H 2 O、0.01％ MnSO 4 The high-temperature-resistant degradation microbial inoculum can be obtained by adjusting the water content to 58-62% (preferably 60%) in a solid matrix serving as a nutrient component, uniformly stirring, carrying out aerobic composting fermentation for 5-7 d (preferably 5 d) at 44-46 ℃ (preferably 45 ℃), and periodically stirring and ventilating (preferably every 60min for 1 min).
The effective viable count in the high-temperature resistant degradation microbial inoculum prepared by the invention can reach 2.87 multiplied by 10 11 cfu/g-microbial inoculum with water content less than or equal to 30 percent, which is convenient for long-term familiesStored. The microbial inoculum can improve the quantity and activity of thermophilic microorganisms in a composting system, and effectively solves the bottleneck problems of slow temperature rise, low fermentation temperature, long decomposition period, poor product quality and the like of the household kitchen waste composting.
Therefore, the method for composting the household kitchen waste by using the high-temperature-resistant degradation microbial inoculum comprises the following steps:
the high-temperature-resistant degradation microbial inoculum is added into household kitchen waste composting equipment, the water content of the material is controlled to be 50% -70% (preferably, the initial mass ratio of the kitchen waste to the microbial inoculum is 2:1), the kitchen waste is poured into the equipment in the morning and evening every day, the mixture is stirred and turned over for 1min, and ventilation and aeration are timely carried out (preferably, an automatic air inlet valve is arranged on the composting equipment, and the air inflow can be adjusted according to the change of the environmental temperature). Through the linkage effects of mechanical stirring, aeration oxygen supply, material conversion and water removal, microorganisms are in an optimal temperature and humidity oxygen environment, and the efficient degradation and decomposition promotion of the microbial inoculum on kitchen waste are realized. If the phenomenon that the temperature of the pile layer is reduced because kitchen waste is not added into the equipment for a long time or the operation management is improper occurs, the composting can be restarted and the rapid temperature rise can be realized by adding the high-temperature resistant microbial inoculum into the equipment.
Wherein, the linkage effect of mechanical stirring, aeration oxygen supply, material conversion and water removal is concretely realized as follows: fully mixing kitchen waste and the microbial inoculum by stirring and turning the piles, and maintaining the porosity at 35% -50%; sufficient oxygen is provided for the pile body through ventilation and aeration, so that microorganisms are ensured to maintain high organic matter degradation activity; the microorganisms convert macromolecular organic matters into micromolecular compounds through secreting amylase, protease, lipase and cellulase, release biological heat, enable the stack to quickly heat up, further promote high temperature resistant bacteria to take a dominant position, and exert degradation effect to a great extent; wherein, a large amount of water generated by organic matter degradation can be converted into water vapor to be removed from the composting system under the high temperature condition, thereby ensuring that the microorganism metabolism is in the optimal humidity.
The specific technical effects of the screening preparation process and application of the high-temperature-resistant degradation microbial inoculum disclosed by the invention are further shown by the following examples.
Example 1
In the embodiment, the gellan gum culture medium for separating and culturing high temperature resistant is modified, and the defect that the gellan gum culture medium is easy to crack in a large amount of high-temperature water evaporation environment is overcome, and the method comprises the following steps of:
10.0-20.0 g/L gellan gum+0-10 mL 20mM CaCl 2 The solution or 10.0-20.0 g/L agar is respectively used as coagulant, deionized water is directly added to prepare a solid culture medium, the solid culture medium is placed in an incubator at 80 ℃ and 90 ℃ for 3d culture, the shape change of the culture medium is observed, and the optimal formula of the coagulant is explored.
The medium was softer than jelly after cooling to solidification and had completely changed to liquid at less than 75℃in comparison to 10.0g/L gellan gum and 10.0g/L agar, both with gellan gum and agar. The need to increase the coagulant concentration was demonstrated, and 20mM CaCl was added 2 The solution acts as a thickener (curing agent).
