Electrochemical stimulation of microbial reductive dechlorination of pentachlorophenol using solid-state redox mediator (humin) immobilization

Accepted Manuscript Electrochemical Stimulation of Microbial Reductive Dechlorination of Pentachlorophenol Using Solid-State Redox Mediator (Humin) Immobilization Dongdong Zhang, Chunfang Zhang, Zhiling Li, Daisuke Suzuki, Daisuke D. Komatsu, Urumu Tsunogai, Arata Katayama PII: DOI: Reference:

S0960-8524(14)00611-7 http://dx.doi.org/10.1016/j.biortech.2014.04.071 BITE 13367

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

27 February 2014 13 April 2014 21 April 2014

Please cite this article as: Zhang, D., Zhang, C., Li, Z., Suzuki, D., Komatsu, D.D., Tsunogai, U., Katayama, A., Electrochemical Stimulation of Microbial Reductive Dechlorination of Pentachlorophenol Using Solid-State Redox Mediator (Humin) Immobilization, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech. 2014.04.071

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.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Electrochemical Stimulation of Microbial Reductive Dechlorination of Pentachlorophenol Using Solid-State Redox Mediator (Humin) Immobilization

Dongdong ZHANG a, Chunfang ZHANG b, Zhiling LI b, Daisuke SUZUKI b, Daisuke D. KOMATSU c, Urumu TSUNOGAI c, Arata KATAYAMA a,b a

Department of Civil Engineering, Graduate School of Engineering, Nagoya University,

Chikusa, Nagoya 464–8603 Japan; b

EcoTopia Science Institute, Nagoya University, Chikusa, Nagoya 464–8603 Japan;

c

Graduate School of Environmental Studies, Nagoya University, Chikusa, Nagoya 464–

8601 Japan;

Abstract: Immobilized solid-phase humin on a graphite electrode set at –500 mV (vs. standard hydrogen electrode) significantly enhanced the microbial reductive dechlorination of pentachlorophenol as a stable solid-phase redox mediator in bioelectrochemical systems (BESs). Compared with the suspended system, the immobilized system dechlorinated PCP at a much higher efficiency, achieving 116 mol Cl– g–1 humin d–1. Fluorescence microscopy showed a conspicuous growth of bacteria on the negatively poised immobilized humin. Electron balance analyses suggested that the electrons required for microbial dechlorination were supplied primarily from the humin-immobilized electrode. Microbial 

Corresponding author. Tel: +81 52 7895856; Fax: +81 52 7895857. E-mail address: a–

[email protected]–u.ac.jp (A. Katayama)

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

community analyses based on 16S rRNA genes showed that Dehalobacter and Desulfovibrio grew on the immobilized humin as potential dechlorinators. These findings extend the potential of BESs using immobilized solid-phase humin as the redox mediator for in situ bioremediation, given the wide distribution of humin and its efficiency and stability as a mediator. Keywords: Bioelectrochemical system; Solid-phase electron mediator; Humin; Redox mediator immobilization; Pentachlorophenol 1. Introduction Bioelectrochemical systems (BESs), in which the cathodes are employed as direct electron donors for the microbial reduction of oxidized contaminants in subsurface environments, have been attracting attention as a promising technology with environmental benefits (Aulenta et al., 2007; Thrash et al., 2007). BESs decrease the needs of energy as well as organic matter (as an electron donor) in comparison with conventional biological methods. The cathode is set at a negative potential that is sufficient to support anaerobic respiration but too high for significant hydrogen production (Lovley, 2011). In BESs, dissolved redox mediators, such as methyl viologen (MV) and anthraquinone-2,6disulfonate (AQDS) act to facilitate electron transfer between the cathodes and microorganisms, and have been studied as a strategy for fine-tuning environmentally relevant microbial metabolisms such as dechlorination (Aulenta et al., 2007, 2010). Dissolved redox mediators, reversibly oxidized and reduced, accelerate reactions by lowering the activation energy (Liu et al., 2012), resulting in the enhancement of microbial transformation of pollutants. In naturally occurring humic substances (HSs), dissolved humic and fulvic acids have also been reported as redox-active organic macromolecules

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

with reversible redox sites (Ratasuk and Nanny, 2007). However, continuous dosing of dissolved redox mediators would be required in the environmental application of BESs, resulting in a reduced performance (Cervantes et al., 2011; Guo et al., 2007). Thus, an insoluble solid-phase redox mediator is needed to eliminate this problem by effective retention of the mediators within the system. The application of redox mediator immobilization on BESs has been carried out, successfully immobilizing MV on a glassy carbon electrode using a Nafion polymer for the dechlorination of trichloroethylene (Aulenta et al., 2007). Other candidates as insoluble redox mediators for BESs are observed in the immobilized mediator-assisted microbial degradation of azo dyes: active carbon (Cardenas-Robles et al., 2013), quinone redox mediators immobilized on polymeric matrixes (Guo et al., 2007), on anion-exchange resins (AERs) (Cervantes et al., 2010), on composites of polypyrrole (Wang et al., 2009) and on metal oxide nanoparticles (Alvarez, et al., 2010). Naturally occurring redox mediators— dissolved humic acids—have also been immobilized on AERs for the successful dechlorination of carbon tetrachloride as well as the degradation of azo dyes (Cervantes et al., 2011). However, there have been no reports on the dehalogenation of highly halogenated aromatic compounds using a solid-phase redox mediator. The redox mediating functions of solid-phase HSs were neglected until 2010 when enhancement by solid-phase HSs was observed for microbial iron reduction (Roden et al., 2010). However, the function of humin, the insoluble fraction of HSs under any pH condition, is still disregarded. We found for the first time that various humins obtained from soils and sediments functioned as solid-phase redox mediators in the microbial reductive dehalogenation of highly halogenated aromatic compounds, pentachlorophenol

