Method for increasing speed of reducing hexavalent chromium in water by bacteria through ferriporphyrin complex

Abstract

The invention belongs to the field of water treatment, and particularly relates to a method for increasing the speed of reducing hexavalent chromium in water by bacteria through a ferriporphyrin complex, which comprises the following steps: under an anaerobic condition, the bacteria and the ferriporphyrin complex are co-cultured and then are mixed and cultured with hexavalent chromium-containing water, and the hexavalent chromium in the water is reduced by the bacteria during the mixed culture period; the bacterial strain is Shewanella oneidensis MR-1, and the bacterial strain is Swanella oneidensis MR-1. The iron porphyrin complex is 5, 10, 15, 20-tetra (4-carboxyl phenyl) iron porphyrin, and the iron porphyrin complex is 5, 10, 15, 20-tetra (4-carboxyl phenyl) iron porphyrin. The method provided by the invention has the advantages of remarkable effect and simplicity in operation, and a new idea is provided for improving the heavy metal pollution remediation rate of the dissimilatory metal reducing bacteria. In addition, the ferriporphyrin complex can be obtained in an artificial synthesis mode, and large-scale application of the technical scheme is promoted.

Description

Technical Field

[0001] The invention belongs to the field of water treatment, and in particular relates to a method for improving the rate of bacteria reducing hexavalent chromium in water by using an iron porphyrin complex. Background Art

[0002] Chromium is widely used in industries such as electroplating, leather making, chemical industry, and printing and dyeing, which directly leads to the generation of a large amount of chromium-containing wastewater. If these chromium-containing wastewaters are discharged directly without effective treatment, they will pose a considerable threat to water bodies, soil, and ecosystems. Therefore, although the governance technology and regulatory system have made great progress, chromium is still one of the main heavy metals that cause environmental pollution, and its governance and prevention and control issues are still 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 a very strong biological toxicity and migration ability, and is the main source of biological toxicity of chromium-containing wastewater; in contrast, the toxicity and teratogenicity of trivalent chromium are far less than hexavalent chromium, and trivalent chromium has a lower solubility in neutral and weakly alkaline environments, and is easier to precipitate and fix. Therefore, the main idea of ​​treating chromium-containing wastewater is to reduce the hexavalent chromium in it to trivalent chromium, which is less toxic and easy to fix.

[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 additional chemical agent consumption caused by traditional chemical treatment, but also reduces the risk of secondary pollution, and has considerable application potential. Shewanella oneidensis MR-1 is one of the most widely studied model strains of DMRB, which can reduce hexavalent chromium to trivalent chromium. The bacterium can not only reduce hexavalent chromium with enzymes inside the cell, but also use a unique extracellular electron transfer pathway to transfer intracellular electrons to extracellular hexavalent chromium, thereby achieving extracellular reduction of hexavalent chromium. The process of extracellular reduction of hexavalent chromium circumvents the rate limitation of hexavalent chromium transmembrane transfer, and at the same time can reduce the toxic effects of hexavalent chromium and its reduction products in the cell. However, the natural extracellular electron transfer rate of wild-type DMRB is generally low, which restricts the rate of bacterial extracellular reduction of hexavalent chromium and severely limits the overall hexavalent chromium bioreduction rate. Therefore, increasing the extracellular electron transfer rate between Shewanella oneidensis MR-1 and hexavalent chromium is a very valuable way to increase the hexavalent chromium reduction rate.

[0004] The prior art provides a variety of methods for hybridizing DMRB with non-biological materials to enhance the extracellular electron transfer capacity, thereby increasing the hexavalent chromium reduction rate, including in-situ nanomaterial modification of cells and exogenous addition of conductive media, but there are generally problems such as complex operation, difficulty in large-scale application, and weak improvement effect. For example, patent CN 117821323 A discloses a method for modifying Shewanella cells with biogenic silver nanoparticles to enhance the extracellular electron transfer capacity and promote hexavalent chromium reduction, but the operation method is relatively complicated and requires microorganisms to generate attached nanoparticles in situ under a specific culture environment; patent CN 113149230A discloses a treatment method for adding wood biochar electron transfer to enhance microbial reduction of hexavalent chromium, using wood biochar as an electron transfer medium between bacteria and hexavalent chromium to increase the electron transfer rate to increase the hexavalent chromium reduction rate, but the improvement effect is weak. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for improving the rate of bacterial reduction of hexavalent chromium in water using an iron porphyrin complex. The method is simple to operate and can significantly improve the extracellular electron transfer rate of Shewanella Oneida, thereby improving its rate of reducing hexavalent chromium in water.

