CN112680373B - Bacillus mucilaginosus and method for preparing bio-organic fertilizer by using same through solid state fermentation - Google Patents
Bacillus mucilaginosus and method for preparing bio-organic fertilizer by using same through solid state fermentation Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to Bacillus mucilaginosus (Bacillus mucilaginosus) M-1 with high-efficiency potassium-decomposing function and growth promoting capability, wherein the preservation number is as follows: CGMCC No.19428 and a method for preparing a biological organic fertilizer by using the same through solid state fermentation. The method for preparing the bio-organic fertilizer by solid state fermentation comprises the following steps: (1) activating the bevel seeds; (2) culturing liquid seeds; (3) pretreating a substrate; (4) and (5) solid state fermentation culture. The invention relieves the problems of viscous fermentation liquor, low viable count and low spore rate of the traditional bacillus mucilaginosus liquid fermentation, adopts a solid state fermentation method, simultaneously ferments and produces bacillus mucilaginosus and secondary decomposition of organic fertilizer by taking cow dung as a substrate, finally forms the biological organic fertilizer, and has simple process and easy implementation.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to bacillus mucilaginosus and a method for preparing a biological organic fertilizer by using the bacillus mucilaginosus through solid state fermentation.
Background
Bacillus mucilaginosus (also called Bacillus mucilaginosus) can decompose potassium and silicon in minerals such as feldspar, mica and the like, promote the transformation of ineffective phosphorus and potassium in soil, increase the supply of phosphorus and potassium in soil and improve the yield of crops. On one hand, the Bacillus mucilaginosus is fermented by adopting the traditional process liquid, so that the number of viable bacteria is too small and the spore rate is low; on the other hand, the problem of concentration and drying of the fermentation liquor in the production of the bacillus mucilaginosus solid powder is that the production becomes an obstacle to the development of enterprises and industries.
In addition, in the production of the traditional biological organic fertilizer, a biological agent and the organic fertilizer are two independent processes, and finally, the biological agent and the organic fertilizer are mixed to prepare the fertilizer; in the composting process of the organic fertilizer, a large amount of nitrogen loss can occur.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the bacillus mucilaginosus and the method for preparing the bio-organic fertilizer by using the bacillus mucilaginosus through solid state fermentation, wherein the solid state fermentation method is adopted, cow dung is used as a substrate to simultaneously ferment and produce the bacillus mucilaginosus and the secondary decomposition of the organic fertilizer, and the bio-organic fertilizer is finally formed.
The invention is realized by the following technical scheme:
provides a Bacillus mucilaginosus (Bacillus mucilaginosus) M-1 with high-efficiency potassium-decomposing function and growth-promoting capability, and the preservation number is as follows: CGMCC No. 19428.
The Bacillus mucilaginosus M-1 is obtained by separating and purifying soil in a vegetable garden, the partial sequence of 16S rDNA of a strain is determined, and the Bacillus mucilaginosus M-1 is determined by phylogenetic analysis and is named as M-1. The morphological characteristics of the strain are as follows: on the nitrogen-free solid culture medium bacteria, bacterial colonies are raised and are in a glass hemispherical shape, the surface is wet, glossy and transparent, and the colloid is thick and elastic; the thallus is rod-shaped, two ends of the thallus are blunt and round, and the size of the thallus is 4-7 mu m multiplied by 1-1.4 mu m; after culturing for 30h in a starch-containing culture medium, a large number of spores are formed in the thallus, wherein the spores are mesogenic or subtenomic, have an oval shape, and have the size of about 1.5-1.7 mu m multiplied by 2.8-3.4 mu m.
The strain is preserved in the China general microbiological culture Collection center, and the preservation address is as follows: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The preservation date is 2020, 2 months and 19 days.
The bacillus mucilaginosus M-1 is applied to biological fertilizers.
