Innovative self-powered submersible microbial electrolysis cell (SMEC) for biohydrogen production from anaerobic reactors

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 7 2 7 e2 7 3 6

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Innovative self-powered submersible microbial electrolysis cell (SMEC) for biohydrogen production from anaerobic reactors Yifeng Zhang, Irini Angelidaki* Department of Environmental Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark

article info

abstract

Article history:

A self-powered submersible microbial electrolysis cell (SMEC), in which a specially

Received 30 November 2011

designed anode chamber and external electricity supply were not needed, was developed

Received in revised form

for in situ biohydrogen production from anaerobic reactors. In batch experiments, the

15 February 2012

hydrogen production rate reached 17.8 mL/L/d at the initial acetate concentration of

Accepted 16 February 2012

410 mg/L (5 mM), while the cathodic hydrogen recovery (RH2 ) and overall systemic

Available online 24 February 2012

coulombic efficiency (CEos) were 93% and 28%, respectively, and the systemic hydrogen

Keywords:

with acetate and buffer concentration. The highest hydrogen production rate of 32.2 mL/L/

Microbial electrolysis cell

d and YH2 of 1.43 mol-H2/mol-acetate were achieved at 1640 mg/L (20 mM) acetate and

Microbial fuel cell

100 mM phosphate buffer. Further evaluation of the reactor under single electricity-

In situ hydrogen production

generating or hydrogen-producing mode indicated that further improvement of voltage

Self-powered

output and reduction of electron losses were essential for efficient hydrogen generation. In

Submersible

addition, alternate exchanging the electricity-assisting and hydrogen-producing function

Anaerobic reactors

between the two cell units of the SMEC was found to be an effective approach to inhibit

yield (YH2 ) peaked at 1.27 mol-H2/mol-acetate. The hydrogen production increased along

methanogens. Furthermore, 16S rRNA genes analysis showed that this special operation strategy resulted same microbial community structures in the anodic biofilms of the two cell units. The simple, compact and in situ applicable SMEC offers new opportunities for reactor design for a microbial electricity-assisted biohydrogen production system. ª 2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Hydrogen has a future potential as environmentally acceptable energy carrier, but most hydrogen produced in the world today is from fossil fuels, resulting in the uncontrolled release of greenhouse gases contributes to climate change (Benemann, 1996). Therefore, alternative technologies that produce hydrogen from waste with less environmental impacts are required.

Microbial electrolysis cell (MEC) has been demonstrated to be a potential technology for high-efficiency biological hydrogen production from wastes (Cheng and Logan, 2007; Liu et al., 2005a; Logan et al., 2008; Lee et al., 2009; Rozendal et al., 2007; Sun et al., 2008). The MEC is developed on the base of microbial fuel cell (MFC), which utilizes microorganisms as catalysts to mediate direct conversion of chemical energy stored in organic matter or bulk biomass into electricity (Logan, 2009; Rabaey et al., 2005a; Zhang et al., 2011a; Zhang

* Corresponding author. Tel.: þ45 45251429; fax: þ45 45932850. E-mail address: [email protected] (I. Angelidaki). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2012.02.038

