Estudio eléctrico y topográfico de apéndices bacterianos en la nanoescala

Abstract

Recently the property of some bacteria to exchange electrons with non-soluble electron acceptors, such as minerals, has been discovered. In addition, some of these bacteria can use electrodes as the final electron acceptor. This phenomenon is called Extracellular Electron Transfer (EET) and it can be done through several mechanisms, especially through conductive bacterial nanowires. The main objective of this thesis is the investigation of the polarization properties of such electrochemically active bacteria and their appendages as the polarization plays a key role in EET. The curiosity also stems out from the increasing interest in using such bacteria as a biosensing platform. Specifically, I have studied two types of bacteria, Shewanella oneidensis MR-1 and cable bacteria, from the Desulfobulbaceae family. With this purpose, I have used the Electrostatic Force Microscopy (EFM) technique, which measures the electrostatic force using a nanometric probe, combined with finite element simulations to obtain the polarization properties of bacterial nanowires. The electrostatic force depends mainly on the geometry and dielectric constant of the probe-sample system. In order to do that, first, I have developed a way to obtain the dimensions of objects without damaging them, avoiding any physical contact, by measuring the electrostatic force. I have tested this technique on silver nanowires and bacterial flagella. In this way, I have been able to optimize the EFM technique to nanowire-like biological samples at the nanoscale. Secondly, I have analyzed one of the S. oneidensis appendages, the flagella, and I have compared their properties with the flagella of a non-electrochemically active bacteria, the Pseudomonas aeruginosa. I have obtained a dielectric constant of εSo = 4.3 ± 0.6 for S. oneidensis and εPa = 4.5 ± 0.7 for P. aeruginosa, similar results for both bacteria. In addition, this value corresponds to the dielectric constant of proteins (εr ~ 4) measured with the same technique, in agreement with the fact that flagella are composed of flagellin protein monomers. Later, I have studied the electrical properties of another S. oneidensis appendages, the outer membrane extensions (OMEs), responsible for the extracellular electron transfer. In this analysis, I have obtained a relatively low value of the dielectric constant (εOME = 3.7 ± 0.7) corresponding to a combination of lipids (εr ~ 2) and proteins (εr ~ 4). However, considering that the conduction mechanism of such OMEs is through electron hopping, and electrons are localized, these results do not contradict the literature. I have also studied the electrical properties of the cable bacteria, especially the fibers that are along this filamentous bacterium. The dielectric constant of the fibers was εr = 7 ± 1. However, this result is not compatible with the conductivity reported in the literature. Therefore, a core-shell model was proposed with a conductive core of h ~ 10 – 20 nm. Subsequently, I have finished the nanoscale analysis performing qualitative EFM measurements in liquid over living S. oneidensis bacteria and rehydrated bacteria. Finally, I have connected these nanoscale measurements in dry conditions with macroscale measurements in living S. oneidensis using a microfluidic device that I designed, fabricated and characterized at the Denmark Technical University (DTU) in Copenhagen. The microfluidic device was used to perform two-electrode impedance measurements. In these measurements, the impedance experiences an abrupt change for f ~ 102 – 103 Hz when bacteria were in anaerobiosis. However, further experiments are needed to explain this phenomenon.

Algunas bacterias pueden intercambiar electrones con aceptores de electrones no solubles, como los minerales. Este fenómeno se llama Transferencia de Electrones Extracelulares (EET) y se puede hacer a través de varios mecanismos, especialmente a través de nanocables bacterianos conductores. El objetivo principal de esta tesis es la investigación de las propiedades de polarización de bacterias electroquímicamente activas y sus apéndices. Específicamente, he estudiado dos tipos, Shewanella oneidensis MR- 1 y cable bacteria. Para ello, he utilizado la microscopía de fuerza electrostática (EFM), que mide la fuerza electrostática utilizando una sonda nanométrica, combinada con simulaciones por elementos finitos. La fuerza electrostática depende principalmente de la geometría y la constante dieléctrica del sistema sonda-muestra. Para hacerlo, he desarrollado una técnica para obtener las dimensiones de los objetos evitando el contacto físico con la muestra, midiendo la fuerza electrostática. He probado esta técnica en nanohilos de plata y flagelos bacterianos, optimizándola para muestras biológicas filamentosas en la nanoescala. Después, he analizado los flagelos de S. oneidensis y he comparado sus propiedades con los flagelos de Pseudomonas aeruginosa, una bacteria no electroquímicamente activa, obteniendo similares resultados (εr~4.5). También he estudiado las extensiones de membrana externa (OMEs) de S. oneidensis, responsables del EET, obteniendo un valor bajo de la constante dieléctrica (εOME=3.7±0.7). Sin embargo, considerando que el mecanismo de conducción de tales OMEs es a través de salto de electrones, donde los electrones están localizados, estos resultados no contradicen la literatura. También he estudiado la cable bacteria, especialmente las fibras que se encuentran a lo largo de esta bacteria filamentosa. He obtenido un modelo núcleo-caparazón para las fibras con un núcleo conductor de h~10–20nm compatible con los resultados experimentales. Posteriormente, he realizado mediciones cualitativas de EFM en líquido sobre S. oneidensis viva y rehidratada. Finalmente, he realizado mediciones en la macroescala en S. oneidensis vivo usando un dispositivo microfluídico que diseñé, fabriqué y caractericé en la Universidad Técnica de Dinamarca (DTU), Copenhague. El dispositivo se ha utilizado para realizar mediciones de impedancia de dos electrodos. En estas mediciones, la impedancia experimenta un cambio abrupto para f~102–103Hz cuando las bacterias estaban en anaerobiosis.