CN116463234A - Straw degrading bacterium HXB17 and application thereof - Google Patents
Straw degrading bacterium HXB17 and application thereof Download PDFInfo
- Publication number
- CN116463234A CN116463234A CN202210603772.9A CN202210603772A CN116463234A CN 116463234 A CN116463234 A CN 116463234A CN 202210603772 A CN202210603772 A CN 202210603772A CN 116463234 A CN116463234 A CN 116463234A
- Authority
- CN
- China
- Prior art keywords
- straw
- hxb17
- straws
- degradation rate
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000010902 straw Substances 0.000 title claims abstract description 83
- 230000000593 degrading effect Effects 0.000 title claims abstract description 17
- 241000894006 Bacteria Species 0.000 title claims abstract description 16
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 19
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 19
- 241000194103 Bacillus pumilus Species 0.000 claims abstract description 15
- 241000209140 Triticum Species 0.000 claims abstract description 9
- 235000021307 Triticum Nutrition 0.000 claims abstract description 9
- 240000008042 Zea mays Species 0.000 claims abstract description 9
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 9
- 244000075850 Avena orientalis Species 0.000 claims abstract description 8
- 240000006162 Chenopodium quinoa Species 0.000 claims abstract description 8
- 235000009419 Fagopyrum esculentum Nutrition 0.000 claims abstract description 8
- 240000008620 Fagopyrum esculentum Species 0.000 claims abstract 2
- 238000004321 preservation Methods 0.000 claims description 8
- 235000007319 Avena orientalis Nutrition 0.000 claims description 7
- 235000007558 Avena sp Nutrition 0.000 claims description 4
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims 1
- 235000009973 maize Nutrition 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 38
- 238000006731 degradation reaction Methods 0.000 abstract description 38
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 abstract description 8
- 235000005822 corn Nutrition 0.000 abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 16
- 239000002609 medium Substances 0.000 description 15
- 239000012153 distilled water Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 239000002689 soil Substances 0.000 description 9
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 241000219051 Fagopyrum Species 0.000 description 6
- 239000001913 cellulose Substances 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 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 5
- 239000008103 glucose Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- OFOBLEOULBTSOW-UHFFFAOYSA-L Malonate Chemical compound [O-]C(=O)CC([O-])=O OFOBLEOULBTSOW-UHFFFAOYSA-L 0.000 description 3
- 239000004677 Nylon Substances 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 3
- 229920001778 nylon Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 101710111935 Endo-beta-1,4-glucanase Proteins 0.000 description 2
- 108010032083 Glucan 1,4-beta-Glucosidase Proteins 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 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 2
- 239000007640 basal medium Substances 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 229940059442 hemicellulase Drugs 0.000 description 2
- 108010002430 hemicellulase Proteins 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 2
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 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 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010876 biochemical test Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 238000004382 potting Methods 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- PRWXGRGLHYDWPS-UHFFFAOYSA-L sodium malonate Chemical compound [Na+].[Na+].[O-]C(=O)CC([O-])=O PRWXGRGLHYDWPS-UHFFFAOYSA-L 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a straw degrading bacterium HXB17 and application thereof. The straw degrading bacterium provided by the invention is Bacillus pumilus (Bacillus pumilus) HXB17. The registration number of the strain is CCTCC M2022682 in China center for type culture collection. Experiments prove that when the strain HXB17 is used for treating various straws for 30 days, the degradation rate of potato straws is 44.2%, the degradation rate of oat straws is 45.44%, the degradation rate of buckwheat straws is 44.92%, the degradation rate of quinoa straws is 40.52%, the degradation rate of corn straws is 42.08%, and the degradation rate of wheat straws is 45.32%.
Description
Technical Field
The invention relates to the field of straw degradation, in particular to straw degradation bacteria HXB17 and application thereof.
