Validation of effective roles of non-electroactive microbes on recalcitrant contaminant degradation in bioelectrochemical systems

Accepted Manuscript Validation of effective roles of non-electroactive microbes on recalcitrant contaminant degradation in bioelectrochemical systems Yingfeng Xu, Zhipeng Ge, Xueqin Zhang, Huajun Feng, Xianbin Ying, Baocheng Huang, Dongsheng Shen, Meizhen Wang, Yuyang Zhou, Yanfeng Wang, Hanqing Yu PII:

S0269-7491(18)35140-6

DOI:

https://doi.org/10.1016/j.envpol.2019.03.036

Reference:

ENPO 12308

To appear in:

Environmental Pollution

Received Date: 16 November 2018 Revised Date:

10 March 2019

Accepted Date: 10 March 2019

Please cite this article as: Xu, Y., Ge, Z., Zhang, X., Feng, H., Ying, X., Huang, B., Shen, D., Wang, M., Zhou, Y., Wang, Y., Yu, H., Validation of effective roles of non-electroactive microbes on recalcitrant contaminant degradation in bioelectrochemical systems, Environmental Pollution (2019), doi: https:// doi.org/10.1016/j.envpol.2019.03.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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Validation of effective roles of non-electroactive microbes on recalcitrant

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contaminant degradation in bioelectrochemical systems

3 Yingfeng Xua, Zhipeng Gea, Xueqin Zhangb, Huajun Fenga, Xianbin Yinga, Baocheng

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Huangc, Dongsheng Shena, Meizhen Wanga∗, Yuyang Zhoua, Yanfeng Wanga,d,

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Hanqing Yuc

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Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, School

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of Environmental Science and Engineering, Zhejiang Gongshang University,

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Hangzhou 310012, China;

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Advanced Water Management Centre, The University of Queensland, St Lucia, QLD 4072, Australia；

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CAS Key Laboratory of Urban Pollutant Conversion, Department of

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State Key Laboratory of Pollution Control and Resource Reuse, School of the

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Chemistry, University of Science & Technology of China, Hefei 230026, China;

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Environment, Nanjing University, Nanjing 210023, China

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∗

Corresponding author. E-mail addresses: [email protected] (M. Wang). 1

ACCEPTED MANUSCRIPT ABSTRACT

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Bioelectrochemical systems (BESs) have been widely investigated for recalcitrant

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waste treatment mainly because of their waste removal effectiveness. Electroactive

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microbes (EMs) have long been thought to contribute to the high effectiveness by

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interacting with electrodes via electron chains. However, this work demonstrated the

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dispensable role of EMs for enhanced recalcitrant contamination degradation in BESs.

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We revealed enhanced p-fluoronitrobenzene (p-FNB) degradation in a BES by

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observing a defluorination efficiency that was three times higher than that in

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biodegradation or electrochemical processes. Such an improvement was achieved by

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the collaborative roles of electrode biofilms and planktonic microbes, as their

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individual contributions to p-FNB degradation were found to be similarly stimulated

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by electricity. However, no bioelectrochemical activity was found in either the

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electrode biofilms or the planktonic microbes during stimulated p-FNB degradation;

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because no biocatalytically reductive or oxidative turnovers were observed on cyclic

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voltammetry curves. The non-involvement of EMs was further proven by the similar

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microbial community evolution for biofilms and planktonic microbes. In summary,

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we proposed a mechanism for indirect electrical stimulation of microbial metabolism

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by electrochemically generating the active mediator p-fluoroaniline (p-FA) and

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further degradation by a sequential combination of electrochemical p-FNB reduction

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and biological p-FA oxidation by non-EMs.

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Capsule: This work unveils a dominant role of non-electroactive microbes rather than

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electroactive microbes on enhanced p-fluoronitrobenzene (p-FNB) removal in BESs.

