Electricity generation from dissolved organic matter in polluted lake water using a microbial fuel cell (MFC)

Biochemical Engineering Journal 71 (2013) 57–61

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Electricity generation from dissolved organic matter in polluted lake water using a microbial fuel cell (MFC) Yan-Rong He a , Xiang Xiao a,b,∗ , Wen-Wei Li a,∗∗ , Pei-Jie Cai a , Shi-Jie Yuan a , Fang-Fang Yan a , Meng-Xing He a , Guo-Ping Sheng a , Zhong-Hua Tong a , Han-Qing Yu a a b

Department of Chemistry, University of Science & Technology of China, Hefei 230026, China School of Environment, Jiangsu University, Zhenjiang 212013, China

a r t i c l e

i n f o

Article history: Received 2 June 2012 Received in revised form 13 October 2012 Accepted 23 November 2012 Available online 1 December 2012 Keywords: Lake water Microbial fuel cell (MFC) Bioremediation Biodegradation Protein Microbial growth

a b s t r a c t A microbial fuel cell (MFC) was explored as a pretreatment method to remove dissolved organic matter (DOM) from polluted lake water and simultaneously generate electricity. After the MFC treatment, the total organic carbon concentration in the raw lake water was reduced by 50%, the physicochemical nature of DOMs was substantially altered. Protein-like substances in lake water were utilized as a major substrate for the MFC, while humic-like substances were refractory to the biodegradation. A further investigation into the bovine serum albumin utilization in an MFC confirms that the electricity generation was closely associated with the removal of protein-like substrates. Toxicity assessment by Salmonella typhimurium Sal94 indicates that the genotoxic agents in the polluted lake water were almost completely removed after the MFC treatment. This approach of coupling microbially-catalyzed electricity generation with DOM removal may offer a potential avenue for energy-efficient bioremediation of lake water. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Water shortage and environmental pollution are raising global concern. In China, a considerable part of freshwater is from lake [1], but 55.8% of the lake waters fail to meet the standard of source water supply [2]. In most lakes, there is a high level of dissolved organic matter (DOM), which is refractory to biodegradation. The situation is further exacerbated by the frequently occurrence of algal bloom in recent years [3], which leads to exhausting of dissolved oxygen, increased death of aquatic lives, and thus raised level of the DOM. Generally, the algal biomass in lakes can be removed though salvage, but the organic substances and potential genotoxic agents produced by algal would persist in water. Both physicochemical processes, such as chlorination, ozonation, membrane filtration, and biological processes have been adopted for treatment of polluted lake water [4–6]. Especially, an integrated application of different processes, such as the combination of ozonation and biofiltration, ozonation and membrane reactor, exhibited good performances [7,8]. Nevertheless, the

∗ Corresponding author at: Department of Chemistry, University of Science & Technology of China, Hefei 230026, China. Fax: +86 551 3606698. ∗∗ Corresponding author. Fax: +86 551 3601592. E-mail addresses: [email protected] (X. Xiao), [email protected] (W.-W. Li). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.11.016

overall treatment and energy efficiencies are yet to be improved to make these processes practically viable. Microbial fuel cell (MFC), being widely recognized as a promising wastewater treatment technology, is capable of recovering electricity from organic pollutants to partially offset the treatment cost [9,10]. It has been demonstrated that MFC could effectively utilize not only degradable organics but also some biorefractory compounds such as p-nitrophenol as substrate [11,12]. Thus, it is expected that MFC might also facilitate removal of DOMs in polluted lake water by utilizing them for electricity generation or altering their genotoxicity, so that further purification of the water can be achieved in subsequent polishing steps. However, lake water is somewhat different from normal wastewaters. First, the organic content is very low even compared to low-strength wastewater like domestic wastewater. Secondly, the sources of organic matter in lake water are more diverse and complex. At last, the breakout of algae bloom would result in the production and accumulation of genotoxic substances in lake water. Therefore, it is still to be found out whether MFC can effectively utilize polluted lake water as substrate and how it would alter the composition of lake water. Therefore, in this study the treatment of polluted lake water by a MFC was investigated with the following three objectives: (1) to explore the feasibility of abating pollution and altering DOM compositions of polluted lake water using a MFC; (2) to find out whether the potential genotoxic agents in the polluted lake water could be significantly reduced after the MFC treatment; and (3) to elucidate

