CN114736837A - Development and application of composite microbial inoculum for degrading kitchen waste - Google Patents
Development and application of composite microbial inoculum for degrading kitchen waste Download PDFInfo
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- 239000010806 kitchen waste Substances 0.000 title claims abstract description 41
- 230000000593 degrading effect Effects 0.000 title claims abstract description 40
- 239000002131 composite material Substances 0.000 title claims abstract description 27
- 238000011161 development Methods 0.000 title claims abstract description 14
- 239000002068 microbial inoculum Substances 0.000 title claims description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 40
- 230000000813 microbial effect Effects 0.000 claims abstract description 25
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 13
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 10
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 10
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 10
- 230000015556 catabolic process Effects 0.000 claims abstract description 9
- 238000006731 degradation reaction Methods 0.000 claims abstract description 9
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- 239000000243 solution Substances 0.000 claims description 18
- 229920002472 Starch Polymers 0.000 claims description 17
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- 235000019698 starch Nutrition 0.000 claims description 17
- 238000012216 screening Methods 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 239000004006 olive oil Substances 0.000 claims description 8
- 235000008390 olive oil Nutrition 0.000 claims description 8
- 241000194108 Bacillus licheniformis Species 0.000 claims description 7
- 241000194107 Bacillus megaterium Species 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000004367 Lipase Substances 0.000 claims description 6
- 102000004882 Lipase Human genes 0.000 claims description 6
- 108090001060 Lipase Proteins 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 239000004365 Protease Substances 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 6
- 239000001913 cellulose Substances 0.000 claims description 6
- 229920002678 cellulose Polymers 0.000 claims description 6
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 229940088598 enzyme Drugs 0.000 claims description 6
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 235000019421 lipase Nutrition 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 235000020183 skimmed milk Nutrition 0.000 claims description 6
- 239000004382 Amylase Substances 0.000 claims description 5
- 102000013142 Amylases Human genes 0.000 claims description 5
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 5
- 235000019418 amylase Nutrition 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 230000000844 anti-bacterial effect Effects 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 238000013329 compounding Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 239000000839 emulsion Substances 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims description 3
- 235000019198 oils Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 238000011218 seed culture Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 229960004441 tyrosine Drugs 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 2
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000005764 inhibitory process Effects 0.000 abstract description 4
- 241000191967 Staphylococcus aureus Species 0.000 abstract description 3
- 238000005260 corrosion Methods 0.000 abstract description 3
- 230000007797 corrosion Effects 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 15
- 238000001179 sorption measurement Methods 0.000 description 5
- 239000004372 Polyvinyl alcohol Substances 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 229920002451 polyvinyl alcohol Polymers 0.000 description 4
- 238000003912 environmental pollution Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000002845 discoloration Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
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- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000009264 composting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000010791 domestic waste Substances 0.000 description 1
- 239000010794 food waste Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
Abstract
The invention discloses development and application of a composite microbial agent for degrading kitchen waste, and belongs to the technical field of kitchen waste degradation. The bacterial strains (Bacillus belgii and Bacillus subtilis) contained in the composite microbial agent have obvious inhibition effect on mixed bacteria (mucor, staphylococcus aureus and the like), after the composite microbial agent is put into a kitchen waste processor for 24 hours, no acid corrosion odor is generated around the kitchen waste processor, the composite microbial agent has high viable bacteria content and good metabolic effect, can efficiently degrade main components in the kitchen waste, and the 24-hour degradation rate can reach more than 95%.
Description
Technical Field
The invention belongs to the technical field of kitchen waste degradation, and particularly relates to development and application of a complex microbial inoculant for kitchen waste degradation.
Background
The kitchen waste is a general term of food waste and kitchen waste, is an important component of municipal domestic waste, and has the characteristics of high water content, high organic matter occupation ratio, high salt content, easiness in biodegradation and the like. At present, the kitchen waste treatment technology in China mainly comprises the traditional treatment technology and the resource treatment technology. The traditional treatment technology mainly comprises a landfill method, an incineration method and the like. The landfill method has extremely low resource treatment level, causes great resource waste due to large sewage quantity, difficult sewage treatment and poor stability of the pile body, increases the incineration cost due to high water content of the kitchen waste, causes incomplete combustion in the incinerator, and causes environmental pollution. The resource treatment technology mainly comprises aerobic composting, feed treatment, anaerobic fermentation and the like, and is mainly a biological treatment technology which degrades organic matters in the stored garbage by utilizing the natural growth and reproduction of microorganisms. Finally, the purpose of resource reuse is achieved. However, the quality of the strains for treating the kitchen waste is uneven at present, and the bacteria content of some complex microbial inoculums is not up to standard, so that the biological fermentation treatment period is long, and the organic matters in the kitchen waste are rotten along with strong sour and rotten taste due to transverse growth of mixed bacteria in the treatment process, thereby causing environmental pollution.
