CN108330085B - Potassium-decomposing strain suitable for low-temperature environment and fermentation method for preparing microecological preparation based on same - Google Patents

Potassium-decomposing strain suitable for low-temperature environment and fermentation method for preparing microecological preparation based on same Download PDF

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CN108330085B
CN108330085B CN201810086647.9A CN201810086647A CN108330085B CN 108330085 B CN108330085 B CN 108330085B CN 201810086647 A CN201810086647 A CN 201810086647A CN 108330085 B CN108330085 B CN 108330085B
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potassium
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liquid
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soil
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CN108330085A (en
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周海燕
田云
肖红仕
林元山
潘虎
白军平
达娃
次顿
王翀
管桂萍
周辉
刘虎虎
卢向阳
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Hunan Agricultural University
Institute of Animal Husbandry and Veterinary Medicine of Tibet Academy of Agriculture and Animal Husbandry Sciences
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Hunan Agricultural University
Institute of Animal Husbandry and Veterinary Medicine of Tibet Academy of Agriculture and Animal Husbandry Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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    • C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention discloses a potassium-dissolving strain suitable for low-temperature environment and a fermentation method for preparing a microecological preparation based on the same, wherein the strain is identified as bacillus, the strain number is BX, the strain is preserved in China general microbiological culture Collection center, and the preservation number is CGMCC No. 14769. The strain can effectively decompose insoluble inorganic mineral substances such as potassium feldspar, apatite and the like, increase the content of available nutrients in crop soil, promote the growth and development of crops, has a strong potassium decomposition effect in a low-temperature environment, can be used for soil improvement in high altitude and northern cold areas, and has a good application value in the aspects of preparing bio-organic fertilizers, soil conditioners and the like.

Description

Potassium-decomposing strain suitable for low-temperature environment and fermentation method for preparing microecological preparation based on same
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a potassium-decomposing strain suitable for a low-temperature environment and a fermentation method for preparing a microecological preparation based on the same.
Background
The potassium is one of three essential nutrient elements for crops, and can promote the growth and development of crops, and can raise the disease resistance and stress resistance of crops. Therefore, the potassium element is processed into a large amount of potassium fertilizer to be applied to agriculture. The soil is a natural potassium reservoir, the potassium content is very rich, but more than 95 percent of potassium exists in silicate minerals such as potassium feldspar or mica in a slow-release form and cannot be directly absorbed and utilized by crops, so that the soil is rich in potassium and deficient in potassium.
On one hand, because the potassium resource which is convenient to utilize in China is deficient and the distribution is extremely uneven, the production and the use of the potassium fertilizer are seriously restricted, and more than 90 percent of the potassium fertilizer applied every year depends on import. According to the general survey data of soil, about 70% of cultivated lands in China are lack of potassium, 45% of cultivated lands are seriously lack of potassium, and the proportion of nitrogen, phosphorus and potassium in fertilizers in China is 1: 0.4: 0.16, much smaller than 1: 0.5: 0.4, which have become important factors limiting the improvement of yield and quality of crops. The currently commonly used chemical potash fertilizer has high cost and low utilization rate, causes certain damage to the ecological environment and influences the quality of agricultural products. Therefore, the environment-friendly silicate bacterial fertilizer with low development cost has great theoretical and practical significance for improving the soil micro-domain environment and developing green and ecological agriculture.
On the other hand, the insoluble potassium aluminosilicate mainly comprising potassium feldspar in China is quite abundant and widely distributed, and has mineral reserves of nearly 100 hundred million tons in Heilongjiang, Liaoning, Hebei, Henan, Jiangsu, Anhui, Hubei, Shaanxi, Sichuan, Yunnan, Guangdong, Guangxi and other provinces. Therefore, the effective development of potassium feldspar resources and the extraction of potassium from the potassium feldspar resources to prepare the potassium fertilizer are important ways for solving the current situation of the shortage of soluble potassium resources in China. Researchers have proposed various methods around development and utilization of insoluble potassium ore resources to prepare potash fertilizers so far, and the methods are broadly divided into a high-temperature method and a wet method, and the methods have the defects of high energy consumption, large amount of ingredients, high production cost, serious environmental pollution and the like, so that large-scale industrial production cannot be realized.
