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Alternating Current Influences Anaerobic Electroactive Biofilm Activity Xin Wang,† Lean Zhou,† Lu Lu,‡ Fernanda Leite Lobo,‡ Nan Li,§ Heming Wang,‡ Jaedo Park,∥ and Zhiyong Jason Ren*,‡ †

MOE Key Laboratory of Pollution Processes and Environmental Criteria/Tianjin Engineering Center of Environmental Diagnosis and Contamination Remediation, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China ‡ Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, Boulder, Colorado 80309, United States § Tianjin Key Lab of Indoor Air Environmental Quality Control, School of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China ∥ Department of Electrical Engineering, University of Colorado Denver, Denver, Colorado 80204, United States S Supporting Information *

ABSTRACT: Alternating current (AC) is known to inactivate microbial growth in suspension, but how AC influences anaerobic biofilm activities has not been systematically investigated. Using a Geobacter dominated anaerobic biofilm growing on the electrodes of microbial electrochemical reactors, we found that high frequency AC ranging from 1 MHz to 1 kHz (amplitude of 5 V, 30 min) showed only temporary inhibition to the biofilm activity. However, lower frequency (100 Hz, 1.2 or 5 V) treatment led to 47 ± 19% permanent decrease in limiting current on the same biofilm, which is attributed to the action of electrohydrodynamic force that caused biofilm damage and loss of intercellular electron transfer network. Confocal microscopy images show such inactivation mainly occurred at the interface between the biofilm and the electrode. Reducing the frequency further to 1 Hz led to water electrolysis, which generated gas bubbles that flushed all attached cells out of the electrode. These findings provide new references on understanding and regulating biofilm growth, which has broader implications in biofouling control, anaerobic waste treatment, energy and product recovery, and general understanding of microbial ecology and physiology.

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INTRODUCTION Biofilm formation is a ubiquitous phenomenon in nature and engineering systems. Undesired biofilm growth may cause a variety of problems such as biofouling, microbially influenced corrosion, infection, and product contamination.1,2 Biofilms can also be beneficial when they are used to remove contaminants in water and wastewater or for bioenergy production.3,4 Protected in the structured and self-produced polymeric matrix in biofilm, bacterial cells are able to survive hostile environments,5 so the inactivation or control of biofilm growth generally requires a more complicated method than controlling the growth of suspension cells. Electrochemical methods have been demonstrated effective in inactivating bacterial growth since the 1970s.6 Direct current (DC) is commonly used to apply an electric field force and produce highly active disinfectants such as •OH, O3, H2O2, etc. When wastewater is mixed with seawater, Cl2 can be generated as a low cost disinfectant to minimize storage and transport of toxic and unstable chemicals.7 However, the electrolysis process during DC treatment normally accelerates corrosion on conductive pipeline or other surfaces, which requires expensive corrosion protection measures. The DC treatment may also © 2016 American Chemical Society

introduce unwanted toxic chemicals on the reaction surface during surface reactions. In contrast, AC current has several advantages over DC by changing the current direction periodically. For a symmetrical AC, the pure charge in a complete cycle is zero, which minimizes chemical reactions especially at high frequency. This limits corrosion reactions on the conductive surface and redox reactions in the solution. Super high voltage, such as 13−35 kV amplitude, and low frequency AC (5−15 V amplitude, 1−10 Hz) can kill bacteria because super high voltage AC provides a strong electric field force,8,9 and low frequency AC generates disinfectants like DC current.10 However, low voltage AC for disinfection has been reported to be more attractive because of its safe and economic nature. Recent studies demonstrated that weak AC electric field forces (0.1−4.5 V/cm) without any Ohmic current or formation of free radicals inhibited the growth of planktonic Staphylococcus aureus and Pseudomonas aeruginosa,11 but most Received: Revised: Accepted: Published: 9169

February 16, 2016 July 21, 2016 August 3, 2016 August 3, 2016 DOI: 10.1021/acs.est.6b00813 Environ. Sci. Technol. 2016, 50, 9169−9176

