Method for the microbial degradation of oil-rich coal using bacillus anthracis

Abstract

The application discloses a method for degrading oil-rich coal by using bacillus anthracis as microorganisms, relates to the technical field of coal microbial degradation, and collects coal samples in a mining area for grinding and screening, and performs oxidation pretreatment on the coal samples by using a nitric acid solution to obtain oxidized coal; the surface microorganisms are eluted by oscillation and ultrasonic washing of the sterile buffer solution, and the sterile oxidized coal is obtained; the medium with the sterile oxidized coal as the only carbon source is used to enrich target strains, and active strains are screened through preliminary screening and rescreening to obtain Bacillus anthracis YL2; the sterile oxidized coal, the Bacillus anthracis YL2 strain and appropriate medium are mixed, and constant-temperature shaking culture is performed; after the degradation experiment is completed, the supernatant is obtained by centrifugal filtration; the residual coal is washed to remove the surface mycelium, dried, weighed, and the degradation rate is calculated. The application solves the problems of low degradation efficiency, single catalytic mechanism, low product resource value and no precise basis for process optimization of traditional microbial degradation strains, and helps the green, low-carbon, efficient and large-scale resource utilization of oil-rich coal.

Description

Technical Field

[0001] This invention relates to the field of coal microbial degradation technology, and in particular to a method for using Bacillus anthracis as a microorganism to degrade oil-rich coal. Background Technology

[0002] Currently, the development of rich oil-bearing coal in China mainly adopts traditional processes such as ground pyrolysis, direct liquefaction, and high-temperature dry distillation. These processes require stringent high-temperature and high-pressure reaction conditions, resulting in high energy consumption, high equipment maintenance costs, complex processes, and large emissions of carbon and pollutants. Furthermore, traditional technologies suffer from low resource utilization rates, difficult byproduct disposal, heavy environmental burdens, and poor economic and ecological benefits, severely hindering the large-scale and sustainable development of the rich oil-bearing coal industry. Therefore, the development of new green alternative conversion technologies is urgently needed. Microbial degradation conversion technology aligns with the requirements of dual-carbon development. Leveraging its significant advantages such as ambient temperature and pressure reaction, low construction and operation costs, minimal secondary pollution, and good targeted resource conversion effects, it breaks through the core bottlenecks of traditional processes, opening up a new path for the low-carbon utilization of rich oil-bearing coal. Microbial degradation technology for rich oil-bearing coal possesses outstanding advantages such as mild reaction conditions, green and low-carbon environmental protection, high resource utilization efficiency, and low operating costs. It is one of the main methods for the clean and efficient treatment of rich oil-bearing coal, effectively promoting the synergistic improvement of energy security, economic development, and ecological protection in the development of rich oil-bearing coal.

[0003] Existing microbial degradation technologies for coal have significant shortcomings and deficiencies in practical applications. Specifically, regarding the technical problems this invention aims to solve: First, existing conventional degradation strains generally have low degradation efficiency for oil-rich coal. Most strains struggle to achieve a coal degradation rate of 60% over long-term degradation cycles, resulting in poor degradation efficiency that fails to meet the efficiency requirements of industrial resource conversion and severely limits the large-scale application of the technology. Second, existing strains have a single mechanism for coal degradation, relying heavily on single enzyme systems. They lack multi-enzyme synergistic catalytic systems such as lignin peroxidase, esterase, and laccase, making it difficult to efficiently catalyze key reactions such as the breaking and demethylation of aromatic structures, alkyl side chains, carbon-carbon bonds, and carbon-oxygen bonds in oil-rich coal. Their ability to disrupt the dense macromolecular structure of oil-rich coal is limited, leading to insufficient coal depolymerization and low conversion rates of high-value-added chemicals. Third, existing technologies do not clearly define the binding mechanism between key functional enzymes of the strains and typical coal substrates, making it difficult to accurately elucidate the core catalytic pathways of coal degradation. This hinders targeted optimization of the degradation process, resulting in a disordered composition of degradation products, a low proportion of target high-value-added products, and a significant reduction in resource utilization value.

[0004] In summary, the lack of a dedicated degradation strain that is suitable for oil-rich coal substrates, has high degradation efficiency, strong multi-enzyme synergistic catalytic ability, and a clearly defined mechanism of action is currently the main technical bottleneck for the industrialization of coal microbial green degradation technology. Therefore, screening and obtaining a dedicated strain that can efficiently degrade oil-rich coal and clarifying its degradation mechanism and application scenarios is of great significance for breaking through the limitations of existing technologies and realizing the efficient and green resource utilization of oil-rich coal. Summary of the Invention

[0005] To address the technical challenges of low degradation efficiency of oil-rich coal by bacterial strains, a single mechanism of action in coal degradation, limited ability to disrupt the dense macromolecular structure of oil-rich coal, insufficient coal depolymerization, low conversion rate of high-value-added chemicals, disordered composition of degradation products, low proportion of target high-value-added products, and low resource utilization value, this invention discloses a method for the degradation of oil-rich coal using Bacillus anthracis as a microorganism. This method solves the industry pain points of low degradation efficiency, single catalytic mechanism, low resource utilization value of products, and lack of precise basis for process optimization in traditional microbial degradation strains. It promotes the green, low-carbon, efficient, and large-scale resource utilization of oil-rich coal, while taking into account the needs of energy security and ecological green development.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for using Bacillus anthracis as a microorganism to degrade oil-rich coal includes the following steps:

[0008] Step 1: Preparation and oxidation pretreatment of coal samples

[0009] Coal samples were collected from the mining area, quickly wrapped and sealed with plastic wrap, packaged in sample bags, and then ground and screened. Industrial analysis, elemental analysis, and tar content were determined according to national standards.

