Electricity generation from cattle dung using microbial fuel cell technology during anaerobic acidogenesis and the development of microbial populations

Waste Management 32 (2012) 1651–1658

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Electricity generation from cattle dung using microbial fuel cell technology during anaerobic acidogenesis and the development of microbial populations Guang Zhao a,⇑, Fang Ma a, Li Wei a, Hong Chua b, Chein-Chi Chang c, Xiao-Jun Zhang d a

State Key Lab of Urban Water Resources and Environment (SKLUWRE), School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China Department of Civil and Structural Engineering, The Hong Kong Polytechnic University, Hong Kong c Department of Chemical, Biochemical, and Environmental Engineering, University of Maryland, Baltimore County, MD 21250, USA d Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China b

a r t i c l e

i n f o

Article history: Received 1 July 2011 Accepted 22 April 2012 Available online 16 May 2012 Keywords: Anaerobic digestion MFC Acidogenesis Cattle dung Microbial community

a b s t r a c t A microbial fuel cell (MFC) was constructed to investigate the possible generation of electricity using cattle dung as a substrate. After 30 days of operation, stable electricity was generated, and the maximum volumetric power density was 0.220 W/m3. The total chemical oxygen demand (TCOD) removal and coulombic efficiency (CE) of the MFC reached 73.9 ± 1.8% and 2.79 ± 0.6%, respectively, after 120 days of operation. Acetate was the main metabolite in the anolyte, and other volatile fatty acids (VFAs) (propionate and butyrate) were present in minor amounts. The PCR–DGGE analysis indicated that the following five groups of microbes were present: Proteobacteria, Bacteroides, Chloroflexi, Actinobacteria and Firmicutes. Proteobacteria and Firmicutes were the dominant phyla in the sample; specifically, 36.3% and 24.2% of the sequences obtained were Proteobacteria and Firmicutes, respectively. Clostridium sp., Pseudomonas luteola and Ochrobactrum pseudogrignonense were the most dominant groups during the electricity generation process. The diversity of archaea dramatically decreased after 20 days of operation. The detected archaea were hydrogenotrophic methanogens, and the Methanobacterium genus disappeared during the periods of stable electricity generation via acidogenesis. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The depletion of fossil fuel sources and environmental pollution caused by their combustion has driven many researchers to seek carbon-neutral renewable energy alternatives in recent years (Ter Heijne et al., 2007). Anaerobic digestion is a highly promising technology for converting biomass waste into biogas, and it has received considerable attention worldwide. Methane production can be used for heat and electrical energy or as a transportation fuel. These alternative energy sources are also economically attractive. It is estimated that 300 million tons of animal waste and 700 million tons of crop stalk waste are generated annually in China. The efficient disposal of these biomass waste types would have considerable environmental benefits and could be used to produce renewable energy. It is known that two-phase anaerobic digestion systems permit much higher organic loads in the digester and are readily controlled, which ensures stable system operation (Mata-Alvarez et al., 2000; Demirer and Chen, 2005). The hydrolysisacidification phase is generally considered to be the rate-limiting step in biogas production (Bouallagui et al., 2004; Veeken and Hamelers, 1999). Therefore, exploring the microbial populations ⇑ Corresponding author. Tel./fax: +86 451 86283805. E-mail address: [email protected] (G. Zhao). 0956-053X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.wasman.2012.04.013

that develop during anaerobic hydrolysis is important. An understanding of these microbes can be used to increase the rate of hydrolysis-acidification and the overall efficiency of anaerobic digestion. Furthermore, electricity generation by a microbial fuel cell (MFC) utilizing renewable carbon sources can provide an alternative to fossil fuels (Logan et al., 2006). Recently, the use of MFC technology has been researched for many applications, such as the wastewater treatment of domestic sewage (Rabaey et al., 2008; Lu et al., 2009; Sun et al., 2008). However, no study has yet reported the use of cattle dung to generate electricity in the hydrolysis-acidification phase of a two-phase anaerobic digestion for the production of biogas. Cattle dung contains several easily degradable substrates as well as several that are difficult to hydrolyze, such as cellulose and hemicellulose. Cattle dung can be utilized by fermentable bacteria and electricity-producing microorganisms. The following is a typical electrode reaction with acetate as an example substrate:

