CN107325984B - Bacterial strains, microbial agents and uses thereof - Google Patents
Bacterial strains, microbial agents and uses thereof Download PDFInfo
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- CN107325984B CN107325984B CN201710564394.7A CN201710564394A CN107325984B CN 107325984 B CN107325984 B CN 107325984B CN 201710564394 A CN201710564394 A CN 201710564394A CN 107325984 B CN107325984 B CN 107325984B
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- 230000000813 microbial effect Effects 0.000 title claims abstract description 41
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 27
- 239000010902 straw Substances 0.000 claims abstract description 47
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 40
- 238000004321 preservation Methods 0.000 claims abstract description 18
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 17
- 241000006381 Bacillus flexus Species 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 7
- 239000002054 inoculum Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 239000012752 auxiliary agent Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 3
- 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 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000004927 clay Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 235000001727 glucose Nutrition 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
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- 235000019764 Soybean Meal Nutrition 0.000 claims description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 claims description 2
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 claims description 2
- 239000004455 soybean meal Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 239000000454 talc Substances 0.000 claims description 2
- 229910052623 talc Inorganic materials 0.000 claims description 2
- 235000015099 wheat brans Nutrition 0.000 claims description 2
- 239000005995 Aluminium silicate Substances 0.000 claims 1
- 239000005909 Kieselgur Substances 0.000 claims 1
- 125000000217 alkyl group Chemical group 0.000 claims 1
- 235000012211 aluminium silicate Nutrition 0.000 claims 1
- 238000001914 filtration Methods 0.000 claims 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-M naphthalene-1-sulfonate Chemical compound C1=CC=C2C(S(=O)(=O)[O-])=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-M 0.000 claims 1
- 239000003415 peat Substances 0.000 claims 1
- 229940088417 precipitated calcium carbonate Drugs 0.000 claims 1
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- 230000015556 catabolic process Effects 0.000 abstract description 18
- 238000006731 degradation reaction Methods 0.000 abstract description 18
- 239000002689 soil Substances 0.000 abstract description 17
- 240000008042 Zea mays Species 0.000 abstract description 8
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 abstract description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 abstract description 8
- 235000005822 corn Nutrition 0.000 abstract description 8
- 244000005700 microbiome Species 0.000 abstract description 5
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 230000035558 fertility Effects 0.000 abstract description 2
- 239000003864 humus Substances 0.000 abstract description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 24
- 241000193388 Bacillus thuringiensis Species 0.000 description 8
- 229940097012 bacillus thuringiensis Drugs 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 230000004580 weight loss Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 241000589876 Campylobacter Species 0.000 description 3
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- 235000012036 Vanilla tahitensis Nutrition 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
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- 230000001737 promoting effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000003899 bactericide agent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 235000009566 rice Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000010455 vermiculite Substances 0.000 description 2
- 229910052902 vermiculite Inorganic materials 0.000 description 2
- 235000019354 vermiculite Nutrition 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- 238000007605 air drying Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000006229 carbon black Substances 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 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 description 1
- 238000005260 corrosion Methods 0.000 description 1
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- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000005669 field effect Effects 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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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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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of microorganism application, and particularly relates to a bacterial strain, a microbial agent and application thereof. The strain is Bacillus flexus A1, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has the preservation number: CGMCC NO. 14088; the preservation time is as follows: year 2017, month 5 and day 4. The microbial inoculum containing the strain is particularly suitable for the rapid degradation of northern corn straws, plays an important role in the field in-situ field returning of crop straws, and simultaneously has the functions of increasing soil humus and improving soil fertility.
Description
Technical Field
The invention relates to the technical field of microorganism application, and particularly relates to a bacterial strain, a microbial agent and application thereof.
Background
The crop straw resources in China are rich, the number is huge, the varieties are various, and the annual crop straw output is 7-8 hundred million tons. Since the straws are not easy to decay in a short period of time and have low utilization efficiency, most straws are abandoned or burnt, and the crop straws become obstacles in production in areas mainly in the planting industry. Therefore, the straw is incinerated throughout vast rural areas, thereby causing environmental pollution and serious waste of organic resources. According to statistics, the crop straws used as returning field and organic fertilizer in China only account for about 20 percent of the total amount, the incineration accounts for 17 percent, and 50 percent of the crop straws are not utilized.