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(PCP) and tetrabromobisphenol A (Zhang and Katayama, 2012; Zhang et al., 2013). It is noteworthy that dissolved humic acids did not maintain the microbial dechlorination of PCP although humin did and the redox activity of humin was stable under various chemical and heat treatments (Zhang and Katayama, 2012). These findings and the ubiquity of humin suggested that this naturally occurring material might be useful as a solid-phase redox mediator in BESs for dehalogenating halogenated aromatic compounds. Although cases not requiring a redox mediator have been reported, such as the dechlorination of 2chlorophenol by Anaeromyxobacter and of PCP by a mixed culture (Strycharz et al., 2010; Huang et al., 2012; Liu et al., 2013), the dehalogenation of highly halogenated aromatic compounds is known to usually require soils or sediments (Yoshida et al., 2007; Nelson et al., 2011), and that these substrates could be replaced by redox-mediating humin (Zhang and Katayama, 2012; Zhang et al., 2013). The dechlorination of polychlorinated biphenyls has also been stimulated electrochemically in the presence of sediments (Chun et al., 2013). Therefore, it is important to study solid-phase humin as a redox mediator for BESs dehalogenating highly chlorinated aromatic compounds. Here, a BES system with humin as the solid-phase redox mediator was applied to the reductive dechlorination of PCP. PCP was extensively used for decades in agriculture and industry because of its large spectrum of applicability and low cost, but it has been banned due to its high toxicity to the immune, nervous and endocrine systems, kidneys, lungs and liver, and suspected carcinogenicity (Proudfoot, 2003). Although bioremediation through reductive dechlorination in combination with anaerobic aromatic ring oxidation has been proposed as a cost-effective strategy for cleaning up PCP under anaerobic conditions (Li et al., 2010; Li et al., 2013), it is worthwhile enhancing the dechlorination step using BESs.

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Further studies are required to develop anaerobic BESs using solid-phase redox mediators, which could be applicable to anaerobic conditions such as contaminated groundwater and sediments. Here, we report the successful employment of solid-phase humin as a redox mediator immobilized on a graphite electrode in a BES in enhancing the reductive dechlorination of PCP. Furthermore, the microbial community on the immobilized humin was investigated, based on 16S rRNA genes. 2. Materials and Methods 2.1 Physicochemical Characterization of Humin Humin was extracted from sediment in the Arako River, Aichi Prefecture, Japan. Humin was obtained from the sediment as described before (Zhang and Katayama, 2012) and provided as the freeze-dried form. The humin contained 9.04% carbon, 1.74% hydrogen, 0.58% nitrogen and 75.96% ash. It had a significant content of iron (3.01 mg Fe g–1 humin), while low concentrations of Ni, Co, Cu, Zn, Mn, As and Cr were detected (lower than 0.7 mg g–1 humin). Cyclic voltammetry analysis was performed as described in the Supplementary Material. 2.2 Source Culture The PCP-dechlorinating humin culture used here was originally enriched from a soildependent PCP-to-phenol dechlorinating culture and had been maintained by 5% (vol/vol) transfer using an anaerobic medium with suspended solid-phase humin as a substitute for soil (Zhang and Katayama, 2012). The anaerobic humin-suspending medium was composed of 20 mL of mineral medium, 0.3 g of freeze-dried humin, 0.2 m filter-

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

sterilized 10 M formate, a vitamin solution (Holliger et al., 1998) and 20 M PCP, all flushed with nitrogen gas. The culture was incubated at 30 °C for 7 d, and 1 mL of culture was serially transferred to the new anaerobic humin-suspending medium when the PCP level had decreased significantly. The mineral medium consisted of (per L): 1.0 g of NH4Cl; 0.05 g of CaCl2·2H2O; 0.1 g of MgCl2·6H2O; 0.4 g of K2HPO4; 1 mL of trace element SL10 solution; 1 mL of Se/W solution; and 15 mM MOPS buffer (pH 7.2) (Widdel et al., 1983). PCP and its metabolites in the culture were analyzed using a gas chromatography– mass spectrometry system (Shimadzu QP5050, Kyoto, Japan) equipped with a DB-5MS column (J&W Scientific, Folsom, CA, USA) (Yoshida et al., 2007). Concentration of hydrogen in the headspace was determined using a highly sensitive continuous-flow isotope ratio mass spectrometry system in nanomolar quantities as described by Komatsu et al. (2011) and detailed in the Supplementary Material. 2.3 BES Setup The BES used in this study was a dual-chambered system, consisting of two gastight borosilicate glass bottles, physically separated by a 5.3 cm2 proton exchange membrane (Nafion 117, DuPont, Wilmington, DE, USA). The membrane was pretreated by boiling in H2O2 (30%), then in 0.5 M H2SO4, and finally in deionized (DI) water, each for 1 h, and then immersed in DI water prior to being used. Side arms sealed with Teflon-faced butyl rubber stoppers and aluminum crimp seals were located laterally. The working electrode (cathode) was a 5 mm diameter graphite rod (effective surface: 6.48 cm2) (Tokai Carbon, Tokyo, Japan) or a humin-immobilized graphite rod. The new graphite electrodes were soaked in 1 M HCl prior to use and were washed in 1 M HCl and 1 M NaOH after each use

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

to remove possible metal and biomass contamination. The humin-immobilized electrode was prepared by coating the graphite rod (5 mm in diameter and 4 cm long) uniformly with 0.02 g of humin powder (dry weight), using a silver epoxy (SE) (ITQ Chemtronics, Kennesaw, GA, USA). A control electrode was coated only with SE. The modified electrodes were soaked in DI water prior to being used. The anode was a spiral of platinum wire 0.8 mm in diameter and 1 m long (Nilaco, Tokyo, Japan). The reference electrode was a saturated Ag/AgCl electrode (+200 mV vs. a standard hydrogen electrode, SHE) (HX-R8, Hokuto Denko Inc., Osaka, Japan) and was introduced into the cathode chamber. The electrodes and stoppers were sterilized by immersing them in 5 M HCl for 5 min, rinsing in ethanol, and drying fully on a clean bench before setting. Electrochemical potentiostatic measurements were performed using a potentiostat (HSV-110, Hokuto Denko Inc., Osaka, Japan). 2.4 BES Experiments Solid-phase humin was examined as the redox mediator for microbial anaerobic PCP dechlorination using two BESs. One was a suspended system where the cathode was the graphite rod, and solid-phase humin was suspended in the cathode chamber. The other was an immobilized system using the humin-immobilized electrode, where the suspended humin was provided initially in the cathode chamber (phase I), and removed later (phase II). The cathode chamber in both systems initially contained 200 mL of the mineral medium with 3 g of suspended humin, and the anode chamber contained only the mineral medium, all flushed with nitrogen gas prior to the experiment. A filter-sterilized vitamin solution was added to the cathode chamber. PCP and formate were also added to the cathode chamber to give final concentrations of 20 M and 10 mM, respectively. The PCP-spiked cathode

