Composite bactericide for mine wastewater and use thereof

Abstract

The application discloses a composite bactericide and application, which comprises cetyltrimethylammonium bromide, 5-chloro-2-methyl-4-isothiazolin-3-one, tetramethylammonium phosphonium sulfate, benzalkonium chloride, wherein the mass concentration of the cetyltrimethylammonium bromide is 0.2-0.5 mg / L, the mass concentration of the tetramethylammonium phosphonium sulfate is 0.4-1 mg / L, the mass concentration of the 5-chloro-2-methyl-4-isothiazolin-3-one is 0.2-0.5 mg / L, and the mass concentration of the benzalkonium chloride is 0.2-0.5 mg / L. The composite bactericide can simultaneously effectively kill Acidithiobacillus ferrooxidans A. ferrooxidans and common desulfurization vibrio D. vulgaris with a sterilization rate of 98%, and has a synergistic sterilization effect.

Description

Technical Field

[0001] This invention relates to the field of compound bactericides, specifically to a compound bactericide for use in mine wastewater and its applications. Background Technology

[0002] Corrosion and damage of materials caused or promoted by the life activities of microorganisms within the biofilm on the material surface are called microbial corrosion.

[0003] ferrothiobacillus ( A. ferrooxidans ) are a type of Gram-negative, short rod-shaped acidophilic bacteria that metabolize ferrous oxide (Fe2+) 2+ It obtains energy through chemoautotrophic metabolism by reacting with reducing sulfides or other substances. The optimal growth temperature for this bacterium is 25-35℃, and its pH range is 2.0-3.5. It is widely distributed in acidic mining environments. *Thiobacillus ferrooxidans* can corrode steel. *Thiobacillus ferrooxidans* (… A. ferrooxidans ) and common desulfurization vibrio ( D. vulgaris This will accelerate the corrosion process of Q235 steel, resulting in a reduction in the mechanical properties and service fatigue life of structural components. Therefore, it is necessary to control the corrosion of *Thiobacillus ferrous oxide* (FPO). A. ferrooxidans ) and common desulfurization vibrio ( D. vulgaris Appropriate measures should be taken to suppress them to avoid corrosion of Q235 steel. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a compound bactericide for use in mine wastewater and its application, wherein the compound bactericide is capable of simultaneously inhibiting ferrous acidophilic thiobacillus. A. ferrooxidans and common desulfurization Vibrio D. vulgaris It effectively sterilizes, with a sterilization rate of up to 98%.

[0005] This invention is achieved through the following technical solution: A composite bactericide for use in mine wastewater comprises hexadecyltrimethylammonium bromide, 5-chloro-2-methyl-4-isothiazolin-3-one, tetramethylolphosphine sulfate, and benzalkonium chloride, wherein the mass concentration of hexadecyltrimethylammonium bromide ranges from 0.2 to 0.5 mg / L, the mass concentration of tetramethylolphosphine sulfate ranges from 0.4 to 1 mg / L, the mass concentration of 5-chloro-2-methyl-4-isothiazolin-3-one ranges from 0.2 to 0.5 mg / L, and the mass concentration of benzalkonium chloride ranges from 0.2 to 0.5 mg / L. This composite bactericide is used for acidic mine wastewater.

[0006] It consists of 0.2~0.5 mg / L hexadecyltrimethylammonium bromide, 0.4~1 mg / L tetrahydroxymethylphosphoric acid, 0.2~0.5 mg / L 5-chloro-2-methyl-4-isothiazolin-3-one, and 0.2~0.5 mg / L benzalkonium chloride.

[0007] It is composed of hexadecyltrimethylammonium bromide, tetramethylphosphoric acid sulfate, 5-chloro-2-methyl-4-isothiazolin-3-one, benzalkonium chloride, and sodium dodecyl sulfate, wherein the mass concentration of hexadecyltrimethylammonium bromide ranges from 0.2 to 1 mg / L, the mass concentration of tetramethylphosphoric acid sulfate ranges from 0.4 to 1 mg / L, the mass concentration of 5-chloro-2-methyl-4-isothiazolin-3-one ranges from 0.2 to 0.5 mg / L, the mass concentration of benzalkonium chloride ranges from 0.2 to 0.5 mg / L, and the mass concentration of sodium dodecyl sulfate ranges from 0.4 to 1 mg / L.

[0008] The mass concentrations of hexadecyltrimethylammonium bromide, tetrahydroxymethylphosphonic acid, 5-chloro-2-methyl-4-isothiazolin-3-one, benzalkonium chloride, and sodium dodecyl sulfate are 0.2 mg / L and 0.4 mg / L respectively.

[0009] A compound bactericide inhibits the growth of ferrous acidophilic thiobacillus in mine wastewater. A. ferrooxidans and common desulfurization Vibrio D. vulgaris The uses of.

[0010] The use of a compound bactericide in preventing microbial corrosion of Q235 steel.

