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Anodic biofilm in single-chamber microbial fuel cells cultivated under different temperatures
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Anodic biofilm in single-chamber microbial fuel cells cultivated under different temperatures Lihong Liu a, Olga Tsyganova a, Duu-Jong Lee a,b,*, Ay Su c, Jo-Shu Chang d,e,f, Aijie Wang a, Nanqi Ren a a
State Key Laboratory of Urban Water Resources and Environments (SKLUWRE), School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China b Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan c Fuel Cell Center, Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan d Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan e Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan f Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan
article info
abstract
Article history:
The microorganisms in anodic biofilms of a microbial fuel cell (MFC) oxidize substrates to
Received 3 December 2011
generate electrons, protons, and metabolic products. This study started up two single-
Received in revised form
chamber MFCs at different temperatures (25 C for MFC A and 15 C for MFC B); after
15 March 2012
successful startup, the cell temperatures were swapped. The MFC A had peak voltage at
Accepted 16 March 2012
540 mV at 25 C, which was decreased rapidly as fed substrate was consumed. At 15 C, the
Available online 17 April 2012
MFC A yielded a nearly constant voltage of 500 mV over complete feed cycle. Conversely, the MFC B produced higher maximum power than MFC A, and can deliver nearly constant
Keywords:
voltage over the entire feed cycle at either 15 or 25 C. Electrochemical analysis revealed
Microbial fuel cell (MFC)
that the MFC B had lower internal resistance than MFC A, with the former having much
Microbial community
lower anodic resistance than the latter. Microbial analysis showed that the MFC started up
Anodic biofilm
at low temperatures had anodic biofilm enriched with psychrophilic bacteria Simplicispira
Psychrophilic bacteria
psychrophila LMG 5408(T)[AF078755] and Geobacter psychrophilus P35(T)[AY653549]. This study suggests the strategy to promote the development of anodic biofilms at low temperatures that are capable of yielding electricity at constant voltage. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
In operation of microbial fuel cell (MFC), microorganisms on anode oxidize substrates to generate electrons, protons, and other metabolic products while the potential at cathode surface reduces terminal electron acceptor with received protons and electrons [1]. Efforts were made to enhance MFC performance and to reduce manufacturing costs [2e5]. Torres
et al. [6] discussed the extracellular electron transfer kinetics for the anode-respiring bacteria (ARB). Wang et al. [7] noted the growth status of cells Escherichia coli affected the power production from an MFC. MFC was used to enhance overall energy gain following dark fermentation step [8,9]. The MFC was adopted in largescale wastewater treatment [10,11] and waste treatment [12e14]. Kong et al. [15] studied the effects of different electron
* Corresponding author. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan. Fax: þ886 2 23625632. E-mail address: [email protected] (D.-J. Lee). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.03.084
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receivers at cathodes on the overall cell performance. Torres et al. [16] noted that the anode potential affects the microbial community in anodic biofilms of an MFC. This study started up two single-chamber MFC at two different temperatures, 15 and 25 C. After successful startup, the cell temperatures were swapped. Waston and Logan [18] noted that running the MFC at increment of 1 mV s1 and variable external resistances at fixed time intervals of 20 min cannot eliminate the power overshoot. However, running the MFC at a fixed resistances for full batch cycle (1e2 d) fully eliminate the occurrence of power overshoot. Practical MFC systems acquire sufficiently high power density to drive external loads and have capability to adapt to change in external loads. With change in external load the power overshoot can occur in MFC operation. The cell performances and electrode characteristics were monitored using increment of 1 mV s-1. The microbial communities in anodic biofilms were studied.
2.
Materials and methods
2.1.
Inoculum and MFC
Two single-chambered MFC (A and B) were prepared for testing. Cylinder of diameter 6 cm and length 5 cm was used, making working volume of 110 ml. The anode was made of carbon fiber brush (40 mm diameter and 40 mm length; T70012 K, Toray Industries Co. Ltd., Japan), and air cathode was made by carbon cloth (W0S1002, CoTech Co., Ltd., 19.6 cm2) with 0.5 mg cm-2 Pt catalyst on water side and a carbon/polytetrafluoroethylene (PTFE) diffusion layer on air side [17]. The anode and cathode were connected to an external resistance (1000 U). Reference Ag/AgCl electrode (type 217, XianRen Industries Co., Shanghai, China) was installed into the anodic chamber for conducting electrochemical measurements. The anaerobic sludge collected from a primary clarifier effluent in Harbin Wenchang wastewater treatment plant was used as inoculum. The MFCs were fed with medium containing (per liter) NaAc 2.0 g, NH4Cl 0.62 g, KCl, 0.26 g, NaH2PO4 4.9 g, Na2HPO4 9.15 g, mineral solution 12.5 ml, and Wolfe’s vitamin solution 5 ml, with pH adjusted to 7.0. The MFC A was first operated at 25 C for 36 d, then the reactor temperature was dropped to 15 C for another 20 d. Conversely, the reactor B was first operated at 15 C for 25 d, then the reactor temperature was increased to 25 C for another 18 d.
2.2.
Electrochemical analysis
The MFC were operated for at least three full cycles with stable cell performance before electrochemical analysis was conducted. Linear sweeping voltammetry (LSV) was performed with a potentiostat/galvanostat (CHI 440, CH Instrument Inc., Austin, TX, USA). The voltage and current were recorded by LSV at a scan rate of 1 mV/s and the power output was calculated by Vcell Icell. Current density and power density were calculated based on cathodic area (19.6 cm2). For analyzing the electrochemical response on the anode, a threeelectrode system was set up in the anodic compartment. The polarization curve, namely, the current density against the
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potential (vs. Ag/AgCl/sat’d KCl), was obtained by LSV at a scan rate of 1 mV s1. The electrochemical impedance spectroscopy (EIS) experiments were performed at the end of tests. ZahnerTM IM6ex potentiostat-AC frequency analyzer equipment was used for the EIS experiments, and the results were analyzed using Thales1 software. The frequency of the AC signal was varied from 100 kHz to 10 mHz with an amplitude of 5 mV. Impedance experiments were performed under galvanostatic closed circuit conditions at 400 mA for the mature biofilms. The initial electrical potential for anode tests was at 0.5 V, while that for cathode tests was at þ0.25 V. To ensure steady state during galvanostatic operation, the MFC was allowed to equilibrate for 10 min between each current setting before applying the AC signal.
2.3.
16S rRNA gene clone library
Anode carbon filter brush was cut for the total genomic DNA extraction using a UNIQ-10 DNA Isolation Kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, Japan) according to the manufacturer’s instructions. The bacterial 16S rRNA gene clone libraries were constructed by using universal primer sets 27F (50 -AGAGTTTGATCC TGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-30 ). PCR amplification was performed following the below conditions: 5 min of denaturation at 94 C, followed by 35 cycles at 94 C for 45 s, 55 C for 45 s and 72 C for 90 s, with a final extension at 72 C for 10 min. The PCR products were purified on a 1% agarose gel, extracted with a UNIQ-10 gel-extraction kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China), then ligated to vector pMD19 and cloned into E. coli DH5a competent cells following the manufacturer’s protocol. Hundred plasmids containing positive insert from this sample were sequenced using an ABI 3730XL sequencer (Applied Biosystems, Foster, CA) with 27F primer. 16S rRNA gene Sequences were analyzed using the BLASTN search tools (http://www.ncbi.nlm.nih.gov/blast) and EzTaxon server [19]. Alignments with different 16S rRNA gene sequences from GenBank were performed using Clustal X 1.8.3 with default settings. Phylogenesis was analyzed with MEGA version 4.0 software, and distances were calculated using the Kimura 2 parameter distance model. A phylogenetic tree was built using the neighbor-joining method. Each dataset was bootstrapped 1000 times [20].
