1. A heavy metal tolerant microbial strain is characterized in that the preservation number of the heavy metal tolerant microbial strain is CGMCC No.22233, and the heavy metal tolerant microbial strain is identified as Pseudomonas taiwanensis (Pseudomonas taiwanensis).

2. Use of a heavy metal tolerant microbial strain according to claim 1 for precipitation hardening of Cu, Cr, Cd, Pb and Zn in heavy metal contaminated soil.

Background

Heavy metals are typical soil pollutants, have the characteristics of concealment, difficult degradation, poor mobility, easy enrichment and the like, and can influence the microbial system, ecological species and microbial process of soil, thereby influencing the structure and function of an ecological system. The heavy metal pollution of soil is the key and difficult point of ecological management of soil, and the current remediation method for the heavy metal of the soil mainly reduces the mobility and the bioavailability of the pollutants by removing or fixing the pollutants, and the traditional remediation method comprises the following steps: firstly, an engineering treatment method mainly comprising soil removal, soil turning and surface soil removal; secondly, chemical fixing technology mainly comprising precipitation, adsorption, complexation, oxidation reduction and the like of phosphates, clay minerals and organic matters; and thirdly, bioremediation mainly comprising plant extraction, fixation, volatilization and filtration. However, these remediation methods are costly, long lasting, large in work volume, and low in soil reuse. Therefore, how to carry out in-situ remediation of heavy metal contaminated sites is a hot spot field which is widely concerned at home and abroad in recent years.

Disclosure of Invention

The present invention provides a heavy metal tolerant microbial strain and its use in view of the above problems.

In order to achieve the purpose, the invention adopts the following technical scheme:

the Pseudomonas is preserved in China general microbiological culture Collection center of Committee for culture Collection of microorganisms at 23.4.2021, the preservation address is No. 3 of West Lu No. 1 of North Chen of the Chaoyang region in Beijing, the preservation number is CGMCC No.22233, and the Pseudomonas is identified as the Pseudomonas taiwanensis (Pseudomonas taiwanensis). The strain is selected from soil in cunquan copper tailing area, is rod-shaped, can produce flagella in the periphery, can move and is a gram-positive bacterium. The colony surface is rough, opaque, yellowish and aerobic. The optimal growth conditions are as follows: the pH value is 7.2-7.5, and the temperature is 30-35 ℃. The culture medium is LB culture medium (NaCl 10g/L, peptone 10g/L, yeast powder 5g/L solid culture medium also need to add agar powder 12g/L, pH7.4).

The separation method of the pseudomonas comprises the following steps:

preparing soil bacterium suspension and separating strains: sieving the sample soil sample with 100 mesh sieve, removing large blocks of sandy soil, weighing 10g of soil sample, adding sterile water containing 90mL, shaking for about 30min to uniformly mix the microorganisms in the soil with the water sample, preparing the concentration of soil suspension to be 0.1g/L, and taking the soil suspension into a super clean benchDiluting with concentration gradient, sucking 1mL of 0.1g/L soil suspension with sterilized gun head, adding into 9mL of sterile water, and diluting 10% in this way^-6。

Gradient plate configuration of heavy metal copper: 200mL of LB solid medium is prepared, simultaneously, a proper amount of heavy metal is respectively added into the prepared 100mL of liquid medium, 1.2g of agar is added, and the mixture is sterilized for 20min at 121 ℃. After sterilization, the culture dish is generally placed at a height of about 5mm on one side, about 10mL of melted medium without metal ions is poured, the culture dish is placed back to a horizontal position after solidification, then a medium containing metal ions in a first volume is poured, and the culture dish is placed for one day to obtain the heavy metal gradient plate.

Sucking 1mL of soil suspension, adding the soil suspension into a heavy metal gradient plate, coating, culturing at 37 ℃ for 24-48h, observing the growth condition of colonies, selecting colonies with different morphologies and good growth at a place with higher heavy metal concentration, and carrying out streak separation and purification on an LB solid culture medium containing heavy metals until a single colony with consistent colony characteristics is screened;

transferring the single colony obtained above to LB slant culture medium, culturing in 37 deg.C incubator for 24h, taking out all slant test tubes, and storing in 4 deg.C refrigerator. Selecting slant strains obtained after separation and purification, respectively inoculating the slant strains in LB liquid culture media containing different heavy metals (Cu, Cr, Cd, Pb and Zn), and determining strains which grow well in the LB liquid culture media containing the heavy metals as primary selection strains.

