Honeker Linnea K, Root Robert A, Chorover Jon, Maier Raina M
Department of Soil, Water, and Environmental Science, P.O. Box 210038, University of Arizona, Tucson, AZ 85721, United States.
J Microbiol Methods. 2016 Dec;131:23-33. doi: 10.1016/j.mimet.2016.09.018. Epub 2016 Sep 29.
Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (p<0.05). Of the bacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate.
由于人为活动导致的环境中金属（类金属）污染是一个全球性问题。要了解污染物的归宿，需要阐明从分子尺度到田间尺度影响金属（类金属）形态的生物和非生物因素。需要改进方法来评估微观尺度的过程，例如在植物组织、微生物细胞和金属（类金属）之间的生物地球化学界面上发生的过程。在此，我们提出一种先进的方法，该方法将荧光原位杂交（FISH）与基于同步加速器的多能量微聚焦X射线荧光微探针成像（ME μXRF）相结合，以检查用于植物稳定化含金属矿山尾矿的植物根表面细菌与金属（类金属）的共定位情况。使用SytoBC核酸染料和针对细菌域及特定菌群（α-变形菌、γ-变形菌或放线菌）的FISH探针，在一小段根上观察细菌。然后使用ME μXRF分析同一根区域中砷和铁的元素分布及金属（类金属）形态。使用ImageJ软件对FISH和ME μXRF图像进行比对，以关联微生物学和地球化学结果。共定位定量分析结果表明，根表面与铁氧化物斑块共定位的砷的比例（重叠分数为0.49±0.19）显著高于与细菌共定位的比例（0.072±0.052）（p<0.05）。在与金属（类金属）共定位的细菌中，以其金属耐受性而闻名的放线菌与砷和铁的相关性均高于α-变形菌或γ-变形菌。该方法证明了将这些微观技术相结合如何能够扩展我们对根、金属（类金属）和微生物之间微观尺度相互作用的理解，这些信息应能改进金属（类金属）形态和归宿的机理模型。
