Real-Time Imaging Revealed That Exoelectrogens from Wastewater

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Real-time Imaging Revealed Exoelectrogens from Wastewater Are Selected at the Center of Gradient Electric Field Qing Du, Quanhua Mu, Tao Cheng, Nan Li, and Xin Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01468 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Environmental Science & Technology

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Date:

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Real-time Imaging Revealed Exoelectrogens from Wastewater

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Are Selected at the Center of Gradient Electric Field

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Jun 12, 2018

Submitted to: Environmental Science & Technology

Qing Du1, Quanhua Mu2, Tao Cheng 1, Nan Li 3 and Xin Wang 1 * 1

MOE Key Laboratory of Pollution Processes and Environmental Criteria / Tianjin Key Laboratory of Environmental Remediation and Pollution Control / College of Environmental Science and Engineering, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China 2

Bioengineering Program, Department of Chemical and Biological Engineering, Hong Kong University of Science and Technology, Hong Kong, China 3

School of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China *Corresponding Author: Phone: (86)18722292585; fax: (86)22-23501117; E-mail: [email protected]

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TOC Art

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1

ACS Paragon Plus Environment


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Abstract

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Exoelectrogens acclimated from the environment are the key of energy recovery from

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waste in bioelectrochemical systems. However, it is still unknown how these bacteria

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were selectively enriched on the electrode. Here we confirmed for the first time that

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the electric field (EF) intensity selected exoelectrogens from wastewater using an

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integrated Electro-visual system with gradient EF. Under operating conditions (I =

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3×10-3A), the EF intensity on the working electrode ranged from 6.00 V/cm in the

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center to 1.08 V/cm at the edge. Thick biofilm (88.9 µm) with spherical pink

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aggregates were observed in the center, while the color became grey at the edge (33.8

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µm). The coverage of the biofilm also increased linearly with EF intensity from 0.42

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at the edge (12 mm to the center) to 0.78 in the center. Biofilm in the center contained

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76 % Geobacter, which was 25 % higher than that at the edge (60 %). Geobacter

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anodireducens was the main species induced by the EF (50 % in the center vs. 24 % at

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the edge). These results improved our fundamental knowledge on exoelectrogen

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acclimation and mixed electroactive biofilm formation, which has broader

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implications on energy recovery from waste and general understanding of microbial

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ecology.

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INTRODUCTION

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Exoelectrogens are increasingly being recognized as important in pollutants removal

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due to their unique ability of direct extracellular electron transfer.1-3 When electrons

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produced from their metabolisms were pumped through an artificial circuit by the

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redox potential gradient or external power, electrical energy or valuable chemicals can 2


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be produced from waste biomass directly.4 These bacteria usually grow in biofilm

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where independent cells are adhered by extracellular polymeric substances to form a

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three-dimensional structure. Kinetic analysis showed that the long-range electron

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transfer through the biofilm matrix is the only viable mechanism for the current

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density > 10 A/m2 in bioelectrochemical systems (BESs),5 although the intercellular

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electron transfer pathway is still under debate.3,

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Geobacter sulfurreducens,9 Geobacter anodireducens10 and Shewanella oneidensis,11

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the current densities varied with different strains, and the performance is mainly

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determined by the electroactivity of each strain. However, the mixed community

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usually produced a higher and more stable current than pure cultures, which has a

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great engineering perspective12. Exoelectrogens, such as Geobacter spp., can be

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acclimated up to 75 % from < 5% (wastewater) in 3-7 d feeding with acetate.13, 14

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Most studies only report the microbial community succession with time.15-17 The

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mechanism involved in electroactive bacteria acclimation from mixed consortia is

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most intriguing, however, largely unknown.

6-8

For pure cultures, such as

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Electroactive bacteria cannot be acclimated on a disconnected electrode. The

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polarized and conductive surface is believed to be vital for the selective growth of

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exoelectrogens in BES. When an electric field (EF) was imposed to cancer cells, it

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had been observed that some living cells can migrate towards electrodes,18 indicating

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a biological response under external EF.19-21 May the growth of exoelectrogens from

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wastewater be induced by EF? How different bacteria response to EF when they

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attach to the anode? So far as we known, there’s no information about this, probably 3



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because the microbial community in wastewater is really complicated and can be

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easily varied in several hours.

