CN113061062A

CN113061062A - Method for preparing short peptide-growth-promoting bacterium compound fertilizer by dechromization fermentation of wet blue dander and application

Info

Publication number: CN113061062A
Application number: CN202110301702.3A
Authority: CN
Inventors: 刘彦; 王海燕; 刘晨; 王文靖; 张晓伟; 卢欣雨; 赵胜宗
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2021-07-02
Anticipated expiration: 2041-03-22
Also published as: CN113061062B

Abstract

The invention provides a short peptide-growth-promoting bacterium compound fertilizer prepared by dechromization fermentation of wet blue dander and application thereof in planting crops such as wheat, alfalfa and highland barley. The wet blue scurf is subjected to dechromization treatment to be used as a raw material for microbial fermentation, and bacillus pumilus is used for fermenting the dechromized wet blue scurf to obtain dechromized wet blue scurf fermentation liquor rich in free amino acid and polypeptide, namely the dechromized wet blue scurf fermentation liquor is used for preparing the oligopeptide-growth-promoting bacteria compound fertilizer, and is applied to crops such as wheat, alfalfa, highland barley and the like. Wherein the Bacillus pumilus BA06 has a preservation number of CGMCC NO. 0518. The invention organically integrates the conventional amino acid organic fertilizer and the growth-promoting bacterial manure, and has various effects of nutrition, growth promotion, disease resistance and the like on crops.

Description

Method for preparing short peptide-growth-promoting bacterium compound fertilizer by dechromization fermentation of wet blue dander and application

Technical Field

The invention relates to a method for preparing a short peptide-growth-promoting bacteria compound fertilizer by dechromization fermentation of wet blue dander, and relates to the short peptide-growth-promoting bacteria compound fertilizer and application thereof, belonging to the technical field of organic liquid fertilizer production processes.

Background

The tanning industry produces tens of thousands of tons of solid chromium-containing waste each year, with wet blue swarf accounting for approximately 60%. The blue wet dander (containing 2-4% of chromium element and 90% of protein) is rich in protein resources, so that the blue wet dander has high recycling value, but because the blue wet dander is difficult to degrade, the traditional treatment mode (landfill or incineration) can not only cause the waste of trivalent chromium and protein resources, but also generate the pollution of trivalent chromium.

Chemical processes (acid process, alkaline process) and biological processes (enzymatic process and microbiological process) are the main modes for degrading the tanning waste. The chemical method mainly utilizes stronger acid reagent or alkaline reagent to hydrolyze the tanning waste under severe conditions, and the method not only has large energy consumption and corrosion on equipment, but also has certain pollution to the environment.

In recent years, with the increasing emphasis on environmental issues, the use of biotechnology to treat waste has received much attention. The leather-making waste is treated by the enzyme method and the microorganism method according to the same principle, and the hydrolysis action of enzyme on the substrate is utilized, however, the microorganism method does not need to add enzyme independently, but depends on the enzyme generated by the self metabolism of microorganism to hydrolyze the substrate. Compared with an enzyme method, a microbial fermentation method is the main mode for preparing organic fertilizers in agriculture. After the skin protein is degraded by an enzyme method, the components of the product are single, the polypeptide is mainly contained, and the product contains a large amount of plant nutrients such as free amino acid and polypeptide, and bioactive substances such as plant endogenous enzymes, antibacterial active substances and plant growth regulators (cytokinin, indoleacetic acid and the like) generated by microbial metabolism and beneficial to plant growth through microbial fermentation, particularly plant probiotic fermentation.

The blue wet leather scraps are collagen-containing wastes with high chromium content, and in the aspect of resource utilization of the blue wet leather scraps in agriculture at present, the blue wet leather scraps are subjected to dechromization and then are hydrolyzed into micromolecular peptides and amino acid to prepare the amino acid organic fertilizer. In the aspect of degradation by microbial fermentation, although reports of degrading chrome leather scraps by actinomycetes, penicillium, pseudomonas aeruginosa, penicillium rugosum and the like are adopted at home and abroad, the good degradation effect is not achieved. Not only the degradation efficiency is not high, but also special high chromium-resistant strains need to be screened, and in addition, the degradation solution contains high-concentration chromium, which cannot meet the requirements of agricultural application.

Disclosure of Invention

In order to solve the problems in the prior art, the application provides a method for preparing a short peptide-growth-promoting bacteria compound fertilizer by dechromization fermentation of blue wet dander, the short peptide-growth-promoting bacteria compound fertilizer and the application thereof in planting crops such as wheat, alfalfa and highland barley. The blue wet dander is subjected to dechromization treatment to be used as a raw material for microbial fermentation, and agricultural probiotics are utilized to ferment the dechromized blue wet dander to obtain dechromized blue wet dander fermentation liquor rich in free amino acid and polypeptide, the dechromized blue wet dander fermentation liquor is used for promoting growth of bacterial manure, and the blue wet dander fermentation liquor is applied to crops such as wheat, alfalfa, highland barley and the like.

On one hand, the application provides a method for preparing a short peptide-growth-promoting bacteria compound fertilizer by dechromization fermentation of wet blue dander, which comprises the following steps:

1. dechromisation of wet blue dander

Specifically, the method for dechromizing blue wet dander by adopting an alkali-first acid-second combination method comprises the following steps:

firstly, performing alkali treatment, wherein in the step, the blue wet dander is soaked in alkali liquor and is mechanically stirred, so that alkali gradually permeates into the blue wet dander to perform dechromization reaction.

Wherein the alkali liquor is obtained by adding 6-8g of calcium oxide into water; the alkali consumption accounts for 8-10% of the mass of the blue dander; the alkali treatment time is 8-12 h; the alkali treatment temperature is normal temperature.

And then carrying out suction filtration to obtain blue wet leather scraps after alkali dechromization treatment.

And then carrying out acid treatment, wherein in the step, the alkali dechromization blue wet leather scraps which are subjected to suction filtration and dehydration are placed in an acid solution for soaking.

Wherein the acid solution is hydrochloric acid aqueous solution; the liquid ratio of the acid solution dosage is 1:20 based on the weight of the wet blue leather after the alkali dechromization treatment; the concentration of the acid solution is 0.6-0.9 mol/L; the acid treatment time is 30-35 min.

Finally, the wet blue scurf is obtained by suction filtration.

Furthermore, the blue wet leather scraps are chromium-containing leather waste materials generated by shaving, grinding and trimming after a tanning working section. The chromium content is about 3 percent, and the requirements of microbial degradation and agricultural application can be met only by dechromization treatment.

2. Fermenting and degrading the dechromized blue wet dander to obtain blue wet dander fermentation liquor, namely the oligopeptide-growth-promoting bacteria compound fertilizer

Specifically, the method comprises the following steps:

firstly, selecting a single bacillus pumilus colony which grows well by plate streaking, inoculating the single bacillus pumilus colony into a sterilized liquid seed culture medium, culturing the single bacillus pumilus colony in a constant-temperature oscillating water bath for not less than 24 hours under the stirring conditions of 34-37 ℃ and 120-150r/min, and taking seed liquid for later use.

Then, mixing the dechromized blue wet dander and a fermentation culture medium according to a solid-to-liquid ratio of 1: 5-1:25, mixing, adjusting pH to 7.0-12.0, and sterilizing at 121 deg.C for 20 min.

After the fermentation culture medium is cooled, inoculating seed liquid with an inoculum size of 0.5-4%, and culturing at 27-47 deg.C for not less than 24 hr in a constant temperature shaking water bath.

More specifically, the bacillus pumilus is bacillus pumilus BA06 with the preservation number of CGMCC NO.0518, which can be seen in ZL00120641.9 and is an alkaline protease producing strain separated from natural environment.

The strain is bacillus pumilus and belongs to growth-promoting bacteria commonly used in agriculture.

Wherein the seed culture medium comprises beef extract 3.0g, peptone 10.0g, NaCl 5.0g, and water 1000mL, and pH is adjusted to 8.0 with sodium hydroxide.

The fermentation medium comprises the following components: 2.5g of yeast powder, 5.0g of glucose and K₂HPO₄ 14.0g，KH₂PO₄ 6.0g，

NaCl 5.0g，MgSO₄·7H₂0.2g of O, 1.0g of sodium citrate, 2.0g of ammonium sulfate, 20.0g of agar and 1000ml of water, and the pH value is adjusted to 8.0 by using sodium hydroxide.

Wherein, the ratio of the mass of the dechromized blue wet dander to the volume of the fermentation medium is called solid-liquid ratio.

Preferably, the age of the bacillus pumilus is 24 hours, the solid-to-liquid ratio is 1:15, the inoculation amount is 0.5%, the fermentation temperature is 37 ℃, the pH value is 9.0, and the fermentation time is 48 hours.

On the other hand, the application provides a short peptide-growth-promoting bacterium compound fertilizer prepared by dechromization fermentation of wet blue dander, and the compound fertilizer is prepared by adopting the method.

On the other hand, the application also provides application of the short peptide-growth-promoting bacterium compound fertilizer in planting of wheat, alfalfa and highland barley. Wherein the concentration of free amino acid in the fermentation liquor of the dechromized blue wet dander is 20-100 mg/L.

Furthermore, the short peptide-growth-promoting bacterium compound fertilizer is used in the planting of wheat, alfalfa and highland barley, and is used as a seed soaking liquid, a water culture liquid or a pot culture fertilizer.