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

chamber was inoculated with 10 mL of the PCP-dechlorinating humin culture and polarized at –500 mV (vs. SHE) after 2 h of equilibration. The BESs were incubated with continuous magnetic stirring at 30 °C for 13 d (phase I). Control experiments for the immobilized system were carried out in parallel to examine the effects of: (1) cathode polarization (open circuit control); (2) microbial action (abiotic control); (3) humin (non-humin control); and (4) SE (SE-coated electrode control). Details of the control experiments are described in the Supplementary Material (Table S1). After 13 d of incubation, the suspended humin was removed from the immobilized system and the open circuit control under anaerobic conditions in a glove chamber (COY7450000, COY, MI, USA). The medium was changed to the mineral medium containing formate (2 mM) and PCP (20 M) but without humin; then the immobilized system and the control were incubated at 30 °C under continuous magnetic stirring (phase II). The cathode of the immobilized system was polarized at –500 mV (vs. SHE) during incubation. All of the experiments were performed in triplicate. 2.5 DNA Extraction Cultures were sampled from the immobilized system, the suspended system and the open circuit control. Three sampling points were chosen in this study: humin-suspended culture; immobilized humin scraped from the humin-immobilized electrode; and the supernatant culture (without humin). The supernatant culture was taken after the system had remained undisturbed for 1 d. Genomic DNA was extracted using an Isoplant DNA Extraction Kit (Nippon Gene Co., Tokyo, Japan) according to the manufacturer’s protocol. 2.6 Denaturing Gradient Gel Electrophoresis (DGGE) Analysis

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

The bacterial 16S rRNA genes were amplified with the universal primers of 357f with a GC clamp and 517r (Muyzer et al., 1993). DGGE analysis of the polymerase chain reaction (PCR) products was performed as described by Yoshida et al. (2007). The DNA bands were excised from the DGGE gel and subcloned with pGEM-T Easy Vector (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Sequence similarity searches were performed in the GenBank data library using the BLAST program. 2.7 Analysis of 16S rRNA Gene Clone Library The bacterial 16S rRNA genes were cloned and sequenced. The PCR amplification was performed using the primer combination of 27f and 1492r (Dojka et al., 1998) and highfidelity KOD FX Neo enzyme (Toyobo, Osaka, Japan). The PCR products were cloned using a pCR®8/GW/TOPO®TA cloning kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The sequences of the 16S rRNA inserts from the recombinant clones were determined by the Dragon Genomics Center of Takara Bio (Yokkaichi, Mie, Japan) using primers GW1, 341f and GW2. Sequences were analyzed using the Ribosomal Database Project classifier (http://rdp.cme.msu.edu/classifier/classifier.jsp), and similarity searches were performed in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) using the BLAST database. The tree was constructed using CLUSTAL X and MEGA 4.0 software, implementing the neighbor-joining method. 2.8 Measurement of Microbial Growth on Immobilized Humin The microbial cells adhered on the immobilized humin were observed using an epifluorescence microscope (BX50W1, Olympus, Tokyo, Japan) equipped with a WIG cube (excitation 520–550 nm, emission > 580 nm) and a digital camera DP7 (Olympus). Small pieces of the immobilized humin scraped from the electrode were stained with

9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

ethidium bromide with minor modifications (Zhang and Katayama, 2012). Q-PCR targeting bacterial 16S rRNA genes was carried out for estimating the populations of Sulfurospirillum, Dehalobacter, Desulfovibrio and total bacteria, and was performed as described in the Supplementary Material. 3. Results 3.1 Microbial Dechlorination of PCP Using Electrical Reducing Power and Solid Humin Figure 1 shows the time course of PCP dechlorination in the immobilized system, the suspended system and the open circuit control. After 6 d, all of the PCP in the immobilized and suspended systems was dechlorinated to monochlorophenol (MCP) and phenol, while in the open circuit control, PCP was degraded to MCP and phenol only after 7 d. Setting at a negative potential in the immobilized and suspended systems resulted in a shorter start-up time. In phase I, the electrochemical enhancement of reductive dechlorination was observed in both the immobilized and suspended systems in terms of the number of additions and dechlorination rate of PCP. The immobilized system maintained an especially high dechlorinating activity until the fourth addition of PCP; after only 1 d, 20 M PCP was degraded to MCP or phenol, then the activity became weaker after the fifth addition. In contrast, the dechlorinating activities in the suspended system and the open circuit control weakened only after the third and second additions of PCP, respectively (Figure 1B, C). The immobilized system achieved the highest removal rate of 7.8 mol Cl– g–1 humin d–1 after the second PCP addition (on day 8), while the suspended system achieved 2.3 mol

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Cl– g–1 humin d–1 on day 8 after the first PCP addition and the open circuit control produced 1.8 mol Cl– g–1 humin d–1 on day 10. The dechlorination of PCP was significantly enhanced by polarization in the immobilized system. Various control experiments were carried out, as shown in the Supplementary Material (Fig. S1). None of the abiotic controls showed dechlorination activity regardless of the presence or absence of humin and/or SE coating on the electrode, suggesting that the PCP dechlorination could be attributed to microbial activity. These results also suggest that there was no direct chemical or electrochemical reaction between PCP and any electrode. The absence of activity of the biotic control with the SE-coated electrode and without humin, and the well-maintained activity with suspended humin proved that SE was not involved in the microbial dechlorination of PCP and was not inhibitory to the PCP-dechlorination activity. The lack of activity in the biotic controls without humin either immobilized or suspended showed that the microbial dechlorination of PCP required the presence of humin. The characteristics of humin as a solid-phase redox mediator were demonstrated by the cyclic voltammograms (Supplementary Fig. S2). These suggested that the dechlorination of PCP in the immobilized system was a microbially catalyzed process and that humin was required for this, probably as a redox mediator. To examine the effect of H2 as an electron donor, which might be produced by a negatively poised electrode at –500 mV (vs. SHE), the dechlorination activity of the immobilized system was examined under conditions lacking H2 and with reduced humin. After all PCP had been dechlorinated on day 6, the cathode chamber of the immobilized system was disconnected from the potentiostat and bubbled with N2 gas, then 20 M of

