Relationship Between Surface Chemistry, Biofilm Structure and Electron Transfer in Shewanella Anodes
Authors: Artyushkova Kateryna , Cornejo Jose Alberto , Santoro Carlo , Roizman Dan , Marsili Enrico , Atanassov Plamen 
Published: February 2020

A better understanding of how anode surface properties affect growth, development, and activity of electrogenic biofilms has great potential to improve the performance of bioelectrochemical systems such as microbial fuel cells. Carbon-based materials are widely used for electrode materials and can vary in surface properties such as morphology, chemistry, available surface area and wettability. Attachment surface charge and wettability (hydrophilicity/hydrophobicity) may be particularly important to the growth, development and properties of bacterial biofilm and optimization of bio-electrochemical systems performance. We previously reported the importance of hydrophilic moieties and surface morphology on bacterial attachment, dynamics of biofilm formation, and MFC performance.[1] The influence of different functional groups on biofilm properties, and, therefore, subsequent MFC performance is still not well understood.
A combination of spectroscopic, microscopic and electrochemical techniques was used to evaluate how electrode surface chemistry influences morphological, chemical, and functional properties of S. oneidensis MR-1 biofilms, to develop improved electrode materials and structures.
Confocal laser scanning microscopy (CLSM) is a powerful method providing morphological parameters of biofilm in three-dimensions, as it allows acquisition of images of fully hydrated biofilms at high spatial resolution in lateral and vertical directions. Imaging processing of CLSM 3D volumes of biofilm provides a set of comprehensive parameters that describe heterogeneous biofilm morphology in three dimensions. In this study, we relate surface chemistry and biofilm areal, volumetric and textural parameters properties to electron transfer rates and efficiency.
In the first system, we used self-assembled monolayers (SAMs) as electrodes to systematically evaluate well-defined surface chemistries’ influence on MFC performance. SAM-modified gold electrodes were used to eliminate the morphology effects associated with conventional 3-D structured carbonaceous electrodes. [2]
In the second system, we extend this approach for biofilms grown on fibrous 3-D carbonaceous materials under different applied potentials using the same biofilm.

Two types of metrics can be extracted from 3D CLSM data sets extracted as demonstrated in Figure 1. From binary volumes, we have calculated biovolume, which is a total number of pixels within the volume having a value of 0 due to present bacteria, and porosity which is the ratio of pixels having a value of 1, i.e. where no bacteria are present, to a total number of pixels. Physical dimensions of biofilm clusters were expressed as the average run length which is the average number of consecutive biomass pixels representing cell cluster in a given direct in the 3d volume. Euler number is an important topological characteristic which is related to connectivity of biofilm formed. The second set of metrics is derived from grayscale intensity is texture parameters. The first parameter is entropy which measures the degree of randomness in the image. The increase in a number of cell clusters due to growth results in more complex textures and more heterogeneous images as shown in the example in Figure 1, which is reflected in higher entropy. Uniformity of the image is related to the orderliness of the structure and is sensitive to change in disorder. Homogeneous images with fewer repeated patterns have higher uniformity while frequent and repeated patterns of pixel clusters as shown in example results in has higher uniformity.
For the model SAM-based system, we demonstrate that positively charged, highly functionalized hydrophilic surfaces are optimal for growth of a uniform biofilm with the smallest cluster size and inter-cluster diffusion distance correlating to the most efficient electron transfer.
Moreover, we followed the evolution of biofilm morphology during its growth and after electrode polarization for both flat and fibrous systems.
We propose metrics describing the loss of biomass, change in inter-cell properties and overall biofilm morphology, based on quantifying biofilm properties from 3D CLSM images directly related to biofilm electron transfer efficiency and stability.
1. Santoro, C., et al., The effects of carbon electrode surface properties on bacteria attachment and start up time of microbial fuel cells. Carbon, 2014. 67(0): p. 128-139.
2. Artyushkova, K., et al., Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes. Biointerphases, 2015. 10(1).

Figure 1