A plurality of provinces in northern areas of China are agricultural provinces, and the demand for straw treatment is more urgent. The northern area has cold weather in autumn and winter, which is not beneficial to the degradation of lignin and cellulose of the corn stalks. Therefore, breaking through the technical bottleneck of large-scale and rapid decomposition and returning of the straws under the condition of adverse climate in northern areas is the problem to be solved urgently in realizing the utilization of the straws in northern areas of China. The method is one of important development trends for solving the problem of straw degradation in northern China.
The straw degradation microorganism is utilized to degrade the straws of corn, wheat and rice and return the straws to the field in situ, so that the method has great application potential. The screening research of the compound bacterial system has also been reported, and the products related to the straw fermentation bacterial agent are very few, and no system is formed, and no stable and efficient straw rapid degradation bacterial flora is produced and used. The rapid straw decomposition technology is not effectively solved, and the efficient utilization of straw resources is limited.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
An aspect of the present invention is to provide a bacterial strain having a cellulose degradation function, which has a high low temperature resistance and a very good cellulose degradation function.
The invention also aims to provide a microbial agent containing the bacterial strains, wherein the mixed microbial agent has obviously stronger cellulose decomposing capacity than any single bacterial strain, and can be well applied to straw degradation.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
according to one aspect of the invention, the bacterial strain is campylobacter A1, which is preserved in China general microbiological culture Collection center (CGMCC), and the preservation numbers are as follows: CGMCC NO. 14088; the preservation time is as follows: year 2017, month 5 and day 4.
The Latin school name of the strain is Bacillus flexus, the Chinese school name is Bacillus flexus, and the strain name is A1.
The strain has a high-efficiency straw degradation function and is high in low-temperature tolerance.
The separated and purified campylobacter has round, flat, grey-white, opaque, sticky, medium-sized, unsmooth surface and irregular edges.
The microbial agents containing the campylobacter A1 also belong to the scope of the present application. For the strain, considering the straw degradation efficiency in application, transportation possibly and other reasons, the strain needs to be expanded and cultured into a microbial agent form to expand the application range of the strain.
Preferably, the microbial agent further comprises one, two or all of the following strains:
bacillus thuringiensis A2 with the preservation number of CGMCC NO. 14089;
vanilla bacillus B2 with the preservation number of CGMCC NO. 14090; and
bacillus flexus D3 with the preservation number of CGMCC NO. 14091;
the phages are all preserved in the China general microbiological culture Collection center, and the preservation time is as follows: year 2017, month 5 and day 4.
In some embodiments, the microbial agent comprises the bacillus curvatus a1 and the bacillus thuringiensis a 2;
in some embodiments, the microbial agent comprises the bacillus curvatus B2 and the bacillus vanilloides D3;
in some embodiments, the microbial agent comprises the bacillus curvatus a1 and the bacillus curvatus D3;
in some embodiments, the microbial agent comprises the bacillus curvatus a1, the bacillus thuringiensis a2, and the bacillus vanilloidis B2;
in some embodiments, the microbial agent comprises the bacillus curvatus a1, the bacillus thuringiensis a2, and the bacillus curvatus D3;
in some embodiments, the microbial agent comprises bacillus curvatus a1, bacillus vanilloidis B2, and bacillus curvatus D3.
Preferably, the microbial agent comprises, in terms of viable count, the mixing ratio of bacillus curvatus A1, bacillus thuringiensis A2, bacillus vanilloidis B2 and bacillus curvatus D3 in the microbial agent is (1-5): (1-5).
More preferably, the agent is a mixture of Bacillus curvatus A1, Bacillus thuringiensis A2, Bacillus vanicus B2 and Bacillus curvatus D3 in equal ratio.
