CN115404178A - Application of lysine bacillus in hexavalent chromium reduction - Google Patents
Application of lysine bacillus in hexavalent chromium reduction Download PDFInfo
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- CN115404178A CN115404178A CN202110597873.5A CN202110597873A CN115404178A CN 115404178 A CN115404178 A CN 115404178A CN 202110597873 A CN202110597873 A CN 202110597873A CN 115404178 A CN115404178 A CN 115404178A
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- 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
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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
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/20—Heavy metals or heavy metal compounds
- C02F2101/22—Chromium or chromium compounds, e.g. chromates
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Abstract
The invention discloses a bacterial strain Lysinibacillus sp.HST-98 with hexavalent chromium reduction capability, and the preservation number is CCTCC NO: M2021361. In the face of increasing hexavalent chromium pollution, bioreduction has proven to be an eco-friendly remediation method. The strain HST-98 can better reduce Cr (VI). The strain HST-98 grew and reduced Cr (VI) well even if the Cr (VI) concentration increased to 250 mg/L. After reaction conditions are optimized, the optimal pH value of reduction is 8-9, the optimal temperature is 36 ℃, and the optimal electron donor is sodium lactate. Cu (copper) 2+ 、Co 2+ 、Mn 2+ Iso-coexisting metal ionThe seed favors the reduction of Cr (VI) and Zn 2+ 、Ni 2+ 、Cd 2+ The coexistence of metal ions is not favorable for the reduction of Cr (VI). The reduction of HST-98 is mediated primarily by intracellular enzymes. In a word, lysinibacillus sp.HST-98 has great application potential in the field of restoration of hexavalent chromium polluted sites.
Description
Technical Field
The invention relates to an application of lysine bacillus in Cr (VI) reduction, belonging to the technical field of environmental microorganisms.
Background
The rapid development of society has led to the rapid consumption of natural resources. Heavy metals are widely used in the industrial field, but some heavy metals cause serious pollution to the environment. The half-life of heavy metals is long and the degradation is difficult. They interfere with certain functions of proteins and nucleic acids after absorption by the body. The leather tanning industry, the electroplating industry, the stainless steel industry, the dyeing industry and the pigment synthesis industry consume large amounts of chromium salts. Many industrial wastes containing chromium salts, such as sludge and sewage, are discharged directly into water, air and soil without any treatment, causing ecological damage.
Chromium is generally considered a toxic substance that can enter the human body by breathing, ingestion and direct skin contact. Cr (III) is an element required by the human body because it plays an important role in maintaining normal physiological activities. However, excessive intake is harmful to the human body. The lack of Cr (III) in food results in growth retardation, diabetic hyperinsulinemia, hypercholesterolemia and enhanced atherosclerosis. Cr (III) is present in different parts of the human body and accumulates in the hair to the highest level. Studies have shown that Cr (VI) is 100-1000 times more toxic than Cr (III). It can lead to skin ulceration, allergy and canceration. In addition, cr (VI) can bind to double-stranded ribonucleotides and interfere with their normal physiological activities, such as replication and repair. Cr (VI) readily migrates through the cell wall, which contributes to its high toxicity to the cell, and free radicals produced by the reduction process damage cellular DNA. Cr (III) is less cytotoxic to cells and may be due to its low permeability to cells. Cr (VI) is absorbed by the body and converted to Cr (III) which is then excreted in the urine, but some forms of the complex may be retained in the body for years. The plant root system can absorb and accumulate chromium compounds. In each part of the plant, the chromium accumulation is highest in the roots, followed by leaves and fruits. High concentrations of chromium can cause damage such as inhibition of plant growth, leaf chlorosis and necrosis.