Compared with the prior art, the technical scheme provided by the invention has the following advantages:

the invention selects the bacillus pumilus strain which has the capability of efficiently producing collagen degradation enzymes and has the functions of promoting plant growth and biocontrol, carries out fermentation hydrolysis of dechromization dandruff, utilizes fermentation liquor rich in amino acid to prepare microbial fertilizer, organically integrates the conventional amino acid organic fertilizer and the growth-promoting bacterial fertilizer, and exerts multiple effects of nutrition, growth promotion, disease resistance and the like on crops.

The dechromization method of the invention has obvious effect on dechromization and deashing of blue wet dander.

The mercury, arsenic, cadmium, lead and chromium contents in the short peptide-growth-promoting bacteria compound fertilizer prepared by the dechromization fermentation of the wet blue dander completely meet the limit requirements of the national agricultural standard (NY 1110-2010) on water-soluble fertilizers; the total amino acid content in the fermentation liquor is 1g/L, and the fermentation liquor mainly contains amino acids required by plants such as proline, alanine, histidine and the like; in addition, the total peptide content in the fermentation liquor is 13.21g/L, and the molecular weight of the protein is smaller and is below 14.4 kda. Therefore, the dechromization blue wet dander fermentation liquid meets the basic requirements of being used as a biological organic slow-release fertilizer.

The short peptide-growth promoting bacteria compound fertilizer prepared by the dechromization fermentation of the wet blue dandruff has obvious pregermination effect on small seeds with deficient self nutrition, such as alfalfa seeds.

The short peptide-growth promoting bacteria compound fertilizer prepared by the dechromization fermentation of the wet blue dander has obvious promotion effect on the growth of wheat. The wheat seedlings cultured by the fermentation liquor have obviously improved plant height, chlorophyll content, dry and wet weight and protein content.

The oligopeptide-growth promoting bacteria compound fertilizer prepared by the dechromization fermentation of the wet blue dandruff can obviously promote the growth of the highland barley, and the plant height, the leaf area and the like of the highland barley are obviously improved.

The oligopeptide-growth-promoting bacterium compound fertilizer prepared by the dechromization fermentation of the wet blue dander has obvious promotion effects on seed germination and plant growth, and has wide application prospects in agricultural production as a biological organic slow-release fertilizer.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

In FIG. 1, FIG. 1a shows the blue wet dander before and after dechromization, and FIG. 1b shows the infrared spectra of the fresh hide, the blue wet dander and the dechromized blue wet dander; FIG. 2 is a graph showing the growth of Bacillus pumilus; FIG. 3 is a graph showing the effect of solid-liquid ratio on the hydrolysis of wet dechromized blue dander; FIG. 4 is a graph of the effect of inoculum size on hydrolysis of wet dander; FIG. 5 is a graph of the effect of temperature on hydrolysis of wet deschrome blue flakes; FIG. 6 is a graph of the effect of pH on hydrolysis of wet dander of dechromized blue; FIG. 7 is a graph of the effect of fermentation time on hydrolysis of wet dander; FIG. 8 shows the molecular weight distribution of proteins in the fermentation broth; FIG. 9 shows germination rates of alfalfa seeds and wheat seeds treated with different concentrations of seed dip; FIG. 10 is the height of the wheat seedlings; FIG. 11 shows the root growth of wheat seedlings; FIG. 12 shows the leaf length and leaf width of highland barley seedlings.

Detailed Description

As the background art shows, the blue wet leather scraps are collagen-containing waste with high chromium content, and in the aspect of resource utilization of the blue wet leather scraps in agriculture, the blue wet leather scraps are subjected to dechromization and then are hydrolyzed into small molecular peptides and amino acid to prepare the amino acid organic fertilizer. In the aspect of degradation by microbial fermentation, although reports of degrading chrome leather scraps by actinomycetes, penicillium, pseudomonas aeruginosa, penicillium rugosum and the like are adopted at home and abroad, the good degradation effect is not achieved. Not only the degradation efficiency is not high, but also special high chromium-resistant strains need to be screened, and in addition, the degradation solution contains high-concentration chromium, which cannot meet the requirements of agricultural application. Therefore, the chromium-removed leather scraps are subjected to dechromization treatment and then added into the culture medium, so that the biomass of microorganisms and the degradation capability of the leather scraps can be obviously improved. The organic bacterial fertilizer is prepared by fermenting dechromization dander by adopting plant growth-promoting bacteria with high-efficiency protein degradation capability, and the report is not found at present. In order to solve the problems in the prior art, the application provides a method for preparing a short peptide-growth-promoting bacteria compound fertilizer by dechromization fermentation of blue wet dander, the short peptide-growth-promoting bacteria compound fertilizer and application thereof in planting of wheat, highland barley and alfalfa.

The method comprises the steps of performing base-first acid-second acid combination on wet blue leather scraps of tanning wastes to remove chromium from the wet blue leather scraps, taking the chromium-removed wet blue leather scraps as a raw material for microbial fermentation, and performing fermentation treatment on the chromium-removed wet blue leather scraps by utilizing agricultural probiotic bacillus pumilus to obtain the short peptide-growth-promoting bacteria compound fertilizer rich in free amino acid and polypeptide.

The short peptide-growth-promoting bacterium compound fertilizer is used in the planting of wheat, alfalfa and highland barley, and is diluted to a certain concentration to be used as a seed soaking liquid, a water culture liquid or a pot culture fertilizer.

The foregoing is the basic idea of the present application, and the contents of the present application are further illustrated by the following examples and experimental examples.

Example 1

A method for preparing a short peptide-growth-promoting bacterium compound fertilizer by dechromization fermentation of wet blue dander comprises the following steps:

1. dechromisation of wet blue dander

firstly, performing alkali treatment, wherein wet blue leather scraps are soaked in an alkali solution, and the alkali solution gradually permeates into the blue leather scraps under the action of mechanical stirring force.

Wherein the alkali solution is a calcium oxide aqueous solution; the concentration of the alkali solution is 6-9g/L, preferably 9g/L, wherein the dosage of the alkali solution is 10% of the mass of the blue leather scraps; the alkali treatment time is 8-12h, in the embodiment, 12h is preferable; the alkali treatment temperature is normal temperature.

And then filtering to obtain blue wet leather scraps after the alkali dechromization treatment.

And then acid treatment is carried out, wherein in the step, the filtered wet blue leather scraps after the alkali dechromization treatment are soaked in an acid solution.

Wherein the acid solution is hydrochloric acid aqueous solution; the liquid ratio of the acid solution is 1:20 based on the weight of wet blue leather scraps after the alkali dechromization treatment;

the concentration of the acid solution is 0.6-0.9mol/L, and in the embodiment, the concentration is preferably 0.9 mol/L; the pickling time is 30-35min, preferably 35min in this example.

And (5) performing suction filtration to obtain dechromized scurf.

In this example, the wet blue hide chips are chrome-containing leather waste produced by shaving, buffing and trimming after the tanning section. The chromium content is about 3 percent, and the requirements of microbial degradation and agricultural application can be met only by dechromization treatment.

Specifically, the method comprises the following steps:

firstly, selecting a single bacillus pumilus colony which grows well by plate streaking, inoculating the single bacillus pumilus colony into a sterilized liquid seed culture medium, culturing the single bacillus pumilus colony in a constant-temperature oscillating water bath for 24 hours under the conditions of stirring at 37 ℃ and 120r/min to obtain the bacillus pumilus, and taking a seed solution for later use.

Then, mixing the dechromized blue wet dander and a fermentation culture medium according to a solid-to-liquid ratio of 1:15, mixing, adjusting the pH value to 9.0, and placing in a sterilizing pot for sterilizing at 121 ℃ for 20 min.

After the fermentation medium is cooled, inoculating the seed liquid with the inoculation amount of 0.5%, and then culturing for 48h in a constant-temperature oscillating water bath at 37 ℃ under the condition of 120 r/min.

NaCl 5.0g，MgSO₄·7H₂0.2g of O, 1.0g of sodium citrate, 2.0g of ammonium sulfate and 2.0g of agarFat 20.0g, water 1000ml, adjusted to pH 8.0 with sodium hydroxide.

Examples 2 to 23 are similar to example 1, with the difference that the relevant parameters. See table 1 for details of the parameters below.

Table 1 table of parameters of examples

Example 24

A short peptide-growth-promoting bacteria compound fertilizer prepared according to the method.

Example 25

And (3) centrifugally sterilizing the short peptide-growth-promoting bacterium compound fertilizer (dechromization blue wet dandruff fermentation liquor), and diluting to ensure that the content of free amino acid in the diluted dechromization blue wet dandruff fermentation liquor is 20-120mg/L, preferably 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L and 120 mg/L.

It is applied in agricultural planting as seed soaking liquid, water culture liquid or pot planting fertilizer.

Experimental example:

experimental example 1: physicochemical index test before and after blue wet scurf dechromization

The physical and chemical index tests of the wet blue flakes before and after dechromization by the method of example 1 showed the following results:

the blue wet dander is dechromized by an acid-base combination method, the color of the blue wet dander is changed from green to white, but the blue wet dander still exists in a solid state, which indicates that a large amount of chromium elements contained in the blue wet dander are removed after the blue wet dander is dechromized, the dechromized blue wet dander does not cause destructive hydrolysis to a fiber structure of the blue wet dander, and the dechromized blue wet dander still keeps a complete collagen fiber structure.

