CN113333460B - Method for in situ remediation of heavy metal contaminated soil - Google Patents

Abstract

The invention discloses a method for in-situ remediation of heavy metal polluted soil, and relates to the technical field of soil remediation. The method utilizes Enterococcus LZU-1 with calcifying effect (the deposit number is CGMCC 22622) to induce and solidify the soil contaminated with heavy metals such as arsenic. It can effectively reduce the bioavailability of heavy metals such as arsenic, so as to hinder the migration of heavy metals to the surrounding soil, plants, groundwater and other environmental media, so that the heavy metal-contaminated soil can be repaired in situ. The method has the advantages of simple process, convenient operation, low processing cost, wide application range and no secondary pollution.

Description

Method for in situ remediation of heavy metal contaminated soil

technical field

The invention relates to the technical field of soil remediation, in particular to a method for in-situ remediation of heavy metal-contaminated soil.

Background technique

The process of urbanization, industrialization, mining and agricultural intensification has intensified the release of heavy metals to the environment. More and more heavy metals directly or indirectly endanger human health through the atmosphere, soil, water and other media. Arsenic (As) is a toxic metalloid element with teratogenic, carcinogenic and mutagenic properties. Heavy metals and other pollutants in the soil enter the human body and animals through the food chain. produce very high toxicity.

Soil is the basis of agricultural production. For areas with arid, little rainfall and water shortages, sewage irrigation was once an important method to alleviate soil drought, but it is also one of the main sources of soil heavy metal pollution such as As, Pb, Cd, Cr, and Zn. , Arsenic ranks in the forefront of soil pollutants in sewage irrigation areas. We urgently need to remediate and remediate heavy metal-contaminated soils such as As, Pb, Cd, Cr, Zn, etc., at least to find an efficient, economical, and environmentally friendly remediation technology that can reduce its bioavailability and prevent its migration. into the surface, groundwater and plants.

At present, there have been many related reports on the use of bioremediation technology to remediate As-contaminated soils, and the hyperaccumulator Centipede grass has been used in many arsenic-contaminated areas. However, phytoremediation has certain limitations. Many plants are difficult to survive in arid and harsh environments, and the phytoremediation cycle is long and the efficiency is low. The harvested plants may cause secondary pollution problems. Therefore, these shortcomings make us urgently need to find an economical, efficient, practical, and non-secondary pollution-free soil in-situ remediation technology.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for in-situ remediation of heavy metal-contaminated soil, which aims to effectively reduce the bioavailability of heavy metal ions, so that the in-situ remediation of heavy metal composite contaminated soil is achieved.

The present invention is realized in this way:

The present invention provides a method for in situ repairing heavy metal contaminated soil, which utilizes Enterococcus LZU-1 bacterial solution with calcification to induce and solidify the heavy metal contaminated soil; wherein, the heavy metal is selected from As, Pb, Cd, Cr, At least one of Zn; the deposit number of Enterococcus LZU-1 is CGMCC 22622.

The invention has the following beneficial effects: the inventor creatively uses Enterococcus LZU-1 to in-situ repair heavy metal contaminated soil such as As by inducing and solidifying, which can effectively reduce the bioavailability of heavy metals such as arsenic, thereby preventing heavy metals from entering the soil. The migration of surrounding soils, plants, groundwater and other environmental media enables in-situ remediation of heavy metal-contaminated soils.

It should be noted that the repair method provided by the present invention has the advantages of simple process, convenient operation, low processing cost, wide application range, and no secondary pollution.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the growth curve of Enterococcus LZU-1 in different arsenic concentrations;

Figure 2 is a schematic diagram of the solidification effect of Enterococcus LZU-1 on exchangeable arsenic in farmland soil under different arsenic pollution gradients;

Figure 3 is a schematic diagram of the solidification effect of Enterococcus LZU-1 on exchangeable lead in farmland soil under different lead pollution gradients.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The embodiments of the present invention provide a method for in-situ repairing of heavy metal-contaminated soil such as arsenic, which utilizes Enterococcus LZU-1 to perform in-situ repair of arsenic-contaminated soil by inducing solidification. 1 The bacterial solution can significantly reduce the bioavailability of heavy metals such as arsenic, hinder the migration of heavy metals to the surrounding soil, plants, groundwater and other environmental media, so that the heavy metal-contaminated soil can be repaired in situ.

It should be noted that the Enterococcus LZU-1 bacterial liquid with calcifying effect is enriched and cultured to obtain a certain concentration of Enterococcus LZU-1 bacterial liquid, and then secondary culture is carried out in a medium containing a calcium source, so that The bacterial liquid has a calcifying effect.

