A method for the reduction and fixation of Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model

Abstract

The present invention discloses a method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model: S1, preparing a mixed functional bacterial community electrolyte consisting of conductive bacteria and metal-reducing functional bacterial communities, wherein the conductive bacteria is Shewanella, and the metal-reducing functional bacterial communities include one or more of Bacillus cereus, Ralstonia pietermaritzii, and Paenibacillus alvei; S2, enriching an electroactive biofilm: pouring the prepared mixed functional bacterial community electrolyte into an electrolytic cell in a three-electrode bioelectrochemical system, and applying a positive potential of 100 to 300 mV; S3, reducing and fixing Cr(VI): after generating a mature anode biofilm with a stable current output, pouring out the electrolyte in the electrolytic cell, adding a new electrolyte containing Cr(VI), and applying a negative potential of 500 to 0 mV to the working electrode, so that the Cr(VI) in the electrolyte is reduced and fixed.

Description

Technical Field

[0001] The present invention relates to the technical field of harmless treatment of hazardous wastes, and in particular to a method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model. Background Art

[0002] Hazardous metals in waste streams generated by anthropogenic activities, such as chromium (VI) and copper (II) in electroplating wastewater, can cause severe pollution to surrounding water bodies, posing a serious threat to ecosystems and human health. Chromium and copper are both redox-sensitive metals whose toxicity lies in their oxidation states, with the highest valence states of Cr(VI) and Cu(II) being more toxic than other valence states. In the environment, chromium exists primarily in two forms: Cr(VI) and Cr(III). Because Cr(III) and Cu(0) are less toxic and insoluble at neutral pH, reducing Cr(VI) to Cr(III) and Cu(II) to Cr(0) is an effective strategy for detoxifying such wastewaters.

[0003] Microbial reduction of heavy metals has prompted the development of a variety of biological approaches for the treatment of various contaminated water systems, but their drawback is the need for a continuous source of organic carbon. Therefore, it is necessary to develop more environmentally sustainable and energy-efficient remediation technologies to treat these wastewaters. Microbial electrochemical systems are clean electrochemical bioreactors that promote the bioelectrochemical reduction of heavy metals using heavy metals as electron acceptors, electroactive bacteria capable of extracellular electron transfer (EET) as biocatalysts, and solid-state electrodes as electron donors. However, the reduction rate of Cr(VI) in microbial electrochemical systems is largely limited by the bidirectional input and output capabilities of the microorganisms.

[0004] Shewanella oneidensis can utilize a variety of electron acceptors for extracellular respiration and can reduce high-valent heavy metals through different electron transfer pathways, thereby promoting the immobilization of heavy metals. Electroactive microorganisms can form mutually beneficial symbiotic relationships through interspecies electron transfer, thereby jointly completing metabolic processes that one microorganism cannot complete alone. Through interspecies electron transfer, the electroactive bacteria Shewanella and chromium-reducing functional bacteria can form a biocathode through interspecies mutualism. The chromium-reducing functional bacteria provide the electroactive bacteria with the necessary organic small molecules, while Shewanella accelerates interspecies electron transfer through membrane proteins or electron mediators. Chinese patent CN106754456B discloses a microbial electricity generation system and microbial fuel cell containing this mixed bacterial community. Chinese patent application CN114409057A discloses a method for reducing and degrading enrofloxacin using a biocathode co-metabolism system. The prior art does not address the biocathode removal performance of bioelectrochemical systems based on the mutualistic behavior of microorganisms based on interspecies electron transfer. Therefore, the construction of a novel and efficient biocathode for heavy metal removal is of great significance. Summary of the Invention

[0005] The purpose of the present invention is to address the above problems and provide a method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model, comprising the following steps:

[0006] S1. Preparing a mixed functional bacterial community electrolyte composed of conductive bacteria and metal-reducing functional bacteria: the conductive bacteria is Shewanella oneidensis, and the bacteria in the metal-reducing functional bacteria community include one or more of Bacillus cereus, Ralstonia pickettii, and Paenibacillus alvei; the conductive bacteria and the metal-reducing functional bacteria are inoculated into a nutrient solution at an inoculum rate of 1-2% (v / v) to obtain a mixed functional bacterial community electrolyte; the nutrient solution includes a phosphate buffer solution, vitamins, trace minerals, and an organic carbon source;

[0007] S2. Enriching the electroactive biofilm: Pour the prepared mixed functional bacterial community electrolyte into the electrolytic cell of the three-electrode bioelectrochemical system, and apply a positive potential of 100-300 mV to the working electrode to enrich the electroactive biofilm on the working electrode; preferably, the positive potential is 150-250 mV;

[0008] S3. Cr(VI) reduction and fixation: After generating a mature anode biofilm with a stable current output, turn off the instrument, pour out the electrolyte in the electrolytic cell, add new electrolyte containing Cr(VI), and apply a negative potential of -500 to 0 mV to the working electrode to reduce and fix the Cr(VI) in the electrolyte. The preferred negative potential is -400 to -200 mV.

[0009] The present invention provides another method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model, comprising the following steps:

[0010] S1. Preparing a mixed functional bacterial community electrolyte composed of conductive bacteria and metal-reducing functional bacteria: the conductive bacteria is Shewanella oneidensis, and the bacteria in the metal-reducing functional bacteria community include one or more of Bacillus cereus, Ralstonia pickettii, Paenibacillus alvei, Lysinibacillus sp, Clostridium sphenoides, and Sedimentibacter sp; inoculating the conductive bacteria and the metal-reducing functional bacteria into a nutrient solution at an inoculum rate of 1-2% (v / v) to obtain a mixed functional bacterial community electrolyte; the nutrient solution includes a phosphate buffer solution, vitamins, trace minerals, and an organic carbon source;

[0011] S2. Enriching the electroactive biofilm: Pour the prepared mixed functional bacterial community electrolyte into the electrolytic cell of the three-electrode bioelectrochemical system, and apply a positive potential of 100-300 mV to the working electrode to enrich the electroactive biofilm on the working electrode; preferably, the positive potential is 150-250 mV;

[0012] S3. Cr(VI) reduction and fixation: After generating a mature anode biofilm with a stable current output, turn off the instrument, pour out the electrolyte in the electrolytic cell, add new electrolyte containing Cr(VI), and apply a negative potential of -500 to 0 mV to the working electrode to reduce and fix the Cr(VI) in the electrolyte. The preferred negative potential is -400 to -200 mV.

