A solid bacterial agent that can be used to repair electroplating contaminated sites, and a method and application thereof

Abstract

The invention discloses a solid bacterial agent and a method and application thereof for remediation of electroplating contaminated sites, belonging to the field of microbial remediation technology. The preparation method of the solid bacterial agent includes mixing polyvinyl alcohol with sodium alginate powder, adding activated carbon, and obtaining a second mixed solution; the chromium-reducing bacterial agent containing Microbacterium sp. Cr2203-1 and Lysinibacillus sp. Cr2203-2 is evenly mixed with the second mixed solution, and then a saturated boric acid solution of calcium chloride with a concentration of 20-50% is dripped at 0-4°C to obtain a solid bacterial agent by embedding. The bacterial community in the present invention has an efficient chromium reduction efficiency, can completely reduce 50mg / L hexavalent chromium within 24 hours, and can be unaffected in the presence of nickel (30-50) mg / L and copper (50-100) mg / L to perform hexavalent chromium reduction. Removing hexavalent chromium in the environment with microorganisms has the advantages of low cost, long effective period, environmental friendliness, stable effect, etc., which is suitable for long-term treatment of a large amount of hexavalent chromium-contaminated environments.

Description

Technical Field

[0001] The invention belongs to the technical field of biodegradation treatment, and specifically relates to the enrichment of chromium-reducing bacteria that can be used for the restoration of electroplating contaminated sites and the application of the enrichment of chromium-reducing bacteria in a liquid phase contaminated system for bioreduction treatment. Background Art

[0002] Over the past 40 years, the electroplating industry has played an important role in China's economic development and has become an important process in the automotive, manufacturing, home appliance and electronics industries. According to statistics, there are nearly 20,000 electroplating plants in my country, of which more than 40% are concentrated in the Yangtze River Delta and the Pearl River Delta. The most widely used processes are zinc plating, copper plating, nickel plating and chromium plating. However, my country's electroplating enterprises generally have outdated equipment, backward processes, and low waste treatment rates, resulting in the electroplating industry generating about 400 million tons of wastewater each year, including heavy metals and 50,000 tons of solid waste, making electroplating plants an important source of heavy metal pollution.

[0003] Chromium is a typical heavy metal pollutant produced by the electroplating industry. It is highly toxic and has strong "three-hazard" effects. It is one of the five heavy metals that are under national key control. At present, the use of microorganisms to control hexavalent chromium pollution has certain development prospects. By utilizing the growth activities of microorganisms, hexavalent chromium is used as the energy source required for its production. At the same time, organic matter in the environment is used as a carbon source. Under the action of chromium reductase, hexavalent chromium is reduced to trivalent chromium with lower toxicity and weaker mobility. Because its final metabolite is low in toxicity, the cost is lower than that of chemical treatment methods, and the ecological risk of using in situ indigenous microorganisms is low, so how to achieve efficient microbial reduction of hexavalent chromium is of great significance.

[0004] At the same time, in the electroplating pollution environment, in addition to the highly toxic hexavalent chromium, there is also complex pollution of nickel and copper heavy metals, and their presence will inhibit the reduction of hexavalent chromium by indigenous microorganisms. Therefore, it is of great significance to study the efficient removal of hexavalent chromium by chromium-reducing bacteria when hexavalent chromium and other heavy metals exist at the same time.

[0005] In the existing technology, there is no hexavalent chromium-reducing bacteria that can tolerate nickel and copper heavy metals. The main application is to treat single hexavalent chromium pollution or adsorb single heavy metals. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a solid bacterial agent that can be used for the remediation of electroplating contaminated sites and a method and application thereof.

[0007] The specific technical solutions adopted by the present invention are as follows:

[0008] In the first aspect, the present invention provides a method for preparing a solid bacterial agent that can be used for remediation of electroplating contaminated sites, as follows:

[0009] The completely dissolved polyvinyl alcohol is mixed evenly with sodium alginate powder to obtain a first mixed solution; activated carbon is added to the first mixed solution to obtain a second mixed solution; a chromium-reducing bacterial agent containing Microbacterium sp. Cr2203-1 and Lysinibacillus sp. Cr2203-2 is mixed evenly with the second mixed solution in a volume ratio of (1-2):10, and then a saturated boric acid solution of calcium chloride with a concentration of 20-50% is added dropwise at 0-4° C. to encapsulate and obtain a solid bacterial agent;

[0010] The Microbacterium Cr2203-1 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 13, 2022, with the accession number CCTCC No: M2022878; the Lysinibacillus Cr2203-2 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 13, 2022, with the accession number CCTCC No: M2022879.