3.
Results and discussion
3.1.
MFCs performance
Fig. 1(a) shows the V-t curves for MFC A. After feeding, the cell voltage was rapidly increased to a peak value, then gradually dropped to <100 mV in next 5e8 d. The peak voltage gradually increased from 540 mV to over 600 mV after five cycles of operation, indicating formation of mature anodic biofilm in MFC. On the 36 d the reactor temperature was suddenly shifted to 15 C, hence producing distinct reactor performance. The cell voltage was suddenly increased from 320 mV to 525 mV, and was maintained at around 500 mV over the
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complete cycle. The cycle time was shortened to 5e6 d. Restated, comparing with that at 25 C, the MFC performance would not drop over time at 15 C. Fig. 1(b) shows the V-t curves for MFC B. After feeding, the cell voltage was rapidly increased to a peak value of approximately 570 mV, and maintained around that value after all acetate was consumed. This cell performance resembled that for MFC A operated at 15 C (37e55 d). Interestingly, when the cell temperature was increased to 25 C on d 26 for MFC B, the cell performance was not changed much. Restated, the cell performance at 25 C would be similar to that at 15 C if the reactor was started at low temperature. This observation was not consistent with the behavior noted for MFC A (Fig. 1(a)).
3.2.
Electrode characteristics
Fig. 2 shows the polarization curves the MFC obtained by LSV at 1 mV s1.
For MFC A starting at 25 C, the open-circle voltage (OCV) was 802 mV (Fig. 2(a)). Reducing external load yielded an increase in electrical current and reduction in cell voltage. The mild initial drop in voltage indicates the presence of activation losses for the present MFC at approximately 270 mV. When reaching V ¼ 276 mV, the power density peaked at 260 mW m-2. Further decrease in external loading reduces the power density. The short circuit current density (Iscc) was 1.79 Am2. The slope of V-I curve estimates the internal resistance (Rin) of the MFC of 212 U. No cell power shoot proposed by Nien et al. [21] and Liu et al. [22] was noted for the present MFC. When temperature of MFC A was reduced from 25 to 15 C, the corresponding OCV and Iscc were slightly reduced (Fig. 2(b)). In particular, the Rin was reduced from 212 to 155U, and the maximum power density was declined from 260 to 205 mW m2. For MFC B starting at 15 C, the open-circle voltage (OCV) was 791 mV and the activation loss was 280 mV (Fig. 2(c)), both are close to those shown in Fig. 2(a) (MFC A at 25 C). However, the internal resistance Rin was much lower (107 U), thus yielding a much higher maximum power density (591 m Wm2 at 362 mV) and Iscc (2.8 Am2). When reactor temperature was increased to 25 C, the OCV, Iscc and maximum power density were dropped to values similar to those for MFC A (Fig. 2(d)). It is noticeable that the Rin was increased from 107 U to 172 U. Nyquist plots for MFC A at 25 C revealed that the timedependent ohmic resistance for anode was negligibly small (Fig. 3(a)); while that for cathode was about 7 U (real axis intercept at high frequency end) (Fig. 4(a)). Reducing cell temperature to 15 C increased the ohmic resistance of anode to 14U (Fig. 3(b)); while that for cathode remained unchanged (7U) (Fig. 4(b)). The magnitude of Nyquist arc estimates the electrochemical polarization resistance of the electrode [23]. The polarization resistances for anode of MFC A were 177 U and 82 U at 25 C and 15 C, respectively. On the cathodes, the corresponding polarization resistances were 49 U and 47 U at 25 C and 15 C, respectively. Hence, the anode contributed principally the electrochemical polarization resistance of the MFC A, while reducing cell temperature would decrease anodic but the cathodic resistance. Nyquist plots for MFC B at 15 C revealed that the timedependent ohmic resistance for anode was small (Fig. 3(c)); while that for cathode was still about 7 U (Fig. 4(c)). Increase cell temperature to 25 C increased the ohmic resistance of anode to 14U (Fig. 3(d)); while that for cathode remained unchanged (7U) (Fig. 4(d)). The polarization resistances for anode of MFC B were 103 U and 106 U at 15 C and 25 C, respectively. On the cathodes, the corresponding polarization resistances were 97 U and 90 U at 15 C and 25 C, respectively. Hence, both the anode and cathode contributed to the electrochemical polarization resistance of the MFC B, while reducing cell temperature would not decrease the polarization resistances for both electrodes.
3.3.
Microbial analysis
The DGGE fingerprints and band intensities for biofilm samples collected from the anode surfaces (MFC A and MFC B) indicate the microbial communities on these electrodes (Fig. S1).
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Fig. 2 e Polarization curve and power density curve at different reactor temperatures. (a) MFC A: 25 C on d 36 d; (b) MFC A: 15 C on d 55. (c) MFC B: 15 C on d 25; (d) MFC B: 25 C on d 43.
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Fig. 3 e Nyquist plots for anodes at different reactor temperatures. (a) MFC A: 25 C on d 36 d; (b) MFC A: 15 C on d 55. (c) MFC B: 15 C on d 25; (d) MFC B: 25 C on d 43.
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Fig. 4 e Nyquist plots for cathodes at different reactor temperatures. (a) MFC A: 25 C on d 36 d; (b) MFC A: 15 C on d 55. (c) MFC B: 15 C on d 25; (d) MFC B: 25 C on d 43.
The 16S rRNA gene clone libraries for anodic biofilm of MFC A yielded 30 operational taxonomic units (OTUs), and that for anodic biofilm of MFC B produced 32 OTUs based on 100 random selected clone sequences (Tables 1 and 2). Ribotypes were identified phylogenetically and were grouped by phylum or in the case of Protebacteria, class, using Global Alignment for Sequence Taxonomy approach. The total frequency for a given phylogenic group was calculated (Fig. 5). The dominant phyla were Actinobacteria, Alphaproteobacteria, Anaerolineae, Bacillales, Bacteroidia, Betaproteobacteria, Clostridia, Cytophagia, Deltaproteobacteria, Erysipelotrichi, Gammaproteobacteria, Lactobacillales, Sphingobacteria, Synergistia, and Victivallales. The difference between MFC A and MFC B was on the deltaproteobacteria (7% for MFC A and 20% for MFC B) and gammaproteobacteria (21% for MFC A and 8% for MFC B). The microbial communities for MFC started at different temperatures had distinct strains on anodes. In particular, the psychrophilic bacteria Simplicispira psychrophila LMG 5408(T) [AF078755] and Geobacter psychrophilus P35(T)[AY653549] were much more in clone accounts for MFC B than MFC A.
3.4.
