CN113481191B - Immobilized particle embedded with selenite reducing bacteria and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an immobilized particle embedded with selenite reducing bacteria, a preparation method and application thereof. The method comprises the following steps, S1: preparing selenite reducing bacteria stock solution; s2: adding an adsorption material into the selenite reducing bacteria stock solution to obtain an embedding body solution; s3: mixing and heating a certain amount of polyvinyl alcohol, sodium alginate and water to form an embedding agent solution, and then cooling the embedding agent solution to 25-35 ℃; s4: uniformly mixing the embedding body solution obtained in the step S2 and the embedding agent solution obtained in the step S3, and then dropwise adding the mixture into a cross-linking agent for reaction until the mixture is solidified into a spherical colloidal microbial inoculum with the particle size of 2-4 mm; s5: and (5) freeze-drying the colloidal microbial inoculum obtained in the step (S4) to obtain immobilized particles embedded with the selenite reducing bacteria. The immobilized particles embedded with selenite reducing bacteria can obviously improve the effective utilization rate of selenium in soil and improve the selenium-rich effect of crops.
Description
Technical Field
The invention relates to the technical field of soil planting, in particular to a preparation method of immobilized particles embedded with selenite reducing bacteria.
Background
Selenium (Se) is one of essential trace elements of human body, is an important component of various enzymes and proteins, has the effects of resisting oxidation, detoxifying, resisting cancer, enhancing immunity and the like, and has very important significance for human health.
The selenium in the soil comprises seven types of water-soluble state, ion-exchange state, carbonate combined state, humic acid combined state, iron-manganese oxide combined state, strong organic combined state and residue state, wherein the water-soluble state and the ion-exchange state are easily absorbed by plants, and other states are difficult or impossible to be absorbed by the plants, so that the selenium resource is wasted.
Disclosure of Invention
The invention aims to provide immobilized particles embedded with selenite reducing bacteria, which can improve the effective utilization rate of selenium in soil and improve the selenium enrichment effect of crops, and a preparation method and application thereof, aiming at the defects in the prior art.
The invention relates to a preparation method of immobilized particles embedded with selenite reducing bacteria, which comprises the following steps,
s1: preparing selenite reducing bacteria stock solution;
s2: adding an adsorption material into the selenite reducing bacteria stock solution to obtain an embedding body solution;
s3: mixing and heating a certain amount of polyvinyl alcohol, sodium alginate and water to form an embedding agent solution, and then cooling the embedding agent solution to 25-35 ℃;
s4: uniformly mixing the embedding body solution obtained in the step S2 and the embedding agent solution obtained in the step S3, and then dropwise adding the mixture into a cross-linking agent for reaction until the mixture is solidified into a spherical colloidal microbial inoculum with the particle size of 2-4 mm;
s5: and (5) freeze-drying the colloidal microbial inoculum obtained in the step (S4) to obtain immobilized particles embedded with the selenite reducing bacteria.
Further, in step S2, the mass-to-volume ratio of the adsorption material to the selenite-reducing bacteria stock solution is: 1 to 3: 100-300 g/mL; in the step S3, the mass-to-volume ratio of the polyvinyl alcohol to the sodium alginate to the water is 24-40 g: 1-3 g: 400-600 mL; in step S4, the volume ratio of the embedding body solution to the embedding agent solution is 1-3.
Further, the specific steps of step S1 are: simultaneously inoculating the selenite reducing bacteria into a selenite reducing bacteria culture medium according to the proportion of 1 percent, culturing for 16h at the temperature of 30 ℃ and the speed of 150r/min to reach the late logarithmic growth stage, centrifuging for 10min in a high-speed centrifuge at 8000r/min, collecting thalli, and adding 200mL of physiological saline to prepare a selenite reducing bacteria stock solution.
Further, the culture medium for selenite reduction bacteria comprises NH 4 Cl 0.1g/L、NaCl 0.1g/L、MgSO 4 ·7H 2 0.1g/L of O, 0.15g/L of yeast powder and 0.5g/L of tryptone; the pH value of the culture medium of the selenite reducing bacteria is 7.0.
Further, the cross-linking agent comprises saturated boric acid solution and CaCl 2 And (3) solution.
Further, the adsorption material comprises corncob powder, straw powder or peanut shell powder.
Further, the adsorbing material is corncob meal.
