CN117778256A - Screening of cadmium-resistant siderophore-producing bacillus dyslysines, preparation of microbial inoculum and application of microbial inoculum - Google Patents
Screening of cadmium-resistant siderophore-producing bacillus dyslysines, preparation of microbial inoculum and application of microbial inoculum Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses screening of cadmium-resistant siderophore-producing bacillus lysine-deforming, preparation of a microbial agent and application thereof, and relates to the field of microorganisms, wherein the method comprises the following specific formulas: the weight percentages of the components are as follows: 10 to 18 weight percent of decomposed agricultural byproducts, 10 to 20 weight percent of calcium magnesium phosphate fertilizer, 5 to 15 weight percent of biochar, 5 to 15 weight percent of rice bran and 2.0 to 3.0 percent of microbial fermentation liquor; according to the screening, microbial inoculum preparation and application of the bacillus dyslysines of the cadmium-resistant siderophores, through bacillus dyslysines WY-C9 fermentation liquor, on one hand, the microorganism can carry out surface adsorption by virtue of hydroxyl (-OH), carbonyl (-CO-), amido (-CO-NH-), phosphate (-PO 43-) and other groups on the cell wall, and carry out complexation such as extracellular complexation and extracellular precipitation and fix heavy metal cadmium in soil through metabolites of the microorganism.
Description
Technical Field
The invention relates to a microbial technology, in particular to screening of cadmium-resistant siderophore-producing bacillus dyslysii, preparation of a microbial inoculum and application thereof.
Background
Soil heavy metal pollution, particularly Cadmium (Cd) pollution, has become one of the most widely polluted areas and the most harmful environmental problems in China. The soil pollutant point position standard exceeding rate of the cultivated land in China is 19.4%, the pollution type is mainly inorganic, the inorganic pollutant standard exceeding rate is 82.8% of the total standard exceeding rate, and the main inorganic pollutants comprise heavy metals such as cadmium, mercury, arsenic, copper, lead, chromium, zinc, nickel and the like, wherein the point position standard exceeding rate of the cadmium is as high as 7.0%. Cadmium is a heavy metal pollutant with strong toxicity, has long-term, concealed and irreversible pollution to soil, has extremely high hazard degree, can cause soil degradation, crop yield and quality reduction, can endanger human health and life through direct contact, food chains and other ways, and can lighten or eliminate heavy metal pollution in farmland soil, which is a typical farmland ecological system, is a soil resource with great economic significance, and has a significant role in grain safety in China. Therefore, the problem of cadmium pollution in cultivated lands is a globally important strategic requirement for environmental safety of agricultural products, drinking water and living, and sustainable development of social economy. The traditional heavy metal pollution restoration method mainly comprises a phytoremediation method, an electrodynamic remediation method, a leaching method, a thermal desorption method, a vitrification method, a fixed stabilization method and the like, wherein the method has high cost and huge engineering quantity, and breaks the original physical and chemical structures of soil, so that the method is not suitable for long-term treatment of large-area heavy metal pollution.
The microbial remediation is taken as a novel green environment-friendly remediation technology, although microorganisms cannot degrade and destroy heavy metals, the migration and conversion of metals in the environment can be influenced by changing the chemical or physical characteristics of the microorganisms, the microbial remediation is simple in culture, the corresponding preparation is low in cost and has the advantages of no secondary pollution, large remediation potential and permanent treatment effect, the soil structure and regional biological communities can be protected, good ecological environment can be created, and the microbial remediation becomes a hotspot for research of countries around the world.
However, at present, due to different repairing environments, more microorganism types with more powerful functions are further required to be screened according to the corresponding environments, and proper microorganism carriers or adjuvants are explored, and the two are complemented with each other to repair the environments, so that the advantages of the two are combined, and a more outstanding effect can be exerted in practical application.
Disclosure of Invention
The invention aims to provide screening, microbial inoculum preparation and application of cadmium-resistant siderophore-producing carrier-deformed lysine bacillus, so as to solve the defects in the prior art.
