CN107287129B - Sulfate reducing bacteria capable of settling heavy metals and application thereof - Google Patents
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Abstract
The invention relates to a sulfate reducing bacterium capable of settling heavy metals and application thereof, wherein the sulfate reducing bacterium is separated from a paddy field. The strain is preserved in China Center for Type Culture Collection (CCTCC) No. M2015243 and named as sulfate reducing bacteria SRB-L (Desulvibrio sp.SRB-L) in 19 th 4 th 2015, has good heavy metal settling property and can be used for removing heavy metal ions in soil and wastewater.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a sulfate reducing bacterium SRB-L (Desulfovibrio sp.SRB-L) capable of settling heavy metals and application thereof in removing heavy metal ions in a water body. The strain was deposited in the China center for type culture Collection on 19 th 4 th 2015 with the following addresses: china, wuhan university, zip code: 430072, telephone: (027)68754052, fax: (027)68754833, E-mail: CCTCC @ whu. edu. cn, the preservation number of which is CCTCC NO: m2015243.
Background
Sulfate-Reducing bacteria SRB (Sulfate-Reducing bacteria SRB) are a type of prokaryotic microorganisms widely existing in nature, Sulfate is used as an electron acceptor, organic acid is consumed, high-reactivity sulfide is generated, the SRB method is used for treating acid mine wastewater (AMD) by utilizing a natural sulfur circulation principle, and the method achieves the purpose of Reducing SO through the metabolic characteristics of microorganisms4 2-The purposes of improving the pH value of the wastewater and removing heavy metals are achieved.
With the rapid development of social economy, the industrialization degree of China is greatly improved, but the environmental problems along with the economic development are increasingly serious. At present, the urban domestic sewage treatment has been technically mature and applied, but the treatment of industrial wastewater, particularly wastewater containing high-concentration sulfate and heavy metal ions, is still a puzzling technical problem. At present, the waste water containing only sulfate and no COD is mainly treated by non-destructive techniques, such as chemical precipitation and electrodialysis, but the methods are not economical and are easy to generate secondary pollution. Sulfate-reducing bacteria can be used to solve this type of wastewater treatment problem. The sulfate reducing bacteria are anaerobic heterotrophic bacteria, have strong vitality and are widely present in soil, river water, sea water and other anaerobic amphibious environments caused by microbial decomposition. SRB is a kind of bacteria with diversified forms and nutrition, takes organic matters as an energy source and an electron donor for biochemical metabolism, and reduces the organic matters by taking sulfate as an electron acceptor through dissimilation. By utilizing the characteristic, the method is widely applied to the treatment of the waste water containing sulfate, the waste water containing heavy metal ions and the like. The biological precipitation effect of sulfate reducing bacteria on heavy metals in acidic wastewater is researched by the Wangfang and the like, and the obtained result shows that the sulfate reducing bacteria on Zn2+、Cu2+、Cd2+Zn when the initial ion content is 60mg/L, 40mg/L and 10mg/L respectively2+、Cu2+The precipitation rate of (2) was 99% and Cd2+The precipitation rate of (2) was 94%. Results of researches on mixed sulfate reducing bacteria and heavy metal wastewater treatment by the same by the aid of the Wanghui and the like show that Cd2+When the initial ion content is more than 10mg/L, the removal rate at the end of the reaction is less than 90%. In these studies, it is common that sulfate-reducing bacteria have poor ability to handle some common heavy metal ions. Aiming at the problem of heavy metal pollution of mines in the current stage, a sulfate reducing bacterium with the capacity of efficiently treating common heavy metal ions is urgently needed.
Disclosure of Invention
The invention relates to a sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) capable of efficiently settling heavy metal ions, which is separated from a paddy field. The culture is preserved in China center for type culture Collection in 2015 4-19 days, and the preservation number is CCTCC NO: m2015243, entitled sulfate reducing bacteria SRB-L (Desulfovibrio sp.
