A microbial remediation method for heavy metal contaminated wastewater

Abstract

The present invention belongs to the technical field of industrial wastewater treatment and discloses a microbial remediation method for heavy metal contaminated wastewater, specifically: using Vibrio natriegens to remove Cu in wastewater. 2+ and / or Cr 6+ The present invention first discovered that Vibrio natrii can be used as a biosorbent to remove heavy metals, especially Cu 2+ Cr 6+ removal, can be used to repair different Cu 2+ Cr 6+ It can be used in wastewater with a high degree of pollution, and Vibrio natrium grows quickly and has a low cultivation cost. It can be seen that the use of Vibrio natrium to repair heavy metal pollution has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of industrial wastewater treatment, and specifically relates to a method for treating wastewater containing Cu 2+ and / or Cr 6+ Microbial remediation method for heavy metal contaminated wastewater. Background Art

[0002] With the development of industry, the environmental problems and harm to the human body caused by heavy metal wastewater are becoming increasingly serious. For example, chromium ion and copper ion pollution can seriously harm animal development, plant growth and human health. Currently, common methods for wastewater heavy metal remediation mainly include physical, chemical and biological remediation methods. Among them, biological remediation methods mainly refer to methods of removing heavy metals through the flocculation, absorption, accumulation and enrichment of microorganisms or plants. Compared with physical and chemical remediation methods, biological remediation methods have the advantages of simple equipment, no secondary pollution, a wide range of cheap materials, and economical and efficient use. It is a heavy metal wastewater treatment method with great development potential and has broad application prospects.

[0003] Among bioremediation methods, the use of microorganisms to treat heavy metal contaminants in wastewater plays an important role. However, microbial remediation of wastewater still faces problems such as incomplete metal removal and long microbial growth time. To address these shortcomings, existing technologies often enhance the effectiveness of microbial remediation through strain domestication or the construction of genetically engineered bacteria. However, domestication is time-consuming, genetically engineered bacteria are expensive, and pose biosafety risks. Therefore, the search for microorganisms that can efficiently remove heavy metals and grow rapidly for bioremediation of wastewater remains a pressing need.

[0004] Vibrio natriegens is a Gram-negative bacterium belonging to the genus Vibrio. In recent years, it has been widely used as a chassis cell. Its advantages include diverse substrate utilization, rapid metabolic rate, lack of pathogenicity to humans, ease of genetic manipulation, and ease of expressing exogenous proteins, showing promising application prospects in synthetic biology. However, its role as a biosorbent for heavy metal removal has not been explored. Summary of the Invention

[0005] In response to the problems in the prior art, the present invention discovered for the first time that Vibrio natrii can be used as a biosorbent to remove heavy metal ions from wastewater. Based on this discovery, the present invention developed a method for remediating heavy metal contaminated wastewater using Vibrio natrii, which can quickly and effectively remove Cu ions from wastewater. 2+ and Cr 6+ , especially the highly toxic Cr 6+ , can be completely removed instantly.

[0006] The technical solutions of the present invention are as follows:

[0007] A microbial remediation method for heavy metal contaminated wastewater, specifically: using Vibrio natrii to remove Cu in wastewater 2+ and / or Cr 6+ .

[0008] Furthermore, in the above-mentioned microbial remediation method, the Vibrio natriuresis is first activated and expanded to obtain a suspension of Vibrio natriuresis that has grown to a stable phase, and then the suspension is added to the wastewater for a removal reaction.

[0009] Furthermore, the method for activating Vibrio natriuresis is as follows: the frozen Vibrio natriuresis is inoculated into a recovery medium and incubated at 30-37°C and 150-250 rpm / min to obtain an activated bacterial solution; wherein the formula of the recovery medium is preferably: LB broth 20-25 g·L -1 , sodium chloride 3-5g·L -1 .