[0006] The present invention provides a method for improving the rate of bacterial reduction of hexavalent chromium in water by using an iron porphyrin complex, comprising the following steps: 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. The bacteria is Shewanella oneidensis MR-1; The iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl)iron porphyrin.

[0007] Preferably, the co-cultivation is carried out in a culture medium, and the bacterial cell density OD during the co-cultivation is 600 The initial value is 0.2~5, and the content of iron porphyrin complex is 0.02~0.6g / L.

[0008] Preferably, the pH value during the co-cultivation is 6.5-10.

[0009] Preferably, the co-cultivation temperature is 15-45°C.

[0010] Preferably, the stirring rate of the co-culture is 100-200 rpm.

[0011] Preferably, after mixing with the hexavalent chromium-containing water, the initial hexavalent chromium concentration of the mixed system is 0.05-0.5 mmol / L.

[0012] Preferably, after mixing with the hexavalent chromium-containing water, the bacterial cell density OD of the mixed system is 600 The initial value is 0.2~2, and the content of iron porphyrin complex is 0.02~0.2g / L.

[0013] Preferably, after mixing with the hexavalent chromium-containing water, the pH value of the mixed system is 6.5-10.

[0014] Preferably, the temperature of the mixed culture is 15-45°C.

[0015] Preferably, the stirring rate of the mixed culture is 100~1000rpm.

[0016] Compared with the prior art, the present invention provides a method for improving the rate of bacteria reducing hexavalent chromium in water by using an iron porphyrin complex, comprising the following steps: under anaerobic conditions, bacteria and an iron porphyrin complex are co-cultured and then mixed with hexavalent chromium-containing water, and the hexavalent chromium in the water is reduced by bacteria during the mixed culture; the bacteria is Shewanella oneidensis MR-1; the iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl) iron porphyrin. The present invention utilizes Shewanella oneidensis MR-1 and a specific iron porphyrin complex (5,10,15,20-tetrakis(4-carboxyphenyl) iron porphyrin) for mixed co-culture to obtain a bacteria-material co-culture that enhances extracellular electron transfer, and the co-culture can efficiently reduce hexavalent chromium in water, thereby achieving the purpose of significantly improving the hexavalent chromium reduction rate of Shewanella oneidensis. The method provided by the present invention has the advantages of significant effect and simple operation, and provides a new idea for improving the rate of DMRB repair of heavy metal pollution. 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0018] Figure 1 1 is a graph showing the properties of the TCPP(Fe) powder synthesized in Example 1 of the present invention; wherein A is a Fourier infrared spectrum of TCPP(Fe), and B is a UV-visible absorption spectrum of TCPP(Fe) and TCPP; Figure 2It is a graph showing the test results of the rate of reducing hexavalent chromium in water by the bacteria-material co-culture solution provided in Example 2 of the present invention and the results of the control group; Figure 3 3 is a result diagram of the experiment on the influence of different experimental conditions on the reduction of hexavalent chromium in water by bacteria-material co-culture solution provided in Example 3 of the present invention; wherein, A is a detection result diagram of the rate of Cr(VI) in water reduced by bacteria-material co-culture solution at different TCPP(Fe) dosages, B is a detection result diagram of the rate of Cr(VI) in water reduced by bacteria-material co-culture solution at different bacteria dosages, C is a detection result diagram of the rate of Cr(VI) in water reduced by bacteria-material co-culture solution at different temperatures, D is a detection result diagram of the rate of Cr(VI) in water reduced by bacteria-material co-culture solution at different initial Cr(VI) concentrations, E is a detection result diagram of the rate of Cr(VI) in water reduced by bacteria-material co-culture solution under different pH environments, and F is a detection result diagram of the pH value of the reaction solution during the E experiment; Figure 4 It is a diagram showing the effect of reducing hexavalent chromium in water by a bacteria-material co-culture solution under the optimized experimental conditions provided in Example 4 of the present invention; wherein A is a detection result of the rate of reducing hexavalent chromium in water by a bacteria-material co-culture solution under the optimized experimental conditions and a result diagram of a control group, B is a linear fitting result diagram calculated based on A, and C is a pseudo-first-order reaction kinetic result diagram of hexavalent chromium reduction in a solution calculated based on A and B; Figure 5 This is a diagram showing the effect of reducing hexavalent chromium in water by a ΔmtrA mutant bacteria-material co-culture solution under the optimized experimental conditions provided in Example 5 of the present invention. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The present invention provides a method for improving the rate of bacterial reduction of hexavalent chromium in water by using an iron porphyrin complex, comprising the following steps: 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. The bacteria is Shewanella oneidensis MR-1; The iron porphyrin complex is 5,10,15,20-tetrakis(4-carboxyphenyl)iron porphyrin.

