CN112723921A - Bacillus amyloliquefaciens YA289 and amino acid fertilizer obtained by degrading same - Google Patents

Bacillus amyloliquefaciens YA289 and amino acid fertilizer obtained by degrading same Download PDF

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CN112723921A
CN112723921A CN202110150574.7A CN202110150574A CN112723921A CN 112723921 A CN112723921 A CN 112723921A CN 202110150574 A CN202110150574 A CN 202110150574A CN 112723921 A CN112723921 A CN 112723921A
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amino acid
nitrogen
content
bacillus amyloliquefaciens
fermentation
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余炼
李雨霖
邓雨萍
张梦菲
刘小玲
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Guangxi University
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    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05C—NITROGENOUS FERTILISERS
    • C05C11/00—Other nitrogenous fertilisers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

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Abstract

The invention belongs to the technical field of microbial degradation and application, and researches show that a dominant strain bacillus amyloliquefaciens YA289 is used for fermenting prawn leftovers, and the loss of nitrogen in the fermentation process is mainly realized through NH3Volatilization of NH3The volatile loss of nitrogen accounts for 19 to 42 percent of the total nitrogen. Therefore, the change of the nitrogen source is reflected by detecting the change of the ammonia nitrogen content of the fermentation liquor. The research result shows that under the condition of the optimal optimization process, the content of amino acid nitrogen and polypeptide is respectively improved by 142.4 percent and 13.14 percent compared with the content before optimization, the content of ammonia nitrogen is reduced by 1.5 percent compared with the content before optimization, and no obvious unpleasant odor exists. The prepared amino acid fertilizer can be absorbed and utilized by plants, and can be prepared into an amino acid liquid fertilizer, so that the yield and the quality of crops can be improved, the pesticide residue can be reduced, and the like.

