CN115261372A - Solid microbial inoculum for repairing electroplating polluted site and method and application thereof - Google Patents
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Abstract
The invention discloses a solid microbial inoculum for repairing an electroplating polluted site and a method and application thereof, belonging to the technical field of microbial remediation. The preparation method of the solid microbial inoculum comprises the steps of mixing polyvinyl alcohol and sodium alginate powder, and adding activated carbon to obtain a second mixed solution; uniformly mixing a chromium reducing agent containing Microbacterium sp Cr2203-1 and Bacillus lysinibus sp Cr2203-2 with the second mixed solution, and then dripping a calcium chloride saturated boric acid solution with the concentration of 20-50% at 0-4 ℃ to embed to obtain the solid microbial agent. The flora has high chromium reduction efficiency, can completely reduce 50mg/L hexavalent chromium within 24 hours, and can reduce hexavalent chromium unaffected in the presence of 30-50mg/L nickel and 50-100mg/L copper. The method for removing hexavalent chromium in the environment by using microorganisms has the advantages of low cost, long validity period, environmental friendliness, stable effect and the like, and is suitable for treating the environment polluted by a large amount of hexavalent chromium for a long time.
Description
Technical Field
The invention belongs to the technical field of biodegradation treatment, and particularly relates to enrichment of chromium reduction flora for repairing an electroplating polluted site and application of the enrichment of chromium reduction flora to biological reduction treatment in a liquid-phase pollution system.
Background
In the past 40 years, the electroplating industry has played an important role in the economic development of china and has become an important process in the automotive, manufacturing, household appliance and electronic industries, among others. According to statistics, nearly 20000 electroplating plants exist in our country at present, more than 40% of the plants are concentrated on long triangles and bead triangles, and the most extensive processes are galvanizing, copper, nickel and chromium. However, the common electroplating enterprises in China have old equipment, backward process and low three-waste treatment rate, so that the electroplating industry generates about 4 hundred million tons of wastewater each year, wherein the wastewater contains heavy metals and 50,000 tons of solid wastes, and the electroplating plants become important heavy metal pollution sources.
Chromium is a typical heavy metal pollutant generated in the electroplating industry, has high toxicity and strong 'three-cause' effect, and belongs to one of five major heavy metals which are mainly controlled by the state. At present, the treatment of hexavalent chromium polluted environment by using microorganisms has certain development prospect. The growth activity of microorganisms is utilized, hexavalent chromium is taken as an energy source required by the production of the hexavalent chromium, and meanwhile, organic matters in the environment are taken as a carbon source, and the hexavalent chromium is reduced into trivalent chromium with lower toxicity and weaker mobility under the action of chromium reductase. Because the final metabolite has low toxicity, the cost is lower compared with the chemical treatment method, and the ecological risk of using the indigenous microorganisms in situ is low, it is significant how to achieve efficient microbial reduction of hexavalent chromium.
Meanwhile, in the electroplating polluted environment, besides high-toxicity hexavalent chromium, composite pollution of nickel and copper heavy metals also exists, and the existence of the composite pollution can inhibit the reduction of hexavalent chromium by indigenous microorganisms, so that the research on the simultaneous existence of hexavalent chromium and other heavy metals by using chromium reducing bacteria has very important significance for the efficient removal of hexavalent chromium.
In the prior art, no hexavalent chromium reducing flora capable of tolerating nickel-copper heavy metals exists at present. The main application is mainly to treat single hexavalent chromium pollution or to adsorb single heavy metal.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a solid microbial inoculum for repairing an electroplating polluted site as well as a method and application thereof.
