0 50 100 150 200 250 300 350 400 t=0 t=1h t=24h t=48h t=120h t=144h t=168h t=192h t=216h t=0h t=24h t=48h t=72h t=96h t=0h t=24h t=48h t=72h t=96h t=120h t=144h t=168h t=192h t=216h t=240h t=264h t=288h t=312h t=336h t=360h t=0h t=24h t=144h t=168h t=192h t=220h t=0 t=24h t=48h t=72h t=96h t=168h t=0 t=24h t=48h t=72h t=96h t=168h CCV (mV) Time (hours) Voltage generation with MFC-C vs MFC-U MFC-C MFC-U E. G. Di Domenico 1 , G. Petroni 3 , D. Mancini 2 , L. Di Palma 2 , A. Geri 3 , F. Ascenzioni 1 1 Biology aŶd BiotechŶology Charles DarwiŶ DepartŵeŶt, Via dei Sardi 70, 00185, SAPIENZA University of Rome, Italy. 2 Chemical Engineering Materials and Environment Department, Via Eudossiana 18, 00184, SAPIENZA University of Rome, Italy. 3 Astronautic, Electric and Energetic Department, Via delle Sette Sale 12/b, 00184, SAPIENZA University of Rome, Italy. 0 100 200 300 400 500 600 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 CCV (mV) Time (days) P (mW) Voltage and Power generation with the MFC-starter P (mW) CCV (mV) Merge Dead Bacteria Live Bacteria PEM Anode Cathode 16SrRNA G. sulfurreducens 16SrRNA MFC-Starter G. sulfurreducens E.Coli H2O LIVE/DEAD ASSAY DNA EXTRACTION AND PCR Electricity Production and Nitrogen Removal from digestate by Microbial Fuel Cells Digestate MFC-C MFC-U G. sulfurreducens E. coli RsaI HinfI 250bp 500bp 750bp 1000bp 1500bp Digestate MFC-C MFC-U G. sulfurreducens E. coli Introduction Digestate, residual from biogas power plant, is a nutrient-rich substance produced by anaerobic digestion that can be used as a fertiliser. All the nitrogen (2.3-4.2 kg/tonne, as well as phosphates, 0.2-1.5 kg/tonne, and potassium, 1.3- 5.2 kg/tonne) presents in the feedstock will remain in the digestate as none is present in the biogas. Therefore, in order to reduce the severe impact that high nitrogen concentrations have on regulating the eutrophic level of natural environment, the treatment of digestate is frequently mandatory (Nitrates Directive 91/676/CEE) prior its sprinkling over soil. Usually, a two steps process achieves biological nitrogen removal by nitrification (aerobic oxidation of ammonia to nitrite and nitrate) and denitrification (reduction of nitrate to nitrogen gas). More recently, the concept of nitrogen removal model has been changed since the confirmation of the anaerobic ammonium oxidation (anammox) process, where autotrophic oxidation process converts ammonia to N 2 using nitrite as the electron acceptor. It has been shown that nitrification and denitrification can occur into the microbial fuel cell (MFC) cathodic compartment leading to simultaneous nitrogen and organic carbon removal in a single reactor (Virdis et al., Water Res., 2010; Yu et al., Water Res., 2010); in addition, these cells may also produce electrical energy (He et al., Environ. Sci. Technol., 2009). The goal of our study was the simultaneous removal of carbon and nitrogen (anammox) in an MFC reactor fed with a real digestate at the anodic chamber. The electroactivity of G. sulfurreducens was monitored over time by electrical potential measurements in a closed circuit, with a digital multimeter (Fluke 87V). After ten days the potential difference reached 478.5 mV corresponding to a current of 2.7 mA and a power of 1.2 mW. The reactors were re-inoculated with fresh medium when the potential difference dropped under 20% of the maximum peak. The polarization curve and power density-current curve with OCV 748 mV, was observed in regime of power overshoot. The polarization curve, power density-current curve and power generation were measured using various external resistors in the range of 0/1000Ω. In the first phase of the trial was set up a two chamber MFC H-type (MFC-Starter) in which the anodic and the cathodic chamber were separated by a proton exchange membrane (PEM - Nafion 117 DuPont™) and the graphite electrodes (Goodfellow Cambridge Ltd.) were connected to an external resistance (180 Ω). The anodic chamber, operated in fed-batch mode in a temperature-controlled room (30 °C), was inoculated with a pure culture of Geobacter sulfurreducens (DSM12127, repository DSMZ) and filled to 250 ml with a synthetic medium containing acetate as the electron donor. To confirm the presence of G. sulfurreducens, microbiological and molecular analysis were carried out. A small portion anode was stained with the Live/Dead assay (BacLight, Invitrogen). An aliquot of the bacteria, detached mechanically from bioanode, was used for a DNA extraction. PCR reactions revealed the existence of a G. sulfurreducens population over the bioanode. 