煤炭的微生物转化是实现煤炭清洁转化、多元增值、安全高效的有效途径。然而，由于煤炭结构和生物转化机理的复杂性，以及传统分子生物学方法的局限性，目前对煤炭微生物转化中降解和液化机理的理解尚不深入。为此，本文以蒙东褐煤和原位采集菌株作为基本研究对象，采用实验研究、数据分析、工程设计等研究方法，系统地分析了菌株的生物特性和褐煤的理化性质，对比了褐煤生物降解和液化前后的微观结构，深入探讨了生物降解和液化褐煤过程中的蛋白质组学，揭示了褐煤环境下生物响应规律和降解液化原因，并解析了生物降解和液化褐煤结构的作用机理。此外，还对褐煤的生物液化产物进行了全组分分析，提出了一种褐煤的原位微生物流态化开采构想。本研究不仅为煤炭微生物转化机理研究提供了理论数据，也为探索煤炭的微生物流态化开采提供了新的思路。本文主要研究成果如下：

（1）从原位煤层中筛选出一株能够降解和液化褐煤的真菌菌株，通过形态学观测和内转录间隔区测序，该菌株被鉴定为镰刀菌属，命名为Fusarium sp. NF01或镰刀菌NF01。研究发现，培养基中碳源的改变会显著影响菌株的生物特性。为探究菌株生物特性及其对褐煤的生物降解和液化情况，配制了不同碳源（G：葡萄糖；M：麦芽糖；SGC：葡萄糖酸钠；SGT：谷氨酸钠）的基本培养基（MM），对比了菌株的生长曲线、菌液pH-t曲线、显/褪色反应，并对褐煤理化性质（氧化预处理、元素组成、微观形貌、官能团、自由基）、生物降解率、生物液化量进行了测定分析。结果表明：当培育MM-SGC时，菌株生长良好，培养液呈现碱性，生物降解率和液化量最大，分别为42.96%和2.2 mL，预测分泌漆酶和降解木质素的过氧化物酶；此外，褐煤经硝酸预处理，表面变得更加粗糙，且结构裂隙发育，元素含量和官能团改变明显，自由基浓度减小；

（2）基于蛋白质组学分析，揭示了Fusarium sp. NF01培育于MM-G中响应褐煤环境规律和降解原因，解析了生物降解褐煤结构的作用机理。采用稳定同位素标记蛋白质组串联质谱（TMT）技术，对有/无褐煤影响的菌株分泌蛋白质组进行了测试对比，共鉴定出显著差异表达蛋白62个。其中，上调蛋白20个，下调蛋白42个。结合生物信息学数据分析，对前5个显著差异上调蛋白质进行功能/通路注释和富集分析，揭示了菌株主要采用细胞防御、褐煤降解、解毒以及代谢调节等多蛋白调控策略去响应褐煤培养环境的规律，并结合生物产酶特性，建立了漆酶为靶点蛋白的假设；通过漆酶的验证试验和抑制试验，确定了Fusarium sp. NF01能够分泌漆酶，且伴随有褐煤时，漆酶活性水平更高，活性为24.6982 U/L；同时，分泌的漆酶是褐煤生物降解的主要原因。在此研究基础上，重点剖析了漆酶催化含羟基类化合物（如酚类化合物）的机理，结合菌株降解产物，推测了漆酶降解褐煤结构中发生化学键破坏位置和漆酶降解其过程，得到了菌株降解褐煤的作用机理；

（3）基于蛋白质组学分析，揭示了Fusarium sp. NF01培育于MM-SGC中响应褐煤环境规律和液化原因，解析了生物液化褐煤结构的作用机理。采用稳定同位素标记蛋白质组的TMT技术和生物信息学分析对菌株在有/无褐煤条件下的蛋白质组进行了测试对比，共鉴定出显著差异表达蛋白18个。其中，上调蛋白8个，下调蛋白10个。通过对前3个显著差异上/下调蛋白质的功能/通路注释和富集分析，揭示了菌株主要采用营养物质的转运和合成、质膜的主动调节、免疫的优化、细胞损伤和死亡的抑制、生长迟缓和代谢不良等多级蛋白质策略去响应褐煤环境的规律，并结合菌液碱性特征，选择上调蛋白中最显著的亚精胺合成酶为靶点蛋白。进一步利用生物胺的靶向检测和抑制多胺下的褐煤生物液化试验，阐明了菌株通过分泌亚精胺合成酶催化腐胺（Put）和脱羧化S-腺苷甲硫氨酸（dc-Sam）生物合成了亚精胺，且分泌的多胺，即亚精胺（占比65%，含量128.903 ug/g）、腐胺（占比20.4%，含量40.541 ug/g）、精胺（占比8.4%，含量16.616 ug/g），是褐煤生物液化的主要原因。同时，推断了菌株合成Put和dc-Sam的代谢途径。在此研究基础上，重点剖析了多胺物解离基团（氨基、亚氨基和氢氧根离子）与含有羧基、醛基、羰基等化合物的化学反应机理，结合菌株液化产物，推测了多胺液化褐煤结构中发生化学键破坏位置和液化过程，得到了菌株液化褐煤的作用机理；