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and Angelidaki, 2011). An MEC can be easily switched from an MFC by excluding oxidants (e.g., O2) from the cathode and providing a certain amount of electricity. MECs have two main advantages over other biological hydrogen production processes. First, various organic substrates can be used as fuel, including cellulose, glucose, glycerol, acetic acid, sewage sludge and varied wastewaters (Cheng and Logan, 2007; Logan et al., 2008; Sakai and Yagishita, 2007; Zhang et al., 2009, 2011b,c). Second, non-fermentable substrates (e.g., acetate, butyrate), which are byproducts of dark fermentation due to thermodynamical limitations, can be completely oxidized, resulting in high H2 yields (Liu et al., 2005a; Logan et al., 2008). Although MECs technology has been described for some time now (Cheng and Logan, 2007), this technology has not moved from bench scale operation. Several limitations such as high internal resistance, low H2 recovery efficiency, large electrode spacing and difficulty of scaling up need to be solved before field application (Cheng and Logan 2007; Lee et al., 2009; Logan et al., 2008). It has been recognized that improvement of the reactor design could significantly contribute to overcome some of the present limitations. Therefore, several different architectures of MECs, including typical H-type, cube-shaped, membrane-less single chamber and up-flow single chamber reactors, have been developed for hydrogen generation (Cheng and Logan, 2007; Hu et al., 2008; Lee et al., 2009; Liu et al., 2005a). Despite the fact that researchers have intensively looked for new configurations, current MECs are not suitable for in situ utilization of organic matter for hydrogen production. Therefore, a novel reactor design that can be applied directly to existing anaerobic reactors or natural anaerobic environments is required for simplifying the construction, operation and maintenance of MECs. Reducing electricity supply is another key issue for the successful application of MECs. Although the voltage supply (0.2e0.8 V) is much lower in MECs compared to water electrolysis process (1.8e2.0 V), the energy consumption is still high, especially in long-term operation (Logan et al., 2008). It has been recently proposed that MFC could be a renewable power source for MEC. Sun et al. (2008) demonstrated an MECeMFC-coupled system, composed by a single-chamber MFC and a two-chamber MEC, which was successfully used for hydrogen production without other power supply. Although the integration of MFC and MEC shows promising perspectives, more efforts are needed to make it more efficient and cost-effective. One of the main concerns is system simplification. The idea behind is why using two separate reactors, if one could merge electricity generation and hydrogen production into a single, compact device to remove some construction-costly parts such as the anode chamber of MFCs and MECs. However, there is still no related report on improvement of the architecture of MECeMFC-coupled system. In this study, an innovative self-powered submersible MEC (SMEC) was developed for in situ hydrogen production. In such a novel design, dedicated anode chambers which are indispensable in the conventional MFCs and MECs, were not required, which makes the SMEC, applicable to existing or natural anaerobic environments (e.g., anaerobic reactors) without additional constructions. Furthermore, the SMEC was composed by only two jointed cathode chambers, one for

electricity generation, while the other for hydrogen production, thereby omitting the demand of external power supply. The SMEC performance in terms of hydrogen production, electricity supply, organic matter removal, inhibition of methanogenesis and evolution of microbial community was investigated. This new system may offer a promising avenue for practical hydrogen recovery and organic waste treatment.

2.

Material and methods

2.1.

SMEC reactor and operation

The SMEC, which was made of nonconductive polycarbonate plates, was a rectangular reactor composed of two cathode chambers (inside dimensions 3 cm  3 cm  1 cm, for each) (Fig. 1), The two cathode chambers were separated with a polycarbonate plate. The whole reactor was assembled with twelve stainless screws and sealed with rubber gaskets to prevent leakage. The sandwich structured membrane electrode assembly (MEA) was placed at the end of each cathode chamber (Fig. 1). In this special design, SMEC can function as two cells, one for hydrogen production while the other one for power supply (Cell 1 and Cell 2, as shown in Fig. 1). The cathode chamber of the electricity-assisting cell was connected to the open air by plastic tubes; no special aeration was employed. The cathode chamber of the hydrogen-producing cell was connected to a gas bag (capacity of 250 mL) by plastic tubes for hydrogen collection. The anode electrode was non-wet-proofed carbon paper (3  3 cm, Toray carbon paper, E-TEK division, USA). The cathode electrode was made of carbon paper coated with Pt (3  3 cm, 0.5 mg/cm2 with 20% Pt, E-TEK division, USA). The anode and cathode electrode were separated with a proton exchange membrane (PEM) (Nafion 117, DuPont Co., USA), and hot-pressed together as an MEA (Min and Logan, 2004). Electrical connections and pretreatment of electrodes were done as previously described (Min and Angelidaki, 2008). The SMEC was submersed in an anaerobic glass reactor (total volume of 1000 mL), which was covered by a rubber stopper having several openings for liquid and gas samples. 500 mL of wastewater (Primary clarifier, Lyngby Wastewater Treatment Plant, Denmark) was firstly amended with 410 mg/ L of acetate and then filled into the reactor as the inoculum and fuel. Following inoculation and stable power generation, the wastewater medium was replaced with the nutrient medium containing (in 1 L): NaH2PO4$H2O, 844 mg; Na2HPO4$H2O, 550 mg; NH4Cl, 310 mg; KCl, 130 mg; CaCl2, 10 mg; MgCl2$6H2O, 20 mg; FeCl2, 5 mg; (NH4)6Mo7O24, 3 mg; MnCl2$4H2O, CuSO4$5H2O, 1 mg; 1 mg; CoCl2$6H2O, 1 mg; ZnCl2, 1 mg; AlCl3, 0.5 mg; H3BO3, 1 mg; NiCl2$6H2O, 0.1 mg. The acetate concentration in the medium was 410 mg/L (5 mM) except as indicated, where the acetate was varied to be 820 and 1640 mg/L (10 and 20 mM respectively). The initial pH of the solution was adjusted to 7.0 with 1 N NaOH or HCl. The Cell 1 and Cell 2 of the SMEC were firstly operated separately at electricity generation mode (with 1000 U resistor) for three months. After that, the SMEC was ready for the tests (each cell had open circuit voltage of 0.65 V), and then the Cell 1 and Cell 2 were connected in series with a 10 U resistor. During

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assisting cell is then converted to hydrogen-producing cell, and vice versa), unless otherwise specified. All batch tests were carried out in duplicate at room temperature (20  5  C).

