CN112852644B - For producing CO by fermenting crop straws2And uses thereof - Google Patents
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- 230000000593 degrading effect Effects 0.000 claims abstract description 14
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- 238000000855 fermentation Methods 0.000 abstract description 11
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- 244000005700 microbiome Species 0.000 abstract description 4
- 241000223259 Trichoderma Species 0.000 abstract 1
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- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- Engineering & Computer Science (AREA)
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- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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Abstract
The invention relates to the field of microorganisms, in particular to a method for producing CO by fermenting crop straws2The Trichoderma longibrachiatum strain YM32425 has a preservation number of CGMCC NO. 15092. The application is used for degrading crop straws. The Trichoderma strain YM32425 of the invention can degrade crop straws and produce CO2High yield and CO production2The characteristic of long period can solve the problem of CO generated in the middle and later periods of the straw fermentation of the facility greenhouse2And the problem of insufficiency.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to a method for producing CO by fermenting crop straws2Trichoderma longibrachiatum (Trichoderma longibrachiatum) strain YM 32425.
Background
The agricultural organic waste mainly comprises crop straws, livestock and poultry manure and the like. A great deal of CO is released by burning the crop straws2And heat, and cause soil organic matter to decrease. The straws are randomly discarded and decomposed by microorganisms to generate a large amount of CO2And CH4. Part of the livestock and poultry manure is discharged randomly, which causes serious pollution to the ecological environment and threatens the health of people and livestock, and is decomposed by microorganisms to release a large amount of CO2、CH4And N2And (4) O gas. In conclusion, the burning and discarding of crop straws and the random discharge of livestock and poultry manure are important agricultural carbon emission sources in China. The efficient recycling of agricultural organic wastes is an important way for reducing the emission of agricultural carbon in China. Meanwhile, facility cultivation in China develops rapidly, and is one of the countries with the largest facility cultivation area in the world. However, in the greenhouse, atmospheric CO2Blocked exchange, CO in the shed2The concentration is generally less than half of that of the outdoor environment, the photosynthesis and the carbon fixation are seriously influenced, the carbon and nitrogen metabolism is disordered, the diseases are serious, the pesticide residue is large, the nitrate content is high, and the yield and the quality of agricultural products are influenced.
CO fermentation by using agricultural organic waste2Fertilization is an effective measure for fixing carbon in plants and storing carbon in soil. The invention patent ZL200410024965.0 provides greenhouse CO fermentation by utilizing agricultural organic wastes2A method for fertilizing. However, the existing organic wastes are adopted to ferment CO2Fertilization technique, usually one-time feeding to maintain greenhouse CO2The concentration of more than 800 mul/L is only about 2 weeks, and the problem of insufficient gas production in the middle and later stages of fermentation occurs. The reason is mainly related to the fermentation inoculum. Thus, screening for the sustained degradation of agricultural organic wastes to produce CO2The strain solves the problem of CO generation in the middle and later stages of fermentation2And the problem of insufficiency.
The prior application of the trichoderma longibrachiatum is as follows:
2017104245158 Notification of preparation and application of Trichoderma longibrachiatum DQ2 wettable powder: the Trichoderma longibrachiatum DQ2 wettable powder can promote fertilizer absorption and crop growth, is non-toxic to human and livestock, and does not pollute the environment.
2017114446878 entitled "A Trichoderma longibrachiatum strain and its use", discloses: trichoderma longibrachiatum (Trichoderma longibrachiatum) TL1 with a preservation number of CCTCC NO. M2017184. The strain has strong inhibiting effect on pathogenic fungi of the clubroot of the hot pickled mustard tuber, and particularly has obvious preventing and treating effect on the clubroot of the Fuling hot pickled mustard tuber.
2012103019138 Notification of the invention, namely the reconstruction of saponin components in pseudo-ginseng by Trichoderma longibrachiatum: the method uses Trichoderma longibrachiatum Rifai to ferment the panax notoginseng, so that the composition and the content of the saponin in the panax notoginseng are obviously changed, and the reconstruction of the effective components of the panax notoginseng is realized.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for producing CO by fermenting and degrading crop straws with Trichoderma longibrachiatum strain YM324252The method of the invention is used for carrying out microbial degradation on crop straws to generate CO2Has the effect of producing CO2High yield and CO production2Long cycle.
In order to solve the technical problems, the invention provides Trichoderma longibrachiatum (Trichoderma longibrachiatum) YM32425, wherein the preservation number is CGMCC NO. 15092.