[0010] The coal sample was pretreated with nitric acid solution, washed with deionized water, and dried to obtain sterile oxidized coal.

[0011] Step 2, strain isolation, purification and identification

[0012] Oxidized coal pretreated with nitric acid was subjected to agitation with sterile buffer, ultrasonic elution to remove surface microorganisms, and then serially diluted.

[0013] The target strain was enriched in a culture medium with oxidized coal as the sole carbon source. The diluted solution was spread on plates for culture, and characteristic single colonies were picked and repeatedly streaked for purification.

[0014] Active strains were screened based on growth status and functional indicators through primary and secondary screening, followed by physiological and biochemical tests, and molecular biological identification by amplifying the 16S rRNA gene. The resulting strain was named Bacillus anthracis YL2.

[0015] Step 3, Coal Biodegradation

[0016] Sterilize the oxidized coal in a high-pressure steam sterilizer for 15 minutes to obtain sterile oxidized coal. Then, mix it with Bacillus santhracis YL2 and an appropriate amount of culture medium, and incubate at 25-45°C. o Culturing in a constant temperature shaker at 130-140 r / min for 1-14 days;

[0017] After the degradation experiment, the supernatant was obtained by centrifugation and filtration; the residual coal was washed multiple times with distilled water to remove surface hyphae, dried and weighed, and the degradation rate was calculated.

[0018] Further, in step 1, the concentration of the nitric acid solution is 8 mol / L. The coal sample and the nitric acid solution are placed in a beaker and reacted in a fume hood for 48 h. Then, the sample is washed with deionized water until the pH reaches 6-7, and then dried in a drying oven at 105℃ for 8 h.

[0019] Furthermore, the strain obtained in step 2 exhibits the following morphology on LB medium: round colonies, moist, smooth, with neat edges, and pale yellow in color; the bacterial cells are rod-shaped and 2 µm × 1.5 µm in size.

[0020] Furthermore, in step 3, the OD of Bacillus anthracis YL2 600 =0.5~1.0.

[0021] Further, in step 3, the optimal single-factor conditions are: when the liquid culture medium volume is 50 mL, the bacterial culture volume is 10 mL, the coal addition amount is 0.4 g, the degradation time is 10 d, the culture temperature is 35 ℃, and the culture is carried out in a shaker.

[0022] Furthermore, based on the results of the single-factor experiments, the BBD model was used to study the four factors affecting the degradation rate: degradation time, bacterial culture volume, coal addition amount, and temperature, and the interaction between these factors was analyzed. The multivariate quadratic response surface regression model obtained after regression fitting of the above four influencing factors is as follows:

[0023] Y=-1624.57315+173.475X1+28.92917X2+78.15741X3+37.635X4-0.2875X1 X2-1.08333X1X3+0.04X1X4-0.083333X2X3-0.03X2X4-0.15X3X4-8.49583X1 2 -1.208333X2 2 -76.34259X3 2 -0.53633X4 2 ;

[0024] Where X1 is the degradation time, X2 is the bacterial solution volume, X3 is the amount of coal added, and X4 is the temperature;

[0025] The R-value of the quadratic regression coding equation 2 =0.9723, with a corrected coefficient of determination of 0.9400.

[0026] Furthermore, analysis using SAS 8.0 software, based on response surface and contour plots as well as regression equation analysis, revealed that the optimal conditions for the YL2 strain to degrade oil-rich coal were: degradation time 10.09 days, bacterial culture volume 10.32 mL, coal addition 0.4 g, and temperature 35.11 ℃. Simulation verification experiments showed that the YL2 strain achieved a degradation rate of 76.5124% for oil-rich coal, with a confidence level as high as 95%.

[0027] The beneficial effects of this invention are that, compared with the prior art, it has the following advantages:

[0028] (1) In view of the shortcomings of existing degradation strains that rely on a single enzyme system for catalysis and lack a multi-enzyme synergistic catalytic system, which cannot effectively destroy the dense structure of oil-rich coal macromolecules, have weak catalytic ability for key pyrolysis reactions, and have low conversion rate of high-value-added chemicals, the Bacillus anthracis used in this invention can construct a multi-enzyme synergistic catalytic system, effectively activate the synergistic effect of multiple functional enzymes such as lignin peroxidase, esterase, and laccase, and efficiently catalyze the core reactions such as the breaking of aromatic structure, alkyl side chain, carbon-carbon bond, carbon-oxygen bond and demethylation of oil-rich coal, so as to achieve full depolymerization of oil-rich coal macromolecules and greatly improve the conversion efficiency of high-value-added chemicals.

[0029] (2) In view of the problems that the existing technology does not clearly define the binding mechanism between functional enzymes and coal substrates and the core catalytic pathway is unclear, resulting in disordered degradation product composition, low proportion of target product and inability to accurately optimize degradation process, the present invention can clarify the binding mechanism and core catalytic pathway between the key functional enzymes of Bacillus anthracis and typical substrates of oily coal, providing precise theoretical support for targeted optimization of oily coal degradation process and regulation of degradation product composition, effectively increasing the proportion of target high value-added products and maximizing the exploitation value of oily coal resource utilization.

