CN111235076A

CN111235076A - Bacillus anethoides, microbial inoculum thereof and application thereof in heavy metal remediation field

Info

Publication number: CN111235076A
Application number: CN202010243541.2A
Authority: CN
Inventors: 单宝龙; 胡著然; 郭静; 王丽荣; 刘晓晓; 孔德荣; 张文娟; 申小冉; 朱自然; 董现义
Original assignee: Shandong Greenblue Biotechnology Co ltd
Current assignee: Shandong Greenblue Biotechnology Co ltd
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-06-05
Anticipated expiration: 2040-03-31
Also published as: CN111235076B; WO2021196735A1; KR20210124232A

Abstract

The invention belongs to the technical field of heavy metal soil remediation, and particularly relates to Bacillus anethoides, a microbial inoculum thereof and application thereof in the field of heavy metal remediation. The invention obtains a Bacillus subtilis P46 from heavy metal contaminated soil by screening, wherein the Bacillus subtilis P46 is a phosphate solubilizing bacterium, is nontoxic to plant growth when applied to crop culture and has a certain promotion effect; the strain has good degradation effect on cadmium and lead, and does not affect the stability of soil while repairing heavy metal ions. At present, related reports about Bacillus aetheratus are few, and the invention provides a strain with good phosphate solubilizing and heavy metal repairing effects, which is applied to heavy metal soil repairing and has good application prospect in improving the growth condition of crops.

Description

Bacillus anethoides, microbial inoculum thereof and application thereof in heavy metal remediation field

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a Bacillus subtilis P46, a microbial inoculum thereof and application thereof in the field of soil heavy metal remediation.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The world faces food, resources and environmental problems closely related to soil. Since the 20 th 20 s of the 20 th century, the production of metals has increased significantly due to the development of industry, and the problem of environmental pollution caused by heavy metals has also been raised. In many developing and developed countries, soil pollution is facing the problem of severely hindering agricultural production. With the development of social modernization and production, the problem of soil pollution is becoming more serious, and thus people have attracted extensive attention.

Among the heavy metals commonly noted in environmental sciences are mercury, cadmium, nickel, chromium, vanadium, arsenic, selenium, iron, manganese, copper, zinc, lead, and the like. Some of the heavy metals are essential trace elements for organisms, and others are environmental pollutants, and currently, cadmium (Cd), lead (Pb), copper (Cu), zinc (Zn), and mercury (Hg) are widely studied. In agricultural production, heavy metal pollution not only reduces soil fertility, but also causes the quality of produced agricultural products to be inconsistent with the requirements of ecological safety and harms human and livestock health, thereby having great harmfulness.

Meanwhile, the soil pollution remediation technology is rapidly developed, particularly the bioremediation technology is developed, and a more efficient and green means is provided for the remediation of heavy metal pollution. Bioremediation is an environment-friendly technology, which combines scientific technology with natural life processes and utilizes life activities of bacteria, fungi, green plants and the like to restore polluted environments. The microorganism has the characteristics of low cost, low energy consumption, no secondary pollution, high efficiency and the like in the remediation of the heavy metal contaminated soil. Meanwhile, part of microorganisms have good adaptive capacity and cell mechanism, and can absorb various heavy metals, shorten the repair time and achieve good heavy metal soil transformation effect. Meanwhile, microorganisms can participate in regulating and controlling nutrient circulation in soil, and the heavy metal conversion speed of the soil is improved. Therefore, the method has great potential for repairing the heavy metal contaminated soil by utilizing some fungi, bacteria, actinomycetes and the like. Among various microorganisms, sulfate reducing bacteria and phosphate solubilizing bacteria have become a hotspot for researching the microorganisms to restore the soil heavy metals at present due to higher soil heavy metal tolerance and heavy metal restoration potential.

Disclosure of Invention

The invention obtains a Bacillus subtilis P46 with the functions of phosphate solubilizing and heavy metal restoration by screening from heavy metal contaminated soil. The Bacillus gastrodiae has good phosphate-solubilizing, plant growth promoting and heavy metal restoration capacity in soil, and has obvious restoration effect on metal lead and cadmium in soil.

