A biocatalytic method for enhancing arsenic methylation and volatilization by Clostridium and its application

Abstract

The present invention belongs to the field of microbial technology, and discloses a biocatalytic method for enhancing arsenic methylation and volatilization by Clostridium and its application. Specifically, it discloses the application of resazurin in improving the arsenic methylation ability of bacteria. The present invention first proposes that resazurin can improve the functions of bacteria (such as Clostridium sporogenes) in the generation and volatilization of methylarsenic and cell adsorption. Experiments have proved that the exogenous addition of resazurin can significantly improve the methylation and volatilization effects of Clostridium sporogenes on inorganic trivalent arsenic, and can provide an effective means for the remediation of arsenic-contaminated soil and water bodies.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a biocatalytic method for enhancing arsenic methylation and volatilization by Clostridium and its application. Background Art

[0002] Arsenic is a toxic metalloid element commonly present in the environment, mainly existing in the forms of inorganic trivalent and pentavalent arsenic. Except for the natural high-arsenic background in some areas, human activities such as fossil fuel development, ore smelting, and pesticide use can also increase the arsenic in the environment, thereby increasing the health risks brought by human arsenic exposure. Arsenic-methylating functional microorganisms can convert inorganic trivalent arsenic into less toxic methylarsenic and volatile arsenic gas, reducing the concentration of available arsenic, which helps to remove arsenic from water bodies. Therefore, using microbial arsenic methylation can provide an environmentally friendly and sustainable remediation technology for arsenic-contaminated water bodies. Arsenic methylation and volatilization, as a detoxification mechanism of microorganisms, are catalyzed by the intracellular S-adenosylmethionine (SAM) methyltransferase ArsM, and the synthesis of the methyl donor SAM requires the consumption of ATP to drive. The efficiency of arsenic methylation is affected by the internal arsenic conversion process in cells (such as cell arsenic efflux, etc.) and external environmental factors (such as redox potential and organic matter concentration), which limits the application effect of arsenic volatilization in the microbial remediation technology of arsenic. Therefore, it is urgent to propose effective means to improve arsenic volatilization in arsenic-contaminated groundwater and soil environments.

[0003] Chinese invention patent CN114480230A provides a Clostridium sporogenes with the function of fermentative hydrogen production and synchronous anaerobic arsenic methylation, but this strain has certain deficiencies in arsenic methylation and volatilization. Therefore, this application is proposed. Summary of the Invention

[0004] The purpose of the first aspect of the present invention is to provide the application of resazurin in improving the arsenic methylation ability of bacteria.

[0005] The purpose of the second aspect of the present invention is to provide a method.

[0006] The purpose of the third aspect of the present invention is to provide a method.

[0007] The purpose of the fourth aspect of the present invention is to provide a reagent that can improve the arsenic methylation and volatilization functions of bacteria.

[0008] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0009] The first aspect of the present invention provides the application of resazurin in any one of (1) to (12):

[0010] (1) Improving the arsenic methylation ability of bacteria;

[0011] (2) To prepare a product for enhancing the methylation ability of bacteria to arsenic;

[0012] (3) To enhance the arsenic volatilization performance of bacteria;

[0013] (4) To prepare a product for enhancing the arsenic volatilization performance of bacteria;

[0014] (5) To enhance the arsenic adsorption of bacteria;

[0015] (6) To prepare a product for enhancing the arsenic adsorption of bacteria;

[0016] (7) To promote the growth of bacteria;

[0017] (8) To prepare a product for promoting the growth of bacteria;

[0018] (9) To inhibit the hydrogen production activity of bacteria;

[0019] (10) To prepare a product for inhibiting the hydrogen production activity of bacteria;

[0020] (11) To inhibit the production of fermentation metabolites of bacteria;

[0021] (12) To prepare a product for inhibiting the production of fermentation metabolites of bacteria;

[0022] The fermentation metabolites include acetic acid and / or lactic acid.