[0010] Furthermore, the method for expanding the culture of Vibrio natriuresis is as follows: the activated Vibrio natriuresis is inoculated into an expansion culture medium and expanded at 30-37°C and 150-250 rpm / min; wherein the formula of the expansion culture medium is preferably: ammonium sulfate 3-5 g·L -1 , sodium chloride 10-15g·L -1 , potassium dihydrogen phosphate 0.5-1g·L -1 , dipotassium hydrogen phosphate 0.5-1g·L -1 , 3-(N-morpholine)propanesulfonic acid 17-21g·L -1 , D-glucose 8-10g·L -1 , magnesium sulfate heptahydrate 0.1-0.25g·L -1 Calcium chloride 0.01-0.02g·L -1 , ferrous sulfate heptahydrate 15-16.4 mg·L -1 , manganese sulfate monohydrate 5-10 mg·L -1 , copper sulfate pentahydrate 0.2-0.4 mg·L -1 , zinc sulfate heptahydrate 0.5-1mg·L -1 , nickel chloride hexahydrate 0.01-0.02 mg·L -1 In addition, the pH of the expansion medium is preferably 7-8, and the expansion culture is carried out under this condition for 5-7 hours.

[0011] Furthermore, in the above-mentioned microbial remediation method, the mass ratio of the amount of Vibrio natrii added (dry weight) to the chromium in the wastewater is preferably (1-10):1; under this condition, Vibrio natrii can complete the degradation of Cr in a short time. 6+ Complete removal of .

[0012] Furthermore, in the above-mentioned microbial remediation method, the mass ratio of the amount of Vibrio natrii added (dry weight) to the copper in the wastewater is preferably (0.2-1):1; under this condition, Vibrio natrii can effectively remove Cu. 2+ Has good adsorption effect.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention utilizes the biological metabolism of Vibrio natriuresis to remove hexavalent chromium ions and divalent copper ions in wastewater, has the advantages of simple operation, environmental protection and low consumption, and is suitable for the remediation of wastewater areas contaminated by heavy metals to varying degrees;

[0015] (2) Vibrio natrii grows quickly, and the optimized culture medium can make it grow to the stable phase in a short time (5-7 h), effectively reducing the time cost of microbial culture;

[0016] (3) Vibrio natriuresis that has grown to the stable phase can instantly remove highly toxic hexavalent chromium ions and divalent copper ions, and can remove 100% of the hexavalent chromium ions with a sufficient bacterial dosage. This shows that the repair method provided by the present invention is extremely efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a growth curve of Vibrio natriuresis in Example 1 at different culture medium pH values.

[0018] Figure 2 The different dosages of Vibrio natrii in Example 2 were used to determine the effect of Cr on the wastewater. 6+ Graph of analysis results for removal effects.

[0019] Figure 3 The different dosages of Vibrio natrii in Example 3 were used to determine the effects of different dosages of Vibrio natrii on Cu in wastewater. 2+ Graph of analysis results for removal effects. DETAILED DESCRIPTION

[0020] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0021] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0022] The Vibrio natriegens bacteria used in the following examples were purchased from DSMZ (German Collection of Microorganisms and Cell Culture, Braunschweing, Germany).

[0023] Example 1 Effect of pH of the Expanded Culture Medium on the Growth Curve of Vibrio natriuresis

[0024] The experimental process of this example is as follows:

[0025] (1) Prepare the recovery medium. The specific formula of the recovery medium is: LB broth 25g·L -1 , sodium chloride 5g·L -1 ; After preparation, sterilize at 121℃ for 20 minutes.

[0026] (2) Prepare expansion culture medium. The specific formula of expansion culture medium is: ammonium sulfate 5g·L -1 , sodium chloride 15g·L -1 , potassium dihydrogen phosphate 1g·L -1 , dipotassium hydrogen phosphate 1g·L -1 , 3-(N-morpholino)propanesulfonic acid 21g·L -1 , D-glucose 10g·L -1 , magnesium sulfate heptahydrate 0.25g·L -1 , calcium chloride 0.01g·L -1 , ferrous sulfate heptahydrate 16.4 mg·L -1 , manganese sulfate monohydrate 10 mg·L -1 , copper sulfate pentahydrate 0.3 mg·L -1 , zinc sulfate heptahydrate 1mg·L -1 , nickel chloride hexahydrate 0.02 mg·L -1 The pH of the expansion medium was then adjusted to 5.5, 7.5, 9.5, and 11.5, respectively. The medium was then sterilized at 121°C for 20 min (glucose was sterilized separately).