Aiming at the technical effects, the invention provides the following technical scheme:

in the first aspect of the invention, a strain of Bacillus subtilis P46, which has been deposited in China general microbiological culture Collection center (CGMCC) in 3 months and 2 days in 2020, addresses are as follows: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC NO. 19444.

Bacillus aetherotis was first isolated in 14 years and identified as a new species. At present, the research aiming at the Bacillus aegypti in the field is relatively blank, and the biological activity and whether the Bacillus aegypti has a probiotic function are not clear. The Bacillus subtilis P46 provided by the invention is a strain with good phosphate-solubilizing capability, and can help to improve soil fertility and promote plant growth. In addition, the phosphate solubilizing bacteria also have good passivation capability on heavy metals, and under the condition of single use, the phosphate solubilizing bacteria have the degradation rate on heavy metal cadmium in soil of more than 60 percent, and the degradation rate on metal lead of more than 80 percent.

The Bacillus subtilis strain P46 has a 16S rDNA sequence shown in SEQ ID NO: 1.

The strain is separated from sewage around an electroplating plant, the bacterial morphology of the strain meets the characteristics of Bacillus, the identity of the strain with the known strain Bacillus tianshanensis strain YIM 13235 (98%) can reach 98% through genome sequencing comparison, the strain is identified as the Bacillus nervosa, and the strain is named as the Bacillus tianshen (Bacillus tianshanensis strain) P46.

The morphological characteristics of the Bacillus subtilis P46 are as follows:

the characteristics of the thallus are as follows: the size of the strain cell is 0.3-0.5 Mum multiplied by 1.4-3.0 Mum, which is rod-shaped and gram-positive; the spores are oval, the size is 0.6-0.9 μm multiplied by 1.0-1.5 μm, and the spores have motility.

Colony characteristics: the colony is round or irregular, the edge is irregular, the surface has no wrinkles and roughness, and the colony is dirty white and has no bulge.

The physiological and biochemical characteristics of the Bacillus subtilis P46 are as follows:

can utilize glucose, xylose, arabinose and the like, can hydrolyze molasses, and has the function of phosphate solubilizing; the growth rate was highest in the culture at 30 ℃ and did not grow under anaerobic conditions.

In a second aspect of the present invention, there is provided a bacterial agent comprising a culture of the Bacillus subtilis P46 and/or the bacterium of the first aspect.

The dosage form of the microbial inoculum is liquid microbial inoculum, powder or granules; further is a water suspending agent, a dispersible oil suspending agent, a wettable powder or a water dispersible granule.

Preferably, the microbial inoculum also comprises an agriculturally and pharmaceutically acceptable auxiliary material, and the agriculturally and pharmaceutically acceptable auxiliary material is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder, a defoaming agent, an antifreeze agent, a thickening agent, a filler and a solvent. The invention has no special limitation on the sources of the auxiliary materials acceptable in the agricultural pharmacy, and the like, and generally adopts the products sold in the market.

In a third aspect of the invention, the bacterial strain of the first aspect and the microbial inoculum of the second aspect are applied to the heavy metal soil remediation.

In a fourth aspect of the invention, a complex microbial inoculum is provided, which consists of Bacillus subtilis P46 and Bacillus subtilis P30 or a culture thereof; the Bacillus subtilis (Bacillus subtilis strain) P30 has been preserved in China general microbiological culture Collection center (CGMCC) in 3, month and 2 of 2020, and has the address as follows: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC NO. 19443.

The beneficial effects of one or more of the above technical solutions are as follows:

1. the Bacillus anethoides is a new genus, related research is almost blank, and the research result provides reference experience for the discovery and application of the Bacillus anethoides.

2. The Bacillus nervosa has various probiotic effects, including decomposing phosphorus in soil to provide nutrients for plant growth, promoting plant growth, and passivating and repairing various metal elements in soil, and has good application prospect when applied to repairing polluted farmlands.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph showing the effect of the culture conditions described in example 6 on the culture of P46;

wherein, FIG. 1A is a line graph showing the influence of temperature and pH value on the number of viable bacteria;

FIG. 1B is a line graph showing the influence of temperature and pH on the number of spores;

FIG. 1C is a line graph showing the influence of the rotation speed and time on the number of viable bacteria;

FIG. 1D is a line graph showing the effect of rotational speed and time on the number of spores.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, aiming at the defects in the prior art, the invention provides Bacillus anethoides, a microbial inoculum thereof and application thereof in the field of heavy metal remediation.