In order to achieve the above object, the present invention provides the following technical solutions: screening of cadmium-resistant siderophore-producing bacillus dyslysines, preparation of microbial inoculum and application thereof, and comprises the following specific formulas:
the weight percentages of the components are as follows: 10 to 18 weight percent of decomposed agricultural byproducts, 10 to 20 weight percent of calcium magnesium phosphate fertilizer, 5 to 15 weight percent of biochar, 5 to 15 weight percent of rice bran and 2.0 to 3.0 percent of microbial fermentation liquor;
the preparation of the microbial fermentation broth is as follows:
s1, strain activation: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium, and performing activation culture for 24-48 h at the temperature of 25-35 ℃;
solid NB seed medium: 10g/L of peptone, 5g/L of beef extract, 10g/L of sodium chloride, 1L of water, 2% of agar and natural pH;
s2, preparing seed liquid: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium into the liquid seed culture medium, and shake culturing for 24-48 h at 25-35 ℃ to prepare seed liquid;
liquid seed medium (NB): 5-10g/L peptone, 5-10g/L beef extract, 5-10g/L sodium chloride, 1L water and natural pH;
s3, fermenting in a seed tank; inoculating the seed liquid obtained in the step 2 into a seed tank according to the inoculum size of 5-8% of the volume percentage of the culture medium to be inoculated, wherein the seed liquid uses NB culture medium; introducing sterile air and stirring, culturing for 24-36 h at 25-35 ℃, and stirring for 150-250 rpm at a ventilation rate of 1-2 Vols/vol.min, wherein after fermentation, the content of the lysine-modified bacillus WY-C9 in the microbial fermentation broth is: 1X 10 6 ～1×10 8 CFU/ml；
S4, culturing in a production fermentation tank
Inoculating the fermentation liquor of the seed tank into the fermentation tank according to the inoculum size of 5-8% of the volume percentage of the culture medium, introducing sterile air and stirring, culturing for 24-36 h at 28-35 ℃, and the ventilation rate is 1-2 Vols/vol.min, the stirring rotating speed is 180-250rpm, and the content of the lysine-modified bacillus WY-C9 in the microbial fermentation liquor after the fermentation is finished: 1X 10 8 ～1×10 9 CFU/ml；
1L of fermentation tank culture medium formula: liquid fermentation medium: molasses 10-20g/L, soytone 2-7g/L, KH 2 PO 4 0.1-1.5g/L，K 2 HPO 4 0.1-2.2g/L，MnSO 4 ﹒H 2 O 0.01-0.2g/L，pH6.5-8.0；
S5, mixing the components in sequence during preparation of the microbial inoculum.
As a further optimization of the invention, the screening of the lysine bacillus strains comprises the following steps:
s1, collecting soil samples in heavy metal polluted farmland, and carrying the soil samples back to a laboratory by using a sterile paper bag to separate and screen strains;
s2, separating bacterial strain by adopting a gradient dilution method, weighing 10g of soil sample, putting into a triangular flask filled with 90ml of sterile water, and vibrating on a shaking table for 30min to obtain the dilution degree of 10 –1 Is a soil suspension of (a);
s3, taking 6 glass test tubes filled with 4.5ml of sterile water, and respectively marking the dilution degree as 10 –2 ，10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 ；
S4, sucking 0.5ml from the soil suspension with the dilution of 10-1, and adding the soil suspension into the marked dilution of 10 –2 Is sufficiently vibrated and uniformly mixed to obtain the glass test tube (15 multiplied by 150 mm) with the dilution of 10 –2 A soil dilution;
s5, sequentially carrying out gradient dilution according to a 10-time method, wherein the obtained dilutions are respectively 10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 Sequentially and respectively sucking 0.1mL of the soil diluents into a sterile 96-well cell culture plate, adding 0.1mL of improved R2A liquid culture medium into each well, fully and uniformly mixing, and sequentially adding a proper amount of non-sterile materialsBacterial 100mg/ml CdSO 4 Solutions, preparation of Cd-containing solutions, respectively 2+ The concentration is 25mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L of mixed bacterial liquid;
s6, placing the cell culture plate in a microplate constant-temperature oscillator, culturing at 35 ℃ and 150 revolutions per minute, taking out the cell culture plate every 6 hours, and measuring the OD of each hole by using a spectrophotometer 600 To the existing OD 600 Initial OD 600 >1.2, picking mixed bacterial liquid by using a sterile inoculating needle, streaking on NB solid culture medium to obtain pure culture, picking single bacterial colony, microscopic examination, and transferring to an inclined plane for preservation at 4 ℃.
As a further optimization of the invention, the strain WY-C9 is in Cd 2+ Strain screening culture medium with concentration of 25-200mg/L for normal growth, improved R2A culture medium: yeast leaching 0.1-0.5g, peptone 0.1-0.8g, casein hydrolysate 0.1-0.6g, glucose 0.1-0.7g, soluble starch 0.1-0.5g, KH 2 PO 4 0.1-0.5g，MgSO 4 0.01-0.05g, sodium pyruvate 0.1-0.6g, water 1000ml, pH 7.2+ -0.2 (R2A solid medium additionally added with 15g agar);
beef extract peptone medium (NB): 2-5g of beef extract, 5-10g of peptone, 2-5g of NaCl, and pH=7.0-7.2;
bacterial siderophore assay medium (CAS): 20% glucose solution 0.5-2ml,10% Casein amino acid solution 3-5ml,1mol/LCaCl 2 0.05-0.8ml，1mol/LMgSO 4 0.8-4ml, 5-20ml of phosphate buffer solution (pH 6.8), 10-20ml of dye liquor (prepared from chrome azure and hexadecyl trimethyl ammonium bromide) and 60-80ml of 1.8% agar;
siderophore quantitative Medium (MKB): 5.0g/L casein amino acid, 15mL/L glycerol, 2.5g/L potassium dihydrogen phosphate, 2.5g/L magnesium sulfate heptahydrate, pH7.2.