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Environmental Science & Technology

that were disconnected for 30 min were operated as the control. After electrochemical tests, reactors were reconnected to the potentiostat as described above and operated at 0 V for 12 h, which aimed to allow biofilm recovery. The 12 h recovery time was determined based on the doubling time of 6 h for Geobacter sulf urreducens in biofilm.21 Electrochemical Characterizations. Cyclic voltammetry (CV) was performed before and after AC treatments as well as at the end of 12 h recovery. Working, counter, and reference electrodes were the same as aforementioned. The potential range for CV was determined to be −0.6 to 0 V vs Ag/AgCl according to previous results,19 and the scan rate was 1 mV/s. The first derivative CV (DCV) was derived from turnover CV to determine the changes in each peak value after AC treatments. The main oxidation peak in DCV was fitted to Guassian function to separate overlapped peaks. Nonturnover CV was performed in PBS without growth media. The electrochemically active molecule coverage (Γ, mol/cm2) was Q calculated as Γ = nFA , where Q (C) is the charge calculated from the oxidation peak of baseline subtracted nonturnover CV; n is the number of electrons; F is Faraday constant (96500 C/mol); and A (cm2) is the area of electrode.22 All potentials mentioned in this paper are versus Ag/AgCl (3 M KCl) except as noted. Microbial Community and Biofilm Characterization. Biofilm samples were collected by wiping the surface of the electrode using a sterile pledget. The sample DNAs were extracted using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., CA, U.S.) using standard protocols, and 16S rRNA at the hypervariable V1−V3 region was amplified by forward primer 8F and reverse primer 533R, as described previously.23 High throughput 454 GS-FLX pyrosequencing bioinformatics analysis was performed using the method described previously.24 The spatial topography and changes of bacterial cellular permeability in biofilm were observed by fluorescent staining using a LIVE/DEAD BacLight Bacterial Viability Kit (L7007, ThermoFisher Scientific Inc., U.S.). Biofilms on the graphite plates were stained in situ in the dark for 15 min in 50 mM PBS and rinsed two times in 50 mM PBS before being examined using confocal laser scanning microscopy (CLSM; Biorad, Radiance 2100 MP, U.S.).25 The viability of each layer was calculated as the ratio of viable to total cells according to pixel counting.26 Layer-scanned images were stacked and analyzed using the ImageJ (http://imagej.net/) software.

studies so far limited the scope to planktonic bacteria inactivation. To the best of our knowledge, there is no report showing how low voltage AC affects the activity of naturally formed biofilms. In this study, we investigated for the first time how AC current acted on anaerobic electroactive biofilm that led to different levels of biofilm inhibition. The use of electroactive biofilm dominated by Geobacter species provides a unique platform and surrogate for understanding the effects of AC current on biofilms because while the electrohydrodynamic force generated by AC acts on all cells only the electroactive biofilm responses can be monitored in situ and in real time by electrochemical methods. Electrons from electroactive bacteria transfer across cell wall and outer membranes via mediators, cytochromes, or conductive appurtenances,12−14 which can be easily detected via the anodic current with a poised potential. Electrons can travel tens of microns in the electroactive biofilm network, though the exact mechanisms are still under investigation.13,15 The advancement of electrochemical tools used in these studies enables direct characterizations of electroactive biofilm activities under changing environmental conditions such as changes in toxicity or organic loadings.16,17 This study uses a similar concept but focuses on the effects of AC current on biofilm inhibition. In conjunction with the in situ electrochemical analysis, confocal microscopy was used to depict the changes of biofilm structure under different AC conditions.18