The Bacillus subtilis P46 has a 16S rDNA sequence shown in SEQ ID NO. 1.

Preferably, the preparation method of the Bacillus anethoides powder comprises the following steps: inoculating the Bacillus subtilis P46 into a slant culture medium for culture; taking the cultured inclined plane, taking the strain under the aseptic condition, inoculating the strain into a seed liquid culture medium, and performing shake culture to obtain a first-stage seed liquid; carrying out amplification culture on the primary seed liquid to obtain a secondary seed liquid; and (3) taking 0.8-1.2% of inoculation amount to measure secondary seed liquid, inoculating the secondary seed liquid into a liquid fermentation culture medium, collecting fermentation liquid when the spore rate reaches more than 90%, adding light calcium carbonate, and performing spray drying to obtain bacterial powder.

More preferably, in the slant culture, the strain is inoculated on a solid slant culture medium in a loop and cultured for 24-30 h at 37 ℃.

Further preferably, the primary seed culture is performed at 37 ℃ and 180rpm for 18-24 h with shaking.

More preferably, the expanding culture is performed by inoculating the primary seed solution into 500 mL-1000 mL of seed liquid culture medium with an inoculation amount of 1% (v/v), and performing shake culture at 37 ℃ for 12-14 h to prepare the secondary seed solution.

Further preferably, in the step of spray drying, 0.67 to 1.00% (mass percent) of light calcium carbonate is added into the fermentation liquor.

Further preferably, the seed culture medium formula is as follows: 4.0% molasses, 2.0% soybean meal, 0.7% K₂HPO₄、0.3％KH₂PO₄，0.15％(NH₄)₂SO₄0.05 percent of sodium citrate, 0.01 percent of magnesium sulfate and the balance of water, adjusting the pH value to 6.8, and sterilizing for 30min at the temperature of 121 ℃.

More preferably, the solid slant culture medium is an NB synthetic liquid culture medium added with 1.5-2.0% agar powder.

Further preferably, the formula of the liquid fermentation medium is as follows: 40% molasses, 2.0% soybean meal, 0.7% K₂HPO₄、0.3％KH₂PO₄，0.15％(NH₄)₂SO₄0.05% sodium citrate, 0.01% magnesium sulfate and the balance of water, and the pH is adjusted to 6.8.

In a second aspect of the present invention, there is provided a bacterial agent comprising the Bacillus subtilis (strain) P46 and/or a culture of the bacterium of the first aspect.

Preferably, the formulation of the microbial inoculum according to the second aspect is liquid microbial inoculum, powder or granules; further is a water suspending agent, a dispersible oil suspending agent, a wettable powder or a water dispersible granule.

Preferably, the Bacillus subtilis strain P30 has a 16S rDNA sequence shown in SEQ ID NO. 2.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

EXAMPLE 1 screening of bacterial species

1. Materials and methods

1.1 sample

11 sample materials such as soil and water sample around an electroplating plant.

1.2 screening Medium

Strain nutrient medium: NB is synthesized into a culture medium, and 1.5% of agar is added on the basis of the NB culture medium to obtain the solid culture medium.

Screening medium for phosphate solubilizing strains (NBRIP): glucose 1.0%, Ca₃(PO₄)₂0.5％、MgCl₂0.5 percent, 0.5 percent of peptone, 2.0 percent of agar powder, pH 7.2-7.4, sterilizing at 121 ℃ for 30 min.

1.3 preliminary screening of samples

Primary screening of phosphate solubilizing bacteria: sampling 1 g (1ml) of soil sample and water sample obtained from electroplating plant, mixing, placing into a triangular flask filled with 99ml of sterile physiological saline, shaking and culturing for 2h at 30 ℃ and 180r/min in a constant temperature shaking table. Obtaining suspension, diluting continuously, and taking 1ml to obtain 10^-3-10^-5Diluting, taking 10^-3-10^-50.1ml of each diluent is added to an NBRIP culture medium plate for dilution and coating to separate a single colony, the single colony with a phosphate-solubilizing ring is picked for separation and purification, and the slant is stored in a refrigerator for later use.

