Direct electricity production from subaqueous wetland sediments and banana peels using membrane-less microbial fuel cells

Industrial Crops & Products 128 (2019) 70–79

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Direct electricity production from subaqueous wetland sediments and banana peels using membrane-less microbial fuel cells

T

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Yunlong Yanga,b, , Ershu Lina, Shuqian Suna, Huan Chenb, Alex T. Chowb,c a

College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China Baruch Institute of Coastal Ecology & Forest Science, Clemson University, Georgetown, SC, 29442, USA c Department of Environmental Engineering and Earth Sciences, Clemson University, Anderson, SC, 29625, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Subaqueous wetland sediment Banana peel Microbial fuel cell Electricity

Subaqueous wetland sediments (SWSs) and banana peels (BPs), respectively used as anaerobic inoculums and raw materials, were valorized for electricity generation in membrane-less and biocathode-containing microbial fuel cells (MLBC-MFCs). The maximum current density was 78.2 mA/m2 for banana peel extracts (BPE) and 91.3 mA/m2 for anaerobically fermented banana peel extracts (ABPE). The polarization data indicated that Ohmic resistances were high that were 3.5, 4.2 and 9.3 kΩ when MLBC-MFCs were fed with ABPE, BPE and sodium acetate (SA), respectively. The ultraviolet-visible absorption spectroscopy coupled with three-dimensional fluorescence spectroscopy was used to explore the mechanisms of generating electricity. Results showed that SWSs provided functional microorganisms and a source of organic matter, while BPs were more helpful for the growth of microorganisms than SA and the microorganisms attached on the cathode primarily functioned as biocatalysts. Overall, this study highlights the simplicity and scalability of MLBC-MFCs equipped with SWSs and BPs.

1. Introduction In recent years, microbial fuel cells (MFCs) have been attracting wide attention since they can harness cleaner electricity directly from organic substrates. Conventionally, MFCs consist of a biological anode and an abiotic cathode separated by an ion exchange membrane that is easily fouled. Meanwhile, a metal catalyst that could deactivate with time and needs to be regenerated during the operation(Zhao et al., 2006), is usually combined with the abiotic cathode to transfer electrons efficiently, which would undoubtedly give rise to a higher cost. Such drawbacks, however, can be overcome by membrane-less and biocathode-containing microbial fuel cells (MLBC-MFCs), in which aerobic microorganisms are used as biocatalysts to assist oxygen reduction. Thanks to their striking advantages, MLBC-MFCs have been utilized to produce electrical energy from various wastes including synthetic wastewater (Aldrovandi et al., 2009; Ghangrekar and Shinde, 2007; Wang et al., 2013; Zhu et al., 2011, 2013), azo dye Acid Orange 7-containing wastewater (Thung et al., 2015) and domestic sewage (Zhang et al., 2016a). As an extremely important ecosystem, wetlands play a vital role in protecting our environments due to their capability of water purification and ground water recharge, both of which are closely related to

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functional microorganisms including bacteria and fungi. These microbes were found to be able to inhabit anywhere in wetlands ecosystem like plants, river waters and sediments, out of which sediments usually possess a huge number of microorganisms. For example, Cao et al found that Proteobacteria and Bacteroidetes dominate and account for 39.4–61.5%. Planctomycetes, Acidobacteria, Nitrospirae, Verrucomicrobia, Actinobacteria, Gemmatimonadetes, and Firmicutes as important phyla of bacteria are also largely found in the studied wetlands sediments (Cao et al., 2017). Likewise, similar results were documented in another literature (Pang et al., 2016). Notably, many of microbes mentioned above encompass genera of exoelectrogenic microbes such as Rhodopseudomonas (McGrath and Harfoot, 1997), Clostridium (Park et al., 2001), Geothrix (Bond and Lovley, 2005) and Corynebacterium (Liu et al., 2010) belonging to Proteobacteria, Firmicutes, Acidobacteria and Actinobacteria, respectively, which implies that wetland sediments could become a potent inoculum for the anaerobic area of MFCs. However, to the author’s knowledge, no studies so far have employed subaqueous wetland sediments as the anaerobic inoculum to generate electricity in MFCs. Banana peel is one of the important wastes generated in large quantities due to banana fruit consumption. Banana peel contributes about 40% of total weight of the fresh banana fruit (Anhwange, 2008),

Corresponding author at: College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China. E-mail address: [email protected] (Y. Yang).

