Disclosure of Invention
The first object of the present invention is to provide a strain of methanotrophic bacteria (Methylomonas sp.) C1-2, which strain C1-2 was deposited at the microorganism strain collection (GDMCC) of Guangdong province at 2024, under the address of Guangzhou city martyr, no. 100, no. 59, post code 510070, accession number GDMCC No, 64255.
The second object of the invention is to provide the application of the methanotrophic bacterium C1-2 in reducing methane emission.
The methanotrophic bacteria C1-2 can grow by utilizing methane under the condition of low concentration methane (500, 1000 ppm), and are suitable for methane emission reduction in main emission sites such as paddy fields, landfill sites, anaerobic digester wastewater, oil and gas wells and the like.
Preferably, the use scenario for reducing methane emissions includes paddy fields, landfill sites, anaerobic digester wastewater or oil and gas wells.
Preferably, the methane content in the air above the use scene is equal to or greater than 500ppm.
The third object of the invention is to provide the application of the methanogen C1-2 in mineralization of organic phosphorus.
The methanotrophic bacteria C1-2 grow on a methanol-lecithin culture medium, form an obvious phosphorus dissolving ring and have the capacity of mineralizing organic phosphorus.
Preferably, the mineralized organic phosphorus is an organic phosphorus that is not directly available to plants in the soil, to an available effective phosphorus.
The fourth object of the invention is to provide the application of the methanotrophic bacteria C1-2 in preparing phosphate-dissolving bacteria or methane emission reducing bacteria.
The fifth object of the invention is to provide a phosphate solubilizing microbial agent comprising methanotrophic bacteria C1-2.
The sixth object of the present invention is to provide a methane-emission reducing microbial agent comprising methanotrophic bacteria C1-2.
The invention adopts a continuous enrichment method to separate and purify a strain of methanotrophic bacteria (Methylomonas sp.) C1-2 from paddy soil, and identifies the strain C1-2 as a new species of the genus Methylomonas according to morphological characteristic analysis, systematic development analysis and genome related index calculation results. The methanotrophic bacteria C1-2 can grow by using low-concentration methane as the only carbon source and energy source, and have application value in the aspect of reducing methane emission at a main methane emission point. The methanotrophic bacteria C1-2 can mineralize organic phosphorus and have application value in the aspect of preparing growth-promoting microbial agents. The methanotrophic bacteria C1-2 have application value in reducing the emission of greenhouse gas methane and mineralizing organic phosphorus.
Methane-oxidizing bacteria (Methylomonas sp.) C1-2 were deposited at the microorganism culture Collection (GDMCC) of Guangdong province at 1 month 9 of 2024 under the address of No. 59 building 5 of Dai 100 in martyr of Guangzhou province, post code of 510070 and deposit number of GDMCC No:64255.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1 isolation and purification of Strain C1-2
1. Enrichment of methane-oxidizing bacteria
The flooded paddy soil was collected from Meng-screened Zhenman Niu Cun (N21°57'53' ', E100°16'31 '') in Menghai county, xishuangbanna, yunnan province. About 5g of the aqueous soil was placed in a 120mL glass bottle, 20mL of NMS liquid medium (10 mL/L of phosphate buffer solution containing KNO31g/L,MgSO4·7H2O 1g/L,CaCl2 0.2g/L,Fe-EDTA 7.6mg/L,Na2MoO4·2H2O 0.26mg/L, trace element solution at pH 6.8, 10mL/L of solvent water) was added, and the mixture was sealed with a rubber stopper and an aluminum cap. Subsequently, 20% of the headspace gas was replaced with methane, and the glass flask was placed in a 30℃incubator with shaking for 1 week.
Culturing for 1 week to obtain the first generation enrichment solution. Standing the first generation enrichment liquid, taking the upper layer bacterial liquid according to the volume ratio of 1:200 after the clay layer and the bacterial liquid are obviously layered, transferring the upper layer bacterial liquid into a 120mL glass bottle filled with 20mL of fresh NMS liquid culture medium for continuous culture, wherein the headspace gas content and enrichment condition are the same as those of the first enrichment. After 1 week of culture, the culture solution is observed to be obviously turbid, and then the second generation enrichment solution is obtained. And subculturing the second generation enrichment liquid for 1 week according to the same method, wherein the obtained culture liquid with higher turbidity degree is the third generation enrichment liquid.
2. Separation, purification and preservation of methane-oxidizing bacteria C1-2
100. Mu.L of the third-generation enrichment solution is uniformly coated with NMS solid culture medium (agar 1.5% and the rest components are the same as the liquid culture medium), and then a solid flat plate is subjected to cap removal and placed in a closed dryer to separate methanotrophic bacteria. The air content of the top of the dryer is the same as that of enrichment culture, and the whole system is placed in light-shielding culture at 30 ℃. After 5d of cultivation, the desiccator was opened in an ultra clean bench, and single colonies on the separation plate were picked up to purify methane-oxidizing bacteria in a new NMS solid medium, and the purification plate was also placed in a closed desiccator with 20% methane for the headspace gas. The purified single colonies were picked up and transferred to liquid NMS medium, and the strain was preserved at-80℃at a final DMSO concentration of 10%, thereby obtaining strain C1-2.