2.2.

Electrochemical analysis and calculations

The voltage (V) was monitored with 30 min intervals using a multimeter (Model 2700, Keithley Instruments, Inc., Cleveland, OH, USA) linked to a differential multiplexer (Model 7701, Keithley Instruments, Inc.). Acetate was measured by gas chromatography with FID detection (Agilent, 6890) as previously described (Zhang et al., 2009). H2 was analyzed by a GC-TCD fitted with a 4.5 m  3 mm s-m stainless column packed with Molsieve SA (10/80). CH4 was analyzed with a GCTCD fitted with column of 1.1 m  3/16 “Molsieve 137 and 0.7 m 1/4” chromosorb 108. pH was measured with a PHM 210 pH meter (Radiometer). System performance of the SMEC was evaluated in terms of volumetric hydrogen production rate based on the total liquid volume of the anaerobic reactor, the cathodic hydrogen recovery (RH2 ), the overall systemic Coulombic efficiency (CEos), and the hydrogen yield of SMEC (YH2 ). The current intensity (I ) was calculated according to the Ohm’s law, I ¼ V/ R, where V is the voltage and R is the resistance. Current density (J ) was calculated as J ¼ I/A, where A is the projected surface area of the anode electrode (0.0009 m2). RH2 is calculated as RH2 ¼ nH2 =nTh , where nH2 is the moles of hydrogen harvested, nH2 ¼ VH2 =RT; and nTh is the theoretical number of hydrogen moles that could be produced from the measured total current in the circuit, nTh ¼ Q/2F. Q is the total Coulombs calculated by integrating the current over time, VH2 is the measured hydrogen volume, R is the gas constant, T is the absolute temperature, and F is Faraday’s constant. CEos and YH2 are respectively calculated as CEos ¼ nTh/(4ns) and YH2 ¼ nH2 =ns , where ns is the moles of acetate consumed in the anaerobic reactor. The CEos is calculated based on the assumption that 8 mol of electrons are produced from1 mol of acetate (Sun et al., 2008). For single MEC and MFC studies, CEs are calculated as previous described (Cheng and Logan, 2007; Zhang et al., 2009).

2.3.

Fig. 1 e Schematic diagram (A) and an image (B) of the SMEC. Numbered items: 1. Plastic tube; 2. Cover plate (polycarbonate); 3. MEA; 4. Cathode chamber (Cell 1); 5. Screw; 6. Cover plate; 7. Cathode chamber (Cell 2); 8. Polycarbonate plate; 9. Rubber gasket; 10. The other piece of MEA, but cannot be seen from current view.

operation, the cathode chamber of the cell for power supply was open to the air, while the other one for hydrogen generation was first purged with N2 and connected to an aluminum foil gas collecting bag (capacity of 250 mL). After each batch, the function of Cell 1 and Cell 2 was exchanged (the electricity-

Microbial community analysis

To explore the microbial communities established in such newly designed reactor and how it responded to the operation strategy of alternate function exchange, the biofilm attached on the anodes was sampled at the end of experiments by scraping the electrode surface with a sterilized scalpel. Total DNA extraction, PCR-DGGE and 16S rDNA analysis were done as previously described (Zhang et al., 2009). Dominant bands were sequenced (Macrogen, Netherlands). Sequences were subjected to Basic Local Alignment Search Tool (BLSAT) and Ribosomal Database Project analysis. Nucleotide sequences have been deposited in the GenBank database and are available under accession numbers JF272703 to JF272711. The fluorescent in situ hybridization (FISH) analysis was performed as previously described (Amann et al., 1995). Two domain specific oligonucleotide probes, including EUB338 (targeting Bacteria) and ARC915 (targeting Archaea), were used. Prior to hybridization, biofilm samples were dispersed by mild