The addition of 20.0g/L gellan gum and 20.0g/L agar both made the medium stiffer than the addition of 10.0g/L agar, and without the addition of 20mM CaCl 2 In the case of the solution as a thickener, the setting ability of the gellan gum of 20.0g/L is better. Comparison without addition of 20mM CaCl 2 Culture medium of the solution with 20mM CaCl 2 The solution can be used as a thickening agent to improve the melting point of the culture medium to a certain extent, wherein the effect is most obvious when the adding amount is 5 mL. However, according to the results shown in Table 1, 20mM CaCl 2 The optimal addition amount of the solution may be between 1 and 5mL, so that the optimal conditions need to be continuously searched in the interval.
TABLE 1 comparison of media melt levels
As shown in Table 2, it is possible to maintain a minimum of 20mM CaCl, which is a 20.0g/L gellan gum medium having a good coagulation effect and a water retention capacity at a high temperature of 80℃and 90 ℃ 2 The amount of solution added was 4mL. Therefore, the optimal proportion of the high temperature resistant culture medium coagulant is 20.0g/L gellan gum+4 mL/L20 mM CaCl 2 A solution.
TABLE 2 comparison of media melt levels
Inoculating high temperature resistant bacteria with known properties into gellan gum with concentration of 20.0g/L and CaCl with concentration of 4mL/L and 20mM 2 The solution replaces 20.0g/L agar in the traditional solid culture medium to be used as the high temperature resistant LB solid culture medium (pH 7.4, including 20.0g/L gellan gum, 4 mL/L20 mM CaCl) 2 Solution, 10.0g/L NaCl, 10.0g/L tryptone and 5.0g/L yeast extract), and culturing for 2d in a constant temperature incubator at 55 ℃ in an inverted way, and observing that the culture medium has growth activity and the properties are not obviously changed, the novel gellan gum culture medium formula obtained in the embodiment can be used for culturing high temperature resistant strains.
Example 2
In the embodiment, directional separation and screening are carried out on strains which can be used for preparing the high-temperature-resistant degradation microbial inoculum for household kitchen garbage compost, and the specific steps are as follows:
(1) Environmental genome analysis of kitchen waste compost high-temperature period sample
And (5) extracting DNA of the sample at the high-temperature stage of the aerobic composting of the kitchen waste, and carrying out PCR amplification. Based on the amplified 16S region characteristics, a small fragment library was constructed and was double-ended sequenced (Paired_End) based on the Illumina NovaSeq sequencing platform. And performing Reads splicing filtration, OTUs (Operational Taxonomic Units) clustering, and performing species annotation and abundance analysis. The results showed that at the gate level firmics (42.18%), actionobacteria (25.45%) and Proteobacteria (11.84%) predominated in the samples; at the class level, bacillus (37.72%), actionobacteria (24.65%) and gammaproteobacter (7.04%) predominate; at the genus level, bacillus (11.92%), nocardiopsis (3.90%) and Saccharomonospora (3.68%) predominate. According to literature reports, firmics has good stress resistance at high temperature due to a special cell structure, and most Bacillus under the gate is non-pathogenic bacteria with high organic matter degradation capability, so that the bacteria compound high-temperature degradation resistant microbial inoculum can be directionally separated and screened.
(2) Isolation and purification of high temperature resistant strains
After the sample of kitchen waste aerobic composting high temperature stage is extracted by aseptic water, the leaching solution is diluted into 10 by a 10-time dilution method -3 To 10 -8 Dilution, suction 10 -6 、10 -7 、10 -8 200 mu L of each dilution is added with high temperature resistant beef extract peptone solid culture medium (pH 7.4 comprising 20.0g/L gellan gum, 4 mL/L20 mM CaCl) suitable for Bacillus growth 2 Solution, 5.0g/L NaCl, 10.0g/L tryptone, 3.0g/L beef extract) and culturing for 2d in an incubator at 55 ℃ in an inverted way until colonies are grown; single colonies were picked, streaked repeatedly and cultured at 55deg.C until a pure culture was obtained. 9 strains of high temperature resistant bacteria are obtained through separation in the steps, and the strains are numbered as H-CD-1, H-CD-2, H-CD-3, H-CD-4, H-CD-5, H-CD-6, H-CD-7, M-FW-7 and M-FW-17.
