A method for biosynthesis of selenium nanomaterials by methanosarcina barkeri and application thereof

Abstract

A method for biosynthesis of selenium nanomaterials by using Methanosaeta harundinacea, comprising the following steps: step 1, activating and expanding culture of Methanosaeta harundinacea under anaerobic conditions to obtain a culture system; step 2, adding sodium selenite to the culture system obtained in step 1 to make the final concentration of sodium selenite in the system 0.01-1.0 mM, and carrying out biological reduction reaction under anaerobic conditions for 24 hours to obtain a reaction mixture; and step 3, separating the reaction mixture obtained in step 2 to obtain selenium nanomaterials. The present application utilizes the anaerobic metabolic characteristics of Methanosaeta harundinacea to reduce the highly toxic sodium selenite into stable and biocompatible nanoselemium materials, and explores the influence of concentration change on the synthesis effect.

Description

Technical Field

[0001] This invention relates to the fields of nanobiotechnology and environmental microbiology, specifically to a method for biosynthesizing selenium nanomaterials using methanogenic taeniae and its applications. Background Technology

[0002] Nano-selenium (SeNPs) have broad application prospects in biomedicine (such as antibacterial and anticancer), photoelectric sensing, catalysis, and agricultural selenium supplementation due to their unique physicochemical properties and bioactivity. Currently, the preparation methods for nano-selenium are mainly divided into physical, chemical, and biological methods. Physical and chemical methods (such as vapor deposition and liquid-phase reduction) typically require high temperatures, high pressures, or the use of toxic reducing agents (such as hydrazine hydrate and sodium borohydride), which are not only energy-intensive and costly but also prone to environmental pollution, and the resulting nanoparticles have poor biocompatibility.

[0003] Biosynthesis has attracted widespread attention due to its green, mild, and sustainable characteristics. Existing technologies have reported the preparation of nano-selenium using the reduction of sodium selenite by aerobic bacteria (such as *Pseudomonas* and *Bacillus*), fungi, or plant extracts. For example, Zhang et al. (Process Biochemistry, 2023, 130: 481-491) synthesized nano-selenium using *Shewanella oneidensis* MR-1, with a reaction cycle as long as 7 days. This method suffers from drawbacks such as large nano-selenium particle size, wide size distribution, and long synthesis cycle. More importantly, there is very little research on the application of methanogenic bacteria (such as *Pasteurella multocida*) under strictly anaerobic conditions in the synthesis of nanomaterials.

[0004] Methanosarcina barkeri is a typical strict anaerobic archaea that is widely found in freshwater sediments, anaerobic digesters, and paddy soils. It has a complex electron transport chain and multiple reductase systems in its methanogenic metabolism.

[0005] Currently, research on this strain mainly focuses on methanogenic metabolism and anaerobic digestion processes. Its application in the reduction of exogenous electron acceptors (such as sodium selenite) and the targeted synthesis of nanomaterials has not yet been reported domestically or internationally. Summary of the Invention

[0006] To overcome the above technical problems, the present invention aims to provide a method for biosynthesizing selenium nanomaterials using Pasteurella multocida and its application. This method utilizes the anaerobic metabolic characteristics of Pasteurella multocida to reduce highly toxic sodium selenite into stable, biocompatible nanomaterials, and explores the effect of concentration changes on the synthesis effect.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for biosynthesizing selenium nanomaterials using Pasteurella multocida, comprising the following steps; Step 1: Strain activation and scale-up culture: Methanosarcina barkeri strain was inoculated into modified MS anaerobic medium and cultured statically at 37°C under anaerobic conditions (N2:CO2 = 80:20, v / v) until the logarithmic growth phase to obtain seed culture. Step 2: Induced synthesis reaction: Transfer the seed culture obtained in step 1 to fresh modified MS anaerobic medium at an inoculation rate of 5% to 10% by volume; then add sterile filtered sodium selenite (Na2SeO3) stock solution; maintain the anaerobic state of the system and let it stand at 37°C in the dark for 24 hours to obtain the reaction mixture; observe and record the color change every 6 hours, and at the same time take samples to determine the residual concentration of sodium selenite in the culture medium; Step 3: Product observation and collection: During the reaction, the culture medium under different concentration gradients was observed to gradually change from light yellow to different shades of red, indicating the formation of selenium nanoparticles. The culture medium was centrifuged at 8000-10000 rpm for 10-15 minutes at 4℃, the precipitate was collected, and the precipitate was washed three times with sterile deionized water to obtain selenium nanomaterials.

