CN101066897A - A K3 strain capable of dissolving calcium phosphate in soil and its microbial organic fertilizer - Google Patents

Abstract

The present invention relates to agricultural production technology, and is especially one kind of K3 bacterial strain capable of dissolving calcium phosphate in soil and microbial fertilizer produced with the K3 bacterial strain. The bacterium strain with powerful calcium phosphate dissolving capacity is separated from limy soil, used in liquid fermentation, and mixed with specific organic carrier to produce phosphate decomposing microbial organic fertilizer, which contains phosphate decomposing microbes in 5x10<7> /g, total nitrogen in 4 wt%, total potassium in 1-2 wt%, total phosphorus in 1-2 wt% and organic matter in 35 wt%. Test shows that the phosphate decomposing microbial organic fertilizer can provide phosphorous nutrients in the effect similar to chemical phosphate fertilizer, and the phosphate decomposing bacterium can reach soluble phosphorus concentration in calcium phosphate solution up to 643 mg/l.

Description

A K3 strain capable of dissolving calcium phosphate in soil and its microbial organic fertilizer

1. Technical field

The invention discloses a bacterial strain K3 capable of dissolving calcium phosphate in soil and the microbial organic fertilizer produced therefrom, which belongs to the technical field of agricultural intensive production and is specially used for overcoming the phosphorus deficiency problem of crops on calcareous soil.

2. Background technology

After the chemical phosphorus fertilizer is applied to the soil, the crops of the current season can only use about 15%, and about 85% of the phosphorus fertilizer is fixed in the soil and gradually ages and loses its effectiveness. Even the crops of the next season can hardly use the phosphorus fertilizer applied in the previous season. . Therefore, farmers have to apply chemical phosphate fertilizers every season. In this way, on the one hand, a large amount of phosphate rock resources are exhausted. It is predicted that the existing high-grade phosphate rock resources in my country will be completely exhausted in only 30 years according to the current development and utilization speed. On the other hand, On the one hand, farmers apply phosphorus fertilizers every season, so that there is a lot of accumulated phosphorus in the soil, but it is difficult for plants to use it. Therefore, inventing a microbial or microbial organic fertilizer that can dissolve the ineffective phosphorus in the soil and make it effective is a very important part of the management of soil phosphorus and phosphorus fertilizers in my country.

There are two types of phosphorus bacteria in the soil according to their mode of action, one is inorganic phosphorus bacteria, and the other is organic phosphorus bacteria. The former produces organic acids (such as citric acid, lactic acid, malic acid, etc.) through the life activities of bacteria. These organic acids release phosphorus by sequestering calcium in calcareous soils and lowering the pH to destabilize the calcium phosphate in the soil, thereby converting inorganic calcium-phosphorus (except in acidic soils) compounds in the soil that are ineffective to plants into effective inorganic phosphorus. It is one of the development directions of microbial organic fertilizer to mobilize ineffective phosphorus in soil by using some special-function microorganisms. Another type of phosphorus bacterial fertilizer (organophosphorus bacteria) is to decompose organic phosphorus compounds in the soil that are not effective for plants into inorganic phosphorus that can be absorbed by plants through the secretion of phosphatase by microorganisms. This type of phosphorus bacterial fertilizer is applied to high organic matter content. The effect is more pronounced on the soil.

On the other hand, the solid organic waste in my country's agricultural product processing industry and the livestock and poultry manure after large-scale breeding not only seriously pollute the environment, but also greatly waste raw materials that can be used as organic fertilizers and microbial organic fertilizer products; a large amount of Nutrient resources (C, N, P, K, S and trace elements) are lost outside the soil-plant system, which obviously weakens the ability of my country's agricultural sustainable development. The only way to maximize the return of the nutrients taken from the soil by harvesting crops to the soil is to make these solid organic wastes into commercial organic fertilizers and then apply them to the soil. If these solid organic wastes are fermented at high temperature to synthesize high-grade organic compost, and then used as the organic carrier of agricultural functional bacteria, the microbial organic fertilizer produced has clear functions and will have a good application prospect.