The blue dander of example 1 was tested for chromium content and ash content before and after dechroming, and the test results are shown in table 2.

TABLE 2 indexes of main components before and after dechromization of blue wet dander

Test sample	pH value	Ash content/%)	Chromium content/(mg/g dry weight)
				Blue wet leather scraps	3.5	7.37	26.282
Dechromised blue wet dander	7.0	0.26	0.819

Therefore, after the method disclosed by the invention is adopted for dechromization, the chromium content is reduced to 0.819mg/g from the original 26.282mg/g, and is reduced by 96.88%; the ash content of the blue wet scurf is reduced to 0.26% from the original 7.37%, and is reduced by 96.47%, which shows that the optimized acid-base combined dechroming method has obvious effects on dechroming and deashing of the blue wet scurf.

Referring to FIG. 1, FIG. 1a shows the form of blue wet dander before and after dechromization, and FIG. 1b shows the measured infrared spectra of fresh hide, blue wet dander and dechromized blue wet dander.

Referring to fig. 1a, it can be seen that the blue wet dander is dechromized by the acid-base combination method, the color of the blue wet dander changes from green to white, but the blue wet dander still exists in a solid state, which indicates that a large amount of chromium contained in the blue wet dander is removed after the dechromization treatment, and the dechromization treatment does not cause destructive hydrolysis to the fiber structure of the blue wet dander, and the dechromized dander still maintains a complete collagen fiber structure.

Referring to FIG. 1b, there are top-down IR spectra of fresh hide, dechromized blue wet dander and blue wet dander, because the main components of the sample are all proteins, all have the most characteristic IR absorption peaks of protein molecules, i.e. a strong and broad N-H absorption peak around 3400cm-1 and absorption peaks of amide I, amide II and amide III bands between 1700-1300 cm-1. The infrared spectrogram of the dechromized blue wet leather scraps is basically completely superposed with the infrared spectrogram of the fresh leather, which shows that the blue wet leather scraps are basically restored to the fresh leather state after being dechromized by the method. The infrared spectrum of the fresh skin and the dechromized blue wet skin scraps is 1400cm^-1There is an absorption peak in the vicinity, which is the infrared characteristic peak of the O-H bending vibration in-COOH, probably due to the destruction of the group by the action of chrome tanning (binding of the chrome element to the carboxyl group), and after the dechroming treatment, the-COOH group is restored, so that the absorption peak reappears. In addition, only the wet blue dander is 1118cm^-1The blue wet scurf contains a certain amount of lactone, acetal or some complexes (the vibration of a ligand can generate infrared absorption here, the blue wet scurf is subjected to dechromization treatment, the substances are basically removed, so the dechromized blue wet scurf has no corresponding absorption peak.

2. Experimental example 2 test of age of inoculated bacteria

According to the method of example 1, single colonies of Bacillus pumilus which grew well on streaks were picked up, inoculated into the sterilized liquid seed medium (20mL) of example 1, and cultured in a constant temperature shaking water bath at 37 ℃ and 120 r/min. Sampling every 4 hours, using distilled water as blank control, measuring OD value of the seed liquid at 600nm with ultraviolet spectrophotometer, and stopping sampling until OD value is basically unchanged. The inoculation age of Bacillus pumilus was determined by plotting the growth curve of Bacillus pumilus as OD and incubation time, see FIG. 2.

As can be seen from FIG. 2, the OD value increases slowly from 0 to 4 hours, which indicates that the Bacillus pumilus is in the lag phase; the OD value is increased rapidly within 4-24 h and is increased in a nearly linear manner, which indicates that the Bacillus pumilus is in a logarithmic growth phase at the moment; after 24 hours of culture, the OD value varied slightly, indicating that Bacillus pumilus is in the stationary phase. Since the strains in the late logarithmic phase not only have vigorous growth and metabolic capability, but also the number of the strains is basically highest, the strains grow for 24 hours and are used as the optimal inoculation age of the bacillus pumilus.

Experimental example 3 fermentation broth testing

3.1 determination of free amino groups in the fermentation broths

The determination of free amino groups in the fermentation broth was carried out using the following method:

1. preparation of test solutions

(1) Preparation of ninhydrin solution 0.5g of ninhydrin was weighed out accurately, dissolved in absolute ethanol and brought to volume in a 100mL brown volumetric flask.

(2) Preparation of acetic acid-sodium acetate buffer solution 120.0g of sodium acetate is weighed, 4.0mL of anhydrous acetic acid or glacial acetic acid is added, and water is added to a volumetric flask with a constant volume of 1000 mL.

(3) Preparation of potassium iodate solution 0.3g of potassium iodate was accurately weighed, dissolved in 90mL of distilled water, and then 60mL of absolute ethanol was added and mixed well.

(4) Preparation of Standard solutions

Accurately weighing 0.1g of glycine in a 100mL brown volumetric flask for constant volume, then respectively taking 0, 1, 2, 3, 4 and 5mL of glycine solution in 6 volumetric flasks with 100mL for constant volume, and numbering according to the concentration from small to large in sequence.

2. Determination of free amino groups

(1) Drawing of standard curve

Accurately sucking 1mL of the 6 groups of standard solutions, respectively placing the 6 groups of standard solutions in 6 colorimetric tubes, sequentially adding 1mL of buffer solution and 1mL of ninhydrin solution, sealing, shaking uniformly, heating in a boiling water bath for 15min, taking out, and placing in cold water for cooling for 15 min. After cooling, 5mL of potassium iodate solution was added to each cuvette and the volume was adjusted to 10mL with distilled water. And (3) adjusting the wavelength of the ultraviolet-visible spectrophotometer to 568nm, adjusting the absorbance of the blank sample in the No.0 colorimetric tube to 0, and then sequentially measuring the absorbance of other 5 groups of samples. And (3) drawing a standard curve by taking the content of free amino as a horizontal coordinate and the absorbance as a vertical coordinate, and obtaining a linear regression equation.

3. Determination of free amino groups in fermentation broth

Diluting the fermentation liquor to a certain extent, accurately sucking 1mL of the fermentation liquor, placing the fermentation liquor in a colorimetric tube, sequentially adding 1mL of buffer solution and 1mL of ninhydrin solution, sealing, shaking up, heating in a boiling water bath for 15min, taking out, and placing in cold water for cooling for 15 min. After cooling, 5mL of potassium iodate solution was added to each cuvette and the volume was adjusted to 10mL with distilled water. The absorbance of the fermentation liquor is measured in the same way as that of the standard liquor. And converting the absorbance of the fermentation liquor into the corresponding free amino content according to the relationship between the free amino content and the absorbance in the standard curve.

3.2 analysis of fermentation products by determination of the principal Components

And (3) after the dechromized blue wet dander is fermented, measuring the main components of the fermentation liquor.

3.2.1 determination of the heavy Metal content

(1) Cadmium, chromium and lead content determination fermentation liquor is digested by a nitric acid-perchloric acid wet method in DB 33/T647-2007, and then the content of four elements of cadmium, chromium and lead in a sample is determined by an ICP-OES method.

(2) Measuring the content of mercury and arsenic according to the atomic fluorescence spectrometry method in NY/T1978-2010.

3.2.2 determination of free amino acids

Putting 400 mu L of fermentation liquor and 100 mu L of 10% sulfosalicylic acid into a small centrifugal tube with a filter membrane, uniformly mixing, placing in a refrigerator, standing and refrigerating for 60min at 2-8 ℃, then placing in a centrifugal machine, centrifuging for 15min at 5000r/min, and taking supernatant, and centrifuging for 5min at 5000r/min again. And after the centrifugation is finished, taking the supernatant, and diluting the fermentation liquor according to the approximate concentration of the free amino acid in the fermentation liquor so that the concentration of the free amino acid is between 50 and 250 nmol/mL. After the sample is diluted, the amino acid is analyzed on an A300 full-automatic amino acid analyzer after being filtered by a 0.22um filter membrane.

3.2.3 determination of polypeptide content in fermentation broth

And (3) after the fermentation of the dechromized blue wet dander is finished under the optimal fermentation condition, determining the polypeptide content in the fermentation liquor by adopting a biuret method.

1. Reagent preparation

(1) The standard solution is prepared into a standard solution of 5mg/mL by using standard bovine serum albumin.

(2) Biuret reagent 1.5g CuSO4 & 5H2O and 6g potassium sodium tartrate were dissolved in 500mL of water, 300mL of 10% sodium hydroxide solution was added with stirring, and diluted to 1L with water.

2. Drawing of standard curve

Taking 6 test tubes, respectively adding 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of standard protein solution, supplementing to 1mL with water, and adding 4mL of biuret reagent. After shaking sufficiently, the mixture was allowed to stand at 25 ℃ for 30min, and absorbance was measured at 540 nm. And drawing a standard curve of the polypeptide content by taking the bovine serum albumin content as an abscissa and the absorbance as an ordinate, and obtaining a linear regression equation.

