CN114621904A

CN114621904A - Micrococcus and application thereof

Info

Publication number: CN114621904A
Application number: CN202210516544.8A
Authority: CN
Inventors: 刘振
Original assignee: Institute of Geographic Sciences and Natural Resources of CAS
Current assignee: Institute of Geographic Sciences and Natural Resources of CAS
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2022-06-14
Anticipated expiration: 2042-05-13
Also published as: CN114621904B

Abstract

The invention discloses micrococcus and application thereof, and belongs to the technical field of microorganisms. The micrococcus was named YRD202203CF and classified asMacrococcus spThe strain is preserved in China general microbiological culture Collection center in 2022, 3 months and 11 days, wherein the preservation address is No. 3 of Xilu No.1 of Beijing, facing Yang, and the preservation number is CGMCC No. 2450. The invention discloses that the micrococcus has the capability of forming stable carbon-containing minerals by utilizing various exogenous substances and has the function of fixing atmospheric CO for the first time₂The method has the capability of forming the carbon-containing mineral by utilizing oxalate, especially has very important engineering application prospect for deeply understanding a microbial carbon sequestration mechanism of the saline-alkali soil, regulating and controlling the formation process of inorganic carbon in the saline-alkali soil and relieving climate change.

Description

Micrococcus and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to micrococcus and application thereof.

Background

In recent years, with the deep research of the academic world on the ecosystem service function of saline-alkali soil, a large amount of soil inorganic carbon is stored in the saline-alkali soil. Because saline-alkali soil is stressed by saline-alkali, only a few salt-tolerant plants can grow, and because of environmental limitation, the vegetation is rare, the carbon fixation capability of the saline-alkali soil is worth deeply discussing. Soil microorganisms play an important role in the soil carbon conversion process as an engine of the geochemical cycle. The deep research on soil microorganisms has very important significance for reasonably judging the carbon process of the saline-alkali soil and the formation mechanism of inorganic carbon in the saline-alkali soil. Especially, under the background of global change, the function of the microorganisms for forming the carbon-containing mineral substances is known in detail, and the method has extremely important potential application value.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides micrococcus and application thereof, and discloses the capacity of micrococcus separated from saline-alkali soil for generating carbonate minerals by using various substances for the first time.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

providing a micrococcus, named YRD202203CF, classified and namedMacrococcus spThe strain is preserved in China general microbiological culture Collection center (CGMCC) at 11

days

3 and 3 months 2022, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, and the preservation number is CGMCC No. 2450.

Further: the sequence of the micrococcus 16S rDNA gene is shown in SEQ ID No. 1.

Further: micrococcus is from saline-alkali soil.

Further: the screening method of micrococcus comprises the following steps: diluting saline-alkali soil, taking soil diluent, inoculating the soil diluent into an inorganic salt MSM culture medium containing calcium oxalate, and culturing and separating to obtain micrococcus.

The invention also provides the application of micrococcus in the metabolism of calcium oxalate to form carbon-containing minerals.

The invention also provides application of micrococcus in inducing generation of carbonate minerals.

The invention also provides the application of micrococcus in fixing atmospheric carbon dioxide.

Further: carbonaceous minerals include, but are not limited to, carbonaceous inorganic compounds, such as carbonates.

Further: carbonates include, but are not limited to, calcium carbonate and allotropes of calcium carbonate, such as vaterite, calcite, aragonite.

The invention has the beneficial effects that:

the invention discloses for the first timeMicrococcus separated from saline-alkali soil has the capability of utilizing various substances to generate carbonate minerals, and can improve the carbon reserve potential of soil and fix atmospheric CO for deeply understanding the soil carbon conversion process₂The method has the advantages of reducing the greenhouse effect of the atmosphere and relieving global warming, and has important scientific significance.

Drawings

FIG. 1 is a scanning electron microscope image of the surface of a soil thin layer of an experimental group in example 2 of the present invention;

FIG. 2 is an electron microscope scanning image of the surface of a control group soil thin layer in example 2 of the present invention;

FIG. 3 is a graph of the energy dispersive diffraction analysis of the minerals on the surface of the soil thin layer of the experimental group in example 2 of the present invention;

FIG. 4 is a graph of the energy dispersive diffraction analysis of the mineral on the surface of the soil thin layer of the control group in example 2 of the present invention;

FIG. 5 is an electron micrograph of a precipitate obtained in example 3 of the present invention;

FIG. 6 is an energy dispersive diffraction analysis chart of the precipitate in example 3 of the present invention.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions as recommended by the manufacturer's instructions.