Description

Bacillus amyloliquefaciens YA289 and amino acid fertilizer obtained by degrading same
Technical Field
The invention relates to the technical field of microbial degradation and application, in particular to bacillus amyloliquefaciens YA289 and an amino acid fertilizer obtained by degrading the same.
Background
The penaeus vannamei boone has the advantages of delicious meat, high nutritional value and the like, and gradually becomes a main penaeus vannamei boone breeding variety in China, and the breeding amount can account for more than 70% of the total prawn breeding amount. The processing mode of the south America white shrimps in China generally adopts a mode of removing frozen shrimps such as shrimp heads, shrimp shells and the like to export, and about 45 percent of waste can be generated in the production process. These wastes usually contain nutrients such as protein and astaxanthin, and improper disposal of these wastes pollutes the environment and wastes resources. The microbial fermentation method utilizes the metabolism of microorganisms, not only can change the quality of protein, but also can generate substances such as short peptides, amino acids and the like which are easy to digest and utilize, so that processing waste is converted into a fermentation product with high added value.
The amino acid nitrogen can quickly detect the hydrolysis degree of protein in food, and the amount of the amino acid nitrogen and the amount of polypeptide can be jointly used as an index for observing the protein hydrolysis condition after shrimp head fermentation. Meanwhile, leftovers rich in organic matters and the like are easy to generate malodorous gases including NH in the degradation process of the organic matters in the fermentation process3Sulfur compounds, amines and the like odors and N2O and the like, and the generation of these gases causes environmental pollution and a reduction in product quality.
Disclosure of Invention
In order to overcome the defect that the prior art lacks a method for preparing a biological fertilizer with rich amino acid nitrogen and pleasant smell by microbial degradation of prawn leftovers, the invention mainly aims to provide the application of the screened dominant strain bacillus amyloliquefaciens YA289 in preparation of the biological fertilizer with amino acids.
The second purpose of the invention is to provide a preparation method of the amino acid biological fertilizer.
The purpose of the invention is realized by the following technical scheme:
application of bacillus amyloliquefaciens YA289 in preparation of amino acid biofertilizer.
The research of the invention finds that the bacillus amyloliquefaciens YA289 can obviously improve the content of amino acid nitrogen and polypeptide by fermenting the raw materials rich in protein, and simultaneously obviously reduce the generation amount of ammonia nitrogen, thereby avoiding the odor after fermentation.
The bacillus amyloliquefaciens YA289 sequence used by the invention is uploaded to NCBI, and the accession numbers are as follows: GCA _ 000196735.1.
Preferably, the bacillus amyloliquefaciens YA289 is used for increasing the content of amino acid nitrogen and polypeptide in the biological fertilizer and reducing the content of ammonia nitrogen.
Therefore, preferably, the invention also provides the application of the bacillus amyloliquefaciens YA289 in reducing the content of ammoniacal nitrogen in amino acid biofertilizer.
The invention also provides an amino acid biological fertilizer, which is obtained by fermenting the prawn leftovers by the bacillus amyloliquefaciens YA 289.
Specifically, the leftovers of the prawns are mainly heads of the prawns.
Preferably, the fermentation process of the bacillus amyloliquefaciens YA289 is as follows: inoculating the bacterial suspension of the bacillus amyloliquefaciens YA289 into a sterilized prawn leftover culture medium, and performing shake culture and fermentation.
The sterilized prawn leftover culture medium is prepared by mixing prawn leftovers and water.
More preferably, the conditions for fermenting the bacillus amyloliquefaciens YA289 are as follows: the material-liquid ratio is 1: 7, NaCl adding amount of 5g/L, inoculation amount of 5 percent, pH6.2, rotation speed of 180rpm, 37 ℃ and 36 hours.
Compared with the prior art, the invention has the following beneficial effects:
the research of the invention finds that the dominant strain bacillus amyloliquefaciens YA289 is used for fermenting the leftovers of the prawns, and the loss of nitrogen in the fermentation process is mainly realized through NH3Volatilization of NH3The volatile loss of nitrogen accounts for 19 to 42 percent of the total nitrogen. Therefore, the change of the nitrogen source is reflected by detecting the change of the ammonia nitrogen content of the fermentation liquor. The research result shows that under the condition of the optimal optimization process, the content of amino acid nitrogen and polypeptide is respectively improved by 142.4 percent and 13.14 percent compared with the content before optimization, the content of ammonia nitrogen is reduced by 1.5 percent compared with the content before optimization, and no obvious unpleasant odor exists. The prepared amino acid fertilizer can be absorbed and utilized by plants, and can be prepared into an amino acid liquid fertilizer, so that the yield and the quality of crops can be improved, the pesticide residue can be reduced, and the like.
Drawings
FIG. 1 shows the effect of different carbon sources on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 2 is a graph showing the effect of different nitrogen sources on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 3 shows the effect of different NaCl addition levels on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 4 shows the effect of different inoculum sizes of bacteria liquid on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 5 is the effect of different pH values of the medium on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in the fermentation samples;
FIG. 6 shows the effect of different feed liquid ratios on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 7 is a graph showing the effect of different fermentation times on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 8 is a graph showing the effect of different fermentation temperatures on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples;
FIG. 9 shows the effect of different fermentation speeds on the content of amino acid nitrogen, polypeptide and ammonia nitrogen in fermentation samples.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
EXAMPLE 1 activation of Bacillus amyloliquefaciens YA289 and preparation of a bacterial suspension
(1) Activation of bacterial species
The bacillus amyloliquefaciens YA289 was inoculated on ISP2 plates, and the plates were placed in an incubator at 37 ℃ for 24 h.
(2) Preparation of the bacterial suspension
The 1-ring activated cells were inoculated into a 250mL Erlenmeyer flask containing 100mL of LB liquid medium, cultured at 37 ℃ and 200rpm for 24 hours, and the culture broth in the logarithmic phase was centrifuged (4000r/min, 10 min). After the collection of the cells, the cells were resuspended in 0.9% sterile physiological saline and the OD of the suspension was adjusted600Around 0.7.
