CN113444645A - Multifunctional composite fermentation inoculant and preparation method and application thereof - Google Patents
Multifunctional composite fermentation inoculant and preparation method and application thereof Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 80
- 230000004151 fermentation Effects 0.000 title claims abstract description 80
- 239000002131 composite material Substances 0.000 title claims abstract description 31
- 239000002054 inoculum Substances 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims abstract description 83
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 21
- 240000006439 Aspergillus oryzae Species 0.000 claims abstract description 20
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims abstract description 20
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 20
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 20
- 241000194107 Bacillus megaterium Species 0.000 claims abstract description 20
- 241000194103 Bacillus pumilus Species 0.000 claims abstract description 20
- 241000589776 Pseudomonas putida Species 0.000 claims abstract description 20
- 241001465752 Purpureocillium lilacinum Species 0.000 claims abstract description 20
- 241000187747 Streptomyces Species 0.000 claims abstract description 20
- 241000223259 Trichoderma Species 0.000 claims abstract description 18
- 241000881860 Paenibacillus mucilaginosus Species 0.000 claims abstract description 17
- 239000007633 bacillus mucilaginosus Substances 0.000 claims abstract description 17
- 241000235646 Cyberlindnera jadinii Species 0.000 claims abstract description 16
- 239000004310 lactic acid Substances 0.000 claims abstract description 14
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 14
- 239000010802 sludge Substances 0.000 claims abstract description 11
- 239000003895 organic fertilizer Substances 0.000 claims abstract description 10
- 239000010806 kitchen waste Substances 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims description 124
- 239000001963 growth medium Substances 0.000 claims description 75
- 229920001817 Agar Polymers 0.000 claims description 46
- 239000008272 agar Substances 0.000 claims description 46
- 230000001580 bacterial effect Effects 0.000 claims description 40
- 238000012258 culturing Methods 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 230000003321 amplification Effects 0.000 claims description 25
- 239000008367 deionised water Substances 0.000 claims description 25
- 229910021641 deionized water Inorganic materials 0.000 claims description 25
- 238000009630 liquid culture Methods 0.000 claims description 25
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 25
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 22
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000012136 culture method Methods 0.000 claims description 20
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 20
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 20
- 230000004913 activation Effects 0.000 claims description 18
- 238000011068 loading method Methods 0.000 claims description 18
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 15
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 15
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 12
- 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 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000015278 beef Nutrition 0.000 claims description 10
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 10
- 229940041514 candida albicans extract Drugs 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 10
- 239000000284 extract Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 238000011049 filling Methods 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 6
- 239000002699 waste material Substances 0.000 claims description 6
- 229920002261 Corn starch Polymers 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 5
- 239000012880 LB liquid culture medium Substances 0.000 claims description 5
- 229930195725 Mannitol Natural products 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000008120 corn starch Substances 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 5
- 239000011702 manganese sulphate Substances 0.000 claims description 5
- 235000007079 manganese sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 239000000594 mannitol Substances 0.000 claims description 5
- 235000010355 mannitol Nutrition 0.000 claims description 5
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 5
- 229920000053 polysorbate 80 Polymers 0.000 claims description 5
- 239000004323 potassium nitrate Substances 0.000 claims description 5
- 235000010333 potassium nitrate Nutrition 0.000 claims description 5
- 229920001592 potato starch Polymers 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- 235000015393 sodium molybdate Nutrition 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 229960004793 sucrose Drugs 0.000 claims description 5
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 5
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 2
- 239000010815 organic waste Substances 0.000 abstract description 12
- 239000003337 fertilizer Substances 0.000 abstract description 11
- 239000010902 straw Substances 0.000 abstract description 10
- 210000003608 fece Anatomy 0.000 abstract description 9
- 239000010871 livestock manure Substances 0.000 abstract description 9
- 244000144972 livestock Species 0.000 abstract description 7
- 244000144977 poultry Species 0.000 abstract description 7
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 244000000010 microbial pathogen Species 0.000 abstract description 2
- 230000004044 response Effects 0.000 abstract description 2
- 239000002068 microbial inoculum Substances 0.000 description 16
- 241000588724 Escherichia coli Species 0.000 description 8
- 244000052616 bacterial pathogen Species 0.000 description 8
- 241000186660 Lactobacillus Species 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229940039696 lactobacillus Drugs 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 238000001514 detection method Methods 0.000 description 5
- 239000005416 organic matter Substances 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 239000010813 municipal solid waste Substances 0.000 description 4
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 238000012364 cultivation method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 238000005507 spraying Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 241000499912 Trichoderma reesei Species 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- 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
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- 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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Engineering & Computer Science (AREA)
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Abstract
The invention relates to a multifunctional composite fermentation inoculant, a preparation method and an application thereof, wherein the multifunctional composite fermentation inoculant comprises the following components in parts by weight: 5-15 parts of bacillus megaterium, 3-12 parts of bacillus licheniformis, 4-15 parts of bacillus amyloliquefaciens, 5-17 parts of bacillus pumilus, 8-20 parts of pseudomonas putida, 5-15 parts of bacillus mucilaginosus, 1-5 parts of aspergillus niger, 3-10 parts of aspergillus oryzae, 1-8 parts of paecilomyces lilacinus, 3-12 parts of trichoderma, 3-10 parts of streptomyces jingyangensis, 2-8 parts of candida utilis and 3-12 parts of lactic acid bacteria. Compared with the prior art, the zymophyte can be matched with each other in each stage of organic waste fermentation; the microbial organic fertilizer has the advantages of quick response, high temperature tolerance, high efficiency and pathogenic microorganism killing, can be used for preparing biological organic fertilizers by fermenting traditional raw materials such as livestock and poultry manure and straws, can also be used for fermenting kitchen waste, municipal sludge and the like to form organic fertilizer raw materials, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a composite microbial inoculum for fermentation of various organic wastes and a preparation method thereof.