(3) Screening of efficient degradation strains of organic matters
After the 9 strains of high temperature resistant bacteria obtained by separation are respectively subjected to shaking culture for 48 hours at 55 ℃ in fermentation culture solution of amylase, protease, lipase and cellulase, the activities of the amylase, the protease, the lipase and the cellulase are respectively measured (as shown in the following table 3), and 5 strains of H-CD-1, H-CD-5, H-CD-6, M-FW-7 and M-FW-17 which have relatively high organic matter degradation capability and complementary functions are selected from the strains as candidate strains for preparing the high temperature resistant degradation bacteria.
TABLE 3 high temperature resistant Strain amylase, protease, lipase and cellulase Activity
Wherein, the four enzyme fermentation culture solutions have the following formula:
amylase fermentation broth: pH 7.0, including 10.0g/L soluble starch, 5.0g/L tryptone, 3.06g/L KH 2 PO 4 、2.50g/L(NH 4 ) 2 SO 4 、0.21g/L CaCl 2 ；
Protease fermentation broth: pH 7.0, including 4.0g/L casein, 0.5g/LMgSO 4 ·7H 2 O、0.36g/L KH 2 PO 4 、1.07g/L Na 2 HPO 4 ·12H 2 O、0.16g/L NaCl、0.05g/L Tryptilase、0.014g/L ZnCl 2 、0.002g/L CaCl 2 、0.002g/L FeSO 4 ；
Lipase fermentation broth: pH 7.4-7.6, comprising 12mL/L olive oil polyvinyl alcohol emulsion, 10.0g/L tryptone, 10.0g/L NaCl, 5.0g/L yeast powder, 1.0g/L MgSO 4 ·7H 2 O、0.5g/L KH 2 PO 4 、0.5g/L K 2 HPO 4 ；
Cellulase fermentation culture solution: pH 7.0-7.2, including 7.0g/L CMC-Na, 3.0g/L tryptone, 0.5g/L MgSO 4 ·7H 2 O、2.0g/L(NH 4 ) 2 SO 4 、1.0g/L KH 2 PO 4 、0.5g/L NaCl、0.1g/LCaCl 2 、0.01g/L FeCl 3 。
The degradation reaction of the four enzyme activity tests is carried out at 55 ℃, and the specific method is as follows:
amylase activity: referring to the colorimetric method of 3, 5-dinitrosalicylic acid mentioned in soil enzyme and research method thereof, the enzyme amount required for hydrolyzing 1mL of original enzyme solution for 30min to produce 1mg of maltose is taken as an enzyme activity unit (U);
protease activity: referring to the measurement of protease activity in appendix D of agricultural microbial agent Standard (GB 20287-2006), the amount of enzyme required to hydrolyze casein to produce 1 μg tyrosine in 1mL of as-is enzyme solution for 1min is one enzyme activity unit (U);
lipase activity: referring to the measurement of lipase activity in the general test method for Industrial enzyme preparation (QB/T1803-1993), the enzyme amount required for 1 mu mol of fatty acid production by hydrolyzing fat with 1mL of the intact enzyme solution at 40℃and pH 7.5 for 1min is one enzyme activity unit (U);
cellulase activity: referring to the measurement of cellulase activity in appendix D of "Standard for agricultural microbial agents" (GB 20287-2006), the amount of enzyme required to hydrolyze cellulose to 1. Mu.g of glucose in 1mL of the enzyme solution as it is one enzyme activity unit (U).
(4) Inter-strain antagonism test
And (3) carrying out antagonism test on 5 strains of high-temperature resistant degradation bacteria obtained by screening by adopting a filter paper sheet method, uniformly coating one strain of bacteria on a high-temperature resistant LB solid culture medium, placing the filter paper sheet soaked in bacterial liquid of the other strain of bacteria on a coated flat plate, culturing for 2 days in an inverted mode at 55 ℃, and judging whether the two strains of bacteria antagonize or not according to whether a bacteriostasis zone is generated around the filter paper sheet. Complementary strains which are not antagonistic and have high starch, fat, cellulose and protein degradation capability respectively are combined with H-CD-1, H-CD-5, H-CD-6 and M-FW-7 to be used for preparing the high temperature resistant degradation microbial inoculum.