[0023] In some embodiments of the present invention, the bacteria include at least one of Clostridium sporogenes, Bacillus, Shewanella, Cytophagaceae bacteria, chitinophagous bacteria, Achromobacter xylosoxidans.

[0024] In some preferred embodiments of the present invention, the bacteria are Clostridium sporogenes and / or Bacillus.

[0025] In some more preferred embodiments of the present invention, the bacteria are Clostridium sporogenes (deposit number: GDMCC No: 62212) and / or Bacillus (deposit number GDMCC No: 62092).

[0026] In some embodiments of the present invention, the arsenic is trivalent arsenic.

[0027] In some embodiments of the present invention, the arsenic includes arsenous acid and / or arsenite.

[0028] In some embodiments of the present invention, the methylation in (1)-(2) includes monomethylation, dimethylation and / or trimethylation.

[0029] The second aspect of the present invention provides a reagent or kit, comprising resazurin, glucose and tryptone.

[0030] In some embodiments of the present invention, when the reagent or kit is used, the reaction system contains 1 to 600 μM resazurin.

[0031] In some preferred embodiments of the present invention, when the reagent or kit is used, the reaction system contains 50 to 500 μM resazurin.

[0032] In some embodiments of the present invention, when the reagent or kit is used, the reaction system contains 5 to 20 mM glucose and 0.5 to 3 g / L tryptone.

[0033] In some embodiments of the present invention, when the reagent or kit is used, the reaction system contains 7 to 15 mM glucose and 0.5 to 2 g / L tryptone.

[0034] In some embodiments of the present invention, when the reagent or kit is used, the reaction system contains 10 to 15 mM glucose and 1 to 2 g / L tryptone.

[0035] The third aspect of the present invention provides a method, including the step of placing resazurin, the reagent or kit of the second aspect of the present invention, in an environment containing arsenic and bacteria;

[0036] The method includes any one of (a1) to (a3):

[0037] (a1) A method for improving the methylation ability of bacteria to arsenic;

[0038] (a2) A method for improving the volatilization performance of bacteria to arsenic;

[0039] (a3) A method for improving the adsorption of bacteria to arsenic.

[0040] In some embodiments of the present invention, the methylation includes mono-methylation, di-methylation and / or tri-methylation.

[0041] In some embodiments of the present invention, the arsenic is inorganic trivalent arsenic.

[0042] In some embodiments of the present invention, the arsenic includes arsenous acid and / or arsenite.

[0043] In some embodiments of the present invention, the environment is a culture medium, soil or water body.

[0044] In some embodiments of the present invention, the concentration range of the inorganic trivalent arsenic in the environment and water body is 0 to 200 μmol / L.

[0045] In some embodiments of the present invention, the culture medium includes NBAF culture medium.

[0046] In some embodiments of the present invention, the carbon and nitrogen sources in the culture medium include at least one of glucose, trypsin, and methionine.

[0047] In some embodiments of the present invention, the carbon and nitrogen sources in the culture medium are glucose and trypticase.

[0048] In some embodiments of the present invention, the bacteria include at least one of Clostridium sporogenes, Bacillus, Shewanella, Cellulophila, Chitinophage, and Achromobacter xylosoxidans.

[0049] In some preferred embodiments of the present invention, the bacteria are Clostridium sporogenes and / or Bacillus.

[0050] In some more preferred embodiments of the present invention, the bacterium is Clostridium sporogenes (deposit number: GDMCC No: 62212) and / or Bacillus (deposit number: GDMCC No: 62092).

[0051] A fourth aspect of the present invention provides a method comprising the steps of treating bacteria using resazurin, the reagent or the kit of the second aspect of the present invention;

[0052] The method includes any one of (b1) to (b3):

[0053] (b1) a method for promoting bacterial growth;

[0054] (b2) a method for inhibiting hydrogen production activity of bacteria;

[0055] (b3) A method for inhibiting the production of fermentation metabolites of bacteria.

[0056] In some embodiments of the present invention, the fermentation metabolites include acetic acid and / or lactic acid.