Traditional chromium remediation strategies include physical and chemical methods, where physical remediation methods include adsorption, such as granular activated carbon, electrodialysis, membrane filtration, head sealing, photocatalysis, and soil washing. And the chemical repair method comprises the use of sulfur dioxide, na2S2O5, feSO4, na2SO3 and limestone. One advantage of conventional methods is the simple, fast and efficient metal recovery of these processes. The chemical method has disadvantages in that a large amount of energy and raw materials are used, resulting in excessive treatment cost and low treatment efficiency. In addition, the Cr (VI) value in the wastewater is too low to be treated by a chemical method, and secondary pollution is caused by using a large amount of chemical raw materials. The low efficiency and high energy consumption of the subsequent transport of the sludge resulting from the chemical treatment to the specific treatment plant also limits its widespread use. Bioremediation refers to a technique that utilizes organisms to reduce or detoxify harmful pollutants and convert them into harmless substances. Biological methods include the use of bacteria, fungi, algae, and plants to detoxify Cr (VI). In contrast to conventional methods, bioremediation methods have the advantage of low energy and material requirements and no secondary pollution, which makes the method more economical, environmentally friendly and safe.
Disclosure of Invention
The invention provides a strain with Cr (VI) reducing capability, which can reduce Cr (VI) into Cr (III) with much less toxicity, thereby greatly reducing the toxic effect of chromium on the environment.
The invention relates to a lysine bacillus with Cr (VI) reducing capability, which comprises the following preparation steps:
the method comprises the steps of collecting heavily polluted chromizing factory soil, removing miscellaneous stone weeds and preserving the soil by using an aseptic sampling bag.
Weighing 1g of the soil in 100ml of LB liquid medium with the final concentration of Cr (VI) of 100mg/L, and culturing for 12h.
Thirdly, diluting and coating the culture solution on an LB flat plate containing Cr (VI), selecting a single colony to test Cr (VI) tolerance capability and reducing capability according to the color and morphological characteristics of the colony, and finally obtaining the lysine bacillus with stronger Cr (VI) reducing capability. The strain is delivered to China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan, china at 12.4.2021, the preservation period is thirty years, and the strain is preserved for five years after receiving a request for providing a culture sample before the preservation period. The culture was tested by the viability collection at 19 days 4 months 2021 and was found to be viable. The name of the preservation culture is Lysinibacillus sp.HST-98, and the preservation number is CCTCC NO: M2021361.
The lysine bacillus can completely reduce 100mg/L Cr (VI) under aerobic conditions, and the reduction rate gradually decreases along with the increase of the concentration of the Cr (VI).
The maximum tolerance concentration of the lysine bacillus to Cr (VI) is 250mg/L, and the Cr (VI) reduction rate at the maximum tolerance concentration is 59%.
The optimal conditions for reducing Cr (VI) by the lysine bacillus are as follows: pH = 8-9 and temperature 36 ℃.
Among the many carbon sources tested, sodium lactate was the most suitable carbon source for B.lysii. In addition, many coexisting ions such as Cu in commonly contaminated water bodies 2+ ,Co 2+ And Mn 2+ Has effect in promoting the reduction of Cr (VI) by the bacteria, and Zn 2+ ,Ni 2+ And Cd 2+ But has the inhibiting effect on the reduction of Cr (VI) by the bacteria.
The reduction of Cr (VI) by lys bacillus is mainly intracellular enzyme mediated and the reduction products are mainly accumulated extracellularly.
Drawings
FIG. 1 shows OD of HST-98 strain at different Cr (VI) concentrations 600 。
FIG. 2 is a graph showing the effect of initial Cr (VI) concentration on the Cr (VI) reduction ratio.
FIG. 3 is a graph showing the effect of pH on Cr (VI) reduction.
FIG. 4 is a graph of the effect of temperature on Cr (VI) reduction.
FIG. 5 shows the effect of electron donors on Cr (VI) reduction.
FIG. 6 shows the effect of coexisting heavy metal ions on Cr (VI) reduction.
FIG. 7 shows the distribution of Cr in the reaction system after the reduction of Cr (VI).
FIG. 8 shows the effect of different denaturation treatments on the reduction of Cr (VI) by strain HST-98.