Discussion
As Sec. 3.2 shows, the polarization resistance of anode contributed principally to the internal resistance for MFC A; while the polarization resistances for both anodic and cathodic biofilms determined the internal resistance for MFC
B. As the MFC A was started up at 25 C, the anodic polarization resistance was high (177 U). When the cell operational temperature was reduced to 15 C, the anodic polarization resistance was correspondingly dropped (82 U), however, the biofilms were not capable of producing excess electricity. Conversely, as MFC B was started up at 15 C, the anodic polarization resistance was low (103 U). The inoculum, reactor geometry and feeds were identical for MFC A and MFC B. The community analysis in Sec 3.3 revealed that the anodic biofilms incubated at different temperature had distinct microbial structure. Hence, we speculated that the noted performance differences between the MFC A and MFC B were attributable to the different microbial strains in the biofilms. Torres et al. [2] proposed that anodic resistance is contributed by four series reactions including mass transfer (Rmt), substrate utilization (Rsu), extracellular electron transfer (Ret) and charge transfer (Rct): Ranode ¼ Rmt þ Rsu þ Ret þ Rct
(1)
As commonly expected, the rates transfer of substrate (1/Rmt) would increase with temperature. For instance, Lu et al. [24] showed that a microbial electrolysis cells (MECs) with the psychrophilic bacterium Geobacter psychrophilus would produce lower power at reduced temperatures than at room temperature. In the present study, the cell performance was on the contrary higher at reduced temperature than at room temperature. EI-Naggar et al. [25] observed the conducting
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Table 1 e Similarity of the 16S rRNA gene sequences obtained from clone library of anode brush of MFC A and their closely matched species. Name of Clone the clone account
MFC-R20 MFC-R2 MFC-R9 MFC-R12 MFC-R25 MFC-R30 MFC-R10 MFC-R11 MFC-R22 MFC-R1 MFC-R3 MFC-R5 MFC-R6 MFC-R14 MFC-R17 MFC-R21 MFC-R27 MFC-R28 MFC-R8 MFC-R16 MFC-R23 MFC-R26 MFC-R15 MFC-R18 MFC-R19 MFC-R4 MFC-R24 MFC-R29 MFC-R31 MFC-R7 MFC-R13 MFC-R32
1 1 1 1 8 1 4 2 1 8 2 3 2 1 2 1 22 1 1 1 1 1 2 6 1 19 1 1 1 1 1 1
Phylogenetic division (clone account)
Actinobacteria(3)
Alphaproteobacteria(10)
Bacillales(6) Bacteroidia(1) Betaproteobacteria(42)
Clostridia(4)
Cytophagia(2) Deltaproteobacteria(7) Gammaproteobacteria(21)
Lactobacillales(1) Synergistia(2) Victivallales(1)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.)
Similarity
Genebank accession number
Gordonia humi CC-12301(T)[FN561544] Aciditerrimonas ferrireducens IC-180(T)[AB517669] Atopobium parvulum DSM 20469(T)[CP001721] Bradyrhizobium liaoningense 2281(T)[AF208513] Rhodopseudomonas faecalis gc(T)[AF123085] Telmatospirillum siberiense 26-4b1(T)[AF524863] Bacillus anthracis ATCC 14578(T)[AB190217] Bacillus cereus ATCC 14579(T)[AE016877] Parabacteroides gordonii MS-1(T)[AB470343] Achromobacter denitrificans DSM 30026(T)[Y14907] Acidovorax defluvii BSB411(T)[Y18616] Alcaligenes faecalis subsp. parafaecalis G(T)[AJ242986] Alicycliphilus denitrificans K601(T)[CP002657] Comamonas granuli Ko03(T)[AB187586] Diaphorobacter nitroreducens NA10B(T)[AB064317] Ottowia pentelensis RB3-7(T)[EU518930] Simplicispira limi EMB325(T)[DQ372987] Simplicispira psychrophila LMG 5408(T)[AF078755] Anaerovorax odorimutans NorPut(T)[AJ251215] Desulfonispora thiosulfatigenes DSM 11270(T)[Y18214] Proteiniclasticum ruminis D3RC-2(T)[DQ852338] Sedimentibacter hydroxybenzoicus JW/Z-1(T)[L11305] Cytophaga fermentans ATCC 19072(T)[M58766] Geobacter chapellei 172(T)[U41561] Geobacter psychrophilus P35(T)[AY653549] Acinetobacter lwoffii DSM 2403(T)[X81665] Raoultella ornithinolytica JCM 6096(T)[AJ251467] Stenotrophomonas daejeonensis MJ03(T)[GQ241320] Trichococcus flocculiformis DSM 2094(T)[AJ306611] Aminomonas paucivorans DSM 12260(T)[CM001022] Synergistes sp. RMA 16406(T)[EU476081] Victivallis vadensis ATCC BAA-548(T)[ABDE02000010]
97.9 89.1 86.7 86.3 99.5 96.1 100 100 86.7 99.6 98.3 99.3 97.1 96.6 96.9 96.7 98.3 97.7 94.7 81.5 99.6 93.6 83.3 97.0 96.6 99.5 99.1 85.2 99.5 88.1 88.1 94.5
JN792255 JN792237 JN792244 JN792247 JN792260 JN792265 JN792245 JN792246 JN792257 JN792236 JN792238 JN792240 JN792241 JN792249 JN792252 JN792256 JN792262 JN792263 JN792243 JN792251 JN792258 JN792261 JN792250 JN792253 JN792254 JN792239 JN792259 JN792264 JN792266 JN792242 JN792248 JN7922265
Table 2 e Similarity of the 16S rRNA gene sequences obtained from clone library of anode brush of MFC B and their closely matched species. Clones
MFC-L-clone6 MFC-L-clone8 MFC-L-clone19 MFC-L-clone5 MFC-L-clone29 MFC-L-clone20 MFC-L-clone22 MFC-L-clone23 MFC-L-clone30 MFC-L-clone24 MFC-L-clone2 MFC-L-clone3 MFC-L-clone11 MFC-L-clone14 MFC-L-clone26 MFC-L-clone28 MFC-L-clone33
Clone account
1 2 1 1 1 1 1 4 1 2 1 8 1 1 24 3 1
Phylogenetic division (clone account)
Actinobacteria（4）
Anaerolineae(1) Sphingobacteria(1) Alphaproteobacteria(7)
Bacteroidia(2) Betaproteobacteria(47)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.) Brooklawnia cerclae BL-34(T)[DQ196625] Candidatus Microthrix parvicella 17[X89561] Patulibacter minatonensis KV-614(T)[AB193261] Bellilinea caldifistulae GOMI-1(T)[AB243672] Sphingobacterium kitahiroshimense 10C(T)[AB361248] Pseudaminobacter defluvii THI 051(T)[D32248] Rhodobacter aestuarii JA29(T)[AM748926] Rhodopseudomonas faecalis gc(T)[AF123085] Sphingomonas oryziterrae YC6722(T)[EU707560] Rikenella microfusus ATCC 29728(T)[L16498] Alcaligenes faecalis subsp. parafaecalis G(T)[AJ242986] Alicycliphilus denitrificans K601(T)[CP002657] Comamonas granuli Ko03(T)[AB187586] Diaphorobacter oryzae RF3(T)[EU342381] Simplicispira limi EMB325(T)[DQ372987] Simplicispira psychrophila LMG 5408(T)[AF078755] Thauera terpenica 58Eu(T)[AJ005817]
Similarity
Genebank accession number
93.9 99.6 88.8 83.2 81.9 96.5 96.1 99.4 46.9 82.1 90.3 96.9 83.4 95.9 98.3 97.3 98.1
JN792209 JN792211 JN792222 JN792208 JN792232 JN792223 JN792225 JN792226 JN792233 JN792227 JN792205 JN792206 JN792214 JN792217 JN792229 JN792231 JN792236
(continued on next page)
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Table 2 e (continued ) Clones
MFC-L-clone1 MFC-L-clone18 MFC-L-clone10 MFC-L-clone7 MFC-L-clone25 MFC-L-clone12 MFC-L-clone16 MFC-L-clone17 MFC-L-clone13 MFC-L-clone15 MFC-L-clone21 MFC-L-clone31 MFC-L-clone32 MFC-L-clone4 MFC-L-clone9
Clone account
6 2 3 1 1 2 8 10 1 1 6 1 1 1 1
Phylogenetic division (clone account)
Clostridia(5)
Cytophagia(2) Deltaproteobacteria(19)
Erysipelotrichi(1) Gammaproteobacteria(8)
Synergistia(2)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.)
