CN110982727B - Stenotrophomonas maltophilia strain and application thereof - Google Patents
Stenotrophomonas maltophilia strain and application thereof Download PDFInfo
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- CN110982727B CN110982727B CN201910804250.3A CN201910804250A CN110982727B CN 110982727 B CN110982727 B CN 110982727B CN 201910804250 A CN201910804250 A CN 201910804250A CN 110982727 B CN110982727 B CN 110982727B
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/02—Other organic fertilisers from peat, brown coal, and similar vegetable deposits
Abstract
The invention discloses a stenotrophomonas maltophilia strain and application thereof, the stenotrophomonas maltophilia strain is named as stenotrophomonas maltophilia GZU-Stm01, is preserved in the China Center for Type Culture Collection (CCTCC) in 2019, 1 month and 21 days, and has the preservation number of CCTCC NO: m2019059. The Stenotrophomonas maltophilia GZU-Stm01 can effectively dissociate insoluble phosphorus, potassium, silicon, calcium, sulfur and other components in coal gangue and convert the insoluble phosphorus, potassium, silicon, calcium, sulfur and other components into nutrient substance components absorbed by plants, such as available phosphorus, available potassium, available silicon, exchangeable calcium, available sulfur and the like. Particularly, GZU-Stm01 can effectively dissociate the insoluble phosphorus in the coal gangue to make the insoluble phosphorus become effective phosphorus, which provides a source of available phosphorus resources, and GZU-Stm01 has good dissociation effect on other phosphorus-containing minerals, so that the coal gangue can be doped with other phosphorus-containing minerals such as low-grade phosphorus ore, and the phosphorus content in the mineral microbial compound fertilizer using the coal gangue as a basic raw material can be further improved.
Description
Technical Field
The invention relates to a strain and application thereof, in particular to a stenotrophomonas maltophilia strain and application thereof.
Background
The coal resources in China are rich, the distribution is wide, the varieties are complete, the coal occupies the main position in the energy structure in China, and the coal plays a significant role in the development of economy in China. The coal gangue is a solid waste with low carbon content and harder than coal, which is generated in the coal mining and washing processes and accounts for about 15-20% of the original coal yield. At present, coal gangue is the largest industrial waste, and because of no substantial application, hundreds of millions of tons of coal gangue are estimated to be discarded in each coal production area in China throughout the year and are mostly stacked in an open-air manner to form a plurality of coal gangue dumps. After the coal gangue piled in the open air is washed and drenched by rainwater, harmful substances in the coal gangue are gradually dissolved and flow into soil, surface water or underground water along with the rainwater, so that serious pollution is caused.
The coal gangue contains rich nutrient components required by plant growth, such as organic matters, phosphorus, potassium, silicon, calcium, sulfur and the like. However, phosphorus, potassium and other components in the coal gangue mostly exist in the form of insoluble minerals, and cannot be directly absorbed and utilized by plants. Due to various reasons and technical limitation, the coal gangue is not effectively utilized, so that the resource is left unused and the environment is greatly polluted.
The stenotrophomonas maltophilia (Stenotrophormonastra) can be widely applied to various coal gangue types, and can effectively dissociate insoluble phosphorus, potassium, silicon, calcium, sulfur and other components in the coal gangue and change the insoluble phosphorus, potassium, silicon, calcium, sulfur and other components into nutrient components which can be absorbed by plants, such as available phosphorus, available potassium, available silicon, exchangeable calcium, available sulfur and the like.
Experiments show that stenotrophomonas maltophilia has a phosphate solubilizing effect on coal gangue superior to that of the traditional phosphate solubilizing strain bacillus megaterium (Bacillus megaterium), and GZU-Stm01 is an excellent phosphate solubilizing bacterium.
The method has the advantages of simple and reliable process, low cost and environmental friendliness, and is a new scheme for green utilization of the coal gangue by utilizing microbial bacteria to dissociate the coal gangue and release nutritional ingredients in the coal gangue. The coal gangue is dissociated by microbial bacteria, and nutrient components available for plants are released, so that the microbial compound fertilizer taking the coal gangue as a basic raw material is created.
Therefore, the field needs to further develop more new strains for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.
Disclosure of Invention
The invention aims to provide a stenotrophomonas maltophilia strain and application thereof. The strain can dissociate phosphorus, potassium, silicon, calcium and/or sulfur in the coal gangue, and the method is simple, good in effect, low in cost and environment-friendly.
The technical scheme of the invention is as follows: a stenotrophomonas maltophilia strain is a stenotrophomonas maltophilia, is named as stenotrophomonas maltophilia GZU-Stm01, is preserved in the China center for type culture Collection of university of Wuhan, in 2019 and 1 month and 21 days, and has the preservation number of CCTCC NO: m2019059.
