CN114230402A - Efficient decomposed liquid bacterial fertilizer and preparation method and application thereof - Google Patents
Efficient decomposed liquid bacterial fertilizer and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a high efficiencyThe bacterial fertilizer contains bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum, eupenicillium corticum, myceliophthora thermophila and pediococcus pentosaceus, and the effective viable count is more than or equal to 1 multiplied by 109cfu/mL. The invention has the advantages that the prepared bacterial manure liquid contains sufficient nitrogen source required by the growth and metabolism of microorganisms, and the straw decomposition efficiency can be effectively improved without additionally supplementing the nitrogen source during application; on the other hand, various metabolic pathways of different strains and complementation on different degrading enzyme systems are fused, pediococcus pentosaceus can effectively degrade intermediate metabolites of straw lignocellulose, such as low-molecular oligosaccharides, lactic acid, ethanol and the like, and relieve the repression effect caused by product accumulation in the straw carbon metabolism process.
Description
Technical Field
The invention belongs to the field of organic material decomposing agents or liquid bacterial manure, and particularly relates to a high-efficiency decomposing liquid bacterial manure and a preparation method and application thereof.
Background
As the related ministerial committees of the state issue policies such as straw burning prohibition and comprehensive resource utilization successively, the mode taking straw returning as priority is developed rapidly. The straws are agricultural byproducts with very high fiber component content, wherein the difficultly decomposed macromolecular chemical components, namely cellulose, hemicellulose and lignin, have compact structure, high complex polymerization degree, stable property and strong decomposition resistance, so that the decomposition rate of the returned straws under natural conditions is slow, and a large amount of insufficiently decomposed straws can cause the problems of aggravation of plant diseases and insect pests, influence on the growth of next-stubble crops and the like. The straw is biologically converted by utilizing related functional bacteria in nature, and the straw biological conversion agent has the characteristics of high degradation efficiency, safety, no pollution and the like.
At present, most of commercial microbial agents in the market are compounded by functional bacteria for degrading lignocellulose, and the problems of poor system stability, non-lasting effect and the like generally exist. When the straw is subjected to biotransformation by using microorganisms, because the carbon-nitrogen ratio of the straw is high, in the process of straw carbon transformation, the microorganisms need to absorb nitrogen to maintain the growth and metabolism of the straw, while the microbial inoculum is usually sprayed on the surface of the straw, the available nitrogen is limited to the release of the straw nitrogen, the utilization of the nitrogen in soil is relatively difficult, and the problem of weak colonization of functional degradation bacteria after the microbial inoculum is applied is caused; on the other hand, the functional degradation bacteria can be subjected to a repression effect caused by accumulation of metabolites in the straw degradation process, and due to lack of lignocellulose intermediate metabolite degradation bacteria, imbalance in the straw carbon conversion process is easily caused, so that the straw decomposition function is difficult to be continuously exerted.
Disclosure of Invention
The invention mainly aims at the problem that the components of the straw microbial inoculum lack nitrogen sources and lignocellulose intermediate metabolic bacteria, and provides a preparation method of the high-efficiency decomposed liquid bacterial fertilizer, on one hand, the bacterial fertilizer liquid already contains quick-acting and slow-release nitrogen sources required by microorganism growth and metabolism, and the decomposition efficiency of the straw can be effectively improved without additionally supplementing the nitrogen sources during application; on the other hand, various metabolic pathways of different strains and complementation on different degrading enzyme systems are fused, pediococcus pentosaceus can effectively degrade intermediate metabolites of straw lignocellulose, such as low-molecular oligosaccharides, lactic acid, ethanol and the like, and relieve the repression effect caused by accumulation of the metabolites.