https://doi.org/10.1016/j.indcrop.2018.10.070 Received 25 April 2018; Received in revised form 20 October 2018; Accepted 23 October 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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10–20 ng of genomic DNA. The amplification program consisted of a denaturing step at 94 °C for 3 min, followed by 25 cycles where the first 5 cycles comprised: denaturation at 94 °C for 30 s, annealing at 45 °C for 20 s and elongation at 65 °C for 30 s; the subsequent 20 cycles were composed of: denaturation at 94 °C for 20 s, annealing at 55 °C for 20 s and elongation at 72 °C for 30 s, and then a final elongation stage at 72 °C for 10 min. The concentration of DNA was determined in a Qubit 3.0 fluorometer (Invitrogen) and the sequencing of resultant amplicons was performed on a MiSeq sequencer in a 2 × 300 bp paired-end run. Raw data retrieved from Illumina sequencing after barcode and index removal was sequentially processed through the following software: Cutadapt (1.2.1), Pear (0.9.6) and Prinseq (0.20.4), and were checked for chimeras within the UCHIME algorithm (Edgar et al., 2011). The Quantitative Insights into Microbial Ecology (QIIME) at 97% identity level was used to cluster the high-quality reads into operational taxonomic units (OTUs), and the representative sequence of each OTU was assigned taxonomy with RDP Classifier (Caporaso et al., 2010). R-software and the Vegan (2.0–10) package were used to estimate relative abundances between samples and to calculate community diversity.

and thus it is estivated that the banana industry produces more than 57.6 million metric tons of banana peels annually (Ahmad and Danish, 2018). The main constituents of banana peel are cellulose, pectin, chlorophyll and low molecular weight species, and some other substances including lipids (1.7%), proteins (0.9%), crude fiber (31%) and carbohydrates (59%) were also found (Munagapati et al., 2018). Several tons of banana peels are produced every day in fruit market and household garbage, creating a severe environmental problem because of anaerobic digestion of the biomass mentioned above. Accordingly, in order to bring an additional economic gain for the agricultural industry and alleviate the local environmental pressure, researchers have been trying to develop effective methods to convert banana peels into useful materials, such as adsorbents (Munagapati et al., 2018), indigenous medium (Kindo et al., 2016), supercapacitors (Zhang et al., 2016b), semiconductor nanoparticles (Bisauriya et al., 2018), methane production (Odedina et al., 2017), nanocomposites (Pelissari et al., 2017) and so on. In comparison, utilizing banana peels to generate electricity in MFCs was scarcely reported (Lalitha and Kanakaraju, 2015). In the current study, subaqueous wetland sediments were first combined with banana peels to generate electrical energy through MLBC-MFCs, with overall objectives being (1) to investigate the feasibility of using subaqueous wetland sediments as the anaerobic inoculum for MFCs, (2) to compare electricity generation efficiencies between raw banana peels and anaerobically fermented banana peels and (3) to explore mechanisms of generating electricity by ultravioletvisible absorption spectroscopy (UV–vis) coupled with three-dimensional fluorescence spectroscopy (3D-EEM).

2.2. Inoculum and medium Subaqueous wetland sediments (SWSs), surface soil (0–5 cm) and saltmarsh wetland water (SWW) were collected from saltmarsh wetlands (33°21′12. 60″ N, 79°11′44. 00″ W) located in Georgetown Counties, South Carolina, USA. In order to provide a relatively beneficial environment for indigenous microorganisms involved in SWSs, SWW was used during the entire experiment. As media, sodium acetate (SA), BPE and ABPE were respectively added into SWW to investigate the efficiency of generating electricity by banana peels, and the pure SWW was used as a control in each run.

2. Materials and methods 2.1. Banana peels 2.1.1. Pretreatment and components analysis The fresh banana peel was flushed with distilled water three times and immediately ground with a grinder. To gain dissolved carbon source as much as possible, the resultant banana peel was transferred to a flask containing distilled water with a ratio of banana peel to water being 50 g/100 mL and then cultured overnight at room temperature in an oscillator with a rotation speed of 180 rpm. Afterwards, banana peel mixtures were subjected to a centrifugation of 5000×g for 20 min at room temperature. After pellets were removed, the supernatant fraction, namely banana peel extracts (BPE) were stored at 4 °C for further use. For the anaerobically fermented banana peel extracts (ABPE), the procedure was almost the same as that mentioned above, except that the mixture (50 g/100 mL) was fermented anaerobically in a sealed flask at room temperature for one month. The components of banana peels before and after anaerobic fermentation were subjected to detailed analysis by ICAS Testing Technology Service Company (Shanghai, China).

