Online Monitoring of Electrochemically Active Biofilm Developing Behavior by Using EQCM and ATR / FTIR

It is crucial to study the bacterial attaching behavior for revealing information of electrochemically active biofilm on the interface of liquid/electrode in microbial fuel cells (MFCs). In the current study, mixed-culture from waste water as model bacteria was investigated for gaining more sight onto relationship between current and biomass or onto bioadhesion mechanism at the molecular level using Electrochemical Quartz Crystal Microbalance (EQCM) and Attenuated Total Reflection-SEIRAS (ATR-SEIRAS) combined with electrochemistry. From the frequency shift using Sauerbrey equation via EQCM the maximum cells mass of 11.5 μg/cm was estimated for the mature biofilm. Notably, the highest current density of 110 μA/μg·cm occurred before maximum biomass coming, which indicated that mature biofilm may not be an optimal state for enhancing power output of MFCs. Furthermore, the sensed biomass attached on electrode for mature biofilm will be constant for more than 40 h ∗ Corresponding author, Current address: College of Life Sciences, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi Province, P.R. China, 712100, E-mail: lydiayliu@yahoo.com 2 even under depletion of substrate, implying that cells attached on surface surrounded by filamentous materials and extracellular polymeric substances (proteins, polysaccharides, humic substances, etc) packed together can tolerant lacking nutrients. On the other hand, using ATR-SEIRAS techniques the obvious adsorbed water structure change during biofilm formation on electrode surface was observed and showed that the absorption bands linked to bacterial adsorption increased. It can be concluded that water adsorption accompanies the adsorbed bacteria and the cells number attaching on the electrode increased with time. Especially, the direct contact of bacteria and electrode via outer-membrane protein can be confirmed via series spectra at amideIand amideII modes and water movement from negative bands displacing by adsorbed bacteria. Our study provided supplementary information about the interaction between the microbes and solid electron acceptors beyond traditional electrochemistry. It is expected to explore more information on electrochemically active microbial kinetics for improvement of their competence and understanding of attaching mechanisms in nature using our approach. Introduction Traditional biofilm research based on microbial cell aggregates has long history on studying its bioblocking, biocorrosion, and also on advantages such as treating wastewater using biofilm fluidized bed reactors. A biofilm can be defined as a complex coherent structure of cells and extracellular polysaccharide structures to form spontaneously large and dense granules, or grow/attach on solid surfaces. Recently, biofilms have another remarkable function as biocatalysts in renewable energy of microbial fuel cells (MFCs). The distinct differences of biofilms in MFCs by 3 comparison to the traditional biofilms are that they are electrochemically active by using the solid electrode as electron acceptor in some step of bacterial metabolism. Electrochemically active biofilm development on electrode surfaces has critical influence on MFC application. At present, much effort has been made to provide new insights into electron transfer processes occurring at the electrode/biofilm interface by combining some modern analytic technologies with traditional methods. The unique technique of scanning tunnel microscope has successfully exhibited the presence of conductive pili nanowires (Gorby et al. 2006; Reguera et al. 2005; Reguera et al. 2006) which have been involved or may play key function in the electron transfer through anode G. sulfurreducens with biofilm even more than 50 μm thick (Malvankar et al.). The identification of secreted or metabolized self-mediator or intermediator e.g. phenazine (Hernandez 2004) or flavins in shewanella species (Marsili et al. 2008) has been realized by combining electrochemistry, biology, and typical analytical chemistry techniques including high-performance liquid chromatography and chromatography-mass spectrometry, etc. Besides, genetic engineering can identify the complicated outer-membrane system making up of hundreds of cytochromes types (Busalmen et al. 2008; Lovley 2008; Myers and Myers 2000) and find that c type of cytochrome in biofilm will be in charge of the electron transfer step. Furthermore, for molecular function group identification, spectroscopy is one of most important methods to give information about molecular structure aspects. In this respect, the interface between G. sulfurreducens cells and electrodes was studied using a spectro-electrochemical approach by attenuated total reflection-SEIRAS (ATR-SEIRAS) (Busalmen et al. 2008). Most importantly, Bond group advanced a nondestructive in situ linking technology of electrochemical and UV-visible spectroscopy through which the function of redox cytochrome c in biofilm in situ was