FIG. 9 shows the residual Cr (VI) after resting cells and CFS reduction.
FIG. 10 shows the residual Cr (VI) after CFE and CD reduction.
Detailed Description
In order to make the invention more comprehensible, specific examples are provided below as an illustration.
Example 1: separation of Cr (VI) reducing bacteria
The sample was chromized from a long-term contaminationCollected from the area surrounding the plant. To isolate the target strain, 1g of soil was added to the enrichment medium. After 12h, the medium was diluted to 10 -1 ，10 -2 ，10 -3 ，10 -4 ，10 -5 And 10 -6 The strain is smeared on an LB plate (solid culture medium contains 1.5 to 1.7 percent of agar), and the Cr (VI) is 100mg/L. Finally, the cells were incubated at 37 ℃ on a shaker. After multiple purifications, all isolates were submitted to 16S rRNA gene sequencing for identification.
Example 2: testing of Cr (VI) tolerance
The adjustment of the Cr (VI) concentration in the liquid medium is accomplished by adding a Cr (VI) mother liquor at a high concentration. Adding a certain amount of Cr (VI) mother liquor to newly prepared LB culture medium to ensure that the final concentration of Cr (VI) in the culture medium is 100mg/L. All the isolated strains were inoculated into a newly prepared liquid LB medium, respectively, and then cultured on a shaker at 37 ℃ and 200rpm until logarithmic phase. The bacterial liquid of the above isolated strain in the logarithmic phase was inoculated into LB liquid medium with Cr (VI) concentration of 100mg/L, respectively, at an inoculation ratio of 1%, and then cultured overnight. If the bacterial cells grow well after overnight culture, the strain has good tolerance to Cr (VI). On the contrary, the strain does not have good Cr (VI) tolerance. All strains with good Cr (VI) tolerance were selected for the next Cr (VI) reduction test.
Example 3: test of Cr (VI) reducing ability
The adjustment of the Cr (VI) concentration in the prepared liquid medium was carried out by adding a Cr (VI) mother liquor at a high concentration. The above-mentioned tolerant strains were tested for their reducing power at Cr (VI) concentrations of 100mg/L and 200mg/L by adding an amount of Cr (VI) mother liquor to a fresh LB medium to make the final Cr (VI) concentrations in the medium 100mg/L and 200mg/L, respectively. Then according to the inoculation ratio of 1%, the bacterial suspension of the tolerant strain in the logarithmic phase is respectively inoculated into LB culture medium with Cr (VI) concentration of 100mg/L and 200mg/L, and is placed into a shaker at 37 ℃ and 200rpm for 48h. After 48h, 1ml of the bacterial suspension was removed from the above liquid medium containing Cr (VI) to test its reducing power. The Cr (VI) concentration in the culture medium is determined by a dibenzoyl dihydrazide color development method. If the Cr (VI) concentration is reduced, the strain is proved to have Cr (VI) reduction capability. Otherwise, the isolated strain is proved not to have the Cr (VI) reduction capability. The strain with the strongest Cr (VI) reduction capability and named HST-98 is selected for the next step of experiment.
Example 4: investigation of Minimum Inhibitory Concentration (MIC)
LB liquid culture media with Cr (VI) concentration gradients of 50, 100, 150, 200 and 250mg/L respectively are prepared to search the maximum Cr (VI) tolerance of the strain with the strongest Cr (VI) reduction capability. 1ml of strain HST-98 was inoculated into LB liquid medium with the above gradient of Cr (VI) concentration, and the medium was cultured overnight on a shaker at 37 ℃ and 200 rpm. If the bacterial cells grow well after overnight culture, the strain has good tolerance to the concentration gradient of Cr (VI). Otherwise, the strain does not have good tolerance to Cr (VI) in the concentration gradient. The maximum Cr (VI) tolerance of strain HST-98 was recorded based on the growth of the cells. The results are shown in FIG. 1.