(5) Identification of species
And (3) delivering the 4 alternative strains to the Guangdong province microorganism strain collection center for strain identification to obtain a 16SrDNA sequence, and carrying out BLAST data alignment on the sequence at NCBI. Based on the sequencing results, the pathogenicity and other physiological and biochemical characteristics of each strain were reviewed through BacDive platform and China general microbiological culture Collection center website, and H-CD-1 (Aspergillus sp.), H-CD-5 (Pueribacillus sp.), H-CD-6 (Bacillus licheniformis ), M-FW-7 (Bacillus subtilis, bacillus subtilis) were non-pathogenic bacteria.
Finally, H-CD-1 (Aerobacillus sp.) was deposited at the Cantonese collection of microorganisms and cell cultures under the accession number GDMCC No.62364 at 4 and 8 of 2022; H-CD-5 (Pueribacillus sp.) was deposited at the Cantonese microorganism strain collection at 4 and 8 of 2022 under the accession number GDMCC No.62363; H-CD-6 (Bacillus licheniformis ) was deposited at the Cantonese province microorganism strain collection at 4/8 of 2022 under the accession number GDMCC No.62362; M-FW-7 (Bacillus subtilis ) was deposited at the Cantonese microorganism strain collection at 4.8 of 2022 under the accession number GDMCC No.62361.
Example 3
In the embodiment, a high-temperature degradation resistant microbial inoculum for household kitchen waste composting is prepared, and the preparation method specifically comprises the following steps:
(1) Strain expansion culture
H-CD-1 (Aspergillus sp.), H-CD-5 (Pueribacillus sp.), H-CD-6 (Bacillus licheniformis), and M-FW-7 (Bacillus subtilis) were inoculated into LB liquid medium (pH 7.4, comprising 10.0g/L NaCl, 10.0g/L tryptone, and 5.0g/L yeast extract), respectively, and cultured with shaking at 55℃and 180rpm for 2 days to obtain four bacterial liquids.
(2) Solid composite microbial agent fermentation
Sterilizing 2kg corn cob (particle size 1-2 cm) and 3kg bamboo chip (passing through 4 mesh sieve) with 121 deg.C high pressure steam for 30min, mixing, and adding 250g corn starch, 250g soybean meal, 25g (NH) 4 ) 2 SO 4 、25g KH 2 PO 4 、5g NaCl、5g MgSO 4 ·7H 2 O、0.5g MnSO 4 In a solid matrix serving as a nutrient component, H-CD-1 (bacillus sp.), H-CD-5 (Pueribacillus sp.), H-CD-6 (Bacillus licheniformis) and M-FW-7 (Bacillus subtilis) bacterial liquid are sprayed into the solid matrix according to the mass ratio of 1:1:1:1, the water content of the solid matrix is regulated to 60%, the solid matrix is uniformly stirred, and the solid matrix is subjected to aerobic composting fermentation for 5d at 45 ℃ and is stirred and ventilated for 1min every 60min, so that the high-temperature-resistant degradation bacterial agent is obtained, and the water content is measured to be less than or equal to 30%.
(3) Determination of effective viable count of microbial inoculum
And (3) counting the effective viable count of the prepared high-temperature degradation resistant microbial agent by referring to a serial dilution method in agricultural microbial agent (GB 20287-2006). The effective viable count of the composite microbial inoculum is measured to be 2.87 multiplied by 10 11 cfu/g-microbial inoculum, wherein the number of viable bacteria of the Aerobacillus sp.effective is 8.34×10 10 cfu/g-microbial inoculum, pueribacillus sp. Effective viable count is 3.27X10% 10 cfu/g-microbial inoculum, bacillus licheniformis effective viable count is 5.71×10 10 cfu/g-microbial inoculum, bacillus subtilis effective viable count is 4.69×10 10 The cfu/g-microbial inoculum is higher than the technical indexes of the effective viable count of the products in organic material decomposing inoculant (NY 609-2002) and agricultural microbial inoculum (GB 20287-2006).