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p-fluoronitrobenzene (p-FNB) degradation effectiveness was stimulated in BESs

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Electrode biofilms and planktonic microbes contributed similarly to effectiveness

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stimulation

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Electroactive microbes were excluded in electrical stimulation for enhanced

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p-FNB degradation

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Non-electroactive microbes indirectly stimulated by electricity for enhanced

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p-FNB degradation

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Keywords: Electroactive microbes; Non-electroactive microbes; Electrode biofilms;

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Planktonic microbes

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1. Introduction More and more organic recalcitrant wastes are produced by the rapid

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development of industry and agriculture, and social concerns over recalcitrant

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contaminants are increasingly raised due to the recalcitrance and persistence of these

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wastes in the environment (Huang et al., 2011a). In addition, the presence of

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recalcitrant wastes has high environmental and health risks owing to their toxicity

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and potential hazardous health effects on living organisms (Vilar et al., 2017). Thus,

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wastewater containing recalcitrant contaminants should be properly treated before

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being discharged into environments.

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The development of conventional strategies for treatments of recalcitrant

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contaminants in wastewater, in terms of physical removal and chemical degradation,

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is limited by high operating costs and the generation of secondary pollutants (Ayoub

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et al., 2010; He et al., 2017; Samsudeen and Matheswaran, 2018). Biological

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treatments are attractive due to their low-cost and environmentally friendly nature

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(Cheng and Hu, 2017; Zhang et al., 2017), but they are normally limited by kinetic

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inefficiency because of the anti-biodegradability of recalcitrant wastes (Huang et al.,

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2011a).

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Alternatively, the bioelectrochemical system (BES) has been developed to be

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promising for treating recalcitrant wastes (Kumar et al., 2017; Zhang et al., 2017).

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This type of system has become prevalent recently by featuring a higher waste

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removal effectiveness than conventional biological processes (Huang et al., 2011b; 4

ACCEPTED MANUSCRIPT Zhang et al., 2013; Wang et al., 2011). Its advantage is normally attributed to the

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biocatalytic roles of selectively amended (Strycharz et al., 2008; Strycharz et al., 2010)

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or specifically evolved electroactive microbes (EMs) (Wang et al., 2011; Pham et al.,

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2009), which are characterized by interacting with electrodes via extracellular

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electron transfer chains (Rozendal et al., 2008). The biocatalytic role of EMs on

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recalcitrant contaminant activation and degradation in BESs has been intensively

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studied using electrochemical characterizations such as cyclic voltammetry (CV)

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(Feng et al., 2014; Wang et al., 2011), and biological analyses such as 16Ss rRNA

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sequencing (Patil et al., 2012; Jiang et al., 2018). Moreover, evidences supporting

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these BES findings have been strengthened by the use of electroactive pure cultures in

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the degradation of recalcitrant contaminants (Strycharz et al., 2008; Liang et al., 2014).

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However, consensus on the effective roles of EMs may overestimate their

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contributions to waste degradation in BESs; in particular, in most relevant studies,

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mixed cultures are used as biocatalysts and EMs and non-EMs may evolve and

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co-exist simultaneously (Wang et al., 2016; Feng et al., 2016). In these cases, do EMs

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contribute more than non-EMs to increasing the removal effectiveness of recalcitrant

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wastes?

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Although some EMs are reported to transfer electrons to electrodes via the

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diffusion of molecules, direct interactions between EMs and electrodes through a

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conductive matrix are more likely preferred in commercial BESs for practical use

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because they enables higher kinetic rates of electron transfer (Borole et al., 2011). 5

ACCEPTED MANUSCRIPT Thus, biofilms formed on electrode surfaces depending on the colonization of EMs

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are normally thought to be the predominant contributors to recalcitrant waste removal

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in BESs (Wang et al., 2011; Huang et al., 2012). In contrast, the roles of planktonic

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microbes (normally existing as a suspended culture) seem to be much less attractive

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as they are either excluded from systems intentionally (Huang et al., 2012) or kept but

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rarely evaluated with regard to their contributions to waste degradation (Jiang et al.,

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2016). Such faith in the advantageous catalysis of EMs may have long covered the

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contributing roles of planktonic microbes and mislead our understanding of the

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dispensability of electrode biofilms.

