Coal desulfurization genetically modified microbial composite inoculant and application thereof

Abstract

The application provides a coal desulfurization genetically modified microbial composite inoculant and application thereof, and belongs to the technical field of microorganisms.The preservation number of the Pseudomonas putida-coal-09 is CGMCC No.35656.The microbial composite inoculant comprises the Pseudomonas putida-coal-09 and the Ferrooxidation Bacteria.The experiment shows that the desulfurization rate of the Pseudomonas putida-coal-09 to fine coal samples reaches 79.49%, the desulfurization rate of the Pseudomonas putida-coal-09 to coarse coal reaches 66.49%, and the desulfurization rate of the microbial composite inoculant to fine coal samples reaches 84.55%.In conclusion, the Pseudomonas putida-coal-09 and the microbial composite inoculant can efficiently remove sulfur in coal, and lay a foundation for the application of the microbial desulfurization technology.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a genetically modified coal desulfurization microbial compound agent and its application. Background Technology

[0002] Coal is the world's most important fossil fuel, but the sulfur in it transforms into pollutants such as SO2 and SO3 during combustion, leading to acid rain and posing a serious threat to the ecological environment and human health. Countries around the world have successively introduced strict sulfur emission control policies to promote the development of coal desulfurization technologies.

[0003] Currently, traditional desulfurization technologies include physical and chemical methods. Physical desulfurization utilizes the differences in density and surface properties between sulfur minerals and coal for separation, but it can only remove inorganic sulfur such as pyrite and is ineffective against organic sulfur, with a desulfurization rate typically <50%. Chemical desulfurization uses strong acids / alkalis or high-temperature oxidation to decompose sulfur compounds, removing some organic sulfur and achieving a desulfurization rate of 70%~90%. However, it suffers from problems such as high cost (large acid and alkali consumption, high energy consumption), secondary pollution (difficult wastewater and exhaust gas treatment), and coal quality damage (high temperature leads to a decrease in calorific value).

[0004] Microbial coal desulfurization is a novel desulfurization method with significant advantages such as low energy consumption, low cost, strong environmental friendliness, less secondary pollution, and the ability to remove both organic and inorganic sulfur. However, the desulfurization effect is directly related to the performance of the microbial strain. Therefore, developing a microbial strain with high desulfurization efficiency is the primary problem to be solved in the application of microbial desulfurization technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a *Pseudomonas putida*-coal-09 strain capable of efficiently removing sulfur from coal.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a *Pseudomonas putida* ( Pseudomonas putida The *Pseudomonas putida*-coal-09 has the accession number CGMCC No. 35656.

[0008] This invention provides a method for improving the coal desulfurization effect of bacterial strains based on genetic modification, comprising:

[0009] Using *Desulfovibrio* genomic DNA as a template, the *sox* gene cluster was amplified to obtain the target fragment. The primers for amplifying the *sox* gene cluster included an upstream F primer and a downstream R primer. The nucleotide sequence of the upstream F primer is shown in SEQ ID. 4. The nucleotide sequence of the downstream R primer is shown in SEQ ID NO. 5.

[0010] The target fragment is ligated into a backbone vector to obtain a recombinant plasmid;

[0011] The recombinant plasmid was transformed into a *Pseudomonas putida* strain to obtain a recombinant strain; the recombinant strain included *Pseudomonas putida*-coal-09.

[0012] This invention provides a microbial preparation comprising the above-described Pseudomonas putida-coal-09.

[0013] This invention provides a method for preparing the microbial preparation described in the above technical solution, comprising:

[0014] Microbial preparations were obtained by culturing *Pseudomonas putida*-coal-09 in a culture medium.

[0015] Preferably, the culture temperature is 15℃~40℃; and the pH value of the culture medium is 4.0~9.0.

[0016] This invention provides a microbial compound agent, comprising *Pseudomonas putida*-coal-09 and *Thiobacillus ferrooxidans* as described in the above technical solution.

[0017] This invention provides the application of the above-described Pseudomonas malodorans-coal-09, the above-described microbial preparation, the above-described microbial preparation prepared by the above-described preparation method, or the above-described microbial compound agent in coal desulfurization.

[0018] This invention provides a method for coal desulfurization, characterized by comprising:

[0019] Coal is mixed with a microbial agent containing *Pseudomonas malodorans*-coal-09 as described in the above technical solution, or a microbial compound agent containing the above technical solution, for coal desulfurization.

[0020] Preferably, the temperature for coal desulfurization is 15℃~40℃.

[0021] Preferably, the mass-to-volume ratio of coal to the microbial preparation or the microbial compound agent is (20~400) g: (30~80) mL.

[0022] Preferably, the particle size of the coal is ≤3mm.

[0023] The beneficial effects of this invention are:

[0024] This invention provides a *Pseudomonas putida* ( PseudomonasThe *Pseudomonas putida*-coal-09 strain, with accession number CGMCC No. 35656, was isolated from coal samples in this invention. After genetic modification, *Pseudomonas putida*-coal-09 was obtained. This strain exhibits high desulfurization efficiency and good temperature and pH adaptability. The results of the embodiments of this invention show that *Pseudomonas putida*-coal-09 achieves a desulfurization rate of 79.49% when treating fine coal samples that have passed through a 120-mesh sieve. When treating coarse coal with a diameter of 1-3 mm, *Pseudomonas putida*-coal-09 achieves a desulfurization rate of 66.49%. In summary, the *Pseudomonas putida*-coal-09 provided by this invention can efficiently remove sulfur from coal, laying the foundation for the application of microbial desulfurization technology.

