CN113333460B - Method for in-situ remediation of heavy metal contaminated soil - Google Patents
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Abstract
The invention discloses a method for in-situ remediation of heavy metal contaminated soil, and relates to the technical field of soil remediation. The method utilizes enterococcus LZU-1 (with preservation number of CGMCC 22622) bacterial liquid with calcification to induce and solidify the heavy metal polluted soil such as arsenic and the like. The biological effectiveness of heavy metals such as arsenic and the like can be effectively reduced, so that the migration of the heavy metals to surrounding soil, plants, underground water and other environment media is hindered, and the soil polluted by the heavy metals is repaired in situ. The method has the advantages of simple process, convenient operation, low treatment cost, wide application range, no secondary pollution and the like.
Description
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
The release of heavy metals to the environment is aggravated in the process of urbanization, industrialization, mining industry and agricultural intensification, and more heavy metals are harmful to human health directly or indirectly through media such as atmosphere, soil, water and the like. Arsenic (As) is a toxic metalloid element, has teratogenic, carcinogenic, mutagenic properties, and pollutants such As heavy metals in soil enter human and animal bodies through food chains, and generates great toxicity to organ functions after being enriched to a certain value.
Soil is the basis of agricultural production, and for areas with drought, little rain and water resource shortage, sewage irrigation is an important method for relieving soil drought, but is also one of main sources of heavy metal pollution of soil such As As, Pb, Cd, Cr, Zn, and the like, and arsenic is ahead of the middle position of soil pollutants in a sewage irrigation area. The method has the advantages that the method is urgently needed to treat and repair the heavy metal polluted soil such As As, Pb, Cd, Cr, Zn and the like, and at least a high-efficiency, economical, practical and environment-friendly repair technology capable of reducing the biological effectiveness of the heavy metal polluted soil is found first, so that the heavy metal polluted soil is prevented from migrating to the earth surface, underground water and plant bodies.
At present, a lot of reports are related to the remediation of As-polluted soil by using a bioremediation technology, and the hyper-enriched plant ciliate desert-grass is used in a lot of arsenic-polluted areas. However, phytoremediation has certain limitations, many plants are difficult to survive in a drought harsh environment, the phytoremediation period is long, the efficiency is low, and the harvested plants may cause secondary pollution problems and the like. Therefore, the defects make the in-situ soil remediation technology which is economical, efficient, practical and free of secondary pollution urgently needed.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for in-situ remediation of heavy metal contaminated soil, which aims to effectively reduce the bioavailability of heavy metal ions and enable the heavy metal complex contaminated soil to be in-situ remedied.
The invention is realized in the following way:
the invention provides a method for in-situ remediation of heavy metal contaminated soil, which utilizes enterococcus LZU-1 bacterial liquid with calcification to carry out induction solidification on the heavy metal contaminated soil; wherein the heavy metal is at least one of As, Pb, Cd, Cr and Zn; the preservation number of the enterococcus LZU-1 is CGMCC 22622.
The invention has the following beneficial effects: the inventor creatively carries out in-situ remediation on heavy metal composite contaminated soil such As As by the enterococcus LZU-1 in an induction and solidification mode, and can effectively reduce the bioavailability of heavy metals such As arsenic, so that migration of heavy metals to surrounding soil, plants, underground water and other environment media is hindered, and the heavy metal contaminated soil is subjected to in-situ remediation.
The repairing method provided by the invention has the advantages of simple process, convenience in operation, low treatment cost, wide application range, no secondary pollution and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a graph of the growth of enterococcus LZU-1 in culture medium with different arsenic concentrations;
FIG. 2 is a schematic diagram showing the solidification effect of enterococcus LZU-1 on exchangeable arsenic in farmland soil under different arsenic pollution gradients;
FIG. 3 is a schematic diagram of the solidification effect of enterococcus LZU-1 on exchangeable-state lead in farmland soil under different lead pollution gradients.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The embodiment of the invention provides a method for in-situ remediation of heavy metal contaminated soil such as arsenic, which is characterized in that enterococcus LZU-1 is used for carrying out in-situ remediation on the arsenic contaminated soil in an induction and solidification mode, and the inventor finds that the enterococcus LZU-1 bacterial liquid with calcification can obviously reduce the biological effectiveness of the heavy metal such as arsenic, and prevent the heavy metal from migrating to the surrounding soil, plants, underground water and other environment media, so that the heavy metal contaminated soil is subjected to in-situ remediation.