1.4 Re-screening of strains

Rescreening phosphate solubilizing bacteria: and respectively inoculating the preliminarily screened phosphate solubilizing bacteria into 250mL triangular flasks (liquid containing 50mL) filled with NBRIP liquid culture medium, culturing at the rotation speed of 180rpm and the temperature of 37 ℃ for 24h, observing the color change and the viable count of the culture medium, and recording the time.

1.5 instruments

A constant-temperature water bath kettle; an incubator; an optical microscope.

2. Results

2.1 preliminary screening of the results

As shown in Table 1, in this example, 78 strains were isolated from 11 samples, and 17 strains of phosphate solubilizing bacteria were preliminarily selected by selecting the ratio of the phosphate solubilizing transparent circles to the amplification rate of the selective culture.

TABLE 1 preliminary screening results

2.2 rescreening results

As shown in Table 2, the strains P30, P46, S3 and S5 exhibited the best adsorption efficiency of cadmium.

TABLE 2 rescreening results

Note: 1. and (2) preparing: indicating that it is not detectable; 2. p7: is acinetobacter defluorinatum screened from soil; 3. p10: is Klebsiella screened from soil; 4. p30: is bacillus subtilis screened from sewage; 5. p46: is selected from Bacillus aegerens in soil; 6. p51: is the bacillus subtilis screened from sewage.

After the disease bacteria and fungi are removed from the 17 phosphorus dissolving bacteria, strains with a phosphorus dissolving effect of more than 50% are screened for a shake flask experiment, wherein four strains with a better cadmium removing effect are shown in table 2, and the P46 strain has the best cadmium removing effect.

Example 2 identification and safety test of bacterial species

1. Materials and methods

1.1 bacterial species

After primary screening and secondary screening, 4 strains with high heavy metal degradation performance are screened out from microorganisms separated from samples such as electroplating plant soil, water samples and the like, and the serial numbers are P30, P46, S3 and S5 respectively.

1.2 morphological and molecular identification of bacteria

Unless otherwise stated, general morphological and molecular tests were carried out according to Bergey's Manual of bacteria identification (9 th edition) and manual of microbiology experiments.

1.3 safety test

The phytotoxicity of bacteria is detected by using 'pearl' tomato seeds and 'crystal' lettuce seeds. The seeds were soaked in 2% concentration of single bacterial liquid, mixed bacterial liquid (1:1:1) and distilled water (control group) for 10 minutes. Then washing with distilled water, uniformly placing in a culture dish paved with wet filter paper, and placing in a constant temperature incubator with the temperature set to 24 ℃. And recording the germination rate after 3-4 days, and recording the weight of the roots and stems of the seedlings after the test group and the control group are dried respectively after 10 days. Phytotoxicity was judged by observing the degree of delay in seed germination and the growth of seedlings.

2. Results

2.1 identification of the species

Through morphological identification of bacteria, the bacillus belongs to bacillus, and the cells are rod-shaped, gram-positive bacteria, terminal spores and flagellum-free. Meets the characteristics of the Bacillus aegerens, and is primarily determined as the Bacillus aegerens.

And (3) selecting a single colony, inoculating the single colony into an NB liquid culture medium, performing shake culture at 37 ℃ and 180rpm for 24 hours, and extracting the genomic DNA of the strain from 1-5ml of bacterial liquid by using a bacterial genomic DNA extraction kit. The 16S rRNA universal primers 8f (5'-agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') amplified the genomic DNA of the strain and sent to Shanghai Biotech for sequencing. The BLAST software compares and analyzes that the identity of the strain can reach 98 percent with the following strains: bacillus tianshenii strain YIM 13235 (98%).

And (3) determining that the strain belongs to the Bacillus aegypti by integrating sequencing and morphological identification results.

2.2 safety test

TABLE 3 influence of the bacteria selected on germination percentage and seedling growth of tomato and lettuce seeds

As can be seen from Table 3, the two strains P30 and P46 have obvious effects of promoting the germination rates of tomatoes and lettuce seeds and have certain effects of promoting the growth of tomatoes and lettuce seedlings. P30 belongs to Bacillus subtilis, is a known strain with plant growth promoting effect, and P46 shows growth promoting effect equivalent to P30. Example 3 liquid shaking test for repairing heavy metal cadmium and lead by each strain

1. Materials and methods

1.1 bacterial species

P30、P46

1.2 culture Medium

NB liquid synthetic medium, 121 ℃, sterilization for 30 min.