The invention also provides the application of the Trichoderma longibrachiatum YM 32425: for degrading crop straw to produce CO2。
The strain YM32425 of the invention has a preservation name of Trichoderma longibrachiatum, and the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.15092, preservation time 2017, 12 months and 15 days.
The trichoderma longibrachiatum strain YM32425 is trichoderma longibrachiatum separated from farmland soil, can be used for degrading crop straws and has CO production effect2High yield and CO production2The characteristic of long period can solve the problem of CO generated in the middle and later periods of the straw fermentation of the facility greenhouse2And the problem of insufficiency.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows that four degrading bacteria generate CO for straw and pig manure matrix2The influence of (a);
FIG. 2 shows that four degrading bacteria generate CO for corn straw and pig manure substrate2The influence of (c).
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
it is common knowledge that all media of the present invention need to be autoclaved at 121 ℃ before use.
Example 1 screening of microbial strains degrading cellulose and lignin
Sodium carboxymethyl cellulose culture medium: sodium carboxymethylcellulose 10g, (NH4)2SO4 4g、KH2PO4 2g、MgSO4·7H20.5g of O, 1.0g of peptone, 15g of agar and 0.5g of deoxysodium cholate, and water is added until the volume is 1000 mL.
Guaiacol potato dextrose agar medium: 200g of potato, 20g of glucose and 15g of agar, and adding water to 1000 mL. Sterilizing at 121 deg.C under high pressure, and adding 0.4g of filtered and sterilized guaiacol, wherein the content of guaiacol is 0.04%.
Rose potato dextrose agar medium: 200g of potato, 20g of glucose, 15g of agar, 0.1g of streptomycin sulfate, 0.3g of chloramphenicol, 0.02g of rose bengal and water till 1000 mL.
Separating from paddy field and vegetable field soil such as Hangzhou, Wenzhou, Quzhou and the like on a rose red potato glucose agar culture medium according to a conventional dilution plate method to obtain a plurality of test strains.
Screening cellulose degrading strains by adopting a sodium carboxymethylcellulose culture medium:
culturing the test strains on a potato glucose agar culture medium plate for 5d at 28 ℃, punching colony discs from the edges of the colonies by using a puncher, inoculating 1 colony disc to the center of a sodium carboxymethyl cellulose culture medium plate, and culturing for 4d at 28 ℃. Using 1 mg. mL-1Covering the culture medium plate with Congo red dye solution, standing for 30min, and adding 1 mol. L-1And (5) washing with NaCl for 1 hour, and judging whether the test strain has the capacity and strength of degrading cellulose or not through the size of the hydrolysis transparent ring.
Screening lignin degrading strains by adopting a guaiacol potato glucose agar culture medium:
culturing the test strains on a potato glucose agar culture medium plate for 5d at 28 ℃, punching colony discs from the edges of the colonies by using a puncher, inoculating 1 colony disc to the center of the guaiacol potato glucose agar culture medium plate, and culturing for 5d at 28 ℃. And judging whether the test strain has the capacity and the strength of degrading lignin or not according to the size of the reddish-brown color-changing ring.
The strain YM32425 has the best capacity of degrading cellulose and lignin, and thus the strain is preserved.
The deposit information of YM32425 is as follows:
the preservation name is Trichoderma longibrachiatum, the preservation unit is: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.15092, preservation time 2017, 12 months and 15 days.
Example 2 fermentation culture of Trichoderma longibrachiatum strain YM32425
Potato dextrose agar medium (PDA): 200g of potato, 20g of glucose and 15g of agar, and adding water to 1000 mL.
Potato Dextrose Broth (PDB): 200g of potato and 20g of glucose, and adding water to 1000 mL.
Inoculating Trichoderma longibrachiatum (Trichoderma longibrachiatum) YM32425 on a PDA plate, and culturing at 28 deg.C for 5d with 12h light/dark alternation; cutting a fungus cake with the diameter of 5mm by using a puncher; inoculating 5 blocks of the bacterial cake into 200ml of PDB culture solution, and culturing at 28 ℃ and 150rpm for 7d to obtain bacterial liquid.
Example 3 fermentation substrate formulation adjustment and optimization
Drying and crushing corn stalks and straws (5 mm, the water content is less than 15%), drying and crushing pig manure (1 mm, the water content is less than 20%).
Trichoderma longibrachiatum strain YM32425 and Bacillus subtilis strain D4; phanerochaete chrysosporium (Phanerochaete chrysosporium) strain ACCC 30414, purchased from China center for agricultural microbial cultures Collection; chaetomium globosum (Chaetomium globosum) strain Cg2, self-isolated.