[0030] (3) This method relies on the excellent degradation performance of Bacillus anthracis to overcome the drawbacks of high energy consumption, high pollution, high cost and low utilization rate of traditional oil-rich coal pyrolysis and liquefaction processes, as well as the industrialization shortcomings of existing microbial degradation technologies. It realizes the green, efficient and low-cost targeted conversion of oil-rich coal at room temperature and pressure, promotes the upgrading of the comprehensive utilization efficiency of oil-rich coal resources, helps the coal industry to achieve green, low-carbon and high-quality development, and takes into account the multiple development needs of energy supply, industrial economic improvement and ecological environmental protection. Attached Figure Description

[0031] Figure 1This is an identification diagram of the bacterial strains in an embodiment of the present invention;

[0032] Figure 2 This is a diagram illustrating the degradation effect of bacteria on oil-rich coal in an embodiment of the present invention.

[0033] Figure 3 These are the results of single-factor experiments in the embodiments of the present invention;

[0034] Figure 4 The response surface and contour lines of the interaction between the four factors in this embodiment of the invention on the degradation rate are shown.

[0035] Figure 5 This illustrates the changes in enzyme activity during microbial degradation in this embodiment of the invention.

[0036] Figure 6 This is the molecular docking result of the interaction between thymol and lignin peroxidase in the embodiments of the present invention;

[0037] Figure 7 The results of molecular docking between thymol and catechol 2,3-dioxygenase in the embodiments of the present invention;

[0038] Figure 8 The results of molecular docking between thymol and laccase in the embodiments of the present invention;

[0039] Figure 9 The images show the total ion chromatogram and component distribution diagram of the degradation products in this embodiment of the invention.

[0040] Figure 10 3D-EEM images of degradation solutions at different number of days in this embodiment of the invention;

[0041] Figure 11 These are NMR images of different coal samples in the embodiments of the present invention;

[0042] Figure 12 The images show the infrared spectra of different coal samples in the embodiments of the present invention. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] To address the problem that existing conventional degrading strains have poor degradation efficiency for oily coal and the degradation rate is difficult to reach 60% over a long period of time, which cannot meet the efficiency requirements for industrial-scale conversion, this invention aims to screen and obtain an anthrax bacillus strain that is adapted to the characteristics of oily coal, significantly improve the degradation conversion rate of oily coal, break through the technical limitations of existing strains in degradation efficiency, and meet the efficiency standards for industrial applications.

[0045] This invention discloses a method for using Bacillus anthracis as a microorganism to degrade oil-rich coal, specifically:

[0046] 1. Experimental Procedure

[0047] 1.1 Preparation of coal samples

[0048] Coal samples were collected from the Hanshuiquan mining area in Xinjiang. After collection, they were quickly wrapped and sealed with plastic wrap, then packaged in sample bags, ground, and screened. Industrial analysis, elemental analysis, and tar content were determined according to national standards. The results are shown in Table 1. Nitric acid oxidation pretreatment increased coal tar yield, with oxidized coal showing a 1.57% increase compared to oil-rich coal, indicating that oxidation treatment optimized coal usability. In industrial analysis, the moisture and ash content of oxidized coal were lower than those of oil-rich coal, while the volatile matter content was higher, indicating that oxidation treatment reduced impurities and increased volatile components. Elemental analysis showed that the O and N content of oxidized coal increased, while the C and S content decreased, confirming that nitric acid oxidation increases oxygen-containing functional groups and removes sulfur, providing more metabolic substrates for bacterial degradation.

[0049] Table 1. Industrial analysis, elemental analysis, and tar yield of coal samples.

[0050]

[0051] 1.2 Isolation, purification and identification of strains

[0052] Coal samples were collected under aseptic conditions. Surface microorganisms were eluted by shaking with sterile buffer and sonication, followed by serial dilution. Target strains were first enriched using a medium with coal powder as the sole carbon source. The diluted samples were then plated and cultured. Characteristic single colonies were repeatedly streaked for purification, and morphological identification was completed by observing colony morphology and cell staining. Active strains were screened based on growth status and functional indicators through primary and secondary screening. Physiological and biochemical tests were then performed, and the 16S rRNA gene was amplified for molecular biological identification to ultimately determine the strain species. Superior strains were preserved by low-temperature slant culture and glycerol cryopreservation for future use.

[0053] 1.3 Coal Biodegradation Process

[0054] 0.4g sterile oil-rich coal and 12mL OD 600Mix 1.0g of Bacillus anthracis YL2 (abbreviated: YL2) bacterial suspension with an appropriate amount of culture medium, and incubate at 35°C. o The samples were cultured in a constant-temperature shaker at 140 rpm. After the degradation experiment, the supernatant was obtained by centrifugation and filtration. The residual coal was washed multiple times with distilled water to remove surface hyphae, dried, weighed, and the degradation rate was calculated. A blank control group (coal sample + culture medium) and a bacterial suspension control group (bacterial suspension + culture medium) were set up. Each experiment was performed three times to eliminate interference.

[0055] The effects of four factors—degradation time, bacterial culture volume, coal addition amount, and temperature—on the degradation rate were investigated. The optimal process conditions for microbial degradation of oil-rich coal were determined using single-factor experiments and response surface methodology.

[0056] 1.4 Determination of enzyme activity

[0057] Lignin peroxidase oxidizes resveratrol to produce veratral, and the product exhibits a characteristic absorption peak at 310 nm. The activity of lignin peroxidase can be characterized by changes in absorbance. Unit definition: One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 nmol of resveratrol per minute per mL of liquid sample.