The enterococcus LZU-1 strain having calcification is obtained by enrichment culture of enterococcus LZU-1 strain at a certain concentration, and secondary culture in a culture medium containing calcium source to make the strain have calcification.
The method for in-situ remediation of arsenic-contaminated soil provided by the embodiment of the invention comprises the following steps:
s1, enrichment culture
Enrichment culture comprises inoculating enterococcus LZU-1 into nutrient broth with pH of 7-7.2 for culturing to allow enterococcus LZU-1 to grow rapidly.
Specifically, the enterococcus LZU-1 used in the invention can be obtained by separating, purifying and identifying from an arid region environment through a conventional method, and the specific separation and purification method is the prior art, and can refer to the separation method of urease-producing bacteria mentioned in "effect and mechanism of mineralizing and repairing Cd and Pb polluted soil by urease-producing bacteria" by Chuihong et al. BLAST analysis of the obtained 16S rDNA gene of the ureaplasma urealyticum shows that the ureaplasma urealyticum is Enterococcus and has close genetic relationship with Enterococcus sp.123py (NCBI: txid1095537), and the similarity of the 16S rDNA gene of the ureaplasma urealyticum and the Enterococcus sp.123py reaches 99.66 percent. Therefore, the strain is named as Enterococcus sp.LZU-1, the gene sequence of the strain is submitted to GenBank to obtain the accession number of MZ 021475, meanwhile, the strain is preserved in the China general microbiological culture Collection center to obtain the preservation number of CGMCC 22622, and the preservation date is 2021, 5 and 31 days.
Further, the nutrient broth used for the enrichment culture is not limited, and an existing culture medium can be used.
In some embodiments, the nutrient broth used for enrichment culture comprises peptone, beef extract, sodium chloride, and water; the concentration of the peptone is 8-12g/L, the concentration of the beef extract is 2-4g/L, and the concentration of the sodium chloride is 4-6 g/L. By further controlling the type of the culture medium and the concentration of each component, the enterococcus LZU-1 can be rapidly enriched and grown.
Specifically, the concentration of peptone may be 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, and may be any value between adjacent concentration values; the concentration of the beef extract can be 2g/L, 3g/L, 4g/L, and can also be any value between adjacent concentration values; the concentration of sodium chloride may be 4g/L, 5g/L, 6g/L, or may be any value between adjacent concentration values.
In a preferred embodiment, the culture temperature of the enrichment culture is 25-35 ℃, and the culture time is 12-30 h; the rotation speed is kept at 100-150rpm/min during the culture process. By further controlling the culture temperature and time and matching with the components of the culture medium, a bacterium solution of enterococcus LZU-1 with larger concentration is obtained after culture.
Specifically, the culture temperature may be 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, or any value between adjacent temperature values. The incubation time may be 20h, 22h, 25h, 27h, 30h, or any value between adjacent time values.
S2, secondary culture
The secondary culture process is to inoculate the bacteria liquid after the enrichment culture into nutrient broth containing urea and calcium chloride for culture. The enterococcus LZU-1 is domesticated in the culture process to generate the self-adaptive capacity, so that the repair effect of the enterococcus LZU-1 on arsenic-polluted soil can be further improved.
Further, in the nutrient broth used for the secondary culture, the concentration of urea was 50-70g/L and the concentration of calcium chloride was 30-50 mM/L. The calcification effect can be improved by further controlling the concentration of urea and calcium chloride, and the repair effect of enterococcus LZU-1 on arsenic-polluted soil can be further improved.
Specifically, the concentration of urea can be 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, and can also be any value between adjacent concentration values; the concentration of calcium chloride may be 30mM/L, 35mM/L, or 40mM/L, or may be any value between adjacent concentration values.
In a preferred embodiment, the culture temperature of the secondary culture is 25-35 ℃, the culture time is 5-7 days, the rotation speed is kept at 100-150rpm/min during the culture process, the temperature, the time and other parameters of the secondary culture are further controlled so that the enterococcus LZU-1 has better calcification capability, and the arsenic-polluted soil is subjected to in-situ remediation in an induction solidification mode.
Specifically, the culture temperature for the secondary culture may be 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 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 calcified bacterial solution is 0.8-1.0. OD600 is a conventionally understood parameter, and OD600 is a standard method for following the growth of microorganisms in liquid culture, using a culture solution without adding a culture solution as a blank solution, and then quantifying the culture solution containing bacteria after the culture.