3. Contents of the invention

Technical problem The object of the present invention is to develop a kind of bacterium that can dissolve calcium phosphate in soil and the microbial organic fertilizer that it is made of, and the inactive state phosphorus (calcium phosphate) in the calcareous soil is converted into part available state phosphorus, thereby improves crop phosphorus. Nutrients, save a lot of phosphate rock resources, and ensure the sustainable development of my country's agricultural production.

Technical solutions

1. Physiological and biochemical characteristics of Phosphobacteria K3 strain

A K3 bacterial strain capable of dissolving calcium phosphate of the present invention is characterized in that the bacterial strain is Pseudomonas aeruginosa, and was preserved in China Microbial Cultures Preservation Committee General Microorganisms on May 14, 2007. Center, the strain preservation number is CGMCC NO.2045, the main biological characteristics are G ^- , the bacteria are rod-shaped, facultative anaerobic; catalase, oxidase, indole test, methyl red reaction are positive, The VP test is negative; gelatin can be used and litmus milk can be coagulated, but starch and citrate cannot be used; glucose, sucrose and lactose fermentation do not produce gas. The query number of the 16S rDNA of the K3 strain in Genban is: EF064786.

2. Microbial organic fertilizer produced by phosphorus-solubilizing bacteria K3 strain

The fertilizer contains more than 5× ¹⁰⁷ /g of phosphorus-dissolving microorganisms; the total nitrogen content is 4%, of which more than 90% is organic nitrogen; the total potassium content is 1-2%; the total phosphorus content is 1-2%. The content is 35%.

3, the method for producing microbial organic fertilizer with K3 bacterial strain comprises:

1) select the phosphorus-solubilizing bacterium K3 strain described in claim 1, first carry out liquid fermentation production, the conditions of its fermentation production are: pH growth range 5.0～8.0, culture temperature range 25～45 ℃, liquid culture medium is LB enrichment, The fermentation period is 18-30 hours, and the bacterial content of the K3 strain in the fermentation broth is ≥1× ¹⁰¹⁰ /ml;

2) Organic carrier production of phosphorus-solubilizing bacteria: the organic high-nitrogen fertilizer produced by the method described in ZL2004 1 0014773.1 according to the patent "Using agricultural solid waste to make organic high-nitrogen fertilizer and its fertilizer products" obtained by this research group as an organic carrier;

3) The production of organic fertilizer of phosphorus-solubilizing bacteria K3 bacterial strain: the above-mentioned organic carrier is added 5% fermented liquid of phosphorus-solubilizing bacteria K3 bacterial strain by weight ratio After fully mixing the fermented liquid of phosphorus-solubilizing bacteria and the organic carrier, place it under natural conditions for 3-5 days, during which the number of phosphorus-solubilizing bacteria will multiply from about 1×10 ⁷ /g to 1×10 ⁸ /g, and the number of phosphorus-solubilizing bacteria in the final product will reach more than 5×10 ⁷ /g, and the water content will be less than 30%. , the total nitrogen content reaches 4% (organic nitrogen is greater than 90%), and the organic matter content reaches 35%, reaching the national microbial organic fertilizer product standard.

In the above-mentioned production method, the optimal temperature for the fermentation and production of phosphate solubilizing bacteria is 37.5°C. In the microbial fermentation production method, enriching the medium LB refers to adding ammonium nitrate, ammonium sulfate, soybean powder, etc. to the LB.

Beneficial effect

The invention discloses a microbial organic fertilizer capable of dissolving calcium phosphate in soil and a production method thereof. The specific organic carrier is made of agricultural solid organic waste, and then mixed with the fermentation liquid of phosphorus-dissolving bacteria to make a microbial organic fertilizer. The product is the same as Compared with the products currently on the market, it has the following advantages:

1) Fertilizer products contain high-efficiency strains that dissolve soil calcium phosphate, and their phosphorus-dissolving effect is very significant. The content of this high-efficiency strain in fertilizers reaches more than 5×10 ⁷ /g. After being applied to the soil, it can quickly form a dominant group, and in survive in the soil.