[0011] This invention constructs ferrous acidophilic thiobacillus separately. A. ferrooxidans The synergistic system of compound bactericides: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.03 mg / L CIT, and common desulfurized Vibrio. D. vulgaris The synergistic system of compound bactericides: 0.2 mg / L CTAB + 0.4 mg / L THPS + 0.2 mg / L CIT + 0.2 mg / L 1227, further enhanced by constructing ferrous acidophilic thiobacillus. A. ferrooxidans and common desulfurization Vibrio D. vulgaris The synergistic effect system of the two compound bactericides ultimately yielded a system that can simultaneously and synergistically inhibit ferrous acidophilic thiobacillus. A. ferrooxidans and common desulfurization Vibrio D. vulgaris Highly efficient, low-concentration compound bactericide.

[0012] This invention obtains a second composite fungicide scheme by balancing the synergistic effect of SDS-CIT and the antagonistic effect between SDS-CTAB-THPS-1227: it consists of hexaalkyltrimethylammonium bromide, tetrahydroxymethylphosphonic acid, 5-chloro-2-methyl-4-isothiazolin-3-one, benzalkonium chloride, and sodium dodecyl sulfate.

[0013] Therefore, this invention also provides a method for screening compound bactericides: first constructing *Thiobacillus ferrous acidophilus*. A. ferrooxidans The synergistic system of compound bactericides: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.03 mg / L CIT; then, common desulfurization Vibrio bacteria were constructed. D. vulgaris The synergistic system of compound bactericides: 0.2 mg / L LCTAB + 0.4 mg / L THPS + 0.2 mg / L CIT + 0.2 mg / L 1227, finally constructing ferrous acidophilic thiobacillus. A. ferrooxidans and common desulfurization Vibrio D. vulgaris The synergistic system of the two compound bactericides was obtained, ultimately achieving the simultaneous and synergistic inhibition of two types of ferrous acidophilic thiobacilli. A. ferrooxidans and common desulfurization Vibrio D. vulgaris The final compound bactericide.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: The compound bactericide of the present invention can simultaneously target ferrous acidophilic thiobacillus. A. ferrooxidans and common desulfurization Vibrio D. vulgaris It effectively sterilizes, achieving a sterilization rate of up to 98%, and has a synergistic sterilization effect, effectively controlling... A. ferrooxidans and D. vulgaris Microbial corrosion of Q235 steel caused by this. Attached Figure Description

[0015] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings: Figure 1 To determine the SO4 levels in different systems after adding different compound bactericides 2- Concentration (a) and SO4 2- Reduction rate (b) plot; Figure 2 SEM images of the surface morphology of Q235 steel after immersion in different systems for 10 days: (a) D. vulgaris (b) A3B3C3; (c) A3B3C1; (d) Aseptic system; Figure 3 Here is a rendering of Example 1: (a) A. ferrooxidans (b) Appearance of the culture medium; D. vulgaris (c) Appearance of the culture medium; (d) Corrosion rate of Q235 steel in acidic mine wastewater after different treatments; Figure 4 Fe for Example 2 2+ and SO4 2- Concentration change; Figure 5 Example 2 shows the corrosion rate of Q235 steel in acidic mine wastewater. Figure 6 Fe for Example 3 2+ and SO4 2- Concentration change; Figure 7 Fe for Comparative Example 1 2+ and SO4 2- Concentration change; Figure 8 Fe for Comparative Example 2 2+ and SO4 2- Concentration change; Figure 9 Appearance of DSM 63 medium after adding compound bactericide: (a) increased by 5 times; (b) increased by 10 times. Figure 10 Fe for Comparative Example 5 2+ and SO4 2- Concentration change; Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0017] Materials and Methods

[0018] 1.2.1 Experimental Apparatus and Reagents A. ferrooxidans The culture medium was 9K liquid medium, consisting of solutions A and B. Solution A consisted of 3g (NH4)2SO4, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.1g KCl, and 0.01g Ca(NO3)2, dissolved in 600 mL of ultrapure water. Solution B consisted of 44.2g FeSO4·7H2O, dissolved in 400 mL of ultrapure water. The pH of both solutions A and B was adjusted to 2.0 using a 20% H2SO4 solution. Solution A was sterilized at 121℃ for 20 min, and solution B was sterilized by filtration through a 0.22 μm microporous membrane. After solution A cooled to room temperature, solutions A and B were mixed before use.

[0019] D. vulgaris The culture medium was DSM 63, composed of three solutions: A, B, and C. Solution A consisted of 0.5 g / L K₂HPO₄, 1.0 g / L NH₄Cl, 1.0 g / L Na₂SO₄, 0.1 g / L CaCl₂·2H₂O, 2.0 g / L MgSO₄·7H₂O, 2.0 mL / L sodium lactate, 1.0 g / L yeast extract, and 1.0 mg / L resazurite. Solution B consisted of 0.1 g sodium thioglycolate and 0.1 g vitamin C dissolved in 10 mL of ultrapure water. Solution C consisted of 0.5 g FeSO₄·7H₂O dissolved in 10 mL of ultrapure water. The pH of solution A was adjusted to 7.2 with 2 mol / L NaOH solution, then high-purity nitrogen was bubbled through it for 30 min to remove oxygen. Finally, the solution was autoclaved at 121°C for 20 min. To prevent the oxidation and degradation of ferrous sulfate heptahydrate and vitamin C, solutions B and C were sterilized by filtration through a 0.22 μm microporous membrane. After solution A cooled to room temperature, 980 mL of solution A was mixed with solutions B and C for later use.

