富里酸驱动发酵梭菌砷甲基化的代谢机制解析

Metabolic mechanism of fulvic acid-driven arsenic methylation by fermentative Clostridium

微生物介导砷甲基化是砷生物地球化学循环中的重要过程.已有研究表明,腐殖质添加能促进稻田土壤厌氧微生物砷甲基化,但其关键机制尚不明确.本研究以砷污染稻田土壤中分离的一株砷甲基化功能厌氧发酵梭菌Clostridium sporogenes LHA6为对象,探究富里酸对梭菌细胞代谢活性的影响,并解析其与砷甲基化效率的内在关系.结果表明,相比对照处理,特定浓度(0.5~2 g·L^(-1))富里酸处理中菌株LHA6的水溶态和细胞结合态甲基砷生成量分别增加25.7%~84.4%和39.8%~60.4%,挥发性甲基砷产物增加0.68~1.86倍,主要产物形态为二甲基砷及其氢化物.综合电化学、代谢物和基因表达定量等方法证实:富里酸可作为胞外电子受体,抑制梭菌胞内发酵产氢活性,增加NADH/NAD+比率;同时,富里酸存在时梭菌显著上调S-腺苷甲硫氨酸(SAM)合成相关基因转录活性,增强SAM合成代谢通量,很可能是富里酸促进梭菌产生甲基砷及其氢化物的关键机制.本研究系统揭示了富里酸调控梭菌细胞代谢,进而强化其砷甲基化及挥发的砷解毒机制,获得了对砷的生物地球化学循环的新认知,有望为土壤砷污染控制提供新思路.

Microbially mediated methylation of arsenic(As)plays a critical role in the As biogeochemical cycle.Previous studies have shown that humic substances can promote microbial As methylation in anaerobic paddy soil,however,the associated microbial mechanism for this process remains unclear.Here,Clostridium sporogenes LHA6,a paddy soil-derived anaerobic As-methylating bacterium,was used to probe the linkage between fulvic acid-one of typical components of humic substances‒and metabolic activity and fermentative metabolism of strain LHA6 and its As methylation efficiency.Anaerobic As methylation microcosms evidence that fulvic acid(particularly 0.5~2 g·L^(-1))could significantly increase dissolved and cellbound methylated As from strain LHA6 by 25.7%~84.4%and 39.8%~60.4%,respectively,leading to 0.68~1.86-fold increase of methylated arsine products,compared with the control.The detected dominant As species were dimethylarsenate and volatile dimethylarsine.Combing electrochemical analysis with metabolite and gene transcript quantification,we found that fulvic acid mainly function as extracellular electron acceptor to inhibit intracellular hydrogen production activities and to increase the NADH/NAD+ratio in strain LHA6.Further,fulvic acid was shown to significantly upregulate the transcription of S-adenosylmethionine(SAM)synthesis-related genes,which may enhance SAM synthesis flux,contributing to an increase of As methylation and arsine generation in strain LHA6.Collectively,this finding highlights the microbial mechanism underlying the stimulatory effect of fulvic acid on anaerobic microbial As methylation and volatilization,which expands our understanding of the biogeochemical cycle of As and potential bioremediation of As in the contaminated soil.