Iron mineral-microorganism biofilm, its preparation method and application in degradation of pollutants in dark environment

Abstract

The application belongs to the field of new materials, pollution treatment and microbial technology, and particularly relates to an iron mineral-microbial biofilm, a preparation method thereof and application of the biofilm in degradation of pollutants in a dark environment. The iron mineral-microbial biofilm provided by the application comprises a sheet-shaped biofilm formed by bacillus megaterium and iron minerals dispersed in the biofilm. The iron mineral-microbial biofilm converts photo-generated electrons into stable chemical energy storage, continuously releases electrons in a dark environment, and realizes efficient degradation of pollutants in the dark environment.

Description

Technical Field

[0001] This invention belongs to the fields of new materials, pollution control and microbial technology, specifically relating to an iron mineral-microbial biofilm, its preparation method and its application in the degradation of pollutants in dark environments. Background Technology

[0002] Groundwater, as a vital global source of freshwater and drinking water, sustains ecosystem balance and supports human socio-economic development. Pollutants such as petroleum hydrocarbons, chlorinated solvents, heavy metals, and emerging persistent contaminants (e.g., antibiotics, PFAS) enter the groundwater environment through various pathways, creating complex and persistent groundwater pollution problems. Currently used groundwater remediation technologies include extraction, in-situ chemical oxidation / reduction, bioremediation, air disturbance, and natural attenuation. However, these technologies often face bottlenecks such as low efficiency, high cost, and the risk of secondary pollution when dealing with low-permeability formations and highly stable compounds. Photocatalysis, as a green and advanced oxidation process, has demonstrated unique advantages in groundwater remediation in recent years. However, traditional photocatalysis relies on continuous illumination, and its failure in the saturation zone due to limited light penetration severely restricts the continuous removal of pollutants such as antibiotics from groundwater. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide an iron mineral-microbial biofilm, its preparation method and its application in the degradation of pollutants in dark environments. The iron mineral-microbial biofilm provided by the present invention has excellent photoelectric conversion ability and high specific capacitance characteristics, which can realize light energy storage and continuously release electrons in dark environments, thereby efficiently degrading pollutants in dark environments.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] The present invention provides an iron mineral-microbial biofilm, comprising a sheet-like biofilm formed by Bacillus megaterium and iron minerals dispersed in the biofilm.

[0006] Preferably, the iron mineral includes one or more of hematite, magnetite, ferric hydroxide, and ferrous sulfide.

[0007] This invention also provides a method for preparing the iron mineral-microbial biofilm described in the above technical solution, comprising the following steps:

[0008] Iron minerals, purified Bacillus megaterium powder, and sterile distilled water were mixed and co-cultured to obtain the iron mineral-microbial biofilm.

[0009] Preferably, the dry weight ratio of the iron mineral and the purified Bacillus megaterium powder is 1-2:1-5.

[0010] Preferably, the co-culture temperature is 20–30°C; and the co-culture time is 1–7 days.

[0011] The present invention also provides the application of the iron mineral-microbial biofilm described in the above technical solution or the iron mineral-microbial biofilm prepared by the preparation method described in the above technical solution in the degradation of pollutants in dark environments.

[0012] The present invention also provides a method for degrading pollutants in a dark environment, comprising the following steps:

[0013] After photocharging the iron mineral-microbial biofilm, it is mixed with a pollutant-containing medium and degraded in a dark environment.

[0014] The iron mineral-microbial biofilm is the iron mineral-microbial biofilm described in the above technical solution or the iron mineral-microbial biofilm prepared by the preparation method described in the above technical solution.

[0015] Preferably, the contaminant is an antibiotic; the antibiotic includes chloramphenicol and / or tetracycline hydrochloride.

[0016] Preferably, the medium includes soil or water; when the medium is water, the concentration of pollutants in the pollutant-containing medium is 1-15 mg / L, and the ratio of the mass of the iron mineral-microbial biofilm to the volume of the pollutant-containing medium is (2-6) mg:25 mL.

[0017] Preferably, the photocharging is performed by irradiating the iron mineral-microbial biofilm with light; the power of the light irradiation is 50-500W.