1.3 method and instrument for measuring content of heavy metal cadmium

The determination method comprises the following steps:

(1) preparing NB liquid culture medium, adding heavy metal cadmium (to make the heavy metal cadmium content of the culture medium be 5mg/L), and sterilizing for later use.

(2) Setting 2 treatments, respectively inoculating 1ml of bacterial liquid of corresponding strains, wherein the 2 treatments are respectively as follows:

treatment 1: inoculation of only P30;

and (3) treatment 2: only P46 was inoculated while 3% nano-adsorption clay was added. The same amount of sterile water was used as a blank for the experiment instead of the inoculum.

(3) The inoculated flask was shake-cultured at 37 ℃ and 180rpm for 3 days.

(4) Centrifuging each treatment after shaking culture (rotating speed of 5000 revolutions), and taking the supernatant to determine the content of the heavy metal cadmium.

Instruments and measurement standards: the flame atomic absorption method is used for determination, and the specific operation refers to GB 15618-1995.

2 test contents and results

1) The concentration of the initial heavy metal cadmium is 5mg/L

And (4) conclusion:

the actual concentration of the heavy metal cadmium in the treatment 1 is 1.9595mg/L, and the actual degradation rate of the heavy metal is 60.81%;

the actual concentration of the heavy metal cadmium in the treatment 2 is 1.9755mg/L, and the actual degradation rate of the heavy metal is 60.49%.

2) The initial concentration of heavy metal lead was 50mg/L

As a result:

the actual concentration of the heavy metal lead in the treatment 1 is 8.1220mg/L, and the actual degradation rate of the heavy metal is 83.76%;

the actual concentration of the heavy metal lead in the treatment 2 is 7.9500mg/L, and the actual degradation rate of the heavy metal is 84.10%.

And (4) conclusion:

the transverse comparison in example 1 proves that P46 has excellent viability and cadmium repairing capability in the phosphate solubilizing bacteria screened from the same batch. In this example, the shaking flask experiment was performed using a conventional culture medium, and the experimental conditions are closer to the actual industrial conditions. The data show that the repair efficiency of the strain P46 on heavy metal lead can reach more than 83%, and the removal effect on cadmium can reach 60% on average.

Example quadruple Metal remediation potting test

In the embodiment, a composite microbial inoculum is provided, which is formed by mixing Bacillus subtilis P46 and Bacillus subtilis P30 in a ratio of 1:1 of viable bacteria.

1. The method for measuring the content of heavy metal cadmium and lead in soil comprises the following steps:

the method comprises the following steps of (1) measuring the content of total cadmium and total lead in the heavy metal in the soil by using a graphite furnace atomic absorption spectrophotometry, wherein the specific operation is referred to GB/T17141;

the method for measuring the different morphological contents of the heavy metals cadmium and lead in the soil refers to a BCR continuous extraction method.

The BCR continuous extraction method is to utilize different extractants specified in international standard to extract Cd from soil²⁺、Pb²⁺Extracting the forms, quantitatively detecting the extracting solution, and extracting Cd in different forms in the soil by comparing with a BCR method²⁺、Pb²⁺Content, simple analysis of repairing effect of microorganism and final repairing stability, 2, heavy metal repairing potted plant test method

Inoculating phosphate solubilizing bacteria P30 and P46 in LB culture medium, culturing at constant temperature of 37 deg.C at 160rpm for 2 days, and making the obtained bacterial thallus agglomerate into 3.0-5.0 × 10⁹CFU/mL bacterial suspension. The Cd polluted soil with different concentration gradients is respectively loaded in each flowerpot at 200 g/pot,two experimental groups are set, namely a non-bacterium-adding group (CK group) and a single compound bacterium liquid (P group, wherein the ratio of the number of viable bacteria of P46 and P30 is 1: 1). And (3) adding no bacteria into the CK group, adding 1mL of compound bacteria liquid into the P group, adding water into each group until the water content of the soil is 60% -70%, then uniformly stirring the soil samples in all flowerpots by using a wood shovel, ensuring that the added bacteria are uniformly distributed in the soil, and enabling three groups to be parallel. And after the bacteria are added into the soil for one month, measuring the content of heavy metals such as cadmium and lead in exchangeable, oxidizable, reducible and residual soil states. The initial exchangeable state content of the heavy metal in the soil is set to be 2ml/kg, 5ml/kg, 10ml/kg, 20ml/kg and 50 ml/kg.