Background
In recent years, with the development of industrial and agricultural production and the improvement of living standard of people, the yield of various organic wastes such as straws, livestock and poultry manure, kitchen waste, aquatic product processing waste, municipal sludge and the like is increased day by day, and great environmental protection pressure is formed. On the other hand, farmland soil has various problems of hardening, acidification and the like due to long-term excessive application and unreasonable cultivation of chemical fertilizers and pesticides, and needs a large amount of organic fertilizers. The fermentation treatment of the organic wastes into organic fertilizers is an important means for connecting and solving the two problems. The microbial agents for fermenting the organic wastes are many, most of the microbial agents are used for fermenting traditional raw materials such as livestock and poultry manure and straws, the tolerable temperature is generally lower than 65 ℃, and the bacteria can be killed when the temperature is too high. And most of the microbial inoculums are only suitable for fermentation of traditional raw materials, and when the microbial inoculums are used for fermentation of kitchen waste, municipal sludge, aquatic product processing fertilizers and the like, the microbial inoculums usually take effect slowly and have poor fermentation effect due to different components. Meanwhile, kitchen waste, aquatic product processing waste and the like may contain a large amount of germs, and the germs, parasitic ova and the like can be well killed only by higher temperature and quick effect. Meanwhile, as the components are complex, more strains are required to be matched, and a good fermentation effect can be achieved.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the multifunctional composite fermentation microbial inoculum which has quick response and can efficiently kill pathogenic microorganisms and can be used for fermenting various organic wastes such as traditional raw materials of livestock and poultry manure and straw mixtures, municipal sludge, kitchen garbage and the like.
The purpose of the invention can be realized by the following technical scheme: a multifunctional composite fermentation inoculant comprises the following components in parts by weight: 5-15 parts of bacillus megaterium, 3-12 parts of bacillus licheniformis, 4-15 parts of bacillus amyloliquefaciens, 5-17 parts of bacillus pumilus, 8-20 parts of pseudomonas putida, 5-15 parts of bacillus mucilaginosus, 1-5 parts of aspergillus niger, 3-10 parts of aspergillus oryzae, 1-8 parts of paecilomyces lilacinus, 3-12 parts of trichoderma, 3-10 parts of streptomyces jingyangensis, 2-8 parts of candida utilis and 3-12 parts of lactic acid bacteria.
Furthermore, the preservation numbers of the strains are respectively Bacillus megaterium CGMCC1.234, Bacillus licheniformis CGMCC1.6510, Bacillus amyloliquefaciens CGMCC1.7463, Bacillus pumilus CGMCC1.7456, Pseudomonas putida CGMCC1.1003, Bacillus mucilaginosus CGMCC1.2326, Aspergillus niger CGMCC3.15297, Aspergillus oryzae CGMCC1711, Paecilomyces lilacinus CGMCC2780, Trichoderma reesei CGMCC3.4004, Streptomyces jingyangensis ACCCC 40126, Candida utilis CGMCC2.615 and lactobacillus CGMCC 1.3114.
A method for preparing multifunctional composite zymophyte agent comprises culturing each bacteria, mixing the obtained bacteria liquid to obtain the product.
Further, the culture method of the bacillus megaterium, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus pumilus and the pseudomonas putida comprises the following steps:
placing the mother bacteria in an improved LB culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain a first-grade seed bacterial colony;
then inoculating the first-level seed colonies into an agar-free LB liquid culture medium, wherein the liquid loading amount is 150ml/500ml, and culturing for 30-36 hours at the temperature of 30-36 ℃ and at the speed of 110-220 r/min to obtain a second-level seed liquid;
putting the secondary seed liquid into a fermentation tank for amplification culture;
the improved LB culture medium comprises the following components in percentage by weight: 0.8-1.2% of peptone, 0.8-1.2% of beef extract, 0.3-0.6% of yeast extract, 8-12% of glucose, 0.1-0.3% of monopotassium phosphate, 0.08-0.12% of sodium chloride, 0.1-0.2% of corn starch, 1.8-2.2% of agar and the balance of deionized water.
Further, the culture method of bacillus mucilaginosus is as follows: firstly, performing primary activation culture, namely putting parent bacteria into a culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed colonies; then, inoculating the first-level seed bacterial colony into the liquid culture medium without agar, wherein the liquid filling amount is 150ml/500ml, and culturing for 30-36 hours under the conditions of 30-36 ℃ and 110-220 r/min to obtain a second-level seed liquid; putting the secondary seed liquid into a fermentation tank for amplification culture;
the culture medium comprises the following components in percentage by weight: 0.02-0.025% of monopotassium phosphate, 0.002-0.003% of sodium molybdate, 1.8-2.2% of mannitol, 0.07-0.1% of dipotassium phosphate, 0.08-0.12% of ferric chloride, 0.08-0.12% of calcium carbonate, 0.02-0.03% of magnesium sulfate heptahydrate, 1.8-2.2% of agar and the balance of deionized water.
Further, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Paecilomyces lilacinus were cultured by the following method:
firstly, performing primary culture of each bacterium, and placing parent bacteria in an improved PDA culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed colonies;
then carrying out liquid culture: inoculating the activated and cultured colony in the PDA culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 30-36 hours at 34 ℃ and 110-220 r/min to obtain a secondary seed solution;
the second-stage seed liquid is put into a fermentation tank for amplification culture;
the improved PDA culture medium comprises the following components in percentage by weight: 15-20% of potato flour, 5-7% of bran, 2-2.5% of cane sugar, 0.25-0.35% of monopotassium phosphate, 0.14-0.18% of magnesium sulfate heptahydrate, 2-2.5% of agar and the balance of deionized water.