The method for extracting potassium from potassium feldspar by utilizing the microbiological method has the advantages of low energy consumption, low cost, no pollution, mild conditions, simple method and the like, and is known as the most promising potassium feldspar potassium extraction method. The silicate bacteria are also called potassium bacteria, and can decompose aluminosilicate minerals such as potassium feldspar, mica and the like, and release soluble potassium from the aluminosilicate minerals, so that the aluminosilicate minerals can be absorbed and utilized by plants. In addition, the metabolites of the compound, such as hormone substances, polysaccharide, organic acid and the like, can inhibit the growth of pathogenic bacteria and improve the soil micro-domain environment, thereby being beneficial to the nutrient absorption of crops, enhancing the disease resistance of the crops, increasing the crop yield and improving the crop quality. Therefore, silicate bacteria with high potassium-dissolving activity are screened, the potassium-dissolving performance of the silicate bacteria is researched, and a certain theoretical and material basis can be provided for preparation of silicate bacteria fertilizers.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a potassium-solubilizing strain suitable for a low-temperature environment and a fermentation method for preparing a microecological preparation based on the strain.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the high-efficiency potassium-decomposing strain capable of adapting to the low-temperature environment is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms in Beijing in 2017, 9 and 28, the preservation number is CGMCC No.14769, the strain is named as Bacillus BX strain, and the strain belongs to Bacillus.
Wherein the suitable growth temperature of the potassium-decomposing strain is 18-37 ℃, and the suitable potassium-decomposing temperature is 20-37 ℃.
Preferably, the optimal growth temperature of the potassium-solubilizing strain is 35-37 ℃, and the optimal potassium-solubilizing temperature is 28-30 ℃.
The colony surface of the potassium-solubilizing strain is rough and opaque, and the colony color is dirty white or yellowish; gram-positive bacteria; glucose, starch, sucrose, maltose, lactose, glucomannan, galactose can be utilized; can decompose glucose to produce pyruvic acid, decompose tryptophan to form indole, hydrolyze starch, and has lecithin enzyme hydrolysis ability.
The potassium-decomposing strain has no symbiotic characteristic with crops and is harmless to the crops and the crop environment. The content of potassium available for plants in the soil is increased by degrading insoluble aluminosilicate inorganic mineral matters in the soil.
The solid fermentation method for preparing the microecological bactericide based on the potassium-solubilizing strain comprises the following steps:
(1) selecting potassium bacteria BX, inoculating into L B liquid culture medium, shake culturing at 37 deg.C, and culturing to obtain bacterial liquid OD600Obtaining potassium-decomposing bacterium culture solution as 0.5;
(2) transferring the potassium bacteria culture solution into a fermentation tank, and performing ventilation culture at 37 ℃ for 20-28 h, preferably 24h to obtain strain liquid seeds;
(3) inoculating strain seed solution into sterilized solid testa Tritici at high temperature, the mass ratio of strain seed solution to solid testa Tritici is 8-12%, preferably 10%, culturing at 25-37 deg.C for 24-48 hr until viable count reaches 4.0 × 1010cfu/g to obtain a microbial inoculum meeting the requirement of viable count;
(4) drying the microbial inoculum until the water content is less than or equal to 5 percent to obtain the potassium bacteria microecological microbial inoculum; preferably at 50 deg.C.