150,03 Ω 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 0 100 200 300 400 500 600 700 800 900 1000 1100 Power, mW Rest [Ω] Power Generation Two-chamber reactors, separated by cation exchange membrane and fed with the liquid phase of a digestate were assembled: one containing a bioanode conditioned with G. sulfurreducens (MFC-C - Conditioned) and the second with a sterile anode (MFC-U - Unconditioned). The reactors operated under batch feeding for two months during which they were monitored daily. Following inoculation the MFC-C, showed a rapid increase of the electrical performance reaching, after 48 hours, a maximum power of 0.6 mW – 240 mW/m 2 - to 346.8 mV. At the same time, the MFC-U did not show an appreciable electrical activity; nonetheless, following a 28 days (the time required for anode colonization) the MFC-U showed acceptable electrical performance (the maximum power transfer 0.4 mW – 172.2 mW/m 2 - at 359.4 mV). MFC fed with digestate Start-up phase From an area closed to the anode, samples of planktonic bacteria were harvested. Since biofilm is a dynamic structure in which the bacteria pass from a phase of adhesion to a phase of dispersion, it is reasonable to hypothesize that the bacteria near the anode reflect the microbial composition of the anode itself. DNA extracts from the digestate, the MFC-C and MFC-U were used to analyze the microbial diversity. Microbial communities analysis Digestate MFC–U MFC-C E.coli Geobacter Pseudomonas Burkholderia DNA E.coli (+) H 2 O 250bp 500bp 750bp 1000bp 1500bp 2000bp 2500bp 3000bp Random Amplified Polymorphic DNA (RAPD) Restriction Fragment Analysis Digestate MFC-C MFC-U G. sulfurreducens E. coli H 2 O 16SrRNA G. sulfurreducens PCR for G.sulfurreducens The RAPD and the restriction fragments analysis indicate that the microbial community in the two reactors is similar to that in the digestate. The analysis of the profiles from the MFCs and from G. sulfurreducens highlights the presence of similarities. The existence of a G. sulfurreducens community in the digestate and in the MFCs was confirmed by PCR. Further analyzes, such as cloning and sequencing of 16SrRNA gene will provide more information about the microbial composition in the digestate and the MFCs. Concluding remarks In this study, for the first time in our knowledge, a real digestate, coming from cow manure and agricultural waste, was used to power the anodic compartment of an MFC. We showed that the resident bacterial community present in the digestate revealed good performance in terms of carbon and nitrogen removal. Additionally, we also showed that the digestate is a source of electroactive bacteria that, when placed in the MFC configuration, provide the same performance of G. sulfurreducens in terms of electricity production and metabolism. Pollutants removal efficiency Electrochemical performance The polarization curve, with the respective open circuit voltage (OCV) and the power performance curve, with the maximum power (Pmax). From the point of maximum power, the optimal voltage (DEopt) and optimal current (iopt) can be deduced. 0 5 10 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 current (mA) Current generation MFC-C MFC-U 0 1 2 3 4 0 1 2 3 4 5 6 7 current (mA) time (days) MFC-C MFC-U The current was obtained starting from an OCV of 1 h; the insert show the values of the current with a external resistance fixed at 180 Ω. 0 50 100 150 200 250 300 350 400 450 500 0 100 200 300 400 500 600 700 800 900 1000 1100 0 100 200 300 400 500 600 700 800 Power density, mW/m 2 Current density, mA/m 2 Voltage, mV Voltage and maximum power generation Voltage Power density Both the reactors showed a good removal of organic matter (up to 60-65%), corresponding to a coulombic efficiency of 19,55% and 15,58% respectively (upper panel). Samples from both the reactors were also collected for the nitrogen removal analysis. The results revealed the same nitrogen removal power which runs to about 40%. 0% 10% 20% 30% 40% 50% 60% 70% 0 1 2 3 4 5 CE e COD (%) Time (days) Coulombic Efficiency and COD removal (%) CE MFC-U CE MFC-C COD MFC-U COD MFC-C 0,4 0,5 0,6 0,7 0,8 0,9 0 1 2 3 4 5 N (g/l) Time (days) Nitrogen removal MFC-C MFC-U Pmax iopt ΔEopt 0 100 200 300 400 500 600 700 800 0 20 40 60 80 100 120 140 160 180 200 0 200 400 600 800 1000 Voltage, mV Power density, mW/m 2 Current density, mA/m 2 Voltage and maximum power generation MFC-U Power density Voltage Pmax iopt ΔEopt 0 100 200 300 400 500 600 700 800 0 50 100 150 200 250 300 0 200 400 600 800 1000 1200 1400 Voltage, mV Power density, mW/m 2 Current density, mA/m 2 Voltage and maximum power generation MFC-C Power density Voltage Electrochemical Impedance Spectroscopy (EIS) with electrical circuit of the Randles cell for the MFC-C and MFC-U respectively.