[0058] Catechol 2,3-dioxygenase is a copper-containing oxidase that, under certain temperature and pH conditions and in the presence of aerobic conditions, catalyzes the oxidation of catechol to produce colored substances. The absorbance of the colored substances at 410 nm per unit time is positively correlated with the enzyme activity. The OD value of the reaction system is changed at 410 nm using a spectrophotometer, and the activity of catechol 2,3-dioxygenase is determined by the change in OD value.

[0059] Laccase breaks down the substrate 2,2'-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abbreviated as ABTS) to generate ABTS radicals. The absorption coefficient of ABTS radicals at 420 nm is much greater than that of the substrate ABTS. The laccase activity can be calculated by measuring the rate of increase of ABTS radicals.

[0060] 1.5 Molecular docking

[0061] Based on the results of infrared, nuclear magnetic resonance, and XRD measurements, as well as the findings of Shi et al., thymol was selected as the model compound for oil-rich coal. The structures of lignin peroxidase, catechol 2,3-dioxygenase, and laccase were obtained from the RCSB protein database and used as docking models. Detailed information is shown in Table 2. Docking experiments were performed using the Lamarckian genetic algorithm (LGA) in AutoDock 4.2. The alkaline protease structure (PDB ID: 1AKL) was retrieved from the RCSB protein database as the docking model. Hydrogen atoms and Gasteiger charges were added to the alkaline protease using the Auto Dock tool. The single bond of benzoic acid was set to be rotatable. By setting the x, y, and z dimensions of the grid box to 126, 102, and 126 respectively, and the grid spacing to 0.375 Å, it was ensured that the binding sites of the enzyme and thymol, after carrying Gasteiger charges, covered the main cavity of the enzyme. LGA parameters were: GA population size, 150; maximum number of energy assessments, 2.5 × 10⁻⁶. 6 Algebra, 2.7 × 10 3 A total of 100 runs were performed. The lowest energy consistency was selected based on their rank and score. Pymol was used to visualize docking posture and the interaction between thymol and alkaline protease.

[0062] Table 2 Information on the three enzymes

[0063]

[0064] 2. Structure and Discussion

[0065] 2.1 Strain Identification

[0066] like Figure 1 As shown, (a) plate culture; (b) 3D SEM; (c) agarose gel electrophoresis; (d) phylogenetic tree; from Figure 1 (a) Based on the plate culture characteristics, the target strain exhibited distinct colony morphology on LB medium: round colonies, moist and smooth surface, regular edges, and pale yellow color. The cell morphology was observed using SEM. Figure 1 (b). From Figure 1 (b) The cells are clearly shown to be rod-shaped with a size of 2µm × 1.5µm.

[0067] Results were obtained by agarose gel electrophoresis and 16S rDNA gene sequence homology comparison. Figure 1 (c) and 1(d). From Figure 1(d) It can be seen that the bacteria share 100% homology with B. anthracis. Therefore, this strain is named Bacillusanthracis YL2 (abbreviation: YL2). These microscopic morphological characteristics are not only a key supplement to the accurate identification of the bacterial species, but also provide intuitive microscopic structural evidence for subsequent research on the interaction mechanism between the bacterial species and oil-rich coal.

[0068] 2.2 Degradation effect

[0069] like Figure 2 The diagram shows the degradation effect of YL2 on oil-rich coal, where (a) bacteria + oxidized coal; (b) bacteria + raw coal; (c) bacteria; (d) oxidized coal; (e) raw coal; and (f) culture medium. Figure 2 (a) has a better degradation effect than Figure 2 (b) This indicates that oily coal pretreated with nitric acid oxidation is more easily degraded by bacteria. This is because oxidation pretreatment increases the number of oxygen-containing functional groups (such as carboxyl and hydroxyl groups) in the coal, providing bacteria with more attachment sites and metabolic substrates, thus reducing the difficulty of degradation. In addition, the pure bacterial control group (c) and the pure coal sample groups (d) and (e) showed no obvious coal degradation-related characteristics, further eliminating the interference of bacterial metabolites and coal dissolved in the culture medium on the experimental results, highlighting the substrate specificity of bacterial degradation of oily coal.

