CN114804371A - Bioremediation method for heavy metal polluted environment - Google Patents
Bioremediation method for heavy metal polluted environment Download PDFInfo
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 28
- 229910052793 cadmium Inorganic materials 0.000 claims abstract description 34
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- 239000007788 liquid Substances 0.000 claims description 9
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- 238000009630 liquid culture Methods 0.000 claims description 8
- 239000002689 soil Substances 0.000 claims description 8
- 239000002351 wastewater Substances 0.000 claims description 8
- 229910052802 copper Inorganic materials 0.000 claims description 6
- 229910052759 nickel Inorganic materials 0.000 claims description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 5
- 229910017052 cobalt Inorganic materials 0.000 claims description 5
- 239000010941 cobalt Substances 0.000 claims description 5
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 4
- 238000005067 remediation Methods 0.000 claims description 4
- 241000366859 Cupriavidus taiwanensis Species 0.000 claims description 3
- 239000002068 microbial inoculum Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 241000040415 Myotis adversus taiwanensis Species 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims 2
- 239000004332 silver Substances 0.000 claims 2
- 238000001179 sorption measurement Methods 0.000 abstract description 9
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- 230000000694 effects Effects 0.000 description 5
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Environmental & Geological Engineering (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Water Supply & Treatment (AREA)
- Chemical & Material Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Hydrology & Water Resources (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Soil Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention provides a bioremediation method of heavy metal polluted environment. The invention provides a Cd high-tolerance strain-Cuophilus taiwan X1, which is characterized in that heavy metal (especially Cd) in the environment is removed by contacting a strain X1 with the heavy metal polluted environment and simultaneously utilizing an extracellular adsorption and intracellular aggregation method, so that bioremediation of the heavy metal polluted environment is realized. The invention provides important strain resources for bioremediation of heavy metal polluted environments (particularly cadmium-containing sewage).
Description
Technical Field
The invention relates to a heavy metal pollution remediation technology, in particular to a bioremediation method of a heavy metal polluted environment.
Background
Cadmium (Cd) is not only a main pollutant affecting the environmental quality of agricultural soil in China, but also a main pollutant in wastewater generated in non-ferrous metal smelting, mining and chemical manufacturing. Cadmium affects the kidneys, liver and bones due to the effects of bioaccumulation, and poses a great threat to human health. Meanwhile, the high-concentration cadmium not only influences the growth of crops and reduces the yield of the crops, but also seriously threatens the quality and safety of agricultural products. In China, strict limit standards are established for Cd concentration in surface water environment quality standards (GB3838-2002, Cd of I-type water is less than or equal to 0.001mg/L), farmland irrigation water quality standards (GB5084-2021, total Cd is less than or equal to 0.01mg/L), sanitary standards for domestic drinking water (GB5749-2006, Cd is less than or equal to 0.005mg/L), underground water quality standards (GB14848-2017, Cd of I-type water is less than or equal to 0.0001mg/L) and sewage comprehensive discharge standards (GB8978-1996, total Cd is less than or equal to 0.1 mg/L).
The existing common Cd pollution remediation technologies comprise physical and chemical methods such as a redox method, an immobilization method, an ion exchange method and the like, but the methods have the problems of low efficiency, high treatment cost, easy generation of secondary pollution and the like. The method for treating Cd pollution in the environment by using a bioremediation method, particularly a microbial remediation method, is considered to be an efficient, economic and green technical method. The microbes can remove Cd pollution in the environment by using modes such as surface EPS adsorption, intracellular aggregation and the like. However, the currently screened strains have weak Cd tolerance and low removal efficiency, and are difficult to meet the requirements of actual Cd-containing sewage treatment.
Disclosure of Invention
The invention aims to provide a bioremediation method of heavy metal polluted environment.
In order to achieve the purpose, the invention provides an application of the staphyloferra X1 in heavy metal polluted environment restoration, wherein the preservation number of the staphyloferra X1(Cupriavidus taiwanensis X1) is CCTCC NO: M2010233. Strain X1 can be found in CN 111647592A.
In the present invention, the heavy metals include, but are not limited to, cadmium, copper, cobalt, nickel, manganese, preferably cadmium.