Further, streptomyces jingyangensis is cultured by the following method: firstly, performing primary activation culture, and placing the mother bacteria in an improved Gao's first culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed bacterial colonies; then carrying out liquid culture: inoculating the first-level seed colonies into the Gao's first liquid culture medium without agar, filling the liquid in the culture medium with the liquid volume of 150ml/500ml, and culturing the liquid for 20-30 hours under the conditions of 30-36 ℃ and 110-220 r/min to obtain a second-level seed liquid; the second-stage seed liquid is put into a fermentation tank for amplification culture;
the improved Gao's I culture medium comprises the following components in percentage by weight: 1.2-2.2% of soluble starch, 0.1-0.15% of potassium nitrate, 0.04-0.06% of sodium chloride, 0.04-0.06% of dipotassium phosphate, 0.001-0.002% of ferrous sulfate, 0.028-0.032% of calcium carbonate, 0.04-0.06% of magnesium sulfate heptahydrate, 1.8-2.2% of agar and the balance of deionized water.
Further, the lactic acid bacteria were cultured by the following method: firstly, performing first-stage activation culture, and placing parent bacteria in an improved MRS culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain first-stage seed bacterial colonies; then inoculating the first-level seed colonies into the MRS liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 4-5 days at 37 ℃ and 110-220 r/min to obtain a second-level seed liquid; and carrying out two times of expanding and matching on the second-stage seed liquid to obtain a fourth-stage seed liquid. Then the fourth-level seed liquid is put into a fermentation tank for amplification culture.
The improved MRS culture medium comprises the following components in percentage by weight: 0.8-1.2% of peptone, 0.8-1.2% of beef extract, 0.5-0.8% of yeast extract, 1.8-2.2% of glucose, 0.18-0.22% of dipotassium hydrogen phosphate, 0.5-0.8% of sodium acetate, 0.18-0.22% of ammonium citrate, 0.05-0.07% of magnesium sulfate heptahydrate, 0.02-0.03% of manganese sulfate, 0.1-0.15% of Tween 80, 2-2.4% of agar and the balance of deionized water.
The multifunctional compound fermentation inoculant is applied to the fermentation of bio-organic fertilizers of traditional raw materials or the fermentation of kitchen waste, aquatic product processing waste and municipal sludge to prepare organic fertilizer products.
Compared with the prior art, the invention has the following beneficial effects:
1) the bacillus such as bacillus megaterium, bacillus licheniformis, bacillus amyloliquefaciens, bacillus pumilus and bacillus mucilaginosus in the invention is a common organic fertilizer fermentation bacterium, can also quickly mineralize nutrient substances in organic wastes, and has the functions of fixing nitrogen, dissolving phosphorus, dissolving potassium and the like; pseudomonas putida can eliminate toxic substances such as hydrogen sulfide and ammonia; aspergillus niger, Aspergillus oryzae, Trichoderma, Paecilomyces lilacinus and other fungi can decompose organic matter and produce various pathogenic bacteria inhibiting matters, and Aspergillus niger may chelate heavy metal; the candida utilis can remove impurities such as urea or nitrate; the lactobacillus decomposes the saccharides to produce acid and adjusts the pH value; the streptomyces jingyangensis is actinomycetes, can secrete a plurality of substance antagonistic pathogenic bacteria, and can be used for fermentation of a mixture of livestock and poultry manure and straws which are traditional raw materials and fermentation of a plurality of organic wastes such as municipal sludge, kitchen garbage and the like due to synergistic effect among the bacteria.
2) After the strains are compounded and optimized, the effect taking speed is high, the temperature can reach more than 45 ℃ within 1-2 days, the high temperature required by the fermentation of the organic fertilizer can reach more than 55 ℃ within 2-3 days, and the fermentation time cost is saved. When the microbial inoculum is used for fermenting organic wastes with more pathogenic bacteria or mixed bacteria, the main fermentation bacteria in the microbial inoculum can resist the high temperature of more than 70 ℃, so that the pathogenic bacteria can be killed efficiently, and the high-temperature fermentation period is safer and is easy to manage; and the bacteria survive in the fermented product, so that the fertilizer efficiency of the product is improved.
3) The composite microbial inoculum has wide application, and can be used for fermenting straw, livestock and poultry manure, kitchen waste, aquatic product processing waste, municipal sludge and other organic wastes and preparing organic fertilizer.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
The mother bacteria used for culturing the bacteria related to the following embodiments are all commercial or published strains, for example, the published strains with the preservation numbers of respectively bacillus megaterium CGMCC1.234, bacillus licheniformis CGMCC1.6510, bacillus amyloliquefaciens CGMCC1.7463, bacillus pumilus CGMCC1.7456, pseudomonas putida CGMCC1.1003, bacillus mucilaginosus CGMCC1.2326, aspergillus niger CGMCC3.15297, aspergillus oryzae CGMCC1711, paecilomyces lilacinus CGMCC2780, trichoderma CGMCC3.4004, streptomyces jingyangensis ACCCC 40126, candida utilis CGMCC2.615 and lactobacillus CGMCC1.3114 can be selected as the mother bacteria.
Example 1
A multifunctional composite fermentation inoculant comprises the following components in parts by weight: 10 parts of bacillus megaterium, 8 parts of bacillus licheniformis, 10 parts of bacillus amyloliquefaciens, 10 parts of bacillus pumilus, 10 parts of pseudomonas putida, 10 parts of bacillus mucilaginosus, 3 parts of aspergillus niger, 8 parts of aspergillus oryzae, 5 parts of paecilomyces lilacinus, 6 parts of trichoderma, 6 parts of streptomyces jingyangensis, 4 parts of candida utilis and 8 parts of lactic acid bacteria.
The multifunctional composite zymophyte agent is in a composite bacteria liquid state, namely, the bacteria are weighed according to the weight parts and used as mother bacteria, the bacteria liquid is obtained by respectively culturing the mother bacteria, and the bacteria liquid is uniformly mixed, so that the multifunctional composite zymophyte agent product is obtained.
The strains are all the existing strains, and the preservation numbers are respectively Bacillus megaterium CGMCC1.234, Bacillus licheniformis CGMCC1.6510, Bacillus amyloliquefaciens CGMCC1.7463, Bacillus pumilus CGMCC1.7456, Pseudomonas putida CGMCC1.1003, Bacillus mucilaginosus CGMCC1.2326, Aspergillus niger CGMCC3.15297, Aspergillus oryzae CGMCC1711, Paecilomyces lilacinus CGMCC2780, Trichoderma CGMCC3.4004, Streptomyces jingyangensis ACCCC 40126, Candida utilis CGMCC2.615 and lactobacillus CGMCC 1.3114.
