CN112538449B - Alcaligenes faecalis DY-8 and application thereof in removal of heavy metal cadmium - Google Patents
Alcaligenes faecalis DY-8 and application thereof in removal of heavy metal cadmium Download PDFInfo
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Abstract
The invention discloses an Alcaligenes faecalis DY-8 strain and application thereof in removing heavy metal cadmium, wherein the preservation number of the Alcaligenes faecalis DY-8 strain is CCTCC No: m2020786. The strain is obtained by screening and separating from the soil of the high-cadmium polluted paddy field, and the pH value of a culture medium reaches 8.25-8.75 in a stable phase. The strain is used for leaching cadmium-contaminated soil, the removal rate of weak acid cadmium, iron-manganese combined cadmium and total cadmium is 65-75%, the removal rate of organic combined cadmium is 50-60%, and the pH value of the soil is obviously improved by 1-2.5 units. The strain realizes the treatment of cadmium-polluted soil under alkaline conditions, relieves the soil acidification problem commonly existing at present while removing the content of heavy metal cadmium, and has great application prospect in the aspect of solving the soil cadmium pollution and acidification problems.
Description
Technical Field
The invention belongs to the technical field of soil heavy metal treatment by microorganisms, and particularly relates to an alcaligenes faecalis DY-8 and application thereof in efficient leaching of heavy metal cadmium in soil.
Background
In recent years, the health problem caused by heavy metal cadmium pollution is becoming more serious, and the problem becomes an environmental safety problem which is widely concerned all over the world. The farmlands in 11 provinces and 25 regions of China are polluted by Cd of different degrees, and the cadmium pollution area reaches 1300 kilohm2. Research shows that the cadmium entering farmlands in China annually reaches 1417 tons, except 178 tons output through various ways annually, 87 percent of cadmium is retained in soil, and according to the speed, the soil of all farmlands in China is expected to exceed the current standard of 0.3mg/kg within 20 years. Therefore, the problem of treating the cadmium pollution of the soil is not easy to solve.
At present, the problems of cadmium pollution in soil mainly comprise a traditional physical and chemical remediation method, a novel high-enrichment plant extraction technology and a microorganism remediation method. The microorganism is widely concerned by people due to the characteristics of species diversity, functional diversity, easy culture, high growth speed and the like, and the method for treating the cadmium pollution of the soil by utilizing the microorganism is considered to be a remediation method with wide application prospect. The microbial treatment of the cadmium-polluted soil is to remove cadmium from the soil by utilizing the direct action of microorganisms or the indirect action of generated metabolites, so that the cadmium pollution problem can be thoroughly solved, and the microbial treatment method has great advantages compared with the traditional remediation technology.
In general, soil pH is one of the most important factors affecting cadmium activity in soil, and the lower the pH, the higher the cadmium activity. Research shows that under an acidic condition, the effective cadmium content is increased by 35-37% when the pH value is reduced by one unit. And the increase of the pH value promotes the conversion of the soluble cadmium to the residue cadmium, so that the heavy metal cadmium in the soil is immobilized. Thus, the removal of cadmium from soil by lowering the soil pH is a conventional means, however this will exacerbate the soil acidification problem. The soil acidification problem is also a soil problem which is urgently needed to be solved at present. The alcaligenes is applied to the soil cadmium pollution treatment, so that cadmium can be efficiently removed under the alkaline condition, soil acidification can be relieved, and the alcaligenes has great application prospect in the aspect of solving the problems of cadmium pollution and acidification of soil.
Disclosure of Invention
The invention mainly aims to provide the Alcaligenes faecalis DY-8 and application thereof aiming at the problems and the defects in the prior art, the Alcaligenes faecalis strain not only can convert insoluble cadmium into soluble cadmium and realize the aim of quickly and efficiently removing the cadmium content in soil, but also has a remarkable improvement effect on the soil acidification problem.
The object of the present invention is achieved as follows.
An Alcaligenes faecalis DY-8 strain with a preservation number of CCTCC No: m2020786. Is preserved in China Center for Type Culture Collection (CCTCC) at 27.11.2020, with the preservation address of Wuhan university in Wuhan city, Hubei province.