[0057] In some embodiments of the present invention, the bacteria include at least one of Clostridium sporogenes, Bacillus, Shewanella, Cellulophila, Chitinophage, and Achromobacter xylosoxidans.

[0058] In some preferred embodiments of the present invention, the bacteria are Clostridium sporogenes and / or Bacillus.

[0059] In some more preferred embodiments of the present invention, the bacterium is Clostridium sporogenes (deposit number: GDMCC No: 62212) and / or Bacillus (deposit number: GDMCC No: 62092).

[0060] The beneficial effects of the present invention are:

[0061] The present invention first proposes that resazurin can improve the functions of bacteria (such as Clostridium sporogenes) in arsenic methylation, volatilization, and adsorption. Experiments have proven that the exogenous addition of resazurin can significantly improve the methylation and volatilization of inorganic trivalent arsenic by Clostridium sporogenes.

[0062] Specifically, compared with the blank, 50 - 500 μM resazurin can significantly promote the formation of water-soluble methylarsenic by Clostridium sporogenes, and it increases with the increase in concentration. Among them, the concentrations of water-soluble dimethylarsenic and trimethylarsenic in the 500 μM treatment are 2.3 times and 2.7 times higher than those of the blank, respectively. The proportion of dimethylarsenic in total methylarsenic is 95.6% - 96.6%. The production of volatile arsenic (mainly dimethylarsenic hydride, accounting for 90.8% - 94.9% of total volatile arsenic) also shows a significant increasing trend with the addition of resazurin. There is no significant difference in volatile arsenic between the 100 μM resazurin and 300 μM treatments, and the volatile arsenic in the 500 μM treatment is 3.2 times higher than that of the blank. The presence of resazurin significantly increases the concentration of cell-adsorbed arsenic by 1.4 - 2.1 times compared with the blank treatment (0.47 μg / L). There is no significant difference in cell-adsorbed methylarsenic between the 100 μM resazurin and 300 μM treatments, and the proportion of dimethylarsenic in total adsorbed methylarsenic in all treatments is 67.7% - 83.——4%.

[0063] 50 - 500 μM resazurin significantly promotes the growth of Clostridium sporogenes, increasing by 11.7% - 18.3%. For fermentation metabolites, the presence of resazurin reduces the lactic acid production, and it decreases with the increase in resazurin concentration. The lactic acid production is the lowest when the resazurin concentration is 500 μM, which is 52.5% lower than that of the blank treatment. For the fermentation product acetic acid, the acetic acid production in the resazurin treatment group decreases with the increase in resazurin concentration, and it decreases by 21.1% - 38.8% compared with the group without resazurin treatment. The acetic acid production under the 500 μM resazurin treatment is the lowest. The cumulative hydrogen production in the resazurin treatment decreases with the increase in resazurin concentration, and it decreases by 5.3% - ——5% compared with the group without resazurin treatment (12.3 mM). The cumulative hydrogen amount is the lowest (9.2 mM) under the condition of 500 μM resazurin.

[0064] It can be seen that Clostridium sporogenes can accelerate growth and arsenic volatilization under the condition of exogenous addition of resazurin, and has application potential in carbon-neutral arsenic pollution remediation.

[0065] The method provided by the present invention can efficiently improve the arsenic methylation and volatilization rate of Clostridium, and can provide an effective means for the remediation of arsenic-polluted soil and water bodies, and has the characteristics of simple operation, economy, and practicality. Description of the Drawings

[0066] Figure 1 It is a diagram showing the influence results of the carbon-nitrogen organic substrate components of the culture medium on the formation of water-soluble methylarsenic by C. sporogenes LHA6.

[0067] Figure 2 Effect diagrams of resazurin (RZ) at different concentrations on arsenic methylation and volatilization of C. sporogenes LHA6; among them, (a) is the change of dimethylarsine concentration in aqueous solution with culture time, (b) is the change of trimethylarsine concentration in aqueous solution with culture time, (c) is the composition and concentration of volatile methylarsenic after 72 h of culture, and (d) is the composition and concentration of cell-bound methylarsenic after 72 h of culture.