[0021] In the method provided by the present invention, the specific process of co-cultivation preferably includes: mixing bacterial culture, inorganic salt solution of iron porphyrin complex and carbon source to obtain a bacteria-material mixed solution; then shaking culture and reaching a certain anaerobic level to obtain a bacteria-material co-cultivation solution.

[0022] In the method provided by the present invention, the components of the bacterial culture liquid 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) iron porphyrin (TCPP(Fe)) and an inorganic salt solution; the inorganic salt solution should provide the pH conditions, osmotic pressure and trace nutrients required to maintain the growth of Shewanella oneidensis MR-1; the carbon source is preferably an organic matter that can be used for the metabolism of Shewanella oneidensis MR-1; the certain anaerobic level preferably needs to meet the requirement of Shewanella oneidensis MR-1 to transfer electrons to extracellular hexavalent chromium.

[0023] In the method provided by the present invention, the bacterial culture liquid is preferably obtained according to the following steps: Shewanella oneidensis MR-1 is cultured in 2×YT medium until the logarithmic phase, collected by centrifugation, washed, and then dispersed with an inorganic salt solution to obtain Shewanella oneidensis MR-1 culture liquid.

[0024] 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)iron porphyrin in the inorganic salt solution to obtain a uniform dispersion.

[0025] In the method provided by the present invention, the carbon source is preferably lactate, more preferably sodium lactate.

[0026] In the method provided by the present invention, when the co-culture is performed, the bacterial cell density OD in the co-culture system is 600 The initial value is preferably 0.2-5, specifically 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.

[0027] In the method provided by the present invention, when the co-cultivation is carried out, the content of the iron porphyrin complex in the co-cultivation system is preferably 0.02-0.6 g / L, specifically 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.

[0028] In the method provided by the present invention, when the co-cultivation is carried out, the initial content of the carbon source in the co-cultivation system is preferably 0.02-0.5 mol / L, specifically 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.

[0029] In the method provided by the present invention, when the co-culture is carried out, the pH value of the co-culture system is preferably 6.5-10, specifically 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0030] In the method provided by the present invention, the co-culture temperature is preferably 15-45°C, specifically 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C.

[0031] In the method provided by the present invention, the stirring rate of the co-culture is preferably 100-200 rpm, specifically 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or 200 rpm.

[0032] In the method provided by the present invention, the co-cultivation time is preferably not less than 0.5 h, more preferably 0.5-3 h, specifically 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h or 3 h.

[0033] In the method provided by the present invention, the product of the co-culture of the bacteria and the iron porphyrin complex is mixed with water containing hexavalent chromium to obtain an anaerobic biological reduction system.

[0034] 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, specifically 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.

[0035] In the method provided by the present invention, the bacterial cell density OD of the anaerobic bioreduction system is 600 The initial value is preferably 0.2-2, specifically 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.

[0036] In the method provided by the present invention, the iron porphyrin complex content of the anaerobic biological reduction system is preferably 0.02-0.2 g / L, specifically 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.

[0037] In the method provided by the present invention, the carbon source content of the anaerobic biological reduction system is preferably 0.02-0.2 mol / L, specifically 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.

[0038] In the method provided by the present invention, the pH value of the anaerobic biological reduction system is preferably 6.5-10, specifically 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

[0039] In the method provided by the present invention, the temperature for culturing the anaerobic bioreduction system is preferably 15-45°C, specifically 15°C, 20°C, 25°C, 30°C, 35°C, 40°C or 45°C.

