Method for enhancing rate of bacteria to reduce hexavalent chromium in water by iron porphyrin complex

Abstract

The present invention belongs to the field of water treatment, and particularly relates to a method for enhancing the rate of hexavalent chromium reduction in water by bacteria using an iron porphyrin complex, which comprises the following steps: under anaerobic conditions, bacteria are co-cultured with an iron porphyrin complex and then mixed with water containing hexavalent chromium for culturing, and the hexavalent chromium in the water is reduced by the bacteria during the mixed culturing; the bacteria are Shewanella oneidensis Shewanella oneidensis MR-1; the iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl) iron porphyrin. The method provided by the present invention has the advantages of remarkable effect and simple operation, and provides a new idea for enhancing the rate of heavy metal pollution remediation by dissimilatory metal-reducing bacteria. In addition, the iron porphyrin complex involved in the present invention can be obtained by artificial synthesis, which is conducive to promoting the large-scale application of the technical solution of the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of water treatment, and particularly relates to a method for enhancing the rate of hexavalent chromium reduction in water by iron porphyrin complex with bacteria. Background Art

[0002] Chromium is widely used in industries such as electroplating, leather making, chemical industry, and printing and dyeing, directly resulting in a large amount of chromium-containing wastewater. If these chromium-containing wastewaters are directly discharged without effective treatment, they will pose a considerable threat to water bodies, soil, and ecosystems. Therefore, although treatment technologies and regulatory systems have made great progress, chromium is still one of the main heavy metals causing environmental pollution, and its treatment and prevention remain the focus and difficulty in the field of environmental protection. Hexavalent chromium and trivalent chromium are the two main valence states of chromium in nature. Among them, hexavalent chromium has relatively strong biological toxicity and migration ability, and is the main source of the biological toxicity of chromium-containing wastewater; in contrast, the toxicity and teratogenicity of trivalent chromium are much smaller than those of hexavalent chromium, and trivalent chromium has a small solubility in neutral and weakly alkaline environments and is more likely to precipitate and be fixed. Therefore, the main idea for treating chromium-containing wastewater is to reduce hexavalent chromium in it to trivalent chromium with low toxicity and easy fixation.

[0003] Dissimilatory Metal Reducing Bacteria (DMRB) can use metal atoms inside and outside the cell as electron acceptors to complete their own metabolic processes. When treating chromium-containing wastewater, DMRB can reduce hexavalent chromium to reduce biological toxicity while degrading organic pollutants through metabolism. Using DMRB to treat chromium-containing wastewater not only avoids the consumption of additional chemical agents caused by traditional chemical treatment but also reduces the risk of secondary pollution, showing considerable application potential. Shewanella oneidensis MR-1 is one of the most widely studied model strains among DMRB, which can reduce hexavalent chromium to trivalent chromium. This bacterium can not only reduce hexavalent chromium with enzymes intracellularly but also use a unique extracellular electron transfer pathway to transfer intracellular electrons to extracellular hexavalent chromium to achieve extracellular reduction of hexavalent chromium. The process of extracellular reduction of hexavalent chromium circumvents the rate limitation of hexavalent chromium transmembrane transport and can also reduce the toxic effects of hexavalent chromium and its reduction products generated inside the cell. However, the natural extracellular electron transfer rate of wild-type DMRB is generally low, which restricts the rate of extracellular reduction of hexavalent chromium by bacteria and seriously limits the overall biological reduction rate of hexavalent chromium. Therefore, enhancing the extracellular electron transfer rate between Shewanella oneidensis MR-1 and hexavalent chromium is a valuable way to increase the rate of hexavalent chromium reduction.

[0004] The prior art provides various methods for hybridizing DMRB with non-biological materials to enhance extracellular electron transfer ability and thus improve the hexavalent chromium reduction rate, including in-situ modification of cells with nanomaterials and exogenous addition of conductive media, etc. However, there are generally problems such as complex operations, difficulty in large-scale application, and weak improvement effects. For example, Patent CN 117821323 A discloses a method for modifying Shewanella cells with biogenic silver nanoparticles to enhance extracellular electron transfer ability and promote hexavalent chromium reduction. However, the operation method is relatively complex, and microorganisms need to generate attached nanoparticles in-situ under specific culture environments; Patent CN 113149230A discloses a treatment method for enhancing microbial reduction of hexavalent chromium by adding lignocellulose biochar. Using lignocellulose biochar as an electron transfer medium between bacteria and hexavalent chromium to increase the electron transfer rate to improve the hexavalent chromium reduction rate, but the improvement effect is weak. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for enhancing the rate of bacteria reducing hexavalent chromium in water by iron porphyrin complex. This method is simple to operate, can significantly improve the extracellular electron transfer rate of Shewanella oneidensis, and thus improve its rate of reducing hexavalent chromium in water.