[0018] This invention provides an iron mineral-microbial biofilm, comprising a sheet-like biofilm formed by Bacillus megaterium and iron minerals dispersed within the biofilm. Unlike traditional microbial biofilm complexes where redox pseudocapacitor structures are formed at the interface, the Bacillus megaterium biofilm of this invention can partially reduce Fe(III) in the iron mineral to Fe(II). Fe(II) can then hybridize with N, P, and other elements in the biofilm to form a doped structure, continuously absorbing light to store photoelectrons and releasing electrons in the dark, thereby improving photoelectric conversion efficiency, increasing the capacitance of the iron mineral-microbial biofilm, and further enhancing the catalytic degradation performance of pollutants in the dark. This overcomes the problem of low capacitance in existing technologies (traditional microbial biofilm complex interfaces) and has promising application prospects. Attached Figure Description

[0019] Figure 1Current density diagrams of Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, α-Fe2O3 of Comparative Example 1, and purified Bacillus megaterium powder of Comparative Example 2 under alternating light and dark conditions.

[0020] Figure 2 The electron accumulation diagrams are for the Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, α-Fe2O3 in Comparative Example 1, and purified Bacillus megaterium powder in Comparative Example 2. The left side shows the electron accumulation and release amounts under different numbers of light-dark alternation cycles, and the right side shows the total electron accumulation and release amounts after 3 light-dark alternation cycles.

[0021] Figure 3 The graph shows the residual concentration of antibiotics after degradation in Application Example 1 and Comparative Application Examples 1 and 2.

[0022] Figure 4 The graph shows the residual concentration of antibiotics after degradation in Application Example 2 and Comparative Application Examples 2-3. Detailed Implementation

[0023] The present invention provides an iron mineral-microbial biofilm, comprising a sheet-like biofilm formed by Bacillus megaterium and iron minerals dispersed in the biofilm.

[0024] Unless otherwise specified, the present invention does not have special requirements on the source of raw materials used, and commercially available products well known to those skilled in the art can be used.

[0025] In one embodiment, the iron mineral includes one or more of hematite (α-Fe2O3), magnetite (Fe3O4), ferric hydroxide (γ-FeOOH), and ferrous sulfide (FeS), with hematite (α-Fe2O3) or ferric hydroxide (γ-FeOOH) being a specific example.

[0026] This invention constructs a pseudocapacitive interface between iron minerals and Bacillus megaterium, and converts photogenerated electrons into stable chemical energy through Fe(II) / Fe(III) redox pairs for storage. It continuously releases electrons in the dark environment to achieve the degradation of chloramphenicol and tetracycline hydrochloride in groundwater under no-light conditions, thus breaking through the core problem of "light supply interruption-metabolic chain disruption".

[0027] This invention also provides a method for preparing the iron mineral-microbial biofilm described in the above technical solution, comprising the following steps:

[0028] Iron minerals, purified Bacillus megaterium powder, and sterile distilled water were mixed and co-cultured to obtain the iron mineral-microbial biofilm.

[0029] As one implementation method, the preparation method of the purified Bacillus megaterium (B. megaterium) bacterial powder is as follows: After centrifuging the Bacillus megaterium bacterial solution to remove the supernatant, the resulting precipitate is washed and freeze-dried sequentially to obtain the purified Bacillus megaterium bacterial powder. The Bacillus megaterium bacterial solution is the bacterial solution after 24 hours of culture; the culture is a liquid culture; the centrifugation temperature is 4–10°C, specifically 4°C in this embodiment; the centrifugation speed is 3000–8000 rpm, specifically 4000–8000 rpm in this embodiment; the time is 2–10 min, specifically 5 min in this embodiment; the washing reagent is sterile ultrapure water; the washing is performed 3–5 times, specifically 5 times in this embodiment; the centrifugation conditions are repeated for each washing; the freeze-drying temperature is 4–10°C, specifically 4°C in this embodiment; the time is 2–10 min, specifically 5 min in this embodiment. This invention does not have a special limitation on the source of the Bacillus megaterium; ordinary commercially available products can be used.

[0030] As one implementation method, the purified Bacillus megaterium powder has a moisture content of ≤5wt%, and in a specific embodiment, it is 1wt%.

[0031] In one embodiment, the dry weight ratio of the iron mineral and the purified Bacillus megaterium powder is 1-2:1-5, and in specific embodiments it is 1:2, 1:1 or 2:1.

[0032] In one embodiment, the dry weight content of Bacillus megaterium in the mixture obtained by mixing the iron mineral, the purified bacterial powder of Bacillus megaterium, and sterile distilled water is 20-85 wt%, and in a specific embodiment it is 50-70 wt%.

[0033] In one implementation, the co-culture temperature is 20–30°C, specifically 25°C in this embodiment; the co-culture time is 1–7 days, specifically 3–7 days in this embodiment.