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Here we designed an online system to investigate the influence of EF on the

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formation of electroactive biofilm from wastewater. There are two bottlenecks for the

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design of this experiment, including 1) the real-time imaging and 2) the comparison of

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biofilm at different EF intensity. For the first point, it is not easy to online monitor the

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formation of the transparent biofilm without any chemical / physical interruption. So

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far, several electro- and spectroelectro- chemical techniques have been developed for

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bioﬁlm imaging and analysis, such as scanning electron microscopy,22 atomic force

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microscopy23 and confocal laser scanning microscopy.24 Wide-field microscopy seems

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to be the best choice because it has advantage of true color imaging with minimized

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intervention to living cells.25,26 However, the transparent biofilm is hard to be focused

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in reflection imaging system, especially at the initial stage of biofilm formation. Here

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we designed a new Electro- visual system to overcome this problem by introducing an

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additional Halogen light source. For the second point, the electroactive biofilm

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formation from mixed culture is hard to be compared at different electric field

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intensity in parallel reactors, because it is reported that the microbial community is

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not the same even the operational condition is exactly the same in replicate reactors.27

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Here we used a much larger working electrode (310 mm2) than the counter electrode

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(20 mm2) to produce a gradient electric field in the same reactor, which is different

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from most of previous designs with equal or larger counter electrode than the working

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electrode. In this gradient electric field, the biofilm as well as microbial community 4


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acclimated at different EF intensity can be examined in the same reactor to rule out

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the difference in inoculation.

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In our system, the growth of electroactive biofilm was monitored starting from the

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inoculation of wastewater in a gradient EF. These results will help to understand how

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exoelectrogens be selected from the environment.

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MATERIALS AND METHODS

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Electro-visual system

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The Electro-visual system included two parts: the Electrochemical System and the

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Imaging System. The configuration of the entire system was shown in Figure 1.

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Real-time electrochemical signals from the BES and optical images of biofilms on the

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working electrode were simultaneously recorded in the computer.

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The Electrochemical System was a three-electrode anaerobic reactor controlled by

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a potentiostat (CHI 660E, CH Instrument, China). The 7 mL reactor (32 mm in inside

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diameter, 66 mm in outside diameter and 40 mm in height, AiDa, Tianjin, China,

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Pending Chinese Patent No. CN201710327839.X, Figure S1) was made of

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polyfluortetraethylene (PTFE) and rubber with excellent sealing performance. A piece

107

of tin indium oxide (ITO) glass (38×38×0.7 mm) was fixed on the transparent bottom

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as both working electrode (32 mm in diameter, 800 mm2 of actual working area) 28, 29

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and imaging window. A copper cylindrical firing pin sealed in PTFE was designed to

110

connect the ITO electrode (dry connection) to the external circuit. The connection of

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firing pin to ITO was good because the etched circle right appeared in the center but

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not towards the connecting point (Figure S3). A Pt filament electrode rolled to a 5



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diameter of 5 mm (evolute shape, 20 mm2 of working area) was fixed on the top

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straightly facing to the center of the ITO electrode as the counter electrode. Ag/AgCl

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(4 M KCl, 0.205 V versus standard hydrogen electrode) reference electrode was

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inserted close to the working electrode. The distance between the working and counter

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electrodes was fixed at 2 mm. Prior to use, the ITO anode was immersed in acetone

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solution for 1 hour, cleaned in ethanol overnight and rinsed with deionized water.

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The Imaging System was a wide-field reflection microscope (IX73, Olympus,

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Japan) located under the reactor. In our preliminary tests, it is not possible to direct

121

focus on the thin layer of biofilm because bacterial cells are transparent. An additional

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Halogen light source (incident angle of 75°, Figure S2) was employed to achieve a

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high resolution, stereoscopic and real-color imaging through total reflection and dark

124

field imaging.