[0070] 2.3 Determination of process conditions

[0071] 2.3.1 Analysis of Single-Factor Experiment Results

[0072] like Figure 3 As shown, (a) bacterial culture volume; (b) coal addition amount; (c) degradation time; (d) temperature; and (e) culture method. Bacterial culture volume, coal addition amount, degradation time, temperature, and culture method all significantly affected the lignite degradation rate. As the bacterial culture volume increased from 2 mL / 50 mL to 10 mL / 50 mL, the degradation rate increased from 12.4% to 76.1%. After further increasing the bacterial culture volume, the degradation rate remained relatively stable. Figure 3 (a) indicates that an appropriate inoculum size can increase the number and metabolic activity of microorganisms, while excessive inoculum size has a limited effect on promoting degradation, possibly due to limitations in the bacterial growth environment. The amount of coal added shows a trend of first increasing and then decreasing with the degradation rate. Figure 3 (b) The degradation rate reached its maximum (76.1%) when the coal addition was 0.4 g / 50 mL. Further increasing the coal addition led to a significant decrease in the degradation rate, possibly because excessively high coal slurry concentration inhibited the growth of strain YL2. With prolonged degradation time, the lignite degradation rate continued to increase and stabilized after day 10. Figure 3(c) Extending the degradation time did not significantly change the degradation rate, indicating that the easily degradable components in the system were basically consumed, the degradation process entered a plateau phase, and the nutrients in the culture medium were completely consumed by YL2. Temperature has a significant impact on microbial degradation. Figure 3 (d) The highest degradation rate (76.1%) was obtained at 35℃. Increasing or decreasing the temperature decreased the degradation rate, indicating that the growth and metabolism of the strain were most vigorous at this temperature. Furthermore, the degradation rate under shaker culture conditions was significantly higher than that under static culture conditions. Figure 3 (e) indicates that sufficient oxygen supply and good mass transfer conditions are conducive to full contact between microorganisms and coal particles, thereby promoting the biodegradation of lignite. Therefore, the optimal single-factor conditions were determined to be: bacterial culture volume 10 mL / 50 mL, coal addition amount 0.4 g / 50 mL, degradation time 10 days, culture temperature 35℃, and shaker culture, providing experimental basis for subsequent process optimization.

[0073] 2.3.2 Analysis of Response Surface Experiment Results

[0074] The results of the single-factor experiments show that degradation time, bacterial volume, coal addition amount and temperature are important factors affecting the degradation rate. Therefore, based on the results of the single-factor experiments, the BBD model was used to study these four factors affecting the degradation rate and to analyze the interaction between the factors. The degradation rate was used as the experimental response value for optimization. The experimental design is shown in Table 3 and the results are shown in Table 4.

[0075] Table 3 Response Surface Experimental Design

[0076]

[0077] Table 4. Response Surface Experimental Design and Results

[0078]

[0079] Based on the results of the BBD experimental design, the following multivariate quadratic response surface regression model was obtained after regression fitting of four influencing factors: degradation time, bacterial culture volume, coal addition amount, and temperature:

[0080] Y=-1624.57315+173.475X1+28.92917X2+78.15741X3+37.635X4-0.2875X1 X2-1.08333X1X3+0.04X1X4-0.083333X2X3-0.03X2X4-0.15X3X4-8.49583X1 2 -1.208333X2 2 -76.34259X3 2 -0.53633X4 2 ;

[0081] The R-value of the quadratic regression coding equation 2 =0.9723, indicating that the model fits the experimental results and predicted values ​​well within the experimental range; the corrected coefficient of determination is 0.9400, indicating that the equation has a high degree of fit. The BBD experimental design and result model are shown in Table 5 and Figure 4 As shown, the model parameters indicate that the model can replace the actual values ​​for process optimization and prediction of the degradation rate of lignite by bacteria.

[0082] Table 5. Analysis of Variance for the Regression Model

[0083]

[0084] Note: *Significant (P<0.05), **Highly significant (P<0.01)

[0085] Analysis of the response surface, contour plots, and regression equations revealed that the optimal conditions for the degradation of oil-rich coal from Dananhu by YL2 bacteria were: degradation time of 10.09 days, bacterial solution volume of 10.32 mL, coal addition of 0.4 g, and temperature of 35.11℃. Simulation experiments conducted under these optimized conditions showed that the degradation rate of oil-rich coal from Dananhu by YL2 strain was 76.5124%, with a confidence level as high as 95%. Therefore, this model can effectively predict the degradation rate of oil-rich coal by YL2 bacteria.

[0086] 2.4 Analysis of Enzyme Activity Results

[0087] Figure 5 This study demonstrates the dynamic changes in the activities of key enzymes during the microbial degradation of low-rank coal, including (a) total protease, (b) lignin peroxidase, (c) catechol 2,3-dioxygenase, and (d) laccase. This provides direct evidence for elucidating the mechanism of synergistic degradation of oil-rich coal by bioenzymes. During coal biodegradation, the A450 value initially increases and then levels off, representing the degradation kinetics of lignite and consistent with the trend of total protease activity. Figure 5 (a). These results indicate that the degradation process of oil-rich coal is associated with lignin peroxidase, catechol 2,3-dioxygenase, and laccase produced by YL2 bacteria.

[0088] Depend on Figure 5 (b) It can be seen that the activity of lignin peroxidase during coal biodegradation shows a trend of first increasing and then decreasing, reaching a peak of 43.43 U / mL on day 7, significantly higher than the control group (coal-free system), and the activity is maintained for a longer time. This indicates that the coal system can enhance the secretion of lignin peroxidase by YL2, efficiently breaking the aromatic ring side chains and ether bonds in coal, and achieving the initial depolymerization of coal macromolecules. Figure 5As shown in (c) and 5(d), the activities of catechol 2,3-dioxygenase and laccase exhibited the same trend, reaching their maximum values ​​on day 10, at 5.99 U / mL and 558.89 U / mL, respectively. This indicates that YL2 bacteria can effectively degrade intermediate products generated from coal depolymerization, opening aromatic rings into easily metabolized aliphatic compounds, thus promoting the continuous degradation process. Therefore, in the early stage of coal biodegradation, lignin peroxidase plays a major role, while in the middle and later stages, catechol 2,3-dioxygenase and laccase play a more significant role. The three enzymes synergistically degrade oil-rich coal, improving biodegradation efficiency.