Background
The crop straw is rich in straw resources, has annual yield of 9 hundred million tons, is mainly from three large grain crops of corn, rice and wheat, and is secondarily from potato, beans, rape, cotton and other cereal crop straws. The comprehensive utilization rate of straw resources in China is less than 40% each year, and more than 60% of straw is burnt, randomly piled or used as living fuel. Some crop plant diseases and insect pests hibernate on the straw, if the straw is directly returned to the field, the straw cannot be degraded before sowing the next crop, which is not beneficial to the growth of the next crop and aggravates the occurrence of plant diseases and insect pests. Accelerating the degradation of the straw, and some insect dormancy can die along with the degradation of the straw, thereby greatly reducing the occurrence of insect diseases. The straw is a byproduct such as a stalk, a leaf and the like from crops, contains nutrient elements required by plant growth, and can return the nutrients to soil for the crop growth by degradation, so that the straw becomes an important fertilizer.
The straw fiber structure is broken by utilizing the microbial degradation capability, coarse fibers in the straw are decomposed into micromolecular substances, and organic matters and the fiber structure are degraded and converted into CO 2 The water, the mineral substances and the heat not only promote the growth of crops, prevent and treat plant diseases and insect pests and fertilize the soil and improve the organic matter content of the soil, but also has the advantages of no environmental pollution, simplicity and easiness in implementation and high straw utilization rate. The establishment of a perfect straw field treatment, collection and storage and transportation system is an effective technical approach for realizing land cultivation, improving the quality of cultivated land and establishing high-yield and stable-yield farmlands.
Disclosure of Invention
One of the purposes of the present invention is to provide a Bacillus pumilus HXB17 useful for straw degradation.
The invention also aims to provide an application of the straw degrading bacterium HXB17 in degrading potato, oat, buckwheat, corn, quinoa and wheat straws.
The invention is realized by the following technical scheme:
taking a soil sample from the ground surface 5-10 cm away from a potato field, primarily screening microorganisms in the soil by using a Congo red staining method with cellulose as a target, carrying out a liquid fermentation test on potato straws by using the screened cellulose degrading bacteria, measuring the degradation rate of the straws, measuring the activity of cellulase, identifying the screened strains by morphology, physiological biochemistry and molecular biology, and carrying out a potting prevention test on potato, oat, buckwheat, corn, quinoa and wheat straws by using the strain HXB17. To obtain a strain of straw degrading bacteria, bacillus pumilus (Bacillus pumilus).
The Bacillus pumilus is preserved in China center for type culture Collection, and the preservation unit address: eight paths of 299 in Wuchang district of Wuhan, hubei province, the preservation date is: 2022, 05, 19, classification naming: bacillus pumilus with a preservation number of CCTCC M2022682.
The invention has the advantages that:
the straw degrading bacterium is Bacillus pumilus (Bacillus pumilus) HXB17. The bacterial HXB17 is used for treating various straws for 30d, the degradation rate of potato straws is 44.20%, the degradation rate of oat straws is 45.44%, the degradation rate of buckwheat straws is 44.92%, the degradation rate of quinoa straws is 40.52%, the degradation rate of corn straws is 42.08%, and the degradation rate of wheat straws is 45.32%. The Bacillus pumilus HXB17 provided by the invention is used for rapidly returning straws to the field, is safe to people and has no pollution to the environment, and has good development and application prospects.
Preservation description:
the invention discloses Bacillus pumilus HXB17 which is preserved in China Center for Type Culture Collection (CCTCC) in the year of 2022 and the month of 05 and 19, wherein the preservation address is eight-path 299 Chinese type culture collection in Wuchang district of Wuhan, hubei province, the registration number of the preservation center is CCTCC M2022682, the strain HXB17 is abbreviated in the invention, and the strain is verified to survive.