CH3 COO þ 2H2 O ! 2CO2 þ 7Hþ þ 8e

ð1Þ

Methane production can originate from two sources in the presence of acetate: carbon dioxide and hydrogen. In the first of the following two pathways, methane is produced by acetoclastic methanogenesis, and in the second, it is produced by hydrogenotrophic methanogenesis:

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C2 H4 O2 ! CH4 þ 2CO2

ð2Þ

4H2 þ CO2 ¼ CH4 þ 2H2 O

ð3Þ

utilized cattle dung at 30 °C. The characteristics of the fresh cattle dung are listed in Table 1. 2.2. Analytical methods

In this work, we focused on a two-chamber MFC system that utilizes cattle dung as the substrate over a period of 120 days. The objective of this study was to demonstrate that electricity can be generated by this system run in a fed-batch mode. We also examined the variation in VFA production during electricity generation, and we investigated the bacterial and archaeal community structures of the anodic biofilm using denaturing gradient gel electrophoresis (DGGE). 2. Materials and methods 2.1. Design and operation of the reactor The MFC was composed of Plexiglas and consisted of two chambers separated by a proton-exchange membrane (PEM; NafionTM117, DuPont Co., DE, USA; Fig. 1). The anolyte was stirred with a blade stirrer (400 rpm) for 5 min every 2 h. The total volume of the MFC was 15 L, and the reactor was equipped with dual anodes made of carbon brushes. The brushes were embedded in graphite granule electrodes (with a diameter of 1–5 mm, 0.5 X/ granules, Jiuxin Carbon Goods Co., Jilin, China). A fixed external resistance of 1000 X was connected via the electrodes through copper wires. Potassium permanganate buffer was used for the cathode electrolytes and was replaced once per week. The experiment was conducted in fed-batch mode under mesophilic conditions (35 ± 1 °C), and the system operated continuously for 120 days. Fresh cattle dung was collected from a dairy farm in Heilongjiang Province, China. The initial total solids (TS) content was 8%, and the inoculum consisted of a biogas slurry that was taken from a semi-continuous, large-scale, anaerobic digester that

The total solids concentration (TS), volatile solids concentration (VS), pH, total organic carbon (TOC), and total Kjeldahl nitrogen (TKN) were determined using standard methods (APHA, 1998). Volatile fatty acids (VFAs) content was determined using gas chromatography (7890A GC-System, Agilent Technologies, USA). The biogas was collected in glass bottles, and its total volume was recorded at 24 h intervals. Scanning electron microscopy (SEM) anodes made of carbon fiber were gently placed on a silicon wafer. The samples were fixed in 2% glutaraldehyde and 1% osmium tetroxide and then dehydrated in increasing concentrations of ethanol. The fixed samples were dried using liquid CO2 and coated with platinum and palladium for 3 min at 100 V and 100 mA. Scanning electron microscopy (Quanta200, FEI, USA) analysis was then performed. 2.3. MFC calculation The MFC was continuously monitored using a voltage data acquisition system (RBH8251, Shanghai Rui Bohua Instrument Co., Ltd.). The current (I) was calculated at a given resistance (R) using the voltage (V) and the relationship I = V/R. The power density (P) was calculated as P = IV/A, where A (m3) is the anode volume. The reactor was equipped with a Ag/AgCl reference electrode (RE-5B, BASi, Ningbo, Jiangsu Province, China) for measuring the cathodic and anodic electrodes potential. Coulombic efficiency was calculated by dividing observed coulombs by theoretical coulombs. The theoretical coulomb was calculated based on the TCOD consumed due to complete substrate oxidation (Kim et al., 2005). Internal resistance (Rint) was determined from the slope of the polarization curves (Cheng et al., 2006a).

Fig. 1. Schematic diagram of the bioreactor.