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Therefore, the aim of this work is to validate the effect of non-EMs on

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recalcitrant contaminant removal in BESs. To achieve this goal, the fluorine-bearing

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recalcitrant pollutant p-fluoronitrobenzene (p-FNB) was chosen to investigate its

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degradation dependence on EMs and non-EMs in BESs. The contributions and

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mechanisms of electrode biofilms and planktonic microbes for p-FNB removal and

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defluorination were evaluated.

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2. Materials and methods

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2.1. Reactor configuration

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Single chamber reactors used in this work were made from glass bottles with a

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6.5 cm outer diameter, a 12.5 cm height and an active volume of 130 mL. The top

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port of each bottle was sealed by a rubber stopper, which was vertically pierced with

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two titanium wires (1 mm in diameter) for electrical collection. Two pieces of 6

ACCEPTED MANUSCRIPT graphite felt (3 cm × 3.5 cm × 0.1 cm), fixed at the end of each titanium wire by

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conductive glue, were used as electrodes. The distance between electrodes was 1 cm.

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On the top of the rubber stopper, the protruding wires were connected to a voltage

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power supplier (Hangzhou Siling Electronic Equipment Co., China) to supply

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electrical input to the reactors. Two groups of duplicate reactors were operated

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without microbial inoculum as an electrochemical control (EC) or in an open circuit

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mode as a biological control (BC).

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2.2. Microbial inoculation and reactor operations

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Aerobic seeding sludge (4000 mg L-1) obtained from an industrial wastewater

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treatment plant (Zhejiang Yongtai Technology Co., Zhejiang, China) was used as the

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inoculum. p-FNB (98% pure; Aladdin Chemical Co., Shanghai, China) was added to

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the reactor at an initial concentration of 0.4 mmol L-1 as previously described (Feng et

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al., 2016), and with additional sucrose (400 mg L-1) as a supporting substrate.

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Unless otherwise noted, all reactors were operated in batch mode with a

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hydraulic retention time of 2 d and ambient temperature of 30 ± 2 °C. The medium

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(synthetic wastewater) consisted of modified M9 electrolytes (3.4 g L-1 K2HPO4, 4.4 g

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L-1 KH2PO4, 0.1 g L-1 NH4Cl, 0.5 g L-1 NaCl, and 0.1 g L-1 MgSO4 7H2O) and trace

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elements as previously described (Feng et al., 2014). The medium was changed

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occasionally

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degradation-relevant microbes. An external power source (1.4 V) was supplied to

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reactors to provide sustainable electrical stimulation. Liquid samples were collected at

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p-FNB

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and defluorination rate increases were observed throughout the incubation cycles, and

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a triply repeatable rate was regarded as an indicator of the stabilization of the reactors.

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2.3. Tests on the individual roles of electrode biofilm and planktonic microbes

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To evaluate the individual roles of electrode biofilms and planktonic microbes on

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p-FNB degradation, the evolved biofilm-electrodes (including bioanodes and

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biocathodes) in stabilized BES reactors were gently separated from the suspending

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sludge for downstream tests. Two groups of batch tests were then conducted: in

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Group I, the biofilm-electrodes (BE) were moved into new single-chamber reactors to

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construct biofilm based BESs; Group II, biofilm-electrodes were excluded from BESs,

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and two pieces of new abiotic electrodes were inserted into the residual suspending

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sludge (SS) to form planktonic microbe-based BESs. The new reconfigured BESs in

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the different test groups were operated with a closed circuit of 1.4 V or an open circuit;

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and were fed with different contaminants (p-FNB or p-FA). Additionally, p-FNB

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electrolyzed for 24 hours as the target pollutant was added to BE (O-BE1) and SS

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(O-SS1), and then the BESs were operated with a hydraulic retention time of 2 d in an

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open circuit. All operational series can be found in Table 1.

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2.4. Sampling and analyses

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In the stable operation cycle, liquid samples were collected from each reactor at

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predetermined times, and then filtered (0.22 µm) for the analysis of p-FNB,

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p-fluoroaniline (p-FA) and fluoride ions. Once the p-FNB removal rate and 8

ACCEPTED MANUSCRIPT defluorination rate stabilized (a difference of less than 5% in three consecutive

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batches), a CV test was performed on the anode and cathode biofilms and on the

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planktonic microbe-based reactors. At the end of all experiments, microbial samples

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were collected from the biofilms and suspending sludge for community analysis.