3. Content and results of the experiments

TABLE 4 potted plant test one month later, 4-valence heavy metal cadmium content determination results

TABLE 5 determination of lead content in heavy metals of 4 valence states sampled one month after potting test

And (4) conclusion:

as can be seen from the data in tables 4 and 5, the complex microbial inoculum consisting of the phosphate solubilizing bacteria P30 and P46 can achieve good repairing effect on lead and cadmium with different concentrations, and the repairing efficiency can reach more than 98% on average.

Example 5 stability test of heavy Metal remediation microbial inoculum

Four groups of soil samples (50mg/kg CK and 50mg/kg P) repaired in example 4 are divided into a plurality of components by 2g and added into a 50mL polyethylene centrifuge tube. In order to study the influence of different environmental conditions on the improved soil components, the stability of the microorganisms for repairing the heavy metal pollution of the soil under different conditions is further discussed. First of allThe method is used for judging the influence of pH on the repair stability and comprises the following specific steps: adding 40mL of acid-base solution with different pH values of 3-9 adjusted by NaOH and HCl into a tube, shaking at 150rpm at room temperature for 18 +/-2 h, centrifuging at 4000g for 15min, and measuring Cd in the soil supernatant²⁺And (4) content. The second group is used for judging the influence of different metal cations on the repair stability, and the specific steps are as follows: to analyze pure NaNO₃，KNO₃，Ca(NO₃)₂Dissolving the medicine in deionized water to prepare 0.01mol/L Na⁺，K⁺And Ca²⁺Adding 40mL of metal cation salt solution into a bottle, shaking at 150rpm for 18 +/-2 h, centrifuging at 4000g for 15min, and measuring Cd in the soil supernatant²⁺And (4) content.

TABLE 7 Effect of PH on soil stability after remediation

TABLE 8 influence of heavy metal cations on the soil stability after remediation

The results in Table 7 show that the pH of the soil changes the formed stable Cd morphology to some extent, especially in H⁺When present in large amounts, CdS or Cd₂(PO₄)₃The generated products may be oxidized or decomposed, so that the stability of the microbial repair result is reduced, and the products are released back to the environment for the second time. In alkaline environment, the microbial repair product can still keep stable and is not easy to elute and release to the environment. Whereas the P group showed a passivation effect to reduce the activity with respect to the CK group.

From the results comparing the stability of the three metal cations to soil, Ca²⁺Cd that solutions can extract²⁺At the most, this is probably due to Ca²⁺With Cd²⁺Has the same valence, and can generate lattice exchange, coordination complex and the like to lead Cd during ion exchange²⁺The phenomenon of activation from the stable state to the exchangeable state, while the P group shows a decrease with respect to the CK groupPassivation effect of low activity.

Soil acid-base and metal cations can cause the stability of the microbial remediation result to be reduced, but the influence on the stability is small and can be ignored in practical application.

EXAMPLE 6 optimization of the culture Medium and culture conditions

Placing the P46 strain in different temperatures for shake cultivation for 48 hours, wherein the strain can grow in an environment of 10-40 ℃, and the strain grows fastest in an environment of 30-40 ℃; and (3) placing the strain in different initial pH value environments for shake cultivation for 48 hours, wherein the strain can grow in the environment with the pH value ranging from 3.5 to 8.5, and grows fastest in the environment with the pH value ranging from 6.5 to 7.5. Culturing at 37 deg.C and pH 6.8 with shaking at 120rpm, 150rpm, and 180rpm, and collecting culture solution at 12 th, 24 th, 36 th, 48 th, and 60 th positions for counting. The optimal culture conditions of the Bacillus anethoides P46 are as follows: pH: 6.8; the temperature is 37 ℃, the stirring speed is 180r/min, and the culture time is 48 h.