11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

PCP was added. Figure 1A (inset) shows that the dechlorination rate (4.3 mol Cl– g–1 humin d–1) was still significantly higher than that of the suspended system and the open circuit control, although there was a slight decrease that might have resulted from nonsustained reduction of humin by the negatively poised electrode compared with the closed circuit condition (5.5 mol Cl– g–1 humin d–1). The results clearly suggest that the microbial dechlorination of PCP was linked to electrochemically reduced humin but it did not depend on hydrogen. Moreover, a highly-sensitive hydrogen measurement showed that the hydrogen production rate was only at a rate of 0.288 ± 0.053 nmol h–1 and 0.185 ± 0.119 nmol h–1 from the graphite electrode only (poised at –500 mV vs. SHE) and from the humin-immobilized graphite electrode (poised at –500 mV vs. SHE), respectively. The hydrogen production rate was much slower than the rate of PCP dechlorination (0.98 μmol Cl- h-1 in the immobilized system), which also allowed to exclude the contribution of H2 derived by the electrolysis of water to the enhancement of the microbial dechlorination of PCP as an electron donor. In phase II, all the suspended humin was removed from the immobilized system (Fig. 1A) and the open circuit control (Fig. 1C). Both conditions had 2 mM of formate. The immobilized system dechlorinated 20 M of PCP to MCP or phenol within 7 d, whereas negligible formation of PCP dechlorinatin products was observed in the open circuit control (Fig. 1A, C). These results suggest that the dechlorination was electrochemically aided through reduction of the immobilized humin. However, 2 mM of formate was not enough to reduce the immobilized humin in the open circuit control by microbial action. The dechlorination rate per unit weight of humin was 116 mol Cl– g–1 humin d–1 (0.36 mol

12

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Cl– cm–2 d–1 or 11.6 mol Cl- L–1 d–1), which was higher by 15-fold than that of the immobilized system in phase I. After 27 d, the dechlorination activity of the immobilized system was almost lost. Upon addition of formate (2 mM) after 30 d, the dechlorination activity was rejuvenated to a similar level (166 mol Cl– g–1 humin d–1). These results show the long-term stability of humin as a redox mediator. They also suggest that the carbon source was essential for the maintenance of PCP dechlorination activity. 3.2 Changes of Current in BESs The current level decreased and reached a stable value with humin reduced at the beginning (until day 6). Then, a conspicuous enhancement in the current was observed whenever PCP was added to both systems (Supplementary Fig. S3), which was considered to be utilized for PCP dechlorination. Based on these results, the current utilization proportion was calculated by dividing the current enhancement measured (in coulombs) by the number of electrons required for dechlorination according to the dechlorination metabolites (in coulombs), as shown in Figure 2. In phase I, the current utilization proportion was low in both the immobilized and suspended systems, although it increased slowly from 2.5% to 22.9% in the immobilized system and from 13.7% to 28.4% in the suspended system. Most of the electrons were considered to be donated by formate in phase I. The similar values between the two systems probably arose from little colonization by the PCP-dechlorinating bacteria onto the immobilized humin. The gradual increase of the current utilization proportion suggested an adaptation of bacteria to a system wherein electrons are supplied by the electrode through humin.

13

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

In phase II, the current utilization proportion of the immobilized system increased and reached 70.8% (Fig. 2A) after discarding the suspended humin, when only the immobilized humin functioned as a redox mediator. The electrons required for the microbial dechlorination of PCP were more dependent on the electrochemical reduction in the immobilized system, compared with the suspended system. This could be attributed to very little loss of the electricity by shuttling electrons and by the efficient reduction of immobilized humin by the electrode. 3.3 Microbial Community Analysis Based on 16S rRNA Genes The microbial communities in the immobilized system and open circuit control (Fig. 3A) and cultures transferred twice with/without PCP or humin (Fig. 3B) were characterized by PCR–DGGE targeted to the 16S rRNA genes of the domain Bacteria. Two major fragments, 2 and 8, which were identified as belonging to Dehalobacter sp. and Desulfovibrio sp., became very strong in the immobilized humin of the immobilized system but disappeared in the twice-transferred cultures to which PCP was not added. The immobilized humin reduced by the electrode contributed to the growth of humin-oxidizing microorganisms using PCP as an electron acceptor in the cathode chamber, and weakened the growth of humin-reducing microorganisms on the immobilized humin. Therefore, we regard Dehalobacter sp. and Desulfovibrio sp. as the microorganisms that depended for their growth on the oxidization of humin and reductive dechlorination of PCP in this culture. This was similar to the result observed with adaptive evolution for the enhanced reduction of uranium at a biocathode when the electrode was placed in uranium-contaminated subsurface sediments (Gregory and Lovley, 2005). In addition, band 7, identified as Coriobacteriaceae, became weak in immobilized humin. It might be that the humin-

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

reducing microorganism was responsible for the reduction of humin coupling to the oxidation of formate in this culture. Coriobacteriaceae-related species have been reported to be able to obtain energy for growth via anaerobic respiration, oxidizing hydrogen, formate or lactate for reducing oxidized compounds (Anderson et al., 2000). In all of the supernatant samples and in cultures transferred without humin, almost all of the bands became weak or disappeared. The microorganisms in this culture might need the solidphase humin as a habitat in which to grow and work. For further characterization of the microorganisms involved in the dechlorination of PCP, a full-length 16S rRNA gene library was constructed using the DNA samples extracted from the immobilized humin on day 30 and humin-suspended culture on day 13 in the immobilized system. This contained a total of 90 and 82 nonchimeric clones, respectively. As shown in Figure 4, Dehalobacter, Sulfurospirillum and Desulfovibrio were detected as the potential dehalogenators. Two operational taxonomic units, immo-6 and sus-5, belonged to the same genus and were classified as different Desulfovibrio species, with a level of sequence similarity <90%. The closest relatives of immo-6 and sus-5 are D. vulgaris and Desulfovibrio sp. LG-2009, respectively. The Dehalobacter clones contained sequences that were nearly identical (99% identity over 1480 bases) to Dehalobacter sp. FTH1 strain. Dehalobacter sp., Sulfurospirillum sp. and Desulfovibrio sp. were detected more frequently in the immobilized humin than in the humin-suspended culture. Six clones of Coriobacteriaceae were identified in the humin-suspended culture but none in the immobilized humin. The results of the library analysis were in good agreement with that of the DGGE analysis. 3.4 Microbial Growth on the Immobilized Humin