Table 1 Characteristics of the fresh cattle dung. Type of analysis

Total solids (% Wet)

Volatile solids (% TS)

Total organic carbon (%TS)

Total Kjeldahl nitrogen (%TS)

Phosphorus (% TS)

Potassium (% TS)

Fresh cattle dung

17.2 ± 2.6

75.8 ± 1.8

27.6 ± 2.9

1.7 ± 0.3

0.3 ± 0.1

0.6 ± 0.1

The parameter mean and standard deviation comprised three samples of cattle dung.

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Genomic DNA was extracted from the anodic biofilm sample using a Fast DNA SPIN Kit for Soil preparation (Qbiogene Inc., USA) according to the manufacturer’s instruction. The crude DNA extracts were purified using the Wizard DNAÒ clean-up system (Promega, WI, USA). The V2–V3 regions of the bacterial 16S rRNA genes were amplified for DGGE using the universal bacterial primers 101GC-F and 534-R (Nubel et al., 1996). Amplification was carried out in a 50 ll reaction volume, which contained 50 ng of genomic DNA, 1X reaction buffer, 3 mM MgCl2, 10 pmol of each primer, 0.4 mM of each dNTP and 1.25 U of Taq DNA polymerase. The amplification conditions consisted of an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 95 °C for 45 s, 57 °C for 30 s, and 72 °C for 45 s and finally an elongation step at 72 °C for 8 min. The V2–V3 regions of archaeal 16S rRNA genes were amplified for DGGE using the primers A109 (T)-F and 515GC-R (Grosskopf et al., 1998). Amplification was carried out in a 50 ll reaction volume, which contained 100 ng of genomic DNA, 1X reaction buffer, 3 mM MgCl2, 20 pmol of each primer, 0.4 mM (each) dNTP, and 1.25 U of Taq DNA polymerase. The amplification conditions consisted of an initial denaturation step at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s and finally an elongation step at 72 °C for 3 min. All of the PCR reactions were conducted using a GeneAmp PCR System 9700 (Applied Biosystems, CA, USA). DGGE was performed using 35–55% and 30–50% gradients for the bacterial and archaeal amplicons, respectively (Ben-Amor et al., 2005; Roest et al., 2005). The gels were run for 16 h at 75 V in 1X TAE buffer at 60 °C and stained using silver nitrate. PCR products of the expected size (409–433 bp) were cloned in a pGEM-T Easy vector and transformed into Escherichia coli JM109 competent cells (Promega, WI, USA) according to the manufacturer’s protocol. The plasmid inserts were amplified using M13F (50 -GTAAAACGACGGCCAG-30 ) and M13R (50 -CAGGAAACAGCTATGAC-30 ), and the cloned genes were sequenced in an automated 3730 DNA analyzer using the M13F primer (Applied Biosystems, CA, USA). Sequence similarity searches were performed using the BLAST network service of the NCBI database. Sequence alignments were performed using Clustal-X software (Koichiro et al., 2007). Mega 4.0 software was used to construct phylogenetic trees based on the neighbor-joining method (Larkin et al., 2007). The branches were tested using bootstrap analysis (1000 iterations). The GenBank accession numbers for the bacterial 16S rRNA gene sequences are HQ142902–HQ142909, HQ180355–HQ180379, and HQ231785–HQ231793.

3. Results 3.1. Performance of the MFC and TCOD removal efficiency The MFC was continuously operated in fed-batch mode for 120 days. After start-up, the electricity generation was found to be markedly increased on the third day, at which time the cell voltage output reached 520 mV (Fig. 2). This increase likely occurred because the cattle dung contained some easily degradable substrates (e.g., glucose, acetate) that could be utilized by electricity producing microorganisms, thus accelerating electricity generation. After 30 days of operation, stable electricity could be generated, and the cell voltage output ranged between 900 and 1000 mV over the subsequent 60 days. After approximately 40 days of operation, the cell voltage output remained stable, and various resistances were recorded to establish the relationship between

1000 800

Voltage (mV)

2.4. 16S rRNA gene amplification, DGGE (denaturing gradient gel electrophoresis) and sequence analysis

