CN113322211A - LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof - Google Patents
LX-88 bacterium with good selenium conversion and Se (0) oxidation capacity and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural microorganisms, and particularly discloses an LX-88 strain with good selenium conversion and Se (0) oxidation capacity and application thereof, wherein the preservation number of the strain is CCTCC NO: m2021542. The strain can convert SeMet into selenocysteine (SeCys2), methylselenocysteine (MSeCys) and the like, has oxidation effect on elemental selenium, and the strain LX-88 can reduce Se (IV) and can reduce Se (IV) into red nano selenium (SeNPs), SeCys2 and MSeCys. Therefore, the strain LX-88 can be used as an industrial microorganism and applied to the production and preparation of SeCys2 and MSeCys; can be used as a model microorganism for researching the metabolic conversion of organic selenium and Se (IV); can be prepared into a plant selenium-rich microbial inoculum and can be used as a microorganism for Se (0) oxidation research.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and relates to an LX-88 strain with good selenium conversion and Se (0) oxidation capability and application thereof, wherein the LX-88 strain is Croceibacteroumsp.
Background
Selenium (Se) is in the VI main group of the periodic Table of the elements, and the Se has four valences of Se (-II), Se (0), Se (IV) and Se (VI) in natureThe state forms exist. Se (-II) is mainly organic selenium, and is commonly found in organisms, such as selenocysteine (SeCys), selenomethionine (SeMet), and monomethyl selenium (CH)3-Se), dimethylselenium (CH)3-Se-CH3) And methylselenocysteine (SeMeCys), and the like. Se (IV) and Se (VI) are mainly inorganic selenium, mostly present in non-living organisms, and usually present as oxyanions, such as selenite (SeO)3 2-) And selenate (SeO)4 2-) It is the main source of selenium absorbed and utilized by plants. The chemical elemental selenium Se (0) is less common in nature and most exists in selenium-rich ores. Wherein selenite has the strongest toxicity among the four valence compounds, while elemental selenium is basically nontoxic.
The metabolism of selenium by microorganisms mainly comprises the transportation, reduction, oxidation, assimilation, methylation and the like of selenium. In general, the research on microbial selenium metabolism is clear in the assimilation and reduction mechanism of selenium, and the assimilation mechanism of selenium in fungi and bacteria (including archaea) is elucidated; specific selenate reductase is also found in gram-negative bacteria and gram-positive bacteria, but specific selenite reductase or other selenite reduction key genes are not found at present. Methylation of selenium also finds some key genes and enzymes. However, microorganisms for selenium oxidation are poorly understood, and there is a lack of systematic studies on the mechanism of selenium oxidation by microorganisms to date.
Selenium can be assimilated in microbial cells to selenoamino acids, including SeMet, SeCys2 and selenocysteine (Sec, U), and further participate in the synthesis of selenoproteins. Selenium is covalently bonded to amino acids in SeMet, SeCys2 and Sec. Selenocysteine is the 21 st amino acid and the only amino acid which can not be recycled to synthesize selenoprotein, is completely degraded and converted into selenium in a reduction state under the action of selenocysteine Lyase (Lyase), and the synthesis of selenocysteine is mainly directly related to products of four genes of selA, selB, selC and selD.
At present, most of organic selenium or selenium amino acid is chemically synthesized and extracted, the technical difficulty is high, the cost is high, and the synthesis of the organic selenium by utilizing microbial transformation is an economic and effective mode and a green production mode. The method for strengthening the selenium enrichment of plants has various methods, and the most widely application is the methods of applying selenium in soil, spraying selenium on leaf surfaces, dressing seeds and the like. The soil contains elemental selenium, selenite, selenate, organic selenium and the like, and plants mainly absorb the selenite and the selenate. Therefore, the method is a safe, reliable and low-cost way for adding the microorganisms into the soil to dissolve and convert the selenium or the selenium oxide so as to increase the absorption of the plants to the selenium.
The strain LX-88 provided by the invention belongs to a strain of a genus Croceibacter of the family Erythrobacteraceae. The first model strain of this genus was reported in 2019 by Liu et al, and there was no extensive study on its application. For bacteria of the family of rhodobacteriaceae, there are currently reports on the use for the preparation of flocculants; can promote the growth of plants, improve the salt tolerance of the plants and have good ecological and social benefits; can tolerate high salt, efficiently degrade nitrate, and has higher salt-tolerant denitrification characteristics and the like.
No strain of the genus Croceibacter of the family Rhodobacter (Erythrobacteraceae) has been reported to function in the present invention.
Disclosure of Invention
The invention aims to provide a strain of Croceibacter sp.LX-88, wherein the Tibetan number of the rhodobacter is CCTCC NO: m2021542.