125

BES operation and ITO etch

126

All BESs were inoculated with the supernatant (pre-precipitated for 1 h) of domestic

127

wastewater from preliminary sedimentation tank (3 mL). The pre-precipitation was

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used to remove the large flocs in wastewater that may influence the imaging and the

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evenness of inoculation. The cell density of the inoculum measured by OD600 was ~

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0.2. The inoculum was then diluted in 7 mL of electrolyte. The electrolyte contents 50

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mM phosphate buffer solution (PBS, pKa=7.2, pH=7.1, conductivity of ~7.65 mS/cm,

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containing Na2HPO4, 4.58 g/L; NaH2PO4, 2.13 g/L; NH4Cl, 0.31 g/L; KCl, 0.13 g/L,

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trace minerals, 25 mL/L and vitamin solution 10 mL/L) and 2 g/L of acetate13. Before

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connecting to circuits, the electrolyte was vigorously flushed with N2/CO2 gas (N2: 6


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CO2=4:1 [vol/vol]) for 30 min to remove dissolved oxygen. The reactor was

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continuously fed by sterilized 2 g/L acetate at a rate of 100 µL/h through anaerobic

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hose (Tygon R-3603, Saint-Gobain, France) using an injection pump (LSP01-2A,

138

Longer Pump, China). Current densities were measured by chronoamperometry every

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100 s at -0.2 V vs. Ag/AgCl. All tests were operated in triplicate at room temperature.

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We select -0.2 V because: 1) it is reported to be the optimal potential for electroactive

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biofilm growth on ITO and 2) ITO was electrochemically stable before and after

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biofilm formation at this potential (Figure S3). 26, 28

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Two types of etched ITO glasses were prepared (Ailian, Tianjin, China) with

144

conductive areas of 310 mm2 (ITO-1) and 20 mm2 (ITO-2, the same as the counter

145

electrode) in the center. Both etched ITO glasses were assembled in BES reactors to

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compare the maximum current density using the same inoculum.

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Electric field simulation and measurement

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A reactor was designed by modifying the BES reactor to measure the induction field.

149

Briefly, a Pt wire was fixed in the center (fixed probe) of the counter electrode while

150

keeping them isolated from each other. Another moving Pt wire (moving probe)

151

installed on the cap can slide following a track from the center to the edge (Figure S4).

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The potential differences between two probes were measured at different distances.

153

According to Ohm’s law, the electric field intensity through a bulk material is

154

E=kI/(σWh),18 where I (mA) is the electric current flowing through the bulk material

155

and the electrolyte in the reactor, σ (mS/mm) is the conductivity of the electrolyte, W

156

(mm) is the distance between two Pt wires, h (mm) is the distance between working 7



157

electrode and counter electrode (2 mm in this study), k is the coefficient. Simulation

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of the EF in the BES was performed using R language. The fluidic density and

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electrical conductivity of solution were set as 1000 kg/m3 and 12.12 ±0.2 mS/cm.

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Actual EF intensity inside microchannel was measured as follows. Potential

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differences (∆V, mV) among the two Pt wires were measured by a potentiostat

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(PGSTAT 302N, Metrohm, Switzerland) and a multimeter (Model 189, Fluke, U.S.).18

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The EF was then calculated by dividing ∆V with the distance between the platinum

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wires.

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Biofilm real-color photography and 3D-strap CLSM imaging

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The biofilm morphology was monitored by the Electro-visual system since

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inoculation. The three coordinate points (a, b, c) on the ITO working electrode were

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marked as the focus points to record optical images (Figure 3B). The locations of the

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three points were 2 mm, 6 mm and 10 mm to the center. Images (videos) of biofilm

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developed on points a, b and c were recorded every day, and all data were exported to

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a computer. The current produced by the same electrode was simultaneously recorded.

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Biofilm on the ITO glass was stained in situ with a LIVE/DEAD BacLight

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Bacterial Viability Kit (L13152, ThermoFisher Scientific Inc., U.S.). Confocal laser

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scanning microscopy (CLSM, LSM880, Zeiss, Germany) was used to image the

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spatial topography (3D biofilm images) of the biofilm at the end of 30 d. A strip of the

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biofilm (length = 34 mm, width = 0.25 mm，about 8.5 mm2) was fast scanned from the

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center to the edges using 10× objective. The biofilm coverage was calculated

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according to the pixel counting.30-32 Layer-scanned images were stacked and analyzed 8


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using ZEN software.33 The biofilm thickness at each location was directly obtain from

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3D images.