The Alcaligenes faecalis DY-8 is separated from soil of a certain heavily cadmium-polluted farmland in Hunan Tan City, Hunan province, and the strain screening process is as follows:
(1) adding a fresh cadmium-polluted soil sample into sterilized deionized water in a proportion of 1% -10%, oscillating for 10-30 minutes on a vortex oscillator to obtain a microorganism suspension, and sequentially diluting 1mL of suspension to 10 times of the suspension by a gradient of 10 times-4，10-5，10-6，10-7，10-8And (4) doubling.
(2) Adding 2% (w/v) agar powder into a culture medium, sterilizing at 115 ℃ for 30 minutes, pouring a flat plate, transferring the diluted microorganism suspension onto a solid NB culture medium for coating, culturing at 25-35 ℃ for 12-48 hours, selecting a single colony, and carrying out multiple streaking to separate pure bacteria.
(3) Inoculating the strain separated from the solid culture medium into a liquid culture medium for culture to obtain a bacterial liquid, wherein the culture conditions of the strain are as follows: the temperature is 25-35 ℃, the initial pH value is the natural pH, the rotating speed is 170-180 rpm, and the culture time is 2-7 days.
The liquid culture medium is Nutrient Broth (NB) culture medium, and the culture medium comprises: 10.0g/L of tryptone, 5g/L of yeast extract powder, 10.0g/L of sodium chloride and 1000mL of deionized water, wherein the pH value is 7.0 +/-0.1.
(4) Inoculating the strain separated from the solid culture medium into a liquid culture medium, naturally adjusting the pH value, and obtaining a bacterial liquid after culturing at 30 ℃ and 180rpm for 3 days. Adding 100mL of bacterial liquid into 10g of cadmium-polluted soil, carrying out shaking for 5 days at 30 ℃ and at the rotating speed of 180rpm, comparing the leaching effect of each strain on cadmium in the soil, and screening to obtain DY-8 with the highest leaching efficiency on cadmium.
BLAST sequence alignment and homology analysis were performed, and phylogenetic trees were constructed using MEGA 7.0 software by the adjacency method. The results show that the strain DY-8 has 97% homology with the sequence of Alcaligenes faecalis.
The Alcaligenes faecalis DY-8 has a high growth speed, shows beige in a flat plate, shows a migrating shape on the surface of the strain, shows orange yellow in a liquid culture medium, and can quickly grow within the range of pH 6-8 and 25-35 ℃.
The culture medium of the strain is nutrient broth NB culture medium.
The culture conditions of the strains are as follows: the temperature is 25-35 ℃, the rotating speed is 170-180 rpm, the initial pH value is 7.0-7.5, and the culture time is 2-7 days.
The pH value of the cultured bacterial liquid is 8.05-8.85.
The invention provides application of the Alcaligenes faecalis DY-8 strain to removal of heavy metal cadmium.
The invention uses Alcaligenes faecalis (Alcaligenes faecalis) DY-8 to convert insoluble cadmium into soluble cadmium ions for leaching.
Furthermore, the method is used for leaching heavy metal cadmium in the soil. In particular to leaching weak acid cadmium, ferro-manganese combined cadmium and organic combined cadmium.
The application is to mix and leach the bacteria liquid and the cadmium contaminated soil, and then recover the leachate. The method specifically comprises the following steps:
the concentration is 1x108～1x109Adding the bacterial liquid per mL into the cadmium-polluted soil, and treating at the temperature of 25-35 ℃ and the rotating speed of 170-180 rpm; treating cadmium-contaminated soil with bacterial liquid for 10-12 hours, standing for 12-24 hours, taking out supernatant, and then adding fresh bacterial liquid again for treatmentAnd (4) repeating the treatment for 3-7 times.
The strain is obtained by screening and separating the high-cadmium polluted paddy soil, and the pH value of a culture medium reaches 8.25-8.75 in a stable phase. The strain is used for leaching cadmium-contaminated soil, the removal rate of weak acid cadmium, iron-manganese combined cadmium and total cadmium is 65-75%, the removal rate of organic combined cadmium is 50-60%, and the pH value of the soil is obviously improved by 1-2.5 units.
