CN115287219A - Straw degradation composite bacterial TW and application thereof - Google Patents
Straw degradation composite bacterial TW and application thereof Download PDFInfo
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- CN115287219A CN115287219A CN202210603773.3A CN202210603773A CN115287219A CN 115287219 A CN115287219 A CN 115287219A CN 202210603773 A CN202210603773 A CN 202210603773A CN 115287219 A CN115287219 A CN 115287219A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention relates to a straw degradation composite bacterial system TW and application thereof, belonging to the field of straw degradation. The efficient straw degradation composite bacterial system TW provided by the invention comprises Bacillus subtilis TXB2 and Bacillus velezensis WXB10. The registration numbers of China center for type culture collection management are CCTCC M2022679 and CCTCC M2022680 respectively. The straw degradation rate of the straw degradation composite bacterial system TW for treating each straw for 30d is 60.08%, 58.76%, 53.88%, 34.16%, 31.56% and 50.32% for potato, oat, buckwheat, quinoa, corn and wheat.
Description
Technical Field
The invention relates to the field of straw degradation, in particular to a straw degradation composite bacterial system TW and application thereof.
Background
China is a big agricultural country, has rich straw resources with 9 hundred million t annual output, mainly comes from three large grain crops of corn, rice and wheat, and then is straws of potatoes, beans, rape, cotton and other cereal crops. The comprehensive utilization rate of straw resources is less than 40% every year in China, and more than 60% of straws are burnt and randomly stacked or used as living fuel. Some dormancy bodies of crop diseases and insect pests overwinter on the straws, if the straws are directly returned to the field, the dormancy bodies cannot be degraded before the next crop is sown, the growth of the next crop is not facilitated, and the occurrence of the diseases and insect pests is aggravated. The straw degradation is accelerated, and some diseases and insect dormant bodies die along with the straw degradation, so that the occurrence of diseases and insect pests can be greatly reduced. The straws are byproducts from stalks, leaves and the like of crops, contain nutrient elements required by plant growth, and can return the nutrients to soil for the growth of crops by straw degradation, so that the straws become important fertilizers.
The fiber structure of the straws is crushed by utilizing the microbial degradation capability, crude fibers in the straws are decomposed into micromolecular substances, and organic matters and the fiber structure are degraded and converted into CO 2 Water, minerals and heat. According to the report, the cellulase activity of a single strain is lower than that of a compound strain, the degradation capability of the compound strain is stable and reliable, and the compound strain has higher practical application value. The straw is quickly degraded, the crop growth can be promoted, the plant diseases and insect pests can be prevented and controlled, the soil is fertilized, the organic matter content of the soil is improved, the environment is not polluted, the implementation is simple and easy, and the utilization rate of the straw is improved. A more complete straw field treatment, collection, storage and transportation system is established, and straw in-situ returning is promoted, so that the method is an effective technical way for realizing land cultivation, improving the quality of cultivated land and establishing a high-yield and stable-yield farmland.
Disclosure of Invention
The invention aims to provide a composite bacterial line TW for straw degradation, which comprises Bacillus subtilis and Bacillus velezensis.
The invention also aims to provide application of the straw degradation composite bacterial system TW in degradation of potato, oat, buckwheat, corn, quinoa and wheat straws.
The invention is realized by the following technical scheme:
taking a soil sample from a potato field, and carrying out primary screening on the straw degrading bacteria by using a Congo red dyeing method to obtain 2 cellulose degrading bacteria. Carrying out a potato straw liquid state fermentation test on the screened cellulose degrading bacteria, obtaining strains TXB2 and WXB10 with high degradation efficiency through the straw degradation rate, measuring the cellulase activity of the strains, constructing a straw degrading composite bacterial system, and carrying out a pot culture degradation effect test on potato, oat, buckwheat, corn, quinoa and wheat straw by the composite bacterial system.