[0040] In the method provided by the present invention, the stirring rate of the anaerobic bioreduction system for cultivation is preferably 100-1000 rpm, specifically 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm or 1000 rpm.

[0041] The method provided by the present invention is simple to operate and can significantly increase the extracellular electron transfer rate of Shewanella Oneida, thereby increasing 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: (1) The present invention first discovered that TCPP(Fe) has the ability to increase the rate of hexavalent chromium reduction in water by Shewanella oneidensis MR-1 under anaerobic conditions; and first proposed a method for adding TCPP(Fe) to increase the extracellular electron transfer rate and the extracellular reduction rate of hexavalent chromium by Shewanella oneidensis MR-1, which provides a new idea for the control of heavy metal pollution.

[0042] (2) The technical solution provided by the present invention has the advantages of simple operation and significant effect. The present invention enhances the ability of bacteria to reduce hexavalent chromium by adding TCPP (Fe) in advance and co-culturing with bacteria, which is easy to operate; under optimized experimental conditions, the pseudo-first-order reaction kinetic constant of the technical solution provided by the present invention for reducing hexavalent chromium is 6.27 times that of pure bacteria, and the improvement effect is significant.

[0043] (3) The TCPP(Fe) used in the technical solution provided by the present invention is completely obtained by chemical synthesis method, which is conducive to promoting the large-scale application of the present invention.

[0044] For the purpose of greater clarity, the invention is described in detail through the following examples.

[0045] The Shewanella oneidensis MR-1 bacteria and its ΔmtrA mutant used in the following examples are all from laboratory storage. The ΔmtrA mutant has the mtrA gene knocked out, effectively blocking the extracellular electron transfer pathway of the bacteria, and thus can effectively inhibit the extracellular reduction of hexavalent chromium by the bacteria.

[0046] Example 1 The synthesis method of TCPP(Fe) was developed, and its basic properties were tested to verify its successful synthesis.

[0047] (1) Synthesis of TCPP(Fe) powder: Step 1: Purification of pyrrole 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 condensed liquid, collect the remaining liquid (>6 mL) into a stoppered graduated test tube to obtain purified pyrrole, and refrigerate at 4 °C for later use.

[0048] Step 2: Synthesize TCPP-Me Under nitrogen, 6.9 g of methyl p-formylbenzoate was dissolved in 100 mL of propionic acid in a 500 mL three-necked round-bottom flask, and then 3.0 mL of pyrrole purified in step 1 was added dropwise into the round-bottom flask using a constant pressure separatory funnel. The mixed solution was condensed and refluxed at 150°C for 18 h. After the reaction product was cooled to room temperature, about 1.8 g of TCPP-Me purple crystals were obtained by suction filtration using qualitative filter paper.

[0049] Step 3: Inserting the central metal atom to synthesize TCPP(Fe)-Me In a 250mL single-mouth round-bottom flask, take 1.50g of ferrous chloride tetrahydrate and 0.450g of TCPP-Me obtained in step 2, and mix them in 60mL of DMF. Then react at 160°C for at least 10h. When the reaction cools to room temperature, add 150mL of ultrapure water and mix thoroughly, then filter with a 0.45μm organic phase filter membrane several times. Collect the filter cake obtained by filtration, dissolve it in 100mL of chloroform, then filter it again, and extract the collected filtrate three times with 250mL of 1mol / L hydrochloric acid and three times with ultrapure water, and finally place it in a fume hood and evaporate it naturally to obtain a solid material TCPP(Fe)-Me.

[0050] Step 4: Removal of ester group to synthesize TCPP(Fe) Take 480mg of TCPP(Fe)-Me obtained in step 3, dissolve it in 20mL of tetrahydrofuran and 20mL of methanol, and mix it with 20mL of KOH solution (1.8g of KOH dissolved in 20mL of ultrapure water) in a 250mL single-mouth round-bottom flask, and reflux it at 85°C for at least 12h. After the reaction solution cools to room temperature, the mixed solution is evaporated to about 20mL at a temperature above 85°C. Then add 100mL of ultrapure water, stir evenly, and then adjust the pH with 1mol / L hydrochloric acid until no solid precipitates. Finally, collect the obtained solid by centrifugation at 11500rpm, wash it three times and freeze-dry it to obtain TCPP(Fe) with the structure shown in the following formula: .

