A strain for degrading coal gangue, its cultivation method and application

Abstract

The invention discloses a strain for degrading coal gangue, a cultivation method and an application thereof. The strain for degrading coal gangue is classified and named as Arthrobacter oryzae Arthrobacter oryzae YJ1#, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.32157. Its optimal culture conditions are: culture temperature 30°C, culture speed 200 r / min, and culture time 48h. The coal gangue degradation strain can produce cracks in the coal gangue and then crack it, and decompose the organic matter in it into humic acid and other inorganic substances. Using this strain to degrade coal gangue can not only solve the environmental pressure caused by the large-scale stacking of coal gangue, but also enhance its application potential for resource recycling.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of a strain for degrading coal gangue, and in particular to a strain for degrading coal gangue, a culturing method and application thereof. Background Art

[0002] A large amount of coal gangue solid waste is generated during the coal mining process. A large amount of coal gangue cannot be consumed and can only be stored on site, which occupies a large amount of land and destroys the local ecosystem, causing serious soil degradation and vegetation destruction, and even affecting people's health. At present, the main treatment measures for coal gangue are: physical measures and chemical measures. Physical measures are mainly used as roadbed materials, backfilling goafs and fertilizers, etc.; but the heavy metal components in them may seep out, causing potential harm to the soil and human health. Chemical measures are mainly used as building materials, power generation and extraction of rare elements, etc., and there are also problems of pollution and waste. The above measures are difficult to achieve a sustainable development model for coal gangue solid waste.

[0003] The method of using microorganisms to degrade coal gangue can effectively decompose the organic matter in coal gangue into humic acid and other inorganic substances, which not only helps to reduce the environmental pressure caused by the stacking of coal gangue, but also provides new impetus for environmental protection and economic development through recycling and reuse, and realizes the sustainable development of comprehensive utilization of coal gangue. However, most of the methods of microbial degradation of coal gangue are still in the theoretical exploration or laboratory stage, with low efficiency and lack of strains and application solutions with high practical value. Summary of the invention

[0004] The purpose of the present invention is to provide a strain for degrading coal gangue, a cultivation method and application thereof, which can crack coal gangue and decompose the organic matter therein into humic acid and other inorganic substances, which can not only solve the environmental pressure caused by the large-scale stacking of coal gangue, but also enhance its application potential for resource recycling. The purpose of the present invention is achieved through the following technical solutions:

[0005] A strain for degrading coal gangue, the strain for degrading coal gangue is classified and named as Arthrobacter oryzae ( Arthrobacter oryzae )YJ1#, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32157 and the deposit date October 9, 2024. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. Arthrobacter oryza The homology is 99.29%.

[0006] The coal gangue degrading strain is isolated from coal gangue. The strain is a Gram-positive obligate aerobic bacterium, the strain is light yellow in color, the colony is round, the surface is raised and smooth, and it adheres to the culture medium in an adhesive manner. The bacterial body is rod-shaped and has a length of 1.0 μm to 5.0 μm. Figure 1 shown.

[0007] The method for culturing the coal gangue-degrading strain is as follows: the logarithmic growth phase of the coal gangue-degrading strain is 48 h, and the optimal culture conditions are: culture temperature 30 °C, culture speed 200 r / min, and culture time 48 h.

[0008] The separation and screening method for the bacterial strain that degrades coal gangue comprises the following steps:

[0009] Step 1: Take 10 g of coal sample within 10-50 cm below the surface of the gangue mountain and oscillate it in 100 mL of inorganic salt culture medium at a constant temperature of 25-35 ° C, an oscillation frequency of 150-220 r / min, and an oscillation time of 48 h to obtain a bacterial enrichment solution;

[0010] Step 2: Prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The diluted solutions of different dilutions were spread on LB solid medium and cultured in a constant temperature incubator at 25-35°C for 48-72h;

[0011] Step 3: The microorganisms grown on the above LB solid medium were cultured in 100 mL of liquid inorganic salt medium, 1.0 g of coal gangue was added, and the culture was shaken at a temperature of 28-32°C and a rotation speed of 180-200 rpm for 48 hours;

[0012] Step 4: Take the culture solution after shaking culture and transfer it to a conical flask containing 100 mL of fresh inorganic salt culture medium at a 20% inoculation volume and continue shaking for 48 hours. The shaking temperature is 28-32°C and the rotation speed is 180-200 rpm. Set 20 acclimation cycles in this way;

