Atrazine biodegradation agent

Abstract

This invention discloses an atrazine biodegradable agent. The biodegradable agent is composed of bio-derived manganese trioxide and NaIO4; the final concentrations of the bio-derived manganese trioxide and NaIO4 in the atrazine degradation solution are 10±2 g / L and 1 mmol / L, respectively.

Description

Technical Field

[0001] This invention belongs to the fields of applied microbial technology and environmental remediation, and specifically relates to an atrazine biodegradable agent. Background Technology

[0002] Atrazine is a broad-spectrum herbicide belonging to the triazine class of compounds. It is a synthetic organic nitrogen heterocyclic compound with strong herbicidal activity. Atrazine primarily exerts its herbicidal effect by inhibiting the activity of enzymes involved in pigment synthesis within plants. It inhibits chlorophyll production, preventing photosynthesis. Atrazine also suppresses plant growth processes, hindering normal development. Due to its broad-spectrum activity, atrazine can kill many different types of weeds and is therefore widely used in agriculture.

[0003] Atrazine is a persistent organic pollutant with high stability, making it difficult to degrade naturally. Therefore, once atrazine enters soil or water bodies, it may persist for a long time and pose a potential risk to the environment.

[0004] Manganese oxides, as the second strongest oxidizing agent on Earth after oxygen, are widely distributed in terrestrial soils, freshwater lakes, and marine sediments. The oxidation of Mn(II) in nature, including both biological and abiotic pathways, continuously drives the formation and evolution of manganese oxides in the Earth's crust. Generally, the biological pathway of Mn(II) oxidation is considered closely related to the physiological and metabolic activities of microorganisms on the Earth's surface. Manganese oxides obtained by microbial oxidation of Mn(II) are defined as biogenic manganese oxides (BMOs). Compared with chemically synthesized manganese oxides, biogenic manganese oxides have slightly lower crystallinity, more octahedral vacancies in their crystal structure, a higher valence state of manganese, and a higher redox potential. Therefore, biogenic manganese oxides possess strong adsorption and oxidation characteristics and are considered a natural, pollution-free, economical, and efficient adsorbent and oxidant for heavy metals and organic pollutants. They show greater application potential than chemically synthesized manganese oxides in the treatment of heavy metal and organic pollutant wastewater. Currently, evidence for bacterial oxidation of Mn(II) to form manganese oxides mainly comes from marine and lacustrine environments. Research on bacterial-mediated Mn(II) oxidation primarily focuses on a few model strains, such as *Cladosporium argentiflorum*. Leptothrix discophora SS-1 and SP-6), *Pseudomonas putida* ( Pseudomonas putida MnB1 and GB-1), and Bacillus ( Bacillussp. SG-1, etc. However, their Mn(II) oxidation products are all high-valence hexagonal shale manganese dioxide, whose heavy metal adsorption capacity and organic matter degradation capacity are not as good as those of low-valence ferromanganese trioxide. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide an atrazine biodegradable agent.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0007] The atrazine biodegrading agent is composed of bio-derived manganese trioxide and NaIO4; the final concentrations of the bio-derived manganese trioxide and NaIO4 in the atrazine degradation solution are 10 g / L and 1 mmol / L, respectively, preferably 10 g / L and 1 mmol / L.

[0008] Preferably, the bio-derived manganese trioxide is produced by the manganese-oxidizing bacterium *Providenciasp. LLDRA6*. This *Providenciasp.* strain was deposited on December 10, 2018, at the China Center for Type Culture Collection, Wuhan, China, with accession number CCTCC NO: M 2018876, and named... Providencia sp. LLDRA6.

[0009] More preferably, the bio-derived manganese trioxide is produced by the manganese-oxidizing bacterium Providenciasp.LLDRA6 of the genus Providenciasp.LLDRA6 according to the following steps:

[0010] (1) After activating the Providencia strain, inoculate it into LB liquid medium with a final manganese concentration of 50±5mM at an inoculation rate of 0.2±0.05% and culture it in a constant temperature shaker at 35℃ and 180±5rpm for 5±1d; the Providencia strain is Providencia sp.LLDRA6.