[0025] This invention provides a microbial compound inoculant, including *Pseudomonas putida* (…). Pseudomonas The microbial compound agent contains *C. coal-09* and *Thiobacillus ferrooxidans*. This microbial compound agent exhibits highly efficient desulfurization, significantly improving desulfurization efficiency. The results of the embodiments of this invention demonstrate that using this microbial compound agent, compared to using a single agent, can more efficiently improve the desulfurization effect of coal, achieving a desulfurization rate of over 84%. In summary, the microbial compound agent provided by this invention can efficiently remove sulfur from coal.

[0026] Preservation Certificate

[0027] Pseudomonas putida-coal-09, classified and named as follows: Pseudomonas putida It was deposited on August 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35656. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a single colony plate image of strain MY-09;

[0030] Figure 2 This is a graph showing the coal desulfurization results of the strains screened in Example 2;

[0031] Figure 3 This is an optical microscope image of strain MY-09;

[0032] Figure 4 The growth curve of *Pseudomonas putida* MY-09;

[0033] Figure 5 The growth curves of *Pseudomonas putida* MY-09 under different pH conditions are shown.

[0034] Figure 6 The growth curves of *Pseudomonas putida* MY-09 under different culture temperature conditions;

[0035] Figure 7 The graph shows the sulfur content of residual coal in each treatment group in Example 5.

[0036] Figure 8 The graph shows the desulfurization rate results for each treatment group in Example 5;

[0037] Figure 9 The graph shows the sulfur content of residual coal in each treatment group in Example 6;

[0038] Figure 10 The graph shows the desulfurization rate results for each treatment group in Example 6;

[0039] Figure 11 The graph shows the sulfur content of residual coal in each treatment group in Example 7.

[0040] Figure 12 The graph shows the desulfurization rate results for each treatment group in Example 7;

[0041] Figure 13 Figure 8 shows the results of residual coal sulfur content in each treatment group in Example 8;

[0042] Figure 14 The graph shows the desulfurization rate results for each treatment group in Example 8;

[0043] Figure 15 The graph shows the sulfur content of residual coal in each treatment group in Comparative Example 2.

[0044] Figure 16 The graph shows the desulfurization rate results for each treatment group in Comparative Example 2.

[0045] Figure 17 The graph shows the sulfur content of residual coal in each treatment group of Comparative Example 3.

[0046] Figure 18 The graph shows the desulfurization rate results for each treatment group in Comparative Example 3. Detailed Implementation

[0047] This invention provides a *Pseudomonas putida* ( Pseudomonas putida The Pseudomonas 1,000-coal-09, has the accession number CGMCC No. 35656.

[0048] The *Pseudomonas putida*-coal-09 described in this invention is derived from *Pseudomonas putida* MY-09 isolated from the environment and obtained through genetic modification. The *Pseudomonas putida*-coal-09 exhibits a significantly improved coal desulfurization efficiency compared to *Pseudomonas putida* MY-09, increasing from 40.34% to 79.49%. The *Pseudomonas putida*-coal-09 is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 35656.

[0049] The *Pseudomonas putida* MY-09 strain described in this invention is a strain isolated and purified from coal samples. In this invention, the nucleotide sequences of the 16S rDNA of *Pseudomonas putida* MY-09 and *Pseudomonas putida*-coal-09 are shown in SEQ ID NO. 3. *Pseudomonas putida* MY-09 and *Pseudomonas putida*-coal-09 are Gram-negative bacteria, short rod-shaped, approximately 0.7-1.1 μm × 2.0-4.0 μm in size. *Pseudomonas putida* MY-09 and *Pseudomonas putida*-coal-09 are cultured on NB medium. The lag phase is approximately 0-2 hours, the logarithmic phase is approximately 2-10 hours, and the stationary phase is 10-30 hours. After 30 hours of culture, they begin to die due to nutrient depletion. The *Pseudomonas putida* MY-09 and *Pseudomonas putida*-coal-09 can adapt to a pH range of 4.0–9.0, with an optimal pH range of 6.5–7.5. The *Pseudomonas putida* MY-09 and *Pseudomonas putida*-coal-09 can adapt to a temperature range of 15–40°C, with an optimal temperature range of 25–35°C.

[0050] This invention provides a method for improving the coal desulfurization effect of bacterial strains based on genetic modification, comprising:

[0051] Using *Desulfovibrio* genomic DNA as a template, the *sox* gene cluster was amplified to obtain the target fragment. The primers for amplifying the *sox* gene cluster included an upstream F primer and a downstream R primer. The nucleotide sequence of the upstream F primer is shown in SEQ ID. 4. The nucleotide sequence of the downstream R primer is shown in SEQ ID NO. 5.

[0052] The target fragment is ligated into a backbone vector to obtain a recombinant plasmid;

[0053] The recombinant plasmid was transformed into a *Pseudomonas putida* strain to obtain a recombinant strain; the recombinant strain included *Pseudomonas putida*-coal-09.