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Microbial community analysis

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Biofilm samples from three regions (outside lane, middle lane and inside lane) on ITO

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were collected by scraping the surface using a sterile blade，shown in Figure 3B. DNA

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was extracted using the Soil Genomic DNA Kit (CW2091S, ComWin Biotech, China)

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according to standard protocols. The hypervariable V4 region of 16S rDNA was

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amplified by PCR with the universal primer set including the forward primer 515F

187

(5’-GTGCCAGCMGCCGCGGTAA-3’)

and

the

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(5’-GGACTACHVGGGTWTCTAAT-3’).

The

amplicons

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determined by Illumina MiSeq sequencing platform in Novogene (Beijing, China).

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The sequencing data were then processed using the DADA2 pipelinee,34 including

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trimming, sequencing error correction, filtering, abundance estimation and taxonomy

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assignment. DADA2 is an open-free-source package based on the divisive amplicon

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denoising algorithm (DADA) for correcting Illumina-sequenced amplicon errors and

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infers sample sequences exactly (at 1 nucleotide resolution). DADA2's residual

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error rate can be as low as 2.46 × 10−8. Due to the precision of the sequence

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identification, it is possible to assign the sequences to the species level, with high

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confidence. In our study, the sequences that were assigned to Geobacter

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sulfurreducens and Geobacter anodireducens were further confirmed by aligning with

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the reference sequence using NCBI blast, and the identities were all 100% (data not

200

shown). R language was used for visualization of the results. 9


reverse were

primer

806R

subsequently


201

RESULTS

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EF intensity distribution the surface of the working electrode

203

EF intensity (E) on the surface of the working electrode was simulated at two

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conditions: the clean electrode (I = 1.65× 10-4 A) and the electrode covered by mature

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biofilm (I = 3×10-3A). As shown in Figure 2, the growth of biofilm enhanced the E

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values by nearly two orders of magnitude. E was uniform over a diameter < 2.5 mm

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because the working electrode and counter electrode were straightly faced, and the

208

distance was relatively small. E gradually decreased from 0.16 V/cm at the center to

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0.03 V/cm at the edge for the clean electrode, comparing to 6.00 V/cm (the center)

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and 1.08 V/cm (the edge) for bioanode. The E at the center was more than 5 times

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larger than that at the edge. Specifically, the ratio of the E values at the three selected

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points, a, b and c, were 3.5:1.7:1.

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In order to verify the above simulated E values, EF intensity was measured at

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eight points under each condition. Results showed that the measured E values well

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fitted with simulated values (Figure 2, lower part). Pearson correlation coefficients of

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the measured and simulated values were >0.96 under both conditions (Figure S5A and

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S5B).

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The growth of mixed electroactive biofilm

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The biofilm growth was monitored in 10 days on three coordinate points (a, b, c),

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representing the center, middle and edge of the working electrode (Figure 3 and S6).

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Scattered bacterial cells randomly landed onto the working electrode after wastewater

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inoculation. These bacteria gradually colonized on the surface and formed clusters in 10


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the initial 3 days. These clusters became more and more crowded, and spherical pink

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aggregates emerged in the center on day 5. The pink color was probably due to the

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accumulation of cytochromes. The biofilm rapidly expanded and stacked layer by

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layer from day 6 to day 9. Finally, the thickness of the biofilm reached 75 µm (point a)

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on day 10. The biofilm at three points shared the same trend of growth over time.

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Three strategies were taken to ensure anaerobic environment within the reactor and

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thus exclude the possibility of leaked oxygen affecting biofilm formation: 1) ITO

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glass was sealed inside the reactor; 2) the working area of ITO electrode was large

231

enough (~600 mm2) and 3) the height of outer seal of the reactor was >10 mm.

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The current density exponentially increased to 0.32 A/m2 at 1.5 d, which was

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slightly higher than 0.15 A/m2 of G. sulfurreducens under similar condition.28 The

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maximum current density of 0.80 A/m2 was obtained at 2.3 d (Figure 3D). However,

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at this time (day 2 to 3), the biofilm was forming clusters, far from the “mature stage”.

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Surprisingly, the current density declined with the growth of biofilm. After 6.1 d, the

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current density was stabilized to 0.47 A/m2, half value of the maximum.