Further, the raw materials of the culture medium used for the enrichment culture and the secondary culture can be the same, and both comprise 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 4-6 g/L.
S3, induction curing
The induction solidification is to mix and culture the enterococcus LZU-1 bacterial liquid with calcification and arsenic contaminated soil for 15-20 days at room temperature, such as 15-25 deg.C. Arsenic in the arsenic-polluted soil can be fixed under the calcification effect of the enterococcus LZU-1 through mixed culture, and the arsenic-polluted soil is repaired in situ, so that the migration rate of the arsenic-polluted soil to surrounding soil, underground water or plant bodies is reduced.
Specifically, the culture time may be 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or any value between adjacent time values.
Furthermore, the volume of the enterococcus LZU-1 bacteria liquid corresponding to each gram of heavy metal contaminated soil is 0.3-0.6 mL. The amount of the enterococcus LZU-1 bacterial solution used is determined depending on the degree of soil contamination, 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 by the embodiment of the present invention is mainly soil contaminated by arsenic, lead, cadmium, and the like, the total amount of heavy metals in the contaminated soil is the sum of exchangeable state, carbonate bound state, iron-manganese oxide bound state, organic bound state, and residue state, the pH is 6.5 to 7.5, and arsenic is taken as the main pollutionElement with total arsenic concentration not less than 30mg/kg-1。
The features and properties of the present invention are described in further detail below with reference to examples.
The enterococcus LZU-1 used in the following examples was enterococcus LZU-1 isolated and purified from the arid region; the heavy metal contaminated soil treated in the following examples was taken from 1000m of both banks of the east China gully basin of silver, California, Gansu, and the total arsenic concentration in the contaminated soil was determined to be 30mg/kg-360 mg/kg.
Example 1
The embodiment provides a method for in-situ remediation of heavy metal contaminated soil, which comprises the following steps:
(1) enrichment culture of enterococcus LZU-1
Enterococcus LZU-1 was inoculated into nutrient broth, cultured overnight (12h) at 30 deg.C in a shaker at 130rpm, and the OD of the broth was obtained600Is 0.98. The nutrient broth culture medium comprises the following components: 1L of deionized water, 10g of peptone, 3g of beef extract and 5g of sodium chloride, and the pH is 7.0 +/-0.2.
(2) Second culturing
Adding 60g of urea and 5.8804g (40mM) of CaCl into 1L of the bacterial liquid obtained by enrichment in the step (1)2.2H2O, a repairing agent bacterial solution containing 60g/L urea and 40mM calcium chloride was obtained, and cultured for 6 days at 30 ℃ on a shaker at 130 rpm.
(3) Weighing 21g of heavy metal contaminated soil into a 100mL conical flask, adding 9mL of the repairing agent bacterial liquid obtained in the step (2) into the conical flask, uniformly mixing with the soil, and standing at room temperature of 25 ℃ for 20 days.
Example 2
The embodiment provides a method for in-situ remediation of heavy metal contaminated soil, which comprises the following steps:
(1) enrichment culture of enterococcus LZU-1
Enterococcus LZU-1 was inoculated into nutrient broth and incubated overnight (24h) at 25 ℃ on a shaker at 100 rpm. The nutrient broth culture medium comprises the following components: 1L of deionized water, 8g of peptone, 2g of beef extract and 4g of sodium chloride, and the pH is 7.0 +/-0.2.
(2) Second culturing
Adding 50g of urea and 4.41g (30mM) of CaCl into 1L of the bacterial liquid obtained by enrichment in the step (1)2.2H2O, a repairing agent-containing bacterial solution containing 60g/L urea and 40mM calcium chloride was obtained and cultured on a shaker at 30 ℃ and 100rpm for 5 days.
(3) And (3) weighing 21g of heavy metal contaminated soil into a 100mL conical flask, adding 9mL of the repairing agent bacterial liquid obtained in the step (2) into the conical flask, uniformly mixing with the soil, and standing at room temperature of 25 ℃ for 20 days.
Example 3
The embodiment provides a method for in-situ remediation of heavy metal contaminated soil, which comprises the following steps:
(1) enrichment culture of enterococcus LZU-1
Enterococcus LZU-1 was inoculated into nutrient broth and incubated overnight (30h) at 30 ℃ on a shaker at 150 rpm. The nutrient broth culture medium comprises the following components: 1L of deionized water, 12g of peptone, 4g of beef extract and 6g of sodium chloride, and the pH is 7.0 +/-0.2.
