w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 2 0 e7 1 3 0

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Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells Shun’ichi Ishii a,*, Shino Suzuki a, Trina M. Norden-Krichmar a,1, Angela Wu a, Yuko Yamanaka a, Kenneth H. Nealson a,b, Orianna Bretschger a a b

J. Craig Venter Institute, San Diego, CA, USA University of Southern California, Los Angeles, CA, USA

article info

abstract

Article history:

Microbial fuel cells (MFCs) are devices that exploit microorganisms as “biocatalysts” to

Received 1 April 2013

recover energy from organic matter in the form of electricity. MFCs have been explored as

Received in revised form

possible energy neutral wastewater treatment systems; however, fundamental knowledge

15 July 2013

is still required about how MFC-associated microbial communities are affected by different

Accepted 22 July 2013

operational conditions and can be optimized for accelerated wastewater treatment rates.

Available online 20 October 2013

In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater

Keywords:

treatment: 1) MFC operation using a 750 U external resistor (0.3 mA current production); 2)

Microbial fuel cell

set-potential (SP) operation with the anode electrode potentiostatically controlled to

Potentiostatic operation

þ100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero

Anode biofilm

current generation). For all reactors, primary clarifier effluent collected from a municipal

Microbial community dynamics

wastewater plant was used as the sole carbon and microbial source. Batch operation

16S rRNA clone analysis

demonstrated nearly complete organic matter consumption after a residence time of 8e12 days for the MFC condition, 4e6 days for the SP condition, and 15e20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species related to family Desulfobulbaceae and Geobacteraceae are correlated with the electricity generation in the biofilm and may be key players for optimizing wastewater treatment rates and energy recovery in applied MFC systems. ª 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. 10355 Science Center Drive, San Diego, CA 92121, USA. Tel.: þ1 858 200 1841; fax: þ1 858 200 1801. E-mail address: [email protected] (S. Ishii). 1 Current address: Scripps Translational Science Institute, The Scripps Research Institute, La Jolla, CA 92037, USA. 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.07.048

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 2 0 e7 1 3 0

1.

Introduction

Conventional wastewater treatment is an energy intensive process. In the United States, wastewater treatment plants use an average of 1200 kWh per million gallons of wastewater treated; and approximately 60% of this energy consumption is associated with aeration processes including activated sludge and flotation thickening (Water Environment Federation, 2009). Decreasing total energy consumption during wastewater treatment is an important goal that may be accomplished by developing new treatment methods and technologies that can decrease the amount of aeration required during wastewater treatment. Microbial fuel cells (MFCs) have been studied as an efficient wastewater treatment technology, because relative to conventional wastewater treatment processes, MFC systems have the benefit of reducing overall operational costs because aeration is not needed (Rozendal et al., 2008). MFC systems exploit microbial extracellular electron transport (EET) processes to solid electrode for directly recovering energy as electricity during the degradation of organic matter contained in wastewater and/or sludge (Pant et al., 2010; Rulkens, 2008; Huang and Logan, 2008). In addition, secondary sludge production can be reduced because the growth of secondary biomass is limited under anaerobic MFC conditions (Logan et al., 2006). Recently we demonstrated long-term MFC operation using only primary clarifier effluent from a municipal wastewater treatment plant as the sole microbial resource and substrate (Ishii et al., 2012b). During each repeat-batch process, turbidity of the wastewater notably decreased, and 86% of chemical oxygen demand (COD) was removed after an 8e13 day residence time. On average, the operational current generation was 0.2e0.3 mA, and the coulombic efficiency (electron recovery) was w25%; however, the anode polarization curves indicated that the operating current was strongly limited by slow cathodic reactions resulting in an increased internal resistance of the air-cathode MFC system (Ishii et al., 2012b; Fan et al., 2008). Other groups have also reported that improving MFC cathodes and minimizing electrode spacing in MFC reactors (Liu et al., 2005; Zhang et al., 2011; Fan et al., 2007) enables faster wastewater treatment and better electricity recovery (Logan et al., 2006; Liu and Logan, 2004). On the other hand, the electrogenic microbial communities and their specific functionalities are rarely considered in these practical evaluations of MFC wastewater treatment, despite the critical importance of maintaining and optimizing biocatalytic activity at the MFC anode (Pant et al., 2010; Rulkens, 2008; Huang and Logan, 2008; Ishii et al., 2012a; Shimoyama et al., 2009; White et al., 2009). Conventional wastewater treatment processes, including activated sludge, contain an overwhelming amount of microbial diversity and the metabolic roles for each microbe are unknown. Unstable input of organic matter from variable sewage flows and seasonal changes also have a confounding effect on trying to understand, and control, microbial populations during wastewater treatment. Because of these obstacles, microbial ecosystems in wastewater treatment processes are often treated as a “black box” (Allison and

