腐殖质促进水稻土砷还原的化学与微生物机制

Organic matter is a key factor controlling arsenic reduction and release from anoxic paddy soils. Humic substances (HS) constitute a major fraction of soil organic matter, which are also important redox-active compounds in nature. However, the effect and mechanism of HS on arsenic reduction in paddy soil are still unclear. In this project, anaerobic microcosms were set up with the typical arsenic-contaminated paddy soils in China and amendments of three extracted paddy soil humic fractions (i.e., fulvic acid, humic acid, and humin) to investigate the chemical and microbial mechanism of arsenic reduction in paddy soil. By determining the reaction kinetics of chemical (reduced humic substances) and microbial arsenic reduction facilitated by HS, the reaction-kinetic model was then established to quantify the relative contribution of chemical and microbial process to arsenic reduction and to reveal the underling relationship between the important chemical properties of HS and arsenic reduction. Furthermore, the extracted humic fractions were used as the sole substrate to enrich the dominant arsenic-reducing microorganisms following repeated subculturing. A metatranscriptomic approach was then used to identify the functional microbes and reveal the molecular mechanism of arsenic reduction in paddy soil. The innovation for this project is to elucidate the scientific links between HS and arsenic reduction in paddy soil in the perspective of the coupling of arsenic and HS cycles. We expect that the results of this study can provide a theoretical basis for the directed regulation of arsenic pollution in rice paddy field in China.

有机质是控制厌氧水稻土中砷还原释放的主要因素。腐殖质是土壤有机质的主要组分，也是自然界中重要的氧化还原活性物种。然而，有关腐殖质对水稻土砷还原过程的影响和具体机制尚不明确。本项目以我国典型砷污染稻田土壤为研究对象，提取水稻土腐殖质的不同组分富里酸、腐殖酸和胡敏素，建立厌氧微宇宙模拟体系，探讨了腐殖质影响稻田砷还原的化学与微生物机制。通过测定稻田土壤腐殖质的化学（还原态腐殖质）与微生物砷还原反应动力学，建立体系的反应动力学模型，定量评估化学与微生物过程对砷还原的贡献，揭示腐殖质关键化学性质与砷还原之间的内在联系；进一步以腐殖质为唯一底物，多次富集培养和驯化，获得优势砷还原功能菌群，采用宏转录组学技术揭示腐殖质促进稻田砷还原的关键功能微生物及还原机制。其创新性在于从砷循环与腐殖质循环耦合的视角，深入阐明水稻土腐殖质与砷还原转化间的科学本质。可为我国稻田砷污染过程的定向调控提供理论依据。

有机质是调控厌氧水稻土中砷还原释放的关键因素，腐殖质是土壤有机质的重要组分。然而，有关腐殖质对水稻土砷还原过程的影响与分子机制尚不明确。本项目首先以我国华南地区典型砷污染稻田土壤为研究对象，建立水稻土-腐殖质厌氧微宇宙体系比较分析了不同来源腐殖酸（HA），包括植物来源腐殖酸（JHA），腐殖酸钠盐（aHA）及泥炭土壤提取的腐殖酸（PHA）对淹水稻田微生物砷还原转化的影响。表明添加腐殖酸主要提高异化砷还原基因（arrA）丰度进而促进稻田土壤微生物砷还原；后续研究中我们以土壤提取腐殖酸PHA为研究对象，利用稳定同位素探针技术（SIP）结合宏基因组测序解析腐殖质作用下代谢活性异化砷还原微生物的物种多样性及代谢机制。主要发现为：1）添加HA富集的具有代谢活性的异化砷还原微生物主要为芽孢杆菌（Bacillus）、短芽孢杆菌（Brevibacillus）、脱亚硫酸菌（Desulfitobacterium）、梭菌纲（Clostridia）以及酸杆菌门（Acidobacteria）的类群；2）添加HA提高代谢活性异化砷还原微生物基因组中arrA基因丰度；3）酸杆菌门（Acidobacteria）和梭菌纲（Clostridia）微生物类群基因组中检测到与电子传递pcc和mtr系统相关的基因丰度较高；最后我们通过添加腐殖质对水稻土中潜在砷还原微生物类群富集驯化，进而借助组学技术对不同腐殖质组分驯化体系中砷还原微生物多样性、群落组成及代谢机制进行深入解析。表明与16S rDNA群落解析相比，16S rRNA更能反映土壤复杂体系微生物群落变化。RNA水平上富集的潜在活性砷代谢微生物类群为AKIW659、Candidatus_Solibacter和厌氧粘细菌属（Anaeromyxobacter），芽孢杆菌属（Bacillus）和固氮弧菌属（Azoarcus），其相对丰度总和占总细菌的40-60%。

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