2.3. Microbial fuel cell setup and operation Membrane-less and biocathode-containing microbial fuel cells (MLBC-MFCs) were two-phase (sediment and water) transparent plexiglass column reactors with a diameter of 10 cm and a height of 30 cm (Fig. 1), in which an 8 cm SWSs layer was placed at the bottom as solid phase while SWW (inoculated with a certain amount of surface soil) plus substrates was used as liquid phase. According to our previous study (Dai et al., 2015), the electrodes distance was fixed at about 10 cm, where the anodes were buried about 3 cm below the solid-liquid interface and the cathodes floated on the water surface (the height of water column was about 7 cm). In these MFC reactors, both anodes and cathodes were made of a 16 cm2 carbon felt (Fuel Cell Store, Texas, USA) with a good capability to adsorb functional microorganisms owing to its relatively large specific surface area. To enrich functional microbes on both anodes and cathodes as soon as possible, sodium acetate, ammonium chloride and disodium hydrogen phosphate (C:N:P in molar ratio was 100:50:1) were used as substrates during the start-up of MLBC-MFCs, which could not only promote the growth of electroactive microorganisms and the biofilm formation but also ensure a common starting point for the subsequent experiments. The voltage output across a 1 kΩ resistor was recorded automatically very 2 min by voltage loggers Keithley 2700 (Keithley Instruments Inc., Cleveland, OH, USA). All MFCs were operated at room temperature and pHs were maintained at 7.0–7.5 with 1 M HCl or 1 M NaOH. Samples were taken immediately from oxic and anaerobic area of MFCs, respectively, after each run finished.

2.1.2. Sample collection and DNA extraction After anaerobic fermentation finished, samples were collected by an aseptic syringe with a needle able to pierce into the rubber stopper, and were immediately stored at −20 °C to characterize the microbial population involved in the anaerobic fermentation of banana peels. Genomic DNA was extracted with an E.Z.N.A™ Mag-Bind Soil DNA Kit (Omega-Biotek) following the manufacturer’s protocol. 2.1.3. Amplicon sequencing and data analysis The 16S rRNA gene of bacteria was amplified with the barcoded V3V4 universal primers: 341 F (5′−CCCTACACGACGCTCTTCCGATCTGCCTACGGGNGGCWGC AG-3′) and 805R (5′-GACTGGAGTTCCTTGGCACCCGAGAATTCCAGACTACH VGGGTATCTAATCC-3′). PCR reactions were conducted in a volume of 30 μL, containing 15 μL 2×Taq master Mix, 1 μL of each primer and

2.4. Calculations The following equations were used: U = R × I, P = U × I and E = P × t, where U, R, I, P, E ant t are indicative of the voltage in volts, the resistance in ohms, the current in amperes, the power in watts, the 71

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Fig. 1. Schematic figure of MLBC-MFC used in this study.

freshness index (FIX) and humification index (HIX) were determined according to a previous literature (Jason et al., 2010). FI was calculated as the ratio of fluorescence signals at Em 470 and Em 520 nm, at E x 370 nm. HIX was determined by dividing the area under the emission spectra 435–480 nm by the peak area 300–345 nm, at E x 254 nm. FIX was expressed as the ratio of the fluorescence signal at Em 380 nm divided by the maximum signal between Em 420 and 435 nm, at E x 310 nm.

total electrical energy in joules and the time in seconds, respectively. To calculate the total electrical energy per gram substrate, E was divided by the number of substrates (in the form of dissolved organic carbon) consumed in MFCs. A resistance decade box ranging from 50 to 100,000 Ω was used to determine the internal resistance of the system by first plotting the voltage versus the current and then calculating on the basis of the regression line slope resulting from the above data.

