CN112813008A - Preparation method and application of low-temperature efficient corn straw degrading microbial inoculum - Google Patents

Preparation method and application of low-temperature efficient corn straw degrading microbial inoculum Download PDF

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CN112813008A
CN112813008A CN202110217463.3A CN202110217463A CN112813008A CN 112813008 A CN112813008 A CN 112813008A CN 202110217463 A CN202110217463 A CN 202110217463A CN 112813008 A CN112813008 A CN 112813008A
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CN112813008B (en
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高聚林
张赛楠
于晓芳
韩升才
青格尔
胡树平
马达灵
屈佳伟
王志刚
孙继颖
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Inner Mongolia Agricultural University
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Abstract

The invention relates to the field of microorganisms, and particularly discloses a preparation method and application of a low-temperature high-efficiency corn straw degradation microbial inoculum, wherein the microbial inoculum is prepared by combining 2 single strains of bacteria, the 2 single strains of bacteria are respectively Pseudomonas (Pseudomonas sp) and Acinetobacter (Acinetobacter sp), the preservation numbers are respectively CGMCC No.20521 and CGMCC No.20522, the invention takes a specifically-composed strain as a strain source sample, uses a culture medium adapted to the strain source sample for fermentation and propagation, uses a bacterial solution fermented under the optimal fermentation condition to be matched with an appropriate carrier and a protective agent, the powdery microbial inoculum is prepared by a freeze drying technology, can degrade the corn straws under the low temperature condition, has simple preparation method, lower cost and short production period, can solve the problem of inconvenient farming production caused by the incapability of degrading the straws under the low temperature condition after returning the straws to the field in the north.

Description

Preparation method and application of low-temperature efficient corn straw degrading microbial inoculum
Technical Field
The invention relates to the field of preparation of microbial agents, in particular to a preparation method and application of a low-temperature high-efficiency corn straw degrading microbial agent.
Background
The straw returning is one of effective measures for improving the soil fertility, the field crop straws are mechanically crushed and then deeply turned in situ to return to the field, and the method has the advantages of reducing the fertilizer using amount, increasing organic matters, improving the soil structure, reducing the environmental pollution and the like. However, the temperature of the harvested corns in the cold and dry areas in the north of China is quickly reduced to enter winter, the returned straws cannot be fully degraded due to the low temperature in winter, the seeding quality, the emergence rate, the farming operation and other problems in the spring of the next year are affected, and the popularization of the technology is seriously affected.
Corn is used as the first large grain crop in China, and spring corn in the north is the largest production area of corn in China, and the in-situ returning of the straws is severely limited by low temperature. In order to improve the straw returning rate, researchers adopt various ways to treat corn straws, wherein the way of degrading the straws by microorganisms is generally accepted. However, the growth process of the microorganism is greatly influenced by the environment, most of the screened and found straw-degrading microorganisms only have the capability of partially degrading the straw under the conditions of normal temperature or high temperature, and are not suitable for being used in the low-temperature environment in winter in the north. Therefore, the development of a microbial inoculum capable of efficiently degrading the corn straws under the low-temperature condition becomes an urgent problem to be solved, and the method has important significance for the popularization of the straw returning technology.
Disclosure of Invention
The invention aims to provide a preparation method and application of a low-temperature efficient corn straw degrading microbial inoculum, so as to realize the seasonal efficient degradation of in-situ returning corn straws in northern cold regions.
In order to realize the purpose of the invention, the main components of the low-temperature high-efficiency degradation microbial inoculum of the corn straws comprise 2 single bacterial strains: pseudomonas and Acinetobacter, wherein the bacillus strain is selected from the group consisting of Pseudomonas bacteria,
the pseudomonas is preserved in China general biological center of culture Collection of microorganisms, and the address of the preservation unit is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: pseudomonas sp, with the preservation number of CGMCC No. 20521;
the acinetobacter is preserved in China general biological center of culture Collection of microorganisms, and the preservation unit address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: acinetobacter Acinetobacter sp, with the preservation number of CGMCC No. 20522.
In the microbial inoculum, 2 strains of bacteria are obtained by separating soil of scientific and technological park of professional technical institute of inner Mongolia university;
the microbial inoculum is prepared by the following steps:
activating strains: several of the above individual bacterial strains stored in a refrigerator at-80 ℃ were taken out, inoculated on LB agar medium (tryptone 10g, yeast extract 5g, sodium chloride 10 g), and subjected to inverted culture in an incubator at 28 ℃ for 30 hours.