Drawings
FIG. 1 variation of degradation rate of potato straw
FIG. 2 variation of degradation rate of oat straw
FIG. 3 variation of degradation rate of buckwheat straw
FIG. 4 change in degradation rate of quinoa straw
FIG. 5 variation of degradation rate of corn straw
FIG. 6 variation of wheat straw degradation rate
Detailed Description
The following examples are presented to better illustrate the invention but do not limit the scope of the invention. The test methods in the following examples are conventional methods unless otherwise specified. The materials used in the examples described below, unless otherwise specified, are commercially available from conventional biochemicals.
The medium in the following examples:
LB solid medium: peptone 10g, yeast extract powder 5g, naCl 10g, agar 15g, distilled water 1000mL.
LB liquid medium: peptone 10g, yeast extract powder 5g, naCl 10g, distilled water 1000mL.
PDA medium: peeled potato 200g, agar 18g, glucose 20g, distilled water 1000mL.
Culture medium No. 1, gao: mgSO (MgSO) 4 ·7H 2 O 0.5g，FeSO 4 ·7H 2 O 0.01g，KNO 3 1g，NaCl 0.5g，K 2 HPO 4 0.5g, 20g of soluble starch, 15g of agar and 1000mL of distilled water.
Sodium carboxymethyl cellulose medium: CMC-Na 20g, NH 4 NO 3 1g，KH 2 PO 4 1.5g，MgSO 4 ·7H 2 O 0.5g，Na 2 HPO 4 2.5g, yeast extract powder 0.5g, peptone 2.5g, agar 15g, distilled water 1000mL.
Enzyme-producing medium: peptone 3g, (NH) 4 ) 2 SO 4 3g，KH 2 PO 4 1g，MgSO 4 ·7H 2 O0.2 g, naCl 5g, distilled water 1000mL.
Nutrient solution: (NH) 4 ) 2 SO 4 1.4g，KH 2 PO 4 2g，MgSO 4 ·7H 2 O 0.3g，CaCl 2 0.3g，FeSO 4 ·7H 2 O 7.5mg，MnSO 4 ·H 2 O 2.5mg，ZnSO 4 2mg，CoCl 2 3mg, 0.3mg of urea and 1000mL of distilled water.
Potato straw fermentation medium: 2g of 1-2 cm long potato straw and 50mL of nutrient solution.
Carbon source measurement basal medium: KH (KH) 2 PO 4 2.38g，K 2 HPO 4 ·3H 2 O 5.65g，(NH 4 ) 2 SO 4 2.64g，MgSO 4 ·7H 2 O 1g，CuSO 4 ·5H 2 O 6.4mg，ZnSO 4 ·7H 2 O 1.5mg，FeSO 4 ·7H 2 O 1.1mg，MnCl 2 ·7H 2 O7.9 mg, agar 15g, distilled water 1000mL.
Nitrogen source determination basal medium: glucose 10g, K 2 HPO 4 ·3H 2 O 1g，MgSO 4 ·7H 2 O 5g，NaCl 2 5g，FeSO 4 ·7H 2 O10 mg, agar 15g, distilled water 1000mL.
Malonate test medium: 3g of sodium malonate, 1g of yeast extract, 2g of NaCl, (NH) 4 ) 2 SO 4 2g，KH 2 PO 4 0.4g，K 2 HPO 4 ·3H 2 O0.6 g, bromothymol blue 25mg, distilled water 1000mL, pH 7.4.
Methyl red test medium: peptone 7.0g, glucose 5g, naCl 5g, distilled water 1000mL.
Starch hydrolysis test medium: 2g of soluble starch, 3g of beef extract, 5g of peptone, 2.5g of glucose, 18g of agar, 1000mL of distilled water and pH 7.0.
Fat hydrolysis test medium: peptone 10g, caCl 2H 2 O0.1 g, agar 17g, distilled water 1000mL, pH 7.4.
NA medium: beef extract 3g, peptone 5g, glucose 2.5g, agar 18g, distilled water 1000mL, pH 7.0.
