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Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula
Accepted Manuscript Title: Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula Authors: Xiao Min Li, Ka Yu Cheng, Jonathan W.C. Wong PII: DOI: Reference:
S1359-5113(12)00370-4 doi:10.1016/j.procbio.2012.10.001 PRBI 9681
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
28-2-2012 6-10-2012 8-10-2012
Please cite this article as: Li XM, Cheng KY, Wong JWC, Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula, Process Biochemistry (2010), doi:10.1016/j.procbio.2012.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Process Biochemistry
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Bioelectricity production from acidic food waste leachate using
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microbial fuel cells: Effect of microbial inocula
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Xiao Min Li a, Ka Yu Cheng b, and Jonathan W.C. Wong a,*
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a
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of Biology, Hong Kong Baptist University, Hong Kong SAR b
CSIRO Land and Water, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Underwood Avenue, Floreat, WA6014, Australia
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Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department
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* Corresponding author. Tel.: +852 3411 7056; fax: +852 3411 2355.
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E-mail address: [email protected] (J.W.C. Wong).
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ABSTRACT
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The effects of three different inocula (domestic wastewater, activated sludge, and
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anaerobic sludge) on the treatment of acidic food waste leachate in microbial fuel cells
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(MFC) were evaluated. A food waste leachate (pH 4.76; 1,000 mg chemical oxygen
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demand (COD)/L) was used as the substrate. The results indicate that the leachate itself
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can enable electricity production in an MFC, but the co-addition of different inocula
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significantly reduces the start-up time (approximately 7 days). High COD and volatile
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fatty acids removal (> 87 %) were obtained in all MFCs but with only low coulombic
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efficiencies (CE) (14-20 %). The highest power (432 mW/m3) and CE (20 %) were
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obtained with anaerobic sludge as the co-inoculum. Microbial community analysis
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(PCR-DGGE) of the established biofilms suggested that the superior performance of the
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anaerobic sludge-MFC was associated with the enrichment of both fermentative
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(Clostridium sp. and Bacteroides sp.) and electrogenic bacteria (Magnetospirillum sp.
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and Geobacter sp.) at the anode.
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Keywords: bioelectrochemical systems; anaerobic digestion; waste-to-energy; acidic
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condition; microbial community
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1. Introduction Food waste constitutes the largest component of municipal solid waste (MSW) in
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many urbanized societies (30-55 % by weight). For example, in Hong Kong, there is
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approximately 3,200 tons of food waste produced every day, constituting 35 % of the
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total MSW, according to the Environmental Protection Department of Hong Kong. In
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2010, more than 34 million tons of food waste was generated in the United States, of
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which less than three percent was recovered and recycled, according to the U.S.
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Environmental Protection Agency [1]. Disposing of food waste in landfills is an
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unsustainable waste management practice, as the food waste still represents a valuable
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source of nutrients and energy. It has been estimated that approximately 2,030 trillion
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BTU (2,142 petajoule) of energy were embedded in the generated food waste (up to 27
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% of the total edible food) in the United States in 2007 [2].
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Anaerobic digestion is a proven technology for organic waste treatment and is able
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to recover the embedded energy in the organic waste (e.g., food waste) as renewable
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biogas (methane). To maximize biogas production, the hydrolysis and fermentation
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stages are usually separated from the final methanogenic stage using two-stage
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anaerobic digesters. However, the overall process is often limited by the detrimental
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acidic condition (pH < 5) produced by the accumulation of volatile fatty acids (VFAs)
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[3,4].
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Recently, microbial fuel cell (MFC) technology has been suggested to complement
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anaerobic digestion for bioenergy recovery [5-7]. MFCs are bioelectrochemical systems
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that can convert chemical energy stored in an organic substrate directly into electrical
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energy. Electrochemically active microorganisms are involved in this process to
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catalyze the oxidation of organic compounds using an insoluble electrode (anode) as an
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electron acceptor [8,9]. Unlike anaerobic digestion, MFCs are particularly suitable for
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treating low strength soluble organics such as VFAs and are less susceptible to
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unfavorable conditions (e.g., low temperature (≤ 20 °C) and low pH). Because the food
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waste leachate produced from the hydrolysis and acidogenic stages is rich in VFAs [10],
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it may be practical to use MFCs to covert VFAs in food waste leachate directly into
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electricity instead of loading the leachate to a final methanogenic reactor for methane
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production.
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In fact, a variety of organic feedstocks have been shown to drive electricity
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production in MFCs; these feedstocks range from simple organics, such as glucose [11],
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acetate, butyrate [12], ethanol and methanol [13], to complex substrates, such as yogurt,
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starch, protein and meat processing wastewater [14-17]. A mixture of synthetic VFAs
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and VFAs from the fermentation of glucose and food waste has been reported to be
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removed with concomitant electricity production in an MFC [18-21]. Furthermore, the
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use of MFCs as a secondary energy recovery step in fermentative hydrogen (H2)
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production processes has been evaluated using cereal wastewater and vegetable waste as
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substrates [22,23]. However, the anodic pH conditions in the aforementioned studies
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were maintained at approximately neutral by dosing buffers or alkaline substances,
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which are costly and non-sustainable. Arguably, operating an MFC under anodic
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conditions in which the pH is not controlled or buffered (low pH) is preferred for
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practical food waste leachate treatment.
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The aim of this study was to investigate the use of acidic food waste leachate
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produced from the hydrolysis and acidogenic stage of an anaerobic digester as an MFC
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feedstock for electricity generation. The effects of three commonly used MFC inoculum
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sources, namely, (i) domestic wastewater, (ii) activated sludge, and (iii) anaerobic
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sludge, on the MFC performance (power generation and substrate removal) were
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compared. The acclimated microbial communities in different treatments are also
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reported.
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2. Materials and methods
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2.1. Food waste leachate
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Food waste leachate collected from a leach bed reactor was used throughout the
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entire experiment and stored at 4 °C to avoid compositional changes before use. As
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previously reported [10], the total and working volume of the leach bed reactor were 6.4
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and 4.6 L, respectively. The leach bed reactor was provided with a perforated plate at
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the bottom for leachate collection. On the top of the perforated plate, 0.5 kg of an
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acid-washed and oven-dried sand bed was placed with a nylon screen support to
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facilitate the percolation of the leachate. The leach bed reactor was initially fed with a
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synthetic food waste mixture, which consisted of 350 g/kg of bread, 250 g/kg of rice,
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250 g/kg of cabbage, and 150 g/kg of pork. The pH, electrical conductivity, total
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chemical oxygen demand (COD), and total VFAs of the leachate were 4.76, 3.58 mS/cm,
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52,200 mg/L, and 12,700 mg/L, respectively.
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2.2. Microbial inocula
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Three types of inocula, (i) municipal wastewater from a primary clarifier, (ii)
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activated sludge, and (iii) anaerobic sludge from an anaerobic digester, were compared,
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the total COD concentrations of which were 753 mg/L, 1,064 mg/L, and 14,056 mg/L,
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respectively. All the inocula were collected from a domestic wastewater treatment plant
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in Shek Wu Hui, New Territories, Hong Kong.
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2.3. Reactor configuration and operation The MFCs consisted of two equal rectangular chambers separated by a cation
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exchange membrane (6.8 × 6.8 cm, Qianqiu Group Co., Ltd., Zhejiang, China). Each
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cell chamber had a working volume of 75.6 ml (6.0 × 6.0 × 2.1 cm). Both the anode and
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cathode were constructed of carbon felt (4.5 × 4.5 × 0.5 cm each, Liaoyang Jingu
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Carbon Fibre Sci-Tech Co., Ltd., Tianjin, China). A titanium wire was inserted into the
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carbon felt to allow electrical contact with the external circuit. The carbon felt was
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pretreated according to a previous report [24]. The anode potentials were measured
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against a saturated calomel reference electrode (SCE, 242 mV against the standard
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hydrogen electrode, SHE), which was inserted into the anode chamber. The cathode
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potentials were calculated as the sum of the anode potential and the cell voltage. The
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reported electrode potentials refer to values against SCE.
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Four MFC settings were operated simultaneously: (1) Control-MFC, food waste
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leachate only; (2) W.w.-MFC, food waste leachate and wastewater; (3) AS-MFC, food
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waste leachate and activated sludge; and (4) AnS-MFC, food waste leachate and
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anaerobic sludge. To start up the reactors, the anodic half cells were fed with inocula
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and leachate (with a COD ratio of 1:10) and operated in batch mode for three batch
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cycles (hereafter known as Run 1 to 3). Thereafter, all suspended inoculum residues that
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were unable to be retained as a biofilm at the anode were discarded, and only the food
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waste leachate was replenished in the anodic chambers. The leachate used in each run
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was diluted with deionized water to approximately 1,000 mg COD/L. All cathodic
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chambers were fed with air-saturated deionized water to standardize the initial cathodic
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condition in all treatments. Because diffusion of ionic species across the ion exchange
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membrane between the anolyte and the catholyte would occur after the catholyte
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renewal, all the catholytes were ionically conductive throughout the experimental runs.