Determination of polypeptide content in fermentation liquor

Adding 2mL of 15% trichloroacetic acid into 2mL of fermentation liquor, uniformly mixing, standing for 10min, and centrifuging for 10min at a speed of 5000 r/min. 1mL of the supernatant diluted moderately was added with 4mL of biuret reagent, shaken well, allowed to stand at 25 ℃ for 30min, and then absorbance was measured at 540 nm. And converting the absorbance of the fermentation liquor into the corresponding polypeptide content according to the linear relation between the polypeptide content and the absorbance in the standard curve.

3.2.4 distribution of protein molecular weight in fermentation broth

Fermenting the dechromized blue wet dander under the optimal condition, sampling every 12h after the fermentation is started, and determining the distribution of the protein molecular weight in the fermentation solution at different time points by adopting an SDS-PAGE gel electrophoresis method. The concentration of the concentrated gel is 5 percent, and the concentration of the separation gel is 15 percent. The formulation of the gum is shown in table 3 below:

TABLE 3 SDS-PAGE gel formulation

Composition (I)	15% separation gel	5% concentrated gum
			Ultrapure water (mL)	2.0	2.1
30% acrylamide (mL)	10.0	0.5
			1MTris-HCl,pH8.8(mL)	7.6	—
1M Tris-HCl,pH6.8(mL)	—	0.38
			10％SDS(mL)	0.2	0.03
10％APS(mL)	0.2	0.03
			TEMED(mL)	0.008	0.003

After the glue preparation is finished, respectively taking a proper amount of Marker and the processed sample by using a micro-injector, injecting the Marker and the processed sample into a sample injection groove, switching on a power supply, setting the voltage to be 80V, and adjusting the voltage to be 150V when the sample enters the separation glue. And ending the electrophoresis when the bromophenol blue dye is about 1-2 cm away from the edge of the gel. After electrophoresis is finished, the power supply is turned off, the film is taken out, and the film is dyed and decolored to obtain clear protein bands.

Using the above procedure, the free amino group contents of examples 2 to 6, 7 to 11, 12 to 16, 17 to 22, and 23 were measured, respectively, and the results are shown in FIGS. 3 to 7, respectively.

The data in FIG. 3 (example 2 to example 6) show that the solid-to-liquid ratio is between 1:5 and 15, and the content of free amino groups in the fermentation liquor is obviously increased along with the increase of the solid-to-liquid ratio. When the solid-liquid ratio is 1:15, the content of free amino in the fermentation liquor is 49.00 percent and 15.36 percent higher than that of free amino when the solid-liquid ratio is 1:5 and 1:10 respectively, and probably because the solid-liquid ratio is too small, the organic nutrient concentration in the fermentation liquor is too high, the viscosity of the fermentation liquor is too high, and the dissolved oxygen is insufficient, so that the growth and metabolism of microorganisms are inhibited. When the solid-liquid ratio exceeds 1:15, the increase of the free amino content in the fermentation liquor tends to be smooth, and a platform area appears. It is shown that the solid-liquid ratio is too large, which does not promote the increase of the product amount, but increases the load burden and water consumption of the fermentation tank.

As can be seen from FIG. 4 (examples 7 to 11), the fermentation time is in the range of 6-24 h, and the larger the inoculation amount is, the higher the content of free amino groups in the fermentation liquid is; and when the fermentation time reaches 24 hours, the inoculation amount is different, and the content of free amino in the fermentation liquor is almost the same. The reason for this phenomenon is probably that the larger the inoculation amount is, the more the number of live bacteria in the fermentation broth is, the higher the content of free amino group is, and as the fermentation time is prolonged, the number of live bacteria in the fermentation broth with different inoculation amounts tends to be the same, so that the difference of the content of free amino group in the fermentation broth with different inoculation amounts is smaller and smaller. Therefore, the continuous increase of the inoculation amount on the basis of the inoculation amount of 0.5 percent does not increase the final free amino content in the fermentation liquid, but wastes the seed liquid and increases the fermentation cost.

FIG. 5 (examples 12 to 16) shows that at the same fermentation time point, the free amino group content in the fermentation broths of different fermentation temperatures does not increase with increasing temperature, wherein the free amino group content in the fermentation broths is relatively low at too high a temperature (47 ℃) or too low a temperature (27 ℃), in particular the latter, which indicates that low temperatures are detrimental to the metabolic activity of Bacillus pumilus strains; however, it is not necessary for the temperature to be as high as possible, for example, the free amino group content at 42 ℃ is significantly below 37 ℃.

The experimental result shows that the content of free amino generated by fermenting the dechromized blue wet dander by the bacillus pumilus at 37 ℃ is the highest, which indicates that the temperature is the optimal temperature for degrading the dechromized blue wet dander by the bacillus pumilus strain.

From FIG. 6 (examples 17 to 22), it is easy to find a similar rule with the effect of fermentation temperature, that is, the content of free amino groups in the fermentation broth does not increase with the increase of pH value when the fermentation is performed at different pH values, the content of free amino groups in the fermentation broth increases with the increase of pH value when the pH value is between 7 and 9, and the content of free amino groups in the fermentation broth significantly decreases with the increase of pH value when the pH value exceeds 10, and especially the fermentation process is basically stopped when the pH value reaches 12. Wherein pH is too high (10) or too low (7), and the content of free amino groups in the fermentation broth is relatively low, especially the former, which indicates that too high pH is not favorable for the metabolic activity of the Bacillus pumilus strain; however, it is not the case that the lower the pH, the better, for example, the free amino group content at pH 7 is significantly lower than 9.

The experimental result shows that the content of free amino generated by fermenting the dechromized blue wet dander by the bacillus pumilus at the pH value of 9 is the highest, which indicates that the pH value is the optimum pH value for degrading the dechromized blue wet dander by the bacillus pumilus strain.

As can be seen from FIG. 7 (example 23), after fermentation for 12-24 h, the content of free amino groups in the fermentation broth rapidly rises, at this time, the metabolism of the Bacillus pumilus strain is very active, and the strain gradually secretes extracellular protease to decompose the dechromized blue wet dander from macromolecular protein into free amino acid and polypeptide with small molecular weight; and fermenting for 24-48 h, wherein the content of free amino in the fermentation liquor slowly rises, and the content of free amino in the fermentation liquor reaches the highest value in 48 h. At this time, although the metabolic rate of the Bacillus pumilus strain is slowed down, the rate of producing amino acids and polypeptides is still far higher than the metabolic consumption rate of the strain, so the content of free amino groups is continuously increased; but the content of free amino in the fermentation liquid slowly decreases after fermentation for 48-72 hours. At the moment, the biological degradation of the dechromized blue wet dander is basically finished, the micromolecule substances generated by degradation are continuously consumed as the nutrient substances of the bacterial strains, and the product repression effect is generated, so that the content of free amino in the fermentation liquor is continuously reduced.

In conclusion, when the Bacillus pumilus strain is fermented for 48 hours, the content of free amino groups in the fermentation liquid is the highest, and the fermentation effect is the best.

By optimizing the fermentation conditions of the dechromized blue wet dander, the optimized fermentation conditions are as follows: the strain age is 24h, the solid-to-liquid ratio is 1:15, the inoculation amount is 0.5%, the temperature is 37 ℃, the pH value is 9.0, and the fermentation time is 48h, namely the strain age is the example 1.

The heavy metal content, the free amino acid content and the polypeptide content of the dechromized blue wet dandruff fermentation broth of example 1 and the protein molecular weight of example 23 were measured by the above methods, respectively.

Determination of heavy Metal content

The contents of mercury, arsenic, cadmium, lead and chromium in the dechromized blue wet dander fermentation broth are shown in the following table 3.

TABLE 3 heavy metal content in fermentation broths

	Hg	As	Cd	Pb	Cr
						Content (mg/kg)	0	0	0	0	2.91

As can be seen from Table 3, the contents of mercury and arsenic are not detected in the dechromized blue wet dander fermentation broth by adopting the atomic fluorescence spectrometry, the contents of cadmium and lead are also not detected by adopting the ICP-OES method, the content of chromium is only 2.91mg/Kg, and the heavy metal content completely meets the limit requirements of the Ministry of agriculture standards NY 1110-2010 on water-soluble fertilizers, namely, the content of mercury is less than or equal to 5mg/Kg, the content of arsenic is less than or equal to 10mg/Kg, the content of cadmium is less than or equal to 10mg/Kg, the content of lead is less than or equal to 50mg/Kg, and the content of chromium is less than or equal.

Measurement results of free amino acids

The types and contents of free amino acids in the dechromized blue wet dander fermentation broth are shown in table 4 through the determination of an A300 full-automatic amino acid analyzer.

TABLE 4 free amino acid content in fermentation broths

Kind of amino acid	Alanine	Cysteine	Histidine	Proline	Total amino acids
						Content (mg/L)	169.65	254.47	127.23	460.47	1011.81

Proline is an amino acid required by most plants and can improve the stress resistance of the plants; cysteine, alanine and histidine are also amino acids required by plants, wherein cysteine has the effects of resisting oxidation and maintaining cell functions, alanine can increase synthesis of chlorophyll, and histidine can regulate plant stomatal opening and provide a precursor of carbon skeleton hormone.

As can be seen from Table 4, when the A300 full-automatic amino acid analyzer is used for measuring the content of free amino acids in the dechromized blue wet dander fermentation liquid, the content of the free amino acids in the fermentation liquid is proline at the highest content, and cysteine, alanine and histidine at the second highest content, so that the quick nutrition supply can be provided for the growth of plants. The total free amino acid content in the fermentation liquor reaches 1g/L, and the fermentation liquor can be diluted by at least 10 times for plant planting according to the application concentration of the current commercial amino acid fertilizer of 100 mg/L.