[0027] (3) Activation of Vibrio natriegens strains. Take out the frozen tube of Vibrio natriegens strain stored in the -80℃ freezer. Use an inoculation loop to poke a small amount of strain with crushed ice in the clean bench and inoculate it into a sterilized test tube. The test tube is pre-filled with 5mL of sterilized recovery medium.

[0028] (4) Place the test tube in a constant temperature shaker at 37°C and 200 rpm / min and incubate. After 12 hours, the incubation ends and a clear white bacterial solution is visible in the test tube, indicating that the activated bacterial solution has been obtained.

[0029] (5) Scale up the culture of the Vibrio natrium strain. After the strain is successfully activated, add 2.5% (v / v) activated bacterial solution to a conical flask containing expansion medium of different pH values. Scale up the culture at 37°C and 200 rpm for 7 hours. Measure the growth curve at different points during the growth process.

[0030] The growth curves of Vibrio natriuresis under different pH conditions are as follows Figure 1 As shown, it is shown that Vibrio natriuresis grows rapidly under suitable culture conditions and reaches a stable period in about 5 hours, indicating that Vibrio natriuresis has the advantage of low culture time cost as a microbial remediation material.

[0031] Example 2: Effect of Sodium Vibrio on the Removal of Hexavalent Chromium Ions in Wastewater

[0032] The experimental process of this example includes the following steps:

[0033] (1) Prepare a seed suspension of Vibrio natriuresis that has grown to the stationary phase.

[0034] The Vibrio natriegens strain cryopreservation tube stored in a -80 ℃ refrigerator was taken out, and a small amount of strain with crushed ice was pierced with an inoculating loop in a clean bench and inoculated into a sterilized test tube, which was pre-filled with 5 mL of sterilized recovery medium. The test tube was placed in a constant temperature shaker at 37 ℃ and 200 rpm / min and incubated. After 12 hours of incubation, a clear white bacterial liquid was visible in the test tube, indicating that the activated bacterial liquid was obtained. After the strain was successfully activated, 2.5% (v / v) of the activated bacterial liquid was inoculated into a conical flask containing an expansion medium, and the culture was expanded at 37 ℃ and 200 rpm / min for 7 hours to prepare a seed suspension. The recovery medium and expansion medium used in this example were the same as those in Example 1, and the pH of the expansion medium was 7.5.

[0035] (2) Simulating high concentration of Cr 6+ Wastewater, adsorption experiment was carried out in a conical flask. The specific experimental process is as follows: at an initial concentration of 100 mg / L Cr 6+ The seed suspension prepared in step (1) was added to the solution so that the amount of Vibrio natrii was 0.1, 0.25, 0.5, 0.75, and 1 g (dry weight), respectively. The solution was placed in a constant temperature incubator at 37° C. and 200 rpm / min for removal experiment.

[0036] (3) Samples were taken at fixed time points and centrifuged at 8000 rpm and 4°C for 10 min. The Cr content in the supernatant was determined using the diphenylcarbazide colorimetric method. 6+ concentration.

[0037] Test results such as Figure 2As shown in the figure, it can be seen that Vibrio natrii 6+ It has a good removal effect. Different dosages of Vibrio natrii can completely remove Cr in different time periods. 6+ , and with the increase of the concentration of Vibrio natrii, Cr was completely removed. 6+ The time is getting shorter and shorter.

[0038] Example 3: Effect of Sodium Vibrio on Removal of Divalent Copper Ions in Wastewater

[0039] The experimental process of this example includes the following steps:

[0040] (1) Prepare a seed suspension of Vibrio natriuresis that has grown to the stationary phase.

[0041] The Vibrio natriegens strain cryopreservation tube stored in a -80 ℃ refrigerator was taken out, and a small amount of strain with crushed ice was pierced with an inoculating loop in a clean bench and inoculated into a sterilized test tube, which was pre-filled with 5 mL of sterilized recovery medium. The test tube was placed in a constant temperature shaker at 37 ℃ and 200 rpm / min and incubated. After 12 hours of incubation, a clear white bacterial liquid was visible in the test tube, indicating that the activated bacterial liquid was obtained. After the strain was successfully activated, 2.5% (v / v) of the activated bacterial liquid was inoculated into a conical flask containing an expansion medium, and the culture was expanded at 37 ℃ and 200 rpm / min for 7 hours to prepare a seed suspension. The recovery medium and expansion medium used in this example were the same as those in Example 1, and the pH of the expansion medium was 7.5.

