CN108676755B - Microbial liquid fertilizer containing bacillus and preparation method and application thereof - Google Patents
Microbial liquid fertilizer containing bacillus and preparation method and application thereof Download PDFInfo
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- CN108676755B CN108676755B CN201810562169.4A CN201810562169A CN108676755B CN 108676755 B CN108676755 B CN 108676755B CN 201810562169 A CN201810562169 A CN 201810562169A CN 108676755 B CN108676755 B CN 108676755B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a liquid microbial fertilizer containing bacillus, which contains 2.0 × 10 viable count8cfu/ml~2.5×108cfu/ml fermentation liquid of bacillus, wherein the bacillus is a mixed bacterium of bacillus licheniformis and bacillus subtilis, and the bacillus licheniformis is bacillus licheniformis (C: (C))Bacillus licheniformis) W205, the strain is preserved in the China general microbiological culture Collection center on 8 th 2016 with the preservation number as follows: CGMCC No. 12817; the Bacillus subtilis is Bacillus subtilis (B.) (Bacillus subtilis) W309, the strain is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms at 2016, 8 months and 8 days, and the preservation number is as follows: CGMCC No. 12815. Also discloses a preparation method and application thereof in tea tree planting. The microbial liquid fertilizer provided by the invention has the advantages of simple preparation method, low cost, simple use and no environmental pollution, can obviously improve the yield, taste and quality of tea leaves, and has important significance for promoting the tea production and the sustainable development of ecological tea gardens.
Description
Technical Field
The invention relates to the technical field of agricultural fertilizers, in particular to a microbial liquid fertilizer containing bacillus and a preparation method and application thereof.
Background
As a main tea-producing country in the world, the tea consumption in China is rapidly increased in recent years, and reaches 161.32 ten thousand tons in 2013, which is 225 percent higher than 2001. Meanwhile, the global tea consumption increased from 298.58 ten thousand tons in 2001 to 482.1 ten thousand tons in 2013. The market demands a great deal of tea, and the large-area planting and the quality improvement of the tea are directly promoted. At present, China has become the fastest growing and most potential tea market worldwide. Improving the tea quality and increasing the tea yield are two key technical problems of improving the market competitiveness of tea products and increasing the economic benefits of tea manufacturing enterprises. Fertilizing is one of the most important agricultural measures for improving the yield and the quality of tea and promoting the sustainable development of tea gardens. In the prior art, a plurality of reports are reported on the research of tea fertilization, but the applied fertilizers mainly comprise chemical fertilizers, organic fertilizers and leguminous green fertilizers. However, with the blind use of chemical fertilizers in tea gardens and the increasingly prominent ecological problems and environmental problems caused by unreasonable fertilization modes, the development of environment-friendly and efficient fertilizers capable of promoting the sustainable development of tea gardens is of great importance and urgency.
The microbial fertilizer is a living fertilizer, is a novel fertilizer in twenty-first century, and is an organic microbial agent with low carbon, pure nature, no toxicity, no harm and no pollution. The biological fertilizer is mainly completed by the metabolism of the life activities of a large number of beneficial microorganisms, and has the functions of improving the soil fertility, increasing the number and the activity of the beneficial microorganisms in the soil, improving the soil properties, preventing the soil from hardening, improving the soil fertility, water retention and cold resistance, enhancing the disease resistance of crops, increasing organic matters in the soil, preventing the invasion of pathogenic bacteria, reducing the growth of plant diseases and insect pests, promoting the growth of crops, improving the yield of the crops, improving and reducing the quality of agricultural products and the like. Microbial fertilizers are classified into solid and liquid forms. The liquid microbial fertilizer is favored by the tea planting industry by the advantages of instant dissolution, uniformity, instant use, sprinkling irrigation and irrigation, difficult moisture absorption and caking and the like. However, the research on promoting tea planting by microbial fertilizers in the prior art generally focuses on promoting soil fertility and improving yield, and the research on promoting tea quality is relatively simple in function and relatively less. In addition, the technicians in the industry find that in the process of applying the liquid microbial fertilizer, the liquid fertilizer has the problems of poor stability, obvious reduction of the number of viable bacteria in the liquid fertilizer along with the increase of storage time and the reduction of the efficacy.