[0013] Step 5: Inoculate the final domesticated bacterial solution into LB solid medium and culture it in a constant temperature incubator at 25-35°C for 24-48 hours;

[0014] Step 6: Take the bacterial colony to verify the degradation performance;

[0015] Step 7: Select the single colony with the best degradation effect and continue to inoculate it into LB solid culture medium. After repeated streaking and purification culture, the obtained single colony is the coal gangue degradation strain;

[0016] The inorganic salt culture medium comprises: 2.8 g / L Na2HPO4·12H2O, 1 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.053 g / LMgCl2, 0.05 g / LCaCl2·4H2O, 0.002 g / L FeSO4·7H2O, 0.02 mg / L CoCl2·6H2O, 0.01 mg / L ZnSO4·7H2O, 0.003 mg / L mnCl2·4H2O, and the pH is adjusted to 7.3.

[0017] The LB solid culture medium contains: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 16 g / L agar, and the pH is adjusted to 7.3.

[0018] Further, the degradation performance verification: a single colony cultured on LB solid medium for 48 hours was shaken and cultured in 50mL inorganic salt medium for 24h~48h. The control group was 0.5g coal gangue and 50mL inorganic salt medium, and the experimental group was 0.5g coal gangue, 1ml bacterial solution and 50mL inorganic salt medium, cultured in a shaker at a culture temperature of 28~32°C and a rotation speed of 180~200rpm for 3~4 days, and the humic acid content in the coal gangue solution after degradation was determined by volumetric method; if the humic acid content in the solution is higher, it means that the degradation effect is more significant, and the single colony is a colony with the performance of degrading coal gangue.

[0019] The method for culturing a coal gangue-degrading strain comprises inoculating the coal gangue-degrading strain into a liquid culture medium under the following culture conditions: culture temperature of 30°C, culture speed of 200 r / min, and culture time of 48 h.

[0020] The method for culturing a coal gangue-degrading strain comprises inoculating the coal gangue-degrading strain into a solid culture medium, wherein the solid culture medium comprises 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 16 g / L agar, adjusting the pH to 7.3, culturing at 30°C, and culturing for 48 h.

[0021] Application of coal gangue degrading strains in coal gangue cracking.

[0022] Further, the following steps are included:

[0023] Step 1): Prepare microbial culture and polish coal gangue: The microbial culture is prepared by amplifying the coal gangue-degrading strain and culturing it to the logarithmic growth phase. The OD of the culture is 600 The value is 1.0~2.0; the surface of the coal gangue is polished smooth by an electric grinding machine;

[0024] Step 2): Take the microbial liquid and add it dropwise to the surface of coal gangue, culture it at a temperature of 25-32°C for 3-4 days and then dry it.

[0025] The microscopic morphology of the coal gangue without bacterial solution and the coal gangue block with bacterial solution was observed by scanning electron microscope (SEM), and the change of minerals was analyzed by X-ray diffraction (XRD). The microbial bacterial solution loosens and decomposes the coal gangue, forming cracks to achieve cracking of the coal gangue, wherein the size of the cracks is 1.5-5.5 μm. The microbial bacterial solution changes the minerals in the coal gangue and achieves cracking of the coal gangue.

[0026] The application of the coal gangue degrading strain in extracting humic acid.

[0027] Further, the following steps are included:

[0028] Step (1): preparing a microbial degradation solution: the bacterial solution is prepared by amplifying the coal gangue degradation strain and culturing it to the logarithmic growth phase. The OD of the bacterial solution is 600 The value is 1.0~2.0;

[0029] Step (2): Performing microbial decomposition and extraction to form a humic acid solution: inoculating the bacterial solution and coal gangue into an inorganic salt culture medium, with the ratio of bacterial solution to coal gangue being 10 mL bacterial solution / g coal gangue, and culturing for 3 to 6 days in a shaking incubator at a culture temperature of 30° C. and a rotation speed of 200 rpm to obtain a humic acid solution.

[0030] The microbial liquid decomposes the coal gangue to form a humic acid solution to extract the humic acid in the coal gangue. The humic acid content in the solution is determined by a volumetric method.

[0031] The advantages and beneficial effects of the present invention are:

[0032] 1. The coal gangue degradation strain provided by the present invention is Arthrobacter oryzae ( Arthrobacter oryzae )YJ1# strain can be used as a new type of microbial strain for degrading coal gangue. It can crack coal gangue and efficiently dissociate humic acid. It can not only solve the problem of large-scale storage of coal gangue that cannot be digested, but also prepare coal gangue-based artificial soil or nutrient soil with good performance, thereby realizing the resource recycling of coal gangue.