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Unless otherwise stated, a fixed external resistance (1,000 Ω) was used to facilitate the
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comparison of the current production in different treatments. The reactors were operated
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in batch mode at 28 ± 2 °C.
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2.4. Chemical analysis
COD (including both soluble and particulate) was determined using a standard
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dichromate oxidation (open reflux method) method [25]. For the VFA analysis, the
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liquid samples were filtered through a 0.45 µm mixed cellulose ester membrane filter,
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and then, 0.9 ml of the filtrate was transferred to amber GC vials and mixed with 0.1 ml
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of formic acid before analysis. An HP 6890 Series gas chromatograph (Hewlett Packard)
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was employed with a flame ionization detector and an Econo-Cap EC-1000 (15 m ×
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0.53 mm × 1.20 μm) column. Nitrogen was used as a carrier gas, whereas air and
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hydrogen were used for combustion. Samples (1.0 μl) were analyzed using a
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temperature program beginning with an initial temperature of 75 °C for 1 min,
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increasing to 180 °C at a rate of 6 °C/min, then increasing to 230 °C at a rate of
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10 °C/min, and finally maintaining 230 °C for 5 min to ensure complete VFA
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volatilization. Concentrations were determined using a standard curve obtained by
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injecting standard solutions of acetic acid, propionic acid, butyric acid, iso-butyric acid,
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valeric acid, iso-valeric acid and hexanoic acid. The sum of these seven VFA
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concentrations is reported as the total VFAs.
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2.5. Microbial community analysis
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Biofilms were harvested from the carbon felt anode of different MFCs for microbial
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community analysis after all systems were operated for over three months. Genomic
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DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN) according to the
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manufacturer’s instructions. Denaturing gradient gel electrophoresis (DGGE) analysis
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focused on the V3 region of the 16S rRNA gene, which was amplified using separate
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primers to target bacterial sequences. Amplification used the primers 805R (5'-GAC
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TAC CAG GGT ATC TAA TCC-3') and GC-341F (5'-CGC CCG CCG CGC GCG GCG
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GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CA-3'),
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containing the GC clamp on the amplified 16S rDNA template. PCR mixtures contained
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12.5 μl of 2× PCR Master Mix (Promega), 1 µl of each primer and 10 ng of DNA
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extraction product, and sterile Milli-Q water was added to a final volume of 25 μl. The
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samples were amplified in a PTC-200 (Bio-Rad Laboratories, Hercules, CA) with an
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initial denaturation of DNA for 5 min at 94 °C followed by 33 cycles of 30 s at 94 °C,
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20 s at 55 °C, and 45 s at 72 °C with a final extension for 7 min at 72 °C. Blank controls
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were performed throughout all steps.
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DGGE was performed with a DCode universal mutation detection system (Bio-Rad
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Laboratories, Hercules, CA). Approximately 1 µg of PCR product per lane was loaded
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onto 6.5 % (wt/vol) polyacrylamide (37.5:1 acrylamide:bisacrylamide) gels in a 1× TAE
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buffer with a denaturing gradient ranging from 40 % to 60 %. Denaturation of 100 %
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corresponds to 7 M urea and 40 % (vol/vol) deionized formamide. Gel electrophoresis
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was performed at 60 °C for 14 h at 75 V. The gel was subsequently stained in 1× TAE
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buffer containing a 1:100,000 dilution of RedSafe nucleic acid staining solution
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(iNtRON, 20,000×) for 1 h before being photographed on a blue light transilluminator.
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Ten DGGE bands were excised for sequencing.
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A small portion of each selected band was excised from the DGGE gel using a
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sterile scalpel blade and immediately added to a premixed gel elution buffer at 4 °C
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overnight. PCR reactions of identical composition were conducted as mentioned above,
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and the PCR products were analyzed by DGGE. The position of the extracted DNA
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bands was then compared to the original gel using BioNumerics software. After
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confirming the presence of target bands, the bands were excised, amplified and analyzed
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by DGGE. This cycle was repeated until a single dominant band was obtained from
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each extracted band. The selected bands were then sent to the Hong Kong Genome
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Research Facility for sequencing. Sequence analyses were performed using the online
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BLAST software tool (http://www.ncbi.nlm.nih.gov).
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2.6. Data collection and calculation
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To quantify the cell performance throughout the experiments, the potential
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difference between the anode and cathode (i.e., cell voltage, V) was recorded every
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minute using a data acquisition system (PicoLog 1216, Pico Technology,
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Cambridgeshire, UK) connected to a personal computer. Current density (I) and power
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density (P) normalized to the volume of the anodic chamber were calculated according
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to I = V R−1 and P = V2 R−1, respectively, where R is the external resistance. When all
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MFCs were acclimatized for steady power outputs, polarization and power density
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curve analysis was performed to compare the MFC performance in different treatments.
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This analysis was performed by adjusting the external resistance from 10 to 40,000 Ω,
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and pseudo-steady state voltage was recorded at each resistance level. The internal
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resistance of the MFC was obtained from the slope of the linear region of the
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polarization curve according to Logan et al. [8]. Coulombic efficiency (CE) in the
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fed-batch mode was estimated according to the formula shown below, where M is the
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molecular weight of oxygen (32), F is Faraday’s constant (96,485 C), b represents the
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number of electrons exchanged per mole of oxygen (4), vAn is the volume of liquid in
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the anode compartment (0.075 L), and ∆COD is the change in COD over a period of
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time (tb) for each run [8].
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3. Results and Discussion
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3.1. Effect of the inocula on the MFC electrical output
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The power output of the described MFCs over the experimental period is shown in
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Fig. 1. MFCs amended with additional inocula (W.w-MFC, AS-MFC, and AnS-MFC)
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began to produce electricity after 7 days, whereas the control demonstrated a
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remarkably slower start-up (after 12 days). This result indicates that the food waste
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leachate is able to serve as an inoculum for electricity generation but the co-addition
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domestic wastewater, activated sludge, and anaerobic sludge accelerates the start-up
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process.
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During Run 2 and 3, renewals of food waste leachate and inocula in all MFCs (at
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day 28 and day 43) resulted in immediate increases in power outputs in all treatments,
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indicating that all MFCs had developed electrochemically active biofilms at the anode.
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Subsequent leachate renewal in Run 4 and 5, again, resulted in a sharp, reproducible
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increase in power density in all treatments (Fig. 1). The maximal power outputs
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obtained from Run 4 and 5 were 195.4 ± 18.3, 453.9 ± 10.4, 316.1 ± 6.0 and 445.6 ±
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15.2 mW/m3 for the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC, respectively
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(Table 1), and followed a descending order of W.w.-MFC ≈ AnS-MFC > AS-MFC >
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Control-MFC.
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[Fig. 1.]
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[Table 1]
3.2. COD and VFA removal
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An increase in the COD removal efficiency was observed in all MFCs as the
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electricity outputs increased gradually from day 0 to 58 (Runs 1-3) (data not shown).
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After the initial two-month enrichment, all MFCs demonstrated high COD and VFA
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removal efficiencies of 87-92 % and 89-96 %, respectively (Runs 4 and 5) (Table 1). Fig.
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2 illustrates the changes in the individual VFA concentrations in different treatments
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during a batch cycle (Run 4). In all treatments, neither propionic acid nor valeric acid
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was detected, whereas acetic acid, butyric acid, and hexanoic acid were found to be the
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major species. In general, all VFAs were gradually removed from the anolyte of all
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MFCs during the current production cycle (Figs. 1 and 2). However, hexanoic acid was
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found to be the dominant residual VFA (4-11 % of the initial VFAs), suggesting that
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long-chain VFAs might be less degradable as compared with short-chain VFAs in the
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tested systems. The significant increase of accumulated acetate in the AnS-MFC (D) as
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compared with the Control-MFC (A), W.w.-MFC (B), and AS-MFC (C) from day 4 to
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day 8 may imply that the acetate production rate was higher than that of the acetate
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consumption in the AnS-MFC (Fig. 2), since during each batch cycle, acetate could be
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produced from the fermentable compounds in the leachate by the fermenters, and the
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produced acetate could be anodically oxidized by the electrogenic microorganisms to
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generate electricity.
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[Fig. 2.]
