CN115537349B - Straw degrading bacterium and application thereof - Google Patents
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- 239000010902 straw Substances 0.000 title claims abstract description 102
- 241000894006 Bacteria Species 0.000 title claims abstract description 39
- 230000000593 degrading effect Effects 0.000 title claims description 28
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 7
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 238000000855 fermentation Methods 0.000 claims description 6
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- 229920002678 cellulose Polymers 0.000 claims description 5
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- 239000011780 sodium chloride Substances 0.000 claims 1
- 239000002689 soil Substances 0.000 abstract description 20
- 244000068988 Glycine max Species 0.000 abstract description 11
- 235000010469 Glycine max Nutrition 0.000 abstract description 11
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 abstract description 11
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- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 abstract description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004021 humic acid Substances 0.000 abstract description 4
- 239000011574 phosphorus Substances 0.000 abstract description 4
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 4
- 239000011591 potassium Substances 0.000 abstract description 4
- 229910052700 potassium Inorganic materials 0.000 abstract description 4
- 230000035558 fertility Effects 0.000 abstract description 3
- 230000008635 plant growth Effects 0.000 abstract description 2
- 240000007594 Oryza sativa Species 0.000 abstract 1
- 229940075612 bacillus cereus Drugs 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
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- 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 5
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- 238000009331 sowing Methods 0.000 description 1
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- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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- 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
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
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- 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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- 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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Abstract
本发明提供了一株秸秆降解菌W118，分类名称为蜡样芽孢杆菌Bacilluscereus.，保藏编号：CGMCC No.23973，保藏日期：2021年11月25日，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心。菌株W118在4℃下对秸秆降解率在15天内达到27％；该菌株分别与水稻、玉米和大豆秸秆共同直接还田后，秸秆降解率分别达到50％，43％、38％。该菌株与秸秆共同直接还田的土壤中的速效磷、有效钾、腐殖酸和有机质含量与仅秸秆直接还田的土壤相比均明显增加。第二年种植作物后，作物产量均有所增加。W118菌剂和秸秆共同还田有利于改良土壤肥力，培肥土壤，有利于植物生长，增加作物产量。
The invention provides a straw-degrading bacterium W118, the classification name is Bacilluscereus., the preservation number: CGMCC No. 23973, the preservation date: November 25, 2021, the preservation unit: China Microbial Culture Collection Management Committee Microbiology Center. The straw degradation rate of strain W118 reached 27% within 15 days at 4°C; after this strain was returned directly to the field with rice, corn and soybean straw, the straw degradation rates reached 50%, 43% and 38% respectively. The contents of available phosphorus, available potassium, humic acid and organic matter in the soil where this strain and straw were directly returned to the field were significantly increased compared with the soil where only straw was directly returned to the field. Crop yields all increased after the crops were planted in the second year. The return of W118 inoculant and straw to the fields is beneficial to improving soil fertility, fertilizing the soil, conducive to plant growth and increasing crop yields.
Description
The invention relates to the technical field of biology, in particular to a low-temperature straw degrading bacterium and application thereof.
Background
Straw returning is an effective measure capable of improving straw utilization rate, effectively providing needed nutrition for crop growth and development and improving soil fertility, and is also a main mode of straw recycling. However, the straw which is directly returned to the field is easily affected by factors such as temperature and soil texture. Especially, the lower air temperature of the northern area after harvesting season leads to the difficult degradation of the straw. If the degradation is not timely, the sowing, seedling emergence and growth of the next crop can be affected. At present, the action temperature of the straw degrading bacteria is mostly 20-50 ℃, and the straw degrading bacteria is difficult to use in the cold environment in winter in the north.
Disclosure of Invention
In view of the above problems, the invention provides a strain capable of efficiently degrading straw under low temperature, and the treated straw can be returned to the field to effectively improve the physical and chemical properties of soil and increase the crop yield.
The invention provides a straw degrading bacterial strain W118, which is classified by the name of Bacillus cereus (Bacillus cereus.) and has the preservation number: CGMCC No.23973, date of preservation: 2021, 11, 25, deposit unit: china general microbiological culture Collection center (CGMCC), address: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.
The straw degrading bacteria W118 provided by the invention are gram positive bacteria, can normally grow in a sodium carboxymethyl cellulose culture medium, are circular, white and opaque in colony morphology on a solid culture medium, have a rough surface, are waxy, and have certain expansibility at the edge; after a period of incubation, the strain turns pale pink due to pigment production. The W118 strain is rod-shaped under an optical microscope and a scanning electron microscope, and the microscopic morphology is shown in figure 1.
The GenBank accession number of the 16SrDNA sequence of the straw degrading bacterium W118 is MW911450.