Y.-R. He et al. / Biochemical Engineering Journal 71 (2013) 57–61

the mechanisms of DOM removal and genotoxicity reduction in such an MFC system. 2. Experimental 2.1. Reactor construction and operation A single-chamber air-cathode MFC with the same configuration as described by Zang et al. [13] was used for the experiment. The cathode electrode was made of Pt-loaded carbon paper (2 cm × 2 cm, 2 mg/cm2 ; GEFC Co., China). Activated carbon fiber, fabricated as described by Zhang et al. [14] was used as anode electrode. The bioanode was cultivated in another air-cathode MFC inoculated with anaerobic sludge, and acetate was used as substrate. A stable voltage output was obtained after 2-months cultivation, indicating that a consortium of electrochemically active microorganisms was enriched on the carbon fiber. Enrichment medium contained (in 1 l of 50 mM phosphate buffer, pH 7.0): NH4 Cl, 310 mg; KCl, 130 mg; CaCl2 , 10 mg; MgCl2 ·6H2 O, 20 mg; NaCl, 2 mg; FeCl2 , 5 mg; CoCl2 ·2H2 O, 1 mg; MnCl2 ·4H2 O, 1 mg; AlCl3 , 0.5 mg; (NH4 )6 Mo7 O24 , 3 mg; H3 BO3 , 1 mg; NiCl2 ·6H2 O, 0.1 mg; CuSO4 ·5H2 O, 1 mg; ZnCl2 , 1 mg, and CH3 COONa, 100 mg as substrate. Water samples were collected from Chaohu Lake, the China’s fifth largest freshwater lake. This lake is usually under severe contamination because of blue-green algae bloom. The water samples were filtered through gauzes to remove algal biomass and then centrifuged for 10 min at 6000 rpm and 4 ◦ C. Aliquots of 400 ml supernatants were added into the MFC anode chamber, which was then purged with N2 and sealed. A resistor of 1000  was connected to the circuit. The MFC was operated under 28 ◦ C and the voltage across the resistor was recorded continuously using an on-line data acquisition system. The anodic solution was sampled every 2 or 3 days for analysis. To get a better insight into the relationship between DOM decomposition and electricity generation, a model protein, bovine serum albumin (BSA), was also used as substrate for MFC. The reactor setup and operation were the same as above except for the use of BSA to replace acetate. Water samples were taken at the predetermined time intervals for analysis. 2.2. Chemical analysis The concentration of total organic carbon (TOC) of water samples was measured with a TOC analyzer (VCPN, Shimadzu Co., Japan). The liquor samples were centrifuged at 12,000 rpm for 5 min, and the supernatant fluid was diluted to a range of 1–1000 mg/l with distilled water for TOC analysis. BSA concentration was determined using the modified Lowry methods with chicken egg albumin as the standard [15]. Three-dimensional excitation-emission matrix (EEM) fluorescence spectra of the water samples before and after the MFC treatment were measured using a luminescence spectrometry (LS55, Perkin-Elmer Co., USA). The emission spectra from 250 to 700 nm at 0.5 nm increment repeatedly and the excitation wavelengths from 240 to 500 nm spaced by 10 nm intervals in the excitation domain were measured. Excitation and emission slits were both maintained at 10 nm, and the scanning speed was set at 1200 nm/min for all measurements. A 290 nm emission cutoff filter was used. The EEM fluorescence spectra data were processed and plotted using MatLab 7.0 (MathWorks Inc., USA). The molecular weight (MW) distribution of water samples was measured using a gel permeation chromatography (GPC) (Waters 1515, Waters Co., USA) coupled with a UV absorbance detector. Deionized water was used as the eluent at a flow rate of 1.0 ml/min.

