Biofilm Structure of Geobacter sulfurreducens by Helium Ion Microscopy

Abstract

Microbial communities form biofilms on material surfaces in a multitude of ecosystems, from the root hairs of a plant to the human gut. The hallmarks of an established biofilm include (1) the attachment of microbial cells to a surface, (2) production of extracellular polymeric substance, (EPS) (3) a complex structure or "architecture," and (4) the ability to exchange genetic information between cells. [1] Geobacter sulfurreducens forms unique, electrically conductive biofilms, a property that can be exploited in production and design of microbial fuel cells. In this work, examine biofilm formation, and biofilm properties of Geobacter sulfurreducens using a Scanning Electron Microscope (SEM) as well as a Helium Ion Microscope (HIM). SEM is a high-resolution imaging technique used for characterization of a broad variety of materials. However, in order to image highly insulating, soft biological materials, the samples must be coated for charge compensation. These (typically) metallic coatings create a homogenous surface and may cloak true biological behavior and material contrast in the micrograph. In the case of Geobacter sulfurreducens, metal coating precludes detailed investigation of microbial attachment, presence of EPS, and fine surface details that may elucidate the mechanisms behind architecture formation and genetic material exchange. Recently introduced HIM, offers more flexibility in investigating biological samples, as highly insulating sample can be imaged sui generis, without the use of a conductive coating. [2] This opens new pathways to capturing high resolution spatial details of biofilm formation and biofilm properties. Furthermore, high-resolution HIM imaging reveals true surface details of Geobacter sulfurreducens, such as flagella or pilin typically inaccessible by SEM. Finally, the effects of different sample preparation strategies for SEM and HIM will be illustrated and discussed [3].

Bio-imaging with the helium-ion microscope: A review

“…HIM was used to visualise the nanostructure of the bacterial colonies; however, no micrographs with sub-20 nm resolution are shown. The second publication is an extended abstract by Belianinov et al on the investigation of the biofilm structure of Geobacter sulfurreducens by HIM [79]. The helium-ion micrograph of the bacterium shown in this abstract displays the importance of an appropriate preparation of the sample.…”

“…HIM imaging has already been used to study numerous microorganisms including bacteria [6,18,19,60,79,80], bacteriophages [17,18,81], yeast [82], protozoa [83][84][85], and microalgae [46]. The highly resolved and contrast-rich views of the tiniest creatures that were obtained in these studies are discussed in the following.…”

“…To date, there are not many studies which have applied HIM to study microbial Fe(III) reduction. The study by Belianov et al [79], who imaged a biofilm of the Fe(III)-reducing bacterium Geobacter sulfurreducens, is the only published example, to the best of our knowledge.…”