Example 5: effect of different Cr (VI) initial concentrations on hexavalent chromium reduction ratio
LB liquid culture medium with initial Cr (VI) concentrations of 50, 100, 150, 200 and 250mg/L respectively is prepared, 1ml of log phase bacterial suspension of the strain HST-98 is inoculated into the liquid culture medium, and then the liquid culture medium is placed in a shaking table at 37 ℃ and 200rpm for culture. At set time intervals, a certain amount of the bacterial suspension was removed from the above culture medium to determine the concentration of Cr (VI) remaining in the liquid medium. The Cr (VI) reduction rate at each sampling time can be calculated according to the ratio of the Cr (VI) to the initial Cr (VI) concentration. Three replicates of each set were tested. The results are shown in FIG. 2.
Example 6: effect of different pH on Cr (VI) reduction Rate
According to the results of example 5, LB liquid medium with an initial Cr (VI) concentration of 200mg/L was prepared, and the pH of the above medium was adjusted to 5, 6, 7, 8,9 and 10, respectively. Then, 1ml of log phase bacterial suspension of the strain HST-98 was inoculated into the above liquid medium, and cultured on a shaker at 37 ℃ and 200 rpm. At set time intervals, a certain amount of the bacterial suspension was removed from the above culture medium to determine the concentration of Cr (VI) remaining in the liquid medium. The Cr (VI) reduction rate at each sampling time can be calculated according to the ratio of the Cr (VI) to the initial Cr (VI) concentration. Three replicates of each set were tested. The results are shown in FIG. 3.
Example 7: effect of different temperatures on the Cr (VI) reduction Rate
According to the results of example 6, LB liquid medium with an initial Cr (VI) concentration of 200mg/L and a pH of 8 was prepared, and 1ml of a logarithmic phase bacterial suspension of the strain HST-98 was inoculated into the above liquid medium. The above culture medium was subjected to shake cultivation at 20 deg.C, 24 deg.C, 28 deg.C, 32 deg.C, 36 deg.C and 40 deg.C, respectively. At set time intervals, an amount of the bacterial suspension was removed from the culture medium to determine the residual Cr (VI) concentration in the liquid medium. The Cr (VI) reduction rate at each sampling time can be calculated according to the ratio of the Cr (VI) to the initial Cr (VI) concentration. Three replicates of each set were tested. The results are shown in FIG. 4.
Example 8: effect of different Electron donors on the Cr (VI) reduction Rate
According to the results of example 7, LB liquid medium with an initial Cr (VI) concentration of 200mg/L and a pH of 8 was prepared, and to the above liquid medium were added electron donors such as sodium lactate, sucrose, glucose, lactose, sodium pyruvate, glycerol, fructose and sodium acetate, respectively. Then, 1ml of a logarithmic phase suspension of the strain HST-98 was inoculated into the above medium and cultured on a shaker at 37 ℃ and 200 rpm. At set time intervals, a certain amount of the bacterial suspension was removed from the above culture medium to determine the concentration of Cr (VI) remaining in the liquid medium. The Cr (VI) reduction rate at each sampling time can be calculated according to the ratio of the Cr (VI) to the initial Cr (VI) concentration. Three replicates of each set were tested. The results are shown in FIG. 5.
Example 9: influence of coexisting ions of different metals on the reduction rate of Cr (VI)
According to the results of example 8, LB liquid medium with an initial Cr (VI) concentration of 200mg/L, a sodium lactate as an electron donor and a pH of 8 was prepared, and heavy metal ions Co were introduced into the medium separately 2+ ，Cd 2+ ，Cu 2+ ，Ni 2+ ，Zn 2+ And Mn 2+ . Then 1ml of the strain was addedThe logarithmic phase suspension of HST-98 was inoculated into the above medium and cultured on a shaker at 37 ℃ and 200 rpm. At set time intervals, a certain amount of the bacterial suspension was removed from the above culture medium to determine the concentration of Cr (VI) remaining in the liquid medium. The Cr (VI) reduction rate at each sampling time can be calculated according to the ratio of the Cr (VI) to the initial Cr (VI) concentration. Three replicates of each set were tested. The results are shown in FIG. 6.