Example 1

Isolation and identification of strain YRD202203 CF:

taking soil of typical saline-alkali soil of Shandong yellow river delta as target soil, the specific screening steps are as follows:

1. collecting 0-10 cm deep soil1.0 g of collected soil is added into 9 mL of sterilized water, and the mixture is shaken for 10 min to prepare a soil solution. 1 mL of the soil suspension was taken, placed in 9 mL of sterilized water, and the soil solution was diluted. This operation was repeated to dilute the soil solution to 10 deg.f^-3And (4) doubling. 100 μ L of the diluted soil solution was inoculated into MSM medium containing calcium oxalate, and cultured at 25 ℃ for 7 days.

Specifically, the medium comprises the following components: na (Na)₂HPO₄·2 H₂O，3.5 g/L； KH₂PO₄，1.0 g/L；(NH₄) ₂SO₄，0.5 g/L；MgCl₂·6 H₂O，0.1 g/L；Ca(NO₃)₂·4 H₂O, 0.05 g/L; 1 mL/L of trace element solution; CaC₂O₄2 g/L; 1.5% agar, pH 7.25.

The formula of the trace element solution is as follows: FeSO₄·7H₂O，0.4 g/L；ZnSO₄·7H₂O，0.1 g/L；MnCl₂· 4H₂O，0.3 g/L；H₃BO₃，0.3 g/L；CuCl₂·2H₂O，0.1 g/L；NiCl₂·6H₂O，0.2 g/L；NaMoO₄，0.3 g/L；CoCl₂·6H₂O，0.1 g/L；Na₂SeO₄·2H₂O，0.05 g/L。

2. And (4) picking a single colony growing on the culture medium, coating the single colony on an LB culture medium, and further separating and purifying. Strain YRD202203CF was obtained.

3. 16S rDNA sequencing of strain YRD202203 CF: the universal primer 27F and the 1492R primer are adopted to perform PCR amplification on the genome DNA of the strain YRD202203CF, the amplified product is sequenced, and the sequencing result is shown as SEQ ID No. 1.

SEQ ID No.1 is shown below:

GGCGGGGTGCTATAATGCAAGTCGAGCGAACAGACGAGAGTGCTTGCACTCTCTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATCTAGCTTCGCATGAAGCGATAGTGAAAGACGGTTCTGCTGTCACTTATAGATGGACCCGCGGTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACGTTAGTAACTGAACGTACCTTGACGGTACCTTACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTCTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGGGGGTTTCCGCCCCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTAGAGATAGAGCTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCATCATTAAGTTGGGCACTCTAGAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCAGCAAAACCGCGAGGTCAAGCAAATCCCATAAAACCATTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCTTTTAGGAGCTAGCCGTCGAAGGTGTCAGAG

BLAST analysis was performed using NCBI database based on the 16S rDNA gene sequence of the strain, and the results showed that the 16S rDNA gene sequence of the strain was homologous to (and homologous to) MicrococcusMacrococcus caseolyticus strain 19Msa1047 homology 99%, GenBank: CP 080013.1), and finally determining the strain to be micrococcus by combining the morphological, structural and physiological and biochemical characteristics of the strain YRD202203CF, and the strain is named YRD202203 CF.

Example 2

Strain YRD202203CF formed carbonaceous minerals using refractory organics (calcium oxalate):

1. and (3) acidizing, neutralizing, leaching and drying the saline-alkali soil to obtain the soil without inorganic carbon. And taking 10 g of treated soil sample, adding 200 mg of calcium oxalate powder serving as a carbon source substance and 20 mg of polyethylene glycol serving as a soil binder, uniformly mixing, placing the mixed sample in 50 mL of deionized water, and oscillating on a vortex oscillator for 5 min to prepare suspension. A200. mu.L suspension was placed in a 35 mm diameter petri dish and dried in a dust-free environment, at which time a thin layer of soil was formed in the petri dish. The thin layer is an observation interface for observing the conversion of the calcium oxalate into the carbon-containing minerals by the microorganisms.

2. Sterilizing a culture dish containing a soil thin layer for 30 min at 121 ℃ to remove the pollution of mixed bacteria, adding 2 mL of sterilized deionized water to soak the soil thin layer into water, inoculating the strain YRD202203CF into the culture dish, and inoculating the inactivated strain to a control group under the same conditions. The petri dish was capped and incubated at room temperature for 3 months.