[0054] This invention uses *Vibrio desulfurans* genomic DNA as a template to amplify the *sox* gene cluster and obtain the target fragment. The source of the *Vibrio desulfurans* genomic DNA is not particularly limited; any conventional *Vibrio desulfurans* genomic DNA in the art can be used. This embodiment describes the preparation of recombinant strains using *Vibrio desulfurans* genomic DNA purchased from Beina Chuanglian Biotechnology Co., Ltd. (BNCC number 372827). As an optional implementation of this invention, the primers for amplifying the *sox* gene cluster include an upstream F primer and a downstream R primer; the nucleotide sequence of the upstream F primer is shown in SEQ ID 4; the nucleotide sequence of the downstream R primer is shown in SEQ ID NO. 5. The target fragment amplified using the upstream F primer and downstream R primer contains EcoRI / HindIII restriction sites, facilitating subsequent preparation of recombinant plasmids and recombinant vectors. This invention does not particularly limit the amplification method; any conventional amplification method in the art that can amplify the target fragment is acceptable.

[0055] After obtaining the target fragment, the present invention ligates the target fragment into a backbone vector to obtain a recombinant plasmid. As an optional embodiment of the present invention, the backbone vector includes the pBBR1MCS-5 plasmid. The present invention does not specifically limit the ligation method; any conventional ligation method in the art can be used.

[0056] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into a *Pseudomonas putida* strain to obtain a recombinant strain. The present invention does not have a specific limitation on the source of the *Pseudomonas putida* strain; any conventional *Pseudomonas putida* strain in the art can be used. As an optional embodiment of the present invention, the *Pseudomonas putida* strain can be *Pseudomonas putida* MY-09. The present invention does not have a specific limitation on the transformation method; any conventional transformation method in the art can be used. As an optional embodiment of the present invention, the transformation method can be electroporation. As an optional embodiment of the present invention, the recombinant strain includes *Pseudomonas putida*-coal-09. The *Pseudomonas putida*-coal-09 prepared by the method described above significantly improves the coal desulfurization effect of *Pseudomonas putida*.

[0057] This invention provides a microbial preparation comprising the above-described Pseudomonas putida-coal-09.

[0058] The present invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: culturing *Pseudomonas putida*-coal-09 in a culture medium to obtain the microbial preparation.

[0059] This invention does not specifically limit the type of culture medium; any conventional culture medium in the art can be used. As an optional embodiment of this invention, the culture medium can be LB medium or NB medium. As an optional embodiment of this invention, the culture temperature can be 15℃~40℃, or 15℃, 20℃, 25℃, 30℃, 35℃, or 40℃; the pH value of the culture medium can be 4.0~9.0, or 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. As an optional embodiment of this invention, the culture process can be accompanied by oscillation; the oscillation speed can be 120~220 r / min, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 r / min.

[0060] This invention provides a microbial compound agent comprising *Pseudomonas putida* and *Thiobacillus ferrooxidans* as described in the above-mentioned technical solution. This invention does not specifically limit the source of the *Thiobacillus ferrooxidans*; any conventional *Thiobacillus ferrooxidans* in the art can be used. This invention does not specifically limit the mixing ratio of the two bacteria; any conventional mixing ratio in the art can be used. As an optional embodiment of this invention, the preparation method of the microbial compound agent can be: mixing *Pseudomonas putida*-coal-09 culture medium and *Thiobacillus ferrooxidans* culture medium to obtain the microbial compound agent. This invention does not specifically limit the preparation method of the *Pseudomonas putida*-coal-09 culture medium; any conventional culture method in the art that enables *Pseudomonas putida*-coal-09 to grow normally can be used. This invention does not specifically limit the preparation method of the *Thiobacillus ferrooxidans* culture medium; any conventional culture method in the art that enables *Thiobacillus ferrooxidans* to grow normally can be used. As an optional embodiment of this invention, the OD of the *Pseudomonas putida*-coal-09 culture medium... 600 The value can be 1.0; the OD600 value of the *Thiobacillus ferrooxidans* culture medium can be 1.5. After obtaining *Pseudomonas putida*-coal-09 culture medium and *Thiobacillus ferrooxidans* culture medium, the present invention mixes the *Pseudomonas putida*-coal-09 culture medium and the *Thiobacillus ferrooxidans* culture medium. As an optional embodiment of the present invention, the volume ratio of the mixed *Pseudomonas putida*-coal-09 culture medium and the *Thiobacillus ferrooxidans* culture medium can be (1~1.35):(1~5), or it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1.35:1 or 1.25:1. The present invention can directly use the mixed system of *Pseudomonas putida*-coal-09 culture medium and *Thiobacillus ferrooxidans* culture medium as a microbial compound inoculant.

[0061] This invention utilizes the aforementioned microbial composite agent to more significantly desulfurize coal. Compared to using only a single agent for coal desulfurization, the use of this microbial composite agent significantly improves the desulfurization effect.