The straw degradation bacterial system comprises: bacillus subtilis (Bacillus velezensis) and Bacillus subtilis (Bacillus subtilis).
The Bacillus subtilis is preserved in China center for type culture collection and management, and the preservation unit address is as follows: eight-way No. 299 in Wuchang area of Wuhan city, hubei province, with preservation date: year 2022, 05, 19, category name: the Bacillus subtilis has a preservation number of CCTCC M2022679.
The Bacillus velezensis is preserved in China center for type culture collection and management, and the preservation unit address is as follows: eight-way No. 299 in Wuchang area of Wuhan city, hubei province, with preservation date: 19/05/2022, category name: the Bacillus velezensis has a preservation number of CCTCC M2022680.
The invention has the advantages that:
according to the invention, the straw degradation rate of each straw is 60.08%, 58.76%, 53.88%, 34.16%, 31.56% and 50.32% respectively by treating each straw with the straw degradation composite bacterial system TW for 30 days. The straw degradation composite bacterial TW provided by the invention is used for rapidly degrading straws, has stable effect, is safe to people and livestock, does not pollute the environment, and has good development and application prospects.
Deposit description:
the Bacillus subtilis TXB2 (Bacillus subtilis) is preserved in China center for type culture collection and management (CCTCC) in 2022, 05 and 19 days, the preservation address is eight-way No. 299 China center for type culture collection and management in Wuchang district, wuhan, hubei province, the registration number of the preservation center is CCTCC M2022679, and the Bacillus subtilis TXB2 is called as strain TXB2 for short in the invention and survives through inspection. Bacillus belgii (WXB 10Bacillus velezensis) is preserved in China center for type culture collection and management (CCTCC) in 2022, 05 and 19 days, the preservation address is eight-way No. 299 China center for type culture collection and management in Wuchang district, wuhan, hubei, the registration number of the preservation center is CCTCC M2022680, and the strain WXB10 is called as strain in the invention for short, and the strain is survived after inspection.
Drawings
FIG. 1 variation of degradation Rate of Potato stalks
FIG. 2 variation of oat straw degradation rate
FIG. 3 shows the change of the degradation rate of buckwheat straw
FIG. 4 shows the change of straw degradation rate of quinoa
FIG. 5 variation of corn stover degradation rate
FIG. 6 variation of wheat straw degradation rate
Detailed Description
The following examples are intended to better illustrate the invention without limiting its scope. The test methods in the following examples are conventional methods unless otherwise specified. The materials used in the following examples are commercially available from conventional biochemicals, unless otherwise specified.
The method for separating and screening straw degrading bacteria specifically comprises the following steps
The media in the following examples:
LB solid medium: 10g of peptone, 5g of yeast extract powder, 10g of NaCl, 15g of agar and 1000mL of distilled water.
LB liquid medium: 10g of peptone, 5g of yeast extract powder, 10g of NaCl and 1000mL of distilled water.
PDA culture medium: 200g of peeled potato, 18g of agar, 20g of glucose and 1000mL of distilled water.
Gao 1 medium: mgSO (MgSO) in vitro 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 culture 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, and distilled water 1000mL.
Enzyme production culture medium: peptone 3g, (NH) 4 ) 2 SO 4 3g，KH 2 PO 4 1g，MgSO 4 ·7H 2 O0.2g, naCl 5g and 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 potato straws 1-2 cm long and 50mL of nutrient solution.
Carbon source determination of basal medium: KH (Perkin Elmer) 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 assay 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: 7.0g of peptone, 5g of glucose, 5g of NaCl and 1000mL of distilled water.
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 0.1g of O, 17g of agar and 1000mL of distilled water, and the pH value is 7.4.
NA culture medium: 3g of beef extract, 5g of peptone, 2.5g of glucose, 18g of agar and 1000mL of distilled water, and the pH value is 7.0.