In order to solve the above technical problems, a first objective of the present invention is to provide a high efficiency decomposing liquid bacterial fertilizer, which contains three high efficiency decomposing bacteria capable of secreting cellulase and xylanase: stenotrophomonas sp.WS6-1, Achromobacter sp.WS6-2 and Pediococcus pentosaceus YC2, and the effective viable count in the bacterial manure is more than or equal to 1 × 109 cfu/mL;
the Stenotrophomonas (Stenotrophoromonas sp.WS6-1) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation address is as follows: china, Wuhan university, the preservation number is: CCTCC No. M20191060;
the Achromobacter sp.WS6-2 strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation address is as follows: china, Wuhan university, the preservation number is: CCTCC No. M20191061;
the Pediococcus pentosaceus (Pediococcus pentosaceus YC 2) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation address is as follows: china, Wuhan university, the preservation number is: CCTCC No. M20191062. When the liquid bacterial manure is applied, no additional nitrogen source is needed to be supplemented
The efficient decomposition liquid bacterial manure provided by the invention is further set to contain eupenicillium crustaceum which is purchased from Beijing Beinanna institute of Biotechnology, and the resource number of the eupenicillium crustaceum is BNCC 146720. The cold-resistant eupenicillium crustaceum is adopted, the metabolic pathway and complementation on different degrading enzyme systems can effectively relieve the repression effect caused by accumulation of metabolites, and the decomposition speed is high.
The efficient decomposition liquid bacterial fertilizer provided by the invention is further set to contain one or more of bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum and myceliophthora thermophila.
The efficient decomposition liquid bacterial fertilizer provided by the invention is further set to be that bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum and myceliophthora thermophila in the bacterial fertilizer agent are purchased from Beijing Beinanna Chuanglian biotechnology research institute, the resource number of the bacillus subtilis is BNCC188080, the resource number of the streptomyces roseoflavus is BNCC228869, the resource number of the trichoderma harzianum is BNCC336568, and the resource number of the myceliophthora thermophila is BNCC 186098.
The second purpose of the invention is to provide a preparation method of the high-efficiency decomposed liquid bacterial fertilizer, which comprises the following steps:
step 1, strain activation
Taking a proper amount of pure culture from a slant seed-preserving culture medium according to aseptic operation, and inoculating stenotrophomonas and achromobacter to an LB solid plate; the inoculated flat plate is inverted and horizontally placed in an incubator at the temperature of 26-30 ℃ for culture, and when a single bacterial colony grows out, spores produced by fungi are stable and uniform in size;
step 2, seed culture
Under the aseptic condition, picking single colonies of the activated stenotrophomonas and achromobacter in the step 1 by using inoculating loops, and respectively inoculating the single colonies to an LB liquid culture medium; after inoculation, carrying out shaking culture on each strain at 26-30 ℃ and 150r/min by using a shaking table, and determining the culture time according to the growth curve of each strain to obtain strain seed liquid at the middle and later logarithmic growth stages;
step 3, expanded culture
Transferring the seed solution obtained in the step (2) into a liquid culture medium of a corresponding type according to the inoculation amount of 10-20% (V/V), performing propagation expansion, adjusting the inoculation time and the culture time according to the growth rate of each strain of bacteria so as to obtain a bacterial solution with basically consistent concentration at the same time, and performing centrifugal concentration on the bacterial solution subjected to propagation expansion still according to aseptic operation;
step 4, mixed fermentation
And (3) centrifugally concentrating and collecting the bacteria/spores of the strain in the step (3), preparing the bacteria/spores into heavy suspension with consistent concentration by using sterile water, mixing according to a ratio of 1:1, inoculating the mixed bacteria liquid into a liquid fermentation culture medium, and naturally fermenting for about 10 days at room temperature to obtain the liquid bacterial fertilizer with stable property and function and effective viable count of more than or equal to 1 x 109 cfu/mL.