Further, the invention provides a method for degrading straw by utilizing straw degrading bacteria W118, which is characterized by comprising the following steps: coating a microbial inoculum of straw degrading bacteria W118 on the surface of straw, and standing at 4 ℃ for 15 days; wherein the viable count of straw degrading bacteria W118 in the microbial inoculum is 1 multiplied by 10 8 CFU/mL; the microbial inoculum is used in the following amount: each kilogram of straw is correspondingly coated with 1mL of microbial inoculum.
Preferably, the microbial inoculum is subjected to activation treatment before use, and the specific mode is as follows: straw degrading bacteria W118 for preparing the microbial inoculum are activated and cultured in peptone cellulose culture medium at 30 ℃ for 12 hours.
Preferably, the microbial inoculum of the straw degrading bacteria W118 is fermentation liquor containing W118 viable bacteria.
In addition, the technical scheme of the invention further comprises the following steps: the application of the straw degrading bacteria W118 in straw returning is provided.
Preferably, the application method of the straw degrading bacteria W118 in straw returning comprises the following steps: directly ploughing and returning the straw sprayed with the microbial inoculum of the straw degrading bacteria W118 to the field at the outdoor environment temperature of-10 to-20 ℃; wherein the viable count of straw degrading bacteria W118 in the microbial inoculum is not less than 1×10 8 CFU/mL, the use amount of the microbial inoculum is as follows: each kilogram of straw is correspondingly coated with 1mL of microbial inoculum.
The strain W118 provided by the invention has a straw degradation rate of 27% in 15 days at 4 ℃.
The strain W118 provided by the invention can be directly returned to the field together with rice, corn and soybean straws respectively in the Heilongjiang province in winter, and the degradation rates of the straws respectively reach 50%,43% and 38% (shown in figure 4).
After the W118 microbial inoculum is added, the contents of quick-acting phosphorus, effective potassium, humic acid and organic matters in the soil which is directly returned to the field together with the straw are obviously increased compared with the soil which is directly returned to the field by the straw. After the crops are planted in the second year, the crop yield is increased. The W118 microbial inoculum and the straw are returned to the field together, so that the soil fertility is improved, the soil is fertilized, the plant growth is facilitated, and the crop yield is increased.
The straw degrading bacteria W118 and the straw can be directly returned to the field together, so that the straw can be fully decomposed at low temperature, the cost is low, the physical and chemical properties of the soil can be effectively improved after application, and the crop yield is increased.
Description of the drawings:
FIG. 1 microscopic morphology of strain W118; a and b are the forms under an optical microscope and an electron microscope, respectively.
FIG. 2 phylogenetic tree of strain W118.
FIG. 3W 118 shows the absorbance at 48hOD nm and the degradation rate of straw for 15 days at 4 ℃.
FIG. 4 degradation rate of the W118 microbial inoculum and different straws after returning to the field together.
The invention provides a straw degrading bacterial strain W118, which is classified as Bacillus cereus (Bacillus cereus) with the preservation number: CGMCC No.23973, date of preservation: 2021, 11, 25, deposit unit: china general microbiological culture Collection center (CGMCC), address: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.
Detailed Description
The technical scheme of the invention is further described below by combining specific embodiments. The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
1. Isolation and identification of Strain W118
(1) Sampling and culturing: taking soil in a severe cold environment, diluting the soil by 10 times with sterile water, adding the diluted soil into a sterilized enrichment medium, and carrying out shaking culture for 72 hours by a shaking table;
(2) And (3) primary screening: taking the enriched supernatant, diluting and coating on a sodium carboxymethylcellulose solid medium. The coated plates were placed in an incubator at 4℃and incubated at constant temperature.
(3) Strain purification: single colonies were selected for rescreening in peptone cellulose liquid medium (PCS) containing straw, followed by 3-4 streaking cycles on sodium carboxymethyl cellulose solid medium to obtain single colonies of strain W118.
The main components of the culture medium used in the above steps are as follows:
enrichment medium: naNO 3 0.5g/L、KH 2 PO 4 1g/L、MgSO 4 0.5g/L、KCl 0.5g/L、Fe 2 (SO 4 ) 3 Trace amount and 10g/L of straw.
Sodium carboxymethyl cellulose medium: sodium carboxymethylcellulose (CMC-Na) 10g/L, peptone 5g/L, yeast extract 0.5g/L, KH 2 PO 4 1.5g/L、MgSO 4 0.2g/L, naCl g/L, pH is 7.0.
Peptone cellulose medium (PCS medium): 5g/L of straw, 5g/L, naCl g/L, caCO of peptone 3 2g/L, 3g/L of yeast extract.