600 400 200 0 0

20

40

60

80

100

120

140

Time (day) Fig. 2. Electricity voltage outputs of the MFC.

the resistance and the voltage. The power density was obtained from our previous study (Zhao et al., 2010). The maximum open circuit voltage (OCV) was 1180 mV, and the highest volumetric power density (Pmax) was 0.220 W/m3. The internal resistance (Rint) of the cell was 96 X. After 120 days of operation, the TCOD removal efficiency reached 73.9 ± 1.8%, and the CE reached 2.79 ± 0.6% (Table 2). The low CE obtained was influenced by many factors, including the configuration of the reactor and the substrate type. In the present study, the low CE is most likely because the dominant bacteria in the anode removed COD but did not generate electricity. To improve CE going forward, the structure of the bioreactor should be improved to decrease the internal resistance. Many studies have indicated that increasing the volume of the MFC results in a low CE obtained (Liu et al., 2009; Hu, 2008). 3.2. VFA variations and biogas production The hydrolysis-acidification of cattle dung in the anode chamber is critical for biogas production. Therefore, pH variations during acidogenesis affect the stability of two-phase anaerobic digestion systems. Terminal fermentation VFAs likely include acetic acid and butyric acid when the pH is below 5.5 or above 6.0. The pH decreased from 7.15 to 6.52 after 25 days of operation and then remained stable between 6.2 and 6.5 (Fig. 3). Electricity generation was also stable over this time. However, even small accumulations of VFAs could decrease the pH. Thus, the concentration of VFAs has been found to reflect the metabolic status of anaerobic digestion (Björnsson et al., 2000; He et al., 2008). In this study, the main VFAs found during the acidogenesis of cattle dung were acetate, butyrate and propionate; other acids were virtually absent (Fig. 3). During the phases of continuous electricity generation by the hydrolysis–acidification process (between 0 and 50 days), the acetate and butyrate concentrations gradually increased, but the propionate concentration remained low. During acidogenesis, the maximum concentrations of acetate and butyrate reached 2059.7 and 369.1 mg/L, respectively. It is known that acetate is readily degraded into methane by Methanobacterium, which improves methane production. Furthermore, higher propionate concentrations dramatically inhibit the activity of acidogenic bacteria and methanogens (Barredo and Evison, 1991; Hu and Chen, 2007). However, after 50–120 days of fermentation, the acetate and propionate concentrations exhibited opposite trends; the acetate concentration was found to decrease rapidly, and the propionate concentration was found to increase gradually. The maximum propionate concentration was 383.4 mg/L during the electricity production process. The increase occurred because propionate could be used as a fuel to generate electricity and promote electricity production (Jang et al., 2010). Many studies have

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Table 2 Evolution of TCOD removal and coulombic efficiencies of the MFC over time. Operation time (d) 100

120

63.2 ± 1.5 2.58 ± 0.4

68.6 ± 1.1 2.66 ± 0.3

73.9 ± 1.8 2.79 ± 0.6

3000

Propionic acid pH

7.0 6.5

2500

6.0

2000

5.5 1500

pH

VFA concentation (mg/L)

Butyric acid Acetic acid

0 day

7.5

3500

70 days

80

49.5 ± 1.4 2.36 ± 0.3

20 days

60

38.2 ± 1.2 2.13 ± 0.3

0 day

40

15.6 ± 0.8 1.73 ± 0.2

70 days

20

20 days

TCOD removal efficiency (%) Coulombic efficiency (%)

5.0

1000

4.5

500

4.0

0

3.5 20

40

60

80

100

120

Time (day) Fig. 3. Variation of pH and VFA over operation.

100 Biogas

Methane

80

300

60 200 40 100

20

0

Methane in biogas (%)

-1

Biogas productivity (mL d )

400

0 0

2

4

6

8

10

12

Time (day)

a

b

Fig. 6. Denaturing gradient gel electrophoresis (DGGE) profiles of the V2–V3 region of the bacterial (a) and archaeal (b) 16S rRNA gene amplified with the following primer pairs: 101F-GC and 534R and 109F-GC and 515R.