The invention adopts the following specific technical scheme:
in a first aspect, the invention provides a preparation method of a solid microbial inoculum for repairing an electroplating polluted site, which comprises the following specific steps:
uniformly mixing the completely dissolved polyvinyl alcohol and sodium alginate powder to obtain a first mixed solution; adding activated carbon into the first mixed solution to obtain a second mixed solution; uniformly mixing a chromium reducing microbial inoculum containing Microbacterium sp Cr2203-1 and Bacillus lysinibus sp Cr2203-2 with the second mixed solution according to the volume ratio of (1-2) to (10), and then dripping a calcium chloride saturated boric acid solution with the concentration of 20-50% at the temperature of 0-4 ℃ to embed to obtain a solid microbial inoculum;
the Microbacterium Cr2203-1 is preserved in Wuhan, wuhan university, china center for type culture Collection in 2022, 13.06.13.D., with the preservation number of CCTCC No: M2022878; the lysine bacillus Cr2203-2 is preserved in Wuhan, university of Wuhan and China center for type culture Collection in 2022 at 06/13, with the preservation number of CCTCC No. M2022879.
Preferably, the polyvinyl alcohol is at 70 to 80 ℃ to achieve complete dissolution.
Preferably, the mass ratio of the polyvinyl alcohol to the sodium alginate powder is 1 (1-5).
Preferably, the concentration of the activated carbon in the second mixed solution is 3 to 5g/L.
Preferably, the content of the Cr2203-1 of the Microbacterium in the chromium reducing microbial inoculum is 3-10.2%, and the content of the Cr2203-2 of the lysine bacillus is 62-89.2%.
Preferably, the preparation method of the chromium reducing microbial inoculum comprises the following steps:
s1: adding 2g of tryptone, 1g of yeast extract and 2g of sodium chloride into 200mL of water, adjusting the pH to 7 by using HCl, increasing the pressure to 103.4kPa, maintaining the temperature at 121.3 ℃ for 15-30 minutes, and sterilizing at high temperature and high pressure to prepare a bacterial basal medium; adding 0.17-0.56g of potassium dichromate into the bacteria basal culture medium to prepare a chromium-containing culture medium containing 300-1000mg/L hexavalent chromium;
s2: adding electroplating site contaminated soil into the chromium-containing culture medium according to the proportion of 10-40g (l) 11L, culturing for 3 days at 35 ℃ and 120rpm, and taking supernatant liquid to obtain an initial inoculation source;
s3: and (3) inoculating the initial inoculation source into a bacterial basic culture medium containing 30-50mg/L of nickel, 50-100mg/L of copper and 50-300mg/L of hexavalent chromium to reduce the hexavalent chromium until the flora has the function of reducing the hexavalent chromium and the hexavalent chromium of 50-300mg/L is completely reduced, and enriching to obtain the chromium reducing microbial inoculum.
In a second aspect, the invention provides a solid microbial inoculum which is obtained by the preparation method in any one of the first aspect and can be used for repairing an electroplating pollution site.
In a third aspect, the invention provides an application of the solid microbial inoculum in the second aspect in treating contaminated soil, and specifically, the contaminated soil containing 50-300mg/L hexavalent chromium and the solid microbial inoculum are added into a bacteria basal medium under the conditions of 35 ℃ and 120 rpm.
In a fourth aspect, the invention provides an application of the solid microbial inoculum of the second aspect in treating contaminated soil, and specifically, the contaminated soil containing 50-300mg/L hexavalent chromium, 30-50mg/L nickel or 50-100mg/L copper and the solid microbial inoculum are added into a bacteria basal medium at 35 ℃ and 120 rpm.
Preferably, the bacterial basic culture medium of the third aspect or the fourth aspect is prepared by the following method:
2g of tryptone, 1g of yeast extract and 2g of sodium chloride are added into 200mL of water, the pH value is adjusted to 7 by HCl, the pressure is increased to 103.4kPa, the mixture is maintained at the temperature of 121.3 ℃ for 15 to 30 minutes, and the mixture is sterilized at high temperature and high pressure to prepare a bacterial basal medium.
Compared with the prior art, the invention has the following beneficial effects:
1) The solid microbial inoculum has high efficiency of reducing hexavalent chromium, and can completely reduce hexavalent chromium of 50mg/L within 24 hours.
