Bioelectrochemistry 117 (2017) 34–39

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Bioelectrochemistry

journal homepage: www.elsevier.com/locate/bioelechem

Biofilm promoted current generation of Pseudomonas aeruginosa

microbial fuel cell via improving the interfacial redox reaction
of phenazines
Ya-Juan Qiao, Yan Qiao ⁎, Long Zou, Xiao-Shuai Wu, Jian-Hua Liu
Faculty of Materials & Energy, Chongqing Key Laboratory for Advanced Materials & Technologies of Clean Energies, Southwest University, Chongqing 400715, China

a r t i c l e i n f o a b s t r a c t

Article history: Bacteria biofilm plays a key role in current generation of microbial fuel cells (MFCs), especially for the start-up
Received 19 December 2016 stage. However, the detailed mechanism of the biofilm promoting the power generation is not very clear so
Received in revised form 6 April 2017 far, especially for those exoelectrogens who rely on the self-excreted electron mediators for extracellular electron
Accepted 26 April 2017
transfer. In this work, a biofilm formation inhibitor—sodium houttuyfonate (SH) is used to build a “non-biofilm”
Available online 23 May 2017
anode of Pseudomonas aeruginosa (P. aeruginosa) without affecting the bacteria growth during the MFC opera-
Keywords:
tion. According to the comparison results of the “non-biofilm” anode and biofilm-covered anode on current gen-
Microbial fuel cell eration, phenazines concentration variation and anodic electrocatalysis, the biofilm on the anode not only
Pseudomonas aeruginosa provides plenty of bacterial cells for catalysis but also promotes the interfacial phenazine redox reaction through
Biofilm on anode accumulating the self-generated mediators on anode for fast interfacial electron transfer. This work proves that
Phenazine accumulation the biofilm assisted electron mediator accumulation will benefit such kind of exoelectrogens to sustain sufficient
Current generation electron mediators for extracellular electron transfer.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction anode is much lower than that of the place far away from the anode dur-
ing the MFC operation [13]. It is possible that the biofilm on the anode
Microbial fuel cells (MFCs) emerge as a challenging technology with results in the gradient of phenazines concentration in the anolyte. To ex-
potential to accomplish simultaneously wastewater treatment and elec- plore whether the biofilm affect the phenazines distribution in the an-
tricity production in recent years [1–3]. The bottleneck for the low odic chamber, it is necessary to build a “non-biofilm” P. aeruginosa
power density of an MFC should be the activation losses in anode, MFC for further analysis. In previous reports, some researchers used di-
which is related to the slow kinetics of the interfacial electron transfer alysis bag-wrapped electrodes [14] or gel beads-entrapped iron oxides
between bacteria cells and anode [4–6]. In most of the MFCs, the biofilm [15] to avoid the direct contact of the exoelectrogens with the solid elec-
formation is essential to the constant current generation, no matter tron acceptors for mediated electron transfer investigation. However,
what kind of extracellular electron transfer pathway is involved [7–9]. the dialysis bag will affect the diffusion or distribution of the phenazines
However, for the MFCs catalyzed by the exoelectrogens relying on and the gel beads will decrease the accessible surface area of the elec-
their self-mediated electron transfer, the function of the biofilm to trode. In this case, a biofilm inhibitor that is non-toxic or low toxic to
anode electrocatalysis was rarely discussed before. P. aeruginosa cells will be more suitable for the purpose.
Pseudomonas aeruginosa (P. aeruginosa) is a typical exoelectrogen Alginate is a secretory exopolysaccharide, consisting of variable
strain that only uses self-generated electron mediators (phenazines) amounts of 1,4-linked-D-mannuronate and its α-L-guluronate, func-
for extracellular electron transfer [10–12]. At the same time, the strains tions as extracellular matrix in biofilm formation of P. aeruginosa cells
in this genus are quite easy to form biofilms. In our previous work, we [16–17]. Sodium houttuyfonate (SH) is one of the main ingredients in
found that the MFC operation promoted not only the phenazines excre- the volatile oil of Houttuynia cordata Thunb [18], which can inhibit P.
tion but also the biofilm formation, both of which could facilitate the in- aeruginosa biofilm formation by depressing the expression of alginate
terfacial electron transfer between the bacteria cells and the anode. It is biosynthesis genes (algD and algR) [19]. Meanwhile, the SH is not
also interesting that the phenazines concentration surrounding the very efficient on the inhibition of cell growth for P. aeruginosa (the min-
imum inhibitory concentration is 512 μg mL−1) [20]. In this case, it is
⁎ Corresponding author at: Faculty of Materials & Energy, Southwest University,
possible to use a low concentration of SH to inhibit the biofilm forma-
Chongqing 400715, China. tion of P. aeruginosa without significant effect on the cell growth. In
E-mail address: [email protected] (Y. Qiao). this work, the SH with optimal concentration is introduced as a biofilm