The stenotrophomonas maltophilia GZU-Stm01 is isolated from weathered potassium ore and nearby soil in a potassium ore producing area.
Application of the stenotrophomonas maltophilia strain in preparation of products for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.
A product for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, wherein the active ingredient of the product comprises or is the stenotrophomonas maltophilia GZU-Stm 01.
A preparation method of a product for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue adopts the stenotrophomonas maltophilia GZU-Stm01 as an active ingredient or one of the active ingredients for preparing the product.
A method for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue adds and/or uses the products during the dissociation process.
A microbial inoculum for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue comprises or is the stenotrophomonas maltophilia GZU-Stm01 as an active ingredient.
The microbial inoculum also comprises conventional components for preparing the microbial inoculum.
The application of the strain in dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue is provided.
The invention has the advantages of
1. The invention relates to a Stenotrophomonas maltophilia strain GZU-Stm01 separated, purified and cultured from completely weathered potassium ores and nearby soil in a potassium ore producing area, wherein the Stenotrophomonas maltophilia strain is a new strain which can effectively decompose insoluble phosphorus, potassium, silicon, calcium, sulfur and the like in coal gangue and can be changed into effective phosphorus, quick-acting potassium, effective silicon, exchangeable calcium, effective sulfur and the like serving as nutrient components absorbed by plants. The strain has good dissociation effect, can effectively dissociate various components in the coal gangue, particularly has good effect on the dissociation of the indissolvable phosphorus in the coal gangue, and has the phosphorus-dissolving effect superior to that of the traditional Bacillus megaterium.
2. The invention adopts microbial dissociation, and has simple operation and low cost.
3. The environment hazard brought by the coal gangue can be relieved by the development and utilization of the coal gangue resource, the created microbial compound fertilizer can provide effective phosphorus required by plants, the strain is introduced into the soil, the problems of soil hardening and the like caused by long-term use of the fertilizer can be avoided, and the use efficiency of phosphorus resources in the soil is improved. Compared with the prior chemical method for treating the coal gangue, the microbial dissociation method is more environment-friendly.
Drawings
FIG. 1 is a growth curve of GZU-Stm01 strain of the present invention;
FIG. 2 is an appearance diagram of a colony of the GZU-Stm01 strain of the present invention;
FIG. 3 is a morphological diagram of GZU-Stm01 cells of the present invention;
FIG. 4 is a phylogenetic tree established by the GZU-Stm01 strain based on partial 16S rRNA gene sequences.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Examples of the invention
Example (b):
isolation of stenotrophomonas maltophilia GZU-Stm01 comprising the steps of:
1. isolation and screening of functional Strain GZU-Stm01
1) Collection of samples
(1) The sample is collected from completely weathered potassium ore and nearby soil in a certain potassium ore production area, the completely weathered potassium ore sample is selected, and the collected sample is filled into a collection bag;
(2) crushing and screening the collected samples, and collecting the samples which pass through 100 meshes;
(3) the samples were stored in a refrigerator at 4 ℃ until use.
2) Screening of functional strains
(1) Accurately weighing 1g of sample into a sterilized 250mL conical flask, and adding 99mL of sterile water;
(2) oscillating for 30min in a constant temperature shaking table at about 37 ℃ at 170 r/min;
(3) the control group was prepared by adding 100mL of sterile water to a 250mL Erlenmeyer flask;
(4) oscillating for 30min, collecting supernatant, and gradually diluting to 10-3，10-4，10-5，10-6，10-7。
(5) Coating 100uL of each concentration on 3 phosphate solubilizing bacteria screening solid culture media respectively, wherein the blank is a flat plate coated with sterile water;
(6) putting the flat plate in a 30 ℃ constant temperature incubator for 3-4 days;
(7) streaking and inoculating single colonies with different appearance characteristics on a phosphate-solubilizing bacteria screening solid culture;
(8) after the strains grow out, observing the purification condition of the strains through primary appearance morphology observation and microscopic morphology observation, and repeating the steps for many times until a single strain is obtained and different strains are distinguished;
(9) gram staining the purified strain;
(10) culturing the primarily screened purified strains, dissociating coal gangue, determining the effective phosphorus content in the coal gangue, confirming the phosphorus dissolving effect of the strains, and screening the strains with good phosphorus dissolving effect.
2. Drawing of growth curves of functional strains
1) And (4) preparing seed liquid. Inoculating the strain in LB liquid culture medium with inoculating loop, and culturing at 30 deg.C to logarithmic phase.
2) And (4) inoculating. Inoculating 10mL of the seed solution into 200mL of liquid LB culture medium, mixing uniformly, and respectively placing 5mL of mixed solution into 18 marked sterile test tubes.