[0033] 2. Arthrobacter oryzae ( Arthrobacter oryzae ) The YJ1# strain is a self-degrading strain for coal gangue, which is suitable for the modification and reuse of coal gangue. This strain has good environmental tolerance and strong degradation ability. At the same time, it can avoid the impact of exogenous strains on the environment and has great research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0035] Figure 1 This is the YJ1# colony morphology and SEM scanning electron microscope image provided in the examples of this application;

[0036] Figure 2 It is the phylogenetic tree of YJ1# provided in the examples of the present application;

[0037] Figure 3 This is the growth curve of YJ1# provided in the examples of the present application;

[0038] Figure 4 A microscopic scanning electron microscope image of cracks formed in YJ1# during the degradation of coal gangue provided in the embodiment of the present application;

[0039] Figure 5 The XRD spectra of the coal gangue before and after efficient degradation in the embodiment of the present application;

[0040] Figure 6 This is a bar chart of YJ1# humic acid yield provided in the examples of this application. DETAILED DESCRIPTION

[0041] Example 1

[0042] A strain for degrading coal gangue, the strain for degrading coal gangue is classified and named as Arthrobacter oryzae ( Arthrobacter oryzae )YJ1#, deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No.32157 and the deposit date October 9, 2024. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. Arthrobacter oryzae The homology is 99.29%.

[0043] Arthrobacter oryzae YJ1#16S rRNA gene sequence:

[0044]

[0045] The coal gangue-degrading strain is isolated from coal gangue. The strain is a Gram-positive obligate aerobic bacterium, the strain is light yellow in color, the colony is round, the surface is raised and smooth, and it adheres to the culture medium in a sticky manner. The bacterial body is rod-shaped and has a length of 1.0 μm to 5.0 μm. Figure 1 shown.

[0046] In this embodiment, the separation and screening of the coal gangue-degrading strain includes the following steps:

[0047] Step 1: Isolation and screening of microorganisms from coal gangue, specifically including the following steps:

[0048] The samples used in this experiment are mainly from coal gangue. The coal gangue was taken from Longwanggou Coal Mine in Zhungeer Banner, Inner Mongolia.

[0049] Step 1: Take 10 g of coal sample within 10-50 cm below the surface of the gangue mountain and oscillate it in 100 mL of inorganic salt culture medium at a constant temperature of 25-35 ° C, an oscillation frequency of 150-220 r / min, and an oscillation time of 48 h to obtain a bacterial enrichment solution;

[0050] Wherein, the liquid culture medium is an inorganic salt culture medium, 2.8g / L Na2HPO4·12H2O, 1g / L KH2PO4, 0.5g / L(NH4)2SO4, 0.053g / LMgCl2, 0.05g / LCaCl2·4H2O, 0.002g / L FeSO4·7H2O, 0.02mg / LCoCl2·6H2O, 0.01mg / L ZnSO4·7H2O, 0.003mg / LMnCl2·4H2O, and the pH is adjusted to 7.3.

[0051] Step 2: Prepare 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The diluted solutions of different dilutions were spread on LB solid medium and cultured in a constant temperature incubator at 25-35°C for 48-72h;

[0052] The solid culture medium is LB solid culture medium, containing 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, and 16 g / L agar, and the pH is finally adjusted to 7.3.

[0053] Step 3: The microorganisms grown on the above LB solid medium were cultured in 100 mL of liquid inorganic salt medium, 1.0 g of coal gangue was added, and the culture was shaken at a temperature of 28-32°C and a rotation speed of 180-200 rpm for 48 hours;

[0054] Step 4: Take the culture solution after shaking culture and transfer it to a conical flask containing 100 mL of fresh inorganic salt culture medium at a 20% inoculation volume and continue shaking for 48 hours. The shaking temperature is 28-32°C and the rotation speed is 180-200 rpm. Set 20 acclimation cycles in this way;

[0055] Step 5: Inoculate the final domesticated bacterial solution into LB solid medium and culture it in a constant temperature incubator at 25-35°C for 24-48 hours;

[0056] Step 6: Take the bacterial colony to verify the degradation performance;

[0057] The verification of the degradation performance of the strain comprises the following steps:

[0058] a. Take a single colony cultured on LB solid medium for 48 hours and shake culture it in 50 mL inorganic salt medium for 24 hours to 48 hours;

[0059] b. The control group was 0.5 g of coal gangue and 50 mL of inorganic salt medium, and the experimental group was 0.5 g of coal gangue, 1 ml of bacterial solution and 50 mL of inorganic salt medium, and cultured in a shaker at a temperature of 28-32°C and a rotation speed of 180-200 rpm for 3-4 days;

[0060] c. Use the volumetric method to determine the humic acid content in the coal gangue solution after degradation.