3. And after the culture is finished, observing the change conditions of the carbon-containing minerals on the soil thin layers of the experimental group and the control group by adopting a scanning electron microscope and an energy dispersion diffraction analysis method. As shown in FIGS. 1 and 2, the thin soil layer of the experimental group was found to have many minerals on its surface and various shapes, and the hemispherical structure was found, while the surface of the control group was smooth, as determined by SEM analysis. Energy dispersive diffraction analysis (fig. 3 and 4) was performed on the representative points on fig. 1 and fig. 2, and the compositions of the minerals on the surfaces of the experimental group and the control group (table 1) both contained carbon, oxygen, calcium, magnesium, aluminum, silicon and other elements, while the carbon content of the experimental group was significantly higher than that of the control group, i.e. the strain YRD202203CF could indeed form carbon-containing minerals.

TABLE 1 surface mineral element composition of experimental and control groups

Example 3

The strain YRD202203CF utilizes easily available organic matters (organic matters rich in carbon and nitrogen) to generate carbonate:

100 mL of liquid culture medium is prepared, placed in a 250 mL triangular flask, and sterilized at 121 ℃ for 30 min. Specifically, the liquid medium comprises the following components: 10 g/L of tryptone, 5 g/L of yeast extract, 10 g/L of sodium chloride and 5 g/L of calcium chloride.

Inoculating the strain YRD202203CF into the culture medium by using an inoculating needle, inoculating the inactivated strain as a control group, keeping the conditions consistent, and culturing at room temperature. Whether precipitates are generated or not is observed every other week, precipitates are generated in the experimental group is observed at week 4, and the process is cultured for 60 days in order to obtain enough precipitates for subsequent analysis. After the completion of the culture, the precipitates formed in the experimental group were collected and treated with a hydrogen peroxide solution to remove the microbial residues and the like, thereby obtaining 0.2265 g of the precipitated substances in total, whereas the control produced no precipitate. And (4) performing electron microscope scanning and energy dispersion diffraction analysis on the precipitate.

The results are shown in fig. 5, and the analysis by scanning electron microscope shows that the precipitate generated by the experiment is hemispherical, and has porous imprints thereon, which is a direct evidence of the action of the microorganism; as shown in fig. 6 and table 2, according to the energy dispersive diffraction analysis, the main components of the precipitate were carbon, oxygen, calcium, and were analyzed as calcium carbonate by atomic percent and weight percent, indicating that strain YRD202203CF has the ability to produce carbon-containing minerals. Furthermore, it was found by X-ray diffraction analysis that the calcium carbonate formed, which was mainly composed of 40% vaterite and 60% calcite, demonstrated that strain YRD202203CF was indeed able to produce calcium carbonate crystals, and that the crystal types were diverse.

TABLE 2 carbon-containing mineral element composition analysis Table (number of weight and atom)

Element(s)	Weight percent (%)	Atomic percent (%)
			C	16.83	25.13
O	55.88	62.65
			Ca	27.29	12.21
Total amount of	100.00	100.00

Example 4

Strain YRD202203CF fixes atmospheric carbon dioxide by forming carbonaceous minerals:

the medium was prepared and inoculated with the strain YRD202203CF by the procedure of example 3. The experimental group is that the culture medium is placed in¹³CO₂Culturing in the environment to mark CO in the atmosphere₂The direction of arrival; the control group is placed in¹³CO₂Culturing in a marked environment. After the culture is finished, the precipitate is treated by hydrogen peroxide solution, organic matters are removed by oxidation, and the carbon-containing minerals are measured¹³C abundance value.

The results are shown in Table 2, and the experimental groups are¹³CO₂In the environment of carbon-containing minerals¹³The C abundance value is 870.89 ‰, which is significantly higher than-11.59 ‰ of the control group, and indicates that part of C in the carbon-containing minerals in the experimental group is derived from atmospheric CO₂. The results also show that the pH of the culture medium of the control group is 6.81, while the pH of the culture medium of the experimental group is 8.90, and the concentration of the ammonium ions of the experimental group is obviously higher than that of the control group. The results show that the strain YRD202203CF generates ammonium ions through decomposition of nitrogenous organic matters, the ammonium ions are hydrolyzed to be alkaline, the pH of the solution is promoted to be increased, and the liquid culture medium is alkaline. CO in the atmosphere₂Dissolving into alkaline liquid medium to form HCO₃ ^-The process continues to result in "CO₂—HCO₃ ^-—CO₃ ^2-The "equilibrium shifts to the right, which in turn leads to the formation of carbonate minerals. The above results show that it is possible to obtain,the strain YRD202203CF can convert atmospheric CO into CO₂The carbon in the carbon-containing mineral is converted into carbon in the carbon-containing mineral and is preserved in the carbon-containing mineral.

