CN107815433B - Compound microbial agent and application thereof in producing fulvic acid by fermenting humic acid lignite - Google Patents
Compound microbial agent and application thereof in producing fulvic acid by fermenting humic acid lignite Download PDFInfo
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Abstract
The invention discloses a compound microbial agent and application thereof in producing fulvic acid by fermenting humic acid lignite, wherein the compound microbial agent consists of bacillus subtilis with the preservation number of CGMCC NO.12905, trichoderma harzianum with the preservation number of CGMCC NO.13762 and penicillium oxalicum with the preservation number of CGMCC NO. 13763; the application method of the compound microbial agent comprises the following steps: mixing humic acid lignite with auxiliary materials, adding the composite microbial agent, adding water into a base material, controlling the humidity to be 60%, standing and decomposing for 7-14 days, then adding water, uniformly stirring, standing at room temperature for 2 hours, and separating supernatant (containing fulvic acid). The invention has the advantages that: (1) the composite microbial agent provided by the invention is used for fermenting humic acid lignite to generate fulvic acid, and compared with a chemical method, the method does not generate pollutants and is more environment-friendly; (2) the waste can be used as organic fertilizer, so that the sustainable utilization with green, high efficiency and high quality is realized.
Description
Technical Field
The invention relates to a microbial agent and application thereof, in particular to a compound microbial agent and application thereof in producing fulvic acid by fermenting humic acid lignite, and belongs to the technical field of microorganisms.
Background
The fulvic acid can be widely applied to the aspects of industry, agriculture, medicine, pasture and the like. Taking the application in agriculture as an example, fulvic acid can promote the growth of plants, control the opening degree of stomata on leaf surfaces of crops, reduce transpiration, resist drought, improve the stress resistance of crops, increase yield and improve the quality of crops.
At present, fulvic acid is produced mainly by a chemical method and is mainly extracted from weathered coal. China has abundant lignite resources, but lignite combustion has low thermal efficiency and is easy to cause environmental pollution. The traditional chemical method for extracting fulvic acid from lignite is low in extraction efficiency, the activity of the extracted fulvic acid is poor, the flocculation limit is low, and secondary pollutants polluting the environment can be generated in the extraction process.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the compound microbial agent and the method for producing the fulvic acid by fermenting the humic acid lignite by using the compound microbial agent.
In order to achieve the above object, the present invention adopts the following technical solutions:
the compound microbial agent is characterized by consisting of bacillus subtilis with the preservation number of CGMCC number 12905, trichoderma harzianum with the preservation number of CGMCC NO.13762 and penicillium oxalicum with the preservation number of CGMCC NO. 13763.
The compound microbial agent is characterized in that in the compound microbial agent, the spore content of bacillus subtilis with the preservation number of CGMCC NO.12905 is more than or equal to 20 hundred million/g, the spore content of trichoderma harzianum with the preservation number of CGMCC NO.13762 is more than or equal to 5 hundred million/g, and the spore content of penicillium oxalicum with the preservation number of CGMCC NO.13763 is more than or equal to 8 hundred million/g.
The compound microbial agent is applied to fermentation of humic acid lignite to produce fulvic acid.
The application is characterized in that the application method comprises the following steps: mixing humic acid lignite with auxiliary materials, adding the composite microbial inoculant in claim 1, adding water into a base material, controlling the humidity to be 60%, standing and decomposing for 7-14 days, then adding water, uniformly stirring, standing at room temperature for 2 hours, and separating supernatant, wherein the supernatant contains fulvic acid.
The application is characterized in that the auxiliary materials are wheat bran and/or soybean meal.
The application is characterized in that the mixing ratio of the humic acid lignite, the wheat bran, the soybean meal and the compound microbial agent is 7-10: 0-3: 0-3: 1 to 3.
Or the application method comprises the following steps: mixing humic acid lignite with the composite microbial inoculant in claim 1, adding water into a base material, controlling the humidity to be 60%, standing for 7-14 days for decomposition, then adding water, uniformly stirring, standing for 2 hours at room temperature, and separating supernatant, wherein the supernatant contains fulvic acid.
The application is characterized in that the mixing ratio of the humic acid lignite to the composite microbial agent is 7-10: 1 to 3.
The invention has the advantages that:
(1) the composite microbial agent provided by the invention is used for fermenting humic acid lignite to generate fulvic acid, and compared with a chemical method, the method does not generate pollutants and is more environment-friendly;
(2) the waste can be used as organic fertilizer, so that the sustainable utilization with green, high efficiency and high quality is realized.