The culture solution of the primary selected strain is centrifuged, and the concentration of heavy metals in the supernatant is measured, so that the precipitation efficiency of the LQCu strain is more than 85%.

The application of a heavy metal tolerant microbial strain is used for precipitation and solidification of Cu, Cr, Cd, Pb and Zn in heavy metal contaminated soil.

Compared with the prior art, the invention has the following advantages:

1) no secondary pollution is caused; 2) the cost is low; 3) good ecological restoration effect and can promote soil fertility.

Drawings

FIG. 1 shows the heavy metal concentrations of control and spiked supernatants after incubation in liquid medium;

FIG. 2 shows the soluble heavy metal content in soil of a contaminated soil irrigation area after spraying control, kaolin, white carbon black and diatomite wettable powder;

FIG. 3 shows the soluble heavy metal content in soil of a sewage irrigation land after organic microbial agents of control, chicken manure, corn straw and turfy soil are applied;

FIG. 4 shows the content of soluble heavy metals in soil of sewage irrigation land after straw inoculants with different diameters are applied.

Detailed Description

Example 1

Preparing soil bacterium suspension and separating strains: sieving a sample soil sample by a 100-mesh sieve, removing large blocks of sandy soil, weighing 10g of the soil sample, adding sterile water containing 90mL of the soil sample, shaking for about 30min to uniformly mix microorganisms in the soil with a water sample, preparing a soil suspension with the concentration of 0.1g/L, taking the soil suspension in a super clean bench for concentration gradient dilution, sucking 1mL of 0.1g/L soil suspension by using a sterilizing gun head, adding the soil suspension into 9mL of sterile water, and diluting 10g of the soil suspension in the manner^-6。

Gradient plate configuration of heavy metal copper: 200mL of LB solid medium is prepared, simultaneously, a proper amount of heavy metal is respectively added into the prepared 100mL of liquid medium, 1.2g of agar is added, and the mixture is sterilized for 20min at 121 ℃. After sterilization, the culture dish is generally placed at a height of about 5mm on one side, about 10mL of melted medium without metal ions is poured, the culture dish is placed back to a horizontal position after solidification, then a medium containing metal ions in a first volume is poured, and the culture dish is placed for one day to obtain the heavy metal gradient plate.

Sucking 1mL of soil suspension, adding the soil suspension into a heavy metal gradient plate, coating, culturing at 37 ℃ for 24-48h, observing the growth condition of colonies, selecting colonies with different morphologies and good growth at a place with higher heavy metal concentration, and carrying out streak separation and purification on an LB solid culture medium containing heavy metals until a single colony with consistent colony characteristics is screened;

transferring the single colony obtained above to LB slant culture medium, culturing in 37 deg.C incubator for 24h, taking out all slant test tubes, and storing in 4 deg.C refrigerator. Selecting slant strains obtained after separation and purification, respectively inoculating the slant strains in LB liquid culture media containing different heavy metals (Cu, Cr, Cd, Pb and Zn), and determining strains which grow well in the LB liquid culture media containing the heavy metals as primary selection strains.

The culture solution of the primary selected strain was centrifuged, and the concentration of heavy metals in the supernatant was measured (as shown in fig. 1), and it was found that the precipitation efficiency of all LQCu strains was 85% or more.

The Pseudomonas is preserved in China general microbiological culture Collection center of Committee for culture Collection of microorganisms at 23.4.2021, the preservation address is No. 3 of West Lu No. 1 of North Chen of the Chaoyang region in Beijing, the preservation number is CGMCC No.22233, and the Pseudomonas is identified as the Pseudomonas taiwanensis (Pseudomonas taiwanensis). The strain is selected from soil in cunquan copper tailing area, is rod-shaped, can produce flagella in the periphery, can move and is a gram-positive bacterium. The colony surface is rough, opaque, yellowish and aerobic. The optimal growth conditions are as follows: the pH value is 7.2-7.5, and the temperature is 30-35 ℃.