Measurement of polypeptide content

After trichloroacetic acid is used for removing protein molecules in the fermentation liquor, a biuret method is adopted, and the polypeptide content in the fermentation liquor at the end of fermentation (48h) is measured to be 13.21 g/L.

It can thus be seen that: the dechromized blue wet dander fermentation liquor not only contains free amino acid with higher concentration, but also contains a large amount of polypeptide substances. The polypeptide can obviously improve the quality of crops, increase the yield of the crops and enhance the stress resistance of the crops like amino acid, but compared with the amino acid, the polypeptide has slow release fertilizer efficiency and can provide nutrition for plants more durably. The dechromization blue wet dander fermentation liquor prepared by the experiment contains quick-acting amino acid and slow-release polypeptide, and is hopeful to be used as a plant nutrient solution with balanced and rich nutrition.

Distribution of protein molecular weight in fermentation broth

Two groups of fermentation solutions, namely 5% dechromized blue scurf (wet weight), 1% inoculation amount, 20% dechromized blue scurf (wet weight) and 4% inoculation amount, are prepared respectively, sampling is carried out every 12 hours within 0-96 hours, the difference of the hydrolytic capacities of the two fermentation solutions is compared through SDS-PAGE, and the change of the molecular weight of the protein is observed.

Comparing the two electrophorograms, it can be seen that the band of FIG. 8 is clearer and the color development is deeper. The analysis reason is that the experimental group has high dechromized blue scurf content, namely high dechromized blue scurf protein content, so that a relatively obvious strip display can be realized. In combination with the amino acid content profile, it was found that different amounts of dechromized blue dander had no significant effect on the hydrolysis curve of the protein, and therefore, the larger difference from the other samples was due to the fact that the total protein content in the fermentation broth was twice that of the other samples. The actual effect of the experimental group of fig. 8b should be comparable to that of the experimental group of fig. 8 a. Therefore, 5% of dechrominated blue dander and 1% of Bacillus pumilus inoculation amount are selected as final hydrolysis conditions for the reason of saving raw materials and the like. Under the condition, the dechromized blue wet dander is degraded into amino acid and small molecular weight polypeptide after strain fermentation treatment for 48 hours, free amino acid in a large amount in fermentation liquor can provide rapid and balanced nutrition guarantee for plants in a rapid growth period, and the rich polypeptide content can provide continuous slow-release nutrition supply for plant growth.

Mercury, arsenic, cadmium and lead elements are not detected in the dechromized blue wet dander fermentation liquor, the content of chromium is 2.91mg/Kg and is lower than the maximum limit of 50mg/Kg of agricultural standard chromium, and the content of heavy metal in the dechromized blue wet dander fermentation liquor reaches the national standard of Ministry of agriculture.

The free amino acids in the fermentation liquor mainly comprise proline, cysteine, alanine and histidine, wherein the content of proline is the highest, and the content of cysteine, alanine and histidine is the next highest. The content of total free amino acid in the fermentation liquor reaches a higher concentration of 1 g/L.

The total peptide content in the fermentation liquor measured by a biuret method is 13.21g/L, which indicates that the dechromized blue wet scurf fermentation liquor not only contains high-concentration free amino acid, but also contains a large amount of polypeptide substances.

No obvious protein band exists in the fermentation liquor between the molecular weight of 66-14.4 kda, which indicates that the dechromized blue wet dander is basically degraded into free amino acid and polypeptide substances with smaller molecular weight, and the molecular weight is below 14.4 kda.

The experimental results show that fermentation liquor rich in free amino acids and polypeptides, which is generated by fermenting the dechromized blue wet dander, is possible to be used as a biological organic slow-release fertilizer to promote the growth of plants.

Experimental example 4 application of dechromised blue wet dander fermentation broth in crop planting

4.1 application experiment of dechromized blue wet dander fermentation liquor as seed soaking agent

The following experiments were carried out using the dechromised blue wet dander fermentation broth of example 1

Selecting small-particle alfalfa seeds and large-particle wheat seeds as germination experimental materials, and soaking the seeds by using dechromization blue wet dander fermentation liquor with different dilution concentrations so as to obtain the optimal fermentation liquor concentration for promoting the efficient germination of the seeds and the optimal germination effect.

Experiment of using dechromised blue wet dander fermentation liquor as alfalfa seed soaking agent

And (3) performing centrifugal sterilization on the dechromized blue wet dandruff fermentation liquor, and then diluting to ensure that the free amino acid content of the diluted fermentation liquor is 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L and 120mg/L respectively. Selecting 100 alfalfa seeds with uniform size, and soaking the alfalfa seeds respectively in fermentation liquids (the dosage is 5 times of the weight of the seeds) with different amino acid contents at 25 ℃ for 12 h; and (3) placing the soaked seeds in a surface dish with filter paper in order (the filter paper is wetted by fermentation liquor with corresponding amino acid content), then placing the surface dish in a constant-temperature constant-humidity incubator at the temperature of 25 ℃ and the humidity of 60% for culture, and observing and recording the germination condition of the seeds. The blank control uses tap water instead of dechromized blue wet dander fermentation liquor as a seed soaking agent, and other operations are the same. The germination rate and the germination growth rate were calculated according to the following formulas, respectively.

In the formula: n is the number of sprouts; 100-total number of seeds. The germination growth rate (%) ═ R1-R2. R1 — daily germination (%); r2-germination (%) in the first 24 h.

Experiment of using dechromized blue wet dander fermentation liquor as wheat seed soaking agent

And (3) performing centrifugal sterilization on the dechromized blue wet dandruff fermentation liquor, and then diluting to ensure that the free amino acid content of the diluted fermentation liquor is 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100mg/L respectively. Selecting 100 wheat seeds with uniform size, and soaking the wheat seeds respectively with fermentation liquids (the dosage is 5 times of the weight of the seeds) with different amino acid contents at 25 ℃ for 12 h; and (3) placing the soaked seeds in a surface dish with filter paper in order (the filter paper is wetted by fermentation liquor with corresponding amino acid content), then placing the surface dish in a constant-temperature constant-humidity incubator at the temperature of 25 ℃ and the humidity of 60% for culture, and observing and recording the germination condition of the seeds. Tap water is used as a seed soaking agent for replacing dechromized blue wet dander fermentation liquor in the blank control group, and other operations are the same.

4.2 application experiment of dechromization blue wet dander fermentation liquor as wheat seedling water culture nutrient solution

And (3) performing centrifugal sterilization on the dechromized blue wet dandruff fermentation liquor, and diluting for later use to ensure that the free amino acid content of the diluted fermentation liquor is respectively 20mg/L, 40mg/L, 60mg/L, 80mg/L and 100 mg/L. Weighing 4g of wheat seeds with uniform size, adding tap water with the weight 5 times that of the seeds, and soaking at 25 ℃ for 12 hours; the soaked seeds are placed in a watch glass filled with filter paper in order, then the watch glass is placed in a constant-temperature and constant-humidity incubator with the temperature of 25 ℃ and the humidity of 60% and cultured with tap water, when the nutrition of the wheat seeds is completely consumed, the residual water in the watch glass is poured out, a certain amount of diluted fermentation liquor is added, the fermentation liquor just submerges the root of the wheat seedling, then the wheat seedling is placed in the constant-temperature and constant-humidity incubator to be continuously cultured (a proper amount of tap water is added in the culture process, the root of the wheat seedling is just submerged), the plant height of the wheat is observed and recorded every 7 days, and the growth condition of the wheat is photographed and recorded. The blank control group and the positive control group respectively use tap water and a commercially available water-soluble fertilizer containing amino acid (concentration is 100mg/L) to replace the dechromized blue wet dander fermentation liquor, and the other operations are the same. And (4) harvesting seedlings after fertilizing and culturing for two weeks, and measuring quality parameters such as dry weight, wet weight, plant height, root system state, protein content, chlorophyll content and the like of the wheat seedlings.

Measurement of height of wheat seedling

After the wheat seedlings are fertilized, cultured for two weeks and harvested, the distance from the stem base to the highest leaf of the wheat seedlings is measured by a ruler. And (4) measuring the total length of the adventitious roots of the wheat seedlings and counting the number of lateral roots. After the wheat seedlings are fertilized, cultured for two weeks and harvested, the root length of the wheat seedlings is measured and the number of lateral roots of the wheat seedlings is counted. Root length was measured directly with a ruler.

Method for measuring chlorophyll content of wheat seedlings

After the wheat seedlings are fertilized and cultured for two weeks, the chlorophyll content of the wheat seedlings is measured.

Accurately weighing 0.5g of wheat leaves, mashing, homogenizing, adding 100mL of ethanol and acetone mixed extract (V: V is 1:1), standing at room temperature in a dark place, and extracting for 5 h; after extraction, filtering, and detecting filtrate; the absorbance values of the filtrates were measured at 645nm and 663nm, respectively, using the extractant as a blank solution. The chlorophyll a, chlorophyll b and total chlorophyll content in the sample were calculated according to the following formulas.