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MATERIALS AND METHODS Reactors and Biofilm Enrichment. Each bioelectrochemical reactor consisted of a cylindrical glass container (net volume of 100 mL) and a polytetrafluoroethylene lid. The reactors were well sealed by both ground glass and silicone gel at the interface of container and lid. A graphite plate (diameter of 6 mm, area of 0.28 cm2), a platinum plate (1 cm2), and an Ag/AgCl electrode (3 M KCl, AgCl saturated, +0.210 V versus standard hydrogen electrode, 25 °C) were fixed on the lid and used as the working, counter, and reference electrodes in each reactor, respectively.19 The spacing between working and counter electrodes was 1 cm. All these materials were purchased from Aida Hengsheng Technology Co. Ltd., Tianjin, China. Graphite plate electrodes were cleaned by 1 M HCl and 1 M NaOH before polished using α-Al2O3 powders (diameters from 1.5 μm to 50 nm) and then rinsed with deionized water. All reactors were operated at 25 °C. The reactors were inoculated by the effluent of an air− cathode microbial fuel cell that was operated for 1 year with an external resistance of 10 Ω (65%, v/v). The culture medium contained 1 g/L of NaAc, 50 mM phosphate buffer solution (PBS, NaH2PO4·2H2O 3.32 g/L; Na2HPO4·12H2O 10.32 g/L; NH4Cl 0.31 g/L; KCl 0.13 g/L), 5 mL/L of vitamin solution, and 12.5 mL/L of trace mineral solution.20 It was sparged with N2 for 30 min to remove oxygen before filling into reactors. A multichannel potentiostat (CHI 1000B, CH Instrument, Shanghai, China) was employed to poise the anode potential at 0 V (versus Ag/AgCl) to enrich electroactive biofilm. AC Treatment. When the current generated by the reactors stabilized, different external AC currents were applied to the respective reactors. AC frequencies of 1 MHz, 10 kHz, 1 kHz, 100 Hz, and 1 Hz and amplitudes of 5 V (10 V of peak to peak voltage) or 1.2 V were applied to the working and counter electrodes for 30 min using a 4 M Hz sweep/function generator (Model 188-S-1257, Wavetek Co., CA, United States). Reactors

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RESULTS AND DISCUSSION Biofilm Acclimation and Community Structure. When the medium with inoculum was filled into bioelectrochemical reactors, anode potential was poised at 0 V versus Ag/AgCl. Linear current increase was observed after a short lag phase (∼40 h, Figure S1), and then the increase slowed when the current density reached 3 A/m2. A stable current plateau was maintained at 89 ± 3 h with current densities of 4.0 ± 0.2 A/ m2. Previous studies reported a similar growth pattern by Geobacter sulf urreducens, which is a model species of exoelectrogenic bacteria. G. sulfurreducens on the anode grew at a constant doubling time when current density was below 3 A/m2, and beyond this point, the current trended to a stable value.21 CVs and AC treatments were performed when currents reached to the stable value, which was defined as the end of biofilm enrichment. 9170