A method for preparing a bio-organic fertilizer by using Bacillus mucilaginosus M-1 solid state fermentation comprises the following steps:
(1) activating the slant seeds: inoculating bacillus mucilaginosus M-1 to a nutrient agar slant culture medium in a sterile manner, and culturing at the temperature of 37 +/-2 ℃ for 36-48 h to obtain slant seeds;
(2) culturing liquid seeds: inoculating the slant seeds obtained in the step (1) into a liquid culture medium, and culturing at 37 +/-1 ℃ for 36-48 h to obtain liquid seeds;
(3) pre-treating the substrate: crushing a material substrate, adjusting the moisture content to 55-60%, placing the mixed material in a fermenter, heating to 75 +/-3 ℃, and quickly performing harmless treatment and decomposition at a high temperature for 8-10 hours until the odor disappears;
(4) solid state fermentation culture: inoculating 5-10 parts by weight of the liquid seeds obtained in the step (2) into the pretreated substrate obtained in the step (3), culturing at 37 +/-2 ℃ for 5-7 days to obtain a fermentation product, and drying the fermentation product until the water content is less than or equal to 15% to obtain the final bio-organic fertilizer.
Further, the material matrix comprises the following components in parts by weight: 50-60 parts of cow dung, 20-30 parts of mushroom residues, 10-20 parts of sawdust and 2-4 parts of molasses.
Further, the pH value of the M-1 liquid seeds inoculated in the step (4) is 6.5-7.5.
A biological organic fertilizer prepared by a method for preparing the biological organic fertilizer by solid state fermentation.
Furthermore, the content of viable count of bacillus mucilaginosus M-1 in the bio-organic fertilizer is 1.2 multiplied by 109More than one per gram.
The invention has the beneficial effects that:
the method has the advantages that the solid-state fermentation method is adopted, cow dung is used as a main substrate, and the Bacillus mucilaginosus is simultaneously fermented and produced, so that the secondary decomposition of the organic fertilizer is realized, the nitrogen loss of the organic fertilizer is reduced, and the quick-acting potassium in the organic fertilizer is activated. The use of the biological organic fertilizer can improve and maintain soil fertility, convert nutrient elements, reduce the use of chemical fertilizers, improve the utilization rate of the chemical fertilizers, promote the growth of crops, resist soil-borne diseases, purify the environment and balance an ecological system. Not only can not cause new pollution, but also can control and improve the environment and generate long-term good ecological effect.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1:
isolation of potassium-solubilizing bacteria
The separation process generally comprises the following steps:
step one, preparing an Alexander bauov culture medium: the components of the composition comprise: na (Na)2HPO4 2g，FeCl30.005g，MgSO4·7H2O 0.5g，CaCO30.1g, 3g of sucrose, 1g of potassium feldspar powder (washed five times by deionized water), 1000mL of distilled water, pH 7.0, 20g of agar and 20min of sterilization at 121 ℃.
Step two, preliminary screening of potassium-decomposing bacteria: inoculating the enrichment strain to an Alexander bauov culture medium solid culture medium, culturing in a constant-temperature incubator at 37 ℃ for 10 days, and observing whether a transparent ring is formed on the culture medium and the size of the transparent ring. The diameters of the hydrolysis ring and the bacterial colony are measured by a ruler, and the relative size of the potassium dissolving capacity of the bacterial strain can be judged according to the diameter ratio of the hydrolysis ring to the bacterial colony. The strain with the largest hydrolysis loop was selected and designated strain M-1.
Example 2:
molecular identification of 16S rDNA sequence of strain M-1
The strain QF-101 genome DNA is extracted by a Chelex method, and the 16S rDNA gene full-length sequence of the strain QF-101 genome DNA is subjected to PCR amplification by using 16S rDNA universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR product was subjected to 1% agarose gel electrophoresis, and the target DNA fragment was recovered using a SanPrep column DNA gel recovery kit (Sangon Biotech, Order NO. B518131) after the target band was cut. Sequence analysis of the DNA fragments was performed using 3730xl DNA Analyzer (Applied Biosystems), and BigDyeTerminator v3.1(Applied Biosystems) was used as a sequencing kit.