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Martiny, 2008; Gernaey et al., 2004) even though some efforts have tried to elucidate microbial functions using systematic approaches (Daims et al., 2006; Whiteley et al., 2006). Researchers investigating the practical role of MFCs in wastewater treatment have begun to study the microbial populations involved with electrogenic processes (Ishii et al., 2012b; Yu et al., 2012; Jong et al., 2011; Patil et al., 2009; Sun et al., 2010); however, strategies for optimizing EET-related microbial activities during MFC wastewater treatment still remain unclear. To begin characterizing the microbial functions in wastewater-fed MFC biofilms, we recently conducted a novel stimulus-induced metatranscriptomics approach to identify electrogenic microbes and genes associated with increased current production (Ishii et al., 2013). Increased current production, resulting from increased EET rates, was induced by set-potential (SP) conditions that made the electrode surface a more electropositive (better) electron acceptor (Wagner et al., 2010). We also evaluated the microbial functional response to decreased EET rates achieved by open-circuit (OC) operation, which stopped electron flow through the circuit and limited EET-activity at the electrode. The most significant gene expression responses measured after 45 min of exposure to those EET stimuli occurred in only two microbial groups in the anode biofilm, family Desulfobulbaceae and order Desulfuromonadales, which clearly suggests that those taxa were associated with EET reactions to the electrode (Ishii et al., 2013). The short-term genetic responses suggest the importance of electrogenic functions in an anode-associated biofilm; however, the longer-term impacts of applied EET stimuli are unknown for both microbial community dynamics and wastewater treatment kinetics. To these ends, here we report the results from long-term operation of three parallel wastewater-fed MFC reactors held to MFC, SP, and OC conditions. The objective of these studies was to compare how different MFC operational conditions impacted long-term energy recovery, wastewater treatment rates, and microbial community dynamics for systems exposed solely to municipal wastewater effluents. An analysis of the combined results suggests that highly defined operating conditions will induce accelerated wastewater treatment rates and enhanced energy recovery. Further, the key microorganisms involved with these processes can be consistently selected and maintained in MFC systems.

2.

Materials and methods

2.1.

MFC configuration and operation

Three single-chamber, air-cathode MFCs were used for municipal sewage wastewater treatment under three different operational conditions. The MFCs were bottle-type reactors (350 ml in capacity), with two joined anode electrodes made of carbon cloth (7 cm  3 cm, or 84 cm2 total projected surface area per reactor; TMIL, Japan) (Ishii et al., 2012b, 2008). The air-cathode was made with a 30 wt% wetproofed carbon cloth (type B-1B, E-TEK) coated with

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platinum (0.5 mg/cm2), Nafion, and PTFE as described elsewhere (Cheng et al., 2006). The air-cathode was placed at the side port, providing a total projected cathode surface area (one side) of 4.9 cm2. After sterilization of the fully assembled MFCs, the chambers were filled with municipal wastewater collected from the primary clarifier at the North City Water Reclamation Plant (San Diego, USA) without any pretreatment except the mechanical removal of grit, rags and scum (Meyer, 2009). The average COD concentration associated with these raw effluents was w250 mg/L (Ishii et al., 2012b; Meyer, 2009). The sole inoculum source consisted of microorganisms present in the primary clarifier effluents. The reactors was gently mixed with a magnetic stirrer, and incubated at room temperature (22  C  3  C) throughout the duration of testing (Ishii et al., 2012b). During MFC operation, the anode and cathode electrodes were connected with an external resistor of 750 U. During SP operation, an Ag/AgCl reference electrode (þ200 mV vs SHE, RE-5B, BASi) was placed in the side port of the reactor, and the anode surface potential was controlled to þ100 mV vs SHE using a potentiostat (HA-151A, Hokuto Denko). Before starting the SP operation, the electrogenic microbial community established in MFC reactor (described as W3 community in the previous work (Ishii et al., 2012b)) was inoculated, and operated 80 days under MFC operation. During OC operation, the anode and cathode electrodes were not connected. Cell voltages across the resistor for the MFC reactor, open circuit voltages for the OC reactor, and electric currents for the SP reactor were recorded every 30 min using a voltage recorder (GL200A, Graphtec). The corresponding electric current was calculated using Ohm’s law (V ¼ IR) for the MFC reactor. When the electric current for the MFC and SP reactors decreased or when open circuit voltage for the OC reactor decreased due to depletion of the organic matter in the wastewater, the anode solution was fully discarded and the reactor was refilled with either the fresh wastewater collected that day or with aged wastewater that had been stored at 4  C. This repeat-batch process occurred over 400 days. Each wastewater sample introduced to the reactor included the naturally occurring microorganisms and various chemical compounds, no filtration or additional pretreatment was conducted. When the current generation decreased due to thick biofilm formation on the air-cathode, the cathodic biofilm was mechanically removed or a new air-cathode was replaced to recover the cathode performance.

2.2.

Polarization analyses

In order to obtain anode polarization curves without cathodic reaction limitation, an Ag/AgCl reference electrode (þ200 mV vs SHE, RE-5B, BASi) was placed in the side port of the MFC and OC reactors, and linear sweep voltammetry analyses of the anode electrodes were conducted using a potentiostat (Reference 600, Gamry) (Ishii et al., 2012b; Tsujimura et al., 2001). The anode potential was swept from open circuit anode potential to þ300 mV vs SHE at a scan rate of 0.5 mV/s and the corresponding anodic current, resulting from the biofilm, was recorded.