2.5. Mechanisms for electricity generation 2.6. Analytical methods To explore the mechanisms for electricity generation, the ultraviolet-visible absorption spectroscopy (UV–vis) coupled with three-dimensional fluorescence spectroscopy was utilized to analyze the dissolved organic matter (DOM) of water samples from both oxic and anaerobic area of MFCs. Optical indices reflecting the DOM composition, specific UV absorbance at 254 nm (SUVA254 in L mg-C−1 m-1) and the E2/E3 ratio, were calculated following a previous study (Wang et al., 2015). Fluorescence excitation − emission matrices (EEMs) from 3-D spectrofluorometry were analyzed by fluorescence regional integration (FRI) and the raw EEM was corrected for instrument-dependent effects, inner-filter effects, and Raman effects and standardized to Raman’s units (normalized to Raman peak at E x 350 nm) (Wang et al., 2015). The corrected EEM was divided into five operationally defined regions based on Simpson's rule (Zhou et al., 2013): I (tyrosine-like), II (tryptophan-like), III (fulvic acid-like), IV (soluble microbial byproductlike) and V (humic acid-like). The percentage of fluorescence response in each region (Pi,n) was expressed as the proportion of area-normalized volume in region i to the entire region. Some fluorescence-based indices like fluorescence index (FI),

Prior to any analysis, all water samples were filtrated by pre-rinsed 0.45 μm polyethersulfone filters. The UV–vis absorption was conducted by a Shimadzu UV-1800 spectrophotometer (scan range: 200–700 nm), while the EEM was carried out by a Shimadzu spectrofluorometer RF5301 (emission wavelength, abbreviated as Em: 200–550 nm; excitation wavelength, abbreviated as Ex: 220–450 nm; slit width: 5 nm). The pH was measured with an Accumet XL60 dual channel pH/Ion/ Conductivity meter. The dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were determined using a Shimadzu TOC/TN analyzer (SM 5310B).

2.7. Statistical analyses Differences among voltage output groups (SA, BPE, ABPE and control) were evaluated through the student’s t-test.

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Fig. 2. Voltage output over time after the addition of SA (a), BPE (b) and ABPE (c). SA: sodium acetate; BPE: banana peel extracts; ABPE: anaerobically fermented banana peel extracts.

3. Results

Table 1 Summary of MFCs parameters on SA, BPE and ABPE. The voltage was recorded with a 1 kΩ resistor. SA: sodium acetate; BPE: banana peel extracts; ABPE: anaerobically fermented banana peel extracts.

3.1. MFCs performance After the start-up of MFCs, sodium acetate (SA), banana peel extracts (BPE) and anaerobically fermented banana peel extracts (ABPE) were respectively used to generate electrical energy and results were shown in Fig. 2. All voltages increased gradually after substrates addition with the maximum voltage being 59 mV for SA, 125 mV for BPE and 146 mV for ABPE (Fig. 2 and Table 1), and a lag phase of 40–60 h before voltages became relatively steady could be seen. As for three controls, there was a similar changing trend in voltages and all of them were around 30 mV. Also, maximum current densities, maximum power densities and internal resistances for different substrates could be observed (Fig. 3 and Table 1). For example, the maximum current density was 36.9 mA/m2 for SA, 78.2 mA/m2 for BPE and 91.3 mA/m2 for ABPE, while the internal resistance was 9.3 kΩ for SA, 4.2 kΩ for BPE and 3.5 kΩ for ABPE.

Substratea

Max voltage (mV)

Max power density (mW/ m2)

Max current density (mA/ m2)

Internal resistance (kΩ)b

SA BPE ABPE

59 125 146

5.3 11.3 13.1

36.9 78.2 91.3

9.3 4.2 3.5

a Substrates were normalized as DOC concentration, which is 395 mg/L for SA, 304 mg/L for BPE and 331 mg/L for ABPE. b The internal resistance was determined based on the slope of voltages versus currents with resistance ranging from 50 to 100,000 Ω.

and 13.5 J/g, respectively, suggesting that there was no significant difference between these two substrates, but it was higher than that generated by SA (data not shown). This result was unexpected since SA, as an organic carbon source with a low molecular weight, is supposed to be consumed easily by microorganisms, whereas banana peels usually contain lots of those substances with a high molecular weight such as polysaccharides and lipids, both of which are relatively hard to be