(4) Determination of microbial agent enzyme Activity
The enzyme activity of the high-temperature-resistant degrading bacterial agent obtained by the compounding was measured by referring to the enzyme activity test method in example 2. The high temperature resistant microbial agent compounded by four strains is measured to have the amylase activity of 38.65U/g-microbial agent, the protease activity of 92.48U/g-microbial agent, the lipase activity of 52.15U/g-microbial agent and the cellulase activity of 77.89U/g-microbial agent, which are higher than the technical index of the enzyme activity of the product in agricultural microbial agent (GB 20287-2006), and are higher than the simple addition of the single-strain enzyme activity, thus indicating that the four strains have good synergistic effect.
Example 4
In this embodiment, a simulated degradation test of household kitchen waste is performed on the high-temperature-resistant degradation microbial inoculum prepared in the embodiment 2, and the method specifically comprises the following steps:
the high-temperature-resistant degradation microbial inoculum is added into household kitchen waste composting equipment (the initial mass ratio of kitchen waste to microbial inoculum is 2:1), the water content of the material is controlled to be 50% -65%, the kitchen waste is poured in the morning and evening every day, then stirred and turned over for 1 minute, and ventilation and aeration are performed in time (an automatic air inlet valve can be arranged on the composting equipment for ventilation and aeration, and the air inflow can be adjusted according to the change of the environmental temperature). Through the linkage effects of mechanical stirring, aeration oxygen supply, material conversion and water removal, microorganisms are in an optimal temperature and humidity oxygen environment, and the efficient degradation and decomposition promotion of the microbial inoculum on kitchen waste are realized. If the phenomenon that the temperature of the pile layer is reduced because kitchen waste is not added into the equipment for a long time or the operation management is improper occurs, the composting can be restarted and the rapid temperature rise can be realized by adding the high-temperature resistant microbial inoculum into the equipment.
(1) Control test setup
9.65kg of kitchen waste which is manually sorted and crushed to have the particle size of 15-80 mm by scissors is respectively put into two small kitchen waste composting reactors provided with a constant temperature heating system, a ventilation aeration system, a stirring and turning system, a heat preservation system and a timing system, wherein 2kg of high temperature resistant degradation bacteria agent is added as a test group, 2kg of sterilized solid matrix is added, the initial water content of the materials is controlled to be 70%, and the heating, ventilation aeration and stirring and turning at 45 ℃ are all set to be operated for 1min every 4 hours; the reactor was run continuously for 10 days, during which the reactor temperature was measured at 9:00, 15:00, 21:00 a day, the pH and Germination Index (GI) of the material seeds were measured every 2 days, the organic matter (starch, protein, fat, cellulose) content of the material on day 0 and 10 was measured, and the number of refractory bacteria in the material at the high temperature stage of composting (day 2) was measured.
(2) Sample detection method
Temperature: the temperature of the center and the two sides of the pile body is measured by a digital display thermometer, and the average value is obtained.
pH value: putting a plurality of glass beads into a 250mL conical flask, adding 20.0g of fresh materials and 200mL of distilled water, and oscillating for 2 hours at 25 ℃ and 150 r/min; taking out and standing for 30min, suction filtering, and collecting leaching solution; taking a proper amount of leaching liquid, and measuring the pH value of the leaching liquid by using a pH meter; each set of samples was run in triplicate.
Seed Germination Index (GI): placing a piece of filter paper in a culture dish, adding 5.0mL of distilled water (blank control) or leaching solution (treatment group), then placing 10 radish seeds, closing a cover, placing in a dark condition at 25 ℃ for 3d, and measuring the germination number and root length of the seeds; each sample required 4 replicates and the seed Germination Index (GI) was calculated using the following formula:
organic matter content: the determination of the starch content in the sample adopts an anthrone colorimetric method, the determination of the crude protein content adopts a Kjeldahl nitrogen determination method, the determination of the crude fat content adopts a Soxhlet extraction method, and the determination of the crude fiber content adopts a normal form washing method.