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60

initial 24 h, and then this yield slightly declined, while a final yield of 1.05 mol-H2/mol-acetate was achieved. This is a 31% of the maximum theoretical yield (3.04 mol-H2/mol-acetate), which is calculated based on the complete conversion of the energy stored in acetate (upper heating value of 870.28 kJ/mol) to hydrogen (285.83 kJ/mol) (Cheng and Logan, 2007; Sun et al., 2008). The RH2 was 87 e 98%, indicating the high efficiency of the cathodic hydrogen recovery from current. The CEos was relatively low and ranging from 24 to 36% during 168 h batch operation, which indicated that a large part of electrons have been consumed by other mechanisms (e.g., biomass generation, anaerobic degradation, aerobic respiration) than hydrogen generation (Rozendal et al., 2007). The contribution of methanogenesis to the low CEos could be excluded as no CH4 was detected both in the headspace of anaerobic reactor and the cathodes of the SMEC, which could be due to the alternate function exchange between cells (discussed later). In a previous MECeMFC-coupled system (Sun et al., 2008), the hydrogen production rate was 2.2 mL/L/d, which was much lower than the value (17.8 mL/L/d) observed in this study at the same phosphate buffer concentration (10 mM). Additionally, the CEos, RH2 , YH2 here were comparable to that obtained in their system (30%, 94%, 1.1 mol-H2/mol-acetate, respectively), even though more favorable condition (e.g., temperature of 30  C) was adopted in their study. However, it should be noticed that the hydrogen production rate was still low compared with conventional MECs (Logan et al., 2008). The relatively lower voltage supplied by the SMEC itself was probably the main reason. As shown in Fig. 3, the input voltage of hydrogen-producing cell (average of 0.327 V) was close to the output voltage of electricity-assisting cell (average of 0.336 V). The average circuit current was 0.9 mA (1000 mA/m2), which could be an explanation for the relatively higher hydrogen production rate here than that in previous MECeMFC system where only 0.25 mA (156 mA/m2) was observed (Sun et al. 2008). The above results show the feasibility of the SMEC for in situ and self-powered hydrogen production from anaerobic reactors.

2

3.2. Comparison with conventional MEC and MFC operation

ultrasonic pretreatment (0.5 min pulsed) for 2 min and suspended in PBS buffer. Ethanol was also added to reach a final concentration of 50%. DAPI was used for total microbial cell quantification. All hybridizations were performed as simultaneous dual color hybridizations. Images were acquired with an epifluorescence microscope (Nikon Corporation, Japan) equipped with a 100 W mercury lamp, a 100/1.25 an oil objective and appropriate filter for FITC (Croma Technology Corp., USA) and Cy3 (Nikon Corporation, Japan). For each sample, 25 microscopic fields were analyzed.

3.

Results and discussion

3.1.

System performance

CE (%)

A

100

100

75

90 CE

50

80

RH2

25

70

0 0

B

RH2 (%)

Hydrogen was continuously produced in one of the cathode chamber that was used for microbial electrolysis in the SMEC. An example of one cycle of hydrogen production and system performance in terms of CEos, RH2 and YH2 is shown in Fig. 2. The hydrogen volume increased gradually with reaction time and reached to 62.3 mL at the end of operation (168 h), resulting in an average hydrogen production rate of 17.8 mL/L/ d. The hydrogen yield was 1.27 mol-H2/mol-acetate in the

50

100 Time (hour)

150

200

80

1.5

40

1

20

0.5

0

0

50

100 Time (hour)

150

200

-1

YH2

60

YH2 (mol-H 2 mol-acetate )

Hydrogen volume (ml)

Hydrogen volume

0

Fig. 2 e Performance of the SMEC at 10 mM of phosphate buffer: (A) Coulombic efficiency (CE ) and cathodic hydrogen recovery (RH2 ); (B) hydrogen volume and hydrogen yield (YH2 ). Error bars are based on the duplicate batch experiments.

SMEC system is actually based on the cooperation between the hydrogen-producing and electricity-assisting cells. Better understanding the performance of each part will help to optimize the whole system. Thus the performance of SMEC reactor operated at typical MEC and MFC mode was investigated, respectively. In MFC mode, the hydrogen-producing cell (e.g., Cell 1) was disconnected (open circuit), while the electricity-assisting cell (e.g., Cell 2) was connected with a resistor of 373 U instead, which was equal to the external resistance calculated based on the data shown in Fig. 3. As shown in Fig. 4A, an average voltage of 0.39 V was generated and stable for 268 h. Correspondingly, the CE was ranging from 39 to 65%, and reached to 53% for the whole process. It is clear that the output voltage and CE increased after replacing the hydrogen-producing cell with an equivalent resistor. The results, on the one hand, suggest that the mechanism for powering a hydrogen-producing cell might be more complex than that of powering a resistor. The electric potential and

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 7 2 7 e2 7 3 6

Fig. 3 e Circuit current, output voltage of electricityassisting cell, and input voltage of hydrogen-producing cell during operation.