The results of the two-way sequencing were spliced using SeqMan (DNASTAR Lasergene) to obtain a near-full-length 16S rRNA gene sequence. The 16S rRNA gene sequence results were aligned with the GenBank database using an EzBioCloud identification service (https:// www.ezbiocloud.net/identification) and NCBI Nucleotide BLAST (https:// BLAST. NCBI. nlm. nih. gov/BLAST. cgi) and a well-named model strain was selected as a reference.
The strain M-1 was identified as Bacillus mucilaginosus (Bacillus mucoarginosus). The morphological characteristics of the strain are as follows: on the nitrogen-free solid culture medium bacteria, bacterial colonies are raised and are in a glass hemispherical shape, the surface is wet, glossy and transparent, and the colloid is thick and elastic; the thallus is rod-shaped, two ends of the thallus are blunt and round, and the size of the thallus is 4-7 mu m multiplied by 1-1.4 mu m; after culturing for 30h in a starch-containing culture medium, a large number of spores are formed in the thallus, wherein the spores are mesogenic or subtenomic, have an oval shape, and have the size of about 1.5-1.7 mu m multiplied by 2.8-3.4 mu m.
Example 3:
qualitative test of potassium-dissolving capacity of strain M-1
Culturing the culture solution of the strain M-1 for 48h with the liquid loading of a 250mL triangular flask being 50mL, and taking the fermentation liquid sterilized at 121 ℃ for 30min as a blank control. Weighing 12.5g potassium feldspar powder, adding into the culture solution and the inactivated culture solution respectively, performing 3 parallel treatments, culturing at 37 deg.C and 160rpm, and determining the content of soluble potassium by potassium tetraphenylborate gravimetric method on day 5 and day 10.
TABLE 1 qualitative test of potassium-decomposing ability of strain M-1
| 5 days of potassium (mg/L) | Potassium-releasing amount (mg/L) for 10 days | |
| Blank control | 2.381 | 4.912 |
| Strain M-1 | 30.735 | 58.975 |
The strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.19428 and is named as Bacillus mucilaginosus (Bacillus mucoarginosus) M-1. The bacillus subtilis has strong potassium-dissolving capacity and growth-promoting capacity, and the potassium-dissolving amount in a culture solution can reach 30.735mg/L and 58.975m g/L in 5d and 10d respectively.
The strain bacillus mucilaginosus M-116S rDNA sequence
Strain bacillus mucilaginosus M-116S rDNA sequence
AGAGTTTGATCCTGGCTCAGGACGACCGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGAGCACTTCGGTGCTTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTGTAAGATCGGGATAACTACCGGAAACGGTAGCTAAGACCGGATAGCTGGTTTCGGTGCATGCCGGAATCATGAAACACGGGGCAACCTGTCGCTTACGGATGGGCCTGCGGCGCATTAGCTAGTTGGCGGGGTAATGGCCCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGCAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGCCAGGGAAGAATGTCGTGGAGAGTAACTGCTCTGCGAATGACGGTACCTGAGAAGAAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTCTTTTAAGTCTGGTGTTTAAGCCCGGGGCTCAACCCCGGTTCGCACCGGAAACTGGAAGACTTGAGTGCAGGAGAGGAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCGCGAAAACGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGGGTTTCGATACCCTTGGTGCCGAAGTAAACACAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTCTGAAAACCCTAGAGATAGGGCCCTCCTTCGGGACAGAGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGACTTTAGTTGCCAGCATTGAGTTGGGCACTCTAGAGTGACTGCCGGTGACAAACAGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGAAGCGAAGTCGCGAGATGGAGCGAATCCTTAGAAGCCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATACCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTACAACACCCGAAGCCGGTGGGGTAACCCGTAAGGGAGCCAGCCGTCGAAGGTGGGGTAGATGAATGGGGTGAAGTCGTAACAAGGTAACC
Example 4:
growth promotion test of strain M-1 fermentation liquor
The growth promotion of the strain M-1 fermentation liquor adopts a wheat water culture method, wheat seeds with uniform size are used as test objects, 25 seeds are uniformly placed in each plate, 3 parallel tests are set, the test is repeated for 4 times, and the blank is a clear water control; the number of viable bacteria in clear water adjusting strain M-1 fermentation liquor is 106The treatment 1 is the inactivation of the strain M-1; the treatment 2 is the strain M-1 viable bacteria fermentation liquor. After inoculation, indoor illumination culture is carried out, illumination is carried out for 10h every day, dark culture is carried out for 14h, and random arrangement is carried out. After the wheat had been grown for 40 days on a permanent-planting basis, the plants and kernels were cut with scissors, the fresh weight of the aerial parts was measured, and 10 plants were taken out of each plate and the height of the plants was measured. Test results show that the fresh weight of the inactivated strain M-1 fermentation liquor is increased by 4.21% and the plant height is increased by 8.46% compared with the fresh weight of the control ground; compared with the contrast ground fresh weight, the live bacteria-containing fermentation liquor increases 15.5%, the plant height increases 18.58%, the plant height and ground fresh weight of wheat can be improved, and the live bacteria-containing fermentation liquor has a promoting effect on the growth and development of wheat.