The method for in-situ remediation of arsenic-contaminated soil provided in the embodiment of the present invention includes the following steps:

S1, enrichment culture

The enrichment culture is to inoculate Enterococcus LZU-1 in a nutrient broth with a pH value of 7-7.2 for culture, so that Enterococcus LZU-1 can grow rapidly.

Specifically, the Enterococcus LZU-1 used in the present invention can be isolated and purified from the arid area environment by conventional methods. The separation method of urease-producing bacteria mentioned in "Effects and Mechanisms of Remediation of Cd and Pb Contaminated Soil by Mineralization". BLAST analysis of the 16S rDNA gene of the obtained urease-producing bacteria showed that it was Enterococcus, which was closely related to Enterococcus sp.123py (NCBI: txid1095537), and their 16S rDNA gene similarity was 99.66%. Therefore, the strain was named Enterococcus sp.LZU-1, and its gene sequence was submitted to GenBank to obtain accession number MZ 021475. At the same time, it was deposited in the General Microbiology Center of China Microorganism Culture Collection and Management Committee, and the accession number was CGMCC22622. The deposit date is May 31, 2021.

Further, the nutrient broth medium used for enrichment culture is not limited, and existing medium can be used.

In some embodiments, the nutrient broth medium used in enrichment culture includes peptone, beef extract, sodium chloride and water; the concentration of peptone is 8-12g/L, the concentration of beef extract is 2-4g/L, The concentration of sodium chloride is 4-6g/L. Enterococcus LZU-1 can be rapidly enriched and grown by further controlling the type of medium and the concentration of each component.

Specifically, the concentration of peptone can be 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, or any value between adjacent concentration values; the concentration of beef extract can be 2g/L , 3g/L, 4g/L, or any value between adjacent concentration values; the concentration of sodium chloride can be 4g/L, 5g/L, 6g/L, or between adjacent concentration values any value of .

In a preferred embodiment, the culturing temperature of the enrichment culture is 25-35° C., and the culturing time is 12-30 h; the rotation speed is maintained at 100-150 rpm/min during the culturing. By further controlling the culturing temperature and time, the composition of the medium is matched to obtain the bacterial liquid of Enterococcus LZU-1 with a larger concentration after culturing.

Specifically, the culture temperature can be 25°C, 28°C, 30°C, 32°C, 35°C, or any value between adjacent temperature values. The incubation time can be 20h, 22h, 25h, 27h, 30h, or any value between adjacent time values.

S2, secondary cultivation

The process of secondary culture is to inoculate the bacterial liquid after enrichment culture in a nutrient broth containing urea and calcium chloride for culture. Enterococcus LZU-1 was acclimated during the cultivation process to generate self-adaptive ability, which could further improve the remediation effect of Enterococcus LZU-1 on arsenic-contaminated soil.

Further, in the nutrient broth used in the secondary culture, the concentration of urea is 50-70 g/L, and the concentration of calcium chloride is 30-50 mM/L. By further controlling the concentrations of urea and calcium chloride, the effect of calcification can be improved, which is beneficial to further improve the remediation effect of Enterococcus LZU-1 on arsenic-contaminated soil.

Specifically, the concentration of urea can be 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, or any value between adjacent concentration values; the concentration of calcium chloride can be 30mM/L L, 35mM/L, and 40mM/L may be any value between adjacent concentration values.

In a preferred embodiment, the culture temperature of the secondary culture is 25-35° C., the culture time is 5-7 days, and the rotational speed is maintained at 100-150 rpm/min during the culture process. By further controlling the temperature and time of the secondary culture, etc. The parameters are used to enable Enterococcus LZU-1 to have better calcification ability, and to carry out in situ remediation of arsenic-contaminated soil by inducing solidification.

Specifically, the culture temperature of the secondary culture can be 25°C, 28°C, 30°C, 32°C, 35°C, or any value between adjacent temperature values. The culture time may be 5.0 days, 5.5 days, 6.0 days, 6.5 days, 7.0 days, or any value between adjacent time values.

Specifically, the OD600 of the bacterial liquid with calcification is 0.8-1.0. OD600 is a commonly understood parameter, and OD600 is a standard method for tracking the growth of microorganisms in liquid cultures. The culture medium without bacteria solution is used as a blank solution, and then the culture solution containing bacteria after quantification is quantified.