Further, in step S4, the cross-linking agent is stirred at a certain rotation speed, a 50mL syringe is used to suck the mixed solution of the embedding body and the embedding agent, and the mixed solution is dripped into the cross-linking agent at a speed of 2 drops per second to obtain the colloidal microbial inoculum, the stirring is continued after the dripping is finished, and the colloidal microbial inoculum is soaked in the cross-linking agent for 20 to 28 hours after the stirring is finished.
Further, the specific steps of step S5 are: the colloidal microbial inoculum is put into a refrigerator at minus 80 ℃ for freezing for 4h and then put into a freeze dryer for freeze drying under the conditions that the temperature is minus 50 ℃, the air pressure is 10Pa and the time is 48h.
An immobilized particle embedded with selenite reducing bacteria is prepared by the preparation method.
The immobilized particles embedding selenite reducing bacteria are applied to selenium-rich soil.
The immobilized particles for embedding selenite reducing bacteria are prepared by taking selenite reducing bacteria liquid and an adsorption material as an embedding body and taking polyvinyl alcohol and sodium alginate as embedding agents, and the prepared particles can obviously improve the effective utilization rate of selenium in soil and improve the selenium enrichment effect of crops.
Drawings
FIG. 1 is a diagram of a finished product of immobilized particles embedded with selenite-reducing bacteria of the present invention;
FIG. 2 is a selenite reduction curve of immobilized selenite-reducing bacteria-embedded particles prepared using corncob meal as the adsorbent material;
FIG. 3 is an selenite reduction curve of immobilized particles embedded with selenite-reducing bacteria, which is manufactured by using straw powder as an adsorbing material;
FIG. 4 is a selenite reduction curve of immobilized particles embedding selenite-reducing bacteria fabricated using peanut shell meal as the adsorbent material;
FIG. 5a is a graph showing the variation of water soluble selenium content in the soil in experiments 1-4;
FIG. 5b is a graph showing the change in ion-exchanged selenium content in the soil of experiments 1-4;
FIG. 5c is a graph showing the change in the content of selenium in carbonate bound state in the soil of experiments 1-4;
FIG. 5d is a graph showing the change in the humic acid bound selenium content in the soil of experiments 1 to 4;
FIG. 5e is a graph showing the change in the content of FeMnO bound selenium in the soil of experiments 1-4;
FIG. 5f is a graph of the change in the content of strongly organically bound selenium in the soil in experiments 1-4;
FIG. 5g is a graph showing the change in the amount of selenium in the soil in the residue state in experiments 1-4;
fig. 6 is a statistical plot of the selenium content in the shanghai green planted in experiments 1, 3 and 4.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.
Example 1:
(I) material preparation:
1. materials: selenium-rich soil collected from Jiangzhong Tang Zhejiang province and Zhejiang province; selenite reducing bacteria, which is selected from Jiang Zheng Hua City in Zhejiang province; polyvinyl alcohol (PVA); sodium Alginate (SA); corncob meal, caCl 2 Boric acid, selenite and 0.9% physiological saline.
2. Culture medium:
selenite reducing bacteria liquid culture medium: NH (NH) 4 Cl 0.1g/L；NaCl 0.1g/L；MgSO 4 ·7H 2 O is 0.1g/L; 0.15g/L of yeast powder; tryptone 0.5g/L,the pH of the medium was 7.0. Sterilizing at high temperature and high pressure.
3. Instruments and devices:
an electric heating constant temperature incubator, a Sanyo full-automatic high-pressure sterilization pot, a pH meter, an ultra-clean workbench, an AFS-9700 double-channel atomic fluorescence photometer, a magnetic stirrer, a freeze dryer, an ultra-low temperature refrigerator and a high-speed refrigerated centrifuge.
(II) preparing an immobilized selenium-rich microbial agent:
culturing selenite reducing bacteria stock solution: simultaneously inoculating the selenite reducing bacteria into a selenite reducing bacteria culture medium according to the proportion of 1%, and culturing for 16h at 30 ℃ and 150r/min to reach the late logarithmic growth stage. Centrifuging at 8000r/min in a high speed centrifuge for 10min to collect thallus, and adding 200mL of physiological saline to obtain selenite reducing bacteria stock solution.