In a second aspect, the invention provides a bioremediation method of a heavy metal polluted environment, which comprises the step of contacting the environment polluted by heavy metal with the cuprophilus taiwanensis X1.
The heavy metal polluted environment refers to water or soil polluted by heavy metal.
Third aspect of the inventionThe invention provides a method for removing and enriching cadmium element (Cd) in cadmium-containing wastewater 2+ ) The method comprises adding the cuprophilus taiwanensis X1 into cadmium-containing wastewater, and performing shake culture at 16-42 ℃ under the condition of 50-200 rpm.
Preferably, the culture is carried out at 37 ℃ under 140 rpm for 72h with shaking.
The concentration of cadmium in the wastewater is 5-20mg/L, preferably 5, 10, 20 mg/L. In a fourth aspect, the invention provides a method for removing and enriching cadmium element (Cd) in cadmium-polluted soil 2+ ) The method of (4), adding a microbial inoculum or a bacterial suspension of the cuprophilus taiwanensis X1 into the cadmium-contaminated soil.
The preparation method of the bacterial suspension comprises the following steps: the bacterial quantity is 10 5 -10 8 CFU (preferably 10) 7 CFU) of the culture medium is inoculated into 100mL of liquid culture medium, and the culture medium is subjected to constant-temperature shaking culture for 8h at 37 ℃ under the condition of 140 r/min, so as to obtain the bacterial suspension.
Preferably, the liquid medium is LB medium (containing tryptone 10g/L, yeast extract 5g/L and NaCl 5 g/L).
In a fifth aspect, the invention provides an application of the cuprophilus taiwanensis X1 in preparation of a heavy metal polluted environment repairing agent.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention provides a Cd high-tolerance strain- -Cuophilus taiwan X1, which can remove Cd in the environment by using extracellular adsorption and intracellular aggregation methods simultaneously, and provides important strain resources for bioremediation of cadmium-containing sewage.
In the second gradient plate test, the maximum tolerance concentration of the strain X1 to Cd was 2.73 mM. Under liquid culture conditions, the maximum Cd tolerance concentration of strain X1 was 3 mM.
And (III) the strain X1 is crosslinked or embedded in the immobilized material, so that the tolerance of the strain X1 to the complex environment is improved, and the toxic effect of pollutants in the complex environment on the strain is reduced. The produced microbial inoculum can be directly used for repairing polluted soil and water body.
(IV) Strain X1 vs Cd 2+ Cd in water with concentration of 5, 10mg/L 2+ The removal efficiency of (a) was 70% and 47%, respectively, wherein intracellular aggregation containedThe amount is higher than the extracellular adsorption content.
And (V) the strain X1 can simultaneously resist various heavy metals, including copper, cobalt, nickel and manganese, and has stronger adaptability in actual environment.
Drawings
FIG. 1 is a scanning electron micrograph of the strain X1 of the present invention.
FIG. 2 is a graph showing the determination of the Cd tolerance concentration of strain X1 in the preferred embodiment of the present invention.
FIG. 3 is a graph showing the amount of Cd removed by the strain X1 at different Cd concentrations in the preferred embodiment of the present invention.
FIG. 4 is a graph showing the amount of Cd removed by strain X1 at different temperatures in a preferred embodiment of the invention.
FIG. 5 is a graph showing the amount of Cd removed by strain X1 at different initial bacterial loads in a preferred embodiment of the invention.
FIG. 6 is a graph showing the extracellular adsorption amount and the intracellular accumulation amount of the strain X1 at 5mg/L cadmium content in the preferred embodiment of the present invention.
FIG. 7 shows the Cd tolerance of strain X1 under liquid culture conditions in a preferred embodiment of the invention.
FIG. 8 shows the maximum concentration of other heavy metals (e.g., Cu, Co, Ni, Mn) tested by strain X1 in the preferred embodiment of the present invention.
Detailed Description
The invention provides a strain for efficiently removing and enriching cadmium in water, and the strain is used for bioremediation of a cadmium-containing water environment.
The invention adopts the following technical scheme:
the invention provides a bacterial strain for efficiently removing and enriching cadmium (Cd) in water, namely a cuprophus taiwanensis X1(Cupriavidus taiwanensis X1), which is preserved in China center for type culture collection (CCTCC NO: M2010223) with the preservation date of 9-15 months in 2010. Strain X1 can be found in CN 111647592A.