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3.3. Coulombic efficiencies and pH changes Similar to other studies that use complex substrates such as real wastewater as the
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MFC feedstock, the CE values obtained in this study were low (13 to 20 %) (Table 1)
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[26]. It has been suggested that high CE can be obtained with acetic acid as the main
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electron donor [18], whereas a high level of butyric acid in the medium can result in low
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CE [19]. In our experiment, butyric acid accounted for more than 50 % of the total
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VFAs in the food waste leachate, whereas acetic acid was less than 12 % (Fig. 2). Such
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a unique VFA composition of the tested food waste leachate might account for the
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observed low CE. On the other hand, substrate consumption via other competing
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metabolic processes, such as fermentation and methanogenesis, might also explain the
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low CE [17]. Among all treatments, the W.w.-MFC and AnS-MFC had significantly
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higher CE when compared with the Control-MFC and AS-MFC (Table 1), indicating
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that the anodic biofilms established with the co-additions of domestic wastewater and
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anaerobic sludge were more capable of utilizing the food waste leachate for electricity
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production in the MFCs.
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A gradual increase in the anolyte pH was observed in the Control-MFC (Run 4) (Fig.
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3), which is contradictory to the commonly observed anolyte acidification due to anodic
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COD oxidation [27,28]. Nevertheless, the increase in the pH in this condition may be
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due to the loss of VFAs in the anolyte via non-bioelectrochemical oxidation (e.g.,
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oxygen intrusion from the catholyte). It has been reported that VFA oxidation leads to
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an increase in the pH of VFA-laden manure slurries [29]. However, the observed
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increase in the pH is unlikely to have resulted from the release of ammonia through the
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hydrolysis of proteins in the food waste leachate, as the amount of ammonia in the
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anolyte was found to decrease over time (data not shown) [16]. At the beginning of the
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batch cycle, 12.6 mg/L of ammonia was detected in the anolyte, and its concentration
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gradually decreased until it became undetectable (< 0.1 mg/L) at the end of the cycle.
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Alternatively, the decrease in the pH observed in the W.w.-MFC, AS-MFC, and
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AnS-MFC before day 8 might also be due to the formation of other acidic compounds,
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such as amino acids and long-chain fatty acids, from the food waste leachate. However,
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further studies are required to determine the formation of these compounds during the
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process.
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3.4. Polarization characteristics
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The effects of different inocula on the MFC performance were compared based on
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the polarization behavior and maximal power output recorded after all MFCs were
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acclimatized with the food waste leachate (Fig. 4). All MFCs produced an open circuit
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voltage (OCV) of approximately 400 mV, which is consistent with other studies using
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fermenting vegetable waste effluents as the MFC substrate but under neutral conditions
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[23]. Among all treatments, the Control-MFC was more susceptible to polarization (i.e.,
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deviation of the cell voltage from the OCV) compared with other treatments receiving
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additional microbial inoculum (Fig. 4a). This result suggests that the external inoculum
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is able to establish a better anodic biofilm for electricity production. The distinctive
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polarization patterns observed in different treatments are also noteworthy. At a current
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density < 2000 mA/m3, the polarization followed an order of AnS-MFC > W.w.-MFC >
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AS-MFC, whereas at a current density > 2000 mA/m3, the order was AnS-MFC >
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AS-MFC > W.w.-MFC. Such a change was due to an abrupt polarization in the
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W.w.-MFC at a current density of approximately 2200 mA/m3 and may be explained by
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the so-called “power overshoot” phenomenon in which the cell resistance suddenly
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increased, leading to decreases in power density as external loads decrease [30].
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Resistance in the electroactive bacteria for substrate utilization was proposed to induce
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MFC power overshoot [31]; therefore, it is important to understand the microbial
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community attached on the anode.
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The internal resistances of the W.w.-MFC, AnS-MFC, AS-MFC, and Control-MFC
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were 1137, 1174, 1918, and 2384 Ω, respectively (Fig. 4a), and the respective maximal
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power outputs were 363, 432, 267, and 251 mW/m3 (Fig. 4b). The maximum power
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density of the AnS-MFC was higher than that of the W.w.-MFC even though they both
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had a similar internal resistance. No clear relationship between internal resistance and
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power output was noted in the tested systems. The internal resistances measured in this
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study were based on the use of a polarization curve method, which only reflects the
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ohmic resistance of the MFC. While it is generally expected that the power density of an
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MFC is indirectly related to the internal resistance, other factors such as the affinity of
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the anodic biofilm for the substrate (electron donor) and the anode (electron acceptor)
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may also affect the current production and hence the power. These factors may explain
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the observed independency between power and internal resistance in the W.w.-MFC and
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AnS-MFC.
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At open circuit, the anode potentials for all MFCs were similar (-372 ± 9.15 mV)
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according to Fig. 5. All the anode potentials increased (became less negative) slightly
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with the current density, particularly in the lower current density range (0-1000 mA/m3),
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but became unsteady at higher current densities in the Control-MFC, W.w.-MFC, and
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AS-MFC. The increase in the anode overpotential at high current densities suggests that
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the established anodic bacteria were unable to transfer electrons to the anode at a
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sufficient rate to maintain the current [12]. Because the AnS-MFC had the lowest anode
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overpotential and was able to sustain the highest current output among all treatments,
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anaerobic sludge appeared to be the most suitable inoculum. [Fig. 4.]
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[Fig. 5.]
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3.5. Microbial communities
To understand the effect of the various co-inocula on the anode community structure
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in the food waste leachate-treated MFCs, the anode-attached biofilms established in
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different MFCs were characterized using the PCR-DGGE technique (Fig. 6). A number
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of bands excised from the gels were processed for sequence analysis (Table 2). In
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general, bacteria of the phylum Proteobacteria were dominant in all MFCs. A similar
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finding was reported by Freguia et al. [18] with their anodic biofilm community that
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was enriched with mixed-VFAs. Bands 1 and 2 were found in all MFCs, whereas bands
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4 and 5 were predominant in the W.w-MFC. Additionally, bands 3, 9, and 10 were also
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found in the AnS-MFC.
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Clostridium sp. (band 1) is capable of producing H2 in addition to acetone, butanol,
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and ethanol from sugar, glucose or starch under anaerobic conditions [32]. Liang et al.
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[33] reported that Clostridium sp. is the predominant microbial species found in an
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acidic (pH 2-6) anaerobic H2-producing sequencing batch reactor. Bacteroides sp. (band
341
3) is known to be able to produce H2 fermentatively and to produce electricity in MFCs
342
[34]. Aside from VFAs, the food waste leachate used in this study also contained other
15 Page 15 of 30
343
organic matter (collectively measured as COD). The fermentation of this organic matter
344
in the food waste leachate might be enhanced by Clostridium sp. and Bacteroides sp.,
345
especially in MFCs co-inoculated with activated sludge and anaerobic sludge. Geobacter sp. (band 5) have been widely described for their capacity to perform
347
direct electron transfer and are commonly found in MFC microbial communities
348
particularly when acetate is provided as the electron donor [35]. Magnetospirillum sp.
349
(band 4) has been reported to be able to use fermentation end products, such as acetate
350
and ethanol, as carbon sources and electron donors for heterotrophic growth, and can be
351
grown chemolithotrophically with hydrogen as the electron donor [36]. The presence of
352
Magnetospirillum sp. in the W.w.-MFC and AnS-MFC may facilitate current generation
353
by anodically oxidizing the fermentation end products produced by the fermenters.
M
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The enrichment of both fermentative (Clostridium sp. and Bacteroides sp.) and
355
electrogenic species (Magnetospirillum sp. and Geobacter sp.) in the anodic community
356
of the AnS-MFC may explain why this treatment, in particular, was able to more
357
efficiently use the leachate for electricity production. However, further research is
358
needed to elucidate the interaction between non-electrogenic and electrogenic bacteria
359
for food waste leachate treatment using MFCs.
Ac ce
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ed
354
360
[Fig. 6.]
361
[Table 2]
362 363
4. Conclusions
364
Acidic food waste leachate can serve as an inoculum for electricity generation in
365
MFCs, but the co-addition of domestic wastewater, activated sludge or anaerobic sludge
366
accelerates the MFC start-up process. Efficient COD and VFA removal (> 87 %) were
16 Page 16 of 30
367
achieved in all MFCs, but only low CE (14-20 %) were obtained using the food waste
368
leachate as the MFC substrate. Among all treatments, the highest power output and CE
369
were obtained with anaerobic sludge as the co-inoculum, and this result might be due to
370
the enrichment of both fermentative and electrogenic bacteria at the anode over time.
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373
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[18] Freguia S, The EH, Boon N, Leung KM, Keller J, Rabaey K. Microbial fuel cells operating on mixed fatty acids. Bioresour Technol 2010;101:1233–1238.
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[19] Teng SX, Tong ZH, Li WW, Wang SG, Sheng GP, Shi XY, et al. Electricity
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[20] Jeong CM, Choi JDR, Ahn YH, Chang HN. Removal of volatile fatty acids (VFA)
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by microbial fuel cell with aluminum electrode and microbial community
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identification with 16S rRNA sequence. Korean J Chem Eng 2008;25:535–541.
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[21] Choi JDR, Chang HN, Han JI. Performance of microbial fuel cell with volatile fatty acids from food wastes. Biotechnol Lett 2011;33:705–714.