Disclosure of Invention
The invention aims to: the development and application of the complex microbial inoculant for degrading the kitchen waste are provided for solving the problems that the existing complex microbial inoculant has unqualified bacteria content, causes long biological fermentation treatment period, and transverse mixed bacteria in the treatment process, causes corruption of organic matters in the kitchen waste, and causes environmental pollution along with strong acid and rot taste.
In order to achieve the purpose, the invention adopts the following technical scheme:
a composite microbial agent for degrading kitchen waste is prepared by compounding Bacillus belgii, Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium, wherein the Bacillus belgii: b, bacillus subtilis: b, bacillus licheniformis: the ratio of the bacillus megaterium is (1-3): (1-3): (1-3): (0.5-2), the bacillus subtilis mainly secretes protease, the bacillus megaterium mainly secretes lipase, the bacillus licheniformis mainly secretes amylase, and the bacillus beleisi mainly has an antibacterial effect and can effectively inhibit the growth of miscellaneous bacteria such as mucor.
Development and application of a complex microbial inoculant for degrading kitchen waste specifically comprise the following steps:
s1, enrichment medium formula:
s2, screening a culture medium;
s3, taking a sample in the kitchen waste processor, respectively adding the sample into 100mL of each single strain enrichment culture medium (the inoculation amount is 5% by mass, namely 5g of the sample is inoculated into 100mL of the culture medium) under an aseptic condition, placing the sample in a shaking table for shake culture for 7d (200rpm, 37 ℃), then taking 1mL of culture solution to be inoculated into each single strain enrichment culture medium again, carrying out enrichment culture again, and repeating for three times;
s4, after the culture solution of each single strain subjected to enrichment culture repeatedly for three times is subjected to gradient dilution, each single strain is adopted to produce enzyme to screen a plate culture medium for plate coating experiment, strains with strongest enzyme production of each single strain are screened (namely, the strains with the largest transparent ring or color-changing ring at the periphery of the strains on a coating plate are selected, liquid enrichment culture and plate dilution coating are repeatedly carried out for three times until pure colonies are obtained), the obtained pure strains are inoculated to an improved glucose peptone inclined plane test tube culture medium to be cultured (at 37 ℃) until bacterial lawn is formed, and the obtained pure strains are stored in a refrigerator at 4 ℃ for later use;
s5, contacting a third-party detection mechanism, and performing 16S rRNA detection on each separated single strain to determine the genus;
and S6, respectively carrying out enrichment and propagation on each single strain, preparing a seed culture solution of the composite microbial inoculum, then carrying out inoculation, starting mixed fermentation, and producing the composite microbial inoculum.
As a further description of the above technical solution:
in the S1, multiple strains are required to be used, namely starch degrading bacteria, protein degrading bacteria, fat degrading bacteria and cellulose degrading bacteria.
As a further description of the above technical solution:
the formula of the starch degrading bacteria is 10g/L of peptone, 10g/L of soluble starch, 5g/L, NaC15 g/15 g/L of beef extract powder, 20g/L of agar and 0.125g/L of Triflozin blue (60%), the pH value needs to be kept between 7.0 and 7.2 in the culture process, screening is carried out in the culture process, the Triflozin blue and the soluble starch are stable blue after being bonded by electrostatic non-specific adsorption, the adsorption force is weakened due to molecular reduction after the starch is hydrolyzed by amylase, the Triflozin blue is liberated, the colour is deepened due to the adsorption of the liberated Triflozin blue by the surrounding unhydrolyzed starch, and a colorless and transparent hydrolysis ring is formed in a starch hydrolysis area.
As a further description of the above technical solution:
the formula of the protein degrading bacteria is 100g/L of skimmed milk powder, 10g/L of glucose and 20g/L of agar, the PH value needs to be kept at 7.0 in the culture process, screening is carried out in the culture process, and after the skimmed milk is degraded by protease, colorless and transparent degradation rings can be generated around colonies.
