Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10(4) bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10(5) bacteria mg(−1) of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.

本研究开发了一种酶联免疫过滤测定法（enzyme-linked immunofiltration assay, ELIFA），用于直接且特异性地定量氧化亚铁硫杆菌（Thiobacillus ferrooxidans）在硫化矿物表面的附着情况。该方法源于酶联免疫吸附试验，依托滤膜开展实验——滤膜可截留矿物颗粒，以便后续在微孔板中进行免疫酶促反应。本研究使用的多克隆抗血清以氧化亚铁硫杆菌DSM 583为免疫原制备，可识别硫杆菌属（Thiobacillus）细菌表面的细胞抗原。该抗血清与ELIFA可实现附着细菌的直接定量，且灵敏度极高：每块微孔板孔可检出10⁴个细菌。在粒径56~65 μm的黄铁矿样品中，细菌附着量的平均值约为每毫克黄铁矿10⁵个细菌。氧化亚铁硫杆菌在黄铁矿表面的几何覆盖率介于0.25%~2.25%之间，这表明氧化亚铁硫杆菌会附着于黄铁矿表面具备特定电化学或表面特性的明确有限位点。实验证实ELIFA可适配不同水平的黏附量检测，因此该方法可用于构建氧化亚铁硫杆菌在不同理化性质的各类硫化矿物表面的黏附等温线，以解析细菌与矿物表面的相互作用机制。