[0020] Other major experimental reagents are shown in Table 1-1.

[0021] Table 1-1 Main Experimental Reagents Potassium dichromate <![CDATA[K2CrO7]]> Superior Pure Chengdu Kelong Chemical Co., Ltd. Sodium dodecyl sulfate <![CDATA[C 12 H 25 SO4Na]]> Analytical Pure Chengdu Kelong Chemical Co., Ltd. cetyltrimethylammonium bromide <![CDATA[C 19 H 42 B r N]]> Analytical Pure Chengdu Kelong Chemical Co., Ltd. Isothiazolinone <![CDATA[C4H4ClNOS]]> Analytical Pure Shanghai Maclean Biochemical Co., Ltd. benzalkonium chloride <![CDATA[C 17 H 30 ClN]]> Analytical Pure Shanghai Maclean Biochemical Co., Ltd. Sodium diphenylamine sulfonate <![CDATA[C 12 H 10 NNaO3S]]> Analytical Pure Chengdu Kelong Chemical Co., Ltd. Tetramethylthion <![CDATA[C4H 13 O8PS]]> Analytical Pure Shanghai Maclean Biochemical Co., Ltd. Propidium iodide phosphoric acid <![CDATA[H3PO4C 27 H 34 I2N4]]> Analytical purity superior grade purity Chengdu Kelong Chemicals Co., Ltd. Jiangsu Kaiji Biotechnology Co., Ltd. 1.2.2 Minimum concentration for bacteriostatic inhibition by a single bactericide 1.2.2.1 Screening Concentration Gradients for Single Fungicides Five preliminary experiments were conducted for each fungicide at five concentration gradients: 1 mg / L, 10 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L. A. ferrooxidans and D. vulgaris Antibacterial assay. The inoculum size of the culture medium was 5%. Based on the preliminary experimental results, seven concentration gradients with smaller ranges were set up for each fungicide, and each concentration was tested in triplicate to screen out the minimum inhibitory concentration (MIC) of each fungicide. All fungicides were filtered through a 0.22 μm filter membrane for sterilization before addition, and their concentrations are shown below: The concentration gradient of hexadecyltrimethylammonium bromide (CTAB) was as follows: 0.01 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, and 10 mg / L.

[0022] The concentration gradients of sodium dodecyl sulfate (SDS) were as follows: 0.5 mg / L, 1 mg / L, 3 mg / L, 5 mg / L, 10 mg / L, 30 mg / L, and 50 mg / L.

[0023] The 5-chloro-2-methyl-4-isothiazolin-3-one CIT concentration gradient was divided into two groups, targeting... A. ferrooxidans Sterilization: 0.05 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 50 mg / L, 100 mg / L; Targeting D. vulgaris Sterilization: 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L.

[0024] The concentration gradients of benzalkonium chloride 1227 were as follows: 20 mg / L, 50 mg / L, 70 mg / L, 90 mg / L, 110 mg / L, 130 mg / L, and 150 mg / L.

[0025] The concentration gradients of tetrahydroxymethylphosphoric acid (THPS) were as follows: 20 mg / L, 50 mg / L, 70 mg / L, 90 mg / L, 110 mg / L, 130 mg / L, and 150 mg / L.

[0026] Each sterilization experiment included a blank liquid culture medium without bacteria and a pure bacterial solution without bactericide as a control, with three replicates. (Targeting...) A. ferrooxidans The sterilization experiment involved periodically measuring the OD value, pH value, and Fe content of the culture medium in each experimental group. 2+ Concentration determines the minimum inhibitory concentration (MIC) of the bactericide. (This is relevant to...) D. vulgaris In the sterilization experiment, all experimental groups used anaerobic culture. The appearance color of the culture medium, conductivity, and SO4 levels were observed. 2- Concentration determines the minimum inhibitory concentration of the bactericide.

[0027] 1.2.2.2 Bacterial activity detection Bacterial redox capacity is a crucial indicator of bacterial activity. By detecting the content of key oxidized / reduced ions and calculating their oxidation or reduction rates, the activity level of bacteria can be determined. For *Thiobacillus ferrous acidophilus*, the key ion is Fe. 2+ For common desulfurization vibrio, the key ion is SO42-. 2- The specific measurement method is based on existing technology.

[0028] 1.2.2.3 Detection of conductivity in culture medium Since live bacteria carry a negative charge, changes in the conductivity of the bacterial culture can reflect changes in bacterial cell membrane permeability. Therefore, a conductivity meter (DDS-307, Shanghai Leici) was used to measure the conductivity of each group of culture media to check bacterial activity. 15 mL of culture media from each experimental group was taken and placed in a 25 mL sterile beaker for conductivity measurement.