Disclosure of Invention
The invention aims to provide a microbial liquid fertilizer containing bacillus and a preparation method and application thereof, and aims to solve the problems of single function and poor storage stability of the conventional microbial liquid fertilizer.
The invention aims to realize the technical scheme that the liquid fertilizer containing the bacillus is a microbial liquid fertilizer containing 2.0 × 10 viable bacteria8cfu/ml～2.5×108The Bacillus is a mixed bacterium of Bacillus licheniformis (Bacillus licheniformis) W205 which is separated from farmland soil of longevity mountain, countryside and village in Shanghai province of Jinan, Fuzhou province, the strain is preserved in the general microorganism center of China Committee for culture Collection of microorganisms 8 months 8 days 2016, and the preservation number is as follows: CGMCC No. 12817; the thallus of the strain is rod-shaped, the length of the thallus is 2.0-3.0 mu m, and the width of the thallus is 0.3-0.5 mu m; the Bacillus subtilis is Bacillus subtilis W309 separated from farmland soil of shorea village and countryside village in Jinan area of Fuzhou city, Fujian province, and the strain is separated in 20168 months and 8 days, the microbial inoculum is preserved in the China general microbiological culture Collection center with the preservation number as follows: CGMCC No.12815, the thallus is rod-shaped, the length is 1.0-3.0 μm, and the width is 0.5-0.8 μm.
The fermentation broth is prepared by inoculating Bacillus subtilis in fermentation medium, fermenting at 33 deg.C for 12 hr, inoculating Bacillus licheniformis, and fermenting at 33 deg.C for 72 hr to obtain Bacillus with viable count of 2.0 × 108cfu/ml～2.5×108The fermentation medium is a liquid medium which is prepared by taking starch wastewater of a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0 and sterilizing.
Organic acid can be added into the liquid fertilizer to increase the storage stability of the liquid fertilizer; the adding mass of the organic acid is 0.01-2.0% of the mass of the liquid fertilizer, and the preferable mass is 0.06%; preferably, the organic acid is any one of formic acid, acetic acid or citric acid. The viable count of the liquid fertilizer added with the organic acid can be stably kept for 3 months at normal temperature (25 ℃).
The invention also discloses a preparation method of the microbial liquid fertilizer containing the bacillus, which comprises the following steps:
(a) inoculating bacillus licheniformis into L B culture medium, performing shaking culture at 33 ℃ for 24h at 180r/min in a constant temperature shaking table to obtain bacillus licheniformis liquid seeds, wherein the bacillus licheniformis is bacillus licheniformis (Bacillus licheniformis) W205, and the strain is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 8 and 12817;
(b) respectively inoculating bacillus subtilis to L B culture media, and performing shaking culture at 180r/min for 24h in a constant-temperature shaking table at 33 ℃ to prepare bacillus subtilis liquid seeds, wherein the bacillus subtilis is bacillus subtilis W309, is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 8 and 35 days, and has the preservation number of CGMCC No. 12815;
(c) inoculating the bacillus subtilis liquid seeds into a fermentation culture medium according to the inoculation amount of 3% by mass volume, aerating, fermenting for 12 hours at 33 ℃, then inoculating the bacillus licheniformis liquid seeds into the fermentation culture medium according to the inoculation amount of 3% by mass volume, aerating, mixing and fermenting at 33 ℃, supplementing the fermentation culture medium in the fermentation process, fermenting for 72 hours, and collecting fermentation liquor to obtain the microbial liquid fertilizer containing bacillus.