The seed liquid is prepared byThe activated strains are respectively inoculated into 2 conical flasks (the volume of the conical flask is 150ml, the liquid LB culture medium is 50 ml) filled with liquid seed culture medium, and the conical flasks are placed in a shaking table for cultivation (the parameters of the shaking table are set as the temperature is 28 ℃ and the rotating speed is 150 r/min). Until the appearance of each bacterial liquid is turbid and thorough, and the growth curve (OD) of the strain600nmValue) is in logarithmic growth phase and the number of viable bacteria is the maximum, the seed liquid is used.
The invention also aims to provide a preparation method of the microbial agent for degrading the corn straws, so as to obtain the microbial agent capable of efficiently degrading the corn straws under the low-temperature condition:
the technical problem to be solved by the invention is realized by adopting the following method scheme: respectively activating 2 single bacteria, then shaking the activated single bacteria to prepare seed liquid, inoculating the seed liquid into a fermentation tank for mixed fermentation at the optimal bacterial ages (the optimal bacterial ages of the 2 single bacteria are respectively 4h and 8 h), mixing the obtained bacterial liquid with a proper amount of carrier and protective agent by utilizing the optimal fermentation conditions, and freeze-drying the obtained bacterial liquid in a freeze dryer to obtain the powdery microbial inoculum.
Preferably, in the preparation method of the microbial inoculum, fermentation conditions of a fermentation tank are as follows: the total liquid loading of the 5L fermentation tank is 4L (400 ml bacterial liquid + 3600ml LB culture medium); the total inoculation amount of 2 single bacteria is 8-10%% (200 ml each, 400ml total) of the liquid loading amount; the fermentation temperature is 28 ℃; the initial pH value of the bacterial liquid is a natural value; the ventilation volume is 1L/min; the stirring speed is 300 r/min; fermenting and culturing for 28 h.
Preferably, the carrier is corn straw powder (screened by a 60-mesh sieve) 0.15 g/ml.
Preferably, the protective agent is 0.15g/ml of starch.
Preferably, when the microbial agent of the corn straws is applied, 0.02g of ammonium phosphate and 0.01g of urea are applied to each gram of the microbial agent.
Preferably, in the method for preparing the microbial inoculum, the ratio of the bacterial liquid to the filler (the carrier and the protective agent are collectively referred to as the filler) is 10:3 (ml/g).
Preferably, in the preparation method of the microbial inoculum, the ratio of the carrier to the protective agent is 1:1 (g/g).
Preferably, in the preparation method of the microbial inoculum, the filler needs to be sterilized at high temperature (110-.
Further, in the preparation method of the microbial inoculum, the microbial inoculum is obtained by the following freeze drying steps:
step 1: and (6) pre-freezing. The pre-freezing temperature is-40 deg.C, the temperature changing time is 5min, and the duration time is 180 min.
Step 2: and (5) sublimation and drying. A first stage of sublimation drying: the temperature is-10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.18 mbar; a second stage of sublimation drying: the temperature is 0 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.20 mbar; a third stage of sublimation drying: the temperature is 10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.21 mbar; a fourth stage of sublimation drying: the temperature is 40 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.22 mbar.
And step 3: and (5) resolving and drying. The temperature was 40 ℃, duration 180min and vacuum 0.22 mbar.
Preferably, the freeze dryer charge is 3100 ± 20 g.
The invention also aims to provide the application of the microbial agent in the degradation of corn straws in a field.
In the field application of the microbial agent, the corn straws are cut into corn straws with the length of 5 +/-0.5 cm.
In field application of the microbial agent, every 30g of straws are subpackaged in gauze bags with the aperture of 5 mm and buried underground.
In the field application of the microbial agent, the buried depth of the straw is 25 +/-5 cm.
The invention has the beneficial effects that: according to the preparation method of the corn straw low-temperature high-efficiency degradation microbial inoculum, the bacteria with specific components are taken as a bacteria source sample, the culture medium adapted to the bacteria source sample is used for mixed fermentation, the bacteria liquid obtained under the optimal fermentation condition is matched with a proper and proper amount of carrier and protective agent, and the bacteria liquid is placed in a freeze dryer for freeze drying to prepare the microbial inoculum. The method can efficiently degrade the corn straws under the low-temperature condition, is simple and convenient, has lower cost, provides technical support for preparation of microbial inoculum capable of degrading the corn straws under the low-temperature condition, and has important significance for popularization of the straw returning technology in the north.
