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%).