TABLE 2 influence of the M-1 Strain fermentation broth on the hydroponic aerial parts of wheat
TABLE 3 influence of Strain M-1 fermentation broth wheat hydroponics strain height
Example 5:
solid state fermentation production of bio-organic fertilizer and common organic fertilizer fermentation production
A method for preparing a bio-organic fertilizer by solid state fermentation comprises the following specific steps:
(1) activating the slant seeds: inoculating bacillus mucilaginosus M-1 on a nutrient agar slant culture medium under an aseptic condition, and culturing at 37 ℃ for 36h to obtain a slant seed:
(2) culturing liquid seeds: inoculating the slant seeds into a liquid culture medium, and culturing at 37 ℃ for 36h to obtain liquid seeds;
(3) pre-treating the substrate: selecting the following components in parts by weight: 55 parts of cow dung, 20 parts of mushroom residues, 15 parts of wood chips and 2 parts of molasses, and after the material substrates are mixed and crushed, adjusting the water content to about 60% and the pH value to 7.5, placing the mixed material in a closed fermentor with built-in turning equipment, heating the material to 75 ℃ through external equipment, and quickly harmlessly treating the material at a high temperature for 9 hours until the rotten odor disappears to finish the pretreatment of the material;
(4) solid state fermentation culture: inoculating 5% of the liquid seeds obtained in the step (2) into the substrate pretreated in the step (3) according to the weight ratio, and culturing at 37 ℃ for 5d to obtain a fermentation product: and (4) drying the fermentation product after granulation until the water content is less than or equal to 15% to obtain the bio-organic fertilizer.
And fermentation of common organic fertilizer: and (3) taking the pretreated solid matrix with the same mass fraction as that in the step (3), culturing at 37 ℃ for 5d to obtain a fermentation product, and drying the fermentation product after granulation until the water content is less than or equal to 15% to obtain the common organic fertilizer.
TABLE 4 influence of the fermentation broth of the strain M-1 on the fermentation nutrients of compost
Compared with the common organic fertilizer without the added bacteria, the total nitrogen of the biological organic fertilizer produced by adding the strain M-1 is improved by 32.9 percent, and the quick-acting potassium is improved by 23.6 percent, which shows that the strain M-1 has the functions of nitrogen preservation, potassium dissolution and total nutrient improvement of the organic fertilizer in the solid state fermentation process of the matrix.
The organic matter content of the finally obtained biological organic fertilizer is 61.5 percent through detection, the total nutrient is 9.22 percent, and the viable count of bacillus mucilaginosus M-1 can reach 1.2 multiplied by 109More than 83 percent of spores per gram, pH7.6 which is higher than the agricultural industry standard bioorganic fertilizer (NY884-2012) and meets the requirements of agricultural microbial agents (GB 20287-2006). The invention solves the problems of difficult fermentation of the traditional bacillus mucilaginosus liquid, viscous requirement of fermentation liquid, low viable count and low spore rate, simultaneously realizes the decomposition of bacillus mucilaginosus and organic fertilizer by simultaneously fermenting and producing the bacillus mucilaginosus and the organic fertilizer by taking cow dung as a substrate, finally forms the biological organic fertilizer, and has simple process and easy implementation.