Adding organic acid into the fermentation liquor in the step (c) in order to increase the efficacy stability of the microbial liquid fertilizer; the adding mass of the organic acid is 0.01-2.0% of the mass of the fermentation liquor, and the preferable mass is 0.06%; the organic acid is one of formic acid, acetic acid or citric acid; the viable count of the liquid fertilizer added with the organic acid can be stably kept for 3 months at normal temperature (25 ℃).
The L B culture medium comprises 10 g/L of peptone, 5 g/L of yeast extract and 10 g/L of sodium chloride, the pH value is 6.0-8.0, and the sterilization is carried out for 20min at 121 ℃.
The fermentation medium is a liquid medium prepared by taking starch wastewater of a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to be 6.0-8.0, and sterilizing at 121 ℃ for 20min, wherein the COD of the starch wastewater is 18000-22000 mg/L.
The fermentation culture medium is supplemented for the first time in 24h of fermentation, the supplementation volume is 40-60% of the volume of the original fermentation liquid, and the fermentation culture medium is supplemented for the second time in 56h of fermentation, and the supplementation volume is 20-40% of the volume of the fermentation liquid before supplementation.
Experiments prove that the microbial liquid fertilizer provided by the invention can improve the tea yield, the tea quality, the soil environment and the microbial flora, and can be applied to tea planting production.
In tea planting, the method for applying the microbial liquid fertilizer comprises the steps of adjusting the pH value of the microbial liquid fertilizer to 7.0 by using a sodium hydroxide solution with the concentration of 6 mol/L before use, and diluting the microbial liquid fertilizer by 50 times by using the fermentation medium, wherein the recommended application rate is 1500L/mu.
According to the invention, the mixed Bacillus microbial liquid fertilizer is prepared by screening Bacillus licheniformis (Bacillus licheniformis) W205 and Bacillus subtilis (Bacillus subtilis) W309, and is applied to a tea garden, and the fertility effect is good through comparison; the soil of the tea garden can be obviously improved; the content of organic matters and humic acid in the tea garden soil is obviously improved, and the improvement ranges are 109.1% and 103.1% respectively; the contents of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the tea garden soil can be obviously improved by 86.6 percent, 98.8 percent and 39.6 percent respectively; the diversity of the tea garden soil microbial communities can be obviously improved; the method can obviously increase the yield of the tea, improve the quality of the tea, and obviously increase the contents of tea water extract, free amino acid and aroma substances, and particularly, the method obviously reduces the content of caffeine in the tea by 22.4 percent. It is known that dry tea leaves contain a certain amount of caffeine. The high content of caffeine causes the tea to become bitter; in addition, caffeine can excite the central nervous system of people and enhance the excitation of cerebral cortex, and especially, insomnia is easy to cause by a large number of people drinking tea at night. The liquid fertilizer provided by the invention can reduce caffeine in tea, remarkably improve the taste of tea and solve the problem of easy insomnia when drinking tea. In addition, the invention effectively solves the problems of short storage time and fast fertilizer efficiency attenuation of the liquid fertilizer in the industry by adding the organic acid into the liquid fertilizer. The preparation method is simple, low in cost and simple to use, is safe to people and livestock, does not cause environmental pollution, and has important significance for promoting tea production and sustainable development of ecological tea gardens.
Drawings
FIG. 1 is an electron microscope scan of Bacillus licheniformis (Bacillus licheniformis) W205.
FIG. 2 is an electron micrograph of Bacillus subtilis W309.
FIG. 3 is a PCoA analysis diagram of difference of soil microbial community structures of a CK group and a DKYF group tea garden.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. In the following examples, unless otherwise specified, the culture medium and experimental conditions in the examples were those of the conventional ones. The test materials, reagents and the like used in the examples are commercially available unless otherwise specified.