The M.taiwanensis X1 is gram-negative bacteria (figure 1), and grows rapidly on LB agar medium, and the colony is light-colored, convex, circular, complete in edge, and 0.5-1.0mm in diameter. The growth temperature range is 16-42 deg.C, pH range is 4-11, optimum temperature is 37 deg.C, and optimum pH is 7.0.
The strain X1 is a Cd high-tolerance strain, and can be used for Cd in a gradient plate test 2+ The maximum tolerated concentration of (2.73 mM).
The strain X1 can remove and enrich Cd in water by extracellular adsorption and intracellular aggregation 2+ For Cd 2+ Cd in water with concentration of 5, 10mg/L 2+ The removal efficiency of (a) was 70% and 47%, respectively, and the maximum enrichment factor was 1092 times.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Example 1 tolerance of Strain X1 to Cd
1. Placing the disposable flat plate on a glass rod at an inclination angle of about 5 degrees, pouring about 10mL of LB solid culture medium which is melted in advance, horizontally placing the flat plate after the culture medium is solidified, pouring the melted LB solid culture medium with the equal volume concentration of 10mM of cadmium chloride to fill the flat plate, and placing for one day to obtain the cadmium chloride-containing gradient flat plate.
2. Draw 100. mu.L of OD 600 A bacterial suspension of 0.8 was applied to the plate and spread evenly, the plate was incubated in an incubator at 37 ℃ and the growth of strain X1 was observed and recorded and the tolerated concentration calculated.
The result of the gradient plate shows that the Minimum Inhibitory Concentration (MIC) of cadmium to the strain X1 is 2.73 +/-0.08 mM, and the strain X1 responds to high-concentration Cd 2+ When the environment was stressed, it showed a sufficiently strong growth ability (FIG. 2).
Example 2 different Cd 2+ Effect of concentration on cadmium removal ability of Strain X1
Preparing Cd-containing materials with final concentrations of 5mg/L, 10mg/L and 20mg/L respectively 2+ LB liquid Medium, Experimental group addition of OD 1% of Total reaction volume 600 When the suspension was changed to 0.8X 1, no suspension was added to the control. Culturing the sample in constant temperature shaking table at 37 deg.C and 150r/min, sampling at 0, 12, 24, 36, 48, 72, 96, 120, 144h to extract appropriate amount of culture medium with strain X1, and centrifuging at 8000r/min with high speed freezing centrifugeCentrifuging for 10min, taking appropriate amount of supernatant, diluting with sterile deionized water, filtering with 0.22 μm water phase filter membrane, and performing flame atomic absorption spectrometry to remove Cd 2+ The concentration was measured.
The results are shown in FIG. 3, where strain X1 contained 5mg/L, 10mg/L and 20mg/L Cd 2+ Cd in LB liquid medium 2+ The maximum removal amounts of (b) are 3.5mg/L, 4.7mg/L and 3.9mg/L, respectively, corresponding to Cd 2+ The removal rates were 70%, 47%, and 19.5%, respectively.
Example 3 Effect of different temperatures on the cadmium removal ability of Strain X1
The final concentration of the prepared Cd-containing material is 5mg/L 2+ LB liquid Medium, OD addition of 1% Total reaction volume 600 The samples were activated and cultured in constant temperature shaking tables at three different temperatures of 16 ℃, 25 ℃ and 37 ℃ respectively, while the medium without the bacterial suspension was used as a blank control group, which was X1 bacterial suspension of 0.8. Sucking a certain amount of liquid culture medium for growing the strain X1 at 0, 12, 24, 36, 48, 72, 96, 120 and 144h, centrifuging for 10 minutes by using a freezing high-speed centrifuge 8000r/min, and taking supernatant to dilute with sterile deionized water. Filtering a sample by a 0.22 mu m aqueous phase filter membrane, and then using a flame atomic absorption spectrometer to remove Cd in the sample solution 2+ The concentration was measured.