2) The solid microbial inoculum can simultaneously reduce hexavalent chromium in the environment and nickel and copper in the tolerant environment.
3) Compared with the prior art, the method for removing hexavalent chromium in the environment by using the microbial solid microbial inoculum has the advantages of low cost, long validity period, stable degradation effect, environmental friendliness and the like, and is suitable for treating the environment with hexavalent chromium and nickel-copper composite pollution for a long time.
Drawings
FIG. 1 is a data graph of hexavalent chromium reduction performed by the solid microbial inoculum of the present invention;
FIG. 2 is a graph of the reduction efficiency of hexavalent chromium with different concentrations by the solid microbial inoculum according to the invention;
FIG. 3 is a hexavalent chromium reduction efficiency graph under nickel-copper mixed pollution by the solid microbial inoculum in the invention;
FIG. 4 composition classes (phylum levels) of the solid microbial inoculum and different starting hexavalent chromium concentrations of the present invention;
FIG. 5 illustrates the solid microbial inoculum and the composition classes (at levels) for different starting hexavalent chromium concentrations in the present invention;
FIG. 6 is a diagram showing the reduction efficiency of hexavalent chromium in soil under nickel-copper mixed pollution by the solid microbial inoculum.
Biological material preservation
The Microbacterium sp Cr2203-1 is preserved in Wuhan, wuhan university, china center for type culture Collection in 2022, 06 months and 13 days, with the preservation number of CCTCC No: M2022878; the Bacillus lysinate Cr2203-2 is preserved in Wuhan, wuhan university and China center for type culture Collection in 13 months at 2022, with the preservation number of CCTCC No: M2022879.
Detailed Description
The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.
In the invention, the initial inoculation source is the flora enriched in the bacterial basic culture medium containing 300-1000mg/L hexavalent chromium by utilizing the polluted soil. The contaminated soil is obtained in the following manner: collecting polluted soil (collected from a shut-down electroplating workshop of a home power machinery plant) of a waste electroplating site without leak-proof measures, culturing the polluted soil in a bacterial basic culture medium containing 300-1000mg/L hexavalent chromium for 3 days, and then inoculating the supernatant flora into the bacterial basic culture medium containing 50-300mg/L hexavalent chromium and 30-50mg/L nickel or 50-100mg/L copper until the hexavalent chromium is completely reduced, thereby obtaining the flora with stable strain content. The flora contains the following strains calculated according to the content ratio: lysine bacillus (lysine bacillus sp.) Cr2203-2 accounts for 62-89.2 percent, and Microbacterium bacillus (Microbacterium sp.) Cr2203-1 accounts for 3-10.2 percent, which are proved to have the functions of hexavalent chromium reduction and nickel copper heavy metal tolerance, and the balance is non-enrichment object mixed bacteria.
The enrichment method of flora specifically comprises the following steps:
s1: 2g tryptone, 1g yeast extract and 2g sodium chloride were added to 200mL water, pH adjusted to 7 with HCl and pressure increased to 103.4kPa (1.05 kg/cm)2) Maintaining at 121.3 deg.c for 15-30 min, high temperature and high pressure sterilizing to prepare bacteria culture medium; adding 0.17-0.56g of potassium dichromate into the bacteria basal culture medium to prepare a chromium-containing culture medium containing 300-1000mg/L hexavalent chromium;
s2: adding electroplating site contaminated soil into the chromium-containing culture medium according to the proportion of 10-40g (l) 11L, culturing for 3 days at 35 ℃ and 120rpm, and taking supernatant liquid to obtain an initial inoculation source;
s3: and (2) inoculating the initial inoculation source to a bacteria basic culture medium containing 30-50mg/L of nickel or 50-100mg/L of copper and 50-300mg/L of hexavalent chromium (namely the bacteria basic culture medium contains 50-300mg/L of hexavalent chromium and 30-50mg/L of nickel or 50-300mg/L of hexavalent chromium and 50-100mg/L of copper) to perform hexavalent chromium reduction until the flora has the function of reducing hexavalent chromium and 50-300mg/L of hexavalent chromium is completely reduced, and enriching to obtain the chromium reducing microbial inoculum.