The microbial inoculum is preferably a liquid inoculum in which a culture solution is preferably a liquid medium such as LB liquid medium, or further added with nutrients such as glucose, thiamine, biotin, and nicotinic acid, and preferably, in order to achieve a good cellulose degradation effect, the number of viable bacteria of each strain is not less than 1 × 107CFU/ml, preferably, the number of viable bacteria per strain is 1 × 107-16CFU/ml, more preferably 1 × 108-15CFU/ml, alternatively 1 × 109CFU/ml，1×1010CFU/ml，1×1011CFU/ml，1×1012CFU/ml，1×1013CFU/ml，1×1014CFU/ml, etc.
Preferably, the preparation method of the microbial agent comprises the following steps:
the invention provides a preparation method based on the liquid microbial agent, which comprises the following steps:
inoculating the strain into a liquid culture medium for culturing to obtain a liquid microbial inoculum;
optionally, the liquid inoculum is filtered and/or concentrated.
The whole microbial agent has simple preparation method, can be stored for a short time and is convenient to transport, and can be directly added into soil according to a certain proportion when in use, so that the use is very convenient.
The microbial inoculum is preferably a solid inoculum prepared by adding a solid carrier and/or an auxiliary agent to the strain.
Preferably, the solid carrier includes one or more of grass carbon, bran powder, wheat bran, kaolin, light calcium carbonate, diatomite, white carbon black, talcum powder, fine sand and clay.
Preferably, the microbial agent is one or a mixture of two or more of sucrose, glucose, peptone, soybean meal, sodium dodecylbenzenesulfonate and sodium alkylnaphthalenesulfonate polycondensates.
The invention provides a preparation method based on the solid microbial agent, which comprises the following steps:
and mixing the purified strain with a solid carrier and/or an auxiliary agent to obtain the strain.
The application of the strain or the microbial inoculum in straw digestion.
The microbial inoculum can be used for degradation and in-situ returning of corn, wheat and rice straws.
Compared with the prior art, the invention provides the microbial strain for efficiently degrading the straws and the microbial agent containing the strain, wherein the microbial agent mainly comprises four microbial strains, is particularly suitable for fast degradation of the corn straws in the north, and has an important function in field in-situ returning of the crop straws to the field. Meanwhile, the fertilizer has the functions of increasing soil humus and improving soil fertility.
The Bacillus flexus strain provided by the invention has the strain name of A1 and the preservation number of CGMCC NO. 14088;
the Bacillus thuringiensis provided by the invention has a strain name of A2 and a preservation number of CGMCC NO. 14089;
the bacterial strain name of the vanilla Bacillus (Bacillus vanillica) provided by the invention is B2, and the preservation number is CGMCC NO. 14090;
the strain name of the Bacillus flexus provided by the invention is D3, and the preservation number is CGMCC NO. 14091;
the strains are all preserved in China general microbiological culture Collection center, and the preservation addresses are as follows: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation time is as follows: year 2017, month 5 and day 4. The strains were detected as viable by the depository at 2017, 5 and 4 days.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.
Example 1
This example provides a method for screening individual microorganism strains and a method for combining microbial agents.
1. Experimental Material
1.1 strains and soil
Test strains: the bacterial seed bank stores cellulose-degrading bacteria.
Black soil in northeast: 0-20 cm of surface soil in the field of Jilin princess mountains.
1.2 culture Medium
1.2.1 bacterial culture Medium: 3g of beef extract, 10g of peptone, 5g of NaCl, 16-18 g of agar and 1L of deionized water.
1.2.2 bacterial fermentation Medium (YPD Total nutrient Medium): dissolving yeast extract/yeast powder 10g/L, peptone 20g/L in 1L deionized water, pH 7.0.
1.2.3 cellulose degrading bacteria culture medium: KH (Perkin Elmer)2PO40.5g、MgSO40.25g, peptone 2g, sodium carboxymethylcellulose (CMC)1.88g, Congo red 0.2g, agar 16-18 g, deionized water 1L, and pH 7.0.
