Biological nano-selenium nutrient as well as preparation method and application thereof

Abstract

The invention provides a biological nano-selenium nutrient as well as a preparation method and application thereof, and belongs to the technical field of heavy metal treatment. The biological nano-selenium nutrient disclosed by the invention is obtained by microorganism synthesis and separation; the microorganisms are selenium-enriched saccharomycetes; the biological nano-selenium nutrient is derived from biosynthesis, and compared with a traditional chemical synthesis nano-material, the preparation process of the biological nano-selenium has the advantages of low energy consumption and few byproducts, and can be used for heavy metal treatment in multiple fields.

Description

Technical Field

[0001] The present invention relates to the technical field of heavy metal treatment, and in particular to a biological nano-selenium nutrient and a preparation method and application thereof. Background Art

[0002] With the acceleration of industrialization and urbanization, heavy metal pollution has become a serious global problem. Traditional heavy metal pollution control technologies include chemical precipitation, ion exchange, and physical adsorption. Traditional heavy metal pollution control methods, such as chemical precipitation, are often limited by the selectivity of the reaction between the precipitant and the heavy metal ions. Some heavy metals are difficult to completely convert into precipitates and remain. When chemically treating heavy metal pollution, the large amount of chemicals used may introduce new pollutants. In practical applications, some physical adsorption methods have difficulty adjusting the adsorbent performance in real time according to the degree of pollution.

[0003] At the same time, nanotechnology is booming, and nanomaterials have shown unique advantages in the field of pollution control. Nanoselenium has a stronger interaction with heavy metals than traditional selenium materials due to its special size effect and quantum effect. Biosynthetic nanoselenium combines the green and sustainable characteristics of biotechnology, while meeting the needs of heavy metal pollution control and conforming to the trend of environmental protection. Early research focused on the preparation and basic property exploration of nanoselenium. In recent years, it has gradually shifted to the practical application of biological nanoselenium in complex pollution scenarios, and is committed to overcoming many difficult problems from laboratory to large-scale engineering applications. Summary of the Invention

[0004] The purpose of the present invention is to provide a bio-nanoselenium nutrient and its preparation method and application. The bio-nanoselenium nutrient in the present invention is derived from biosynthesis. Compared with traditional chemically synthesized nanomaterials, the preparation process of bio-nanoselenium has low energy consumption and few by-products, and can be used for heavy metal treatment in multiple fields.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a bio-nano selenium nutrient, which is synthesized and isolated by microorganisms;

[0007] The microorganism is selenium-enriched yeast.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned biological nano-selenium nutrient, comprising the following steps:

[0009] S1. Sample collection: Collect samples from environments rich in selenium and microbial diversity and store them for processing.

[0010] S2. Strain Isolation: Dilute and spread the collected sample evenly on a selective culture medium containing selenate or selenite as the sole selenium source, maintaining an appropriate temperature and humidity. Incubate for 2-7 days, observing the growth of the colonies, and selecting well-growing individual colonies with distinct morphologies for further purification and culture.

[0011] S3. Strain Identification: Identify the individual colonies obtained from the initial screening to determine their species. Combined with physiological and biochemical characterization, identify dominant strains with high selenium conversion capabilities.

[0012] S4. Seed solution preparation: The identified dominant strain was inoculated into a conical flask containing liquid seed culture medium at 1-5 V / V%, and cultured with shaking at an appropriate temperature and speed for 12-24 hours to obtain vigorous growth and sufficient bacterial count.

[0013] S5. Biotransformation: The seed solution is inoculated into a biosynthesis medium at 5-10 V / V% to maintain a suitable growth environment;

[0014] S6. Centrifugation collection: After the biotransformation is completed, the culture medium is transferred to a high-speed centrifuge and centrifuged at 5000-10000 rpm for 10-30 minutes to collect the precipitate;

[0015] S7. Washing and dispersion: Add an appropriate amount of sterile water or buffer solution to the precipitate, gently shake to resuspend the precipitate, centrifuge again, wash 2-3 times, then add an appropriate amount of dispersant, and sonicate to form a stable nano-selenium dispersion;

[0016] The dominant strains in S3 are Candida utilis and Saccharomyces cerevisiae; the conversion efficiency of organic selenium of the dominant strains is over 80%.