Example 6:
application test of bio-organic fertilizer
The bacillus mucilaginosus bio-organic fertilizer prepared by the method is used as a base fertilizer for field planting experiments of tomatoes (with quality being doubled) in Boxing county in Shandong, and the planting density is 960 plants per mu. On the basis of applying 30kg of calcium superphosphate base fertilizer, four groups of habitual fertilization CK1, common cow dung organic fertilizer CK2, biological organic fertilizer Treat1, biological organic fertilizer Treat2 and the like are arranged in a test way, 3 groups are repeated, and the area of a cell is 0.05 mu (48 plants). Applying 15% compound fertilizer for 1 time during planting period; the other cultivation measures such as fertilization time, irrigation, pesticide spraying and the like are the same. Picking is started after the ripe fruits are ripe, and the picking is finished within 20 days.
TABLE 5 application test arrangement (Kg/mu) of bio-organic fertilizer in tomatoes
TABLE 6 influence of the Bio-organic fertilizer on tomato growth
The results show that the growth potential, quality, fruit setting rate and yield of the treatment 4 are the best, but the differences between the treatment 3 and the treatment 4 are small, the treatment 3 is superior to conventional fertilization CK1 and common organic fertilizer CK2, and the treatment 3 is the best from the perspective of input and output. The soil applied with the bio-organic fertilizer is loose, and the incidence of diseases of plants is low.
Of course, the above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, and will not be described herein again; the above embodiments are merely for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention has been described in detail with reference to the preferred embodiments, and those skilled in the art should understand that changes, modifications, additions or substitutions which are made by those skilled in the art within the spirit of the present invention are also within the scope of the claims of the present invention.
Claims (7)
1. Bacillus mucilaginosus (Bacillus mucilaginosus) M-1 with potassium-dissolving function and growth-promoting capability has the preservation number: CGMCC No. 19428.
2. The use of Bacillus mucilaginosus M-1 as claimed in claim 1 in biofertilizers.
3. A method for preparing a bio-organic fertilizer by using the bacillus mucilaginosus M-1 solid state fermentation of claim 1, which is characterized in that: the method comprises the following steps:
(1) activating the slant seeds: inoculating bacillus mucilaginosus M-1 to a nutrient agar slant culture medium in a sterile manner, and culturing at the temperature of 37 +/-2 ℃ for 36-48 h to obtain slant seeds;
(2) culturing liquid seeds: inoculating the slant seeds obtained in the step (1) into a liquid culture medium, and culturing at 37 +/-1 ℃ for 36-48 h to obtain liquid seeds;
(3) pre-treating the substrate: crushing a material substrate, adjusting the moisture content to 55-60%, placing the mixed material in a fermenter, heating to 75 +/-3 ℃, and quickly performing harmless treatment and decomposition at a high temperature for 8-10 hours until the odor disappears;
(4) solid state fermentation culture: inoculating 5-10 parts by weight of the liquid seeds obtained in the step (2) into the pretreated substrate obtained in the step (3), culturing at 37 +/-2 ℃ for 5-7 days to obtain a fermentation product, and drying the fermentation product until the water content is less than or equal to 15% to obtain the final bio-organic fertilizer.
4. The method for preparing the bio-organic fertilizer by solid state fermentation according to claim 3, characterized in that: the material matrix comprises the following components in parts by weight: 50-60 parts of cow dung, 20-30 parts of mushroom residues, 10-20 parts of sawdust and 2-4 parts of molasses.
5. The method for preparing the bio-organic fertilizer by solid state fermentation according to claim 3, characterized in that: the pH value of the M-1 liquid seed inoculated in the step (4) is 6.5-7.5.
6. A bio-organic fertilizer prepared by using the method for preparing the bio-organic fertilizer by solid state fermentation according to any one of claims 3 to 5.
7. The bio-organic fertilizer according to claim 6, characterized in that: the content of viable count of bacillus mucilaginosus M-1 in the bio-organic fertilizer is 1.2 multiplied by 109More than one per gram.
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