[0011] (2) Centrifuge the LB liquid culture after step (1) at 2000±500 rpm, remove the supernatant, and obtain a mixture of bacteria and manganese trioxide;

[0012] (3) The mixture of bacteria and manganese trioxide was centrifuged and washed at 8000±500 rpm;

[0013] (4) Add phenol to the cleaned mixture of bacteria and manganese trioxide and sonicate for 50±5 min at a power of 200±50W;

[0014] (5) Add the same volume of chloroform as the added phenol to the mixture after ultrasonic treatment in step (4), and sonicate for 20±5 min at a power of 200±50W.

[0015] (6) Add methanol and deionized water to the mixture after ultrasonic treatment in step (5). The volume ratio of the added methanol, deionized water and chloroform added in step (5) is 12:(2-4):(4-6). After ultrasonic treatment for 10±2 min at a power of 200±50W, centrifuge at 2000±500rpm, remove the supernatant, and obtain the sediment.

[0016] (7) The sediment was centrifuged at 8000±500 rpm to clean it;

[0017] (8) Adjust the pH of the washed sediment to 3.0 and shake it on a shaker at 35℃ and 180±5rpm for 0.5±0.1h;

[0018] (9) The sediment treated in step (8) is centrifuged at 8000±500 rpm until the pH of the supernatant is neutral.

[0019] (10) Add sodium hypochlorite to the sediment after centrifugation in step (9) and shake it on a shaker at 35°C and 180±5 rpm for 4±0.5 h;

[0020] (11) The sediment treated in step (10) was centrifuged and washed at 8000±500 rpm and then dried at 60±5℃ to obtain pure manganese trioxide.

[0021] Preferably, the degradation time of the degrading agent is 120-168 hours.

[0022] This invention constructs an advanced oxidation system for degrading atrazine, in which the concentration ratio of bio-derived manganese trioxide to NaIO4 is 10±2 g / L:1 mmol / L. Furthermore, this invention discovers that only NaIO4 can act as an activator for bio-derived manganese trioxide.

[0023] This invention utilizes manganese-oxidizing bacteria of the genus Providence. Providencia Based on the ability of bio-derived manganese trioxide produced by sp.LLDRA6 to degrade atrazine, sodium periodate was used to further activate the bio-derived manganese trioxide to construct an advanced oxidation system, which significantly enhanced the ability of bio-derived Mn2O3 to degrade atrazine. Attached Figure Description

[0024] Figure 1The images are physical samples and SEM images of manganese trioxide from biological and chemical sources; (A) Physical sample of manganese trioxide from biological source; (B) Physical sample of manganese trioxide from chemical source; (C) SEM image of manganese trioxide from biological source; (D) SEM image of manganese trioxide from chemical source;

[0025] Figure 2 These are XRD characterization diagrams of manganese trioxide from biological and chemical sources;

[0026] Figure 3 The study compared the effects of bio- and chemically derived manganese trioxide on the degradation of atrazine, with an initial concentration of atrazine of 1 mg / L.

[0027] Figure 4 The effects of biologically and chemically derived manganese trioxide on the degradation of atrazine under different activators are: (A) sodium bisulfite; (B) sodium persulfate; (C) potassium persulfate; (D) sodium hypochlorite; (E) sodium percarbonate; (F) sodium periodate. The initial concentration of atrazine is 1 mg / L.

[0028] Figure 5 The study compared the degradation abilities of atrazine by biological and chemically derived manganese trioxide after reducing the initial concentration of atrazine and then using sodium periodate as an activator. The initial concentration of atrazine was 0.1 mg / L. Detailed Implementation

[0029] ① Manganese carbonate; ② Phenol; ③ Chloroform; ④ Methanol; ⑤ Sodium hypochlorite.

[0030] LB medium: 5 g yeast extract, 10 g peptone, 5 g sodium chloride, 1 L deionized water, pH adjusted to 7.2-7.4.