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[22] Oh SE, Logan BE. Hydrogen and electricity production from a food processing
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wastewater using fermentation and microbial fuel cell technologies. Water Res
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[23] Mohanakrishna G, Mohan SV, Sarma PN. Utilizing acid-rich effluents of
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fermentative hydrogen production process as substrate for harnessing bioelectricity: An integrative approach. Int J Hydrog Energy 2010;35:3440–3449.
[24] Liu L, Li FB, Feng CH, Li XZ. Microbial fuel cell with an azo-dye-feeding cathode. Appl Microbiol Biotechnol 2009;85:175–183.
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[25] APHA, Standard methods for the examination of water and wastewater, 21st ed.
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American Public Health Association, American Water Works Association and
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municipal,
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Electroanalysis 2010;22:832–843.
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food,
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[27] Cheng KY, Ho G, Cord-Ruwisch R. Anodophilic biofilm catalyzes cathodic oxygen reduction. Environ Sci Technol 2010;44:518–525.
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[28] Harnisch F, Schroder U, Scholz F. The suitability of monopolar and bipolar ion
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exchange membranes as separators for biological fuel cells. Environ Sci Technol
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2008;42:1740–1746.
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[29] Paul JW, Beauchamp EG. Relationship between volatile fatty acids, total ammonia, and pH in manure slurries. Biological Wastes 1989;29:313–318.
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[30] Logan BE. Essential data and techniques for conducting microbial fuel cell and
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other types of bioelectrochemical system experiments. ChemSusChem 2012;5:
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988–994.
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[31] Nien PC, Lee CY, Ho KC, Adav SS, Liu LH, Wang AJ, Ren NQ, Lee DJ. Power
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overshoot in two-chambered microbial fuel cell (MFC). Bioresour Technol
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2010;102:4742–4746.
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[32] Andersch W, Bahl H, Gottschalk G. Level of enzymes involved in acetate, butyrate,
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acetone and butanol formation by Clostridium acetobutylicum. Eur J Appl
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Microbiol Biotechnol 1983;18:327–332.
458
[33] Liang DW, Shayegan SS, Ng WJ, He JZ. Development and characteristics of
459
rapidly formed hydrogen-producing granules in an acidic anaerobic sequencing
460
batch reactor (AnSBR). Biochem Eng J 2010;49:119–125.
461
[34] Kim GT, Webster G, Wimpenny JWT, Kim BH, Kim HJ, Weightman AJ. Bacterial
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community structure, compartmentalization and activity in a microbial fuel cell. J
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463 464 465
Appl Microbiol 2006;101:698–710. [35] Bond DR, Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003;69:1548–1555. [36] Thrash JC, Ahmadi S, Torok T, Coates JD. Magnetospirillum bellicus sp. Nov., a
467
novel dissimilatory perchlorate-reducing Alphaproteobacterium isolated from a
468
bioelectrical reactor. Appl Environ Microbiol 2010;76:4730–4737.
cr
ip t
466
469
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pt
ed
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21 Page 21 of 30
Figure legends
471
Fig. 1. Power density (mW/m3) profiles of different food waste leachate-fed MFCs
472
co-inoculated with various microbial inocula (Control: without co-inoculum; W.w.:
473
domestic wastewater; AS: activated sludge; AnS: anaerobic sludge) over the course of
474
five batch cycles (Runs 1-5). For Runs 1-3, both co-inoculum and food waste leachate
475
were renewed in each cycle. For Runs 4 and 5, only food waste leachate was renewed.
476
Fig. 2. A profile of the concentration of VFAs over a current-producing batch cycle
477
(Run 4) in the Control-MFC (A), W.w.-MFC (B), AS-MFC (C), and AnS-MFC (D).
478
Fig. 3. Change in the anolyte pH over a current-producing batch cycle (Run 4) in the
479
Control-MFC (A), W.w.-MFC (B), AS-MFC (C), and AnS-MFC (D).
480
Fig. 4. Voltage (a) and power density (b) as a function of current density obtained over a
481
batch cycle (Run 4) from the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC.
482
Fig. 5. Electrode potentials (vs. SCE) as a function of current density obtained over Run
483
4 from the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC.
484
Fig. 6. DGGE profiles obtained from the mature anodic biofilms established in different
485
MFC reactors. Control: Control-MFC; W.w.: W.w.-MFC; AS: AS-MFC; and AnS:
486
AnS-MFC.
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470
22 Page 22 of 30
Table 1.
488
Maximal power density, COD removal, VFA removal, and coulombic efficiency (CE) in
489
different food waste leachate-fed MFCs inoculated with various microbial inocula
490
(Values are the mean ± standard deviation of the data obtained over Runs 4 and 5). Maximal power COD removal VFA removal
CE (%)
density (%)
(%)
89.96 ± 0.64
14.36 ± 2.01
91.60 ± 1.05
20.27 ± 2.06
94.30 ± 0.26
13.65 ± 1.11
96.61 ± 0.89
20.26 ± 0.33
195.43 ± 18.25
91.27 ± 1.09
W.w.-MFC
453.90 ± 10.44
90.97 ± 0.08
AS-MFC
316.12 ± 5.95
92.21 ± 1.10
AnS-MFC
445.61 ± 15.17
87.13 ± 2.30
M
an
Control-MFC
us
(mW/m3)
cr
Treatments
ip t
487
Control-MFC: without co-inoculum; W.w.-MFC: domestic wastewater; AS-MFC:
492
activated sludge; and AnS-MFC: anaerobic sludge.
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491
23 Page 23 of 30
493
Table 2.
494
Overview of the sequencing results of the excised bands from the DGGE analysis (Fig.
495
6).
DGGE DGGE
Sequence
Closest
similarity match
accession
(%)
number
Phylum
ip t
band
accession
cr
band
Closest match
number
JX193892
Clostridium acetobutylicum HP7
98
FM994940.1
Firmicutes
2
JX193893
Burkholderia vietnamiensis RPB3
99
HQ606073.1
Proteobacteria
3
JX193894
Uncultured Bacteroides sp. J3
99
DQ168847.1
Bacteroidetes
4
JX193895
Uncultured Magnetospirillum sp.
98
FJ823930.1
Proteobacteria
5
JX193896
Geobacter pickeringii G13
97
DQ145535.1
Proteobacteria
6
JX193897
Novosphingobium nitrogenifigens Y88
100
DQ448852.1
Proteobacteria
99
FJ570212.1
Acidobacteria
ed
M
an
us
1
Uncultured Acidobacteria bacterium
7 JX193898
pt
A6YA20RM JX193899
Uncultured Aeromonas sp. ASP-21
98
EF679186.1
Proteobacteria
9
JX193900
Burkholderia sp. C527
99
HQ704709.1
Proteobacteria
96
EF613974.1
Acidobacteria
Ac ce
8
Uncultured Acidobacteria bacterium
10
JX193901
NGA53
496
24 Page 24 of 30
496
Fig. 1.
Run4
Run5
500 400
cr
300
ip t
Control-MFC W.w.-MFC AS-MFC AnS-MFC
us
200 100
an
Power density (mW/m3)
Enrichment (Run 1 to 3)
0 20
40
60
80
M
0
Time (days)
Ac ce
pt
ed
497
25 Page 25 of 30
Fig. 2.
300 Acetate Iso-butyrate Butyrate Iso-valerate Hexanoate
ABCD
ip t
ABCD
150 ABCD
100
cr
200
us
VFAs (mg/L)
250
an
50 0 4
8 Time (days)
13
Ac ce
pt
ed
M
0
ABCD
26 Page 26 of 30
Fig. 3.
6.5
cr
5.5
us
pH
6.0
ip t
Control-MFC W.w.-MFC AS-MFC AnS-MFC
an
5.0
4.5 4
8
12
M
0
Ac ce
pt
ed
Time (days)
27 Page 27 of 30
Fig. 4.
500 Control-MFC W.w.-MFC AS-MFC AnS-MFC
300
cr
200 100
us
Voltage (mV)
400
1000
2000
3000 Current density (mA/m3)
M
0
an
0
600
4000
(b)
ed
Control-MFC W.w.-MFC AS-MFC AnS-MFC
500
pt
400 300
Ac ce
Power density (mW/m3)
ip t
(a)
200 100
0
0
1000 2000 3000 Current density (mA/m3)
4000
28 Page 28 of 30
Fig. 5.
100 Control-MFC (Cathode) W.w.-MFC (Cathode)
0
AS-MFC (Cathode)
ip t
-100
cr
Control-MFC (Anode) W.w.-MFC (Anode)
-200
us
AS-MFC (Anode) AnS-MFC (Anode)
-300
an
Potential (mV)
AnS-MFC (Cathode)
-400 0
600
1200
1800
2400
3000
3600
Ac ce
pt
ed
M
Current density (mA/m3)
29 Page 29 of 30
Fig. 6. Highlights
Control
W.w.