Example 1 acquisition and characterization of Bacillus licheniformis (Bacillus licheniformis) W205 and Bacillus subtilis W309
1. Strain isolation
Weighing 5g of soil sample in a sterilized centrifuge tube, adding 5 times of sterile water, filtering the soil, inoculating the filtrate in L B plate culture medium, culturing at constant temperature at 37 ℃ until bacterial colonies appear, selecting bacteria with good growing edges by using an inoculating needle, inoculating the bacteria on a new L B plate culture medium, after the newly inoculated endophyte grows into bacteria, selecting the endophyte at the edges, streaking and culturing, repeatedly purifying in the way, obtaining 18 single bacterial colonies in total, and selecting two single bacterial colonies, wherein the two single bacterial colonies are named as W205 and W309 respectively.
The L B plate culture medium comprises peptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, agar 15 g/L, pH 7.0, and is sterilized at 121 ℃ for 20 min.
2. Identification of strains
(1) And (3) observing the shape of the thallus: observing under a scanning electron microscope, the thallus of the strain W205 is rod-shaped, the length is 2.0-3.0 μm, and the width is 0.3-0.5 μm, as shown in figure 1; the thallus of the strain W309 is rod-shaped, the length is 1.0-3.0 μm, and the width is 0.5-0.8 μm; as shown in fig. 2.
(2) Physiological and biochemical characteristics of strain W205 and strain W309: the strain W205 and the strain W309 can grow in a culture medium containing glucose, fructose and sucrose as carbon sources; the results of the starch hydrolysis test were positive and are shown in Table 1.
TABLE 1 part of the physiological and biochemical characteristics of Strain W205 and Strain W309
| Experimental project | Results | Experimental project | Results | Experimental project | Results | Experimental project | Results |
| Glucose | + | Fructose | + | Sucrose | + | Starch hydrolysis | + |
(4) The molecular biology identification of the strain W205 and the strain W309 comprises the steps of extracting the whole genome of the strain W205 and the strain W309 by using a bacterial whole genome rapid extraction kit, carrying out PCR by using primers F9-27 (5'-GAGTTTGATCCTGGCTCA G-3') and R1525-1539(5 ' -AGAAAGGAGGTGATCCAGCC-3), and then carrying out sequencing analysis, comparing a sequencing result by B L AST, identifying the strain W205 as Bacillus licheniformis (Bacillus licheniformis) W205, storing the strain in China general microbiological culture collection center at 2016, 8 and 8 days, wherein the storage number is CGMCC No.12817, the nucleotide sequence is shown as SEQ ID NO:1, identifying the strain W309 as Bacillus subtilis W309, storing the strain in China general microbiological culture collection center at 2016, 8 and 8 days, wherein the storage number is CGMCC No.12815, the nucleotide sequence is shown as SEQ ID NO:2, and the northern Asian culture Collection center No. 100101, Beijing university institute No. 3, Japan society for Japan microbial culture Collection.
Example 2 preparation of a microbial liquid fertilizer
(1) L B culture medium is prepared from peptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L by adjusting pH to 7.0 and sterilizing at 121 deg.C for 20 min.
The preparation method of the fermentation medium comprises the steps of taking starch wastewater of a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0, and sterilizing at 121 ℃ for 20min to obtain the product.
(2) Inoculating Bacillus licheniformis (Bacillus licheniformis) W205 obtained in example 1 onto L B culture medium, and shake culturing at 37 deg.C and 180r/min for 24h to obtain Bacillus licheniformis liquid seed;
(3) inoculating the Bacillus subtilis W309 obtained in example 1 to L B culture medium, and performing shaking culture at a constant temperature of 37 ℃ for 24h at a speed of 180r/min in a shaking table to prepare Bacillus subtilis liquid seeds;
(4) inoculating bacillus subtilis liquid seeds into a fermentation culture medium in a fermentation tank according to the inoculation amount of 3% by mass volume, aerating, fermenting for 12 hours at 33 ℃, then inoculating bacillus licheniformis liquid seeds into the fermentation culture medium in the fermentation tank, aerating, fermenting for 72 hours at 33 ℃, supplementing the fermentation culture medium for the first time in 24 hours of fermentation, wherein the supplemented liquid volume is 55% of the volume of the fermentation liquid before supplementation, supplementing the fermentation culture medium for the second time in 56 hours of fermentation, wherein the supplemented liquid volume is 35% of the volume of the fermentation liquid before supplementation, and detecting that the total number of viable bacillus contained in the fermentation liquid is 2.23 × 10 when the fermentation is carried out for 72 hours8cfu/ml, and collecting fermentation liquor to obtain the microbial liquid fertilizer.