Number of high temperature resistant microorganisms: bacteria in the material were counted by plate culture at 55℃with reference to serial dilution in agricultural microbial inoculum (GB 20287-2006).
(3) Test results
The stack temperature change during the test is shown in figure 1. After composting starts for 1d, the test group enters a high temperature period (more than or equal to 45 ℃), and the control group enters the high temperature period after 2 d; the highest temperature of the test group can reach 62.57 ℃, and is obviously higher than the highest temperature 50.67 ℃ (p=0.014) of the control group; the duration of the high temperature period of the test group is close to 2d, and the stack temperature of the control group is maintained for 1d only in the high temperature period. Therefore, the high-temperature-resistant degradation microbial inoculum can enable kitchen waste compost to be quickly started to rise in temperature, effectively improve the temperature of the compost and prolong the duration of a high-temperature period.
The pH change of the materials during the test is shown in FIG. 2. Along with compostingThe pH value of the material is gradually increased, and the pH value of the material in the test group is higher than that in the control group. Typically, the increase in the pH of the compost material is due to degradation of nitrogen-containing organics to NH 4 + And small molecular organic acid degradation, so that the addition of the high-temperature-resistant degradation bacteria agent can be presumed to improve the degradation effect of organic matters of kitchen waste.
The variation of Germination Index (GI) of the seed extract during the test is shown in FIG. 3. After composting started, the GI of the test group increased rapidly, exceeding 100% after 3d, whereas the control group exceeded 100% after 5d, with significant differences (p=0.000 on day 2, p=0.029 on day 3); at the end of composting, the GI of the test group reached 150.4% and the control group only 114.6%. Therefore, the addition of the high-temperature-resistant degradation microbial inoculum can obviously reduce the toxicity of kitchen waste and promote composting.
The degradation effect of organic matters of kitchen waste is shown in the following table 4. Through a composting test for 10 days, the degradation rates of starch, crude protein, fat and cellulose in the kitchen waste of the test group are 92.90%, 43.55%, 98.80% and 29.23%, and the degradation rates of starch, crude protein, fat and cellulose in the kitchen waste of the control group are only 80.54%, 28.57%, 92.34% and 11.98%. Therefore, the high-temperature-resistant degradation microbial inoculum has obvious promotion effect on degradation of organic matters such as starch, protein, fat, cellulose and the like in kitchen waste.
Table 4 degradation effect of high temperature resistant degradation microbial inoculum on organic matters in kitchen waste
According to the measurement, the number of the high-temperature resistant bacteria in the test group in the high-temperature period (day 2) of the compost can reach 6.24 multiplied by 10 7 cfu/g-material, whereas the control group was only 8.06X10 6 cfu/g-material, wherein the high temperature resistant bacteria community structure of the test group is more single than that of the control group, and the bacteria agents with high temperature resistance degradation comprise bacteria sp, pueribacillus sp, bacillus licheniformis and Bacillus subtilis which are dominant bacteria, the number of which is not less than 1.89 multiplied by 10 6 cfu/g-material. Therefore, the high-temperature resistant degradation microbial agent can improve the corresponding high-temperature resistance by adjusting the microbial community structure at the high-temperature stage of compostingThe bacterial quantity and activity promote the degradation of organic matters such as starch, protein, fat, cellulose and the like in kitchen garbage, and further promote the rapid decomposition of compost.
The above embodiment is only a preferred embodiment of the present invention, but it is not intended to limit the present invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, all the technical schemes obtained by adopting the equivalent substitution or equivalent transformation are within the protection scope of the invention.
Claims (7)
1. A high-temperature-resistant degradation microbial inoculum for household kitchen waste compost is characterized in that: the high-temperature resistant degradation microbial inoculum is prepared fromAeribacillus sp.、Pueribacillussp. Bacillus licheniformisBacillus licheniformis) And bacillus subtilis @Bacillus subtilis) Compounding and assembling; the saidAeribacillussp.4.8 of 2022 deposited with the cantonese province microorganism strain collection under the accession number GDMCC No.62364; the saidPueribacillussp.24.2022 deposited with the cantonese microbiological strain collection center under the accession number GDMCC No.62363; the bacillus licheniformis is [ ]Bacillus licheniformis) Deposited with the Guangdong province microorganism strain collection at 2022, 4 and 8 under the accession number GDMCC No.62362; the bacillus subtilis is [ ]Bacillus subtilis) Deposited under the accession number GDMCC No.62361 at 4 and 8 of 2022 in the China center for type culture Collection of microorganisms, guangdong province.