Another object of the invention is to provide the use of the strain croceibacter sp.lx-88 in the oxidation of inorganic selenium.
It is also an object of the present invention to provide the use of the strain croceibacter sp.lx-88 for the reductive transformation of inorganic selenium.
A final object of the invention is to provide the use of the strain croceibacter sp.lx-88 for the transformation of organic selenium.
In order to achieve the purpose, the invention adopts the following technical measures:
the applicant selects and separates a strain LX-88 with the capabilities of selenomethionine conversion, oxidation of elemental selenium and Se (IV) reduction conversion from selenium-rich soil in Enshi of Hubei province in China, and the applicant names the strain as Croceibacter sp.LX-88, the strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation in 2021, 5 and 17 days, and the preservation number is CCTCC NO: m2021542, classification name: lx-88, address: wuhan university in Wuhan, China.
The culture and morphological characteristics of the strain are as follows:
the strain LX-88 has the cellular morphology of a rod, terminal flagella and two convex ends, and has the length of 0.5-0.7 mu m and the width of 0.2-0.4 mu m. The culture was carried out for 72h at 28 ℃ on R2A agar plate medium, and white colonies were formed, which were stippled, flat and had intact and smooth edges.
The application of the strain Croceibacter sp.LX-88 in organic selenium conversion comprises the step of converting selenomethionine into selenocysteine or methyl selenocysteine by using the strain.
Use of the strain croceibacter sp.LX-88 for the oxidation of inorganic selenium, comprising the oxidation of elemental selenium to water-soluble selenite using the strain.
The application of the strain Croceibacter sp.LX-88 in inorganic selenium reduction comprises reducing Se (IV) into red nano selenium (SeNPs), SeCys2 and/or MSeCys by using the strain.
The application of the strain Croceibacter sp.LX-88 in the preparation of the selenium-rich agent for plants comprises the step of preparing the selenium-rich agent by taking the strain as one or the only effective component of the effective component, and the selenium-rich agent is scattered into soil, so that the selenium enrichment of the plants can be promoted, insoluble elemental selenium can be oxidized into selenite which can be absorbed and utilized by the plants, the selenium form in the soil can be converted, and the selenium element can be more effectively utilized by the plants.
Compared with the prior art, the invention has the following advantages:
(1) the strain LX-88 obtained by the invention has the functions of converting selenomethionine into selenocysteine and/or methylselenocysteine; reducing and converting Se (IV) into red nano selenium (SeNPs), SeCys2 and MSeCys; the insoluble elementary selenium is oxidized into the capacity of the plant to absorb the utilized selenite, thereby promoting the selenium enrichment of the plant; is the supplement to the very limited selenomethionine conversion and selenium oxidation strain resources.
(2) The strain LX-88 can be used as an industrial microorganism to produce and prepare selenocysteine and methylselenocysteine, and can be used as a model microorganism for selenium oxidation and selenium conversion to research a molecular mechanism of selenium oxidation and a molecular metabolic mechanism of selenium conversion.
(3) The invention provides a new way for producing and preparing selenocysteine and methylselenocysteine by using beneficial microorganisms, and the new way is green, economic and environment-friendly.
(4) The invention provides a novel method for promoting selenium enrichment of plants by using beneficial microorganisms, and the method is safe, green and environment-friendly.
Drawings
FIG. 1 is a schematic representation of the growth curve of strain LX-88 in R2A medium supplemented with different selenium elements;
wherein: control represents the growth curve of the strain LX-88 without adding selenium in the culture medium;
100 μ M SeMet represents the growth curve of the strain LX-88 after the addition of 100 μ MSeMet to the medium;
the 100. mu.M Se (IV) represents the growth curve of the strain LX-88 after the addition of the Se (IV) to the medium to a final concentration of 100. mu.M;
0.1g L-1se (0) indicates that 0.1g L is added to the medium to the final concentration-1Growth curve of the strain LX-88 after Se (0). FIG. 2 is a graph showing the effect of strain LX-88 on the transformation of SeMet;
wherein: control represents a blank Control without addition of strain LX-88 plus SeMet;
SeMet represents a curve of the change of the concentration of SeMet in the system with time after the addition of the strain LX-88;
SeCys2 shows the time course of the concentration of SeCys2 in the system after addition of strain LX-88;
the MSeCys represents the curve of the concentration of MSeCys in the system as a function of time after the addition of the strain LX-88.
FIG. 3 is a schematic diagram of the oxidation of elemental selenium by strain LX-88;
wherein: 0.1g L-1Se (0) represents control treatment without adding bacteria;
0.1g L-1se (0) LX-88 represents the change curve of Se (IV) concentration in the system with time after the strain LX-88 is added.