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2.5. Analytical methods

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The concentrations of p-FNB and p-FA in the samples were analyzed using a

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high-performance liquid chromatograph (HPLC, e2695, Waters Corp., USA) with a

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C18 column. The liquid chromatograph used water and methanol (3/7, v/v) as the

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mobile phase at 1 mL min-1 and a column temperature of 35°C. The injection volume

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of each sample was 10 µL.

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The fluoride ion concentration was measured with a Metrohm 882 compact IC

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plus ion chromatograph (Metrohm AG, Herisau, Switzerland) using an anion AS1-HC

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(Metrosep A Supp 4-250/4.0) analytical column with a sodium carbonate/sodium

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bicarbonate mobile phase at a flow rate of 1.0 mL min-1. All groups consisted of three

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independent replicates and all data are presented as the average ± standard deviation

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(SD). Differences among groups were identified by analysis of variance using SPSS

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(version 22), and a p <0.05 was considered statistically significant. The p-FNB

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removal ratio (R p-FNB) and the defluorination ratio (RF) were calculated as previously

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described (Feng et al., 2016). The change in the defluorination ratio was calculated as

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follows:

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(1)

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Where C0(F) is the concentration of F- (mmol L-1) in the original state, and C(F) is the

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concentration of F- in the change state.

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Cyclic voltammetry (CV) was performed using an electrochemical workstation

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(Biologic VSP, Claix, France) equipped with a three-electrode system. All potentials

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in the study are quoted relative to the Ag/AgCl reference electrode. CV was

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conducted at 10 mV s-1 for all groups. Before each test, O2 was eliminated by smooth

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N2 sparging.

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The genomic DNA of the biofilm and planktonic microbes were extracted using

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an extraction kit (DR4011; Bioteke Corporation, Beijing, China) according to the

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manufacturer’s instructions. The 16S rRNA gene of the extracted DNA was amplified

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using

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(50-GGACTACATCGACGGGTATTCTAAT-30) primer set (Zhang et al. 2015). The

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bacterial communities were investigated by Illumina high-throughput sequencing,

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which was conducted by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China)

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(Wang et al. 2016). Originally derived OTU data were analysed, and a final microbial

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community heatmap was drawn using RStaudio software (Version 1.0.153).

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3. Results and discussion

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3.1. p-FNB removal and defluorination in the BESs

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(50-ACTCCTACGGGAGGCAGCAG-30)

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ACCEPTED MANUSCRIPT As soon as the reactors stabilized, the p-FNB degradation efficiency was

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enhanced in the BES compared to that in the EC and BC (Fig. 1a-c). More

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specifically, 100% of the p-FNB could be removed in 10 h in the BES, while it took

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more than 500 h and 35 h, respectively, in the EC and BC. Similarly, the

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defluorination efficiencies in the three systems also significantly varied (p<0.05), and

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the BES exhibited the highest defluorination efficiency of 80.3%, much higher than

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the efficiencies of 33.5% and 40.4% in the EC and BC, respectively (Fig. 1a-c). This

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result was further consolidated by the first-order kinetics model of fluoride ion release

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in systems (Table S1). The constant defluorination rate in the BES (0.033 h-1) was

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approximately 3 times higher than that in the EC and BC. As the fluoride ions

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absorbed by the graphite felt electrodes were negligible and p-FNB removal caused

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by adsorption effects of electrodes (approximately 25%) could be excluded from the

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subsequent batch trials (Fig. S1), these results demonstrated the considerably

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enhanced p-FNB degradation in the BES. Such an enhancement was achieved by the

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coexistence of electrode biofilm and suspending sludge, yet their synergetic

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contribution warranted further investigation.

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3.2. p-FNB removal and defluorination in BE and SS

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Under a closed circuit with a voltage supply of 1.4 V, the biofilm electrode

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(C-BE) and suspending sludge (C-SS) could achieve p-FNB removal and

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defluorination individually with similar performances (Fig. 1d and g). p-FNB was

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completely removed within 15 h by both systems, and defluorination efficiencies of 11

ACCEPTED MANUSCRIPT 53.1% ± 3.6% (corresponding to a rate constant of 0.015 h-1) and 54.7% ± 2.4% (a

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rate constant of 0.017 h-1) were achieved in 2 days in the C-SS and C-BE, respectively.