[0006] The present invention provides a method for enhancing the rate of bacteria reducing hexavalent chromium in water by iron porphyrin complex, including the following steps:

[0007] Under anaerobic conditions, the bacteria are co-cultured with the iron porphyrin complex and then mixed with water containing hexavalent chromium for culture. During the mixed culture, the hexavalent chromium in the water is reduced by the bacteria;

[0008] The bacteria are Shewanella oneidensis MR-1;

[0009] The iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl) iron porphyrin.

[0010] Preferably, the co-culture is carried out in a culture medium. When carrying out the co-culture, the initial value of the bacterial cell density OD 600 is 0.2 - 5, and the content of the iron porphyrin complex is 0.02 - 0.6 g / L.

[0011] Preferably, the pH value when carrying out the co-culture is 6.5 - 10.

[0012] Preferably, the temperature of the co-culture is 15 - 45 °C.

[0013] Preferably, the stirring rate of the co-culture is 100 - 200 rpm.

[0014] Preferably, after mixing with the chromium(VI)-containing water, the initial concentration of chromium(VI) in the mixed system is 0.05 - 0.5 mmol / L.

[0015] Preferably, after mixing with the chromium(VI)-containing water, the bacterial cell density OD 600 of the mixed system has an initial value of 0.2 - 2, and the content of the iron porphyrin complex is 0.02 - 0.2 g / L.

[0016] Preferably, after mixing with the chromium(VI)-containing water, the pH value of the mixed system is 6.5 - 10.

[0017] Preferably, the temperature of the co-culture is 15 - 45 °C.

[0018] Preferably, the stirring rate of the co-culture is 100 - 1000 rpm.

[0019] Compared with the prior art, the present invention provides a method for enhancing the rate of chromium(VI) reduction in water by bacteria using an iron porphyrin complex, which includes the following steps: under anaerobic conditions, bacteria and an iron porphyrin complex are co-cultured and then mixed with chromium(VI)-containing water for co-culture, and chromium(VI) in the water is reduced by the bacteria during the co-culture; the bacteria is Shewanella oneidensis MR-1; the iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin iron. The present invention uses Shewanella oneidensis MR-1 and a specific iron porphyrin complex (5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin iron) for mixed co-culture to obtain a bacteria-material co-culture that enhances extracellular electron transfer. This co-culture can efficiently reduce chromium(VI) in water, thereby achieving the purpose of significantly enhancing the chromium(VI) reduction rate of Shewanella oneidensis. The method provided by the present invention has the advantages of remarkable effect and simple operation, providing a new idea for enhancing the rate of heavy metal pollution remediation by DMRB. In addition, the iron porphyrin complex involved in the present invention can be obtained by artificial synthesis, which is beneficial to promoting the large-scale application of the technical solution of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0021] Figure 1It is the property test result diagram of the synthesized TCPP(Fe) powder provided in Example 1 of the present invention; among them, A is the Fourier infrared spectrum diagram of TCPP(Fe), and B is the ultraviolet-visible light absorption spectrum diagrams of TCPP(Fe) and TCPP;

[0022] Figure 2 It is the detection result of the reduction rate of hexavalent chromium in water by the bacteria-material co-culture solution provided in Example 2 of the present invention and the result diagram of the control group;

[0023] Figure 3 It is the result diagram of the experiment on the influence of different experimental conditions on the reduction of hexavalent chromium in water by the bacteria-material co-culture solution provided in Example 3 of the present invention; among them, A is the detection result diagram of the reduction rate of Cr(Ⅵ) in water by the bacteria-material co-culture solution under different dosages of TCPP(Fe), B is the detection result diagram of the reduction rate of Cr(Ⅵ) in water by the bacteria-material co-culture solution under different dosages of bacteria, C is the detection result diagram of the reduction rate of Cr(Ⅵ) in water by the bacteria-material co-culture solution at different temperatures, D is the detection result diagram of the reduction rate of Cr(Ⅵ) in water by the bacteria-material co-culture solution under different initial Cr(Ⅵ) concentrations, E is the detection result diagram of the reduction rate of Cr(Ⅵ) in water by the bacteria-material co-culture solution under different pH environments, and F is the detection result diagram of the pH value of the reaction solution during the E experiment;