The Alcaligenes faecalis DY-8 provided by the invention is short in culture period, simple in culture medium components and low in cost, can quickly convert insoluble cadmium into soluble cadmium, realizes the purpose of efficiently removing the cadmium content in soil, can act under neutral or slightly alkaline conditions, has a remarkable improvement effect on the soil acidification problem, and has a wide application prospect in the aspect of soil cadmium pollution remediation.
Drawings
FIG. 1 shows the morphology of strain (Alcaligenes faecalis) DY-8 in solid and liquid media;
FIG. 2 is a phylogenetic tree of strain (Alcaligenes faecalis) DY-8;
FIG. 3 shows the change of total cadmium content in soil after the strain (Alcaligenes faecalis) DY-8 is used for treating soil.
FIG. 4 shows the change of cadmium morphological content in soil after the strain (Alcaligenes faecalis) DY-8 treatment;
FIG. 5 shows the morphological leaching rate of cadmium in soil treated by strain (Alcaligenes faecalis) DY-8.
FIG. 6 shows the change in pH of the supernatant during the soil treatment with the strain (Alcaligenes faecalis) DY-8.
Detailed Description
The invention is further illustrated by, but is not limited to, the following examples.
Example 1: separation, purification and identification of strain (Alcaligenes faecalis DY-8)
The strain is obtained by separating and purifying soil samples collected from a certain severe cadmium-polluted rice field in Hunan Tan City of Hunan province. The method comprises the following specific steps: adding 10g of fresh cadmium-contaminated soil sample into 100mL of sterilized deionized water, and placing the mixture in a vortex oscillatorThe suspension of the microorganism is obtained by shaking for 15 minutes, and 1mL of the suspension is sequentially diluted to 10 times of gradient-4，10-5，10-6，10-7，10-8And (4) doubling. Weighing 3.6g of Nutrient Broth (NB) culture medium into a conical flask, adding 4g of agar powder and 200mL of deionized water, keeping the natural pH value, sterilizing at 115 ℃ for 30 minutes, pouring a flat plate, transferring 100 microliters of microorganism suspension onto a solid culture medium for coating, culturing at 30 ℃ for 24-48 hours, and selecting a single colony for multiple streaking separation. After all isolated single cells were cultured for 1-2 days, the cells on the plates were collected and stored at-80 ℃ with 30% sterile glycerol.
Inoculating the strain separated from the solid culture medium into a liquid culture medium, naturally adjusting the pH value, rotating at 30 ℃ and 180rpm, and culturing for 3 days to obtain a bacterial liquid. Adding 100mL of bacterial liquid into 10g of cadmium-polluted soil, carrying out shaking for 5 days at 30 ℃ and at the rotating speed of 180rpm, comparing the leaching effect of each strain on cadmium in the soil, and screening to obtain DY-8 with the highest leaching efficiency on cadmium.
Strain DY-8 was picked from a fresh solid medium into a centrifuge tube, DNA was extracted using a bacterial genome extraction kit, and PCR amplification was performed using 16S universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTACCTTGTTACGACTT-3'). The PCR products were sequenced by Shanghai Biometrics, spliced using Biedit software, then uploaded to the NCBI database (https:// www.ncbi.nlm.nih.gov /), subjected to BLAST sequence alignment and homology analysis, and used to construct phylogenetic trees using MEGA 7.0 software by the neighbor method (FIG. 2). The results show that the strain DY-8 has 97% homology with the sequence of Alcaligenes faecalis.
The 16S rDNA sequencing results were as follows:
example 2 Total cadmium and effective cadmium removal Effect of Strain (Alcaligenes faecalis) DY-8 on cadmium contaminated soil
Soil samples were collected from a rice field with a certain heavy cadmium pollution in Hunan Poultan City:and (3) adopting 0-15 cm of plough layer soil, removing plant residues, fully mixing uniformly, air-drying at room temperature, grinding and sieving by a 100-mesh sieve for later use. The solid-liquid ratio is 1: 10(w/v), setting an experimental group (DY-8) and a control group (CK) in the experiment, wherein the experimental group adopts the bacterial concentration of 8 multiplied by 108each/mL of the bacterial solution, deionized water with the same pH (8.45) as the bacterial solution was added to the control group, and the leaching was performed under the same conditions, 3 replicates for each group.