Preparing immobilized particles embedding selenite reducing bacteria: weighing 10g of corncob meal, adding into the selenite reducing bacteria stock solution, placing in a constant temperature incubator at 30 ℃, and shaking at 150r/min for 30min. Weighing 50g of boric acid, adding the boric acid into 100mL of water, stirring the boric acid for 5min by using a glass plate to dissolve the boric acid as much as possible, standing the mixture for 30min, taking a supernatant to obtain a saturated boric acid solution, and adding 4g of CaCl into the saturated boric acid solution 2 The required cross-linking agent is prepared. 80g of polyvinyl alcohol and 7.5g of sodium alginate are weighed into a 2L beaker, added into 800mL of water and heated at 120 ℃ for 20min to form the colloidal embedding agent. And (3) when the embedding agent is cooled to 35 ℃, adding the selenite reducing bacteria stock solution added with the corncob powder into the colloidal embedding agent, and stirring for 10min to uniformly mix the selenite reducing bacteria stock solution and the colloidal embedding agent. And (3) adding 100mL of cross-linking agent into a glass evaporation pan with the diameter of 100mm, placing the glass evaporation pan on a magnetic stirrer, setting the rotating speed of the magnetic stirrer to be 600r/min, and dropwise adding the mixed solution of the embedding agent and the selenite reducing bacteria stock solution into the cross-linking agent at the speed of 2 drops per second by using a 50-min injector to form the selenium-rich composite microbial agent. And after the dropwise addition is finished, the microbial inoculum is continuously stirred on a magnetic stirrer for 1h to ensure that the balls are uniform, and after the stirring is finished, the microbial inoculum is soaked in the cross-linking agent for 24h to ensure that the structure of the microbial inoculum is stable. After 24h, the cross-linking agent was poured off and the inoculum was washed 3 times with 0.9% physiological saline. Placing the microbial inoculum inFreezing the mixture in a refrigerator at the temperature of minus 80 ℃ for 4 hours, and then putting the mixture into a freeze drier for freeze drying to obtain the immobilized particles embedded with the selenite reducing bacteria, wherein the conditions are that the temperature is minus 50 ℃, the air pressure is 10Pa, and the time is 48 hours. FIG. 1 is a diagram of the immobilized particles embedding selenite-reducing bacteria of example 1.
Example 2: the corncob powder of example 1 was replaced with straw powder, and immobilized particles in which selenite-reducing bacteria were embedded were prepared in the same manner as in example 1.
Example 3: the corncob meal in example 1 was replaced with peanut shell meal, and immobilized particles in which selenite-reducing bacteria were embedded were prepared in the same manner as in example 1.
The functional ratio of the immobilized particles embedding selenite-reducing bacteria of the three different adsorbing materials of examples 1-3 was compared
100mL of selenite reducing bacteria culture medium is prepared, 0.1mM selenite solution and 1g of three immobilized selenium-rich microbial agents with different adsorption materials are added, and the mixture is cultured in a constant temperature shaking table at 30 ℃ and 150 r/min. Samples were taken at 12h, 1d, 2d, 3d, 4d, 5d, 6d and 7d, respectively. The growth condition of the microorganisms is monitored by measuring protein, and the consumption of selenite is measured to monitor the selenite reduction function of the microbial inoculum.
As can be seen from FIG. 2, the immobilized microbial agent prepared by using corncob meal as an adsorbing material has a selenite reduction rate of 67.35% under the selenite condition with the concentration of 1 Mm/L.
As can be seen from FIG. 3, the immobilized microbial inoculum prepared by using straw powder as the adsorbing material has a reduction rate of 61.33% to selenite under the selenite concentration of 1 Mm/L.
As can be seen from FIG. 4, the immobilized microbial inoculum prepared by using peanut shell powder as the adsorbing material has a selenite reduction rate of 52.81% under the selenite concentration of 1 Mm/L.
Through comparison of three different adsorbing materials, the corn cob powder serving as the adsorbing material has the best effect.
Performance test of application of immobilized particles embedding selenite reducing bacteria in selenium-rich soil
Experiment 1: 1kg of selenium-rich soil was placed in a flowerpot with a diameter of 16cm and a height of 14cm, 0.5% of the immobilized particles embedded with selenite-reducing bacteria prepared in example 1 was added, and after stirring, 3 well-grown seedlings of Shanghai green were transplanted for 30 days.
Soil of the Shanghai Qinggen line was taken for determination of seven forms of selenium at five time periods of 0 day, 5 days, 10 days, 15 days and 30 days, respectively.