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Their performances were greatly suppressed without the assistance of electricity. With

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an HRT of 2 days, only 62.1% ± 2.3% and 57.6% ± 4.3% of p-FNB was removed,

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accompanied by defluorination efficiencies of 17.1% ± 1.9% and 13.8% ± 2.9% in the

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O-SS and O-BE, respectively (Fig 1e and h). It is widely recognized that a biofilm

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growing on the electrode surface is more likely stimulated for enhanced contaminant

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removal (Wang et al., 2011; Huang et al., 2012), while our tests demonstrated that the

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suspended organisms in the medium, such as planktonic cells, could also be

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stimulated by electricity for enhanced p-FNB degradation. This finding can be further

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confirmed by either the 48.2% increase in the defluorination efficiency once electrical

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stimulation was increased by doubling the anode and cathode electrode areas in the

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C-SS (Fig. 2a); or the 34.1% decreased in the defluorination efficiency once the

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biomass concentration of suspending sludge in C-SS was reduced by three-quarters

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(Fig. 2b). Together, these results prove that the suspending sludge plays an important

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role in p-FNB degradation in BESs.

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It was noteworthy that the stimulated p-FNB degradation achieved by the

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individual role of suspending sludge was almost the same as that achieved by the

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individual role of biofilm, indicating that planktonic microbes and electrode biofilms

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made comparable contributions to p-FNB degradation in the BESs. This finding is

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further supported by the observation of similar levels of defluorination efficiency in 12

ACCEPTED MANUSCRIPT O-SS1 (63.4% ± 0.7%) and O-BE1 (59.1% ± 1.2%) (Fig. 1f and i). In comparison to

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O-SS and O-BE, much higher defluorination efficiencies were observed in O-SS1 and

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O-BE1, also implying that electrochemical or bioelectrochemical production of the

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intermediate p-fluoroaniline (p-FA) was essential to the facilitated p-FNB

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defluorination in both electrode biofilm- and planktonic microbe-based BESs (Feng et

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al., 2014).

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To prove this hypothesis, p-FA was added to replace p-FNB as the targeted

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pollutant in the biofilm-electrode and planktonic microbe-based BESs. Similar rates

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of p-FA removal and defluorination were achieved under both closed and open circuit

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conditions (Fig. 3). Inconsistent with the electrical role of stimulating p-FNB

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degradation, p-FA degradation stimulated by electricity in the BES was limited. This

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result implies that EMs were not positively involved enhancing p-FA degradation.

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3.3. Electroactivity characteristics of BE and SS

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Both the electrochemical cathode and cathodic biofilm exhibited strong

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reduction peaks at a potential of approximately -0.68 V vs Ag/AgCl, (Fig. 4a) at

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which p-FNB was reduced to p-FA as previously reported (Wang et al., 2016).

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Transformation of –NO2 to –NH2 in BESs was previously reported to be catalysed by

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EMs (Feng et al., 2014). However, the lack of an obvious shift in reduction potential

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and the lack of significant current intensity variation regardless of the presence of

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biofilm or planktonic microbes, demonstrated that nitro reduction of p-FNB did not

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make a bioelectrochemical contribution (Fig. 4a). No reduction peaks were observed

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in the electrochemical or bioelectrochemical defluorination for p-FNB (Fig. 4a) or

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p-FA (Fig. 4c), revealing the unfeasibility of a reduced defluorination process. Similarly, non-turnover of p-FNB oxidation was not observed in the CV curves of

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either the purely electrochemical anodes or the bio-anodes with biofilm or planktonic

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cells (Fig. 4b), suggesting the importance of p-FA production as an active mediator to

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stimulate p-FNB oxidation. This result was supported by oxidation peaks on CV

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curves with p-FA amendment (Fig. 4d). However, no oxidative turnover caused by

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biocatalysis in terms of electrode biofilm or suspending sludge was detected,

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demonstrating the exclusion of EMs involving in oxidative p-FNB metabolism.