DOI: 10.1021/acs.est.6b00813 Environ. Sci. Technol. 2016, 50, 9169−9176

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model, as previously described.28 The onset potential was near −0.450 V vs Ag/AgCl, and the maximum limiting current density was 4.1 ± 0.2 A/m2. After peak separation, the first derivative curve showed two minor peaks (centered at −0.489 ± 0.015 and −0.428 ± 0.011 V) and one main peak (centered at −0.363 ± 0.003 V) in the oxidation curve. In addition, one main peak was centered at −0.364 ± 0.002 V (n = 8, measured in different reactors) in the reduction curve (Figure 1A). Since the peak with the most negative potential (named shoulder peak) was 2−10 times lower than peak 1 and 10−20 times lower than peak 2 in height, it was not taken into comparison after AC treatment. AC treatment over a frequency ranged from 1 MHz to 1 Hz for 30 min indeed decreased biofilm electrochemical activity in terms of current densities and peak heights in DCV curves (Figure S3). When the frequency was higher than 1 kHz, the decreases in both limiting current and peaks in DCV curves were less than 40% with an average of ∼22%, whereas decreases in the no treatment control were less than 6%. It is clear that the maximum oxidation peak, reduction peak, and peak 2 (separated from the maximum oxidation peak) in DCV followed the same trend as the limiting current, showing that these peaks were from the dominate redox species in bacterial extracellular electron transfer. As a minor peak that 2−3 times lower than peak 2 in oxidation curve, the peak 1 decreased by 4−10% after treatment except that of 10 kHz (27 ± 7%), which was comparable with 5% in the control. The shoulder peak centered at −0.489 ± 0.015 V was also weakened but still visible after these treatments (Figure S3). Previous studies showed that extracellular electron transfer of Geobacter species conducted via multiheme outer membrane cytochromes such as OmcB, OmcE, and OmcS) and conductive pili. 28,29 For example, the model strain of Geobacter sulf urreducens showed the main redox peak centered at −0.363 ± 0.003 V, which was close to the formal potential of PpcA (−0.356 V)30 within the OmcS and OmcZ potential ranges. PpcA was a periplasmic cytochrome that was proposed as the gate to exchange electrons between outer membrane cytochromes and the electrode.29 OmcS was an outer membrane cytochrome localized on pili with a potential ranged from −0.570 to 0.250 V,31,32 while OmcZ was an highly expressed extracellular cytochrome in biofilm with potentials from −0.630 to −0.270 V.33,34 Even though pyrosequencing analysis at genus level showed that Geobacter was dominant on the anode biofilm, as shown in Figure S1, the biofilm was a mixed culture with different bacterial species, so the main peak observed could be from a mixture of different cytochromes or other electron carriers that cannot be accurately identified based on electrochemical signals. While this study shows the proof-of-concept, the exact effects of AC current on extracellular electron transfer can be conducted in pure culture condition with detailed physiology characterizations. Comparing with minor peaks observed (the shoulder peak and peak 1), the main redox peak was considered as the key point at the interface of bacteria and anode, because it has a more positive potential (−0.363 ± 0.003 V) than −0.489 ± 0.015 V and −0.428 ± 0.011 V, as aforementioned. Therefore, AC treatment with high frequency (>1 kHz) likely acted on the electrochemically active proteins or secretions associated with electron exchange between the electrode and bacteria. For cytochromes with more negative potentials such as peak 1, they were less affected due to the protection of cytochromes with a more positive potential that were able to buffer electrons.

Biofilm samples for community analysis were collected from three individual batch reactors with the exact same enrichment process. Based on pyrosequencing results, samples 1, 2, and 3 showed the same genus level of identification with Geobacter species occupying 98% of the total populations (Figure S2). This can be partially explained by the inoculum source because the biofilm was enriched from the effluent of a well-acclimated microbial fuel cell fed with acetate. Anaerobic Gram negative Flavobacterium (1% in sample 1 and 0.4% in sample 2) and Oscillibacter (0.4% in sample 2 and 0.5% in sample 3) affiliated with Bacteroidetes and Firmicutes families were primary species except Geobacter in all samples (Figure S2). This finding is consistent with the literature: Geobacter dominated in biofilm produces high current in bioelectrochemical systems.27 Electroactive Biofilm Inhibition by AC Current. Turnover CV was performed when stable current was obtained. Similar sigmoidal waves shown in Figure 1A indicate that all biofilms enriched on anodes followed a similar electron transfer

Figure 1. (A) Turnover CV profile changes of the anodes under 1 MHz, 10 kHz, 1 kHz, 100 Hz, and 1 Hz AC treatments and no AC control (before treatment, after treatment, and recovery after 12 h). (B) The different percentages of biofilm responses in terms of the limiting current and DCV oxidation curve peak heights. The blank fraction (top) of each column shows the unaffected percentage of each biofilm function parameter; the solid fraction (middle) shows the recoverable percentage 12 h after AC treatment; the shaded fraction (bottom) shows the percentage of biofilm with permanent damage. Peaks 1 and 2 were separated from the main peak. The inset image shows electrolysis occurred at 1 Hz with bubbles generated. 9171