AS
AnS
ip t
1
cr
2
4 5
an
6
us
3
M
7
9 10
Ac ce
pt
ed
8
1. Electricity was produced from acidic food waste leachate using microbial fuel cells (MFCs) 2. Domestic wastewater, activated sludge and anaerobic sludge were compared as MFC co-inoculum. 3. Anaerobic sludge was the best due to the enrichment of fermentative/ electrogenic bacteria. 4. Food waste leachate alone could already serve as a MFC inoculum for bioelectricity production.
30 Page 30 of 30
S1359-5113(12)00370-4 doi:10.1016/j.procbio.2012.10.001 PRBI 9681
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
28-2-2012 6-10-2012 8-10-2012
Please cite this article as: Li XM, Cheng KY, Wong JWC, Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula, Process Biochemistry (2010), doi:10.1016/j.procbio.2012.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Process Biochemistry
2
Bioelectricity production from acidic food waste leachate using
4
microbial fuel cells: Effect of microbial inocula
ip t
3
5
Xiao Min Li a, Ka Yu Cheng b, and Jonathan W.C. Wong a,*
cr
6
a
9
11
of Biology, Hong Kong Baptist University, Hong Kong SAR b
CSIRO Land and Water, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Underwood Avenue, Floreat, WA6014, Australia
M
10
Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department
an
8
us
7
12
* Corresponding author. Tel.: +852 3411 7056; fax: +852 3411 2355.
14
E-mail address: [email protected] (J.W.C. Wong).
pt Ac ce
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13
1 Page 1 of 30
ABSTRACT
16
The effects of three different inocula (domestic wastewater, activated sludge, and
17
anaerobic sludge) on the treatment of acidic food waste leachate in microbial fuel cells
18
(MFC) were evaluated. A food waste leachate (pH 4.76; 1,000 mg chemical oxygen
19
demand (COD)/L) was used as the substrate. The results indicate that the leachate itself
20
can enable electricity production in an MFC, but the co-addition of different inocula
21
significantly reduces the start-up time (approximately 7 days). High COD and volatile
22
fatty acids removal (> 87 %) were obtained in all MFCs but with only low coulombic
23
efficiencies (CE) (14-20 %). The highest power (432 mW/m3) and CE (20 %) were
24
obtained with anaerobic sludge as the co-inoculum. Microbial community analysis
25
(PCR-DGGE) of the established biofilms suggested that the superior performance of the
26
anaerobic sludge-MFC was associated with the enrichment of both fermentative
27
(Clostridium sp. and Bacteroides sp.) and electrogenic bacteria (Magnetospirillum sp.
28
and Geobacter sp.) at the anode.
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Keywords: bioelectrochemical systems; anaerobic digestion; waste-to-energy; acidic
31
condition; microbial community
32
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30
2 Page 2 of 30
32
1. Introduction Food waste constitutes the largest component of municipal solid waste (MSW) in
34
many urbanized societies (30-55 % by weight). For example, in Hong Kong, there is
35
approximately 3,200 tons of food waste produced every day, constituting 35 % of the
36
total MSW, according to the Environmental Protection Department of Hong Kong. In
37
2010, more than 34 million tons of food waste was generated in the United States, of
38
which less than three percent was recovered and recycled, according to the U.S.
39
Environmental Protection Agency [1]. Disposing of food waste in landfills is an
40
unsustainable waste management practice, as the food waste still represents a valuable
41
source of nutrients and energy. It has been estimated that approximately 2,030 trillion
42
BTU (2,142 petajoule) of energy were embedded in the generated food waste (up to 27
43
% of the total edible food) in the United States in 2007 [2].
M
an
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33
Anaerobic digestion is a proven technology for organic waste treatment and is able
45
to recover the embedded energy in the organic waste (e.g., food waste) as renewable
46
biogas (methane). To maximize biogas production, the hydrolysis and fermentation
47
stages are usually separated from the final methanogenic stage using two-stage
48
anaerobic digesters. However, the overall process is often limited by the detrimental
49
acidic condition (pH < 5) produced by the accumulation of volatile fatty acids (VFAs)
50
[3,4].
Ac ce
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44
51
Recently, microbial fuel cell (MFC) technology has been suggested to complement
52
anaerobic digestion for bioenergy recovery [5-7]. MFCs are bioelectrochemical systems
53
that can convert chemical energy stored in an organic substrate directly into electrical
54
energy. Electrochemically active microorganisms are involved in this process to
55
catalyze the oxidation of organic compounds using an insoluble electrode (anode) as an
3 Page 3 of 30
electron acceptor [8,9]. Unlike anaerobic digestion, MFCs are particularly suitable for
57
treating low strength soluble organics such as VFAs and are less susceptible to
58
unfavorable conditions (e.g., low temperature (≤ 20 °C) and low pH). Because the food
59
waste leachate produced from the hydrolysis and acidogenic stages is rich in VFAs [10],
60
it may be practical to use MFCs to covert VFAs in food waste leachate directly into
61
electricity instead of loading the leachate to a final methanogenic reactor for methane
62
production.
cr
ip t
56
In fact, a variety of organic feedstocks have been shown to drive electricity
64
production in MFCs; these feedstocks range from simple organics, such as glucose [11],
65
acetate, butyrate [12], ethanol and methanol [13], to complex substrates, such as yogurt,
66
starch, protein and meat processing wastewater [14-17]. A mixture of synthetic VFAs
67
and VFAs from the fermentation of glucose and food waste has been reported to be
68
removed with concomitant electricity production in an MFC [18-21]. Furthermore, the
69
use of MFCs as a secondary energy recovery step in fermentative hydrogen (H2)
70
production processes has been evaluated using cereal wastewater and vegetable waste as
71
substrates [22,23]. However, the anodic pH conditions in the aforementioned studies
72
were maintained at approximately neutral by dosing buffers or alkaline substances,
73
which are costly and non-sustainable. Arguably, operating an MFC under anodic
74
conditions in which the pH is not controlled or buffered (low pH) is preferred for
75
practical food waste leachate treatment.
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63
76
The aim of this study was to investigate the use of acidic food waste leachate
77
produced from the hydrolysis and acidogenic stage of an anaerobic digester as an MFC
78
feedstock for electricity generation. The effects of three commonly used MFC inoculum
79
sources, namely, (i) domestic wastewater, (ii) activated sludge, and (iii) anaerobic
4 Page 4 of 30
80
sludge, on the MFC performance (power generation and substrate removal) were
81
compared. The acclimated microbial communities in different treatments are also
82
reported.
84
2. Materials and methods
85
2.1. Food waste leachate
ip t
83
Food waste leachate collected from a leach bed reactor was used throughout the
87
entire experiment and stored at 4 °C to avoid compositional changes before use. As
88
previously reported [10], the total and working volume of the leach bed reactor were 6.4
89
and 4.6 L, respectively. The leach bed reactor was provided with a perforated plate at
90
the bottom for leachate collection. On the top of the perforated plate, 0.5 kg of an
91
acid-washed and oven-dried sand bed was placed with a nylon screen support to
92
facilitate the percolation of the leachate. The leach bed reactor was initially fed with a
93
synthetic food waste mixture, which consisted of 350 g/kg of bread, 250 g/kg of rice,
94
250 g/kg of cabbage, and 150 g/kg of pork. The pH, electrical conductivity, total
95
chemical oxygen demand (COD), and total VFAs of the leachate were 4.76, 3.58 mS/cm,
96
52,200 mg/L, and 12,700 mg/L, respectively.
98
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97
cr
86
2.2. Microbial inocula
99
Three types of inocula, (i) municipal wastewater from a primary clarifier, (ii)
100
activated sludge, and (iii) anaerobic sludge from an anaerobic digester, were compared,
101
the total COD concentrations of which were 753 mg/L, 1,064 mg/L, and 14,056 mg/L,
102
respectively. All the inocula were collected from a domestic wastewater treatment plant
103
in Shek Wu Hui, New Territories, Hong Kong.