[0062] This invention provides the application of *Pseudomonas putida*-coal-09, the microbial preparation described in the above-mentioned technical solutions, the microbial preparation obtained by the preparation method described in the above-mentioned technical solutions, or the microbial compound agent described in the above-mentioned technical solutions in coal desulfurization. *Pseudomonas putida*-coal-09 or a microbial preparation containing *Pseudomonas putida*-coal-09 of this invention exhibits good coal desulfurization effects, with desulfurization rates reaching 66% to 80%. The results of the embodiments of this invention show that *Pseudomonas putida*-coal-09 achieves a desulfurization rate of 79.49% when treating fine coal samples passing through a 120-mesh sieve; and a desulfurization rate of 66.49% when treating coarse coal of 1-3 mm. The *Pseudomonas putida*-coal-09 provided by this invention can be used for coal desulfurization, and the desulfurization effect is good, comparable to that of chemical desulfurization methods. The present invention combines the aforementioned *Pseudomonas putida*-coal-09 with *Thiobacillus ferrooxidans* to obtain a microbial composite agent, which has a more efficient coal desulfurization effect, with a desulfurization rate of 84.55%.

[0063] The present invention provides a method for coal desulfurization, comprising: mixing coal with a microbial preparation containing the aforementioned *Pseudomonas malodorans*-coal-09 or a microbial compound preparation containing the aforementioned microbial preparation, and performing coal desulfurization.

[0064] As an optional embodiment of the present invention, the microbial preparation containing *Pseudomonas putida*-coal-09 can be a culture medium of *Pseudomonas putida*-coal-09; the OD of the culture medium of *Pseudomonas putida*-coal-09... 600The value can be 1.5~2.5, or 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5. In this invention, the method for preparing the culture medium of *Pseudomonas putida*-coal-09 includes: culturing *Pseudomonas putida*-coal-09 in a culture medium to obtain a culture medium of *Pseudomonas putida*-coal-09. When culturing *Pseudomonas putida*-coal-09 in the culture medium, the inoculum amount of *Pseudomonas putida*-coal-09 is 2% (v / v) of the culture medium volume. The culture medium can be NB medium or LB medium. The culture temperature can be 15℃~40℃, or 15℃, 20℃, 25℃, 30℃, 35℃, or 40℃. The culture process can be accompanied by shaking; the shaking speed can be 180 r / min. The incubation time can be 8-24 hours, or even 12 hours. After incubation, a culture medium of *Pseudomonas putida*-coal-09 is obtained. In this invention, the composition and preparation method of the microbial compound agent have been specifically described in the above scheme and will not be repeated here.

[0065] After obtaining the microbial preparation containing *Pseudomonas putida*-coal-09 or the microbial compound agent, the present invention mixes coal with the microbial preparation containing *Pseudomonas putida*-coal-09 or the microbial compound agent. In the present invention, the particle size of the coal is ≤3mm, or can be 1~3mm, or ≤120 mesh; the mass-to-volume ratio of the coal to the microbial preparation containing *Pseudomonas putida*-coal-09 or the microbial compound agent can be (20~400)g:(30~80)mL, or 20g:30mL, 20g:40mL, 20g:50mL, 20g:60mL, 20g:70mL, 20g:80mL, or 250g:50mL. As an optional embodiment of the present invention, when using a microbial preparation containing *Pseudomonas putida*-coal-09 or the microbial compound agent for coal desulfurization, Tween 80 can also be added to the desulfurization system; the amount of Tween 80 added can be 0.2%~0.8%, or 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, and 0.8%. Adding Tween 80 to the coal desulfurization system allows for better binding of the microbial agent with the coal, thereby improving the coal desulfurization effect.

[0066] As an optional embodiment of the present invention, the temperature for coal desulfurization can be 15℃~50℃, or 15, 20, 25, 30, 35, 40, 45 or 50℃; the time for coal desulfurization can be 3~30 days, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days; the coal desulfurization process is accompanied by oscillation; the rotation speed of the oscillation can be 120~220 r / min, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210 or 220 r / min.

[0067] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1: Isolation and purification of bacteria from coal samples

[0069] (1) Weigh 10g of each of the 7 coal samples from the Yellow River Coal Mine in Wuhai, place them in 50mL of sterile distilled water, shake to mix, and soak for 3 days.

[0070] (2) Preparation of culture media: LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH adjusted to 7.0, sterilized at 121℃ for 15 min. LB solid medium: 15 g / L agar powder added to LB liquid medium.

[0071] (3) Take 1 mL of the extract from (1) and inoculate it into 20 mL of LB liquid medium. Incubate at 37°C and 180 r / min for 24 h.

[0072] (4) Take 50µL of culture medium and streak and spread it on an LB solid plate, and incubate it upside down at 37℃ for 24h.

[0073] (5) Pick a single colony and incubate it in 2 mL of LB liquid medium at 37°C and 200 r / min for 4 h. Then, take 0.5 mL of the colony and inoculate it in 20 mL of LB liquid medium at 37°C and 180 r / min for 24 h.

[0074] (6) Repeat the streaking and spreading process multiple times until all colonies on the plate have the same morphology, thus obtaining a single pure bacterial strain. Mix the bacterial solution with 50% glycerol solution at a volume ratio of 1:1 and store it in a -20℃ refrigerator for later use. Pick a single colony for Gram staining and observation.

[0075] Results: A total of 15 single colonies were isolated and purified, and 10 mL of each colony was stored for later use. Figure 1This is a single colony plate image of strain number MY-09.