Similarity
Genebank accession number
Achromobacter denitrificans DSM 30026(T)[Y14907] Hydrogenophaga palleronii DSM 63(T)[AF019073] Clostridium sticklandii DSM 519(T)[FP565809] Caldicoprobacter oshimai JW/HY-331(T)[AB450762] Sedimentibacter hydroxybenzoicus JW/Z-1(T)[L11305] Cytophaga fermentans ATCC 19072(T)[M58766] Geobacter chapellei 172(T)[U41561] Geobacter psychrophilus P35(T)[AY653549] Desulfovibrio termitidis HI1(T)[X87409] Erysipelothrix inopinata 1 MF-EP02(T)[AJ550617] Pseudomonas geniculata ATCC 19374(T)[AB021404] Stenotrophomonas acidaminiphila AMX19(T)[AF273080] Stenotrophomonas maltophilia ATCC 13637(T)[AB008509] Aminomonas paucivorans DSM 12260(T)[CM001022] Cloacibacillus evryensis RMA 16406(T)[EU476081]
99.5 99.4 99.3 86.6 91.9 83.3 96.7 96.6 99.1 90.7 99.0 99.8 91.7 88.3 88.1
JN792204 JN792221 JN792213 JN792210 JN792228 JN792215 JN792219 JN792220 JN792216 JN792218 JN792224 JN792234 JN792235 JN792207 JN7922212
Fig. 5 e Phylum distributions for taxonomically assigned tags from the anode biofilm of MFC. (a) MFC A, (b) MFC B.
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bacterial nanowires, and Torres et al. [2] concluded that the nanowires are the major way to pass electrons. However, Nien et al. [21] proposed that Rsu >> Ret. Hence, for the present MFC tests, the change in (1/Rsu) likely led to the noted enhanced cell performances at reduced temperature. This speculation counts on the important roles of the psychrophilic bacteria Simplicispira psychrophila and Geobacter psychrophilus enriched in anodic biofilms at reduced temperatures. In operation of an MFC the anodic microorganisms oxidize substrates to generate electrons, protons, and metabolic products. In a batch cycle the substance in cell chamber is continuously decreased, leading to a rapidly declined voltage as noted in MFC A at 25 C (Fig. 1(a)) consumed by anodic strains. A working electrical cell should provide a constant voltage regardless of the environmental change. In this regard, to operate the MFC B is superior to MFC A since the former yielded a nearly constant voltage over the entire cycle. Additionally, the MFC A, started at 25 C, could deliver a nearly constant voltage over a cycle at 15 C, though the maximum power density yielded was lower than the MFC B at 15 C. This study suggests the strategy to promote the development of anodic biofilms at low temperatures that are capable of yielding electricity at constant voltage. Experimental results also support the hypothesis that the startup of anodic biofilms could determine the cell performance.
4.
Conclusions
Two single-chamber MFCs were started up at 15 C or 25 C. The cell temperatures were swapped after successful startup. The MFC started up at 15 C (MFC B) could yield higher maximum power density than that at 25 C (MFC A). Additionally, the MFC B could deliver electricity at nearly constant voltage (540e560 mV) over the complete feed cycle at two studied temperatures. Electrochemical analysis revealed that the MFC B had lower anodic resistance than MFC A, which was attributable to the enriched psychrophilic bacteria S. psychrophila LMG 5408(T) and G. psychrophilus P35(T) in anodic biofilms. Experimental results support the hypothesis that the startup of anodic biofilms could determine the cell performance.
Acknowledgements The authors gratefully acknowledge funding from Project 51176037 supported by National Nature Science Foundation of China and partial supports by State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology.
Appendix A. Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.ijhydene.2012.03.084.
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references
[1] Logan BE. Microbial fuel cell. New Jersey: John Wiley & Sons; 2008. [2] Torres CI, Marcus AK, Lee HS, Parameswaran P, KrajmalnikBrown R, Rittmann BE. A kinetic perspective on extracellular electron transfer by anode-respiring bacteria. FEMS Microbiol Rev 2010;34:3e17. [3] Liu H, Logan BE. Electricity generation using an air-cathode single chamber microbial fuel cell in the presence and absence of a proton exchange membrane. Environ Sci Technol 2004;2004(38):4040e6. [4] Martin E, Savadogo O, Guiot SR, Tartakovsky B. The influence of operational conditions on the performance of a microbial fuel cell seeded with mesophilic anaerobic sludge. Biochem Eng J 2010;51:132e9. [5] Mohan Y, Das D. Effect of ionic strength, cation exchanger and inoculum age on the performance of microbial fuel cells. Int J Hydrogen Energy 2009;2009(34):7542e6. [6] Rismani-Yazdi H, Carver SM, Christy AD, Tuovinen OH. Cathodic limitations in microbial fuel cells: an overview. J Power Source 2008;180:683e94. [7] Wang CT, Chen WJ, Huang RY. Influence of growth curve phase on electricity performance of microbial fuel cell by Escherichia coli. Int J Hydrogen Energy 2010;2010(35): 7217e23. [8] Perera KRJ, Ketheesan B, Gadhamshetty V, Nirmalakhandan N. Fermentation biohydrogen production: evaluation of net energy gain. Int J Hydrogen Energy 2010; 2010(35):12224e33. [9] Sharma Y, Li B. Optimizing energy harvest in wastewater treatment by combining anaerobic hydrogen producing biofermentor (HPB) and microbial fuel cell (MFC). Int J Hydrogen Energy 2010;2010(35):3789e97. [10] Jiang DQ, Curtis M, Troop E, Scheible K, McGrath J, Hu BX, et al. A pilot-scale study on utilizing multi-anode/cathode microbial fuel cells (MAC MFCs) to enhance the power production in wastewater treatment. Int J Hydrogen Energy 2011;2011(36):876e84. [11] Jiang DQ, Li XA, Raymond D, Mooradan J, Li BK. Power recovery with multi-anode/cathode microbial fuel cells suitable for future large-scale applications. Int J Hydrogen Energy 2010;2010(35):8683e9. [12] Vazquez-Larios AL, Solorza-Feria O, Vazquez-Huerta G, Esparza-Garcia F, Rinderknecht-Seijas N, Poggi-Varaldo HM. Effects of architechtural changes and inoculums type on internal resistance of a microbial fuel cell designed for the treatment of leachates from the dark hydrogenogenic fermentation of organic solid wastes. Int J Hydrogen Energy 2011;2011(36):6199e209. [13] Goud RK, Babu PS, Mohan SV. Canteen based composite food waste as potential anodic fuel for bioelectricity generation in single chambered microbial fuel cell (MFC): bioelectrochemical evaluation under increasing substrate loading condition. Int J Hydrogen Energy 2011;2011(36): 6210e8. [14] Cusick RD, Kiely PD, Logan BE. A monetary comparison of energy recovery from microbial fuel cells and microbial electrolysis cells fed winery or domestic wastewaters. Int J Hydrogen Energy 2010;2010(35):8855e61. [15] Kong XY, Sun YM, Yuan ZH, Li D, Li LH, Li Y. Effect of cathode electron-receiver on the performance of microbial fuel cells. Int J Hydrogen Energy 2010;2010(35):7224e7. [16] Torres CI, Krajmalnik-Brown R, Marcus AK, Wanger G, Gorby YA, Rittmann BE. Selecting anode-respiring bacteria based on anode potential: phylogenic, electrochemical, and microscopic characterization. Environ Sci Technol 2009;43:9515e24.