5 Page 5 of 30
104 105
2.3. Reactor configuration and operation The MFCs consisted of two equal rectangular chambers separated by a cation
107
exchange membrane (6.8 × 6.8 cm, Qianqiu Group Co., Ltd., Zhejiang, China). Each
108
cell chamber had a working volume of 75.6 ml (6.0 × 6.0 × 2.1 cm). Both the anode and
109
cathode were constructed of carbon felt (4.5 × 4.5 × 0.5 cm each, Liaoyang Jingu
110
Carbon Fibre Sci-Tech Co., Ltd., Tianjin, China). A titanium wire was inserted into the
111
carbon felt to allow electrical contact with the external circuit. The carbon felt was
112
pretreated according to a previous report [24]. The anode potentials were measured
113
against a saturated calomel reference electrode (SCE, 242 mV against the standard
114
hydrogen electrode, SHE), which was inserted into the anode chamber. The cathode
115
potentials were calculated as the sum of the anode potential and the cell voltage. The
116
reported electrode potentials refer to values against SCE.
ed
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106
Four MFC settings were operated simultaneously: (1) Control-MFC, food waste
118
leachate only; (2) W.w.-MFC, food waste leachate and wastewater; (3) AS-MFC, food
119
waste leachate and activated sludge; and (4) AnS-MFC, food waste leachate and
120
anaerobic sludge. To start up the reactors, the anodic half cells were fed with inocula
121
and leachate (with a COD ratio of 1:10) and operated in batch mode for three batch
122
cycles (hereafter known as Run 1 to 3). Thereafter, all suspended inoculum residues that
123
were unable to be retained as a biofilm at the anode were discarded, and only the food
124
waste leachate was replenished in the anodic chambers. The leachate used in each run
125
was diluted with deionized water to approximately 1,000 mg COD/L. All cathodic
126
chambers were fed with air-saturated deionized water to standardize the initial cathodic
127
condition in all treatments. Because diffusion of ionic species across the ion exchange
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6 Page 6 of 30
membrane between the anolyte and the catholyte would occur after the catholyte
129
renewal, all the catholytes were ionically conductive throughout the experimental runs.
130
Unless otherwise stated, a fixed external resistance (1,000 Ω) was used to facilitate the
131
comparison of the current production in different treatments. The reactors were operated
132
in batch mode at 28 ± 2 °C.
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128
134
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133
2.4. Chemical analysis
COD (including both soluble and particulate) was determined using a standard
136
dichromate oxidation (open reflux method) method [25]. For the VFA analysis, the
137
liquid samples were filtered through a 0.45 µm mixed cellulose ester membrane filter,
138
and then, 0.9 ml of the filtrate was transferred to amber GC vials and mixed with 0.1 ml
139
of formic acid before analysis. An HP 6890 Series gas chromatograph (Hewlett Packard)
140
was employed with a flame ionization detector and an Econo-Cap EC-1000 (15 m ×
141
0.53 mm × 1.20 μm) column. Nitrogen was used as a carrier gas, whereas air and
142
hydrogen were used for combustion. Samples (1.0 μl) were analyzed using a
143
temperature program beginning with an initial temperature of 75 °C for 1 min,
144
increasing to 180 °C at a rate of 6 °C/min, then increasing to 230 °C at a rate of
145
10 °C/min, and finally maintaining 230 °C for 5 min to ensure complete VFA
146
volatilization. Concentrations were determined using a standard curve obtained by
147
injecting standard solutions of acetic acid, propionic acid, butyric acid, iso-butyric acid,
148
valeric acid, iso-valeric acid and hexanoic acid. The sum of these seven VFA
149
concentrations is reported as the total VFAs.
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150 151
2.5. Microbial community analysis
7 Page 7 of 30
Biofilms were harvested from the carbon felt anode of different MFCs for microbial
153
community analysis after all systems were operated for over three months. Genomic
154
DNA was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN) according to the
155
manufacturer’s instructions. Denaturing gradient gel electrophoresis (DGGE) analysis
156
focused on the V3 region of the 16S rRNA gene, which was amplified using separate
157
primers to target bacterial sequences. Amplification used the primers 805R (5'-GAC
158
TAC CAG GGT ATC TAA TCC-3') and GC-341F (5'-CGC CCG CCG CGC GCG GCG
159
GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CA-3'),
160
containing the GC clamp on the amplified 16S rDNA template. PCR mixtures contained
161
12.5 μl of 2× PCR Master Mix (Promega), 1 µl of each primer and 10 ng of DNA
162
extraction product, and sterile Milli-Q water was added to a final volume of 25 μl. The
163
samples were amplified in a PTC-200 (Bio-Rad Laboratories, Hercules, CA) with an
164
initial denaturation of DNA for 5 min at 94 °C followed by 33 cycles of 30 s at 94 °C,
165
20 s at 55 °C, and 45 s at 72 °C with a final extension for 7 min at 72 °C. Blank controls
166
were performed throughout all steps.
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152
DGGE was performed with a DCode universal mutation detection system (Bio-Rad
168
Laboratories, Hercules, CA). Approximately 1 µg of PCR product per lane was loaded
169
onto 6.5 % (wt/vol) polyacrylamide (37.5:1 acrylamide:bisacrylamide) gels in a 1× TAE
170
buffer with a denaturing gradient ranging from 40 % to 60 %. Denaturation of 100 %
171
corresponds to 7 M urea and 40 % (vol/vol) deionized formamide. Gel electrophoresis
172
was performed at 60 °C for 14 h at 75 V. The gel was subsequently stained in 1× TAE
173
buffer containing a 1:100,000 dilution of RedSafe nucleic acid staining solution
174
(iNtRON, 20,000×) for 1 h before being photographed on a blue light transilluminator.
175
Ten DGGE bands were excised for sequencing.
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8 Page 8 of 30
A small portion of each selected band was excised from the DGGE gel using a
177
sterile scalpel blade and immediately added to a premixed gel elution buffer at 4 °C
178
overnight. PCR reactions of identical composition were conducted as mentioned above,
179
and the PCR products were analyzed by DGGE. The position of the extracted DNA
180
bands was then compared to the original gel using BioNumerics software. After
181
confirming the presence of target bands, the bands were excised, amplified and analyzed
182
by DGGE. This cycle was repeated until a single dominant band was obtained from
183
each extracted band. The selected bands were then sent to the Hong Kong Genome
184
Research Facility for sequencing. Sequence analyses were performed using the online
185
BLAST software tool (http://www.ncbi.nlm.nih.gov).
an
2.6. Data collection and calculation
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186 187
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176
To quantify the cell performance throughout the experiments, the potential
189
difference between the anode and cathode (i.e., cell voltage, V) was recorded every
190
minute using a data acquisition system (PicoLog 1216, Pico Technology,
191
Cambridgeshire, UK) connected to a personal computer. Current density (I) and power
192
density (P) normalized to the volume of the anodic chamber were calculated according
193
to I = V R−1 and P = V2 R−1, respectively, where R is the external resistance. When all
194
MFCs were acclimatized for steady power outputs, polarization and power density
195
curve analysis was performed to compare the MFC performance in different treatments.
196
This analysis was performed by adjusting the external resistance from 10 to 40,000 Ω,
197
and pseudo-steady state voltage was recorded at each resistance level. The internal
198
resistance of the MFC was obtained from the slope of the linear region of the
199
polarization curve according to Logan et al. [8]. Coulombic efficiency (CE) in the
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9 Page 9 of 30
fed-batch mode was estimated according to the formula shown below, where M is the
201
molecular weight of oxygen (32), F is Faraday’s constant (96,485 C), b represents the
202
number of electrons exchanged per mole of oxygen (4), vAn is the volume of liquid in
203
the anode compartment (0.075 L), and ∆COD is the change in COD over a period of
204
time (tb) for each run [8].
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tb
Cb
0
(1)
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205
M Idt
Fb An COD
3. Results and Discussion
208
3.1. Effect of the inocula on the MFC electrical output
an
207
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206
The power output of the described MFCs over the experimental period is shown in
210
Fig. 1. MFCs amended with additional inocula (W.w-MFC, AS-MFC, and AnS-MFC)
211
began to produce electricity after 7 days, whereas the control demonstrated a
212
remarkably slower start-up (after 12 days). This result indicates that the food waste
213
leachate is able to serve as an inoculum for electricity generation but the co-addition
214
domestic wastewater, activated sludge, and anaerobic sludge accelerates the start-up
215
process.
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M
209
216
During Run 2 and 3, renewals of food waste leachate and inocula in all MFCs (at
217
day 28 and day 43) resulted in immediate increases in power outputs in all treatments,
218
indicating that all MFCs had developed electrochemically active biofilms at the anode.
219
Subsequent leachate renewal in Run 4 and 5, again, resulted in a sharp, reproducible
220
increase in power density in all treatments (Fig. 1). The maximal power outputs
221
obtained from Run 4 and 5 were 195.4 ± 18.3, 453.9 ± 10.4, 316.1 ± 6.0 and 445.6 ±
222
15.2 mW/m3 for the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC, respectively
10 Page 10 of 30
223
(Table 1), and followed a descending order of W.w.-MFC ≈ AnS-MFC > AS-MFC >
224
Control-MFC.
225
[Fig. 1.]
226
[Table 1]
3.2. COD and VFA removal
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228
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227
An increase in the COD removal efficiency was observed in all MFCs as the
230
electricity outputs increased gradually from day 0 to 58 (Runs 1-3) (data not shown).
231
After the initial two-month enrichment, all MFCs demonstrated high COD and VFA
232
removal efficiencies of 87-92 % and 89-96 %, respectively (Runs 4 and 5) (Table 1). Fig.