Example 2 identification of Strain C1-2
1. Morphological observation of strains
Bacterial strain C1-2 showed pale pink colonies on NMS solid medium containing 20% methane headspace gas, and the whole colony was round (FIG. 1A), and the single cells of bacterial strain C1-2 were observed to be in the shape of short rods with a size of 1-1.5 μm×1-4 μm under a phase contrast microscope (FIGS. 1B and 1C).
2. Strain identification based on 16S rRNA gene sequence
Scraping fresh thalli growing on NMS solid culture medium for 3 days, adding a small amount of sterile water, placing at 90 ℃ for 10min, centrifuging, and taking the supernatant as a template for PCR reaction. The PCR reaction system (25. Mu.L) was 12.5. Mu.L of buffer, 1. Mu.L of each of the upstream and downstream primers (27F/142R, 27F:5'-AGAGTTTGATCCTGGCTCAG-3'; 142R: 5 '-GGTTACCTTGTTACGACTT-3'), 1. Mu.L of template, and 9.5. Mu.L of sterile deionized water. The reaction procedure was 95℃for 5min,95℃for 30s,56℃for 30s,72℃for 90s,30 cycles, 72℃for 5min. The PCR products were subjected to agarose electrophoresis to determine that no errors occurred and then submitted to Jin Weizhi Biotechnology Inc for sequencing.
The 16S rRNA gene sequences were BLAST aligned at EzBioCloud (https:// www.ezbiocloud.net /). The results showed that strain C1-2 had the highest similarity to model strain Methylomonas rapida MP 1.1T, with a 97.79% similarity in 16S rRNA gene sequence. The 16S rRNA gene sequence is shown as SEQ ID NO. 1. Phylogenetic tree was constructed in MEGA using strain C1-2 and its closely related species, which indicated that the most recent source species of strain C1-2 was M.rapida MP1T, and that C1-2 represents a potential new species in the genus Methylomonas Methylomonas (FIG. 2).
3. Strain identification based on genome sequencing
Bacterial strain C1-2 grown in NMS solid medium (20% methane) was collected and genomic DNA was extracted by CTAB method. The 16S rRNA gene is amplified by using the extracted genome DNA as a template and a 27F/1492R primer, and is subjected to sequencing comparison and is delivered to the Meji biological medicine science and technology Co-Ltd for genome sequencing. And after sequencing and machine data quality control, assembling by SPAdes, evaluating genome quality by checkM, and selecting the assembly result with the maximum N50 as a strain genome sketch sequence. The average nucleotide similarity ANI and hybridization values dDDH between the genomes of the strain C1-2 and its closely related species were calculated using software FastANI and web page tool GGDC, respectively. The results in Table 1 show that the ANI values between the genome of strain C1-2 and the Methylomonas closely related species are 74.38-79.95% (95-96% of the species division threshold) and dDDH values are 18.8-22.2% (70% of the species division threshold). In summary, strain C1-2 was identified as a new species of Methylomonas based on morphological feature analysis, phylogenetic analysis, and genome-related index calculations.
TABLE 1 ANI and dDDH values for strains C1-2 and its closely related model strains
Through the morphological and molecular biological identification, the strain C1-2 is identified as a new species of the genus Methylomonas, named methane-oxidizing bacteria (Methylomonas sp.) C1-2, and is deposited with the Guangdong province microorganism strain collection (GDMCC) on the 1 st month 9 th year 2024, address: guangzhou city martyr, university No. 100, building No. 59, post code: 510070, deposit number GDMCC No:64255.
Methane oxidizing bacteria oxidize methane into methanol under the action of methane monooxygenase, and currently known key enzymes for methanogenesis are divided into two types of granular methane monooxygenase genes (pmoA) and soluble methane monooxygenase genes (mmoX). Genome annotation results show that the strain C1-2 contains two methane monooxygenase genes containing pmoA and mmoX simultaneously, and the protein sequences of the genes are shown as SEQ ID NO.2 and SEQ ID NO. 3.