The microbial agent disclosed by the invention is used for degrading the corn straws in a field, the degradation time is 10 and 5 days in 2019 and 11 and 15 days in 2019, the total degradation time is 40 days, the degradation rate reaches 32.3%, and the percentage is improved by 14.42% compared with that of a blank control.
Drawings
FIG. 1 shows the OD values and viable cell counts of the pseudomonads of the present invention at different fermentation times.
FIG. 2 shows the OD values and viable cell counts of Acinetobacter of the present invention at different fermentation times.
FIG. 3 shows the pH change of the fermentation in the bacterial fermentation tank of the present invention.
FIG. 4 is a graph showing the DO value change in the fermentation in the bacterial fermentor of the present invention.
FIG. 5 is a graph showing the change in OD value and viable cell count in the fermentation in the bacterial fermentation tank of the present invention.
FIG. 6 is a graph of the degradation rate of corn stover under various treatment conditions according to the present invention.
FIG. 7 is a field blank map of the present invention.
FIG. 8 is a diagram of the application of the microbial inoculum of the field corn stover of the invention.
FIG. 9 is a diagram of a commercial decomposition agent applied to field corn stover in accordance with the present invention.
Detailed Description
The invention discloses a preparation method and application of a low-temperature high-efficiency corn straw degrading microbial inoculum. In order to make the above-mentioned technical features, objects and advantages of the present invention more apparent, the present invention will be described in more detail and fully hereinafter. Where specific operations or conditions are not indicated in the description, they are carried out in accordance with conventional operations or conditions; the reagents or instruments used are not specified in detail and are all available from commercially available conventional sources. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The invention is further illustrated by the following examples.
Example 1: fermentation of the bacterial liquid of the invention
(1) The source and species of the strain are as follows:
the 2 bacterial strains are separated from the soil of the scientific park of the professional technical institute of inner Mongolia university of agriculture;
the 2 single bacterial strains were: pseudomonas and Acinetobacter, wherein the bacillus strain is selected from the group consisting of Pseudomonas bacteria,
the pseudomonas is preserved in China general biological center of culture Collection of microorganisms, and the address of the preservation unit is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: pseudomonas sp, with the preservation number of CGMCC No. 20521;
the acinetobacter is preserved in China general biological center of culture Collection of microorganisms, and the preservation unit address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: acinetobacter Acinetobacter sp, with the preservation number of CGMCC No. 20522.
(2) The formula of the culture medium is as follows:
LB liquid medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1L of distilled water;
LB agar medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 1L of distilled water and 15g of agar.
(3) Activating strains:
2 individual bacterial strains stored in a refrigerator at-80 ℃ are taken out, respectively inoculated in an LB agar culture medium and inversely cultured in an incubator at 28 ℃ for 30 hours.
(4) Preparing a seed solution:
under aseptic conditions, the activated strains are respectively inoculated into 2 conical flasks (the volume of the conical flask is 150ml, and the volume of the conical flask is 50 ml) filled with LB liquid culture medium, and the conical flasks are placed in a shaking table for culture. The parameters of the shaking table are set as follows: the temperature is 28 ℃, and the rotating speed is 150 r/min. Until the appearance of each bacterial liquid is turbid and thorough, and the growth curve (OD) of the strain600nmValue) is in logarithmic growth phase and the number of viable bacteria is the maximum, the seed liquid is used.
The results of the age studies for 2 species are shown in the figure (FIGS. 1 and 2), and pseudomonadsStrain growth curves (OD) of bacteria and acinetobacter in shake flask fermentation for 10h and 12h600nmValue) is in logarithmic growth phase and the number of viable bacteria is the largest, namely the two are respectively 10h and 12h in optimal bacterial age, and the seed liquid can be used as the seed liquid after being respectively fermented for 10h and 12 h.