The culture methods of the above-mentioned bacteria are respectively as follows:
(1) the culture method of the bacillus megaterium, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus pumilus, the pseudomonas putida and the candida utilis comprises the following steps:
putting the mother bacteria into an improved LB culture medium to be cultured for 32 hours at 32 ℃ to obtain a first-class seed colony; the improved LB culture medium comprises the following components in percentage by weight: peptone 1.0%, beef extract 1.0%, yeast extract 0.5%, glucose 10%, potassium dihydrogen phosphate 0.2%, sodium chloride 0.1%, corn starch 0.15%, agar 2.0%, and the balance of deionized water.
Then, inoculating the first-class seed bacterial colony into an agar-free LB liquid culture medium, wherein the liquid loading amount is 150ml/500ml, and culturing for 32 hours at the temperature of 32 ℃ and at the speed of 150 r/min to obtain a second-class seed liquid;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain bacillus megaterium solution, bacillus licheniformis solution, bacillus amyloliquefaciens solution, bacillus pumilus solution and pseudomonas putida solution.
(2) The culture method of the bacillus mucilaginosus comprises the following steps: firstly, performing primary activation culture, and placing parent bacteria in a culture medium to culture for 32 hours at 32 ℃ to obtain primary seed colonies; then inoculating the first-class seed bacterial colony into the liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 32 hours at the temperature of 32 ℃ and at the speed of 150 r/min to obtain a second-class seed liquid; and (4) putting the secondary seed liquid into a fermentation tank for amplification culture to obtain the colloidal bacillus liquid.
The culture medium comprises the following components in percentage by weight: 0.022% of monopotassium phosphate, 0.0025% of sodium molybdate, 2.0% of mannitol, 0.08% of dipotassium phosphate, 0.1% of ferric trichloride, 0.1% of calcium carbonate, 0.028% of magnesium sulfate heptahydrate, 2.0% of agar and the balance of deionized water.
(3) The culture method of Aspergillus niger, Aspergillus oryzae, Trichoderma and Paecilomyces lilacinus comprises the following steps:
firstly, respectively carrying out primary culture on each bacterium, and placing parent bacteria in an improved PDA culture medium to be cultured for 32 hours at 32 ℃ to obtain primary seed colonies; the improved PDA culture medium comprises the following components in percentage by weight: 18% of potato flour, 6% of bran, 2.3% of sucrose, 0.30% of monopotassium phosphate, 0.16% of magnesium sulfate heptahydrate, 2.3% of agar and the balance of deionized water;
then carrying out liquid culture: inoculating the colony subjected to the activation culture into the PDA culture medium without agar, wherein the liquid loading is 150ml/500ml, and culturing at 34 deg.C and 150 rpm for 32 hr to obtain secondary seed solution;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain Aspergillus niger bacterial liquid, Aspergillus oryzae bacterial liquid, Trichoderma bacterial liquid and Paecilomyces lilacinus bacterial liquid.
(4) The cultivation method of streptomyces jingyangensis is as follows: firstly, performing primary activation culture, and placing parent bacteria in an improved Gao's first culture medium to be cultured for 32 hours at 32 ℃ to obtain primary seed colonies; then carrying out liquid culture: inoculating the first-class seed colony into the Gao's first liquid culture medium without agar, filling liquid in the culture medium with the volume of 150ml/500ml, and culturing for 25 hours at the temperature of 32 ℃ and at the speed of 150 r/min to obtain a second-class seed liquid; the second-stage seed liquid is put into a fermentation tank for amplification culture to obtain Jingyang streptomyces liquid;
the improved Gao's I culture medium comprises the following components in percentage by weight: 2.0 percent of soluble starch, 0.12 percent of potassium nitrate, 0.05 percent of sodium chloride, 0.05 percent of dipotassium phosphate, 0.0015 percent of ferrous sulfate, 0.030 percent of calcium carbonate, 0.05 percent of magnesium sulfate heptahydrate, 2.0 percent of agar and the balance of deionized water.
(5) The culture method of the lactic acid bacteria comprises the following steps: firstly, performing primary activation culture, and putting parent bacteria in an improved MRS culture medium to culture for 32 hours at 32 ℃ to obtain primary seed colonies; then inoculating the first-level seed bacterial colony into the MRS liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 4-5 days at 37 ℃ and 150 rpm to obtain a second-level seed liquid; and carrying out two times of expanding and matching on the second-stage seed liquid to obtain a fourth-stage seed liquid. And then, putting the four-stage seed liquid into a fermentation tank for amplification culture to obtain the lactic acid bacteria liquid.
The improved MRS culture medium comprises the following components in percentage by weight: peptone 1.0%, beef extract 1.0%, yeast extract 0.6%, glucose 2.0%, dipotassium hydrogen phosphate 0.20%, sodium acetate 0.6%, ammonium citrate 0.20%, magnesium sulfate heptahydrate 0.06%, manganese sulfate 0.025%, tween 80 0.12%, agar 2.2%, and the balance of deionized water.
Example 2
A multifunctional composite fermentation inoculant comprises the following components in parts by weight: 5 parts of bacillus megaterium, 3 parts of bacillus licheniformis, 4 parts of bacillus amyloliquefaciens, 5 parts of bacillus pumilus, 20 parts of pseudomonas putida, 15 parts of bacillus mucilaginosus, 1 part of aspergillus niger, 3 parts of aspergillus oryzae, 1 part of paecilomyces lilacinus, 12 parts of trichoderma, 10 parts of streptomyces jingyangensis, 2 parts of candida utilis and 3 parts of lactic acid bacteria.
The multifunctional composite zymophyte agent is in a composite bacteria liquid state, namely, the bacteria are weighed according to the weight parts and used as mother bacteria, the bacteria liquid is obtained by respectively culturing the mother bacteria, and the bacteria liquid is uniformly mixed, so that the multifunctional composite zymophyte agent product is obtained.