Example 3 application of microbial liquid fertilizer to tea production
The test is carried out in Hangzhou county flood pool tea field (with an elevation of about 1000 m) in Longyan City of Fujian province, the variety of the tea tree is Tieguanyin, and the tree age is 12 years. The planting mode is close planting in ridge planting, and the planting density is 3000-. The physical and chemical index parameters of the initial soil of the tea garden are as follows: the pH value is 5.8, the total nitrogen is 0.203%, the total phosphorus is 0.026%, the total potassium is 1.12%, the alkaline hydrolysis nitrogen is 138.1mg/kg, the available phosphorus is 106.7mg/kg, the quick-acting potassium is 297mg/kg, the organic matter is 45.0g/kg, and the humic acid is 17.1 g/kg.
The test was divided into two groups: blank control group (CK group) and test group (DKYF group).
Wherein clear water is applied to a blank control group (CK group) at the application rate of 1500L/mu;
the test group (DKYF group) applied the liquid fertilizer for microorganisms prepared in example 2 of the present invention, which was diluted 50-fold with fermentation medium before use at an application rate of 1500L/mu.
Each treatment area is 0.5 mu, each treatment is repeated for 4 times, and the foliar spraying and furrow application are carried out in 2016, 8 months, 10 days and 9 months, 1 day, wherein the foliar spraying amount and the furrow application amount respectively account for about 50 percent. Soil and tea leaves are picked in 2016, 10 and 20 days, and the tea leaves are processed according to the Tie Guanyin green-making process to prepare dry tea.
And (4) detecting the microbial flora of the soil in the tea gardens and the yield and quality of the tea leaves treated differently.
1. The detection method comprises the following steps:
the tea water extract is determined according to GBT 8305 and 2013 tea water extract determination;
the total amount of free amino acids is determined according to GBT 8314 and 2013 total amount of free amino acids in tea;
the tea polyphenol determination refers to a method for detecting the contents of tea polyphenol and catechins in tea leaves in GBT 8313-2008;
the caffeine determination refers to GBT 8312 and 2013 Thea caffeine determination;
the aroma extraction method comprises the steps of (1) sample processing, wherein 10.0g of a ground tea sample is weighed in a 150m L triangular flask by adopting an HS-SPME method, 2mg/m L ethyl decanoate (internal standard) 25 mu L and boiling distilled water 100m L are added, a top hollow threaded cover of a silica gel spacer is covered tightly, the stirring speed is 450rpm, the mixture is balanced in a 50 ℃ oven for 5min, an extraction head is inserted into the triangular flask to be adsorbed above the liquid surface of the tea soup for 40min, the extraction temperature is 50 ℃, finally, the mixture is desorbed at 230 ℃ by a GC-MS injection port for 5min, (2) gas chromatography-mass spectrum conditions are that a chromatographic column is HP-5MS (30m 8650.25 mm NI ×.25 mu m film thickness), a carrier gas is high-purity helium, a sample inlet temperature is 230 ℃, a pulse is not distributed on the sample inlet, the sample inlet is L, the flow rate of the chromatographic column is 1m L/mass spectrum interface temperature, the chromatographic column interface temperature is 250 ℃, the ion energy mode is 230 ℃, the ion energy is further, the sample inlet is 58min, the sample inlet temperature is kept for 70 min, the peak area of the sample inlet is kept in a GC-MS analysis mode, the peak area is kept in combination with a GC-MS analysis method, the GC-MS analysis is kept for 5 ℃, the peak area of a GC-MS analysis is kept in a GC-MS analysis method, the standard temperature analysis method, the area of the GC-MS analysis is kept for 5min, the area of the peak area is kept in combination of the area of a GC-MS analysis is kept for 5min, the standard spectrum analysis, the area is kept in the area of.