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Spatial structure of the biofilm

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Planktonic cells precipitated uniformly on a, b and c at the beginning. However, the

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topography significantly changed when the biofilm was mature on day 10. Spherical

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pink aggregates covered most area of point a (0.64 clusters/mm2), while sparsely

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distributed at point b (0.20 clusters/mm2) and c (0.05 clusters/mm2). Blanket-like

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biofilm containing large bacteria (~10 µm in length) were enriched at both point b and

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c, and the color gradually changed from pink to grey. A wide-view image of the 11



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mature biofilm at points of a and b also showed this color gradient and topographic

246

change (Figure 3C). However, these differences tended to be eliminated after

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long-term operation. For example, the thickness at point a and b respectively reached

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87 ± 1 µm and 81 ± 1 µm, while point c was 70 ± 2 µm after 30 days.

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Strip scanning along the radius using CLSM showed a denser and richer biomass

250

in the center than that at the edge (Figure 4A), which was in accordance with the

251

results of the true color images in Figure 3A. The overall landscape of the biofilm on

252

the electrode was like a mushroom, with density decreasing from the center to the

253

surrounding area. The enlarged photos of biofilm at point a, b and c clearly

254

demonstrated this trend (Figure 4A). The biofilm coverage was highest in the center

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(0.78), and drastically dropped to 0.42 (12 mm to the center). When extended further

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to the edge, the coverage was close to zero. Similarly, the biofilm thickness was

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highest in the center (88.9 µm), which was 2.6 times higher than the edge (12 mm to

258

the center, 33.8 µm). Both the biofilm coverage and biofilm thickness decreased

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linearly (y=-0.0168x+0.7958, R2=0.9762; y=-1.8076x+91.54, R2=0.982, respectively).

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Microbial community in the biofilm

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Geobacter spp., the model exoelectrogen, accounted for 3.9 % in the inoculum

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(wastewater). However, the percentage increased to > 60 % in the mature biofilm.

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Community analysis based on three samples collected from the center, middle and

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edge showed that the percentage of Geobacter genus increased from the edge (60 %)

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to the middle (71 %) and the center (76 %) (Figure 5A).

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Acetobacterium spp, an anaerobic genus fomenting sugar and alcohol into acetates,

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was also found to be enriched in the biofilm. In wastewater its abundance was < 0.1 %,

268

while in the biofilm the abundance was > 5 %, 50 times higher. Interestingly, its

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abundance reversely decreased from the edge (13 %) to the middle (9 %) and the

270

center (6 %).

271

On the contrary, Arcobacter, Sulfurospirillum, Acinetobacter, Sulfurimonas,

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Pseudomonas and Macellibacteroides, abundant in wastewater, were observed to be

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depleted in the biofilm. For instance, Arcobacter was 31 % in wastewater, and sharply

274

decreased to < 1 % in the anode biofilm. Noticeably, all of these genera were aerobic

275

(Figure 5A).

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Species level analysis further revealed that Geobacter anodireducens35 and

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Geobacter sulfurreducens36 were enriched (> 24 %) but distributed differently. G.

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sulfurreducens uniformly distributed on the anode biofilm (26 ± 2 %), while G.

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anodireducens increased from 24 % at the edge to 50 % in the center (Figure 5B and

280

Figure S7).

281

Currents of etched ITO electrodes

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Based on our observation of the high bacterial density and exoelectrogen percentage

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at the center of the working electrode, we hypothesized that the center of the working

284

electrode contributed most of the current output. The actual working area of ITO-1

285

was 15 times bigger than ITO-2 which was shown in Figure 6A. ITO-1 reached the

286

high current peak after 90 h, much faster than ITO-2 (150 h). However, they both got

287

the similar peak current of ~1.70 mA (Figure 6B). Extending the area of working 13



288

electrode did not improve the current output, although it accelerated the start of the

289

BES.

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Discussion

291

Selection of exoelectrogens by EF intensity

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Multiple evidences in this study demonstrated that exoelectrogens can be selected

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from mixed consortia by EF intensity. Both the true color images and the CLSM

294

showed that exoelectrogens concentrated in the center of the electrode. In the

295

strip-scanning CLSM images, the biofilm coverage in each view field was quantified,

296

and it turned out that the biofilm coverage was linearly correlated with and EF

297

intensity (Pearson’s R=0.897, P10

319

µm) bacteria appeared at the edge. In addition, there was a clue that the growth

320

direction of bacteria pointing to the center of the EF in the mature film (Figure S8).