Chlorophyll a content (mg/g) ═ 12.72 × A663-2.59 × A645 × v/(1000 × m)

Chlorophyll b content (mg/g) ═ 22.88 × A645-4.67 × A663 × v/(1000 × m)

The total chlorophyll content (mg/g) ═ 8.05 × A663+20.29 × A645 × v/(1000 × m)

In the formula: v-total volume of extract in milliliters (mL); m-fresh weight of sample in grams (g).

Determination of dry and wet weight and water content of wheat seedling plant

After the wheat seedlings are fertilized and cultured for two weeks, the wheat seedlings are pulled up with roots, and the roots are removed, weighed and counted as wet weight M; and (3) putting the weighed part in a constant-temperature drying oven for deactivation of enzymes for 0.5h at the temperature of 100 ℃, and then drying the part at the temperature of 80 ℃ to constant weight, wherein the constant weight result is dry weight m. The moisture content of the plant is then calculated according to the following formula:

in the formula: w-moisture content (%) of the wheat seedling plant; m, wet weight (g) of wheat seedling plants; m is the dry weight (g) of the wheat seedling.

Determination of protein content in wheat leaves

After the wheat seedlings are fertilized and cultured for two weeks, the wheat seedling leaves are taken for measuring the protein content.

Sample treatment: accurately weighing 0.2-2 g of dried wheat leaves into a 100mL nitrogen determination bottle, adding 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid, placing the bottle in a small funnel after shaking slightly, placing the bottle on an electric furnace, heating and carbonizing until the liquid is blue-green, clear and transparent, and continuing to heat for 0.5 h. The flask was taken down and left to cool, 20mL of water was added, and after left to cool, the volume was determined to 100 mL. Meanwhile, a blank experiment is carried out, wheat leaves are not added in the blank experiment, and other operations are the same.

And (4) after the sample is processed, determining the protein content by adopting a Kjeldahl method. The protein content of the sample was calculated according to the following formula.

Wherein X is the protein content of the sample in grams per hundred grams dry matter (g/100g dry matter); v1-volume of the sample solution consumed sulfuric acid standard titration solution in milliliters (mL); v2 — volume of reagent blank spent sulfuric acid standard titration solution in milliliters (mL); v3 — volume of digest aspirated in milliliters (mL); c is the concentration of a sulfuric acid standard titration solution, and the unit is mol per liter (mol/L); 0.0140-1 mL sulfuric acid [ c (1/2H2SO4) ═ 1.000mol/L ] standard titration solution equivalent mass of nitrogen in grams (g); 6.25-Nitrogen as a factor for protein.

4.3 application experiment of dechromization blue wet dander fermentation liquor for highland barley pot experiment fertilizer

Taking half of the dechromized blue wet dandruff fermentation liquor, the bacillus pumilus bacterial liquid and the dechromized blue wet dandruff enzymolysis liquid to chelate metal ions, and leaving the other half not to chelate. Then, the solution was diluted 1000X, 200X, 50X and 10X, and a commercially available organic water-soluble fertilizer (Yingluo Volpofen) was used as a control, and the concentration was prepared according to the instruction. About 20 highland barley seeds were sown per pot, 200ml fertilizer was poured into each pot at the indicated dilution, and 3 pots were repeated for each treatment. Topdressing is carried out for 1 time on the 15 th day after sowing, and the using amount is the same. And measuring the height and the width of the first leaf on the 22 th day after sowing, and recording the growth condition of the highland barley.

The following are experimental results of the above experiments

Application research result of dechromization blue wet dander fermentation liquor as seed soaking agent

The status of the alfalfa seeds and wheat seeds before germination was: the individuals of the two seeds are very different, and a single wheat seed is about 20 times heavier than a single alfalfa seed, so that the wheat seed is richer in nutrient storage than the alfalfa seed. The dechromization blue wet scurf fermentation liquor is used as a seed soaking agent of the two seeds, so that the optimal seed soaking concentration of the corresponding seeds can be obtained, and the influence of the dechromization blue wet scurf fermentation liquor on the germination of different nutrition storage seeds can be inspected.

Experimental result of taking dechromization blue wet dander fermentation liquor as alfalfa seed soaking agent

Soaking alfalfa seeds with the dechromization blue wet dander fermentation liquid with different concentrations, and observing the germination conditions of the alfalfa seeds after the soaking treatment with the fermentation liquid with different concentrations.

Alfalfa seed germination percentage statistics

After 3 days of germination, the statistical results of the germination rates of the alfalfa seeds treated by the fermentation liquor with different concentrations through seed soaking are shown in the table below.

TABLE 5 statistical results of alfalfa seed germination

As can be seen from table 5: the germination rate of the alfalfa seeds increases along with the time, and the germination growth rate is reduced after reaching a peak in 48 hours, which indicates that 24-48 hours are the fastest germination period of the alfalfa seeds. Compared with the control, the germination percentage of the fermentation liquor with the concentration of 20mg/L is equivalent to that of the control, and the germination percentage of the other concentrations is obviously increased compared with that of the control. And the germination rate and the germination growth rate of the alfalfa seeds treated by soaking the fermentation liquor with different concentrations are increased along with the increase of the concentration of the dechromized blue wet dandruff fermentation liquor, the germination rate and the germination growth rate reach the highest value when the concentration reaches 80mg/L, and are reduced after the concentration exceeds 80mg/L, statistical data show that the fermentation liquor with proper concentration can really play a role in accelerating germination, but the germination of the alfalfa seeds can be inhibited when the concentration is too high.

After 72h of culture, the germination of seeds in the control group and the experimental group is over half, but the germination growth rate and the ratio at 48h are reduced. Compared with the control, the germination rate of the fermentation liquor with the concentration of 20mg/L is equivalent to that of the control, and the germination rates of the rest concentrations are higher than that of the control, which shows that the fermentation liquor has the function of promoting the germination of the alfalfa seeds. Meanwhile, the germination rate of the alfalfa seeds subjected to seed soaking treatment by the fermentation liquor with different concentrations is also found to increase along with the increase of the concentration of the dechromized blue wet scurf fermentation liquor, the germination rate is increased to 80mg/L most obviously, and the germination rate is reduced after the fermentation liquor exceeds 80mg/L, which indicates that the seed germination can be inhibited by overnutrition.

After 72 hours of culture, almost 1/3 seeds do not germinate, and the germination rate of the fermentation liquor with the concentration of 80mg/L also reaches 76 percent, so the germination rate can be improved by properly prolonging the time. The highest germination rate is still the alfalfa seeds treated by soaking the seeds in the fermentation liquor of 80mg/L, the germination rate is 76 percent, which is 28.81 percent higher than that of the alfalfa seeds treated by soaking the seeds in water, and the germination accelerating effect is the best when the concentration is 80 mg/L. Therefore, the optimal germination accelerating concentration of the fermentation liquor for promoting the germination of the alfalfa seeds is 80mg/L, and the maximum germination growth rate of the seeds is 48 h.

Experimental result of fermentation liquor as wheat seed soaking agent

Soaking alfalfa seeds with the dechromization blue wet dander fermentation liquid with different concentrations, and observing the germination conditions of the wheat seeds subjected to the soaking treatment with the fermentation liquid with different concentrations.

Statistics of wheat seed germination rate

After 3 days of germination, the statistical results of the germination rates of the wheat seeds treated by seed soaking at different concentrations are shown in the following table.

TABLE 6 statistical results of wheat seed germination

The statistical data in table 6 show that the germination growth rate of the wheat seeds treated by different seed soaking treatments is the highest in 24h, and rapidly decreases with the time, which indicates that 0-24 h is the rapid growth period of the germination of the wheat seeds. When the fermentation broth is cultured for 24h, the germination rates of 20mg/L fermentation broth seed soaking are the same as those of a blank control group and are 87%, and when the seed soaking concentration exceeds 20mg/L, the germination rates of wheat seeds are gradually reduced along with the increase of the seed soaking concentration (the same trend is shown in 48h and 72h of culture) unlike alfalfa seeds, which shows that the higher concentration fermentation broth seed soaking treatment has a certain inhibition effect on the germination of the wheat seeds, and the higher the fermentation broth concentration is, the stronger the inhibition effect is, and the inhibition effect is probably related to the structure of the wheat seeds. The wheat seeds are large and have thick cotyledons and enough nutrition for germination, and the extra application of the high-nutrition protein fermentation liquor can cause the excessive nutrition during the germination of the seeds, so that the growth vigor of the seeds is reduced, the germination is inhibited, and the inhibition effect is stronger when the concentration is higher.

The statistical data of 48h and 72h of germination show that the germination rates of 72h and 48h of culture have almost no obvious change, which indicates that the germination of the wheat seeds is basically finished at 48 h. And the germination rate of the wheat seeds is reduced along with the increase of the concentration of the fermentation liquor, the germination rate of the wheat seeds is 1.05 percent higher than that of a blank control group only when the concentration is 20mg/L, and the rest concentrations are lower than that of the blank control group, which shows that the germination accelerating effect of the fermentation liquor on the wheat seeds is not obvious in the concentration range used in the experiment.

Comparison of the effects of dechromised blue Wet dander fermentation broths on the germination of alfalfa and wheat seeds

At the end of germination, the germination rates of alfalfa seeds and wheat seeds as a function of seed soaking concentration are shown in FIG. 9.