Based on the above potassium saltThe liquid fermentation method for preparing the microecological preparation by the bacterial strain is to inoculate the potassium-dissolving bacterial strain to a liquid culture medium, ferment and culture for 20-28 h, preferably 24h, at 37 ℃ and 180rpm, and obtain fermentation liquor for soil potassium dissolving, namely the potassium-dissolving bacteria microecological bactericide; or drying the fermentation liquor to obtain a dried substance for soil potassium dissolution, namely the potassium-dissolving bacterium microecological bactericide; the liquid culture medium comprises the following components in parts by weight: glucose 8-12, MgSO4·7H2O 0.3—0.7，Na2HPO41.5—2.5，CaCO30.3 —0.7，FeCl20.008-0.012 wt% of potassium feldspar powder, 0.8-1.2 wt% of potassium feldspar powder (the potassium feldspar powder passes through a 40-mesh sieve, is soaked in deionized water overnight, then is repeatedly washed by deionized water to remove soluble potassium in the potassium feldspar powder, and is dried at 50 ℃), 950-1000 wt% of deionized water, and the pH value is 7.0-7.5.
Preferably, the liquid culture medium consists of the following components in parts by weight: glucose 10, MgSO4·7H2O 0.5，Na2HPO42，CaCO30.5，FeCl20.01, potassium feldspar powder 1, deionized water 1000, and pH 7.2.
The potassium-dissolving strain can be used for preparing a biological fermentation organic fertilizer, and can also be used for preparing a soil conditioner, an improver or a soil loosening agent. When in use, the high-efficiency potassium-decomposing bacteria can be prepared into granules, powder, solution, suspension or sticks, and can be mixed with organic fertilizer for use or used independently to improve the rhizosphere microbial environment.
The potassium bacteria BX is screened from soil in a highland barley planting area of Tibet valley, a soil sample is properly diluted and then coated in a screening culture medium, bacterial colonies with high activity potassium-dissolving capacity are separated, and the bacterial colonies are inoculated into a test tube inclined plane to obtain a plurality of potassium-dissolving bacteria; performing shake flask liquid fermentation for re-screening, and determining potassium-decomposing activity to obtain a strain BX with highest potassium-decomposing activity; the potassium-dissolving activity is determined by measuring K in the culture solution before and after fermentation+The evaluation was carried out by inoculating the potassium-decomposing bacterium BX in 100m L liquid medium, culturing at 37 ℃ and pH 7.4 under shaking at 160rpm for 2 days, collecting 10ml of the culture, and adding 2ml of 6% H2O2Digesting for 1h in boiling water bath, and performing atomic absorption spectroscopyPhotometric determination of K in digestive juices+And calculating the decomposition rate of the bacterial strain on the potash feldspar ore powder.
Compared with the prior art, the invention has the beneficial effects that:
1. the Bacillus BX strain and the potassium-dissolving microbial inoculum prepared by fermenting and culturing the Bacillus BX strain can effectively decompose insoluble aluminosilicate inorganic mineral substances such as potassium feldspar, apatite and the like, promote nutrient elements such as insoluble potassium, phosphorus, silicon, magnesium and the like to be converted into soluble nutrients, increase the content of the effective nutrients in soil, promote the growth and development of crops and improve the yield. Has extremely important significance and application value for fully exerting the ecological fertility of soil, keeping the balance of agricultural ecological environment and the like.
2. The potassium-decomposing microbial inoculum provided by the invention does not discharge pollutants to the environment in the production process, and is an environment-friendly fertilizer; the preparation method of the potassium-decomposing inoculant provided by the invention is simple in production process and high in production efficiency, can realize industrial mass production, and has good economic effect and social effect.