2.3.

Chemical analyses

Chemical oxygen demand (COD) was determined using a potassium chromide assay according to the manufacturer’s instructions (Orion CODHP0, Thermo Scientific). Coulombic efficiency, CE (%), was calculated as CE ¼ Cp/Cth  100, where Cp (C) is the total charge passed during a single batch, and Cth (C) is the theoretical amount of charge allowable from a complete COD decrease (assuming that reducing 1 mol of oxygen requires the transfer of four electrons). Volatile suspended solid (VSS), turbidity, nitrate-N, nitrite-N, ammoniumN, sulfate, and heavy metal concentrations were determined in accordance with US EPA and state of California requirements at CRG Marine Laboratories, Inc (Torrance CA, USA).

2.4.

Microbial composition analysis

Total DNA was extracted from the biofilm associated with the carbon cloth anodes or from suspended cells in anolyte solution using the UltraClean Soil DNA Isolation Kit (MO bio) according to manufacturer instructions. The 16S rRNA clone libraries are constructed using universal primers U27f (50 AGAGTTTGATCCTGGCTCAG-30 ) and U1492r (50 -GGTTACCT TGTTACGACTT-30 ) (DeLong, 1992), and sequenced using primer U907r (50 -CCGYCAATTCMTTTRAGTTT-30 ) (Watanabe et al., 2001) as described previously (Ishii et al., 2012b). Sequences of the partial 16S rRNA genes were aligned to each other using CLC Genomics Workbench version 5.0 (CLCbio), and assigned to phylotypes (classified as an operational taxonomic unit, >99% cut-off). Searches for related 16S rRNA gene sequences from the nr database were conducted using the BLAST program (Karlin and Altschul, 1990). Identification of chimeric sequences, taxonomic classification, and statistical analyses including multidimensional scale (MDS) plot, rarefaction analysis, Chao1 richness, Shannon’s index, Simpson diversity index, and Sørensen similarities among the bacterial communities were performed as described previously (Ishii et al., 2012b). Canonical correspondence analysis was performed using XLSTAT (Addinsoft) to describe the correlations between community composition and environmental factors (ter Braak, 1986). The nucleotide sequences reported in this paper have been deposited in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases under accession numbers KC860516 to KC860606 for the OC reactor, KC860607 to KC860718 for the SP reactor, and KC860719 to KC860761 for the MFC reactor.

3.

Results and discussion

3.1.

MFC/SP/OC long-term operations

The three different operational conditions fed solely with primary clarifier effluent from a municipal wastewater plant showed different current-generating trends over the 400 day operation in repeat-batch mode (Fig. 1). The results of preliminary MFC operation from startup to day 263 are described in our previous work (Ishii et al., 2012b), and the currentgenerating trend, using a 750 U external resistor, is shown in Supplementary Fig. S1. The external resistance was chosen

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A

(0) (2) MFC3 MFC4

(22 ) MFC5

(4 2 ) MFC6

MFC 0.5 Current (mA)

0.4 0.3 0.2 0.1 0.0 0

B

50

(0) MFC3

100

( 8) SP1 MFC

150

200

250 Time (day)

(23) (27) SP2 SP3

300

350

400

450

(38) SP4

SP

500

(71) SP5

MFC

SP

Current (mA)

5 4 3 2 1 0 0

50

C

100

150

(8) OC1

200

250 Time (day)

300

350

400

(1 1 ) OC2

450

500

( 17) OC3 0.5

800

0.4

600

0.3

400

0.2

200

0.1

Current (mA)

Open circuit voltage (mV)

OC 1000

0.0

0 0

50

100

150

200

250 Time (day)

300

350

400

450

Fig. 1 e Electricity generation under three operational conditions on air-cathode MFCs fed with primary clarifier effluents. A, MFC operation; B, Set potential operation; C, Open circuit operation. Operational conditions are shown above the panels as MFC for microbial fuel cell operation, SP for set potential operation, and OC for open circuit operation. Filled arrowheads above the panels indicate anode biofilm sampling for DNA extraction and the sample ID. Numbers of wastewater feeds are described in parentheses above the sample IDs. Open arrowheads indicate air-cathode replacement to the fresh electrodes. Filled arrows inside the panels indicate effluent sample collection for chemical composition analysis.

based on power curve measurements and was the resistance required to achieve maximum power density in the aircathode MFC (Ishii et al., 2012b). We continued the repeatedbatch operation of the MFC reactor for another 491 days, and the current-generating trend was similar to the previous 263day operation (Fig. 1A). During long-term MFC operation, current generation decreased as a result of increased internal resistance due to dense cathode biofilm formation (Yang et al., 2009). Current generation was restored to previously observed amounts after either cathode biofilm removal by mechanical treatment (day 58 and 345) or air-cathode replacement (day 118 and 241).