[0061] Step 7: Select the single colony with the highest humic acid content in the gangue solution and continue to inoculate it into the LB solid culture medium. After repeated streaking and purification, the obtained single colony is the gangue-degrading strain Arthrobacter oryzae. Arthrobacter oryzae YJ1#;

[0062] The strain is light yellow in color, with round colonies, a raised, smooth surface, and sticky adhesion to the culture medium. The bacteria are rod-shaped and 1.0 μm to 5.0 μm in length. Figure 1 shown.

[0063] Step 2: Identification of bacterial species. The strains obtained were compared with known sequences in the gene bank (Gen Bank) established by the National Center for Biotechnology Information (NCBI, http: / / www.Ncbi.nlm.nih.gov) of the United States using the local alignment search tool (Blast). Based on the 16S rDNA sequencing results, a phylogenetic tree was constructed using MEGA4.1 software. This strain is a Gram-positive obligate aerobic bacterium, and its 16S rDNA gene sequencing results are shown in SEQ ID NO.1. The phylogenetic tree constructed based on the gene sequencing results is shown in Figure 2 The bacteria screened were confirmed to be of the genus Arthrobacter and named Arthrobacter oryzae Arthrobacter oryzae YJ1# was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 9, 2024, and the registration number of the collection center is CGMCC No.32157.

[0064] Step 3: Growth characteristics of YJ1#. Figure 3 The logarithmic growth period of YJ1# bacteria is 6 to 48 hours, and it enters the stable period after 48 hours.

[0065] Embodiment 2:

[0066] The application of the coal gangue degrading strain in cracking coal gangue comprises the following steps: In this embodiment, the strain is isolated by the separation and screening method of the coal gangue degrading strain in embodiment 1.

[0067] Step 1): Prepare microbial culture liquid and coal gangue: The microbial culture liquid is prepared by amplifying the coal gangue-degrading strain Arthrobacter oryzae and culturing it to the logarithmic growth phase. The OD of the culture liquid is 600 The value is 1.5~2.0; the gangue is firstly ground by an electric grinding machine to make its surface smooth and then ground by a small rock cutting machine, the thickness of the gangue slice is 1.0~2.0mm, and the diameter of the gangue slice is 0.5~1.0cm;

[0068] Step 2): Add microbial liquid to form cracks: The microbial liquid addition includes the steps of taking a small amount of bacterial liquid in the logarithmic growth period and dropping it on the surface of the coal gangue, culturing it in a microbial incubator at a culture temperature of 30°C for 3 days and then drying it, scanning the coal gangue slices without the bacterial liquid and the coal gangue slices with the bacterial liquid by electron microscope (SEM) to observe the microscopic morphology, and analyzing the changes in minerals by X-ray diffraction (XRD).

[0069] The microbial liquid loosens and decomposes the coal gangue to form cracks to achieve cracking of the coal gangue, wherein the size of the cracks is 1.5-5.5 μm. Figure 4 The coal gangue-degrading strains of the present application examples form cracks on the coal gangue, thereby further cracking the coal gangue. Figure 5 This is the XRD spectrum of the coal gangue degrading strain in the embodiment of the present application before and after decomposing coal gangue powder.

[0070] Embodiment 3:

[0071] The application of the coal gangue degrading strain in extracting humic acid comprises the following steps: In this embodiment, the strain is isolated by the separation and screening method for coal gangue degrading strains of embodiment 1.

[0072] Step (1): preparing microbial degrading bacterial solution and coal gangue: the microbial bacterial solution is prepared by amplifying the coal gangue degrading strain Arthrobacter oryzae, and the OD of the bacterial solution is 600 The value is 1.5~2.0; the coal gangue is crushed by a crusher;

[0073] Step (2): Perform microbial decomposition and extraction to form a humic acid solution: The microbial decomposition comprises the following steps:

[0074] A. Take 6 250 mL conical flasks, add 100 mL of inorganic salt culture medium, and sterilize in a high pressure steam autoclave at 121°C for 20 min.

[0075] B. After sterilization, 5 mL of bacterial solution in the logarithmic growth phase and 0.5 g of coal sample were inoculated into the inorganic salt culture medium and cultured in a shaker at a temperature of 30°C and a rotation speed of 200 rpm for 1-6 days.