As a further optimization scheme of the invention, screening of the spawn siderophores;
qualitative screening of spawn iron carrier production capacity: after activating the selected Cd-resistant strain, dipping a bacterial solution by using a sterile inoculating loop, inoculating the bacterial solution onto a CAS siderophore plate, culturing for 16-24 hours at a constant temperature of 35 ℃, if siderophores are not produced, enabling a CAS solid culture medium to be blue as a contrast, and if the bacterial strain siderophores are produced, enabling the CAS solid culture medium to be changed into a visible yellow hydrolysis ring;
quantitative screening of spawn iron carrier production capacity: sucking 1mL of bacterial suspension of the strain to be detected, inoculating the bacterial suspension into an MKB liquid culture medium, and culturing for 48 hours at 28 ℃ and 180 r/min; the culture broth was centrifuged for 10min (1200 r/min) and 200. Mu.L of supernatant (200. Mu.L of uninoculated MKB liquid medium was added for the measurement of the reference value (Er)) was taken according to 1:1 with CAS detection solution; after reacting for 1h at normal temperature, the enzyme-labeled instrument measures the OD value (E) of the wavelength at 630nm, if no siderophore is produced, the CAS liquid culture medium is blue as compared with the control, if the strain produces siderophores, the CAS liquid culture medium changes to orange, the ratio of E/Er is used for representing the relative content of siderophores in a sample, the smaller the value is, the stronger the capacity of the strain for producing siderophores is represented, the ratio of (Er-E)/Er is used for representing the activity unit of siderophores in the sample, and the higher the activity unit is, the stronger the siderophore producing capacity is;
and taking bacterial liquid growing to a logarithmic phase from the strain obtained by screening, centrifugally collecting bacterial bodies, and extracting genome DNA by using the kit.
Compared with the prior art, the screening, microbial inoculum preparation and application of the bacillus dyslysiae strain resistant to the cadmium siderophore provided by the invention are characterized in that the bacillus dyslysiae WY-C9 fermentation liquor is modified, on one hand, the microorganism can carry out surface adsorption by virtue of hydroxyl (-OH), carbonyl (-CO-), amido (-CO-NH-), phosphate (-PO 43-) and other groups on the cell wall, and the metabolite of the microorganism is used for carrying out extracellular complexation, extracellular precipitation and other complexation and fixing heavy metal cadmium in soil.
In soil, the strain has strong colonization capability, can effectively passivate heavy metals in soil, and reduces the absorption of plants.
The microbial inoculum prepared based on the lysine-modified bacillus WY-C9 can respectively reduce the effective cadmium in the soil of the heavy metal polluted paddy field by 52.2%, the cadmium content in the soil by 54.8% and the yield of the paddy by 4.8%, and meanwhile, the microbial inoculum has the functions of improving the physical and chemical properties of the soil and the like, and has great popularization and application values.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to these drawings for a person having ordinary skill in the art.
FIG. 1 is a schematic diagram showing growth conditions of a strain of Bacillus dysarius WY-C9 according to an embodiment of the present invention under different cadmium concentration culture conditions;
FIG. 2 is a schematic diagram showing the growth of a lysine-modified bacillus strain WY-C9 in a siderophore-producing plate (CAS);
FIG. 3 is a schematic diagram showing the growth curve of a strain WY-C9 of Bacillus dysarius according to the example of the present invention.
Detailed Description
In order to make the technical scheme of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings.