The obtained Microbacterium Cr2203-1 is preserved in China center for type culture Collection in 2022, 06 months and 13 days, with the preservation number of CCTCC No. M2022878; the obtained lysine bacillus Cr2203-2 is preserved in China center for type culture Collection in 2022, 06 months and 13 days, and the preservation number is CCTCC No. M2022879.
Embedding the bacterial liquid formed by the flora by using sodium alginate, polyvinyl alcohol, graphite and calcium chloride saturated boric acid solution to form the solid microbial inoculum. The method comprises the following specific steps:
completely dissolving polyvinyl alcohol at 70-80 ℃, and uniformly mixing the completely dissolved polyvinyl alcohol and sodium alginate powder according to the mass ratio of 1 (1-5) to obtain a first mixed solution; adding activated carbon into the first mixed solution to obtain a second mixed solution, wherein the concentration of the activated carbon in the second mixed solution is 3-5g/L; uniformly mixing a chromium reducing microbial inoculum containing 3-10.2% of Microbacterium sp Cr2203-1 and 62-89.2% of lysine Bacillus sp Cr2203-2 with the second mixed solution according to the volume ratio of (1-2) to 10, and then dripping a calcium chloride saturated boric acid solution with the concentration of 20-50% at 0-4 ℃ to embed to obtain the solid microbial inoculum.
The deposited microbacterium Cr2203-1 and the deposited bacillus lysine Cr2203-2 are applied to a specific embodiment and are prepared into solid microbial inoculum for soil remediation to show the technical effect.
Examples
Completely dissolving polyvinyl alcohol at 70-80 ℃, and uniformly mixing the completely dissolved polyvinyl alcohol with sodium alginate powder according to the mass ratio of 1:5 to obtain a first mixed solution; adding activated carbon into the first mixed solution to obtain a second mixed solution, wherein the concentration of the activated carbon in the second mixed solution is 3g/L; uniformly mixing a chromium reducing agent containing 10.2% of Microbacterium sp Cr2203-1 and 89.2% of lysine bacillus sp Cr2203-2 with the second mixed solution according to the volume ratio of 1:5, and then dripping a calcium chloride saturated boric acid solution with the concentration of 25% at 4 ℃ to embed to obtain the solid microbial agent.
The enrichment method of the flora comprises the following specific steps:
s1: 2g tryptone, 1g yeast extract and 2g sodium chloride were added to 200mL water, pH adjusted to 7 with HCl and pressure increased to 103.4kPa (1.05 kg/cm)2) Maintaining at 121.3 deg.C for 30 min, and sterilizing at high temperature and high pressure to obtain bacteria basic culture medium; adding a certain mass of potassium dichromate into a bacteria basal culture medium to prepare a chromium-containing culture medium containing 50, 100, 150 and 200mg/L hexavalent chromium;
s2: adding electroplating site polluted soil (collected from a shut-down electroplating workshop of a family power machinery plant) into the chromium-containing culture medium according to the proportion of 40g (1L), culturing for 3 days at 35 ℃ and 120rpm, and taking supernatant liquid to obtain an initial inoculation source;
s3: inoculating the initial inoculation source into a bacteria basic culture medium containing heavy metals to obtain six culture media with different concentrations and contents of the heavy metals, wherein the six culture media are as follows:
a first medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 30mg/L nickel;
a second medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 40mg/L nickel;
third medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 50mg/L nickel;
fourth medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 50mg/L copper;
fifth medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 75mg/L copper;
sixth medium: a bacteria basal culture medium containing 50mg/L hexavalent chromium and 100mg/L copper;
and then, carrying out hexavalent chromium reduction in six culture media until the flora has the function of reducing hexavalent chromium and 50mg/L hexavalent chromium is completely reduced, and enriching to obtain the chromium reducing microbial inoculum.