2) Fertilizer products are rich in organic carbon and organic nitrogen, which can provide a very superior growth environment for phosphorus-dissolving functional bacteria, and greatly improve the ability of foreign functional microorganisms to colonize and function in the soil.

3) The laboratory culture results show that the phosphorus bacteria can make the soluble phosphorus concentration in the calcium phosphate solution reach 643mg/L, which is more than 4 times higher than the latest foreign reported results.

4) The source of raw materials for this product is wide and abundant, the cost of raw materials is very low, the production process is simple, the investment in fixed assets is small, and the consumption of electricity and water is very little in the production process, so the product price is low and the pertinence is strong. The total cost of the microbial organic fertilizer for phosphate-dissolving bacteria does not exceed 600 yuan, while the market price of this kind of special-purpose microbial organic fertilizer is relatively high, generally around 1,000 yuan/ton, and it is very scarce in the market at present, so this product has Strong social, environmental and economic vitality.

5) The results of field biological tests show that after applying this product on corn, soybeans and other crops, phosphorus-solubilizing bacteria can multiply rapidly, form a dominant group in the soil, and significantly increase the available phosphorus in the soil. Apply 100 kilograms of this microbial organic fertilizer, and the crop effect that it provides phosphorus nutrition is equivalent to the chemical phosphate fertilizer (75 kilograms superphosphate) of 10 kilograms (P ₂ O ₅ ) per mu, the chemical nitrogen fertilizer (18 kilograms) of 8 kilograms (N) kg of urea), 1-2 kg (K ₂ O) of chemical potassium fertilizer (3 kg of potassium chloride). The input cost for farmers to apply the microbial organic fertilizer is 100 yuan, while the input cost for applying the above-mentioned chemical fertilizer is 120 yuan. Not only that, after applying the microbial organic fertilizer product of the present invention, the quality and flavor of the produced agricultural products are greatly improved, and the ecological environment and soil quality are greatly improved.

4. Description of drawings

Figure 1 Isolation and screening flow chart of highly efficient phosphorus-solubilizing bacteria K3 strain

Figure 2 Obtained high-efficiency phosphorus-solubilizing bacteria K3 strain by isolation and screening of PVK solid medium

Fig. 3 liquid fermentation culture technological process of phosphate-solubilizing bacteria

5. Specific implementation

(1) Acquisition of Phosphorus Solubilizing Bacteria

Select the well-grown plant rhizosphere from the calcareous phosphorus-deficient soil to collect soil and plant roots, and save the samples at low temperature (4-15°C) and bring them back to the laboratory; adopt a series method to utilize PVK-improved solid plate selective medium (glucose 10g) , tricalcium phosphate 5g, ammonium sulfate 0.5g, sodium chloride 0.2g, magnesium sulfate 0.1g, potassium chloride 0.2g, yeast extract 0.5g, manganese sulfate 0.002g, ferrous sulfate 0.002g, 0.4% bromophenol blue 6ml, agar 18g, distilled water 1000ml, pH natural) at 37.5°C for 36-48 hours to obtain a single colony of phosphorus-solubilizing bacteria, and select the strain with a larger ratio of phosphorus-dissolving circle to colony circle for further shaker screening; use NBRIP liquid medium (glucose 10g, tricalcium phosphate 5g, magnesium chloride 5g, magnesium sulfate 0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, distilled water 1000ml, pH7.0.) and phosphate rock powder improved medium (glucose 10g, phosphate rock Powder 5g, magnesium chloride 5g, magnesium sulfate 0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, distilled water 1000ml, pH7.0) obtain phosphate solubilizing bacteria fermentation liquid under the condition of 37.5 degree of cultivation 18-24 hours, measure fermentation liquid and Phosphorus content in the bacteria, 4 strains with higher phosphorus dissolving capacity were selected for pot experiments; corn was used for pot experiments on phosphorus-deficient calcareous soil. Superphosphate, potassium dihydrogen phosphate, fermented broth of phosphorus-dissolving bacteria, and inactivated fermented broth of phosphorus-dissolving bacteria were used instead of fertilization. After 30-35 days of greenhouse cultivation, the total phosphorus content of the plant and the available phosphorus content in the soil were measured. The robustness, total phosphorus content of the plant, and soil available phosphorus content were used to screen high-efficiency phosphorus-solubilizing strains, and the K3 strain was obtained. The genus and species were determined through physiological, biochemical and 16sRNA molecular identification, and the strains were freeze-dried and stored at -20°C.