The preparation method of the high-efficiency decomposed liquid bacterial fertilizer provided by the invention is further set as that in the step 1, the strain activation also comprises the steps of taking a proper amount of pure culture from a slant strain-preserving culture medium according to aseptic operation, and inoculating the bacillus subtilis to an LB solid plate; inoculating Trichoderma harzianum, Penicillium crusum and myceliophthora thermophila to a PDA solid plate; inoculating Streptomyces roseoflavus to a Gao's No. 1 solid plate; and (3) inoculating lactic acid bacteria to the basic solid culture medium, inverting the inoculated flat plate, horizontally placing the flat plate in an incubator at the temperature of 26-30 ℃ for culture, and allowing the bacteria to grow a single bacterial colony and the fungi to produce spores stably and uniformly.
The preparation method of the high-efficiency decomposed liquid bacterial fertilizer provided by the invention is further set as that in the step 2, under the aseptic condition, the seed culture also comprises the step of selecting a single bacterial colony from the bacillus subtilis activated in the step 1 by using an inoculating loop, and inoculating the single bacterial colony to an LB liquid culture medium; adding a small amount of sterile water into a trichoderma harzianum, eupenicillium crustosum and myceliophthora thermophila solid flat plate, gently scraping lawn by using an inoculating loop to wash off spores, filtering by using sterile cotton to remove hyphae, and inoculating to a PDA liquid culture medium; inoculating streptomyces roseoflavus to a Gao's No. 1 liquid culture medium containing glass beads; inoculating lactobacillus to MRS liquid culture medium; and (3) after inoculation of each strain, carrying out shaking culture at 26-30 ℃ and 150r/min in a shaking table, and determining the culture time according to the growth curve of each strain to obtain strain seed liquid in the middle and later logarithmic growth stages.
The preparation method of the high-efficiency decomposition liquid bacterial manure provided by the invention is further set as follows, wherein an LB liquid culture medium: 10g of peptone, 10g of beef extract, 5g of sodium chloride, 1000mL of distilled water, and pH 6.5-7, and 20g of agar is added when a solid culture medium is prepared;
PDA culture medium: 200g of peeled potato cooking juice 1000ml, sucrose 20g, monopotassium phosphate 3g, magnesium sulfate 1.5g and vitamin B110 mg, wherein the pH value is natural; when preparing a solid culture medium, 20g of agar is added;
gao's No. 1 medium: 20g of soluble starch, 1g of potassium nitrate, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.01 g of ferrous sulfate, 1000mL of distilled water and pH 7.4-7.6, and 20g of agar is added when preparing a solid culture medium.
The preparation method of the high-efficiency decomposition liquid bacterial manure provided by the invention is further set as follows, wherein the liquid fermentation culture medium comprises the following components: 0.5% tryptone, 0.5% NaCl, 0.2% CaC03, 0.1% yeast powder, 0.3% urea and 1000mL of corn straw leaching liquor, wherein the pH value is natural, and the corn straw leaching liquor is sterilized for 20min at 121 ℃.
The third purpose of the invention is to provide the application of the liquid bacterial manure for efficient decomposition, the liquid bacterial manure is applied to the rapid and efficient decomposition of the corn straws, and a nitrogen source is not required to be additionally supplemented when the liquid bacterial manure is applied.
Has the beneficial effects.
1. The high-efficiency decomposition liquid bacterial fertilizer provided by the invention contains three high-efficiency decomposition bacteria capable of secreting cellulase and xylanase: stenotrophomonas sp.WS6-1, Achromobacter sp.WS6-2 and Pediococcus pentosaceus YC2, which are screened and preserved by the laboratory, have no antagonistic action among the strains, meet the assembly conditions and improve the degradation capability to cellulose.
2. The efficient decomposition liquid bacterial fertilizer provided by the invention is prepared by compounding and fermenting the liquid bacterial fertilizer through microbial bacteria, the effective viable count is more than or equal to 1 multiplied by 109cfu/mL, the efficient decomposition liquid bacterial fertilizer has stable property and function and high decomposition speed of corn straws, and has higher market popularization and application values.
