A method for the combined degradation of low-rank coal by fungi and bacteria, a method for detecting its degradation rate, and a method for evaluating the environmental safety of the degradation liquid

Abstract

The present invention belongs to the technical field of microbial transformation of coal, and provides a method for the combined degradation of low-rank coal by fungi and bacteria, a method for detecting the degradation rate, and a method for evaluating the environmental safety of the degradation liquid. The fungus is Cunninghamella elegans F11, and the bacteria is Bacillus B1; the specific method of the combination of fungi and bacteria is: F11 is inoculated into a PDA solid culture medium, inverted and cultured at a constant temperature of 30°C for 5 days, and the mycelium is spread on the surface of the culture medium for use; B1 is inoculated into an LB solid culture medium, inverted and cultured at a constant temperature of 30°C for 2 days, and the colony is spread on the plate for use; the already cultured F11 and B1 are transferred to the sterilized LB liquid culture medium, and the culture is shaken at a constant temperature of 30°C and 150rpm for 4 days, and then sterilized nitric acid is added to oxidize the coal, and the culture is continued at 30°C and 150rpm until the absorbance A450 is stable. The degradation effect is determined according to the content of heavy metals in the degradation liquid, and the higher the heavy metal content, the higher the degradation rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial conversion of coal, and specifically relates to a method for degrading low-rank coal by combining fungi and bacteria, a method for detecting its degradation rate, and a method for evaluating the environmental safety of degradation liquid. Background Art

[0002] my country has 5.5 billion mu of degraded soil that needs to be repaired. In the context of reducing the use of chemical fertilizers, humic acid has become a recognized green and efficient soil conditioner. Weathered coal and lignite are currently the main raw materials for producing humic acid. Coal can be degraded under the action of aerobic microorganisms. Studies have shown that the main product of aerobic microbial degradation of coal is humic acid. Compared with chemical extraction methods that require strong acids and alkalis, biological methods have the advantages of being green and mild.

[0003] my country is rich in coalbed methane resources, with geological resources of about 26 trillion cubic meters of coalbed methane at a depth of less than 2,000 meters. Coal can produce gas under the action of anaerobic microorganisms. The basic principle is that anaerobic microorganisms use part of the organic matter in coal to produce methane, thereby increasing the production of coalbed methane and converting coal into low-carbon clean energy. In order to promote the degradation of coal and increase the yield of biogas, domestic and foreign scholars have tried various pretreatment methods, such as microbial pretreatment, external electric field enhancement, hydrogen peroxide oxidation pretreatment, supercritical carbon dioxide extraction, sodium hydroxide pretreatment, nitric acid oxidation pretreatment, etc. White rot fungi and Pseudomonas commonly used in microbial pretreatment are also common strains in aerobic microbial degradation of coal.

[0004] Heavy metals refer to metals with an atomic weight > 55. Heavy metals can pollute the environment and accumulate in the human body through the food chain, thus endangering human health. Heavy metals are still found in coal in all provinces and regions of my country. The health risk of coal-bearing aquifers caused by heavy metals is greater than that of other aquifers. A certain amount of heavy metals are released into the soil during the stacking of coal gangue mountains, and heavy metal pollution of the atmosphere is caused during the burning of coal. Issues such as the transformation and emission of heavy metals during coal processing and utilization require special attention. The adsorption of metal elements by bacteria and fungi, and the promotion of the release of iron, manganese and cadmium in coal gangue by microorganisms all indicate that the changes in heavy metals during coal degradation need to be paid attention to.

[0005] In summary, the study of coal microbial degradation can promote the clean and low-carbon utilization of coal, which is of great significance for achieving the "dual carbon" goal, especially the aerobic microbial degradation of coal, which can be used as a pretreatment step for coal microbial gasification, or as an independent process to produce soil conditioners and other products. Studying the changes in heavy metal content during coal microbial degradation, conducting environmental pollution assessment and human health assessment are of certain significance for understanding the environmental impacts that may occur during the application of technology.

[0006] Existing research shows that microorganisms that can degrade coal include: bacteria such as: Bacillus spp. such as Bacillus polymyxa ( Paenibacillus polymyxa )、Bacillus megaterium ( Bacillus megaterium ), Bacillus pumilus ( Bacillus pumilus ), Bacillus subtilis ( Bacillus subtilis ), Bacillus cereus ( Bacillus cereus ), Bacillus thuringiensis ( Bacillus thuringiensis ), etc., Streptomyces ( Streptomyces ) such as Streptomyces flavoscens ( Streptomyces flaveolus ), Streptomyces sarcomycinicus ( Streptomyces zoamyceticu ), Streptomyces chlorosporus ( Streptomyces vridosporus ), Streptomyces luteus ( Streptomyces flavovirens ), Streptomyces xitangii ( Streptomyces setonii )、Streptomyces thomsoniae( Streptomyces badius ), Pseudomonas species such as Pseudomonas putida ( Pseudomonos putida ), Pseudomonas sphaeroides ( Rhodopseudomonas spheroids ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Staphylococcus aureus ( Staphylococcusaureus ), Corynebacterium bovis of the genus Corynebacterium ( Corynebacterium renale ), Escherichia coli ( Escherichia coli ), Arthrobacter spp. Arthrobacter ), Flavobacterium ( Flavobacterium ) etc. Pseudomonas ( Pseudomonas ) can use the organic components in hard coal as the only carbon source and energy source for its growth, and the hard coal partially dissolves to form a colored liquid.

[0007] Fungi associated with coal degradation have evolved from the original Versicolor ( polyporousversicolor )、Mountain Porites ( Poriamonticolar ) expanded to the present Phanerochaete chrysosporium ( Phanerochaete chrysosporium )、Candida ( Candida sp.), Candida ( Candida mycoderma )、Aspergillus( Aspergillus ), Aspergillus terreus ( Aspergillus terreusl )、Aspergillus terreus Aspergillus terricola ), Paecilomyces albus ( Simplicillium lanosoniveum )、Penicillium( Penicillium )、Penicillium wilt( Penicillium citrinum )、Penicillium funiculosum( Penicillium fumiculosum ), Penicillium wackerii Penicillium waksmanii )、Penicillium decumbentum Penicillium decumbens )、Penicillium chrysogenum( Penicillium citreoviride Biourge ), Trichoderma koningii ( Trichoderma pseudokoningii )、Trichoderma viride ( Trichoderma viride ), Fusarium / Fusarium spp. ( Fusariumsp.)、Mucor ( Mucor )、Brown rot fungi( brownrot fungus ), Versicolor versicolor / Colored Coriolus versicolor ( Trametes versicolor ), Neurospora crassa / Red bread mold ( Neurospore )、Rhizopus ( Rhizopus ) etc. polyporousversicolor ) and Pleurotus eryngii ( Poriamonticolar ) can oxidize lignite into black droplets, and the generated droplets can dye the SMB culture medium black.

[0008] There are two types of bacterial fermentation. Pure bacterial fermentation studies the degradation of coal by a single fungus or bacteria, while mixed bacterial fermentation studies the degradation of coal by a combination of fungi and bacteria. Pure bacterial fermentation involves only a single bacterial species, and the action process is clear, which is the most common mode of action.

[0009] In order to improve the efficiency of coal degradation by microorganisms, it is often considered to mix different strains of bacteria. However, there is no consensus on the effect of mixing.