[0051] (2) The Fourier transform infrared spectrum of TCPP(Fe) powder synthesized by the above method was tested. The results are as follows: Figure 1 As shown in A, the key functional groups correspond to the functional groups in the structure of TCPP(Fe), which partially proves that the TCPP(Fe) powder was successfully synthesized.

[0052] (3) Testing the UV-visible absorption spectrum of TCPP(Fe) powder synthesized by the above method: The TCPP(Fe) powder and TCPP standard prepared by the above method were dispersed in N,N-dimethylformamide, respectively, and two solutions were obtained by ultrasonic dispersion and then scanned to obtain the absorption spectrum in the range of 300-800 nm. Figure 1 As shown in B, the change in the UV-visible absorption spectra of TCPP standard and the TCPP(Fe) powder synthesized above is manifested in the blue shift of the B band absorption peak, and the absorption peaks of the Q band are reduced from 4 to 2, which is consistent with the change law of introducing metal atoms into the center of the TCPP porphyrin ring, once again proving that the synthesis of TCPP(Fe) powder is successful.

[0053] Example 2 A bacteria-material co-culture solution was prepared and used to reduce hexavalent chromium in water.

[0054] Step 1: preparing an inorganic salt solution dispersion of an iron porphyrin complex: ultrasonically dissolving the TCPP(Fe) synthesized by the method described in Example 1 with a sterilized inorganic salt solution free of dissolved oxygen to obtain an inorganic salt solution dispersion of an iron porphyrin complex having a certain content of TCPP(Fe).

[0055] The inorganic salt solution includes: 8.6mmol / L ammonium chloride, 0.5mmol / L magnesium sulfate heptahydrate, 10mmol / L potassium dihydrogen phosphate, 10mmol / L potassium dihydrogen phosphate, 1.7mmol / L ammonium sulfate and 10mL / L trace element concentrate; the specific formula of the trace element concentrate is (per liter): 1.5g nitrilotriacetic acid (NTA), 0.1g manganese chloride tetrahydrate, 0.3g ferric sulfate heptahydrate, 0.17g cobalt chloride hexahydrate, 0.1g zinc chloride, 0.04g copper sulfate pentahydrate, 0.005g potassium aluminum sulfate dodecahydrate, 0.005g boric acid, 0.09g sodium molybdate, 0.12g nickel chloride, 0.02g sodium tungstate dihydrate and 0.01g sodium selenate; when preparing the inorganic salt solution, it is necessary to adjust the pH to 7.2~7.4 with 4mol / L sodium hydroxide or 4mol / L hydrochloric acid solution after mixing the components. The prepared inorganic salt solution is sterilized after passing nitrogen gas to remove dissolved oxygen to obtain a sterile inorganic salt solution free of dissolved oxygen.

[0056] Step 2: Prepare the bacterial solution of Shewanella oneidensis MR-1: Mix the bacterial solution of Shewanella oneidensis MR-1 transferred normally with 30vol% glycerol in a volume ratio of 1:1 in a container and store it at -80℃. When using, add the stored bacterial solution to 2×YT medium at a volume ratio of 1:20, activate it and transfer it at a volume ratio of 1:1000. After the transferred bacteria are cultured at 30℃ and 150rpm to the logarithmic phase, centrifuge and wash twice with a sterilized inorganic salt solution without dissolved oxygen, and finally resuspend to obtain the bacterial solution of Shewanella oneidensis MR-1 for use. The above culture is carried out under aerobic conditions.

[0057] Step 3: Prepare different bacteria-material co-culture solutions. In an anaerobic operating box, use an anaerobic glass bottle to mix the inorganic salt solution dispersion of the iron porphyrin complex, the bacterial solution of Shewanella oneidensis MR-1 and sodium lactate, and then seal it to obtain 19 mL of bacteria-material mixed solution, which contains a cell density OD 600 The concentration of the mixture was 0.21 of Shewanella oneidensis MR-1, 0.053 g / L TCPP(Fe) and 21 mmol / L sodium lactate, and the pH was 7.2-7.4; the sealed bacteria-material mixed solution was co-cultured 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".