[0008] Step 4: Product Characterization and Validation To confirm that the obtained red precipitate was selenium nanoparticles, reaction solutions with two representative concentrations of 0.25 mM and 1.0 mM were selected for characterization. The color changes under the 0.25 mM and 1.0 mM concentration conditions were recorded using photographs. In step 1, the *Paecilomyces pasteurellii* strain is inoculated into a modified MS anaerobic medium and cultured statically at 37°C under anaerobic conditions until the logarithmic growth phase to obtain a seed culture. Specifically, the modified MS anaerobic medium is DSMZ Medium 120, wherein the vitamin solution concentration in DSMZ Medium 120 is adjusted to ten times the original concentration, and no sodium sulfide (Na2S·9H2O) is added. The original concentration was Wolin vitamins in the form of 10× concentrate, added to the culture medium at a ratio of 1:1000 (v / v) (i.e., 1 ml of stock solution was added per liter of culture medium). The anaerobic conditions are N2:CO2 = 80:20, v / v.

[0009] Step 2 specifically involves transferring the seed solution obtained in step 1 to fresh modified MS anaerobic medium at an inoculation rate of 5% to 10% by volume; subsequently, adding sterile filtered sodium selenite (Na2SeO3) stock solution to maintain the anaerobic state of the system and obtaining the reaction mixture. The final concentration of sodium selenite is 0.25 mM or 1.0 mM.

[0010] The residual concentration of sodium selenite in the culture supernatant at different time points under concentrations of 0.1 mM, 0.25 mM and 1.0 mM was determined by colorimetry, and concentration change curves were plotted.

[0011] Following the method described in step 3, crude nano-selenium products at concentrations of 0.25 mM and 1.0 mM were collected, observed using a scanning electron microscope (SEM), and analyzed for elemental composition using energy dispersive spectroscopy (EDS).

[0012] In step 1, the modified MS anaerobic culture medium is specifically DSMZ Medium 120, wherein the concentration of the vitamin solution is adjusted to ten times that of the original formula, and no sodium sulfide (Na2S·9H2O) is added.

[0013] The methanogenic octopus strain described is a wild-type isolate with equivalent reducing capacity.

[0014] In step 2, the sodium selenite mother liquor is a pre-prepared standard solution of Na2SeO3 with different concentration gradients (0mM, 0.01mM, 0.05mM, 0.1mM, 0.25mM, 1.0mM), and 0.1mL of each solution is added to each culture system.

[0015] In the preferred embodiment, the methanosarcina barkeri is used as the standard strain.

[0016] The optimal concentration of sodium selenite in the culture system was 0.25 mM or 1.0 mM, used to investigate the effect of concentration on the synthesis effect.

[0017] The separation method described in step 3 is as follows: the reaction mixture obtained in step 2 is centrifuged at 8000-10000 rpm for 10-15 minutes at 4-10℃, the precipitate is collected, and then the precipitate is washed 3 times with sterile deionized water.

[0018] The preferred reaction time for the nano-selenium biosynthesis reaction is 24 hours. During this time, the resulting selenium nanomaterials are spherical or near-spherical particles with a particle size distribution of 80-200 nm and uniform particle dispersion. The pH is controlled at 6.8-7.2 during the reaction to avoid pH fluctuations affecting cell activity and product stability.

[0019] The resulting selenium nanomaterials can be applied in fields such as antibacterial agents, antioxidants, functional food additives, drug carriers, or environmental pollutant treatment.

[0020] Observation frequency: Color changes are recorded every 6 hours.

[0021] The beneficial effects of this invention are: (1) This invention utilizes the anaerobic metabolic reduction ability of Pasteurella methanogens. The entire process can be carried out under normal temperature, normal pressure, and neutral pH conditions to reduce sodium selenite to nano-selenium without using any organic solvents or toxic reducing agents.

[0022] (2) Based on the efficient reduction characteristics of sodium selenite by Pasteurella methanogene, the present invention can observe the initial color change within 6 hours and complete the synthesis of nano selenium within 24 hours.

[0023] (3) The present invention can achieve bioconversion under the condition of sodium selenite concentration of 0.01 to 1.0 mM, and is suitable for the treatment of selenium-containing wastewater and the recovery of selenium resources.

[0024] (4) Extracellular polymers (EPS) secreted by bacteria and proteins released by cell lysis can be coated on the surface of nano-selenium in situ, giving it stability. Attached Figure Description

[0025] Figure 1 The images show the color changes of Pasteurella multocida culture broth after reacting for 24 hours under sodium selenite concentrations of 0.25 mM and 1.0 mM.

[0026] Figure 2 The bar chart shows the change in residual sodium selenite concentration in the culture medium over time under different concentrations of sodium selenite (0.25 mM and 1.0 mM).