In order to verify the effect of the solid microbial inoculum obtained in this example, the following experiment was also performed.
The obtained solid microbial inoculum is added into a bacterial basic culture medium containing 50mg/L hexavalent chromium, the reaction is carried out under the conditions of 35 ℃ and 120rpm, 50mg/L hexavalent chromium can be completely reduced within 20 hours, as shown in figure 1, and the solid microbial inoculum has the reduction effect of hexavalent chromium.
The obtained solid microbial inoculum is added into a bacterial basic culture medium containing 50, 100, 150 and 200mg/L hexavalent chromium, the reaction is carried out at 35 ℃ and 120rpm, 200mg/L hexavalent chromium can be completely reduced within 96h, as shown in figure 2, and the microbial inoculum has a wider range of hexavalent chromium reduction concentration.
The obtained solid microbial inoculum is added into the first to sixth culture media, the reduction rate of hexavalent chromium is observed when nickel and copper respectively exist, as shown in figure 3, the existence of nickel has no influence on the reduction rate of hexavalent chromium within 12h, and the existence of copper has a promoting effect on the reduction rate of hexavalent chromium. The existence of the two has no inhibiting effect on the reduction rate of hexavalent chromium, which indicates that the solid microbial inoculum has the hexavalent chromium reduction function under the pollution of various metals.
Extracting DNA of the solid microbial inoculum, amplifying 16SrRNA, sending to a sequencing company for sequencing, and determining the components of the microbial inoculum, as shown in figures 4 and 5.
The resulting solid microbial agent was added to actual contaminated soil (containing 1220mg/kg hexavalent chromium, 30mg/kg nickel, 198mg/kg copper), and a bacterial culture (soil: bacterial culture = 10ml) was added, and a reaction was carried out under conditions of 35 ℃ and 120rpm, as shown in fig. 6. As can be seen from the figure, the solid microbial inoculum can completely reduce hexavalent chromium within 7 days, which shows that the solid microbial inoculum has a high-efficiency treatment function on the actual soil polluted by various metals.
Therefore, the solid microbial inoculum prepared by the invention has high chromium reduction efficiency, can completely reduce 50mg/L hexavalent chromium within 24 hours, and can reduce hexavalent chromium unaffected in the presence of 30-50mg/L nickel and 50-100mg/L copper. The method for removing hexavalent chromium in the environment by using microorganisms has the advantages of low cost, long validity period, environmental friendliness, stable effect and the like, and is suitable for treating the environment polluted by a large amount of hexavalent chromium for a long time.
The above-described embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.
Claims (10)
1. A preparation method of a solid microbial inoculum for repairing an electroplating polluted site is characterized by comprising the following steps:
uniformly mixing the completely dissolved polyvinyl alcohol and sodium alginate powder to obtain a first mixed solution; adding activated carbon into the first mixed solution to obtain a second mixed solution; uniformly mixing a chromium reducing microbial inoculum containing Microbacterium sp Cr2203-1 and Bacillus lysinibus sp Cr2203-2 with the second mixed solution according to the volume ratio of (1-2) to (10), and then dripping a calcium chloride saturated boric acid solution with the concentration of 20-50% at the temperature of 0-4 ℃ to embed to obtain a solid microbial inoculum;
the Microbacterium Cr2203-1 is preserved in China center for type culture Collection in 2022, 06 months and 13 days, with the preservation number of CCTCC No. M2022878; the lysine bacillus Cr2203-2 is preserved in China center for type culture Collection in 2022, 06 months and 13 days, and the preservation number is CCTCC No. M2022879.
2. The method for preparing the solid microbial inoculum capable of being used for repairing the electroplating pollution site according to claim 1, wherein the polyvinyl alcohol is completely dissolved at 70-80 ℃.