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

The microbial growth on the immobilized humin was observed using epifluorescence microscopy (Supplementary Fig. S4). A small number of the microbial cells were attached to the humin in the immobilized system on day 6, and the number had clearly increased by day 30. Microorganisms scarcely adhered to the immobilized humin in the open circuit control on day 20. Thus, the humin-immobilized electrode poised at a negative potential favored microbial growth on its surface. To determine whether PCP dechlorination was coupled to microbial growth, changes in copy numbers of the 16S rRNA genes were tracked by Q-PCR, targeting Sulfurospirillum, Dehalobacter, Desulfovibrio and the total bacteria (Supplementary Fig. S5). All bacteria increased on the immobilized humin set at a negative potential from day 6 to 30. They increased by 5.4 times, 7.6 times, 10.3 times and 35.3 times in Sulfurospirillum, Dehalobacter, Desulfovibrio and total bacteria, respectively. This was in agreement with the results of epifluorescence microscopy. The number of bacteria was 1.19 ± 0.2  1010 copies g–1 humin (suspended humin) and 1.94 ± 0.3  1010 copies g–1 humin (immobilized humin) in immobilized system on day 6. Many more bacteria (P < 0.05 by Tukey’s Least Significant Difference test) adhered to the immobilized humin (5.91 ± 1.8  1011 copies g–1 humin) than on the suspended humin (1.29 ± 0.4  1011 copies g–1 humin) on day 13, and the number increased to 6.85 ± 2.4  1011 copies g–1 humin on the immobilized humin on day 30. 4. Discussion We have successfully established a BES in which humin was immobilized on a graphite electrode as a solid-phase redox mediator for electrochemical enhancement of the microbial

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

dechlorination of PCP. Various control experiments—an open circuit control, an abiotic control and a control lacking humin—and comparisons between the intact graphite rod electrode, the SE-coated electrode and humin-immobilized electrode, as well as the results in hydrogen-depleted conditions and the insufficient H2 production from the electrodes (500mV vs SHE), confirmed that the microbial dechlorination of PCP was enhanced by the direct electron transfer from the cathode via the solid-phase humin to the PCPdechlorinating bacteria. It should be noted that humin worked as a stable mediator for the long term as shown by the enhanced activity after 30 d of polarization with the supplementation of formate. Our BES using solid-phase humin as a redox mediator also enhanced iron reduction by Shewanella, a bacterium that has the ability to utilize solidphase iron oxide directly without a mediator (Supplementary Fig. S6). These findings suggest that this BES system using solid-phase humin has a great potential to enhance various microbial reactions in the long term. Further study is warranted to develop remediation technology based on the BES using solid-phase humin. Although humin worked as a solid-phase redox mediator to enhance the microbial dechlorination of PCP in both the immobilized and suspended systems, the immobilized system achieved much higher efficiency per unit weight of humin. The capacity of solidphase humin was also higher than that of an immobilized dissolved redox mediator, methyl viologen immobilized on a glassy carbon electrode set at –500 mV (vs. SHE) for the reductive dechlorination of trichloroethene (Aulenta et al., 2007). The maximum reductive dechlorination rate per unit working electrode surface area of the methyl viologen electrode has been reported as 0.19 mol Cl– cm–2 d–1. The rate was lower than that of immobilized

17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

system in phase II dechlorinating PCP in this study (0.36 mol Cl– cm–2 d–1), indicating a high potential of humin as a solid-phase redox mediator for the bioremediation of oxidized pollutants using BESs. Two cases of PCP dechlorination in a BES system without an electron mediator have been reported, in which the cathodes are employed as direct electron donors (Huang et al., 2012; Liu et al., 2013). The system capacity was indicated as a degradation rate of PCP of 23.7 mol L–1 d–1 under simultaneous aerobic and anaerobic degradation pathways in a tubular microbial fuel cell (Huang et al., 2012). However, the cathode was under aerobic conditions and could not be applied to totally anaerobic conditions such as contaminated groundwater and sediments. Another study reported a dechlorination rate per unit working electrode surface area of 0.53 mol Cl– cm–2 d–1 in a potentiostat mode in a BES system (Liu et al., 2013). In the present study, the dechlorination rate was 0.36 mol Cl– cm–2 d–1 (or 11.6 mol Cl- L–1 d–1) in the immobilized system during phase II, where just 0.02 g of immobilized humin was present as a redox mediator and bacteria adhered on it as the dechlorinators. This BES system showed a PCP dechlorination capacity similar to the previous studies lacking a redox mediator. Given the capacity of humin in mediating multiple microbial redox reactions for a long time, humin has high potential as a mediator in bioremediation technology. Cathode with very limited effective surface area was used in this study. Increasing the amount of immobilized humin by increasing the surface area of the electrode would enhance the capacity of the immobilized system. Humin also served as a microbial habitat in our BES system. The total microbial population was much higher in the immobilized than in the suspended humin. The specific