[0013] Preferably, in any of the above technical solutions, in step S1, OD 600 The conductive bacteria with a value of 0.5-2 and the culture liquid of the metal-reducing functional bacteria are inoculated into the nutrient solution at an inoculum rate of 1-2% respectively; preferably, the metal-reducing functional bacteria include Bacillus cereus.

[0014] Preferably, in any of the above technical solutions, the effective viable bacteria count of Bacillus cereus in the metal-reducing functional bacteria accounts for 80% or 85% or 90% or 95% or more of the effective viable bacteria count in the metal-reducing functional bacteria;

[0015] Preferably, the metal-reducing functional bacteria further include Paenibacillus alvei, and the effective viable bacteria count of Paenibacillus alvei accounts for 0.5-10% or 0.8-5% or 0.8-1.5% of the effective viable bacteria count in the metal-reducing functional bacteria;

[0016] It is further preferred that the metal-reducing functional bacteria group also includes one or more of Ralstonia piezoelectrica, Bacillus lysinicola, Clostridium wedge-like, and Bacillus sedimentatum, wherein the effective viable bacteria count of Ralstonia piezoelectrica accounts for 0.1%-1% or 0.1-0.5% of the effective viable bacteria count in the metal-reducing functional bacteria group, the effective viable bacteria count of Bacillus lysinicola accounts for 0.1-10% or 0.3-5% or 0.5-2% or 0.7-1% of the effective viable bacteria count in the metal-reducing functional bacteria group, the effective viable bacteria count of Clostridium wedge-like accounts for 0.1%-1% or 0.1-0.5% of the effective viable bacteria count in the metal-reducing functional bacteria group, and the effective viable bacteria count of Bacillus sedimentatum accounts for 0.1%-1% or 0.1-0.4% of the effective viable bacteria count in the metal-reducing functional bacteria group.

[0017] Preferably, in any of the above technical solutions, in step S1, the ratio of the culture medium is: take 1L of phosphate buffer solution, add 10-15mL of trace mineral solution, 5-10mL of vitamin solution, and add an organic carbon source, and the concentration of the organic carbon source in the culture medium is 15-20mM; the organic carbon source is selected from glucose, pentose, hexose, cellobiose, lactic acid, acetic acid, formic acid, and amino acids;

[0018] Preferably, the organic carbon source is glucose and / or lactic acid;

[0019] Preferably, the ratio of the culture solution is: take 1 L of phosphate buffer solution, add 12.5 mL of trace mineral solution, 10 mL of vitamin solution, and the concentration of the organic carbon source in the culture solution is 20 mM.

[0020] Preferably, in any of the above technical solutions, the vitamin solution contains the following components: 1.5-2.5 mg / L vitamin H, 1.5-2.5 mg / L vitamin B9, 9.5-10.5 mg / L pyridoxine hydrochloride, 4.5-5.5 mg / L vitamin B1, 4.5-5.5 mg / L vitamin B2, 4.5-5.5 mg / L niacin, 4.5-5.5 mg / L D-(+) calcium pantothenate, 0.08-0.12 mg / L vitamin B12, 4.5-5.5 mg / L p-aminobenzoic acid and 4.5-5.5 mg / L lipoic acid; preferably contains: 2.0 mg / L vitamin H, 2.0 mg / L vitamin B9, 10.0 mg / L pyridoxine hydrochloride, 5.0 mg / L vitamin B1, 5.0 mg / L vitamin B2, 5.0 mg / L niacin, 5.0 mg / L D-(+) calcium pantothenate, 0.1 mg / L vitamin B12, 5.0 mg / L p-aminobenzoic acid, and 5.0 mg / L lipoic acid;

[0021] The preparation method of the trace mineral solution is as follows: first, 1.3-1.7 g of nitrilotriacetic acid is dissolved in distilled water, and the pH is adjusted to 6-7. Then, the following components are added in weight: 2.5-3.5 g of MgSO4·7H2O, 0.4-0.6 g of MnSO4·H2O, 0.8-1.2 g of NaCl, 0.08-0.12 g of FeSO4·7H2O, 0.08-0.12 g of CoCl2·6H2O, 0.08-0.12 g of CaCl2, 0.08-0.12 g of ZnSO4·7H2O, 0.008-0.012 g of CuSO4·5H2O, 0.08-0.12 g of AlK(SO4)2·12H2O, 0.008-0.012 g of H3BO3, and 0.008-0.012 g of Na2MoO4·2H2O, stir evenly, fully dissolve, and dilute to 1000mL;

[0022] The phosphate buffer solution contains the following components: Na2HPO4·2H2O 3-3.6 g / L, NaH2PO4·12H2O 10-10.5 g / L, NH4Cl 0.2-0.4 g / L and KCl 0.1-0.16 g / L, with a pH value of 6.7-7.3.

[0023] Preferably, in any of the above technical solutions, the working electrode of the three-electrode microbial electrochemical system is a carbon cloth electrode, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum electrode;

[0024] The electrolyte is aerated with inert gas before being poured into the electrolytic cell to remove dissolved oxygen.