[0017] Preferably, the Candida utilis is the strain described in the accession number CCTCC M 2010090; the Saccharomyces cerevisiae is the strain described in the accession number CGMCC No. 20553.

[0018] Preferably, the environment in S1 that is rich in selenium and has rich microbial diversity is, for example, selenium-rich soil, selenium-rich mine wastewater, and wetlands around selenium mines.

[0019] Preferably, other components of the selective culture medium in S2 may include conventional carbon sources, nitrogen sources, inorganic salts and growth factors.

[0020] Preferably, the liquid seed culture medium in S4 contains selenate or selenite as the sole selenium source.

[0021] Preferably, the selenium source concentration in the culture medium in S5 is 1-10 mM.

[0022] In a third aspect, the present invention provides the application of the above-mentioned biological nano-selenium nutrient in the treatment of heavy metal pollution.

[0023] Preferably, the bio-nanoselenium nutrient can be used to treat soil and water polluted by heavy metals.

[0024] Preferably, the bionanoselenium nutrient can reduce heavy metals in food.

[0025] Preferably, the bio-nanoselenium nutrient can assist in the discharge of heavy metals from the human body.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The bio-nanoselenium nutrient in this invention is derived from biosynthesis. Compared to traditional chemically synthesized nanomaterials, its preparation process consumes less energy and produces fewer byproducts. At pollution remediation sites, its mild action prevents significant disturbances to soil structure and aquatic ecosystems, facilitating in-situ remediation and maintaining the natural balance of ecosystems, in line with current environmental sustainability principles.

[0028] 2. On the one hand, the biological nano-selenium nutrient in the present invention has antioxidant properties, which can provide additional protection for organisms when dealing with oxidative stress damage caused by heavy metal pollution; on the other hand, it works synergistically with microorganisms, organic materials, etc. to activate microbial communities in the soil, promote a benign cycle of soil ecology, and at the same time enhance the adsorption and conversion capabilities of heavy metals, achieving multiple functions in one.

[0029] 3. The bio-nanoselenium nutrient in this invention can be customized to synthesize bio-nanoselenium with specific targeted functions based on different heavy metal pollution scenarios by adjusting microbial culture conditions and nano-selenium modification. For example, tailoring the characteristics of acid mine drainage, which is rich in heavy metals, to prepare an adapted nano-selenium system can accurately and efficiently remove complex heavy metal combinations such as copper, zinc, and arsenic, thereby improving the targeted and effective treatment. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.

[0036] The reagents and equipment used in the following examples of the present invention are all commercially available.

[0037] Example

[0038] 1. Microbial screening and cultivation

[0039] 1. Cultivation:

[0040] The selected strain was the Candida utilis described in the accession number CCTCC M 2010090.

[0041] 1.1 When initially isolating the strain, culture on plates at 30°C is recommended. This temperature is suitable for the growth of most common mesophilic microorganisms with selenium conversion potential, allowing for the acquisition of a large number of colonies in a relatively short period of time, facilitating initial screening.

[0042] 1.2 If after 3-4 days of incubation, the number of colonies is low or no target characteristic colonies are found, try adjusting the temperature within the range of 25-28°C or 32-37°C to accommodate the growth requirements of microorganisms from specific environmental sources. For example, for samples collected from high-altitude selenium-rich wetlands, lowering the temperature to 25-28°C may lead to new discoveries; for samples collected from selenium-rich soil near geothermal areas, raising the temperature to 35-37°C may be recommended.

[0043] 1.3 Cultivation time:

[0044] 1.4 During the first three days, observe the changes in colony morphology, size, color, and other characteristics regularly every day, and record the initial growth status.

[0045] 1.5 From the 4th to the 7th day, focus on strains that grow slowly but have clear colony edges and dense texture, which may indicate high selenium conversion efficiency. If necessary, the period can be extended to 10 days to ensure that potential excellent strains are not missed.

[0046] 1.6 Seed liquid culture time:

[0047] 1.7 In addition to the conventional 12-24 hour shaking culture, samples can be taken at 16 and 20 hours to test the bacterial concentration (e.g., by measuring the optical density at 600 nm using a spectrophotometer).