[0034] This invention uses a co-culture method to prepare iron mineral / B. megaterium biofilms. The operation is simple and the reaction conditions are mild, close to room temperature, which is conducive to large-scale production.

[0035] In one implementation method, after co-culturing, the process further includes: sequentially performing solid-liquid separation, washing, and freeze-drying on the co-cultured mixture to obtain an iron mineral-microbial biofilm; the solid-liquid separation is performed by centrifugation; the centrifugation temperature is 4–10°C, specifically 4°C in this embodiment, the rotation speed is 4000–8000 rpm, specifically 6000 rpm in this embodiment, and the time is 5–15 min, specifically 10 min in this embodiment; the washing reagent is water; the number of washing cycles is 3–5, specifically 3 in this embodiment; the washing is performed by washing centrifugation; the washing centrifugation temperature is 4–10°C, specifically 4°C in this embodiment, the rotation speed is 4000–8000 rpm, specifically 6000 rpm in this embodiment, and the time for each washing centrifugation is 2–5 min, specifically 5 min in this embodiment; the freeze-drying temperature is 4–10°C, specifically 4°C in this embodiment, and the time is 24–36 h, specifically 36 h in this embodiment. This invention removes airborne bacteria through washing.

[0036] The present invention also provides the application of the iron mineral-microbial biofilm described in the above technical solution or the iron mineral-microbial biofilm prepared by the preparation method described in the above technical solution in the degradation of pollutants in dark environments.

[0037] The present invention also provides a method for degrading pollutants in a dark environment, comprising the following steps:

[0038] After photocharging the iron mineral-microbial biofilm, it is mixed with a pollutant-containing medium and degraded in a dark environment.

[0039] The iron mineral-microbial biofilm is the iron mineral-microbial biofilm described in the above technical solution or the iron mineral-microbial biofilm prepared by the preparation method described in the above technical solution.

[0040] In one embodiment, the medium includes soil or water, specifically water; the water is groundwater; the pollutant is an antibiotic; the antibiotic includes chloramphenicol and / or tetracycline hydrochloride, specifically chloramphenicol or tetracycline hydrochloride; when the medium is water, the concentration of the pollutant in the pollutant-containing medium is 1-15 mg / L, specifically 10 mg / L in this embodiment; the mass ratio of the iron mineral-microbial biofilm to the volume of the pollutant-containing medium is (2-6) mg:25 mL, specifically 2 mg:25 mL in this embodiment.

[0041] In one implementation, the photocharging process involves irradiating the iron mineral-microbial biofilm with light; the light source used for irradiation is a mercury lamp; the power of the irradiation is 50–500W, specifically 300W in this embodiment; and the irradiation time is 20–120 minutes, specifically 20–60 minutes in this embodiment.

[0042] As one implementation method, the dark environment is a light-protected environment.

[0043] In one embodiment, the degradation is carried out under stirring conditions; the stirring rate is 10-400 rpm, specifically 300 rpm in this embodiment; the degradation temperature is 20-30°C, specifically 25°C in this embodiment; and the degradation time is 2-30 min, specifically 20 min in this embodiment.

[0044] In one embodiment, when the medium is water, the degradation process further includes: performing solid-liquid separation on the degraded liquid; the solid-liquid separation is filtration; and the pore size of the filter membrane used for filtration is 0.45 μm.

[0045] The iron mineral-microbial biofilm prepared by this invention can achieve photo-to-electric conversion and light energy storage, and release electrons in the dark to degrade organic pollutants such as antibiotics, showing good application prospects.

[0046] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] Preparation of S1.B. megaterium powder

[0049] The B. megaterium bacterial culture (B. megaterium strain purchased from China General Microbiological Culture Collection Center) that had been cultured in liquid for 24 h was centrifuged at 8000 rpm for 5 min at 4 °C, the supernatant was discarded, and the resulting solid was then washed 5 times with sterile ultrapure water (the centrifugation conditions were repeated each time). The resulting solid was then freeze-dried at 4 °C for 5 min to obtain purified bacterial powder (moisture content of 1 wt%).

[0050] Preparation of S2.Fe2O3 / B. megaterium biofilm

[0051] α-Fe2O3 was mixed with purified Bacillus megaterium powder at a dry weight ratio of 1:2. 100 mL of sterile distilled water was added, and the resulting mixture contained 66 wt% Bacillus megaterium. The mixture was co-cultured in a reactor at 25 °C for 7 days to form a dense biofilm. The resulting mixture was centrifuged at 6000 rpm for 10 min at 4 °C, then washed and centrifuged three times at 6000 rpm with water at 4 °C, with each washing and centrifugation lasting 5 min. The solid particles were collected.