Identification of strains: the W118 strain was subjected to extraction of fungal genomic DNA, and PCR amplification was performed by bacterial 16SrDNA universal primers (27F and 1492R) using the extracted DNA as a template. PCR products were sequenced by Beijing Oriental Dingsheng (China). After Blast homology sequence alignment analysis in NCBI's GeneBank database, phylogenetic tree was constructed, with strain W118 clustered with Bacillus cereus (FIG. 2). The GeneBank accession number of the W118 strain on NCBI is MW911450.
2. Strain growth assay
Strain W118 was inoculated into PCS medium and cultured at 4 ℃. The OD value of the strain was varied from 0 to 48h as shown in FIG. 3.
3. Weight loss rate determination
1) Determination of straw degradation (weight loss)
Weighing straws with certain mass, and adding water to keep the water content of the straws to be 60% -65%; coating the fermentation liquor containing W118 viable bacteria on the surface of straw, wherein each gram of straw is correspondingly coated with 0.1mL of microbial inoculum, and the viable bacteria number of straw degrading bacteria W118 in the microbial inoculum is 1 multiplied by 10 8 CFU/mL; the straw coated with the microbial inoculum is placed at 4 ℃ for 15 days, then residual thalli and fermentation liquor on the straw are washed off by using 1% mixed acid, the straw is dried to constant weight at 60 ℃, then the weight is weighed, and the weight loss rate is calculated according to the mass of the straw before and after degradation. The weight loss rate is calculated using the following formula:
straw weight loss ratio= (straw weight after cultivation (g) -initial weight (g))/initial weight (g) ×100%.
The results show that: the weight loss rate of the straw reached 27% after 15 days of liquid fermentation at 4 ℃ (shown in figure 3).
2) Outdoor returning to field weight loss rate test
The test method comprises the following steps:
(1) Weighing 50g of rice straw, corn straw and soybean straw respectively (multiple parts of each straw are repeatedly weighed), putting each part of straw into a nylon mesh bag respectively, drying in an oven at 85 ℃ for 6 hours, and weighing;
(2) Adding a proper amount of water into rice straw, corn straw and soybean straw in the mesh bag, keeping the water content of the straw to be 60% -65%, and then spraying a microbial inoculum of straw degrading bacteria W118 (fermentation liquor containing W118 viable bacteria) onto the three straws; each kilogram of straw is correspondingly coated with 1mL of microbial inoculum, and the W118 viable count in the microbial inoculum is not less than 1 multiplied by 10 8 CFU/mL；
(3) Uniformly burying the net bags filled with the straw into test cells in the month of October (average outdoor environment temperature is-15 ℃), burying 9 net bags in each cell, randomly sampling in the next year, taking out 3 net bags from each group, cleaning with clear water, drying and weighing. And calculating the weight loss rate of the straw.
The measurement and calculation results show that: the degradation rates of rice straw, corn straw and soybean straw after being treated by the straw degrading bacteria W118 microbial inoculum respectively reach 50%,43% and 38% (shown in figure 4).
4. Returning to field test
After harvesting crops in winter (October), carrying out field returning tests on paddy fields, corn fields and soybean fields under outdoor field conditions in eastern region of Heilongjiang province. In the rice field, the treatment of turning over and returning the W118 microbial inoculum and rice straw together is used as a test group (RCT), the treatment of returning the straw without microbial inoculum directly to the field is used as a control group (RK), and the soil nutrients, the crop growth condition and the crop yield are measured in the key period of crop growth in the next year. The same test of the control group is carried out in a corn field and a soybean field, wherein the test group (CCT) in the corn field is that the microbial inoculum and the corn straw are returned to the field together, and the control group (CK) is that only the corn straw is returned to the field; the test group (SCT) in the soybean field is the microbial inoculum and the soybean straw which are returned together, and the control group (SK) is the soybean straw which is returned only. Soil nutrients, crop growth conditions and crop yields of each test group and control group were measured during the critical period of crop growth in the next year.
The test results show that: soil nutrients required by crops in the tillering stage, such as soil organic matters, quick-acting potassium, available phosphorus and humic acid, are obviously increased after the microbial inoculum is added, as shown in table 1: in the tillering stage, compared with the RK group, the RCT group has 20.26 percent, 17.94 percent, 36.3 percent and 25.04 percent of organic matters, available phosphorus, quick-acting potassium and humic acid respectively increased; the CCT group is respectively increased by 21.68%, 31.41%, 98% and 19.68% compared with the CK group; SCT groups increased by 21.45%, 25.7%, 25% and 43.99%, respectively, compared to SK groups.
The crop growth condition in the second year shows that the crop growth condition of the test group which uses the microbial inoculum and the straw to return to the field together is good and the yield is obviously increased. The crop yields (hundred/thousand kernel weight) of the test group for paddy field, corn field and soybean field were increased by 13.04%, 3.34% and 27.78%, respectively.
TABLE 1 influence of W118 microbial inoculum on nutrient content and crop yield of different types of straw direct returning soil
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