Fig. 4. Biogas production and methane concentration in the anode.

discussed the relationships between VFAs and electricity production (Xing et al., 2009). Low propionate concentration is beneficial to methanogenesis and maintains a stable anaerobic digestion environment. The biogas production and the methane concentration in the biogas are shown in Fig. 4. Biogas production persisted for only

8 days, and the maximum production was 285 mL/d. The methane concentration in the biogas was 55–60%. These results suggest that electricity generation competed with the fermentation, methanogenesis or other biological processes. This competition may have resulted in the low CE and biogas production. He et al. (2005) investigated the treatment of wastewater, which contains ferment-

Fig. 5. Scanning electron micrographs of microbes on the carbon fibers obtained from the anode chamber.

G. Zhao et al. / Waste Management 32 (2012) 1651–1658

able substrates and alternative electron acceptors, and found CE values that ranged from 0.7 to 8.1% (He et al., 2005). 3.3. Analysis of microbial communities After 2 months of operation, microbes were found to be attached to the surface of the carbon brush at the anode (Fig. 5). The development of bacteria and archaea were investigated using PCR–DGGE on days 0 (cattle dung), 20 and 70 of acidogenesis. All of the bands were amplified from the V2–V3 region of the bacterial 16S rRNA gene using the universal primers 101F-GC and 534R

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(Fig. 6a). The representative band had a strong intensity and was excised from the DGGE gel. The complete sequences of 33 DNA fragments were successfully obtained, and the bands’ phylogenetic relationships are shown in Fig. 7a and Table 3. The microbial community’s diversity and band intensity increased on days 20 and 70 of the MFC operation (Fig. 6a) compared to day 0. The sequences obtained from the DGGE of the PCR-amplified 16S rRNA gene fragments exhibited great phylogenetic diversity. Five groups were identified: Proteobacteria, Bacteroides, Chloroflexi, Actinobacteria and Firmicutes. Proteobacteria (36.3%) were the dominant microbes, followed by Firmicutes (24.2%). Of

Fig. 7a. A phylogenetic tree demonstrates the relationships among the thirty-three predominant band sequences from the anode bacteria. The scale bar represents 0.05 inferred substitutions per nucleotide position.

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Fig. 7b. A phylogenetic tree demonstrates the relationship among the nine predominant band sequences from the anode archaea. The scale bar represents 0.05 inferred substitutions per nucleotide position.

Table 3 Dominant DGGE sequences of the 16S rRNA V2–V3 region from the anode at various time. Band

Accession no.

Closest GenBank identification

Identity (%)

Isolation source

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

HQ142902 HQ142903 HQ142904 HQ142905 HQ142906 HQ142907 HQ142908 HQ142909 HQ180355 HQ180356 HQ180357 HQ180358 HQ180359 HQ180360 HQ180361 HQ180362 HQ180363 HQ180364 HQ180365 HQ180366 HQ180367 HQ180368 HQ180369 HQ180370 HQ180371 HQ180372 HQ180373 HQ180374 HQ180375 HQ180376 HQ180377 HQ180378 HQ180379

Uncultured Porphyromonadaceae bacterium (EU073788) Uncultured Alpha proteobacterium (FM252759) Pseudomonas knackmussii (HM007154) Pseudomonassp. hyss58 (FJ613311) Trichococcus collinsii (EF111215) Variovorax sp. SGM115 (GU181268) Uncultured Clostridium sp. (AY330127) Uncultured Clostridia bacterium (EU551089) Uncultured Trichococcus sp. (GQ390392) Pseudomonas luteola (EU048332) Bacillus subtilis subsp. Subtilis (GU191901) Uncultured bacterium (CU922992) Ochrobactrum pseudogrignonense (FJ859687) Uncultured bacterium (CU926896) Uncultured bacterium (GU926368) Uncultured Chloroflexi bacterium (AY921658) Pseudomonas sp. 13BT (AF386741) Uncultured anaerobic bacterium (AY953220) Uncultured Clostridium sp. (GQ129976) Uncultured Ruminococcaceae bacterium (FJ542925) Enhydrobacter sp. M2T3B1 (GQ246700) Uncultured bacterium (CU926953) Uncultured rumen bacterium (GU120126) Bacillus thuringiensis (HM068889) Uncultured anaerobic bacterium (AY953150) Uncultured delta proteobacterium (EF662754) Mesorhizobium sp. WG (AF156710) Pseudomonas sp. XLDN4-9 (AY278245) Alcaligenes sp. BBTR16 (EF471233) Uncultured bacterium (DQ419579) Uncultured bacterium (CR933228) Agrobacterium tumefaciens (GU902302) Microbacterium hominis (EU977655)