The SP reactor fed with exactly the same wastewater as the other reactors showed significantly higher current generation and more rapid treatment times than those of the MFC and OC reactors (Fig. 1B). Before starting the SP operation, we operated the reactor under MFC condition for 81 days, resulting in a similar current generation range of 0.2e0.3 mA to the original MFC reactor. After starting the SP condition where the anode surface potential was controlled to þ100 mV vs SHE, the current generation was almost ten-times greater than that observed for the MFC condition. The controlled anode potential of þ100 mV vs SHE was chosen because this potential corresponded with the maximum current density observed

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from previously collected anode polarization curves (Ishii et al., 2012b). In order to clarify the differences between MFC and SP conditions, the operation of the SP reactor was shifted to MFC condition several times, and decreases in current generation to 0.2e0.3 mA under MFC condition were observed. These results indicate that the range of current generation related to microbial EET rates was defined by the operational condition of either MFC condition or SP condition. Operation of the OC reactor with zero current production was conducted in a separate reactor without any preoperation in MFC mode, but fed with the same wastewater to the MFC and SP reactors (Fig. 1C). The OC reactor served as a negative control to analyze the contribution of current generation to both wastewater treatment efficiency and enrichment of the electrogenic microbes. The open circuit cell voltage was observed to be around 800 mV, which is a reasonable range for our air-cathode MFC systems (Ishii et al., 2012b) as described below Section 3.3.

3.2.

COD removal and coulombic efficiency

Removal of organic matter from the wastewater was measured by removal of chemical oxygen demand (COD). In representative repeat-batch cycles, the COD consistently decreased to similar levels (w80% COD removal ratio) in all three reactors, but the COD removal trends were different in the three operational conditions (Fig. 2). The SP reactor showed the most rapid COD removal and higher current generation within 3 days of treatment time. The associated COD removal rates for the batch operation were found to be 8.4 g-COD m-2-anode surface area-d1 and 0.20 g-COD L-1reactor volume-d1, which was two-times faster than the rates observed for the MFC operation. On the other hand, the

300 250

Current (mA)

4

200 3 150 2

SP

100

1

50 MFC

OC

Chemical oxygen demand (mg/L)

5

0

0 0

3

6

9

12

15

18

Time (day) Fig. 2 e Exemplary current generation and substrate consumption rates under three operational conditions. Typical batch cycle under MFC operation with 750 U external resistance (MFC, squares, black lines), set potential operation (SP, circle, red lines), and open circuit operation (OC, diamonds, blue lines). The solid lines indicate electric current (mA). The dashed lines indicate chemical oxygen demand (mg/L). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

OC reactor showed slower COD removal than the MFC and SP reactors, which indicates that the electricity generation under the current producing operational conditions facilitated improved COD removal from the wastewater. Electron recovery was calculated as coulombic efficiency (Logan, 2008), and found to be stable at 23  4% for MFC operation, which suggests that approximately 75% of electrons associated with the COD degradation were consumed by competitive reactions such as oxygen respiration, sulfate or nitrate reduction, methanogenesis, and/or biosynthesis (Ishii et al., 2013). Table 1 shows the chemical components measured in the primary clarifier effluents and the anolyte effluents from the three different reactors. These comparisons show that anaerobic electron acceptors such as sulfate and nitrite were not reduced during the MFC/OC batch cycle, while sulfate degradation was observed during the SP batch cycle. These data suggest that sulfate reduction occurred simultaneously with higher current generation in the SP reactor; however, sulfate and nitrate were not used as the preferential terminal electron acceptors in the other two reactors. In the MFC/OC reactors, COD degradation was likely coupled with oxygen reduction at the cathode interface where thick biofilms at the cathode surface removed the oxygen permeating across the air-breathing cathode and maintained anaerobic conditions in the reactors. The concentrations of

Table 1 e Chemical composition of primary clarifier, MFC, SP, and OC effluents. Primary clarifier effluenta COD (mg/L)e 316 67 VSS (mg/L)e Turbidity 70 (NTU) Nitrate-N 0.08 (mg/L) Nitrite-N NDf (mg/L) Ammonia-N 37.4 (mg/L) Sulfate 236 (mg/L) Heavy metal conc. (mg/L) Fe 1913 Al 434 Mn 103 Cu 68 a

MFC effluentb 48 17 14

SP effluentc 51 17 15

OC effluentd 35 15 17

0.08

0.06

0.07

0.05

0.05

0.22

25.9

40.3

17.8

248.1

114.0

240.8

806 118 256 19

672 140 95 21

1230 84 115 15

Means were calculated from primary clarifier effluent samples analyzed on 5/5/2009, 8/4/2009, and 10/6/2009 (Meyer, 2009). b The MFC effluent was analyzed using a mixture of samples collected on day 81 (8/7/2009), day 105 (8/31/2009), and day 119 (9/ 14/2009). c The SP effluent was analyzed using a mixture of samples collected on day 107 (9/2/2009), day 113 (9/8/2009), and day 119 (9/ 14/2009). d The OC effluent was analyzed using a mixture of samples collected on day 24 (8/17/2009) and day 40 (9/2/2009). e COD ¼ chemical oxygen demand, and VSS ¼ volatile suspended solid. f Not determined.