3) And (5) culturing. The inoculated test tubes are placed in a shaker at 37 ℃ and 170r/min for cultivation, and are respectively taken out for corresponding cultivation time.
4) And (6) measuring. And (3) performing photoelectric turbidimetry with the sample to be detected at a wavelength of 600nm by using an uninoculated LB liquid culture medium as a blank.
As shown in FIG. 1, the measurement results showed that 0 to 10 hours were a lag phase, 10 to 30 hours were a logarithmic phase, 30 to 45 hours were a stationary phase, and 45 hours and thereafter, an apoptosis phase.
3. Identification of functional strains
1) Appearance morphology of colony
As shown in FIG. 2, it is clear that the strain appears yellowish on LB solid medium, is opaque and round, slightly raised, has regular edges, smooth and moist surface, and is glossy, and the colony size is about 1 mm.
2) Gram stain, cell morphology
As shown in FIG. 3, it can be seen that the strains are gram-negative bacilli, arranged singly or in pairs.
3) Identification of strains
(1) Extraction of bacterial DNA
Selecting a single colony of a purified strain to culture in a conical flask filled with LB liquid culture medium to obtain pure strain fermentation liquor, extracting DNA by using a bacterial genome extraction kit (centrifugal column type, Beijing Biotechnology Co., Ltd.), performing Polymerase Chain Reaction (PCR) by using the DNA as a template, amplifying 16S R RNA by using Taq DNA polymer, and amplifying by using general primers of 2F (5'-AGAGTTTGATCCTGGCTCAGGATGA-3') and 1492R (5'-TACGGCTACCTTGTTACGACTTAGC-3'), wherein the PCR amplification reaction system and the amplification reaction conditions are shown in Table 1.
TABLE 1 PCR amplification reaction System and amplification reaction conditions
1.0% agarose gel is prepared by 1 XTBE buffer solution, 40 mul PCR amplification product is added, 8 mul 2000bp DNA Marker is added, electrophoresis is carried out for 45min under the voltage of 110V and the current of 70mA, and the imaging result of the gel is observed.
(2) Homology alignment
The PCR amplification product is purified by a gel recovery kit, sent to a sequencing company (Shanghai Lifei Biotechnology Co., Ltd.) for sequencing, the measured sequence is compared on NCBI (national center for Biotechnology information) by using Standard Nucleotide Blast, and phylogenetic analysis is carried out by using software such as ClustalW and MEGA5.05 based on an adjacency Method (Neighbor Joining Method) to construct a phylogenetic tree (figure 4) so as to obtain the classification information of strains.
As can be seen from FIG. 4, GZU-Stm01 bacterium and Stenotrophomonas maltophilia (Stenotrophoromonas maltophilia) clustered in the same branch, and GZU-Stm01 was identified as Stenotrophomonas maltophilia (Stenotrophoromonas maltophilia) in combination with physiological and biochemical characteristics and morphological observation.
(3) Physiological and biochemical experiment
The strain was identified as stenotrophomonas maltophilia by a series of biochemical experiments, referring to Bergey's Manual of bacteria identification (8 th edition) and Manual of identification of common bacteria systems (Dongxu bead et al, 1999).
4. Determination of dissociation capability of strain to coal gangue
1) The coal gangue comprises the following main components: c11.02%, SiO2 39.41％，Al2O3 17.01％，Fe2O316.02％,TiO2 2.21％，CaO 5.76％，MgO 1.60％，K2O 1.78％，Na2O 1.47％，P2O51.61%, S0.83%, H0.71%, N0.56%, ash 80.03%.
2) The four main factors affecting coal gangue dissociation are: bacterial lysis amount, dissociation time, gangue particle size and system pH value. Aiming at target coal gangue, single-factor experiments of GZU-Stm01 and the bacillus megaterium are firstly carried out, the optimal single-factor conditions of all bacteria are respectively searched, and then the optimal conditions of GZU-Stm01 and the bacillus megaterium for dissociating the coal gangue are searched through designing orthogonal experiments.
3) Dissociation comparison experiments were carried out with respect to coal gangue having a composition of 1) using GZU-Stm01 and Bacillus megaterium.
4) The dissociation experiment procedure was as follows: taking the crushed coal gangue with the specified mesh number, placing the crushed coal gangue in a temperature-controlled stirrer, and controlling the concentrations of GZU-Stm01 and the bacillus megaterium liquid to be: 6.0X 1016-6.5×1016cfu/mL, the ratio of the two strains of bacteria to the coal gangue is 1:1, the bacteria liquid is sprayed under continuous stirring, the mixture is uniformly stirred and turned over once every 2 to 3 hours, the temperature is controlled to be not higher than 30 ℃, and the coal gangue is dissociated for several days.
