Interfacing anoxic Shewanella oneidensis biofilms with electrically conducting nanostructures
Authors: Klein Edina , Wurst René , Rehnlund David , Gescher Johannes 
Published: July 2020

&lt;p&gt;&lt;em&gt;Shewanella oneidensis&lt;/em&gt; MR1 is the best understood model organism with regards to dissimilatory metal reduction and extracellular electron transfer onto carbon electrodes in bioelectrochemical systems (BES)&lt;sup&gt;1&lt;/sup&gt;. However, under anoxic conditions &lt;em&gt;S. oneidensis&lt;/em&gt; is known to form very thin biofilms resulting in low current density output. In contrast, another exoelectrogenic model organism &lt;em&gt;Geobacter surfurreduscens&lt;/em&gt; can form electroactive biofilms up to 100 &amp;#181;m in thickness. This organism is known for its ability to transport electrons over a long range (&gt; 10 &amp;#181;m) along a network of protein filaments, called microbial nanowires. Although still controversial, it was recently reported that OmcS has a special importance for the conductivity of these nanowires&lt;sup&gt;2&lt;/sup&gt;. One of the key differences between &lt;em&gt;G. surfurreduscens&lt;/em&gt; and &lt;em&gt;S. oneidensis&lt;/em&gt; lies in how cell-to-cell electronic communication occurs, which dictate the range of electronic communication between distant cells. &lt;em&gt;S. oneidensis&lt;/em&gt; relies on direct cell-to-cell communication via electron transfer between outer membrane cytochromes or via soluble redox active flavins that are secreted by the cells&lt;sup&gt;3&lt;/sup&gt;. Our research is based on the question, what if the &lt;em&gt;S. oneidensis&lt;/em&gt; biofilm formation could be improved by introducing an artificial electronic network, similar to the native microbial nanowires for &lt;em&gt;G. sulfurreducens&lt;/em&gt;?&lt;/p&gt;
&lt;p&gt;We hypothesize that synthetic biofilms containing conductive nanostructure additives would allow &lt;em&gt;S. oneidensis&lt;/em&gt; to build multilayer thick biofilms under anoxic conditions on solid electron acceptors. To answer this question of how conductive materials affect the formation of anoxic &lt;em&gt;S. oneidensis&lt;/em&gt; biofilms, we integrated both biological and synthetic conductive nanostructures into these biofilms. As biological additive, the &lt;em&gt;c&lt;/em&gt;-type cytochrome OmcS purified from&lt;em&gt; G. sulfurreducens&lt;/em&gt; was utilized. As synthetic additives, both commercially available biotinylated gold nanorods and in-house electrochemically synthesized metal nanostructures were added to anoxic &lt;em&gt;S.&amp;#160;oneidensis&lt;/em&gt; biofilms.&lt;/p&gt;
&lt;p&gt;Cultivation and characterization of the biofilms was performed using our newly developed microfluidic bioelectrochemical platform. Microbial cultivation with the aid of microfluidic flow chambers has a great potential to form biofilms on an easy to handle laboratory scale with simultaneously ongoing multianalytical analysis&lt;sup&gt;4&lt;/sup&gt;. In our bioelectrochemical microfluidic, system &lt;em&gt;S. oneidensis&lt;/em&gt; biofilms can be grown under anoxic conditions using an anode as sole electron acceptor. The growth behavior and bioelectrochemical performance was evaluated by a combination of electrochemical techniques (chronoamperometry, electrochemical impedance spectroscopy, cyclic voltammetry) and optical analyses (confocal laser scanning microscopy and optical coherence tomography). The strategy of conductive nanostructured additives for improved electroactive biofilm formation could be an important tool for other exoelectrogenic microorganisms in order to exploit their physiological abilities for biotechnology.&lt;/p&gt;
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