[0029] 1.2.2.4 Live / Dead Bacteria Staining Detection The dyes used for bacterial staining were cyanine green (SYTO9) and propidium iodide (PI). The staining characteristics of cyanine green are described in section 3.3.2.1. Propidium iodide cannot penetrate intact cell membranes; it can only infiltrate dead bacteria with damaged cell membranes, where it emits red fluorescence after intercalating into the DNA double strand. Therefore, after staining with a mixture of cyanine green and propidium iodide, the live / dead bacteria can be determined based on the green / red fluorescence. The specific method involves centrifuging the bacterial culture (10000 r / min, 5 min) after adding bactericide and incubating for a predetermined time, discarding the supernatant, and then adding 100 μL of a mixed dye mixture of cyanine green (5 μM) and propidium iodide (2 μg / mL) to stain the bacteria. After staining at 37℃ in the dark for 15 min, 2 mL of the reaction solution is added to 1 mL of sterile physiological saline, centrifuged twice more, discarding the supernatant, and washing the precipitate with deionized water to remove excess dye. Finally, add 50 μL of physiological saline, gently shake to disperse the precipitate, prepare a bacterial suspension, and take 10 μL to add to a glass slide and cover with a coverslip. Observe using a CLSM. The excitation wavelength and emission wavelength of the sample to be tested are 535 nm and 615 nm, respectively.

[0030] 1.2.3 Screening of multi-component compound bactericides After determining the minimum inhibitory concentration (MIC) of a single bactericide, this concentration was appropriately reduced, and then comprehensive experiments were conducted on the combination of bactericides in pairs. The results were analyzed by observing the appearance, color, conductivity, and Fe content of the culture medium. 2+ SO4 2- The sterilization effect was examined by methods such as live / dead bacteria staining, and the sterilization rate was calculated (Equation 1-1).

[0031]

[0032] In the formula, N0 is the number of bacteria in the culture medium before sterilization, CFU / mL; N1 is the number of bacteria in the culture medium after sterilization, CFU / mL.

[0033] The antibacterial effect of compound bactericides can also be measured by partial inhibitory concentration indexes (PIIs). FICI ) indicates that, through calculation FICI To confirm whether the compound bactericides have a synergistic effect, the calculation method is shown in Equation 1-2.

[0034]

[0035] In the formula, [A] and [B] are the concentrations of the two bactericides in the compound preparation, respectively, in mg / L; MIC A MIC BThese are the minimum inhibitory concentrations (MICs) of the two bactericides when used alone. MIC ), mg / L. If the minimum compound bactericidal concentration is FICI If the concentration is <0.8, the two fungicides have a synergistic effect when combined; if 0.8 ≤ FICI If the concentration is ≤1.2, then the combination of the two fungicides has no irrelevant effect; if FICI >1.2, the two fungicides, when combined, exhibit antagonistic effects (Greene et al., 2006). In this article... MIC It is an abbreviation for minimum inhibitory concentration, and MIC is an abbreviation for microbial corrosion.

[0036] Based on the effective antibacterial properties of the two-component combination, respectively... A. ferrooxidans and D. vulgaris Three fungicides were selected from each group to establish a multi-component fungicide compound system.

[0037] 1.2.3.1 Establish A. ferrooxidans Multi-component bactericide compound system Multiple preliminary experiments revealed that only when THPS was 260 mg / L and the concentration of 1227 was 300 mg / L could it have the desired effect. A. ferrooxidans A significant antibacterial effect was observed. However, when CTAB, SDS, and CIT were all applied at 50 mg / L, no bacteria grew in the culture media during the bacterial growth cycle, demonstrating a clear antibacterial effect. Since excessively high concentrations of bactericides can put pressure on the ecological environment and increase economic costs, [further action is needed]. A. ferrooxidans The antibacterial experiment selected three bactericides, CTAB, SDS, and CIT, to further determine their minimum inhibitory concentrations.

[0038] A. ferrooxidans For Fe 2+ The bio-oxidative activity of this bacterium is an important biochemical characteristic, which can be detected by measuring the Fe content in the bacterial culture. 2+ Concentration is used to determine bacterial viability. In three different bactericide systems, when CTAB was 0.01–0.1 mg / L, SDS was 0.5–1 mg / L, and CIT was 0.05–0.1 mg / L, Fe... 2+ The content decreased to zero within 3-7 days, indicating the presence of viable bacteria within that concentration range. (Fe) 2+ Oxidation. During a 7-day growth cycle, when the fungicide concentration is not less than 0.5 mg / L CTAB, 3 mg / L SDS, and 1 mg / L LCIT, Fe... 2+ The concentration was basically the same as that of the sterile system, indicating that there were no live bacteria affecting Fe. 2+Oxidation occurs. Based on the analysis of the changes in the appearance, OD value, and pH value of the culture medium, 0.5 mg / L CTAB, 3 mg / L SDS, and 1 mg / L CIT are the minimum inhibitory concentrations for each individual bactericide.