[0052] The above solid particles were freeze-dried at 4°C for 36 hours to obtain Fe2O3 / B. megaterium biofilm, which was then stored for later use.

[0053] Example 2

[0054] The difference from Example 1 is that the dry weight ratio of α-Fe2O3 to the purified Bacillus megaterium powder is 1:1.

[0055] Example 3

[0056] The difference from Example 1 is that the dry weight ratio of α-Fe2O3 to the purified Bacillus megaterium powder is 2:1.

[0057] Example 4

[0058] The difference from Example 1 is that α-Fe2O3 in Example 1 is replaced with γ-FeOOH, and the dry weight ratio of γ-FeOOH to the purified Bacillus megaterium powder is 1:1.

[0059] Example 5

[0060] The difference from Example 1 is that α-Fe2O3 in Example 1 is replaced with γ-FeOOH, and the dry weight ratio of γ-FeOOH to the purified Bacillus megaterium powder is 2:1.

[0061] Example 6

[0062] The difference from Example 1 is that α-Fe2O3 in Example 1 is replaced with γ-FeOOH, and the dry weight ratio of γ-FeOOH to the purified Bacillus megaterium powder is 1:2.

[0063] Comparative Example 1

[0064] α-Fe2O3 was used as a comparative example.

[0065] Comparative Example 2

[0066] The purified Bacillus megaterium powder prepared in Example 1 was used as a comparative example.

[0067] Comparative Example 3

[0068] γ-FeOOH was used as a comparative example.

[0069] Application Example 1

[0070] S1. Place 2 mg of the Fe2O3 / B. megaterium biofilm prepared in Example 2 into a colorimetric tube with a rotor and irradiate it under a 300W mercury lamp for 0 min, 20 min and 60 min respectively.

[0071] S2. Turn off the light source and add 25 mL of 10 mg / L chloramphenicol or tetracycline hydrochloride solution to the Fe2O3 / B. megaterium biofilm that has been pre-illuminated for different times;

[0072] S3. The resulting mixed solution was reacted under conditions of avoiding light, rotating at 300 rpm, and at 25°C, and samples were taken at regular intervals at different reaction times;

[0073] S4. After sampling, filter the sample using a 0.45 μm filter membrane to collect the filtrate, and transfer it to a liquid chromatography vial for detection.

[0074] Application Example 2

[0075] The difference from Application Example 1 is that the Fe2O3 / B. megaterium biofilm prepared in Example 2 used in Application Example 1 is replaced with the FeOOH / B. megaterium biofilm prepared in Example 4.

[0076] Comparative Application Example 1

[0077] The difference from Application Example 1 is that the Fe2O3 / B. megaterium biofilm prepared in Example 1 was replaced with α-Fe2O3 of Comparative Example 1.

[0078] Comparative Application Example 2

[0079] The difference from Application Example 1 is that the Fe2O3 / B. megaterium biofilm prepared in Example 1 was replaced with purified Bacillus megaterium powder from Comparative Example 2.

[0080] Comparative Application Example 3

[0081] The difference from Application Example 1 is that the Fe2O3 / B. megaterium biofilm prepared in Example 1 was replaced with γ-FeOOH in Comparative Example 3.

[0082] Performance testing

[0083] (1) The photoelectrochemical properties and capacitance characteristics of the Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, α-Fe2O3 in Comparative Example 1, and purified Bacillus megaterium powder in Comparative Example 2 were tested using a potentiostat.

[0084] Figure 1The graphs show the current density of the Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, and the purified Bacillus megaterium powders of Comparative Example 1 (α-Fe2O3) and Comparative Example 2 under alternating light and dark conditions. The left graph shows the Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, and the right graphs show the α-Fe2O3 of Comparative Example 1 and the Bacillus megaterium of Comparative Example 2. Figure 1 It can be seen that when α-Fe2O3 and B. megaterium with different dry weight ratios are cultured together, the photocurrent density gradually increases with the increase of the number of light-dark cycles, indicating that the biofilm has the ability to continuously charge; however, α-Fe2O3 and B. megaterium alone do not have the ability to charge.