Further, the raw materials of the medium used in the enrichment culture and the secondary culture can be the same, including peptone, beef extract, sodium chloride and water. The concentration of peptone is 8-12g/L, the concentration of beef extract is 2-4g/L, and the concentration of sodium chloride is 4-6g/L.

S3, induced curing

The induction solidification is to mix and cultivate the calcified Enterococcus LZU-1 bacterial liquid and the arsenic-contaminated soil for 15-20 days, and the culture temperature can be room temperature, such as 15-25°C. Through mixed culture, the arsenic in the arsenic-contaminated soil can be fixed under the calcification of Enterococcus LZU-1, and the arsenic-contaminated soil can be repaired in situ, thereby reducing its migration rate to the surrounding soil, groundwater or plants.

Specifically, the culture time can be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or any value between adjacent time values.

Further, the volume of Enterococcus LZU-1 bacterial solution corresponding to each gram of heavy metal-contaminated soil is 0.3-0.6 mL. The dosage of Enterococcus LZU-1 bacterial liquid is determined according to the degree of soil pollution, and may not be limited to the above range.

It should be noted that the heavy metal-contaminated soil targeted by the remediation method provided in the embodiment of the present invention is mainly polluted soil such as arsenic, lead, and cadmium, and the total amount of heavy metals in the polluted soil is exchangeable, carbonate-bound, iron and manganese The sum of oxide bound state, organic bound state and residue state, pH is between 6.5-7.5, arsenic is the main pollution element, and its total arsenic concentration can be ≥30mg·kg ^-1 .

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

It should be noted that the Enterococcus LZU-1 used in the following examples is from Enterococcus LZU-1 obtained by separation and purification in arid area environment; the heavy metal polluted soil treated in the following examples is taken from Baiyin, Gansu Province The total concentration of arsenic in the polluted soil was determined to be 30mg/kg-360mg/kg within 1000m on both sides of the Dongdagou watershed in the city.

Example 1

The present embodiment provides a method for in-situ remediation of heavy metal-contaminated soil, including:

(1) Enrichment culture of Enterococcus LZU-1

Enterococcus LZU-1 was inoculated into the nutrient broth medium, and cultured overnight (12 h) in a shaker at 30° C. and 130 rpm, and the OD ₆₀₀ of the bacterial liquid was 0.98. The formula of the nutrient broth medium is: 1 L of deionized water, 10 g of peptone, 3 g of beef extract, 5 g of sodium chloride, and the pH is 7.0±0.2.

(2) Secondary cultivation

Add 60g urea and 5.8804g (40mM) CaCl ₂ .2H ₂ O to 1L of the bacterial solution enriched in step (1) to obtain a repairing agent bacterial solution containing 60g/L urea and 40mM calcium chloride, at 30°C, Incubate for 6 days in a shaker at 130 rpm.

(3) Weigh 21 g of heavy metal contaminated soil into a 100 mL conical flask, add 9 mL of the remediation agent bacterial solution obtained in step (2) to the conical flask and mix with the soil evenly, and place it at room temperature of 25°C for 20 days .

Example 2

The present embodiment provides a method for in-situ remediation of heavy metal-contaminated soil, including:

(1) Enrichment culture of Enterococcus LZU-1

Enterococcus LZU-1 was inoculated into a nutrient broth medium and cultured overnight (24 h) in a shaker at 25°C and 100 rpm. The formula of the nutrient broth medium is: 1 L of deionized water, 8 g of peptone, 2 g of beef extract, 4 g of sodium chloride, and the pH is 7.0±0.2.

(2) Secondary culture

Add 50g urea and 4.41g (30mM) CaCl ₂ .2H ₂ O to 1L of the bacterial solution enriched in step (1) to obtain a repairing agent bacterial solution containing 60g/L urea and 40mM calcium chloride, at 30°C, Incubate for 5 days in a shaker at 100 rpm.

(3) Weigh 21 g of heavy metal contaminated soil into a 100 mL conical flask, add 9 mL of the remediation agent bacterial solution obtained in step (2) to the conical flask and mix with the soil evenly, and place it at room temperature of 25°C for 20 days .

Example 3

The present embodiment provides a method for in-situ remediation of heavy metal-contaminated soil, including:

(1) Enrichment culture of Enterococcus LZU-1

Enterococcus LZU-1 was inoculated into a nutrient broth medium and cultured overnight (30 h) in a shaker at 30°C and 150 rpm. The formula of the nutrient broth medium is: 1 L of deionized water, 12 g of peptone, 4 g of beef extract, 6 g of sodium chloride, and the pH is 7.0±0.2.

