CN110982727B - A strain of Stenotrophomonas maltophilia and its application - Google Patents

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

A strain of Stenotrophomonas maltophilia and its application

technical field

The invention relates to a strain and its application, in particular to a Stenotrophomonas maltophilia strain and its application.

Background technique

my country is rich in coal resources, widely distributed and complete in varieties. Coal occupies a major position in my country's energy structure and plays a pivotal role in my country's economic development. Coal gangue is a solid waste with low carbon content and harder than coal produced in the process of coal mining and washing, accounting for about 15%-20% of the original coal output. At present, coal gangue is the largest industrial waste. Since it has not been substantively used, it is estimated that hundreds of millions of tons of coal gangue have been abandoned in various domestic coal-producing areas over the years. After the coal gangue piled up in the open air, after being washed by rain, the harmful substances will be gradually dissolved and flow into the soil, surface water or groundwater with the rain, causing serious pollution.

Coal gangue is rich in nutrients required for plant growth, such as organic matter, phosphorus, potassium, silicon, calcium, sulfur, etc. However, most of the phosphorus, potassium and other components in coal gangue exist in the form of insoluble minerals, which cannot be directly absorbed and utilized by plants. Due to various reasons and technical limitations, coal gangue has not been effectively used, resulting in idle resources and huge pollution to the environment. The research and development of coal gangue recycling technology is of great importance for developing new uses of coal gangue and protecting the environment and ecology. realistic meaning.

The Stenotrophomonas maltophilia of the present invention can be widely applied to various types of coal gangue, can effectively dissociate insoluble phosphorus, potassium, silicon, calcium, sulfur and other components in the coal gangue, and convert them into It becomes the nutrients that plants can absorb such as available phosphorus, available potassium, available silicon, exchangeable calcium, and available sulfur.

Experiments show that Stenotrophomonas maltophilia has a better phosphorus-resolving effect on coal gangue than the traditional phosphorus-resolving strain Bacillus megaterium. GZU-Stm01 is an excellent phosphorus-resolving bacteria.

The use of microbial bacteria to dissociate coal gangue to release the nutrients therein has the advantages of simple and reliable process, low cost, and environmental friendliness. It is a new scheme for green utilization of coal gangue. The gangue is dissociated by microbial bacteria, and the nutrients available to plants are released, and a microbial compound fertilizer with coal gangue as the basic raw material is created.

It can be seen that more new strains need to be further explored in the art for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Stenotrophomonas maltophilia strain and use thereof. The strain of the invention can dissociate phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, and the method has the advantages of simple method, good effect, low cost and environmental friendliness.

Technical solution of the present invention: a Stenotrophomonas maltophilia strain, belonging to the genus Stenotrophomonas maltophilia, named Stenotrophomonas maltophilia GZU-Stm01, and preserved in China on January 21, 2019 · Wuhan · Chinese Type Culture Collection Center of Wuhan University, the deposit number is CCTCC NO: M2019059.

The aforementioned Stenotrophomonas maltophilia strain, the Stenotrophomonas maltophilia GZU-Stm01, was isolated from weathered potash mines and nearby soils in potash mining areas.

Application of the aforementioned Stenotrophomonas maltophilia strain in preparing a product 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, the active ingredients of the product include or the aforementioned Stenotrophomonas maltophilia GZU-Stm01.

A preparation method of a product for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, using the aforementioned Stenotrophomonas maltophilia GZU-Stm01 as an active ingredient or active ingredient for preparing the product one.

A method for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, adding and/or using the aforementioned products during the dissociation.

An inoculum for dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue, the active components of the inoculum include or the aforementioned Stenotrophomonas maltophilia GZU-Stm01.

In the aforementioned inoculum, the inoculum also includes the conventional ingredients for preparing the inoculum.

Application of the aforementioned strain in dissociating phosphorus, potassium, silicon, calcium and/or sulfur in coal gangue.

The beneficial effects of the present invention

1. The present invention separates, purifies, and cultivates the Stenotrophomonas maltophilia GZU-Stm01 GZU-Stm01 from the fully weathered potash mine and the nearby soil in the potash mine area, which is a new strain and can It effectively decomposes insoluble phosphorus, potassium, silicon, calcium, sulfur and other components in coal gangue, and becomes a strain of nutrients such as available phosphorus, available potassium, available silicon, exchangeable calcium, and available sulfur as nutrients absorbed by plants. And the bacterial strain of the invention has good dissociation effect, can effectively dissociate various components in coal gangue, especially the dissociation of insoluble phosphorus in coal gangue, and has good effect, and the effect of dissociating phosphorus is better than that of traditional Bacillus megaterium (Bacillus megaterium).