2. The high-temperature-resistant degradation microbial inoculum for household kitchen waste composting, as claimed in claim 1, characterized in that: the high-temperature resistant degradation microbial inoculum isAeribacillus sp.、Pueribacillus sp.、Bacillus licheniformisAndBacillus subtilisthe solid composite microbial inoculum is prepared by mixing and doping the microbial inoculum into a solid matrix according to the mass ratio of 1:1:1:1 and carrying out aerobic fermentation.
3. A household appliance as claimed in claim 1The high-temperature-resistant degradation microbial inoculum for kitchen waste compost is characterized in that: the total effective viable count in the high-temperature degradation resistant microbial inoculum is 1 multiplied by 10 11 ~4×10 11 cfu/g of microbial inoculum, whereinAeribacillussp, the effective viable count is not less than 8×10 10 cfu/g of the microbial inoculum,Pueribacillussp, the effective viable count is not less than 3×10 10 cfu/g of the microbial inoculum,Bacillus licheniformisthe effective viable count of the strain is not less than 5×10 10 cfu/g of the microbial inoculum,Bacillus subtilisthe effective viable count of the strain is not less than 4×10 10 cfu/g microbial inoculum.
4. A preparation method based on the high-temperature-resistant degradation microbial agent according to any one of claims 1 to 3, which is characterized in that: the preparation method comprises the following steps:
respectively putting the above-mentionedAeribacillus sp.、Pueribacillus sp.、Bacillus licheniformis、Bacillus subtilisInoculating the strain into an LB liquid culture medium, and performing shake culture for 2-3 d to obtain four bacterial liquids; based on a solid matrix obtained by uniformly mixing sterilized corncobs, bamboo scraps and nutritional ingredients, respectively spraying the four bacterial liquids into the solid matrix, adjusting the water content of the solid matrix to 58-62%, uniformly stirring, carrying out aerobic composting fermentation at 44-46 ℃ for 5-7 d, and periodically stirring and ventilating to obtain the high-temperature-resistant degradation bacterial agent.
5. The method for preparing the high-temperature degradation resistant microbial inoculum according to claim 4, which is characterized in that: the mass ratio of the four bacterial liquids sprayed into the solid matrix is 1:1:1:1.
6. The method for preparing the high-temperature degradation resistant microbial inoculum according to claim 4, which is characterized in that: the nutritional ingredients contained in the solid matrix comprise 5% corn starch, 5% soybean meal, 0.5% (NH) 4 ) 2 SO 4 、0.5% KH 2 PO 4 、0.1% NaCl、0.1% MgSO 4 ·7H 2 O and 0.01% MnSO 4 The percentages are mass percentages of the corresponding components in the total mass of the solid matrix.
7. The application method of the high-temperature-resistant degradation microbial inoculant in household kitchen waste compost based on any one of claims 1-3 is characterized by comprising the following steps: the high-temperature-resistant degradation microbial inoculum is added into household kitchen waste composting equipment, and the porosity of the material is maintained at 35% -50% by periodically stirring and turning; through regular ventilation and aeration, sufficient oxygen is provided for the pile body, and the activities of high amylase, protease, lipase and cellulase are ensured to be maintained by microorganisms; the metabolism of microorganisms is utilized to convert macromolecular organic matters into micromolecular compounds including carbon dioxide and water, and simultaneously, biological heat is released, so that the temperature of a stack body is quickly raised, and high temperature resistant bacteria are further promoted to take a dominant position, and the degradation effect of each strain is exerted; and a large amount of water generated by organic matter degradation can be converted into water vapor and removed from a composting system under the high-temperature condition generated by heating up a pile body, so that the water content of the material is ensured to be maintained within the optimal humidity range of microbial metabolism of 50% -65%.
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