w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 2 0 e7 1 3 0

several inorganic heavy metals were also significantly decreased in each reactor process (Table 1); however, it is unknown if the metals served as electron acceptors for the microbial respiration, or if abiotic adsorption affected the metal concentrations in the treated effluents. In the SP reactor, the coulombic efficiency increased to 58  4% for the repeat-batch operation, which suggests that the improved electron flux to the solid electrode in the anodic microbial community is correlated to an overall increase in metabolic activity and therefore improved treatment rates. The correlation between current generation, coulombic efficiency, and treatment rates under the three operations is summarized in Table 2. The results clearly indicate that higher current generation contributed to better electron recovery and more efficient wastewater treatment. In this study, the anode potential was controlled extrinsically to increase the microbial EET flow using a potentiostat with energy input (White et al., 2009; Wagner et al., 2010; Aelterman et al., 2008), which only maintained the anodic half reaction and so the system was not subject to proton diffusion or cathodic polarization limitations. Using this SP approach, we were able to reveal the maximum system performance for these reactors that would result if the anodic electrogenic community is able to achieve a maximum EET rate during the consumption of organic matter. Our results indicate that the established EET-active community can reproducibly treat primary clarifier effluents in w3 days, while producing a current density of w1000 mA/m2, achieving a COD removal rate of 8.4 g-COD m2 d1 (per-anode area), and w60% electron recovery. These findings are important for informing future system designs so that anodeecathode ratios, reactor sizes, and hydraulic retention times can be optimized for maximizing anodic biocatalytic (EET) activity for rapid wastewater treatment and energy recovery in practical MFC systems. From the practical application point of view, the perreactor COD removal rate in the SP reactor was w0.20 gCOD L1 d1, which is approximately ten-times slower than conventional activated sludge wastewater treatment processes (Kushwaha et al., 2011; Grau et al., 1975); however, our results suggest that either the addition of more anode electrodes to the same volume of MFC reactor, or featuring optimized microbial communities to increasing the microbial

Table 2 e Correlation between current generation and organic matter degradation.

MFCb SPc OCd a

Current (mA)a

Coulombic efficiency (%)

Treatment time (day)

0.32  0.04 3.12  0.19 0.00  0.00

23  4 58  4 00

10.0  3.0 4.8  1.5 18.3  4.4

Averaged maximum current production. Means  SD during day 60e143 in Fig. 1A and day 225e278 in Figure S1 under MFC operation. c Means  SD during day 144e158 and day 176e185 in Fig. 1B under set potential operation. d Means  SD during day 7e117 in Fig. 1C under open circuit operation. b

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activity by 10-times will significantly improve treatment rates per reactor volume, similar to what is observed for conventional wastewater treatment processes. As described above, the SP operation needs energy input for controlling the anode potential and accelerating the EET flow and treatment times in the reactor. Using the treatment rates and effluent volumes described above, and power requirements for potentiostatic conditions from potentiostat specifications, we approximated the energy input required for the SP reactor to be 0.02 kWh L1 d1. Using the average reported energy demand for wastewater treatment plants (Water Environment Federation, 2009) and the treatment rates and volumes reported for NCWRP (Meyer, 2009), we estimated an energy consumption rate of 0.10 kWh L1 d1. These calculations suggest that the energy requirements for SP conditions may be competitive with conventional technologies. However, the energy required per mg-COD treated is higher for SP conditions (2.21  103 kWh mg-COD1) than activated sludge (1.05  103 kWh mg-COD1), because treatment rates in the SP reactors still require optimization, as suggested above.

3.3. Electrochemical features of three different operations To analyze the limiting factors associated with energy recovery and wastewater treatment rates in the reactors, we measured anode and cathode polarization curves by linear sweep voltammetry for all three reactors (Fig. 3). The polarization curves for the MFC reactor clearly showed that the cathode polarization strongly affects the whole MFC system, similar to previously reported results (Ishii et al., 2012b; Fan et al., 2008); however, the anode polarization curve showed that anodic limiting (maximum) current was 10-times higher than the observed operational current. This result indicates that the potential microbial current-generating properties are considerably greater than that found for the MFC operating condition. The potentiostatic SP operation maintained a constant electropositive anode potential with higher current generation, which enabled the maximum biocatalytic activity of the anodic electrogenic community independent of system limitations over the long-term operation (Ishii et al., 2008; Tsujimura et al., 2001). The SP reactor showed a slightly different anode polarization curve from the MFC reactor. The open circuit anode potential in the SP reactor (200 mV vs SHE) was more electropositive than the MFC condition (265 mV vs SHE), and the SP maximum current was only slightly increased relative to the MFC reactor (Fig. 3). The observed maximum current of approximately 4.5 mA may be a result of diffusion limitations due to low substrate concentration at the biofilm surface (the COD was 260 mg/L), low ionic conductivity in the anolyte (1.7 mS/cm), and/or similar biocatalytic EET capabilities between the MFC and SP microbial communities. On the other hand, the anode polarization curve for the OC reactor showed significantly lower polarization activity. This result suggests that the microbial electrogenic activity of the biofilm was low in the OC reactor. Improving the reactor design may lead to achieving the anodic maximum current without the energy input needed for