[0011] Preferably, the polyvinyl alcohol is completely dissolved at 70-80°C.

[0012] Preferably, the mass ratio of the polyvinyl alcohol to the sodium alginate powder is 1:(1-5).

[0013] Preferably, the activated carbon concentration in the second mixed solution is 3-5 g / L.

[0014] Preferably, the content of Microbacterium Cr2203-1 in the chromium-reducing bacteria agent accounts for 3% to 10.2%, and the content of Lysinibacillus Cr2203-2 accounts for 62% to 89.2%.

[0015] Preferably, the preparation method of the chromium-reducing bacteria agent is as follows:

[0016] S1: 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride are added to 200 mL of water, the pH is adjusted to 7 with HCl, the pressure is increased to 103.4 kPa, and the temperature is maintained at 121.3° C. for 15 to 30 minutes for high temperature and high pressure sterilization to prepare a bacterial basal medium; 0.17-0.56 g of potassium dichromate is added to the bacterial basal medium to prepare a chromium-containing medium containing 300-1000 mg / L hexavalent chromium;

[0017] S2: adding the contaminated soil from the electroplating site to the chromium-containing culture medium at a ratio of 10-40 g:1 L, culturing at 35° C. and 120 rpm for 3 days, and taking the supernatant to obtain an initial inoculum source;

[0018] S3: The initial inoculum source is inoculated into a bacterial basal culture medium containing 30-50 mg / L nickel, 50-100 mg / L copper and 50-300 mg / L hexavalent chromium for hexavalent chromium reduction, until the bacterial community has the function of reducing hexavalent chromium and 50-300 mg / L hexavalent chromium is completely reduced, thereby enriching and obtaining a chromium-reducing bacterial agent.

[0019] In a second aspect, the present invention provides a solid bacterial agent that can be used for the remediation of electroplating contaminated sites and is obtained by any of the preparation methods described in the first aspect.

[0020] In a third aspect, the present invention provides an application of the solid bacterial agent described in the second aspect in treating contaminated soil. Specifically, contaminated soil containing 50-300 mg / L hexavalent chromium and the solid bacterial agent are added to a bacterial basal culture medium under the conditions of 35°C and 120rpm.

[0021] In a fourth aspect, the present invention provides an application of the solid bacterial agent described in the second aspect in treating contaminated soil. Specifically, contaminated soil containing 50-300 mg / L hexavalent chromium, 30-50 mg / L nickel or 50-100 mg / L copper and the solid bacterial agent are added to a bacterial basal culture medium under the conditions of 35°C and 120rpm.

[0022] As a preferred embodiment of the third aspect or the fourth aspect, the preparation method of the bacterial basal culture medium is as follows:

[0023] Add 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride to 200 mL of water, adjust the pH to 7 with HCl, increase the pressure to 103.4 kPa, maintain the temperature at 121.3°C for 15 to 30 minutes for high temperature and high pressure sterilization to prepare a bacterial basal culture medium.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The solid bacterial agent of the present invention has a high efficiency in reducing hexavalent chromium and can completely reduce 50 mg / L of hexavalent chromium within 24 hours.

[0026] 2) The solid bacterial agent of the present invention can simultaneously reduce hexavalent chromium in the environment and tolerate nickel and copper in the environment.

[0027] 3) Compared with the prior art, the present invention uses microbial solid bacterial agents to remove hexavalent chromium in the environment, which has the advantages of low cost, long effective period, stable degradation effect, and environmental friendliness, and is suitable for long-term treatment of environments with hexavalent chromium and nickel-copper composite pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Data diagram of hexavalent chromium reduction by solid bacterial agent in the present invention;

[0029] Figure 2 The solid bacterial agent of the present invention performs hexavalent chromium reduction efficiency diagram at different concentrations;

[0030] Figure 3 The hexavalent chromium reduction efficiency diagram of the solid bacterial agent in the present invention under nickel-copper mixed pollution;

[0031] Figure 4 The composition categories (gate levels) of the solid bacterial agents and different starting hexavalent chromium concentrations in the present invention;

[0032] Figure 5 The composition categories (genus level) of the solid bacterial agent and different initial hexavalent chromium concentrations in the present invention;

[0033] Figure 6 A graph showing the efficiency of reducing hexavalent chromium in soil under mixed nickel-copper pollution using the solid bacterial agent of the present invention.