Weighing 10g of the soil sample into a 250mL conical flask, respectively adding isovolumetric deionized water and bacterial liquid with the same pH value (8.45) into a control group and an experimental group, oscillating for 12h at 30 ℃ and 170rpm, taking out, standing for 12h at room temperature, taking out supernatant, adding fresh bacterial liquid and deionized water with the same volume as the first time, and repeating for 5 times. After leaching, centrifuging the soil sample to collect residues, air-drying the residues, and passing the air-dried residues through HNO3/HF/HClO4The digestion method is used for determining the total cadmium content in the soil, and the result is shown in figure 3.
The results show that: after 5 days of treatment, the total cadmium removal rate of the soil of the strain (Alcaligenes faecalis) DY-8 treatment group is 56.89% higher than that of the control group, and is 66.39% and 9.50% respectively (figure 3), which shows that the strain (Alcaligenes faecalis) DY-8 has a good removal effect on the total cadmium in the soil and has feasibility for repairing the cadmium soil.
Example 3: cadmium morphological change of cadmium-contaminated soil treated by strain (Alcaligenes faecalis) DY-8
The soil sample leached in example 2 was subjected to BCR continuous extraction to determine soil cadmium morphology, and the result is shown in fig. 4. The cadmium in the soil used in the experiment mainly exists in a weak acid state cadmium (44.44mg/kg) and a ferro-manganese combined state cadmium (31.28mg/kg), and the contents of an organic combined state (2.31mg/kg) and a residue state (0.534mg/kg) are lower. After the strain (Alcaligenes faecalis) DY-8 is used for treating five times, the contents of four cadmium morphologies in the soil of a control group and the soil of an experimental group are respectively as follows: the control group was 39.91mg/kg (weakly acidic), 28.51mg/kg (iron-manganese bound), 2.15mg/kg (organically bound), 0.520mg/kg (residual), the treated group was 14.61mg/kg (weakly acidic), 10.22mg/kg (iron-manganese bound), 0.953mg/kg (organically bound), 0.327mg/kg (residual). The cadmium removal efficiency of each form in the soil after the control group is treated for five times is respectively as follows: 10.19% (weak acid state), 8.86% (iron-manganese binding state), 6.93% (organic binding state), 2.62% (residue state); the cadmium removal efficiency of each form in the soil after the experimental group is treated for five times is respectively as follows: 67.12% (weak acid state), 67.33% (iron-manganese bound state), 58.74% (organic bound state), 38.76% (residue state), see FIG. 5. The removal efficiency of weak acid cadmium, iron-manganese combined cadmium, organic combined cadmium and residue cadmium in the experimental group after five times of treatment is obviously higher than that of the control group, and the strain (Alcaligenes faecalis) DY-8 greatly reduces the risk of bioavailable cadmium.
Example 4: pH change of supernatant during cadmium-contaminated soil treatment with the strain (Alcaligenes faecalis DY-8) was measured by pH change of supernatant during leaching in example 2, and the results are shown in FIG. 6. The pH value of the supernatant of the control group slowly rises along with the number of leaching days and is finally maintained at about 5.5; and in the process of treating soil by the strain (Alcaligenes faecalis) DY-8, the pH of the supernatant fluid is increased from 5.8 to about 8.0, which shows that the strain has a remarkable effect of improving the pH of the soil.