The method for culturing the sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) capable of settling the heavy metal comprises the following steps:
liquid culture: strain of BacillusInoculating into a culture tube containing liquid culture medium (sodium lactate 2.7ml, NH)4Cl 1g、Na2SO4 1g、MgSO4·7H2O 0.1g、K2HPO4 0.5g、Fe(NH4)2(SO 4)·7H2O 0.65g、CaCl20.1g, ascorbic acid 0.5g, H2O1000 ml, pH 7.0), the culture medium is filled, excess air is removed, the culture medium is black after standing culture at 35-37 ℃ for 7 days, and the bottle cap is opened, so that the odor of the eggs is generated.
Solid culture: the strain was spread and inoculated on a solid culture plate (solid medium: sodium lactate 2.7ml, NH)4Cl 1g、Na2SO4 1g、MgSO4·7H2O 0.1g、K2HPO4 0.5g、Fe(NH4)2(SO 4)·7H2O 0.65g、CaCl20.1g, ascorbic acid 0.5g, 2% agar, H2O1000 ml and PH 7.0), placing the inoculated solid culture plate in an anaerobic tank, placing a microaerophilic gas generating bag in the tank, quickly covering the anaerobic tank tightly, and after standing and culturing for 7 days at 35-37 ℃, the bacterial colony of the SRB on the culture medium is black and has the odor of a rotten egg.
The sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) is characterized as follows:
(1) morphological characteristics:
the thallus is rod-shaped or arc-shaped, the length of the thallus is about 1.5-3.5um, and gram-negative bacteria are obtained.
(2) The culture characteristics are as follows:
the strain is cultured on double-layer plate culture medium or single-layer microaerophilic plate culture medium at 37 deg.C for 7 days to obtain milky round colony with diameter of 1-2mm and smooth and wet colony surface, and Fe is added2+The colonies of (4) appear black in the medium. In a double-layer plate medium, colonies are embedded in the medium, and colonies on a single-layer microaerophilic plate medium are on the surface of the medium.
(3) Physiological and biochemical characteristics of the strain:
available SO of strain4 2-As a final electron acceptor, willOrganic matters are used as a carbon source and an electron donor for cell synthesis, and SO is simultaneously added4 2-Reducing the sulfur-containing compound into a sulfur-containing compound.
SRB-L (Desulfovibrio sp.) is capable of reducing sulfate metabolism to produce H2S, and H2S reacts with many heavy metals such as lead, zinc, cobalt, and copper to form lead sulfide, zinc sulfide, cobalt sulfide, and copper sulfide, and is removed.
SRB-L (Desulfovibrio sp. SRB-L) is facultative anaerobe, the oxygen demand for growth is 5% -10%, the optimal growth PH is 7, and the optimal growth temperature is 37 ℃.
(4) Genetic characteristics:
amplification and sequencing primers:
bacterial liquid PCR system:
2.5. mu.l of sulfate-reducing bacteria SRB-L DNA
10xBuffer:5μl
dNTP:4μl
TLHJ-F:0.25μl
TLHJ-R:0.25μl
Taq:0.5μl
ddH2O:37.5μl
The amount of the template added was 2. mu.l, and the amount of the template loaded was 1.1. mu.l
A forward primer: TLHJ-F5 '-AGAGTTTGATCCTGGCTCAGA-3'
Reverse primer: TLHJ-R5 '-CAACTTCATGCAGTCGAGTTG-3'
Extracting total DNA of sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L), carrying out PCR amplification on a 16s rDNA gene of the strain by using primers TLHJ-F and TLHJ-R, and carrying out sequencing by using the primers TLHJ-F and TLHJ-R, wherein the sequence of the 16s rDNA gene of the strain is shown as SEQ ID NO: 1 is shown.
The 16s rDNA gene sequence of sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) was compared to the known sequences of GenBank using BLAST software at http:// blast.ncbi. nlm. nih. gov/blast.cgi. The results show that the sequence homology of the sulfate reducing bacteria SRB-L (Desulfovibrio sp.SRB-L) and the Desulfovibrio (Desulfovibrio sp.F116S ribosomal RNA gene, partial sequence) reaches 99 percent, and the sulfate reducing bacteria SRB-L (Desulfovibrio sp.SRB-L) is proved to belong to the Desulfovibrio.
The sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) has good heavy metal settling performance. Has the capability of removing heavy metal ions in soil and wastewater. 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding 40mg/L heavy metal ion Cu2+The sedimentation rate of the nickel reaches 98 percent, and the nickel content of the nickel is 10mg/L for heavy metal ions Ni2+The sedimentation rate of the iron ore reaches 61.95 percent, and the iron ore can treat 250mg/L heavy metal ions Fe2+The sedimentation rate of the catalyst reaches 96.9 percent, and the catalyst is suitable for 70mg/L heavy metal ions Cd2+The sedimentation rate of the zinc oxide reaches 78.9 percent, and the zinc oxide can treat 70mg/L of heavy metal ions Zn2+The sedimentation rate of (2) was 97.9%.
Sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) for Cu2+、Ni2+、Fe2+、Cd2+And Zn2+Has higher precipitation efficiency, is obviously superior to the sulfate reducing bacteria reported in the prior literature, and can be used for treating water and soil with high-concentration heavy metal ions.
Drawings
FIG. 1 gram stain picture of sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L)
FIG. 2 shows the electron microscope pictures of sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L): s-3400; voltage: 5.00 kV; magnification: 13.3mm × 7.00 kSE; a scale: 5.00 mu m
The specific implementation mode is as follows:
example 1 enrichment of sulfate-reducing bacteria from soil, wastewater, Rice field
(1) Collecting a sample: when collecting soil samples, firstly digging surface soil, removing upper surface soil, collecting soil samples below 5cm of the soil surface, filling the collected soil samples into a self-sealing bag, and removing air in the self-sealing bag so as to ensure the anaerobic environment required by sulfate reducing bacteria. When collecting waste water and a sample in a paddy field, the sample is collected at the bottom of the waste water and the paddy field, preferably, the waste water and a water-mud mixture at the bottom of the paddy field are collected together, and then the mixture is filled into a bottle with a cover to exhaust the air in the bottle, so that the anaerobic state required by sulfate reducing bacteria is ensured.
(2) And (3) enriching the sample: and (3) treating the collected sample, taking a little, putting the sample into an SRB liquid culture medium, enriching the thallus, wherein the color of the liquid culture medium turns black after 7-8 days, and the odor of the stinky egg exists after a cover is opened, thus indicating that the thallus is successfully enriched.
(3) And (3) culturing thalli: diluting the successfully enriched bacterial liquid with sterile water to 10-1、10-2、10-3、10-4、10-5Coating 5 plates, placing in an anaerobic tank, placing a microaerophilic gas generating bag in the tank, quickly covering the anaerobic tank, and standing at 35-37 deg.C for 7 days to obtain black SRB colony on the culture medium with odor of egg.
(4) Picking out single bacterial colony: diluting the bacterial liquid which grows in the liquid culture medium after the colony is picked up to 10 percent by using sterile water-1、10-2、10-3、10-4、10-5And (3) coating the solution with a solid culture medium plate, placing the solution in an anaerobic tank, placing a microaerophilic gas generating bag in the tank, quickly covering the anaerobic tank tightly, and after standing and culturing at 35-37 ℃ for 7 days, enabling SRB bacterial colonies on the culture medium to be black and have the odor of rotten eggs. Picking up the colonies with the black color, putting the colonies into a liquid culture medium, and performing solid-liquid alternate culture.
(5) And (3) purification: diluting the bacterial liquid which grows in the liquid culture medium after solid-liquid alternate culture to 10 percent by using sterile water-1、10-2、10-3、10-4、10-5And (3) marking with a solid culture medium plate, placing in an anaerobic tank, placing a microaerophilic gas generating bag in the tank, quickly covering the anaerobic tank, and performing static culture at 35-37 ℃ for 7 days to obtain black SRB bacterial colonies with egg odor. Single colonies that turned black were picked.
(6) And (4) preservation: purified sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) were stored in glycerol at-80 ℃.
Example 2 characterization of sulfate-reducing bacteria
(1) And (3) observing thallus morphology and performing dyeing reaction:
inoculating sulfate reducing bacteria, culturing in a solid culture medium at constant temperature for a proper time, and performing staining microscopic examination.