[0085] Figure 2 The images show electron accumulation diagrams of the Fe2O3 / B. megaterium biofilms prepared in Examples 1-3, α-Fe2O3 in Comparative Example 1, and purified Bacillus megaterium powder in Comparative Example 2. The left side shows the electron accumulation and release amounts under different numbers of light-dark cycles, while the right side shows the total electron accumulation and release amounts after three light-dark cycles. Figure 2 It can be seen that as the dry weight ratio of B. megaterium increases, the amount of electron accumulation in the Fe2O3 / B. megaterium biofilm increases under light and the amount of net charge accumulation under alternating light and dark conditions.

[0086] (2) Corresponding to Case 1, the residual concentration of antibiotics in Comparative Application Examples 1 and 2 was determined by high performance liquid chromatography (HPLC) and quantitative analysis was performed using the external standard method. The chromatographic conditions were as follows: the separation column was XDB-C18, the mobile phase was methanol:ultrapure water = 60:40, the detector was an ultraviolet detector (UV), the detection wavelength was 278 nm, the flow rate was 1.0 mL / min, the injection volume was 10 μL, the column temperature was maintained at 30℃, and the retention time of chloramphenicol was 3.8 min. The detection conditions for tetracycline hydrochloride were as follows: mobile phase: acetonitrile:ultrapure water = 35:65, detection wavelength: 280 nm, flow rate: 1.0 mL / min, injection volume: 10 μL, column temperature: 30 °C, and retention time: approximately 3.4 min. The results of photocharging of the Fe₂O₃ / B. megaterium biofilm, α-Fe₂O₃, and purified B. megaterium powder prepared in Example 2 for the degradation of chloramphenicol (CPL) and tetracycline hydrochloride (TCH) under dark conditions are as follows: Figure 3 As shown.

[0087] Depend on Figure 3It was found that when the Fe2O3 / B. megaterium biofilm was continuously exposed to light for 20 min, and then the light source was turned off and pollutants were introduced, the degradation efficiency of HCT reached 12%, while that of CPL was 15%. When the light exposure time was extended to 60 min, under dark conditions, the degradation efficiencies of HCT and CPL reached 20% and 22%, respectively, representing increases of 66.7% and 46.7%.

[0088] (3) Corresponding to Example 2, the residual concentration of antibiotics in Comparative Application Examples 2-3 was determined by high performance liquid chromatography (HPLC), and quantitative analysis was performed using the external standard method. The chromatographic conditions were the same as above. The results of photocharging of FeOOH / B. megaterium biofilm, γ-FeOOH, and purified B. megaterium powder prepared in Example 4 for the degradation of chloramphenicol (CPL) and tetracycline hydrochloride (TCH) under dark conditions are as follows: Figure 4 As shown.

[0089] Depend on Figure 4 It can be seen that when FeOOH / B. megaterium biofilm was continuously exposed to light for 20 min, and then the light source was turned off and pollutants were introduced, the degradation efficiency of HCT reached 8.5%, while the degradation efficiency of CPL was 11%. When the light exposure time was extended to 60 min, under dark conditions, the degradation efficiencies of HCT and CPL reached 10% and 12%, respectively.

[0090] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for degrading pollutants in a dark environment, characterized in that, Includes the following steps: After the iron mineral-microbial biofilm is exposed to light, it is mixed with a medium containing pollutants and degraded in a dark environment. The iron mineral-microbial biofilm comprises a sheet-like biofilm formed by Bacillus megaterium and iron minerals dispersed in the biofilm; The contaminant is an antibiotic.

2. The method according to claim 1, characterized in that, The iron minerals include one or more of hematite, magnetite, ferric hydroxide, and ferrous sulfide.

3. The method according to claim 1 or 2, characterized in that, The method for preparing the iron mineral-microbial biofilm includes the following steps: Iron minerals, purified Bacillus megaterium powder, and sterile distilled water were mixed and co-cultured to obtain the iron mineral-microbial biofilm.

4. The method according to claim 3, characterized in that, The dry weight ratio of the iron mineral and the purified Bacillus megaterium powder is 1~2:1~5.

5. The method according to claim 3, characterized in that, The co-culture temperature is 20~30℃; the co-culture time is 1~7 days.

6. The method according to claim 1, characterized in that, The antibiotics include chloramphenicol and / or tetracycline hydrochloride.

7. The method according to claim 1, characterized in that, The medium includes soil or water; when the medium is water, the concentration of pollutants in the pollutant-containing medium is 1~15 mg / L, and the ratio of the mass of the iron mineral-microbial biofilm to the volume of the pollutant-containing medium is (2~6) mg:25 mL.

8. The method according to claim 1, characterized in that, The power of the illumination is 50~500W.

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