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-300

Electrode potential (mV) vs SHE

MFC -200 -100 Cathode (Wastewater)

0

SP

+100 Cathode (Medium)

+200 +300

OC

0

1

2

3

4

5

Current (mA) Fig. 3 e Anode polarization curves as determined by linear sweep voltammetry. The anode polarization curves of electricity-generating community enriched under three different operations; MFC operation at day 137 (MFC, black line), set potential operation at day 137 (SP, red line), and open circuit operation at day 70 (OC, blue line). The cathode polarization curve was determined at day 137 for MFC reactor (Cathode e Wastewater, green dashed line). The cathode polarization curve with a basal salt medium is also depicted (Cathode e Medium, light green line), which is referenced from a previous publications using the same reactors (Ishii et al., 2008). Arrows and solid circles indicate operational currents and the associated anodic potentials. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

SP operation. For example, the low ionic conductivity of wastewater (1.7 mS/cm) greatly increases the ohmic loss in the system. Higher ionic conductivity medium (6.8 mS/cm) in exactly the same reactor system demonstrated much better performance as measured by the cathode polarization curve (Fig. 3). This implies that system improvements including minimizing electrode spacing (Liu et al., 2005; Fan et al., 2007; Shimoyama et al., 2008), modifying the cathodic catalyst (Zhang et al., 2011), and increasing ionic strength by salt addition will enable greater current generation with faster wastewater treatment without any energy input.

3.4.

Microbial community composition

Microbial community composition was assessed periodically for the three different operational conditions. We constructed 16S rRNA gene clone libraries of anode biofilm samples (described in Fig. 1 and Supplementary Fig. S1) and suspended cells in the raw primary clarifier effluents (Ishii et al., 2012b).

The phylotypes in the communities were classified as operational taxonomic units (OTUs, grouped using >99% cut-off value), and the alpha diversity indexes, such as Shannon’s diversity index, Simpson diversity index, and Chao-1 richness, are summarized in Supplementary Table S1. The diversity indexes indicate that all anode biofilms in the three reactors revealed highly diverse microbial communities during the long-term operations, although only SP5 community was slightly less diverse than other communities. This trend to maintain highly diverse microbial communities is often observed in wastewater treatment reactors, where different microbes and chemicals are repeatedly introduced and chemicals are treated by the broader biocatalytic activities of the diverse microbial communities (Ishii et al., 2012b; Daims et al., 2006; Wagner et al., 2002; Sanapareddy et al., 2009; Godon et al., 1997; Nelson et al., 2011). The phylum- or class-level community dynamics clearly indicates that specific microbial communities were established and maintained under the three different conditions (MFC, SP, and OC) (Fig. 4A). As reported previously (Ishii et al., 2012b), class Deltaproteobacteria and phylum Bacteroidetes were relatively abundant at the early stages of electrogenic anode biofilm formation in the MFC reactor (MFC1-MFC4); although, classes Betaproteobacteria, Gammaproteobacteria, and Epsilonproteobacteria were relatively abundant in the primary clarifier effluents which were exposed to the anodic community intermittently (PC1, PC2, and PC4). The community composition within the electrogenic biofilm under the MFC condition was relatively stable throughout the subsequent 400-day MFC operation in this study (MFC5 and MFC6), and occupied by Deltaproteobacteria phylotypes at a 50% relative frequency. Compared to the community dynamics in the MFC reactor, the higher current-generating SP reactor showed a different trend. The longer term SP operation clearly revealed the increase of Deltaproteobacteria relative frequency that achieved almost 80% in the SP5 community, suggesting that the Deltaproteobacteria strains were able to adapt to the electropositive anode potential, and may have contributed to the higher current generation in the SP reactor. On the other hand, the OC reactor maintained diverse phylum/class-level phylogeny in the biofilm during the 400-day operation with zero current production. The OC communities showed approximately 20% relative frequency of Deltaproteobacteria phylotypes during the enrichment, which indicates that some of the Deltaproteobacteria strains may not have been related to electricitygenerating EET reaction in the reactors.

3.5.

Deltaproteobacterial population dynamics

In order to identify electrogenic Deltaproteobacteria strains in the MFC and SP reactors, we divided Deltaproteobacteria populations into family-level taxa (Fig. 4B). The higher resolution community analysis revealed that family Desulfobacteraceae phylotypes, which have been mainly reported as dissimilatory sulfate reducing bacteria (DSRB) (Klein et al., 2001), were observed within all three operational conditions in similar frequencies, suggesting that this taxon was not specifically associated with current generation within the microbial communities.