18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

dechlorination rates based on the 16S rRNA gene copy number were compared between the two systems with the highest current utilization proportion. They were 1.3  10–16 mol Cl– copy–1 d–1 in the immobilized system in phase II and 7.5  10–18 mol Cl– copy–1 d–1 in the suspended system after the third PCP addition. The specific dechlorination rate was more than 17-fold higher in the immobilized system. This enhancement was attributed to the higher bacterial population density as well as the increase in specific activity on the huminimmobilized electrode. The electrons required for PCP dechlorination came primarily from the immobilized humin reduced electrochemically, as shown by the highest current utilization in phase II of the immobilized system. While, fomate was considered to work primarily as the carbon source for bacterial growth rather than electron donor. In the suspended system, most of the electrons required for PCP dechlorination were suggested to come from formate due to low current utilization. The PCR–DGGE results showed that Dehalobacter and Desulfovibrio grew effectively on the humin-immobilized electrode set at a negative potential but disappeared in the twicetransferred cultures to which PCP was not added, whereas Sulfurospirillum always appeared and dominated in all of the samples, regardless of the addition of PCP. The copy number of the 16S rRNA gene increased greatly in the immobilized humin. Dehalobacter has been characterized as an obligate halorespiring bacterium that grows on the reductive dechlorination of chlorinated compounds (Nelson et al., 2011; Yoshida et al., 2009; Yoshida et al., 2013). Our previous study on a soil-dependent PCP-to-phenol dechlorinating culture indicated the involvement of Gram-positive bacteria with PCP-dechlorinating

19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

activity, probably in the phylum Firmicutes (Yoshida et al., 2007). That finding is consistent with results that Dehalobacter was associated with the dechlorination of PCP in the present PCP-dechlorinating humin culture. Desulfovibrio has also been verified as a dechlorinating bacterium (Sun et al., 2000). The electron transfer system of syntrophically grown Desulfovibrio vulgaris has also been reported (Walker et al., 2009). The cloning library results in the present study showed that D. vulgaris could only be identified in the immobilized humin. Therefore, we consider that Dehalobacter and Desulfovibrio were involved in the reductive dechlorination of PCP. The results also indicate direct electron transfer from reduced humin to these bacteria for dechlorination. Desulfovibrio sp. might coexist syntrophically by coupling electron transfer to Dehalobacter sp. for dechlorination of PCP in this immobilized system. To our knowledge, this is the first report on BESs using solid-phase humin as a redox mediator for the dechlorination of highly chlorinated aromatic compounds. There are many examples of the microbial dehalogenating activity requiring solid components of soils or sediments (Yoshida et al., 2007; Nelson et al., 2011; Zhang and Katayama, 2012; Zhang et al., 2013), many of which are considered to require a solid redox mediator (Zhang and Katayama, 2012; Zhang et al., 2013). Our BES model using solid-phase humin also enhanced the iron reduction of Shewanella, which can directly transfer electrons to external solid iron oxide. Electron transfer from the cathode to microorganisms is probably a ratelimiting step in BESs in many cases. The humin-immobilized electrode appeared to facilitate electron transfer regardless of the dependency of microorganisms on the redox mediator. In particular, it is important to note that only a small amount of immobilized humin (0.1 mg to 1 mL of contaminant solution) on the electrode could maintain

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

dechlorination activity. This would be convenient for the further enhancement of activity and for practical applications. Thus, humin immobilized as a solid-phase redox mediator in BESs extends the potential application of these systems for the in situ remediation of anaerobic environments, such as sediments and groundwater. Further study is warranted to elucidate the mechanism of electron transfer between humin and bacteria. This study is also the first to report that Dehalobacter and/or Desulfovibrio are involved in the reductive dechlorination of PCP and that electricity can enhance their growth. This adds to the body of research on the metabolism of Dehalobacter and the mechanisms involved in this PCPdechlorinating humin culture. 5. Conclusions Humin was immobilized for the first time here on a graphite electrode as a stable solidphase redox mediator and significantly enhanced the microbial reductive dechlorination of PCP. Electron balance analysis and hydrogen measurement in nanomolar quantities demonstrated that the dechlorination was stimulated by electrochemically reduced humin rather than hydrogen. Based on the 16S rRNA genes analysis, a conspicuous growth of Dehalobacter and Desulfovibrio on the negatively poised immobilized humin implied that they were involved in the reductive dechlorination of PCP. The results also indicate direct electron transfer from reduced humin to these bacteria for dechlorination. Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research (23310055, 23658272) and a university grant for ―Design of cascade utilization system for unused biological resources in the Tokai area‖ from the Ministry of Education, Culture, Sports,

21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Science and Technology in Japan, and by the Sekisui Chemical Grant Program for Research on Manufacturing Based on Innovations Inspired by Nature. Appendix A. Supplementary data The detailed experimental procedures of hydrogen determination at nanomolar levels, cyclic voltammetry, microbial reduction of FeOOH and the results for the dechlorination activity of the BES controls (Table S1, Figure S1), cyclic voltammograms (Figure S2), the current change at cathode (Figure S3), epifluorescence microscopy (Figure S4), Q-PCR (Figure S5), and the microbial reduction of FeOOH (Table S2, Figure S6).

References 1. Alvarez, L.H., Perez-Cruz, M.A., Rangel-Mendez J.R., Cervantes, F.J., 2010. Immobilized redox mediator on metal-oxides nanoparticles and its catalytic effect in a reductive decolorization process. J. Hazard. Mater. 184, 268–272. 2. Anderson, R.C., Rasmussen, M.A., Jensen, N.S., Allison, M.J., 2000. Denitrobacterium detoxificans gen. nov., sp. nov., a ruminal bacterium that respires on nitro compounds. Int. Syst. Evol. Microbiol. 50, 633–638. 3. Aulenta, F., Catervi, A., Majone, M., Panero, S., Reale, P., Rossetti, S., 2007. Electron transfer from a solid-state electrode assisted by methyl viologen sustains efficient microbial reductive dechlorination of TCE. Environ. Sci. Technol. 41, 2554–2559. 4. Aulenta, F., Di Maio, V., Ferri, T., Majone, M., 2010. The humic acid analogue anthraquinone-2,6-disulfonate (AQDS) serves as an electron shuttle in the electricity-driven microbial dechlorination of trichloroethene to cis-dichloroethene. Bioresour. Technol. 101, 9728–9733.