(2) Fermentative production of phosphate-solubilizing bacteria strains

1. Seed culture of phosphobacteria K3 strain

Seed medium (shake flask culture): medium LB enriched, steam sterilized at 121°C for 20 minutes;

Seed culture conditions: 5-10% inoculation amount, 37.5 degrees, 150-200 rpm; culture period 18-25 hours.

2. Fermentative production of bacterial strains (expanded culture): LB-enriched medium is used for high-density expanded culture to obtain high-concentration fermented liquid (bacteria content ≥ 1×10 ¹⁰ cells/ml).

Fermentation medium: medium LB is enriched (the enriched main ingredients are ammonium nitrate, ammonium sulfate and soybean powder, etc.), steam sterilized at 121°C for 20 minutes;

Fermentation conditions: 5-10% inoculum size, 80-100 rpm, culture temperature 37.5°C, pH 7.0-7.5, fermentation period 20-25 hours. The dissolved oxygen is stable at 100%-60%. When the dissolved oxygen starts to rise and gradually tends to ⁷⁵ %, it indicates that the fermentation has entered a flat period and the tank can be put in. The density fermented bacteria liquid is stored at low temperature for later use.

3. Production of Phosphorus Solubilizing Bacteria K3 Strain Bio-Organic Fertilizer

Phosphorus solubilizing bacteria and organic carrier (according to Chinese patent "the method that utilizes agricultural solid waste to make and its fertilizer product ", patent number: the organic high-nitrogen fertilizer produced by the method described in ZL2004 1 0014773.1 is organic carrier) fully mix (add according to The phosphate-solubilizing bacteria fermented liquid of 5% by weight,) the mixture after mixing loses moisture to water content less than 30% under natural conditions, and this process needs about 3-5 days time, wherein the phosphorus-solubilizing bacteria quantity will be from 1 * 10 About ⁷ /g to 1×10 ⁸ /g, the number of phosphorus-solubilizing bacteria in the final product reaches more than 5×10 ⁷ /g, the total nitrogen content reaches 4% (organic nitrogen is greater than 90%), and the organic matter content reaches 35%, the product index reaches the national microbial organic fertilizer standard. Product packaging, stored in a cool, dry place.

Table 1 Some characteristics of microbial organic fertilizer product of the present invention Total organic matter (%) Soluble organic matter (%) Total organic nitrogen (%) Total phosphorus P ₂ O ₅ (%) Total potassium K ₂ O(%) PH (water extraction) The number of living bacteria of phosphate-solubilizing bacteria K3 (unit/g) 35.8 9.52 4.12 1.65 1.13 6.8 5×10 ⁷

Table 1 shows that the organic matter content is 35.8% in the product produced by the technology of the present invention and production process, and contains a very high proportion of soluble organic matter (9.52%) and a very high organic nitrogen content (4.12%). The material basis for the massive reproduction of phosphorus-solubilizing bacteria in the soil; at the same time, these large amounts of organic carbon and organic nitrogen also significantly promote the biological activity of soil microorganisms after being applied to the soil, thereby greatly increasing the conversion rate of soil nutrients, so that crops can be grown throughout the growth period. In a good nutritional state; the number of viable phosphate-solubilizing bacteria in the product reaches 5×10 ⁷ . The above characteristics have fully illustrated the feasibility of the special phosphorus-dissolving microbial organic fertilizer manufactured by the technology and production process of the present invention.