5) The content of each index of the coal gangue after dissociation is measured, and the result is shown in table 2. As can be seen from Table 2, the overall effect of the GZU-Stm01 on dissociating coal gangue is better than that of the Bacillus megaterium, and the main indexes such as available phosphorus, quick-acting potassium and the like are better than that of the Bacillus megaterium.
Table 2 orthogonal design experimental results
SEQUENCE LISTING
<110> Guizhou university China tobacco general Co, Guizhou province Co
<120> stenotrophomonas maltophilia strain and application thereof
<130> 2019
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1425
<212> DNA
<213> Stenotrophomonas maltophilia (Stenotrophoromonas maltophilia)
<400> 1
actaccatgc agtcgacggc agcacaggag agcttgctct ctgggtggcg agtggcggac 60
gggtgaggaa tacatcggaa tctacttttt cgtgggggat aacgtaggga aacttacgct 120
aataccgcat acgacctacg ggtgaaagca ggggaccttc gggccttgcg cgattgaatg 180
agccgatgtc ggattagcta gttggcgggg taaaggccca ccaaggcgac gatccgtagc 240
tggtctgaga ggatgatcag ccacactgga actgagacac ggtccagact cctacgggag 300
gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatac cgcgtgggtg 360
aagaaggcct tcgggttgta aagccctttt gttgggaaag aaatccagcc ggctaatacc 420
tggttgggat gacggtaccc aaagaataag caccggctaa cttcgtgcca gcagccgcgg 480
taatacgaag ggtgcaagcg ttactcggaa ttactgggcg taaagcgtgc gtaggtggtt 540
gtttaagtct gttgtgaaag ccctgggctc aacctgggaa ctgcagtgga aactggacga 600
ctagagtgtg gtagagggta gcggaattcc cggtgtagca gtgaaatgcg tagagatcgg 660
gaggaacatc catggcgaag gcagctacct ggaccaacac tgacactgag gcacgaaagc 720
gtggggagca aacaggatta gataccctgg tagtccacgc cctaaacgat gcgaactgga 780
tgttgggtgc aatttggcac gcagtatcga agctaacgcg ttaagttcgc cgcctgggga 840
gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagta 900
tgtggtttaa ttcgatgcaa cgcgaagaac cttacctggc cttgacatgt cgagaacttt 960
ccagagatgg attggtgcct tcgggaactc gaacacaggt gctgcatggc tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgtc cttagttgcc 1080
agcacgtaat ggtgggaact ctaaggagac cgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaag tcatcatggc ccttacggcc agggctacac acgtactaca atggtgggga 1200
cagagggctg caagccggcg acggtaagcc aatcccagaa accccatctc agtccggatt 1260
ggagtctgca actcgactcc atgaagtcgg aatcgctagt aatcgcagat cagcattgct 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg agtttgttgc 1380
accagaagca ggtagcttaa ccttcgggag ggcgctgcca cgggg 1425
Claims (8)
1. Stenotrophomonas maltophilia(Stenotrophomonas maltophilia )A strain characterized by: is stenotrophomonas maltophilia, is named as stenotrophomonas maltophilia GZU-Stm01, is preserved in the China center for type culture Collection in 1 month and 21 days in 2019, and has the preservation number of CCTCC NO: m2019059.
2. Use of a stenotrophomonas maltophilia strain according to claim 1 for the preparation of a product that dissociates phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.
3. An article of manufacture for dissociating phosphorus, potassium, silicon, calcium, and/or sulfur in a coal gangue, comprising: the active ingredient of the product comprises or is stenotrophomonas maltophilia GZU-Stm01 of claim 1.
4. A preparation method for dissociating products of phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue is characterized in that: the stenotrophomonas maltophilia GZU-Stm01 according to claim 1 is used as an active ingredient or one of the active ingredients for preparing the product.
5. A method for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue is characterized in that: adding and/or using the preparation described in claim 4 during the dissociation.
6. A microbial inoculum for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, which is characterized in that: the active ingredient of the microbial inoculum comprises GZU-Stm01 stenotrophomonas maltophilia according to claim 1.
7. The microbial inoculum of claim 6, wherein: the microbial inoculum also comprises conventional components for preparing the microbial inoculum.
8. Use of the strain according to claim 1 for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.
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| CN114368997B (en) * | 2021-04-30 | 2022-09-09 | 四川农业大学 | Development and utilization method of insoluble potassium ore resources |
| CN114561319B (en) * | 2021-04-30 | 2022-08-19 | 四川农业大学 | Potassium-decomposing bacteria and potassium-decomposing bacterial fertilizer |
| WO2023071880A1 (en) * | 2021-10-26 | 2023-05-04 | 北京一凹生态科技有限公司 | Pseudomonas fluorescens n1 and use thereof |
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