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for 5 V and 100 Hz the drop of electrochemical activities maintained at 47 ± 19% in limiting current and 56 ± 10% in DCV peaks except the oxidation peak (86 ± 6%), which suggests that more than half of the electrochemical activity was still inhibited. Similar to the result in Figure 1B, the oxidation peak in DCV showed more sensitivity at the most sensitive part at this condition (Figure S3). When the amplitude decreased to 1.2 V, the losses in limiting current, DCV oxidation peak, and reduction peak after 12 h were 74 ± 17%, 84 ± 8%, and 82 ± 10%, respectively, which are comparable with the results obtained at 5 V (Figure S4). This suggests the frequency not the amplitude serves as a key parameter affecting electrochemical activity recovery. No electrochemical activity was found after 12 h of recovery after 1 Hz treatment, suggesting the biofilm was not able to recolonize on the anode after water electrolysis with gas bubble and hydroxyl radical generation. The inactivation of biofilm was further confirmed by nonturnover CV. We selected 10 kHz and 100 Hz as examples of high and low frequencies, respectively (Figure 2). In total, four redox couples centered at −0.435 V (E1), −0.371 V (E2),

Furthermore, electrons transferred during AC treatment can also cause the periodical movement of ions at high frequency, which can act on bacterial cells by electrohydrodynamic flow (EHF) and electroosmotic flow (EOF), though the strength of these forces is closely associated with the frequency and solution characteristics.35 Compared with high frequency treatment, low frequency AC treatment (100 and 1 Hz) showed much stronger inhibition of bacterial electrochemical activity. Limiting current and peaks in DCV decreased more than 80% for 100 Hz and 100% for 1 Hz (Figure 1B). The typical sigmoidal wave in oxidation curve changed to a straight line after 100 Hz treatment, corresponding to the no peak line in oxidation part of DCV (Figure S3), illustrated that the oxidation part of CV was more sensitive than the reduction part. When the amplitude decreased from 5 to 1.2 V at 100 Hz, CVs in Figure S4 showed that limiting currents decreased by 98 ± 2% with very limited recovery after 12 h. Because 1.2 V is below water electrolysis voltage, this finding indicates that hydroxyl radical oxidation of biofilm was not a main mechanism of activity loss under 100 Hz. Previous studies discussed that in an AC electric field, EHF and EOF can form an electrohydrodynamic force at a critical frequency, which is generally below a few hundred Hertz depending on the particle size and electrolyte content.36 This electrohydrodynamic force forms fluid convection within the biofilm on the electrode surface, which is believed caused expansion and damage of the biofilm structure. When a higher frequency is used (≥1 kHz), the imbalance between EHF and EOF results in no fluid convection so the effectiveness is low, as shown in Figure 1B.37 When 5 V and 1 Hz frequency was applied to the biofilm, the whole CV curve became a straight line without any peaks shown in either oxidation or reduction curves. This indicates that no electrochemical activity of the biofilm was detected. In the meantime, fine bubbles were produced on the anode surface as soon as the 1 Hz AC was applied, indicating water electrolysis occurred (Figure 1B). This suggests that the biofilm and its components are oxidized by the hydroxyl radicals generated during electrolysis.11 These findings further support the hypothesis that the biofilm inhibition mechanisms varied depending on AC frequencies and amplitudes. Biofilm recovery and bacterial viability analysis results also support the hypothesis. However, more defined studies are needed to fully reveal the interactions of different mechanisms under different conditions. Biofilm Activity Recovery and Electroactive Molecule Coverage. Because biofilm is capable of self-repairing after interruption, limiting current and DCV peaks all increased back to certain levels after 12 h of AC treatment except 1 Hz (Figures 1 and S3). Figure 1B shows the difference between the initial activity loss and the recovered fraction in different conditions. For each column, the blank fraction from the top until the solid fraction indicates the percentage of electrochemical activities that were not affected by AC treatment. The solid fraction (middle) shows the recoverable percentage 12 h after AC treatment, and the shaded fraction (bottom) shows the percentage of biofilm with long-term or “permanent” damage. For AC frequencies higher than 1 kHz, approximately 22 ± 5% of the initial 40 ± 3% drop were recovered after 12 h, indicating a relative ∼60% of the inhabitation was temporal and recoverable. However, lower frequency treatments caused long-term damage to the electrochemical activity of the biofilm, because