[0039] Antibacterial experiments were conducted on a combination of CTAB, 3 mg / L SDS, and 1 mg / L CIT, based on the concentration of a single bactericide. The Fe content of the culture medium was measured after applying the combined bactericide. 2+ Based on the concentration and viable bacterial count, the conclusions are as follows: the 0.3 mg / L CTAB + 0.08 mg / L CIT combination group and the 0.5 mg / L SDS + 0.05 mg / L CIT combination group have the following effects on... A. ferrooxidans The sterilization rate reached 99.8%.

[0040] The partial inhibitory concentration index (FICI) was used to evaluate the efficacy of compound bactericides. For the aforementioned bactericides that effectively inhibit bacterial growth... A. ferrooxidans The compound fungicides 0.3 mg / L CTAB + 0.08 mg / L CIT and 0.5 mg / L SDS + 0.05 mg / L CIT are calculated according to Equation 6-2:

[0041] The calculation results are 0.68 < 0.8 and 0.22 < 0.8. According to... FICI According to the evaluation criteria, both the CTAB + CIT combination and the SDS + CIT combination have synergistic effects.

[0042] Two compound formulations were obtained through orthogonal experiments, range analysis, and mean analysis. These two formulations were then added to the culture medium for Fe... 2+ The optimal formulation was determined by oxidation and corrosion weight loss experiments: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.03 mg / L CIT.

[0043] 1.2.3.2 Establishment D. vulgaris Multi-component bactericide compound system Multiple preliminary antibacterial experiments revealed that only when CTAB concentration was 500 mg / L and SDS concentration was 650 mg / L could the antibacterial effect be achieved. D. vulgaris This concentration, while exhibiting antibacterial effect, is significantly too high. Excessive application of fungicides not only poses potential harm to the ecological environment but also increases economic costs. Therefore, it is crucial to... D. vulgaris Three bactericides, THPS, 1227 and CIT, were selected for the bactericidal experiment.

[0044] 1. By using three fungicides at different concentrations... D. vulgaris SO4 in culture medium2- Concentration detection experiments concluded that when CIT is 5–40 mg / L, THPS is 20–70 mg / L, and 1227 is 10–70 mg / L, SO42- 2- The content gradually decreases to zero over 3-7 days, and the lower the concentration of the fungicide, the lower the SO4 content. 2- The faster the content decreases, the more viable bacteria are growing within that concentration range. 2- It is reduced. Within 10 days, when THPS is not less than 90 mg / L, 1227 is not less than 90 mg / L, and CIT is not less than 60 mg / L, SO42- 2- The concentration was consistent with that of the sterile system, meaning there were no live bacteria affecting SO4. 2- It produces a reducing effect.

[0045] Based on the concentration of individual bactericides, THPS, CIT, and 1227 were paired and mixed in three ways: THPS+CIT, THPS+1227, and CIT+1227. The proposed mixed concentrations are shown in Table 1-2. Antibacterial experiments were conducted: after adding different concentrations of the compound bactericides and culturing for 10 days, the SO4 in the culture medium of each system was... 2- concentration.

[0046] Table 1-2 Combination of Three Fungicides Table 6-7 The compound concentration of three bactericides 1# 10 10 10 20 10 20 2# 10 20 10 40 10 40 3# 10 40 10 60 10 60 4# 30 10 30 20 20 20 5# 30 20 30 40 20 40 6# 30 40 30 60 20 60 7# 50 10 50 20 40 20 8# 50 20 50 40 40 40 9# 50 40 50 60 40 60 In the three compound bactericide systems, when the concentration ratios of the bactericides were: (1) THPS (mg / L) + CIT (mg / L): 30+40, 50+10, 50+20, 50+40, (2) THPS (mg / L) + 1227 (mg / L): 50+20, 50+40, 50+60, (3) CIT (mg / L) + 1227 (mg / L): 40+20, 40+40, 40+60, the SO4 in the culture medium was... 2- No significant decrease in concentration was observed; the concentration was similar to that of the sterile system, indicating that no viable bacteria were present to react with SO4. 2- It produces a reducing effect.

[0047] Based on the above results, a synergistic effect analysis of the fungicides was conducted: the combined fungicide with added THPS / CIT at 50 mg / L THPS + 10 mg / L CIT... FICI THPS+CIT =0.72<0.8, this combination is suitable for... D. vulgaris The inhibitory effect showed a synergistic effect; when THPS / 1227 compound fungicide was added, at 50 mg / L THPS + 20 mg / L 1227, the inhibition was synergistic. FICI THPS+1227 =0.78 < 0.8, showing a synergistic effect; while CIT and 1227 combined... FICI CIT+1227 The values ​​are all greater than 0.8, indicating no synergistic effect.

[0048] 2. To maximize the bactericidal effect, THPS, CIT, and 1227 fungicides were selected for a ternary compound (so that THPS and CIT, and THPS and 1227 can exert the best synergistic effect). Through factor influence experiments, an orthogonal experiment was conducted. The factors and levels of the compound fungicides are shown in Tables 1-3. Each row of levels constitutes an experimental condition. A three-factor, three-level orthogonal experiment L9 (3 3 ).