[0089] 2.5 Analysis of Molecular Docking Results

[0090] like Figures 6-8 The molecular docking results of lignin peroxidase, catechol 2,3-dioxygenase, and laccase with thymol were presented, elucidating the binding mode and interaction mechanism of thymol with these three enzymes. Figure 5 (a) Docking posture of thymol and lignin peroxidase, 5(b) Binding orientation of thymol and lignin peroxidase, 5(c) Action site of thymol and lignin peroxidase, 5(d) Interaction between thymol and lignin peroxidase.

[0091] 6(a) Docking posture of thymol and catechol 2,3-dioxygenase; 6(b) Binding orientation of thymol and catechol 2,3-dioxygenase; 6(c) Action site of thymol and catechol 2,3-dioxygenase; 6(d) Interaction between thymol and catechol 2,3-dioxygenase; 7(a) Docking posture of thymol and laccase; 7(b) Binding orientation of thymol and laccase; 7(c) Action site of thymol and laccase; 7(d) Interaction between thymol and laccase.

[0092] Depend on Figures 6-8 It is known that thymol can be stably embedded in the active pockets of three proteases, and its overall conformation is highly compatible with the hydrophobic cavity of the enzyme, providing a spatial basis for its catalytic reaction. Figure 5-7 This indicates that the benzene ring and hydroxyl group of thymol are completely enclosed by the lattice structure of the active site, with no obvious exposure, confirming that its binding orientation is reasonable, its steric hindrance is small, and it possesses the structural conditions for stable binding. Secondly, the binding of the three enzymes to thymol is driven by multiple intermolecular forces working together, including hydrogen bonds, hydrophobic interactions, electrostatic interactions, and other non-covalent interactions, which together stabilize the complex conformation and regulate the binding behavior.

[0093] The binding energies of thymol with lignin peroxidase, catechol 2,3-dioxygenase and laccase are shown in Table 6, with binding energies of -4.15 kcal / mol, -3.75 kcal / mol and -4.03 kcal / mol, respectively.

[0094] Table 6. Comparison of binding energies of thymol with three enzymes (unit: kcoal / mol)

[0095]

[0096] The binding energies are all negative, indicating that intermolecular interactions and docking processes can occur spontaneously. Thymol has the highest binding energy with lignin peroxidase, indicating a stronger binding effect and the most stable structure, suggesting that thymol is more easily degraded by lignin peroxidase.

[0097] 2.6 Characterization of Degradation Products

[0098] (2.6.1) Temperament Analysis

[0099] according to Figure 9 It was found that after degradation by YL2, various small-molecule products, including oxygen-containing organic matter, alkanes, aromatics, and alkenes, were detected in the degradation liquid of oxidized coal. Among them, oxygen-containing compounds accounted for the highest proportion, reaching 57.77%. The degradation of oil-rich coal by the YL2 strain is mainly based on the depolymerization of aliphatic structures and partial aromatic ring cleavage, which can convert the organic matter in the coal into low-carbon-number small-molecule substances. At the same time, a large number of oxygen-containing organic compounds are present in the products. Combined with the infrared and nuclear magnetic resonance characterization results, it was confirmed that the degradation process is accompanied by alkyl side chain oxidation, hydroxylation, and carboxylation reactions, further generating alcohols, esters, phenols, and amides. Aromatics and phenols are all present in the form of low-ring small molecules, indicating that the aromatic structure and heteroatom structure only undergo mild cleavage, without deep cleavage. Overall, GC-MS directly proves from the product composition level that YL2 can achieve efficient depolymerization of large molecules in oil-rich coal through selective cleavage of carbon chains and oxidation of side chain functional groups, generating high-value-added products mainly composed of small-molecule oxygen-containing organic matter and alkanes.

[0100] (2.6.2) 3D-EEM fluorescence spectroscopy analysis

[0101] The degradation effect of YL2 bacteria on oil-rich coal was analyzed using 3D-EEM fluorescence spectroscopy. Based on previous studies, the 3D-EEM fluorescence spectrum was divided into five distinct regions. Region I (Ex=200-250nm / Em=240-330nm) is associated with tyrosine-like substances; Region II (Ex=200-250nm / Em=330-380nm) corresponds to tryptophan-like components; Region III (Ex=200-250nm / Em=380-600nm) represents fulvic acid-like substances; Region IV (Ex=250-420nm / Em=240-380nm) reflects soluble microbial metabolites; and Region V (Ex=250-420nm / Em=380-600nm) is characterized by humic acid-like components.

[0102] Figure 10The 3D-EEM fluorescence spectra of the degradation solution at different days are shown, including (a) 1 day; (b) 5 days; (c) 10 days; and (d) 0 days. The initial sample exhibits high fluorescence intensity in regions I, II, and IV. Compared with the control group... Figure 10 (d) Compared to the previous method, after one day of adding coal, the fluorescence intensity in zones I, II, and IV decreased significantly. This indicates that in the early stages of degradation, YL2 preferentially utilized easily degradable tyrosine-like and tryptophan-like substances and soluble microbial metabolites in the solution, and easily degradable small-molecule organic matter in the system was preferentially consumed. As the degradation time increased, the fluorescence signals in zones I, II, and IV continued to decrease and almost disappeared completely, while the fluorescence intensity in zone V increased significantly. This indicates that in the later stages of degradation, YL2 bacteria mainly transformed the organic matter in oil-rich coal, and the macromolecular structure of oil-rich coal was gradually destroyed, generating more humic acid-like substances. The 3D-EEM fluorescence spectral parameters are shown in Table 7. The fluorescence index (FI) characterizes the source of soluble organic matter (DOM) in the solution. According to Table 7, when the degradation day is 0, FI = 1.911 > 1.9, indicating that the DOM is mainly produced by the YL2 strain. As the degradation time increases, the FI value is between 1.4 and 1.9, indicating that the DOM is a product of the YL2 strain degrading oil-rich coal. The HIX value reflects the degree of humification of DOM. An HIX value < 4 indicates that the DOM is not highly humified, is mainly autogenic, and is easily utilized by microorganisms. Table 7 shows that the HIX value increases with increasing degradation time, indicating an increase in the content of humic acid-like substances in the degradation products.