Example 1 soil sample Collection and separation and screening of straw-degrading bacteria
Soil samples are taken from potato fields in Tokto county and Wuchuan county, which are called and are expensive in inner Mongolia, 5-10 cm away from the ground surface. Weighing 10g of soil sample, adding into 250mL of triangular flask containing 90mL of sterile water, placing in a constant-temperature shaking incubator at 28 ℃ and 180r/min, shaking for 30min, standing for 20min, taking 1mL of supernatant under aseptic condition, performing gradient dilution, and diluting to 10% -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 And (3) taking 0.1mL of each of the three concentrations, coating and separating the three concentrations on an LB solid culture medium, culturing the three concentrations on 3 plates in a constant-temperature incubator at 28 ℃ until bacterial colonies grow out, and selecting strains with different forms to perform repeated streaking purification to obtain pure strains.
Screening straw degrading bacteria in a Congo red dyeing test, respectively inoculating purified strains to a sodium carboxymethylcellulose culture medium by adopting a dot inoculation method, inoculating 3 dots to each flat plate, repeating the steps for 3 times, culturing for 72h in a constant-temperature incubator at 28 ℃, adding a proper amount of 1mg/mL Congo red solution for dyeing for 30min, discarding dye liquor, adding a proper amount of 1mol/L NaCl solution for decoloring for 20min, observing and measuring the diameter (D, cm) of a bacterial colony and the diameter (D, cm) of a transparent ring around the bacterial colony, and calculating the value of D/D.
As a result, 2 strains of bacteria all have transparent rings on the Congo red staining test plate, the ratio (D/D) of the diameter D of the transparent rings to the diameter D of the bacterial colonies is 5.53 and 6.22 respectively, and the bacteria have certain cellulose degradation capacity. After primary screening, 2 strains are obtained and named as TXB2 and WXB10.
Congo red dyeing test results
Example 2 determination of the degradation rate of strains TXB2 and WXB10 on potato straw
Strains TXB2 and WXB10 are inoculated into LB liquid culture medium and the preparation concentration is 1X 10 7 Inoculating the CFU/mL bacterial suspension into a potato straw fermentation culture medium according to the volume of 10%, taking non-inoculated bacteria as blank control, repeating for 3 times, culturing in a constant-temperature oscillation 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 straws.
Degradation rate = (M) 1 -M 2 )/M 1 ×100％
Wherein M is 1 For initial straw mass, M 2 The quality of the degraded straw is shown.
The results show that the degradation rates of the potato straws of the strains TXB2 and WXB10 are respectively 55.48 percent and 57.73 percent when the strains are treated for 35d, and are higher than those of the control group.
Degradation effect of potato straw liquid state fermentation
Example 3 cellulase Activity assay
TXB2 and WXB10 strains were inoculated into LB liquid medium at a concentration of 1X 10 7 CFU/mL bacterial suspension, in vivoInoculating the bacterial suspension into an enzyme production culture medium respectively with the inoculation amount of 9 percent of volume ratio, culturing for 7d at 28 ℃ and 180r/min, taking the bacterial suspension every 24h, using a commercial kit purchased from Sores biotech, suzhou, and determining the activity of the hemicellulase, the endo-beta-1, 4-glucanase, the exo-beta-1, 4-glucanase and the beta-glucosidase of the strain according to the Sores enzyme activity determination instruction of Suzhou. Taking 2mL of bacterial suspension, carrying out ultrasonic disruption for 15s at an interval of 10s, repeating for 30 times, carrying out centrifugation at 12000rpm for 10min at 4 ℃, and taking a supernatant to be tested.
As a result, both strains TXB2 and WXB10 can produce hemicellulase, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase, and the hemicellulase, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase of the strain TXB2 have the maximum enzymatic activities respectively: 29.94U/mL, 289.93U/mL, 533.7U/mL, and 4.78U/mL. The hemicellulase, endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase and beta-glucosidase activity maximum values of the strain WXB10 are respectively as follows: 32.17U/mL, 238.75U/mL, 478.48U/mL, and 4.82U/mL.