[0024] Figure 4 It is the effect diagram of the reduction of hexavalent chromium in water by the bacteria-material co-culture solution under the optimized experimental conditions provided in Example 4 of the present invention; among them, A is the detection result of the reduction rate of hexavalent chromium in water by the bacteria-material co-culture solution under the optimized experimental conditions and the result diagram of the control group, B is the linear fitting result diagram calculated according to A, and C is the pseudo-first-order reaction kinetic result diagram of the reduction of hexavalent chromium in the solution calculated according to A and B;

[0025] Figure 5 It is the effect diagram of the reduction of hexavalent chromium in water by the ΔmtrA mutant bacteria-material co-culture solution under the optimized experimental conditions provided in Example 5 of the present invention. Detailed implementation manners

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] The present invention provides a method for enhancing the reduction rate of hexavalent chromium in water by bacteria with an iron porphyrin complex, comprising the following steps:

[0028] Under anaerobic conditions, the bacteria are co-cultured with an iron porphyrin complex and then mixed with hexavalent chromium-containing water for cultivation. During the mixed cultivation, hexavalent chromium in the water is reduced by the bacteria;

[0029] The bacteria are Shewanella oneidensis MR-1;

[0030] The iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin iron.

[0031] In the method provided by the present invention, the specific process of the co-cultivation preferably includes: mixing a bacterial solution, an inorganic salt solution of an iron porphyrin complex, and a carbon source to obtain a bacteria-material mixed solution; then culturing with shaking and reaching a certain anaerobic level to obtain a bacteria-material co-cultivation solution.

[0032] In the method provided by the present invention, the components of the bacterial solution preferably include: Shewanella oneidensis MR-1 and an inorganic salt solution; the components of the inorganic salt solution of the iron porphyrin complex preferably include: 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin iron (TCPP(Fe)) and an inorganic salt solution; the inorganic salt solution should provide the pH conditions, osmotic pressure, and trace nutrients required for the growth of Shewanella oneidensis MR-1; the carbon source is preferably an organic substance that can be utilized by the metabolism of Shewanella oneidensis MR-1; the certain anaerobic level preferably needs to meet the requirement for Shewanella oneidensis MR-1 to transfer electrons to extracellular hexavalent chromium.

[0033] In the method provided by the present invention, the bacterial solution is preferably obtained according to the following steps: culturing Shewanella oneidensis MR-1 in a 2×YT medium until the logarithmic phase, centrifuging and collecting, washing, and then dispersing with an inorganic salt solution to obtain a Shewanella oneidensis MR-1 bacterial solution.

[0034] In the method provided by the present invention, the inorganic salt solution of the iron porphyrin complex is preferably obtained according to the following steps: ultrasonically dispersing the powder of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin iron in an inorganic salt solution to obtain a uniform dispersion.

[0035] In the method provided by the present invention, the carbon source is preferably lactate, more preferably sodium lactate.

[0036] In the method provided by the present invention, when performing the co-culture, the bacterial cell density OD in the co-culture system 600 The initial value is preferably 0.2 to 5, and specifically may be 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.7, 1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5.

[0037] In the method provided by the present invention, when performing the co-culture, the content of the iron porphyrin complex in the co-culture system is preferably 0.02 to 0.6 g / L, and specifically may be 0.02 g / L, 0.025 g / L, 0.03 g / L, 0.035 g / L, 0.04 g / L, 0.045 g / L, 0.05 g / L, 0.055 g / L, 0.06 g / L, 0.065 g / L, 0.07 g / L, 0.075 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, 0.5 g / L, 0.55 g / L or 0.6 g / L.

[0038] In the method provided by the present invention, when performing the co-culture, the initial content of the carbon source in the co-culture system is preferably 0.02 to 0.5 mol / L, and specifically may be 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.07 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L or 0.5 mol / L.

[0039] In the method provided by the present invention, when performing the co-culture, the pH value of the co-culture system is preferably 6.5 to 10, and specifically may be 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0040] In the method provided by the present invention, the temperature of the co-culture is preferably 15 to 45 °C, and specifically may be 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C or 45 °C.

[0041] In the method provided by the present invention, the stirring rate of the co-culture is preferably 100 to 200 rpm, and specifically may be 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or 200 rpm.