http://dx.doi.org/10.1016/j.bioelechem.2017.04.003
1567-5394/© 2017 Elsevier B.V. All rights reserved.
 Y.-J. Qiao et al. / Bioelectrochemistry 117 (2017) 34–39 35

formation inhibitor to build a “non-biofilm” anode to explore the func- sequentially dehydrated with increasing concentration of ethanol
tions of the anode biofilm on the interfacial electron transfer in P. (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min each, followed by vacu-
aeruginosa MFCs. The possible mechanism behind the biofilm promoted um dried at room temperature for 12 h [24]. For each sample, more than
current generation performance is also discussed. ten micrographs were captured.

2. Material and methods 2.6. Electrochemical measurements

2.1. Bacteria culture For the anodic half-cell analysis, a piece of 1 cm ∗ 1.5 cm carbon cloth
was used as working electrode, saturated calomel electrode (SCE) was
P. aeruginosa (ATCC 9027) was cultivated in 5 mL Luria–Bertani (LB) used as the reference electrode and titanium plate as the counter elec-
broth (Tryptone 10 g L− 1, Yeast extract 5 g L−1, Sodium chloride trode [25]. Cyclic Voltammetry (CV) and Square Wave Voltammetry
10 g L−1) overnight with shaking at 37 °C. Then 2 mL bacterial suspen- (SWV) experiments were conducted by using a potentiostat (CHI660E,
sion was inoculated into 200 mL of M9 buffer (Na2HPO4, 6 g L−1; Shanghai Chenhua, China). Except as otherwise stated, the
KH2PO4, 3 g L−1; NH4Cl, 1 g L−1; NaCl, 0.5 g L−1; MgSO4, 1 mM; and votammograms were recorded with a scan rate of 10 mV s−1. All tests
CaCl2, 0.1 mM) with 20 g L−1 glucose [13] and incubated at 37 °C with were conducted at room temperature and repeated at least three
shaking until the optical density at 600 nm (OD600) reached about 1.0. times to make sure the reliability.
For the real-time phenazine concentration analysis, a cavity micro-
2.2. MFC operation electrode (CME) consisting of a glass tube containing a platinum wire
of 50 μm diameter with typical tip depths of 20–30 μm was prepared
Classic H-shaped MFC device that consisting of two 120 mL glass as previous report [13,26]. The CME was placed into the anodic chamber
flasks separated through a proton exchange membrane (PEM, Nafion
117, Dupont, Wilmington, DE) was used in this work. The P. aeruginosa
cells were harvested by centrifugation at 4 °C (6000 rpm, 5 min) and re-
suspended in 100 mL M9 buffer with 5 g L−1 glucose. Different concen-
trations (0 μg mL−1, 9.37 μg mL−1, 18.75 μg mL−1, 37.5 μg mL−1) of SH
(NICPBP, Beijing, China) were added into the 100 mL anolyte to inhibit
the biofilm formation. The cell suspension was purged with nitrogen
gas for 30 min to remove the dissolved oxygen before being transferred
to the anodic chamber [21]. The cathodic chamber was filled with
50 mM K3[Fe(CN)6] as terminal electron acceptor in 0.1 M phosphate
buffered saline (PBS; pH 7.4). Carbon fiber brush electrodes were used
as both anode and cathode. A 1.5 kΩ external resistor was loaded into
the circuit for MFC operation. The projection area of the anode was
used for current density calculation.

2.3. Phenazine concentration and cell density analyses of the anolyte

For phenazine concentration and cell density analyses, 0.5 mL

anolyte was taken from anodic chamber at different discharging time.
The cell density was determined at OD600 nm with a UV–vis spectro-
photometer (UV-2550, Japan). Then the anolyte was centrifuged at
10000 rpm (3 min) to obtain the supernatant for phenazine concentra-
tion evaluation according to the absorption peak at 370 nm [13].