Example 4 identification of strains TXB2 and WXB10
Morphological identification
Coating the strain in an LB solid culture medium by a dilution method to grow a single colony, wherein the single colony of the strain WXB10 is milky white, and has a dry and rough surface, folds, opaqueness and irregular edges; TXB2 colonies are milky white, opaque, irregular in edges, wrinkled and sunken in the middle.
Physiological and biochemical identification
The test refers to the handbook of identifying common bacteria systems, and the physiological and biochemical indexes of the strain, such as the utilization of carbon sources and nitrogen sources, the utilization of malonate, methyl red test, starch hydrolysis, oxidase test, catalase test and the like are measured. 2 strains of bacteria are gram-positive bacteria, can utilize malonate, generate catalase and cannot generate oxidase. The strain TXB2 can decompose starch and fat, can use glucose and mannitol as a sole carbon source, and can be used as a sole nitrogen source such as ammonium nitrate and ammonium sulfate. The strain WXB10 can utilize mannitol, fructose, glucose, histidine, ammonium nitrate and ammonium sulfate as a unique carbon-nitrogen source and can hydrolyze starch and fat.
Physiological and biochemical characteristics of the strain
Note: "+" indicates positive, and "-" indicates negative
Molecular biological identification
DNA extraction was performed according to the instructions of the TIAN GEN bacterial genome DNA extraction kit (centrifugal column type) (Beijing, china) and purchased from Tiangen Biochemical technology, inc. The PCR primers of strain WXB10 are 27F and 1492R, 7F and 1540R, rpoBF and rpoBR, trpBF and trpBR respectively, the PCR primers of strain TXB2 are 27F and 1492R, 7F and 1540R, rpoBF and rpoBR, tpiF and tpiR respectively, and the sequences of the primers are shown in the following table. 25 μ L of reaction contained: 17 μ L of ddH 2 O, 2.5. Mu.L of 10 XPCR Buffer, 2. Mu.L of dNTP, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer, 0.25. Mu.L of Taq DNA polymerase. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s; the annealing temperature is 30s; 1min at 72 ℃; circulating for 34 times; 10min at 72 ℃. PCR products of 4 genes were detected by 1% agarose gel electrophoresis and sent to Shanghai Biotech for 16S rDNA sequencing. Splicing the obtained 4 gene sequences, comparing the splicing result with a Gen Bank nucleic acid database, selecting a strain sequence with higher similarity for analysis, and constructing a phylogenetic tree by using software MEGA 7.0. Splicing the sequencing results of the 4 PCR products, comparing by using a Blast program in an NCBI database, and analyzing by a MEGA7.0 software phylogenetic tree, wherein the strain WXB10 and the Bacillus velezensis (Bacillus velezensis) are gathered into one, and the accession numbers are (MG 461457), (MF 471767) and (MT 114571) respectively; the strain TXB2 and Bacillus subtilis are gathered together, and the accession numbers are (MT 950332), (JQ 900623), (OK 428684), (EU 056572), (OK 428687) and (MN 900849), respectively.
According to the results of morphological, physiological and biochemical tests and molecular biological identification, the strain WXB10 is identified as Bacillus subtilis (Bacillus velezensis), and the strain TXB2 is identified as Bacillus subtilis (Bacillus subtilis).
Primary primer
Example 5 TXB2 and WXB10 compatibility testing
Preparing bacterial suspension of degrading strains TXB2 and WXB10 with the concentration of 1X 10 7 And CFU/mL, respectively taking 200 mu L of the suspension, uniformly coating the suspension on an LB solid culture medium, placing 7 layers of sterilized filter paper sheets with the diameter of 1cm in the center of the culture medium, respectively inoculating 200 mu L of other treated bacterial suspensions by using a pipette, inoculating every two of the suspensions on the same LB solid culture medium, placing the same LB solid culture medium in a constant-temperature incubator at the temperature of 28 ℃ for culture, repeating the steps for 3 times, and observing whether two strains have antagonism or not.