Embodiment one:
referring to fig. 1-2, a screening method of a cadmium-resistant siderophore-producing bacillus dyslysii, a preparation method of a microbial inoculum and an application thereof are disclosed, wherein the method comprises the following specific formulas:
the weight percentages of the components are as follows: 10 to 18 weight percent of decomposed agricultural byproducts, 10 to 20 weight percent of calcium magnesium phosphate fertilizer, 5 to 15 weight percent of biochar, 5 to 15 weight percent of rice bran and 2.0 to 3.0 percent of microbial fermentation liquor;
the preparation of the microbial fermentation broth is as follows:
s1, strain activation: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium, and performing activation culture for 24-48 h at the temperature of 25-35 ℃;
solid NB seed medium: 10g/L of peptone, 5g/L of beef extract, 10g/L of sodium chloride, 1L of water, 2% of agar and natural pH;
s2, preparing seed liquid: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium into the liquid seed culture medium, and shake culturing for 24-48 h at 25-35 ℃ to prepare seed liquid;
liquid seed medium (NB): 5-10g/L peptone, 5-10g/L beef extract, 5-10g/L sodium chloride, 1L water and natural pH;
s3, fermenting in a seed tank; inoculating the seed liquid obtained in the step 2 into a seed tank according to the inoculum size of 5-8% of the volume percentage of the culture medium to be inoculated, wherein the seed liquid uses NB culture medium; introducing sterile air and stirring, culturing for 24-36 h at 25-35 ℃, and stirring for 150-250 rpm at a ventilation rate of 1-2 Vols/vol.min, wherein after fermentation, the content of the lysine-modified bacillus WY-C9 in the microbial fermentation broth is: 1X 10 6 ～1×10 8 CFU/ml；
S4, culturing in a production fermentation tank
Inoculating the fermentation liquor of the seed tank into the fermentation tank according to the inoculum size of 5-8% of the volume percentage of the culture medium, introducing sterile air and stirring, culturing for 24-36 h at 28-35 ℃, and the ventilation rate is 1-2 Vols/vol.min, the stirring rotating speed is 180-250rpm, and the content of the lysine-modified bacillus WY-C9 in the microbial fermentation liquor after the fermentation is finished: 1X 10 8 ～1×10 9 CFU/ml；
1L of fermentation tank culture medium formula: liquid fermentation medium: molasses 10-20g/L, soytone 2-7g/L, KH 2 PO 4 0.1-1.5g/L，K 2 HPO 4 0.1-2.2g/L，MnSO 4 ﹒H 2 O 0.01-0.2g/L，pH6.5-8.0；
S5, mixing the components in sequence during preparation of the microbial inoculum.
The microorganism in the microbial fermentation broth is bacillus dyslysiae WY-C9 with the preservation number of CGMCCNO.26650;
the specific formula for preparing the microbial agent based on the strain is as follows:
the decomposed agricultural byproducts (crop straw, tea seed cake and pig manure) are high-quality organic fertilizer, the organic matter content of the organic fertilizer is 20-50wt%, and active groups (such as carboxyl, amino, hydroxyl and the like) of the organic fertilizer are easily used as ligands to complex or chelate with heavy metal elements Cd and the like, so that heavy metals in soil are passivated or adsorbed.
The main component of the calcium magnesium phosphate fertilizer comprises Ca 3 (PO 4 ) 2 、CaSiO 3 、MgSiO 3 The main chemical composition of the composition is as follows: mgO is greater than or equal to 15%, siO 2 More than or equal to 22 percent, caO more than or equal to 30 percent, can improve the content of effective calcium, magnesium and silicon in the soil, improve the pH value of the soil, reduce the content of effective cadmium in the soil, and further prevent rice from absorbing excessive heavy metals.
The biochar is mainly prepared from insoluble, stable, highly aromatic and carbon-rich solid substances generated by pyrolysis of agricultural and forestry wastes, organic wastes and the like under the condition of oxygen deficiency, has a loose and porous structure, a large specific surface area, a large amount of negative charges on the surface and higher charge density, is rich in a series of oxygen-containing, nitrogen-containing and sulfur-containing functional groups, has large cation exchange capacity and is a good adsorption material.
The rice bran is mainly prepared from pericarp, seed coat, endosperm, aleurone layer and embryo. In general, rice bran contains on average 15% protein, 16% to 22% fat, 3% to 8% sugar, 10% moisture, and about 125.1KJ/g calories. The main fatty acid in the fat is mostly unsaturated fatty acid such as oleic acid, linoleic acid and the like, and contains high amounts of vitamins, plant alcohols, dietary fibers, amino acids, minerals and the like, thereby being beneficial to the growth and colonization of microorganisms.
