CN112813008B - Preparation method and application of low-temperature high-efficiency corn straw degrading microbial inoculum - Google Patents
Preparation method and application of low-temperature high-efficiency corn straw degrading microbial inoculum Download PDFInfo
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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.
Description
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 modes to treat the corn straws, wherein the mode 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 low-temperature high-efficiency degradation microbial inoculum for the corn straws comprises the following main components of 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: year 2020, 11/08 month, category designation: 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 individual bacterial strains were taken out from the refrigerator at-80 ℃ and inoculated on LB agar medium (tryptone 10g, yeast extract 5g, sodium chloride 10 g), respectively, and cultured in an inverted state in an incubator at 28 ℃ for 30 hours.
The preparation method of the seed liquid is carried out under aseptic conditions, and each activated strain is 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 placed into a shaking table for culture (the parameters of the shaking table are set as the temperature of 28 ℃ and the rotating speed of 150 r/min). Until the appearance of each bacterial liquid is turbid and thorough, and the growth curve (OD) of the strain 600nm Value) is in the para-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 the 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 optimum age (the optimum age of the 2 single bacteria is 4h and 8h respectively), mixing the seed liquid with a proper amount of carrier and protective agent by using the optimal fermentation condition, and freeze-drying the mixture 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% of the liquid loading amount (200 ml of each single bacteria, 400ml of the total 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 300r/min; fermenting and culturing for 28h.
Preferably, the carrier is corn straw powder (screened by a 60-mesh sieve) 0.15g/ml.
Preferably, the protective agent is 0.15g/ml 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.
Preferably, in the method for preparing the microbial inoculum, the ratio of the carrier to the protective agent is 1.
Preferably, in the preparation method of the microbial inoculum, the filler is sterilized at high temperature (110-130 ℃) for 18-21min, and then the filler and the bacterial liquid are compatible and uniformly mixed under aseptic conditions and then are placed in a freeze dryer.
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 180min.
Step 2: and (5) 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 0.18mbar; 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 0.20mbar; 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 0.21mbar; a fourth stage of sublimation drying: the temperature is 40 deg.C, the temperature changing time is 5min, the duration is 30min, and the vacuum degree is 0.22mbar.
And step 3: and (4) resolving and drying. The temperature was 40 ℃, duration 180min and vacuum 0.22mbar.
Preferably, the freeze dryer charge is 3100 ± 20g.
The invention also aims to provide the application of the microbial agent in the degradation of the corn straws in the 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 efficient degradation microbial inoculum, the bacteria with specific components are taken as a bacteria source sample, a culture medium adapted to the bacteria source sample is used for mixed fermentation, the bacteria liquid obtained under the optimal fermentation conditions is matched with a proper and appropriate amount of carriers and protective agents, and the bacteria liquid is placed in a freeze dryer for freeze-drying to obtain 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 preparing the microbial inoculum capable of degrading the corn straws under the low-temperature condition, and has important significance for the 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 between 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 degradation rate is improved by 14.42% compared with 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 liquid LB medium is 50 ml) filled with LB liquid 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 strain 600nm Value) is in the para-growth phase and the number of viable bacteria is the maximum, the seed liquid is used.
The results of the age studies for 2 strains are shown in the figure (FIGS. 1 and 2), and the strain growth curves (OD) of Pseudomonas and Acinetobacter are shown in the figure when they are fermented in shake flasks for 10h and 12h 600nm Value) 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) Bacterial liquid fermentation:
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 flame protection, and putting the cultured seed liquid into a fermentation tank 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 single bacteria). 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 300r/min.
The bacteria liquid fermentation is carried out under the fermentation conditions, and the metabolic change rule of the bacteria in the fermentation tank can be known from figure 3, namely the pH value of the bacteria liquid is rapidly reduced and then slowly increased in the fermentation process, the pH value is rapidly reduced from an initial value of 6.184 to a minimum value of 5.998 from the beginning of the fermentation to 4 hours, and the analysis can be that the pH value is reduced by a large amount of intermediate products such as organic acid and the like 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 acid intermediate products generated in the early stage of fermentation are metabolized, and thalli start to carry out autolysis in the later stage of fermentation to release amino nitrogen substances.
FIG. 4 is the change 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 20h 600nm Value) and the number of viable bacteria gradually rise, 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 28h 10 CFU/ml, then gradually decreased. The bacterial cells are autolyzed in a large amount within 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)
And (3) a protective agent: starch
(2) Preparation method of microbial inoculum
Weighing 7.5g of carrier (corn husk straw powder) and protectant (starch) respectively, mixing in aluminum box culture dish with diameter of 12cm and height of 2cm, covering, fixing with rubber band, and sterilizing in high temperature sterilizing pot (temperature: 121 deg.C, time: 20 min).