2. The present invention adopts microbial dissociation, with simple operation and low cost.

3. The development and utilization of coal gangue resources can alleviate the environmental damage caused by coal gangue. The created microbial compound fertilizer can provide the effective phosphorus needed by plants. Introducing this strain into the soil can also avoid the long-term use of chemical fertilizers. Such as soil compaction and other problems, improve the utilization efficiency of phosphorus resources in the soil. Compared with the current application of chemical methods to treat coal gangue, the microbial dissociation method is more environmentally friendly.

Description of drawings

Fig. 1 is the growth curve of GZU-Stm01 strain of the present invention;

Fig. 2 is the appearance diagram of GZU-Stm01 bacterial strain colony of the present invention;

Fig. 3 is the morphological diagram of GZU-Stm01 cell of the present invention;

Figure 4 is a phylogenetic tree based on the partial 16S rRNA gene sequence of the GZU-Stm01 strain.

Detailed ways

The present invention will be further described below in conjunction with the examples, but not as a basis for limiting the present invention.

Embodiments of the present invention

Example:

The isolation of Stenotrophomonas maltophilia GZU-Stm01 includes the following steps:

1. Isolation and screening of functional strain GZU-Stm01 strain

1) Collection of samples

(1) The sample is collected from the fully weathered potassium mine and the nearby soil in a certain potassium mine area, select the fully weathered potassium mine sample, and put the collected sample into the collection bag;

(2) crush and sieve the collected samples, and collect 100 mesh samples;

(3) Store the samples in a 4°C refrigerator for later use.

2) Screening of functional strains

(1) Accurately weigh 1 g of the sample into a sterilized 250 mL conical flask, and add 99 mL of sterile water;

(2) Shake for 30min in a constant temperature shaker at 170r/min and 37°C;

(3) The control group was added with 100 mL of sterile water in a 250 mL conical flask;

(4) After shaking for 30min, take the supernatant and dilute to 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 .

(5) 100uL of each concentration was respectively applied to 3 phosphate-solubilizing bacteria screening solid medium, and the blank was a flat plate coated with sterile water;

(6) Place the plate in a 30°C constant temperature incubator for 3 to 4 days;

(7) streak and inoculate single colonies with different appearance characteristics on the solid culture of phosphate-solubilizing bacteria screening;

(8) After the strain grows, observe the purification situation of the strain through preliminary appearance and microscopic observation, and if the purification requirement is not met, repeat the process several times until a single strain is obtained, and different strains are distinguished at the same time;

(9) Gram-staining the purified strain;

(10) Cultivate the primary screened and purified strains to dissociate the coal gangue, and determine the effective phosphorus content of the strains to confirm the phosphorus-resolving effect of the strains, and screen out strains with good phosphorus-resolving effects.

2. Drawing of Growth Curves of Functional Strains

1) Preparation of seed solution. The strain was inoculated into LB liquid medium with an inoculation loop, and cultivated to logarithmic phase at 30°C.

2) Inoculation. Take 10 mL of the above seed solution and inoculate it into 200 mL of liquid LB medium, and after mixing, take 5 mL of the mixed solution and put it in 18 sterile test tubes marked on it.

3) Cultivate. The inoculated test tubes were cultured in a shaker at 37°C and 170 r/min, and were taken out for the corresponding incubation times.

4) Measurement. Use uninoculated LB liquid medium as a blank, and conduct photoelectric turbidimetry together with the sample to be tested at a wavelength of 600 nm.

The measurement results are shown in Figure 1. It can be seen from the figure that 0-10h is the lag phase, 10-30h is the logarithmic phase, 30h-45h is the stable phase, and after 45h is the decay phase.

3. Identification of functional strains

1) Colony appearance and morphology

As shown in Figure 2, it can be seen from the figure that the strain is light yellow on LB solid medium, opaque and round, slightly raised, with neat edges, smooth and moist surface, glossy, and the colony size is about 1mm.

2) Gram stain, cell morphology

As shown in Figure 3, it can be seen from the figure that the strains are Gram-negative bacilli, arranged singly or in pairs.