The strains are all the existing strains, and the preservation numbers are respectively Bacillus megaterium CGMCC1.234, Bacillus licheniformis CGMCC1.6510, Bacillus amyloliquefaciens CGMCC1.7463, Bacillus pumilus CGMCC1.7456, Pseudomonas putida CGMCC1.1003, Bacillus mucilaginosus CGMCC1.2326, Aspergillus niger CGMCC3.15297, Aspergillus oryzae CGMCC1711, Paecilomyces lilacinus CGMCC2780, Trichoderma CGMCC3.4004, Streptomyces jingyangensis ACCCC 40126, Candida utilis CGMCC2.615 and lactobacillus CGMCC 1.3114.
The culture methods of the above-mentioned bacteria are respectively as follows:
(1) the culture method of the bacillus megaterium, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus pumilus, the pseudomonas putida and the candida utilis comprises the following steps:
placing the mother bacteria in an improved LB culture medium to be cultured for 36 hours at 30 ℃ to obtain a first-class seed bacterial colony; the improved LB culture medium comprises the following components in percentage by weight: peptone 0.8%, beef extract 0.8%, yeast extract 0.3%, glucose 8%, potassium dihydrogen phosphate 0.1%, sodium chloride 0.08%, corn starch 0.1%, agar 1.8%, and deionized water in balance
Then inoculating the first-class seed bacterial colony into an agar-free LB liquid culture medium, wherein the liquid loading amount is 150ml/500ml, and culturing for 36 hours under the conditions of 30 ℃ and 110 r/min to obtain a second-class seed liquid;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain bacillus megaterium solution, bacillus licheniformis solution, bacillus amyloliquefaciens solution, bacillus pumilus solution and pseudomonas putida solution.
(2) The culture method of the bacillus mucilaginosus comprises the following steps: firstly, performing primary activation culture, and placing parent bacteria in a culture medium to culture for 36 hours at 30 ℃ to obtain primary seed colonies; then inoculating the first-class seed bacterial colony into the liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 36 hours under the conditions of 30 ℃ and 110 r/min to obtain a second-class seed liquid; and (4) putting the secondary seed liquid into a fermentation tank for amplification culture to obtain the colloidal bacillus liquid.
The culture medium comprises the following components in percentage by weight: 0.02% of monopotassium phosphate, 0.002% of sodium molybdate, 1.8% of mannitol, 0.07% of dipotassium phosphate, 0.08% of ferric trichloride, 0.08% of calcium carbonate, 0.02% of magnesium sulfate heptahydrate, 1.8% of agar and the balance of deionized water.
(3) The culture method of Aspergillus niger, Aspergillus oryzae, Trichoderma and Paecilomyces lilacinus comprises the following steps:
firstly, respectively carrying out primary culture on each bacterium, and placing parent bacteria in an improved PDA culture medium to be cultured for 30 hours at 30 ℃ to obtain primary seed colonies; the improved PDA culture medium comprises the following components in percentage by weight: 15% of potato flour, 5% of bran, 2% of sucrose, 0.25% of monopotassium phosphate, 0.14% of magnesium sulfate heptahydrate, 2% of agar and the balance of deionized water;
then carrying out liquid culture: inoculating the colony subjected to the activation culture into the PDA culture medium without agar, wherein the liquid loading is 150ml/500ml, and culturing at 34 deg.C and 110 rpm for 36 hr to obtain secondary seed solution;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain Aspergillus niger bacterial liquid, Aspergillus oryzae bacterial liquid, Trichoderma bacterial liquid and Paecilomyces lilacinus bacterial liquid.
(4) The cultivation method of streptomyces jingyangensis is as follows: firstly, performing primary activation culture, and placing parent bacteria in an improved Gao's first culture medium to be cultured for 36 hours at 30 ℃ to obtain primary seed colonies; then carrying out liquid culture: inoculating the first-class seed colony into the Gao's first liquid culture medium without agar, filling liquid in the culture medium with the volume of 150ml/500ml, and culturing for 30 hours at the temperature of 30 ℃ and the speed of 110 r/min to obtain a second-class seed liquid; the second-stage seed liquid is put into a fermentation tank for amplification culture to obtain Jingyang streptomyces liquid;
the improved Gao's I culture medium comprises the following components in percentage by weight: 1.2% of soluble starch, 0.1% of potassium nitrate, 0.04% of sodium chloride, 0.04% of dipotassium phosphate, 0.001% of ferrous sulfate, 0.028% of calcium carbonate, 0.04% of magnesium sulfate heptahydrate, 1.8% of agar and the balance of deionized water.
(5) The culture method of the lactic acid bacteria comprises the following steps: firstly, performing first-stage activation culture, and placing parent bacteria in an improved MRS culture medium to be cultured for 36 hours at 30 ℃ to obtain first-stage seed bacterial colonies; then inoculating the first-level seed bacterial colony into the MRS liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 4-5 days at 37 ℃ and 110 r/min to obtain a second-level seed liquid; and carrying out two times of expanding and matching on the second-stage seed liquid to obtain a fourth-stage seed liquid. And then, putting the four-stage seed liquid into a fermentation tank for amplification culture to obtain the lactic acid bacteria liquid.
The improved MRS culture medium comprises the following components in percentage by weight: 0.8% of peptone, 0.8% of beef extract, 0.5% of yeast extract, 1.8% of glucose, 0.18% of dipotassium phosphate, 0.5% of sodium acetate, 0.18% of ammonium citrate, 0.05% of magnesium sulfate heptahydrate, 0.02% of manganese sulfate, 0.1% of tween 80, 2% of agar and the balance of deionized water.