TABLE 9 viable count of Bacillus Neurospora P46 on nutrient media of different composition

And (4) surface note:

1. 4.0 percent of cane molasses; 2.0% of soybean meal; KH (Perkin Elmer)₂PO₄0.3％；(NH)₂SO₄0.15 percent; 0.05% of sodium citrate; MgSO (MgSO)₄·7H₂O 0.01％，MnSO₄0.05 percent; the balance of water; the pH value is 7.0-7.2.

2. 6.0 percent of corn flour; 3.5% of soybean meal; 0.3 percent of ammonium sulfate; 0.6 percent of sodium bicarbonate; 0.1 percent of calcium carbonate; pH 7.0.

According to the comprehensive analysis of the viable count and the spore count, the components of the culture medium are finally determined as follows: 6.0 percent of corn flour; 3.5% of soybean meal; 0.3 percent of ammonium sulfate; 0.6 percent of sodium bicarbonate; 0.1 percent of calcium carbonate; the balance of water; pH 7.0.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong blue Biotech Ltd

<120> Bacillus anethoides, microbial inoculum thereof and application thereof in heavy metal remediation field

<130>2010

<160>2

<170>PatentIn version 3.3

<210>1

<211>1458

<212>DNA

<213> Bacillus subtilis P4616S rDNA

<400>1

acctggcggc gtgctataca tgcaagtcga gcgaatggat taagagcttg ctcttatgaa 60

gttagcggcg gacgggtgag taacacgtgg gtaacctgcc cataagactg ggataactcc 120

gggaaaccgg ggctaatacc ggataacatt ttgaaccgca tggttcgaaa ttgaaaggcg 180

gcttcggctg tcacttatgg atggacccgc gtcgcattag ctagttggtg aggtaacggc 240

tcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact gggactgaga 300

cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct 360

gacggagcaa cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg 420

aagaacaagt gctagttgaa taagctggca ccttgacggt acctaaccag aaagccacgg 480

ctaactacgt gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg 540

ggcgtaaagc gcgcgcaggt ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt 600

ggagggtcat tggaaactgg gagacttgag tgcagaagag gaaagtggaa ttccatgtgt 660

agcggtgaaa tgcgtagaga tatggaggaa caccagtggc gaaggcgact ttctggtctg 720

taactgacac tgaggcgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 780

acgccgtaaa cgatgagtgc taagtgttag agggtttccg ccctttagtg ctgaagttaa 840

cgcattaagc actccgcctg gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg 900

ggggcccgca caagcggtgg agcatgtggt ttaatttgaa gcaacgcgaa gaaccttacc 960

aggtcttgac atcctctgac aaccctagag atagggcttc tccttcggga gcagagtgac 1020

aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080

gcgcaaccct tgatcttagt tgccatcatt aagttgggca ctctaaggtg actgccggtg 1140

acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200

acacgtgcta caatggacgg tacaaagagc tgcaagaccg cgaggtggag ctaatctcat 1260

aaaaccgttc tcagttcgga ttgtaggctg caactcgcct acatgaagct ggaatcgcta 1320

gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380

cacaccacga gagtttgtaa cacccgaagt cggtggggta acctttttgg agccagccgc 1440

ctaagggtga caaaatgg 1458

<210>2

<211>1456

<212>DNA

<213> Bacillus subtilis strain P3016S rDNA

<400>2

gcctggcggg gtgctataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt 60

agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120

aaaccggggc taataccgga tgcttgattg aaccgcatgg ttcaattata aaaggtggct 180

tttagctacc acttacagat ggacccgcgg cgcattagct agttggtgag gtaacggctc 240

accaaggcaa cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300

cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360

cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaaactctgt tgttagggaa 420

gaacaagtac cgttcgaata gggcggtacc ttgacggtac ctaaccagaa agccacggct 480

aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540

cgtaaagcgc gcgcaggcgg tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg 600

agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt ccacgtgtag 660

cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct ctggtctgta 720

actgacgctg aggcgcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgagtgcta agtgttagag ggtttccgcc ctttagtgct gcagcaaacg 840

cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960

gtcttgacat cctctgacaa ccctagagat agggcttccc cttcgggggc agagtgacag 1020

gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080

gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac tgccggtgac 1140

aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200

acgtgctaca atgggcagaa caaagggcag cgaagccgcg aggctaagcc aatcccacaa 1260

atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg aatcgctagt 1320

aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380

caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttggagc cagccgccga 1440

aggtgaacag agaggg 1456

Claims

1. A strain of Bacillus subtilis P46, which has been preserved in China general microbiological culture Collection center (CGMCC) in 3 months and 2 days of 2020, addresses as follows: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC NO. 19444.