0.5

100

75

0.3 50 0.2 25

Voltage, MFC mode with 373

0.1

CE, MFC mode

0 0

B

50

100

150 200 Time (hour)

250

0 300

2

200

1.5 Current (mA)

CE (%)

Voltage (V)

0.4

150 Current, MEC mode with 0.65 V Hydrogen volume, MEC mode CE, MEC mode

1

100

0.5

50

0

CE (%) / Hydrogen (mL) ..

A

0 0

50

100 Time (hour)

150

200

Fig. 4 e The performance of SMEC at different operation modes. (A): Voltage output and circuit current generation with time in MFC mode, where external resistance of 373 U was used; (B): Hydrogen production and system CE with external power supply (0.65 V) in MEC mode.

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internal resistance of the hydrogen-producing cell may change with time (e.g., due to substrate consumption) which in return might affect the performance of the electricityassisting cell. On the other hand, the electricity-assisting cell could be further optimized. There was still a large gap between the observed output voltage and the maximum voltage (approx. 0.8 V) reported so far (Freguia et al., 2008; Liu et al., 2010a; Rabaey et al., 2005b). In the light of recent developments in MFCs, the output voltage of the electricityassisting cell has great potential to be improved as high as the maximum value reported so far, which may thereby boost the hydrogen production. The SMEC was also tested in typical MEC mode by disconnecting the electricity-assisting cell (e.g., Cell 2 was open circuit). In order to see how the hydrogenproducing cell works if one day the electricity-assisting cell can produce much higher voltage as high as its OCV, an external voltage of 0.65 V was applied. As shown in Fig. 4B, the hydrogen volume increased gradually with reaction time and reached to 120 mL after 192 h, resulting in a final hydrogen yield of 1.97 mol-H2/mol-acetate. The results indicated that the performance of hydrogen-producing cell can be improved if adequate electricity would be generated by the electricityassisting cell. Nevertheless, it should be noticed that the hydrogen production can be further improved since hydrogen yield up to 3.95 mol-H2/mol-acetate (99% of the theoretical maximum) have been obtained previously with conventional MEC (Cheng and Logan, 2007; Logan et al., 2008). The reason for this big difference could be because the electrode material, reactor size and microbial activity in the present system were not optimized. The CE (58%) was still low even with much higher external voltage supply (0.65 V). It could be due to the growth of aerobic bacteria in the open-circuited Cell 2, as its cathode chamber was still opened to the air with tubes in order to keep the same experimental condition as for the previous test. Using of diffusion layers such as carbon/polytetrafluoroethylene on the air-side of the cathode may adjust the oxygen diffusion into the anode chamber and improve CE, which is our future research goal. The substrate degradation in the above tests was also investigated. As shown in Fig. 5, the acetate removal in selfpowered mode was much faster than that in MFC and MEC mode, reached to 95% within 168 h. In MEC mode, although higher external voltage was applied, completely acetate removal took about 24 h longer, compared with self-powered mode. Acetate removal in MFC mode was even slower, and took about 268 h. The relative faster acetate removal achieved in self-powered mode could be due to the consumption of acetate both in electricity-assisting and hydrogen-producing processes. It was also noticed that the acetate degradation rate of the SMEC was much lower than the sum of single MEC and MFC, which were partly due to the higher voltage (0.65 V) applied in the single MEC mode, and also because the higher voltage generation (0.39 V) in the single MFC mode. The results show the applicability of the SMEC for rapid and in situ waste removal with simultaneous hydrogen production.

3.3.