[0068] Figure 3 Effect diagrams of resazurin (RZ) at different concentrations on the growth and fermentation product formation of C. sporogenes LHA6; among them, (a) is the protein content and the concentrations of short-chain fatty acids acetic acid and lactic acid after 72 h of culture, and (b) is the change of headspace hydrogen concentration with culture time. Specific implementation manners

[0069] The content of the present invention will be further described in detail below through specific examples.

[0070] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0071] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0072] The features and properties of the present invention will be further described in detail below in combination with the embodiments.

[0073] Example 1 Arsenic transformation differences of arsenic-methylating functional Clostridium LHA6 under different carbon and nitrogen source conditions

[0074] Clostridium sporogenes LHA6 (preservation number: GDMCC No: 62212, preserved in the Guangdong Provincial Microbial Culture Collection Center, 59th Building, 100th Yard, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on January 14, 2022, and has been disclosed in the patent document CN114480230A).

[0075] First, to obtain a strain seed solution in the logarithmic growth phase, LHA6 was inoculated at 5% (v / v) into 1 / 4 TSB medium containing 30 mM piperazine-1,4-diethanesulfonic acid (PIPES) buffer (pH = 7.0). The volume of the culture system was 50 mL in a 120 mL vial. After purging with high-purity nitrogen for 30 min, it was sealed to form an anaerobic state, and then incubated statically and protected from light at 30 °C for 30 h to obtain a strain seed solution in the logarithmic growth phase with a cell density reaching 0.8×10 8 ~1.6×10 8 cells / mL. The obtained seed solution was centrifuged at 5000×g for 10 min, and the cells were washed three times with physiological saline and then resuspended in modified NBAF medium. The resuspended seed solution was inoculated at 5% (v / v) into arsenic-containing modified NBAF medium with a sodium arsenite concentration of 30 μM. The components of the arsenic-containing modified NBAF medium include 0.04 g / L of CaCl2·2H2O, 0.1 g / L of MgSO4·7H2O, 1.8 g / L of NaHCO3, 0.4273 g / L of Na2CO3, 0.001 mM of Na2SeO4, 10 mL / L of 100X NB Salts, and 10 mL / L of NB Mineral Elixir. Among them, the composition of 100X NB Salts is as follows: 42 g / L of KH2PO4, 22 g / L of K2HPO4, 20 g / L of NH4Cl, 38 g / L of KCl, and 36 g / L of NaCl. The composition of NB Mineral Elixir is as follows: 2.14 g / L of nitrilotriacetic acid, 0.1 g / L of MnCl2·4H2O, 0.3 g / L of FeSO4·7H2O, 0.17 g / L of CoCl2·6H2O, 0.2 g / L of ZnSO4·7H2O, 0.03 g / L of CuCl2·2H2O, 0.005 g / L of AlK(SO4)2·12H2O, 0.005 g / L of H3BO3, 0.09 g / L of Na2MnO4·2H2O, 0.11 g / L of NiSO4·6H2O, and 0.02 g / L of Na2WO4·2H2O.

[0076] To determine the optimal carbon and nitrogen components of the medium for arsenic methylation by the strain, different organic substrate treatments were set up, including methionine (10 mM), methionine (10 mM) + tryptone (1 g / L), glucose (10 mM), and glucose (10 mM) + tryptone (1 g / L). The volume of the culture system was 50 mL in a 120 mL vial. The pH of the culture system was adjusted to 7.0. After purging with a CO2:N2 (20:80) gas mixture for 30 min, it was sealed and then incubated statically in the dark at 30 °C for 72 h. After the culture, the liquid sample was filtered through a 0.22 μm filter head, and the concentrations of soluble trimethylarsine, dimethylarsine, and monomethylarsine in the solution were determined using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS, PerkinElm).