Dyeing and microscopic examination results: SRB-L (Desulfovibrio sp. SRB-L) is an arc (rod) shaped bacterium, gram negative, and the length of the thallus is about 1.5-3.5 μm.
(2) And (3) identifying the sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) by adopting 16s rDNA comparison analysis:
amplification and sequencing primers:
a forward primer: TLHJ-F5 '-AGAGTTTGATCCTGGCTCAGA-3'
Reverse primer: TLHJ-R5 '-CAACTTCATGCAGTCGAGTTG-3'
Extracting total DNA of a sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) strain, carrying out PCR amplification on a 16s rDNA gene of the strain by using primers TLHJ-F and TLHJ-R, and carrying out sequencing by using the primers TLHJ-F and TLHJ-R, wherein the 16s rDNA gene sequence of the strain is shown as SEQ ID NO: 1 is shown.
The 16s rDNA gene sequence of sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) was compared to the known sequences of GenBank using BLAST software at http:// blast.ncbi. nlm. nih. gov/blast.cgi. The results showed that the sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) has 99% sequence homology with Desulfovibrio F1 strain (Desulfovibrio sp. F116S ribosomal RNA gene, partial sequence), and belongs to the genus Desulfovibrio.
Example 3 Settlement Properties of sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) for heavy Metal ions (1) Settlement Properties of sulfate-reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) for heavy Metal ions Cu2+Settling property of
Preparing copper sulfate mother liquor, adding 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid according to the concentration of 20mg/L, 30mg/L and 40mg/L, and culturing for 7 days. Atomic absorption measurement of Cu in supernatant2+And (4) content.
For Cu2+Atomic absorption measurement of
10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding 40mg/L heavy metal ion Cu2+The sedimentation rate of (2) was 98%. Compared with Cu disclosed in research on condition of optimizing heavy metal ions in sulfate reducing bacteria by response surface method of Xujianpei and the like2The maximum removal rate of 96.62% under the optimal condition is slightly higher.
(2) Sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) heavy metal ion Ni2+Settling property of
Preparing nickel sulfate mother liquor, adding 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid according to the concentration of 2mg/L, 4mg/L,6mg/L,8mg/L and 10mg/L, and culturing for 7 days. Determination of Ni in supernatant by atomic absorption2+And (4) content.
To Ni2+Atomic absorption measurement of
10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding Ni to 10mg/L heavy metal ions2+The sedimentation rate of (2) was 61.95%.
(3) Sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) heavy metal ion Fe2+Settling property of
Preparing ferrous sulfate mother liquor, and respectively adding 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid according to the concentration of 150mg/L, 200mg/L and 250 mg/L. Determination of Fe in supernatant by atomic absorption2+And (4) content.
For Fe2+Atomic absorption measurement of
10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding 250mg/L heavy metal ion Fe2+Is heavyThe reduction rate reaches 96.9 percent.
(4) Sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) heavy metal ion Cd2+Settling property of
Preparing cadmium sulfate mother liquor, adding 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid according to the concentration of 30mg/L, 50mg/L and 70mg/L, and culturing for 7 days. Determination of Cd in supernatant by atomic absorption2+And (4) content.
For Cd2+Atomic absorption measurement of
10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding 70mg/L heavy metal Cd2+The sedimentation rate of (2) reached 78.9%. Compared with the mixed sulfate reducing bacteria and the research on the treatment of heavy metal wastewater by the mixed sulfate reducing bacteria by the aqua regia and the like reported in the literature, the SRB-L bacteria disclosed by the invention have more remarkable removal efficiency of heavy metal ions, and the treatment capacity of the SRB-L (Desulfovibrio sp. SRB-L) of the sulfate reducing bacteria disclosed by the invention on heavy metals is also remarkably higher than that of the reported mixed sulfate reducing bacteria and the bacterial strains in the research literature on the treatment of heavy metal wastewater by the mixed sulfate reducing bacteria.