SEQ ID NO.1 16S rRNA Gene sequence of Strain C1-2
>16S rRNA gene of C1-2
TTACCATGCAGTCGAACGCTGAAGGGTGCTTGCACCCGGATGAGTGGCGGACGGGTG
AGTAATGCATAGGAATCTGCCTATTAGTGGGGGATAACGTGGGGAAACTCACGCTAATA
CCGCATACGATCTACGGATGAAAGCCGGGGACCTTCGGGCCTGGCGCTAATAGATGAG
CCTATGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCGTAGC
TGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATACCGCG
TGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAATAGGAAGGAATACCTATCGG
TTAATACCCGATAGACTGACATTACCTATACAAGAAGCACCGGCTAACTCCGTGCCAGC
AGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGC
GTAGGCGGTTTTTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAACTGCATTT
GATACTGGAAGACTAGAGTTGAGTAGAGGAGAGTGGAATTTCAGGTGTAGCGGTGAA
ATGCGTAGAGATCTGAAGGAACACCAGTGGCGAAGGCGGCTCTCTGGACTCAAACTG
ACGCTGAGGTACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACG
CCGTAAACGATGTCAACTAACTGTTGGGTTCTTAAAGAACTTAGTAGTGGAGCTAACG
TATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAATGAATTGACG
GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTT
ACCTACCCTTGACATCCTCGGAACTTGTCAGAGATGACTTGGTGCCTTCGGGAACCGA
GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCC
GTAACGAGCGCAACCCTTATCCTTAGTTGCCAGCGAGTCAAGTCGGGAACTCTAGGGA
GACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTT
ATGGGTAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGCAGCAAACTCGCGA
GAGCCAGCAAATCCCAAAAAGCCGATCCTAGTCCGGATTGCAGTCTGCAACTCGACTG
CATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCG
GGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGAGGGCG。
SEQ ID NO.2 pMMO_PmoA protein sequence of Strain C1-2
>pMMO_PmoA
MSAHIPISTFKPYRGEKARLARAYDYLILVLALFLFIGSFHLHFALTVGDWDFWVDWKDR
QWWPLVTPLIGITFPAAVQAVLWDKFRLPLGATLCVAALLLGTWVTRVFAYHYWNFFPIN
MVLPATMVPGALVLDTLLMLTNSLTITSIFGGGAFALLFYPTNWPIFGMFHQAVEYHNSQL
TVADLFGFQYIRTGMPEYLRIIERGTLRTYGQYATPLAAFCSALLCSLMYPLWWYIGKWFANTSYLKKI。
SEQ ID NO.3 sMMO_ MmoX protein sequence of Strain C1-2
>sMMO_MmoX
MAISAATKAATDALAINRAPTSVNAQEVHRWLQSFTWDFDKNRTKYPTKYKMANETKE
QFKLIAKEYARMESVKDERQFGSLQDVLTRLDAGNRVHPKWGETMKVASNFLEVGEYN
AIAATGMLWDSASAPEQKNGYLAQVLDEIRHTNQCGYVNYYYSKHYHDPAGHNDARRT
RTIGPLWKGMKRVFSDGFISGDAVECSINLQLVGEACFTNPLIVAITEWAAANGDEITPTVF
LSIETDELRHMANGYQTVVSIANDEAASKYLNTDLNNAFWTQQKYFTPVLGMLFEYGSK
FKVEPWVKTWNRWVYEDWGGIWIGRLGKYGVESPRSLRDAKKDAYWAHHDLFLLAYA
LWPTGFFRLTLPTQEEMDWFEENYPGWYDHYGKIYDEWRARGCEDPNSGFIPLMWFIEN
NHPIYIDRVSQVPFCPSLCKGASTLRVHEWNGKKHSFSDDWGERMWLTEPERYECQNMFEQYAGRELSEVIAEQHGVRSDGKTLIAQPHTNKNGKLWTLDDIKKLNCVFKDPLEAL.
Example 3 growth of strains under different methane concentrations
100. Mu.L of NMS culture solution of strain C1-2 was inoculated as seed solution into a glass bottle containing 20mL of NMS liquid medium, and sealed with a rubber stopper and an aluminum cap. The headspace gas of the glass bottle was set at methane concentrations of 200, 500, 1000, 5000, 50000 and 100000ppm, 3 replicates were set for each group, and the culture was performed at 30℃and 200rpm for 3 days with shaking, during which time the growth of the cells was observed. As a result, it was found that the strain C1-2 was able to grow under low concentration methane conditions (200, 500, 1000, 5000 ppm) and the cells were suspended in the shaking liquid medium as white particles, and under high concentration methane conditions (50000, 100000 ppm) the cells were able to grow well and were suspended in the shaking liquid medium as light pink irregular agglomerates (FIG. 3). The above results indicate that strain C1-2 not only can grow rapidly to consume methane at concentrations comparable to atmospheric methane sources, but also can accumulate biomass rapidly at concentrations of >50000ppm methane.
EXAMPLE 4 characterization of mineralized lecithins of the Strain
10 Mu L of strain C1-2 in logarithmic growth phase is inoculated in NMS-methanol-lecithin solid culture medium (0.5% methanol, 1% lecithin, 1.5% agar, all in mass fraction,% and the rest components are the same as the above liquid culture medium), cultured at 30deg.C for 3d, and strain growth is observed. As a result, it was found that the strain C1-2 grew well on this medium and formed a distinct phosphate ring (FIG. 4).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.