The molecular ecology of arsenic; probing the biogeochemical basis of a humanitarian disaster

饮用和灌溉使用含有高浓度砷的地下水正在毒害全世界数百万人。例如，在西孟加拉邦和孟加拉国，砷含量可以达到每升水毫克的砷含量，这导致了被称为“人类历史上最严重的大规模中毒”。尽管迫切需要关于砷从沉积物释放到水中的机制的基本信息，但这一人道主义灾难的原因仍然存在争议。有几种可能的机制可以释放被沉积物中矿物质吸附的砷，所有这些都有激烈的争论。这些变化包括含水层上部富砷黄铁矿的氧化或沉积物深层还原条件下富砷铁（III）氢氧化物的分解，而水中的其他离子也可以调动吸收的砷，例如磷酸盐或碳酸盐。虽然这些变化是化学的，但越来越多的人一致认为，沉积物中的微生物很可能驱动这些反应。事实上，我们实验室最近的结果表明，专门的地下微生物动员了沉积物中矿物质吸收的有毒砷。在缺氧条件下生长，这些“金属还原细菌”通过吸附的砷(V)的还原以及有机物的氧化来获得能量。在其他实验室研究的支持下，现在有一种共识，即这种形式的微生物代谢在控制世界范围内含水层沉积物中的砷浓度方面起着关键作用。然而，关于负责的细菌的身份的信息很少，并且没有模式生物作为“地球微生物学”研究恒河三角洲含水层中砷还原和动员机制的基础。迫切需要这些信息来支持补救工作或帮助开发更安全的用水方法，因为如果不详细了解原因，显然很难解决环境问题。这项工作的目的是通过对西孟加拉邦含水层的微生物学进行详细和早就应该进行的研究，以及应用最先进的分子生物学技术来识别参与沉积物中砷释放的基因和蛋白质，来解决我们对含水层中砷动员机制理解的局限性。通过为微生物群落提供同位素标记的有机物质（乙酸盐和乳酸盐作为新有机物质的代用物，通过取水和石油作为深层沉积物中的电子供体），我们将分离出当砷被动员时在沉积物中活跃的细菌合成的标记核酸，并使用遗传指纹技术识别这些“活跃”细菌。以及它们所含的相应的减少/调动砷的基因。由于许多砷基因将是新的，我们将在“宏基因组文库”中寻找它们，这些文库将包含来自沉积物的大片段DNA，这些DNA片段编码As(V)还原酶基因和其他高度保守的标记基因，我们可以使用它们来准确识别细菌。这将是宏基因组文库构建和筛选首次在该领域被用于鉴定潜在的新型As(V)呼吸细菌，而不需要培养它们。最后，为了更好地了解这些生物转化在砷循环中的作用，我们还将使用最先进的矿物学和地球化学技术研究矿物相和地下水成分，同时我们还将监测微生物群落及其表达的基因的变化。这将使我们能够对微生物代谢对水态和矿物结合形式的砷的影响形成详细的分子尺度图像。

The use of groundwaters containing high concentrations of arsenic for drinking and irrigation is poisoning millions worldwide. For example, in West Bengal and Bangladesh arsenic levels can reach mg quantities of arsenic per litre of water, and this has led to what has been described as 'the worst mass poisoning in human history'. Despite the urgent need for fundamental information in the mechanism of arsenic release from sediments into water, the causes of this humanitarian disaster remain controversial. Several possible mechanisms may release arsenic sorbed to minerals in the sediments, and all have been debated vigorously. These include changes in the sediment minerals from the oxidation of arsenic-rich pyrite in the upper regions of the aquifers or the breakdown of arsenic-rich Fe(III) oxyhydroxides under reducing conditions deeper in the sediments, while other ions in the water could also mobilise sorbed arsenic e.g. phosphate or carbonate. Although these changes are chemical, there is a growing consensus that microorganisms in the sediments may well drive these reactions. Indeed, recent results from our laboratory have shown that specialist subsurface microorganisms mobilise the toxic arsenic sorbed to minerals in the sediments. Growing in the absence of oxygen, these 'metal-reducing bacteria' gain energy from the reduction of sorbed As(V) coupled to the oxidation of organic matter. With growing support from studies in other laboratories, there is now a consensus that this form of microbial metabolism plays a critical role in controlling arsenic concentrations in aquifer sediments worldwide. There is, however, little information on the identity of the bacteria responsible, and no model organisms on which to base 'geomicrobiological' studies on the mechanism of arsenic reduction and mobilisation in aquifers in the Ganges delta. This information is needed urgently to underpin remediation efforts or help develop safer practices for water use, as it is clearly very difficult to solve an environmental problem without a detailed understanding of the cause. The aim of this work is to address our limitations of the understanding of the mechanism of As mobilisation in aquifers by conducting a detailed and long overdue study of the microbiology of such an aquifer in W. Bengal, alongside the application of state of the art molecular biology techniques to identify the genes and proteins involved in arsenic release from the sediments. By feeding microbial communities with isotopically labeled organic matter (acetate and lactate as proxies for new organic matter drawn into the aquifers by water abstraction and petroleum which is an electron donor in deeper sediments), we will isolate the labeled nucleic acids synthesized from bacteria that are active in the sediments when arsenic is mobilised, and use genetic fingerprinting techniques to identify these 'active' bacteria, and the corresponding arsenic reducing/mobilising genes that they contain. As many of the arsenic genes will be novel, we will look for them in 'metagenomic libraries' which will contain large fragments of DNA from the sediments that encode both the As(V) reductase genes and other highly conserved marker genes that we can use to identify the bacteria accurately. This will be the first time that metagenomic library construction and screening has been used in this field to identify potentially novel As(V)-respiring bacteria without the need to culture them. Finally, so that we can gain a better picture of the role of these biological transformations in the arsenic cycle, we will also study the mineral phases and groundwater composition using state of the art mineralogical and geochemical techniques, while we are monitoring changes in the microbial communities and the genes that they are expressing. This will allow us to develop a detailed molecular-scale picture of the impact of microbial metabolism on the aqueous and mineral-bound forms of arsenic.