[0027] Figure 3 The images show scanning electron microscope (SEM) images and energy dispersive spectroscopy (EDS) spectra of nano-selenium. Detailed Implementation

[0028] The following is a further description of a microbial synthesis method for nano-selenium provided by the present invention.

[0029] Example 1: Biosynthesis of nano-selenium: (1) Activation and expansion culture of strain: The methanogenic octopus strain was inoculated into a modified MS anaerobic medium, namely DSMZ Medium 120, in which the vitamin solution concentration was ten times that of the original formula and no sodium sulfide (Na2S·9H2O) was added; it was statically cultured at 37℃ under anaerobic conditions (N2:CO2=80:20, v / v) until the logarithmic growth phase to obtain the seed culture.

[0030] (2) Induced synthesis of nano-selenium: The seed liquid obtained in step (1) was inoculated into fresh modified MS anaerobic medium at a volume ratio of 5%, and sterile filtered sodium selenite was added to make the concentration of Na2SeO3 in the culture system range of 0.01mM; the reaction was allowed to stand at 37℃ for 24 hours under anaerobic conditions, and the color change was observed and recorded every 6 hours.

[0031] (3) Collection of nano-selenium: The reaction solution was centrifuged at 8000 rpm for 10 minutes at 4℃, the precipitate was collected, and the precipitate was washed 3 times with sterile deionized water to obtain crude nano-selenium product.

[0032] Example 2: Biosynthesis of nano-selenium: (1) Activation and expansion culture of strain: The methanogenic octopus strain was inoculated into a modified MS anaerobic medium, namely DSMZ Medium 120, in which the vitamin solution concentration was ten times that of the original formula and no sodium sulfide (Na2S·9H2O) was added; it was statically cultured at 37℃ under anaerobic conditions (N2:CO2=80:20, v / v) until the logarithmic growth phase to obtain the seed culture.

[0033] (2) Induced synthesis of nano-selenium: The seed liquid obtained in step (1) was inoculated into fresh modified MS anaerobic medium at a volume ratio of 10%, and sterile filtered sodium selenite was added to make the concentration of Na2SeO3 in the culture system range of 1.0mM; the reaction was allowed to stand at 37℃ for 24 hours under anaerobic conditions, and the color change was observed and recorded every 6 hours.

[0034] (3) Collection of nano-selenium: The reaction solution was centrifuged at 10,000 rpm for 15 minutes at 4°C, the precipitate was collected, and the precipitate was washed three times with sterile deionized water to obtain crude nano-selenium product.

[0035] Example 3: Biosynthesis of nano-selenium: (1) Activation and expansion culture of strain: The methanogenic octopus strain was inoculated into a modified MS anaerobic medium, namely DSMZ Medium 120, in which the vitamin solution concentration was ten times that of the original formula and no sodium sulfide (Na2S·9H2O) was added; it was statically cultured at 37℃ under anaerobic conditions (N2:CO2=80:20, v / v) until the logarithmic growth phase to obtain the seed culture.

[0036] (2) Induced synthesis of nano-selenium: The seed liquid obtained in step (1) was inoculated into fresh modified MS anaerobic medium at a volume ratio of 8%, and sterile filtered sodium selenite was added to make the concentration of Na2SeO3 in the culture system range of 0.8mM; the reaction was allowed to stand at 37℃ for 24 hours under anaerobic conditions, and the color change was observed and recorded every 6 hours.

[0037] (3) Collection of nano-selenium: The reaction solution was centrifuged at 9000 rpm for 10-15 minutes at 4℃, the precipitate was collected, and the precipitate was washed 3 times with sterile deionized water to obtain crude nano-selenium product.

[0038] Example 4: Characterization of color change in nano-selenium: The color changes of the reaction solutions synthesized using sodium selenite at different concentrations in Example 1 were observed. For example... Figure 1 As shown, under concentrations of 0.25 mM and 1.0 mM, the color of the culture medium gradually changed from light yellow to red as the reaction time increased, and the color depth under the 1.0 mM condition was higher than that under the 0.25 mM group. Specifically: under the 0.25 mM condition, the culture medium was light red after 6 hours of reaction, the red color deepened after 12 hours, and the color remained stable and uniform from 18 to 24 hours; under the 1.0 mM condition, the culture medium was red after 6 hours of reaction, and dark red after 12 to 24 hours. Under the 0.01 mM, 0.05 mM, and 0.1 mM concentrations, the color of the culture medium also changed to varying degrees within 24 hours.