2.4. Protein assay of the biofilm on the anodes

Before the test, 1 g of carbon fibers was cut down from the carbon
brush electrode each time (three repetitions) and immersed in 1 mL so-
dium hydroxide under 95 °C to dissolve the biofilm from the carbon fi-
bers [22]. Bradford Protein Assay [23] Kit P0006 (Beyotime, Jiangsu,
China) was used for protein concentration determination. Bovine
Serum Albumin (BSA) was dissolved into 8 g L−1 sodium hydroxide solu-
tion to prepare different concentration of standard solutions: 0 μg mL−1,
25 μg mL−1, 50 μg mL−1, 100 μg mL−1, 200 μg mL−1, 300 μg mL−1, 400
μg mL−1, 500 μg mL−1. After cooling at room temperature, 20 μL various
concentration of standard solutions and sample solutions were trans-
ferred to the 96-well plates, mixed with 200 μL Coomassie brilliant blue
G-250 solution. The absorbance was measured at the wavelength of
595 nm with a microplate reader.

2.5. Morphology observation of the biofilm

Scanning electron microscopy (SEM, JEOL JSM-6510LV, Japan) was

used to observe the morphology of biofilm on the anodes (three repeti- Fig. 1. (a) The current generation profiles of different MFCs without or with different
tions). Before examining the morphology of the biofilm, the samples concentration of SH addition. (b) The growth curve of P. aeruginosa in the MFC devices
were immersed in 4% polyoxymethylene solution overnight and without discharge.
36 Y.-J. Qiao et al. / Bioelectrochemistry 117 (2017) 34–39

close to the anode with a platinum-wire counter electrode and a satu- process. After 36 h, a continuous biofilm can be observed to cover
rated calomel reference electrode for real-time monitoring of phena- most of the control anode. However, for the one time SH added one,
zines concentration near the anode during the process of discharge. the cell density on the anode increased quite slowly especially at first
36 h and there are only scattered biofilms on the anode even after
3. Result and discussion 60 h. For the anode with continuous SH addition, no biofilm can be
found even after 180 h. In this case, the current generation of this MFC
3.1. Current generation profiles of the MFCs should be relied on the planktonic bacteria cells and the shuttling
phenazines.
The current generation profiles of the MFCs with different concen- To evaluate the cell amount adhered on the one time SH added and
tration of SH are shown in Fig. 1A. The results show that the addition control anodes, the total protein contents of the removed biofilms were
of SH into the anode significantly inhibits the current generation, espe- measured at different time. The results (Fig. 3) show that the maximum
cially at early stage of the discharge. It is noted that the high concentra- protein content for the SH anode is only around 60% of that for control
tion of SH not only extends the start-up time of the MFC but also anode. This is in accordance with the SEM results. At the same time,
decrease the current density dramatically. The current density of the the amount of the planktonic bacteria cells in SH involved anode is
plateau in 37.5 μg mL− 1 involved MFC (2.4 μA cm− 2) is only about more than that of the control anode (the inset of Fig. 3). It suggests
50% of the MFC without SH (4.8 μA cm−2). Considering the slowly de- that the SH greatly inhibits the formation of biofilm so that most of
composition of SH [27] in the anolyte during the MFC operation, the cur- the cells remain in the anolyte. According to the current generation
rent generation profile of the MFC with continuously addition of 18.75 curves and the time-course biofilm variation profiles, it seems that the
μg mL−1 SH every 36 h was also examined. It is noted that the current more biofilm-covered anode will achieve higher current density. How-
density of this MFC only can reach a steady state of 1.75 μA cm−2 after ever, the interfacial electrons transfer of P. aeruginosa anode only relies
120 h. (Fig. S1). The results suggest that the present of SH in the anolyte on the phenazines, which can shuttle between cells and the anode. To
will restrain the current generation of the MFC especially at high con- understand the function of biofilm in such electron shuttles mediated
centration. To evaluate the effect of SH to the growth curve of the P. electron transfer, it is necessary to explore relationship between the
aeruginosa cells, a time-course variation on the bacterial cell density biofilm formation and the distribution of phenazines in the anodes.
(OD600) of the anolyte (without discharge) is determined (Fig. 1B). It
shows that only the highest concentration (37.5 μg mL−1) of SH 3.3. Phenazines concentration evaluation during MFC operation
shows significant inhibition on the cell growth. The growth curves of
P. aeruginosa cultured in flask with different concentration of SH (Fig. During the P. aeruginosa MFC operation, the blue-green color of the
S2) also suggest the same fact. In this case, the SH with the concentra- anolyte can often be observed (Fig. S4). It is because that one of the
tion of 18.75 μg mL−1 could be used for the following investigation to major phenazine – pyocyanin displays blue-green color and plays key
ensure the notable inhibition on the biofilm formation with negligible role in the extracellular electron transfer of P. aeruginosa [28–30]. In
effect on the bacteria growth. this case, the color change of the anolyte can directly indicate the con-
centration variation of the phenazines. At the same time, the absorption
3.2. Biofilm formation on the anode peak of the UV–vis spectrum at 370 nm can be used to evaluate the con-
centration of the phenazines in the anolyte. From Fig. 4, it is noted that
To evaluate the inhibition effect of the SH on the biofilm formation, for all three groups, the color of the anolyte as well as the concentration
the morphologies of biofilm on the anode were observed with SEM at of phenazines changes according to the discharging time. For the open-
different time during the discharge. From Fig. 2, the cell density on the circuit MFC, the concentration of phenazine increases gradually and al-
carbon fiber is increasing for the control anode and one time SH most reaches a plateau after 90 h. For the operated MFC without SH, the
added one but the SH apparently slows down the biofilm formation phenazine absorption peak intensity increases fast at first 20 h to reach