Detailed Description
Example 1 screening and isolation of Potassium bacteria
Collecting soil samples from potash feldspar ore areas in autonomous areas of Tibet in parts by weight, storing in sterile plastic bags, and immediately separating after air transportation and sending back. Under aseptic operation, 1.0g of soil sample is weighed, dissolved in 9ml of aseptic normal saline, added with glass beads, fully oscillated for 30min at 28 ℃ by a shaking table at 180rpm, diluted in a gradient manner and coated on a prepared separation culture medium plate. And (3) culturing in a thermostat at 28-30 ℃ for 2-3 days, picking up a colony which is edge-finished, moist, viscous, colorless, transparent and elastic by using an inoculating needle, and further separating and purifying. The purified strain is transferred to a screening culture medium plate which takes potassium feldspar as the only potassium source, the strain is cultured for 4 days at the temperature of 28 ℃, and a colony which grows well, is large, smooth, round, transparent, wet and sticky in surface and rich in elasticity is selected for slant preservation.
The primary strain was inoculated in a potassium-releasing assay medium, the plate was covered with sodium tetraphenylborate (1% sodium tetraphenylborate + 2% potassium hydroxide), and 0.04% bromophenol blue was added to develop color, and the periphery of the colony became bright blue, which was the target strain.
The formula of the culture medium is as follows: 10.0g of glucose, KH2PO40.2g， MgSO4·7H2O0.2g，NaCl 0.2g，CaCO35.0g deionized water 1000m L, pH 7.2, screening culture medium glucose 10.0g, MgSO 24·7H2O 0.5g，Na2HPO42g，CaCO30.5g， FeCl20.01g of potassium feldspar powder and 1.0g of potassium feldspar powder (the potassium feldspar powder is sieved by a 40-mesh sieve, soaked in deionized water overnight, then repeatedly washed by the deionized water to remove soluble potassium in the potassium feldspar powder, and dried at 50 ℃), the deionized water is 1000m L, and the pH value is adjusted to 7.2.
Growth medium: glucose 10.0g, MgSO4·7H2O 0.5g，CaCO31.0g，(NH4)2SO41.0g，NaCl0.1g，Na2HPO42.0g potassium feldspar powder 2.5g (same method as above), pH 7.2, deionized water 1000m L.
EXAMPLE 2 measurement of Potassium-decomposing ability of Strain
Inoculating the screened strain in 100m L growth medium, inoculating in potassium-dissolving determination medium at 2% inoculum size, inoculating equivalent inactivated bacteria solution in control group, performing shake culture at 28 deg.C and 160rpm for 5d, collecting strain culture solution 10ml, adding 6% H2O22ml, digesting in boiling water bath for 1h, centrifuging the digestive solution at 4000rpm for 20min, collecting supernatant, and measuring K by atomic absorption spectrophotometry+And (4) calculating the decomposition rate of the strain on the potash feldspar ore powder.
The potassium dissolution rate calculation formula is as follows:
Figure BDA0001562524670000051
among the strains obtained by screening, the BX strain has the highest potassium-dissolving capacity, and K in the fermentation liquor+The concentration was 3.16 times that of the control group.
Example 3 identification of Potassium solubilizing bacteria BX
And (3) measuring the growth condition of the bacterial strain BX in a culture medium with a unique carbon source or a unique nitrogen source, observing the colony morphology, and obtaining the morphological characteristics of the potassium bacteria BX. Bacterial colonies of the bacterial strain BX on a solid culture medium are light yellow and circular, the surface is rough and opaque, the diameter is about 5mm, and the middle part is protruded. The bacterium is in a short rod shape and gram-negative when observed under a microscope. Glucose, starch, sucrose, maltose, lactose, glucomannan, galactose can be utilized; can decompose glucose to produce pyruvic acid, decompose tryptophan to form indole, hydrolyze starch, and has lecithin enzyme hydrolysis ability. Total DNA of the strain BX is extracted by a cracking method, 16s rDNA is amplified for carrying out bacterial molecule identification, and an amplification product is sequenced by Shanghai Mipu Biotech Co., Ltd and is compared with GenBank number homology to determine as Bacillus sp.