Prior to the analysis, the samples were dialyzed with deionized water (2 h each time) for three times, and then filtrated through 0.45-mm acetate cellulose membranes. Electrochemical measurements were conducted using an electrochemical workstation (CHI 660, Chenhua Instrument Co., China). The polarization curve was obtained using linear sweep voltammetry at a scan rate of 1 mV/s [11]. The current density and power density were normalized to the MFC volume of 400 ml. 2.3. Toxicity bioassay Salmonella typhimurium Sal94 (pRecA::LuxCDABE tolCC CmR AmpR ) strain was used to assay the environmental genotoxicity of the water samples [16]. Sal94 strain was cultured overnight in LB medium in a shaking incubator at 26 ◦ C, with 30 ␮g/ml kanamycin added in order to maintain the plasmid. A 2000-fold dilution of the cell suspension was prepared using fresh LB without kanamycin. Water samples collected from the MFC were centrifuged at 12,000 rpm for 5 min. The supernatant of 100 ␮l was mixed and incubated with an equal volume of diluted bacterial solution in 2-ml Eppendorf tube at 26 ◦ C. Sterile distilled water was used as the control. The emitted luminescence was monitored using a luminometer (GloMax 20/20, Promega, USA) for 5 s. Luminescence values are presented as arbitrary relative light units, or as the response ratio of luminescence of the induced sample to that of the blank control [17]. 3. Results 3.1. DOM removal and electricity generation in the MFC For the MFC system, the anode chamber was filled with the polluted lake water and operated under anaerobic conditions. No other external carbon sources were added. A gradual decline of TOC concentration from 62 mg/l to 30 mg/l was achieved in this system during the 45-days operation (Fig. 1). Meantime, a continuous generation of electricity was observed (Fig. 2A). The output voltage varied substantially over time. A relatively high voltage was obtained at the beginning, attributed to the presence of easily degradable components in the DOMs of lake water. With the fast depletion of such easily degradable contents, the voltage dropped rapidly. Then, the less-degradable organics were further utilized to sustain a continuous but slow-declining electricity generation. Similar profiles of voltage and TOC evolution over time were obtained in a repeated experiment, although at a much shorter time span

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(Supplementary material, Figure S1). This difference should be attributed mainly to the different concentrations and structures of DOMs in the fed lake water, which lead to different utilization rates by bacteria. Thus, these results suggest a good repeatability of this system for lake water treatment. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2012.11.016. Fig. 2B illustrates the polarization curve and power density curve of the MFC. The maximum power density reached 164 mW/m3 .

3.2. Changing profiles of DOM compositions in the MFC treatment To track the fate of DOM fractions in the MFC system, the compositions of raw water and the MFC-treated water samples were analyzed using EEM. The EEM fluorescence spectra of the two samples were similar in peak locations, but varied in the fluorescence intensity (Fig. 3). The peak identified at excitation/emission (Ex/Em) of 280/350–358 nm is ascribed to protein-like substances, in which the aromatic amino acid tryptophan gives fluorescence [18]. A second peak was located around Ex/Em of 330–360/416–442 nm, which could be ascribed to visible humic-like substrate [19]. As shown in Fig. 4, the protein-like substances were significantly reduced after the treatment, indicating that such substances had been degraded. However, the fluorescence intensity of the humic-like substances increased only slightly and a visible blue shift (from Ex/Em of 360/442 nm to 330/416 nm) was observed (Fig. 3), suggesting that the composition of the humic-like substances was slightly changed. The MW profiles of DOMs in the treated water samples and the raw lake water control were measured to detect possible DOM decomposition. Fig. 4 shows that the MW peak intensity of the treated water was significantly lower than that of the control, suggesting a substantial decomposition of the DOMs in the lake water to low MW substances after the MFC treatment.

Fig. 3. EEM fluorescence spectra of: (A) the raw lake water and (B) the water sample after the MFC treatment. Data were collected every 10 nm over an excitation range of 240–500 nm and an emission range of 250–700 nm.