The results show that strains TXB2 and WXB10 are compatible and do not antagonize.
Example 6 construction of straw-degrading composite bacterial line
Inoculating 2 strains of the strain into LB liquid culture medium respectively to prepare the strain with the concentration of 1 × 10 7 The bacterial suspension CFU/mL is combined in pairs according to the inoculation amount of 9 percent in volume ratio of 1: 8, 2: 7, 3: 6, 4: 5, 5: 4, 6: 3, 7: 2 and 8: 1 respectively, and is inoculated into an enzyme production culture medium for culture under the culture conditions of 28 ℃ and 180r/min, the activity of endo-beta-1, 4-glucanase, exo-beta-1, 4-glucanase, beta-glucosidase and hemicellulase which are combined in different ratios is determined every 24 hours, and each treatment is repeated for 3 times. And continuously measuring for 5d, and determining the optimal proportion combination according to the maximum enzyme activity of each combination.
As a result, each of the strains TXB2 and WXB10 produced cellulose degrading enzymes at a ratio, and the combination of 5: 4 by volume was the best complex strain combination named TW.
Enzyme production condition of TXB2 and WXB10 in different ratio combinations
Example 7 straw degradation Effect of different crops
Potato, oat, corn, quinoa, wheat, buckwheat and sunflower straw are cleaned under running tap water, dried in an oven at 80 ℃ and cut into short sections of 3-4 cm. Placing 5g of different crop straws in small nylon bags respectively, and using 9mL of the small nylon bags with the concentration of 1 × 10 7 CFU/mL of suspension of the composite strain TW soaked straw, with no inoculation as control CK, repeated 5 times for each treatment. Placing the treated straws in the middle of flowerpots, covering 2.0kg of sterilized soil on each flowerpot, covering 2-3 cm of sterilized soil on each flowerpot, placing 5 small nylon bags of the treated straws in each flowerpot, adding 420mL of tap water (relative humidity is 70-80%), placing the straws in a laboratory for culture, pouring 420mL of tap water every 5d, sampling when culturing for 5d, 10d, 20d and 30d, taking out 1 small nylon bag of straws every time, and determining the straw degradation rate of different straws.
As a result, 30d of each straw was treated by the straw composite bacterial line TW, and the degradation rates of the potato straw, the oat straw, the buckwheat straw, the quinoa straw, the corn straw and the wheat straw were 60.08% (figure 1), 58.76% (figure 2), 53.88% (figure 3), 34.16% (figure 4), 31.56% (figure 5) and 50.32% (figure 6), respectively.
Claims (3)
1. A high-efficiency straw degradation composite bacterial system TW comprises a Bacillus subtilis TXB2 and a Bacillus velezensis WXB10,
bacillus subtilis TXB2 (Bacillus subtilis) is preserved in China center for type culture collection and management, the preservation address is No. 299 of eight branches in Wuchang district, wuhan city, hubei province, the preservation date is 2022 years, 5 months and 19 days, the preservation number is CCTCC M2022679,
the Bacillus belgii WXB10 (Bacillus velezensis) is preserved in China center for type culture collection and management, the preservation address is No. 299 of eight branches in Wuchang district, wuhan city, hubei province, the preservation date is 2022 years, 5 months and 19 days, and the preservation number is CCTCC M2022680.
2. The high-efficiency straw degradation composite bacterial system according to claim 1, wherein the ratio of viable count of Bacillus subtilis TXB2 (Bacillus subtilis) to Bacillus belief WXB10 (Bacillus velezensis) is (1-8) to (8-1), and the optimal ratio is 5: 4.
3. The use of the high efficiency straw-degrading composite bacterial strain TW according to claim 1, which is used for degrading potato, oat, buckwheat, quinoa, corn and wheat straw.
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