[0025] Preferably, in any of the above technical solutions, the metal-reducing functional bacteria are isolated from bacteria in chromium-contaminated sites;

[0026] In step S2, after applying a positive potential for 6-8 days, the current decreases and a new electrolyte is replaced. After the current is continuously and stably output for three cycles, the enrichment of the electroactive biofilm is completed, and a mature anodic biofilm is formed on the working electrode;

[0027] At the end of step S2, enriching the electroactive biofilm, the biomass ratio of Bacillus cereus, Ralstonia pekinensis, Paenibacillus alvei and Shewanella spp. on the biofilm formed is 4-50:3-15:2-10:40-75, preferably 4-40:3-11:2-6:45-75 or 4-10:3-5:2-4:60-75.

[0028] Preferably, in any of the above technical solutions, in step S3, the concentration of the chromium compound in the new electrolyte containing Cr(VI) is 1-100 mg / L, preferably 10-80 mg / L or 10-60 mg / L or 20-50 mg / L or 20-30 mg / L.

[0029] Preferably, in any of the above technical solutions, the new electrolyte containing Cr(VI) further contains other heavy metals, and the other heavy metals are selected from Cu(II), As(V), and Ni(II);

[0030] Preferably, the concentration of other heavy metal compounds in the electrolyte is 1-100 mg / L, preferably 10-80 mg / L, 10-60 mg / L, 20-50 mg / L, or 20-30 mg / L.

[0031] The present invention provides a method for reducing and fixing Cr(VI) using key functional metal-reducing microorganisms based on a mutualistic symbiotic model. The principle is as follows: by constructing a functional bacterial community composed of conductive bacteria and metal-reducing bacteria, the characteristics of their division of labor and cooperation are achieved. The metal-reducing functional bacterial community provides riboflavin, a carrier for electron transfer, to the system, and converts the most commonly used and inexpensive glucose into small molecule acids for use by the electrogenic bacteria Shewanella. Shewanella accelerates interspecies electron transfer in the system through membrane proteins or electron mediators. The present invention selects Shewanella and functional bacterial communities from chromium-contaminated sites to construct a metal-reducing functional bacterial community, expands the carbon source spectrum of the electrogenic bacteria Shewanella, and enhances the interspecies electron transfer efficiency of the functional bacterial community. The biocathode composed of the functional bacterial community can further accelerate the reduction and fixation efficiency of the heavy metals Cr(VI) and Cu(II). The construction of the functional bacterial community can improve the conductivity of the biocathode, and thus is widely applicable to the water quality characteristics of various heavy metal wastewaters.

[0032] The key functional metal-reducing microorganisms based on the mutualistic symbiotic model provided by the present invention and their application in the biocathode solve the following problems:

[0033] (1) The electrogenic bacterium Shewanella has a narrow spectrum of substrates and carbon sources. It can only utilize small molecules such as lactic acid, formic acid, and amino acids, but cannot utilize a wide range of carbon sources—five- and six-carbon sugars such as glucose and xylose. It cannot convert the abundant chemical energy of cellulose or the widely available and inexpensive glucose into electrical energy.

[0034] (2) From the perspective of “system mutualism”, develop an independent, stable, and efficient metal-reducing functional bacterial community;

[0035] (3) The problem of co-cultivation of biocathode functional bacteria. How to make the electrogenic bacteria and metal-reducing bacteria work synergistically to improve interspecies electron transfer;

[0036] (4) It has overcome the technical difficulties in treating low-concentration heavy metal wastewater, and has high economic benefits and strong operability.

[0037] The method described in this paper constructs a symbiotic functional microbial community based on enhanced interspecies electron transfer and metabolite mutualism. It also constructs a ternary system of electrons, biofilm, and Cr(VI). Organic functional groups on the microbial surface at the biocathode are used to reduce and fix the heavy metal Cr(VI). The method is simple, highly operational, and has the potential for large-scale, continuous industrial production, potentially addressing current environmental challenges. This research expands our understanding of interspecies mutualistic symbiosis in functional microorganisms and provides a new strategy for enhancing heavy metal remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the changes in glucose consumption over time for metal-reducing functional bacteria and mixed functional bacteria.

[0039] Figure 2 This is a graph showing the change in lactic acid production of metal-reducing functional bacteria and mixed functional bacteria over time.

[0040] Figure 3 This is a graph showing the changes in acetic acid production over time by metal-reducing functional bacteria and mixed functional bacteria.

[0041] Figure 4 This is a graph showing the temporal changes in the production of the electron mediator riboflavin by metal-reducing functional bacteria and mixed functional bacteria.

[0042] Figure 5 The ampere-time diagram of the electrochemical reactor of metal-reducing functional bacteria and mixed functional bacteria.

[0043] Figure 6Non-turnover CV diagrams of biocathode constructed for metal-reducing functional bacteria and mixed functional bacteria.

[0044] Figure 7 Structural analysis of the microbial communities of different cathode biofilms at the genus level (a) and species level (b).

[0045] Figure 8 The removal rate of Cr(VI) by biocathodes constructed with metal-reducing functional bacteria and mixed functional bacteria.

[0046] Figure 9 The chromium reduction curves within 24 hours for reactors A1, B1-1, and B2-1 are shown.

[0047] Figure 10 The fixation rate of total chromium by biocathodes constructed with metal-reducing functional bacteria and mixed functional bacteria.

[0048] Figure 11 XPS patterns of chromium reduction products of biocathodes constructed with metal-reducing functional bacteria and mixed functional bacteria.

[0049] Figure 12 This is the reduction curve of the composite heavy metals Cr(VI) and As(V).

[0050] Figure 13 This is the reduction curve of the composite heavy metals Cr(VI) and Ni(II). DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0052] In the examples of the present invention, if the specific conditions are not specified, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. All raw materials and reagents used without specifying the manufacturer are conventional products that can be purchased from the market.