(5) Fermenting bacterial liquid:
placing the whole fermentation liquid containing LB liquid culture medium in a high-temperature sterilization pot for sterilization (temperature: 121 ℃, time: 20 min), cooling, opening an inoculation port under the protection of flame, and putting the cultured seed liquid into the fermentation pot for fermentation according to the proportion that the total inoculation amount of 2 single bacteria is 10% of the liquid containing amount (200 ml of each single bacteria, 400ml of the total). The fermentation conditions were: the volume of the fermentation tank is 5L; the total liquid loading is 4L (400 ml bacterial liquid + 3600ml LB liquid culture medium); the fermentation temperature is 28 ℃; the initial pH value of the bacterial liquid is a natural value; the ventilation volume is 1L/min; the stirring speed was 300 r/min.
The bacteria liquid fermentation is carried out under the above fermentation conditions, and the metabolic change rule of the bacteria in the fermentation tank can be known from fig. 3, namely the pH value of the bacteria liquid is rapidly reduced and then slowly increased in the fermentation process, when the fermentation is started to 4 hours, the pH value is rapidly reduced from the initial value of 6.184 to the minimum value of 5.998, and the analysis is that the pH value is reduced by a large amount of intermediate products such as organic acid generated in the early growth process of the bacteria. Then the pH value gradually rises back to 8.500 after 48 h. The pH rise is mainly caused by that acidic intermediate products generated in the early stage of fermentation are metabolized, and thalli start to be autolyzed in a large amount in the later stage of fermentation to release amino nitrogen substances.
FIG. 4 shows the variation trend of dissolved oxygen level (DO value) during fermentation of bacterial liquid. As can be seen from the figure, the dissolved oxygen is rapidly reduced within 0-4h, and the thallus has extremely strong growth and reproduction capability and high oxygen consumption at the stage; the dissolved oxygen slowly decreases within 5-31h and is at a lower level; in the later stage of fermentation for 32-48h, a large amount of thalli are autolyzed, the respiration effect is weakened, and the dissolved oxygen rises rapidly.
As can be seen from FIG. 5, the bacterial liquid concentration (OD) in 20h600nmValue) and the number of viable bacteria gradually increase, and the increasing speed of the number of viable bacteria is relatively slow; after 20h, the viable count of the bacteria liquid is increased rapidly until the maximum viable count reaches 1.45 multiplied by 10 after 28h10CFU/ml, then gradually decreased. The thallus is autolyzed in a large amount for 48 hours, and the number of viable bacteria is reduced. The fermentation time of the bacterial suspension in a 5L fermentation tank was determined to be 28 hours from the results of the fermentation in accordance with the objective of the highest viable cell count with the higher concentration of the bacterial suspension.
Example 2: preparation of microbial inoculum
(1) Test materials
Strain: same as example 1
Carrier: corn stalk powder (60 mesh sieve)
A protective agent: starch
(2) Preparation method of microbial inoculum
Weighing 7.5g of the carrier (corn husk straw powder) and the protective agent (starch) respectively, mixing in an aluminum box culture dish with the diameter of 12cm and the height of 2cm, covering, fixing by a rubber band, placing in a high-temperature sterilization pot (the temperature is 121 ℃, and the time is 20 min), and sterilizing for later use.
After the fermentation of the bacterial liquid is finished, the number of viable bacteria reaches 1.45 multiplied by 1010. And (3) subpackaging the fermented bacterial liquid into the aluminum box culture dish filled with the sterilized fillers every 50ml under the aseptic condition, so that the ratio of the bacterial liquid to the fillers (the carrier and the protective agent are collectively called as the fillers) is 10:3 (ml/g), and the ratio of the carrier to the protective agent is 1:1 (g/g). And then completely mixing the bacterial liquid and the filler, and putting the mixture into a freeze dryer for freeze-drying treatment.
The contained materials are obtained in a freeze dryer through the following steps:
step 1: and (6) pre-freezing. The pre-freezing temperature is-40 deg.C, the temperature changing time is 5min, and the duration time is 180 min.
Step 2: and (5) sublimation and drying. A first stage of sublimation drying: the temperature is-10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.18 mbar; a second stage of sublimation drying: the temperature is-0 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.20 mbar; a third stage of sublimation drying: the temperature is 10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.21 mbar; a fourth stage of sublimation drying: the temperature is 40 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.22 mbar.
And step 3: and (5) resolving and drying. The temperature was 40 ℃, the duration 180min and the vacuum 0 mbar.