[0031] Preparation of bio-derived manganese trioxide: LB liquid culture medium was prepared and sterilized in an autoclave at 121℃ for 20 min. After cooling to room temperature, it was dispensed. Then, filtered and sterilized manganese chloride (MnCl2) solution was added to achieve a final Mn(II) concentration of 50 mM. The culture medium was then activated for 12 h. Providencia sp. LLDRA6 (a strain of manganese-oxidizing bacteria of the genus *Providencens*) was deposited on December 10, 2018, at the China Center for Type Culture Collection, Wuhan, China, accession number CCTCC NO: M2018876, and named... Providenciasp. LLDRA6 was inoculated at a rate of 0.2% (v / v) into LB liquid medium containing 50 mM Mn(II) and cultured continuously for 5 days at 35°C and 180 rpm on a constant temperature shaker. The bacterial / manganese oxide mixture was collected in 50 mL centrifuge tubes and centrifuged at 2000 rpm and room temperature for 10 min. The supernatant was discarded, and the sediment was washed with deionized water and centrifuged. This step was repeated until the supernatant was clear, and the sediment was retained. ② The sediment was sonicated with phenol for 60 min, and then an equal volume of chloroform was added and the mixture was sonicated for another 30 min. Finally, methanol and deionized water were added (the volume ratio of the added methanol, deionized water and the previously added chloroform was 12:(2-4):(4-6), preferably 12:3:5), and the mixture was sonicated again for 30 min. ③ The supernatant was removed by centrifugation, and the sediment was washed with deionized water as in step ①. ④ Resuspend the sediment in pre-prepared HCl solution, acidify the suspension in a 50 mL centrifuge tube to pH 3.0, and shake well on a shaker for 30 min. ⑤ Centrifuge and discard the supernatant, wash the sediment with deionized water as in step ①, until the pH of the supernatant is neutral. ⑥ Add NaClO and shake well on a shaker for 4 h. ⑦ Centrifuge and discard the supernatant, retain the brown precipitate, wash it with deionized water as in step ①. Then dry the brown precipitate and store it at room temperature for later use.

[0032] Preparation of chemically sourced manganese trioxide: Chemically sourced manganese trioxide was prepared by high-temperature decomposition of manganese carbonate. 1.5 g of manganese carbonate was weighed and evenly placed in a ceramic reaction boat (2 × 5 cm). The temperature was raised to 560 °C at a rate of 10 °C / min in a tube furnace and reacted for 6 h, with air continuously purging during the reaction. After the reaction, the product was allowed to cool naturally to obtain a powder. The powder was then ground and sieved through a 0.075 mm stainless steel mesh for later use.

[0033] Weigh a small amount of purified and dried manganese oxide granules and place them in an alcohol solution. Use ultrasound to disperse them into a suspension. Then, a small amount of the suspension is drawn off and dropped onto a copper-plated mesh. After drying at room temperature and vacuum spraying with gold powder, the surface morphology and structure of the manganese oxide are observed using a scanning electron microscope.

[0034] After manganese oxide powder was compressed into tablets, the purified manganese oxide samples were tested using a Bruker D8 Advance X-ray diffractometer (Cu target, Kα source, λ=0.154 nm, Ni filter). The testing conditions were: 2θ scan range of 10... ° ~80 ° Scanning speed 5 ° / min, electron generating current is 40 mA, accelerating voltage is 40 kV.

[0035] ① Sodium chloride; ② Atrazine.

[0036] Atrazine, with a pre-prepared stock solution concentration of 0.5 g / L, was added to an electrolyte solution (10 mM sodium chloride) to achieve a final concentration of 1 mg / L. 0.5 g of purified and dried bio- or chemically derived manganese trioxide was added to 50 mL of the atrazine solution for degradation. A blank control containing only atrazine was used, and each experimental group was repeated three times. The degradation reaction was carried out in the dark on a shaker at 35℃ and 180 rpm. Samples were taken at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. After centrifugation (12000 rpm, 10 min), the supernatant was filtered through a 0.22 µm polyethylene syringe, and the concentration of atrazine was determined by HPLC (Agilent HPLC 1100; C18 reversed-phase column (250 mm × 4.6 mm, 5 µm)).

[0037] ① Sodium chloride; ② Atrazine; ③ Sodium bisulfite; ④ Potassium persulfate; ⑤ Sodium persulfate; ⑥ Sodium hypochlorite; ⑦ Sodium percarbonate; ⑧ Sodium periodate.