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[17] Cheng S, Liu H, Logan BE. Increased performance of singlechamber microbial fuel cell using an improved cathode structure. Electrochem Comm 2005;8:489e94. [18] Waston VJ, Logan BE. Analysis of polarization methods for elimination of power overshoot in microbial fuel cells. Electrochem Comm 2011;13:54e6. [19] Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, et al. EzTaxon: a web-based tool for the identification of prokaryotes based on 16Sribosomal RNA gene sequences. Int J Syst Evol Microbiol 2007;57:2259e61. [20] Tamura K, Dudley J, Nei M. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596e9. [21] Nien PC, Lee CY, Ho KC, Liu LH, Wang AJ, Ren NQ, et al. Power overshoot in Two-Chambered microbial fuel cells (MFC). Bioresour Technol 2011;102:4742e6.
[22] Liu LH, Lee CY, Ho KC, Nien PC, Su A, Wang AJ, et al. Occurrence of power overshoot for two-chambered MFC at nearly steady-state operation. Int J Hydrogen Energy 2011; 36(21):13896e9. [23] Ramasamy RP, Ren ZY, Mench MM, Regan JM. Impact of initial biofilm growth on the anode impedance of microbial fuel cells. Biotechnol Bioeng 2008;2011(101): 101e8. [24] Lu L, Ren NQ, Zhao X, Wang H, Wu D, Xing DF. Hydrogen production, methanogen inhibition and microbial community structures in psychrophilic single-chamber microbial electrolysis cells. Energy Environ Sci 2011;4: 1329e36. [25] El-Naggar MY, Gorby YA, Xia W, Nealson KH. The molecular density of states in bacterial nanowires. Biophys J Biophys Lett 2008;95:L10e2.
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Anodic biofilm in single-chamber microbial fuel cells cultivated under different temperatures Lihong Liu a, Olga Tsyganova a, Duu-Jong Lee a,b,*, Ay Su c, Jo-Shu Chang d,e,f, Aijie Wang a, Nanqi Ren a a
State Key Laboratory of Urban Water Resources and Environments (SKLUWRE), School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China b Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan c Fuel Cell Center, Department of Mechanical Engineering, Yuan Ze University, Taoyuan 320, Taiwan d Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan e Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan f Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan
article info
abstract
Article history:
The microorganisms in anodic biofilms of a microbial fuel cell (MFC) oxidize substrates to
Received 3 December 2011
generate electrons, protons, and metabolic products. This study started up two single-
Received in revised form
chamber MFCs at different temperatures (25 C for MFC A and 15 C for MFC B); after
15 March 2012
successful startup, the cell temperatures were swapped. The MFC A had peak voltage at
Accepted 16 March 2012
540 mV at 25 C, which was decreased rapidly as fed substrate was consumed. At 15 C, the
Available online 17 April 2012
MFC A yielded a nearly constant voltage of 500 mV over complete feed cycle. Conversely, the MFC B produced higher maximum power than MFC A, and can deliver nearly constant
Keywords:
voltage over the entire feed cycle at either 15 or 25 C. Electrochemical analysis revealed
Microbial fuel cell (MFC)
that the MFC B had lower internal resistance than MFC A, with the former having much
Microbial community
lower anodic resistance than the latter. Microbial analysis showed that the MFC started up
Anodic biofilm
at low temperatures had anodic biofilm enriched with psychrophilic bacteria Simplicispira
Psychrophilic bacteria
psychrophila LMG 5408(T)[AF078755] and Geobacter psychrophilus P35(T)[AY653549]. This study suggests the strategy to promote the development of anodic biofilms at low temperatures that are capable of yielding electricity at constant voltage. Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
In operation of microbial fuel cell (MFC), microorganisms on anode oxidize substrates to generate electrons, protons, and other metabolic products while the potential at cathode surface reduces terminal electron acceptor with received protons and electrons [1]. Efforts were made to enhance MFC performance and to reduce manufacturing costs [2e5]. Torres
et al. [6] discussed the extracellular electron transfer kinetics for the anode-respiring bacteria (ARB). Wang et al. [7] noted the growth status of cells Escherichia coli affected the power production from an MFC. MFC was used to enhance overall energy gain following dark fermentation step [8,9]. The MFC was adopted in largescale wastewater treatment [10,11] and waste treatment [12e14]. Kong et al. [15] studied the effects of different electron
* Corresponding author. Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan. Fax: þ886 2 23625632. E-mail address: [email protected] (D.-J. Lee). 0360-3199/$ e see front matter Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2012.03.084
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receivers at cathodes on the overall cell performance. Torres et al. [16] noted that the anode potential affects the microbial community in anodic biofilms of an MFC. This study started up two single-chamber MFC at two different temperatures, 15 and 25 C. After successful startup, the cell temperatures were swapped. Waston and Logan [18] noted that running the MFC at increment of 1 mV s1 and variable external resistances at fixed time intervals of 20 min cannot eliminate the power overshoot. However, running the MFC at a fixed resistances for full batch cycle (1e2 d) fully eliminate the occurrence of power overshoot. Practical MFC systems acquire sufficiently high power density to drive external loads and have capability to adapt to change in external loads. With change in external load the power overshoot can occur in MFC operation. The cell performances and electrode characteristics were monitored using increment of 1 mV s-1. The microbial communities in anodic biofilms were studied.
2.
Materials and methods
2.1.
Inoculum and MFC
Two single-chambered MFC (A and B) were prepared for testing. Cylinder of diameter 6 cm and length 5 cm was used, making working volume of 110 ml. The anode was made of carbon fiber brush (40 mm diameter and 40 mm length; T70012 K, Toray Industries Co. Ltd., Japan), and air cathode was made by carbon cloth (W0S1002, CoTech Co., Ltd., 19.6 cm2) with 0.5 mg cm-2 Pt catalyst on water side and a carbon/polytetrafluoroethylene (PTFE) diffusion layer on air side [17]. The anode and cathode were connected to an external resistance (1000 U). Reference Ag/AgCl electrode (type 217, XianRen Industries Co., Shanghai, China) was installed into the anodic chamber for conducting electrochemical measurements. The anaerobic sludge collected from a primary clarifier effluent in Harbin Wenchang wastewater treatment plant was used as inoculum. The MFCs were fed with medium containing (per liter) NaAc 2.0 g, NH4Cl 0.62 g, KCl, 0.26 g, NaH2PO4 4.9 g, Na2HPO4 9.15 g, mineral solution 12.5 ml, and Wolfe’s vitamin solution 5 ml, with pH adjusted to 7.0. The MFC A was first operated at 25 C for 36 d, then the reactor temperature was dropped to 15 C for another 20 d. Conversely, the reactor B was first operated at 15 C for 25 d, then the reactor temperature was increased to 25 C for another 18 d.