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Considering the much superior defluorination effectiveness in the BES than in the EC,

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a biologically dominated p-FA oxidation process with other electron acceptor

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candidates rather than the anode was expected in the BES. Unlike biological

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respiration of simple organics (such as acetate) interacting with electrodes, due to the

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bio-recalcitrance of contaminants, other energetically favourable electron acceptors

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(especially oxygen) instead of the anode may be favoured by organisms other than

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EMs (such as aerobic microorganisms, methanogens, sulfate-reducing bacteria,

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nitroreducens) (Borole et al., 2011) for recalcitrant contaminant degradation. Thus,

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the trace oxygen residual is a candidate with high potential as an electron acceptor for

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p-FNB degradation, as our previous study has demonstrated the simulating effects of

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limited oxygen supply on the mineralization of p-FNB (Shen et al., 2014). Such an

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oxygen-affinity is likely to be the reason for the lack of electroactivity in biofilms and

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ACCEPTED MANUSCRIPT the considerable contribution of suspending sludge to defluorination in our current

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BES. Therefore, a sequential combination of electrochemical p-FNB reduction and

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biological p-FA oxidation by non-EMs was proposed for p-FNB degradation. The

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more favourable biocompatibility and biodegradability of p-FA stimulated the whole

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kinetics of p-FNB degradation.

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3.4. Microbial communities of BE and SS

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Principal co-ordinates analysis (PCoA) showed that the suspending sludge and

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biofilms of the anode and cathode were separate from the inoculum but grouped

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together, indicating that the community had adapted to p-FNB degradation and that

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the suspending culture featured similar communities as the biofilms (Fig. S2). Such a

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community similarity was confirmed by the Shannon index at the class level, showing

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no differences among the suspending sludge and the biofilms of the anode and

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cathode (p>0.05) (Fig. S3). This similarity indicated that no special characteristics of

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EMs were involved in community evolution.

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To be specific, the dominant communities were highly similar among the

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suspending sludge and biofilms (Fig. 5). The class Saccharibacteria dominated by the

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genus Saccharibacteria increased remarkably from a hardly detectable level to the

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most dominant in the suspending sludge (25.0%), anodic biofilm (11.9%) and

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cathodic biofilm (30.8%). Saccharibacteria was not reported to be electroactive (Li et

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al., 2017); but was reported to be biologically active for recalcitrant pollutant

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degradation (Liang et al., 2015). Similarly, the genus Pseudomonas dominating the

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ACCEPTED MANUSCRIPT class Gammaproteobacteria increased as another dominant strain, from a negligible

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proportion to predominant percentages of 10.4% in the suspending sludge and, 13.5%

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and 15.3% in the anodic biofilm and cathodic biofilm, respectively. Pseudomonas sp.

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has been shown to be highly selected for the biodegradation of p-FNB

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chemically-analogous compounds, such as aromatics (Ning et al., 2017),

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2-chloronitrobenzen (Liu et al., 2005), and nitroaromatics (Kapley et al., 2007; Liang

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et al., 2013). Thus, comparable contributions of the suspending sludge and electrode

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biofilms to p-FNB degradation in the BESs were likely derived from the similar

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community development, with essential roles played by Saccharibacteria and

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Pseudomonas. Although it has been widely reported that the direct current in BESs is

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a selective stress driving microbial community evolution and leading to the

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dominance of functional EMs for bioelectrocatalytic waste removal (Zhang et al.,

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2013; Pham et al., 2009), the non-electroactive development of a contributing

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consortia consisting of Saccharibacteria and Pseudomonas in the current BES was

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likely driven by p-FA, which was electrochemically reduced from p-FNB.

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4. Conclusions

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In summary, our study shows a BES case for p-FNB degradation, in which

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enhanced p-FNB degradation in the BES was not related to electroactive microbes.