3. The preparation method of the solid microbial inoculum for repairing the electroplating pollution site as claimed in claim 1, wherein the mass ratio of the polyvinyl alcohol to the sodium alginate powder is 1 (1-5).
4. The preparation method of the solid microbial inoculum capable of being used for repairing the electroplating pollution site as claimed in claim 1, wherein the concentration of the activated carbon in the second mixed solution is 3-5g/L.
5. The preparation method of the solid microbial inoculum capable of being used for repairing the electroplating pollution site as claimed in claim 1, wherein the content of the Microbacterium Cr2203-1 in the chromium reducing microbial inoculum is 3% -10.2%, and the content of the lysine bacillus Cr2203-2 in the chromium reducing microbial inoculum is 62% -89.2%.
6. The preparation method of the solid microbial inoculum for repairing the electroplating pollution site according to claim 1, wherein the preparation method of the chromium reducing microbial inoculum is as follows:
s1: adding 2g of tryptone, 1g of yeast extract and 2g of sodium chloride into 200mL of water, adjusting the pH to 7 by using HCl, increasing the pressure to 103.4kPa, maintaining the temperature at 121.3 ℃ for 15-30 minutes, and sterilizing at high temperature and high pressure to prepare a bacterial basal medium; adding 0.17-0.56g of potassium dichromate into the bacteria basal culture medium to prepare a chromium-containing culture medium containing 300-1000mg/L hexavalent chromium;
s2: adding electroplating site contaminated soil into the chromium-containing culture medium according to the proportion of 10-40g (l) 11L, culturing for 3 days at 35 ℃ and 120rpm, and taking supernatant liquid to obtain an initial inoculation source;
s3: and (3) inoculating the initial inoculation source into a bacterial basic culture medium containing 30-50mg/L of nickel, 50-100mg/L of copper and 50-300mg/L of hexavalent chromium to reduce the hexavalent chromium until the flora has the function of reducing the hexavalent chromium and the hexavalent chromium of 50-300mg/L is completely reduced, and enriching to obtain the chromium reducing microbial inoculum.
7. A solid microbial inoculum which is obtained by the preparation method of any one of claims 1 to 6 and can be used for repairing an electroplating pollution site.
8. The use of the solid microbial inoculum according to claim 7 in the treatment of contaminated soil, wherein the contaminated soil containing 50 to 300mg/L hexavalent chromium and the solid microbial inoculum are added to a bacterial basal medium at 35 ℃ and 120 rpm.
9. The use of the solid microbial inoculum of claim 7 in the treatment of contaminated soil, wherein the contaminated soil containing 50 to 300mg/L hexavalent chromium, 30 to 50mg/L nickel or 50 to 100mg/L copper and the solid microbial inoculum are added to a bacterial basal medium at 35 ℃ and 120 rpm.
10. The application of the solid microbial inoculum of claim 8 or 9 in the treatment of polluted soil, wherein the preparation method of the bacterial basic culture medium is as follows:
2g of tryptone, 1g of yeast extract and 2g of sodium chloride are added into 200mL of water, the pH value is adjusted to 7 by HCl, the pressure is increased to 103.4kPa, the mixture is maintained at the temperature of 121.3 ℃ for 15 to 30 minutes, and the mixture is sterilized at high temperature and high pressure to prepare a bacterial basal medium.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116986738A (en) * | 2023-09-26 | 2023-11-03 | 包头市生态环境局综合保障中心(包头市生态环境监控监测中心) | Chromium-polluted underground water in-situ chemical and biological combined restoration method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116986738A (en) * | 2023-09-26 | 2023-11-03 | 包头市生态环境局综合保障中心(包头市生态环境监控监测中心) | Chromium-polluted underground water in-situ chemical and biological combined restoration method |
| CN116986738B (en) * | 2023-09-26 | 2023-12-26 | 包头市生态环境局综合保障中心(包头市生态环境监控监测中心) | Chromium-polluted underground water in-situ chemical and biological combined restoration method |
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