3. The invention provides a preparation method of a multi-strain-compounded liquid bacterial fertilizer agent for efficiently decomposing corn straws. The degradation capability of the corn straws is the best, the degradation rate of the corn straws is improved by 35.0 percent compared with that of the micro-organisms, and the degradation rate of the corn straws is improved by 66.3 percent and 76.8 percent compared with that of the Diele and the decomposing agent 150 respectively.
4. The invention provides a preparation method of a multi-strain combined maize straw high-efficiency decomposition liquid bacterial manure agent, which is characterized in that bacterial manure liquid is fused with various metabolic pathways of different strains and complementation on different degradation enzyme systems, pediococcus pentosaceus can effectively degrade intermediate metabolites of straw lignocellulose, such as low-molecular oligosaccharide, lactic acid, ethanol and the like, and the repression effect caused by accumulation of the metabolites is relieved.
Drawings
FIG. 1 is a graph of weight loss data for filter paper.
FIG. 2 is a graph of straw weight loss data.
FIG. 3 is a flow chart of the preparation and application of the high-efficiency decomposed liquid bacterial manure.
The specific implementation mode is as follows:
the present invention will be described in detail and specifically by the following examples, and it should be understood that the following examples are included to provide a better understanding of the present invention and are not intended to limit the scope of the present invention.
The efficient decomposition liquid bacterial fertilizer selects the following 8 effective active microorganisms: bacillus subtilis (A), (B) and (C)Bacillus subtilis) Streptomyces roseoflavus (see)Streptomyces roseoflavus) Trichoderma harzianum (a)Trichoderma harzianum Rifai) Penicillium notatum (A) and (B)Eupenicillium crustaceum) Myceliophthora thermophila (Myceliophthora thermophila) Pediococcus pentosaceus (A)Pediococcus pentosaceus YC2) The number of effective viable bacteria in the bacterial fertilizer agent is more than or equal to 1 multiplied by 109cfu/mL。
Example 1
Preparation and functional verification of corn straw bacterium fertilizer
The key functional strains obtained by self-screening in a laboratory in the decomposition process of stenotrophomonas, achromobacter and lactic acid bacteria and straws, namely bacillus subtilis (biocontrol bacteria), streptomyces roseoflavus (biocontrol bacteria), trichoderma harzianum (biocontrol bacteria), penicillium corticum (cold-resistant type) and myceliophthora thermophila (thermophilic type), are combined pairwise to perform a plate confrontation test, and the results show that all strains do not have antagonistic action and meet the prerequisite condition of combination.
The liquid bacterial manure agent is prepared by the 8 strains of bacteria through strain activation, seed culture, enlarged culture and mixed fermentation.
According to aseptic operation, using sterile water to prepare bacterial manure agents, stenotrophomonas, achromobacter, bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum, eupenicillium crustoides and myceliophthora thermophila amplification culture solutions into bacterial suspension/spore solutions with consistent concentrations, respectively taking 5mL of the bacterial suspension/spore solutions to inoculate into a triangular flask filled with 45mL of enzyme production culture medium, carrying out shaking culture on a shaking table at 30 ℃ and 150r/min, and taking fermentation liquor to determine beta-glucosidase activity, endoglucanase activity, exoglucanase activity, filter paper enzyme activity and xylanase activity at different time intervals.
Definition of enzyme activity: under the specified conditions, the enzyme amount of 1mL of enzyme solution catalyzing substrate hydrolysis to generate 1 mug of reducing sugar per minute is one enzyme activity unit and is recorded as U/mL.
Enzyme production culture medium: 0.5% of peptone, 0.5% of corn straw powder, 0.5% of NaCl, 0.2% of CaC03, 0.1% of yeast powder and 1000mL of distilled water, wherein the pH is natural, and the sterilization is carried out at 121 ℃ for 20 min.