As shown in FIG. 4, in LB liquid medium at a total feed concentration of 5mg/L, strain X1 was added to Cd at 16 ℃, 25 ℃ and 37 ℃ 2+ The removal amounts of (A) were 0.23, 1.99 and 3.50mg/L, respectively, and the removal rates thereof were 4.6%, 39.8% and 70%, respectively. Strain X1 on Cd at 37 DEG C 2+ The best removal effect is shown.
Example 4 Effect of different initial bacterial loads on the cadmium removal ability of Strain X1
Preparation of OD 600 Bacterial suspensions of strain X1 with values of 0.1, 0.5 and 1.0, respectively, were added to a final concentration of 5mg/L containing Cd 2+ Placing the LB liquid culture medium in a constant temperature shaking table at 37 ℃ and 150r/min for culturing, respectively sucking a proper amount of liquid culture medium containing the strain X1 in 0, 12, 24, 36, 48, 72, 96, 120 and 144h, and centrifuging at 8000r/min by using a freezing high-speed centrifugeAfter 10 minutes, the supernatant was diluted with sterile deionized water. Filtering the sample by a 0.22 mu m aqueous phase filter membrane, and then using a flame atomic absorption spectrometer to remove Cd in the sample solution 2+ Concentrations were determined and three parallel control experiments were set up for each experiment.
The results are shown in FIG. 5, where different initial inoculum sizes were applied to the growth of the strain in Cd 2+ The growth influence under the environment is small. When the amount of added bacteria is low (OD) 600 0.1), strain X1 containing 5mg/L Cd 2+ The growth speed of the LB liquid culture medium is the lowest, and the growth speed of the bacteria in the culture medium is improved along with the increase of the adding concentration.
Example 5 ability of Strain X1 to adsorb on the surface and accumulate heavy metal cadmium in the cells
Three Cd-containing materials with different concentrations of 0mg/L, 5mg/L and 20mg/L are prepared 2+ LB liquid Medium to which OD of 1% of the total reaction volume was added 600 After being suspended in 0.8X 1 bacteria, the suspension was put into a shaker at constant temperature of 150r/min at 37 ℃ for activation culture for 72 h. The cultured LB liquid medium containing the strain X1 was centrifuged at 8000r/min at 4 ℃ for 10 minutes in a refrigerated high-speed centrifuge, and the cells were collected. Resuspending and cleaning the collected thallus for three times by using sterile deionized water, and detecting Cd in the supernatant after cleaning 2+ The ion concentration. After discarding the supernatant, 10mL of 1mM EDTA buffer was added to the remaining cells, vortexed sufficiently, and then placed again in a 37 ℃ constant temperature shaker for 30 min. Centrifuging the shaken bacterial suspension at 8000r/min for 10 minutes, sucking 5mL of supernatant fluid to detect Cd 2+ Ion concentration (Cd in solution at this time) 2+ The increased amount can be regarded as the Cd adsorbed and chelated on the surface of the bacteria washing 2+ Amount of). Adding equal volume of concentrated nitric acid into the thallus, digesting the thallus at 121 deg.C for 10min with a microwave digestion instrument, diluting completely digested digestion solution with deionized water to constant volume, and detecting Cd in the solution by using a flame atomic absorption spectrometer 2+ Ion concentration (Cd after digestion) 2+ The increased amount of the Cd is regarded as the Cd adsorbed in the bacteria washing interior 2+ Amount of).
As shown in FIG. 6, the removal of heavy cadmium by the strain X1 was a result of the co-participation of extracellular adsorption and intracellular accumulation. With Cd 2+ The concentration is increased, the intracellular and extracellular adsorption capacity of the strain X1 to heavy metals is changed, and the concentration is 5mg/L Cd 2+ In the experimental group, the strain X1 is used for Cd 2+ The biological adsorption quantity and the biological accumulation quantity are respectively 1.28 plus or minus 0.08mg/g and 1.33 plus or minus 0.12 mg/g; at 20mg/L Cd 2+ In the experimental group, the strain X1 is used for Cd 2+ The bioadsorption amount and the bioaccumulation amount of (2.05. + -. 0.06 mg/g) and (3.11. + -. 0.05 mg/g) are given. At high concentration of Cd 2+ Under the stress condition of (2), the strain X1 is used for Cd 2+ The removal pattern of (a) is dominated by intracellular accumulation. At 5-20mg/L Cd 2+ Intracellular pairing of Cd in thallus in solution 2+ The enrichment factor of the product is up to 209-1092 times.