[0042] (2) Simulating high concentration of Cu 2+ Wastewater, adsorption experiment was carried out in a conical flask. The specific experimental process is as follows: at an initial concentration of 100 mg / L Cu 2+ The seed suspension prepared in step (1) was added to the solution so that the amount of Vibrio natrii was 0.02, 0.04, 0.06, 0.08, and 0.1 g (dry weight), respectively. The solution was placed in a constant temperature incubator at 37° C. and 200 rpm / min for removal experiment.

[0043] (3) Samples were taken at fixed time points and centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was taken and the Cu content in the supernatant was determined using the dicyclohexanone oxalyldihydrazone colorimetric method. 2+ concentration.

[0044] Test results such as Figure 3 As shown in the figure, it can be seen that Vibrio natrii 2+ It has a good removal effect. Different amounts of Vibrio natrii have a better effect on Cu 2+ The adsorption can reach equilibrium within 5 minutes, and with the increase of the concentration of Vibrio natriuresis, the adsorption of Cu 2+The adsorption efficiency is also increasing.

[0045] The upper and lower limits, and interval values ​​of the raw materials, as well as the upper and lower limits, and interval values ​​of the method parameters listed in the present invention can all implement the present invention, and the embodiments are not listed one by one here.

[0046] In summary, the method of the present invention for remediating heavy metal wastewater using Vibrio natriuresis has the advantages of high efficiency, low cost, simple operation, and environmental protection. More importantly, Vibrio natriuresis can instantly remove highly toxic hexavalent chromium ions and divalent copper ions, and can be applied to the remediation of wastewater areas contaminated by chromium ions and copper ions to varying degrees. It can be seen that Vibrio natriuresis has great application potential in removing heavy metals.

[0047] The above description is a preferred embodiment of the present invention, which cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microbial remediation method for heavy metal contaminated wastewater, characterized in that: Using Vibrio natriuresis ( Vibrio natriegens )Removal of Cu from wastewater 2+ and / or Cr 6+ , that is, activating and expanding the Vibrio natriureticus to obtain a Vibrio natriureticus suspension grown to a stable phase, and then adding the suspension into the wastewater for reaction; The expansion culture method comprises: activating Vibrio natrii and inoculating the activated Vibrio natrii into an expansion culture medium and culturing the culture medium at 30-37° C. and 150-250 rpm / min for 5-7 hours; wherein the pH of the expansion culture medium is 7-8 and the formula thereof is: 3-5 g·L ammonium sulfate -1 , sodium chloride 10-15g·L -1 , potassium dihydrogen phosphate 0.5-1g·L -1 , dipotassium hydrogen phosphate 0.5-1g·L -1 , 3-(N-morpholine)propanesulfonic acid 17-21g·L -1 , D-glucose 8-10g·L -1 , magnesium sulfate heptahydrate 0.1-0.25g·L -1 Calcium chloride 0.01-0.02g·L -1 , ferrous sulfate heptahydrate 15-16.4 mg·L -1 , manganese sulfate monohydrate 5-10 mg·L -1 , copper sulfate pentahydrate 0.2-0.4 mg·L -1 , zinc sulfate heptahydrate 0.5-1mg·L -1 , nickel chloride hexahydrate 0.01-0.02 mg·L -1 .

2. The microbial remediation method for heavy metal contaminated wastewater according to claim 1, characterized in that: The activation method of the natriuretic Vibrio natriuretic is as follows: the frozen natriuretic Vibrio natriuretic is inoculated into a recovery culture medium, and incubated at 30-37° C. and 150-250 rpm / min to obtain an activated bacterial solution.

3. The microbial remediation method for heavy metal contaminated wastewater according to claim 2, characterized in that: The formula of the recovery medium is: LB broth 20-25g·L -1 , sodium chloride 3-5g·L -1 .

4. The microbial remediation method for heavy metal contaminated wastewater according to claim 1, characterized in that: The mass ratio of the natriuretic vibrio to chromium is (1-10):

1.

5. The microbial remediation method for heavy metal contaminated wastewater according to claim 1, characterized in that: The mass ratio of the natriuretic vibrio to copper is (0.2-1):1.

Images