2.2.
Electrochemical analysis
The MFC were operated for at least three full cycles with stable cell performance before electrochemical analysis was conducted. Linear sweeping voltammetry (LSV) was performed with a potentiostat/galvanostat (CHI 440, CH Instrument Inc., Austin, TX, USA). The voltage and current were recorded by LSV at a scan rate of 1 mV/s and the power output was calculated by Vcell Icell. Current density and power density were calculated based on cathodic area (19.6 cm2). For analyzing the electrochemical response on the anode, a threeelectrode system was set up in the anodic compartment. The polarization curve, namely, the current density against the
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potential (vs. Ag/AgCl/sat’d KCl), was obtained by LSV at a scan rate of 1 mV s1. The electrochemical impedance spectroscopy (EIS) experiments were performed at the end of tests. ZahnerTM IM6ex potentiostat-AC frequency analyzer equipment was used for the EIS experiments, and the results were analyzed using Thales1 software. The frequency of the AC signal was varied from 100 kHz to 10 mHz with an amplitude of 5 mV. Impedance experiments were performed under galvanostatic closed circuit conditions at 400 mA for the mature biofilms. The initial electrical potential for anode tests was at 0.5 V, while that for cathode tests was at þ0.25 V. To ensure steady state during galvanostatic operation, the MFC was allowed to equilibrate for 10 min between each current setting before applying the AC signal.
2.3.
16S rRNA gene clone library
Anode carbon filter brush was cut for the total genomic DNA extraction using a UNIQ-10 DNA Isolation Kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, Japan) according to the manufacturer’s instructions. The bacterial 16S rRNA gene clone libraries were constructed by using universal primer sets 27F (50 -AGAGTTTGATCC TGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-30 ). PCR amplification was performed following the below conditions: 5 min of denaturation at 94 C, followed by 35 cycles at 94 C for 45 s, 55 C for 45 s and 72 C for 90 s, with a final extension at 72 C for 10 min. The PCR products were purified on a 1% agarose gel, extracted with a UNIQ-10 gel-extraction kit (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China), then ligated to vector pMD19 and cloned into E. coli DH5a competent cells following the manufacturer’s protocol. Hundred plasmids containing positive insert from this sample were sequenced using an ABI 3730XL sequencer (Applied Biosystems, Foster, CA) with 27F primer. 16S rRNA gene Sequences were analyzed using the BLASTN search tools (http://www.ncbi.nlm.nih.gov/blast) and EzTaxon server [19]. Alignments with different 16S rRNA gene sequences from GenBank were performed using Clustal X 1.8.3 with default settings. Phylogenesis was analyzed with MEGA version 4.0 software, and distances were calculated using the Kimura 2 parameter distance model. A phylogenetic tree was built using the neighbor-joining method. Each dataset was bootstrapped 1000 times [20].
3.
Results and discussion
3.1.
MFCs performance
Fig. 1(a) shows the V-t curves for MFC A. After feeding, the cell voltage was rapidly increased to a peak value, then gradually dropped to <100 mV in next 5e8 d. The peak voltage gradually increased from 540 mV to over 600 mV after five cycles of operation, indicating formation of mature anodic biofilm in MFC. On the 36 d the reactor temperature was suddenly shifted to 15 C, hence producing distinct reactor performance. The cell voltage was suddenly increased from 320 mV to 525 mV, and was maintained at around 500 mV over the
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complete cycle. The cycle time was shortened to 5e6 d. Restated, comparing with that at 25 C, the MFC performance would not drop over time at 15 C. Fig. 1(b) shows the V-t curves for MFC B. After feeding, the cell voltage was rapidly increased to a peak value of approximately 570 mV, and maintained around that value after all acetate was consumed. This cell performance resembled that for MFC A operated at 15 C (37e55 d). Interestingly, when the cell temperature was increased to 25 C on d 26 for MFC B, the cell performance was not changed much. Restated, the cell performance at 25 C would be similar to that at 15 C if the reactor was started at low temperature. This observation was not consistent with the behavior noted for MFC A (Fig. 1(a)).
3.2.
Electrode characteristics
Fig. 2 shows the polarization curves the MFC obtained by LSV at 1 mV s1.
For MFC A starting at 25 C, the open-circle voltage (OCV) was 802 mV (Fig. 2(a)). Reducing external load yielded an increase in electrical current and reduction in cell voltage. The mild initial drop in voltage indicates the presence of activation losses for the present MFC at approximately 270 mV. When reaching V ¼ 276 mV, the power density peaked at 260 mW m-2. Further decrease in external loading reduces the power density. The short circuit current density (Iscc) was 1.79 Am2. The slope of V-I curve estimates the internal resistance (Rin) of the MFC of 212 U. No cell power shoot proposed by Nien et al. [21] and Liu et al. [22] was noted for the present MFC. When temperature of MFC A was reduced from 25 to 15 C, the corresponding OCV and Iscc were slightly reduced (Fig. 2(b)). In particular, the Rin was reduced from 212 to 155U, and the maximum power density was declined from 260 to 205 mW m2. For MFC B starting at 15 C, the open-circle voltage (OCV) was 791 mV and the activation loss was 280 mV (Fig. 2(c)), both are close to those shown in Fig. 2(a) (MFC A at 25 C). However, the internal resistance Rin was much lower (107 U), thus yielding a much higher maximum power density (591 m Wm2 at 362 mV) and Iscc (2.8 Am2). When reactor temperature was increased to 25 C, the OCV, Iscc and maximum power density were dropped to values similar to those for MFC A (Fig. 2(d)). It is noticeable that the Rin was increased from 107 U to 172 U. Nyquist plots for MFC A at 25 C revealed that the timedependent ohmic resistance for anode was negligibly small (Fig. 3(a)); while that for cathode was about 7 U (real axis intercept at high frequency end) (Fig. 4(a)). Reducing cell temperature to 15 C increased the ohmic resistance of anode to 14U (Fig. 3(b)); while that for cathode remained unchanged (7U) (Fig. 4(b)). The magnitude of Nyquist arc estimates the electrochemical polarization resistance of the electrode [23]. The polarization resistances for anode of MFC A were 177 U and 82 U at 25 C and 15 C, respectively. On the cathodes, the corresponding polarization resistances were 49 U and 47 U at 25 C and 15 C, respectively. Hence, the anode contributed principally the electrochemical polarization resistance of the MFC A, while reducing cell temperature would decrease anodic but the cathodic resistance. Nyquist plots for MFC B at 15 C revealed that the timedependent ohmic resistance for anode was small (Fig. 3(c)); while that for cathode was still about 7 U (Fig. 4(c)). Increase cell temperature to 25 C increased the ohmic resistance of anode to 14U (Fig. 3(d)); while that for cathode remained unchanged (7U) (Fig. 4(d)). The polarization resistances for anode of MFC B were 103 U and 106 U at 15 C and 25 C, respectively. On the cathodes, the corresponding polarization resistances were 97 U and 90 U at 15 C and 25 C, respectively. Hence, both the anode and cathode contributed to the electrochemical polarization resistance of the MFC B, while reducing cell temperature would not decrease the polarization resistances for both electrodes.