Pulling out the planted Shanghai Qing on the 30 th day, cleaning with clear water, freezing in a refrigerator at-80 deg.C for 2h, taking out, and freeze-drying in a freeze-drying machine at-50 deg.C under 10Pa for 24h. The method is used for measuring the selenium content of Shanghai Qing.
Experiment 2: the Shanghai green is not planted, and immobilized particles for embedding selenite reducing bacteria are not added.
Experiment 3: the immobilized particles embedding selenite-reducing bacteria were not added, and the rest conditions were the same as those in experiment 1.
Experiment 4: the immobilized particles embedded with selenite reducing bacteria in the application 1 are replaced by the immobilized particles without reducing bacteria, and the rest conditions are the same as those of the experiment 1.
As shown in fig. 5a, water soluble selenium in soil: compared with the soil without the 'Shanghai Qing', the water-soluble selenium content of the soil in the planting group is slightly reduced, which indicates that the selenium in the form is utilized by plants in the soil. The water-soluble selenium of the immobilized particle group added with the embedded selenite reducing bacteria is increased to a certain extent, and the addition of the immobilized particles embedded with the selenite reducing bacteria is beneficial to the release of the soluble selenium. The immobilized particles embedded with the selenite reducing bacteria can promote the conversion of selenium in soil to water-soluble selenium which is more beneficial to the absorption and utilization of plants to a certain extent.
As shown in fig. 5b, ion-exchanged selenium: the 'Shanghaiqing' soil without the immobilized particles embedded with selenite reducing bacteria has weak reduction of ion exchange state selenium due to the absorption and transformation effects of plants; the ion exchange state selenium in the 'Shanghai Qing' soil added with the immobilized particles embedding the selenite-reducing bacteria still rises to a certain extent, which indicates that the immobilized particles embedding the selenite-reducing bacteria can possibly convert more selenium in the soil to the ion exchange state; the ion exchange state selenium in the soil added with the blank immobilized microspheres is increased to a certain extent, and presumably the addition of the immobilized microspheres leads to better soil air permeability, is beneficial to the growth of a large amount of soil in-situ aerobic selenium-enriched bacteria and promotes the conversion of selenium in the soil.
As shown in fig. 5c, carbonate bound state: as can be seen from the figure, the carbonate bound selenium in the three types of soil planted with the 'Shanghai Qing' is slightly reduced. The reason is that the absorption of plants enables the carbonate-bound selenium to be converted and supplied to a water-soluble state and an ion exchange state, the reduction amplitude of the microbial inoculum group is the largest, and the reason is presumed that the weak-bound selenium is converted to the water-soluble state and the ion exchange state more due to the action of the microbial inoculum, and the secretion generated by immobilized particles embedding selenite reducing bacteria and the adsorption effect of the bacteria per se enable the carbonate-bound selenium in soil to be converted to an difficultly utilized state.
As shown in fig. 5d, humic acid bound state: as can be seen from the figure, the humic acid combined selenium in the three types of soil planted with the 'Shanghai Qing' shows a certain rising trend, which is probably because the organic matters of the soil are utilized and degraded by microorganisms along with the growth of the plants, the content of the humic acid which is difficult to be utilized in the soil is increased, and the content of the humic acid combined selenium in the soil is further promoted to be increased.
As shown in fig. 5e, the iron-manganese binding state: compared with the blank group, the iron-manganese combined selenium added with the microbial inoculum group is obviously reduced. The added microbial inoculum can promote the transformation of the manganese oxide combined selenium to the selenium which is more beneficial to the absorption and utilization of plants, thereby promoting the selenium enrichment of the plants.
As shown in fig. 5f and 5g, selenium in the strongly organically bound and residual states was the most stable and less readily converted to other types of selenium. There is no significant change in the two forms of selenium as a whole.
Fig. 6 is a statistical plot of the selenium content in the shanghai green planted in experiments 1, 3 and 4.
As shown in fig. 6, the selenium content in the "shanghai qing" is significantly increased after the immobilized particles embedded with selenite-reducing bacteria are added, and the selenium-rich effect is significant, but the selenium content in the "shanghai qing" added with the blank microbial inoculum is also increased to some extent, which may be because the immobilized microspheres play a role of a soil swelling agent, and the air permeability of the soil is increased after the immobilized particles are added into the soil, so that the growth of in-situ aerobic selenium-rich bacteria is facilitated, the effective selenium content in the soil is increased, and the absorption of the "shanghai qing" to the soil selenium is promoted.