3.2. Electrical energy per gram substrate The total electrical energy generated by BPE and ABPE was 11.4 J/g 73

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reaction and 2.70 for the anaerobic effluent after reaction (Fig. 4a). By comparison, there was no major difference in SUVA, FI and FIX. On the basis of Simpson's rule, five operationally defined regions were obtained: I, II, III, IV, V (Fig. 4b). Out of them, regions of I, II and IV increased, whereas regions of III and V decreased. Regardless of increase or decrease, all five regions for the anaerobic effluent changed more remarkably than those for the oxic effluent compared with the influent. Take I and V as examples. The proportion of region I went up from 4.3% for the influent before reaction to 4.7% for the oxic effluent and 11.8% for the anaerobic effluent after reaction. The proportion of region V dropped from 35.5% for the influent before reaction to 35.4% for the oxic effluent and 24.2% for the anaerobic effluent after reaction. The fluorescence EMM of DOM was distinct from each other (Fig. 4c). There were two peaks falling into in the influent before reaction. After MFCs treatment, these two peaks were reduced slightly and two weak peaks appeared in Ex/Em: 220-250/350–380 nm in the oxic effluent of MFCs, but they were all reduced dramatically in the anaerobic effluent of MFCs. 3.3.2. DOM from MLBC-MFCs loaded with SWSs and BPE The optical properties, fluorescence regional integration and 3D excitation emission matrices of the DOM before and after treatment of MFCs fed with BPE are exhibited in Fig. 5. The E2/E3 changed from 4.15 for the influent before reaction to 5.17 for the oxic effluent and to 3.5 for the anaerobic effluent after reaction, while HIX changed from 1.79 for the influent reaction to 4.21 for the oxic effluent and to 4.06 for the anaerobic effluent after reaction (Fig. 5a). However, there was no obvious change in SUVA, FI and FIX before and after MFCs treatment. Five operationally defined regions (I, II, III, IV, V) for water samples were also obtained (Fig. 5b). These regions could be divided into two fractions: one consisting of I, II and IV and the other containing III and V, based on the changing trend. For example, the region IV first decreased from 24% for the influent before reaction to 16.8% for the oxic effluent and then increased to 17.7% for the anaerobic effluent after MFCs reaction. On the contrary, the region V first increased from 19.6% for the influent before reaction to 30.4% for the oxic effluent and then decreased to 28.8% for the anaerobic effluent after MFCs treatment. Fig. 5c shows the fluorescence EEM of DOM from water samples before and after MFCs treatment. Two peaks lying in Ex/Em: 225–275/ 330–350 nm could be found in the influent before reaction, but they seemed to disappear in the oxic effluent after reaction. Additionally, two new peaks located in Ex/Em: 230–350/400–500 nm appeared in the oxic effluent and subsequently became weak in the anaerobic effluent after reaction. 3.3.3. DOM from MLBC-MFCs loaded with SWSs and ABPE Clearly, optical properties, fluorescence regional integration and 3D excitation emission matrices of the DOM from MFCs fed with ABPE shown in Fig. 6 were similar with those presented in Fig. 5. The E2/E3 changed from 3.67 for the influent before reaction to 5.17 for the oxic effluent and to 3.7 for the anaerobic effluent after reaction, while HIX changed from 1.54 for the influent reaction to 7.11 for the oxic effluent and to 4.51 for the anaerobic effluent after reaction (Fig. 6a). Likewise, there was no obvious change in SUVA, FI and FIX before and after MFCs treatment. As shown in Fig. 6b, the region IV first decreased from 33.8% for the influent before reaction to 15.1% for the oxic effluent and then increased to 16.5% for the anaerobic effluent after MFCs reaction. This similar trend could also be found in regions I and II but was just opposite to that in regions III and V. With regard to the fluorescence of EEM, not only were there peaks lying in Ex/Em: 225–275/330–350 from the influent before reaction and peaks located in Ex/Em: 230–350/400–500 nm from the oxic effluent after reaction in MFCs fed with ABPE (Fig. 6c), but also all of them were stronger than those in MFCs fed with BPE (Fig. 5c).

Fig. 3. Voltage (square) and total power (triangle) versus current using variable resistance from 50 to 100,000 Ω. Substrates were SA (a), BPE (b) and ABPE (c). SA: sodium acetate; BPE: banana peel extracts; ABPE: anaerobically fermented banana peel extracts.

degraded by microbes. The detailed reasons responsible for this phenomenon would be discussed in the section of mechanisms study.