According to aseptic operation, using sterile water to prepare bacterial fertilizer agents, stenotrophomonas, achromobacter, bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum, eupenicillium dermatum and myceliophthora thermophila amplification culture solutions into bacterial suspension/spore solutions with consistent concentrations, respectively taking 5mL of the bacterial suspension/spore solutions, inoculating the bacterial suspension/spore solutions into a culture medium which takes straws and filter paper as substrates, carrying out shaking culture on a shaker at 30 ℃ and 150r/min, sampling every 48 hours, adding a proper amount of (5-10 mL) of mixed solution of sulfuric acid and sodium nitrate to remove thalli and insoluble calcium carbonate, sieving the mixed solution through a 80-mesh sieve, gently washing the residual substrates on the sieve with distilled water, drying the mixture in an 80- ℃ oven to constant weight, weighing, calculating the weight loss rate, and enabling 3 groups to be parallel.
The measurement results are shown in table 1, fig. 1 and fig. 2.
TABLE 1 results of enzyme activity measurement
As shown in Table 1 and figures 1 and 2, the enzyme activity of the bacterial manure agent is basically higher than that of a single bacterial strain in the combination after fermentation for 10 days under the same inoculation amount and culture conditions; the filter paper weight loss rate of the bacterial manure agent is 28.0 percent, which is 10.6 percent higher than that of the single bacterial strain myceliophthora thermophila with the best degradation effect on the filter paper in the combination; the weight loss rate of the corn straws reaches 38.85 percent, the weight loss rate is improved by 2.25-4 times compared with that of a single bacterial strain in the combination, and the bacterial fertilizer prepared by combining multiple strains has the advantages of fusing multiple metabolic pathways of different bacterial strains and complementation on different degrading enzyme systems, realizing rapid degradation of the substrate, and showing that the degradation capability of the cellulosic substrate is enhanced after the bacterial strains are combined.
Example 2
Liquid fermentation degradation comparative tests are carried out on the liquid bacterial manure agent prepared by the invention and three commercially available organic material decomposition agents, in the tests, the substrate dosage, the microbial inoculum size, the culture conditions and the like are kept consistent, and 3 times of repetition are set.
Nutrient solution: 0.5% peptone, 0.5% NaCl, 0.2% CaC030.1% yeast powder, natural pH
The test method comprises the following steps: weighing 1g of substrate, adding the substrate into triangular flasks filled with 45mL of nutrient solution, sterilizing at 121 ℃ for 20min, diluting the prepared bacterial fertilizer agent and a commercially available organic material decomposition agent with sterile water, adjusting the diluted bacterial fertilizer agent and the commercially available organic material decomposition agent into suspensions with consistent concentrations, inoculating 5mL of the bacterial fertilizer agent and the commercially available organic material decomposition agent into the suspensions, performing shaking culture at 30 ℃ and 150r/min for 10d, sampling, adding a proper amount of sulfuric acid-sodium nitrate mixed solution into the samples, washing to remove attached bacteria and calcium carbonate, cleaning with distilled water, filtering, and drying in an oven at 70 ℃ to constant weight. The weight loss of the straw was measured by the mass difference method, and the results are shown in Table 2.
TABLE 2 substrate weight loss ratio
Through test comparison, the degradation capability of the bacterial fertilizer agent prepared by the invention on four cellulosic substrates is higher than that of a commercial microbial inoculum Dierle and a decomposition agent 150, the degradation capability on corn straws is the best among different cellulosic substrates, the degradation rate of the corn straws is improved by 35.0% compared with that of a microorganism, and the degradation rate of the corn straws is respectively improved by 66.3% and 76.8% compared with that of the Dierle and the decomposition agent 150.