3.3.
Microbial analysis
The DGGE fingerprints and band intensities for biofilm samples collected from the anode surfaces (MFC A and MFC B) indicate the microbial communities on these electrodes (Fig. S1).
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Fig. 2 e Polarization curve and power density curve at different reactor temperatures. (a) MFC A: 25 C on d 36 d; (b) MFC A: 15 C on d 55. (c) MFC B: 15 C on d 25; (d) MFC B: 25 C on d 43.
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The 16S rRNA gene clone libraries for anodic biofilm of MFC A yielded 30 operational taxonomic units (OTUs), and that for anodic biofilm of MFC B produced 32 OTUs based on 100 random selected clone sequences (Tables 1 and 2). Ribotypes were identified phylogenetically and were grouped by phylum or in the case of Protebacteria, class, using Global Alignment for Sequence Taxonomy approach. The total frequency for a given phylogenic group was calculated (Fig. 5). The dominant phyla were Actinobacteria, Alphaproteobacteria, Anaerolineae, Bacillales, Bacteroidia, Betaproteobacteria, Clostridia, Cytophagia, Deltaproteobacteria, Erysipelotrichi, Gammaproteobacteria, Lactobacillales, Sphingobacteria, Synergistia, and Victivallales. The difference between MFC A and MFC B was on the deltaproteobacteria (7% for MFC A and 20% for MFC B) and gammaproteobacteria (21% for MFC A and 8% for MFC B). The microbial communities for MFC started at different temperatures had distinct strains on anodes. In particular, the psychrophilic bacteria Simplicispira psychrophila LMG 5408(T) [AF078755] and Geobacter psychrophilus P35(T)[AY653549] were much more in clone accounts for MFC B than MFC A.
3.4.
Discussion
As Sec. 3.2 shows, the polarization resistance of anode contributed principally to the internal resistance for MFC A; while the polarization resistances for both anodic and cathodic biofilms determined the internal resistance for MFC
B. As the MFC A was started up at 25 C, the anodic polarization resistance was high (177 U). When the cell operational temperature was reduced to 15 C, the anodic polarization resistance was correspondingly dropped (82 U), however, the biofilms were not capable of producing excess electricity. Conversely, as MFC B was started up at 15 C, the anodic polarization resistance was low (103 U). The inoculum, reactor geometry and feeds were identical for MFC A and MFC B. The community analysis in Sec 3.3 revealed that the anodic biofilms incubated at different temperature had distinct microbial structure. Hence, we speculated that the noted performance differences between the MFC A and MFC B were attributable to the different microbial strains in the biofilms. Torres et al. [2] proposed that anodic resistance is contributed by four series reactions including mass transfer (Rmt), substrate utilization (Rsu), extracellular electron transfer (Ret) and charge transfer (Rct): Ranode ¼ Rmt þ Rsu þ Ret þ Rct
(1)
As commonly expected, the rates transfer of substrate (1/Rmt) would increase with temperature. For instance, Lu et al. [24] showed that a microbial electrolysis cells (MECs) with the psychrophilic bacterium Geobacter psychrophilus would produce lower power at reduced temperatures than at room temperature. In the present study, the cell performance was on the contrary higher at reduced temperature than at room temperature. EI-Naggar et al. [25] observed the conducting
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Table 1 e Similarity of the 16S rRNA gene sequences obtained from clone library of anode brush of MFC A and their closely matched species. Name of Clone the clone account
MFC-R20 MFC-R2 MFC-R9 MFC-R12 MFC-R25 MFC-R30 MFC-R10 MFC-R11 MFC-R22 MFC-R1 MFC-R3 MFC-R5 MFC-R6 MFC-R14 MFC-R17 MFC-R21 MFC-R27 MFC-R28 MFC-R8 MFC-R16 MFC-R23 MFC-R26 MFC-R15 MFC-R18 MFC-R19 MFC-R4 MFC-R24 MFC-R29 MFC-R31 MFC-R7 MFC-R13 MFC-R32
1 1 1 1 8 1 4 2 1 8 2 3 2 1 2 1 22 1 1 1 1 1 2 6 1 19 1 1 1 1 1 1
Phylogenetic division (clone account)
Actinobacteria(3)
Alphaproteobacteria(10)
Bacillales(6) Bacteroidia(1) Betaproteobacteria(42)
Clostridia(4)
Cytophagia(2) Deltaproteobacteria(7) Gammaproteobacteria(21)
Lactobacillales(1) Synergistia(2) Victivallales(1)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.)
Similarity
Genebank accession number
Gordonia humi CC-12301(T)[FN561544] Aciditerrimonas ferrireducens IC-180(T)[AB517669] Atopobium parvulum DSM 20469(T)[CP001721] Bradyrhizobium liaoningense 2281(T)[AF208513] Rhodopseudomonas faecalis gc(T)[AF123085] Telmatospirillum siberiense 26-4b1(T)[AF524863] Bacillus anthracis ATCC 14578(T)[AB190217] Bacillus cereus ATCC 14579(T)[AE016877] Parabacteroides gordonii MS-1(T)[AB470343] Achromobacter denitrificans DSM 30026(T)[Y14907] Acidovorax defluvii BSB411(T)[Y18616] Alcaligenes faecalis subsp. parafaecalis G(T)[AJ242986] Alicycliphilus denitrificans K601(T)[CP002657] Comamonas granuli Ko03(T)[AB187586] Diaphorobacter nitroreducens NA10B(T)[AB064317] Ottowia pentelensis RB3-7(T)[EU518930] Simplicispira limi EMB325(T)[DQ372987] Simplicispira psychrophila LMG 5408(T)[AF078755] Anaerovorax odorimutans NorPut(T)[AJ251215] Desulfonispora thiosulfatigenes DSM 11270(T)[Y18214] Proteiniclasticum ruminis D3RC-2(T)[DQ852338] Sedimentibacter hydroxybenzoicus JW/Z-1(T)[L11305] Cytophaga fermentans ATCC 19072(T)[M58766] Geobacter chapellei 172(T)[U41561] Geobacter psychrophilus P35(T)[AY653549] Acinetobacter lwoffii DSM 2403(T)[X81665] Raoultella ornithinolytica JCM 6096(T)[AJ251467] Stenotrophomonas daejeonensis MJ03(T)[GQ241320] Trichococcus flocculiformis DSM 2094(T)[AJ306611] Aminomonas paucivorans DSM 12260(T)[CM001022] Synergistes sp. RMA 16406(T)[EU476081] Victivallis vadensis ATCC BAA-548(T)[ABDE02000010]
97.9 89.1 86.7 86.3 99.5 96.1 100 100 86.7 99.6 98.3 99.3 97.1 96.6 96.9 96.7 98.3 97.7 94.7 81.5 99.6 93.6 83.3 97.0 96.6 99.5 99.1 85.2 99.5 88.1 88.1 94.5
JN792255 JN792237 JN792244 JN792247 JN792260 JN792265 JN792245 JN792246 JN792257 JN792236 JN792238 JN792240 JN792241 JN792249 JN792252 JN792256 JN792262 JN792263 JN792243 JN792251 JN792258 JN792261 JN792250 JN792253 JN792254 JN792239 JN792259 JN792264 JN792266 JN792242 JN792248 JN7922265