TABLE 3 among the carbonaceous minerals¹³C abundance value

	¹³C abundance value (‰)
		Control group	-11.59‰
Experimental group	870.89‰

The invention discloses that micrococcus separated from saline-alkali soil has the capability of utilizing various substances to generate carbonate minerals for the first time, improves the carbon reserve potential of soil and fixes atmospheric CO for deeply understanding the soil carbon conversion process₂The method has important scientific significance for slowing down the greenhouse effect of the atmosphere and relieving global warming.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> institute of geoscience and resource of Chinese academy of sciences

<120> Micrococcus and application thereof

<141> 2022-05-09

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1451

<212> DNA

<213> Micrococcus YRD202203CF (Macrococcus sp. YRD202203CF)

<400> 1

ggcggggtgc tataatgcaa gtcgagcgaa cagacgagag tgcttgcact ctctgacgtt 60

agcggcggac gggtgagtaa cacgtgggta acctacctat aagactggga taacttcggg 120

aaaccggagc taataccgga taatatctag cttcgcatga agcgatagtg aaagacggtt 180

ctgctgtcac ttatagatgg acccgcggtg tattagctag ttggtgaggt aacggctcac 240

caaggcaacg atacatagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300

gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360

gagcaacgcc gcgtgagtga agaaggtttt cggatcgtaa aactctgttg taagggaaga 420

acaagtacgt tagtaactga acgtaccttg acggtacctt accagaaagc cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggaat tattgggcgt 540

aaagcgcgcg taggcggtct cttaagtctg atgtgaaagc ccccggctca accggggagg 600

gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgtgtagcgg 660

tgaaatgcgc agagatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact 720

gacgctgagg tgcgaaagcg tggggatcaa acaggattag ataccctggt agtccacgcc 780

gtaaacgatg agtgctaagt gttggggggt ttccgcccct cagtgctgca gctaacgcat 840

taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat tgacggggac 900

ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaaatc 960

ttgacatcct ttgacaactc tagagataga gctttcccct tcgggggaca aagtgacagg 1020

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttat ctttagttgc catcattaag ttgggcactc tagagagact gccggtgaca 1140

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgattt gggctacaca 1200

cgtgctacaa tggatggtac aaagggcagc aaaaccgcga ggtcaagcaa atcccataaa 1260

accattctca gttcggattg tagtctgcaa ctcgactaca tgaagctgga atcgctagta 1320

atcgtagatc agcatgctac ggtgaatacg ttcccgggtc ttgtacacac cgcccgtcac 1380

accacgagag tttgtaacac ccgaagccgg tggagtaacc ttttaggagc tagccgtcga 1440

aggtgtcaga g 1451

Claims

1. A micrococcus, characterized in that: the micrococcus is named as YRD202203CF, and the classification is namedMacrococcus sp11.11 days in 2022, 3 months, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang district, and the preservation number is CGMCC No. 2450.

2. The micrococcus of claim 1, wherein: the sequence of the micrococcus 16S rDNA gene is shown in SEQ ID No. 1.

3. The micrococcus of claim 1, wherein: the micrococcus is isolated from saline-alkali soil.

4. The micrococcus of claim 3, wherein: the screening method of micrococcus comprises the following steps: diluting saline-alkali soil, taking soil diluent, inoculating the soil diluent into an inorganic salt MSM culture medium containing calcium oxalate, and culturing and separating to obtain micrococcus.

5. Use of a micrococcus according to any one of claims 1-4 to metabolise calcium oxalate to form a carbonaceous mineral.

6. Use of micrococcus according to claim 5 for the metabolic utilization of calcium oxalate to form carbonaceous minerals, characterized in that: the carbonaceous minerals include, but are not limited to, carbonaceous inorganic compounds.

7. Use of a micrococcus according to any one of claims 1-4 to induce carbonate mineral formation.

8. Use of micrococcus according to claim 7 for inducing carbonate mineral production, wherein: the carbonates include, but are not limited to, calcium carbonate and the allotrope of calcium carbonate.

9. Use of a micrococcus according to any one of claims 1-4 for fixing atmospheric carbon dioxide.