The tea garden soil fertility detection method comprises the following steps: the pH value, organic matter, alkaline hydrolysis nitrogen, available phosphorus, quick-acting potassium, total nitrogen, total phosphorus, total potassium and humic acid content are respectively detected according to NY/T1377 + 2007, NY/T1121.6-2006, soil agricultural chemical analysis method, NY/T1121.7-2006, NY/T889 + 2004, NY/T53-1987, NY/T88-1988, NY/T87-1988 and soil agricultural chemical analysis method.
And (3) detecting microorganisms in the tea garden soil: according to the kit instructions (
SPIN Kit for Soil, MPBIO, USA) to extract total DNA of Soil, and then analyzing the diversity of Soil microorganisms by using 16S rDNA sequencing method and bioinformatic analysis software.
The detection results are as follows:
TABLE 2 yield and physicochemical indices of tea leaves of the test group (DKYF) and of the blank control group (CK group)
Note: results are expressed as mean ± standard deviation (n ═ 4), and the upper right of the same row of data indicates significance compared to the CK group (P < 0.05, independent sample t-test).
As can be seen from Table 2, compared with the CK group, the yield of the tea in the DKYF group is obviously increased (P is less than 0.05), the amplification is 12%, and the application of the microbial liquid fertilizer can obviously improve the yield of the tea. Meanwhile, the results of physical and chemical index detection show that the water extract content and the free amino acid content of the tea in the DKYF group are both obviously increased (P is less than 0.05), the increase of the water extract content and the free amino acid content are respectively 8.9% and 11.8% relative to the CK group, the caffeine content is obviously reduced (P is less than 0.05), the reduction amplitude is 22.4%, the tea polyphenol content is slightly increased, but the difference is not obvious (P is more than 0.05).
TABLE 3 aroma component content of tea leaves of test group (DKYF) and blank control group (CK group)
Note: results are expressed as mean ± standard deviation (n ═ 4), and the upper right of the same row of data indicates significance compared to the CK group (P < 0.05, independent sample t-test).
The test results in Table 3 show that the application of the microbial liquid fertilizer can obviously improve the content of aromatic substances such as trans-2-nonen-1-ol, cis-3-hexenylbutyl ester, hexyl butyrate, 2-methyl butanoic acid leaf alcohol ester, isoamyl hexanoate, hexanoic acid leaf alcohol ester, hexanoic acid hexyl ester, cis-jasmone, trans- β -farnesene, β -ionone, (Z, E) - α -farnesene, nerolidol and hexanaphthestrol (P is less than 0.05), and reduce the content of benzaldehyde, phenylacetaldehyde and linalool (P is less than 0.05).
TABLE 4 physicochemical indices of the soil in the test (DKYF) and blank Control (CK) tea gardens
Note: results are expressed as mean ± standard deviation (n ═ 4), and the upper right of the same row of data indicates significance compared to the CK group (P < 0.05, independent sample t-test).
Table 4 shows that, compared with the CK group, the total nitrogen content, alkaline hydrolysis nitrogen, available phosphorus, quick-acting potassium, organic matter and humic acid content of the tea garden soil in the DKYF group are significantly higher (P is less than 0.05), and the increase of the DKYF group relative to the CK group respectively reaches 90.3%, 86.6%, 98.8%, 39.6%, 109.1% and 103.1%, which indicates that the liquid fertilizer DKYF provided by the invention can significantly improve the utilization rate of the tea garden soil fertilizer and improve the soil fertility.
TABLE 5 microbial diversity of the test (DKYF) and blank Control (CK) tea garden soils
Note: results are expressed as mean ± standard deviation (n ═ 4), and the upper right of the same row of data indicates significance compared to the CK group (P < 0.05, independent sample t-test).