[0010] Since the rise of coal microbial degradation in the 1980s, after nearly 40 years of development, hundreds of microorganisms related to coal degradation have been isolated, and the research covers various types of coal such as lignite, hard coal, weathered coal, and nitric acid oxidized coal. Studies have shown that the higher the oxygen content and the lower the degree of metamorphism, the easier it is to be degraded by microorganisms. The mechanism of coal microbial degradation can be summarized as A alkaline substance mechanism (alkaline substance), B biological oxidase (biological oxidase), C chelating agent (chelating agent), D surfactant (detergent), and E esterase (esterase) mechanism.

[0011] The alkaline substance mechanism is that microorganisms degrade coal by secreting alkaline substances such as ammonia and alkaline peptides to neutralize the carboxylic acid functional groups in coal. The biological oxidase and esterase mechanism is that microorganisms secrete manganese peroxidase, lignin peroxidase, laccase, hydrolase, etc. to promote the breaking of chemical bonds in the coal structure to achieve degradation. The chelating agent mechanism is that biological chelating agents such as ammonium oxalate produced by microorganisms combine with heavy metal cations in coal to destroy metal bridge bonds. The role of surfactants is to reduce the surface tension of coal, and generally do not work alone. The mechanism of microbial action on coal is complex. No matter which microorganism acts on which type of coal, it is often through multiple mechanisms.

[0012] How to judge whether coal has been degraded by microorganisms is the basis of the experiment. Generally speaking, it can be judged by naked eyes or with the help of instruments.

[0013] When coal degradation is carried out on solid culture medium, the degradation phenomenon can be observed with the naked eye. Figure 1 The formation of A-1 to A-3) is the most commonly used judgment standard, but this requires that the coal has a high degree of degradation and the droplets formed are large, otherwise it is difficult to observe. At the same time, this method of observing droplets will be affected by the condensation of water droplets on the surface of the culture medium and cause misjudgment. In addition to observing droplets, studies have shown that the products produced by coal degradation can penetrate into the culture medium and dye the culture medium black. Therefore, it is also possible to observe whether the culture medium is stained black (such as Figure 1 B-1 to B-2) to determine the presence of degradation.

[0014] In liquid culture medium, the absorbance at 450nm, 513nm, 600nm, 650nm, etc. can be measured with a spectrophotometer. By comparing the absorbance of the experimental group containing coal with that of the experimental group without coal, it can be judged whether the coal sample has been degraded. A450, A513, and A600 indicate the formation of humic acid, and A650 indicates the formation of fulvic acid. Studies have shown that the microbial degradation products of coal are acid precipitable, so the supernatant of the experimental group can also be taken, the pH can be adjusted to below 2 to observe whether a large amount of precipitation is generated to determine whether degradation has occurred.

[0015] The determination of the microbial degradation rate of coal can be considered from two perspectives: substrate (coal) and product. There are three common methods.

[0016] The first method is to consider the substrate, i.e. coal, and measure the mass difference of coal before and after the reaction to calculate the degradation rate of coal. This method is simple and direct, so it is often used. However, when the coal powder particles are small, the residual coal after the reaction is difficult to collect, which will lead to large errors in the settlement results, especially when the coal powder is adsorbed and wrapped by fungal hyphae (such as Figure 2 This error is even greater.

[0017] The second method is to consider it from the perspective of the product. As mentioned earlier, the microbial degradation products of coal are acid-precipitable. Therefore, the fermentation liquid after the reaction can be quantitatively collected, precipitated with acid, dried and weighed, and the degradation rate can be measured by comparing the mass of the precipitate to the initial mass of the substrate coal. This method essentially uses the product yield to indirectly reflect the degradation rate of the substrate.

[0018] The third method is also considered from the product perspective. There is a certain linear relationship between the absorbance of the coal degradation liquid and the coal degradation rate. Therefore, the degradation rate can be indirectly reflected by measuring the absorbance. The most commonly used in practice is A450. Compared with the first two methods, measuring the absorbance only requires a small amount of fermentation liquid, so it is more suitable for monitoring the experimental process and used to reflect the stage of the experiment.

[0019] The problem with the above three methods is that when the coal powder particles are fine, the residual coal after the reaction is difficult to collect, resulting in large errors in the calculation results. In addition, both the weight loss method and the precipitation method require that the degradation rate of the coal meet certain requirements, otherwise the mass of the weight loss or precipitation is not enough for accurate quantification. Therefore, in the field of coal biodegradation, the A450 of the degradation liquid has become the main monitoring indicator, and the main instrument relied on is the spectrophotometer. Due to the limitations of the instrument itself, the spectrophotometric method requires that the measurement result be between 0.2 and 0.8. When the absorbance of the sample to be tested is higher than 0.8, it needs to be diluted, especially when the absorbance is less than 0.2, it cannot be accurately quantified. Therefore, a new quantitative method is urgently needed as a supplement.

[0020] Regarding the degradation products, predecessors have conducted research from the aspects of elemental composition, product structure, molecular weight, solubility, etc. Elemental analysis shows that the nitrogen, oxygen, and hydrogen elements of the product are significantly higher than those of the raw coal, while the carbon content is lower than that of the raw coal, indicating that oxidative hydrolysis has occurred. From the infrared spectral characteristics, the structure of the product is similar to that of the raw coal, with an increase in phenol, alcohol, ether or ester compounds in the product, and a possible decrease in the methyl-containing structure. It is generally believed that the molecular weight of the coal product is relatively high, with a molecular weight of about 30~300kDa. The solubility study of the product shows that it is alkali-soluble and acid-precipitated.

[0021] At present, the use of nuclear magnetic resonance spectroscopy, infrared spectroscopy, ultraviolet visible spectroscopy and other means has proved that the biodegradation products of coal are highly similar to humic acid. At present, the production of industrial humic acid is mainly based on chemical extraction and has been widely used in agriculture, animal husbandry, industry and medicine. However, the application research of coal biodegradation products is still mainly focused on agriculture, such as increasing the yield of crops such as soybeans, cabbage, corn, and tomatoes. In addition, the degradation products can also be used as additives for water-coal slurries, as medicines with antioxidant and anti-inflammatory properties, etc. In short, compared with chemically extracted humic acid, the products obtained by microbial dissolution have higher biochemical activity.

[0022] Changes of heavy metals during coal biodegradation: Heavy metals affect microorganisms in two ways: they can damage the structure and function of enzymes, and they are also components of enzymes. Within a suitable concentration range, they can be used as trace elements by microorganisms, but if they exceed a certain concentration, they will be toxic to microorganisms and inhibit their growth and metabolism.

[0023] The influence of microorganisms on heavy metals is also reflected in two aspects. On the one hand, it can promote the transformation of heavy metals or their compounds from bound state to dissolved state, such as bioleaching technology to repair mine soil, bioleaching technology to treat heavy sludge, microbial leaching of copper, etc. On the one hand, it can promote the transformation of heavy metals from dissolved state to bound state, such as the microbial mineralization phenomenon existing in nature, biostabilization method and biofilm method to repair soil pollution.

[0024] In the field of coal biodegradation, the impact of heavy metals on microorganisms is mainly reflected in the use of heavy metals as trace elements for microbial growth. Existing studies have shown that 15 mg / L of Fe 2+ and 0.005 mg / Ni 2+ It promotes the bio-methane production of coal. Research on the influence of microorganisms on heavy metals is mostly focused on the transfer of heavy metal content between the substrate and the fermentation liquid during the reaction process. Existing studies have shown that trace elements such as As, Ba, Co, Ni and Pb are transferred from coal to the fermentation liquid during coal biogasification, resulting in an increase in the mass concentration of trace elements in the fermentation liquid. Zn and Sr are precipitated from coal during the microbial degradation of coal. Microorganisms play an important role in the formation of acidic mine drainage. Metal ions and sulfides in coal gangue dissolve into the water to form acidic mine drainage. Thiobacillus ferrooxidans ( Acidithiobaillus ferroxridans ) promoted the release of three heavy metals in coal gangue.