Example 3
The liquid bacterial manure is applied to rapid and efficient decomposition of rice, wheat and corn straws, the preferable field application temperature range is 5-25 ℃, and in addition, a nitrogen source does not need to be additionally supplemented when the liquid bacterial manure is applied. The application method of the liquid bacterial manure is to spray the liquid bacterial manure on the surface of the straw and perform heap fermentation. The fermentation method adopts conventional fermentation means and devices, and has the advantages of simple operation, convenient use and easy popularization and use.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the examples, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (10)
1. The efficient decomposition liquid bacterial fertilizer is characterized by comprising three efficient decomposition bacteria capable of secreting cellulase and xylanase: stenotrophomonas sp.WS6-1, Achromobacter sp.WS6-2 and Pediococcus pentosaceus YC2, and the effective viable count in the bacterial manure is more than or equal to 1 × 109 cfu/mL;
the Stenotrophomonas (Stenotrophoromonas sp.WS6-1) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation number is as follows: CCTCC No. 20191060;
the Achromobacter sp.WS6-2 strain is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation number is as follows: CCTCC No. 20191061;
the Pediococcus pentosaceus (Pediococcus pentosaceus YC 2) is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 12 months and 18 days, and the preservation numbers are as follows: CCTCC No. 20191062.
2. The efficient decomposition liquid bacterial fertilizer of claim 1, wherein the bacterial fertilizer comprises eupenicillium crustaceum, wherein the eupenicillium crustaceum is purchased from institute of biotechnology, north beijing, and has a resource number of BNCC 146720.
3. The efficient decomposed liquid bacterial fertilizer of claim 1, wherein the bacterial fertilizer further comprises one or more of bacillus subtilis, streptomyces roseoflavus, trichoderma harzianum and myceliophthora thermophila.
4. The efficient decomposed liquid bacterial fertilizer of claim 3, wherein the bacillus subtilis, the streptomyces roseoflavus, the trichoderma harzianum and the myceliophthora thermophila in the bacterial fertilizer agent are purchased from the institute of biotechnology, north beijing, and have the resource number of BNCC188080, the resource number of the streptomyces roseoflavus of BNCC228869, the resource number of the trichoderma harzianum of BNCC336568 and the resource number of the myceliophthora thermophila of BNCC 186098.
5. The preparation method of the high-efficiency decomposed liquid bacterial fertilizer is characterized by comprising the following steps:
step 1, strain activation
Taking a proper amount of pure culture from a slant seed-preserving culture medium according to aseptic operation, and inoculating stenotrophomonas and achromobacter to an LB solid plate; the inoculated flat plate is inverted and horizontally placed in an incubator at the temperature of 26-30 ℃ for culture, and when a single bacterial colony grows out, spores produced by fungi are stable and uniform in size;
step 2, seed culture
Under the aseptic condition, picking single colonies of the activated stenotrophomonas and achromobacter in the step 1 by using inoculating loops, and respectively inoculating the single colonies to an LB liquid culture medium; after inoculation, carrying out shaking culture on each strain at 26-30 ℃ and 150r/min by using a shaking table, and determining the culture time according to the growth curve of each strain to obtain strain seed liquid at the middle and later logarithmic growth stages;
step 3, expanded culture
Transferring the seed solution obtained in the step (2) into a liquid culture medium of a corresponding type according to the inoculation amount of 10-20% (V/V), performing propagation expansion, adjusting the inoculation time and the culture time according to the growth rate of each strain of bacteria so as to obtain a bacterial solution with basically consistent concentration at the same time, and performing centrifugal concentration on the bacterial solution subjected to propagation expansion still according to aseptic operation;
step 4, mixed fermentation
And (3) centrifugally concentrating and collecting the bacteria/spores of the strain in the step (3), preparing the bacteria/spores into heavy suspension with consistent concentration by using sterile water, mixing according to a ratio of 1:1, inoculating the mixed bacteria liquid into a liquid fermentation culture medium, and naturally fermenting for about 10 days at room temperature to obtain the liquid bacterial fertilizer with stable property and function and effective viable count of more than or equal to 1 x 109 cfu/mL.