[0053] Sources of main reagents and materials:

[0054] Shewanella (S. oneidensis) strain MR-1 was used and was commercially available.

[0055] Luria-Bertani (LB) liquid medium is a conventional LB medium.

[0056] Example 1

[0057] 1. Construction of mixed functional bacterial communities

[0058] Constructing a mixed functional bacterial community composed of conductive bacteria and chromium-contaminated site bacteria:

[0059] 1. Prepare conductive bacteria: First, culture Shewanella strain MR-1 aerobically in Luria-Bertani (LB) liquid medium at 37°C for 24 hours with shaking at 180 rpm. Collect cells (bacteria) by centrifugation and wash them multiple times with 0.85% NaCl solution before use. Resuspend the cells in PBS.

[0060] 2. Prepare bacterial flora of chromium-contaminated sites:

[0061] Obtaining functional bacterial communities from chromium-contaminated sites (i.e., metal-reducing functional bacterial communities): Functional bacterial communities were isolated from a chromate slag heap in Pingnan County, Fujian Province, China, using conventional methods. The isolated bacterial communities were cultured in LB liquid medium at 37°C with shaking at 180 rpm for later use. A portion of the bacterial culture was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for bacterial community composition testing. The test results are shown in Table 1 (the percentage values ​​in the table represent the percentage of the effective viable bacteria of each bacteria to the total effective viable bacteria in the metal-reducing functional bacterial community):

[0062] Table 1

[0063]

[0064] As can be seen from Table 1, the dominant bacterial species is Bacillus cereus, accounting for about 95%; followed by Paenibacillus alvei, accounting for about 1%; followed by Bacillus lysinin, Ralstonia pietermaritzii, Clostridium wedge-like, and Bacillus sedimentatus, accounting for about 0.8%, about 0.3%, about 0.3%, and about 0.2%, respectively.

[0065] Take OD 600 1 mL of the functional bacterial flora of the chromium contaminated site with a value of 0.5 was added to 100 mL of culture medium. The culture medium consisted of phosphate buffer solution, vitamins, trace minerals and organic carbon source. The composition of each component was as follows:

[0066] Preparation of vitamin solution: Weigh the following vitamins: vitamin H (also known as biotin, 2.0 mg), vitamin B9 (also known as folic acid, 2.0 mg), pyridoxine hydrochloride (10.0 mg), vitamin B1 (also known as thiamine hydrochloride, 5.0 mg), vitamin B2 (also known as riboflavin, 5.0 mg), niacin (5.0 mg), D-(+) calcium pantothenate (5.0 mg), vitamin B12 (also known as cobalamin, 0.1 mg), p-aminobenzoic acid (5.0 mg) and lipoic acid (5.0 mg) into a beaker, add sterile water to dissolve them, transfer to a volumetric flask, dilute to 1000 mL, and set aside.

[0067] Preparation of trace mineral solution: First, dissolve 1.5g of nitrilotriacetic acid in approximately 0.5L of distilled water, then adjust its pH to 6.5 with 1M KOH solution, and then add the following components by mass: 3.0g MgSO4·7H2O, 0.5gMnSO4·H2O, 1.0g NaCl, 0.1g FeSO4·7H2O, 0.1g CoCl2·6H2O, 0.1g CaCl2, 0.1gZnSO4·7H2O, 0.01g CuSO4·5H2O, 0.1gAlK(SO4)2·12H2O, 0.01g H3BO3 and 0.01gNa2MoO4·2H2O, stir evenly and fully dissolve, then dilute to 1000mL and set aside.

[0068] Phosphate buffer solution: composed of the following components: Na2HPO4·2H2O 3.32 g / L, NaH2PO4·12H2O 10.32 g / L, NH4Cl 0.31 g / L and KCl 0.13 g / L, pH = 7.0.

[0069] Preparation of culture medium: Take 1 L of the above-mentioned phosphate buffer solution, add 12.5 mL of the above-mentioned trace mineral solution, 10 mL of the above-mentioned vitamin solution, and add an organic carbon source (glucose or lactic acid). The concentration of the organic carbon source in the culture medium is 20 mM.

[0070] 3. Preparation of mixed functional bacterial flora:

[0071] Take the previously prepared Shewanella and change the OD 600 1 mL of Shewanella bacterial solution with a value of 0.5 was added to the aforementioned culture solution containing the functional bacterial flora of the chromium-contaminated site to obtain the electrolyte used for subsequent experiments, and the solution was shaken and cultured at 37°C with a rotation speed of 180 rpm.

[0072] The consumption of organic carbon source (glucose in this experiment) by mixed functional bacteria (reactor B1) was measured. The control group contained only functional bacteria from chromium-contaminated sites without Shewanella (reactor A1). The results are as follows: Figure 1 As shown in Figure 2, the control group with only metal-reducing functional bacteria from the chromium-contaminated site consumed 71.45% of the glucose within 24 hours ( Figure 1 In contrast, the substrate utilization rate of the mixed functional bacterial community after compounding increased to 90.15% ( Figure 1 During this process, glucose is rapidly consumed and a certain amount of organic acids accumulates.

[0073] 2. Construction of electrochemical reactor

[0074] Build an electrochemical reactor: A three-electrode bioelectrochemical system is used, including a cubic microbial electrolysis cell device and a constant potential instrument. The electrolysis cell is equipped with three electrodes: the three-electrode system consists of a working electrode (4×5 cm carbon cloth), a reference electrode (saturated calomel electrode) and a counter electrode (2×2 cm platinum mesh electrode). The cubic microbial electrolysis cell device used in this example is the same as that in Example 5 of patent application CN202110517085.0 (publication number CN 113398523 A) Figure 8 The cubic dual-chamber microbial electrolysis cell shown in the figure has essentially the same structure, except that the cubic microbial electrolysis cell device in the present invention does not use a proton exchange membrane to separate the device into chambers I and II, but instead has only one chamber in total, and the working electrode is different. The working electrode of the present invention is a 4×5 cm carbon cloth, and the rest of the structure is the same. The three-electrode bioelectrochemical system is connected to a multichannel potentiostat (CHI1000C, CH Instrument, Shanghai, China) with wires. The device is operated in the same manner as CN202110517085.0.