The viable count of the obtained microbial inoculum after freeze drying is 1.27 multiplied by 1010The survival rate of the live bacteria is 87.59%. (live bacterium survival rate = live bacterium count B/live bacterium count A × 100% where A is the number of live bacteria before freeze-drying and B is the number of live bacteria after freeze-drying)
Example 3: research on field application effect of microbial inoculum
(1) Preparing corn straws:
taking the harvested straws of the corns. The straws are cut into blocks with the length of about 5cm, the completeness of the inner pulp and the outer skin of the straws is ensured, and then every 30g of the straws are subpackaged in red gauze bags with the aperture of 5 mm and are ready to be buried underground.
(2) The implementation mode is as follows:
the method comprises the steps of setting blank control (figure 7), corn straw and the microbial inoculum of the invention (figure 8) and corn straw and the commodity decomposition agent (figure 9) for 3 treatments, and repeating the treatment setting for 5 times. In the implementation process, the external conditions of the first step and the second step are ensured to be consistent, the application amount ratio of the straws to the microbial inoculum is 8001, and 0.6g of ammonium phosphate and 0.3g of urea are applied when each bag of straws (30 g) is applied with the microbial inoculum.
Burying the ground depth of straw: 25 + -5 cm.
Straw burying time: the total time is 40 days from 10 months and 5 days in 2019 to 11 months and 15 days in 2019.
The results are shown in FIG. 9. The degradation rates of the corn straws processed firstly, secondly and thirdly are respectively 17.88%, 32.30% and 24.54%, and the degradation rates of the corn straws and the microbial inoculum of the invention are respectively improved by 14.22% and 7.76% compared with the degradation rates of blank control and the degradation rates of applied commodity decomposition agents, and reach remarkable levels. The microbial inoculum has a certain effect on the degradation of the corn straws.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method of a low-temperature high-efficiency corn straw degradation microbial inoculum is characterized by comprising two bacteria of pseudomonas and acinetobacter;
the pseudomonas is preserved in China general biological center of culture Collection of microorganisms, and the address of the preservation unit is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: pseudomonas sp, with the preservation number of CGMCC No. 20521;
the acinetobacter is preserved in China general biological center of culture Collection of microorganisms, and the preservation unit address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 11/08/2020, under the classification name: acinetobacter Acinetobacter sp, with the preservation number of CGMCC No. 20522;
the preparation method comprises the following steps:
step 1: activating strains: taking out a plurality of individual bacterial strains stored at the temperature of minus 80 ℃, respectively inoculating the bacterial strains to an LB agar culture medium, and inversely culturing the bacterial strains in an incubator at 28 ℃ for 30 hours;
step 2: preparing a seed solution: under the aseptic condition, respectively inoculating each activated strain into 2 conical flasks filled with liquid seed culture medium, and culturing in a shaking table; the parameters of the shaking table are set as follows: the temperature is 28 ℃, and the rotating speed is 150 r/min; when the appearance of each bacterial liquid is turbid and thorough, the growth curve of the strain is in a logarithmic phase and the number of viable bacteria is the maximum, the bacterial liquid is used as seed liquid;
and step 3: fermenting; placing the fermentation liquid containing LB liquid culture medium in a sterilization pot with temperature of 110-; the fermentation conditions were: the volume of the fermentation tank is 5L, the liquid loading amount is 4L, the total inoculation amount of 2 single bacteria is 8-10% of the liquid loading amount, the temperature is 28 ℃, the initial pH value of the bacteria liquid is 6.85 +/-0.05, the ventilation amount is 1L/min, the stirring speed is 300r/min, and the fermentation culture is carried out for 28 h;
and 4, step 4: a formulation; and (3) adding a proper amount of sterilized carrier and protective agent into the fermented bacterial liquid, and freeze-drying in a freeze dryer to prepare the powdery microbial inoculum.
2. The preparation method of the microbial inoculum according to claim 1, wherein the microbial inoculum carrier material is corn husk straw powder, and the protective agent material is starch.
3. The method for preparing a bacterial agent according to claim 1, wherein the ratio of the bacterial liquid to the carrier to the protective agent is 10:3, and the unit is ml/g.
4. The method for preparing the microbial inoculum according to claim 1, wherein the ratio of the carrier to the protective agent is 1:1, and the unit is g/g.