FIG. 4 is a schematic representation of the reduction of Se (IV) by strain LX-88;
wherein: control represents a blank Control without addition of strain LX-88 plus Se (IV);
se (IV) represents a curve of Se (IV) concentration in a system after the strain LX-88 is added along with time;
SeCys2 shows the time course of the concentration of SeCys2 in the system after addition of strain LX-88;
the MSeCys represents the curve of the concentration of MSeCys in the system as a function of time after the addition of the strain LX-88.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments. The methods used in the following examples are conventional methods unless otherwise specified.
Example 1:
separation and identification of strain LX-88
(1) Sample collection: the applicant collected the selenium-rich soil sample used by the invention from the surface soil of the selenium-rich area of the Rishi soil family of Hubei province, autonomous State of the Ministry of the soil family in 2018.
(2) And (3) separating and purifying thalli: weighing 10g of soil sample in a triangular flask filled with 90mL of sterile physiological saline, placing the soil sample in a shaking table at 28 ℃ for half an hour, and sequentially adding 1mL of soil sample into 9mL of sterile physiological saline to gradually dilute the soil sample to 10-1，10-2，10-3，10-40.1mL of each was plated on 0.5mM Se (IV) R2A solid medium, 3 plates per gradient. Culturing at 28 deg.C for one week, picking white single colony, and streaking for purification. After purification, the mixture is preserved in a screwed pipe according to the ratio of 50% of glycerol to bacteria liquid (the volume ratio is 1:1) and preserved at the temperature of minus 80 ℃.
(3) Selenium conversion, oxidation and reduction bacteria screening: the strain is added with 100 mu MSeMet,0.1g L respectively according to the inoculation amount of 1 percent-1Elemental selenium powder and 100 μ M Se (IV) were subjected to shake cultivation at 28 ℃ and 150rpm for 120 hours in R2A liquid medium (medium sterilization treatment), and a fixed-point sampling (supernatant) was performed in the middle. Subjecting the obtained mixture toCentrifuging the clear solution at 12000rpm for 10min, and detecting Se (IV) content and SeMet content or other organic selenium content by using high performance liquid chromatography-hydride generation-atomic fluorescence spectrometer (HPLC-HG-AFS).
If the selenium is added into the group of elemental selenium, the Se (IV) content is obviously increased, which indicates that the bacterium has oxidability on the elemental selenium;
if the SeMet is added into the group, the reduction of the SeMet content or the increase of the content of other organic selenium indicates that the bacterium has the assimilation and transformation capacity on the SeMet.
If the Se (IV) is added into the group, the Se (IV) content is obviously reduced or the content of other organic selenium is increased and red nano particles are generated, which indicates that the bacterium has the capability of reducing and converting the Se (IV).
Finally, a strain is screened out, and the strain has the 3 capabilities.
(4) Classification and identification of the strain LX-88: the obtained strain is subjected to 16S rRNA gene sequencing and a phylogenetic tree thereof is constructed, and the applicant identifies the strain to be in the genus Croceibacter by combining the physiological and biochemical characteristics of the strain. The strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation in 2021, 5 months and 17 days, and the preservation number is CCTCC NO: m2021542, classification name: lx-88, address: wuhan university in Wuhan, China.
The culture and morphological characteristics of the strain are as follows:
the strain LX-88 has the cellular morphology of a rod, terminal flagella and two convex ends, and has the length of 0.5-0.7 mu m and the width of 0.2-0.4 mu m. Culturing at 28 deg.C for 72 hr on R2A agar plate medium to form white colony, marked, flat, and smooth edge
(5) Growth curve: the strains were added to final concentrations of 100. mu. MSeMet, 100. mu. M Se (IV) and 0.1g L, respectively, at 1% inoculum size-1Placing the elemental selenium powder in 50mLR2A liquid culture medium, repeating each treatment for 3 times, shaking-culturing at 28 deg.C and 150rpm, sampling 2mL every 12 hr, measuring thallus growth concentration OD with ultraviolet spectrophotometer600Values, growth curves were plotted and the results are shown in figure 1. The strain LX-88 grows well in the R2A culture medium and can reach a stationary growth phase within 72 hours.
Example 2:
the curve for the transformation of SeMet by the strain LX-88 is:
the method comprises the following specific steps:
(1) 3 bottles of sterilized R2A liquid medium were prepared, 50mL each. SeMet, which had been filter-sterilized at the final concentration, was added each. Log phase of inoculation (OD)600The value is about 0.8) LX-88 bacterial liquid, and the inoculation amount is 1%. 2mL of sample was taken immediately after mixing and this sample was taken as the first sample and placed in a clean centrifuge tube. The mixture was incubated at 28 ℃ on a constant temperature shaker at 150 rpm. Samples were taken every 12 hours and the experiment was set up in triplicate, with no addition of strain LX-88, and only the same final concentration of SeMet in R2A liquid medium as a control; after sampling, the samples were processed as follows.