Inhibition of methanogenesis

Methanogenesis is commonly observed in the anode of MECs after several batches operation (Chae et al., 2010a; Clauwaert

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500

160 Mode switching SMEC mode

400

CH4

120 MFC mode

300

Volume (mL)_

Acetate (mg/L) ))

H2

MEC mode

200

80 5 mM 3rd batch 40

10 mM 4th batch

100

0

0 0

40

80

120 160 Time (hour)

200

240

280

Fig. 5 e Acetate degradation at different operation modes shown in Fig. 4. The samples were taken during the test shown in Fig. 4. In the MFC mode, the hydrogen-producing cell was replaced with an external resistor of 373 U; In the MEC mode, electricity-assisting cell was disconnected (open circuit) and external power (0.65 V) was supplied to the hydrogen-producing cell.

and Verstraete, 2009). Methanogens are quite sensitive to oxygen and complete inhibition has even been observed at dissolved oxygen level below 30 nM (Scott et al., 1983). Thus exposure to air is considered to be an efficient way of controlling their activity and improving hydrogen recovery in MECs (Chae et al., 2010a). As membrane electrode assembly was employed in the SMEC, methanogens could be inhibited in the anode of electricity-assisting cell as a result of oxygen crossover through the membrane (micro-aerobic environment where oxygen level is limited) (Chae et al., 2010b). In addition, it has been reported that aceticlastic methanogens can be inhibited on the anode of MFCs during electricity generation (Hu et al., 2008). According to the above considerations, the most possible methane source was the growth of methanogens on the hydrogen-producing cell side. Therefore, alternate changing the hydrogen-producing cell to electricityassisting cell could be an effective way to inhibit methane production. To confirm above hypothesis, the SMEC reactor was operated without function change (Cell 1 for electricity supply, Cell 2 for hydrogen production) for four batches. As shown in Fig. 6, when acetate concentration of 5 mM was supplied, a little amount of methane (8.4 mL) was detected in the headspace of the anaerobic glass reactor from the 3rd batch, resulting in a decrease of hydrogen production. The methane volume increased to 15 mL after increasing the acetate concentration to 10 mM at the 4th batch. The detected methane could be due to the presence of methanogens on the anode of hydrogen-producing cell. From the 5th batch, the function of Cell 1 and Cell 2 was exchanged. It is clearly shown that the methane production was significantly inhibited, while the hydrogen volume was improved by 29%. The methane production was nearly eliminated after one more batch operation (function exchange again), indicating the

0

200

400

10 mM 5th batch

600 Time (hour)

800

10 mM 6th batch

1000

Fig. 6 e Effect of function exchange on hydrogen production at 10 mM of phosphate buffer. The arrows indicated the substrate replacement. 5 mM or 10 mM means the acetate concentration used in the batch test. The function of the cells was exchanged from 5th batch.

important role of alternate function exchange between the cells of the SMEC on methanogenesis inhibition. Methane was not detected in the cathode of the hydrogen-producing cell in all the tests above, which could be due to the low concentration of methane in the anode, resulting probably in only insignificant diffusion of methane through the PEM membrane. Furthermore, biofilm samples were taken from the anode of Cell 2 at the end of 4th and 6th batch shown in Fig. 6. The samples were analyzed by FISH. The FISH analysis clearly shows that the methanogenic archaea (in green) disappeared after switching the Cell 2 to the electricity-assisting cell (Fig. 7). The results suggest that alternate function exchange between the cells could be an appropriate approach for inhibiting methanogens in the SMEC system. These results have also implications for inhibiting methane production in conventional MECs. Compared with typical air exposure method, this approach has no negative impact on the system. The inhibition of methanogenesis was accomplished with hydrogen production without breaking off the process and opening the reactor. Most importantly, micro-aerobic conditions close to the anode rather than complete exposure to the air was good for suppressing the methanogens specifically without adversely affecting the exoelectrogens, as confirmed by the elevated H2 production. Due to the complexity of this observation, more research such as the long-term operation effect, exchange frequency, and the effect on different methanogens (e.g., hydrogenotrophic) are needed to unravel the precise mechanism.

3.4. System performance at different acetate and buffer concentrations Table 1 shows the system performance at different initial substrate and buffer concentrations. The hydrogen volume

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Fig. 7 e Fluorescent image of anodic biofilm sampled from Cell 2 of SMEC before (AeC) and after (DeF) function exchange. (A) and (D) are image of samples stained with DAPI for total microorganism (blue); (B) and (E) are image of samples hybridized with ARC915 for detecting Archaea (green); (C) and (F) are image of samples hybridized with EUB338 for detecting Bacteria (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

and production rate increased with initial acetate concentration. With an initial acetate concentration of 20 mM and 10 mM phosphate buffer, the mean hydrogen production rate and hydrogen volume reached to 24.1 mL/L/d and 301.2 mL, respectively. Meanwhile, the input/output voltage and current also increased with acetate concentration, which could explain the increase of hydrogen production. However, the maximum hydrogen yield of 1.22 mol-H2/mol-acetate and CE of 33% were obtained with initial acetate concentration of 10 mM, while they ceased to increase when higher acetate concentration was supplied. It could be due to the increase of electron loss (CE decrease) with high substrate concentration, which was well documented in MFC studies (Min et al., 2005; Zhang et al., 2009). When the phosphate buffer concentration was increased to 100 mM and the initial acetate concentration was kept at 20 mM, the average hydrogen production rate increased to 32.2 mL/L/d, and the hydrogen yield was 1.43 mol-H2/mol-acetate, which was nearly 20% higher than