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A

Phylum [Class] abundance (%) 0

20

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20

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PC1 PC2 PC4 MFC1 MFC2 MFC3 MFC4 MFC5 MFC6 SP1 SP2 SP3 SP4 SP5 OC1 OC2 OC3

B

[Alphaproteobacteria] [Betaproteobacteria] [Deltaproteobacteria] [Gammaproteobacteria] [Epsilonproteobacteria] [Clostridia] other Firmicutes Bacteroidetes Acidobacteria Fusobacteria Synergistetes Tenericutes Spirochaetes Chloroflexi Verrucomicrobia Planctomycetes BRC1 OP10 WS3 Caldisericia unclassified

Family abundance within Deltaproteobacteria (%) 40

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PC1 PC2 PC4 MFC1 MFC2 MFC3 MFC4 MFC5 MFC6 SP1 SP2 SP3 SP4 SP5 OC1 OC2 OC3

Desulfobacteraceae Desulfobulbaceae Desulfuromonadaceae Geobacteraceae Desulfomicrobiaceae Syntrophobacteraceae other Deltaproteobacteria none Deltaproteobacteria

Fig. 4 e Taxonomic distribution of 16S rRNA community profile under three operational conditions. The taxonomic profiles were analyzed for the original inoculum and carbon sources of primary clarifier effluents (PC1, PC2, PC4) and the three operational conditions as MFC operation (MFC1 e MFC6), set potential operation (SP1 e SP5), and open circuit operation (OC1 e OC3). A, phylum-level taxonomies where phylum Proteobacteria and Firmicutes are divided into class level taxonomies. B, family-level taxonomies within class Deltaproteobacteria.

Families Desulfuromonadaceae and Geobacteraceae, which are both reported as dissimilatory iron reducing bacteria (DIRB) (Lovley et al., 2004) and/or electrogens in MFC reactors (Logan, 2009; Kiely et al., 2011), were observed only in the currentgenerating MFC and SP reactors. Interestingly, their preferred operation was different between MFC and SP conditions. The Geobacteraceae phylotypes were relatively abundant at the early stage of MFC operation, but their portion decreased with time; however, their frequencies significantly increased under SP conditions during the 400-day operation. Meanwhile, the Desulfuromonadaceae phylotypes were more relatively abundant at later stages of MFC operation (MFC5 and MFC6) and not observed in the later stages of the SP communities (SP4 and SP5). This result suggests that those Geobacteraceae and Desulfuromonadaceae phylotypes were

playing an important role during electricity generation within the MFC and SP communities, but family Geobacteraceae might be more active and efficient (contributing higher EET rates) given a more electropositive electrode surface. In addition to family Geobacteraceae, family Desulfobulbaceae phylotypes also showed increasing trends within the electrogenic communities in both MFC and SP reactors. The relative frequency within the SP community was much higher than that within the MFC community (Fig. 4B), which suggests that the Desulfobulbaceae phylotypes also played an important role for the electricity generation within the communities. Although dissimilatory sulfate reduction is a well-known metabolic function within family Desulfobulbaceae (Klein et al., 2001), solid metal and electrode reduction has also been reported for Desulfobulbus propionicus (Holmes et al.,

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A 30

clarifier effluent revealed that the most significant gene expression changes occurring as a result of changing EET conditions (SP and OC) were associated with family Desulfobulbaceae and order Desulfuromonadales including families Geobacteraceae and Desulfuromonadaceae (Ishii et al., 2013). All these findings support that Desulfobulbaceae phylotypes in the anode biofilms were directly involved with EET-related activity. The phylotype-level dynamics and comparison of the three operational conditions showed the shift of abundant Desulfobulbaceae and Geobacteraceae phylotypes in the SP reactor (Supplementary Fig. S2 and Table S2). The changes of the potential EET-active microbes appear to occur by the strong selective force of the electropositive anode surface potential (þ100 mV vs SHE) in the SP reactor. The phylogenetic positions of the phylotypes were assessed using phylogenetic trees (Supplementary Fig. S3 and S4). The detailed discussion for the electricity generation related microbes can be found in Supplementary Discussion.

SP5

20

SP4

Dim 2

10 SP1

SP3

SP2

0

MFC3 MFC5

MFC4

PC4

MFC2

PC2

MFC1

MFC6 PC1

-10

OC2

OC1

-20

OC3

−20

B

0

20 Dim 1

40

60

1.5

Chloroflexi 1

OC

3.6.