Example 3
A multifunctional composite fermentation inoculant comprises the following components in parts by weight: 15 parts of bacillus megaterium, 12 parts of bacillus licheniformis, 15 parts of bacillus amyloliquefaciens, 17 parts of bacillus pumilus, 8 parts of pseudomonas putida, 5 parts of bacillus mucilaginosus, 5 parts of aspergillus niger, 10 parts of aspergillus oryzae, 8 parts of paecilomyces lilacinus, 3 parts of trichoderma, 3 parts of streptomyces jingyangensis, 8 parts of candida utilis and 3 parts of lactic acid bacteria.
The multifunctional composite zymophyte agent is in a composite bacteria liquid state, namely, the bacteria are weighed according to the weight parts and used as mother bacteria, the bacteria liquid is obtained by respectively culturing the mother bacteria, and the bacteria liquid is uniformly mixed, so that the multifunctional composite zymophyte agent product is obtained.
The strains are all the existing strains, and the preservation numbers are respectively Bacillus megaterium CGMCC1.234, Bacillus licheniformis CGMCC1.6510, Bacillus amyloliquefaciens CGMCC1.7463, Bacillus pumilus CGMCC1.7456, Pseudomonas putida CGMCC1.1003, Bacillus mucilaginosus CGMCC1.2326, Aspergillus niger CGMCC3.15297, Aspergillus oryzae CGMCC1711, Paecilomyces lilacinus CGMCC2780, Trichoderma CGMCC3.4004, Streptomyces jingyangensis ACCCC 40126, Candida utilis CGMCC2.615 and lactobacillus CGMCC 1.3114.
The culture methods of the above-mentioned bacteria are respectively as follows:
(1) the culture method of the bacillus megaterium, the bacillus licheniformis, the bacillus amyloliquefaciens, the bacillus pumilus, the pseudomonas putida and the candida utilis comprises the following steps:
placing the mother bacteria in an improved LB culture medium to be cultured for 30 hours at 36 ℃ to obtain a first-class seed bacterial colony; the improved LB culture medium comprises the following components in percentage by weight: peptone 1.2%, beef extract 1.2%, yeast extract 0.6%, glucose 12%, potassium dihydrogen phosphate 0.3%, sodium chloride 0.12%, corn starch 0.2%, agar 2.2%, and deionized water in balance
Then, inoculating the first-class seed bacterial colony into an LB liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 30 hours at 36 ℃ and 220 r/min to obtain a second-class seed liquid;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain bacillus megaterium solution, bacillus licheniformis solution, bacillus amyloliquefaciens solution, bacillus pumilus solution and pseudomonas putida solution.
(2) The culture method of the bacillus mucilaginosus comprises the following steps: firstly, performing primary activation culture, and placing parent bacteria in a culture medium to culture for 30 hours at 36 ℃ to obtain primary seed colonies; then inoculating the first-class seed bacterial colony into the liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 36 hours at 36 ℃ under the condition of 110 r/min to obtain a second-class seed liquid; and (4) putting the secondary seed liquid into a fermentation tank for amplification culture to obtain the colloidal bacillus liquid.
The culture medium comprises the following components in percentage by weight: 0.025 percent of monopotassium phosphate, 0.003 percent of sodium molybdate, 2.2 percent of mannitol, 0.1 percent of dipotassium phosphate, 0.12 percent of ferric trichloride, 0.12 percent of calcium carbonate, 0.03 percent of magnesium sulfate heptahydrate, 2.2 percent of agar and the balance of deionized water.
(3) The culture method of Aspergillus niger, Aspergillus oryzae, Trichoderma and Paecilomyces lilacinus comprises the following steps:
firstly, respectively carrying out primary culture on each bacterium, and placing parent bacteria in an improved PDA culture medium to be cultured for 30 hours at 36 ℃ to obtain primary seed colonies; the improved PDA culture medium comprises the following components in percentage by weight: 20% of potato flour, 7% of bran, 2.5% of sucrose, 0.35% of monopotassium phosphate, 0.18% of magnesium sulfate heptahydrate, 2.5% of agar and the balance of deionized water;
then carrying out liquid culture: inoculating the colony subjected to the activation culture into the PDA culture medium without agar, wherein the liquid loading is 150ml/500ml, and culturing at 34 deg.C and 220 rpm for 30 hr to obtain secondary seed solution;
and putting the secondary seed liquid into a fermentation tank for amplification culture.
Can respectively obtain Aspergillus niger bacterial liquid, Aspergillus oryzae bacterial liquid, Trichoderma bacterial liquid and Paecilomyces lilacinus bacterial liquid.
(4) The cultivation method of streptomyces jingyangensis is as follows: firstly, performing primary activation culture, and placing parent bacteria in an improved Gao's I culture medium to be cultured for 30 hours at 36 ℃ to obtain primary seed colonies; then carrying out liquid culture: inoculating the first-class seed colony into the Gao's first liquid culture medium without agar, filling liquid in the culture medium with the volume of 150ml/500ml, and culturing for 20 hours at 36 ℃ and 110 r/min to obtain a second-class seed liquid; the second-stage seed liquid is put into a fermentation tank for amplification culture to obtain Jingyang streptomyces liquid;
the improved Gao's I culture medium comprises the following components in percentage by weight: 2.2 percent of soluble starch, 0.15 percent of potassium nitrate, 0.06 percent of sodium chloride, 0.06 percent of dipotassium hydrogen phosphate, 0.002 percent of ferrous sulfate, 0.032 percent of calcium carbonate, 0.06 percent of magnesium sulfate heptahydrate, 2.2 percent of agar and the balance of deionized water.
(5) The culture method of the lactic acid bacteria comprises the following steps: firstly, performing first-stage activation culture, and placing parent bacteria in an improved MRS culture medium to be cultured for 30 hours at 36 ℃ to obtain first-stage seed bacterial colonies; then inoculating the first-level seed bacterial colony into the MRS liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 4-5 days at 37 ℃ and 220 r/min to obtain a second-level seed liquid; and carrying out two times of expanding and matching on the second-stage seed liquid to obtain a fourth-stage seed liquid. And then, putting the four-stage seed liquid into a fermentation tank for amplification culture to obtain the lactic acid bacteria liquid.