Figure 2. Variations of oxidation curves of nonturnover CVs for (A) control, (B) 10 kHz treated, and (C) 100 Hz treated anodes. 9172


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Figure 3. Stacked CLSM images (green dots) of biofilms in (A) control, (B) 1 MHz, (C) 10 kHz, (D) 1 kHz, and (E) 100 Hz reactors. The comparison of biofilm thickness and topography at x direction is shown in part F. The scale bar in each image is 20 μm. (G) The viability of each sample was calculated based on pixel counting. Samples were viability stained, resulting in green live cells and red dead cells. The error bar (±SD) was calculated from three repeated images. The biofilm layer at 0 μm is the biofilm−electrode interface (bottom). f = frequency.

−0.335 V (E3), and −0.283 V (E4) were found in the baseline subtracted CV (Figure S5B). It is believed that the redox couple with a poor oxidation peak centered at −0.435 V referred to peak 1 (−0.428 ± 0.011 V) in turnover DCVs, where it was also weak in oxidation part. The main oxidation peak centered at −0.363 ± 0.003 V in turnover DCVs seemed to be a combination of E2 to E4, which was similar to the main reduction peak of DCVs. The electroactive molecule coverage (Γ) was calculated as 30 ± 6 nmol/cm2, a value similar to the 30 nmol/cm2 obtained at the comparable limiting current (4.5−5 A/m2) with a wastewater-acclimated biofilm.38 After 10 kHz of AC treatment, Γ decreased by 19% from 23 to 19 nmol/cm2 compared to 3% in the control (from 34 to 33 nmol/cm2), which correlates with the ∼22% (AC treated) and 4% (control) decreases obtained in turnover CVs. This further supports the decrease was from the inactivation of electroactive species such as cytochromes. No distinctive changes in peak shape except those minor peaks were observed (Figure 2B). The 100 Hz treatment resulted in nearly twice the amount of decrease in Γ (58%, from 33 to 14 nmol/cm2), and the 4 peaks in the oxidation curve were

combined into a weak and wide peak (Figure 2C). Different from results obtained under 10 kHz, the decrease in Γ was similar to those in the recovered system (47 ± 19% and 56 ± 10%) but lower than that (>80%) obtained just after 100 Hz treatment. This indicates that the inactivation of the biofilm occurred at the electrode−biofilm interface, though it is not the only reason for biofilm inhibition. Biofilm Viability and Thickness Characterization. Fluorescent confocal microscopy was used to directly observe the biofilm changes. It is shown that biofilm covered more than 80% of the anode surface based on pixel counting at the end of biofilm enrichment (89 ± 3 h after inoculation, Figure 3) except for the 1 Hz experiment. No fluorescence signal was detected from the 1 Hz treated anode, indicating no biofilm coverage after treatment. There was no distinct difference between x and y directions of the samples, showing that the biofilm was uniform in z direction or thickness. Based on the stacked CLSM images, all AC treated samples and the control showed smooth biofilm coverage, and the thickness was within 19 ± 2 μm except for the 100 Hz sample (Figure 3A−E). After the 100 Hz AC treatment, the surface of biofilm became rough 9173


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Figure 4. Schematics of possible mechanisms after electroactive biofilm was treated by AC current with different frequencies.