Example 10: distribution of Cr
According to the results of example 8, LB liquid medium with an initial Cr (VI) concentration of 200mg/L, a sodium lactate as an electron donor and a pH of 8 was prepared, and then 1ml of a logarithmic phase suspension of the strain HST-98 was inoculated into the above medium and subjected to shake cultivation at 37 ℃ and 200 rpm. According to the set time interval, taking out quantitative bacteria liquid for analysis. Firstly, the bacterial liquid is centrifuged (4 ℃,12000rpm, 20min), and then the Cr (VI) content in the upper layer liquid is detected. Next, the bacterial pellet was washed three times. After acid digestion, the supernatant was assayed for total Cr and total Cr in the bacterial pellet by ICP-AES. Finally, the content of Cr (III) in the supernatant was obtained by the following calculation. The results are shown in FIG. 7.
Example 11: mechanism of reduction
Various denaturing measures are taken on the enzyme to determine whether the decrease in the medium is caused by reduction or adsorption. First, bacterial cells were collected in log phase by centrifugation (4 ℃,12000rpm, 25min) and 1/3 of the supernatant (CFS) was taken as a further study. The cells were washed with Tris-HCl buffer (25 mM) and then divided into three fractions. The first portion was suspended with buffer (restating cell). The second portion was also suspended in buffer and sonicated for 30min (300 w, sonication for 3 seconds, pause for 3 seconds). After centrifugation, the disrupted Cells (CD) were suspended with 25ml of washing buffer. At the same time, the remaining supernatant was made up to 25ml (CFE). Finally, all the solutions described above were tested for reducing power by adding Cr (VI) mother liquor. The results are shown in FIGS. 8,9 and 10.
Claims (2)
1. A lysine bacillus (Lysinibacillus sp. HST-98) with the number of CCTCC NO: M2021361 in the culture collection center, characterized in that the strain has the potential of reducing toxic heavy metal hexavalent chromium.
2. The use of claim 1, wherein the reducing is characterized by: culturing Lysinibacillus sp.HST-98 to logarithmic growth phase, inoculating into liquid LB culture medium according to the proportion of 1%, wherein the strain can completely reduce Cr (VI) with the concentration of 100mg/L under the aerobic condition with the pH = 8-9 and the temperature of 37 ℃; under the condition that the concentration of Cr (VI) is 250mg/L, the bacteria can also achieve 60 percent of reduction efficiency; among the many carbon sources tested, sodium lactate was the most suitable carbon source; in addition, many coexisting ions such as Cu in commonly contaminated water and soil 2+ ,Co 2+ And Mn 2+ Has promoting effect on the reduction of hexavalent chromium by the bacteria, and Zn 2+ ,Ni 2+ And Cd 2+ But has the inhibiting effect on the reduction of hexavalent chromium by the bacteria.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002281960A (en) * | 2001-03-27 | 2002-10-02 | Marine Biotechnol Inst Co Ltd | Method for reducing hexavalent chromium using microorganism |
| CN103395893A (en) * | 2013-07-29 | 2013-11-20 | 中国地质大学（武汉） | Application of Lysinibacillus sp Cr-6 |
| CN106676045A (en) * | 2017-02-16 | 2017-05-17 | 武汉科技大学 | Antibiotic-resistant hexavalent chromium reducing bacterium and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002281960A (en) * | 2001-03-27 | 2002-10-02 | Marine Biotechnol Inst Co Ltd | Method for reducing hexavalent chromium using microorganism |
| CN103395893A (en) * | 2013-07-29 | 2013-11-20 | 中国地质大学（武汉） | Application of Lysinibacillus sp Cr-6 |
| CN106676045A (en) * | 2017-02-16 | 2017-05-17 | 武汉科技大学 | Antibiotic-resistant hexavalent chromium reducing bacterium and application thereof |
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