The improved MRS culture medium comprises the following components in percentage by weight: peptone 1.2%, beef extract 1.2%, yeast extract 0.8%, glucose 2.2%, dipotassium hydrogen phosphate 0.22%, sodium acetate 0.8%, ammonium citrate 0.22%, magnesium sulfate heptahydrate 0.07%, manganese sulfate 0.03%, tween 80 0.15%, agar 2.4%, and the balance of deionized water.
The performance of the complex microbial inoculum prepared in the above examples 1-3 was tested as follows:
dissolving brown sugar in water according to the weight ratio of 1:10, adding 1/4 brown sugar solution volume of the composite microbial inoculum prepared in the embodiment 1-3 after the brown sugar is completely dissolved, and culturing at room temperature for 24 hours to obtain a working bacterial liquid.
Composite microbial inoculum for fermenting mixture of straws and chicken manure
Mixing the straws and the dry chicken manure according to the weight ratio of 1:1, and adjusting the water content to 45-60%. Spraying the working bacterial liquid according to the proportion of 0.5kg of fermentation raw material per ton and turning over the working bacterial liquid simultaneouslyAnd stacking and mixing uniformly. The result shows that the temperature of the heap is raised to more than 45 ℃ after the heap is sprayed with the bacterial liquid for 1-2 days, the temperature is raised to more than 55 ℃ after 2-3 days, and ventilation fermentation is continued for 15-20 days. The organic matter content in the fermented fertilizer is more than 45 percent, and the total nutrient is (N + P)2O5+K2O) content is more than 5 percent, and viable count is more than 5 hundred million per gram. After 6-8 days from the beginning of fermentation, the detection rate of the escherichia coli is zero and the escherichia coli is completely killed.
The specific results are shown in table 1 below:
| example 1 | Example 2 | Example 3 | |
| Organic matter (%) | 52 | 48 | 57 |
| Total nutrient (%) | 5.3 | 6.2 | 4.8 |
| Number of live bacteria (hundred million/g) | 5.2 | 10.3 | 15 |
| Detection rate of Escherichia coli (cfu/g) | 0 | 0 | 0 |
Secondly, the composite microbial inoculum is used for municipal sludge fermentation
Mixing sludge with water content of 80% treated by a municipal sewage treatment plant with straws according to the weight ratio of 7-8.5: 3-1.5 to obtain a fermentation raw material, wherein the water content of the mixed raw material is 60-65%. Spraying the working bacterial liquid according to the proportion of 1kg of fermentation raw materials per ton, and simultaneously turning and uniformly mixing. The result shows that the temperature rises to more than 45 ℃ after the heap is sprayed with the bacteria liquid for 2-3 days, the temperature reaches more than 55 ℃ after 3-4 days, and ventilation fermentation is continued for 15-20 days. The content of organic matters in the fermented fertilizer is 30-45%, and the total nutrients are (N + P)2O5+K2O) content of about 4-6%, and viable count of more than 0.2 hundred million per gram. 7-9 days after the fermentation, the detection rate of the escherichia coli is zero and the escherichia coli is completely killed.
The specific results are shown in table 2 below:
thirdly, the composite microbial inoculum is used for the fermentation of wet garbage
And squeezing and dehydrating the classified wet garbage to reduce the water content to 60-65% to be used as a fermentation raw material. Spraying the working bacterial liquid according to the proportion of 1kg of fermentation raw materials per ton, and simultaneously turning and uniformly mixing. The result shows that the temperature of the pile is raised to be more than 45 ℃ after the bacterial liquid is sprayed on the pile for 1 to 2 days, the temperature is raised to be more than 55 ℃ after 2 to 3 days, the highest temperature is kept to be more than 70 ℃ for 2 to 3 days, and then ventilation fermentation is continued for 15 to 20 days. The organic matter content in the fermented fertilizer is more than 50 percent, and the total nutrient is (N + P)2O5+K2O) content is about 4-6%, and viable count is more than 2 hundred million per gram. Fermentation ofAfter 10 days, the detection rate of escherichia coli and staphylococcus aureus is zero, and all the escherichia coli and staphylococcus aureus are effectively killed.
The specific results are shown in table 3 below:
| example 1 | Example 2 | Example 3 | |
| Organic matter (%) | 58 | 47 | 62 |
| Total nutrient (%) | 4.3 | 5.8 | 4.2 |
| Number of live bacteria (hundred million/g) | 2.4 | 3.4 | 5.1 |
| Detection rate of Escherichia coli (cfu/g) | 0 | 0 | 0 |
| Staphylococcus aureus (cfu/g) | 0 | 0 | 0 |
The strain disclosed by the invention is compounded and optimized, so that the effect taking speed is high, the temperature can reach more than 45 ℃ within 1-2 days, the high temperature required by organic fertilizer fermentation can reach more than 55 ℃ within 2-3 days, and the fermentation time cost is saved. When the microbial inoculum is used for fermenting organic wastes with more pathogenic bacteria or mixed bacteria, the main fermentation bacteria in the microbial inoculum can resist the high temperature of more than 70 ℃, so that the pathogenic bacteria can be killed efficiently, and the high-temperature fermentation period is safer and is easy to manage; and the main strains survive in the fermented product, thereby improving the fertilizer efficiency of the product. The composite microbial inoculum has wide application, and can be used for fermenting straw, livestock and poultry manure, kitchen waste, aquatic product processing waste, municipal sludge and other organic wastes and preparing organic fertilizer.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. The multifunctional composite fermentation inoculant is characterized by comprising the following components in parts by weight: 5-15 parts of bacillus megaterium, 3-12 parts of bacillus licheniformis, 4-15 parts of bacillus amyloliquefaciens, 5-17 parts of bacillus pumilus, 8-20 parts of pseudomonas putida, 5-15 parts of bacillus mucilaginosus, 1-5 parts of aspergillus niger, 3-10 parts of aspergillus oryzae, 1-8 parts of paecilomyces lilacinus, 3-12 parts of trichoderma, 3-10 parts of streptomyces jingyangensis, 2-8 parts of candida utilis and 3-12 parts of lactic acid bacteria.