[0048] 1.8 When the optical density value reaches between 0.6-0.8, it indicates that the strain is in the vigorous logarithmic growth stage. At this time, the seed liquid inoculation effect is best, and the culture time can be flexibly adjusted according to the test results.

[0049] 2. Biosynthetic Nano-Selenium

[0050] 1. Inoculation volume:

[0051] 1.1 For newly screened and in-depth understood strains, a small-batch test was first conducted using a 3% inoculation to observe the biotransformation startup speed and the initial nano-selenium production.

[0052] 1.2 If the transformation starts slowly and the yield is low, the inoculation amount can be gradually increased to 4%-5% for fast-growing strains. For slow-growing but stable strains, a 2%-3% inoculation amount can be tried and the culture time can be appropriately extended to find the optimal inoculation strategy.

[0053] 2. Selenium source concentration:

[0054] 2.1 When culturing a strain for the first time, start with a selenium source concentration of 1-2 mM and closely monitor the growth status of the microorganism (e.g., by changes in turbidity and pH).

[0055] 2.2 If the microorganism grows normally and begins to show signs of nano-selenium production within 48 hours, the selenium source concentration can be increased by 1-2 mM in the next batch to gradually explore the optimal selenium source concentration range that the strain can tolerate to balance microbial growth and nano-selenium production.

[0056] 3. Shaker speed:

[0057] 3.1 Initially, set the shaker speed to 150-160 rpm. After 24 hours of culture, observe the mixing degree of the culture medium and the morphology of the microbial cells (which can be simply observed under a microscope).

[0058] 3.2 If stratification of the culture medium or slight aggregation of microorganisms is found, the speed can be appropriately increased to 180-200 rpm, and continuous attention should be paid to whether it has an adverse effect on the synthesis of nano-selenium. If problems such as uneven size of nano-selenium particles occur, the speed needs to be adjusted back.

[0059] 4. Cultivation time:

[0060] 4.1 Samples were taken at 24 hours, 48 ​​hours, 60 hours and 72 hours respectively, and the characteristic absorption peak intensity of nano-selenium was detected by UV-visible spectrometer. At the same time, the morphology, size and agglomeration of nano-selenium particles were observed by transmission electron microscopy.

[0061] 4.2 When the absorption peak intensity no longer increases significantly and the nano-selenium particles are stable in morphology and evenly distributed, it is the optimal culture time to avoid excessive culture leading to waste of resources or deterioration of nano-selenium properties.

[0062] 5. pH value control:

[0063] 5.1 The pH value is automatically monitored every 4-6 hours, and the pH value deviates from the optimal range by ±0.5 as the warning range.

[0064] 5.2 When the pH value exceeds the warning range, use a micro-injection pump to slowly add acid and alkali solution for adjustment, and control the addition speed at 0.1-0.2mL / min to ensure that the pH value returns to the optimal range steadily and reduce the impact on microbial growth and nano-selenium synthesis.

[0065] 3. Separation and purification of nano-selenium

[0066] 1. Centrifugal speed and time:

[0067] 1.1 During the first centrifugation, centrifuge at 6000-7000 rpm for 15-20 minutes. This can achieve initial efficient precipitation while avoiding damage to the nano-selenium particles due to excessively high speed.

[0068] 1.2 If further purification is required, increase the speed to 8000-10000 rpm, extend the centrifugation time to 25-30 minutes, and use a low-temperature centrifuge (4°C) to reduce particle thermal motion, reduce agglomeration, and improve the purity of the precipitation.

[0069] 2. Cleaning times:

[0070] 2.1 Wash twice as a routine operation, and take samples after each wash to determine the residual selenium content in the supernatant (e.g., by inductively coupled plasma mass spectrometry).

[0071] 2.2 If the residual selenium content is higher than the predetermined threshold (such as 0.1 mM), an additional cleaning is performed to ensure that the purity of the nano-selenium meets the requirements of subsequent applications.

[0072] 3. Ultrasonic power and time:

[0073] 3.1 First, treat with 200-300W ultrasonic power for 15-20 minutes to make the nano-selenium disperse initially and observe the uniformity of the dispersion.

[0074] 3.2 If agglomeration is found, the ultrasonic power can be increased in stages, increasing by 100W each time, and the ultrasonic time can be extended by 5-10 minutes until a uniform and stable nano-selenium dispersion is obtained. However, it should be noted that the total ultrasonic power should not exceed 500W to prevent excessive breakage of the nano-selenium particles.