[0049]

[0050] Nine systems after adding compound fungicide were cultured for another 10 days. The orthogonal experimental results of the three fungicides are shown in Tables 1-4, and mean and range analyses were performed.

[0051] (1) Mean analysis According to the mean analysis method (see 6.3.3), T can be calculated. A1 T A2 T A3 T B1 T B2 ,…,T C3 The value of . , , The values ​​for T1, T2, and T3 represent the average values ​​of the three rows of data, i.e., the average bactericidal rate of each factor at each level. Based on the average values ​​of the bactericides, level 3 (40 mg / L) is optimal for THPS in the compound bactericide mixture. The analytical method for CIT and 1227 follows the same approach, selecting the levels corresponding to the highest average bactericidal rates of the bactericides for recombination. That is, both CIT and 1227 are set to 20 mg / L. Through mean analysis, the optimal combination for the orthogonal experiment is found to be A3B3C3.

[0052] (2) Range analysis As shown in Table 1-4, R A =11.8, R B =10.6, R C =4.4, indicating that factor A has the greatest impact on the bactericidal rate in the compound combination, and the maximum level value of factor A should be selected, i.e., the third level of 40 mg / L; the range R of factor C is... C=4.4 is the minimum, indicating that factor C has the least impact on the sterilization rate. Therefore, the minimum level value should be selected, i.e., the dosage of factor C at level 1 is 5 mg / L, or 5 mg / L for 1227. Thus, the experimental combination determined through range analysis is A3B3C1.

[0053]

[0054] The A3B3C3 and A3B3C1 samples were subjected to sterilization experiments again, and the corrosion morphology of the samples was observed. SO4 was also detected. 2- The reduction rate was used to determine bacterial activity and identify the optimal combination formulation. The experimental group contained... D. vulgaris Pure bacterial cultures were cultured, and bacterial systems with combinations A3B3C3 and A3B3C1 were added, as well as a sterile system. After 10 days of incubation, the SO4 levels in each system were measured. 2- Concentration and reduction rate, and corrosion morphology detection results of Q235 steel in each system are as follows: Figure 1 and Figure 2 As shown.

[0055] Calculations showed that the sterilization rate of group A3B3C1 was 99.3%, and the sterilization rate of group A3B3C3 was 99.9%. Figure 1 It can be seen that, D. vulgaris SO4 in the system 2- The concentration decreased sharply within 2 days, reaching its minimum on day 7, with a final reduction rate of 97.6%. In the A3B3C1 combination, SO42- was reduced after 10 days. 2- The concentrations of all three compounds decreased slightly to 56.0 mg / L, indicating a decrease in SO42- concentration. 2- The concentration was 48.6 mg / L; the calculated reduction rate of group A3B3C3 was 7.9%, which is closer to the reduction rate of the aseptic system (4.5%), while the reduction rate of group A3B3C1 was 20.1%, higher than that of group A3B3C3. Figure 2 It is evident that the Q235 steel samples in the A3B3C3 and A3B3C1 combinations did not exhibit similar characteristics. Figure 2 (a) The pitting corrosion morphology of the sample is similar to that of the sterile system sample, exhibiting a wavy pattern. Comprehensive analysis of sterilization rate and SO4 content... 2- Based on the reduction rate and corrosion morphology, the optimal formulation was finally determined to be A3B3C3, namely 40 mg / L THPS + 20 mg / L CIT + 20 mg / L 1227.

[0056] Establish a compound system of two multi-component fungicides Fresh wastewater was collected from the washing and screening workshop, sedimentation tank, and drainage outlet. Equal volumes of water samples were mixed, and the trace dilution method was used to detect the pollutants. A. ferrooxidans and D. vulgarisThe number of colonies of the two bacteria was approximately 1.0 × 10⁻⁶. 5 cells / mL, 1.5×10 3 cells / mL D. vulgaris The number was only 0.1% of the experimental colony count. A. ferrooxidans For Fe 2+ The bio-oxidative activity of this bacterium is an important biochemical characteristic, which can be detected by measuring the Fe content in the bacterial culture. 2+ Concentration determines bacterial viability.

[0057] To further illustrate the technical effects of this invention, two types of corrosive bacteria targeting acidic (AMD) mine wastewater from a vanadium-titanium magnetite mine were developed. A. ferrooxidans and D. vulgaris The influence of these two bacteria on the corrosion behavior of Q235 steel was investigated using a compound bactericide to control corrosion. Corrosion weight loss, surface morphology observation, and electrochemical testing were employed to study the effects. A. ferrooxidans and D. vulgaris The corrosion behavior characteristics of Q235 steel were investigated; the corrosion mechanism of these two bacteria on Q235 steel was revealed; the influence of microbial corrosion on the static tensile and low-cycle fatigue properties of Q235 steel was elucidated; and a treatment method targeting... A. ferrooxidans and D. vulgaris A highly efficient and low-consumption compound bactericide. A. ferrooxidans It has a significant accelerating effect on Q235 steel, with a corrosion rate approximately 2 to 3 times that of the sterile system. General corrosion is dominant, with localized pitting corrosion randomly distributed. The main corrosion component is ferrite. A. ferrooxidans The corrosion products of both the aseptic and sterile systems are the same: iron oxides and jaundice. In the ternary system of vanadium-titanium magnetite-microorganisms-Q235 steel, due to bioleaching of the minerals, the energy substance Fe... 2+ The increased bacterial growth rate and biofilm formation accelerate the corrosion of Q235 steel. During this process, the electrochemical corrosion behavior of Q235 steel is responsive to the surface biofilm area; increased biofilm coverage leads to increased impedance and decreased corrosion current density. Bacteria can still cause metal corrosion even without direct adhesion to the sample surface. A. ferrooxidans The corrosion process of Q235 steel is a redox reaction process. A. ferrooxidans Fe 2+ Oxidized to Fe 3+ Fe 3+ with Fe 0 The disproportionation reaction occurs again to produce Fe. 2+ This repeated reaction accelerates the corrosion of Q235 steel.