(2) Secondary culture

70g urea, 7.35g (50mM) CaCl ₂ .2H ₂ O were added to 1L of the bacterial liquid enriched in step (1) to obtain a repairing agent bacterial liquid containing 60 g/L urea and 40 mM calcium chloride. Incubate for 7 days in a shaker at 150 rpm.

(3) Weigh 21 g of heavy metal contaminated soil into a 100 mL conical flask, add 9 mL of the remediation agent bacterial solution obtained in step (2) to the conical flask and mix with the soil evenly, and place it at room temperature of 25°C for 20 days .

Comparative Example 1

This comparative example provides a method for remediating heavy metal contaminated soil in situ. The only difference from Example 1 is that Enterococcus LZU-1 is replaced with Lysinibacillus fusiformis.

Comparative Example 2

This comparative example provides a method for remediating heavy metal contaminated soil in situ. The only difference from Example 1 is that Enterococcus LZU-1 is replaced with a mixture of Bacillus fusiformis and Enterococcus LZU-1.

NOTE: Bacillus fusiformis and Enterococcus LZU-1 were mixed 1:1.

Test Example 1

To test the tolerance study of Enterococcus LZU-1, the nutrient broth is composed of: peptone 10g/L ^-1 , beef extract 3g/L ^-1 , sodium chloride 5g/L ^-1 , and the solvent is disodium hydrogen arsenate ( Na ₂ HAsO ₄ ) and deionized water, pH 7.0±0.2.

Enterococcus LZU-1 was inoculated into the nutrient broth medium prepared by different arsenic concentration solvents prepared from disodium hydrogen arsenate (Na ₂ HAsO ₄ ) and deionized water. The arsenic concentration gradient in the solvent was: 0 mg /L, 100mg/L, 200mg/L, 300mg/L, 400mg/L. The growth curve (OD600) of Enterococcus LZU-1 under different arsenic concentrations is shown in Figure 1 after cultured overnight (12h) in a shaker at 30°C and 130rpm.

It can be seen from Figure 1 that Enterococcus LZU-1 can grow normally when the bioavailable arsenic content is less than or equal to 400 mg/L.

Test Example 2

The remediation effect of the remediation method in Example 1 on heavy metal-contaminated soil was tested, and the test results are shown in FIGS. 2 and 3 .

Test method: in step (3) of Example 1, weigh 21 g of heavy metal contaminated soil into a 100 mL conical flask, in triplicate, each 21 g of soil, respectively take 9 mL of the remediation agent bacterial liquid in step (2), add to The conical flask was mixed with soil evenly, and placed at room temperature of 25°C for 20 days to measure the chemical form changes and solidification rates of As, Pb and other metals in the soil. The chemical forms of each metal were extracted by Tessier continuous extraction method, the content of As in each form was detected by atomic fluorescence spectrometer, and the content of various forms of metals such as Pb was measured by atomic flame absorption spectrometer.

Note: Heavy metal (arsenic is the main pollutant) contaminated soil samples were collected from different points from upstream to downstream within 1000m on both sides of the Dongdagou watershed in Baiyin City, Gansu Province.

In Figures 2 and 3, the abscissas E1 to E7 are the soil sampling points with different metal pollution gradients under natural conditions in Dongdagou, Baiyin City, Gansu Province. The numbers in brackets are the total concentration of As or Pb in the soil of the sampling point respectively. (mg/kg). The histograms of the sampling points in Figure 2 and Figure 3 are from left to right: CK-EX, BV-EX, CK-CAB, BV-CAB, respectively indicating that the repair agent bacteria without the addition of Enterococcus LZU-1 were applied. Exchangeable As or Pb content after the solution, exchangeable As or Pb content after applying the repair agent bacteria solution with Enterococcus LZU-1, carbonate binding after applying the repair agent bacteria solution without Enterococcus LZU-1 As or Pb content in as-state, carbonate-bound As or Pb content after application of the restoration agent bacterial solution added with Enterococcus LZU-1.

The results in Figure 2 show that the concentration of soil exchangeable As was reduced under the action of the remediation agent bacterial solution containing Enterococcus LZU-1, urea and calcium chloride, and the solidification rate was expressed as the reduction rate of exchangeable As.

The results in Fig. 3 show that the concentration of soil exchangeable Pb decreased under the action of the remediation agent bacterial solution containing Enterococcus LZU-1, urea and calcium chloride, and the solidification rate was expressed as the reduction rate of exchangeable Pb.