[0042] In the method provided by the present invention, the co-culture time is preferably not less than 0.5 h, more preferably 0.5 - 3 h, and specifically can be 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h or 3 h.

[0043] In the method provided by the present invention, after the product of co-culturing the bacteria with the iron porphyrin complex is mixed with the hexavalent chromium-containing water, an anaerobic biological reduction system is obtained.

[0044] In the method provided by the present invention, the initial concentration of hexavalent chromium in the anaerobic biological reduction system is preferably 0.05 - 0.5 mmol / L, and specifically can be 0.05 mmol / L, 0.1 mmol / L, 0.15 mmol / L, 0.2 mmol / L, 0.25 mmol / L, 0.3 mmol / L, 0.35 mmol / L, 0.4 mmol / L, 0.45 mmol / L or 0.5 mmol / L.

[0045] In the method provided by the present invention, the bacterial cell density OD of the anaerobic biological reduction system 600 The initial value is preferably 0.2 - 2, and specifically can be 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.

[0046] In the method provided by the present invention, the content of the iron porphyrin complex in the anaerobic biological reduction system is preferably 0.02 - 0.2 g / L, and specifically can be 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L, 0.19 g / L or 0.2 g / L.

[0047] In the method provided by the present invention, the content of the carbon source in the anaerobic biological reduction system is preferably 0.02 - 0.2 mol / L, and specifically can be 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, 0.09 mol / L, 0.1 mol / L, 0.11 mol / L, 0.12 mol / L, 0.13 mol / L, 0.14 mol / L, 0.15 mol / L, 0.16 mol / L, 0.17 mol / L, 0.18 mol / L, 0.19 mol / L or 0.2 mol / L.

[0048] In the method provided by the present invention, the pH value of the anaerobic biological reduction system is preferably 6.5 to 10, and specifically can be 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0049] In the method provided by the present invention, the temperature for culturing the anaerobic biological reduction system is preferably 15 to 45 °C, and specifically can be 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C or 45 °C.

[0050] In the method provided by the present invention, the stirring rate for culturing the anaerobic biological reduction system is preferably 100 to 1000 rpm, and specifically can be 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm or 1000 rpm.

[0051] The method provided by the present invention is simple to operate, can significantly improve the extracellular electron transfer rate of Shewanella oneidensis, and further improve its rate of reducing hexavalent chromium in water. More specifically, the method provided by the present invention has at least the following effects and advantages:

[0052] (1) The present invention first discovers that TCPP(Fe) has the ability to improve the rate of reducing hexavalent chromium in water by Shewanella oneidensis MR-1 under anaerobic conditions; and first proposes a method of adding TCPP(Fe) to improve the extracellular electron transfer rate and extracellular reduction rate of hexavalent chromium of Shewanella oneidensis MR-1, providing a new idea for the treatment of heavy metal pollution.

[0053] (2) The technical solution provided by the present invention has the advantages of simple operation and remarkable effect. The present invention enhances the ability of bacteria to reduce hexavalent chromium by co-culturing with TCPP(Fe) added in advance, which is easy to operate; under optimized experimental conditions, the pseudo-first-order reaction kinetic constant of reducing hexavalent chromium by the technical solution provided by the present invention is 6.27 times that of pure bacteria, and the improvement effect is remarkable.

[0054] (3) TCPP(Fe) used in the technical solution provided by the present invention is completely obtained by chemical synthesis methods, which is beneficial to promoting the large-scale application of the present invention.

[0055] For the sake of clarity, the following is a detailed description through the following examples.

[0056] The Shewanella oneidensis MR-1 bacteria and its ΔmtrA mutant strain used in the following examples were both sourced from laboratory storage. The ΔmtrA mutant strain knocked out the mtrA gene, effectively blocking the extracellular electron transfer pathway of the bacteria, and thus could effectively inhibit the extracellular reduction of hexavalent chromium by the bacteria.

[0057] Example 1

[0058] Synthesis method of TCPP(Fe), and test its basic properties to verify the success of synthesis.

[0059] (1)Synthesis of TCPP(Fe) powder:

[0060] Step 1: Purify pyrrole

[0061] Take 18 mL of pyrrole in a 50 mL single-necked flask, distill and condense it at 160 °C, discard the first 2 - 3 mL of the condensed liquid, and collect the remaining liquid (>6 mL) into a stoppered graduated test tube to obtain purified pyrrole, which is refrigerated at 4 °C for later use.