Table 2 e Similarity of the 16S rRNA gene sequences obtained from clone library of anode brush of MFC B and their closely matched species. Clones
MFC-L-clone6 MFC-L-clone8 MFC-L-clone19 MFC-L-clone5 MFC-L-clone29 MFC-L-clone20 MFC-L-clone22 MFC-L-clone23 MFC-L-clone30 MFC-L-clone24 MFC-L-clone2 MFC-L-clone3 MFC-L-clone11 MFC-L-clone14 MFC-L-clone26 MFC-L-clone28 MFC-L-clone33
Clone account
1 2 1 1 1 1 1 4 1 2 1 8 1 1 24 3 1
Phylogenetic division (clone account)
Actinobacteria（4）
Anaerolineae(1) Sphingobacteria(1) Alphaproteobacteria(7)
Bacteroidia(2) Betaproteobacteria(47)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.) Brooklawnia cerclae BL-34(T)[DQ196625] Candidatus Microthrix parvicella 17[X89561] Patulibacter minatonensis KV-614(T)[AB193261] Bellilinea caldifistulae GOMI-1(T)[AB243672] Sphingobacterium kitahiroshimense 10C(T)[AB361248] Pseudaminobacter defluvii THI 051(T)[D32248] Rhodobacter aestuarii JA29(T)[AM748926] Rhodopseudomonas faecalis gc(T)[AF123085] Sphingomonas oryziterrae YC6722(T)[EU707560] Rikenella microfusus ATCC 29728(T)[L16498] Alcaligenes faecalis subsp. parafaecalis G(T)[AJ242986] Alicycliphilus denitrificans K601(T)[CP002657] Comamonas granuli Ko03(T)[AB187586] Diaphorobacter oryzae RF3(T)[EU342381] Simplicispira limi EMB325(T)[DQ372987] Simplicispira psychrophila LMG 5408(T)[AF078755] Thauera terpenica 58Eu(T)[AJ005817]
Similarity
Genebank accession number
93.9 99.6 88.8 83.2 81.9 96.5 96.1 99.4 46.9 82.1 90.3 96.9 83.4 95.9 98.3 97.3 98.1
JN792209 JN792211 JN792222 JN792208 JN792232 JN792223 JN792225 JN792226 JN792233 JN792227 JN792205 JN792206 JN792214 JN792217 JN792229 JN792231 JN792236
(continued on next page)
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Table 2 e (continued ) Clones
MFC-L-clone1 MFC-L-clone18 MFC-L-clone10 MFC-L-clone7 MFC-L-clone25 MFC-L-clone12 MFC-L-clone16 MFC-L-clone17 MFC-L-clone13 MFC-L-clone15 MFC-L-clone21 MFC-L-clone31 MFC-L-clone32 MFC-L-clone4 MFC-L-clone9
Clone account
6 2 3 1 1 2 8 10 1 1 6 1 1 1 1
Phylogenetic division (clone account)
Clostridia(5)
Cytophagia(2) Deltaproteobacteria(19)
Erysipelotrichi(1) Gammaproteobacteria(8)
Synergistia(2)
The closest type strain or uncultured bacterium clone in GenBank database (GenBank accession no.)
Similarity
Genebank accession number
Achromobacter denitrificans DSM 30026(T)[Y14907] Hydrogenophaga palleronii DSM 63(T)[AF019073] Clostridium sticklandii DSM 519(T)[FP565809] Caldicoprobacter oshimai JW/HY-331(T)[AB450762] Sedimentibacter hydroxybenzoicus JW/Z-1(T)[L11305] Cytophaga fermentans ATCC 19072(T)[M58766] Geobacter chapellei 172(T)[U41561] Geobacter psychrophilus P35(T)[AY653549] Desulfovibrio termitidis HI1(T)[X87409] Erysipelothrix inopinata 1 MF-EP02(T)[AJ550617] Pseudomonas geniculata ATCC 19374(T)[AB021404] Stenotrophomonas acidaminiphila AMX19(T)[AF273080] Stenotrophomonas maltophilia ATCC 13637(T)[AB008509] Aminomonas paucivorans DSM 12260(T)[CM001022] Cloacibacillus evryensis RMA 16406(T)[EU476081]
99.5 99.4 99.3 86.6 91.9 83.3 96.7 96.6 99.1 90.7 99.0 99.8 91.7 88.3 88.1
JN792204 JN792221 JN792213 JN792210 JN792228 JN792215 JN792219 JN792220 JN792216 JN792218 JN792224 JN792234 JN792235 JN792207 JN7922212
Fig. 5 e Phylum distributions for taxonomically assigned tags from the anode biofilm of MFC. (a) MFC A, (b) MFC B.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 1 5 7 9 2 e1 5 8 0 0
bacterial nanowires, and Torres et al. [2] concluded that the nanowires are the major way to pass electrons. However, Nien et al. [21] proposed that Rsu >> Ret. Hence, for the present MFC tests, the change in (1/Rsu) likely led to the noted enhanced cell performances at reduced temperature. This speculation counts on the important roles of the psychrophilic bacteria Simplicispira psychrophila and Geobacter psychrophilus enriched in anodic biofilms at reduced temperatures. In operation of an MFC the anodic microorganisms oxidize substrates to generate electrons, protons, and metabolic products. In a batch cycle the substance in cell chamber is continuously decreased, leading to a rapidly declined voltage as noted in MFC A at 25 C (Fig. 1(a)) consumed by anodic strains. A working electrical cell should provide a constant voltage regardless of the environmental change. In this regard, to operate the MFC B is superior to MFC A since the former yielded a nearly constant voltage over the entire cycle. Additionally, the MFC A, started at 25 C, could deliver a nearly constant voltage over a cycle at 15 C, though the maximum power density yielded was lower than the MFC B at 15 C. This study suggests the strategy to promote the development of anodic biofilms at low temperatures that are capable of yielding electricity at constant voltage. Experimental results also support the hypothesis that the startup of anodic biofilms could determine the cell performance.
4.
Conclusions
Two single-chamber MFCs were started up at 15 C or 25 C. The cell temperatures were swapped after successful startup. The MFC started up at 15 C (MFC B) could yield higher maximum power density than that at 25 C (MFC A). Additionally, the MFC B could deliver electricity at nearly constant voltage (540e560 mV) over the complete feed cycle at two studied temperatures. Electrochemical analysis revealed that the MFC B had lower anodic resistance than MFC A, which was attributable to the enriched psychrophilic bacteria S. psychrophila LMG 5408(T) and G. psychrophilus P35(T) in anodic biofilms. Experimental results support the hypothesis that the startup of anodic biofilms could determine the cell performance.
Acknowledgements The authors gratefully acknowledge funding from Project 51176037 supported by National Nature Science Foundation of China and partial supports by State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology.
Appendix A. Supplementary data Supplementary data related to this article can be found online at doi:10.1016/j.ijhydene.2012.03.084.
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