[0075] The electrolyte (in the present invention, the culture solution with bacteria added) was aerated with N2 for 30 minutes before being added to the electrolytic cell to remove dissolved oxygen.

[0076] Different electrochemical reactors were set up for comparative experiments: reactors A1, B1, and B2. 100 mL of electrolyte containing bacterial culture was placed in a cubic microbial electrolysis cell. All reactors were operated at room temperature with a potential of +200 mV applied.

[0077] Reactor A1: The electrolyte contains only functional bacteria from chromium-contaminated sites, no Shewanella, and glucose is the only organic carbon source;

[0078] Reactor B1: The electrolyte is the mixed functional bacterial community electrolyte containing Shewanella and chromium-contaminated site functional bacteria prepared above, and glucose is the only organic carbon source;

[0079] Reactor B2: The electrolyte is the mixed functional bacterial community electrolyte containing Shewanella and chromium-contaminated site functional bacteria prepared above, and lactic acid is the only organic carbon source;

[0080] Determination of bacterial metabolites, the results are as follows Figure 2 、 3 As shown, in the electrochemical reactor B1, the concentration of organic acid is relatively low and gradually depleted. During this process, the metal-reducing functional bacteria consume glucose and produce small molecular organic acids (lactic acid, acetic acid), etc. Lactic acid is the main organic carbon source of Shewanella, and lactic acid is then used by Shewanella to produce more acetic acid.

[0081] The extracellular electron mediator riboflavin of the bacterial community was measured, and the results were as follows Figure 4 As shown in the figure, in all electrochemical reactors, the concentration of riboflavin reached a high level at 48 h, and then gradually decreased and remained stable. The riboflavin content of the mixed bacterial culture electrochemical reactor B1 could reach 233.64 μg / L, which exceeded the riboflavin content secreted by Shewanella under electrogenic conditions.

[0082] pass Figure 2 and Figure 3 It can be seen that the metal-reducing functional bacteria (A1) play an important role in the glycolysis process, metabolizing glucose to produce lactic acid and acetic acid. In the absence of Shewanella to further utilize lactic acid, the lactic acid content is maintained at around 249.55 mg / L. Figure 4 It can be seen that metal-reducing functional bacteria can secrete and produce the electron mediator riboflavin.

[0083] 3. Construction of the Biocathode

[0084] In a three-electrode bioelectrochemical system, a bacteria-containing electrolyte was placed in an electrolytic cell and incubated at 37°C for 24 hours. A positive potential of +200mV was then applied to the working electrode to enrich the electroactive biofilm on the carbon cloth of the working electrode. After seven days of positive potential application and a decrease in current, the electrolyte was replaced with fresh bacteria-containing electrolyte. After three cycles of continuous and stable current output, a mature anodic biofilm formed on the carbon cloth of the working electrode.

[0085] After generating a mature anodic biofilm with stable current output, a negative potential of -300 mV was applied to the three-electrode microbial electrochemical system, which was acclimated at this potential and 30°C for 1 day, and then the state of the biofilm was characterized electrochemically.

[0086] Figure 5 The ampere-time diagram of the bacterial community is shown. It can be seen that the mixed functional bacterial community electrochemical reactor B1 with glucose as the only carbon source generated current in about 28 hours, with a peak current density of up to 240.9 μA / cm 2 This indicates that after the addition of electrochemically active bacteria (Shewanella), the microbial community structure on the biofilm has evolved, which is more conducive to long-distance electron transfer between species. In the electrochemical reactor A1 with metal-reducing functional bacteria using glucose as the only carbon source, the maximum current density is about 8μA / cm 2 The results showed that the metal-reducing functional bacteria in chromium-contaminated sites have certain electrochemical activity, but their electricity generation capacity is weak.

[0087] Further determination of the non-turnover CV graph of the biocathode constructed by the bacterial community shows that the mixed functional bacterial community electrochemical reactor B1 has two obvious reduction peaks ( Figure 6), which is mainly attributed to direct interspecies electron transfer mediated by flavin-binding OM-Cyts proteins and indirect interspecies electron transfer mediated by riboflavin.

[0088] 4. Microbial Community Structure and Function

[0089] To analyze the differences between the microbial communities in the cathode biofilms, genetic testing was performed on the cathode biofilms from reactors A1, B1, and B2. The bacterial communities on the cathode biofilms were isolated and cultured, and then colonies with different functional characteristics were selected for 16S rRNA gene pyrosequencing. The specific measurement process involved placing the bacteria in sterile centrifuge tubes, labeling them, and storing them on dry ice. DNA extraction and sequencing were performed by Shanghai Majorbio Technology Co., Ltd., with PCR amplification targeting the V3-V4 variable region. All sequencing results were systematically analyzed using the I-Sanger platform on the Majorbio Cloud.