[0058] D. vulgaris The corrosion rate of Q235 steel is less than A. ferrooxidans However, obvious pitting corrosion appeared on the surface of the steel sheet.D. vulgaris The corrosion products of aseptic and sterile systems are different. D. vulgaris The corrosion products of the system are iron oxides and FeS, while those of the sterile system are a mixture of iron oxides and ferric sulfates. After the addition of electron carriers, FAD and RF... D. vulgaris In the medium, the self-oxidation / reduction state transformation promotes electron transfer and accelerates the corrosion of Q235 steel. In vanadium-titanium magnetite... D. vulgaris In the ternary system of Q235 steel, the corrosion rate of Q235 steel is slightly increased compared to that without added minerals, but... D. vulgaris The reducing activity was not affected by the mineral. Combined with product analysis, it is believed that... D. vulgaris As a biological cathode region, SO4 is transferred via extracellular electron transfer. 2- Restore to HS - Further ionization into S 2- Fe dissolved from Q235 steel 2+ FeS precipitate is formed, and the reaction continues, leading to pitting corrosion in Q235 steel.

[0059] Example 1

[0060] The compound bactericide formulation is: 0.5 mg / L CTAB + 1 mg / L THPS + 0.5 mg / L CIT + 0.5 mg / L 1227. After incubation at 30 ℃ for 10 days, the solution in the mine wastewater test bottle did not turn red. Figure 3 -(a)), and no black precipitate appeared ( Figure 3 - (b)); The sterilization test results showed that the sterilization rate was 98%. Comparing the rate of corrosion due to weight loss, it can be seen that the corrosion rate of carbon steel in acidic mine wastewater treated with this composite bactericide is similar to that in mine wastewater after high-pressure sterilization. Figure 3 -(c)). Therefore, this compound bactericide can effectively inhibit A. ferrooxidans and D. vulgaris Growth and control of microbial corrosion of carbon steel caused by these two bacteria.

[0061] Example 2

[0062] The compound fungicide formulation was: 0.2 mg / L CTAB + 0.4 mg / L THPS + 0.2 mg / L CIT + 0.2 mg / L 1227. Experimental results showed that after 10 days of cultivation, no black precipitate appeared in the culture medium, and the concentrations of ferrous and sulfate did not decrease significantly (see...). Figure 4 Following this, a sterilization experiment was conducted, showing a sterilization rate of 98%, indicating that this composite bactericide has excellent antibacterial effect. The weight loss corrosion rate verification experiment showed that the weight loss corrosion rate of Q235 steel at different time points after different treatments by AMD is shown in the figure. Figure 5Based on the redox reaction and the corrosion rate of Q235 steel, it can be seen that this composite bactericide can effectively inhibit... A. ferrooxidans and D. vulgaris To prevent the growth of these two bacteria and control the microbial corrosion of carbon steel equipment.

[0063] Example 3

[0064] A compound fungicide was prepared: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.2 mg / L CIT + 0.4 mg / L THPS + 0.2 mg / L 1227. No color change was observed in the 9K and DSM 63 media. The test results showed (…). Figure 6 The concentrations of ferrous ions and sulfate ions did not change significantly, indicating that no viable bacteria oxidized or reduced the culture medium. This compound bactericide can effectively kill bacteria in mining wastewater. A. ferrooxidans and D. vulgaris .

[0065] Comparative Example 1 A compound bactericide was prepared: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.05 mg / L CIT + 0.04 mg / L THPS + 0.02 mg / L 1227. This compound bactericide was added to the collected mine wastewater. After incubation at 30 ℃ for 10 days, no significant changes were observed in the appearance of the wastewater. The treated wastewater was then added to diluted 9K and DSM 63 media, respectively, and incubated for another 10 days. The ferrous concentration in the 9K liquid medium did not change significantly, decreasing from approximately 4.34 g / L to 4.12 g / L, with an oxidation rate of approximately 5.1%. This result can be attributed to oxidation caused by atmospheric oxygen. A black precipitate appeared in the DSM 63 medium, and a hydrogen sulfide odor was detected upon opening the aluminum cap. The sulfate concentration decreased from 121.5 mg / L to 55.3 mg / L (see...). Figure 7 The reduction rate was approximately 54.5%, indicating that the bactericide failed to inhibit bacterial growth and still had some residue. D. vulgaris The live bacteria produced a reducing effect, failing to achieve the desired effect on the mining wastewater. D. vulgaris Effectively inhibits bacteria.