321

The faster current production on ITO-1 than ITO-2 also supported the migration

322

mechanism. However, due to the large area of the anode surface and the fast growing

323

and overlap of the biofilm in this study, it is not possible to track the migration of one

324

single bacterium. The radial distribution of pink aggregates from the center to the

325

edge was observed during the biofilm formation, which might be a preliminary

326

evidence of biofilm migration (Figure S8). Microbial labelling, in combination with

327

microfluidics technologies, can be applied to reveal the movement of exoelectrogens

328

in the future. For the last mechanism, a transcriptome analysis using pure culture in

329

gradient EF was required.

330

Though noticed long time ago, the response of exoelectrogens to EF has been

331

rarely studied. Previous studies have reported that Geobacter genus tended to 15



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332

accumulate in the most internal layer of the anode biofilm, possibly because the close

333

contact to the anode favored their anode-respiring activity.37,

334

revealed that Geobacter were enriched to the stronger end in gradient EF. It was

335

reported that the biofilm density could affect the produced currents.39 Interestingly,

336

the EF intensities that induced the gradient distribution of exoelectrogens in this study

337

(600 mV/mm, 289 mV/mm and 173.2 mV/mm) were comparable to the that reported

338

to move eukaryotic cells (300 mV/mm).40, 41

339

The growth of electroactive biofilm in gradient EF

340

We observed asynchronous growth of microbial biofilm and current output. The

341

current density declined from the maximum of 0.80 A/m2 (2.3 d) to 0.47 at the end of

342

6 d, but the biofilm consecutively grew in this period (Figure 3). It seemed that the

343

thick biofilm limited the current density since the maximum current was observed

344

with a very thin layer of biofilm, showing a typical diffusion limitation. A thick

345

biofilm may hinder the diffusion of acetate into the biofilm as well as the proton out.

346

It had been reported that a 15~80 µm dead/inactivated inner-core covered by a live

347

outer-layer (10~15 µm) biofilm was formed by Geobacter anodireducens SD-1.42 The

348

inactive inner-core may bring additional resistance for the distant electron transfer in

349

thick biofilm. Therefore, to maintain a thin electroactive biofilm may be beneficial to

350

achieve higher power density in engineering applications.

38

Our study further

351

After 30 days the biofilm thickness was stabilized at 70~90 µm over all position

352

on ITO electrode. The quorum sensing or the limitation of distant electron transfer in

353

the

mixed

biofilm

may

prevent

bacteria

to

form

16


an

over-thick

and

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poor-for-mass-transfer biofilm.

355

Different bacterial response to EF

356

Based on sequencing results, G. anodireducens is likely to be the main bacteria

357

selected by EF. G. anodireducens SD-1, the representative strain of G. anodireducens,

358

was isolated from wastewater, with a genomic similarity of 98 % to G. sulfurreducens

359

PCA.10 Compared to G. sulfurreducens, G. anodireducens has a lower charge transfer

360

resistance and a higher power density. In this study, G. sulfurreducens uniformly

361

distributed at different positions of the working electrode. However, the G.

362

anodireducens relative abundance linearly increased with the EF intensity (Pearson’s

363

R=0.981), probably due to its specific response to EF as electrotaxis. It is very

364

interesting that different strains of Geobacter, G. sulfurreducens and G.

365

anodireducens, exhibited different responses to EF.

366

Acetobacterium genus distributed follow the opposite trend to Geobacter genus on

367

the ITO, increased from the center (6 %) to the edge (13 %). Previous studies have

368

reported the symbiotic growth of Acetobacterium and Geobacter. where

369

Acetobacterium convert sugar or alcohol in wastewater to acetate to feed Geobacter.

370

In our system, acetate was abundant in electrolyte due to the continuous pump feeding,

371

so that the symbiotic growth of Acetobacterium seemed unnecessary. As showed in

372

Figure 5A, the fast colonization of Geobacter induced by EF in the center compressed

373

the percentage of other bacteria, which was one of the most possible reason for the

374

percentage change of Acetobacterium. Therefore, a strong EF can be a powerful tool

375

to exclude non-exoelectrogens from microbial community. 17



376

Implications

377

The electroactive biofilm formation in a gradient EF was monitored by an integrated

378

Electro-visual system. It was directly observed that the exoelectrogens can be selected

379

by EF intensity from mixed inoculum, and the exoelectrogens did not uniformly

380

distribute in gradient EF. A high EF intensity led to a thick and dense biofilm with