[0090] Figure 7 a depicts the structure of the microbial community at the genus level. As can be seen, the biofilm inoculated with metal-reducing functional bacteria (reactor A1) is enriched with Bacillus, Paenibacillus, Lachnoclocstridium, Ralstonia, and Sedimentibacter. Most of these bacteria are Gram-positive bacteria with thicker cell walls. It has been reported that only a subset of Gram-positive bacteria exhibit electrochemical activity (Cui et al. 2020, Hederstedt et al. 2020, Qin et al. 2019). The electrochemical activity of these Gram-positive bacteria is generally lower than that of Gram-negative electrogenic bacteria, such as G. sulfurreducens and S. oneidensis MR-1, because their cell walls are thicker than those of Gram-negative electrogenic bacteria, and cell wall thickness may affect electron transfer properties (Zhao et al. 2021). Additionally, Bacillus, Bacillus pilosus, and Paenibacillus are associated with hydrolysis and fermentation, capable of breaking down glucose and producing acid (Vilas-Boas et al. 2007). Ralstonia has been reported to exhibit high heavy metal transport / detoxification capabilities and has been used to treat heavy metal-contaminated wastewater (Huang et al. 2021). Shewanella became the dominant genus after the addition of S. oneidensis MR-1. Shewanella exhibits remarkable electrochemical capabilities, accelerating cathode electron transfer and thus promoting efficient heavy metal reduction (Jia et al. 2023).

[0091] Species-level microbial community structure ( Figure 7 b) Clearly, the biofilms in the different reactors were enriched primarily with S. oneidensis MR-1, Bacillus cereus 12-2, Paenibacillus alvei DSM29, Ralstonia pickettii, and other species, though the proportions of the individual microorganisms varied significantly. Compared to reactor B2 (4.4%), the proportion of Bacillus cereus 12-2 increased to 49.4% and 36.8% in reactors A1 and B1, respectively. Bacillus cereus can ferment glucose, with lactic acid as the primary metabolite after anaerobic cultivation (Todorova et al. 2019). Furthermore, Ralstonia pickettii can survive and grow in extremely oligotrophic environments and is resistant to heavy metals (Huang et al. 2021). It is worth mentioning that the abundance of S. oneidensis MR-1 in reactors B1 and B2 was approximately 47.9% and 71.5%, respectively, suggesting that biofilm formation on the electrodes can help the extracellular respiratory bacteria S. oneidensis MR-1 gain a survival advantage. The synergistic microbial community composed of electroactive and fermentative bacteria is more conducive to mass transfer and electron transport, resulting in higher reduction efficiency.

[0092] After testing, the bacterial colony structure in each reactor is as follows:

[0093] Reactor B1: The abundance of Bacillus cereus was 36.8%, the abundance of S. oneidensis MR-1 was 47.9%, the abundance of Ralstonia piezoelectrica was 10.2%, and the abundance of Paenibacillus alvei was 5.1%.

[0094] Reactor B2: The abundance of Bacillus cereus was 4.4%, the abundance of S. oneidensis MR-1 was 71.5%, the abundance of Ralstonia piezoelectrica was 3.6%, and the abundance of Paenibacillus alvei was 2.3%.

[0095] Reactor A1: The abundance of Bacillus cereus was 49.4%, the abundance of Ralstonia piezoelectrica was 15%, and the abundance of Paenibacillus alvei was 10%.

[0096] 5. The process of Cr(VI) fixation and mineralization

[0097] Using the cathode biofilm as an electron donor, equal amounts of Cr(VI) and Cu(II) ions were added to measure the chromium reduction and fixation efficiency. The conversion of Cr(VI) to chromium oxide was achieved by utilizing the synergistic effect of the organic functional groups on the microbial surface of the conductive biofilm and Cu(II). The specific operation steps are:

[0098] Following the aforementioned steps, an electroactive biofilm was enriched on the carbon cloth of the working electrode. After a mature anodic biofilm with a stable current output was generated, the instrument was shut down, the electrolyte in the electrolytic cell was poured out, and a new electrolyte containing heavy metals was added to the cell (the other components and proportions of the new electrolyte were the same as those in the old electrolyte, the only difference being the addition of heavy metals). The potentiostat potential was changed, and a negative potential of -300 mV was applied to the working electrode. Five experimental groups were set up:

[0099] Reactor A1: This is the aforementioned reactor A1, except that after a mature anode biofilm is formed, K2Cr2O7 is added to the electrolyte replaced in the electrolytic cell, and the concentration of K2Cr2O7 in the electrolyte is 50 mg / L.

[0100] Reactor B1-1: The same as the aforementioned reactor B1, except that after a mature anode biofilm is formed, K2Cr2O7 is added to the electrolyte replaced in the electrolytic cell, and the concentration of K2Cr2O7 in the electrolyte is 50 mg / L.

[0101] Reactor B1-2: This is the aforementioned reactor B1, except that after a mature anode biofilm is formed, K2Cr2O7 and CuSO4·5H2O are added to the electrolyte replaced in the electrolytic cell. The concentrations of K2Cr2O7 and CuSO4·5H2O in the electrolyte are both 25 mg / L.

[0102] Reactor B2-1: The same as the aforementioned reactor B2, except that after a mature anode biofilm is formed, K2Cr2O7 is added to the electrolyte replaced in the electrolytic cell, and the concentration of K2Cr2O7 in the electrolyte is 50 mg / L.

[0103] Reactor B2-2: This is the aforementioned reactor B2, except that after a mature anode biofilm is formed, K2Cr2O7 and CuSO4·5H2O are added to the electrolyte replaced in the electrolytic cell. The concentrations of K2Cr2O7 and CuSO4·5H2O in the electrolyte are both 25 mg / L.

[0104] Determination of Cr / Cu reduction and fixation efficiency:

[0105] Figure 8 The removal rate of Cr(VI) by the biocathode was determined. Figure 9 The figure below shows the chromium reduction curve within 24 hours of the reactor. In the mixed functional bacterial community electrochemical reactor B1-1 with glucose as the only carbon source, the reduction efficiency of Cr(VI) by the biocathode reached almost 100% within 24 hours. The mixed functional bacterial community can secrete more riboflavin with redox activity during the Cr(VI) reduction process, which can further promote the efficiency of electron transfer. Figure 6 and Figure 8The reduction efficiency of the biocathode was lower when lactate was used as the carbon source (B2-1, 71.3%), indicating that different carbon sources may affect the reducing capacity of the biofilm. This may be attributed to the higher electron transfer efficiency and greater abundance of chromium-reducing bacteria in the mixed functional bacterial community when glucose was used as the carbon source.