Drawings
FIG. 1 is an infrared spectrum of the supernatant separated in example 1;
FIG. 2 is an infrared spectrum of the supernatant separated in example 2;
FIG. 3 is an infrared spectrum of the supernatant separated in example 3;
FIG. 4 is an infrared spectrum of the supernatant separated in example 4.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
First, separating and screening bacillus subtilis
1. Screening of isolated strains
We collected a soil sample from a fermented heap of lignite compost in Leisi, Qingdao and diluted the soil sample by 10%5And (4) doubling.
Selecting an LB culture medium: 10g of peptone, 5g of yeast extract powder, 10g of sodium chloride, 15g of agar, and supplementing water to 1L, wherein the pH value is natural.
And uniformly coating the diluted sample on an LB culture medium, culturing for 12h at 37 ℃, then picking a bacterial colony on the culture medium, and performing purification culture to obtain a strain.
2. Identification of strains
We have identified the strains isolated by the above screening, as follows: the 16SrRNA, gyrA gene and gyrB gene were used for sequencing and alignment in NCBI database.
And (3) comparing the results: the similarity between the gene sequence of the strain obtained by screening and separating and the sequence of Bacillus subtilis in a database reaches more than 99 percent.
According to the gene sequence determination result, the strain obtained by screening and separating is finally determined to be Bacillus subtilis.
3. Deposited strain
In 2016, 8 months and 26 days, the bacillus subtilis obtained by separation and screening is preserved in China general microbiological culture collection center, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, the preservation number is CGMCC NO.12905, and the classification names are as follows: bacillus subtilis (Bacillus subtilis).
Secondly, separating and screening trichoderma harzianum and penicillium oxalicum
1. Screening of isolated strains
We collected samples from a fermented heap of lignite compost in Lexi, Qingdao, and diluted the soil sample by 10%5And (4) doubling.
Selecting a PDA culture medium: potato (peeled) 200g, glucose 20g, agar 15g, distilled water to 1L, natural pH.
And uniformly coating the diluted sample on a PDA culture medium, culturing at 27 ℃ for 24h, then picking a bacterial colony on the culture medium, and performing purification culture to obtain two strains.
2. Identification of strains
We identified the two strains obtained by the screening and separation, which are as follows: the ITS1-ITS5 gene was used for sequencing and alignment in the NCBI database.
And (3) comparing the results: two strains obtained by screening and separating are selected, wherein the similarity of the gene sequence of one strain and the sequence of Trichoderma harzianum in a database reaches more than 99 percent, and the similarity of the gene sequence of the other strain and the sequence of Penicillium oxalicum in the database reaches more than 99 percent.
According to the gene sequence determination result, the two strains obtained by screening and separating are finally determined to be Trichoderma harzianum (Trichoderma harzianum) and Penicillium oxalicum (Penicillium oxalicum), respectively.
3. Deposited strain
In 2017, 3 and 14 days, the Trichoderma harzianum obtained by separation and screening is preserved in the China general microbiological culture collection center, the preservation address is No. 3 of No.1 Homex west Lu of the sunward area in Beijing, the preservation numbers are CGMCC NO.13762 and CGMCC NO.13763 respectively, the classification name of the Trichoderma harzianum is Trichoderma harzianum, and the classification name of the Penicillium oxalicum is Penicillium.
Thirdly, preparing the compound microbial agent
1. Activated bacterial strain
Activating bacillus subtilis with the preservation number of CGMCC NO.12905 in an LB liquid culture medium for 12 hours, and taking the activated strain as an original strain.
Respectively inoculating Trichoderma harzianum with the preservation number of CGMCC NO.13762 and Penicillium oxalicum with the preservation number of CGMCC NO.13763 on a PDA plate culture medium, carrying out dark culture at 27 ℃ for 7d, and taking the cultured strain as an original strain.
2. Preparation of seed liquid
And oscillating the activated bacillus subtilis at 30 ℃ for 15h, and taking the fermented liquid as seed liquid.
Respectively inoculating the activated Trichoderma harzianum stock and Penicillium oxalicum stock on a PDA liquid culture medium, carrying out dark culture at the temperature of 27 ℃ and the rpm of 121 for 4 days, and taking the fermented liquid as a seed liquid.
3. Fermentation culture
1#Fermentation medium: 5-10 g/L yeast extract, 8-16 g/L soluble starch, 4-8 g/L glucose, 8-16 g/L soybean cake powder, 6-12 g/L beef extract and corn8-16 g/L of powder, a proper amount of defoaming agent and the balance of water.
In this embodiment, 1#Fermentation medium: 8g/L of yeast extract, 10g/L of soluble starch, 6g/L of glucose, 12g/L of soybean cake powder, 9g/L of beef extract, 12g/L of corn flour, 2g/L of antifoaming agent and the balance of water.