1.2.4 basic nutrient solution (hertzson medium): 1.0g of monopotassium phosphate, 0.1g of NaCl and NaNO32.5g, 1L deionized water, and natural pH.
Single strain low-temperature activity screening method
2.1 Strain activation and rejuvenation
The colony in the preservation inclined plane tube is picked up by the inoculating loop and inoculated in a bacterial liquid culture medium (1.2.1) without agar, and is cultured for 72 hours at the temperature of 28 ℃ to prepare a single bacterial strain test bacterial liquid.
2.2 Low temperature Activity screening of strains
Adding 40 g of soil into a 250ml triangular shake flask, spraying water to moisten the soil until the water content is 30-50% of the field water retention rate, sealing a breathable film, carrying out moist heat sterilization for 3-5 times, carrying out plate inspection on the soil without viable bacteria by using a bacterial culture medium (1.2.1), inoculating 2ml of test bacteria liquid, culturing for 3 days at 28 ℃, and then carrying out heat preservation for 24 hours at-20 ℃; the temperature is kept at 20 ℃ for 24h for one cycle. After the freeze thawing cycle is carried out for 9 times, 100ml of sterile water is added into a shake flask, the shake flask is vibrated repeatedly (the rotating speed is 170r/min) for 30 minutes to prepare soil suspension, the soil suspension is diluted step by step, and the viable count determination is carried out by a plate counting method. The number of cells in the soil after freeze-thaw cycles>1011The inoculated strain was judged to have high activity at low temperature at cfu/g soil.
TABLE 1
| Inoculating strains | Count after 9 cycles of freeze-thaw cycle (cfu/g. soil) |
| A1 | 1.38×1012 |
| A2 | 2.72×1011 |
| B1 | 9.50×1012 |
| B2 | 1.16×1012 |
| D1 | 6.50×1012 |
| D2 | 5.60×1016 |
| D3 | 1.26×1012 |
2.3 Shake flask method for screening high-efficiency straw degradation bacterial strain
Adding 50mL of basic nutrient solution (1.2.4) and 5g of dried crushed corn straws into a 100mL triangular flask, inoculating high-activity low-temperature strains in the table 1 after sterilization by a wet-heat method, performing shaking culture at 30 ℃ and 170r/min for 10 days, 15 days and 20 days, repeating each treatment for three times, simultaneously performing basic nutrient solution and straw control CK, determining the weight loss rate of the straws cultured for 10 days, 15 days and 20 days, and respectively determining the strains with strong degradation function by increasing the weight loss rate of the straws treated by the inoculated strains by more than 10% compared with the weight loss rate of the straws of the blank control without the inoculated strains.
TABLE 2
TABLE 3
According to tables 2 and 3, the strains with good degradation effect after being inoculated for 10 days comprise A1, A2, B1 and B2, the strains with strong straw degradation function after being inoculated for 15 days comprise B1, B2, D1, D2 and D3, and the high-efficiency straw degradation strains after being inoculated for 20 days comprise A1, A2, B2 and D3.
2.4 Complex strains screening
A combination of A1A 2-type 4B-D strains, i.e., any combination of B1 or B2 strains with D1, D2 or D3 after 2 strains were removed, was set. Mixing the bacterial suspensions of each bacterial strain combination according to the volume ratio of 1: 1, respectively inoculating 6mL of the bacterial suspensions into sterilized vermiculite mixed with 5g of dried straws, keeping the vermiculite in a saturated water absorption state, and culturing for 20 days (25 ℃ day and 20 ℃ night) in an artificial climate chamber; and (4) determining the weight loss rate of the straws at 20d, repeating each time for 3 times, and simultaneously preparing a basic nutrient solution and a straw control CK. The percentage of weight loss ratio and relative blank increase of the inoculated microbial inoculum straw is the decay promoting rate, and the composite bacterial system which is screened is the one with the decay promoting rate more than 10%. As a result of the experiment, 5 groups of complex strains such as A1A2B3D3, A1A2B2D3, A1A2B2D1, A1A2B2D5 and A1A2B2D4 were obtained.