[0066] Comparative Example 2 A compound bactericide was prepared: 0.2 mg / L CTAB + 0.4 mg / L SDS + 0.13 mg / L CIT + 0.2 mg / L THPS + 0.1 mg / L 1227. After incubation at 30 ℃ for 10 days, a black precipitate was observed in the culture flask. The ferrous concentration did not change significantly, remaining at approximately 4.25 ± 0.03 mg / L; the sulfate concentration decreased from 123.3 mg / L to 75.2 mg / L (see...). Figure 8The sulfate reduction rate was 39%, indicating that a small amount of sulfate was still present in the mine wastewater at this time. D. vulgaris Live bacteria.

[0067] Comparative Example 3 A compound bactericide was prepared: 1 mg / L CTAB + 2 mg / L SDS + 0.25 mg / L CIT + 0.2 mg / L THPS + 0.1 mg / L 1227. The results showed no significant decrease in ferrous concentration, but a black precipitate appeared in the culture flask (see...). Figure 9 -(a)), the concentration of sulfate ions is significantly reduced. It is not possible to simultaneously... A. ferrooxidans and D. vulgaris Effectively inhibits bacteria.

[0068] Comparative Example 4 A compound fungicide was prepared: 2 mg / L CTAB + 4 mg / L SDS + 0.5 mg / L CIT + 0.4 mg / L THPS + 0.2 mg / L 1227. However, a significant amount of black precipitate still appeared in the DSM 63 medium (see...). Figure 9 -(b)), cannot be simultaneously applied to A. ferrooxidans and D. vulgaris Effectively inhibits bacteria.

[0069] Comparative Example 5 A compound bactericide was prepared: 0.2 mg / L CTAB + 0.13 mg / L CIT + 0.2 mg / L THPS + 0.1 mg / L 1227. After incubation at 30℃ for 10 days, a black precipitate was observed in the culture flask. The ferrous concentration did not change significantly, remaining at approximately 4.1 ± 0.06 g / L, while the sulfate concentration decreased from 118.5 mg / L to 82.3 mg / L (see...). Figure 10 The reduction rate is approximately 30.5%, at which point a small amount of [unclear - possibly referring to a specific substance or quantity] remains in the mine wastewater. D. vulgaris Live bacteria reduce sulfate ions.

[0070] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound bactericide for use in mine wastewater, characterized in that, The compounds include hexadecyltrimethylammonium bromide, 5-chloro-2-methyl-4-isothiazolin-3-one, tetramethylolphosphine sulfate, and benzalkonium chloride, wherein the mass concentration range of hexadecyltrimethylammonium bromide is 0.2~0.5 mg / L, the mass concentration range of tetramethylolphosphine sulfate is 0.4~1 mg / L, the mass concentration range of 5-chloro-2-methyl-4-isothiazolin-3-one is 0.2~0.5 mg / L, and the mass concentration range of benzalkonium chloride is 0.2~0.5 mg / L.

2. The compound bactericide according to claim 1, characterized in that, It consists of 0.2~0.5 mg / L hexadecyltrimethylammonium bromide, 0.4~1 mg / L tetrahydroxymethylphosphoric acid sulfate, 0.2~0.5 mg / L 5-chloro-2-methyl-4-isothiazolin-3-one, and 0.2~0.5 mg / L benzalkonium chloride.

3. The compound bactericide according to claim 1, characterized in that, It is composed of hexadecyltrimethylammonium bromide, tetramethylphosphoric acid sulfate, 5-chloro-2-methyl-4-isothiazolin-3-one, benzalkonium chloride, and sodium dodecyl sulfate, wherein the mass concentration of hexadecyltrimethylammonium bromide ranges from 0.2 to 1 mg / L, the mass concentration of tetramethylphosphoric acid sulfate ranges from 0.4 to 1 mg / L, the mass concentration of 5-chloro-2-methyl-4-isothiazolin-3-one ranges from 0.2 to 0.5 mg / L, the mass concentration of benzalkonium chloride ranges from 0.2 to 0.5 mg / L, and the mass concentration of sodium dodecyl sulfate ranges from 0.4 to 1 mg / L.

4. The compound bactericide according to claim 3, characterized in that, The mass concentrations of hexadecyltrimethylammonium bromide, tetrahydroxymethylphosphonic acid, 5-chloro-2-methyl-4-isothiazolin-3-one, benzalkonium chloride, and sodium dodecyl sulfate are 0.4 mg / L.

5. The compound bactericide according to any one of claims 1-4 inhibits ferrous acidophilic thiobacillus in mine wastewater. A. ferrooxidans and common desulfurization Vibrio D. vulgaris Its uses.

6. The use of the composite bactericide according to any one of claims 1-4 in preventing microbial corrosion of Q235 steel.

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