(2) Second culturing
Adding 70g of urea and 7.35g (50mM) of CaCl into 1L of the bacterial liquid obtained by enrichment in the step (1)2.2H2O, a repairing agent bacterial solution containing 60g/L urea and 40mM calcium chloride was obtained, and cultured for 7 days at 35 ℃ on a shaker at 150 rpm.
(3) Weighing 21g of heavy metal contaminated soil into a 100mL conical flask, adding 9mL of the repairing agent bacterial liquid obtained in the step (2) into the conical flask, uniformly mixing with the soil, and standing at room temperature of 25 ℃ for 20 days.
Comparative example 1
The comparative example provides a method for in-situ remediation of heavy metal contaminated soil, which is different from the method in example 1 in that: the enterococcus LZU-1 was replaced with Lysinibacillus fusiformis (Lysinibacillus fusiformis).
Comparative example 2
The comparative example provides a method for in-situ remediation of heavy metal contaminated soil, which is different from the method in example 1 in that: the enterococcus LZU-1 is replaced by a mixed bacterium of lysine bacillus fusiformis and enterococcus LZU-1.
Note: lysinibacillus fusiformis and enterococcus LZU-1 were mixed at a ratio of 1: 1.
Test example 1
Test enterococcus LZU-1 tolerance studies, the nutrient broth composition was: peptone 10g/L-1Beef paste 3g/L-15g/L of sodium chloride-1The solvent is disodium hydrogen arsenate (Na)2HAsO4) Prepared with deionized water, and the pH value is 7.0 +/-0.2.
Enterococcus LZU-1 was inoculated to a strain consisting of disodium hydrogen arsenate (Na)2HAsO4) In a nutrient broth culture medium prepared from solvents with different arsenic concentrations and prepared by deionized water, the concentration gradient of arsenic in the solvents is as follows: 0mg/L, 100mg/L, 200mg/L, 300mg/L, 400 mg/L. The cells were incubated overnight (12h) at 30 ℃ on a shaker at 130rpm and growth curves (OD600) of enterococcus LZU-1 at different arsenic concentrations are shown in FIG. 1.
As can be seen from fig. 1: the enterococcus LZU-1 can grow normally when the arsenic content is less than or equal to 400 mg/L.
Test example 2
The remediation method of example 1 was tested for remediation effects on heavy metal contaminated soil, and the test results are shown in fig. 2 and 3.
The test method comprises the following steps: in the step (3) of example 1, 21g of heavy metal contaminated soil is weighed into a 100mL conical flask in triplicate, 9mL of the remediation agent bacterial liquid obtained in the step (2) is respectively taken out of each 21g of soil, added into the conical flask, uniformly mixed with the soil, placed at room temperature of 25 ℃ for 20 days, and the change of the chemical morphology and the solidification rate of metals such As As, Pb and the like in the soil are measured. The chemical forms of the metals are extracted by a Tessier continuous extraction method, the As content of each form is detected by an atomic fluorescence spectrometer, and the content of each form of metals such As Pb is detected by an atomic flame absorption spectrometer.
Note: heavy metal (arsenic is the main pollutant) polluted soil samples are collected from different points from upstream to downstream in the range of 1000m on both banks of east China gully basin of silver city in Gansu province.
In fig. 2 and 3, the abscissa E1 to E7 are soil samples with different metal pollution gradients under natural conditions of the eastern major groove of silver, ca, kansu, and the numbers in parentheses are the total concentration (mg/kg) of As or Pb in the soil of the sample points, respectively. The histograms for the sample points in fig. 2 and 3 are, from left to right: CK-EX, BV-EX, CK-CAB, BV-CAB, respectively: the content of exchangeable As or Pb after applying the repair agent bacterial liquid without adding the enterococcus LZU-1, the content of exchangeable As or Pb after applying the repair agent bacterial liquid with adding the enterococcus LZU-1, the content of carbonate-bound As or Pb after applying the repair agent bacterial liquid without adding the enterococcus LZU-1, and the content of carbonate-bound As or Pb after applying the repair agent bacterial liquid with adding the enterococcus LZU-1.
The results in FIG. 2 show that: the concentration of exchangeable As in the soil is reduced under the action of a repairing agent bacterial liquid containing enterococcus LZU-1, urea and calcium chloride, and the solidification rate is expressed by the reduction rate of exchangeable As.