The above is not relevant and is applicable to the prior art.
While certain specific embodiments of the present invention have been described in detail by way of illustration, it will be understood by those skilled in the art that the foregoing is illustrative only and is not limiting of the scope of the invention, as various modifications or additions may be made to the specific embodiments described and substituted in a similar manner by those skilled in the art without departing from the scope of the invention as defined in the appending claims. It should be understood by those skilled in the art that any modifications, equivalents, improvements and the like made to the above embodiments in accordance with the technical spirit of the present invention shall be included in the scope of the present invention.
Claims (3)
1. A preparation method of immobilized particles embedded with selenite reducing bacteria is characterized by comprising the following steps: which comprises the following steps of (a) preparing,
s1: preparing selenite reducing bacteria stock solution;
s2: adding an adsorption material into the selenite reducing bacteria stock solution to obtain an embedding body solution;
s3: mixing and heating a certain amount of polyvinyl alcohol, sodium alginate and water to form an embedding agent solution, and then cooling the embedding agent solution to 25-35 ℃;
s4: uniformly mixing the embedding body solution obtained in the step S2 and the embedding agent solution obtained in the step S3, and then dropwise adding the mixture into a cross-linking agent for reaction until the mixture is solidified into a spherical colloidal microbial inoculum with the particle size of 2-4 mm;
s5: freeze-drying the colloidal microbial inoculum obtained in the step S4 to obtain immobilized particles embedded with the selenite reducing bacteria;
in the step S2, the mass-to-volume ratio of the adsorption material to the selenite reducing bacteria stock solution is as follows: 1:20g/mL; in the step S3, the mass volume ratio of the polyvinyl alcohol to the sodium alginate to the water is 24-40 g: 1-3 g: 400-600 mL; in the step S4, the volume ratio of the embedding body solution to the embedding agent solution is 1-3;
the specific steps of step S1 are: simultaneously inoculating selenite reducing bacteria into a selenite reducing bacteria culture medium according to the proportion of 1-5%, culturing for 16-24 h under the conditions of 30 ℃ and 150r/min to reach the late logarithmic growth stage, centrifuging for 10min in a high-speed centrifuge at 8000r/min, collecting thalli, and adding 100-300 mL of physiological saline to prepare selenite reducing bacteria stock solution;
the adsorbing material comprises corncob powder, straw powder or peanut shell powder;
the culture medium of the selenite reducing bacteria comprises NH 4 Cl 0.1～0.15 g/L、NaCl 0.1～0.15 g/L、MgSO 4˙ 7H 2 0.1-0.15 g/L of O, 0.15-0.5 g/L of yeast powder and 0.5-1.0 g/L of tryptone; the pH value of the selenite reducing bacteria culture medium is 6.5-7.5;
the cross-linking agent comprises a saturated boric acid solution and 4% -6% of CaCl 2 A solution;
step S4, stirring the cross-linking agent at a certain rotating speed, sucking the mixed solution of the embedding body and the embedding agent by a 50mL injector, dropwise adding the mixed solution into the cross-linking agent at a speed of 2 drops per second to obtain a colloidal microbial inoculum, continuously stirring after dropwise adding is finished, and soaking the colloidal microbial inoculum into the cross-linking agent for 20-28 hours after stirring is finished;
the specific steps of step S5 are: the colloidal microbial inoculum is frozen in a refrigerator at minus 80 ℃ for 4 to 6 hours and then put in a freeze dryer for freeze drying, and the conditions are that the temperature is minus 50 ℃, the air pressure is 10Pa, and the time is 36 to 48 hours.
2. An immobilized particle embedded with selenite-reducing bacteria prepared by the method of claim 1.
3. The use of the immobilized selenite-reducing bacteria-embedding particles of claim 2 in selenium-enriched soil.
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| CN106566790B (en) * | 2016-11-02 | 2018-12-25 | 中国农业大学 | Utilize the method and its application of Agrobacterium tumefaciens biosynthesis nanometer selenium |
| CN109182128A (en) * | 2018-10-16 | 2019-01-11 | 广西壮族自治区农业科学院农业资源与环境研究所 | A kind of strain culturing method of Efficient Conversion Selenium in Soil |
| CN112011476B (en) * | 2020-07-24 | 2022-03-29 | 河北科技大学 | Preparation method of high-strength immobilized microspheres for embedding thiobacillus denitrificans |
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