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Enhanced p-FNB degradation was achieved in the BES, with a first-order kinetics

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constant for the defluorination rate (0.033 h-1) that was approximately 3 times higher

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than that in the EC and BS. The enhancement was achieved by the combined 16

ACCEPTED MANUSCRIPT contributions from both the electrode biofilm and planktonic microbes, with each

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being similarly stimulated by electricity. With voltage input in the BES,

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defluorination efficiencies of 54.7% ± 2.4% and 53.1% ± 3.6% were achieved in 2

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days by the electrode biofilm and the planktonic microbes, respectively, whereas the

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efficiencies were only 13.8% ± 2.9% and 17.1% ± 1.9%, respectively, under open

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circuit conditions. However, validated by CV tests, bioelectrochemical activity of

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EMs occurred neither in electrode biofilms nor in planktonic microbes for -NO2

329

reduction and p-FA oxidation. Enhanced p-FNB degradation in the BES was proposed

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to be achieved by a sequential combination of electrochemical p-FNB reduction and

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biological p-FA oxidation, and these essential roles were played by non-Ems,

332

including a consortia of Saccharibacteria and Pseudomonas.

333 334 Acknowledgments

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This paper was supported by the National Natural Science Foundation of China

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(grant number 51478431); Science and Technology Planning Project from the Science

338

and Technology Department in Zhejiang Province (grant numbers LQ17E080002).

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Table caption

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Table 1. Description of batch tests in biofilm- and planktonic microbe-based BESs

439 Group I (biofilm-electrode (BE) based BESs)

Group II (suspending sludge (SS) based BESs)

Assigned test Electrochemical

Contaminant

parameter

feeding

Assigned test Electrochemical

Contaminant name in this

parameter study

RI PT

name in this feeding

study

closed circuit 1.4 V

0.4 mM p-FNB

C-BE

closed circuit 1.4 V

0.4 mM p-FNB

C-SS

open circuit

0.4 mM p-FNB

O-BE

open circuit

0.4 mM p-FNB

O-SS

effluent from

effluent from EC treating 0.4 mM

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EC treating 0.4

open circuit

O-BE1

open circuit

O-SS1

mM p-FNB for

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p-FNB for 24 h

24 h

0.4 mM p-FA

C-BE2

closed circuit 1.4 V

0.4 mM p-FA

C-SS2

open circuit

0.4 mM p-FA

O-BE2

open circuit

0.4 mM p-FA

O-SS2

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closed circuit 1.4 V

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Fig. 1. p-FNB removal and defluorination performance in different systems (a: BES; b:

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EC; c: BC; d: biofilm-based BES under closed circuit (C-BE); e: biofilm-based BES

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under open circuit (O-BE); f: biofilm-based BES under open circuit with p-FNB

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electrochemically treated for 24 h as influent (O-BE1); g: suspending sludge-based

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BES under closed circuit (C-SS); h: suspending sludge-based BES under open circuit

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(O-SS); i: suspending sludge-based BES under open circuit with p-FNB

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electrochemically treated for 24 h as influent (O-SS1

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Fig. 2. p-FNB removal and defluorination performance in the suspending

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sludge-based BES under closed circuit (a: the areas of the anode and cathode were

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twice the original areas;

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Fig. 3. p-FA removal and defluorination efficiency in biofilm and suspending

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sludge-based BESs under closed circuit or open circuit, with p-FA added replacing

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p-FNB as the targeted compound

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Fig. 4. (a) CVs for the p-FNB reduction on cathodes of the electrode biofilm-based

459

BES (magenta curve), suspending sludge-based BES (blue curve) and electrochemical

460

control system (red curve); (b) CVs for the p-FNB oxidation on anodes of the

461

electrode biofilm-based BES (magenta curve), suspending sludge-based BES (blue

462

curve) and electrochemical control system (red curve); (c) CVs for the p-FA reduction

463

on cathodes of biofilm electrode system (magenta curve), suspending sludge-based

464

BES (blue curve) and electrochemical control system (red curve); (d) CVs for the

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p-FA oxidation on anodes of biofilm electrode system (magenta curve), suspending

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sludge-based BES (blue curve) and electrochemical control system (red curve)

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Fig. 5. Microbial community heatmap analysis of the initial inoculum (seed), evolved

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suspending sludge (SS), evolved biofilm on the anode (BA) and on the cathode (BC)

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