[0075] 4. Quality Inspection and Preservation

[0076] 1. Storage temperature:

[0077] Strictly maintain a refrigerated environment at 4°C. A temperature monitoring alarm can be set in the refrigerator. When the temperature deviates from 4°C±1°C, an alarm will be issued in time and refrigeration faults will be checked.

[0078] 2. Preservative concentration:

[0079] Select the preservative concentration based on shelf life and estimated microbial contamination risk. For a shelf life of less than one month and a relatively clean environment, use 0.02%-0.04% sodium azide. For a shelf life of 3-6 months or an environment with high microbial counts, increase the concentration to 0.06%-0.1% to ensure long-term stability of the nanoselenium dispersion.

[0080] The synthesis conditions and conversion rates are shown in Table 1.

[0081] Table 1

[0082] 1 2 3 First isolation and culture temperature / ℃ 25 30 35 First culture time / d 4 7 10 Seed liquid culture time / h 16 20 24 Inoculation amount / % 1 2 3 Selenium source concentration / nM 1 1.5 2 Shaker speed / rpm 150 180 200 Synthetic selenium culture time 48 60 72 Selenium synthesis rate % = organic selenium content / raw material selenium content 80％ 91％ 95％

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biological nano-selenium nutrient, characterized in that: The biological nano-selenium nutrient is synthesized and separated by microorganisms; The microorganism is selenium-enriched yeast.

2. The method for preparing the bionanoselenium nutrient according to claim 1, characterized in that: The following steps are involved: S1. Sample collection: Collect samples from environments rich in selenium and microbial diversity and store them for processing. S2. Strain Isolation: Dilute and spread the collected sample evenly on a selective culture medium containing selenate or selenite as the sole selenium source, maintaining an appropriate temperature and humidity. Incubate for 2-7 days, observing the growth of the colonies, and selecting well-growing individual colonies with distinct morphologies for further purification and culture. S3. Strain Identification: Identify the individual colonies obtained from the initial screening to determine their species. Combined with physiological and biochemical characterization, identify dominant strains with high selenium conversion capabilities. S4. Seed solution preparation: The identified dominant strain was inoculated into a conical flask containing liquid seed culture medium at 1-5 V / V%, and cultured with shaking at an appropriate temperature and speed for 12-24 hours to obtain vigorous growth and sufficient bacterial count. S5. Biotransformation: The seed solution is inoculated into a biosynthesis medium at 5-10 V / V% to maintain a suitable growth environment; S6. Centrifugation collection: After the biotransformation is completed, the culture medium is transferred to a high-speed centrifuge and centrifuged at 5000-10000 rpm for 10-30 minutes to collect the precipitate; S7. Washing and dispersion: Add an appropriate amount of sterile water or buffer solution to the precipitate, gently shake to resuspend the precipitate, centrifuge again, wash 2-3 times, then add an appropriate amount of dispersant, and sonicate to form a stable nano-selenium dispersion; The dominant strains in S3 are Candida utilis and Saccharomyces cerevisiae; the conversion efficiency of organic selenium of the dominant strains is over 80%.

3. The preparation method according to claim 2, characterized in that The environment in S1 that is rich in selenium and has rich microbial diversity includes, for example, selenium-rich soil, selenium-rich mine wastewater, and wetlands around selenium mines.

4. The preparation method according to claim 2, characterized in that Other components of the selective culture medium in S2 may include conventional carbon sources, nitrogen sources, inorganic salts and growth factors.

5. The preparation method according to claim 2, characterized in that The liquid seed culture medium composition in S4 contains selenate or selenite as the only selenium source.

6. The preparation method according to claim 2, characterized in that The selenium source concentration in the culture medium of S5 is 1-10 mM.

7. Use of the bionanoselenium nutrient according to claim 1 in the treatment of heavy metal pollution.

8. The use according to claim 7, characterized in that The biological nano-selenium nutrient can treat soil and water bodies polluted by heavy metals.

9. The use according to claim 7, characterized in that The biological nano-selenium nutrient can reduce heavy metals in food.

10. The use according to claim 7, characterized in that The biological nano-selenium nutrient can assist in the discharge of heavy metals from the human body.