3) Strain identification

(1) Extraction of bacterial DNA

Pick a single colony of the purified strain into a conical flask equipped with LB liquid medium for cultivation, and after obtaining pure strain fermentation broth, use a bacterial genome extraction kit (spin column type, Tiangen Biochemical Technology Beijing Co., Ltd.) DNA was extracted and used as a template for polymerase chain reaction (PCR), 16S r RNA was amplified using Taq DNA polymer, 2F (5'-AGAGTTTGATCCTGGCTCAGGATGA-3'), 1492R (5'-TACGGCTACCTTGTTACGACTTAGC -3') universal primer for amplification, PCR amplification reaction system and amplification reaction conditions are shown in Table 1.

Table 1. PCR amplification reaction system and amplification reaction conditions

Prepare a 1.0% agarose gel with 1×TBE buffer, add 40 μL of PCR amplification product, add 8 μL of 2000bp DNA Marker, electrophoresis at 110V, current 70mA for 45min, and observe the gel imaging results.

(2) Homology alignment

The polymerase chain reaction amplification products were purified by gel recovery kit, sent to sequencing company (Shanghai Lifei Biotechnology Co., Ltd.) for sequencing, and the measured sequences were used on NCBI (National Center for Biotechnology Information) using Standard Nucleotide Blast was used for comparison, and ClustalW, MEGA5.05 and other software were used to perform phylogenetic analysis based on the Neighbor Joining Method, and a phylogenetic tree was constructed (Fig. 4) to obtain the classification information of bacterial species.

It can be seen from Figure 4 that GZU-Stm01 bacteria and Stenotrophomonas maltophilia are clustered on the same branch. Combined with physiological and biochemical characteristics and morphological observations, GZU-Stm01 was identified as Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia).

(3) Physiological and biochemical experiments

Through a series of biochemical experiments, with reference to "Berger's Bacterial Identification Manual" (8th Edition) and "Common Bacterial System Identification Manual" (Dong Xiuzhu et al., 1999), the strain was identified as Stenotrophomonas maltophilia.

4. Determination of the ability of the strain to dissociate coal gangue

1) The main components of coal gangue used are: C 11.02%, SiO ₂ 39.41%, Al ₂ O ₃ 17.01%, Fe ₂ O ₃ 16.02%, TiO ₂ 2.21%, CaO 5.76%, MgO 1.60%, K ₂ O 1.78% , Na ₂ O 1.47%, P ₂ O ₅ 1.61%, S 0.83%, H 0.71%, N 0.56%, ash 80.03%.

2) The four main factors affecting the dissociation of coal gangue are: the amount of bacteria dissociated, the dissociation time, the particle size of the gangue, and the pH value of the system. Aiming at the target coal gangue, firstly conduct single factor experiments of the above four factors of GZU-Stm01 and Bacillus megaterium to find the best single factor conditions for each bacteria, and then design orthogonal experiments to find the dissociation of GZU-Stm01 and Bacillus megaterium The best conditions for coal gangue.

3) For the composition of 1) coal gangue, GZU-Stm01 and Bacillus megaterium were used to conduct dissociation comparison experiments.

4) The dissociation experiment process is as follows: take the coal gangue that has been crushed with a specified mesh, and in a temperature-controlled agitator, the concentration of GZU-Stm01 and Bacillus megaterium bacteria liquid is controlled at: 6.0×10 ¹⁶ -6.5×10 ¹⁶ cfu/ mL, the dosage ratio of the two strains of bacteria and coal gangue is 1:1, under continuous stirring, spray the bacterial liquid, stir evenly, turn it every 2-3 hours, control the temperature not higher than 30 ℃, dissociate the coal Gangue for several days.

5) Measure the content of each index of coal gangue after dissociation, and the results are shown in Table 2. It can be seen from Table 2 that the overall effect of GZU-Stm01 in dissociating coal gangue is better than that of Bacillus megaterium, and the main indicators such as available phosphorus and available potassium are better than those of Bacillus megaterium.

Table 2 Experimental results of orthogonal design

SEQUENCE LISTING

<110> Guizhou University China National Tobacco Corporation Guizhou Branch

<120> A strain of Stenotrophomonas maltophilia and its application

<130> 2019

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1425

<212> DNA

<213> Stenotrophomonas 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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