[0076] Example 2: Screening and Identification of Desulfurization Strains

[0077] 1. Experimental procedure:

[0078] (1) Take 1 mL of the purified bacterial culture and inoculate it into a 250 mL Erlenmeyer flask containing 60 mL of LB liquid medium. Incubate at 30 °C and 180 r / min for 24 h with constant temperature shaking.

[0079] (2) Take 30g of coal sample that has passed through an 80-mesh sieve, add it to the above bacterial solution and shake well, and continue to treat it at 30℃ and 180r / min for 48h.

[0080] (3) The bacterial solution was filtered, and the remaining coal sample was collected after washing and drying. The sulfur content was measured by an automatic sulfur analyzer, and the desulfurization rate was calculated. The strain with the highest desulfurization rate was selected as the experimental strain for subsequent treatment.

[0081] (4) 500 μL of the strain with obvious desulfurization function was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing identification. The primer sequences were 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO.1); 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2).

[0082] (5) Gram staining was performed on the selected strains for observation. The steps are as follows:

[0083] The smears were fixed by flame, and the bacterial cultures were routinely smeared, dried, and fixed.

[0084] Primary staining: Add crystal violet and stain for 1-2 minutes, then wash with water.

[0085] Mordant: Rinse off residual water with iodine solution, cover with iodine solution for about 1 minute, then wash with water.

[0086] Decolorization: Use filter paper to absorb the residual water on the slide, tilt the slide, and add 95% ethanol with a dropper against a white background to decolorize until the ethanol that flows out is no longer purple. Then wash with water immediately.

[0087] Counterstain: Counterstain with safranin solution for about 2 minutes, then rinse with water.

[0088] Microscopic examination: After drying, observe under an optical microscope with an oil immersion lens. If the bacteria are stained blue-purple, they are Gram-positive bacteria; if they are stained red, they are Gram-negative bacteria.

[0089] 2. Experimental Results:

[0090] (1) A strain with significant desulfurization function was screened, experimental number MY-09, which could reduce the sulfur content of raw coal crushed to ≤80 mesh from 1.81% to 1.08%, with a desulfurization rate of 40.34%. The desulfurization rates of each strain are as follows: Figure 2 As shown.

[0091] (2) The 16S rDNA nucleotide sequence of strain MY-09 is shown in SEQ ID NO.3, specifically as follows:

[0092]

[0093] The 16S rDNA sequencing results of strain MY-09 were compared with those obtained by BLAST on NCBI. Pseudomonas putida With a sequence homology of 99.29%, strain MY-09 was identified as *Pseudomonas putida*. Pseudomonas putida The strain was recorded as *Pseudomonas putida* MY-09.

[0094] (3) The observation results of strain MY-09 are as follows: Figure 3 As shown. After Gram staining, the cells were observed under a light microscope. They appeared red or pink, indicating they are Gram-negative bacteria, short rod-shaped, approximately (0.7~1.1) μm (width) × (2.0~4.0) μm (length). Figure 3 As shown.

[0095] Example 3: Determination of growth characteristics of *Pseudomonas putida* MY-09

[0096] 1. Growth curve determination

[0097] Experimental procedure:

[0098] (1) Take 1 mL of the preserved bacterial culture and inoculate it into 50 mL of NB liquid culture medium. Incubate at 25℃ and 180 r / min for 15 h as seed culture.

[0099] (2) Take 10 mL of seed culture and transfer it into an Erlenmeyer flask containing 400 mL of NB liquid culture medium, and mix well.

[0100] (3) Take 10 mL of the mixture and put it into 12 sterile Erlenmeyer flasks. Incubate at 25°C and 180 r / min with constant temperature shaking.

[0101] (4) Take out the Erlenmeyer flasks at the corresponding times of 2, 4, 6, 8, 12, 24, 26, 32, 48, 60, 72 and 96 hours of cultivation and put them into the refrigerator for storage. Measure their absorbance together after the experiment.

[0102] (5) Using uninoculated NB liquid culture medium as a blank control, the absorbance of the culture medium at different times was measured in chronological order at a wavelength of 600 nm. Growth curves were plotted using absorbance data, as shown below. Figure 4 As shown. Figure 4 This is a growth curve of *Pseudomonas putida* MY-09.

[0103] Figure 4 The results showed that the lag phase of *Pseudomonas putida* MY-09 was about 0-2 hours, the log phase was about 2-10 hours, the stationary phase was 10-30 hours, and it began to die after 30 hours of culture due to the depletion of nutrients.

[0104] 2. Suitable for pH range measurement

[0105] Experimental procedure:

[0106] (1) Take 1 mL of the preserved bacterial culture and inoculate it into 50 mL of NB liquid culture medium. Incubate at 25℃ and 180 r / min for 15 h as seed culture.

[0107] (2) Take 13 sterilized Erlenmeyer flasks containing 50 mL of NB culture medium and adjust their pH to 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 respectively.

[0108] (3) Take 1 mL of seed liquid and inoculate it into 11 culture media in (2), and culture at 25℃ and 180 r / min for 48 h with constant temperature shaking.