(3) Fermentation of
An appropriate amount of the bacterial suspension is inoculated into a 250mL triangular flask containing 100mL of sterilized shrimp head culture medium (shrimp heads: water: 1:8) and cultured in a shaking table under the conditions of natural initial pH, 37 ℃, fermentation time of 48h and rotation speed of 200 rpm.
(4) Single factor experiment
Fermenting according to the conditions of Table 1, centrifuging the fermentation liquid (10000r/min, 5min), and taking out to determine the content of amino acid nitrogen, polypeptide and ammonia nitrogen in each fermentation sample.
TABLE 1
Figure BDA0002932550720000031
Figure BDA0002932550720000041
Firstly, determination of amino acid nitrogen content (GB5009.235-2016)
1. Preparation of Standard Curve
0mL, 0.05mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL of the standard solutions for precise extraction of ammonia nitrogen were placed in 10mL cuvettes, respectively. 4mL of sodium acetate-acetic acid buffer solution (pH 4.8) and 4mL of color developing agent were added to each cuvette, diluted to the scale with water, and mixed well. Heating in 100 deg.C water bath for 15min, taking out, cooling to room temperature, transferring into 1cm cuvette, measuring absorbance at wavelength of 400nm with zero tube as reference, and drawing standard curve or calculating linear regression equation.
2. Measurement of samples
Firstly, diluting a sample to be detected to a proper multiple, and uniformly mixing for later use. 2mL of the sample diluted solution was precisely aspirated into a 10mL colorimetric tube. Add 4mL sodium acetate-acetic acid buffer (pH 4.8) and 4mL color developer, dilute to the mark with water, mix well. Heating in 100 deg.C water bath for 15min, taking out, cooling to room temperature, transferring into 1cm cuvette, and measuring absorbance at wavelength of 400nm with reference to zero tube. And comparing the measurement result with a standard curve for quantification or substituting the measurement result into a linear regression equation to obtain the corresponding nitrogen content, and multiplying the corresponding nitrogen content by the dilution factor of the sample in the whole process to obtain the final result, wherein the unit of the nitrogen content is g/L.
II, determination of polypeptide content
1. Preparation of Standard Curve
A predetermined amount of 20 mg/mL was added to the test tube as shown in Table 2-1And (2) adding less than 1mL of bovine serum albumin solution to 1mL of bovine serum albumin solution by using distilled water, taking 1mL of distilled water as a blank, finally adding 4mL of biuret reagent into each test tube, fully and uniformly mixing, reacting at room temperature for 30min, measuring the light absorption value at 540nm by using an enzyme labeling instrument, and drawing a standard curve.
TABLE 2 preparation of Standard Curve
Figure BDA0002932550720000042
Figure BDA0002932550720000051
2. Measurement of samples
Taking 1mL of fermentation supernatant, adding 1mL of 10% trichloroacetic acid, uniformly mixing, standing for 10min, centrifuging for 15min at 5000 r/min, taking 1mL of supernatant in another test tube, adding 4mL of biuret reagent, uniformly mixing, standing for 30min at room temperature, measuring the light absorption value at 540nm, and measuring the light absorption value by adding 1mL of distilled water and 4mL of biuret reagent as blanks. Comparing with standard curve for quantification, and converting to obtain polypeptide concentration in fermentation supernatant.
Thirdly, measuring the content of ammoniacal nitrogen
1. Preparation of Standard Curve
Adding various reagents according to Table 3, mixing thoroughly, placing into 37 deg.C water bath for color development for 20min, taking out, cooling with water to room temperature, and measuring light absorption value at 637 nm.
TABLE 3 preparation of Standard Curve
Pipe number Solution A/mL Standard solution/. mu.L Water/. mu.L Solution B/mL
0 5 0 100 5
1 5 10 90 5
2 5 20 80 5
3 5 30 70 5
4 5 40 60 5
5 5 50 50 5
6 5 60 40 5
7 5 70 30 5
8 5 80 20 5
9 5 90 10 5
10 5 100 0 5
Standard ammoniacal nitrogen solution: 0.2358g of ammonium sulfate is accurately weighed and dissolved in water, the volume is constant to 100mL, namely the stock solution, the mass concentration of ammoniacal nitrogen is 500 mg.L-1(ii) a Accurately sucking 1mL of stock solution to dilute to 10mL, thus obtaining standard solution, wherein the mass concentration of ammoniacal nitrogen is 50 mg.L-1。
Solution A: 5.00g of phenol was weighed out accurately, dissolved in 400mL of water, and 2.0mL of sodium nitroferricyanide solution (1.25%) was added to make a volume of 500 mL.
Solution B: 2.50g NaOH, 2.0g trisodium citrate and 3.5mL NaClO were weighed out and dissolved in 400mL water to a volume of 500 mL.
2. Measurement of samples
Taking a proper amount of sample (the nitrogen content is 0.50-5.0 mu g), and performing other operations according to the standard curve. The measured result is compared with the standard curve for quantification or is substituted into a linear regression equation to obtain the corresponding ammoniacal nitrogen content.
Fourth, experimental results
1. Plackett-Burman test design
The Plackett-Burman test design was performed as per Table 4, with the results shown in Table 5.
TABLE 4
Figure BDA0002932550720000061
TABLE 5
Figure BDA0002932550720000062
TABLE 6
Figure BDA0002932550720000071
Note: significant levels (p < 0.05); significant level of star (p <0.01)
From the results in table 6, it is clear that A, D, F, G was selected as the main influencing factor to perform the orthogonal experiment.
2. Design of orthogonal experiments
The experiments were performed according to the orthogonal experimental design of table 7, and the experimental results are specifically shown in tables 8 and 9.
TABLE 7
Level of Material to liquid ratio of A D pH Temperature (. degree.C.) G time (h)
1 6 6.2 31 24
2 7 7.2 34 36
3 8 8.2 37 48
TABLE 8
Figure BDA0002932550720000072
Figure BDA0002932550720000081
TABLE 9
Amino acid nitrogen Polypeptides Ammoniacal nitrogen
A2D1F3G2 5.6207 0.3357 3.7235
A2D3F3G2 3.5754 0.3026 3.0977
A3D1F3G2 5.6861 0.1070 74.1846
Before optimization 2.3192 0.2967 3.7785
As can be seen from FIGS. 1 and 2, the addition of carbon and nitrogen sources did not significantly increase the amino acid nitrogen content, and the ammonia nitrogen content was high, so that the carbon and nitrogen sources were not added from the viewpoint of cost saving. Combining the results of Table 8 and Table 9, combination A was selected2D1F3G2Under the selected process conditions (the feed-liquid ratio is 1: 7, the NaCl addition amount is 5g/L, the inoculation amount is 5%, the pH value is 6.2, the rotation speed is 180rpm, 37 ℃ and 36 hours), the contents of amino acid nitrogen and polypeptide are respectively improved by 142.4% and 13.14% and the content of nitrogen is reduced by 1.5% compared with the content before optimization.