As can be seen from fig. 9: when the seed soaking concentration of the dechromization blue wet dander fermentation liquor used in the experiment is 20 mg/L-100 mg/L, the germination rates of the alfalfa seeds increase along with the increase of the seed soaking concentration, are all higher than those of a control group, and reach the maximum value when the germination rates are 80 mg/L; the germination rate of the wheat seeds is reduced along with the increase of the seed soaking concentration, and is equivalent to that of the control group only at the concentration of 20 mg/L. When germination is finished for 72 hours, the maximum germination rate of the alfalfa seeds is 76% (in the fermentation liquor concentration of 80mg/L) which is 28.81% higher than that of the alfalfa seeds treated by water soaking; the germination rate of the large wheat seeds is 95 percent at most (the concentration of the fermentation liquor is 20mg/L), and is only 1.05 percent higher than that of a blank control group. The result shows that the fermentation liquor of the dechromised blue-peel scraps has the functions of obviously accelerating the germination process of small seeds such as alfalfa seeds and strengthening seedlings when being used as a seed soaking agent in the concentration range used in experiments, and has no obvious effect on seed soaking and germination acceleration of large seeds such as wheat.

From the germination time, the germination of the wheat seeds is basically finished in 48 hours, the germination rate reaches 95 percent at most, the germination rate of a control group also reaches 94 percent at the moment, and the germination process is faster, so the significance of promoting the germination by using the fermentation liquor is not great; the germination rate of the alfalfa seeds reaches 76% only after 72 hours, the germination rate of the control group is only 56%, the germination process is relatively slow, and the germination accelerating treatment effect by using fermentation liquor with a certain concentration is very obvious.

Research result of using dechromization blue wet dander fermentation liquor as wheat seedling water culture nutrient solution

Observation result of water culture growth state of wheat seedlings

After one week of seedling culture, the growth state of the wheat seedlings cultured for two weeks by different fertilization treatments is observed. Application concentration of commercial fertilizer: 100mg/L, the same as follows.

Measurement results of height of wheat seedling

After 14 days of cultivation after fertilization, the average height of the wheat seedlings was as shown in FIG. 10.

As can be seen from FIG. 10, the average height of the wheat seedlings after fertilization is higher than that of the wheat seedlings after water culture, which indicates that the organic protein fertilizer can promote the growth of the wheat seedling plants. The plant heights of the experimental group are equal to those of the positive control group except that the fertilization concentration is 20mg/L, the plant heights of the wheat seedlings with the rest concentrations are all higher than those of the positive control group, wherein the experimental group with the same concentration as the positive control group is 100mg/L, and the plant heights of the wheat seedlings with the rest concentrations are 5.96% higher than that of the positive control group, so that under the condition that free amino acids are completely consumed, the positive control group and the 20mg/L experimental group have no excessive nutrition and continue to supply the wheat seedlings for growth, the wheat seedling heights of the experimental group are almost the same as those of the negative control group, and the rest wheat seedlings with the higher fertilization concentration in the experimental group have more micromolecule peptides, polypeptides and the like and can provide source continuous nutrient supply for the plant growth after being decomposed by microorganisms, so that the wheat seedling heights of the experimental group with the fermentation liquor concentration of 40-100 mg/L are obviously higher than those of the control group.

The experimental result shows that the dechromized blue wet dander fermentation solution is richer in nutrient components than the common amino acid fertilizer sold on the market, wherein free amino acid can rapidly promote plant growth, and the polypeptide component plays a role of slow release fertilizer and can maintain longer fertilizer efficiency in the plant growth period. Therefore, the dechromized blue wet dander fermentation liquid has better application prospect as a plant growth biological organic slow-release fertilizer.

Total length determination and lateral root number statistics of adventitious roots of wheat seedlings

The results of root growth of wheat seedlings treated with different fertilizers, compared to hydroponics, after 14 days of cultivation after fertilization are shown in fig. 11.

As can be seen from fig. 11: compared with the water culture, the total length and the number of lateral roots of the wheat seedlings treated by fertilization are increased, which shows that the application of a certain amount of protein fertilizer is beneficial to the growth of the root systems of the wheat seedlings. When the fertilizing concentration of the fermentation liquor is between 20 and 60mg/L, the total length of the adventitious roots and the number of lateral roots of the wheat seedlings are increased along with the increase of the fertilizing amount; when the fertilizing concentration of the fermentation liquor exceeds 60mg/L, the total length of the adventitious roots of the wheat seedlings is shortened, and the number of lateral roots is obviously increased. This is probably because when the nutrient concentration around the root system is relatively low, the wheat seedling will take more nutrients by the growth of adventitious and lateral roots; when the nutrition around the root system is more and more sufficient, the wheat seedling disperses the root system mainly through the proliferation of lateral roots, and the contact area between the root system and nutrient substances is increased, so that the absorption of the nutrition is promoted. Compared with the fermentation liquor fertilization, the root growth condition of the positive control group is similar to that of 60mg/L fermentation liquor, which indicates that the nutrition state of the positive control group is similar to that of 60mg/L fermentation liquor and is different from that of dechromized blue wet scurf fermentation liquor (100mg/L) with the same concentration, otherwise, the nutrition component of the fermentation liquor with the same free amino acid content is higher than that of the common amino acid fertilizer, and the fermentation liquor has a slow release effect.

It can thus be seen that: apply the wet dander zymotic fluid of chromium removal blue of different concentrations, different root system growth states can appear in the wheat seedling, when zymotic fluid fertilization concentration is when 20 ~ 60mg/L, the root system promotes simultaneously through the growth of adventitious root and side root and absorbs the nutrition, and when zymotic fluid fertilization concentration exceeded 60mg/L, the root system mainly absorbs the nutrition through the growth that increases the side root, consequently fertilization concentration should not be too high, otherwise the lodging appears easily for adventitious root short, the flourishing length of wheat seedling is unfavorable for the section of a boiler in addition, can lead to the reduction of labour, so fertilization concentration of zymotic fluid should not be too high.

Experimental data of the total length and the number of lateral roots of the adventitious roots of the wheat seedlings show that 60mg/L of fermentation liquor can well promote the growth of the wheat seedlings in the early stage of jointing and can be well controlled.

Measurement result of chlorophyll content in wheat seedling

A certain amount of fresh wheat leaves are taken, the chlorophyll content is measured according to the standard NY/T3082-2017, and the measurement result is shown in Table 7.

TABLE 7 chlorophyll content of wheat seedlings

Fertilization treatment	a(mg/g)	a increase rate (%)	b(mg/g)	b increase rate (%)	Total (mg/g)	Total gain (%)
							Water (W)	0.906	——	0.366	——	1.271	——
20mg/L	1.275	40.76	0.408	11.60	1.684	32.47
							40mg/L	1.265	39.57	0.398	8.75	1.663	30.80
60mg/L	1.248	37.74	0.404	10.47	1.652	29.99
							80mg/L	1.249	37.90	0.405	10.67	1.654	30.16
100mg/L	1.218	34.41	0.391	6.95	1.609	26.61
							Commercial fertilizer	1.423	57.09	0.489	33.66	1.912	50.47

As can be seen from tables 5-3: compared with the wheat seedlings treated by water, the chlorophyll content of the wheat seedlings treated by fertilization is obviously increased. The main reason is that the fermentation liquor of the dechromized blue wet dander and the commercial fertilizer belong to high-nitrogen fertilizer, and the nitrogen element is an important element for composing chlorophyll, so that the application of a certain amount of nitrogen fertilizer can promote the synthesis of chlorophyll of plants. However, the chlorophyll content of the wheat seedlings treated by the fermentation liquor with different concentrations is changed a little (the chlorophyll a content is about 1.2mg/g, the chlorophyll b content is about 0.4mg/g, and the total chlorophyll content is about 1.6 mg/g), and the change is reduced along with the increase of the concentration. Probably, when the fertilization concentration is high, the new leaves are more and grow fast, and the synthesis and accumulation of chlorophyll are relatively retarded, so that the phenomenon that the chlorophyll content does not increase and decrease along with the increase of the concentration is caused. However, compared with the positive control commercial fertilizer, the difference that the chlorophyll content of the wheat seedlings treated by the fermentation liquor fertilization is lower than that of the positive control group can be found because the commercial fertilizer contains a component for making the leaves green, namely non-protein amino acid-5-aminolevulinic acid, which is a precursor substance for promoting the chlorophyll synthesis, can directly promote the chlorophyll synthesis and prevent the withering and yellowing of the leaves, so that the chlorophyll content of the wheat seedlings treated by the commercial fertilizer fertilization is higher than that of the wheat seedlings treated by the fermentation liquor.

In conclusion, the chlorophyll content of the wheat seedlings can be obviously increased by applying the dechromized blue wet dander fermentation liquor with different concentrations, and the chlorophyll content is the highest when the fertilization concentration is 20 mg/L.

Measurement result of dry and wet weight and moisture content of wheat seedling plant

The weight and moisture content of the plant parts of wheat seeds after one week of conventional seedling culture and then two weeks of fertilization culture are shown in table 8.

