Centimeter-long electrical transport in cable bacteria - structural and electro-optical properties

Abstract

This thesis is the result of the first PhD research project within the research group X-LAB, which has the main objective of performing interdisciplinaryand exploratory research with the aim on a creative and sustainable future. The interdisciplinary research of this PhD project explores the boundary between microelectrobiology and bio- and organic electronics by bridging the gap between different disciplines (biology, physics, chemistry, electronics, ...). Bioelectronics is an upcoming interdisciplinary research field, where biological or biomimetic materials are used as building blocks in microelectronics or electro-optical applications, sensors and bio-energetic systems (e.g. microbial fuel cell). In this thesis, which is the result of a collaboration with the group of prof. dr. ir. Filip Meysman, we study a recently discovered species of bacteria with exceptional electrical properties - the so called cable bacteria. Cable bacteria are intriguing microorganisms; they grow in marine or freshwater sediment and one bacterium (a ”filament”) can reach a length of up to 7 cm. One cable bacterium filament is a single chain of sometimes more than 10.000 cells, growing vertically between oxygen-rich seawater and into sulfide-rich sediment. The uptake of oxygen and sulfide is separated by centimeters distances, although these processes have to be able to exchange electrons to produce energy for the bacterium. It was therefore proposed that conductive fibers reside in the bacteria which transport the electrons over the whole length of the filament. For comparison, electron transport in photosynthesis or in other electrogenic bacteria (bacteria capable of extracellular electron transport) achieves distances in the range of a few nanometers to micrometers. The overall goal of this thesis is to gain insight in the ’Terra Incognita’ of electrical transport in cable bacteria by an exploratory study of the morphological and intrinsic electro-optical properties. An interdisciplinary approach is used with methodologies also used in the study of organic and inorganic semiconductors. This PhD thesis consists of a bundle of manuscripts preceded by a number of introductory chapters. The first chapter describes what cable bacteria are, and how they are able to survive in marine sediment. Electrogenic bacteria - bacteria that are able to conduct electrons over long distances - are used as modelorganisms to better understand electron transport in cable bacteria. Cable bacteria also have a direct effect on their environment. For example, it was discovered that they promote the formation a ’firewall’ which prevents the release of toxic sulfides into the seawater. The introduction ends with some applications of electrogenic bacteria, including power generation and biofuel production. Possibly, cable bacteria could also be used for these and other applications in the future (microbial fuel cells, sensors, ...). Since the discovery of cable bacteria in 2012, only a few articles have further explored their architecture. In paper A of this thesis we measured the overall dimensions of differently sized cable bacteria, and built a quantitative structural model. A unique feature of cable bacteria is that the outer membrane is deformed; the full contour of a cell has ridges which run continuously over the whole length of the filament. The conductive fibers are believed to be embedded in these ridges. By making cross-sections at several locations in a filament, it became clear that fiber-like structures are indeed located in the periplasm (the space between the outer and inner cell membrane), and that they cross the gap between adjacent cells. Between two cells the outer membrane folds inwards, surrounding the radial connections in a cartwheel-like structure. Our colleagues from UAntwerp and TU Delft developed a procedure to remove the membranes and cytoplasm from cable bacteria. This way, they were able to retain the fibers which remained connected to an underlying sheath and at the junctions. This extracted fiber sheath, when proven to be electrically conductive, might be very interesting for future use in bioelectronic applications. Although record-breaking electron transport distances have been attributed to cable bacteria, it has proven difficult to directly measure a current flow through a filament. Normally, it is sufficient to connect an electrogenic bacterium between two conductive electrodes, and subsequently measure a current with an applied voltage. Up to now this has never succeeded for cable bacteria, and it was presumed that their outer membrane was electrically insulating. Paper B in this thesis describes how we were able to measure for the first time the electrical properties, which is a significant breakthrough in electrobiology. The conductivity of the bacteria was observed to decay upon contact with oxygen; after a few hours the bacteria were again electrically insulating. Electrical measurements on single filaments yielded the conductivity of a single fiber (~11.5 S cm -1), which is of the same order of magnitude as some organic semiconductors. Furthermore, we measured a current through a filament of a length of 10.1 mm, which make cable bacteria the biological record holders for long-distance electron transport. We determined that the extracted fiber sheath from the first article is also electrically conductive, making them the primary conductive structures responsible for long-distance electron transport in cable bacteria. Further study of the electrical properties is required to gain insight in the potential of applying these structures in e.g. bio-electronics. The use of cable bacteria in photovoltaics or as (transparent) electrodes depends among other things on the optical properties, such as emission and absorbance spectra. Paper C in this thesis is an exploration of the optical properties of cable bacteria. One result of this study is that cable bacteria fluoresce when removed from their natural environment. The autofluorescence originates from the cell envelope and from the junctions, indicating a relation with the metabolic cycles. We also used a technique which is rarely used in biology, photothermal deflection spectroscopy. It measures the emission of heat after illumination with varying photon energy, yielding a measure for the absorbance of cable bacteria. These investigations yielded the photon emission and photothermal spectra of cable bacteria. Further research is needed to gain insight into the autofluorescence behavior and the possible relation with the electrical properties. This thesis ends with the conclusions and outlook with some future research ideas.