To 1#Adding appropriate amount of water into the fermentation medium to obtain a mixture 1#The initial pH of the fermentation medium is 7 + -5, sterilized at high temperature, and then treated to 1#And (3) inoculating the bacillus subtilis seed solution when the temperature of the fermentation culture medium is reduced to below 30 ℃, then performing fermentation culture under the conditions that the temperature is 30 ℃, the ventilation quantity is 180L/h and the stirring speed is 200rpm, and stopping the fermentation culture when the spore rate in the fermentation solution reaches 30-50%.
2#Fermentation medium: adding water into testa Tritici, controlling humidity at about 20%, and sterilizing at 121 deg.C for 30 min.
Inoculating Trichoderma harzianum seed liquid and Penicillium oxalicum seed liquid to the seed liquid 2#In the fermentation medium, the culture is carried out at 27 ℃ in the dark for 14 d.
4. Post-treatment
And (3) carrying out spray drying on the fermentation liquor of the bacillus subtilis to obtain bacillus subtilis powder.
And drying the fermented trichoderma harzianum culture medium, crushing, and screening by a 100-mesh screen to obtain trichoderma harzianum powder. The detection shows that the content of trichoderma harzianum spores in the powder is more than or equal to 200 hundred million/g.
And drying the penicillium oxalicum culture medium after fermentation, crushing, and sieving by a 100-mesh sieve to obtain penicillium oxalicum powder. Through detection, the content of the penicillium oxalicum spores in the powder is more than or equal to 300 hundred million/g.
5. Mixing
B, mixing bacillus subtilis powder, trichoderma harzianum powder and penicillium oxalicum powder according to the ratio of 1-60: 1-40: 1-70 to obtain the compound microbial agent, wherein the spore content of bacillus subtilis is more than or equal to 20 hundred million/g, the spore content of trichoderma harzianum is more than or equal to 5 hundred million/g, and the spore content of penicillium oxalicum is more than or equal to 8 hundred million/g.
Fourthly, fermentation of humic acid lignite
In this example, we used a complex microbial preparation containing 20 hundred million/g spores of Bacillus subtilis, 5 hundred million/g spores of Trichoderma harzianum, and 8 hundred million/g spores of Penicillium oxalicum.
Example 1
Mixing humic acid lignite and a compound microbial agent according to the proportion of 7: 3, adding water into the base material after mixing, controlling the humidity to be about 60%, then standing and decomposing for 7d, and then mixing the materials according to the volume ratio: water 1: adding water according to the volume ratio of 50, stirring uniformly, standing for 2 hours at room temperature, and separating supernatant.
By infrared spectroscopic analysis, referring to FIG. 1, the supernatant was at 1720cm-1、1620cm-1、 1400cm-1、1250cm-1An absorption peak is formed and is consistent with the absorption spectrum of the fulvic acid, and the supernatant is proved to contain the fulvic acid.
The supernatant containing fulvic acid can be directly used, or dried to obtain fulvic acid powder, and the residue can be further extracted or directly used as fertilizer.
Example 2
Mixing humic acid lignite with auxiliary material wheat bran, and then adding a compound microbial inoculant, wherein the volume ratio of the humic acid lignite to the wheat bran to the compound microbial inoculant is 8: 1: 1, adding water into the base material after mixing, controlling the humidity to be about 60%, then standing and decomposing for 7d, and then mixing the materials: water 1: adding water according to the volume ratio of 50, stirring uniformly, standing for 2 hours at room temperature, and separating supernatant.
By infrared spectroscopic analysis, referring to FIG. 2, the supernatant was at 1720cm-1、1620cm-1、 1400cm-1、1250cm-1An absorption peak is formed and is consistent with the absorption spectrum of the fulvic acid, and the supernatant is proved to contain the fulvic acid.
Example 3
Mixing humic acid lignite with auxiliary material soybean meal, and then adding a compound microbial agent, wherein the volume ratio of the humic acid lignite to the soybean meal to the compound microbial agent is 7: 1: 2, adding water into the base material after mixing, controlling the humidity to be about 60%, then standing and decomposing for 7d, and then mixing the materials: water 1: adding water according to the volume ratio of 50, stirring uniformly, standing for 2 hours at room temperature, and separating supernatant.
By infrared spectroscopic analysis, referring to FIG. 3, the supernatant was at 1720cm-1、1620cm-1、 1400cm-1、1250cm-1An absorption peak is formed and is consistent with the absorption spectrum of the fulvic acid, and the supernatant is proved to contain the fulvic acid.