[0077] The results of methylarsine production by LHA6 in different carbon and nitrogen organic substrate treatments are shown in Figure 1 Figure [X]. The production of dimethylarsine in the glucose treatment was 8.4 μg / L. The amount of dimethylarsine in the glucose + tryptone composite substrate treatment was 31.4 times higher than that in the glucose treatment. A similar change was observed in the production of trimethylarsine. The production of trimethylarsine in the glucose treatment was 0.3 μg / L, which increased to 5.1 μg / L after the composite substrate treatment. There was no significant difference in the production of dimethylarsine and trimethylarsine when methionine was used as the sole substrate compared to the methionine + tryptone and single glucose treatments. These results indicate that the carbohydrate substrate (glucose) combined with the tryptone composite substrate is more conducive to the arsenic methylation process of the LH16 strain compared to the amino acid (methionine) composite substrate. Therefore, the glucose + tryptone composite substrate is recommended as the preferred substrate for the arsenic methylation reaction of LHA6.

[0078] Example 2: Promoting effect of resazurin on arsenic methylation and volatilization by Clostridium LHA6

[0079] After obtaining the seed solution of the strain in the logarithmic growth phase by the method of Example 1, it was inoculated at 5% (v / v) into the arsenic-containing modified NBAF medium, where the concentration of sodium arsenite was 30 μM and glucose (10 mM) + tryptone (1 g / L) was used as the organic substrate. To determine the optimal concentration of resazurin (ZR) in the culture system for arsenic methylation and volatilization, different concentration gradients of resazurin were set up, including 0 μM, 50 μM, 100 μM, 300 μM, and 500 μM. The volume of the culture system was 50 mL in a 120 mL vial. The pH of the culture system was adjusted to 7.0. After purging with a CO2:N2 (20:80) gas mixture for 30 min, it was sealed and then incubated statically in the dark at 30 °C for 72 h.

[0080] Liquid samples were taken at various time points during the cultivation process, and the cells and headspace arsenic gas were collected at the end of cultivation after 72 h for testing cell-adsorbed arsenic and volatile arsenic. All bacterial solutions were centrifuged at 5000×g for 10 min, the supernatant was discarded, and the bacteria were washed 3 times with PIPES buffer. The liquid after digestion with 0.5% nitric acid for 1 h was used to determine the cell-adsorbed methylarsenic. The chemical capture of volatile arsenic compounds was achieved by their reaction with the solid oxidant Ag(NO3). High-purity nitrogen gas (100 kPa) filtered through a 0.22 μm syringe filter was used to blow the headspace of the anaerobic tube for 10 min. The nitrogen outlet was connected to a 1 mL syringe containing high-purity silica gel particles (sigma, Davisil Grade 636, pore size 35–60 mesh particle size) soaked in 10% AgNO3. The silica gel particles after arsenic capture were refluxed and digested in 2 mL of 0.5% (V / V) HNO3 at 90 °C for 1 h for the recovery of volatile arsenic. 5 μL of H2O2 (3%) was added to 0.5 mL of the recovered solution to oxidize CH3AsH2, (CH3)2AsH, and (CH3)3As to monomethylarsenic, dimethylarsenic, and trimethylarsenic, respectively. After all liquid samples were filtered through a 0.22 μm filter, the concentrations of soluble trimethylarsenic, dimethylarsenic, and monomethylarsenic were determined using the above high-performance liquid chromatography-inductively coupled plasma mass spectrometer.