Table 5 shows that compared with the CK group, the microbial community Shannon and Simpson indexes of the tea garden soil in the DKYF group are significantly higher (P is less than 0.05), which indicates that the application of the liquid fertilizer DKYF provided by the present invention can significantly improve the abundance and uniformity of the microbial community in the tea garden soil.
The β diversity of the microbial community can be generally analyzed by adopting a PCoA (Principal Coordinate Analysis) method, the difference of the microbial community of the tea garden soil between a CK group and a DKYF group (each group has 4 repetitions) is analyzed by the PCoA method, and the result is shown in figure 3. as can be seen from figure 3, the DKYF group and the CK group can be obviously distinguished, which shows that the microbial liquid fertilizer provided by the invention can obviously regulate the microbial community structure of the tea garden soil.
The test results show that the microbial liquid fertilizer prepared by the method can obviously increase the soil fertility of the tea garden and the diversity of microbial communities, obviously improve the yield of tea leaves, obviously reduce caffeine in the tea leaves, and has obvious effects of improving the taste of the tea leaves and improving the quality of the tea leaves.
Example 4 storage stability of the microbial liquid fertilizer of the invention
Taking 4 parts of the microbial liquid fertilizer prepared in the example 2, wherein each part is 1kg, and 3 parts of the microbial liquid fertilizer are respectively added with acetic acid, the addition amount of the acetic acid is 0.01 percent, 0.06 percent and 2 percent of the mass of the microbial liquid fertilizer, and the untreated part is a blank control.
4 parts of liquid fertilizer are respectively packaged and stored at normal temperature (25 +/-1 ℃), and the survival number of cells is detected every month. The detection method comprises the following steps: plate counting method; calculating the survival rate of viable bacteria of the microbial liquid fertilizer under different treatments according to the following formula:
the survival rate is × 100% of the viable count of the microbial liquid fertilizer when the microbial liquid fertilizer is stored for N months/the initial viable count of the microbial liquid fertilizer, wherein N is 1, 2, 3 or 4.
TABLE 6 survival rates of liquid microbial fertilizers treated differently
The detection result shows that the survival rate of viable bacteria of the microbial liquid fertilizer can be basically kept stable within 3 months when the microbial liquid fertilizer prepared by the invention is added with acetic acid, and the same technical effect can be achieved by replacing acetic acid with formic acid and citric acid.
In addition, the microbial liquid fertilizer added with the organic acid is diluted and applied by the same method by using a proper amount of sodium hydroxide solution with the concentration of 6 mol/L to adjust the pH value to 7.0 before use, and the technical effect is basically consistent with that of the embodiment 3.
The above are only a few specific embodiments of the present invention. The present invention is not limited to the above embodiments, and 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.
SEQUENCE LISTING
<110> institute of agricultural engineering technology of agricultural academy of sciences of Fujian province
<120> microbial liquid fertilizer containing bacillus and preparation method and application thereof
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<170>PatentIn version 3.3
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Claims (9)
1. The liquid microbial fertilizer containing bacillus is characterized by containing 2.0 × viable bacteria108cfu/ml ~2.5×108cfu/ml of fermentation liquor of bacillus, wherein the bacillus is a mixed bacteria of bacillus licheniformis and bacillus subtilis, and the bacillus licheniformis is bacillus licheniformis (b: (b))Bacillus licheniformis) W205, the strain is preserved in the China general microbiological culture Collection center on 8 th 2016 with the preservation number as follows: CGMCC No. 12817; the bacillus subtilis is bacillus subtilis (A), (B)Bacillus subtilis) W309, the strain is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms at 2016, 8 months and 8 days, and the preservation number is as follows: CGMCC No. 12815.
2. The bacillus-containing microbial liquid fertilizer according to claim 1, wherein an organic acid is added to the liquid fertilizer, and the mass ratio of the organic acid to the liquid fertilizer is 0.01-2.0%.