Example 4 production of a Microecological preparation by solid fermentation of Potassium bacteria Bacillus BX
Selecting potassium bacteria BX, inoculating into 100m L L B liquid culture medium, shake culturing at 37 deg.C, and culturing to obtain bacterial liquid OD600Transferring the potassium bacteria culture solution to a 10m L fermenter, culturing at 37 deg.C under ventilation for 24 hr to obtain liquid seed of strain, inoculating the liquid seed into sterilized 100kg solid testa Tritici at an inoculum concentration of 10% by mass, culturing at 25-37 deg.C for 24-48 hr until the viable count in the solid composite carrier reaches 4.0 × 1010cfu/g. Drying the microbial inoculum meeting the requirement of viable count at 50 ℃ until the water content is less than or equal to 5 percent to obtain the potassium-removing microbial inoculum.
Example 5 production of a Microecological preparation by liquid fermentation of Potassium bacteria Bacillus BX
The liquid culture medium formula is 10.0g of glucose and MgSO4·7H2O 0.5g，Na2HPO42g，CaCO30.5g，FeCl20.01g of potassium feldspar powder, 1.0g of potassium feldspar powder (the potassium feldspar powder is sieved by a 40-mesh sieve, soaked in deionized water overnight, then repeatedly washed by the deionized water to remove soluble potassium in the potassium feldspar powder, and dried at 50 ℃), the deionized water is 1000m L, the pH value is 7.2, the culture temperature is 37 ℃, the rotation speed is 180rpm, and the culture time is 24 hours, so that potassium in the soil can be dissolved by fermentation liquor, or the potassium in the soil can be dissolved by drying the fermentation liquor, so that the dried potassium in the soil can be dissolved by the fermentation liquor.

Claims (4)

1. The high-efficiency potassium-solubilizing strain capable of adapting to the low-temperature environment is characterized by being preserved in the China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.14769, and the strain is named as a Bacillus (Bacillus sp.) BX strain.
2. The solid fermentation method for preparing microecologics based on the potassium-solubilizing strain of claim 1, which comprises the following steps:
(1) selecting potassium bacteria BX, inoculating into L B liquid culture medium, shake culturing at 37 deg.C, and culturing to obtain bacterial liquid OD600Obtaining potassium-decomposing bacterium culture solution as 0.5;
(2) transferring the potassium bacteria culture solution into a fermentation tank, and performing ventilation culture at 37 ℃ for 20-28 h to obtain strain liquid seeds;
(3) inoculating strain seed solution into sterilized solid testa Tritici at high temperature, wherein the mass ratio of the strain seed solution to the solid testa Tritici is 8-12%, culturing at 25-37 deg.C for 24-48 hr until viable count reaches 4.0 × 1010cfu/g to obtain a microbial inoculum meeting the requirement of viable count;
(4) drying the microbial inoculum until the water content is less than or equal to 5 percent to obtain the potassium bacteria microecological microbial inoculum.
3. The liquid fermentation method for preparing the microecological preparation based on the potassium-solubilizing strain of claim 1 is characterized in that the method comprises the steps of inoculating the potassium-solubilizing strain to a liquid culture medium, and carrying out fermentation culture for 20-28 h at 37 ℃ and 180rpm to obtain a fermentation liquid for soil potassium solubilization, namely the potassium-solubilizing microbial ecological agent; or drying the fermentation liquor to obtain a dried substance for soil potassium dissolution, namely the potassium-dissolving bacterium microecological bactericide; the liquid culture medium comprises the following components in parts by weight: glucose 8-12, MgSO4·7H2O 0.3—0.7，Na2HPO41.5—2.5，CaCO30.3—0.7，FeCl20.008-0.012 wt%, potash feldspar powder 0.8-1.2 wt%, deionized water 950-1000 wt% and pH 7.0-7.5.
4. The method of claim 3, wherein saidThe liquid culture medium comprises the following components in parts by weight: glucose 10, MgSO4·7H2O 0.5，Na2HPO42，CaCO30.5，FeCl20.01, deionized water 1000, pH 7.2.
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