After the fermentation of the bacterial liquid is finished, the number of viable bacteria reaches 1.45 multiplied by 10 10 . And (3) subpackaging the fermented bacterium liquid into the aluminum box culture dish filled with the sterilized filler every 50ml under an aseptic condition, so that the ratio of the bacterium liquid to the filler (the carrier and the protective agent are collectively called as the filler) is 10 (ml/g), and the ratio of the carrier to the protective agent is 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 180min.
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 0.18mbar; 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 0.20mbar; 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 0.21mbar; 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 0.22mbar.
And step 3: and (5) resolving and drying. The temperature was 40 ℃, the duration 180min and the vacuum 0mbar.
The viable count of the obtained microbial inoculum after freeze drying is 1.27 multiplied by 10 10 The survival rate of the live bacteria is 87.59 percent. ( Bacterial survival rate = viable count B/viable count a × 100% in the formula: a is viable cell count before freeze-drying, and B is viable cell count 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:
3 treatments of (1) blank control (figure 7), (2) corn straw + microbial inoculum of the invention (figure 8) and (3) corn straw + commodity decomposing inoculant (figure 9) are set, and each treatment is set for 5 times. In the implementation process, the external conditions of the treatment (1) and the treatment (2) are 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. 6. The degradation rates of the corn straws treated in the steps (1), (2) and (3) 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 a blank control and the degradation rates of a commodity decomposition agent, and the degradation rates are all significant. 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 (7)
1. A preparation method of a corn straw degrading microbial inoculum is characterized by comprising the following steps:
step 1: activating strains: taking out two single bacterial strains which are preserved at the temperature of-80 ℃, respectively inoculating the two single bacterial strains into LB agar culture medium, and inversely culturing the two single bacterial strains in an incubator at 28 ℃ for 30 hours, wherein the two single bacterial strains are respectively Pseudomonas (Pseudomonas sp.) and Acinetobacter (Acinetobacter sp.);
the pseudomonas is preserved in China general biological center of culture Collection of microorganisms, and the address of the preservation unit is as follows: the preservation date of No. 3 Xilu Beijing Xiyan No. 1 Beijing, chaoyang district is: the preservation number is CGMCC No.20521 at 11 months 08 in 2020;
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: the preservation number is CGMCC No.20522 at 11 months 08 in 2020;
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; until the appearance of each bacterial liquid is turbid and thorough, the strain growth curve is in the logarithmic phase and the number of viable bacteria is the largest, the bacterial liquid is used as seed liquid;
and step 3: fermenting; placing the fermentation liquid-containing tank with LB liquid culture medium in a sterilization pot at 110-130 deg.C for sterilization for 18-21min, cooling, opening inoculating port under flame protection, and putting the cultured seed liquid into a fermentation tank for fermentation according to the ratio of total inoculum size of two single bacteria of 1:1; the fermentation conditions were: the volume of the fermentation tank is 5L, the liquid loading amount is 4L, the temperature is 28 ℃, the initial pH value of the bacterial liquid is 6.85 +/-0.05, the ventilation amount is 1L/min, the stirring speed is 300r/min, and the fermentation culture is 28h;
and 4, step 4: a formulation; mixing the fermented bacteria liquid with a sterilized carrier and a protective agent, wherein the carrier material is corn husk straw powder, and the protective agent material is starch; the ratio of the bacteria liquid to the carrier to the protective agent is 10; freeze-drying in a freeze dryer to prepare a powdery microbial inoculum;
the ratio of carrier to protective agent is 1.
2. The method for preparing the microbial inoculum according to claim 1, wherein the freeze-drying operation mode of the microbial inoculum is as follows:
step 1: pre-freezing; pre-freezing in a freeze drier at-40 deg.C for 5min and 180min;
and 2, step: sublimation drying; a first sublimation drying stage: the temperature is-10 deg.C, the temperature changing time is 5min, the duration is 30min, and the vacuum degree is 0.18mbar; 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 0.20mbar; 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 0.21mbar; a fourth stage of sublimation drying: the temperature is 40 deg.C, the temperature changing time is 5min, the duration is 30min, and the vacuum degree is 0.22mbar;
and step 3: resolving and drying; the temperature was 40 ℃, the duration 180min and the vacuum 0mbar.
3. The method for preparing a microbial preparation according to claim 1, wherein the charge amount of the freeze dryer is 3100 ± 20g.
4. The application of the corn straw degrading microbial inoculum prepared by the preparation method of claim 1 is characterized in that: the microbial inoculum is spread in corn straws.
5. The application of the corn straw degrading microbial inoculum according to claim 4, wherein the ratio of the corn straw to the microbial inoculum is 800.
6. The use of the corn stalk degradation microbial inoculum according to claim 4, wherein 0.02g ammonium phosphate and 0.01g urea are required to be applied per gram of stalk.
7. The application of the corn straw degrading microbial inoculum according to claim 4, wherein the straw burying depth is 25 +/-5 cm.
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| CN110004082B (en) * | 2019-04-01 | 2022-04-19 | 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) | Bacterial strain suitable for nitrogen and phosphorus pollution regulation and control in facility agriculture and application |
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