[0058] The above-mentioned bacteria-material co-culture solution was heated in a 65°C water bath 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" did not contain TCPP(Fe), and the other components were the same; the bacteria-material co-culture solution of the control group "TCPP(Fe)" did not contain bacterial cells, and the other components were the same.

[0059] Step 4: Use a syringe to mix the potassium dichromate solution containing 4mmol / L hexavalent chromium (simulating chromium-containing wastewater) with the bacteria-material co-culture solution of the above experimental group or control group in an anaerobic glass bottle at a volume ratio of 1:19 to obtain the cell density OD 600 The anaerobic bioreduction system is an anaerobic bioreduction system with a pH of 7.2-7.4 and a TCPP (Fe) content of 0.05 g / L, a sodium lactate content of 20 mmol / L, and a hexavalent chromium content of 0.2 mmol / L. The culture is stirred with a magnetic stirrer. The culture conditions are 15-25°C (ambient temperature) and 400-600 rpm.

[0060] After filtering samples with a 0.22μm water-based mixed fiber filter membrane at regular intervals, the hexavalent chromium concentration in the solution was measured. The results are as follows: Figure 2 As shown: TCPP (Fe) can effectively improve the rate of hexavalent chromium reduction in water by Shewanella oneidensis MR-1, and the part of the improved hexavalent chromium reduction rate comes from the synergistic effect between TCPP (Fe) and bacterial life activities.

[0061] The hexavalent chromium concentration was determined by the diphenylcarbohydrazide method: 0.25% (w / v) diphenylcarbohydrazide (solvent is acetone, prepared and used immediately) and 0.2 mol / L dilute sulfuric acid were prepared respectively, 0.08 mL of 0.25% (w / v) diphenylcarbohydrazide, 0.9 mL of 0.2 mol / L dilute sulfuric acid and 0.05 mL of the sample to be tested were mixed evenly, reacted at room temperature for 15-30 minutes, and the absorbance at a wavelength of 540 nm was measured. A standard curve was drawn before each experiment.

[0062] Example 3 Experiment on the effects of different experimental conditions on the reduction of hexavalent chromium in water by bacteria-material co-culture solution.

[0063] (1) Reduction rate of hexavalent chromium in water by bacteria-material co-culture solution under different TCPP(Fe) additions: According to the method of the experimental group in Example 2, only the TCPP (Fe) dosage 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 the anaerobic bioreduction system with TCPP (Fe) dosage of 100 mg / L, 75 mg / L, 50 mg / L, 25 mg / L, and 10 mg / L was prepared, respectively. The culture was stirred with a magnetic stirrer. The stirring culture conditions were the same as in Example 2. The hexavalent chromium concentration in the solution was measured after sampling on time.

[0064] Test results such as Figure 3 As shown in A, the test results show that the rate of synergistic reduction of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 bacteria is at a high level when the TCPP(Fe) content is between 25 and 100 mg / L.

[0065] (2) The rate of reducing hexavalent chromium in water by bacteria-material co-culture solution at different bacterial contents: According to the method of the experimental group of Example 2, only the bacterial cell density OD in the bacteria-material mixed solution was changed. 600 are 0.53, 0.42, 0.32, 0.21, and 0.11, respectively, to obtain the bacterial cell density OD 600The anaerobic bioreduction systems marked as 0.5, 0.4, 0.3, 0.2, and 0.1 were stirred and cultured using a magnetic stirrer. The stirring and culture conditions were the same as those in Example 2. Samples were taken at regular intervals to measure the hexavalent chromium concentration in the solution.

[0066] Test results such as Figure 3 As shown in B, the test results show that TCPP (Fe) and Shewanella oneidensis MR-1 bacteria synergistically reduce the rate of hexavalent chromium in water at the bacterial cell density OD 600 When the value was not less than 0.2, the results showed that the level was high.

[0067] (3) The rate of reducing hexavalent chromium in water by bacteria-material co-culture solution at different temperatures: The anaerobic bioreduction system was prepared according to the method of the experimental group in Example 2, and the culture was stirred with a magnetic stirrer at a speed of 400-600 rpm. Only the culture temperature was changed to 50°C, 40°C, 30°C, 20°C, and 10°C, respectively. The hexavalent chromium concentration in the solution was measured after sampling on time.