that with 10 mM of phosphate buffer. Correspondingly, the circuit current and output voltage increased to 1.6 mA (1778 mA/m2) and 0.395 V, respectively. The improved hydrogen production at high buffer concentration could be due to the reduction of electrolyte ohmic resistance (Liu et al., 2005b). These results indicated that the SMEC can be operated with broad substrate concentrations, and the system performance can be improved with high buffer concentration.

3.5.

Microbial communities

The microbial community enriched in this innovative reactor was explored for better understanding and optimization of the system by addressing the biological limits. The DGGE profiles of the bacteria community sampled from the anodes of the SMEC (Cell 1 and Cell 2) at the end of experiments are summarized in Fig. 8. Based on migration distance, intensities and similarity between the lanes on DGGE gel, the bands

Table 1 e Effect of initial acetate and buffer concentration on the performance of SMEC. Output Input Hydrogen RH2 CE YH2 Current Current Acetate Buffer H2 (mA) voltage (V) voltage (V) concentration concentration (mL) production (%) (%) (mol-H2/mol-acetate) density rate (mA/m2) (mM) (mM) (mL H2/L/d) 5 10 20 20

10 10 10 100

62.3 147.5 301.2 354.7

17.8 22.7 24.1 32.2

93 93 94 94

28 33 32 38

1.05 1.22 1.21 1.43

1000 1222 1333 1778

0.9 1.1 1.2 1.6

0.336 0.360 0.368 0.395

0.327 0.349 0.356 0.379

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Fig. 8 e DGGE profile of anodic biofilm. L1: cell unit 1; L2: cell unit 2; W: wastewater.

patterns in Cell 1 and Cell 2 are same (similarity 95%), indicating there was no significant difference in bacterial community on the hydrogen-producing cell and electricityassisting cell. Sun et al. (2008) also observed the same bacterial community on the anodes of their MECeMFC-coupled system, although these two anodes did not share the same anodic solutions as this study. The same microbial community of these two anodic biofilm samples could be explained by

the alternating operation between MFC and MEC modes. In addition, the two anodes shared the same solution could be another important reason. Some bands in the acclimatized bacteria consortium were not present in the inoculum, suggesting that some new electrochemically active communities were enriched after operation. It was noticed that the bands 1, 2, 3 and 4 were stronger than other bands, thus their respective microorganisms might play a very important role in electricity and hydrogen production. In order to provide insight into microbial ecology and diversity, nine predominant species extracted from DGGE bands were sequenced. Based on the 16S rDNA gene library results (Table 2), the acclimatized bacteria consortium was composed of Bacteroidia (44.4% of sequences), Flavobacteria (11.1%), Betaproteobacteria (11.1%), Synergistia (11.1%), Clostridia (11.1%) and Gammaproteobacteria (11.1%). The four predominant bacteria (bands 1, 2, 3 and 4) were phylogenetically closely related to Dysgonomonas sp. YFZ1, Uncultured Clostridiales sp. A1345, Uncultured Flavobacterium sp. YFZ31 and Acinetobacter sp. PmeaMuc16, respectively. Among them, Dysgonomonas sp. YFZ1 and Uncultured Flavobacterium sp. YFZ31 have been identified respectively as the dominant bacteria for electricity production in wheat-straw and acetate powered MFCs (Zhang et al., 2009, 2011a). Clostridiales sp. and Acinetobacter sp. have also been identified as possible electrochemically active bacteria in previous MFC and MEC studies (Li et al., 2011; Mehanna et al., 2010). The observation indicates that the acclimatized microbial community was predominated by exoelectrogens. However, we should notice that there were also other bacteria (e.g., band 7 represented bacterium) present in the biofilm, for which it is unknown whether they possess exoelectrogenic activity. Some of them might be non-exoelectrogenic microbes, explaining the coulombic losses. Comparing the 16S rDNA gene libraries in relation to hydrogen production, Bacteroidetes were predominated in biofilm, indicating that the diversity of iron-reducing and potentially relevant microbes for microbial electrolysis hydrogen production might extend the commonly studied

Table 2 e DGGE 16S rDNA band identifications. Sample Wa

3 4 5 6 7 8 9 a b c d

L1 

1 2

Genbank accession no.