[0076] C. Transfer all the extract and residue into a 200 mL volumetric flask, dilute to the mark with distilled water, shake well, filter, and use 50~100 mL of the filtrate for determination. Accurately pipette 5 ml of the filtrate into a 250 mL conical flask. Use a pipette to accurately add 5 mL of 0.4 mol / L potassium dichromate solution and 15 mL of concentrated sulfuric acid, and heat and oxidize in a 100±1℃ water bath for 30 min. Remove and cool to room temperature, dilute to about 100 mL with distilled water, add 3 drops of o-phenanthroline indicator after cooling, and drop with 0.1 mol / L ammonium ferrous sulfate solution until it turns brick red, and calculate the yield of humic acid on different days. The humic acid content in the solution is determined by the volumetric method, i.e. GB∕T 11957-2001 "Method for Determination of Humic Acid Yield in Coal".

[0077] The microbial bacterial liquid decomposes the coal gangue to form a humic acid solution to achieve the extraction of humic acid in the coal gangue. Figure 6 This is a bar chart of humic acid yield of the strain degrading coal gangue in the present application example. Figure 6 It can be seen that after 4 days of degradation under the conditions of temperature of 30°C and rotation speed of 200 rpm, the humic acid yield after degradation was measured to be 38.7%, which was significantly higher than the humic acid yield on other days. This shows that the strain has the best effect after 4 days of degradation.

[0078] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain for degrading coal gangue, characterized in that: The coal gangue degradation strain is classified and named as Arthrobacter oryzae ( Arthrobacter oryzae )YJ1#, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32157 and the deposit date of October 9, 2024.

2. The method for culturing the coal gangue-degrading strain according to claim 1, characterized in that: The coal gangue degradation strain was inoculated into a liquid culture medium, and the culture conditions were: culture temperature 30°C, culture speed 200 r / min, and culture time 48 h.

3. The method for culturing the coal gangue-degrading strain according to claim 1, characterized in that: The coal gangue degradation strain was inoculated into a solid culture medium. The solid culture medium components used were: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 16 g / L agar, the pH was adjusted to 7.3, the culture temperature was 30°C, and the culture time was 48 h.

4. Use of the coal gangue-degrading strain according to claim 1 in cracking coal gangue.

5. The use according to claim 4, characterized in that: The following steps are involved: Step 1): Prepare microbial culture liquid and polish coal gangue. The microbial culture liquid is obtained by expanding and culturing the coal gangue-degrading strain to the logarithmic growth phase. The OD of the culture liquid is 600 The value is 1.0~2.0; the surface of the coal gangue is polished smooth by an electric grinding machine; Step 2): Add microbial liquid drops onto the surface of coal gangue to form cracks.

6. The use according to claim 5, characterized in that: In step 2), the microbial liquid is added dropwise to the surface of the coal gangue, cultured at a temperature of 25-32° C. for 3-4 days and then dried.

7. The use of the coal gangue degrading strain according to claim 1 in extracting humic acid, characterized in that: The bacterial liquid of the coal gangue-degrading strain decomposes the coal gangue to form a humic acid solution to extract the humic acid in the coal gangue.

8. The use according to claim 7, characterized in that: The following steps are involved: Step (1): preparing a microbial degradation bacterial solution, wherein the bacterial solution is obtained by amplifying the coal gangue degradation strain and culturing it to the logarithmic growth phase. The OD of the bacterial solution is 600 The value is 1.0~2.0; Step (2): performing microbial decomposition and extraction to form a humic acid solution, inoculating the bacterial solution and coal gangue into an inorganic salt culture medium, wherein the ratio of bacterial solution to coal gangue is 10 mL bacterial solution / g coal gangue, and culturing for 3 to 6 days in a shaking incubator at a culture temperature of 30° C. and a rotation speed of 200 rpm to obtain a humic acid solution; the inorganic salt culture medium comprises: 2.8 g / L Na2HPO4·12H2O, 1 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.053 g / LMgCl2, 0.05 g / LCaCl2·4H2O, 0.002 g / LFeSO4·7H2O, 0.02 mg / L CoCl2·6H2O, 0.01 mg / L ZnSO4·7H2O, 0.003 mg / L mnCl2·4H2O, and adjusting the pH to 7.

3.

9. The use according to claim 8, characterized in that: In step (2), the cells were cultured for 4 days in a shaker at a culture temperature of 30° C. and a rotation speed of 200 rpm.

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