[0106] Figure 10 Results show the Cr(III) fixation efficiency measured by the biocathodes. In electrochemical reactor B1, a mixed functional bacterial community using glucose as the sole carbon source, the biocathode (B1-1) achieved a 54.1% fixation efficiency within 60 hours. With the synergistic effect of Cu(II), the biocathode (B1-2) achieved a 80.0% fixation efficiency. In both B1-1 and B2-1, where copper ions were absent and only hexavalent chromium was added, the functional bacterial communities exhibited a reduction efficiency of less than 9% within 24 hours and a fixation efficiency of less than 28% within 60 hours.

[0107] Figure 11 The XPS results of the chromium reduction products on the biocathode of the corresponding device B1 show that new peaks appear near 576eV and 587eV in the mixed functional bacterial cathode biofilm. These peaks correspond to Cr 2p 1 / 2 and Cr2p 3 / 2 Characteristic peaks of Cr 2 O 3 The peaks of Cr2O7 can be assigned to 576eV and 587eV, while 2- Cr(VI) can be assigned to 580eV. After adding Cu(II), we can see the peaks of Cu 2p orbital at energy levels of 932eV and 953, which correspond to Cu2p 3 / 2 and 2p 1 / 2 , the valence state of Cu is +1 or 0. In addition, there is a Cu 2+ The peaks of Cu(II) and Cr(III) indicate that part of Cu(II) may combine with Cr(III) to form a stable chromium-containing mineral phase.

[0108] From the above results, it can be seen that the method of reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on the mutualistic symbiotic model of the present invention realizes the fixed mineralization of the chromium element therein.

[0109] Example 2 Fixed mineralization of composite heavy metals

[0110] In order to demonstrate the universality of the method of the present invention, the cathode biofilm formed in Example 1 was used as an electron donor, and equal amounts of Cr(VI) (20 mg / L of K2Cr2O7) and As(V) (20 mg / L of Na3AsO4) ions were added to a new electrolyte, and the Cr(VI) and As(V) reduction rates were measured according to the method in Example 1. Figure 12The results of the biocathode reduction rate of heavy metals showed that in the mixed functional bacterial community electrochemical reactor B1 with glucose as the only carbon source, the biocathode reduction efficiency of Cr(VI) could reach up to 100% within 24 hours, and the reduction efficiency of As(V) could reach 54.2%.

[0111] The same method was used to test the reduction rate of heavy metals when an electrolyte containing 20 mg / L K2Cr2O7 and 20 mg / L Ni(II) (nickel nitrate) was added to the mixed functional bacterial community electrochemical reactor B1 using glucose as the only carbon source. Figure 13 The results show that the reduction efficiency of Cr(VI) by the biocathode can reach up to 78.9% and the reduction efficiency of Ni(II) can reach 60.9% within 24 hours.

Claims

1. A method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model, characterized in that: The steps include: S1, prepare a mixed functional bacterial community electrolyte consisting of conductive bacteria and metal reduction functional bacteria: the conductive bacteria is Shewanella ( S. oneidensis ), the bacteria in the metal-reducing functional bacteria group include Bacillus cereus ( Bacillus cereus ), Ralstonia pinnae ( Ralstonia pickettii ), Paenibacillus alveiformis ( Paenibacillus alvei ) one or more; inoculating conductive bacteria and metal-reducing functional bacteria into the culture solution at an inoculum rate of 1-2% (v / v) to obtain a mixed functional bacteria electrolyte; the culture solution includes a phosphate buffer solution, vitamins, trace minerals and an organic carbon source; S2. Enrichment of electroactive biofilm: Pour the prepared mixed functional bacterial community electrolyte into the electrolytic cell of the three-electrode bioelectrochemical system, and apply a positive potential of 100-300 mV to the working electrode to enrich the electroactive biofilm on the working electrode; S3. Reduction and fixation of Cr(VI): After generating a mature anode biofilm with stable current output, turn off the instrument, pour out the electrolyte in the electrolytic cell, add new electrolyte containing Cr(VI), and apply a negative potential of -500 to 0 mV to the working electrode to reduce and fix the Cr(VI) in the electrolyte.

2. A method for reducing and fixing Cr(VI) by key functional metal-reducing microorganisms based on a mutualistic symbiotic model, characterized in that: The steps include: S1, prepare a mixed functional bacterial community electrolyte consisting of conductive bacteria and metal reduction functional bacteria: the conductive bacteria is Shewanella ( S. oneidensis ), the bacteria in the metal-reducing functional bacteria group include Bacillus cereus ( Bacillus cereus ), Ralstonia pinnae ( Ralstonia pickettii ), Paenibacillus alveiformis ( Paenibacillus alvei ), Bacillus lysinicola ( Lysinibacillus sp ), Clostridium wedge-like ( Clostridium sphenoides ) 、Sedimentary Bacillus( Sedimentibacter. sp ) one or more; inoculating conductive bacteria and metal-reducing functional bacteria into the culture solution at an inoculum rate of 1-2% (v / v) to obtain a mixed functional bacteria electrolyte; the culture solution includes a phosphate buffer solution, vitamins, trace minerals and an organic carbon source; S2. Enrichment of electroactive biofilm: Pour the prepared mixed functional bacterial community electrolyte into the electrolytic cell of the three-electrode bioelectrochemical system, and apply a positive potential of 100-300 mV to the working electrode to enrich the electroactive biofilm on the working electrode; S3. Reduction and fixation of Cr(VI): After generating a mature anode biofilm with stable current output, turn off the instrument, pour out the electrolyte in the electrolytic cell, add new electrolyte containing Cr(VI), and apply a negative potential of -500 to 0 mV to the working electrode to reduce and fix the Cr(VI) in the electrolyte.