Example 1 isolation screening of strains
Soil samples are taken from potato fields in the inner Mongolia and the huge cities and in the Lingell county, and are 5 cm to 10cm away from the ground surface. Weighing 10g of soil sample, adding into a 250mL triangular flask containing 90mL of sterile water, placing into a constant-temperature shaking incubator at 28 ℃ and 180r/min for shaking for 30min, standing for 20min, taking 1mL of supernatant for gradient dilution under the sterile operation condition, and diluting to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 A total of 8 concentrations, each concentration was taken from 0.1mL of each on LB solid mediumCoating and separating, inversely culturing 3 plates with each concentration in a constant temperature incubator at 28 ℃ until bacterial colonies grow out, and picking strains with different forms for repeated streak purification to obtain pure strains.
Screening straw degrading bacteria by Congo red staining test, inoculating the purified bacterial strains onto sodium carboxymethylcellulose culture medium by spot inoculation method, inoculating 3 spots on each flat plate, setting 3 times of repetition, culturing in a constant temperature incubator at 28 ℃ for 72h, adding appropriate amount of Congo red solution of 1mg/mL for 30min, discarding the dye solution, adding appropriate amount of NaCl solution of 1mol/L for decolorizing for 20min, observing and measuring colony diameter (D, cm) and its surrounding transparent ring diameter (D, cm), and calculating D/D value.
As a result, strain HXB17 showed a transparent ring on the Congo red staining test plate, and the ratio (D/D) of the transparent ring diameter D to the colony diameter D was 5.28, having cellulose degrading ability. After preliminary screening, 1 strain was obtained and designated HXB17.
Congo red staining test results
Example 2 determination of degradation Rate of Potato straw by Strain HXB17
Inoculating strain with cellulose degrading ability obtained by primary screening into LB liquid medium to obtain strain with concentration of 1×10 7 Inoculating CFU/mL bacterial suspension into a potato straw fermentation culture medium according to the proportion of 10% by volume, taking non-inoculation as a blank control, repeating for 3 times, culturing in a constant-temperature shaking incubator at 28 ℃ and 180r/min, sampling at 0d, 5d, 15d, 25d and 35d respectively, repeatedly cleaning straw residues subjected to different treatments for 3 times by using sterile water, drying in an oven at 80 ℃ to constant weight, weighing, and calculating the relative degradation rate of the straw.
Degradation rate= (M 1 -M 2 )/M 1 ×100％
Wherein M is 1 For the initial straw quality M 2 Is the quality of the degraded straw.
As a result, the strain HXB17 treated each straw 35d, the residual amount of the potato straw was 0.92g, and the weight loss rate of the straw was 54.13%.
Liquid state fermentation degradation effect of potato straw
Example 3 Strain HXB17 cellulase Activity assay
Inoculating strain to be tested into LB liquid medium to prepare strain with concentration of 1×10 7 The CFU/mL bacterial suspension is inoculated into an enzyme-producing culture medium according to the inoculation amount of 9% of the volume ratio, the bacterial suspension is cultured for 7 days at the temperature of 28 ℃ and at the speed of 180r/min, the bacterial suspension is taken every 24 hours, and the bacterial suspension is purchased from Graves biotechnology Co., ltd, suzhou, china and the activity of hemicellulase, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase of the bacterial strain is measured according to the Suzhou Graves enzyme activity measuring instruction. Taking 2mL of bacterial suspension, carrying out ultrasonic crushing for 15s at intervals of 10s, repeating 30 times, centrifuging at 12000rpm and 4 ℃ for 10min, and taking the supernatant to be tested.
As a result, the hemicellulase, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase activities of strain HXB17 were maximum values, respectively: 31.43U/mL, 302.05U/mL, 509.46U/mL and 5.41U/mL, indicating that the bacterium can produce cellulose degrading enzymes to degrade cellulose.
Example 4 identification of Strain HXB17
Morphological identification
The strain is coated in LB solid medium by dilution method, single colony grows out, bacterial colony HXB17 is round, the surface is glossy and sticky, the cream yellow is opaque, the edge is orderly and slightly convex, the middle of some bacterial colonies is slightly wrinkled, and the middle of some bacterial colonies is slightly concave.