As a further description of the above technical solution:
the formula of the fat degrading bacteria is (NH)4)2SO4 2g/L、K2HPO4 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H20.01g/L of O, 20g/L of agar, 12mL of olive oil emulsion (the ratio of olive oil to 20g/L of polyvinyl alcohol (PVA) is 1:3, the rotating speed is 1000r/min, and the time is 5min), and in the culture process, the PH value is required to be kept between 7.2 and 7.4, 0.012g/L of bromocresol purple is added, the screening is carried out in the culture process, and after the olive oil is hydrolyzed by lipase, a yellow discoloration ring and a transparent ring colony can be generated around the colony.
As a further description of the above technical solution:
after the pH value is adjusted, bromocresol purple is added, and the mixture is continuously stirred during subpackage, so that the oil is uniformly distributed in the culture medium.
As a further description of the above technical solution:
the cellulose degrading bacteria: CMC-Na 5g/L, (NH)4)2SO41g/L (or NaNO)2 1g/L)，Na2HPO4·12H2O 1.2g/L、KH2PO4 0.9g/L、MgSO4·7H20.5g/L, KCl 0.5.5 g/L of O, 0.5g/L, L g/L of yeast extract powder, 0.5g/L of tyrosine, 20g/L of agar, deionized water for dissolution, 0.2g/L of Congo red, wherein the pH value needs to be kept at pH7.0 in the culture process, the screening is carried out in the culture process, a colony producing cellulase grows on a Congo red plate culture medium, and a colorless transparent ring appears around the colony.
As a further description of the above technical solution:
the mass ratio of the inoculation amount of the complex microbial inoculum is 2-3 percent, namely, 2-3kg of the complex microbial inoculum is inoculated to 100kg of kitchen waste.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the composite microbial inoculum of the invention is a liquid composite microbial inoculum, and the content of viable bacteria is as high as 50 x 109—1.0*1010CFU/mL (3-5 times of the same product in the market), 4 strains are mixed fermentation products, reasonable proportion is automatically formed in the whole fermentation process, the proportion of the strains in the composite microbial inoculum is reasonable, the fermentation process is integrally carried out in a fermentation tank, the mixed bacteria pollution is avoided, the fermentation condition is stable, the repeatability is good, the product is stable, the viable bacteria content is high, and the 4 strains have a synergistic effect mutually.
The bacterial strains (Bacillus belgii and Bacillus subtilis) contained in the composite microbial agent have obvious inhibition effect on mixed bacteria (mucor, staphylococcus aureus and the like), after the composite microbial agent is put into a kitchen waste processor for 24 hours, no acid corrosion odor is generated around the kitchen waste processor, the composite microbial agent has high viable bacteria content and good metabolic effect, can efficiently degrade main components in the kitchen waste, and the 24-hour degradation rate can reach more than 95%.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: the composite microbial agent for degrading the kitchen waste is characterized by being prepared by compounding bacillus belgii, bacillus subtilis, bacillus licheniformis and bacillus megaterium in a ratio of (1-3): (1-3): (1-3): (0.5-2), the bacillus subtilis mainly secretes protease, the bacillus megaterium mainly secretes lipase, the bacillus licheniformis mainly secretes amylase, the bacillus belvesii mainly generates an antibacterial effect and can effectively inhibit the growth of other bacteria such as mucor, and the mass ratio of the inoculation amount of the composite microbial inoculum is 2-3%, namely, 100kg of kitchen waste is inoculated with 2-3kg of the composite microbial inoculum.