3. The method according to claim 1 or 2, characterized in that: In step S1, OD 600 Conductive bacteria and metal-reducing functional bacteria culture fluids with a value of 0.5-2 are inoculated into the culture fluid at an inoculum rate of 1-2% respectively; The metal-reducing functional bacteria group includes Bacillus cereus.

4. The method according to claim 3, wherein: The number of effective live bacteria of Bacillus cereus in the metal-reducing functional bacteria group accounts for more than 80% of the effective live bacteria in the metal-reducing functional bacteria group; The metal-reducing functional bacterial group also includes Paenibacillus alvei, and the effective living bacteria count of Paenibacillus alvei accounts for 0.5-10% of the effective living bacteria count in the metal-reducing functional bacterial group.

5. The method according to claim 4, characterized in that: The metal-reducing functional bacteria group also includes one or more of Ralstonia piezoelectrica, Bacillus lysinicola, Clostridium wedge-like, and Bacillus sedimentation, wherein the effective viable bacteria count of Ralstonia piezoelectrica accounts for 0.1%-1% of the effective viable bacteria count in the metal-reducing functional bacteria group, the effective viable bacteria count of Bacillus lysinicola accounts for 0.1-10% of the effective viable bacteria count in the metal-reducing functional bacteria group, the effective viable bacteria count of Clostridium wedge-like accounts for 0.1%-1% of the effective viable bacteria count in the metal-reducing functional bacteria group, and the effective viable bacteria count of Bacillus sedimentation accounts for 0.1%-1% of the effective viable bacteria count in the metal-reducing functional bacteria group.

6. The method according to claim 1 or 2, characterized in that: In step S1, the ratio of the culture medium is: take 1L of phosphate buffer solution, add 10-15 mL of trace mineral solution, 5-10 mL of vitamin solution, and add an organic carbon source, the concentration of the organic carbon source in the culture medium is 15-20 mM; the organic carbon source is selected from glucose, pentose, hexose, cellobiose, lactic acid, acetic acid, formic acid, and amino acids.

7. The method according to claim 6, characterized in that: The organic carbon source is glucose and / or lactate; The ratio of the culture solution is: take 1 L of phosphate buffer solution, add 12.5 mL of trace mineral solution and 10 mL of vitamin solution, and the concentration of the organic carbon source in the culture solution is 20 mM.

8. The method according to claim 6, wherein: The vitamin solution contains the following components: 1.5-2.5 mg / L vitamin H, 1.5-2.5 mg / L vitamin B9, 9.5-10.5 mg / L pyridoxine hydrochloride, 4.5-5.5 mg / L vitamin B1, 4.5-5.5 mg / L vitamin B2, 4.5-5.5 mg / L niacin, 4.5-5.5 mg / L D-(+) calcium pantothenate, 0.08-0.12 mg / L vitamin B12, 4.5-5.5 mg / L p-aminobenzoic acid and 4.5-5.5 mg / L lipoic acid; The preparation method of the trace mineral solution is as follows: first, 1.3-1.7 g of nitrilotriacetic acid is dissolved in distilled water, and the pH is adjusted to 6-7, and then the following components are added: 2.5-3.5 g MgSO4·7H2O, 0.4-0.6 g MnSO4·H2O, 0.8-1.2 g NaCl, 0.08-0.12 g FeSO4·7H2O, 0.08-0.12 g CoCl2·6H2O, 0.08-0.12 g CaCl2, 0.08-0.12 g ZnSO4·7H2O, 0.008-0.012 g CuSO4·5H2O, 0.08-0.12 g AlK(SO4)2·12H2O, 0.008-0.012 g H3BO3 and 0.008-0.012 g Na2MoO4·2H2O, stir evenly, fully dissolve, and dilute to 1000mL; The phosphate buffer solution contains the following components: Na2HPO4•2H2O 3-3.6 g / L, NaH2PO4•12H2O 10-10.5 g / L, NH4Cl 0.2-0.4 g / L and KCl 0.1-0.16 g / L, with a pH value of 6.7-7.

3.

9. The method according to claim 1 or 2, characterized in that: The working electrode of the three-electrode microbial electrochemical system is a carbon cloth electrode, the reference electrode is a saturated calomel electrode, and the counter electrode is a platinum electrode; The electrolyte is aerated with inert gas before being poured into the electrolytic cell to remove dissolved oxygen.

10. The method according to claim 1 or 2, characterized in that: The metal-reducing functional bacteria are isolated from bacteria in chromium-contaminated sites; In step S2, after applying a positive potential for 6-8 days, the current decreases and a new electrolyte is replaced. After the current is continuously and stably output for three cycles, the enrichment of the electroactive biofilm is completed, and a mature anodic biofilm is formed on the working electrode; At the end of step S2, enrichment of the electroactive biofilm, the biomass ratio of Bacillus cereus, Ralstonia pekinensis, Paenibacillus alvei, and Shewanella spp. on the formed biofilm is 4-50: 3-15: 2-10: 40-75.

11. The method according to claim 1 or 2, characterized in that: In step S3, the concentration of the chromium compound in the new electrolyte containing Cr(VI) is 1 to 100 mg / L.

12. The method according to claim 11, wherein: The new electrolyte containing Cr(VI) also contains other heavy metals, which are selected from Cu(II), As(V), and Ni(II); the concentration of the other heavy metal compounds in the electrolyte is 1-100 mg / L.

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