Test Example 3

The remediation effects of the remediation methods in Example 1 and Comparative Examples 1-2 were tested on heavy metal-contaminated soil, and the test results are shown in Table 1.

Table 1 The solidification rate of exchangeable As in polluted soil by the remediation methods of Example 1, Comparative Example 1 and Comparative Example 2

In summary, the present invention provides a method for in-situ repairing of heavy metal-contaminated soil such as arsenic, which utilizes Enterococcus LZU-1 bacterial solution with calcification to induce and solidify heavy-metal-contaminated soil. It can effectively reduce the bioavailability of heavy metals such as arsenic, so as to hinder the migration of heavy metals to the surrounding soil, plants, groundwater and other environmental media, so that the heavy metal-contaminated soil can be repaired in situ. The repairing method provided by the invention has the advantages of simple process, convenient operation, low processing cost, wide application range and no secondary pollution.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (17)

1. a method for repairing heavy metal-contaminated soil in situ, is characterized in that, it utilizes the Enterococcus LZU-1 bacterial liquid with calcification to induce and solidify heavy metal-contaminated soil; Wherein, the heavy metal is selected from at least one of As, Pb, Cd, Cr, Zn; The deposit number of Enterococcus LZU-1 is CGMCC 22622. 2. The method for in-situ repairing heavy metal-contaminated soil according to claim 1, wherein the preparation process of the Enterococcus LZU-1 bacterial liquid with calcification comprises: enriching the Enterococcus LZU-1 After the collective culture, secondary culture is performed in a medium containing a calcium source so that the bacterial solution has a calcifying effect. 3 . The method for in-situ remediation of heavy metal-contaminated soil according to claim 2 , wherein the heavy metal comprises As. 4 . 4. the method for in situ repairing heavy metal contaminated soil according to claim 2, is characterized in that, described enrichment culture is to inoculate described Enterococcus LZU-1 in the 35 ml nutrient broth that pH value is 7-7.2 cultivated in. 5. The method for in-situ repairing heavy metal-contaminated soil according to claim 4, wherein the bacterial liquid inoculation dose is 50-200 μL. 6 . The method for in situ remediation of heavy metal-contaminated soil according to claim 4 , wherein the culture temperature of the enrichment culture is 25-35° C., and the culture time is 12-30 h. 7 . 7 . The method for in-situ repairing heavy metal-contaminated soil according to claim 6 , wherein the rotation speed is maintained at 100-150 rpm/min during the culturing process. 8 . 8. the method for in situ repairing heavy metal polluted soil according to claim 2, is characterized in that, the process of described secondary culture is to inoculate the bacterial liquid after enrichment culture in the nutrient broth containing urea and calcium source to cultivate. 9 . The method for in-situ repairing heavy metal-contaminated soil according to claim 8 , wherein the calcium source is calcium chloride. 10 . 10. The method for in-situ remediation of heavy metal-contaminated soil according to claim 8, characterized in that, in the nutrient broth used in the secondary culture, the concentration of urea is 50-70 g/L, and chlorinated The calcium concentration is 30-50 mM/L. 11. The method for in-situ remediation of heavy metal-contaminated soil according to claim 10, wherein the culture temperature of the secondary culture is 25-35°C, and the culture time is 5-7 days. 12 . The method for in-situ remediation of heavy metal-contaminated soil according to claim 11 , wherein the OD600 of the bacterial solution with calcification is 0.8-1.0. 13 . 13. The method for in-situ remediation of heavy metal-contaminated soil according to claim 10, wherein the raw materials of the medium used in the enrichment culture and the secondary culture all include peptone, beef extract, sodium chloride and water. 14. The method for in-situ repairing heavy metal-contaminated soil according to claim 13, wherein the concentration of the peptone is 8-12 g/L, the concentration of the beef extract is 2-4 g/L, and the chlorine concentration is 2-4 g/L. The concentration of sodium chloride is 4-6g/L. 15. The method for in-situ remediation of heavy metal contaminated soil according to claim 1, wherein the induced solidification is a mixed culture of the Enterococcus LZU-1 bacterial liquid with calcification and the heavy metal contaminated soil for 15-20 sky. 16. The method for in-situ repairing heavy metal-contaminated soil according to claim 15, wherein the volume of each gram of the heavy metal-contaminated soil corresponding to the Enterococcus LZU-1 bacterial solution is 0.3-0.6 mL. 17. The method for in-situ repairing heavy metal-contaminated soil according to claim 16, wherein the volume of each gram of the heavy metal-contaminated soil corresponding to the Enterococcus LZU-1 bacterial solution is 0.45 mL.

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