[0039] Example 5: Determination of sodium selenite concentration change: The residual concentration of sodium selenite in the reaction system was determined by colorimetry. Reaction solution samples from different time points (0h, 6h, 12h, 18h, 24h) were centrifuged at 8000g for 5 minutes, washed twice, and the supernatants were combined and filtered through a filter membrane. 200 μL of the prepared sample was mixed with 100 μL of hydrochloric acid (4M), and then 200 μL of ascorbic acid (1M) was added. After incubation for 10 minutes, the absorbance at 500 nm was measured using a microplate reader, and the residual concentration of sodium selenite was calculated based on the standard curve.

[0040] The measurement results are as follows Figure 2 As shown, although there were slight fluctuations in the data at some time points, the overall trend of sodium selenite residual concentration under both concentration conditions was a significant decrease after 6 hours of reaction. This indicates that *Pasteurella multocida* can effectively reduce sodium selenite to generate nano-selenium.

[0041] Example 6: Scanning electron microscopy observation and energy dispersive spectroscopy analysis: The crude selenium nanoparticles obtained in Example 1 were observed by scanning electron microscopy and energy dispersive spectroscopy. Figure 3(a) is a SEM image (left). Under 0.25 mM conditions, spherical selenium nanoparticles are distributed on and around the bacterial cell surface. The particles are small and well dispersed. (b) Under 1.0 mM conditions, the number of selenium nanoparticles increases significantly, the particle size increases, and some particles are slightly aggregated. Figure 3 The corresponding EDS spectrum (right) shows obvious characteristic peaks of selenium, confirming that the nanoparticles are elemental selenium.

[0042] The selenium nanoparticles obtained by the above method have a particle size of 80–200 nm, are spherical or near-spherical, uniformly dispersed, and have a consistent morphology. The selenium nanoparticles synthesized by this method are suitable for the treatment of selenium-containing wastewater and the recovery of selenium resources.

Claims

1. A method for biosynthesizing selenium nanomaterials using *Pasteurella multocida*, characterized in that, Includes the following steps: Step 1: Activate and expand the culture of Pasteurella multocida under anaerobic conditions to obtain the culture system; Step 2: Add sodium selenite to the culture system obtained in Step 1 to make the final concentration of sodium selenite in the system 0.01-1.0 mM, and carry out the bioreduction reaction under anaerobic conditions to obtain the reaction mixture. Step 3: Separate from the reaction mixture obtained in Step 2 to obtain selenium nanomaterials.

2. The method for biosynthesizing selenium nanomaterials using *Pasteurella multocida* according to claim 1, characterized in that, In step 1, the methanogenic occult cocci strain of Pasteurella multocida is inoculated into a modified MS anaerobic medium and cultured statically at 37°C under anaerobic conditions until the logarithmic growth phase to obtain seed culture; wherein, the modified MS anaerobic medium is specifically DSMZ Medium 120. The anaerobic conditions are N2:CO2 = 80:20, v / v.

3. The method for biosynthesizing selenium nanomaterials using *Pasteurella multocida* according to claim 2, characterized in that, Step 2 specifically involves transferring the seed culture obtained in step 1 to fresh modified MS anaerobic medium at an inoculation rate of 5% to 10% by volume; subsequently, adding sterile filtered sodium selenite (Na2SeO3) stock solution to maintain the anaerobic state of the system. The final concentration of sodium selenite is 0.25 mM or 1.0 mM.

4. The method for biosynthesizing selenium nanomaterials using *Pasteurella multocida* according to claim 1, characterized in that, The separation method described in step 3 is as follows: the reaction mixture obtained in step 2 is centrifuged at 8000-10000 rpm for 10-15 minutes at 4-10℃, the precipitate is collected, and then the precipitate is washed 3 times with sterile deionized water.

5. The method for biosynthesizing selenium nanomaterials using *Pasteurella multocida* according to claim 1, characterized in that, The methanogenic octopus strain described is a wild-type isolate with equivalent reducing capacity.

6. The selenium nanomaterial prepared by the method according to any one of claims 1-5, characterized in that, The selenium nanomaterials are spherical or near-spherical particles with a particle size distribution of 80–200 nm. The particles are uniformly dispersed and their surfaces are coated with extracellular polymers secreted by bacteria and protein bioactive molecules released by cell lysis.

7. The selenium nanomaterial according to claim 6, characterized in that, The particle size of the selenium nanomaterials increases with increasing sodium selenate concentration.

8. The application of the selenium nanomaterial according to claim 6, characterized in that, Selenium nanomaterials have applications in antibacterial agents, antioxidants, functional food additives, drug carriers, and environmental pollutant treatment.

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