[0081] Quantitative analysis results of soluble and volatile methylarsenic products of strain LHA6 under different concentrations of resazurin treatment showed that methylarsenic accumulated continuously during the 72 h cultivation of LHA6, and 33 μg / L of water-soluble dimethylarsenic and 1.3 μg / L of water-soluble trimethylarsenic were generated at 72 h ( Figure 2 in a). In the resazurin treatment group, compared with the blank, the generation of water-soluble methylarsenic by LHA6 was significantly promoted at 50–500 μM and increased with the increase in concentration. The concentrations of water-soluble dimethylarsenic and trimethylarsenic in the 500 μM treatment were 2.3 times and 2.7 times higher than those in the blank, respectively ( Figure 2 in b). The proportion of dimethylarsenic in the total methylarsenic was 95.6%–96.6% in all treatments. The volatile arsenic production of strain LHA6 in the blank treatment was 0.025 μg. The production of volatile arsenic also showed a significant increasing trend with the addition of resazurin. There was no significant difference in volatile arsenic between the 100 μM resazurin and 300 μM treatments, and the volatile arsenic in the 500 μM treatment was 3.2 times higher than that in the blank. Figure 2In c), the main form of volatile methylarsenic in all treatments was dimethylarsine hydride, accounting for 90.8% - 94.9% of the total volatile arsenic. Resazurin significantly increased the concentration of cell-adsorbed arsenic by 1.4 - 2.1 times compared to the blank treatment (0.47 μg / L). There was no significant difference in cell-adsorbed methylarsenic between 100 μM resazurin and the 300 μM treatment. The proportion of dimethylarsenic in the total adsorbed methylarsenic in all treatments was 67.7% - 83.4%( Figure 2 In d), these results indicate that exogenous resazurin can increase the arsenic methylation and volatilization efficiency of LHA6.

[0082] Example 3 Changes in the growth and fermentation products of arsenic-methylating bacterium LHA6 under the influence of resazurin

[0083] This example was used to verify the growth and fermentation products of arsenic-methylating bacterium LHA6 under different resazurin concentration treatment conditions in Example 2, as follows:

[0084] After the cultivation was completed, all the cells were collected, washed 3 times with PIPES buffer, and resuspended in 5 mL. 0.3 mL of the resuspended cell suspension was taken and added to 1 mL of 0.2 M NaOH solution. After shaking at 120 r / min for 30 min at 100 °C, the cell lysate was obtained. The protein concentration in the lysate was measured using a modified Bradford method protein concentration assay kit (Sangon, China) to characterize the cell biomass. Organic acids were detected by high-performance liquid chromatography with an ultraviolet detector (Agilent 1260, USA). A C18 chromatographic column (4.6 mm × 250 mm) was used, the column temperature was 30 °C, the mobile phase was 0.1 M NaH2PO4·2H2O, the pH was adjusted to 3.1, and the flow rate was 1 mL / min.

[0085] Resazurin at 50 - 100 μM and 500 μM significantly promoted the growth of strain LHA6, increasing by 11.7% - 18.3% compared to the blank treatment( Figure 3 In a), resazurin promoted cell growth at lower concentrations (50 - 100 μM), which may be related to AQDS and resazurin enhancing the extracellular electron transfer flux of the cells. For the fermentation metabolites, the presence of resazurin reduced the lactic acid production, and it decreased with the increase in resazurin concentration. The lactic acid production was the lowest when the resazurin concentration was 500 μM, which was 52.5% lower than the blank treatment( Figure 3 In a); for the fermentation product acetic acid, the acetic acid production in the resazurin treatment group decreased with the increase in resazurin concentration, decreasing by 21.1% - 38.8% compared to the group without resazurin treatment. The acetic acid production was the lowest under the 500 μM resazurin treatment( Figure 3In a). During the resazurin treatment, the cumulative hydrogen production decreased with the increase in resazurin concentration, showing a 5.3% - 25% reduction compared to the treatment without resazurin (12.3 mM). Among them, the cumulative hydrogen production was the lowest (9.2 mM) under the condition of 500 μM resazurin ( Figure 3 In b). Further, the hydrogen production was normalized by the protein concentration to reflect the hydrogen production capacity of the strain per unit mass. The results showed that the hydrogen production of strain LHA6 (0.81 - 1.05 mmol / mg) decreased by 17.6% - 36.6% when resazurin was present compared to the treatment without resazurin, indicating that resazurin inhibited the hydrogen production activity of the strain.