6. The method of claim 5, wherein: the step 1, the strain activation also comprises
Taking a proper amount of pure culture from a slant seed-preserving culture medium according to aseptic operation, and inoculating bacillus subtilis to an LB solid plate; inoculating Trichoderma harzianum, Penicillium crusum and myceliophthora thermophila to a PDA solid plate; inoculating Streptomyces roseoflavus to a Gao's No. 1 solid plate; and (3) inoculating lactic acid bacteria to the basic solid culture medium, inverting the inoculated flat plate, horizontally placing the flat plate in an incubator at the temperature of 26-30 ℃ for culture, and allowing the bacteria to grow a single bacterial colony and the fungi to produce spores stably and uniformly.
7. The method for preparing the efficient decomposed liquid bacterial fertilizer as claimed in claim 6, wherein the seed culture of step 2 further comprises the steps of picking a single colony of the bacillus subtilis activated in step 1 by using an inoculating loop under aseptic conditions, and inoculating the single colony to an LB liquid culture medium; adding a small amount of sterile water into a trichoderma harzianum, eupenicillium crustosum and myceliophthora thermophila solid flat plate, gently scraping lawn by using an inoculating loop to wash off spores, filtering by using sterile cotton to remove hyphae, and inoculating to a PDA liquid culture medium; inoculating streptomyces roseoflavus to a Gao's No. 1 liquid culture medium containing glass beads; inoculating lactobacillus to MRS liquid culture medium; and (3) after inoculation of each strain, carrying out shaking culture at 26-30 ℃ and 150r/min in a shaking table, and determining the culture time according to the growth curve of each strain to obtain strain seed liquid in the middle and later logarithmic growth stages.
8. The method for preparing high efficiency decomposed liquid bacterial manure according to claim 7,
LB liquid medium: 10g of peptone, 10g of beef extract, 5g of sodium chloride, 1000mL of distilled water, and pH 6.5-7, and 20g of agar is added when a solid culture medium is prepared;
PDA culture medium: 200g of peeled potato cooking juice 1000ml, sucrose 20g, monopotassium phosphate 3g, magnesium sulfate 1.5g and vitamin B110 mg, wherein the pH value is natural; when preparing a solid culture medium, 20g of agar is added;
gao's No. 1 medium: 20g of soluble starch, 1g of potassium nitrate, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of sodium chloride, 0.01 g of ferrous sulfate, 1000mL of distilled water and pH 7.4-7.6, and 20g of agar is added when preparing a solid culture medium.
9. The method for preparing the efficient decomposition liquid bacterial fertilizer according to claim 7, wherein the liquid fermentation culture medium comprises: 0.5% tryptone, 0.5% NaCl, 0.2% CaC03, 0.1% yeast powder, 0.3% urea and 1000mL of corn straw leaching liquor, wherein the pH value is natural, and the corn straw leaching liquor is sterilized for 20min at 121 ℃.
10. The application of the liquid bacterial manure is characterized in that the liquid bacterial manure is applied to the rapid and efficient decomposition of corn straws, and a nitrogen source is not required to be additionally supplemented when the liquid bacterial manure is applied.
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| CN103031254A (en) * | 2013-01-07 | 2013-04-10 | 鹤壁市人元生物技术发展有限公司 | Efficient straw-decomposing inoculant and preparation method thereof |
| CN103194407A (en) * | 2013-03-21 | 2013-07-10 | 江西农业大学 | Straw-decomposition composite microbial preparation and preparation method thereof |
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| CN110283870A (en) * | 2019-06-21 | 2019-09-27 | 盐城工学院 | A kind of method of double bacterial strains mixed solid fermentation corn stover |
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| CN103194407A (en) * | 2013-03-21 | 2013-07-10 | 江西农业大学 | Straw-decomposition composite microbial preparation and preparation method thereof |
| CN106495761A (en) * | 2016-10-28 | 2017-03-15 | 临沂中磷生物科技有限公司 | A kind of straw decomposing inoculant |
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