4. Field test of phosphorus-solubilizing bacteria K3 strain bio-organic fertilizer

The special phosphorus-dissolving microbial organic fertilizer developed by the present invention is very significant to the yield-increasing effect of corn on calcareous soil (see Table 2), as can be seen from Table 2 if only using organic high-nitrogen fertilizer (organic carrier) corn grain yield on calcareous soil 27.1% higher than the control (Table 2 treatment 3 minus treatment 1 divided by treatment 1 and multiplied by 100%), indicating that the organic fertilizer has a certain yield-increasing effect on corn yield. If only the phosphorus-solubilizing bacteria liquid is used, the grain yield of corn is only 5.7% higher than that of the control (Table 2 handles 4 minus processing 1 and divides it by processing 1, and then multiplied by 100%). The effect of increasing production was not significant. If the microbial organic fertilizer (treatment 5) was applied, the yield of corn kernels was close to that of the conventional fertilization by local farmers, which was 43.2% higher than that of the control. If apply a small amount of urea and potassium chloride in conjunction with the application of microbial organic fertilizers, the corn yield is the highest of all treatments, which is also an increase of 3.9% than the local farmer's conventional fertilization (table 2 treatment 6 minus treatment 2 divided by treatment 2, multiplied by 100%), by processing 6 and processing 2 comparisons, draw 100 kilograms of this microbial organic fertilizer that every mu uses, and the crop effect that it provides phosphorus nutrition is equivalent to every mu using 75 kilograms of superphosphate, 18 kilograms of urea and 3 kilograms of chlorine Potassium chloride), the input cost for farmers to apply this microbial organic fertilizer is 100 yuan, and the input cost for applying the above-mentioned chemical fertilizer is 120 yuan.

Phosphorus-dissolving microbial organic fertilizer of the present invention is to the yield-increasing effect (field test) of corn on calcareous soil of table 2 Processing number processing description Corn grain yield (kg/mu) Total phosphorus absorbed by corn (kg/mu) 1 CK 458.3±26.9 16.9±4.4 2 Regular fertilization by local farmers: urea 45kg/mu + superphosphate 75kg/mu + potassium chloride 20kg/mu 692.8±32.4 25.3±3.9 3 Apply organic carrier (100kg/mu) 582.7±24.7 20.6±3.2 4 Apply phosphorus-dissolving bacteria liquid (per acre uses 5kg phosphorus-dissolving bacteria fermentation liquid) 484.6±46.5 19.6±4.5

5 Application of microbial organic fertilizer (100kg/mu): organic carrier + phosphorus-dissolving bacteria liquid 656.3±37.1 22.0±3.7 6 Application of microbial organic fertilizer (100kg/mu): organic carrier + phosphorus-dissolving bacteria liquid + urea 27kg/mu + potassium chloride 17kg/mu 719.6±26.9 26.3±2.8

Note: All organic fertilizers, phosphate fertilizers and potassium fertilizers are applied to the soil as base fertilizer at one time, and urea is applied to the soil three times (60% of base fertilizer, 20% of top dressing for the first time, and 20% of top dressing for the second time.)

Also find out from table 2, apply the total phosphorus amount that corn absorbs after this microbial organic fertilizer significantly increases, because this soil has the feature that soil total phosphorus content is high and available phosphorus is very low (4.6mg/kg), apply this microbial organic fertilizer Finally, the transformation speed of soil invalid phosphorus into available phosphorus is accelerated, and the total phosphorus absorbed by corn during the whole growth period is significantly increased, thereby ensuring the phosphorus nutrition of corn and making corn high and stable yield.