[0086] The above results indicate that the presence of resazurin inhibits the hydrogen production, as well as the production of fermentation metabolites acetic acid and lactic acid of the strain. Since the redox potential of resazurin (-51 mV) is higher than that of hydrogen (-410 mV), this potential difference helps to increase the extracellular electron transfer flux of Clostridium when resazurin is present. In addition, resazurin belongs to a small molecule lipophilic electron shuttle, which can directly enter the periplasm and interact with periplasmic proteins and inner membrane proteins to affect electron distribution, thus possibly affecting the intracellular carbon metabolic pathway. Therefore, resazurin promotes the arsenic methylation reaction and the formation of volatile methylarsenic by adjusting the intracellular carbon metabolic pathway while inhibiting the fermentation metabolic activity.

[0087] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of resazurin in any one of (1) to (12): (1) Improve the ability of bacteria to methylate arsenic under anaerobic conditions; (2) preparing a product that improves the ability of bacteria to methylate arsenic under anaerobic conditions; (3) Improve the volatilization performance of bacteria to arsenic under anaerobic conditions; (4) preparing a product that improves the volatilization performance of bacteria to arsenic under anaerobic conditions; (5) Improve the adsorption of arsenic by bacteria under anaerobic conditions; (6) preparing products that improve the adsorption of arsenic by bacteria under anaerobic conditions; (7) Promote bacterial growth under anaerobic conditions; (8) preparing products that promote bacterial growth under anaerobic conditions; (9) Inhibit the hydrogen production activity of bacteria under anaerobic conditions; (10) preparing a product for inhibiting the hydrogen production activity of bacteria under anaerobic conditions; (11) Inhibit the production of fermentation metabolites of bacteria under anaerobic conditions; (12) preparing products that inhibit the production of fermentation metabolites of bacteria under anaerobic conditions; The fermentation metabolites include acetic acid and / or lactic acid; The bacterium is Clostridium sporogenes with a deposit number of GDMCC No: 62212; the effective concentration of resazurin in (1) to (4) is 50 to 500 μM; The methylation mentioned in (1) to (2) includes monomethylation, dimethylation and / or trimethylation.

2. The use according to claim 1, characterized in that The arsenic is inorganic trivalent arsenic.

3. The use according to claim 1, characterized in that The arsenic includes arsenous acid and / or arsenite.

4. A method comprising placing resazurin, a reagent containing resazurin, or a kit in an environment containing arsenic and bacteria; The method includes any one of (a1) to (a3): (a1) A method for improving the ability of bacteria to methylate arsenic under anaerobic conditions; (a2) A method for improving the volatilization performance of bacteria for arsenic under anaerobic conditions; (a3) A method for improving the adsorption of arsenic by bacteria under anaerobic conditions; The bacterium is Clostridium sporogenes with a deposit number of GDMCC No: 62212; The reagent or kit containing resazurin further comprises glucose and trypticase; The effective concentration of resazurin in (a1) to (a2) is 50 to 500 μM; The methylation includes monomethylation, dimethylation and / or trimethylation.

5. The method according to claim 4, characterized in that The arsenic is inorganic trivalent arsenic.

6. The method according to claim 5, characterized in that The arsenic includes arsenous acid and / or arsenite.

7. The method according to claim 5, characterized in that The environment is culture medium, soil or water.

8. The method according to claim 7, characterized in that The concentration range of the inorganic trivalent arsenic in the environment and water is 0-200 μmol / L.

9. The method according to claim 7, characterized in that The culture medium includes NBAF medium.

10. The method according to claim 9, characterized in that The carbon and nitrogen sources in the culture medium include at least one of glucose, trypsin, and methionine.

11. The method according to claim 9, characterized in that The carbon and nitrogen sources in the culture medium are glucose and trypticase.

12. A method comprising the step of treating bacteria with resazurin, a reagent containing resazurin, or a kit; The method includes any one of (b1) to (b3): (b1) A method for promoting bacterial growth under anaerobic conditions; (b2) a method for inhibiting hydrogen production activity of bacteria under anaerobic conditions; (b3) a method for inhibiting the production of fermentation metabolites by bacteria under anaerobic conditions; The bacterium is Clostridium sporogenes with a deposit number of GDMCC No: 62212; The reagent or kit containing resazurin further comprises glucose and trypticase; The fermentation metabolites are acetic acid and / or lactic acid.

Images