[0034] Biomaterial Deposit

[0035] The Microbacterium sp. Cr2203-1 of the present invention was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 13, 2022, with a preservation number of CCTCC No: M2022878; the Lysinibacillus sp. Cr2203-2 of the present invention was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 13, 2022, with a preservation number of CCTCC No: M2022879. DETAILED DESCRIPTION

[0036] The present invention is further described and illustrated below in conjunction with the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0037] In the present invention, the initial inoculation source is a bacterial community enriched in a bacterial basic culture medium containing 300-1000 mg / L hexavalent chromium using contaminated soil. The contaminated soil is obtained by collecting contaminated soil without leakage prevention measures in an abandoned electroplating site (collected from the discontinued electroplating workshop of Zhangjiakou Power Machinery Plant), culturing it in a bacterial basic culture medium containing 300-1000 mg / L hexavalent chromium for 3 days, and then inoculating the supernatant bacterial community into a bacterial basic culture medium containing 50-300 mg / L hexavalent chromium and nickel (30-50) mg / L or copper (50-100) mg / L until the hexavalent chromium is completely reduced, and a bacterial community with a stable bacterial content can be obtained. The bacterial community contains the following bacterial species calculated by percentage: Lysinibacillus sp. Cr2203-2 is 62% to 89.2%, Microbacterium sp. Cr2203-1 is 3% to 10.2%, both of which have been proven to have the functions of reducing hexavalent chromium and tolerating nickel and copper heavy metals, and the remainder is miscellaneous bacteria that are not enrichment objects.

[0038] The specific method for enriching the bacterial flora is as follows:

[0039] S1: Add 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride to 200 mL of water, adjust the pH to 7 with HCl, and raise the pressure to 103.4 kPa (1.05 kg / cm 2 ), maintaining a temperature of 121.3° C. for 15 to 30 minutes by high temperature and high pressure sterilization to prepare a bacterial basal culture medium; adding 0.17-0.56 g of potassium dichromate to the bacterial basal culture medium to prepare a chromium-containing culture medium containing 300-1000 mg / L hexavalent chromium;

[0040] S2: adding the contaminated soil from the electroplating site to the chromium-containing culture medium at a ratio of 10-40 g:1 L, culturing at 35° C. and 120 rpm for 3 days, and taking the supernatant to obtain an initial inoculum source;

[0041] S3: inoculating the initial inoculum source into a bacterial basal culture medium containing 30-50 mg / L nickel or 50-100 mg / L copper and 50-300 mg / L hexavalent chromium (i.e., the bacterial basal culture medium contains 50-300 mg / L hexavalent chromium and 30-50 mg / L nickel or contains 50-300 mg / L hexavalent chromium and 50-100 mg / L copper) for hexavalent chromium reduction until the bacterial community has the function of reducing hexavalent chromium and 50-300 mg / L hexavalent chromium is completely reduced, thereby enriching and obtaining a chromium-reducing bacterial agent.

[0042] The Microbacterium Cr2203-1 obtained by the present invention was deposited in the China Center for Type Culture Collection on June 13, 2022, with the accession number CCTCC No: M2022878; the Lysinibacillus Cr2203-2 obtained by the present invention was deposited in the China Center for Type Culture Collection on June 13, 2022, with the accession number CCTCC No: M2022879.

[0043] The bacterial liquid formed by the bacterial colony is embedded with sodium alginate, polyvinyl alcohol, graphite and calcium chloride saturated boric acid solution to form a solid bacterial agent. The details are as follows:

[0044] Polyvinyl alcohol is completely dissolved at 70-80°C, and the completely dissolved polyvinyl alcohol is mixed with sodium alginate powder at a mass ratio of 1:(1-5) to obtain a first mixed solution; activated carbon is added to the first mixed solution to obtain a second mixed solution, and the concentration of activated carbon in the second mixed solution is 3-5g / L; a chromium-reducing bacterial agent containing 3%-10.2% Microbacterium sp. Cr2203-1 and 62%-89.2% Lysinibacillus sp. Cr2203-2 is mixed with the second mixed solution at a volume ratio of (1-2):10, and then a saturated boric acid solution of calcium chloride with a concentration of 20-50% is dropped at 0-4°C to encapsulate and obtain a solid bacterial agent.

[0045] The above-mentioned Microbacterium Cr2203-1 and Lysinibacillus Cr2203-2 are applied to a specific embodiment below, and are made into solid bacterial agents for soil remediation to demonstrate the technical effects that can be achieved.

[0046] Example

[0047] Polyvinyl alcohol is completely dissolved at 70-80°C, and the completely dissolved polyvinyl alcohol is mixed with sodium alginate powder at a mass ratio of 1:5 to obtain a first mixed solution; activated carbon is added to the first mixed solution to obtain a second mixed solution, and the activated carbon concentration in the second mixed solution is 3g / L; a chromium-reducing bacterial agent containing 10.2% Microbacterium sp. Cr2203-1 and 89.2% Lysinibacillus sp. Cr2203-2 is mixed with the second mixed solution at a volume ratio of 1:5, and then a saturated boric acid solution of calcium chloride with a concentration of 25% is dropped at 4°C to encapsulate and obtain a solid bacterial agent.