[0109] (4) Using uninoculated NB liquid culture medium as a blank control, the absorbance of the above-mentioned culture solutions at different pH values ​​was measured at a wavelength of 600 nm. Growth curves were plotted based on the absorbance values, and the results are as follows: Figure 5 As shown. Figure 5 This is a growth curve of *Pseudomonas putida* MY-09 under different pH conditions.

[0110] Depend on Figure 5 It can be concluded that the optimal pH for *Pseudomonas putida* MY-09 is between 6.5 and 7.5, and its adaptability range is 4.0 to 9.0, demonstrating good pH adaptability.

[0111] 3. Temperature range measurement

[0112] Experimental procedure:

[0113] (1) Take 1 mL of the preserved bacterial culture and inoculate it into 50 mL of NB liquid culture medium. Incubate at 25℃ and 180 r / min for 15 h as seed culture.

[0114] (2) Take 9 sterilized Erlenmeyer flasks containing 50 mL of NB medium and inoculate each with 1 mL of seed liquid.

[0115] (3) The samples were cultured at 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃ and 50℃, and 180r / min for 48 hours respectively.

[0116] (4) Using uninoculated NB liquid culture medium as a blank control, the absorbance of the culture solutions at different temperatures was measured at a wavelength of 600 nm. Growth curves were plotted based on the absorbance values, as shown below. Figure 6 As shown. Figure 6 This is a growth curve of *Pseudomonas putida* MY-09 under different culture temperature conditions.

[0117] Depend on Figure 6 It can be concluded that the optimal temperature for *Pseudomonas putida* MY-09 is between 25℃ and 35℃, and its adaptability temperature range is 15℃ to 40℃, indicating good temperature adaptability.

[0118] Example 4: Genetic modification of *Pseudomonas putida* MY-09

[0119] Experimental procedure:

[0120] (1) Cloning of the target gene (sox gene cluster)

[0121] Primer design: Based on the sox gene cluster (containing soxX, soxY, soxZ, and soxA, with a total length of approximately 4.2 kb) sequence of *Desulfovibrio vulgaris*, primers with restriction enzyme sites (EcoRⅠ / HindⅢ) were designed:

[0122] Upstream primer: 5'-CCGGAATTCATGAGTGCGATCGTCG-3' (containing EcoRI site), (SEQ ID NO.4);

[0123] Downstream primer: 5'-CCCAAGCTTTCAGCGGCTGATGTCG-3' (containing HindⅢ site), (SEQ ID NO.5);

[0124] PCR amplification: Using Desulfovibrio genomic DNA (purchased from Beina Chuanglian Biotechnology Co., Ltd., BNCC number: 372827) as a template, the sox gene cluster was amplified. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 4 min, for 30 cycles; and 72℃ final extension for 10 min.

[0125] Product validation: 1% agarose gel electrophoresis was used to detect and recover the target fragment of approximately 4.2 kb.

[0126] (2) Construction of recombinant expression vector

[0127] Double digestion: The target gene fragment and pBBR1MCS-5 plasmid (purchased from Shanghai Sangon Biotech Co., Ltd.) were digested with EcoRI and HindIII, respectively. The digestion conditions were 37℃ for 2 hours, and the digestion products were recovered by electrophoresis.

[0128] Ligation reaction: Mix the target gene and vector at a molar ratio of 3:1, add T4 DNA ligase, ligate overnight at 16℃, and construct the recombinant plasmid pBBR1-sox.

[0129] Transformation and Validation: The recombinant plasmid was transformed into E. coli DH5α (competent cells), plated on LB agar plates containing kanamycin, and incubated at 37°C for 16 h. Single colonies were picked, plasmids were extracted, and verified by double enzyme digestion and sequencing to confirm the correct insertion of the sox gene cluster.

[0130] (3) Transformation of Pseudomonas putida with recombinant plasmids

[0131] Preparation of competent cells: Pseudomonas putida MY09 was inoculated into LB medium and cultured in a shake flask at 30°C until OD600 = 0.6~0.8. After incubation on ice for 30 min, the cells were centrifuged at 4°C and 5000×g for 10 min. The cells were washed three times with pre-cooled 10% glycerol and resuspended in a small amount of 10% glycerol (competent cells).

[0132] Electroporation: Take 100 μL of competent cells + 1 μg of recombinant plasmid pBBR1-sox, add it to an electroporation cuvette (2 mm gap), set the parameters: 1.8 kV, 25 μF, 200 Ω, add 1 mL of LB medium immediately after electroporation, and incubate at 30 °C for 1 h.

[0133] (4) Screening positive clones: Spread the revived bacterial solution on LB plates containing kanamycin (30 μg / mL), incubate at 30℃ for 24h, pick single colonies, and verify the colony PCR using the primers in step (1) to obtain engineered bacteria, named Pseudomonas putida-coal-09, and preserve it with the preservation number CGMCC No.35656.

[0134] (5) The growth characteristics of Pseudomonas putida-coal-09 were determined using the method in Example 3. The growth curve, adaptive pH and adaptive temperature of Pseudomonas putida-coal-09 were consistent with those of the original strain.

[0135] Example 5: Fine coal desulfurization via Pseudomonas putida-coal-09 leaching method

[0136] Experimental procedure:

[0137] (1) The *Pseudomonas putida*-coal-09 bacterial suspension was inoculated into 400 mL of NB liquid medium at an inoculation rate of 2% by volume, and cultured overnight at 25°C and 180 r / min to obtain OD. 600 A culture medium of *Pseudomonas putida*-coal-09 with a value of 1.0.