Fig. 2. Morphologies of the biofilm growing on the anodes in the MFCs at 24 h (a, e, i), 36 h (b, f, j), 60 h (c, g, k) & 180 h (d, h, l). (a–d: without SH; e–h: with one time SH addition; i–l: with
continuous SH addition).
 Y.-J. Qiao et al. / Bioelectrochemistry 117 (2017) 34–39 37

biofilm [13]. Here, the addition of SH inhibits the biofilm formation so

that more phenazines will remain in the anolyte. From the above re-
sults, the biofilm formation on the anode could significantly decrease
the concentration of free phenazines in the anolyte. It is also worth to
note that the current generation performance mainly relies on the bio-
film coverage rather than the concentration of free phenazines in the
anolyte. It perhaps that the phenazines accumulated on the anode sur-
face during biofilm formation rather than the free phenazines serve as
electron shuttles for the interfacial electron transfer.
To prove this speculation, the cyclic voltammograms (CVs) of the
one-time SH added anodes and the control one are presented in Fig.
5a. The CVs of the CMEs, which represent the phenazines amount
surrounded the anode are also shown here to make a comparison (Fig.
5b). From Fig. 5A, both of the control anode and the SH involved
anode shows a pair of redox peaks at around −0.4 V vs. SCE after 56 h
discharge, which are ascribed to the redox reaction of the phenazines
and the peak current is proportional to the concentration of phenazines
on the anode surface [13]. According to Fig. S6A, the redox peak current
of the control anode shows continuously increasing during the first 56 h
and reaches the maximum value of 0.29 μA cm−2 (anodic peak). How-
Fig. 3. Total protein content of the biofilm on the anodes in different MFCs. The inset shows ever, for the SH involved MFC, the redox peaks only can be observed
the concentrations of planktonic bacteria in different MFC anodes. after 32 h and increase very slow (Fig. S6B). At 56 h, the peak current
is only 0.19 μA cm−2, around 65% of the control anode. While according
the maximum value and then decreases to a low level. While for the
MFC with one time SH added, it takes more than 48 h to reach a much
higher plateau (1.245) than that of the operating MFC without SH
(0.724) and the open-circuit MFC (0.717). Further, when SH was contin-
uous added into the anode (Fig. S5), the phenazine absorption peak in
the anolyte will keep increasing to a quite high level (2.26) after 76 h.
Apparently, the increase of the phenazines in the anolyte should be
due to the excretion by the P. aeruginosa cells and the MFC operation
greatly enhances the amount of the excreted phenazines. However,
the decrease of the concentration is not easy to explain since the phen-
azines are very stable in the M9 medium and no decomposition can be
found (Fig. S6). It is possible that the carbon brush anode could adsorb
some phenazines during the MFC operation, which could explain the
slight decrease in the SH continuous added MFC after 108 h. However,
the dramatically decrease of the phenazines in the control one is hard
to attributed to the adsorption on the carbon fiber. According to our pre-
vious report, the reduction of the phenazines in the anolyte might be at-
tributed to the accumulation on the anode surface assisted by the