Example 4
Mixing humic acid lignite with auxiliary materials of wheat bran and bean pulp, and then adding a compound microbial inoculant, wherein the volume ratio of the humic acid lignite to the wheat bran to the bean pulp to the compound microbial inoculant is 8: 0.5: 0.5: 1, adding water into the base material after mixing, controlling the humidity to be about 60%, then standing and decomposing for 7d, and then mixing the materials: water 1: adding water according to the volume ratio of 40, stirring uniformly, standing for 2 hours at room temperature, and separating supernatant.
By infrared spectroscopic analysis, referring to FIG. 4, the supernatant was at 1720cm-1、1620cm-1、1400cm-1、1250cm-1An absorption peak is formed and is consistent with the absorption spectrum of the fulvic acid, and the supernatant is proved to contain the fulvic acid.
The method for producing fulvic acid by fermenting humic acid lignite with the compound microbial inoculant provided by the invention has the following advantages that the volume ratio of the humic acid lignite to wheat bran to soybean meal to the compound microbial inoculant is 7-10: 0-3: 0-3: 1-3, the standing and decomposing time can be properly prolonged to 14 days.
Therefore, the composite microbial agent provided by the invention is used for fermenting humic acid lignite to generate fulvic acid, compared with a chemical method, the composite microbial agent does not generate pollutants, is more environment-friendly, can be used as an organic fertilizer for waste materials, realizes green, efficient and high-quality sustainable utilization, has very positive social significance, and is worthy of popularization and application.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Claims (8)
1. The compound microbial agent is characterized by consisting of Bacillus subtilis with the preservation number of CGMCC NO.12905, Trichoderma harzianum with the preservation number of CGMCC NO.13762 and Penicillium oxalicum with the preservation number of CGMCC NO. 13763.
2. The complex microbial inoculant according to claim 1, wherein the bacillus subtilis with the preservation number of CGMCC No.12905 has a spore content of 20 hundred million/g or more, the trichoderma harzianum with the preservation number of CGMCC No.13762 has a spore content of 5 hundred million/g or more, and the penicillium oxalicum with the preservation number of CGMCC No.13763 has a spore content of 8 hundred million/g or more.
3. The use of the composite microbial inoculant of claim 1 for fermenting humic acid lignite to produce fulvic acid.
4. The application of claim 3, wherein the application method comprises: mixing humic acid lignite with auxiliary materials, adding the composite microbial inoculant of claim 1, adding water into a base material, controlling the humidity to be 60%, standing and decomposing for 7-14 days, then adding water, uniformly stirring, standing at room temperature for 2 hours, and separating supernatant, wherein the supernatant contains fulvic acid.
5. The use according to claim 4, wherein the auxiliary material is wheat bran and/or soybean meal.
6. The application of claim 5, wherein the mixing ratio of the humic acid lignite, the wheat bran, the soybean meal and the compound microbial agent is 7-10: 0-3: 0-3: 1 to 3.
7. The application of claim 3, wherein the application method comprises: mixing humic acid lignite with the composite microbial inoculant as defined in claim 1, adding water into a base material, controlling the humidity to be 60%, standing for decomposing for 7-14 days, then adding water, uniformly stirring, standing for 2 hours at room temperature, and separating supernatant, wherein fulvic acid is contained in the supernatant.
8. The application of claim 7, wherein the mixing ratio of the humic acid lignite and the compound microbial inoculant is 7-10: 1 to 3.
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| KR102154243B1 (en) * | 2019-08-09 | 2020-09-09 | 고해훈 | Extracting method of high purity fulvic acid |
| CN110616246A (en) * | 2019-10-11 | 2019-12-27 | 北京大伟嘉生物技术股份有限公司 | Fermentation method for increasing content of sodium fulvate and microbial agent thereof |
| CN110669679B (en) * | 2019-10-31 | 2021-02-23 | 内蒙古科技大学 | Penicillium oxalicum |
| KR102206122B1 (en) * | 2020-06-23 | 2021-01-20 | 강성식 | Extraction Method of Fulvic Acid with Improved Yield |
| CN111944839A (en) * | 2020-08-05 | 2020-11-17 | 中国矿业大学 | Application of improved ATMT (atactic terminal transferase) constructed gene overexpression Trichoderma harzianum strain in lignite degradation |
| CN115504830B (en) * | 2022-09-21 | 2024-03-01 | 河北萌帮生物科技有限公司 | Microbial fermentation biological humic acid, preparation method and application |
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Address after: 266600 No.2 Kunming Road, Wangcheng Industrial Park, Laixi City, Qingdao City, Shandong Province Patentee after: QINGDAO YIBAINONG FERTILIZER Co.,Ltd. Address before: No. 30, Shenzhen South Road, Wangcheng sub district office, Laixi City, Qingdao City, Shandong Province 266600 Patentee before: QINGDAO YIBAINONG FERTILIZER Co.,Ltd. |