3. The bacillus-containing microbial liquid fertilizer according to claim 2, wherein the organic acid is any one of formic acid, acetic acid and citric acid.
4. The bacillus-containing microbial liquid fertilizer according to claim 1, 2 or 3, wherein the bacillus licheniformis (b) isBacillus licheniformis) The thallus of W205 is rod-shaped, the length is 2.0-3.0 μm, and the width is 0.3-0.5 μm; the Bacillus subtilis (A), (B) and (C)Bacillus subtilis) The W309 bacterial cells are rod-shaped, and have a length of 1.0 to 3.0 μm and a width of 0.5 to 0.8. mu.m.
5. A preparation method of a microbial liquid fertilizer containing bacillus is characterized by comprising the following steps:
(a) inoculating Bacillus licheniformis into L B culture medium, shaking and culturing in a shaking table at 33 deg.C for 24 hr at 180r/min to obtain Bacillus licheniformis liquid seedBacillus licheniformis) W205, the strain was deposited in the China culture Collection in 2016, 8/8The general microbiological center of the management committee, the preservation number is: CGMCC No. 12817;
(b) inoculating Bacillus subtilis to L B culture medium, shaking and culturing at 33 deg.C in a shaking table at 180r/min for 24 hr to obtain Bacillus subtilis liquid seedBacillus subtilis) W309, the strain is preserved in the general microbiological culture collection center of China Committee for culture Collection of microorganisms at 2016, 8 months and 8 days, and the preservation number is as follows: CGMCC No. 12815;
(c) inoculating the bacillus subtilis liquid seeds into a fermentation culture medium in a fermentation tank according to the inoculation amount of 3% by mass volume, aerating, fermenting at 33 ℃ for 12 hours, inoculating the bacillus licheniformis liquid seeds into a fermentation liquid in the fermentation tank according to the inoculation amount of 3% by mass volume, aerating, mixing and fermenting at 33 ℃, supplementing the fermentation culture medium in the fermentation process, fermenting for 72 hours, and collecting the fermentation liquid to obtain the microbial liquid fertilizer containing bacillus.
6. The method for producing a microbial liquid fertilizer according to claim 5, wherein an organic acid is added to the fermentation liquid produced in (c), and the organic acid is added in an amount of 0.01 to 2.0% by mass based on the fermentation liquid, to produce a microbial liquid fertilizer containing Bacillus.
7. The method for preparing a liquid fertilizer containing Bacillus according to claim 6, wherein the organic acid is formic acid, acetic acid or citric acid.
8. The preparation method of the microbial liquid fertilizer according to claim 5, 6 or 7, wherein the fermentation medium is a liquid medium prepared by using starch wastewater from a potato starch factory as a raw material, adding 10-30 g/L of soybean meal, adjusting the pH value to 6.0-8.0, and sterilizing.
9. Use of a liquid microbial fertilizer comprising bacillus as claimed in claim 1, 2 or 3 in the production of tea.
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| CN111528237A (en) * | 2020-05-04 | 2020-08-14 | 江苏春江生物科技有限公司 | Application of microbial fermentation preparation in biological control of tea lesser leafhoppers |
| CN111548968A (en) * | 2020-05-28 | 2020-08-18 | 武汉微康益生菌研究院有限公司 | Bacillus coagulans BC99 and screening method thereof |
| CN112998032B (en) * | 2021-03-02 | 2021-12-17 | 广东省科学院生物工程研究所 | Crop leaf surface spraying type bacillus inoculant and preparation method thereof |
| CN114507623B (en) * | 2022-03-04 | 2023-03-10 | 安徽农业大学 | Bacillus and application thereof |
| CN116063111A (en) * | 2022-11-23 | 2023-05-05 | 河北冀微绿色农业科技有限公司 | Application of bacillus subtilis M-15 in preparation of microbial fertilizer |
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