Wherein, A1 corresponds to Bacillus curvatus A1; a2 corresponds to Bacillus thuringiensis A2 deposited in the present invention; b2 corresponds to Vanilla Bacillus deposited herein B2; d3 corresponds to Bacillus curvatus D3;
TABLE 4
Example 2
The embodiment provides a liquid microbial inoculum and a preparation method thereof, and the preparation method specifically comprises the following steps:
preparing single strain bacterial liquid in a bacterial fermentation culture medium (1.2.2), wherein the bran powder is prepared according to a solid-liquid ratio of 1: 2, adsorbing the single bacterium liquid in proportion to prepare single bacterium agents A1, A2, B2 and D3; and (3) mixing the 4 single bacteria agents in equal proportion, air-drying and bagging to complete the preparation of the composite bacteria agent.
The content of each strain in the obtained liquid microbial inoculum is 1 × 1010-11CFU/ml。
Example 3
In this example, the preparation operation was the same as in example 1, and different from example 1, in this example, the single microbial agents a1, B2, D3 were prepared; 3 single bactericides are mixed according to the ratio of viable count of 1:3:5, and are air-dried and bagged to complete the preparation of the composite bactericide.
Example 4
The embodiment provides a solid microbial inoculum and a preparation method thereof, and the preparation method specifically comprises the following steps:
the liquid microbial inoculum prepared in the embodiment 2 is added into talcum powder according to the proportion that 30 g of talcum powder (used as a solid carrier) is added into 1 kg of liquid microbial inoculum, and the mixture is fully stirred, uniformly mixed, filtered, dried and crushed to obtain the solid microbial inoculum.
Example 5
In this example, the procedure was the same as in example 4, except that in this example, the solid carrier used was a mixture of talc, fine sand and clay in equal proportions.
Experimental example field effect verification
The corn is harvested for 10 months and then crushed, 5 kg of the compound microbial inoculum prepared in the embodiment 2 is uniformly spread on the surface of the straw, the straw is buried in the soil by deeply turning over for 30 cm, meanwhile, the straw sample bag is buried, and the decomposition rate of the straw sample (calculated by the weight loss rate) is measured in the spring of the next year.
TABLE 5
TABLE 6
TABLE 7
As shown in the results in tables 6 and 7, the composite microbial inoculum provided by the invention has good corrosion promoting effect on the buried straws in the field in the growth stage of the corn seedling stage and the large flare stage (5-7 months) in spring of princess mountains and double cities.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. The bacterial strain is Bacillus flexus A1, which is preserved in China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC NO. 14088; the preservation time is as follows: year 2017, month 5 and day 4.
2. A microbial agent comprising the strain of claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent is a liquid microbial agent in which the activity of a strain is presentThe number of the bacteria is more than or equal to 1 × 107CFU /ml。
4. The microbial agent according to claim 2, wherein the microbial agent is a solid microbial agent prepared by adding a solid carrier and/or an auxiliary agent to the strain.
5. The microbial inoculant according to claim 4, wherein the solid carrier comprises one or more of peat, bran powder, wheat bran, kaolin, precipitated calcium carbonate, diatomaceous earth, white carbon, talc, fine sand and clay.
6. The microbial inoculant according to claim 4, wherein the auxiliaries are selected from one or more of sucrose, glucose, peptone, soybean meal, sodium dodecylbenzene sulfonate, sodium lignosulfonate, sodium alkyl naphthalene sulfonate polycondensates.
7. The method for preparing a microbial agent according to claim 3, comprising the steps of:
and inoculating the strain into a liquid culture medium for culture to obtain a liquid microbial inoculum.
8. The method for preparing a microbial agent according to claim 7, further comprising the steps of: filtering and/or concentrating the liquid microbial inoculum.
9. The process for the preparation of a microbial inoculant according to any one of claims 4 to 6, comprising the steps of:
and mixing the purified strain with a solid carrier and/or an auxiliary agent to obtain the strain.
10. The strain of claim 1 or the microbial agent of any one of claims 2 to 6, for use in straw digestion.
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