233
2 illustrates the changes in the individual VFA concentrations in different treatments
234
during a batch cycle (Run 4). In all treatments, neither propionic acid nor valeric acid
235
was detected, whereas acetic acid, butyric acid, and hexanoic acid were found to be the
236
major species. In general, all VFAs were gradually removed from the anolyte of all
237
MFCs during the current production cycle (Figs. 1 and 2). However, hexanoic acid was
238
found to be the dominant residual VFA (4-11 % of the initial VFAs), suggesting that
239
long-chain VFAs might be less degradable as compared with short-chain VFAs in the
240
tested systems. The significant increase of accumulated acetate in the AnS-MFC (D) as
241
compared with the Control-MFC (A), W.w.-MFC (B), and AS-MFC (C) from day 4 to
242
day 8 may imply that the acetate production rate was higher than that of the acetate
243
consumption in the AnS-MFC (Fig. 2), since during each batch cycle, acetate could be
244
produced from the fermentable compounds in the leachate by the fermenters, and the
245
produced acetate could be anodically oxidized by the electrogenic microorganisms to
246
generate electricity.
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11 Page 11 of 30
247
[Fig. 2.]
248 249
3.3. Coulombic efficiencies and pH changes Similar to other studies that use complex substrates such as real wastewater as the
251
MFC feedstock, the CE values obtained in this study were low (13 to 20 %) (Table 1)
252
[26]. It has been suggested that high CE can be obtained with acetic acid as the main
253
electron donor [18], whereas a high level of butyric acid in the medium can result in low
254
CE [19]. In our experiment, butyric acid accounted for more than 50 % of the total
255
VFAs in the food waste leachate, whereas acetic acid was less than 12 % (Fig. 2). Such
256
a unique VFA composition of the tested food waste leachate might account for the
257
observed low CE. On the other hand, substrate consumption via other competing
258
metabolic processes, such as fermentation and methanogenesis, might also explain the
259
low CE [17]. Among all treatments, the W.w.-MFC and AnS-MFC had significantly
260
higher CE when compared with the Control-MFC and AS-MFC (Table 1), indicating
261
that the anodic biofilms established with the co-additions of domestic wastewater and
262
anaerobic sludge were more capable of utilizing the food waste leachate for electricity
263
production in the MFCs.
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250
264
A gradual increase in the anolyte pH was observed in the Control-MFC (Run 4) (Fig.
265
3), which is contradictory to the commonly observed anolyte acidification due to anodic
266
COD oxidation [27,28]. Nevertheless, the increase in the pH in this condition may be
267
due to the loss of VFAs in the anolyte via non-bioelectrochemical oxidation (e.g.,
268
oxygen intrusion from the catholyte). It has been reported that VFA oxidation leads to
269
an increase in the pH of VFA-laden manure slurries [29]. However, the observed
270
increase in the pH is unlikely to have resulted from the release of ammonia through the
12 Page 12 of 30
hydrolysis of proteins in the food waste leachate, as the amount of ammonia in the
272
anolyte was found to decrease over time (data not shown) [16]. At the beginning of the
273
batch cycle, 12.6 mg/L of ammonia was detected in the anolyte, and its concentration
274
gradually decreased until it became undetectable (< 0.1 mg/L) at the end of the cycle.
275
Alternatively, the decrease in the pH observed in the W.w.-MFC, AS-MFC, and
276
AnS-MFC before day 8 might also be due to the formation of other acidic compounds,
277
such as amino acids and long-chain fatty acids, from the food waste leachate. However,
278
further studies are required to determine the formation of these compounds during the
279
process.
cr
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[Fig. 3.]
an
280
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271
282
3.4. Polarization characteristics
M
281
The effects of different inocula on the MFC performance were compared based on
284
the polarization behavior and maximal power output recorded after all MFCs were
285
acclimatized with the food waste leachate (Fig. 4). All MFCs produced an open circuit
286
voltage (OCV) of approximately 400 mV, which is consistent with other studies using
287
fermenting vegetable waste effluents as the MFC substrate but under neutral conditions
288
[23]. Among all treatments, the Control-MFC was more susceptible to polarization (i.e.,
289
deviation of the cell voltage from the OCV) compared with other treatments receiving
290
additional microbial inoculum (Fig. 4a). This result suggests that the external inoculum
291
is able to establish a better anodic biofilm for electricity production. The distinctive
292
polarization patterns observed in different treatments are also noteworthy. At a current
293
density < 2000 mA/m3, the polarization followed an order of AnS-MFC > W.w.-MFC >
294
AS-MFC, whereas at a current density > 2000 mA/m3, the order was AnS-MFC >
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13 Page 13 of 30
AS-MFC > W.w.-MFC. Such a change was due to an abrupt polarization in the
296
W.w.-MFC at a current density of approximately 2200 mA/m3 and may be explained by
297
the so-called “power overshoot” phenomenon in which the cell resistance suddenly
298
increased, leading to decreases in power density as external loads decrease [30].
299
Resistance in the electroactive bacteria for substrate utilization was proposed to induce
300
MFC power overshoot [31]; therefore, it is important to understand the microbial
301
community attached on the anode.
cr
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295
The internal resistances of the W.w.-MFC, AnS-MFC, AS-MFC, and Control-MFC
303
were 1137, 1174, 1918, and 2384 Ω, respectively (Fig. 4a), and the respective maximal
304
power outputs were 363, 432, 267, and 251 mW/m3 (Fig. 4b). The maximum power
305
density of the AnS-MFC was higher than that of the W.w.-MFC even though they both
306
had a similar internal resistance. No clear relationship between internal resistance and
307
power output was noted in the tested systems. The internal resistances measured in this
308
study were based on the use of a polarization curve method, which only reflects the
309
ohmic resistance of the MFC. While it is generally expected that the power density of an
310
MFC is indirectly related to the internal resistance, other factors such as the affinity of
311
the anodic biofilm for the substrate (electron donor) and the anode (electron acceptor)
312
may also affect the current production and hence the power. These factors may explain
313
the observed independency between power and internal resistance in the W.w.-MFC and
314
AnS-MFC.
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302
315
At open circuit, the anode potentials for all MFCs were similar (-372 ± 9.15 mV)
316
according to Fig. 5. All the anode potentials increased (became less negative) slightly
317
with the current density, particularly in the lower current density range (0-1000 mA/m3),
318
but became unsteady at higher current densities in the Control-MFC, W.w.-MFC, and
14 Page 14 of 30
AS-MFC. The increase in the anode overpotential at high current densities suggests that
320
the established anodic bacteria were unable to transfer electrons to the anode at a
321
sufficient rate to maintain the current [12]. Because the AnS-MFC had the lowest anode
322
overpotential and was able to sustain the highest current output among all treatments,
323
anaerobic sludge appeared to be the most suitable inoculum. [Fig. 4.]
325
[Fig. 5.]
cr
324
us
326 327
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319
3.5. Microbial communities
To understand the effect of the various co-inocula on the anode community structure
329
in the food waste leachate-treated MFCs, the anode-attached biofilms established in
330
different MFCs were characterized using the PCR-DGGE technique (Fig. 6). A number
331
of bands excised from the gels were processed for sequence analysis (Table 2). In
332
general, bacteria of the phylum Proteobacteria were dominant in all MFCs. A similar
333
finding was reported by Freguia et al. [18] with their anodic biofilm community that
334
was enriched with mixed-VFAs. Bands 1 and 2 were found in all MFCs, whereas bands
335
4 and 5 were predominant in the W.w-MFC. Additionally, bands 3, 9, and 10 were also
336
found in the AnS-MFC.
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328
337
Clostridium sp. (band 1) is capable of producing H2 in addition to acetone, butanol,
338
and ethanol from sugar, glucose or starch under anaerobic conditions [32]. Liang et al.
339
[33] reported that Clostridium sp. is the predominant microbial species found in an
340
acidic (pH 2-6) anaerobic H2-producing sequencing batch reactor. Bacteroides sp. (band
341
3) is known to be able to produce H2 fermentatively and to produce electricity in MFCs
342
[34]. Aside from VFAs, the food waste leachate used in this study also contained other
15 Page 15 of 30
343
organic matter (collectively measured as COD). The fermentation of this organic matter
344
in the food waste leachate might be enhanced by Clostridium sp. and Bacteroides sp.,
345
especially in MFCs co-inoculated with activated sludge and anaerobic sludge. Geobacter sp. (band 5) have been widely described for their capacity to perform
347
direct electron transfer and are commonly found in MFC microbial communities
348
particularly when acetate is provided as the electron donor [35]. Magnetospirillum sp.
349
(band 4) has been reported to be able to use fermentation end products, such as acetate
350
and ethanol, as carbon sources and electron donors for heterotrophic growth, and can be
351
grown chemolithotrophically with hydrogen as the electron donor [36]. The presence of
352
Magnetospirillum sp. in the W.w.-MFC and AnS-MFC may facilitate current generation
353
by anodically oxidizing the fermentation end products produced by the fermenters.