TABLE 8 weight and moisture content of wheat seedlings

As can be seen from Table 8, the fresh weight of wheat seedlings was increased less at a fertilizing concentration of 20mg/L than that of the control group

The fresh weight of the wheat seedlings treated by fertilization at other concentrations in the test group is obviously increased, and the fresh weight of the wheat seedlings shows an ascending trend along with the increase of the fertilization concentration. The fresh weight gain of the fermentation liquor with the concentration of 100mg/L is the largest and is as high as 45.75%, while the fresh weight gain of the commercial fertilizer is only 8.56%, and the fresh weight gain effect is not as good as that of the 20mg/L fermentation liquor.

The dry weight may reflect the synthesis and accumulation of organic matter by the wheat seedlings. From the above table, it can be seen that the concentration of the fermentation liquid is within the range of 20-100 mg/L, and the higher the concentration is, the more the dry weight is increased. Similarly, the dry weight of wheat seedlings in the rest groups is increased obviously compared with the control group except that the dry weight of the wheat seedlings with the fertilizing concentration of 20mg/L is increased least, and particularly, the dry weight increasing rate is the largest when the fertilizing concentration of the fermentation liquor is 100mg/L and reaches 17.99 percent.

In addition, the moisture content of the wheat seedlings can be determined, and the fertilization has certain influence on the moisture content of the wheat seedlings. Similarly, in addition to the fact that the increase of the water content of the fermentation liquid concentration of 20mg/L is not as great as that of the control group, the water content of the wheat seedlings also tends to increase slightly with the increase of the fermentation liquid concentration, and reaches the highest value at the concentration of 100 mg/L.

In conclusion, the dechromized blue wet dander fermentation liquid has obvious promotion effect on the increase of the dry weight, the wet weight and the moisture content of the wheat seedlings.

Measurement result of protein content of wheat leaf

The protein content of the wheat leaves was measured by kjeldahl method, and the results are shown in table 9.

TABLE 9 protein content of wheat leaves

Fertilization treatment	Protein content (g/100g dry weight)	Protein increment (%)
			Water (W)	53.13	——
20mg/L	57.17	7.60
			40mg/L	61.50	15.76
60mg/L	64.29	21.01
			80mg/L	66.88	25.88
100mg/L	71.04	33.70
			Commercial fertilizer	60.14	13.19

As can be seen from the dry weight protein contents of wheat seedling leaves shown in Table 9, the protein contents of the wheat seedlings with the applied fertilizers were all higher than those of the hydroponics, indicating that the application of the protein-based fertilizers promoted the synthesis of the protein of the wheat seedling leaves. The protein content in wheat seedlings shows a trend of obviously increasing along with the increase of the concentration of the dechromized blue wet dander fermentation liquor, and reaches the highest value of 71.04g/100 dry weight at 100mg/L, the protein content is 33.70 percent higher than that of water culture, and the protein content is 18.12 percent higher than that of a commercial fertilizer. The experimental result shows that the dechromized blue wet dander fermentation liquor is rich in nutrition and can obviously improve the content of protein in wheat seedlings.

Experimental example 6 application research of dechromization blue wet dander fermentation liquid for highland barley pot experiment fertilizer

And (5) observing the growth state of the highland barley seedlings. In 2020, the highland barley pot culture experiment is carried out in winter, about 20 highland barley seeds are sown in each pot, and each treatment is repeated for 3 pots.

And (5) observing the growth condition of the seedlings after 3 weeks of different fertilization treatments on the 22 th day after sowing the highland barley. Observation shows that the growth vigor of the dechromization blue wet scurf fermentation liquor with different dilutions and the highland barley seedlings treated by the pure bacterial liquid is better than that of an unfertilized control (water), and the advantage is more obvious when the concentration is higher (10 x). Further comparison of the sample with the highest concentration of fertilizer and the best growth with the unfertilized control (water) and the control with the application of commercial fertilizer (fertilizer) revealed that the growth was better than not only the unfertilized control (water) but also the commercial fertilizer control (fertilizer) and had better results after chelating metal ions.

Measuring result of leaf length and leaf width of highland barley

After 22 days of cultivation after fertilization, the leaf length/height (from the soil surface to the leaf tip) of the first leaf of each plant and the widest dimension of the leaf were measured separately for all treatments and statistically analyzed (fig. 12). The results show that the growth of the 10 x dilution sample is significantly better than the commercial fertilizer group and the water control group in terms of the length and width (which can represent the leaf area) of the first leaf of the plant, and the remaining dilution is slightly better than or equal to the commercial fertilizer group.

The above experimental results show that: the higher the concentration of the fermentation liquor of the wet dander is, the higher the germination rate of the seeds is, and the fermentation liquor of the wet dander with the concentration of 80mg/L is most suitable for the seed soaking and germination accelerating treatment of the alfalfa seeds and can obviously promote the germination of the alfalfa seeds.

The fermentation liquor of the dechrominated blue wet dander with proper concentration can obviously promote the germination of the small alfalfa seeds.

The addition of a proper amount of dechromized blue wet scurf fermentation liquor has an obvious effect of promoting the growth of wheat seedling plants, and can obviously increase the plant height, chlorophyll content, dry and wet weight and protein content of wheat seedlings.

The application of a proper amount of dechromization blue wet scurf fermentation liquor or bacterial liquid has an obvious promotion effect on the growth of highland barley seedlings, and the leaf area can be obviously increased.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a short peptide-growth-promoting bacterium compound fertilizer by dechromization fermentation of wet blue dander is characterized by comprising the following steps:

1. dechromisation of wet blue dander

Firstly, the alkali treatment is carried out, and then,

in the step, the wet blue leather scraps are soaked in alkali liquor and mechanically stirred, so that alkali gradually permeates into the wet blue leather scraps to perform dechromization reaction;

wherein the alkali liquor is obtained by adding 6-9g of calcium oxide into water;

the alkali consumption accounts for 8-10% of the mass of the blue dander;

the alkali treatment time is 8-12 h;

the temperature of the alkali dechromization treatment is normal temperature;

then filtering to obtain blue wet leather scraps after alkali dechromization treatment;

then carrying out acid treatment;

in the step, the alkali dechromized blue wet dander dehydrated by suction filtration is placed in an acid solution for soaking;

wherein the acid solution is hydrochloric acid aqueous solution;

the liquid ratio of the acid solution dosage is 1:20 based on the weight of the wet blue leather after the alkali dechromization treatment;

the concentration of the acid solution is 0.6-0.9 mol/L;

the acid treatment time is 30-35 min;

finally, carrying out suction filtration to obtain wet dechromized blue leather scraps;

2. fermenting and degrading the dechromized blue wet dander to obtain blue wet dander fermentation liquor, namely the short peptide-growth-promoting bacteria compound fertilizer

Firstly, selecting a single bacillus pumilus colony which grows well by plate streaking, inoculating the single bacillus pumilus colony into a sterilized liquid seed culture medium, culturing the single bacillus pumilus colony in a constant-temperature oscillating water bath for not less than 24 hours under the stirring conditions of 34-37 ℃ and 120-150r/min to obtain the bacillus pumilus, and taking seed liquid for later use;

then, mixing the dechromized blue wet dander and a fermentation culture medium according to a solid-to-liquid ratio of 1: 5-1:25, mixing, adjusting pH to 7.0-12.0, sterilizing at 121 deg.C for 20 min;

after the fermentation culture medium is cooled, inoculating seed liquid with an inoculum size of 0.5-4%, and culturing for no less than 24h in a constant temperature shaking water bath at 27-47 deg.C and 120 r/min;

wherein the seed culture medium comprises beef extract 3.0g, peptone 10.0g, NaCl 5.0g, and water 1000mL, and the pH is adjusted to 8.0 with sodium hydroxide;

NaCl 5.0g，MgSO₄·7H₂0.2g of O, 1.0g of sodium citrate, 2.0g of ammonium sulfate, 20.0g of agar and 1000ml of water, and the pH value is adjusted to 8.0 by using sodium hydroxide;

2. The method for preparing the oligopeptide-growth-promoting bacterium compound fertilizer by the dechromization fermentation of the wet blue dander as claimed in claim 1, which is characterized in that: the bacterial age of the bacillus pumilus is 24 hours, the solid-liquid ratio is 1:15, the inoculation amount is 0.5%, the fermentation temperature is 37 ℃, the pH value is 9.0, and the fermentation time is 48 hours.

3. The method for preparing a short peptide-growth-promoting bacteria compound fertilizer by blue wet dander dechromization fermentation as claimed in claim 1 or 2, wherein the bacillus pumilus is bacillus pumilus BA06 with the preservation number of CGMCC NO. 0518.

4. The method for preparing the oligopeptide-growth-promoting bacterium compound fertilizer by the dechromization fermentation of the wet blue dander as claimed in claim 1, which is characterized in that: the blue wet leather scraps are chrome-containing leather waste generated by shaving, leather grinding and trimming after a tanning working section, and the chrome content of the chrome-containing leather waste is about 3 percent.

5. A short peptide-growth-promoting bacteria compound fertilizer prepared by the method of any one of claims 1 to 4.

6. The short peptide-growth promoting bacteria compound fertilizer prepared by any one of the methods of claims 1 to 4 is used in the planting of wheat, alfalfa and highland barley.

7. The use of the short peptide-growth promoting bacteria compound fertilizer prepared by the method of any one of claims 1 to 4 in the planting of wheat, alfalfa and highland barley is characterized in that: the short peptide-growth-promoting bacterium compound fertilizer is used as a seed soaking liquid, a water culture liquid or a pot culture fertilizer.