5. The method for preparing the microbial inoculum according to claim 1, wherein the freeze-drying operation mode of the microbial inoculum in the step 4 is as follows:
step 1: pre-freezing; pre-freezing at-40 deg.C in a freeze dryer for 5min and 180 min;
step 2: sublimation drying; a first stage of sublimation drying: the temperature is-10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.18 mbar; a second stage of sublimation drying: the temperature is 0 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.20 mbar; a third stage of sublimation drying: the temperature is 10 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.21 mbar; a fourth stage of sublimation drying: the temperature is 40 deg.C, the temperature changing time is 5min, the duration time is 30min, and the vacuum degree is 0.22 mbar;
and step 3: resolving and drying; the temperature was 40 ℃, the duration 180min and the vacuum 0 mbar.
6. The method for preparing a microbial preparation according to claim 5, wherein the charge amount of the freeze dryer is 3100 ± 20 g.
7. The application of the low-temperature high-efficiency corn straw degrading microbial inoculum is characterized in that: the microbial inoculum is spread in the corn straws.
8. The application of the low-temperature high-efficiency corn straw degradation microbial inoculum according to claim 7, wherein the ratio of the corn straw to the microbial inoculum is 800: 1.
9. The application of the low-temperature high-efficiency corn straw degrading microbial inoculum according to claim 7, wherein 0.02g of ammonium phosphate and 0.01g of urea are required to be applied to each gram of straw.
10. The application of the low-temperature high-efficiency corn straw degrading microbial inoculum according to claim 7, wherein the buried depth of the straws is 25 +/-5 cm.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886042A (en) * 2010-06-29 2010-11-17 中国农业科学院农业资源与农业区划研究所 Method for constructing composite bacteria system for decaying maize straws
WO2014040395A1 (en) * 2012-09-17 2014-03-20 北京天安生物科技有限公司 Complex microbial flora and application thereof
CN105967943A (en) * 2016-05-05 2016-09-28 山东省农业科学院农产品研究所 Bio-fertilizer for efficient degradation of atrazine and preparation method thereof
CN110004082A (en) * 2019-04-01 2019-07-12 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) A kind of bacterium bacterial strain and application suitable for the regulation of industrialized agriculture nitrogen and phosphorus pollution
CN112080450A (en) * 2020-09-30 2020-12-15 内蒙古农业大学 Straw degrading bacteria and separation screening method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101886042A (en) * 2010-06-29 2010-11-17 中国农业科学院农业资源与农业区划研究所 Method for constructing composite bacteria system for decaying maize straws
WO2014040395A1 (en) * 2012-09-17 2014-03-20 北京天安生物科技有限公司 Complex microbial flora and application thereof
CN105967943A (en) * 2016-05-05 2016-09-28 山东省农业科学院农产品研究所 Bio-fertilizer for efficient degradation of atrazine and preparation method thereof
CN110004082A (en) * 2019-04-01 2019-07-12 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) A kind of bacterium bacterial strain and application suitable for the regulation of industrialized agriculture nitrogen and phosphorus pollution
CN112080450A (en) * 2020-09-30 2020-12-15 内蒙古农业大学 Straw degrading bacteria and separation screening method

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
SHENGCAI HAN等: "Construction of bacterial consortium for cornstalk degradation in soil", 《HTTPS://WWW.SSRN.COM/》 *
XIN ZHANG等: "Microbial Community Succession is Associated With Corn Straw Degradation in Microbial Consortium M44 During Subculture", 《RESEARCH SQUARE》 *
于加瑞等: "改性秸秆吸附微生物对土壤中石油烃降解及演化特征的影响", 《环境工程学报》 *
刘鑫阳: "添加不同菌剂对玉米秸秆微贮菌群及品质的影响", 《中国优秀博硕士学位论文全文数据库(硕士)工程科技Ⅰ辑》 *
尹萌等: "生物质废弃物发酵过程中菌群多样性及秸秆降解菌的筛选", 《江苏农业学报》 *
张必周等: "低温条件下玉米秸秆降解菌复配效果及降解特性研究", 《北方农业学报》 *
张必周等: "玉米秸秆低温降解菌的分离与鉴定及复配菌降解效果研究", 《玉米科学》 *
张鑫等: "玉米秸秆低温降解复合菌的筛选及其菌种组成", 《农业环境科学学报》 *
童乐等: "以农作物秸秆固定化混合菌去除原油", 《环境工程学报》 *
陶莲等: "青贮发酵对玉米秸秆品质及菌群构成的影响", 《动物营养学报》 *
韩梦颖等: "降解秸秆微生物及秸秆腐熟剂的研究进展", 《南方农业学报》 *

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