Claims (7)

1. Application of bacillus amyloliquefaciens YA289 in preparation of amino acid biofertilizer.
2. The use of claim 1, wherein bacillus amyloliquefaciens YA289 is used for increasing the content of amino acid nitrogen and polypeptides in a biological fertilizer and reducing the content of the amino acid nitrogen.
3. Application of bacillus amyloliquefaciens YA289 in reducing the content of ammoniacal nitrogen in amino acid biofertilizer.
4. An amino acid biofertilizer is characterized by being prepared by fermenting prawn leftovers by using bacillus amyloliquefaciens YA 289.
5. The amino acid biofertilizer of claim 4, characterized in that the fermentation process is: inoculating the bacterial suspension of the bacillus amyloliquefaciens YA289 into a sterilized prawn leftover culture medium, and performing shake culture and fermentation.
6. The amino acid biofertilizer of claim 5, wherein the sterilized prawn leftover culture medium is prepared by mixing prawn leftover with water.
7. The amino acid biofertilizer of claim 6, characterized in that the conditions for fermentation of bacillus amyloliquefaciens YA289 are: the material-liquid ratio is 1: 7, NaCl adding amount of 5g/L, inoculation amount of 5 percent, pH6.2, rotation speed of 180rpm, 37 ℃ and 36 hours.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719383A (en) * 2012-06-29 2012-10-10 武汉工业学院 Bacillus amyloliquefaciens, inoculant and application thereof
CN104591808A (en) * 2014-12-30 2015-05-06 浙江海洋学院 Method for fermenting aquatic product leftovers by mixed culture bacteria
CN112194520A (en) * 2020-10-30 2021-01-08 海口海森元生物科技有限公司 Black soldier fly compound amino acid fertilizer and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719383A (en) * 2012-06-29 2012-10-10 武汉工业学院 Bacillus amyloliquefaciens, inoculant and application thereof
CN104591808A (en) * 2014-12-30 2015-05-06 浙江海洋学院 Method for fermenting aquatic product leftovers by mixed culture bacteria
CN112194520A (en) * 2020-10-30 2021-01-08 海口海森元生物科技有限公司 Black soldier fly compound amino acid fertilizer and preparation method thereof

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Application publication date: 20210430