[0048] Among them, the enrichment method of the bacterial community is as follows:

[0049] S1: Add 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride to 200 mL of water, adjust the pH to 7 with HCl, and raise the pressure to 103.4 kPa (1.05 kg / cm 2 ), maintained at 121.3°C for 30 minutes for high temperature and high pressure sterilization to prepare a basic bacterial culture medium; by adding a certain amount of potassium dichromate to the basic bacterial culture medium, a chromium-containing culture medium containing 50, 100, 150, and 200 mg / L hexavalent chromium was prepared;

[0050] S2: adding contaminated soil from an electroplating site (collected from the discontinued electroplating workshop of Zhangjiakou Power Machinery Plant) to the chromium-containing culture medium at a ratio of 40 g:1 L, culturing at 35° C. and 120 rpm for 3 days, and then taking the supernatant to obtain an initial inoculum source;

[0051] S3: Inoculate the initial inoculum source into a bacterial basal culture medium containing heavy metals to obtain six culture media with different heavy metal concentrations and contents. The six culture media are specifically as follows:

[0052] First culture medium: bacterial basal culture medium containing 50 mg / L hexavalent chromium and 30 mg / L nickel;

[0053] Second culture medium: bacterial basal culture medium containing 50 mg / L hexavalent chromium and 40 mg / L nickel;

[0054] The third culture medium: a bacterial basal culture medium containing 50 mg / L hexavalent chromium and 50 mg / L nickel;

[0055] Fourth culture medium: bacterial basal culture medium containing 50 mg / L hexavalent chromium and 50 mg / L copper;

[0056] The fifth culture medium: a bacterial basal culture medium containing 50 mg / L hexavalent chromium and 75 mg / L copper;

[0057] The sixth culture medium: a bacterial basal culture medium containing 50 mg / L hexavalent chromium and 100 mg / L copper;

[0058] Subsequently, hexavalent chromium reduction was carried out in six culture media until the bacterial community had the function of reducing hexavalent chromium and 50 mg / L hexavalent chromium was completely reduced, thereby enriching and obtaining a chromium-reducing bacterial agent.

[0059] In order to verify the effect of the solid bacterial agent obtained in this example, the following experiment was also conducted.

[0060] The obtained solid bacterial agent was added to the bacterial basal culture medium containing 50 mg / L hexavalent chromium, and the reaction was carried out at 35°C and 120 rpm. Within 20 hours, 50 mg / L hexavalent chromium could be completely reduced. Figure 1As shown in the figure, it can be seen that the solid bacterial agent has the effect of reducing hexavalent chromium.

[0061] The obtained solid bacterial agent was added to the bacterial basal culture medium containing 50, 100, 150, and 200 mg / L hexavalent chromium, and the reaction was carried out at 35°C and 120 rpm. Within 96 hours, 200 mg / L hexavalent chromium could be completely reduced. Figure 2 As shown in the figure, it can be seen that the bacterial agent has a relatively wide concentration range for reducing hexavalent chromium.

[0062] The obtained solid bacterial agent was added to the first to sixth culture media mentioned above, and the reduction rate of hexavalent chromium in the presence of nickel and copper was observed. Figure 3 As shown in the results, it was found that within 12 hours, the presence of nickel had no effect on the reduction rate of hexavalent chromium, while the presence of copper promoted the reduction rate of hexavalent chromium. The presence of both had no inhibitory effect on the reduction rate of hexavalent chromium, indicating that the solid bacterial agent has the function of reducing hexavalent chromium under multiple metal pollution.

[0063] The DNA of the solid bacterial agent is extracted and the 16S rRNA is amplified and sent to a sequencing company for sequencing to determine the composition of the bacterial agent, such as Figure 4 and 5 shown.

[0064] The obtained solid bacterial agent was added to the actual contaminated soil (containing 1220 mg / kg hexavalent chromium, 30 mg / kg nickel, and 198 mg / kg copper), and the bacterial culture solution (soil: bacterial culture solution = 1 g: 10 mL) was added, and the reaction was carried out at 35°C and 120 rpm. Figure 6 As shown in the figure, it can be seen that the solid bacterial agent completely reduced hexavalent chromium in 7 days, indicating that the solid bacterial agent has a high efficiency in treating actual soil contaminated by multiple metals.