Microbial community dynamics

F2 (24.39 %)

Unclassified β-Proteobacteria 0.5

Bacteroidetes

Clostridia

PC

0

Desulfobacterace

MFC SP

ε-Proteobacteria γ-Proteobacteria -0.5

Current

Desulfuromonadaceae

CE

Desulfobulbaceae Geobacteraceae

-1 -2

-1.5

-1

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F1 (69.22 %)

Fig. 5 e Multidimensional scale (MDS) plot and canonical correspondence analysis (CCA) diagram comparing bacterial community dynamics under three operational conditions. A, MDS plot comparing bacterial communities based on family level taxonomy of the primary clarifier effluents (PC1, PC2, PC4, open square) and three operational conditions during the enrichment processes; MFC operations (MFC1 e MFC6, solid circle), set potential operations (SP1 e SP5, open circle), and open circuit operations (OC1 e OC3, solid triangle). B, CCA diagram showing the relationships between two reactor variables (operational current and coulombic efficiency, red arrows), four microbial samples (from the three operational conditions and the primary clarifier effluent, open squares) and each phylum, class or family-level (Deltaproteobacteria only) taxonomy (filled circles). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2004). Recently, filamentous Desulfobulbaceae strains have been identified as an important group for EET-related activity in marine sediments, which couples spatially separate biogeochemical processes via EET reactions (Pfeffer et al., 2012). In addition, our previous metatranscriptomic study of the electrogenic microbial community fed with primary

A multidimensional scale (MDS) plot based on family level taxonomy showed that the MFC, SP and OC communities were substantially different from the original inoculum sources of the primary clarifier effluents (PC) (Fig. 5A). The electrogenic communities (MFC and SP) were taxonomically different from the non-electrogenic community (OC). The early-stage MFC and SP communities were relatively similar; however, longterm SP operation changed the family-level community composition to be very distinct from the MFC communities. This indicates that the electropositive electrode surface impacted the community composition such that the taxonomic composition was associated with the function of generating higher electric current and rapidly degrading organic matter. Sørensen’s similarity coefficients based on OTUs is shown in Supplementary Table S1. These results also indicate the continuity of the electrode-associated community development process with time, as well as the relatively higher similarity between EET-active MFC and SP reactors. Canonical correspondence analysis (CCA) diagram was used to describe correlations between community composition and key reactor performance metrics including current generation and coulombic efficiency (Fig. 5B). Results of this CCA diagram indicate that Geobacteraceae, Desulfuromonadaceae, and Desulfobulbaceae were most associated with current generation and coulombic efficiency. This correlation indicates that long-term higher current-generating SP operation leads to similar results as those reported for stimulusinduced gene expression responses of those microbial families exposed to short-term SP conditions (Ishii et al., 2013). Several taxa were strongly associated with the primary clarifier effluent (PC) or with OC operation; however, those taxa did not associate to the MFC or SP conditions, which indicate that they did not directly contributed to current generation in the MFC/SP reactors. Given that the MFC and SP operations imposed a constant selective pressure relative to microbial EET processes, it can be inferred that the taxonomic differences between the enriched microbial communities were mainly due to the electrode

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potentials and consequent differences in the EET rates. This indicates that the comparison of microbial populations as a function of different reactor conditions enables an understanding about the specific taxonomic groups that are associated with electricity generation in wastewater-fed MFC reactors.

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Science Cluster (Award no. 0918983) and the Roddenberry Foundation.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2013.07.048.

4.

Conclusion

Maintaining electropositive anode electrode potentials increases overall MFC wastewater treatment rates and energy recovery as shown by the SP reactor results. These improved performance metrics are a result of adapting the microbial community taxonomic composition through electrogenic selective pressures and enabling higher biocatalytic activity through potentiostatic controls. The families Geobacteraceae, Desulfuromonadaceae, and Desulfobulbaceae were found to be highly abundant in the best performing (SP) reactor and are postulated to be the most important EET-active microbial groups in wastewater-fed MFC systems. These results indicate that MFC systems with appropriately scaled electrodes may be able to demonstrate treatment rates that are competitive with conventional wastewater treatment technologies (e.g. activated sludge) by maintaining a high rate of EET activity within the biofilm. Additionally, our results suggest that the biological reactions in MFC systems can be easily maintained since the selective pressure of an applied potential (or constant current draw) will ensure reproducible microbial functionality for at least one year of operation. Our findings also suggest that optimized/scaled MFC systems may prove to be cost-competitive with activated sludge wastewater treatment due to the lower required energy input (no aeration requirement), lower production of secondary sludge (Logan et al., 2006), and the opportunity for efficient energy recovery through direct electricity production. Importantly, we have demonstrated that selecting and maintaining an optimized microbial taxonomic composition and species function will lead to accelerated treatment rates and efficient energy recovery. Further improvements to MFC architectures will lower the overall capital cost of these systems (Rozendal et al., 2008), and by coupling optimized system architecture with optimized microbial communities, MFCs may prove to be an ideal technology for wastewater treatment.

Acknowledgment We thank Nancy Coglan (North City Water Reclamation Plant) and her laboratory members for providing samples of the primary clarifier effluent. We thank Kelvin Li for technical assistance with executing the JCVI 16S/18S rRNA Pipeline, Tony Phan and Eric Son for technical assistance with clone library analysis. This work was supported by National Science Foundation- Biotechnology, Biochemical, and Biomass Engineering (NSF-BBBE) (Award no. 0933145), NSF Ecosystem

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