[0103] Table 7 3D-EEM parameters of different degradation solutions

[0104]

[0105] Therefore, the 3D-EEM fluorescence spectroscopy results clearly revealed the dynamic evolution of soluble organic components during the degradation process: in the early stage of degradation, rapid consumption of tyrosine-like substances, tryptophan-like substances, and soluble microbial metabolites predominated, while in the later stage, significant generation and accumulation of humic acid-like substances occurred. This process reflects the degradation and transformation mechanism of oil-rich coal by YL2 bacteria, providing an important basis for evaluating the degradation effect of microorganisms on coal.

[0106] (2.6.3) 13 C-NMR analysis

[0107] Figure 11 Among them, (a) raw coal; (b) oxidized coal; and (c) residual coal, the NMR structural parameters of the coal samples in Table 8 show significant differences in structural characteristics between oil-rich coal, oxidized coal, and residual coal. Regarding the aromatic structure, the total proportion of aromatic carbon (f...)... aThe trend shows that raw coal (63.42%) > residual coal (59.83%) > oxidized coal (52.05%). Nitric acid oxidation reduces fa due to the destruction of aromatic ring side chains in raw coal and the introduction of oxygen-containing functional groups, while microorganisms preferentially degrade aliphatic structures, which increases the relative proportion of aromatic carbon.

[0108] Table 8. NMR parameters of different coal samples

[0109]

[0110] Note: f a Total aromatic carbon; f a C carbonyl carbon; f a ’ Aromatic ring carbon; f al Total fatty acids and carbon; f al * CH3; f al H CH or CH2; f al O CO.

[0111] Carbonyl or carboxyl carbon percentage (f) a C The order of carbon dioxide concentrations (FAC) was: residual coal (4.11%) > oxidized coal (3.33%) > raw coal (1.04%), reflecting the stepwise regulatory effect of nitric acid oxidation pretreatment and YL2 bacterial degradation on oxygen-containing functional groups (especially carbonyl and carboxyl groups) in coal. From raw coal to oxidized coal, FAC increased from 1.04% to 3.33%, mainly due to the strong oxidizing effect of nitric acid. As a strong oxidant, nitric acid preferentially attacks the side chains and heteroatom functional groups (such as hydroxyl and ether bonds) of aromatic rings in coal. By oxidizing and breaking side chain chemical bonds, it converts some aliphatic carbons and aromatic ring edge carbons into oxygen-containing functional groups such as carbonyl (C=O) and carboxyl (-COOH), directly increasing the total amount of carbonyl / carboxyl carbon in coal and providing more metabolic substrates and attachment sites for subsequent microbial degradation.

[0112] From oxidized coal to residual coal, f a C The value increased from 3.33 to 4.11, f a ’ The value increased from 48.72 to 55.72, f alThe decrease from 47.95 to 40.17 is due to the "selective retention" effect of microbial degradation. Enzymes secreted by YL2 bacteria (such as alkaline protease and catechol 2,3-dioxygenase) preferentially decompose aliphatic chains and some easily degradable oxygen-containing functional groups (such as aliphatic hydroxyl groups and ether bonds) in coal. Simultaneously, during degradation, microorganisms may further oxidize other types of carbon (such as the methylene carbon in the aromatic ring side chain) to carbonyl or carboxyl groups through enzymatic reactions. The combined effect of these two processes leads to a significant degradable carbon loss in the coal, while the proportion of more stable -COOH and C=O groups increases, ultimately reducing the f... a ’ and f a C Increase, f al reduce.

[0113] (2.6.4) FTIR analysis

[0114] Figure 12 FTIR analysis of three coal samples revealed the regulatory mechanisms of nitric acid oxidation pretreatment and YL2 degradation on the functional group structure of oil-rich coal. (a) represents the coal sample; (b) represents the structural parameters. Comparison of the characteristic infrared absorption peaks of oil-rich coal, oxidized coal, and microbially degraded coal showed that the peaks were concentrated in the 3400-3450 cm⁻¹ range. -1 1730–1740 cm -1 The peaks at 2920-2930 cm⁻¹ represent the OH stretching vibration peak and the C=O stretching vibration peak, respectively. Their intensities show a trend of > for oxidized coal > for oil-rich coal, indicating that nitric acid oxidation can introduce a large number of oxygen-containing functional groups such as hydroxyl, phenolic hydroxyl, and carboxyl groups into coal, significantly improving the hydrophilicity of the coal surface, providing more active sites for microbial attachment and metabolism, and promoting the biodegradation of coal. Oxidized coal and FYRC show peaks at 2920-2930 cm⁻¹. -1 The weakening of the absorption peak indicates that both nitric acid oxidation and microbial degradation can effectively break the fatty side chains in coal. FYRC is located at 1600-1620 cm⁻¹. -1 The presence of a strong absorption peak indicates that the aromatic structure is not easily degraded by microorganisms. Aal / Aar is used to characterize the length and branching degree of aliphatic side chains. After YL2 degradation, the Aal / Aar value of oxidized coal decreased from 0.118 to 0.107, indicating that the aliphatic side chains in the oxidized coal were degraded by YL2, leading to a reduction in aliphatic side chain branching. Io represents the oxygen enrichment degree of the coal sample. After YL2 degradation, the Io of oxidized coal increased, indicating that the biodegradation process of coal is an oxidation process, possibly involving reactions such as hydroxylation and carboxylation. AR and DOC represent the degree of condensation of the aromatic structure in coal. After YL2 degradation, the DOC value of oxidized coal increased, which may be due to the degradation of side chains in the oxidized coal, leading to an increase in the content of aromatic structures.