3.3. Mechanisms study 3.3.1. DOM from MLBC-MFCs loaded with SWSs and SA Fig. 4 shows optical properties, fluorescence regional integration and 3D excitation emission matrices of the DOM from both oxic and anaerobic areas of MFCs fed with SA. Clearly, there was an obvious decline in E2/E3 and HIX when MFCs operation finished. The E2/E3 of influent before reaction, oxic effluent after reaction and anaerobic effluent after reaction was 5.45, 4.96 and 4.76, respectively, while HIX was 9.45 for the influent before reaction, 9.22 for the oxic effluent after 74

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Fig. 4. Optical properties (a), fluorescence regional integration (b) and 3D excitation emission matrices (c) of water samples before and after MFCs fed with SA. SUVA: specific ultraviolet absorption at 254 nm; E2/E3: UVA at 254 nm divided by UVA at 365 nm; FI: fluorescence index; HIX: humification index; FIX: freshness index.

32.98%. For the genus level, the relative abundance was 59.82% for Lactobacillus, 27.02% for Klebsiella, 3.05% for Kluyvera, 1.11% for Leuconostocand 1.08% for Lactococcus.

3.3.4. Carbon and nitrogen sources in MLBC-MFCs Concentrations of DOC and TDN before and after reaction in MFCs were measured and results are listed in Table 2. When SA was used as carbon source, DOC was 395.1 mg/L for the influent, 105.3 mg/L for the oxic effluent and 263.6 mg/L for the anaerobic effluent. When MFCs were fed with BPE, the DOC of the influent, oxic effluent and anaerobic effluent was 304.5, 107.5 and 161.6 mg/L, respectively, while the corresponding DOC was 331.3, 96.2 and 123.1 mg/L after ABPE substituted for BPE. As far as TND was concerned, the same changing trend as DOC could be observed that the concentration of TDN in the anaerobic effluent remained higher than that in the oxic effluent, no matter what kinds of carbon sources were employed.

4. Discussion 4.1. General performance of MFCs In the current study, subaqueous wetland sediments (SWSs) and banana peels (BPs) were combined to produce electrical energy in membrane-less and biocathode-containing microbial fuel cells (MLBCMFCs). Results confirmed that these two materials have good potentials to be used to produce electrical energy, the reasons responsible for which are (1) SWSs comprise both a huge number of functional microorganisms (Cao et al., 2017) and a variety of solid/liquid carbon sources (Morris et al., 2002) that are all indispensible for electricity production in MFCs and (2) banana peels usually contain various substances (Munagapati et al., 2018) that are easily consumed by microbes. Furthermore, as an anaerobic inoculum, SWSs are supposed to be advantageous over anaerobic sludge since anaerobic sludge tends to convert a substantial number of substrates to biogas and contribute to the growth of a variety of undesirable microorganisms (Sjöblom et al., 2017). As shown in Fig. 2, there was a lag phase of about 40–60 h for all substrates used before the voltage got stable, which is similar with a

3.3.5. Chemical and microbial analysis on banana peel extracts In order to explore the potential mechanisms of generating electricity brought about by the addition of banana peels, some chemical components in banana peel extracts before and after anaerobic fermentation were analyzed (Table S1 and S2) and the microbial diversity analysis for anaerobically fermented banana peel extracts (ABPE) was carried out (Fig. S1). Results showed that most carbon source was converted to lactic acid as it got up to 3800 mg/kg in ABPE. Regarding the microbial diversity, the most predominant phyla were Firmicutes and Proteobacteria, accounting for 64.14% and 35.52%, respectively, while the dominating orders were Lactobacillales and Enterobacteriales, the relative abundances of which were respectively 63.76% and 75

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Fig. 5. Optical properties (a), fluorescence regional integration (b) and 3D excitation emission matrices (c) of water samples before and after MFCs fed with BPE. SUVA: specific ultraviolet absorption at 254 nm; E2/E3: UVA at 254 nm divided by UVA at 365 nm; FI: fluorescence index; HIX: humification index; FIX: freshness index.

from those membrane-containing MFCs (Rabaey et al., 2003; Sjöblom et al., 2017). However, as described above, all materials used in this study were extremely cost-effective owing to utilizing natural resources and MFCs without membranes and catalysts. Most importantly, MLBCMFCs equipped with SWSs and BPs are easily scaled up and therefore are more beneficial for the engineering practice.