98 91 99 99 98 98 98 96 99 99 99 98 99 100 100 91 98 93 99 91 99 99 99 99 99 93 99 99 93 99 97 98 96

Subsurface coal beds enrichment cultures Soil nematodes of various feeding groups Isolation from marine sediment in northeast China The deep-sea sediment of the southwest Pacific The Bogota River Soil sample in Korea Methanogenic landfill leachate bioreactor Anaerobic co-digestion of crops and cow manure

Proteobacteria, Gammaproteobacteria (18.2%) were detected more frequently than Alphaproteobacteria (12.1%) and Betaproteobacteria (6.1%). This result was consistent with previous research that used artificial wastewater as a fuel source and found Gammaproteobacteria and Firmicutes to be the most abundant microbes under copiotrophic conditions (Choo et al., 2006). Furthermore, Kim et al. (2006) observed that Gammaproteobacteria are highly abundant

Sindh agricultural fields Novozymes biologicals production strains Anaerobic digestion of sludge Tea rhizosphere Anaerobic digestion of sludge Anaerobic digestion of sludge Comparative metagenomics of microbial communities Axenic cultures of Aureococcus Anaerobic swine lagoons Clean rooms Eisenia earthworms Fermented bovine products Anaerobic digestion of sludge Sheep fed hay or corn Inhibition of toxins by Bacillus proteases Anaerobic swine lagoons Oxygen-limited bioreactors Chryseomonas sp. XLDN4-9 Swine manure Aerosolization of microorganisms by biogas Anaerobic digestion of sludge Mediterranean agricultural soils Phoenix spacecraft-associated surfaces

bacteria, indicating the possible involvement of Gammaproteobacteria in the production of electricity (Kim et al., 2006). Many microorganisms possess the ability to generate electricity from a variety of biodegradable materials, ranging from pure compounds such as acetate, glucose and ethanol to various organic materials including domestic human, animal and meat-packing wastewaters (Cheng et al., 2006b; Min et al., 2005). The phylum