[0065] It can be seen that the solid bacterial agent prepared by the present invention has a high chromium reduction efficiency, can completely reduce 50 mg / L hexavalent chromium within 24 hours, and can reduce hexavalent chromium without being affected in the presence of nickel (30-50) mg / L and copper (50-100) mg / L. Using microorganisms to remove hexavalent chromium in the environment has the advantages of low cost, long effective period, environmental friendliness, stable effect, etc., and is suitable for long-term treatment of environments with a large amount of hexavalent chromium pollution.

[0066] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A method for preparing a solid bacterial agent that can be used for remediation of electroplating contaminated sites, characterized in that: The details are as follows: The completely dissolved polyvinyl alcohol and sodium alginate powder are mixed evenly to obtain a first mixed solution; activated carbon is added to the first mixed solution to obtain a second mixed solution; and the mixture containing Microbacterium spp. ( Microbacterium sp. )Cr2203-1 and Bacillus lysinicus ( Lysinibacillus sp. ) The chromium-reducing bacterial agent Cr2203-2 is uniformly mixed with the second mixed solution in a volume ratio of (1-2):10, and then a saturated boric acid solution of calcium chloride with a concentration of 20-50% is dropped at 0-4° C. to encapsulate and obtain a solid bacterial agent; The Microbacterium Cr2203-1 was deposited in the China Center for Type Culture Collection on June 13, 2022, with a deposit number of CCTCC No: M2022878; the Lysinibacillus Cr2203-2 was deposited in the China Center for Type Culture Collection on June 13, 2022, with a deposit number of CCTCC No: M2022879; The mass ratio of the polyvinyl alcohol to the sodium alginate powder is 1:(1-5); the concentration of activated carbon in the second mixed solution is 3-5 g / L; The preparation method of the chromium-reducing bacteria agent is as follows: S1: Add 2g of tryptone, 1g of yeast extract and 2g of sodium chloride to 200mL of water, adjust the pH to 7 with HCl, increase the pressure to 103.4kPa, maintain the temperature at 121.3°C for 15-30 minutes for high temperature and high pressure sterilization to prepare a bacterial basal medium; add 0.17-0.56g of potassium dichromate to the bacterial basal medium to prepare a chromium-containing medium containing 300-1000 mg / L hexavalent chromium; S2: adding the contaminated soil from the electroplating site to the chromium-containing culture medium at a ratio of 10-40 g:1 L, culturing at 35° C. and 120 rpm for 3 days, and taking the supernatant to obtain an initial inoculum source; S3: The initial inoculum source is inoculated into a bacterial basal culture medium containing 30-50 mg / L nickel, 50-100 mg / L copper and 50-300 mg / L hexavalent chromium for hexavalent chromium reduction, until the bacterial community has the function of reducing hexavalent chromium and 50-300 mg / L hexavalent chromium is completely reduced, thereby enriching and obtaining a chromium-reducing bacterial agent.

2. The method for preparing a solid bacterial agent that can be used for remediation of electroplating contaminated sites according to claim 1, characterized in that: The polyvinyl alcohol is completely dissolved at 70-80°C.

3. The method for preparing a solid bacterial agent that can be used for remediation of electroplating contaminated sites according to claim 1, characterized in that: The content of Microbacterium Cr2203-1 in the chromium-reducing bacteria agent accounts for 3% to 10.2%, and the content of Lysinibacillus Cr2203-2 accounts for 62% to 89.2%.

4. A solid bacterial agent that can be used for remediation of electroplating contaminated sites, obtained by the preparation method described in any one of claims 1 to 3.

5. An application of the solid bacterial agent according to claim 4 in treating contaminated soil, characterized in that: At 35°C and 120rpm, contaminated soil containing 50-300mg / L hexavalent chromium and solid bacterial agent were added to the bacterial basal culture medium.

6. An application of the solid bacterial agent according to claim 4 in treating contaminated soil, characterized in that: At 35°C and 120rpm, contaminated soil containing 50-300mg / L hexavalent chromium, 30-50mg / L nickel or 50-100mg / L copper and a solid bacterial agent were added to the bacterial basal culture medium.

7. The use of the solid bacterial agent in treating contaminated soil according to claim 5 or 6, characterized in that: The preparation method of the bacterial basal culture medium is as follows: Add 2 g of tryptone, 1 g of yeast extract and 2 g of sodium chloride to 200 mL of water, adjust the pH to 7 with HCl, increase the pressure to 103.4 kPa, maintain the temperature at 121.3°C for 15 to 30 minutes for high temperature and high pressure sterilization to prepare a bacterial basal culture medium.

Images