3.3 Denitrification performance of SAMFCs
The denitrification performance of the SAMFC-20Ω, SAMFC-1000 and SAMFC-20Ω at different stages is investigated. The C/N ratio of the feeding media is firstly optimized by feeding PBS containing 1.64 g L-1 acetate but different nitrate concentration. Figure S2 shows that when the C/N ratio is over 2.4, the SAMFCs achieved the optimum denitrification performance, which was basically in accordance well with the value of 2.2 reported in literature (Dhamole, Nair, D’Souza, Pandit, & Lele, 2015). The denitrification performance tests in following are based on the media with a C/N ratio of 2.4. Figure 4 shows that all the SAMFCs display comparatively poor denitrification performance at initial stage. But a gradual enhancement of denitrification performance was observed along with the rise of acclimation time. For example, In SAMFC-20Ω, the initial nitrate concentration of 200 mg L-1NO3--N drops to 127, 110, 95, 66 and 56 mg L-1 at the 20th, 40th, 60th, 80thand 100th day, respectively, in one hour (Figure 4A1, B1, C1, D1 and E1). The decrease of NO3--N concentration from 200 mg L-1 to lower than 30 mg L-1 takes about 4h at the 20th, 40th day, but 3h at the 80th and 100th day. The similar NO3--N concentration variation trend could also be observed in SAMFC-1000Ω and SAMFC-OC. This gradual enhancement of denitrification rate with the rise of acclimation time in all the SAMFCs is probably ascribed to the gradual maturation of denitrifying biofilms at the anodes which could find the proof from microbial community analysis results in Figure S4.
As shown in Figure 4A2, the SAMFC-20Ω possess a lowkDN of 1.07, which is lower than that of the SAMFC-1000Ω (of 1.26) at the 20th day. However, after acclimation for another 20 days, the kDN of the SAMFC-20Ω increases greatly and is up to 1.57, which surpass that of the SAMFC-1000 of about 1.28 at the 40th day (Figure 4B2). Moreover, Figure 4B2, C2, D2 and E2 reveal that the denitrification rate of the SAMFC-20Ω outperforms that of the SAMFC-1000Ω and SAMFC-OC from the 40th day and achieves a stable denitrification rate with a high kDN of 1.9 after acclimation for 80 days, which is much higher than that of SAMFC-1000Ω and SAMFC-OC with similar kDN of 1.3. The slightly lowerkDN in SAMFC-20Ω at the 20thday compared with that in SAMFC-1000Ω is probably due to the competition of electrons between the denitrification of the denitrifers in the anode and oxygen reduction (ORR) in the air-cathode. In the primary stage of operation, the electrons released by the exoelectrogens at the bioanodes are tend to be accepted by the air-cathode for ORR in the SAMFC-20Ω due to the higher electrode potential than that in the SAMFC-1000Ω, thus shows slightly lower denitrification rate. The SAMFC-20Ω facilitates the accumulation of exoelectrogens which usually are capable of bidirectional electron transfer including release electrons by oxidation of acetate, and accept electrons by reduction of oxygen and denitrification. Therefore, the exoelectrogens can enhance the denitrification performance of SAMFC by (a) direct bioelectrochemical denitrification and (b) promoting the accumulation of denitrifers through catalyzing oxygen reduction to remove the trace oxygen diffused from air-cathode.
3.4 Microbial community analysis
The sum of the relative abundance of the denitrifers at the anodes of SAMFCs is summarized in table S3 and displayed in Figure 5. A great increase on the total abundance of denitrifers can be observed during the 100 days acclimation. The total relative abundance of denitrifers increases from 41.6±6.9%, 42.4±9.2% and 32.6±9.4% for the SAMFC-20Ω, SAMFC-1000Ω and SAMFC-OC at the 20th day, to 65.5±9.8%, 48.3±10.1% and 48.9±8.2% for the SAMFC-20Ω, SAMFC-1000Ω and SAMFC-OC at the 100th day, respectively. In fact, exoelectrogens with the feature of bidirectional electron transfer such as Geobacter , Pseudomonas and Comamonas are the contributor for BEDN, they can not only release electrons by the organic oxidation, but also enable denitrification by accepting electrons from electrode (Gregory, Bond, & Lovley, 2004; Yu, Wu, Cao, Gao, & Yan, 2015). It has to be noted that Geobacter in the anodes of SAMFC-20Ω and SAMFC-1000Ω with relative abundance of 4.2% and 2.1% at the initial stage survive in the denitrification process and remains 2.5% and 0.6% at the 100th day, respectively.Pseudomonas in the anode of SAMFC-20Ω and SAMFC-1000Ω with the relative abundance of 2.2% and 1.5% at the initial stage is preserved during the 100 days acclimation, and became 2.3% and 1.3% at the 100th day, respectively. A high relative abundance of 10% Pseudomonasis enriched in the SAMFC-OC at the initial stage, but could not be detected at the 100th day. The successful inheritance of Geobacter and Pseudomonas from anterior biofilm indicated that exoelectrogens could coexist well with denitrifers during denitrification process despite a tiny drop of the relative abundance was observed. In addition, Comamonas that has already existed in acetate-acclimated biofilm is typical exoelectrogens equipped with the ability of denitrification (H. Huang et al., 2019; Liang et al., 2019). The Comamonas at the initial stage has a low relative abundance of 1~2% in all SAMFCs, its proportion in the SAMFC-20Ω rises remarkably to 14.0% after acclimation for 100 days in the presence of nitrate, but there is no obvious change in the SAMFC-1000Ω and SAMFC-OC. Thus, the notable enrichment of Comamonas in SAMFC-20Ω might highly contribute to the consumption of nitrate. Most critically, the sum of relative abundance for Geobacter ,Pseudomonas and Comamonas at the 100th day is 18.9% in SAMFC-20Ω, which is three times higher than that in SAMFC-1000Ω (5.7%) and nearly five times higher than that in SAMFC-OC (3.8%). The high relative abundance of exoelectrogens in the anodophilic biofilms is considered to be the key factor to endow the SAMFC-20Ω with excellent denitrification performance. As shown in Figure S4, in one aspect the exoelectrogens can directly catalyze the bioelectrochemical denitrification, in another aspect the exoelectrogens are also able to catalyze oxygen reduction reaction, thus they could consume the trace oxygen diffused from the air-cathode and thus promote the propagation of denitrifers, e.g. Alicycliphilu s. That is why a high relative abundance of Alicycliphilu s (18.2±1.8 %) could be observed in the SAMFC-20Ω at the 100th day (as shown in Figure 4), which is much higher than that in the SAMFC-1000 (9.8±1.9%) and SAMFC-OC (5.5±2.1%).