(5) Sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) heavy metal ion Zn2+Settling property of
Preparing zinc sulfate mother liquor, adding 10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid according to the concentration of 30mg/L, 50mg/L and 70mg/L, and culturing for 7 days. Atomic absorption determination of Zn in supernatant2+And (4) content.
For Zn2+Atomic absorption measurement of
| Sample (I) | Initial concentration-Zn2+(mg/L) | Post-precipitation concentration-Zn2+(mg/L) | Ion removal Rate (%) |
| SRB-L | 30 | 1.24 | 95.8 |
| SRB-L | 50 | 1.78 | 96.4 |
| SRB-L | 70 | 1.44 | 97.9 |
10% sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) bacterial liquid, culturing for 7 days, and adding 70mg/L heavy metal ion Zn2+The sedimentation rate of (2) was 97.9%. Compared with the research on the treatment of heavy metal ions by sulfate reducing bacteria in Huangzhi and the like reported in the literature, the sulfate reducing bacteria SRB-L (Desulfovibrio sp.SRB-L) disclosed by the invention has more remarkable removal efficiency of the heavy metal ions, and the tolerance capacity of the sulfate reducing bacteria SRB-L (Desulfovibrio sp.SRB-L) disclosed by the invention to the heavy metal is also remarkably higher than that of the strains in the research literature on the treatment of the heavy metal ions by the sulfate reducing bacteria in Huangzhi and the like.
From the above experimental results, it can be seen that sulfate-reducing bacteria SRB-L have activity against Cu2+、Ni2+、Fe2+、Cd2+And Zn2+Has higher sedimentThe precipitation efficiency is obviously superior to that of sulfate reducing bacteria reported in the existing literature, and the method can be used for treating water and soil with high-concentration heavy metal ions.
Claims (7)
1. A bacterium capable of settling heavy metals is characterized in that the bacterium is sulfate reducing bacteria SRB-L (Desulfovibrio sp. SRB-L) which is preserved in China center for type culture Collection and has the number of CCTCC NO: M2015243.
2. Use of a bacterium capable of heavy metal precipitation according to claim 1, wherein the bacterium is used for removing heavy metal ions from wastewater.
3. Use according to claim 2, characterized in that the heavy metal ion is Cu2 +。
4. Use according to claim 2, characterized in that the heavy metal ion is Ni2+。
5. The use according to claim 2, wherein the heavy metal ion is Cd2 +。
6. Use according to claim 2, wherein the heavy metal ion is Fe2 +。
7. Use according to claim 2, characterized in that the heavy metal ion is Zn2 +。
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| CN107974422A (en) * | 2017-12-22 | 2018-05-01 | 中国科学院天津工业生物技术研究所 | A kind of bacterial strain administered for heavy metal pollution of soil |
| CN109957523B (en) * | 2017-12-25 | 2021-02-23 | 有研工程技术研究院有限公司 | Oligotrophic sulfate reducing bacteria and process for restoring heavy metal pollution of bottom mud of river channel by using oligotrophic sulfate reducing bacteria |
| CN109967519B (en) * | 2017-12-28 | 2021-06-08 | 有研资源环境技术研究院(北京)有限公司 | Microbial remediation method for heavy metal pollution of tailing pond |
| WO2020029149A1 (en) * | 2018-08-09 | 2020-02-13 | 中国石油大学(北京) | Bacterial strain having very strong sulfate reduction ability and use thereof |
| CN111996133A (en) * | 2019-05-27 | 2020-11-27 | 惠博普(武汉)生物环保科技有限公司 | Method for biologically enhancing application of sulfate reducing bacteria |
| CN113118206A (en) * | 2019-12-31 | 2021-07-16 | 有研资源环境技术研究院(北京)有限公司 | Microbial remediation method for heavy metal contaminated soil of ex-service slag field of smelting plant |
| CN112940730A (en) * | 2021-01-15 | 2021-06-11 | 华北地质勘查局五一四地质大队 | Agent for repairing contaminated soil of mine heavy metal tailing pond and using method |
| CN114410559B (en) * | 2022-01-25 | 2023-07-25 | 福建农林大学 | Heavy metal-passivated geobacillus engineering strain and construction method thereof |
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