It can be seen from Table 3 that the number of phosphorus-dissolving bacteria in the soil increased significantly after one season of corn growth with the application of phosphorus-dissolving microbial organic fertilizer, reaching 6× ¹⁰⁴ /g soil, indicating that the phosphorus-dissolving microbial organic fertilizer can indeed Make the foreign phosphorus-dissolving bacteria colonize and multiply in the soil, and play a role. After one season of corn, the available phosphorus in the soil is 7.02mg/kg, while the available phosphorus in the soil of 1, 2, and 3 treatments is below 3.5mg/kg, only the application of phosphorus-dissolving bacteria half of the microbial organic fertilizer treatment, indicating that the phosphorus-dissolving effect of the microbial organic fertilizer is remarkable.

Table 3 Phosphorus-dissolving microbial organic fertilizer of the present invention is on the influence of phosphorus-dissolving bacteria and soil phosphorus content on calcareous soil (soil sample when the pot test ends) Processing number processing description Phosphorus solubilizing bacteria (unit/g soil) Available phosphorus content (mg/kg) Total phosphorus content (%) 1 CK 2×10 ¹ 2.89 0.143 2 Apply organic carrier (100kg/mu) 3×10 ¹ 3.45 0.149 3 Application of Phosphorus Solubilizing Bacteria Liquid 7×10 ² 3.50 0.141 4 Application of microbial organic fertilizer (100kg/mu): organic carrier + phosphorus-dissolving bacteria liquid 6×10 ⁴ 7.02 0.136

Note: All fertilizers were applied to the soil as basal fertilizer at one time, and the experimental crop was corn.

Claims (6)

1, a kind of bacterium K3 bacterial strain of energy dissolving phosphoric acid calcium, it is characterized in that, this bacterial strain is Pseudomonas aeruginosa (Pseudomonas aeruginosa), is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 14th, 2007, and culture presevation number is CGMCC NO.2045.

2, the microorganism organic fertilizer of being produced with the described a kind of bacterium K3 bacterial strain that can dissolve soil calcium phosphate of claim 1.

3, according to the described microorganism organic fertilizer that can dissolve soil calcium phosphate of claim 2, it is characterized in that: contain 5 * 10 in this fertilizer ⁷The phosphate solubilizing microorganism quantity that individual/g is above; Total nitrogen content is 4%, wherein is organonitrogen more than 90%; Full potassium content 1～2%; Content of tatal phosphorus 1～2%; Organic content is 35%.

4, according to claim 2 or the 3 described microorganism organic fertilizers that can dissolve soil calcium phosphate, its production method comprises:

1) phosphate-solubilizing bacteria K3 bacterial strain fermentation liquor is produced: select the described phosphate-solubilizing bacteria K3 of claim 1 bacterial strain for use, carry out liquid fermenting production, the condition of its fermentative production is: pH growth scope 5.0～8.0,25～45 ℃ of culture temperature scopes, liquid nutrient medium is that LB adds richness, fermentation period 18～30 hours, K3 bacterial strain bacteria containing amount 〉=1 * 10 in the fermented liquid that is obtained ¹⁰Individual/ml;

2) the organic carrier production of phosphate-solubilizing bacteria: " utilize agricultural solid residue to make the method and the fertilizer product thereof of organic fertilizer with high nitrogen " according to Chinese patent, the organic fertilizer with high nitrogen that the described method of the patent No.: ZL200410014773.1 is produced is an organic carrier;

3) production of phosphate-solubilizing bacteria K3 bacterial strain organic fertilizer: above-mentioned organic carrier is added 5% phosphate-solubilizing bacteria K3 bacterial strain fermentation liquor by weight, behind the thorough mixing, placed under field conditions (factors) 3-5 days, be the phosphate solubilizing microorganism organic fertilizer product of acquisition.

5, according to the described phosphate solubilizing microorganism organic fertilizer of claim 4, phosphate solubilizing bacteria fermentative production optimum temps is 37.5 ℃ in its production method.

6, according to claim 4 or 5 described phosphate solubilizing microorganism organic fertilizer, substratum LB adds richness and refers to add ammonium nitrate, sulphur ammonium and analysis for soybean powder etc. in LB in its microbial liquid fermentation method for producing.

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