3.2 Bioelectroactivity of anodophilic biofilms
After acclimation of the SAMFCs in medium containing acetate for 30 days, the bioelectroactivity of the biofilms enriched at the bioanodes are measured by CVs and the results are shown in Figure 3. Figure 3A1 shows that all the CVs of anodophilic biofilms acclimated in the presence of acetate (turnover condition) present the typical positive sigmoidal shape (Liang et al., 2019; Pous et al., 2016), indicating that exoelectrogens are established successfully in all the SAMFCs. Obvious differences are observed from the CVs of different SAMFC bioanodes. The CVs of the SAMFC-20Ω bioanodes display a high bioelectrocatalytic oxidation current of 7.8 mA, which is much higher than that of the SAMFC-1000Ω (5.5 mA) and SAMFC-OC (0.3 mA), respectively. These results indicate that the biofilms enrich at the bioanodes of the SAMFC-20Ω possesses higher bioelectrocatalytic activity than that of the SAMFC-1000Ω and SAMFC-OC. It has been reported that the bioelectrocatalytic activity of exoelectrogens at the anode is highly associated with the anode potential and current density (Aelterman, Freguia, Keller, Verstraete, & Rabaey, 2008; Zhao & Chen, 2018). Higher bioelectrocatalytic activity could be obtained at comparatively more positive anode potential and higher current density (Aelterman et al., 2008; Hong, Call, Werner, & Logan, 2011; Jiang et al., 2018), because electron transfer from bacteria to anode is accelerated substantially when SAMFC operated under lower external resistances (Zhu, Tokash, Hong, & Logan, 2013). In this study, the SAMFC-20Ω could deliver comparatively more positive anode potential and higher current density than the SAMFC-1000Ω and SAMFC-OC, thereby a much higher anodic electrocatalytic activity of exoelectrogen biofilms are enriched the bioanodes. Furthermore, the high current density in the SAMFC-20Ω enable anode-respiring bacteria to outcompete aerobic heterotrophs for substrate to release much more electrons (Ren, Zhang, He, & Logan, 2014), thus SAMFC operated under 20Ω is an efficient means to enrich exoelectrogens. The CVs in Figure 3A2 show that the exoelectrogens enriched in medium containing only acetate substrate could also catalyze BEDN, which is in accordance to that of previous reports (K. Y. Cheng et al., 2012) .
When the nitrate is supplied to the SAMFCs accompanying with the acetate, denitrifers are enriched at the bioanodes after acclimating for a period time. The CVs of the anodophilic biofilms at the 20th day are performed in the PBS medium containing only nitrate and shown in Figure 3B2. They all display a negative sigmoidal shape which is extremely accordance with that in ref. (Pous et al., 2016) , proving that anodophilic biofilms could successfully catalyze nitrate reduction (K. Y. Cheng et al., 2012). Notably, the CVs of the SAMFC-20Ω anode display a more negative bioelectrocatalytic reduction current response of-1.15 mA, which is four and eight times as that of the SAMFC-1000Ω (-0.28 mA) and SAMFC-OC (-0.13 mA) bioanodes, respectively, representing a higher BEDN performance. The CVs of the anodophilic biofilms at the 20th day shown in Figure 2B1 display typical positive sigmoidal shape but with increased bioelectrocatalytic oxidation peak current of 11.5, 6.6 and 1.3 mA for the SAMFC-20Ω, SAMFC-1000Ω and SAMFC-OC, respectively, indicating that the bioelectrocatalytic activity of exoelectrogens at the anodes is enhanced even in the presence of denitrifers, indicating the good coexistence of exoelectrogens and denitrifers. Moreover, a higher negative denitrification current is observed for all the SAMFCs at the 100th day (Figure 3C2), demonstrating an increase of BEDN performance with increase of acclimation time. In addition, the enrichment of denitrifers at the anode result in a distinctive “duck head” shape (Figure 3B1 and 3C1) in the CVs of the SAMFC-20Ω anodes measured in the presence of acetate. The CVs measured under non-turnover conditions (absence of substrate) can further characterize the electrochemical features and extracellular electron transfer (EET) modes of the biofilm. The non-turnover CVs of the anodophilic biofilm acclimated by feeding media containing acetate shown in Figure 3A3 present two major clear redox systems with the formal potential ranged from -0.260 to -0.350 V, which is reported to be related to membrane bound c-type cytochromes and also highly consistent with the CV characteristic that described in the literature(Fricke, Harnisch, & Schroder, 2008; F. Harnisch & Freguia, 2012). The two major redox systems at the formal potentials of -0.256 V and -0.360 V in SAMFC-20Ω are basically equal to that of -0.260 V and -0.350 V in SAMFC-1000Ω as well as -0.263 V and -0.347 V in SAMFC-OC. The similar two major clear redox systems with the formal potential ranged from 0.235 to 0.370 V can also be observed in the non-turnover CVs of anodophilic biofilm acclimated by feeding media containing both acetate and nitrate (Figure 3B3 and 3C3), hinting that the bioelectrochemical features and EET modes of the exoelectrogen biofilms is preserved even in the presence of denitrifers.