previous report where a different type of MFC was adopted (Sjöblom et al., 2017), implying that an MFC is a complex system that can be affected by many factors. In general, factors such as the electrode material, the electrode surface area, the distance between the electrodes, the reactor configuration or the proton exchange membrane affect power generation and the performance of MFC (Antonopoulou et al., 2010). However, the internal resistance is known to be a limiting factor to the power output of MFCs as it could be changed by most of the above factors like changing the distance between the electrodes (Logan et al., 2006). Although the maximum power densities obtained in the current study were low (Table 1), it is reasonable to consider that MLBC-MFCs are usually characterized of a higher resistance than those membrane-containing MFCs (Sjöblom et al., 2017; Thygesen et al., 2009), which can also be corroborated by the data of internal resistances shown in Table 1. Nonetheless, compared with the same type of MFCs (Dai et al., 2015), the internal resistances for BPs in this study were relatively low, indicating that the combination of SWSs and BPs is suitable for the electricity generation in MLBC-MFCs. Also, as listed in Table 1, the internal resistance for different substrates followed the order of SA > BPE > ABPE, suggesting that ABPE is more propitious for the MFCs system. Moreover, a voltage output of around 30 mV existed in all controls (Fig. 2), which was most likely ascribed to carbon sources involved in SWSs. Undoubtedly, the total electrical energy per gram of substrates (see Section 3.2) from the present MLBC-MFCs was much lower than that

4.2. Mechanisms for the electricity production To understand the effect of the substrates on the electricity generation in MFCs, a metabolic study was conducted to follow the consumption of the sugars and consumption of organic acids during the operation of the MFCs (Sjöblom et al., 2017), but this does not fit the present study, which is mainly due to the fact that the compositions of both SWSs and BPs are quite complicated. Consequently, the UV–vis absorption and fluorescence excitation emission matrix (EEM) were used to analyze the DOM of water samples before and after MFCs treatment. As a widely used indicator of DOM aromaticity (Karanfil et al., 2002), a higher SUVA represents a higher aromaticity of DOM. Despite not very obvious, the SUVA of BPs (BPE and ABPE) for the oxic effluent was lower than that for the anaerobic effluent (Fig. 5,6a), suggesting that the microorganisms in the anaerobic area of MFCs might produce more metabolites with a large molecular weight like proteins than in the oxic area, which could also be proved by E2/E3. As a proxy of 76

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Fig. 6. Optical properties (a), fluorescence regional integration (b) and 3D excitation emission matrices (c) of water samples before and after MFCs fed with ABPE. SUVA: specific ultraviolet absorption at 254 nm; E2/E3: UVA at 254 nm divided by UVA at 365 nm; FI: fluorescence index; HIX: humification index; FIX: freshness index.

good agreement with a conclusion demonstrated in a study that there are relationships between water quality (or, more specifically, its biodegradable organic matter) and fluorescence in the Peak T fluorescence region (Bridgeman et al., 2013). For oxygen environments, compared to the oxic effluent, two humic-like peaks of A and C (Jason et al., 2010) in the anaerobic effluent were reduced significantly for all substrates, and the same trend could be clearly observed in HIX (Fig. 4, 6a), an index for the relative abundance of humic substances (Ohno, 2002), indicating (1) the anaerobic area of the MFCs used in the current study played a crucial role in generating electricity and (2) the aerobic microorganisms attached on the cathode primarily functioned as biocatalysts. In addition, a common index to differentiate the microbial or terrestrial origin of DOM (Cory and McKnight, 2005) (FI) and an index for the relative abundance of recently produced autochthonous DOM

molecular weight, the E2/E3 inversely correlates with the molecular weight of DOM (Peuravuori and Pihlaja, 1997). Thus, there is no doubt that more intermediates with a relatively large molecular weight were synthesized in the anaerobic area as the E2/E3 in this area was lower than that in the oxic area (Fig. 4, 6a). The fluorescence EEM of DOM showed a strong dependence on substrate characteristics and oxygen environments. For substrate characteristics, there were two tryptophan-like DOM peaks of T1 and T2 (Coble et al., 1998) in the influent of both BPE and ABPE but they did not exist in the SA influent (Fig. 4, 6c), which is completely because of components involved in BPs such as tryptophan and some proteins (Happi Emaga et al., 2007). After MFCs treatment, these two peaks almost disappeared in both oxic and anaerobic effluent, manifesting that they were degraded easily by microorganisms. This result is in

Table 2 Concentrations of DOC and TDN before and after MFCs treatment. SA: sodium acetate; BPE: banana peel extracts; ABPE: anaerobically fermented banana peel extracts. DOC (mg/L)

Influent Oxic effluent Anaerobic effluent

TDN (mg/L)