[0068] Test results such as Figure 3 As shown in C, the test results show that TCPP(Fe) and Shewanella oneidensis MR-1 bacteria synergistically reduce hexavalent chromium in water at a high level under the condition of 20~40℃.

[0069] (4) Reduction rate of hexavalent chromium in water by bacteria-material co-culture solution at different initial hexavalent chromium concentrations: According to the method of the experimental group of Example 2, a bacteria-material co-culture solution was prepared, and potassium dichromate solutions containing 10 mmol / L, 6 mmol / L, 4 mmol / L, 2 mmol / L and 1 mmol / L hexavalent chromium (simulated chromium-containing wastewater) were mixed with the bacteria-material co-culture solution at a volume ratio of 1:19 to obtain an anaerobic bioreduction system 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 hexavalent chromium, respectively. The culture was stirred with a magnetic stirrer. The stirring culture conditions were the same as in Example 2, and the hexavalent chromium concentration in the solution was measured after sampling on time.

[0070] Test results such as Figure 3 As shown in D, the test results show that the synergistic reduction rate of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1 bacteria was at a high level when the initial hexavalent chromium concentration was 50-500 μmol / L.

[0071] (5) Reduction rate of hexavalent chromium in water by bacteria-material co-culture solution at different pH values: According to the method of the experimental group of Example 2, an anaerobic bioreduction system was prepared, and its pH was adjusted 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 alone, to obtain anaerobic bioreduction systems with pH marked as 6.0, 6.5, 7.3, 8.0, and 8.5. The culture was stirred with a magnetic stirrer. The stirring and culture conditions were the same as in Example 2. The hexavalent chromium concentration and pH value in the solution were measured after sampling on time.

[0072] Test results such as Figure 3 As shown in Figures E and F, the test results show that TCPP(Fe) and Shewanella oneidensis MR-1 bacteria synergistically reduce hexavalent chromium in water at a high rate under conditions of pH 6.5-8.5; the pH value is relatively stable during the reaction.

[0073] In summary, based on the experimental results of (1) to (5), it can be seen that changing the experimental conditions can obtain a better rate of synergistic reduction of hexavalent chromium in water by TCPP(Fe) and Shewanella oneidensis MR-1.

[0074] Example 4 According to the method 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 The anaerobic bioreduction system of the experimental group "Optimized MR-1+TCPP(Fe)" and the control group "Optimized MR-1" were prepared, respectively, containing bacterial cell density OD 600 The content of sodium lactate is marked as 0.4 and the content of sodium lactate is marked as 40 mmol / L. The mixture was stirred and cultured under the same conditions as in Example 2, and the concentration of hexavalent chromium in the solution was measured after sampling at regular intervals.

[0075] Test results such as Figure 4 As shown. Figure 4 It can be seen that the technical method provided by the present application can increase the rate of reducing hexavalent chromium in water by Shewanella oneidensis MR-1 to 6.27 times under optimized experimental conditions.

[0076] Example 5 According to the method of the experimental group and the control group "MR-1" in Example 4, only the ΔmtrA mutant of Shewanella oneidensis MR-1 in which the mtrA gene is knocked out is used to replace the wild strain of Shewanella oneidensis MR-1 used in Example 5, and the anaerobic bioreduction systems of the experimental group "ΔmtrA+TCPP(Fe)" and the control group "ΔmtrA" are prepared, respectively, wherein the bacterial cell density OD 600 The content of sodium lactate is marked as 0.4 and 40 mmol / L. The mixture was stirred and cultured under the same conditions as in Example 4, and the hexavalent chromium concentration in the solution was measured after sampling at regular intervals.

[0077] Test results see Figure 5 . Figure 5 This indicates that TCPP(Fe) will not significantly increase the rate of hexavalent chromium reduction by the ΔmtrA mutant bacteria after effectively blocking the extracellular electron transfer pathway. Figure 4 The test results of the wild-type Shewanella oneidensis MR-1 bacteria in the experiment indicate that the improvement of TCPP(Fe) on the ability of wild-type bacteria to reduce hexavalent chromium mainly comes from the enhancement of its ability to reduce hexavalent chromium extracellularly.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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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