[0025] The existing strains have limited coal degradation capabilities and are mostly based on pure bacterial fermentation. There is little research on heavy metals in coal degradation liquid, and the environmental problems such as human health risks that may be caused are unknown. These problems have become bottlenecks restricting the industrialization of coal microbial conversion technology. Therefore, finding new strain resources and exploring mixed bacteria combined fermentation processes can expand coal degradation microbial resources and fermentation processes, analyze heavy metals in coal degradation liquid, grasp the potential environmental impacts such as human health risks and environmental pollution, and take preventive measures in advance to reduce adverse effects. Summary of the invention

[0026] In order to solve the problems that the degradation ability of current strains on coal is limited and mostly pure bacteria fermentation is the main method, the degradation effect is low, and heavy metal detection in the degradation liquid is required, the present invention provides a method for degrading low-rank coal by combining fungi and bacteria, a degradation rate detection method, and an environmental safety evaluation method for the degradation liquid. The degradation effect of coal is determined according to the heavy metal content in the coal biodegradation liquid.

[0027] The technical solution adopted by the present invention to solve the technical problem is: a method for degrading low-rank coal by combining fungi and bacteria, wherein the fungus is Cunninghamella elegans ( Cunninghamella elegans) F11, the bacteria is Bacillus sp. B1; the specific method of fungal bacterial combination is as follows: F11 is inoculated into PDA solid culture medium, cultured at a constant temperature of 30℃ and inverted for 5 days, and the mycelium is used after it covers the surface of the culture medium; B1 is inoculated into LB solid culture medium, cultured at a constant temperature of 30℃ and inverted for 2 days, and the colonies are used after they cover the plate; the cultured F11 and B1 are transferred to the sterilized LB liquid culture medium, and cultured at a constant temperature of 30℃ and 150rpm for 4 days, and then sterilized nitric acid-oxidized coal is added, and the culture is continued at 30℃ and 150rpm until the absorbance A450 stabilizes.

[0028] The ratio of punch access to F11 and B1 is: 3-hole F11: 1-hole B1, 2-hole F11: 2-hole B1, 1-hole F11: 3-hole B1 or 50-hole F11: 1-hole B1.

[0029] The nitric acid-oxidized coal is as follows: low-rank coal is crushed and screened to 80-120 mesh, and dried at 80°C for 48 hours; 150 mL of a nitric acid aqueous solution with a volume ratio of 1:1 is added to 100 g of the crushed and screened coal sample, and the reaction is carried out in room temperature water, while continuously stirring until the yellow smoke disappears, and then the reaction is allowed to stand for 24 hours, the coal is washed with pure water until the pH of the supernatant is greater than 6, and then the coal sample is dried at 80°C for 72 hours.

[0030] A method for detecting the biodegradation rate of coal, utilizing the method of jointly degrading low-rank coal by fungi and bacteria to biodegrade the coal, using a spectrophotometer to measure the absorbance A450 of the degradation liquid at 450nm to determine the stage of coal degradation; after the A450 value is stable, the microbial degradation liquid of the coal is centrifuged, the supernatant is taken to determine the heavy metal Cr, As, Pb content therein, and the biodegradation rate of the coal is determined by the heavy metal content.

[0031] The specific steps for determining the heavy metal content are as follows: after the A450 value of the bacterial solution is stable, the co-culture fermentation solution is centrifuged at 10,000 rpm for 10 minutes, the supernatant is collected, and the contents of Cr, As, and Pb in the supernatant are measured in accordance with the "Determination of 65 Elements in Water Quality by Inductively Coupled Plasma Mass Spectrometry" (HJ 700-2014); the relative size of the degradation rate is determined by the size of the absorbance A450 of the degradation solution and the content of heavy metals Cr, As, and Pb.

[0032] The heavy metal content is determined by atomic fluorescence spectrometry or inductively coupled plasma mass spectrometry (ICP-MS).

[0033] A method for evaluating the environmental safety of a degradation liquid obtained by the combined degradation of low-rank coal by fungi and bacteria. The degradation liquid is the degradation liquid obtained by the combined degradation of low-rank coal by fungi and bacteria. The method for determining the heavy metal content in the degradation liquid is the method for detecting the biodegradation rate of the coal. The contents of Cr, As and Pb in the coal degradation liquid are correlated with the absorbance and linearly fitted. A single factor evaluation method is used to calculate the pollution index of the heavy metals in the coal degradation liquid in combination with the measured concentration of the heavy metals, thereby making an environmental safety evaluation.

[0034] According to the classification of the International Agency for Research on Cancer (IARC), toxic and harmful pollutants in water bodies can be divided into two categories: genotoxicants and somatic toxicants. Genotoxicants include radioactive pollutants and carcinogens, and somatic toxicants include non-carcinogenic chemicals [Li Feixing. Characteristics of rural groundwater pollution and health risk assessment during the dry season [D]. Lanzhou University, 2017.]. Among heavy metals, Cr, Cd and As are carcinogens, while Pb, Hg, Se, Mn, Cu, Zn, Ba, Mo, Ni and Ag are non-carcinogens [Li Feixing. Characteristics of rural groundwater pollution and health risk assessment during the dry season [D]. Lanzhou University, 2017.] [Shi Huanhuan, Pan Yujie, Zeng Min, et al. Source analysis and health risk assessment of heavy metals in groundwater in Leizhou Peninsula [J]. Environmental Science, 2021, 42(09):4246-4256.].

[0035] When considering the human health risks caused by the water environment, we often consider the two aspects of carcinogenic health risks and non-carcinogenic health risks. At present, the "four-step method" model developed by the National Academy of Sciences (NAS) and the United States Environmental Protection Agency (US EPA) is one of the most commonly used health risk assessment models and is widely used in the health assessment of various pollutants in various environmental media [Li Peng, Zhang Bo, Wang Wei, et al. Research progress on health risk assessment based on the "four-step method" [J]. Chinese Population, Resources and Environment, 2016, 26(S1): 553-556]. The model includes four basic steps: hazard identification (hazard source identification), dose-effect assessment (dose-response assessment), exposure assessment and risk characterization [Li Peng, Zhang Bo, Wang Wei, et al. Research progress on health risk assessment based on the "four-step method" [J]. Chinese Population, Resources and Environment, 2016, 26(S1): 553-556][Li Fengyan, Jiang Tianyu, Yu Tao, et al. Research progress on the sources of fluorine in the environment and health risk assessment [J]. Rock and Mineral Testing, 2021, 40(06): 793-807]. The calculation of exposure dose is the basis for the evaluation of carcinogenic health risks and non-carcinogenic health risks.