SA

BPE

ABPE

SA

BPE

ABPE

395.1 ± 2.2 105.3 ± 2.4 263.6 ± 3.1

304.5 ± 2.1 107.5 ± 2.4 161.6 ± 28.6

331.3 ± 10.6 96.2 ± 3.4 123.1 ± 3.5

9.5 ± 0.9 15.3 ± 0.7 52.4 ± 1.7

12.9 ± 0.3 21.4 ± 1 59.9 ± 1.5

13.1 ± 0.2 15.7 ± 0.7 48.3 ± 0.4

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5. Conclusions

(Wilson and Xenopoulos, 2009) (FIX) did not change markedly before and after MFCs reaction, which might be attributed to the complexities of DOM in the system. Likewise, the results of fluorescence regional integration also present a high dependence of the fluorescent DOM composition on the substrate and oxygen, especially for the percent fluorescence responses in region I (tyrosine-like), II (tryptophan-like) and IV (microbial byproduct-like). Although the percentages of these three regions were all high in the influent for both BPE and ABPE (Fig. 5, 6b), it is totally reasonable since BPs are well-known to possess various organic matters, which is more evident for the anaerobically fermented BPs (ABPE) (Katongole et al., 2017). Concerning the oxygen environment, the percentages of three regions in the anaerobic effluent were higher than those in the oxic effluent regardless of SA, BPE and ABPE. This was mainly caused by a more release of amino-acid-like compounds produced in the anaerobic area of MFCs, further verifying that the anaerobic microorganisms in systems were very active and hence were more propitious for the electricity generation. Most interestingly, when MFCs reaction finished, the residual DOC and TDN in the anaerobic effluent were much more than in the oxic effluent (Table 2). This phenomenon is most probably due to the characteristics of SWSs. It has been reported that the activities of some hydrolytic enzymes (β-glucosidase, cellobiohydrolase, β-xylosidase, and N-acetylglucosaminidase) on particles were detected (Jackson and Vallaire, 2007), and various cellulases with different molecular sizes were implicated in cellulose breakdown in wetlands (Yamada and Toyohara, 2012). In other words, some solid organic matters could be decomposed by indigenous hydrolytic enzymes to result in an increase in DOC/TDN, which is consistent with the results presented herein (Table 2). In particular, SA seems to be not suitable for the electrical energy production since the voltage on SA was considerably smaller than on BPE and ABPE (Fig. 2). This result can be explained from the following two points. To begin with, SWSs used in this study are entirely distinct from other inoculums such as domestic wastewater (Thygesen et al., 2009) and anaerobic sludge (Sjöblom et al., 2017), in which some functional microorganisms might have been accustomed to an acetate-containing environment. On the other hand, some components like trace elements and amino acids included in BPs are helpful for the rapid growth of microorganisms and therefore contribute to a more active microbial consortium. Especially for ABPE containing a relatively high concentration of lactic acid (Table S2), it seems to be more advantageous to MLBC-MFCs, for it has been demonstrated that lactate-fed MFCs had the highest peak voltage (Jung and Regan, 2007), maximum power density (Wu et al., 2013) and current density (Flayac et al., 2018). The microbial analysis also further verified that ABPE was more propitious for electricity generation since Lactobacillus and Lactococcus, major parts of the lactic acid bacteria group, not only contributed to the lactic acid production during the anaerobic fermentation of banana peel extracts but also have been proved to be capable of ether improving the performance of MFCs (Wang et al., 2017) or catalyzing electricity generation via mediators such as quinones (Freguia et al., 2009) and flavins (Masuda et al., 2010). As the second most predominant genus, Klebsiella was known to play an important role in MFCs. It was reported that Klebsiella contributes mainly to bioelectricity generation in MFCs with biocathode and therefore could be utilized as a biocatalyst for enhancement of electrical performance in MFC systems (Lee et al., 2016; Qiu et al., 2017). In addition to biocathode, the anode medium supernatant was also electrochemically active, where Klebsiella produced mediators to promote extracellular electron transfer (Xia et al., 2010). As for other genera, both Kluyvera and Leuconostoc were observed in MFCs (Hodgson et al., 2016; Yuan et al., 2013). Overall, though there was no remarkable difference in electrical energy per gram substrate (see Section 3.2) between BPE and ABPE, all the results mentioned above evidenced that ABPE was more beneficial to harness electricity from MLBC-MFCs under our experimental conditions.

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