Inoculating D4 to an NA plate by using a Bacillus subtilis strain D4 and adding nutrient agar NA (the formula is 15g of peptone, 0.5g of beef extract, 5g of sodium chloride, 20g of glucose and 15g of agar and adding water to 1000mL), and culturing at 28 ℃ in a dark environment for 2D; inoculating a ring of thallus to 200mL of NA culture solution (the formula is 15g of peptone, 0.5g of beef extract, 5g of sodium chloride and 20g of glucose, and adding water to 1000mL) by using an inoculating ring, and culturing for 2d at the temperature of 28 ℃ and the rpm of 150 to obtain a bacterial solution.
Culture of Phanerochaete chrysosporium (Phanerochaete chrysosporium) strain ACCC 30414, strain Cg2 of Chaetomium globosum (Chaetomium globosum) strain is equivalent to Trichoderma longibrachiatum strain YM 32425.
The culture medium is composed of rice straw and pig manure or corn stalk and pig manure; the straws and maize straws have low nitrogen content and high C: N content.
Weighing 5g of matrix (calculating the proportion and the use amount of the straw or corn stalk and the pig manure according to the C/N ratio 40/1, the C, N content and the water content of the matrix) in a 500mL triangular flask, and calculating the required water addition amount for adjusting the initial water content of the matrix to be 60% according to the water content of the matrix; the bacterial solutions obtained from different strains were subjected to the following procedures, respectivelyThe operation is as follows: 1mL of the bacterial liquid is added into a proper amount of water, mixed evenly and added into 5g of the matrix. The CO is then characterized in terms of the amount of organic carbon decomposed (mg) in the feed2The amount of (a) released. And (4) calculating the weight of the material after culture sampling, and determining the organic carbon content.
The method comprises the following specific steps:
experiment one, taking 4.3g of straw and 0.7g of pig manure as a substrate, adding about 5.7mL of water, and then adding 1mL of bacterial liquid; the fermentation was carried out at 30 ℃.
The experiment was performed on day 10, 25 of the current year, and ended on day 1, 13 of the following year; the detection of the organic carbon content was carried out according to a Multi N/C3100 total organic carbon analyzer at 6 time points.
The results for the 4 strains are shown in FIG. 1.
Experiment two, taking 3.8g of corn straw and 1.2g of pig manure as a matrix, adding about 5.7mL of water, and then adding 1mL of bacterial liquid; the fermentation was carried out at 30 ℃.
The experiment was performed on day 10, 25 of the current year, and ended on day 1, 13 of the following year; the detection of the organic carbon content was carried out according to a Multi N/C3100 total organic carbon analyzer at 6 time points.
The results for the 4 strains are shown in FIG. 2.
As can be seen from FIGS. 1 and 2, 4 kinds of degrading bacteria were decomposed by Trichoderma longibrachiatum strain YM32425 to produce CO2Best effect, CO generation2Large in amount and longer in duration; CO is produced by adopting corn stalks as a substrate and fermenting the corn stalks as the substrate in comparison with rice straws as the substrate2The effect is best.
In the invention process, Trichoderma longibrachiatum DQ2, Trichoderma longibrachiatum TL1 and Trichoderma longibrachiatum Rifaii are detected according to the first and second experiments, and the results show that: the decomposition amount (mg) of organic carbon in the material at the end of the experiment is substantially equal to Chaetomium globosum.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. Trichoderma longibrachiatum (Trichoderma longibrachiatum) YM32425, characterized by: the preservation number is CGMCC NO. 15092.
2. Use of Trichoderma longibrachiatum (Trichoderma longibrachiatum) YM32425 according to claim 1, characterized in that: for degrading crop straw to produce CO2。
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| CN111334456A (en) * | 2020-03-18 | 2020-06-26 | 吉林省农业科学院 | Preparation method and application of straw decomposition microbial inoculum used under low-temperature condition in north |
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| CN103131639A (en) * | 2011-11-23 | 2013-06-05 | 中国农业科学院作物科学研究所 | Trichoderma longibrachiatum strain and application thereof |
| CN109234173A (en) * | 2018-11-02 | 2019-01-18 | 河北省科学院生物研究所 | A kind of long shoot trichoderma, its solid microbial and application |
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| CN103131639A (en) * | 2011-11-23 | 2013-06-05 | 中国农业科学院作物科学研究所 | Trichoderma longibrachiatum strain and application thereof |
| CN109234173A (en) * | 2018-11-02 | 2019-01-18 | 河北省科学院生物研究所 | A kind of long shoot trichoderma, its solid microbial and application |
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