[0062] Step 2: Synthesize TCPP-Me

[0063] Under a nitrogen atmosphere, dissolve 6.9 g of methyl 4-formylbenzoate in 100 mL of propionic acid in a 500 mL three-necked round-bottom flask. Then, gradually add 3.0 mL of the purified pyrrole obtained in Step 1 dropwise to the round-bottom flask using a constant-pressure dropping funnel. Condense and reflux the mixed solution at 150 °C for 18 h. After the reaction product cools to room temperature, filter it through qualitative filter paper to obtain approximately 1.8 g of purple crystals of TCPP-Me.

[0064] Step 3: Incorporate the central metal atom to synthesize TCPP(Fe)-Me

[0065] In a 250 mL single-necked round-bottom flask, take 1.50 g of ferrous chloride tetrahydrate and 0.450 g of TCPP-Me obtained in Step 2, and mix them in 60 mL of DMF. Then, react at 160 °C for at least 10 h. After the reaction cools to room temperature, add 150 mL of ultrapure water and mix well. Then, filter it multiple times through a 0.45 μm organic phase filter membrane. Collect the filter cake obtained by filtration, dissolve it in 100 mL of chloroform, then filter it again, and extract the collected filtrate three times with 250 mL of 1 mol / L hydrochloric acid and three times with ultrapure water respectively. Finally, place it in a fume hood and let it evaporate naturally to obtain the solid material TCPP(Fe)-Me.

[0066] Step 4: Remove the ester group to synthesize TCPP(Fe)

[0067] Take 480 mg of TCPP(Fe)-Me obtained in step 3, dissolve it with 20 mL of tetrahydrofuran and 20 mL of methanol, and mix it with 20 mL of KOH solution (prepared by dissolving 1.8 g of KOH in 20 mL of ultrapure water) in a 250 mL single-neck round-bottom flask, and reflux at 85 °C for at least 12 h. After the reaction solution is cooled to room temperature, the mixed solution is rotary evaporated to about 20 mL at a temperature above 85 °C. Then add 100 mL of ultrapure water, stir evenly, and then adjust the pH to no solid precipitation with 1 mol / L hydrochloric acid. Finally, centrifuge at 11500 rpm to collect the obtained solid, wash it three times and then freeze-dry it to obtain TCPP(Fe) with the structure shown in the following formula:

[0068] 。

[0069] (2)Test the Fourier transform infrared spectrum of the TCPP(Fe) powder synthesized by the above method. The results are as shown in A in Figure 1 . The key functional groups correspond to the functional groups in the TCPP(Fe) structural formula, partially proving the successful synthesis of the TCPP(Fe) powder.

[0070] (3)Test the ultraviolet-visible absorption spectrum of the TCPP(Fe) powder synthesized by the above method:

[0071] After dispersing the TCPP(Fe) powder synthesized by the above method and the TCPP standard product in N,N-dimethylformamide respectively, ultrasonic dispersion is carried out to obtain two solutions, and then the absorption spectra in the range of 300-800 nm are scanned. The results are as shown in B in Figure 1 . The ultraviolet-visible absorption spectra of the TCPP standard product and the TCPP(Fe) powder synthesized above show that the B-band absorption peak blue-shifts and the number of absorption peaks in the Q-band decreases from 4 to 2, which conforms to the change rule of introducing metal atoms into the center of the TCPP porphyrin ring, further proving the successful synthesis of the TCPP(Fe) powder.

[0072] Example 2

[0073] Prepare a bacteria-material co-culture solution and use it to reduce hexavalent chromium in water.

[0074] Step 1: Prepare an inorganic salt solution dispersion of the iron porphyrin complex: Ultrasonically dissolve the TCPP(Fe) synthesized by the method described in Example 1 with a sterilized inorganic salt solution without dissolved oxygen to obtain an inorganic salt solution dispersion of the iron porphyrin complex with a certain content of TCPP(Fe).