Fig. 4. The change of phenazine absorbance in the anolyte with the increase of discharging
time (a: at open circuit without SH addition; b: discharge without SH addition; c: Fig. 5. (a) CVs of the anodes after discharging for 56 h. The inset shows the value of the
discharge with one time SH addition). The inset photo shows the color change of the anodic peak. (b) CVs of the CMEs measured in the anolyte at 56 h to show the
anolytes. phenazine concentration surround anode. The inset shows the value of the anodic peak.
38 Y.-J. Qiao et al. / Bioelectrochemistry 117 (2017) 34–39

Fig. 6. Mechanism for biofilm-assisted phenazines based interfacial electron transfer in P. aeruginosa MFCs. The SH addition prevent the biofilm formation so that the phenazines remain in
the anolyte to make the anodic chamber shows blue-green color (left). Without SH addition, the phenazines accumulated on the anode surface during the biofilm formation to make a
colorless anodic chamber and increase the interfacial redox peak current of phenazines (right).

to the electrochemical signals on the CMEs, the amount of surrounded for the current generation of those exoelectrogens who rely on the
phenazines for SH involved anode is about two times of the control self-excreted electron mediators. The biofilm assisted electron mediator
one. It suggests that the biofilm on the anode assists the accumulation accumulation will benefit these strains to sustain sufficient electron me-
of phenazines onto the anode surface and keeps them not diffusing to diators for interfacial electron transfer on the anode.
the anolyte, which guarantees the large amount of phenazines at the in-
terface to facilitate the interfacial electron transfer. The comparison re-
4. Conclusions
sults of the MFCs with and without SH are summarized in Table S1. It
can be found that the decrease proportion of the current generation is
In this work, a biofilm formation inhibitor – sodium houttuyfonate
in accordance with the decrease proportion on the anode redox peak
has been used to investigate the effect of the anode biofilm on the cur-
current under the effect of SH. This means the redox reaction of phena-
rent generation performance of P. aeruginosa MFCs. According to the
zines accumulated on the anode surface might directly affect the current
analyses of the anodes, it can be concluded that the biofilm formation
generation profile of the MFCs.
promotes the redox reactions of phenazines on the anode via accumula-
tion and thus enhances the interfacial electron transfer as well as the
3.4. The mechanism about P. aeruginosa biofilm assisted interfacial electron
power generation of the MFCs. No matter what the phenazine concen-
transfer in MFCs
tration is in the anolyte, only the phenazines adhered to the anode sur-
face will directly affect the performance of the P. aeruginosa MFCs.
According to the above analysis, a mechanism about P. aeruginosa
biofilm assisted interfacial electron transfer in the MFC anode is de-
scribed in Fig. 6. When the biofilm formation process is inhibited (left Acknowledgements
side), the phenazines excreted from the planktonic cells will suspend
in the anolyte to make the color of the anode chamber from colorless We gratefully acknowledge to the financial support from National
to green color gradually. While for the normal P. aeruginosa anode, the Natural Science Foundation of China (No. 31200102), the Fundamental
rapid formation of the biofilm makes plenty of cells adhered to the elec- Research Funds for the Central Universities (XDJK2015B018), Institute
trode the excreted phenazines accumulate on the anode surface so that for Clean Energy & Advanced Materials, Southwest University, Chong-
the light green color in the anode only can be observed at the early stage qing, P.R. China, Chongqing Key Laboratory for Advanced Materials
of the discharge. After that, the green color will gradually change to col- and Technologies of Clean Energies.
orless till to the end of the discharge cycle. At the same time, the phen-
azine redox peak current of the biofilm anode are much higher than that Appendix A. Supplementary data
of the anode with scattered bacteria adhesion and thus delivers better
current generation performance in the batch mode MFC. Considering Supplementary data to this article can be found online at http://dx.
that there is no big difference on the phenazine excretion ability for doi.org/10.1016/j.bioelechem.2017.04.003.
these two anodes, the higher power generation is just resulting from
the biofilm assisted accumulation of phenazines on the anode, which
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