However afterwards inspection and analysis

However, afterwards inspection and analysis of the resonator proved a response due to the deposition (scaling) of iron oxide on the inner conductor rather than biofilm formation. This observation actually pointed us in the direction of quite another application for our sensor, i.e., the monitoring of scaling and oxidation process [29]. In order to evoke biofilm formation, our next step was to incubate the system with E. coli and perfuse the resonator with a (standard) feed solution promoting bacteria growth. The key adjustment was however to fill the resonator tube with glass beads to enhance the surface area for bacteria adherence. To show the effect of the presence of glass beads, we run two experiments in parallel, one without (Fig. 7) and the other with glass beads (Fig. 8). Clearly, the presence of glass beads significantly increased the response sensitivity of the resonator to biofilm formation. This is reflected in the change of both resonant frequency and ratio of current amplitude. The results confirm the hypothesis that the AF responses mainly depend on the formation of biofouling on the surface of glass beads and less depend on multiplication of bacterial D609 cost (E. coli in this study) in the feed substrate (see also the earlier). There is also growth of bacteria during biofilm formation. Note that the response shown in Fig. 8 is entirely different from the ‘scaling’ response shown in Fig. 6. Whereas deposition of Fe(OH)3 results in an upward shift of the AF response i.e., towards a higher amplitude at the resonance frequency, biofilm formation shifts the response, first, towards higher resonance frequencies and slightly lower amplitudes, followed by a shift in opposite direction.(discussed later in more detail). This difference points to a different working mechanism responsible for the two different types of responses observed. Fig. 9 delineates the changes of resonant frequency and amplitude ratio separately for the first five resonances in Fig. 8. The experiments ran for four days and each plot has a data point for each day. As can be seen, the changes over time for the resonances were very similar, notably in the case of the resonant frequency with a peak value at day 2. The similarity is valid for the amplitude ratio as well (but to a slightly lesser extent) and with a minimum value at day 2. From these similarities in response changes we conclude that the mechanism responsible is the same for each resonance. An important conclusion as it declassifies other, possible interfering, processes causing similar changes. Fig. 10 correlates the observed change in resonant frequency and the one in amplitude, for the first five resonances and over a time period of four days. The increasing dispersion at higher frequencies (resonances) is evident as the individual data points for each resonance diverge with increasing resonance number. This conclusion is in line with decrease of the quality factor i.e., the broader band with of each resonance relative to its center frequency, with increasing resonance number in the AF plot, see the left panel of Fig. 8. In order to correlate the observed AF responses of the resonators to bacterial growth/biofilm formation, samples were taken simultaneously and used for total organic carbon (TOC), total cell number (TCN), colony forming units (CFU) and adenosine triphosphate (ATP) analysis (Fig. 11; n=3). The increase of TOC over time, obtained exclusively from the bead surface, demonstrates the ‘deposition’ of carbon. The observation that TCN and ATP simultaneously increase renders support for the conclusion that the increase of TOC reflects the presence of (living) bacteria on the bead surface rather than scaling effects due to the deposition of inorganic carbon. The temporal dip at day 3 seen simultaneously in the analysis of ATP, TCN and CFU indicates a ‘real’ effect rather than an artefact. The most plausible reason for this observation is carbon depletion of the feed solution (experiments were performed in a closed system at recycle conditions over the resonators and dummies). The carbon coming free after mass starvation of bacteria cells served as carbon source, resulting in a blooming bacteria culture at day 4.