Sequence listing
<110> university of south-middle school
Institute of agricultural biotechnology, Hunan province
<120> Alcaligenes faecalis DY-8 and application thereof in removal of heavy metal cadmium
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggctaccttg ttacgactt 19
<210> 3
<211> 1398
<212> DNA
<213> Alcaligenes faecalis (Alcaligenes faecalis)
<400> 3
agcgccctcc ttacggttag gctacctact tctggtgaaa cccactccca tggtgtgacg 60
ggcggtgtgt acaagacccg ggaacgtatt caccgcgaca ttctgatccg cgattactag 120
cgattccgac ttcacgcagt cgagttgcag actgcgatcc ggactacgat cgggtttctg 180
agattggctc cccctcgcgg gttggcgacc ctctgtcccg accattgtat gacgtgtgaa 240
gccctaccca taagggccat gaggacttga cgtcatcccc accttcctcc ggtttgtcac 300
cggcagtctc attagagtgc tcttgcgtag caactaatga caagggttgc gctcgttgcg 360
ggacttaacc caacatctca cgacacgagc tgacgacagc catgcagcac ctgtgttccg 420
gttctcttgc gagcacggcc aaatctcttc ggctttccag acatgtcaag ggtaggtaag 480
gtttttcgcg ttgcatcgaa ttaatccaca tcatccaccg cttgtgcggg tccccgtcaa 540
ttcctttgag ttttaatctt gcgaccgtac tccccaggcg gtcaacttca cgcgttagct 600
gcgctactaa ggcctaacgg ccccaacagc tagttgacat cgtttagggc gtggactacc 660
agggtatcta atcctgtttg ctccccacgc tttcgtgtct gagcgtcagt attatcccag 720
ggggctgcct tcgccatcgg tattcctcca catatctacg catttcactg ctacacgtgg 780
aattctaccc ccctctgaca tactctagct cggcagttaa aaatgcagtt ccaaggttga 840
gccctgggat ttcacatctt tctttccgaa ccgcctacac acgctttacg cccagtaatt 900
ccgattaacg cttgcaccct acgtattacc gcggctgctg gcacgtagtt agccggtgct 960
tattctgcag ataccgtcag cagtatcccg tattagggga taccttttct tctctgccaa 1020
aagtacttta caacccgaag gccttcatca tacacgcggg atggctggat cagggtttcc 1080
cccattgtcc aaaattcccc actgctgcct cccgtaggag tctgggccgt gtctcagtcc 1140
cagtgtggct ggtcgtcctc tcaaaccagc tacggatcgt tgccttggtg agcctttacc 1200
ccaccaacta gctaatccga tatcggccgc tccaatagtg agaggtcttg cgatcccccc 1260
ctttcccccg tagggcgtat gcggtattag ccactctttc gagtagttat cccccgctac 1320
tgggcacgtt ccgatatatt actcacccgt ccgccactcg ccgccaagag agcaagctct 1380
ctcgcgctgc cgttcgac 1398
Claims (10)
1. An Alcaligenes faecalis DY-8 strain with a preservation number of CCTCC No: m2020786.
2. Alcaligenes faecalis DY-8 according to claim 1, wherein the culture medium of said strain is nutrient broth NB medium.
3. The Alcaligenes faecalis DY-8 according to claim 2, wherein said strain is cultured under the conditions: the temperature is 25-35 ℃, the rotating speed is 170-180 rpm, the initial pH value is 7.0-7.5, and the culture time is 2-7 days.
4. The Alcaligenes faecalis DY-8 according to claim 3, wherein the pH of the cultured bacterial liquid is 8.05 to 8.85.
5. Use of the strain Alcaligenes faecalis DY-8 according to any of claims 1 to 4 for the removal of the heavy metal cadmium.
6. Use according to claim 5, characterized in that leaching of the heavy metal cadmium into soluble cadmium ions is carried out by means of Alcaligenes faecalis DY-8.
7. The use according to claim 6, for leaching cadmium, a heavy metal, from soil.
8. Use according to claim 5 or 6 for leaching cadmium in weak acid, cadmium in iron manganese bound state and cadmium in organic bound state.
9. The use of claim 5, wherein the leachate is recovered after the mixed leaching treatment of the bacterial liquid and the cadmium-contaminated soil.
10. Use according to claim 9, characterized in that it comprises the following steps:
the concentration is 1x108～1x109Adding the bacterial liquid per mL into the cadmium-polluted soil, and treating at the temperature of 25-35 ℃ and the rotating speed of 170-180 rpm; and (3) treating the cadmium-polluted soil with a bacterial solution for 10-12 hours, standing for 12-24 hours, taking out a supernatant, then adding a fresh bacterial solution again for treatment, and repeating the treatment for 3-7 times.
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