Physiological and biochemical identification
The test refers to the "Manual of identification of common bacterial systems" and is used for measuring the physiological and biochemical indexes of the strain, such as carbon source, nitrogen source utilization, malonate utilization, methyl red test, starch hydrolysis, oxidase test, catalase test and the like. Strain HXB17 is a gram positive bacterium, can be tested positive by using malonate and methyl red, can generate catalase, cannot generate oxidase, and can use lactose, mannitol, potassium nitrate, ammonium nitrate, sodium nitrate, ammonium sulfate, histidine and L-methionine as the only carbon-nitrogen sources.
Physiological and biochemical characteristics of strain HXB17
Note that: "+" indicates positive and "-" indicates negative
Molecular biological identification
The extraction of DNA was performed according to the TIAN GEN bacterial genomic DNA extraction kit (centrifugal column) (Beijing, china) instructions, purchased from Tiangen Biochemical technologies (Beijing) Co. PCR primers for strain HXB17 were 27F and 1492R, 7F and 1540R, trpBF and trpBR, pyrEF and pyrER, respectively, and the primer sequences are shown in the following Table. The 25. Mu.L reaction system contains: 17 mu L ddH 2 O, 2.5. Mu.L of 10 XPCR Buffer, 2. Mu.L of dNTPs, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 0.25. Mu.L of TaqDNA polymerase. PCR reaction conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s; annealing temperature for 30s;72 ℃ for 1min; cycling 34 times; and at 72℃for 10min. The PCR products of the 4 genes were detected by 1% agarose gel electrophoresis and sent to Shanghai Biotechnology for 16S rDNA sequencing. And splicing the obtained 4 gene sequences, comparing the splicing result with a GenBank nucleic acid database, selecting a strain sequence with higher similarity for analysis, and constructing a phylogenetic tree by using software MEGA 7.0. The sequencing results of the 4 PCR products were spliced, aligned using Blast program in NCBI database, and analyzed by MEGA7.0 software phylogenetic tree, strain HXB17 was clustered with Bacillus pumilus (accession number HQ 218993).
The strain HXB17 was identified as Bacillus pumilus (Bacillus pumilus) by combining morphological, physiological and biochemical tests and molecular biological identification results.
Main primer and sequence
Example 5 degradation Effect of Strain HXB17 on different crop straw
Cleaning potato, oat, corn, quinoa, wheat and buckwheat straw under flowing tap water, drying in an oven at 80 ℃, and cutting into short sections of 3-4 cm. 5g of different crop straws are respectively placed in small nylon bags, with a concentration of 1X 10 at 9mL 7 CFU/mL HXB17 bacterial suspension soaked the straw and no bacteria was used as control CK, each treatment was repeated 5 times. Placing the treated straws in the middle of a flowerpot, covering 2.0kg of sterilizing soil on each flowerpot, covering 2-3 cm of sterilizing soil on each flowerpot, placing 5 small nylon bags on each flowerpot, adding 420mL of tap water (relative humidity is 70-80%), placing the straws in a laboratory for cultivation, pouring 420mL of tap water every 5d, sampling when 5d, 10d, 20d and 30d are cultivated, taking out 1 small nylon bag straw each time, and measuring the straw degradation rate of different straws.
As a result, the degradation rate of the potato straw reaches 44.20% (figure 1), the degradation rate of the oat straw reaches 45.44% (figure 2), the degradation rate of the buckwheat straw reaches 44.92% (figure 3), the degradation rate of the quinoa straw reaches 40.52% (figure 4), the degradation rate of the corn straw reaches 42.08% (figure 5), and the degradation rate of the wheat straw reaches 45.32% (figure 6).