M
an
us
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346
The enrichment of both fermentative (Clostridium sp. and Bacteroides sp.) and
355
electrogenic species (Magnetospirillum sp. and Geobacter sp.) in the anodic community
356
of the AnS-MFC may explain why this treatment, in particular, was able to more
357
efficiently use the leachate for electricity production. However, further research is
358
needed to elucidate the interaction between non-electrogenic and electrogenic bacteria
359
for food waste leachate treatment using MFCs.
Ac ce
pt
ed
354
360
[Fig. 6.]
361
[Table 2]
362 363
4. Conclusions
364
Acidic food waste leachate can serve as an inoculum for electricity generation in
365
MFCs, but the co-addition of domestic wastewater, activated sludge or anaerobic sludge
366
accelerates the MFC start-up process. Efficient COD and VFA removal (> 87 %) were
16 Page 16 of 30
367
achieved in all MFCs, but only low CE (14-20 %) were obtained using the food waste
368
leachate as the MFC substrate. Among all treatments, the highest power output and CE
369
were obtained with anaerobic sludge as the co-inoculum, and this result might be due to
370
the enrichment of both fermentative and electrogenic bacteria at the anode over time.
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373
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Ac ce
455
Microbiol Biotechnol 1983;18:327–332.
458
[33] Liang DW, Shayegan SS, Ng WJ, He JZ. Development and characteristics of
459
rapidly formed hydrogen-producing granules in an acidic anaerobic sequencing
460
batch reactor (AnSBR). Biochem Eng J 2010;49:119–125.
461
[34] Kim GT, Webster G, Wimpenny JWT, Kim BH, Kim HJ, Weightman AJ. Bacterial
462
community structure, compartmentalization and activity in a microbial fuel cell. J
20 Page 20 of 30
463 464 465
Appl Microbiol 2006;101:698–710. [35] Bond DR, Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003;69:1548–1555. [36] Thrash JC, Ahmadi S, Torok T, Coates JD. Magnetospirillum bellicus sp. Nov., a
467
novel dissimilatory perchlorate-reducing Alphaproteobacterium isolated from a
468
bioelectrical reactor. Appl Environ Microbiol 2010;76:4730–4737.
cr
ip t
466
469
Ac ce
pt
ed
M
an
us
470
21 Page 21 of 30
Figure legends
471
Fig. 1. Power density (mW/m3) profiles of different food waste leachate-fed MFCs
472
co-inoculated with various microbial inocula (Control: without co-inoculum; W.w.:
473
domestic wastewater; AS: activated sludge; AnS: anaerobic sludge) over the course of
474
five batch cycles (Runs 1-5). For Runs 1-3, both co-inoculum and food waste leachate
475
were renewed in each cycle. For Runs 4 and 5, only food waste leachate was renewed.
476
Fig. 2. A profile of the concentration of VFAs over a current-producing batch cycle
477
(Run 4) in the Control-MFC (A), W.w.-MFC (B), AS-MFC (C), and AnS-MFC (D).
478
Fig. 3. Change in the anolyte pH over a current-producing batch cycle (Run 4) in the
479
Control-MFC (A), W.w.-MFC (B), AS-MFC (C), and AnS-MFC (D).
480
Fig. 4. Voltage (a) and power density (b) as a function of current density obtained over a
481
batch cycle (Run 4) from the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC.
482
Fig. 5. Electrode potentials (vs. SCE) as a function of current density obtained over Run
483
4 from the Control-MFC, W.w.-MFC, AS-MFC, and AnS-MFC.
484
Fig. 6. DGGE profiles obtained from the mature anodic biofilms established in different
485
MFC reactors. Control: Control-MFC; W.w.: W.w.-MFC; AS: AS-MFC; and AnS:
486
AnS-MFC.
cr
us
an
M
ed
pt
Ac ce
487
ip t
470
22 Page 22 of 30
Table 1.
488
Maximal power density, COD removal, VFA removal, and coulombic efficiency (CE) in
489
different food waste leachate-fed MFCs inoculated with various microbial inocula
490
(Values are the mean ± standard deviation of the data obtained over Runs 4 and 5). Maximal power COD removal VFA removal
CE (%)
density (%)
(%)
89.96 ± 0.64
14.36 ± 2.01
91.60 ± 1.05
20.27 ± 2.06
94.30 ± 0.26
13.65 ± 1.11
96.61 ± 0.89
20.26 ± 0.33
195.43 ± 18.25
91.27 ± 1.09
W.w.-MFC
453.90 ± 10.44
90.97 ± 0.08
AS-MFC
316.12 ± 5.95
92.21 ± 1.10
AnS-MFC
445.61 ± 15.17
87.13 ± 2.30
M
an
Control-MFC
us
(mW/m3)
cr
Treatments
ip t
487
Control-MFC: without co-inoculum; W.w.-MFC: domestic wastewater; AS-MFC:
492
activated sludge; and AnS-MFC: anaerobic sludge.
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ed
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23 Page 23 of 30
493
Table 2.
494
Overview of the sequencing results of the excised bands from the DGGE analysis (Fig.
495
6).
DGGE DGGE
Sequence
Closest
similarity match
accession
(%)
number
Phylum
ip t
band
accession
cr
band
Closest match
number
JX193892
Clostridium acetobutylicum HP7
98
FM994940.1
Firmicutes
2
JX193893
Burkholderia vietnamiensis RPB3
99
HQ606073.1
Proteobacteria
3
JX193894
Uncultured Bacteroides sp. J3
99
DQ168847.1
Bacteroidetes
4
JX193895
Uncultured Magnetospirillum sp.
98
FJ823930.1
Proteobacteria
5
JX193896
Geobacter pickeringii G13
97
DQ145535.1
Proteobacteria
6
JX193897
Novosphingobium nitrogenifigens Y88
100
DQ448852.1
Proteobacteria
99
FJ570212.1
Acidobacteria
ed
M
an
us
1
Uncultured Acidobacteria bacterium
7 JX193898
pt
A6YA20RM JX193899
Uncultured Aeromonas sp. ASP-21
98
EF679186.1
Proteobacteria
9
JX193900
Burkholderia sp. C527
99
HQ704709.1
Proteobacteria
96
EF613974.1
Acidobacteria
Ac ce
8
Uncultured Acidobacteria bacterium
10
JX193901
NGA53
496
24 Page 24 of 30
496
Fig. 1.
Run4
Run5
500 400
cr
300
ip t
Control-MFC W.w.-MFC AS-MFC AnS-MFC
us
200 100
an
Power density (mW/m3)
Enrichment (Run 1 to 3)
0 20
40
60
80
M
0
Time (days)
Ac ce
pt
ed
497
25 Page 25 of 30
Fig. 2.
300 Acetate Iso-butyrate Butyrate Iso-valerate Hexanoate
ABCD
ip t
ABCD
150 ABCD
100
cr
200
us
VFAs (mg/L)
250
an
50 0 4
8 Time (days)
13
Ac ce
pt
ed
M
0
ABCD
26 Page 26 of 30
Fig. 3.
6.5
cr
5.5
us
pH
6.0
ip t
Control-MFC W.w.-MFC AS-MFC AnS-MFC
an
5.0
4.5 4
8
12
M
0
Ac ce
pt
ed
Time (days)
27 Page 27 of 30
Fig. 4.
500 Control-MFC W.w.-MFC AS-MFC AnS-MFC
300
cr
200 100
us
Voltage (mV)
400
1000
2000
3000 Current density (mA/m3)
M
0
an
0
600
4000
(b)
ed
Control-MFC W.w.-MFC AS-MFC AnS-MFC
500
pt
400 300
Ac ce
Power density (mW/m3)
ip t
(a)
200 100
0
0
1000 2000 3000 Current density (mA/m3)
4000
28 Page 28 of 30
Fig. 5.
100 Control-MFC (Cathode) W.w.-MFC (Cathode)
0
AS-MFC (Cathode)
ip t
-100
cr
Control-MFC (Anode) W.w.-MFC (Anode)
-200
us
AS-MFC (Anode) AnS-MFC (Anode)
-300
an
Potential (mV)
AnS-MFC (Cathode)
-400 0
600
1200
1800
2400
3000
3600
Ac ce
pt
ed
M
Current density (mA/m3)
29 Page 29 of 30
Fig. 6. Highlights
Control
W.w.
AS
AnS
ip t
1
cr
2
4 5
an
6
us
3
M
7
9 10
Ac ce
pt
ed
8
1. Electricity was produced from acidic food waste leachate using microbial fuel cells (MFCs) 2. Domestic wastewater, activated sludge and anaerobic sludge were compared as MFC co-inoculum. 3. Anaerobic sludge was the best due to the enrichment of fermentative/ electrogenic bacteria. 4. Food waste leachate alone could already serve as a MFC inoculum for bioelectricity production.
30 Page 30 of 30