[0036] Exposure via drinking water Calculation formula: The calculation formula is used to obtain the average daily exposure to a certain heavy metal: ; Where: is the average daily exposure to a heavy metal (mg kg -1 ·d -1 ); Ci is the concentration of a heavy metal (mg / L); IR is the drinking water intake (L / d); BW is the body weight (Kg); EF is the exposure frequency (d / a); ED is the exposure duration (a); AT is the average exposure time (d); [Zhang Yanhong, Tang Yuhong, Chen Junhua, et al. Characteristics of heavy metal pollution in Pingshui River surface water and health risk assessment [J]. Nonferrous Metals (Smelting Part), 2021, (07): 116-125][Xie Hao, Liang Yongping, Li Jun, et al. Distribution characteristics of metal elements in groundwater in Longzici Spring Area and health risk assessment [J]. Environmental Science, 2021, 42(09): 4257-4266].

[0037] Cancer risk through drinking water The calculation formula is used to obtain the carcinogenic health risk of water bodies: ;when When the calculated result is greater than 0.01, it is calculated according to the high exposure amount [Zhang Yanhong, Tang Yuhong, Chen Junhua, et al. Heavy metal pollution characteristics and health risk assessment of Pingshui River surface water [J]. Nonferrous Metals (Smelting Part), 2021, (07): 116-125][Xie Hao, Liang Yongping, Li Jun, et al. Metal element distribution characteristics and health risk assessment of groundwater in Longzici Spring Area [J]. Environmental Science, 2021, 42(09): 4257-4266].

[0038] Non-cancer health risks through drinking water The calculation formula is used to obtain the non-carcinogenic health risk of water bodies: ; Where: is the average annual health risk value of carcinogenic heavy metal element i in drinking water (a -1 ); is the average annual health risk value of carcinogenic heavy metal element i in drinking water (a -1 ); SF i is the carcinogenic slope factor of a chemical carcinogenic metal i (mg kg -1 ·d -1 ); RfD i is the reference dose (mg·kg -1 ·d -1); L is the average human life span; [Zhang Yanhong, Tang Yuhong, Chen Junhua, et al. Heavy metal pollution characteristics and health risk assessment of Pingshui River surface water [J]. Nonferrous Metals (Smelting Part), 2021, (07): 116-125][Xie Hao, Liang Yongping, Li Jun, et al. Distribution characteristics and health risk assessment of metal elements in groundwater of Longzici Spring Area [J]. Environmental Science, 2021, 42(09): 4257-4266].

[0039] The overall health risk assessment model is ; Where: R is the total health risk value (a -1 ); R c and R n are the health risk values ​​of carcinogens and non-carcinogens (a -1 )。 [Zhang Yanhong, Tang Yuhong, Chen Junhua, et al. Heavy metal pollution characteristics and health risk assessment of Pingshui River surface water [J]. Nonferrous Metals (Smelting Part), 2021, (07): 116-125][Xie Hao, Liang Yongping, Li Jun, et al. Distribution characteristics and health risk assessment of metal elements in groundwater in Longzici Spring Area [J]. Environmental Science, 2021, 42(09): 4257-4266].

[0040] Commonly used water pollution assessment methods include single factor assessment method, comprehensive pollution index method, fuzzy comprehensive evaluation method, principal component analysis method, grey correlation coefficient method, etc. Among them, the single factor assessment method is the most widely used in practice because of its simplicity and ease of use. Single factor assessment is to divide the actual concentration of the project of concern by the corresponding standard limit to obtain the pollution index. When the pollution index I≤1, it indicates that the content of the factor does not exceed the standard. When I>1, it indicates that the content of the index exceeds the standard. The calculation formula is as follows: Where, C i is the measured concentration of index i; S i I is the standard limit of index i; i Is the pollution index.

[0041] The higher the degree of oxidation of coal, the easier it is to be used by microorganisms. When fungi, bacteria and their mixture act on nitric acid-oxidized coal (1:1.5), a degradation cycle has only two stages: rapid rise and relative stability. The yields of fungal and bacterial degradation products are 58.17% and 60% respectively. When fungi and bacteria act on nitric acid-oxidized coal (1.5:1), a degradation cycle can be divided into three stages: slow rise, rapid rise and relative stability. The yields of degradation products are 11% and 12.5% ​​respectively. This shows that the higher the degree of oxidation of coal, the easier it is to be used by microorganisms.

[0042] The degradation showed the mechanism of alkaline substances and chelating agents. When fungi acted alone, the pH value and the absorbance value of the degradation solution increased synchronously; when bacteria acted alone, the pH of the degradation solution was in the alkaline range; the lower the pH, the lower the absorbance and product yield, indicating that alkaline substances play an important role in the coal degradation process. The higher the heavy metal content, the higher the degradation rate of the coal, indicating that the chelating agent mechanism may exist in the degradation process.

[0043] Fungi and bacteria have a mutually reinforcing effect when they degrade coal in a mixed manner. In terms of absorbance, the absorbance of the mixed bacteria degradation liquid is the highest at 64.36, the absorbance of fungal degradation is 55.65, and the absorbance of bacterial degradation is 59.47. The absorbance of the mixed bacteria is 15.65% higher than that of fungi and 8.22% higher than that of bacteria. In terms of product yield, the yield of mixed bacteria degradation products is the highest at 67.17%, the yield of fungal degradation products is 58.17%, and the yield of bacterial degradation products is 60%. The mixed bacteria is 15.47% higher than fungi and 11.95% higher than bacteria. From the law of pH changes, it can be seen that mixing promotes the production of alkaline substances, thereby improving the degradation efficiency.

[0044] The mixed fermentation of two strains of bacteria can improve the degradation effect of nitric acid-oxidized coal. The yields of the degradation products of fungi, bacteria, and mixed bacteria were 58.17%, 61%, and 67.17%, respectively.

[0045] The more alkaline substances produced by the mixture of fungi and bacteria, the better the degradation effect of the mixed bacteria. There is a strong correlation between the absorbance and the content of Cr, As, and Pb in the acid precipitation product and the degradation liquid, and the chelate action mechanism is produced during the coal degradation process. There is a balance between the release and utilization of heavy metal Mn in the degradation liquid, and the manganese peroxidase action mechanism is produced during the degradation process.

[0046] The present invention can determine the degradation effect of coal according to the heavy metal content in the biodegradable liquid of coal. It solves the shortcomings of traditional methods. For example, the disadvantage of the weight loss method is that when the coal powder particles are fine, the residual coal after the reaction is difficult to collect, resulting in a large error in the calculation result. Both the weight loss method and the precipitation method require that the degradation rate of coal meets certain requirements, otherwise the mass of the weight loss or precipitation is not enough for accurate quantification. The spectrophotometric method requires that the measurement result is between 0.2 and 0.8. When the absorbance of the sample to be tested is higher than 0.8, it needs to be diluted, especially when the absorbance is less than 0.2, it cannot be accurately quantified. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The black droplets after coal degradation and the culture medium being stained black; in the figure: A-1 to A-3 are black droplets after coal degradation; B-1 to B-2 are culture medium being stained black;

[0048] Figure 2 The coal powder is wrapped by fungal hyphae;

[0049] Figure 3 is the phylogenetic tree of F11 based on 18S rDNA sequence;

[0050] Figure 4 is the phylogenetic tree of B1 based on 16S rDNA sequence;

[0051] Figure 5 The growth of fungi, bacteria and their mixture in LB medium;

[0052] Figure 6 Acid precipitation of coal degradation liquid by fungi, bacteria and their mixtures;

[0053] Figure 7 A450 variation diagram of coal degradation liquid by fungi, bacteria and their mixture;

[0054] Figure 8 The amount of coal degradation products by fungi, bacteria and their mixtures;

[0055] Figure 9 The absorbance at the end of the experiment and the mass change curve of the acid precipitation product;

[0056] Figure 10 is the linear fitting curve between absorbance and mass of acid precipitation product;

[0057] Figure 11 It is the pH change diagram of coal degradation liquid by fungi, bacteria and their mixture;

[0058] Figure 12 The heavy metal content in the degradation solution was jointly degraded by the fungus-bacteria mixture;

[0059] Figure 13 This is the fitting diagram of absorbance value and heavy metal content. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0062] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0063] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0064] 1. Screening of strains: There are a large number of microorganisms in rotten wood and soil in mining areas, and it is relatively easy to separate the microbial groups related to coal degradation from them. Therefore, the present invention collects rotten wood and soil in mining areas from forest farms, uses biological methods to separate and identify microbial species, uses low-rank coal as a substrate, and analyzes and compares the degradation rate of coal samples, product characteristics, and changes in heavy metal content in fermentation liquid through the combined fermentation of fungi and bacteria, finds microorganisms related to coal degradation, explores the regulation method of combined fermentation of fungi and bacteria, and preliminarily understands the environmental pollution and health risks caused by heavy metals.