(2) 2mL of the sample is taken each time, centrifuged at 12000rpm for 10min, and the supernatant is passed through a 0.22 μm filter and stored in a refrigerator at-20 ℃.
(3) The content of SeMet and other organic selenium in the sample is detected by HPLC-HG-AFS of Beijing Jitian instruments Ltd.
(4) The SeMet conversion plot was constructed with the measured concentrations of SeMet and other organic selenium (. mu.M) as ordinate and time as abscissa (FIG. 2).
(5) As shown in fig. 2, the content of secs 2 in the sample supernatant increased and the content of SeMet decreased. From hour 0 to hour 120, the SeCys2 and MSeCys contents increased in the supernatant, while SeMet was essentially assimilated for conversion.
Example 3:
oxidation curve of strain LX-88 on elemental selenium
The method comprises the following specific steps:
(1) 3 bottles of sterilized R2A liquid medium were prepared, 50mL each. Each added to a final concentration of 0.1g L-1Sterilized elemental selenium powder. Log phase of inoculation (OD)600The value is about 0.8) LX-88 bacterial liquid, and the inoculation amount is 1%. 2mL of sample was taken immediately after mixing and this sample was taken as the first sample and placed in a clean centrifuge tube. Standing at 28 deg.C and 150rpm constantlyCulturing in a warm shaking table. Samples were taken every 12 hours and triplicate experiments were set up with no addition of strain LX-88, only the same final concentration of elemental selenium in R2A liquid medium as control; after sampling, the samples were processed as follows.
(2) 2mL of the sample is taken each time, centrifuged at 12000rpm for 10min, and the supernatant is passed through a 0.22 μm filter and stored in a refrigerator at-20 ℃.
(3) The content of Se (IV) in the sample is detected by HPLC-HG-AFS of Beijing Jitian instruments.
(4) To determine the concentration (. mu.gL) of Se (IV)-1) The graph of elemental selenium oxidation was constructed with the ordinate and time the abscissa (fig. 3).
(5) As shown in FIG. 3, the Se (IV) content in the sample supernatant is increased continuously, and the Se (IV) content in the supernatant reaches 260.6 +/-15.73 mu gL until 120 hours-1。
Example 4:
strain LX-88 assimilation utilization Se (IV)
(1) 3 bottles of sterilized R2A liquid medium were prepared, 50mL each. Each of the filter-sterilized Se (IV) was added to a final concentration of 100. mu.M. Log phase of inoculation (OD)600The value is about 0.8) LX-88 bacterial liquid, and the inoculation amount is 1%. 2mL of sample was taken immediately after mixing and this sample was taken as the first sample and placed in a clean centrifuge tube. The mixture was incubated at 28 ℃ on a constant temperature shaker at 150 rpm. Samples were taken every 12 hours and the experiment was set up in triplicate, with no addition of strain LX-88, and only the same final concentration of Se (IV) in liquid medium R2A as a control; after sampling, the samples were processed as follows.
(2) 2mL of the sample is taken each time, centrifuged at 12000rpm for 10min, and the supernatant is passed through a 0.22 μm filter and stored in a refrigerator at-20 ℃.
(3) The content of Se (IV) in the sample is detected by HPLC-HG-AFS of Beijing Jitian instruments.
(4) A Se (IV) assimilation utilization graph (FIG. 4) was constructed with the measured Se concentration (. mu.M) as the ordinate and time as the abscissa.
(5) As shown in fig. 4, the content of Se (iv) in the sample supernatant is gradually reduced, and the Se (iv) can be utilized by the microorganism itself, and can be reduced into red nano-selenium (SeNPs, red particles), secs 2 and msecs.
Claims (7)
1. Separating strainCroceibacteriumsp. the preservation number of the strain is CCTCC NO: m2021542.
2. Use of LX-88 as described in claim 1 as an industrial microorganism.
3. As recited in claim 1Croceibacteriumsp, LX-88 in the preparation of SeCys2 and MSeCys.
4. As recited in claim 1Croceibacteriumsp, LX-88 as an application in selenium metabolic conversion model microorganisms.
5. As recited in claim 1CroceibacteriumUse of sp, LX-88 in the oxidation of selenium.
6. As recited in claim 1Croceibacteriumsp, LX-88 in the preparation of selenium-rich plant microbial inoculum.
7. As recited in claim 1CroceibacteriumApplication of sp, LX-88 in transformation of soil selenium form.
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