[0115] Based on the above analysis, it is evident that the biodegradation process of coal involves the addition of oxygen and the reduction of carbon elements. Therefore, FTIR analysis, from the perspective of functional group evolution, confirms that nitrate oxidation reduces the structural stability of coal by increasing oxygen and opening rings, while the YL2 strain selectively degrades aliphatic chains and some aromatic rings through the secretion of extracellular enzymes, ultimately achieving efficient biodegradation of oil-rich coal. This result is corroborated by the results of enzyme activity, molecular docking, and product characterization.

[0116] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for using Bacillus anthracis as a microorganism to degrade oil-rich coal, characterized in that, Includes the following steps: Step 1: Preparation and oxidation pretreatment of coal samples Coal samples were collected from the mining area, quickly wrapped and sealed with plastic wrap, packaged in sample bags, and then ground and screened. Industrial analysis, elemental analysis, and tar content were determined according to national standards. The coal sample was pretreated with nitric acid solution for oxidation, washed with deionized water, and dried to obtain oxidized coal. Step 2, strain isolation, purification and identification Oxidized coal pretreated with nitric acid was subjected to agitation with sterile buffer, ultrasonic elution to remove surface microorganisms, and then serially diluted. The target strain was enriched in a culture medium with oxidized coal as the sole carbon source. The diluted solution was spread on plates for culture, and characteristic single colonies were picked and repeatedly streaked for purification. Active strains were screened based on growth status and functional indicators through primary and secondary screening, followed by physiological and biochemical tests, and molecular biological identification by amplifying the 16S rRNA gene. The resulting strain was named Bacillus anthracis YL2. Step 3, Coal Biodegradation Sterilize the oxidized coal in a high-pressure steam sterilizer for 15 minutes to obtain sterile oxidized coal. Then, mix it with Bacillus santhracis YL2 and an appropriate amount of culture medium, and incubate at 25-45°C. o Culturing in a constant temperature shaker at 130-140 r / min for 1-14 days; After the degradation experiment, the supernatant was obtained by centrifugation and filtration; the residual coal was washed multiple times with distilled water to remove surface hyphae, dried and weighed, and the degradation rate was calculated.

2. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 1, characterized in that, In step 1, the concentration of nitric acid solution is 8 mol / L. The coal sample and nitric acid solution are placed in a beaker and reacted in a fume hood for 48 h. Then, the sample is washed with deionized water until the pH reaches 6-7, and then dried in a drying oven at 105℃ for 8 h.

3. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 2, characterized in that, The strain obtained in step 2 exhibits the following morphology on LB medium: round colonies, moist, smooth, with neat edges, and pale yellow in color; the bacterial cells are rod-shaped and 2 µm × 1.5 µm in size.

4. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 3, characterized in that, In step 3, the OD of Bacillus anthracis YL2 600 =0.5~1.

0.

5. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 4, characterized in that, Step 3, the optimal single-factor conditions are: when the liquid culture medium volume is 50 mL, the bacterial culture volume is 10 mL, the coal addition amount is 0.4 g, the degradation time is 10 days, the culture temperature is 35 ℃, and the culture is carried out in a shaker.

6. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 4, characterized in that, Based on the results of single-factor experiments, the BBD model was used to study the four factors affecting the degradation rate: degradation time, bacterial culture volume, coal addition amount, and temperature, and the interaction between these factors was analyzed. The multivariate quadratic response surface regression model obtained after regression fitting of the above four influencing factors is as follows: Y = -1624.57315 + 173.475X1 + 28.92917X2 + 78.15741X3 + 37.635X4 - 0.2875X1X2 - 1.08333X1X3 + 0.04X1X4 - 0.083333X2X3 - 0.03X2X4 - 0.15X3X4 - 8.49583X1 2 -1.208333X2 2 -76.34259X3 2 -0.53633X4 2 ; Where X1 is the degradation time, X2 is the bacterial solution volume, X3 is the amount of coal added, and X4 is the temperature; The R-value of the quadratic regression coding equation 2 =0.9723, with a corrected coefficient of determination of 0.9400.

7. The method for using Bacillus anthracis as a microorganism to degrade oil-rich coal as described in claim 5, characterized in that, Analysis using SAS 8.0 software, based on response surface methodology, contour plots, and regression equations, revealed that the optimal conditions for the YL2 strain to degrade oil-rich coal were: degradation time 10.09 days, bacterial culture volume 10.32 mL, coal addition 0.4 g, and temperature 35.11 ℃. Simulation experiments confirmed that the YL2 strain achieved a degradation rate of 76.5124% for oil-rich coal, with a confidence level as high as 95%.

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