and patchy, and the biofilm thickness expanded by 58% to ∼30 μm (Figure 3F). Viability analysis, shown in Figure 3G, revealed that the AC with a frequency higher than 1 kHz only slightly decreased the viability of the bacteria near anode (4 μm) from 89 ± 1% to 85 ± 4%, and the overall viabilities calculated from the four thickness were the same (89 ± 1% for control and 87 ± 2% for 1 MHz to 10 kHz treatments). In contrast, the 100 Hz treatment decreased the viability to 56 ± 5% near the anode (4 μm) and 80 ± 4% for bacteria distant from anode, further supporting the hypothesis that it was mainly a surface reaction. The variation of viability was a result of changes in cell membrane permeability. Red cells generally indicate the membrane integration was compromised. Figure 3 shows bacteria close to the anode had the lowest viability, so they are mostly affected by the AC treatment. The 1 MHz to 1 kHz treatments did not change the cell membrane permeability significantly, while the 100 Hz caused more damage. The value of viability near anode (56 ± 5%) was comparable with the decrease in DCV peaks after recovery (56 ± 10%) and Γ (58%). This result further confirms that biofilm inactivation after 100 Hz treatment was from the damage on bacterial cell membrane, which correlates with a loss of electroactive molecule coverage. More importantly, after the 100 Hz treatment, the biofilm structure was compromised and loosened, which indicates the extracellular electron transfer network was interrupted by the electrohydrodynamic force. Because intercellular electron transfer through conductive networks was considered a critical pathway for long distance electron transfer in biofilm, the expansion likely damaged such network connection (Figure 4).39 This could be the reason why the decrease in limiting current and peaks of DCVs were higher than that of Γ. For the 100 Hz treatment, the whole biofilm was uniformly expanded without any trace of bubble trapped in the biofilm at any scanning layer (data not shown), and no visible bubble was produced on the working/counter electrodes during the 30 min of treatment. This further confirms the gas production or water electrolysis was not significant. Biofilm was completely removed from the anode due to electrolysis after 1 Hz treatment because no signal can be detected by CLSM. Implications. This study investigates for the first time how anaerobic electroactive biofilm was inhibited by different frequencies of alternating currents. It confirms that the inactivation and recovery of biofilm activity can be monitored by the in situ and nonintrusive electrochemical methods, which has wide implications for understanding and controlling biofilm growth. AC with higher frequencies ranging from 1 MHz to 1

kHz only temporarily inhibited the biofilm, while lower frequency of 100 Hz decreased the activity more permanently due to fluid convection in the biofilm driven by the electrohydrodynamic force. In contrast, the removal of biofilm by AC at very low frequency (1 Hz) was due to water electrolysis. For the application of AC for biofilm growth control, higher or lower frequencies showed either low efficacy or toxic byproduct generation. AC at 100 Hz showed efficacy in this study by both inhibiting bacterial activity and loosening biofilm structure, so that the treated biofilm can be easily flushed out from the surface by water. It is also interesting that both energy and peak−peak current decreased with the frequency (Figure S6), where 100 Hz had 12% lower energy consumption than 10 kHz did. While this study focuses on electroactive biofilms, the results have wider implications on other biofilms as well, because the AC electrohydrodynamic force acts on all cells in the biofilm. Similar approaches were used in biosensor studies for organic loading or toxicity monitoring, as the electrical signals of electroactive bacteria can be detected in situ, which can represent the effects on other microbes as well.40 However, more studies should be conducted to understand how nonelectroactive biofilms respond to the electrohydrodynamic force and how to better reveal the effects of such force on the formation, growth, and destruction of different biofilms. Moreover, the preliminary results show that variable frequency AC can be a new approach for the investigation of long distance electron transfer in electroactive biofilms. These findings provide new references on understanding and regulating biofilm growth, which has broader implications in biofouling control, anaerobic waste treatment, energy and product recovery, and general understanding of microbial ecology and physiology.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b00813. Additional tables and figures, details on anode bacterial community, turnover CV and DCV profiles and nonturnover CV profiles (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (303) 492-4137. E-mail: [email protected]. 9174


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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Prof. Xiaobo Yin and Dr. Yaoguang Ma for their help on CLSM. X.W. was supported by the National Natural Science Foundation of China (No. 21577068). F.L.L. and Z.J.R. were supported by the U.S. Office of Naval Research (Award N000141310901).

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