[0038] Atrazine, with a pre-prepared stock solution concentration of 0.5 g / L, was added to an electrolyte solution (10 mM sodium chloride) to achieve final atrazine concentrations of 0.1 mg / L and 1 mg / L, respectively. 0.5 g of purified and dried bio- or chemically derived manganese trioxide was added to 50 mL of the atrazine solution, followed by the addition of 0.1 mol / L of activator to a final activator concentration of 1 mmol / L. The degradation reaction was then carried out, with the atrazine-only solution serving as a blank control. Each experimental group was replicated three times. The reaction was conducted in the dark on a shaker at 35℃ and 180 rpm, with samples taken at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. After centrifuging the sample (12,000 rpm, 10 min), the supernatant was filtered with a 0.22 µm polyethylene syringe, and the concentration of atrazine was determined by HPLC (Agilent HPLC 1100; C18 reversed-phase column (250 mm × 4.6 mm, 5 µm)).

[0039] The morphology of manganese trioxide from biological and chemical sources is as follows: Figure 1 As shown. From Figure 1 As can be seen from A and B, bio-derived manganese trioxide has a brown, granular appearance and moderate hardness. Figure 1 A), while the chemical source of manganese trioxide appears as a black, powdery substance. Figure 1 B). SEM scanning revealed that the bio-derived manganese trioxide consisted of irregularly shaped nanoparticles, which aggregated to form amorphous polymers. The absence of mycelial residue indicated good purity of the bio-derived manganese trioxide. Figure 1 C). SEM images of chemically derived manganese trioxide reveal that it consists of aggregates of many relatively regularly shaped polyhedral particles, exhibiting a slightly more regular morphology than biologically derived manganese trioxide. Figure 1 D).

[0040] The XRD (X-ray diffraction) results show that ( Figure 2 The characteristic diffraction peaks of bio-derived manganese trioxide appear at 2θ = 32.95°, 55.18°, and 65.80°. Comparison using Jade software shows that these three characteristic peaks match those in the standard card library PDF#00-041-1442 (argentite-type Mn2O3), corresponding to the (222), (440), and (622) crystal planes, respectively. The d-values ​​(lattice spacing) for these three crystal planes are 0.2716 nm, 0.16629 nm, and 0.1418 nm, respectively. This indicates that bio-derived manganese trioxide belongs to the weakly crystalline (polycrystalline) argentite-type Mn2O3, with manganese trioxide (Mn2O3) as its main phase. In contrast, all the characteristic diffraction peaks of chemically derived manganese trioxide are completely consistent with those in the standard card PDF#00-041-1442, indicating that chemically derived manganese trioxide has a stronger crystalline structure (single crystal).

[0041] The results of bio- and chemically derived manganese trioxide degrading atrazine separately are as follows: Figure 3 As shown, within 72 hours, the degradation rate of atrazine by bio-derived manganese trioxide was relatively fast, gradually decreasing with increasing reaction time. At 120 hours, the degradation reaction of atrazine gradually approached equilibrium. Finally, at 168 hours, the degradation rate of atrazine by bio-derived manganese trioxide was 44.20%. In contrast, chemically derived manganese trioxide had almost no degradation effect on atrazine; at 168 hours, the degradation rate was only 2.10%. Strictly speaking, this meager 2.10% removal rate should be attributed to the adsorption of chemically derived manganese trioxide itself. Ultimately, the degradation efficiency of bio-derived manganese trioxide was approximately 21 times that of chemically derived manganese trioxide.

[0042] Figure 4 The results show the degradation of atrazine by constructing advanced oxidation systems with bio- and chemically derived manganese trioxide and activators, respectively. Sulfites and persulfates are currently the mainstream activators; therefore, sulfites and persulfates were chosen as the first choices to construct advanced oxidation systems with the two types of manganese trioxide to degrade atrazine. Figure 4As shown in AC, after adding one sulfite (NaHSO3) and two persulfate activators (Na2S2O8 and K2S2O8), the degradation efficiency of atrazine by both bio-derived and chemically derived manganese trioxide (Mtrioxide) advanced oxidation systems was far lower than that by bio-derived Mtrioxide alone. This indicates that these three activators did not promote the degradation of atrazine by bio-derived Mtrioxide. On the contrary, these three activators inhibited the degradation of atrazine by bio-derived Mtrioxide. These results suggest that bio-derived Mtrioxide may not be effective in activating persulfate to generate large amounts of SO4. ·- Or perhaps SO4 ·- A significant increase in [amount] did not positively contribute to the degradation of atrazine. Therefore, we selected three additional activators—NaClO (sodium hypochlorite), SPC (sodium percarbonate), and NaIO4 (sodium periodate)—to further construct the advanced oxidation system. Figure 4 As shown in Figure D, NaClO has a certain degradation effect on atrazine, with a degradation rate of 25.87% ( Figure 4 D), while SPC and NaIO4 showed lower degradation rates for atrazine, at only 8.20% and 7.51%, respectively. Figure 4 E and F). After reacting the three activators with bio-derived and chemically derived manganese trioxide respectively, it was found that only the advanced oxidation system composed of bio-derived manganese trioxide and NaIO4 significantly improved the degradation rate of atrazine. At the end of the reaction at 168 h, the degradation rate of atrazine reached 58.16%.