（4）采用pH值测定仪、水分测定仪、X射线荧光光谱仪、傅立叶变换红外吸收光谱仪、X射线衍射仪、气相色谱-质谱仪、裂解气相色谱-质谱仪、热重分析仪等研究方法，对Fusarium sp. NF01液化褐煤产物的理化性质（pH值、水分、元素）和组成成分进行了深入分析。结果表明：生物液化产物pH值为6.98，呈中性，其成分由86.8%水分、5.92%炭黑、2.55%（氨基酸类物质、腐殖酸、混合烃）、2.39%硅酸盐、1.82%（苯并噻唑、邻苯二甲酰亚胺、4-甲基苯酐、Α-菖蒲醇、没药醇氧化物B、2,3-环氧-3,7-二甲基辛-6-烯醇、菲、蒽、雄烯二酮、芥酸酰胺、棕榈酰胺、油酸酰胺、β-雄甾烯醇）、0.42%碳酸钙、小于0.1的挥发物组成。

Coal bioconversion is an effective approach to achieve clean transformation, diversified value addition, and safe and efficient utilization of coal. However, due to the complexity of coal structure and microbial mechanism, as well as the limitations of traditional molecular biology methods, there are still certain challenges in the in-depth understanding of the enzymatic mechanism during coal degradation. It systematically analyses the biological characteristics of these strains and the physicochemical properties of lignite, comparing microstructural changes pre- and post-biodegradation / bioliquefaction through experimental research, data analysis, and engineering design methods. Building upon this study, we delved into the proteomics of lignite biodegradation and bioliquefaction processes, elucidating the biological response patterns and biodegradation / bioliquefaction mechanisms under lignite environments. We further elucidated the potential mechanistic basis of biodegradation and bioliquefaction's influence on lignite structural modification. Additionally, this study conducted a comprehensive compositional analysis of the bioliquefaction products of lignite and proposed a conceptual framework for in-situ microbial liquefaction mining of lignite. This study not only provides theoretical data for researching the biotransformation mechanism of coal but also offers new insights into the microbial flow-state exploitation of coal. The key contributions of this thesis are summarized as follows.

(1) A fungal strain capable of degrading and liquefying lignite was isolated from an in-situ coal seam. Morphological observation and internal transcribed spacer sequencing identified the strain as a Fusarium species, designated as Fusarium sp. NF01. Carbon source alterations in the culture medium significantly impacted the strain's biological characteristics. To investigate the strain's biological properties and its influence on lignite biodegradation and bioliquefaction, basic media (MM) supplemented with different carbon sources (G: glucose; M: maltose; SGC: sodium gluconate; SGT: sodium glutamate) were prepared. The strain's growth curve, culture pH-t curve, and color fading/recovery reactions were compared. Additionally, lignite's physicochemical properties (oxidative pretreatment, elemental composition, microstructure, functional groups, free radicals), biodegradation rate, and bioliquefaction yield were analyzed. The results indicate that when cultivated in MM-SGC, the strain grows well. The culture medium is alkaline, and the biodegradation rate and liquefaction yield are maximized, reaching 42.96% and 2.2 mL, respectively. It is predicted that this strain secretes laccase and peroxidase, which degrade lignin. Additionally, after pretreatment with nitric acid, the surface of lignite becomes rougher, and structural cracks develop. There are significant changes in element content and functional groups, and the concentration of free radicals decreases.