The screening of the lysine bacillus strain comprises the following steps:
s1, collecting soil samples in heavy metal polluted farmland, and carrying the soil samples back to a laboratory by using a sterile paper bag to separate and screen strains;
s2, separating bacterial strain by adopting a gradient dilution method, weighing 10g of soil sample, putting into a triangular flask filled with 90ml of sterile water, and vibrating on a shaking table for 30min to obtain the dilution degree of 10 –1 Is a soil suspension of (a);
s3, taking 6 glass test tubes filled with 4.5ml of sterile water, and respectively marking the dilution degree as 10 –2 ，10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 ；
S4, sucking 0.5ml from the soil suspension with the dilution of 10-1, and adding the soil suspension into the marked dilution of 10 –2 Glass test tube of (2)(15X 150 mm), and mixing under shaking to obtain a dilution of 10 –2 A soil dilution;
s5, sequentially carrying out gradient dilution according to a 10-time method, wherein the obtained dilutions are respectively 10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 Sequentially and respectively sucking 0.1mL of the soil diluents into sterile 96-well cell culture plates, adding 0.1mL of improved R2A liquid culture medium into each well, fully and uniformly mixing, and sequentially adding a proper amount of sterile 100mg/mL CdSO 4 Solutions, preparation of Cd-containing solutions, respectively 2+ The concentration is 25mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L of mixed bacterial liquid;
s6, placing the cell culture plate in a microplate constant-temperature oscillator, culturing at 35 ℃ and 150 revolutions per minute, taking out the cell culture plate every 6 hours, and measuring the OD of each hole by using a spectrophotometer 600 To the existing OD 600 Initial OD 600 >1.2, picking mixed bacterial liquid by a sterile inoculating needle, streaking in NB solid culture medium to obtain pure culture, picking single colony, microscopic examination, transferring to inclined plane for preservation at 4deg.C
Strain WY-C9 is in Cd2+ concentration 25-200mg/L strain screening culture medium of normal growth, modified R2A culture medium: yeast leaching 0.1-0.5g, peptone 0.1-0.8g, casein hydrolysate 0.1-0.6g, glucose 0.1-0.7g, soluble starch 0.1-0.5g, KH 2 PO 4 0.1-0.5g，MgSO 4 0.01-0.05g, sodium pyruvate 0.1-0.6g, water 1000ml, pH 7.2+ -0.2 (R2A solid medium additionally added with 15g agar);
beef extract peptone medium (NB): 2-5g of beef extract, 5-10g of peptone, 2-5g of NaCl, and pH=7.0-7.2;
bacterial siderophore assay medium (CAS): 20% glucose solution 0.5-2ml,10% Casein amino acid solution 3-5ml,1mol/LCaCl 2 0.05-0.8ml，1mol/LMgSO 4 0.8-4ml, 5-20ml of phosphate buffer solution (pH 6.8), 10-20ml of dye liquor (prepared from chrome azure and hexadecyl trimethyl ammonium bromide) and 60-80ml of 1.8% agar;
siderophore quantitative Medium (MKB): 5.0g/L casein amino acid, 15mL/L glycerol, 2.5g/L potassium dihydrogen phosphate, 2.5g/L magnesium sulfate heptahydrate, pH7.2.
Screening the spawn iron carrier production capacity;
qualitative screening of spawn iron carrier production capacity: after activating the selected Cd-resistant strain, dipping a bacterial solution by using a sterile inoculating loop, inoculating the bacterial solution onto a CAS siderophore plate, culturing for 16-24 hours at a constant temperature of 35 ℃, if siderophores are not produced, enabling a CAS solid culture medium to be blue as a contrast, and if the bacterial strain siderophores are produced, enabling the CAS solid culture medium to be changed into a visible yellow hydrolysis ring;
quantitative screening of spawn iron carrier production capacity: sucking 1mL of bacterial suspension of the strain to be detected, inoculating the bacterial suspension into an MKB liquid culture medium, and culturing for 48 hours at 28 ℃ and 180 r/min; the culture broth was centrifuged for 10min (1200 r/min) and 200. Mu.L of supernatant (200. Mu.L of uninoculated MKB liquid medium was added for the measurement of the reference value (Er)) was taken according to 1:1 with CAS detection solution; after reacting for 1h at normal temperature, measuring a wavelength OD value (E) at 630nm by an enzyme-labeling instrument, if no siderophore is produced, the CAS liquid culture medium is blue as compared with a control, if the bacterial strain produces siderophore, the CAS liquid culture medium is changed into orange yellow, the ratio of E/Er is used for representing the relative content of siderophore in a sample, the smaller the value is, the stronger the siderophore producing capability of the bacterial strain is represented by the ratio of (Er-E)/Er, the higher the siderophore producing capability is represented by the ratio of (Er-E)/Er, and the higher the siderophore producing capability is, as a result, the activity unit of siderophore produced by the WY-C9 bacterial strain in the screened bacterial strain is found to be higher and reach 43.2%;
and taking bacterial liquid growing to a logarithmic phase from the strain obtained by screening, centrifugally collecting bacterial bodies, and extracting genome DNA by using the kit.
Primers 27-F and 1492-R,27-F were designed and synthesized according to conserved regions in bacterial 16 SrDNA: GAGAGTTTGATCCTGGCTCAG;1492-R: AAGGAGGTGATCCARCCGCA.
The pair of primers is used for amplification by taking the extracted genome DNA as a template to obtain a PCR product.
The obtained spliced sequence is compared with the existing 16SrDNA sequence in GenBank database by Blast program, and has high homology of 98% with bacillus dysarius (Lysinibacillus sp.) and the strain WY-C9 is initially identified as bacillus dysarius.
The bacillus dyslysiae WY-C9 disclosed by the invention can be combined with heavy metal cadmium in soil.
The bacillus dyslysiae WY-C9 disclosed by the invention is used for adsorbing heavy metals on the surface by virtue of groups including hydroxyl groups, carbonyl groups, amide groups and phosphate groups on the cell wall.
According to the bacillus dyslysiae WY-C9 disclosed by the invention, heavy metals in soil are fixed in modes of extracellular complexation, extracellular precipitation, adsorption and the like through metabolites of the bacillus dyslysiae WY-C9.
Embodiment two:
referring to fig. 3, test design: the field trials were conducted in the hermountain area of Yiyang city (soil of cadmium pollution type).
(1) Testing;
the test fields were divided into 5 treatments and a control group (CK) without any material applied was set; cell application of 10L of strain WY-C9 fermentation broth (K1); cell application of 10L of inactivated strain WY-C9 fermentation broth (K2); applying 100kg of organic passivating agent per mu, and mixing (K3) according to 15 parts of decomposed agricultural byproducts, 15 parts of calcium magnesium phosphate fertilizer, 12 parts of biochar and 5 parts of rice bran; 100kg of developed microbial agent (S) is applied to each mu, each treatment is repeated for 3 times, 15 cells are added, and the area of the cell for planting rice is 30.0m 2 (5 m.times.6m), and the ridges (0.4 m burial depth) are covered by a thick plastic film with the width of 0.2m for spacing. Field management, topdressing and pest control: the cultivation is carried out in a conventional manner by local farmers.
(2) Selecting Taiyou Yue-occupy late rice variety.
(3) Application method
The application amount is reasonably adjusted according to the soil nutrition condition and pollution condition, the soil can be fully Tian Junyun broadcasted before soil preparation (the soil can be broadcasted after being uniformly mixed with the base fertilizer), and the soil can be planted after tillage, harrowing and soil preparation.
(4) Detecting items
The effective cadmium content of soil, the cadmium content of polished rice and the rice yield.
The result shows (see the attached table 1), after the microbial agent is applied to rice maturity, the effective cadmium in the soil of the heavy metal polluted rice field can be respectively reduced by 52.2%, the cadmium content of the rice is reduced by 54.8%, and the yield of the rice is increased by 4.8%.
The cadmium reduction yield increase effect and the comparative test of the microbial inoculum applied to rice are shown as follows;
wherein CK-blank control, K1-WY-C9 bacteria, K2-inactivated WY-C9, K3-bacteria without bacterial strain WY-C9 fermentation broth, S-bacteria with bacterial strain WY-C9 fermentation broth;
the microorganism fermentation liquor is a lysine-modified bacillus WY-C9 fermentation liquor, on one hand, the microorganism can depend on hydroxyl (-OH), carbonyl (-CO-), amido (-CO-NH-) and phosphate (-PO) on the cell wall 4 3- ) The surface adsorption of the groups, the complexation such as extracellular complexation, extracellular precipitation and the like of the metabolites and the fixation of heavy metal cadmium in soil are carried out;
wherein the sequence of the 16SrDNA of WY-C9 is as follows:
CCCCAATCATCTATCCCACCTTCGGCGGCTGGCTCCAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGACTTTATCGGATTAGCTCCCTCTCGCGAGTTGGCAACCGTTTGTATCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTAAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGTTGTCCCCGAAGGGAAAACTGTATCTCTACAGTGGTCAATGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGATAGTCGCCTTCGCCACTGGTGTTCCTCCAAATCTCTACGCATTTCACCGCTACACTTGGAATTCCACTATCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTAATAAGGTACCGTCAAGGTACAGCCAGTTACTACTGTACTTGTTCTTCCCTTACAACAGAGTTTTACGAACCGAAATCCTTCTTCACTCACGCGGCGTTGCTCCATCAGGCTTTCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGCCCATCCTATAGCGACAGCCGAAACCGTCTT；
the bacillus dysarius WY-C9 bacteria are derived from soil of paddy fields polluted by cadmium at the mud river mouth in the Heshan area of Yiyang city, and are positive in gram staining, aerobic, rod-shaped and spore-producing, and are primarily identified as the bacillus dysarius through 16SrDNA molecular identification and phylogenetic analysis. In soil, the strain has strong colonization capability, can effectively passivate heavy metals in soil, and reduces the absorption of plants. The strain of the bacillus lysine strain WY-C9 (bacillus bataviensis) is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.26650 in the 2 nd 21 nd 2023, has remarkable effect on reducing the effective cadmium and rice cadmium in soil, secretes siderophores to promote plant growth and increases the rice yield.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that modifications may be made to the described embodiments in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive of the scope of the invention, which is defined by the appended claims.
Claims (4)
1. The screening, microbial inoculum preparation and application of the cadmium-resistant siderophore-producing bacillus dyslysines are characterized by comprising the following specific formulas:
the weight percentages of the components are as follows: 10 to 18 weight percent of decomposed agricultural byproducts, 10 to 20 weight percent of calcium magnesium phosphate fertilizer, 5 to 15 weight percent of biochar, 5 to 15 weight percent of rice bran and 2.0 to 3.0 percent of microbial fermentation liquor;
the preparation of the microbial fermentation broth is as follows:
s1, strain activation: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium, and performing activation culture for 24-48 h at the temperature of 25-35 ℃;
solid NB seed medium: 10g/L of peptone, 5g/L of beef extract, 10g/L of sodium chloride, 1L of water, 2% of agar and natural pH;
s2, preparing seed liquid: inoculating the bacillus dysmorphism WY-C9 on the inclined plane of the solid seed culture medium into the liquid seed culture medium, and shake culturing for 24-48 h at 25-35 ℃ to prepare seed liquid;
liquid seed medium (NB): 5-10g/L peptone, 5-10g/L beef extract, 5-10g/L sodium chloride, 1L water and natural pH;
s3, fermenting in a seed tank; inoculating the seed liquid obtained in the step 2 into a seed tank according to the inoculum size of 5-8% of the volume percentage of the culture medium to be inoculated, wherein the seed liquid uses NB culture medium; introducing sterile air and stirring, culturing for 24-36 h at 25-35 ℃, and stirring for 150-250 rpm at a ventilation rate of 1-2 Vols/vol.min, wherein after fermentation, the content of the lysine-modified bacillus WY-C9 in the microbial fermentation broth is: 1X 10 6 ～1×10 8 CFU/m l；
S4, culturing in a production fermentation tank
Inoculating the fermentation liquor of the seed tank into the fermentation tank according to the inoculum size of 5-8% of the volume percentage of the culture medium, introducing sterile air and stirring, culturing for 24-36 h at 28-35 ℃, and the ventilation rate is 1-2 Vols/vol.min, the stirring rotating speed is 180-250rpm, and the content of the lysine-modified bacillus WY-C9 in the microbial fermentation liquor after the fermentation is finished: 1X 10 8 ～1×10 9 CFU/ml；
1L of fermentation tank culture medium formula: liquid fermentation medium: molasses 10-20g/L, soytone 2-7g/L, KH 2 PO 4 0.1-1.5g/L，K 2 HPO 4 0.1-2.2g/L，MnSO 4 ﹒H 2 O 0.01-0.2g/L，pH6.5-8.0；
S5, mixing the components in sequence during preparation of the microbial inoculum.
2. The screening, microbial inoculum preparation and application of the cadmium-resistant siderophore-producing bacillus dyslysines according to claim 1, wherein the screening of the bacillus dyslysines strain comprises the following steps:
s1, collecting soil samples in heavy metal polluted farmland, and carrying the soil samples back to a laboratory by using a sterile paper bag to separate and screen strains;
s2, separating bacterial strain by adopting a gradient dilution method, weighing 10g of soil sample, putting into a triangular flask filled with 90ml of sterile water, and vibrating on a shaking table for 30min to obtain the dilution degree of 10 –1 Is a soil suspension of (a);
s3, taking 6 glass test tubes filled with 4.5ml of sterile water, and respectively marking the dilution degree as 10 –2 ，10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 ；
S4, sucking 0.5ml from the soil suspension with the dilution of 10-1, adding the soil suspension into a glass test tube (15 multiplied by 150 mm) with the marked dilution of 10-2, and fully vibrating and uniformly mixing to obtain the soil dilution with the dilution of 10-2;
s5, sequentially carrying out gradient dilution according to a 10-time method, wherein the obtained dilutions are respectively 10 –3 ，10 –4 ，10 –5 ，10 –6 ，10 –7 Sequentially and respectively sucking 0.1mL of the soil diluents into sterile 96-well cell culture plates, adding 0.1mL of improved R2A liquid culture medium into each well, fully and uniformly mixing, and sequentially adding a proper amount of sterile 100mg/mL CdSO 4 Solutions, preparation of Cd-containing solutions, respectively 2+ The concentration is 25mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L and 250mg/L of mixed bacterial liquid;
s6, placing the cell culture plate in a microplate constant-temperature oscillator, culturing at 35 ℃ and 150 revolutions per minute, taking out the cell culture plate every 6 hours, and measuring the OD of each hole by using a spectrophotometer 600 To the existing OD 600 Initial OD 600 >1.2, picking mixed bacterial liquid by using a sterile inoculating needle, streaking on NB solid culture medium to obtain pure culture, picking single bacterial colony, microscopic examination, and transferring to an inclined plane for preservation at 4 ℃.
3. Root of Chinese characterThe screening, microbial inoculum preparation and application of the cadmium-resistant siderophore-producing strain lysine bacillus as claimed in claim 2, wherein the strain WY-C9 is in Cd 2+ Strain screening culture medium with concentration of 25-200mg/L for normal growth, improved R2A culture medium: yeast leaching 0.1-0.5g, peptone 0.1-0.8g, casein hydrolysate 0.1-0.6g, glucose 0.1-0.7g, soluble starch 0.1-0.5g, KH 2 PO 4 0.1-0.5g，MgSO 4 0.01-0.05g, sodium pyruvate 0.1-0.6g, water 1000ml, pH 7.2+ -0.2 (R2A solid medium additionally added with 15g agar);
beef extract peptone medium (NB): 2-5g of beef extract, 5-10g of peptone, 2-5g of NaCl and pH=7.0-7.2;
bacterial siderophore assay medium (CAS): 20% glucose solution 0.5-2ml,10% Casein amino acid solution 3-5ml,1mol/LCaCl 2 0.05-0.8ml，1mol/LMgSO 4 0.8-4ml, 5-20ml of phosphate buffer solution (pH 6.8), 10-20ml of dye liquor (prepared from chrome azure and hexadecyl trimethyl ammonium bromide) and 60-80ml of 1.8% agar;
siderophore quantitative Medium (MKB): 5.0g/L casein amino acid, 15mL/L glycerol, 2.5g/L potassium dihydrogen phosphate, 2.5g/L magnesium sulfate heptahydrate, pH7.2.
4. The screening of the cadmium-resistant siderophore-producing strain lysine bacillus, the preparation of the microbial inoculum and the application of the microbial inoculum according to claim 2, wherein the screening of the siderophore-producing ability of the strain;
qualitative screening of spawn iron carrier production capacity: after activating the selected Cd-resistant strain, dipping a bacterial solution by using a sterile inoculating loop, inoculating the bacterial solution onto a CAS siderophore plate, culturing for 16-24 hours at a constant temperature of 35 ℃, if siderophores are not produced, enabling a CAS solid culture medium to be blue as a contrast, and if the bacterial strain siderophores are produced, enabling the CAS solid culture medium to be changed into a visible yellow hydrolysis ring;
quantitative screening of spawn iron carrier production capacity: sucking 1mL of bacterial suspension of the strain to be detected, inoculating the bacterial suspension into an MKB liquid culture medium, and culturing for 48 hours at 28 ℃ and 180 r/min; the culture broth was centrifuged for 10min (1200 r/min) and 200. Mu.L of supernatant (200. Mu.L of uninoculated MKB liquid medium was added for the measurement of the reference value (Er)) was taken according to 1:1 with CAS detection solution; after reacting for 1h at normal temperature, the enzyme-labeled instrument measures the OD value (E) of the wavelength at 630nm, if no siderophore is produced, the CAS liquid culture medium is blue as compared with the control, if the strain produces siderophores, the CAS liquid culture medium changes to orange, the ratio of E/Er is used for representing the relative content of siderophores in a sample, the smaller the value is, the stronger the capacity of the strain for producing siderophores is represented, the ratio of (Er-E)/Er is used for representing the activity unit of siderophores in the sample, and the higher the activity unit is, the stronger the siderophore producing capacity is;
and taking bacterial liquid growing to a logarithmic phase from the strain obtained by screening, centrifugally collecting bacterial bodies, and extracting genome DNA by using the kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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|---|---|---|---|---|
| WO2015118516A1 (en) * | 2014-02-10 | 2015-08-13 | Biofil Mikrobiológiai, Géntechnológiai És Biokémiai Kft. | Soil bacteria for inoculating stress soils |
| CN108795808A (en) * | 2018-06-15 | 2018-11-13 | 湖南省微生物研究院 | One plant of Batavia bacillus and its application |
| CN108821908A (en) * | 2018-06-15 | 2018-11-16 | 湖南省微生物研究院 | A kind of microbial inoculum comprising Batavia bacillus HJ-1 and its application |
| CN110777085A (en) * | 2018-07-31 | 2020-02-11 | 上海交通大学 | Cadmium-resistant nightshade growth-promoting rhizosphere lysine bacillus and application thereof |
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|---|---|---|---|---|
| WO2015118516A1 (en) * | 2014-02-10 | 2015-08-13 | Biofil Mikrobiológiai, Géntechnológiai És Biokémiai Kft. | Soil bacteria for inoculating stress soils |
| CN108795808A (en) * | 2018-06-15 | 2018-11-13 | 湖南省微生物研究院 | One plant of Batavia bacillus and its application |
| CN108821908A (en) * | 2018-06-15 | 2018-11-16 | 湖南省微生物研究院 | A kind of microbial inoculum comprising Batavia bacillus HJ-1 and its application |
| CN110777085A (en) * | 2018-07-31 | 2020-02-11 | 上海交通大学 | Cadmium-resistant nightshade growth-promoting rhizosphere lysine bacillus and application thereof |
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| 林梵宇;王润萍;易志伟;雷开宇;产竹华;: "海洋解木糖赖氨酸芽孢杆菌JZ008对重金属Cd~(2+)、Cr~(3+)和Cu~(2+)的吸附作用", 应用海洋学学报, no. 03, 15 August 2018 (2018-08-15) * |
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