[0043] To repeatedly verify the promoting effect of the activator NaIO4, and considering that the concentration of atrazine in the environment did not reach the 1 mg / L concentration set in the above experiment, we reduced the initial concentration of atrazine to 0.1 mg / L. The advanced oxidation system constructed from sodium periodate (NaIO4) and bio- and chemically derived manganese trioxide showed the following effect on the degradation of 0.1 mg / L atrazine: Figure 5 As shown, the advanced oxidation system composed of bio-derived manganese trioxide and NaIO4 achieved a degradation rate of 79.32% within 48 hours, with NaIO4 itself having a negligible impact on atrazine degradation. In contrast, the advanced oxidation system composed of chemically derived manganese trioxide and NaIO4 only achieved a degradation rate of 29.45% for atrazine within 48 hours. These results confirm that bio-derived manganese trioxide can only efficiently degrade atrazine when combined with sodium periodate (NaIO4).

Claims

1. An atrazine biodegradable agent, characterized in that, The biodegrading agent is composed of bio-derived manganese trioxide and NaIO4; the final concentrations of the bio-derived manganese trioxide and NaIO4 in the atrazine degradation solution are 10±2 g / L and 1 mmol / L, respectively; the bio-derived manganese trioxide is produced by the manganese-oxidizing bacterium Providenciasp.LLDRA6 of the genus Providenciasp.LLDRA6 according to the following steps: (1) After activating the Providencia strain, inoculate it into LB liquid medium with a final manganese concentration of 50±5mM at an inoculation rate of 0.2±0.05% and culture it in a constant temperature shaker at 35℃ and 180±5rpm for 5±1d; the Providencia strain is Providencia sp.LLDRA6. (2) Centrifuge the LB liquid culture after step (1) at 2000±500 rpm, remove the supernatant, and obtain a mixture of bacteria and manganese trioxide; (3) The mixture of bacteria and manganese trioxide was centrifuged and washed at 8000±500 rpm; (4) Add phenol to the cleaned mixture of bacteria and manganese trioxide and sonicate for 50±5 min at a power of 200±50W; (5) Add the same volume of chloroform as the added phenol to the mixture after ultrasonic treatment in step (4), and sonicate for 20±5 min at a power of 200±50W. (6) Add methanol and deionized water to the mixture after ultrasonic treatment in step (5). The volume ratio of the added methanol, deionized water and chloroform added in step (5) is 12:(2-4):(4-6). After ultrasonic treatment for 10±2 min at a power of 200±50W, centrifuge at 2000±500rpm, remove the supernatant, and obtain the sediment. (7) The sediment was centrifuged at 8000±500 rpm to clean it; (8) Adjust the pH of the washed sediment to 3.0 and shake it on a shaker at 35℃ and 180±5rpm for 0.5±0.1h; (9) The sediment treated in step (8) is centrifuged at 8000±500 rpm until the pH of the supernatant is neutral. (10) Add sodium hypochlorite to the sediment after centrifugation in step (9) and shake it on a shaker at 35°C and 180±5 rpm for 4±0.5 h; (11) The sediment treated in step (10) was centrifuged and washed at 8000±500 rpm and then dried at 60±5℃ to obtain pure manganese trioxide. Using sodium periodate to activate the bio-derived manganese trioxide, an advanced oxidation system was constructed, which significantly enhanced the ability of bio-derived Mn2O3 to degrade atrazine.

2. The atrazine biodegradable agent as described in claim 1, characterized in that, The final concentrations of the bio-derived manganese trioxide and NaIO4 in the atrazine degradation solution were 10 g / L and 1 mmol / L, respectively.

3. The atrazine biodegradable agent as described in claim 1 or 2, characterized in that, The degradation time of the degradation agent is 120-168 hours.

Images