[0075] Among them, the components of the inorganic salt solution include: 8.6 mmol / L ammonium chloride, 0.5 mmol / L magnesium sulfate heptahydrate, 10 mmol / L dipotassium hydrogen phosphate, 10 mmol / L potassium dihydrogen phosphate, 1.7 mmol / L ammonium sulfate, and 10 mL / L trace element concentrate; among them, the specific formula of the trace element concentrate (per liter) is: 1.5 g nitrilotriacetic acid (NTA), 0.1 g manganese chloride tetrahydrate, 0.3 g ferric sulfate heptahydrate, 0.17 g cobalt chloride hexahydrate, 0.1 g zinc chloride, 0.04 g copper sulfate pentahydrate, 0.005 g potassium alum dodecahydrate, 0.005 g boric acid, 0.09 g sodium molybdate, 0.12 g nickel chloride, 0.02 g sodium tungstate dihydrate, and 0.01 g sodium selenate; when preparing the inorganic salt solution, after mixing each component, adjust the pH to 7.2 - 7.4 with 4 mol / L sodium hydroxide or 4 mol / L hydrochloric acid solution.

[0076] After purging the dissolved oxygen from the prepared inorganic salt solution by passing nitrogen, sterilize it to obtain a sterilized inorganic salt solution without dissolved oxygen.

[0077] Step 2: Prepare the Shewanella oneidensis MR-1 bacterial solution: Mix the bacterial solution of Shewanella oneidensis MR-1 transferred normally with 30 vol% glycerol in a volume ratio of 1:1 in a container, and store it at -80 °C. When using, add the stored bacterial solution to the 2×YT medium in a volume ratio of 1:20, activate it and then transfer it in a volume ratio of 1:1000. After culturing the transferred bacteria at 30 °C and 150 rpm until the logarithmic phase, centrifuge and wash twice with the sterilized inorganic salt solution without dissolved oxygen, and finally resuspend to obtain the Shewanella oneidensis MR-1 bacterial solution for standby. The above cultures are all carried out under aerobic conditions.

[0078] Step 3: Prepare different bacteria-material co-culture solutions. In an anaerobic glove box, mix the inorganic salt solution dispersion of the above iron porphyrin complex, the Shewanella oneidensis MR-1 bacterial solution, and sodium lactate in an anaerobic glass bottle and seal it to obtain a 19 mL bacteria-material mixed solution, which contains Shewanella oneidensis MR-1 with a cell density OD 600 of 0.21, 0.053 g / L TCPP(Fe), and 21 mmol / L sodium lactate, with a pH of 7.2 - 7.4; co-culture the sealed bacteria-material mixed solution at 30 °C and 150 rpm for 1 - 1.5 h to obtain the bacteria-material co-culture solution of the experimental group "TCPP(Fe)+MR-1".

[0079] Heat the above-mentioned bacteria-material co-culture solution in a water bath at 65 °C for 1.5 h to obtain the bacteria-material co-culture solution of the control group TCPP(Fe)+MR-1(Dead); the bacteria-material co-culture solution of the control group "MR-1" does not contain TCPP(Fe), and the other components are the same; the bacteria-material co-culture solution of the control group "TCPP(Fe)" does not contain bacterial cells, and the other components are the same.

[0080] Step 4: Use a syringe to mix the potassium dichromate solution containing 4 mmol / L hexavalent chromium (simulating chromium-containing wastewater) with the above-mentioned bacteria-material co-culture solution of the experimental group or the control group in an anaerobic glass bottle according to a volume ratio of 1:19 to obtain an anaerobic biological reduction system with a cell density OD 600 of 0.2, a TCPP(Fe) content of 0.05 g / L, a sodium lactate content of 20 mmol / L, a pH of 7.2 - 7.4, and containing 0.2 mmol / L hexavalent chromium, and stir and culture with a magnetic stirrer. The culture conditions are 15 - 25 °C (ambient temperature) and 400 - 600 rpm.

[0081] After filtering and sampling at regular intervals with a 0.22 μm aqueous mixed cellulose ester membrane, measure the concentration of hexavalent chromium in the solution. The results are as Figure 2 shown: TCPP(Fe) can effectively improve the rate of hexavalent chromium reduction in water by Shewanella oneidensis MR-1, and the part where the hexavalent chromium reduction rate is increased comes from the synergistic effect between TCPP(Fe) and bacterial vital activities.

[0082] Among them, the hexavalent chromium concentration is measured by the diphenylcarbazide method: Prepare 0.25% (w / v) diphenylcarbazide (the solvent is acetone, freshly prepared) and 0.2 mol / L dilute sulfuric acid respectively. Take 0.08 mL of 0.25% (w / v) diphenylcarbazide, 0.9 mL of 0.2 mol / L dilute sulfuric acid and 0.05 mL of the sample to be measured and mix evenly. React at room temperature for 15 - 30 min, and measure the absorbance at a wavelength of 540 nm. Draw a standard curve before each experiment.