2. The method for preparing the multifunctional compound zymophyte agent as claimed in claim 1, wherein the bacteria are cultured, and the obtained bacteria liquids are mixed to obtain the product.
3. The method for preparing the multifunctional composite fermentation inoculant according to claim 2, wherein the culture method of bacillus megaterium, bacillus licheniformis, bacillus amyloliquefaciens, bacillus pumilus, pseudomonas putida and candida utilis comprises the following steps:
placing the mother bacteria in an improved LB culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain a first-grade seed bacterial colony;
then inoculating the first-level seed colonies into an agar-free LB liquid culture medium, wherein the liquid loading amount is 150ml/500ml, and culturing for 30-36 hours at the temperature of 30-36 ℃ and at the speed of 110-220 r/min to obtain a second-level seed liquid;
putting the secondary seed liquid into a fermentation tank for amplification culture;
the improved LB culture medium comprises the following components in percentage by weight: 0.8-1.2% of peptone, 0.8-1.2% of beef extract, 0.3-0.6% of yeast extract, 8-12% of glucose, 0.1-0.3% of monopotassium phosphate, 0.08-0.12% of sodium chloride, 0.1-0.2% of corn starch, 1.8-2.2% of agar and the balance of deionized water.
4. The method for preparing the multifunctional composite fermentation inoculant according to claim 2, wherein the bacillus mucilaginosus is cultured by the following steps: firstly, performing primary activation culture, namely putting parent bacteria into a culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed colonies; then, inoculating the first-level seed bacterial colony into the liquid culture medium without agar, wherein the liquid filling amount is 150ml/500ml, and culturing for 30-36 hours under the conditions of 30-36 ℃ and 110-220 r/min to obtain a second-level seed liquid; putting the secondary seed liquid into a fermentation tank for amplification culture;
the culture medium comprises the following components in percentage by weight: 0.02-0.025% of monopotassium phosphate, 0.002-0.003% of sodium molybdate, 1.8-2.2% of mannitol, 0.07-0.1% of dipotassium phosphate, 0.08-0.12% of ferric chloride, 0.08-0.12% of calcium carbonate, 0.02-0.03% of magnesium sulfate heptahydrate, 1.8-2.2% of agar and the balance of deionized water.
5. The method for preparing the multifunctional composite fermentation inoculant according to claim 2, wherein the culture method of aspergillus niger, aspergillus oryzae, trichoderma and paecilomyces lilacinus is as follows:
firstly, performing primary culture of each bacterium, and placing parent bacteria in an improved PDA culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed colonies;
then carrying out liquid culture: inoculating the activated and cultured colony in the PDA culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 30-36 hours at 34 ℃ and 110-220 r/min to obtain a secondary seed solution;
the second-stage seed liquid is put into a fermentation tank for amplification culture;
the improved PDA culture medium comprises the following components in percentage by weight: 15-20% of potato flour, 5-7% of bran, 2-2.5% of cane sugar, 0.25-0.35% of monopotassium phosphate, 0.14-0.18% of magnesium sulfate heptahydrate, 2-2.5% of agar and the balance of deionized water.
6. The method for preparing the multifunctional compound fermentation inoculant according to claim 2, wherein the streptomyces jingyangensis is cultured by the following steps: firstly, performing primary activation culture, and placing the mother bacteria in an improved Gao's first culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain primary seed bacterial colonies; then carrying out liquid culture: inoculating the first-level seed colonies into the Gao's first liquid culture medium without agar, filling the liquid in the culture medium with the liquid volume of 150ml/500ml, and culturing the liquid for 20-30 hours under the conditions of 30-36 ℃ and 110-220 r/min to obtain a second-level seed liquid; and putting the secondary seed liquid into a fermentation tank for amplification culture.
7. The preparation method of the multifunctional compound fermentation inoculant according to claim 6, wherein the improved high-yield culture medium comprises the following components in percentage by weight: 1.2-2.2% of soluble starch, 0.1-0.15% of potassium nitrate, 0.04-0.06% of sodium chloride, 0.04-0.06% of dipotassium phosphate, 0.001-0.002% of ferrous sulfate, 0.028-0.032% of calcium carbonate, 0.04-0.06% of magnesium sulfate heptahydrate, 1.8-2.2% of agar and the balance of deionized water.
8. The method for preparing the multifunctional composite fermentation inoculant according to claim 2, wherein the culture method of the lactic acid bacteria is as follows: firstly, performing first-stage activation culture, and placing parent bacteria in an improved MRS culture medium to be cultured for 30-36 hours at 30-36 ℃ to obtain first-stage seed bacterial colonies; then inoculating the first-level seed colonies into the MRS liquid culture medium without agar, wherein the liquid loading amount is 150ml/500ml, and culturing for 4-5 days at 37 ℃ and 110-220 r/min to obtain a second-level seed liquid; and carrying out two times of expanding and matching on the second-stage seed liquid to obtain a fourth-stage seed liquid. Then the fourth-level seed liquid is put into a fermentation tank for amplification culture.
9. The preparation method of the multifunctional complex fermentation inoculant according to claim 8, wherein the improved MRS culture medium comprises the following components in percentage by weight: 0.8-1.2% of peptone, 0.8-1.2% of beef extract, 0.5-0.8% of yeast extract, 1.8-2.2% of glucose, 0.18-0.22% of dipotassium hydrogen phosphate, 0.5-0.8% of sodium acetate, 0.18-0.22% of ammonium citrate, 0.05-0.07% of magnesium sulfate heptahydrate, 0.02-0.03% of manganese sulfate, 0.1-0.15% of Tween 80, 2-2.4% of agar and the balance of deionized water.
10. The application of the multifunctional compound fermentation inoculant according to claim 1, wherein the multifunctional compound fermentation inoculant is applied to biological organic fertilizer fermentation of traditional raw materials, or fermentation of kitchen waste, aquatic product processing waste and municipal sludge to prepare organic fertilizer products.
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