The results in FIG. 3 show that: the concentration of exchangeable Pb in the soil is reduced under the action of a repairing agent bacterial liquid containing enterococcus LZU-1, urea and calcium chloride, and the solidification rate is expressed by the reduction rate of exchangeable Pb.
Test example 3
The repairing methods in example 1 and comparative examples 1 to 2 were tested for the repairing effect on the heavy metal contaminated soil, and the test results are shown in table 1.
TABLE 1 solidification rates of exchangeable As in contaminated soil by remediation methods of example 1 and comparative examples 1 and 2
In summary, the invention provides a method for in-situ remediation of heavy metal contaminated soil such as arsenic, which utilizes the enterococcus LZU-1 bacterial liquid with calcification to induce and solidify the heavy metal contaminated soil. The biological effectiveness of heavy metals such as arsenic and the like can be effectively reduced, so that the migration of the heavy metals to surrounding soil, plants, underground water and other environment media is hindered, and the soil polluted by the heavy metals is repaired in situ. The repairing method provided by the invention has the advantages of simple process, convenience in operation, low treatment cost, wide application range, no secondary pollution and the like.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to 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.
Claims (17)
1. The method for in-situ remediation of the heavy metal contaminated soil is characterized in that the heavy metal contaminated soil is induced and solidified by using a calcified enterococcus LZU-1 bacterial liquid;
wherein the heavy metal is selected from at least one of As, Pb, Cd, Cr and Zn;
the preservation number of the enterococcus LZU-1 is CGMCC 22622.
2. The method for in-situ remediation of heavy metal contaminated soil according to claim 1, wherein the preparation process of the enterococcus LZU-1 bacterial solution with calcification comprises the following steps: and after enrichment culture is carried out on the enterococcus LZU-1, secondary culture is carried out in a culture medium containing a calcium source so as to enable the bacterial liquid to have calcification.
3. The method for in situ remediation of heavy metal contaminated soil according to claim 2, wherein said heavy metal comprises As.
4. The method for in-situ remediation of heavy metal contaminated soil according to claim 2, wherein the enrichment culture is performed by inoculating the enterococcus LZU-1 into 35 ml of nutrient broth with pH value of 7-7.2.
5. The method for in-situ remediation of heavy metal contaminated soil according to claim 4, wherein the inoculation dose of the bacterial liquid is 50 to 200 μ L.
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 ℃, and the culture time is 12-30 h.
7. The method for in situ remediation of heavy metal contaminated soil as claimed in claim 6, wherein the rotation speed is maintained at 150rpm/min during the cultivation process.
8. The method for in-situ remediation of heavy metal contaminated soil according to claim 2, wherein the secondary culture is performed by inoculating the enriched culture broth into a nutrient broth containing urea and a calcium source.
9. The method for in situ remediation of heavy metal contaminated soil according to claim 8, wherein the calcium source is calcium chloride.
10. The method for in situ remediation of heavy metal contaminated soil according to claim 8, wherein in the nutrient broth used for the secondary cultivation, the concentration of urea is 50-70g/L and the concentration of calcium chloride is 30-50 mM/L.
11. The method for in-situ remediation of heavy metal contaminated soil according to claim 10, wherein the secondary culture is carried out at a temperature of 25-35 ℃ for 5-7 days.
12. The method for in-situ remediation of heavy metal contaminated soil according to claim 11, wherein the bacterial solution with calcification has an OD600 of 0.8-1.0.
13. The method for in-situ remediation of heavy metal contaminated soil according to claim 10, wherein the raw materials of the culture medium adopted by the enrichment culture and the secondary culture comprise peptone, beef extract, sodium chloride and water.
14. The method for in-situ remediation of heavy metal contaminated soil according to claim 13, wherein the concentration of peptone is 8-12g/L, the concentration of beef extract is 2-4g/L, and the concentration of sodium chloride is 4-6 g/L.
15. The method for in-situ remediation of heavy metal contaminated soil according to claim 1, wherein the induction solidification is to mix and culture the enterococcus LZU-1 bacterial solution with calcification and heavy metal contaminated soil for 15-20 days.
16. The method for in-situ remediation of soil contaminated by heavy metals according to claim 15, wherein the volume of the enterococcus LZU-1 bacterial liquid per gram of the soil contaminated by heavy metals is 0.3-0.6 mL.
17. The method for in-situ remediation of soil contaminated by heavy metal according to claim 16, wherein the volume of the enterococcus LZU-1 bacterial liquid per gram of the soil contaminated by heavy metal is 0.45 mL.
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