[0138] (2) Take 6 sterilized 250mL Erlenmeyer flasks, add 20g of 120-mesh coal sample and 40mL of overnight culture solution, and seal them.

[0139] (3) Take a sterile 250mL Erlenmeyer flask, add 20g of 120-mesh coal sample and 40mL of NB liquid culture medium, label it as the control group, and seal it.

[0140] (4) The above Erlenmeyer flasks were subjected to constant temperature shaking leaching at 25℃ and 180r / min for 1, 2, 3, 4, 5, and 6 days respectively. Coal samples from the Erlenmeyer flasks were taken out every 24 hours for filtration and drying. The sulfur content of the residual coal was determined, and the desulfurization rate was calculated. The results are as follows: Figures 7-8 As shown. Figure 7 Figure 1 shows the sulfur content of residual coal in each treatment group. Figure 8 The graph shows the desulfurization rate results for each treatment group.

[0141] Figures 7-8 The results showed that the sulfur content in the coal sample decreased with the treatment time. On the sixth day, the sulfur content decreased from 1.81% to 0.37%, the desulfurization amount was 1.44%, and the desulfurization rate was 79.49%.

[0142] Example 6: Desulfurization of crude coal by leaching with *Pseudomonas putida*-coal-09

[0143] Experimental procedure:

[0144] (1) The bacterial suspension of *Pseudomonas putida*-coal-09 was inoculated into 400 mL of NB liquid medium at an inoculation rate of 2% by volume, and cultured overnight at 25°C and 180 r / min to obtain OD. 600 A culture medium of *Pseudomonas putida*-coal-09 with a value of 1.0.

[0145] (2) Take 6 sterilized 250mL Erlenmeyer flasks, add 20g of 1-3mm coal sample and 40mL of overnight culture solution, and seal them.

[0146] (3) Take a sterile 250mL Erlenmeyer flask, add 20g of 1~3mm coal sample and 40mL of NB liquid culture medium, mark it as the control group, and seal it.

[0147] (4) The above Erlenmeyer flasks were subjected to constant temperature shaking leaching at 25℃ and 180r / min for 1, 2, 3, 4, 5, and 6 days respectively. Coal samples from the Erlenmeyer flasks were taken out every 24 hours for filtration and drying. The sulfur content of the residual coal was determined, and the desulfurization rate was calculated. The results are as follows: Figures 9-10 As shown. Figure 9 Figure 1 shows the sulfur content of residual coal in each treatment group. Figure 10 The graph shows the desulfurization rate results for each treatment group.

[0148] Figures 9-10The results showed that when 1-3 mm coarse coal was treated with Pseudomonas putida-coal-09 for six days, the sulfur content in the coarse coal decreased from 1.81% to 0.61%, the desulfurization amount was 1.20%, and the desulfurization rate was 66.49%.

[0149] Example 7: Desulfurization method using a combination of *Pseudomonas putida*-coal-09, *Thiobacillus ferrooxidans*, and Tween 80.

[0150] (1) Pseudomonas putida-coal-09 was inoculated into 400 mL of NB liquid medium at a volume ratio of 2% and cultured at 25°C and 180 r / min until OD. 600 The value was 1.0, resulting in Pseudomonas-coal-09 culture medium; *Thiobacillus ferrooxidans* (DSM14882) was cultured to OD... 600 The value was 1.5, and the culture medium of *Thiobacillus ferrooxidans* was obtained.

[0151] (2) Mix the culture medium of Pseudomonas putida and the culture medium of Thiobacillus ferrooxidans in a volume ratio of 1.35:1.0 to form an engineered bacterial solution, which can also be called a microbial compound bacterial agent.

[0152] (3) Take 6 Erlenmeyer flasks, add 20g of coal sample that has passed through a 120-mesh sieve, 60mL of engineered bacterial solution, and 2% Tween 80 to each flask, place them in a shaker, and incubate them at 25℃ and 180r / min for 6 days. During this period, filter and clean them once a day, dry them, determine the sulfur content, and calculate the desulfurization rate.

[0153] Figures 11-12 The results showed that on the sixth day of treatment with the compound microbial agent, the sulfur content in the coal decreased from 2.21% to 0.34%, the desulfurization amount was 1.87%, and the desulfurization rate was 84.55%.

[0154] Example 8: Microbial compound inoculant simulates natural conditions for desulfurization

[0155] (1) Prepare 500 ml of microbial compound inoculant according to the steps (1) and (2) in Example 7.

[0156] (2) Take 5 catties of coal sample and place it in a 30cm×40cm×15cm turnover box. The particle size of the coal sample is ≤3mm. The coal sample is raw coal purchased from the market and has not been crushed.

[0157] (3) Spray 500 ml of the prepared microbial agent into 5 catties of coal sample.

[0158] (4) The coal sample is turned over every 48 hours. The temperature for coal desulfurization is 15~35℃. The humidity is kept constant during the coal desulfurization process, that is, the humidity during the coal desulfurization process is the same as the initial humidity.

[0159] (5) Take a portion of coal samples every day for filtration and cleaning, dry them, determine the sulfur content, and calculate the desulfurization rate.