Claims (2)
1. A straw degrading bacterium HXB17 is characterized in that the straw degrading bacterium is Bacillus pumilus, and is preserved in China center for type culture Collection of strains, wherein the preservation address is No. 299 of Wuchang district of Wuhan, hubei province, the preservation date is No. 2022, 5 and 19, and the preservation number is CCTCC M2022682.
2. Use of the straw degrading bacterium HXB17 according to claim 1, for degrading potato, oat, buckwheat, quinoa, maize and wheat straw.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210603772.9A CN116463234A (en) | 2022-05-30 | 2022-05-30 | Straw degrading bacterium HXB17 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210603772.9A CN116463234A (en) | 2022-05-30 | 2022-05-30 | Straw degrading bacterium HXB17 and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116463234A true CN116463234A (en) | 2023-07-21 |
Family
ID=87177574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210603772.9A Pending CN116463234A (en) | 2022-05-30 | 2022-05-30 | Straw degrading bacterium HXB17 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116463234A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287217A (en) * | 2022-05-30 | 2022-11-04 | 内蒙古农业大学 | Straw degrading bacterium WXB10 and application thereof |
-
2022
- 2022-05-30 CN CN202210603772.9A patent/CN116463234A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115287217A (en) * | 2022-05-30 | 2022-11-04 | 内蒙古农业大学 | Straw degrading bacterium WXB10 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110669696A (en) | Phosphate solubilizing bacteria and extraction method thereof | |
| CN116396888A (en) | Microbial composite microbial agent suitable for livestock and poultry manure and straw fermentation and preparation method thereof | |
| CN116463234A (en) | Straw degrading bacterium HXB17 and application thereof | |
| CN104560817B (en) | Thermophilic bacillus licheniformis UTM102 for producing phytase and application of thermophilic bacillus licheniformis UTM102 | |
| CN109294951A (en) | The application of one plant of false Xanthomonas campestris and its microorganism formulation in terms of biological compost | |
| CN115287215A (en) | Straw degrading bacterium HXB11 and application thereof | |
| CN115287216A (en) | Straw degrading bacterium TXB2 and application thereof | |
| CN115287218A (en) | Straw degradation composite bacterial system HH and application thereof | |
| CN115287219A (en) | Straw degradation composite bacterial TW and application thereof | |
| CN107937302B (en) | Bacterium D10 for improving effectiveness of heavy metal and application thereof | |
| CN114752538B (en) | Oil tea endophyte with soil improvement function and application thereof | |
| CN115959931A (en) | Method for preparing organic fertilizer by deep fermentation of livestock and poultry manure and straws | |
| CN114717125B (en) | Thermophilic bacillus licheniformis AMCC101380 and application thereof in high-temperature composting of tail vegetables | |
| CN110684699B (en) | Cellulosimicrobium cellulans DGNK-JJ1 and application thereof | |
| CN111518726B (en) | Pseudomonas aeruginosa and screening method and application thereof | |
| CN111662846B (en) | Phosphorus-solubilizing bacterium P5, fermentation product, microbial inoculum and application thereof | |
| CN109749959B (en) | Strain HB161398 with nitrogen fixation activity and application thereof | |
| CN115287217A (en) | Straw degrading bacterium WXB10 and application thereof | |
| CN116121134B (en) | Bacillus terreus in phyllostachys pubescens and application thereof | |
| CN115627247B (en) | Preparation method and application of compound microbial fertilizer | |
| CN114410544B (en) | Acinetobacter cinnarii HZJK-2 and application thereof | |
| CN114317330B (en) | Screening and application of cellulose degrading bacteria | |
| Vijayalakshmi et al. | Morphological and biochemical characterization of Azospirillum isolates from rhizoplane of foxtail millet [Setaria italica (L.) Beauv.] | |
| CN115851502B (en) | Bacillus pseudomycoides, microbial inoculum and liquid bio-organic fertilizer and application thereof | |
| CN114410534B (en) | Cloacibacterium normanense TD35 strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |