CN112625947B - Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis - Google Patents
Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis Download PDFInfo
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- CN112625947B CN112625947B CN202011491064.8A CN202011491064A CN112625947B CN 112625947 B CN112625947 B CN 112625947B CN 202011491064 A CN202011491064 A CN 202011491064A CN 112625947 B CN112625947 B CN 112625947B
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- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 64
- 239000003337 fertilizer Substances 0.000 title claims abstract description 63
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- 239000011574 phosphorus Substances 0.000 title claims abstract description 62
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/10—Solid or semi-solid fertilisers, e.g. powders
- C05G5/12—Granules or flakes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
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- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Pest Control & Pesticides (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Inorganic Chemistry (AREA)
- Soil Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention discloses bacillus subtilis with strong phosphorus solubility, a carbon-based microbial compound fertilizer thereof and application thereof, wherein the bacillus subtilis is preserved in China general microbiological culture Collection center in 04-13.2020, with the preservation number as follows: CGMCC No. 19564. The bacillus subtilis has strong spore production capability and is stable, the content of dissolved inorganic phosphorus reaches 179mg/L, and the highest degradation rate of lecithin reaches 100%. According to the invention, the biomass charcoal prepared from fallen leaves contains high nitrogen, phosphorus and potassium nutrients, the fallen leaf charcoal-based microbial compound fertilizer is obtained by combining the fallen leaf biomass charcoal with bacillus subtilis with strong phosphorus dissolving capacity, the yield of spinach can be improved by 60-64% by applying the fallen leaf charcoal-based microbial compound fertilizer compared with the conventional fertilizer, the soluble sugar and Vc content of spinach can be obviously improved by applying the fallen leaf charcoal-based microbial compound fertilizer compared with the conventional fertilizer without applying the fertilizer, and the nitrate content in spinach is reduced, so that the growth and quality improvement of vegetables are promoted.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bacillus subtilis with strong phosphorus solubility, a carbon-based microbial compound fertilizer thereof and application of the bacillus subtilis with strong phosphorus solubility.
Background
Phosphorus is one of the biologically important nutrient elements. Phosphorus is involved in the structure of many important compounds in plants; phosphorus is involved in many metabolic processes in plants; phosphorus can enhance the stress resistance of plants. The content of total phosphorus in soil in China is high, approximately in the range of 0.44-0.85g/kg, but most phosphorus in the soil exists in insoluble calcium phosphate salt, and the content of effective phosphorus which can be directly absorbed and utilized by plants is low, so that the phosphorus in the soil solution cannot meet the requirement of crops on phosphorus in the season. Since the phosphate fertilizer is applied in 50 years, the indissoluble phosphorus accumulated in soil reaches 6000 million tons, which exceeds the total consumption of the phosphate fertilizer in 10 years in China. The development and effective utilization of the part of phosphorus fixed by soil are problems which are urgently needed to be solved in the agricultural production of China at present.
The microorganism can secrete various physiological active substances to directly stimulate and regulate the growth of plants; produce disease-resistant and stress-resistant effects and indirectly promote the growth of plants. Therefore, the application of the phosphorus-dissolving microbial fertilizer capable of decomposing the insoluble phosphorus in the soil can form a micro-area with sufficient phosphorus supply at the root of crops, improve the phosphorus supply of the crops, and is an important way which accords with the development of modern agriculture and future agriculture. However, most species are difficult to survive after entering the soil due to the difficulty of competing with indigenous microorganisms. The bacillus has the advantages of spore and strong heat resistance and stress resistance in the aspects of fermentation process, formulation processing, storage, propagation and the like. The bacillus subtilis is a common strain of bacillus, but the bacillus subtilis with a phosphorus dissolving effect which can be utilized by people at present is only a part of a very small number, the technology is mature, the quantity is small and few, the bacillus subtilis with high phosphorus dissolving efficiency is screened, effective microbial fertilizers are developed, and the improvement of the soil phosphorus environment has important significance for the development of green agriculture.
Along with the urbanization development of China, the urban greening area is continuously increased, the scale of the garden wastes such as pruned branches, dead wood, fallen leaves and the like generated by the urban garden waste is huge and is increased year by year, and the total amount of the garden wastes generated in suburbs of Beijing is over 400 million tons each year. At present, garden waste is mostly buried and burned as solid garbage, which not only puts a great pressure on urban development and garbage disposal, but also causes environmental pollution and resource waste. The centralized leaf falling of the street trees in spring and autumn brings great trouble to urban sanitation. Fallen leaves are not garbage, contain a large amount of nutrients such as C, N, P, K, and the resource recycling of fallen leaves has become a research hotspot in recent years. In the last decade, the resource utilization approach of garden waste is mainly fermentation composting. However, the composting process still has some problems, such as H2S generated in the fermentation process and the release of peculiar smell; the composting temperature can only reach about 70 ℃, and pathogenic bacteria and the like cannot be killed. The newly developed biomass carbonization technology can pyrolyze waste substances such as fallen leaves and the like under the conditions of high temperature and limited oxygen to generate biomass charcoal, overcomes the problem of compost avoidance, and becomes a new way for resource utilization of garden waste materials such as fallen leaves and the like. However, the value of fallen leaves is not yet fully realized only by converting the fallen leaves into biomass charcoal, and the "fallen leaves root" is the final return of the fallen leaves resource recycling.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the defects of the prior art, the invention aims to solve the technical problem of providing the bacillus subtilis with strong phosphorus solubility.
The invention also aims to solve the technical problem of providing the application of the bacillus subtilis in dissolving organic phosphorus and/or inorganic phosphorus.
The invention also aims to solve the technical problem of providing the carbon-based microbial compound fertilizer.
The invention finally solves the technical problem of providing the application of the carbon-based microbial compound fertilizer in vegetable planting.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a bacillus subtilis with strong phosphorus solubility, which is CNBG-PGPR-1 for short, and is preserved in China general microbiological culture Collection center at 13.04.2020 with the preservation number as follows: CGMCC No.19564, classified name of Bacillus subtilis, preservation address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101.
the invention also comprises the application of the bacillus subtilis in dissolving organic phosphorus and/or inorganic phosphorus.
Wherein, the inorganic phosphorus includes but not limited to tricalcium phosphate, and other inorganic phosphorus can also be suitable.
Wherein the organic phosphorus includes, but is not limited to lecithin, and other organic phosphorus can be used.
The invention also comprises the application of the bacillus subtilis in preparing the carbon-based microbial compound fertilizer.
The invention also comprises a carbon-based microbial compound fertilizer, which is obtained by culturing the bacillus subtilis CNBG-PGPR-1 into a spore form and loading the spore form on biomass carbon.
The biomass charcoal is deciduous charcoal, and the deciduous charcoal biomass charcoal may include, but is not limited to, deciduous leaves of plane tree, deciduous leaves of ginkgo, deciduous leaves of poplar, deciduous leaves of goldenrain tree, and the like.
The biomass charcoal accounts for 20-40% of the total weight of the compound fertilizer, the bacillus subtilis is a fermentation liquid of bacillus subtilis CNBG-PGPR-1 with a spore rate of more than or equal to 80%, and the fermentation liquid accounts for 20-30% of the total weight of the compound fertilizer.
Wherein, the carbon-based microbial compound fertilizer also comprises nitrogen, phosphorus, potassium, bentonite and gypsum.
Wherein, the nitrogen, phosphorus and potassium account for 27-30% of the total weight of the compound fertilizer, and the bentonite and the gypsum account for 13% of the total weight of the compound fertilizer.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the bacillus subtilis CNBG-PGPR-1 has strong spore production capacity and is kept stable, the spore production rate can reach more than 80% after 25 hours, the spore production rate exceeds 90% after the bacteria liquid is stood for 9 days, the spore production rate can still be maintained at 80% -90% within 3d, meanwhile, the 6d is inoculated, the content of dissolved inorganic phosphorus reaches 179mg/L, the 2d is inoculated, the degradation rate of the bacterial strain CNBG-PGPR-1 to lecithin reaches 97%, and the degradation rate of the bacterial strain CNBG-PGPR-1 to lecithin reaches 100% after 6 d.
2. The invention provides the deciduous carbon-based microbial compound fertilizer by adopting the biomass carbon prepared from the deciduous leaves and combining the bacillus subtilis with strong phosphorus-dissolving capacity separated and purified in a laboratory, compared with the conventional fertilizer, the application of the deciduous carbon-based microbial compound fertilizer can still improve the yield of spinach by 60-64%, and simultaneously, compared with the non-application and the conventional fertilizer, the application of the deciduous carbon-based microbial compound fertilizer can obviously improve the content of soluble sugar and Vc in the spinach and reduce the content of nitrate in the spinach, thereby promoting the growth and quality improvement of vegetables.
Drawings
FIG. 1, colony morphology of Bacillus subtilis CNBG-PGPR-1 on plates;
FIG. 2 shows the cell morphology of Bacillus subtilis CNBG-PGPR-1 under a microscope at 100 times;
FIG. 3, phylogenetic tree of Bacillus subtilis CNBG-PGPR-1;
FIG. 4 is a microscopic photograph of spore staining of Bacillus subtilis CNBG-PGPR-1;
FIG. 5 shows the effect of a carbon deciduous microbial compound fertilizer on spinach yield;
FIG. 6 shows the influence of the deciduous carbon-based microbial compound fertilizer on spinach quality.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples using Escherichia coli as a host bacterium, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 isolation and characterization of Bacillus subtilis CNBG-PGPR-1
(1) Sample dilution and coating
Taking 0.5g soil sample (collected from farmland around Nanjing, Jiangsu) in 4.5mL sterile fieldSuspended oscillation in saline water is 10-1Gradient (ten-fold dilution), taking 0.5mL from the dilution and suspending and shaking in 4.5mL sterile physiological saline to obtain 10-2Gradient (hundred fold dilution), and so on until dilution to 10-6And (4) gradient. mu.L of each dilution was pipetted from 6 dilutions, spread on a Nutrient Agar (NA) solid medium plate, prepared in 2 portions in parallel, inverted, and placed in a 30 ℃ incubator for 24-36h with occasional observation.
(2) Strain isolation and purification
Taking out all the plates with the grown bacterial colonies under the same sample, selecting a dilution plate with obvious single bacterial colonies and moderate quantity, picking the bacterial colonies with different forms on the plate to a new NA plate, carrying out partition purification, repeating purification until the single bacterial colonies appear, and then repeating streak culture for at least 5 times.
(3) Dyeing and preservation
Selecting bacterial colonies of a strain to be detected in the central area of a glass slide, carrying out primary dyeing by using ammonium oxalate-crystal violet dye, mordanting by using iodine solution, decoloring by using 75% ethanol and redyeing by using safranin, carrying out microscopic observation, and recording a dyeing result; and (3) picking the single colony which is purified and dyed from an NA solid culture medium plate to a sterile NA liquid culture medium, culturing at 30 ℃ for 24 +/-2 h at 200r/min, shaking uniformly, and adding 60% of sterile glycerol: mixing the bacteria liquid in the ratio of V to V of 1 to 1, subpackaging in aseptic bacteria-protecting tubes, marking and storing in a refrigerator at-80 ℃.
(4) Identification of strains
Taking out the plate containing the strain with the corresponding number from a refrigerator at 4 ℃, selecting a single colony as a DNA template, and carrying out PCR amplification and 16S rDNA sequencing and sequence comparison, wherein the specific operations are as follows:
50 μ L of amplification system:
the 50 μ L system consists of the following five parts: taq enzyme Mix (25. mu.L), forward primer 27F (1. mu.L), reverse primer 1492R (1. mu.L), DNA template (1. mu.L), ddH2O (22. mu.L). Wherein the 27F sequence is: the 5 '-AGA GTT TGA TCM TGG CTC AG-3', 1492R sequence is: 5 '-GGY TAC CTT GTT ACG ACT T-3'. The length of the amplified fragment is 1401bp, and the amplified nucleosideThe sequence is shown in the end page.
Amplification conditions
Pre-denaturation temperature: 105 ℃ C
First-step denaturation: 7min at 95 ℃; 30s at 95 DEG C
And a second step of annealing: 30s at 55 DEG C
And a third step of extension: 90s at 72 DEG C
Cycle number: 30s at 95 ℃ for 33 cycles
The fourth step is finally extended: 5min at 72 DEG C
The fifth step is that: 10min at 12 DEG C
③ agarose gel electrophoresis
Weighing 1g of agarose, dissolving in 100mL of TBE electrophoresis buffer solution, heating by microwave until the solution is clear and transparent, adding 1 per thousand of GelRed nucleic acid dye, shaking uniformly, standing until no bubbles exist, slowly pouring into a gel plate, standing for about 1h, taking out a solidified gel block, placing the gel block in an electrophoresis tank, adding 4 muL of PCR amplification product into each gel hole, running gel for 120V 20min, taking out the gel block, placing the gel block in a gel imager, selecting UV illumination and taking a picture, recording the corresponding sample number with clear strips at 1500bp positions on the gel block, and placing the residual PCR amplification product of the sample in a refrigerator at 4 ℃.
Purification of DNA
The PCR amplification product stored in the refrigerator at 4 ℃ was removed and the PRC product was purified using AxyPrepTM PCR clean Kit nucleic acid purification Kit.
Sequence identification
And (3) sending the heavy suspension DNA obtained in the step to Nanjing engine biotechnology limited company for sequencing, inputting the sequence returned by the mail into a sequence retrieval column of an NCBI website for retrieval, and comparing results to show that the strain provided by the invention is Bacillus subtilis.
The colony morphology of the bacillus subtilis CNBG-PGPR-1(CGMCC No.19564) strain is as follows: the bacterial colony on the surface of the plate culture medium is small, circular, flat, milky, rough and opaque in surface, jagged in edge, uniform in texture, smooth in the surface in the early stage and obvious in surface wrinkle in the later stage. The screened bacillus subtilis CNBG-PGPR-1 is rod-shaped, the cell length is 2-3 microns, the width is 0.7-0.8 microns, and the flagella of the whole body can move. The gram staining result is purple, namely the gram-positive bacteria.
The bacillus subtilis CNBG-PGPR-1 is preserved in China general microbiological culture Collection center in 2020, 04 and 13 months, with the preservation number as follows: CGMCC No. 19564.
Example 2 production of spores in NA Medium by Bacillus subtilis
(1) Preparation of culture medium
Liquid medium a: NB liquid medium: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1L of distilled water.
Liquid medium b: LB liquid medium: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride, 15g of agar and 1L of distilled water.
(2) Spore formation and scale-up culture
A single colony was picked from the plate and placed in a prepared centrifuge tube containing 5mL of liquid medium a or b, and shake-cultured at 30 ℃ and 200 rpm. Sampling from 19h, naturally drying the smear, heating and fixing by flame, dripping 5% malachite green staining solution, heating, letting it emit steam for 3-4 times within 30 seconds, cooling, washing with tap water for 30 seconds, adding 0.5% yellow-sand counterstain, after 3 seconds, washing with water, air drying, and counting by microscopic examination, the result is shown in figure 4. The bacterial trophosome is red, and the spore is green. The bacillus subtilis CNBG-PGPR-1 begins to form spores when being subjected to shaking culture in NB and LB culture media for 19 hours, the spore generation rate can reach more than 80% after 25 hours, and the spore generation rate exceeds 90% after the bacterial liquid is kept stand for 9 days.
When the seed solution is transferred into 50mL of fermentation tanks and then transferred into 1L and 5L of fermentation tanks for amplification culture, the spore production capacity of the bacillus subtilis CNBG-PGPR-1 is kept stable, the spore production rate in 3d can still be maintained at 80% -90%, and the industrial fermentation production requirement can be met.
Example 3 inorganic phosphorus solubilizing ability of Bacillus subtilis CNBG-PGPR-1
(1) Preparation of culture Medium
The inorganic phosphorus liquid culture medium comprises 10g of glucose, 5g of tricalcium phosphate, 0.1g of calcium carbonate, 0.5g of magnesium sulfate, 0.5g of ammonium sulfate, 0.1g of calcium sulfate, 0.005g of ferric chloride and 1L of water.
(2) Inorganic phosphorus dissolving capacity
Taking out a strain CNBG-PGPR-1, freezing the strain CNBG-PGPR-1 in a strain holding tube in a refrigerator at the temperature of-80 ℃, thawing the strain CNBG-PGPR-1 to be fluid on ice, shaking and uniformly mixing the strain CNBG-PGPR-1, picking an appropriate amount of bacterial liquid by using an inoculating loop to perform three-zone lineation on an NA solid flat plate, placing the flat plate in a constant temperature culture for 24 +/-2 hours at the temperature of 30 ℃, picking a single bacterial colony for purification for 1-2 times and performing identification confirmation, picking the single bacterial colony to be activated in an NA liquid culture medium after the single bacterial colony is confirmed to be correct, centrifuging to remove the NA culture medium, suspending bacterial sludge in sterile physiological saline with the same volume, adding a heavy suspension of the strain CNBG-PGPR-1 into an inorganic phosphorus liquid culture medium according to the inoculation amount of 5% of volume, repeating the strain CNBG-PGPR-1 for three times in each treatment group, simultaneously setting a blank control without adding bacterial liquid, and dynamically sampling to determine the phosphorus content in the liquid.
The inorganic phosphorus culture medium takes tricalcium phosphate as a phosphorus source, the addition amount is 999.42mg/L, the determination method refers to the national standard GB17378.4-2007 'phosphomolybdic blue spectrophotometry' to determine inorganic phosphate in the solution, and the result is shown in Table 1. After inoculation of the strain, the phosphorus content of the solution gradually increases with the culture time. When the strain CNBG-PGPR-1 is inoculated for 2d, the content of dissolved inorganic phosphorus is obviously higher than that of a control group, and the amount of dissolved phosphorus is rapidly increased along with the time extension. And 6d, inoculating, and dissolving out inorganic phosphorus with the content of 179 mg/L. The results show that the strain CNBG-PGPR-1 can rapidly degrade inorganic phosphorus and release the inorganic phosphorus to the surrounding environment, and can maintain the release capability for a long time.
TABLE 1 inorganic phosphorus content released in solution (mg/L)
Example 4 organophosphorus solubilizing Capacity of Bacillus subtilis CNBG-PGPR-1
(1) Preparation of a culture medium:
the organophosphorus liquid culture medium comprises 10g of glucose, 2g of lecithin, 5g of calcium carbonate, 0.5g of magnesium sulfate, 0.5g of ammonium sulfate, 0.1g of calcium sulfate, 0.005g of ferric chloride and 1L of water.
(2) Quantification of the degradation Capacity of organic phosphorus
Taking out a strain CNBG-PGPR-1, freezing the strain CNBG-PGPR-1 in a strain-preserving tube in a refrigerator at the temperature of-80 ℃, thawing the strain CNBG-PGPR-1 to be fluid on ice, shaking and uniformly mixing the strain CNBG-PGPR-1, selecting an appropriate amount of bacterial liquid by using an inoculating loop to perform three-zone streaking on an NA solid plate, placing the plate in a constant temperature culture for 24 +/-2 hours at the temperature of 30 ℃, selecting a single bacterial colony for purification for 1-2 times and carrying out identification confirmation, selecting the single bacterial colony to activate the single bacterial colony in an NA liquid culture medium after the single bacterial colony is confirmed to be correct, centrifuging the NA culture medium, re-suspending bacterial sludge in sterile physiological saline with the same volume, and adding the re-suspending liquid of the Bacillus subtilis CNBG-PGPR-1 into an organophosphorus liquid culture medium according to the inoculation amount of 5% of volume ratio. And (4) setting three times for each treatment, simultaneously setting a blank control without adding bacterial liquid, and dynamically sampling to determine the content of the organic phosphorus.
The organophosphorus medium takes soybean lecithin as a phosphorus source, the purity is more than 90%, the addition amount is 5g/L, the determination of the content of organophosphorus in the solution is carried out by referring to the national standard GB/T35867-. After inoculation for 2d, the degradation rate of the strain CNBG-PGPR-1 to lecithin is 97%, and when the strain CNBG-PGPR-1 reaches 100% after 6d, which shows that the strain CNBG-PGPR-1 provided by the invention has strong organic phosphorus degradation capability.
TABLE 2 lecithin degradation ratio (%)
Example 5 Mixed deciduous Biomass charcoal and nutrient determination thereof
Drying the fallen leaves of plane tree, ginkgo biloba, poplar and goldenrain tree in the air, weighing, putting into a custom-made metal can, compacting, putting into a muffle furnace, and preparing the biomass charcoal by adopting a thermal cracking process. The initial furnace temperature is 200 ℃, the temperature is programmed to rise by 10 ℃/min, the final temperature is 500 ℃, the retention time is 1h, and the carbon is naturally cooled to obtain the carbon of the fallen leaves of the French phoenix tree. Weighing and calculating the yield. The total nutrient in the mixed deciduous carbon is determined by digestion with sulfuric acid-hydrogen peroxide, determination of total nitrogen with Kjeldahl method, determination of total phosphorus with alum-molybdenum-yellow method, and determination of total potassium with flame photometry. The yield of the mixed deciduous carbon is 33%, and the solid carbon content is 43.57%. The total nutrients of N, P and K of the mixed deciduous carbon are high, namely 10.03g/kg, 4.28g/kg and 16.5g/kg respectively, and the mixed deciduous carbon is suitable for being made into fertilizer.
Example 6 deciduous charcoal-based microbial Fertilizer
Respectively crushing the deciduous leaf charcoal biomass charcoal, urea, monoammonium phosphate and potassium sulfate and sieving the crushed deciduous leaf charcoal, urea, monoammonium phosphate and potassium sulfate by a 20-mesh sieve; weighing a certain amount of deciduous biomass charcoal, urea, monoammonium phosphate, potassium sulfate, bentonite and gypsum, uniformly stirring, putting into a disc granulator, and adding fermentation liquor of bacillus subtilis CNBG-PGPR-1 with a spore rate of more than or equal to 80% to initial concentration (1.6 +/-0.2) multiplied by 109Slowly spraying the mixture per mL, and uniformly mixing the mixture with the raw materials for granulation. The proportion of each component is as follows by weight percent:
1. 40% of fallen leaf biomass charcoal, 10% of urea, 9% of monoammonium phosphate, 8% of potassium sulfate, 10% of bentonite, 3% of gypsum and 20mL (20%) of bacterial liquid;
2. 30% of deciduous biomass charcoal, 15% of urea, 9% of monoammonium phosphate, 8% of potassium sulfate, 10% of bentonite, 3% of gypsum and 25mL (25%) of bacterial liquid;
3. 20% of fallen leaf biomass charcoal, 15% of urea, 10% of monoammonium phosphate, 12% of potassium sulfate, 10% of bentonite, 3% of gypsum and 30mL (30%) of bacterial liquid.
Placing the formed granules into an electric heating forced air drying oven, drying at 30-40 deg.C (or drying in the sun), and shaping. And after drying and sizing are finished, sieving the granulated fertilizer by using metal sieves with the particle sizes of 1mm and 4.75mm, reserving the fertilizer with the particle sizes of 1mm-4.75mm, bagging and sealing for storage.
The results of the 3 deciduous carbon-based microbial compound fertilizers are detected according to the agricultural industry standard NY/T2321-2013 of the people's republic of China, and are shown in Table 3. According to the formula, the deciduous leaf charcoal-based microbial fertilizer is produced by 20-40% of deciduous leaf biomass charcoal, 20-30% of bacterial liquid, 27-37% of urea, monoammonium phosphate and potassium sulfate, 10% of bentonite and 3% of gypsum, and is organicThe content of the matter (dry basis) is 20 to 36.5 percent, and the effective nutrient is (N + P)2O5+K2O) 15-21%, water 11-13%, pH6.3-6.8, effective viable count 1.8-2.6 hundred million/g, all meet national composite microbial fertilizer standard (NY/T798-2015).
TABLE 3 technical indexes of carbon-based microbial compound fertilizer with different formulas
Example 7 Effect of defoliating charcoal-based microbial Fertilizer on improvement of vegetable yield and quality
A field experiment for evaluating the fertilizer efficiency is carried out in Jiangsu Feng county (34 degrees 37 '53' N, 116 degrees 36 '53' E). The experiment was set up with five treatments, treatment 1 being a no fertilizer Control (CK); the treatment 2 is Chemical Fertilizer (CF), the chemical fertilizer is Strobile compound fertilizer, the nutrient content is N: P2O5∶K2O is 15: 15; treatment 3 was the purified Bacillus subtilis CNBG-PGPR-1 isolated in example 1 at an initial concentration (1.6. + -. 0.2). times.109The spore count per mL is more than or equal to 80 percent; treatment 4 was deciduous charcoal from example 5 (re-sterilized before use); treatment 5 was the deciduous charcoal-based microbial compound fertilizer (CB1, CB2, CB3) of formulations 1-3 in example 6. Three replicates were set for each treatment, each replicate cell area being 20m 2. Fertilizing according to the design, wherein the fertilizing amount of the treatment 2 is 0.8 per mill/kg according to the application amount recommended by a manufacturer, namely 120 kg/mu; the application amount of the 5-defoliation carbon-based microbial fertilizer is 16.8 kg/mu, and is about 70 percent of that of the 2-treatment chemical fertilizer according to the effective nutrient. The application amounts of the bacterial liquid and the biomass charcoal in the treatment 3 and the treatment 4 refer to the treatment 5, and are converted into 5.04L/mu bacterial liquid and 6.52 kg/mu deciduous charcoal according to the maximum content, namely 30 percent bacterial liquid and 40 percent deciduous charcoal. After land leveling, uniformly spreading spinach seeds into the applied soil, watering and covering soil. The spinach is uniformly and conventionally managed in the whole growth period, and is harvested after one month. Each cellSelecting a region of 1m multiplied by 1m for sampling, weighing and calculating the yield, referring to figure 5, determining the soluble sugar content of spinach by adopting an anthrone colorimetric method, determining the soluble protein in spinach by adopting a Coomassie brilliant blue method, determining the nitrate content in spinach by adopting a salicylic acid colorimetric method, and determining the content of vitamin C (Vc) by adopting a titration method, wherein the result is shown in figure 6. Compared with a control without fertilization, the application of the deciduous carbon-based microbial compound fertilizer can improve the yield of the spinach by 1.66-1.73 times; compared with the conventional fertilizer, the method has the advantages that the yield of spinach can be improved by 60-64% under the condition that the effective nutrients are reduced by 30% by applying the chemical fertilizer by applying the deciduous carbon-based microbial compound fertilizer. Meanwhile, compared with the method of applying no fertilizer and the conventional fertilizer, the method has the advantages that the content of soluble sugar and Vc in the spinach is improved and the content of nitrate in the spinach is reduced by applying the deciduous carbon-based microbial compound fertilizer. The results show that the deciduous carbon-based microbial compound fertilizer can obviously improve the yield and the quality of spinach.
Claims (10)
1. Bacillus subtilis (B) capable of dissolving phosphorus stronglyBacillus subtilis) CNBG-PGPR-1, wherein the Bacillus subtilis CNBG-PGPR-1 is preserved in China general microbiological culture Collection center at 04.13.2020 with the preservation number: CGMCC No. 19564.
2. Use of the bacillus subtilis CNBG-PGPR-1 of claim 1 for dissolving organic phosphorus and/or inorganic phosphorus.
3. Use according to claim 2, characterized in that the inorganic phosphorus originates from tricalcium phosphate.
4. Use according to claim 2, characterized in that the organophosphorus phosphorus is derived from lecithin.
5. The use of the bacillus subtilis CNBG-PGPR-1 of claim 1 in preparing a carbon-based microbial compound fertilizer.
6. A carbon-based microbial compound fertilizer, which is obtained by culturing the Bacillus subtilis CNBG-PGPR-1 of claim 1 into a spore form and loading the spore form on biomass charcoal.
7. The carbon-based microbial compound fertilizer according to claim 6, wherein the biomass carbon is deciduous carbon biomass carbon.
8. The carbon-based microbial compound fertilizer as claimed in claim 6, wherein the biomass carbon accounts for 20-40% of the total weight of the compound fertilizer, the bacillus subtilis is a fermentation broth of bacillus subtilis CNBG-PGPR-1 with a spore rate of more than or equal to 80%, and the fermentation broth accounts for 20-30% of the total weight of the compound fertilizer.
9. The carbon-based microbial compound fertilizer according to claim 6, further comprising NPK, bentonite and gypsum, wherein the NPK accounts for 27-37% of the total weight of the compound fertilizer, and the bentonite and gypsum account for 13% of the total weight of the compound fertilizer.
10. The use of the carbon-based microbial compound fertilizer as defined in any one of claims 6 to 9 in vegetable planting.
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