Closest relatives (%Sequence similarityd)



JF272703

Dysgonomonas sp. enrichment culture clone YFZ1 (97%) Uncultured Clostridiales bacterium clone A1345 (94%) Uncultured Flavobacterium sp. YFZ31 (94%) Acinetobacter sp. PmeaMuc16 (86%) Uncultured bacterium clone LL141-8H16 (91%) Uncultured Bacteroidetes bacterium 298 (82%) Uncultured bacterium; 6week9 (93%) Uncultured bacterium clone RW6944 (86%) Uncultured bacterium; MFC-B162-G07

Band

b

L2







JF272704



      

      

JF272705 JF272706 JF272707 JF272708 JF272709 JF272710 JF272711

Classc

Bacteroidia Clostridia Flavobacteria Gammaproteobacteria Bacteroidia Bacteroidia Bacteroidia Betaproteobacteria Synergistia

Inoculum. Existence under the condition. The phylotypes were assigned to phyla based on Ribosomal Database Project II (RDP II) taxonomy classifications. Percent values represent similarities between the associated DGGE band sequence and the closest match sequence from GenBank.

w a t e r r e s e a r c h 4 6 ( 2 0 1 2 ) 2 7 2 7 e2 7 3 6

Geobacter species. In general, the microbial community found in this study was different from that found in previous MEC studies, which were normally predominated by Geobacter, Shewanella, Desulfovibrio, and Anaeromyxobacter species (Chae et al., 2008; Liu et al., 2010b). This distinction was probably attributed to the variation of inoculum, and the interaction between electricity-assisting and hydrogen-producing cells.

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between cells. The simple, compact SMEC showed promising potential for in situ hydrogen production from various anaerobic environments by directly utilizing the electricity harvested from waste.

Acknowledgements 3.6.

Significance of the SMEC system

Development of the SMEC technology is addressing two key problems existed in the electricity-assisted biohydrogen production process: construction costs for upscale operation and high energy costs. In the proposed MFCeMEC system, hydrogen is produced from wastewater without extra electricity supply. Furthermore, this technology reduces demands for electricity storage and diminishes power loss. The SMEC has two following advantages over previous MFCeMEC systems: (i) more simple and compact reactor design; and (ii) unique method for inhibiting methanogens. Before field application of the SMEC, several obstacles such as low voltage output, high internal resistance, proton transfer limitation, electron losses need to be overcome. A matured SMEC should combine the both advantages of MFC and MEC. Thus the recent tremendous developments in MFC and MEC (e.g., cheap and effective electrode materials) have offered a new avenue to overcome these limitations (Freguia et al., 2008; Lee et al., 2009; Liu et al., 2010a). A well-developed SMEC could serve as a replacement or post-processing for conventional anaerobic fermentation technologies for costeffective and efficient hydrogen production.

4.

Conclusion

In this study, we propose a newly designed SMEC that successfully produced hydrogen in situ from anaerobic reactor fed with acetate. Beside no need of extra power supply, the advantage of the SMEC is that no special designed anode chamber is needed, which makes it more compact and easily applied to the existing anaerobic reactors or natural anaerobic environments without additional constructions. With 10 mM of phosphate buffer and 410 mg/L acetate, the hydrogen production rate reached 17.8 mL/L/d and the YH2 peaked at 1.27 mol-H2/mol-acetate, while the RH2 and CE were 93% and 28%, respectively. The hydrogen production increased with acetate and buffer concentration. The highest hydrogen production rate of 32.2 mL/L/d and hydrogen yield of 1.43 molH2/mol-acetate were achieved with 100 mM phosphate buffer and 20 mM acetate. Test of the SMEC in typical MFC and MEC mode indicates that improvement of the electricity generation and decrease of the electron loss are essential for efficient hydrogen generation. Furthermore, alternate function exchange between the cells in the SMEC was found to be an effective approach for methanogens inhibition. The anodic biofilm of Cell 1 and Cell 2 in the SMEC had the same microbial community composition. Further optimization of the system should focus on modification of electrodes and membrane, optimization of the process parameters and investigation of the long-term influence of alternate function exchange

The authors thank Sompong O-Thong for advice on molecular biotechnology work and also thank Hector Garcia for his help with analytical measurements. This study was funded by scholarship from the Technical University of Denmark.

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