Example 2

Influence of different formulations on soil heavy metal remediation effect of pseudomonas taiwanensis

The domesticated Pseudomonas taiwanensis (Pseudomonas taiwanensis) LQCu with high-efficiency precipitation and solidification of heavy metals is propagated in an activation culture medium, kaolin, white carbon black and diatomite are respectively used as wettable powder (the number of the Pseudomonas taiwanensis is 1012/g), the wettable powder is sprayed for 1kg per mu, and the result is shown in figure 2, and the concentration of the heavy metals leached by Cu, Cr, Cd, Pb and Zn water in the soil is reduced by about 10%.

The organic microbial inoculum (the quantity of the pseudomonas taiwanensis is 1011/g) is prepared from chicken manure, corn straws and turfy soil, and the application amount of the organic microbial inoculum per mu is 5 kg. As shown in figure 3, after the organic microbial inoculum taking chicken manure as a carrier is applied, the concentration of heavy metals leached by Cr, Cd, Pb and Zn water in soil is reduced by about 35%, and the content of leached Cu is basically not different; after the organic microbial inoculum with straw and turfy soil as carriers is applied, the concentration of heavy metals leached by Cu, Cr, Cd, Pb and Zn water in the soil is reduced by about 75%, wherein the turfy soil is reduced by about 78%, and the straw is used as the organic carrier in consideration of the suitability of materials.

Example 3

Influence of straw type and size on soil heavy metal remediation effect of pseudomonas taiwanensis

Local common straws, namely corn, sorghum and wheat straws are respectively selected as carriers and are crushed into straws with the lengths of 2mm, 5mm, 8mm and 10mm as the carriers, the quantity of the pseudomonas taiwanensis in the prepared microbial inoculum is 1011/g, and the application amount per mu is still 5 kg. The results are shown in fig. 4, and it is found that the type of the straw does not affect the restoration effect of the microbial inoculum, and the length of the straw affects the precipitation effect of the microbial inoculum on heavy metals, wherein the concentration of heavy metals leached by the Cu, Cr, Cd, Pb and Zn water in soil can be reduced by 75% by preparing the microbial inoculum with the thickness of 2mm and 5mm, and the concentration of heavy metals leached by the Cu, Cr, Cd, Pb and Zn water in soil with the thickness of 8mm and 10mm can be reduced by about 63%.

Example 4

Effect of Pseudomonas taiwanensis Agents on heavy metals in plant seeds and vegetables

The method is characterized in that a microbial inoculum prepared from 2mm straws is applied to a farmland in a certain sewage irrigation area in Taiyuan city, the quantity of monocells in the microbial inoculum is 1011/g, the application amount per mu is still 5kg, and the microbial inoculum is not applied as a reference. Corn and cabbage were planted, and the Cu, Cr, Cd, Pb and Zn contents of corn seeds and cabbage leaves were measured, respectively (as shown in Table 1), Cu (1.5mg/kg), Cr (0.07mg/kg), Cd (0.04mg/kg), Pb (0.04mg/kg) and Zn (6.5mg/kg) of cabbage leaves in the control plots, and Cu (0.35mg/kg), Cr (0.01mg/kg), Cd (0.003mg/kg), Pb (0.003mg/kg) and Zn (2.5mg/kg) of cabbage leaves in the plots to which the microbial inoculum was applied.

Cu (3.2mg/kg), Cr (0.09mg/kg), Cd (0.007mg/kg), Pb (0.03mg/kg) and Zn (12.1mg/kg) of corn seeds in the control plots, and Cu (0.95mg/kg), Cr (0.01mg/kg), Cd (0.002mg/kg), Pb (0.006mg/kg) and Zn (3.6mg/kg) of corn seeds in the plots to which the inoculant was applied.

TABLE 1 heavy metal content (mg/kg) in cabbage leaves and corn seeds in control and inoculum plots

		Cu	Cr	Cd	Pb	Zn
							Chinese cabbage	Control	1.500	0.070	0.040	0.140	6.500
	Microbial inoculum	0.350	0.010	0.003	0.030	2.500
Corn (corn)	Control	3.200	0.090	0.007	0.030	12.10
								Microbial inoculum	0.950	0.010	0.002	0.006	3.600