(2) Through proteomic analysis of Fusarium sp. NF01 cultivated in MM-G, the laws of biological response to lignite environment and the causes of lignite biodegradation were revealed, and the possible biodegradation mechanism of lignite structure was explained. Using tandem mass spectrometry (TMT) with stable isotope labeling, the secreted proteins of strains with and without lignite exposure were compared and tested, and a total of 62 significantly differentially expressed proteins were identified, including 20 upregulated proteins and 42 downregulated proteins. Functional/pathway annotation and enrichment analysis of the top 5 significantly upregulated proteins, combined with bioinformatics data analysis, revealed that the strain mainly employs a multi-protein regulatory strategy involving cell defense, lignite degradation, detoxification, and metabolic regulation to respond to the lignite culture environment. Based on the characteristics of biological enzymes, a hypothesis was established with laccase as the target protein. Laccase verification and inhibition experiments confirmed that Fusarium sp. NF01 can secrete laccase, and its activity level is higher in the presence of lignite, reaching 24.6982 U/L. The secreted laccase is the main factor contributing to the lignite biodegradation. Building upon the previous findings, we further elucidated the mechanism of laccase catalysis for various substrates. By analyzing the biodegradation products, we inferred the specific locations of chemical bond cleavage and the overall degradation process during laccase-mediated lignite degradation. This in-depth analysis led to the formulation of a possible mechanism for lignite degradation by the strain.

(3) Through proteomic analysis of Fusarium sp. NF01 cultivated in MM-G, the laws of biological response to lignite environment and the causes of lignite bioliquefaction were revealed, and the possible bioliquefaction mechanism of lignite structure was explained. Utilizing TMT technology and bioinformatics analysis techniques, the proteomes of the strain under lignite-present and lignite-absent conditions were compared and analyzed. A total of 18 significantly differentially expressed proteins were identified, including 8 upregulated proteins and 10 downregulated proteins. By delving into the functional / pathway annotations and enrichment analyses of the top 3 significantly differentially up/downregulated proteins, we unveiled a multi-level protein strategy employed by the strain to respond to the lignite environment. This strategy encompasses nutrient transport and synthesis, active regulation of the plasma membrane, immune optimization, suppression of cell damage and death, growth retardation, and metabolic impairment. In conjunction with the strain's alkaline characteristics, we selected the most significantly upregulated protein, spermidine synthase, as a target protein for further investigation. To further elucidate the role of bioamines in lignite bioliquefaction, we employed targeted bioamine detection and polyamine-inhibited lignite bioliquefaction assays. These experiments revealed that the strain biosynthesizes spermidine through the secretion of spermidine synthase, catalyzing the conversion of putrescine (Put) and decarboxylated S-adenosylmethionine (dc-Sam). The secreted bioamines, predominantly spermidine (65%, 128.903 ug/g), followed by putrescine (20.4%, 40.541 ug/g) and spermine (8.4%, 16.616 ug/g), were identified as the primary drivers of lignite bioliquefaction. Additionally, we inferred the metabolic pathways involved in the strain's synthesis of Put and dc-Sam. Building upon this research, we delved into the mechanistic details of the chemical reactions between polyamine dissociation groups (amino, imine, and hydroxyl ions) and various substrates. By analyzing the strain's liquefaction products, we inferred the locations of chemical bond cleavage and the overall liquefaction process during polyamine-mediated lignite liquefaction. This in-depth analysis led to the formulation of a possible mechanism for lignite liquefaction by the strain.

(4) A comprehensive array of analytical techniques was employed to conduct both qualitative and quantitative assessments of the physicochemical properties and composition of the liquefied lignite products derived from Fusarium sp. NF01. These methods included pH measurement, moisture determination, X-ray fluorescence spectroscopy, Fourier transform-infrared absorption spectroscopy, X-ray diffraction, gas chromatography-mass spectrometry, pyrolysis gas chromatography-mass spectrometry, and thermogravimetric analysis. The resulting analysis revealed that the bioliquefaction products exhibited a neutral pH value of 6.98. The composition of these products was as follows: 86.8% water, 5.92% carbon black, 2.55% (amino acid substances, humic acid, mixed hydrocarbons), 2.39% silicate, 1.82% (benzo-thiazole, phthalimide, 4-methylphthalic anhydride, α-calamus alcohol, bisabolol oxide B2,3-epoxy-3,7-dimethyloct-6-enol, phenanthrene, anthracene, androstenedione, erucamide, palmitamide, oleamide, β-sitosterol), 0.42% calcium carbonate, and trace amounts of volatile compounds (less than 0.1%).

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