[0065] 1. Isolation and identification of coal-degrading microorganisms: After the rotten wood from Yihoushan Forest Farm and the soil from Jincheng Mining Area in Shanxi Province were retrieved with sterile equipment, the fungi were isolated using Bengal red medium, and the bacteria were enriched and isolated using LB medium according to the conventional operation techniques of microbiological separation. The degradation experiments of the bacterial coal samples were carried out using aniline blue, tannic acid, guaiacol and other colorants for auxiliary screening, until the microorganisms that can degrade coal were obtained. The isolated microbial strains were identified using molecular biological methods.

[0066] Coal sample: Nitric acid oxidized coal: low-rank coal from Yima, Henan, crushed and screened to 80-120 mesh (0.125mm-0.20mm), dried at 80℃ for 48h. Existing studies have shown that the higher the oxygen content and the lower the degree of metamorphism, the easier it is to be degraded by microorganisms. Therefore, oxidants such as nitric acid and hydrogen peroxide are used to pretreat the coal sample. 100g of coal sample and 150mL of (1+1) nitric acid are added to a 500mL beaker. During the reaction, the beaker is placed in room temperature water to prevent the reactants from overflowing. At the same time, it is stirred continuously until the yellow smoke disappears and then allowed to stand for 24h. Pure water is added to the beaker and stirred. After standing, the supernatant is discarded. After repeatedly washing the coal until the pH of the supernatant is >6, the coal sample is dried at 80℃ for 72h for use.

[0067] PDA medium: 24 g of commercially available PDB medium powder, 25 g of agar powder, and 1 L of pure water; LB medium: 10 g of peptone, 10 g of sodium chloride, 5 g of yeast powder, and 1 L of pure water.

[0068] Identification of bacterial species: The DNA of bacteria B1 was extracted using the Shanghai Biotechnology Bacteria Extraction Kit, and the DNA of fungus F11 was extracted using the fungal extraction kit. The bacterial DNA was PCR-amplified using the universal primers 27F / 1492R for 16S rDNA, and the fungal DNA was PCR-amplified using the universal primers NS1 / NS8 for 18S rDNA. The PCR products were sent to Beijing Biomarker Biotechnology Co., Ltd. for sequencing.

[0069] The sequencing results were submitted to the GenBank database of BCBI for BLAST comparison to determine the homology with the reported microbial sequences. The homologous sequences with the highest similarity were downloaded, and multiple sequence alignment was performed using ClustalW in MEGA 6.0. The molecular evolutionary tree was constructed using the neighbor-joining (NJ) method, in which the bootstrap method was selected as the test of phylogeny, and the number of bootstrap replications was 500.

[0070] Species identification results: The phylogenetic tree constructed from the 18S rDNA sequence of F11 is as follows Figure 3 As shown, F11 and Cunninghamella elegans ( Cunninghamella elegans ) are in the same branch with a similarity of 99.45%, so the strain was identified as Cunninghamella elegans ( Cunninghamella elegans ).

[0071] The phylogenetic tree constructed based on the 16S rDNA sequence of B1 is shown in Figure 5 As shown, B1 and Bacillus sp. are in the same branch with a similarity of 100%, so B1 is identified as Bacillus sp.

[0072] 2. Combined degradation of fungi and bacteria

[0073] 1. Inoculum culture: Use an inoculation loop to scrape an appropriate amount of mycelium from the preserved F11 and B1 slopes and transfer them to a centrifuge tube containing 5 mL of sterile water. Fully shake the centrifuge tube to make the bacterial solution uniform. Take 200 µL of the bacterial solution and drop it on the PDA medium and LB solid medium respectively. Use a coating rod to evenly spread it and invert it at a constant temperature of 30°C. After 5 days, the F11 mycelium will cover the plate, and after 2 days, the B1 colony will cover the plate, which will be used as the bacterial source for combined degradation inoculation.

[0074] 2. Combined degradation of fungal bacteria mixture: Add 150mL LB medium to a 250mL triangular flask. After wet heat sterilization, use a hole puncher to inoculate 4 holes of fungi, 3 holes of fungi + 1 hole of bacteria, 2 holes of fungi + 2 holes of bacteria, 1 hole of fungi + 3 holes of bacteria, 4 holes of bacteria, 50 holes of fungi + 1 hole of bacteria, and place in a constant temperature oscillator for constant temperature oscillation culture at 30℃ and 150rpm for 4 days. Add 2g of sterilized nitric acid-oxidized coal (1:1.5), place in a constant temperature oscillator for continuous culture at 30℃ and 150rpm, and take the bacterial solution every 5 days to measure pH and perform full wavelength scanning. No coal was added to the culture medium of the sterile control group. Three parallel samples were made for each group of experiments. After A450 stabilizes, collect and measure the product.

[0075] 3. Co-culture: Under sterile conditions, after the fungi grow mycelium balls and the bacterial solution becomes turbid, add 2g of sterilized coal sample (1:1.5ml / g) to each group of pre-treated sterile coal samples, place in a constant temperature oscillator at 30℃, 150rpm to continue culturing, and take the bacterial solution regularly to measure the absorbance A450. No coal is added to the culture medium of the sterile control group. Three parallel samples are made for each group of experiments.

[0076] 4. Product collection and measurement: After A450 is stable, centrifuge the degradation solution at 10,000 rpm for 10 min, collect the supernatant, and measure the contents of Cr, Cd, As, Mn, and Pb in the supernatant according to the "Determination of 65 Elements in Water Quality by Inductively Coupled Plasma Mass Spectrometry" (HJ 700-2014).

[0077] Since fungi were used to degrade coal and the coal particles were small, in order to avoid errors caused by collecting residual coal, the degradation effect was measured by combining the yield of the acid precipitation product of the degradation liquid with the absorbance of the degradation liquid. The pH value of the supernatant was adjusted to below 2 with hydrochloric acid and precipitated for 24 hours. The supernatant was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was collected and weighed after drying at 80°C for 48 hours.

[0078] 5. Degradation effect determination: Compare the heavy metal contents. The higher the heavy metal content in the experimental group, the higher the degradation rate of the corresponding coal sample.

[0079] 3. Results and Discussion

[0080] 1. Joint degradation rules of fungal-bacterial mixture: Through the screening of liquid coal degradation culture medium, LB that can serve as both fungal and bacterial liquid coal degradation culture medium was selected from a variety of culture media, laying the foundation for the joint degradation of fungi and bacteria.