22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

5. Cardenas-Robles, A., Martinez, E., Rendon-Alcantar, I., Frontana, C., GonzalezGutierrez, L., 2013. Development of an activated carbon-packed microbial bioelectrochemical system for azo dye degradation. Bioresour. Technol. 127, 37–43. 6. Cervantes, F.J., Garcia-Espinosa, A., Moreno-Reynosa, M.A., Rangel-Mendez, J.R., 2010. Immobilized redox mediators on anion exchange resins and their role on the reductive decolorization of azo dyes. Env. Sci. Technol. 44, 1747–1753. 7. Cervantes, F.J., Gonzalez-Estrella, J., Marquez, A., Alvarez, L.H., Arriaga S., 2011. Immobilized humic substances on an anion exchange resin and their role on the redox biotransformation of contaminants. Bioresour. Technol. 102, 2097–2100. 8. Chun, C.L., Payne, R.B., Sowers, K.R., May, H.D., 2013. Electrical stimulation of microbial PCB degradation in sediment. Water Res. 47, 141–152. 9. Dojka, M.A., Hugenholtz, P., Haack, S.K., Pace, N.R., 1998. Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl. Environ. Microbiol. 64, 3869–3877. 10. Gregory, K.B., Lovley, D.R., 2005. Remediation and recovery of uranium from contaminated subsurface environments with electrodes. Environ. Sci. Technol. 39, 8943– 8947. 11. Guo, J., Zhou, J., Wang, D., Tian, C., Wang, P., Uddin, M.S., Yu, H., 2007. Biocatalyst effects of immobilized anthraquinone on the anaerobic reduction of azo dyes by the salttolerant bacteria. Water Res. 41, 426–432.

23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

12. Holliger, C., Hahn, D., Harmsen, H., Ludwig, W., Schumacher, W., Tindall, B., Bazquez, F., Weiss, N., Zehnder, A.J.B., 1998. Dehalobacter restrictus gen. nov. and sp. nov., a strictly anaerobic bacterium that reductively dechlorinates tetra- and trichloroethene in an anaerobic respiration. Arch. Microbiol. 169, 313–321. 13. Huang, L., Chai, X., Quan, X., Logan, B.E., Chen G., 2012. Reductive dechlorination and mineralization of pentachlorophenol in biocathode microbial fuel cells. Bioresour. Technol. 111, 167–174. 14. Komatsu, D.D., Tsunogai, U., Kamimura, K., Konno, U., Ishimura, T., Nakagawa, F., 2011. Stable hydrogen isotopic analysis of nanomolar molecular hydrogen by automatic multi-step gas chromatographic separation. Rapid Commun. Mass Spectrom. 25, 3351– 3359. 15. Li, Z.L., Yang, S.Y., Inoue, Y., Yoshida, N., Katayama, A., 2010. Complete anaerobic mineralization of pentachlorophenol (PCP) under continuous flow conditions by sequential combination of PCP-dechlorinating and phenol-degrading consortia. Biotechnol. Bioeng. 107, 775-785. 16. Li, Z., Inoue, Y., Suzuki, D., Ye, L., Katayama, A., 2013. Long-term anaerobic mineralization of pentachlorophenol in a continuous-flow system using only lactate as an external nutrient. Environ. Sci. Technol. 47, 1534–1541. 17. Liu, D., Lei, L., Yang, B., Yu, Q., Li, Z., 2013. Direct electron transfer from electrode to electrochemically active bacteria in a bioelectrochemical dechlorination system. Bioresour. Technol. 148, 9–14.

24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18. Liu, H., Guo, J., Qu, J., Lian, J., Guo, Y., Jefferson, W., Yang, J., 2012. Biological catalyzed denitrification by a functional electropolymerization biocarrier modified by redox mediator. Bioresour. Technol. 107, 144–150. 19. Lovley, D.R., 2011. Powering microbes with electricity: direct electron transfer from electrodes to microbes. Environ. Microbiol. Rep. 3, 27–35. 20. Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695–700. 21. Nelson, J.L., Fung, J.M., Cadillo-Quiroz, H., Cheng, X., Zinder, S.H., 2011. A Role for Dehalobacter spp. in the reductive dehalogenation of dichlorobenzenes and monochlorobenzene. Environ. Sci. Technol. 45, 6806–6813. 22. Proudfoot, A.T., 2003. Pentachlorophenol poisoning. Toxicol. Rev. 22, 3–11. 23. Ratasuk, N., Nanny, N. A., 2007. Characterization and quantification of reversible redox sites in humic substances. Environ. Sci. Technol. 41, 7844–7850. 24. Roden, E.E., Kappler, A., Bauer, I., Jiang, J., Paul, A., Stoesser, R., Konishi, H., Xu, H.F., 2010. Extracellular electron transfer through microbial reduction of solid-phase humic substances. Nat. Geosci. 3, 417–421. 25. Strycharz, S.M., Gannon, S.M., Boles, A.R., Franks, A.E., Nevin, K.P., Lovley, D.R., 2010. Reductive dechlorination of 2-chlorophenol by Anaeromyxobacter dehalogenans with an electrode serving as the electron donor. Environ. Microbiol. Rep. 2, 289–294.

25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

26. Sun, B., Cole, J.R., Sanford, R.A., Tiedje, J.M., 2000. Isolation and characterization of Desulfovibrio dechloracetivorans sp. nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol. Appl. Environ. Microbiol. 66, 2408–2413. 27. Thrash, J.C., Van Trump, J.I., Weber, K.A., Miller, E., Achenbach, L.A., Coates, J.D., 2007. Electrochemical stimulation of microbial perchlorate reduction. Environ. Sci. Technol. 41, 1740–1746. 28. Walker, C.B., He, Z., Yang, Z.K., Ringbauer Jr., J.A., He, Q., Zhou, J., Voordouw, G., Wall, J.D., Arkin, A.P., Hazen, T.C., Stolyar, S., Stahl, D.A., 2009. The electron transfer system of syntrophically grown Desulfovibrio vulgaris. Appl. Environ. Microbiol. 191, 5793–5801. 29. Wang, J., Li, L., Zhou, J., Lu, H., Liu, G., Jin, R., Yang, F., 2009. Enhanced biodecolorization of azo dyes by electropolymerization-immobilized redox mediator. J. Hazard. Mater. 168, 1098–1104. 30. Widdel, F., Kohring, G.W., Mayer, F., 1983. Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids. III. Characterization of the filamentous gliding Desulfonema limicola gen. nov. sp. nov. and Desulfonema magnum sp. nov. Arch. Microbiol. 134, 286–294. 31. Yoshida, N., Yoshida, Y., Handa, Y., Kim, H.K., Ichihara, S., Katayama, A., 2007. Polyphasic characterization of a PCP-to-phenol dechlorinating microbial community enriched from paddy soil. Sci. Total Environ. 381, 233–242.

