# Novel Microfluidic Septum to Optimize Energy Recovery in Single-Chamber Microbial Fuel Cells
## 2 Materials and Methods
### a-SCMFC Acclimation and Start-Up  
The inoculation of the a-SCMFCs was performed with a mixed microbial consortium, obtained from the third enrichment of marine sediment (10% \(v/v\); marine sediment was collected in La Spezia) [41; 42]. Each enrichment was performed via dissolution into a water-based electrolyte, containing 12 mM of sodium acetate as the carbon energy source.  
The acclimation phase for all a-SCMFCs was monitored for 15 days, evaluating the biofilm formation onto the anode surface, by implementing an external load of 470 \(\Omega\), which was shown to be suitable to induce the formation of biofilm [31; 38]. After the acclimation phase, an external load of 1 k\(\Omega\) was applied to directly evaluate the overall a-SCMFCs' performance. During this phase, named the start-up phase, the electrolyte solution was prepared via dissolution in de-ionized water sodium acetate with a concentration of 12 mM (1 g/L).  
To ensure the optimal operation conditions, ammonium chloride (NH\({}_{4}\)Cl, 0.31 g each, 1 g of sodium acetate) was added to provide a nitrogen source to aid microbial growth, potassium chloride (KCl, 0.13 g/L) as a mineral source, and sodium dihydrogen phosphate (NaH\({}_{2}\)PO\({}_{4}\), 2.450 g/L) to maintain a stable neutral pH. All reagents were purchased from Sigma Aldrich, and the obtained electrolyte solutions were autoclaved prior to use.  
The presence of an IMS leads to the achievement of the best performance compared to that achieved without the IMS. To demonstrate and confirm this consideration, we introduced a parameter called energy recovery, defined by the following Equation (1):  
\[E_{rec}=\frac{\int_{t_{1}}^{t_{2}}P(t)dt}{V_{int}}, \tag{1}\]where \(E_{rc}\) (J/m\({}^{3}\)) is the energy recovery, \(V_{int}\) (m\({}^{3}\)) is the internal volume of the MFCs, and \(\int_{t_{1}}^{t_{2}}P(t)dt\) (J) is the integral of the recovered energy between the initial (\(t_{1}\)) and final (\(t_{2}\)) moments associated with each refill.  
During the experiment, solutions with a reduced amount of sodium acetate were also tested. A different reduced concentration of sodium acetate with respect to the standard one (12 mM of sodium acetate) was proposed to confirm that the voltage output decreases as the concentration of sodium acetate decreases. Through this approach, it was intended to demonstrate the effectiveness of the IMS, which was shown to be suitable for the maximization of the response of IMS cells to changes in sodium acetate concentration. This hypothesis could be confirmed by the fact that the IMS ensured the distribution of fluid flow in a drop-like shape, thus leading to the maximization of the distribution of sodium acetate within the device and, therefore, improvements in the response of the IMS cells.  
The analyzed concentrations were 8 mM (0.67 g/L), 4 mM (0.33 g/L), and 2 mM (0.17 g/L) During this part of the experiment, the composition of the electrolyte medium remained unchanged, except for the ammonium chloride concentration, which was reduced accordingly to preserve the same mass ratio as sodium acetate.  
The a-SCMFCs were operated in fed-batch mode, with the electrolyte being replaced every 48 h to 72 h, in correspondence to which a voltage drop was observed. The fresh electrolyte was replaced when the voltage drop achieved a value close to 0 V.