[0081] like Figure 5As shown, 4 days after inoculation, the pure fungus experimental group (4F) grew mycelium balls, which were uniform in size and basically covered the bottom of the bottle. The culture medium of the mixed fungus and bacteria experimental group (50F1B, 3F1B, 2F2B, 1F3B) became turbid, and no mycelium balls were observed from the bottom of the bottle, but mycelium was visible. The more fungi were inoculated, the more mycelium there was. At this time, nitric acid-oxidized coal powder was added, and the absorbance and pH of the reaction solution were measured regularly. After the absorbance stabilized, the supernatant of the degradation solution was added with acid, and the precipitation was shown in the following figure. Figure 6 As shown, a large amount of precipitate was produced in each experimental group, and a small amount of precipitate was also produced in the sterile control group (LB+nitric acid oxidation).

[0082] 2. Relationship between absorbance and precipitation: Changes in A450 of coal degradation solutions of fungi, bacteria and their mixtures Figure 7 As shown by Figure 7 It can be seen from the final absorbance values ​​that, from large to small, they are 2F2B+coal oxidized with nitric acid+LB (64.36), 3F1B+coal oxidized with nitric acid+LB (61.65), 50F1B+coal oxidized with nitric acid+LB (60.92), 1F3B+coal oxidized with nitric acid+LB (60.59), 4B+coal oxidized with nitric acid+LB (59.47), 4F+coal oxidized with nitric acid+LB (55.65), and coal oxidized with nitric acid+LB (1.82).

[0083] Figure 8 is the amount of coal degradation products by fungi, bacteria and their mixtures, Figure 8 It can be seen that the masses of acid precipitation products are as follows from large to small: 2F2B+coal oxidized with nitric acid+LB (1.38 g), 3F1B+coal oxidized with nitric acid+LB (1.34 g), 50F1B+coal oxidized with nitric acid+LB (1.31 g), 1F3B+coal oxidized with nitric acid+LB (1.25 g), 4B+coal oxidized with nitric acid+LB (1.23 g), 4F+coal oxidized with nitric acid+LB (1.20 g), and coal oxidized with nitric acid+LB (0.03 g).

[0084] The mass of the product in the experimental group minus the control (coal oxidized with nitric acid + LB) was divided by the mass of coal added (2 g), and the yields of acid precipitation products in each group were 2F2B + coal oxidized with nitric acid + LB (67.17%), 3F1B + coal oxidized with nitric acid + LB (65.17%), 50F1B + coal oxidized with nitric acid + LB (63.67%), 1F3B + coal oxidized with nitric acid + LB (61%), 4B + coal oxidized with nitric acid + LB (60%), and 4F + coal oxidized with nitric acid + LB (58.17%).

[0085] The absorbance at the end of the experiment and the mass of the acid precipitation product were plotted. The results are as follows: Figure 9As shown in the figure, the absorbance is consistent with the change curve of the acid precipitation product, and the correlation coefficient obtained by correlation analysis is 0.998, indicating that there is a strong correlation between the two.

[0086] The absorbance and the mass of the acid precipitation product were linearly fitted, such as Figure 10 As shown, the fit R 2 =0.997, indicating that absorbance can indicate the degree of coal degradation. Combining the absorbance and product quality data, it can be seen that the effects of fungi and bacteria alone are not as good as those of mixed bacteria, indicating that fungi and bacteria have a mutually reinforcing effect when they degrade coal in a mixed manner.

[0087] 3. The influence of coal oxidation degree on degradation: Figure 7 It can be seen that when the experiment started, the absorbances of the degradation liquids of the fungal experimental group (4F+LB+nitric acid oxidized coal) and the bacterial experimental group (4B+LB+nitric acid oxidized coal) were 6.66 and 17.08, respectively. On the 4th day, the absorbance values ​​were basically stable. Finally, the maximum absorbances of the fungal experimental groups (4F+LB+nitric acid oxidized coal) and (4B+LB+nitric acid oxidized coal) were 55.65 and 59.47, respectively, and the product yields were 58.17% and 60%, respectively.

[0088] Studies have shown that the higher the degree of oxidation of coal, the easier it is to be used by microorganisms. Nitric acid oxidation can increase the oxygen content in coal. Through the degradation of two types of oxidized coal, nitric acid oxidized coal (1.5:1) and nitric acid oxidized coal (1:1.5), by fungi and bacteria, it can be seen that in addition to the concentration of nitric acid, the mixing ratio of nitric acid and coal will also affect the degree of oxidation of coal.

[0089] 4. Effect of pH value on degradation: pH change law is as follows Figure 11 As shown, the pH of the control group (nitric acid oxidized coal + LB) slowly decreased from 5.33 to 4.46 and then stabilized, while the pH of the experimental group fluctuated in the alkaline range of 7.86-8.72 from the 4th day to the end of the experiment, indicating that fungi, bacteria and their mixtures produced alkaline substances during the experiment. The pH under the action of pure bacteria (4B + nitric acid oxidized coal + LB, 4F + nitric acid oxidized coal + LB) was always slightly lower than that of mixed bacteria (50F1B + nitric acid oxidized coal + LB, 3F1B + nitric acid oxidized coal + LB, 2F2B + nitric acid oxidized coal + LB, 1F3B + nitric acid oxidized coal + LB), indicating that the mixed bacteria produced more alkaline substances than pure bacteria.

[0090] Binding absorbance Figure 7 It can be seen that the degradation rate of the corresponding mixed bacteria is higher than that of pure bacteria. Fungi and bacteria have a mutually promoting effect when they degrade coal in a mixed manner, indicating that alkaline substances play an important role in the coal degradation process.

[0091] When the experiment started, the pH of the fungal experimental group (4F+nitric acid oxidized coal+LB) differed from that of the other experimental groups by about 1 pH unit, while the absorbance was about 1 / 3 of that of the other experimental groups, which also shows that alkaline substances have an important effect on coal degradation.

[0092] 5. Heavy metal content analysis: The heavy metal content in the fungus-bacteria mixture degradation solution of coal is as follows: Fig.12 As shown, Cd was not detected in the degradation liquid, and the contents of Cr, As and Pb elements in the degradation liquid of all experimental groups (50F1B+nitric acid oxidized coal+LB, 3F1B+nitric acid oxidized coal+LB, 2F2B+nitric acid oxidized coal+LB, 1F3B+nitric acid oxidized coal+LB, 4B+nitric acid oxidized coal+LB, 4F+nitric acid oxidized coal+LB) were higher than those in the sterile control group (nitric acid oxidized coal+LB), indicating that the fungus F11, bacteria B1 and their mixture can promote the migration of Cr, As, Mn and Pb in coal into the degradation liquid while degrading coal.

[0093] The contents of Cr, As, Mn and Pb in the degradation solution of the pure bacteria experimental groups (4F + nitric acid oxidized coal + LB, 4B + nitric acid oxidized coal + LB) were lower than those of the mixed bacteria (50F1B + nitric acid oxidized coal + LB, 3F1B + nitric acid oxidized coal + LB, 2F2B + nitric acid oxidized coal + LB, 1F3B + nitric acid oxidized coal + LB), indicating that the mixture of fungus F11 and bacteria B1 has a mutually promoting effect in the migration of heavy metals from coal to the degradation solution. Heavy metals Cr, As, Mn and Pb were all detected in the sterile control group (nitric acid oxidized coal + LB), indicating that even without the degradation of microorganisms, a certain amount of heavy metals will migrate from nitric acid oxidized coal to the degradation solution. This is because the coal treated with strong acid oxidation can be partially dissolved in water even without the action of biological or chemical reagents. The contents of Cr, As and Pb elements in the degradation solution are higher than those in the sterile control group, indicating that fungus F11, bacteria B1 and their mixture have the effect of promoting the migration of Cr, As and Pb in coal to the fermentation solution while degrading coal.