G. Zhao et al. / Waste Management 32 (2012) 1651–1658

Proteobacteria was most frequently detected in eight different MFCs, followed by the phyla Firmicutes and Bacteroidetes (Clauwaert et al., 2008). Chloroflexi were enriched on the anode of a cellulose-fed MFC, indicating that they may be involved in cellulose hydrolysis and electricity production (Ishii et al., 2008) Our analysis indicated that the microbiota in cattle dung, which is a fermentable substrate, differ from those collected during the electricity generation process. Indeed, Firmicutes were the dominant phylum found in cattle dung, while Proteobacteria were the most abundant phylum present during the power generation process. This result is consistent with a previous study that used wastewater as an inoculum in a MFC with dissolved oxygen as the cathode and found Proteobacteria (45%) to be the dominant phylum in the microbial community (Kim et al., 2004). The DGGE analysis showed that the intensities of bands 9, 10, 11, 12, 13, 17 and 24 increased during the stable power generation periods (between 20 and 70 days). The gene sequences were identified as the following species: a bacterium that belonged to Trichococcus sp., Pseudomonas sp., Bacillus subtilis subsp., an unclassified bacterium, Ochrobactrum pseudogrignonense, Pseudomonas sp. and Bacillus sp. The Clostridium sp., Pseudomonas luteola and Ochrobactrum pseudogrignonense were the most dominant groups whose capacity for electricity generation has previously been shown (Park et al., 2001; Zuo et al., 2008; Rabaey et al., 2008). Bands 16, 18, 20, 26 and 29 were the most closely related to an uncultured Chloroflexi bacterium (91% similar), an uncultured anaerobic bacterium (93% similar), an uncultured Ruminococcaceae bacterium (91% similar), an uncultured Deltaproteobacterium (93% similar), and Alcaligenes sp. (93% similar), respectively. These low similarities to known bacteria suggest that new bacterial species maybe present. The microbial community’s diversity increased markedly during electricity production compared to that present in cattle dung. We speculated that when cattle dung was used as a substrate to generate electricity the hydrolysis and electricity generation processes would occur at the same time. Thus, we expected that two or more dominant microbial species would exist on the anode biofilm: one that would be responsible for hydrolysis and one that would be responsible for power generation. The first step was expected to be hydrolysis, during which the bacteria degrade the complex organic matter in cattle dung into simpler compounds. Then, the electricigens were expected to use these simple compounds to produce electricity. Though fermentative bacteria were also able to produce electricity in the MFC, only one third of the electrons were most likely available to produce electricity, whereas two thirds remained in the fermentation products (e.g., acetate, butyrate) (Logan, 2004). Furthermore, VFA components are present in high quantities during methane production, and a high propionate concentration could inhibit methanogen activity (Cabirol et al., 2002). Therefore, it is probable that MFC technology could simultaneously serve the following three functions: production of recycling power, provision of the necessary precursors for conversion into methane, and contribution to the alleviation of excess propionate. In this study, the propionate concentration was less than 20% of the total VFAs during the stable power generation periods (70 days of operation). Based on PCR-DGGE, the archaeal 16S rRNA gene showed nine DNA fragments (Fig. 6b), and the bands’ phylogenetic relationships are shown in Fig. 7b. The diversity of archaea decreased after 20 days of MFC operation. The most intense bands, 3 and 7, were found to belong to the Methanobacterium genus and disappeared during stable power generation. Band 6 was most closely related to Methanocorpusculum parvum (99% similar), which is a new strain that was isolated from cold terrestrial habitats and existed after 70 days of operation. Band 8 was related to Euryarchaeota, which were first found to occur in Admiralty Bay, King George Island.

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Bands 1 and 9 had little similarity to known archaea and may be previously unclassified archaea. The archaea detected are associated with hydrogenotrophic methanogens (Methanobacteriales) and are capable of using H2/CO2. Acetoclastic methanogens, however, were virtually absent. Based on this archaeal community investigation, it can be inferred that hydrogenotrophic methanogens might grow at the beginning of the operation due to the hydrogen produced by fermentative bacteria, not the hydrogen from acetate oxidation. A similar result from phylogenetic studies based on the 16S rRNA and methyl-coenzyme M reductase A (mcrA) genes suggests that the majority of the sequences retrieved from bovine rumens are affiliated with hydrogenotrophic methanogens belonging to Methanomicrobiales and Methanobacteriales (Tajima et al., 2001; Tatsuoka et al., 2004). Furthermore, it has been reported that some methanogens can directly reduce solid iron, suggesting that methanogens may directly contribute to power generation (Bond and Lovley, 2002). Further research is required to verify this possibility.

4. Conclusions This study demonstrated that cattle dung can be used as fuel to generate electricity. After 30 days of operation, stable electricity generation was observed, and after 120 days of operation, the total chemical oxygen demand (TCOD) removal and coulombic efficiency (CE) of the cell reached 73.9 ± 1.8% and 2.79 ± 0.6%, respectively. The main VFAs identified were acetate, butyrate and propionate, with acetate as the dominant metabolite. Biogas production persisted for only 8 days, and the maximum production was 285 mL/d. Microbial community analysis showed that Firmicutes was dominant in cattle dung, while Proteobacteria was the most abundant phylum during the power generation process. The archaea population was dominated by hydrogenotrophic methanogens (Methanobacteriales), which disappeared during the periods of stable power generation by acidogenesis.

Acknowledgements The authors gratefully acknowledge the support of the National Creative Research Group from the National Natural Science Foundation of China (No. 51121062).

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