Example 6 Cd tolerance concentration test of Strain X1 under liquid culture conditions
Cd-containing compounds were prepared at final concentrations of 1mM, 2mM, 3mM, 4mM and 5mM, respectively 2+ LB liquid Medium to which OD was added in an amount of 1% of the total volume of the reaction 600 Putting the strain X1 suspension of 0.8 into a constant-temperature shaking table at 37 ℃, activating and culturing at 150r/min, and recording the strain X1 at different concentrations of Cd 2+ Growth conditions in the medium.
The results are shown in FIG. 7, strain X1 vs Cd 2+ Shows strong resistance. Cd of the strain at 3mM concentration 2+ Can still tolerate Cd in a liquid culture medium 2+ And (4) growing.
Example 7 maximum tolerant concentration test of Strain X1 for other heavy metals (e.g., copper, cobalt, nickel, manganese)
1. Placing the disposable plate on a glass rod at an inclination angle of about 5 deg., pouring about 10mL of LB solid culture medium melted in advance, placing the plate horizontally after the culture medium is solidified, and pouring melted Cu with an equal volume concentration of 10mM 2+ ,10mM Co 2+ ,10mM Ni 2+ And 40mM Mn 2+ The LB solid medium is filled with the flat plate, and after cooling, the flat plate is made into a gradient flat plate with corresponding heavy metal concentration.
2. Draw 100. mu.L of OD 600 A suspension of 0.8X 1 bacteria was applied to the plate and spread evenly, the plate was incubated in a 37 ℃ incubator, growth of strain X1 was observed and recorded and the tolerated concentration was calculated.
The results are as followsFIG. 8 shows strain X1 vs Cu 2+ ,Co 2+ ,Ni 2+ ,Mn 2+ The maximum tolerated concentration of (b) is 6.01. + -. 0.49mM, 4.68. + -. 0.35mM, 6.78. + -. 0.28mM, 17.67. + -. 1.68mM, respectively.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The application of the taiwanese cupropophila X1 in the heavy metal polluted environment remediation is that the preservation number of the taiwanese cupropophila X1(Cupriavidus taiwanensis X1) is CCTCC NO: M2010233.
2. Use according to claim 1, characterized in that the heavy metal is selected from cadmium, copper, cobalt, nickel, manganese, silver, preferably cadmium.
3. The bioremediation method of a heavy metal polluted environment is characterized in that the cuprophilus taiwanensis X1 is contacted with the heavy metal polluted environment.
4. The method according to claim 3, wherein the heavy metal is selected from cadmium, copper, cobalt, nickel, manganese, silver, preferably cadmium.
5. The method according to claim 3 or 4, wherein the environment contaminated by heavy metal is water or soil contaminated by heavy metal.
6. The method for removing and enriching cadmium elements in cadmium-containing wastewater is characterized in that the cuprophilus taiwanensis X1 is added into the cadmium-containing wastewater and is subjected to shaking culture at 16-42 ℃ under the condition of 50-200 r/min.
7. The method according to claim 6, wherein the M.taiwanensis X1 is added to the cadmium-containing wastewater and cultured under shaking at 37 ℃ and 140 rpm for 72 hours.
8. The method according to claim 6 or 7, wherein the concentration of cadmium in the wastewater is 5-20 mg/L.
9. The method for removing and enriching cadmium element in the cadmium polluted soil is characterized in that a microbial inoculum or a bacterial suspension of the taiwan copophile X1 is added into the cadmium polluted soil;
the preparation method of the bacterial suspension comprises the following steps: the bacterial quantity is 10 5 -10 8 Inoculating the CFU of the cuprophus taiwanensis X1 into 100mL of liquid culture medium, and performing constant-temperature shaking culture for 8h at 37 ℃ under the condition of 140 r/min to obtain a bacterial suspension;
preferably, the bacterial load is 10 7 CFU；
Preferably, the liquid medium is LB medium.
10. Application of staphylofenia formosana X1 in preparation of heavy metal polluted environment repairing agent.
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