2. The Bacillus subtilis strain P46 of claim 1, which has the 16S rDNA sequence shown in SEQ ID NO. 1.

3. The Bacillus subtilis (strain) P46 of claim 1, wherein the Bacillus subtilis powder is prepared by the following steps: inoculating the Bacillus subtilis P46 into a slant culture medium for culture; taking the cultured inclined plane, taking the strain under the aseptic condition, inoculating the strain into a seed liquid culture medium, and performing shake culture to obtain a first-stage seed liquid; carrying out amplification culture on the primary seed liquid to obtain a secondary seed liquid; and (3) taking 0.8-1.2% of inoculation amount to measure secondary seed liquid, inoculating the secondary seed liquid into a liquid fermentation culture medium, collecting fermentation liquid when the spore rate reaches more than 90%, adding light calcium carbonate, and performing spray drying to obtain bacterial powder.

4. The Bacillus subtilis strain P46 of claim 3, wherein in the slant culture, a strain loop is inoculated on a solid slant culture medium and cultured for 24-30 h at 37 ℃; or culturing the primary seeds at 37 ℃ under a shaking condition of 180rpm for 18-24 h.

5. The Bacillus subtilis (strain) P46 of claim 3, wherein the first-stage seed solution is inoculated into 500-1000 mL of seed liquid culture medium with an inoculation amount of 1% (v/v) in the enlarged culture, and the shake culture is carried out at 37 ℃ for 12-14 h to prepare a second-stage seed solution;

or in the step of spray drying, 0.67-1.00% (mass percent) of light calcium carbonate is added into the fermentation liquor.

6. The Bacillus subtilis strain P46 of claim 3, wherein the seed culture medium is formulated as follows: 4.0% molasses, 2.0% soybean meal, 0.7% K₂HPO₄、0.3％KH₂PO₄，0.15％(NH₄)₂SO₄0.05 percent of sodium citrate, 0.01 percent of magnesium sulfate and the balance of water, adjusting the pH value to 6.8, and sterilizing for 30min at the temperature of 121 ℃;

or the solid slant culture medium is an NB synthetic liquid culture medium added with 1.5-2.0% of agar powder.

7. The Bacillus subtilis strain P46 of claim 3, wherein the liquid fermentation medium formulation is: 6.0 percent of corn flour; 3.5% of soybean meal; 0.3 percent of ammonium sulfate; 0.6 percent of sodium bicarbonate; 0.1 percent of calcium carbonate; pH 7.0.

8. A bacterial agent, comprising a culture of Bacillus (Bacillus tianshanii strain) P46 and/or a bacterium according to any one of claims 1 to 7; preferably, the dosage form of the microbial inoculum is liquid microbial inoculum, powder or granules; further is water suspending agent, dispersible oil suspending agent, wettable powder or water dispersible granule; preferably, the microbial inoculum also comprises an auxiliary material acceptable in the agricultural pharmacy, and the auxiliary material acceptable in the agricultural pharmacy is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder, a defoaming agent, an antifreeze agent, a thickening agent, a filler and a solvent.

9. Use of the Bacillus subtilis P46 according to any one of claims 1 to 7 and the microbial inoculum according to claim 8 for heavy metal soil remediation.

10. A composite microbial inoculum is characterized by consisting of Bacillus subtilis P46 and Bacillus subtilis P30 or a culture thereof; the Bacillus subtilis (Bacillus subtilis strain) P30 has been preserved in China general microbiological culture Collection center (CGMCC) in 3, month and 2 of 2020, and has the address as follows: the biological preservation number of the Xilu No.1 Hospital No. 3 of the Chaojing Chaoyang district is: CGMCC NO. 19443.