[0094] Correlation analysis of the contents of Cr, As, and Pb in the coal degradation liquid with the absorbance (absorbance on the 16th day) showed that the correlation coefficients were 0.857, 0.821, and 0.873, respectively, indicating that the two were strongly correlated. After linear fitting, Fig.13 As shown in (a, b, c), the fit (R 2 ) are 0.735, 0.610, and 0.763, respectively, that is, the higher the heavy metal content in the coal degradation liquid, the higher the corresponding coal degradation rate. This is because a large number of metal elements are complexed in the molecular structure of coal, and microorganisms can degrade coal by chelating the metal elements in coal. After the metal elements in coal are removed, the coal is more easily used by microorganisms, resulting in a decrease in the molecular weight of coal.

[0095] There are a large number of metal elements complexed in the molecular structure of coal. Microorganisms can degrade coal by chelating the metal elements in coal. Existing studies have confirmed that after the metal elements in coal are removed, coal is more easily utilized by microorganisms, resulting in a decrease in the molecular weight of coal. Studies have shown that the mechanism of microbial degradation of coal mainly includes the action mechanism of alkaline substances, enzymes, chelating agents and surfactants. Among them, only the chelating agent mechanism removes heavy metal ions in coal through chelation of ammonium oxalate secreted by organisms, thereby promoting coal degradation. Combined with the correlation between heavy metals in the degradation solution and absorbance, it can be seen that fungi, bacteria and their mixtures promote coal degradation through the chelating agent mechanism.

[0096] Different from Cr, As, and Pb, the content of Mn in the sterile control group was higher than that in the bacterial experimental group, and the correlation between Mn and absorbance was weak. Fig.13 (d) shows the goodness of fit (R 2 ) is only 0.3. The presence of manganese in the control group indicates that the coal contains this element. On the one hand, the above analysis shows that the coal in the pure bacteria experimental group has been degraded and there is a chelation mechanism, so the Mn element in the coal will be transferred to the degradation liquid, resulting in an increase in the content of Mn. On the other hand, during the microbial separation process, it was found that both fungi F11 and bacteria B1 can fade the aniline blue plate. Aniline blue can indicate the presence of manganese peroxidase, indicating that both strains of bacteria can produce manganese peroxidase. Studies have shown that the reaction of manganese peroxidase requires the participation of Mn, so the manganese in the degradation liquid decreases due to the participation in the reaction. In summary, in the process of coal degradation, due to chelation and the action of manganese peroxidase, there is a balance between the release and utilization of the Mn element.

[0097] The data of Mn content in the sterile control group and the bacterial and fungal experimental groups were analyzed by variance analysis. As shown in Table 1, p>0.05, there was no significant difference between the control group and the experimental group. In summary, in the process of coal degradation, due to chelation and the action of manganese peroxidase, there is a balance between the release and utilization of Mn.

[0098] Table 1: ANOVA analysis of Mn content in sterile control group and pure bacteria experimental group

[0099]

[0100] As shown in Table 2, the Mn content in the mixed bacteria experimental group was higher than that in the sterile control group and was significantly different from that in the mixed bacteria experimental group, indicating that the release of manganese was greater than its utilization after the fungus F11 and the bacteria B11 were mixed, so the content in the degradation liquid increased.

[0101] Table 2: ANOVA analysis of Mn content in sterile control group and mixed bacteria experimental group

[0102]

[0103] 6. Water pollution assessment: When evaluating water pollution with a single factor, the standard limits of the three types of water and supplementary items in the surface water environmental quality standard (GB3838-2002) are taken. The pollution index calculation formula refers to [Li Feixing, Characteristics of rural groundwater pollution and health risk assessment during the dry season, Lanzhou University, 2017]. When the pollution index I≤1, it indicates that the content of the factor does not exceed the standard. When I>1, it indicates that the content of the index exceeds the standard. The calculation formula is as follows: Where, C i is the measured concentration of index i; S i I is the standard limit of index i; i Is the pollution index.

[0104] Direct drinking and skin contact are two important ways for metal elements in water to enter the human body. The health risk through drinking water is 2 to 3 orders of magnitude higher than the other two (respiratory and skin contact). Therefore, this paper mainly considers the health risk of drinking water. The calculation formula is based on the references [Xie Hao et al., Distribution characteristics and health risk assessment of metal elements in groundwater in Longzici Spring Area [J]. Environmental Science, 2021, 42(09): 4257-4266], [Zhang Yanhong et al., Characteristics and health risk assessment of heavy metal pollution in surface water of Pingshui River [J]. Nonferrous Metals (Smelting Part), 2021, (07): 116-125]. The RfD and SF values ​​of chemical substances refer to [Wu Li et al. Change characteristics and health risk assessment of ions and trace elements in biogas [J]. Journal of China Coal Society, 2021, 46(03): 994-1002], Li Feixing, Pollution characteristics and health risk assessment of rural groundwater in dry season, Lanzhou University, 2017], [Zhou Jinmei, Jiang Zhongcheng, Xu Guangli, et al. Distribution of metal elements in groundwater around iron mines and health risk assessment [J]. China Environmental Science, 2019, 39(05): 1934-1944], [Li Peng. Change characteristics of organic matter and heavy metals in coal biogasification and its potential environmental risks [D]. Henan Polytechnic University, 2018].

[0105] The specific values ​​of model parameters RfD and SF are shown in Table 3. The average life span L is 70 years, the average daily water intake IR is 2.2L / d, the exposure frequency EF is 365d / a, the exposure duration ED is 35 years for non-carcinogens and 70 years for carcinogens, the average body weight BW is 60kg, and the average exposure time AT is 12775 days for non-carcinogens and 25550 days for carcinogens.

[0106] Table 3 Model parameter RfD and SF values

[0107]

[0108] 7. Evaluation of heavy metal environmental pollution: The specific contents of heavy metals Cr, As, Mn and Pb in the degradation liquid are shown in Table 4. According to the contents of heavy metals in the degradation liquid and the standard limits of three types of water and supplementary items in the Surface Water Environmental Quality Standard (GB3838-2002), the pollution index is calculated by the single factor evaluation model. The specific data are shown in Table 5. As, Mn and Pb did not exceed the standard in each group of degradation liquid. Cr exceeded the standard or the pollution index was close to 1 in the mixed bacteria degradation liquid. Cr exceeded the standard in the mixed bacteria 1F3B, 2F2B and 50F1B, and the pollution index in the mixed bacteria 3F1B was 0.97. Therefore, when degrading coal with a fungal bacterial mixture, attention should be paid to the possible pollution caused by Cr.

[0109] Table 4 Evaluation indexes and contents of fungal-bacterial mixture combined degradation solution

[0110]

[0111] Table 5 Contamination index of the combined degradation solution of fungi and bacteria mixture

[0112]

[0113] 8. Human health risk assessment: Human health assessment and pollution assessment: Using the human health risk assessment model and single factor evaluation method recommended by the United States Environmental Protection Agency (US EPA), combined with the measured concentrations of heavy metals in coal degradation liquid, the carcinogenic and non-carcinogenic health risk values ​​and pollution index of heavy metals are calculated, and then health risk and pollution assessments are made.