381

spherical pink aggregates, while grey and thin biofilm was observed in the area with

382

low EF intensity. The biofilm grown in the high EF area mainly contributed to the

383

current production. The content of Geobacter anodireducens linearly increased with

384

EF intensity, so that the population of other bacteria such as Acetobacterium was

385

compressed. For the engineering perspective, a strong EF can be applied at the early

386

stage to accumulate exoelectrogens and rule non- exoelectrogens out. Our preliminary

387

results provide new knowledge to the fundamental understand of electroactive biofilm

388

acclimation. It also demonstrates the possibility to regulate the exoelectrogens for the

389

bottom-up design of highly active electroactive biofilm in an engineered system.

390

Supporting Information

391

Additional figures, details on reactor photo, optical path of Electro-visual system, ITO

392

glass etched at different potentials in the reactor, device for measuring electric field

393

intensity, linear fitting of measured-EF intensity and simulated EF intensity, 10 d

394

pictures of biofilm developed at different points, linear fitting of Geobacter

395

anodireducens relative abundance and simulated EF intensity, and true color images

396

(10 × objective) of the biofilm at the center.

397

Notes

18


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The authors declare no competing financial interest.

399

Acknowledgements

400

We appreciate the technical helps of Mr. Yan Wang from Metrohm (electrochemical

401

measurement of EF), Mr. Encai Zhou from Aida Co. Ltd (design of all reactors) and

402

Mr. Junling Du from Olympus (design of optical path). This research work was

403

financially supported by National Natural Science Foundation of China (No.

404

21577068), Tianjin Research Program of Application Foundation and Advanced

405

Technology (18JCZDJC39400) and the Fundamental Research Funds for the Central

406

Universities and 111 program, Ministry of Education, China (T2017002).

407

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521

Figure Captions

522

Figure 1 The scheme of the Electro-visual system, including a wide-field reflection

523

microscope, a continuous flow BES reactor, a co-light source and a control system (a

524

potentiostat and a computer). The photo of the BES reactor and the detailed structure

525

of imaging system were shown in Figure S1 and S2. ITO glass was used as the

526

working electrode.

527

Figure 2 Simulated and measured EF along the diameter in the microreactor. (A)

528

Chronoamperometry, I=1.65×10-4A resembling bare electrode. (B)

529

Chronopotentiometry, I=2×10-3A, resembling biofilm matured stage. The error bars

530

are too small to be showed in this figure.

531

Figure 3 The growth of the electroactive biofilm at different positions on ITO glass.

532

(A) The true-color images (40 × objective) taken by the Electro-visual system at 1 d, 3

533

d, 5 d and 10 d at point a, b and c (shown in B). (B) Positions of the three coordinate

534

points: a, 2mm to the center; b, 6mm; c, 10mm. (C) A wider view sight image (10 ×

535

objective) of the mature biofilm at point a and b, showing the gradient of the biofilm

536

density. (D) The corresponding current density collected by the Electro-visual system

537

in 10 days.

538

Figure 4 The gradient of the biofilm density measured by confocal microscope. (A)

539

The strip scanning images using 10 × objective on ITO glass. The white arrows

540

indicate the scanning direction. The biofilm images at point a, b and c were enlarged.

541

(B) The distribution of the biofilm coverage (in orange color) and thickness (in grey

22


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542

color) along the radius (scanning direction). (C) The linear fitting of biofilm coverage

543

and EF intensity.

544

Figure 5 (A) Taxonomic classification of bacterial communities at different positions

545

of the biofilm on the ITO. (B) Relative abundance of Geobacter anodireducens and

546

Geobacter sulfurreducens in the inoculum (wastewater) and different positions on

547

ITO.

548

Figure 6 (A) The scheme of two microreactors with the same counter electrodes and

549

different etched ITO working electrodes. The working area of ITO-1 and ITO-2 were

550

310 and 20 mm2. (B) Current generation at -0.2 V versus Ag/AgCl in 180 h for ITO-1

551

and ITO-2.

552

23



553 554

Figure 1

24


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555 556

Figure 2

25



557 558

Figure 3

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559 560

Figure 4

561

27



562 563

Figure 5

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Figure 6

29


Real-Time Imaging Revealed That Exoelectrogens from Wastewater

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