[0160] Figures 13-14 The results showed that on the sixth day of treatment with the compound microbial agent, the sulfur content in the coal decreased from 3.25% to 0.49%, the desulfurization amount was 2.75%, and the desulfurization rate was 84.82%.

[0161] Comparative Example 1

[0162] A chemical desulfurization method, the steps of which are as follows:

[0163] (1) Weigh 1.0g of coal sample that has passed through a 120-mesh sieve and moisten it with 1-2mL of 95% ethanol;

[0164] (2) Add 30 mL of 50% hydrochloric acid and soak for 0.5 h and heat to a gentle boil. Then filter with filter paper and rinse three times with deionized water.

[0165] (3) Add 30 mL of 12.5% ​​nitric acid to the residual coal, heat to boiling, maintain boiling for 0.5 h, filter again, and wash the coal sample until neutral;

[0166] (4) Place the filter paper in an oven and dry it at 105°C for 2 hours. Use an automatic sulfur analyzer to determine the total sulfur content and calculate the desulfurization rate.

[0167] Experimental results:

[0168] The initial sulfur content in the coal sample was 4.37%, which decreased to 2.89% after chemical treatment, with a desulfurization rate of 1.48%. The desulfurization effect of the microbial compound agent leaching method (Example 7, with a desulfurization rate of 1.87%) was better than that of the chemical method. Moreover, the chemical desulfurization process is cumbersome, requiring conditions such as heating, hydrochloric acid, and nitric acid, which results in high costs and difficult waste liquid treatment.

[0169] Comparative Example 2

[0170] Single strain of *Thiobacillus ferrooxidans* for coal desulfurization

[0171] (1) Incubate *Thiobacillus ferrooxidans* overnight with shaking until OD reaches 0.5. 600 =1.5.

[0172] (2) Take 5 catties of coal sample and place it in a 30cm×40cm×15cm turnover box. The particle size of the coal sample is ≤3mm. The coal sample is raw coal purchased from the market and has not been crushed.

[0173] (3) Spray 500 ml of cultured Thiobacillus ferrooxidans in 5 catties of coal sample.

[0174] (4) The coal sample is turned over every 48 hours. The temperature for coal desulfurization is 15~35℃. The humidity is kept constant during the coal desulfurization process, that is, the humidity during the coal desulfurization process is the same as the initial humidity.

[0175] (5) Take a portion of coal samples every day for filtration and cleaning, dry them, determine the sulfur content, and calculate the desulfurization rate.

[0176] Figures 15-16 The results showed that after 30 days of treatment with *Thiobacillus ferrooxidans*, the sulfur content in the coal decreased from 3.55% to 1.40%, the desulfurization amount was 2.15%, and the desulfurization rate was 60.45%. Compared with the desulfurization effect of the compound bacterial agent in Example 8, the desulfurization rate was 24.37% lower, and the desulfurization time was longer, reaching 30 days, which is not conducive to engineering applications.

[0177] Comparative Example 3

[0178] Desulfurization experiment after genetic modification of Nocardia mangya

[0179] Nocardia mangya ( Nocardia mangyaensis (Purchased from Beina Chuanglian Biotechnology Co., Ltd., BNCC No.: 361211) After genetic modification using the method of Example 4, coal desulfurization experiments were conducted according to the steps of Example 5. The results are as follows: Figures 17-18 The results showed that after 6 days of treatment, the sulfur content in the coal decreased from 2.59% to 2.47%, the desulfurization amount was 0.125%, and the desulfurization rate was only 4.81%, which was significantly lower than the desulfurization effect of Example 5 on the modified Pseudomonas bacteria (desulfurization rate: 79.49%).

[0180] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of putrid pseudomonas ( Pseudomonas putida )-coal-09, characterized in that, The preservation number of the *Pseudomonas putida*-coal-09 is CGMCC No. 35656.

2. A microbial preparation, characterized in that, Includes the *Pseudomonas putida*-coal-09 as described in claim 1.

3. A method for preparing the microbial preparation according to claim 2, characterized in that, include: Microbial preparations were obtained by culturing *Pseudomonas putida*-coal-09 in a culture medium; the culture temperature was 15℃~40℃; and the pH of the culture medium was 4.0~9.

0.

4. A microbial compound inoculant, characterized in that, Includes *Pseudomonas putida*-coal-09 and *Thiobacillus ferrooxidans* as described in claim 1.

5. The application of the *Pseudomonas putida*-coal-09 of claim 1, the microbial preparation of claim 2, the microbial preparation obtained by the preparation method of claim 3, or the microbial compound agent of claim 4 in coal desulfurization.

6. A method for coal desulfurization, characterized in that, include: Coal is desulfurized by mixing it with a microbial preparation containing *Pseudomonas malodorans*-coal-09 as described in claim 1 or a microbial compound preparation containing the microbial compound preparation as described in claim 4.

7. The method according to claim 6, characterized in that, The temperature for coal desulfurization is 15℃~40℃.

8. The method according to claim 6, characterized in that, The mass-to-volume ratio of coal to the microbial preparation or the microbial compound agent is (20~400) g: (30~80) mL.

9. The method according to claim 6, characterized in that, The coal has a particle size of ≤3mm.

Images