26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

32. Yoshida, N., Ye, L., Baba, D., Katayama, A., 2009. A novel dehalobacter species is involved in extensive 4,5,6,7-tetrachlorophthalide dechlorination. Appl. Environ. Microbiol. 75, 2400–2405. 33. Yoshida, N., Ye, L., Liu, F., Li, Z., Katayama, A., 2013. Evaluation of biodegradable plastics as solid hydrogen donors for the reductive dechlorination of fthalide by Dehalobacter species. Bioresour. Technol. 130, 478-485. 34. Zhang, C.F., Katayama, A., 2012. Humin as an electron mediator for microbial reductive dehalogenation. Environ. Sci. Technol. 46, 6575–6583. 35. Zhang, C.F., Li, Z.L., Suzuki, D., Ye, L.Z., Yoshida, N., Katayama, A., 2013. A humindependent Dehalobacter species is involved in reductive debromination of tetrabromobisphenol A. Chemosphere 92, 1343–1348.

27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure Captions

Figure 1. Time course of microbial dechlorination of PCP at –500 mV (vs. SHE) in the immobilized system (A), suspended system (B) and open circuit control (C). Arrows indicate when PCP was added to the culture. After 13 d (phase II), the suspended humin was discarded, and the solution medium was renewed in the immobilized system and open circuit control. Vertical bars denote the standard deviations of means. The inset figure (top left in A) shows the microbial dechlorination of PCP in the immobilized system after it had been disconnected from the potentiostat and N2 gas had been bubbled into the cathode chamber. Figure 2. The current consumption and electrons required for dechlorination after spiking PCP into cultures in the immobilized system (A) and in the suspended system (B). Current enhancement means the cumulative value of current consumption after PCP spiking of the system; dechlorination electrons mean those required for dechlorination according to dechlorination metabolites; and current utilization proportion is equal to the current enhancement divided by the number of dechlorination electrons. Vertical bars denote the standard deviations of means. The numbers on the x-axis are the number of PCP additions to the system. PCP could be added seven times (the immobilized humin functioned as the only redox mediator for dechlorination of PCP in phase II) to the immobilized system, but only three times to the suspended system. Figure 3. DGGE fragment patterns of the 16S rRNA genes, targeted to the domain Bacteria, amplified from all DNAs in the immobilized system and open circuit control (A), and cultures transferred twice with/without the addition of PCP or humin (B). The long arrow

28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

represents the direction of the gel gradient (40–70%). The bands that migrated to a similar position in the gel were presumed to have similar sequences. The numbers in the DGGE gel represent bands 1 to 8 that were retrieved from the DGGE gel. The DNA sequences of these eight characteristic bands on the DGGE gel were determined. The closest relatives to the DNA sequence of the excised DGGE bands were as follows. Band 1, Sulfurospirillum barnesii (NCBI accession number: CP003333; 99% similarity between the sequence of the band and the closely related reference sequence). Band 2, Dehalobacter sp. (JN051269; 99% similarity). Band 3, Clostridium sp. (AB275141; 96% similarity). Band 4, Clostridium sp. (GU370098; 97% similarity). Band 5, Bacillus sp. (JX434141; 95% similarity). Band 6, Clostridiales bacterium (JQ298670; 97% similarity). Band 7, uncultured Coriobacteriaceae (FR774832; 98% similarity). Band 8, Desulfovibrio sp. (HM754210; 99% similarity). On each DGGE pattern, M indicates the DGGE marker; SC indicates the source culture; SH indicates humin-suspended culture in the immobilized system; IH indicates immobilized humin in the immobilized system; SN indicates supernatant culture in the immobilized system; SHC indicates humin-suspended culture in the open circuit control; IHC indicates immobilized humin in the open circuit control; SNC indicates supernatant culture in the open circuit control and +P, +H, –P, –H indicate the culture transferred twice with PCP, with humin, without PCP or without humin, respectively. Immobilized humin was sampled on day 30 (immobilized system, end of phase II) and on day 20 (open circuit control, end of phase II), while humin-suspended and supernatant cultures were sampled on day 13 (end of phase I). Figure 4. Phylogenetic tree showing the relationships between representative clones in immobilized humin and humin-suspended culture, and close relatives and type strains.

29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Immobilized humin was sampled on day 30 (immobilized system, end of phase II), and humin-suspended culture was sampled on day 13 (immobilized system, end of phase I). The clones are shown in boldface under the name starting from ―immo‖ (immobilized humin) or ―sus‖ (humin-suspended culture). For each clone, the ratio of the frequency of appearance in immobilized humin or humin-suspended culture is shown in parentheses. Reference sequences derived from the GenBank database are shown with their accession numbers in parentheses. The trees were constructed by the neighbor-joining method. Bootstrap values were based on 1000 replications shown at branch points, and the bar represents 2% sequence divergence. δ-prot.: δ-proteobacteria; ε-prot.: ε-proteobacteria; Bactero.: Bacteroidetes

30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 1.

31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 2.

32

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

A

B With electricity Without electricity

M 1 0  m

SC 10 m

SH

IH

–P +H

+P +H

SC

SN SHC IHC SNC

40%

1 1 2 3

2 3

4 4

5 6 5 7 8 7 8

70%

Figure 3.

33

+P –H

–P –H

M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Figure 4.

34

Graphical abstract for BITE

Highlights    

Nature-originated, solid-phase electron mediator (Humin) was employed to BESs. Immobilized Humin on cathode significantly enhanced the microbial dechlorination. Dechlorination was stimulated by electrochemically reduced humin. Dehalobacter and Desulfovibrio are involved in the reductive dechlorination of PCP.