S1, an enrichment medium formula, wherein in the S1, a plurality of strains are required, namely starch degrading bacteria, protein degrading bacteria, fat degrading bacteria and cellulose degrading bacteria;
the formula of the starch degrading bacteria is 10g/L of peptone, 10g/L of soluble starch, 5g/L, NaC15 g/L of beef extract powder, 20g/L of agar and 0.125g/L of Trillix benzene blue (60%), the PH value is required to be kept between 7.0 and 7.2 in the culture process, screening is carried out in the culture process, the Trillix benzene blue and the soluble starch are stable blue due to electrostatic non-specific adsorption and combination, the adsorption force is weakened due to molecular reduction after the starch is hydrolyzed by amylase, the Trillix benzene blue is liberated, the liberated Trillix benzene blue is adsorbed by the surrounding unhydrolyzed starch to deepen the color, a colorless and transparent hydrolysis ring is formed in a starch hydrolysis area, and the Trillix benzene blue has no obvious inhibition effect on the growth of microorganisms;
the formula of the protein degrading bacteria is 100g/L of skimmed milk powder, 10g/L of glucose and 20g/L of agar, the PH value needs to be kept at 7.0 in the culture process, screening is carried out in the culture process, and after the skimmed milk is degraded by protease, colorless transparent degradation rings are generated around colonies;
the formula of the fat degrading bacteria is (NH)4)2SO4 2g/L、K2HPO4 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H20.01g/L of O, 20g/L of agar, 12mL of olive oil emulsion (olive oil: 20g/L polyvinyl alcohol (PVA) is 1:3,rotating at 1000r/min for 5min), and in the culture process, maintaining the pH value between 7.2 and 7.4, adding bromocresol purple at 0.012g/L, screening in the culture process, wherein after olive oil is hydrolyzed by lipase, yellow discoloration ring and transparent ring bacterial colony can be generated around the bacterial colony, adding bromocresol purple after the pH value is adjusted, and continuously stirring during subpackaging to uniformly distribute the oil in a culture medium;
the cellulose degrading bacteria: CMC-Na 5g/L, (NH)4)2SO41g/L (or NaNO)2 1g/L)，Na2HPO4·12H2O 1.2g/L、KH2PO4 0.9g/L、MgSO4·7H20.5g/L, KCl 0.5.5 g/L of O, 0.5g/L, L-tyrosine 0.5g/L of yeast extract powder and 20g/L of agar, the components are dissolved by deionized water, Congo red is 0.2g/L, the pH value is required to be kept at pH7.0 in the culture process, the screening is carried out in the culture process, the colony of the cellulase is grown on a Congo red plate culture medium, and a colorless transparent ring appears around the colony:
s2, screening a culture medium;
s3, taking a sample in the kitchen waste processor, respectively adding the sample into 100mL of each single strain enrichment culture medium (the inoculation amount is 5% by mass, namely 5g of the sample is inoculated into 100mL of the culture medium) under an aseptic condition, placing the sample in a shaking table for shake culture for 7d (200rpm, 37 ℃), then taking 1mL of culture solution to inoculate each single strain enrichment culture medium again, carrying out enrichment culture again, and repeating for three times;
s4, after the culture solution of each single strain subjected to enrichment culture repeatedly for three times is subjected to gradient dilution, each single strain is adopted to produce enzyme to screen a plate culture medium for plate coating experiment, strains with strongest enzyme production of each single strain are screened (namely, the strains with the largest transparent ring or color-changing ring at the periphery of the strains on a coating plate are selected, liquid enrichment culture and plate dilution coating are repeatedly carried out for three times until pure colonies are obtained), the obtained pure strains are inoculated to an improved glucose peptone inclined plane test tube culture medium to be cultured (at 37 ℃) until bacterial lawn is formed, and the obtained pure strains are stored in a refrigerator at 4 ℃ for later use;
s5, contacting a third-party detection mechanism, and performing 16S rRNA detection on each separated single strain to determine the genus;
and S6, respectively carrying out enrichment and propagation on each single strain, preparing a seed culture solution of the composite microbial inoculum, then carrying out inoculation, starting mixed fermentation, and producing the composite microbial inoculum.
The bacterial strains (Bacillus belgii and Bacillus subtilis) contained in the composite microbial agent have obvious inhibition effect on mixed bacteria (mucor, staphylococcus aureus and the like), after the composite microbial agent is put into a kitchen waste processor for 24 hours, no acid corrosion odor is generated around the kitchen waste processor, the composite microbial agent has high viable bacteria content and good metabolic effect, can efficiently degrade main components in the kitchen waste, and the 24-hour degradation rate can reach more than 95%.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. The composite microbial agent for degrading the kitchen waste is characterized by being prepared by compounding bacillus belgii, bacillus subtilis, bacillus licheniformis and bacillus megaterium in a proportion of (1-3): (1-3): (1-3): (0.5-2), the bacillus subtilis mainly secretes protease, the bacillus megaterium mainly secretes lipase, the bacillus licheniformis mainly secretes amylase, and the bacillus beleisi mainly has an antibacterial effect and can effectively inhibit the growth of miscellaneous bacteria such as mucor.
2. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 1 are characterized by comprising the following steps of:
s1, enrichment medium formula:
s2, screening a culture medium;
s3, taking a sample in the kitchen waste processor, respectively adding the sample into 100mL of each single strain enrichment medium under an aseptic condition, wherein the inoculation amount is 5% by mass, namely inoculating 5g of the sample into 100mL of the culture medium, placing the sample in a shaking table for shake culture for 7d, culturing at the temperature of 37 ℃ at 200rpm, then taking 1mL of culture solution, inoculating into each single strain enrichment medium again, carrying out enrichment culture again, and repeating for three times;
s4, after the culture solution of each single strain subjected to enrichment culture repeatedly for three times is subjected to gradient dilution, a plate culture medium is adopted for screening enzyme production of each single strain to carry out a plate coating experiment, the strain with the strongest enzyme production of each single strain is screened out, the obtained pure bacterial colony of each single strain is inoculated to an improved glucose peptone inclined plane test tube culture medium to be cultured until bacterial lawn is formed, and the bacterial lawn is stored in a refrigerator at 4 ℃ for later use;
s5, contacting a third-party detection mechanism, and carrying out 16S rRNA detection on each separated single strain to determine the genus;
and S6, respectively carrying out enrichment and propagation on each single strain, preparing a seed culture solution of the composite microbial inoculum, then carrying out inoculation, starting mixed fermentation, and producing the composite microbial inoculum.
3. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 1, wherein in S1, multiple strains are required to be used, and the strains are starch degrading bacteria, protein degrading bacteria, fat degrading bacteria and cellulose degrading bacteria respectively.
4. The development and application of the complex microbial inoculum for degrading the kitchen waste as claimed in claim 3, wherein the formula of the starch degrading bacteria is 10g/L of peptone, 10g/L of soluble starch, 5g/L, NaC15 g/L of beef extract powder, 20g/L of agar and 0.125g/L of 60% Triben blue, the pH value is required to be kept between 7.0 and 7.2 in the culture process, and the screening is performed in the culture process.
5. The development and application of the composite microbial inoculum for degrading the kitchen waste according to claim 3, wherein the formula of the protein degrading bacteria is 100g/L of skimmed milk powder, 10g/L of glucose and 20g/L of agar, the pH value needs to be kept at 7.0 in the culture process, screening is performed in the culture process, and after the skimmed milk is degraded by protease, colorless and transparent degradation rings are generated around bacterial colonies.
6. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 1, wherein the formula of the fat degrading bacteria is (NH)4)2SO4 2g/L、K2HPO4 1g/L、KCl 0.5g/L、MgSO4·7H2O 0.5g/L、FeSO4·7H20.01g/L of O, 20g/L of agar and 12mL of olive oil emulsion, wherein the pH value is required to be kept between 7.2 and 7.4 in the culture process, 0.012g/L of bromocresol purple is added, screening is carried out in the culture process, and after the olive oil is hydrolyzed by lipase, a yellow discoloring ring and a transparent ring colony can be generated around the colony.
7. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 6, wherein bromocresol purple is added after the pH value is adjusted, and continuous stirring is needed during subpackage so that oil is uniformly distributed in a culture medium.
8. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 1, wherein the cellulose degrading bacteria: CMC-Na 5g/L, (NH)4)2SO41g/L or NaNO2 1g/L、Na2HPO4·12H2O 1.2g/L、KH2PO4 0.9g/L、MgSO4·7H20.5g/L, KCl 0.5.5 g/L of O, 0.5g/L, L-tyrosine 0.5g/L of yeast extract powder, 20g/L of agar, 0.2g/L of Congo red dissolved by deionized water, wherein the pH value needs to be kept at pH7.0 in the culture process, the screening is carried out in the culture process, the colony of the cellulase is grown on a Congo red plate culture medium, and a colorless transparent ring appears around the colony.
9. The development and application of the complex microbial inoculant for degrading kitchen waste according to claim 1, wherein the inoculation amount of the complex microbial inoculant is 2-3% by mass, namely, 2-3kg of the complex microbial inoculant is inoculated to 100kg of kitchen waste.
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| CN110846246A (en) * | 2019-11-06 | 2020-02-28 | 广东省生物资源应用研究所 | Preliminary screening method for bacteria with ammonia nitrogen degradation function |
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| CN113980833A (en) * | 2021-09-23 | 2022-01-28 | 中化农业（临沂）研发中心有限公司 | Bacillus megaterium and application thereof in soil phosphate solubilizing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108949614A (en) * | 2018-06-21 | 2018-12-07 | 华北制药集团爱诺有限公司 | A kind of microbial bacterial agent of Bei Laisi bacillus |
| CN110846246A (en) * | 2019-11-06 | 2020-02-28 | 广东省生物资源应用研究所 | Preliminary screening method for bacteria with ammonia nitrogen degradation function |
| CN112080441A (en) * | 2020-07-03 | 2020-12-15 | 河北工程大学 | Fusarium-resistant Bacillus belgii and separation and screening method thereof |
| CN113980833A (en) * | 2021-09-23 | 2022-01-28 | 中化农业（临沂）研发中心有限公司 | Bacillus megaterium and application thereof in soil phosphate solubilizing |
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