[0083] Example 3

[0084] Experiment on the influence of different experimental conditions on the reduction of hexavalent chromium in water by the bacteria-material co-culture solution.

[0085] (1) Rate of reduction of hexavalent chromium in water by the bacteria-material co-culture solution with different additions of TCPP(Fe):

[0086] According to the method of the experimental group in Example 2, only the dosage of TCPP(Fe) in the bacteria-material mixed solution was changed to 0.105 g / L, 0.079 g / L, 0.053 g / L, 0.026 g / L, and 0.011 g / L, and anaerobic biological reduction systems with TCPP(Fe) dosages of 100 mg / L, 75 mg / L, 50 mg / L, 25 mg / L, and 10 mg / L were respectively prepared. They were stirred and cultured with a magnetic stirrer, and the stirring and culturing conditions were the same as those in Example 2. After sampling on time, the concentration of hexavalent chromium in the solution was measured.

[0087] The test results are as Figure 3 shown in A of

[0088] (2) The reduction rate of hexavalent chromium in water by the bacteria-material co-culture solution at different bacterial contents:

[0089] According to the method of the experimental group in Example 2, only the bacterial cell density OD 600 in the bacteria-material mixed solution was changed to 0.53, 0.42, 0.32, 0.21, and 0.11, and anaerobic biological reduction systems with bacterial cell densities OD 600 labeled as 0.5, 0.4, 0.3, 0.2, and 0.1 were respectively prepared. They were stirred and cultured with a magnetic stirrer, and the stirring and culturing conditions were the same as those in Example 2. After sampling on time, the concentration of hexavalent chromium in the solution was measured.

[0090] The test results are as Figure 3 shown in B of 600 The test results show that the reduction rate of hexavalent chromium in water by the cooperation of TCPP(Fe) and Shewanella oneidensis MR-1 bacteria shows a relatively high level when the bacterial cell density OD

[0091] (3) The reduction rate of hexavalent chromium in water by the bacteria-material co-culture solution at different temperatures:

[0092] An anaerobic biological reduction system was prepared according to the method of the experimental group in Example 2, and it was stirred and cultured with a magnetic stirrer at a stirring speed of 400 - 600 rpm. Only the culture temperatures were respectively changed to 50 °C, 40 °C, 30 °C, 20 °C, and 10 °C. After sampling on time, the concentration of hexavalent chromium in the solution was measured.

[0093] The test results are as Figure 3As shown in C in [reference], the test results show that the rate of synergistic reduction of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 is at a relatively high level under the condition of 20 - 40 °C.

[0094] (4)Rate of reduction of hexavalent chromium in water by the bacteria-material co-culture solution at different initial hexavalent chromium concentrations:

[0095] Prepare the bacteria-material co-culture solution according to the method of the experimental group in Example 2. Mix potassium dichromate solutions (simulating chromium-containing wastewater) containing 10 mmol / L, 6 mmol / L, 4 mmol / L, 2 mmol / L, and 1 mmol / L of hexavalent chromium with the bacteria-material co-culture solution at a volume ratio of 1:19 respectively to obtain anaerobic biological reduction systems with a pH of 7.2 - 7.4 and containing 500 μmol / L, 300 μmol / L, 200 μmol / L, 100 μmol / L, and 50 μmol / L of hexavalent chromium respectively. Stir and culture with a magnetic stirrer, and the stirring and culturing conditions are the same as those in Example 2. Take samples at regular intervals and then measure the concentration of hexavalent chromium in the solution.

[0096] The test results are as Figure 3 shown in D in [reference]. The test results show that the rate of synergistic reduction of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 is at a relatively high level under the condition that the initial concentration of hexavalent chromium is 50 - 500 μmol / L.

[0097] (5)Rate of reduction of hexavalent chromium in water by the bacteria-material co-culture solution at different pH values:

[0098] Prepare the anaerobic biological reduction system according to the method of the experimental group in Example 2. Only adjust its pH to 5.8 - 6.0, 6.3 - 6.5, 7.1 - 7.3, 7.8 - 8.0, 8.3 - 8.5 by adding hydrochloric acid to obtain anaerobic biological reduction systems with pH marked as 6.0, 6.5, 7.3, 8.0, and 8.5. Stir and culture with a magnetic stirrer, and the stirring and culturing conditions are the same as those in Example 2. Take samples at regular intervals and then measure the concentration of hexavalent chromium and the pH value in the solution.