[0114] Exposure via drinking water The calculation formula is used to obtain the average daily exposure to a certain heavy metal: ; Where: is the average daily exposure to a heavy metal (mg kg -1 ·d -1 ); Ci is the concentration of a heavy metal (mg / L); IR is the drinking water intake (L / d); BW is the body weight (Kg); EF is the exposure frequency (d / a); ED is the exposure duration (a); AT is the average exposure time (d);

[0115] Cancer risk through drinking water The calculation formula is used to obtain the carcinogenic health risk of water bodies: ;when When the calculated result is greater than 0.01, it is calculated as high exposure: ;

[0116] Non-cancer health risks through drinking water The calculation formula is used to obtain the non-carcinogenic health risk of water bodies: ; Where: is the average annual health risk value of carcinogenic heavy metal element i in drinking water (a -1 ); is the average annual health risk value of carcinogenic heavy metal element i in drinking water (a -1 ); SF i is the carcinogenic slope factor of a chemical carcinogenic metal i (mg kg -1 ·d -1 ); RfD i is the reference dose (mg·kg -1 ·d -1 ); L is the average life span of human beings;

[0117] The overall health risk assessment model is ; Where: R is the total health risk value (a -1 ); R c and R n are the health risk values ​​of carcinogens and non-carcinogens (a -1 );

[0118] The average annual risk value of individuals caused by heavy metals in the fungus-bacteria mixture degradation liquid is shown in Table 6. The non-carcinogenic health risk values ​​caused by Mn, Pb, etc. are distributed in the range of 9.85×10 -12 ~5.21×10 -10 , which is less than the non-carcinogenic health risk threshold of 1 and is at an acceptable level. The non-carcinogenic health risk of a single heavy metal Pb is greater than that of Mn.

[0119] Table 6: Average annual risk of heavy metals in fungal-bacterial mixture degradation liquid (a -1 )

[0120]

[0121] The cancer risk caused by Cr is 10 -3 ~10 -4 The cancer risk caused by As is between 10 -6 The carcinogenic health risks of Cr and As in each group exceeded the maximum acceptable risk level of 1×10- -6 The carcinogenic health risk of Cr is higher than the maximum acceptable risk level of 5×10 -5 , which is also higher than the maximum acceptable risk level of 1×10 -4The carcinogenic health risk caused by Cr is 2 to 3 orders of magnitude higher than that of As. The carcinogenic health risk of Cr in mixed bacteria degradation liquid is 3 orders of magnitude higher than that of As, and the carcinogenic health risk of Cr in pure bacteria degradation liquid is 2 orders of magnitude higher than that of As.

[0122] The total carcinogenic risk of each group was 6 to 7 orders of magnitude higher than the total non-carcinogenic risk, indicating that the health risks caused by heavy metals mainly come from carcinogenic health risks, and the carcinogenic health risk of mixed bacterial degradation liquid is 1 order of magnitude higher than that of pure bacterial degradation liquid. Even without the action of microorganisms, the mixture of coal and LB medium will also produce carcinogenic health risks.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for degrading low-rank coal by combining fungi and bacteria, characterized in that: The fungus is Cunninghamella elegans ( Cunninghamella elegans ) F11, the bacteria is Bacillus sp. B1; the specific method of fungal bacterial combination is as follows: F11 is inoculated into PDA solid culture medium, inverted and cultured at a constant temperature of 30℃ for 5 days, and the mycelium is used for standby after covering the surface of the culture medium; B1 is inoculated into LB solid culture medium, inverted and cultured at a constant temperature of 30℃ for 2 days, and the colonies are used for standby after covering the plate; the cultured F11 and B1 are transferred to the sterilized LB liquid culture medium, and the culture is shaken at a constant temperature of 30℃ and 150rpm for 4 days, and then sterilized nitric acid-oxidized coal is added, and the culture is continued at 30℃ and 150rpm until the absorbance A450 is stable; the ratio of F11 and B1 inserted into the hole punch is: 3 holes F11: 1 hole B1, 2 holes F11: 2 holes B1, 1 hole F11: 3 holes B1 or 50 holes F11: 1 hole B1.

2. The method for degrading low-rank coal by combining fungi and bacteria according to claim 1, characterized in that: The nitric acid-oxidized coal is as follows: low-rank coal is crushed and screened to 80-120 mesh, and dried at 80°C for 48 hours; 150 mL of a nitric acid aqueous solution with a volume ratio of 1:1 is added to 100 g of the crushed and screened coal sample, and the reaction is carried out in room temperature water, while continuously stirring until the yellow smoke disappears, and then the reaction is allowed to stand for 24 hours, the coal is washed with pure water until the pH of the supernatant is greater than 6, and then the coal sample is dried at 80°C for 72 hours.

3. A method for detecting biodegradation of coal, using the method for degrading low-rank coal by combining fungi and bacteria as described in any one of claims 1-2 to perform biodegradation of coal, characterized in that: The spectrophotometer measures the absorbance A450 of the degradation liquid at 450nm to determine the stage of coal degradation; after the A450 value stabilizes, the microbial degradation liquid of the coal is centrifuged, and the supernatant is taken to determine the content of heavy metals Cr, As, and Pb therein, and the biodegradation of the coal is determined by the level of heavy metal content.

4. A method for detecting coal biodegradation according to claim 3, characterized in that: The specific steps for determining the heavy metal content are as follows: after the A450 value of the bacterial liquid is stable, the co-culture fermentation liquid is centrifuged at 10,000 rpm for 10 minutes, the supernatant is collected, and the contents of Cr, As, and Pb in the supernatant are measured in accordance with the "Determination of 65 Elements in Water Quality by Inductively Coupled Plasma Mass Spectrometry" HJ 700-2014; the relative size of the degradation rate is determined by the content indicators of heavy metals Cr, As, and Pb in the degradation liquid.

5. The method for detecting the biodegradability of coal according to claim 4, characterized in that: The heavy metal content is determined by atomic fluorescence spectrometry or inductively coupled plasma mass spectrometry (ICP-MS).

6. A method for evaluating the environmental safety of a degradation liquid obtained by the combined degradation of low-rank coal by fungi and bacteria, wherein the degradation liquid is the degradation liquid obtained by the combined degradation of low-rank coal by fungi and bacteria as described in any one of claims 1-2, and the method for determining the heavy metal content in the degradation liquid is the method for detecting the biodegradation rate of coal as described in claim 4, characterized in that: The contents of Cr, As and Pb in the coal degradation liquid were correlated with the absorbance and linear fitting was performed. The water pollution situation was evaluated using a single factor evaluation. Combined with the measured concentrations of heavy metals in the coal degradation liquid, the pollution index of heavy metals was calculated, and then an environmental safety evaluation was made.

7. The method for evaluating the environmental safety of the degradation solution of low-rank coal by the combined degradation of fungi and bacteria according to claim 6, characterized in that: The water pollution situation is evaluated by a single factor evaluation, and the pollution index is obtained by dividing the actual concentration of the project of concern by the corresponding standard limit. When the pollution index I≤1, it indicates that the content of the factor does not exceed the standard. When I>1, it indicates that the content of the index exceeds the standard. The calculation formula is as follows: Where, C i is the measured concentration of index i; S i I is the standard limit of index i; i Is the pollution index.

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