[0099] The test results are as Figure 3 shown in E and F in [reference]. The test results show that the rate of synergistic reduction of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 is at a relatively high level under the condition of pH 6.5 - 8.5; the pH value is relatively stable during the reaction process.

[0100] In summary, based on the experimental results of (1) to (5), it can be seen that by changing the experimental conditions, a better synergistic reduction rate of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 can be obtained.

[0101] Example 4

[0102] According to the methods of the experimental group and the control group "MR-1" in Example 2, only the cell density OD of Shewanella oneidensis MR-1 in the bacteria-material mixed solution was changed 600 to 0.42 and the carbon source was sodium lactate at 42 mmol / L. Anaerobic biological reduction systems of the experimental group "condition-optimized MR-1 + TCPP(Fe)" and the control group "condition-optimized MR-1" were respectively prepared, which contained a bacterial cell density OD 600 labeled as 0.4 and the sodium lactate content labeled as 40 mmol / L. Stirring culture was carried out under the same conditions as in Example 2, and the concentration of hexavalent chromium in the solution was measured after sampling on time.

[0103] The test results are as Figure 4 shown. It can be seen from Figure 4 that the technical method provided by this application can increase the reduction rate of hexavalent chromium in water by Shewanella oneidensis MR-1 to 6.27 times under optimized experimental conditions.

[0104] Example 5

[0105] According to the methods of the experimental group and the control group "MR-1" in Example 4, only the wild strain of Shewanella oneidensis MR-1 used in Example 5 was replaced with the ΔmtrA mutant strain of Shewanella oneidensis MR-1 with the mtrA gene knocked out. Anaerobic biological reduction systems of the experimental group "ΔmtrA + TCPP(Fe)" and the control group "ΔmtrA" were respectively prepared, which contained a bacterial cell density OD 600 labeled as 0.4 and the sodium lactate content labeled as 40 mmol / L. Stirring culture was carried out under the same conditions as in Example 4, and the concentration of hexavalent chromium in the solution was measured after sampling on time.

[0106] The test results are shown in Figure 5 . Figure 5 It shows that after effectively blocking the extracellular electron transfer pathway, TCPP(Fe) will not significantly increase the reduction rate of hexavalent chromium by the ΔmtrA mutant strain bacteria. Combining with the attached Figure 4The test results of the wild strain of Shewanella oneidensis MR-1 bacteria in indicate that the enhancement of the ability of the wild strain bacteria to reduce hexavalent chromium by TCPP(Fe) mainly stems from the enhancement of its extracellular ability to reduce hexavalent chromium.

[0107] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for increasing the rate of bacterial reduction of hexavalent chromium in water using an iron porphyrin complex, characterized in that: The following steps are involved: Under anaerobic conditions, bacteria and iron porphyrin complexes are co-cultured and then mixed with hexavalent chromium-containing water. During the mixed culture, the hexavalent chromium in the water is reduced by bacteria. Shewanella Oneida Shewanella oneidensis MR-1; The iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl)iron porphyrin.

2. The method according to claim 1, characterized in that: The co-cultivation is carried out in a culture solution, and the bacterial cell density OD 600 The initial value is 0.2~5, and the content of iron porphyrin complex is 0.02~0.6g / L.

3. The method according to claim 2, characterized in that The pH value during the co-cultivation is 6.5-10.

4. The method according to claim 2, characterized in that: The co-cultivation temperature is 15-45°C.

5. The method according to claim 2, characterized in that: The stirring rate of the co-cultivation is 100-200 rpm.

6. The method according to claim 1, characterized in that After being mixed with the hexavalent chromium-containing water, the initial hexavalent chromium concentration of the mixed system is 0.05-0.5 mmol / L.

7. The method according to claim 1, characterized in that After mixing with the hexavalent chromium-containing water, the bacterial cell density OD of the mixed system 600 The initial value is 0.2~2, and the content of iron porphyrin complex is 0.02~0.2g / L.

8. The method according to claim 1, characterized in that After being mixed with the hexavalent chromium-containing water, the pH value of the mixed system is 6.5-10.

9. The method according to claim 1, characterized in that: The temperature of the mixed culture is 15-45°C.

10. The method according to claim 1, characterized in that The stirring rate of the mixed culture is 100-1000 rpm.

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