3.3. Toxicity evaluation The changes of genotoxicity of the water samples during the MFC operation were evaluated by using a bioluminescent bacterium S. typhimurium Sal94. This strain has been successfully used to evaluate the purification consequence in terms of the potential genotoxic agents present in the polluted lake water [16]. By measuring the bioluminescence (lux) of Sal94 strain, the cytotoxicity and genotoxicity of samples can be sensitively detected [20]. The luminescence profiles of water samples are presented in Fig. 5A. The bioluminescence of Sal 94 was found to be significantly affected by the induction time, and higher values of bioluminescence were observed with the 2-h induction compared with the 1-h and 3-h induction (Fig. 5B). Thus, in this case 2 h was selected as the induction time for the Sal 94 to assay the lake water. The lake water induced a very high luminescence value at the beginning of MFC treatment, indicating a strong inducing ability of the raw lake water. The genotoxicity can be more clearly reflected by response ratio.

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Fig. 2. (A) Voltage output of the MFC and (B) polarization curves of the MFC.

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The luminescence value induced by the water samples was always higher than that of the control, i.e., the response ratio > 1.0. These results confirm a clear SOS inductive effect and a potential genotoxicity activity of the polluted lake water. The continuous decline of the response ratio value in Fig. 5B indicates a weakening trend of the genotoxicity. The response ratio dropped to approaching 1.0 at the end of MFC treatment, indicating that the genotoxic agents in the lake water were almost completely removed in the MFC. 4. Discussion In the present study, an air-cathode MFC was used to treat DOM in polluted lake water. A substantial decomposition of DOM accompanied with electricity generation was achieved in this system. The maximum power density reached 164 mW/m3 , which is relatively lower than those reported in many previous studies [9]. Noting that no extra carbon source but the lake water was supplied in this system, the electricity production was attributed to the utilization of the DOM in the polluted lake water. The biorefractory nature of DOMs, together with the sub-optimal operating conditions, might be the main reason for a low electricity generation in this study. Thus, there is still room to elevate the electricity generation if the system design and operation can be optimized. Moreover, the results of this study imply that a main and more promising application direction of this technology might lie on the efficient remediation and purification of lake water rather than simply on electricity generation. EEM is a rapid, selective and sensitive technique for composition analysis. It has been successfully used to evaluate the characteristics of natural DOMs from various origins, and is commonly used to differentiate humic and protein-like substances

[6,18,21–23]. In this work, the EEM fluorescence spectra analysis indicates that the protein-like substances in the lake water were reduced significantly. Taking into account the protein-like substances degradation, TOC removal and electricity generation, it is reasonable to assume that the protein-like substances was utilized as a major substrate for electricity generation. To further confirm that electricity generation is associated with the removal of protein-like substrates, pure BSA was used as substrate for MFCs. Accompanied with the electricity generation, a rapid decline of BSA concentration was observed (Fig. 6). However, when the external circuit was disconnected, the BSA concentration dropped only a little. It was also found that the drop of voltage lagged behind the BSA concentration decline. These results indicate that the protein was initially broken down into small organic molecules with a concomitant electricity generation, then the small molecules were further utilized by bacteria to sustain the electricity output. This is evidenced by the changing profile of volatile organic acids, which initially increased but then declined over time (data not shown). A similar phenomenon of volatile organic acid change was also observed in previous works [24]. Notably, the concentration of humic-like substances increased slightly and the peak maxima location showed a visible blue shift (Fig. 3), suggesting that the humic-like substances were less degraded and were even produced as soluble microbial products by microorganisms. This result is consistent with a previous report [6]. It should be noted that, because not all of the DOM components could be detected by EEM and GPC, the decomposition of DOMs by MFCs might have not been fully reflected by these measurements. Nonetheless, the results of this study clearly demonstrate a significant change of DOM compositions and TOC removal by the MFC treatment, and imply a high potential of this technology for lake remediation application. According to the genotoxicity tests, the polluted lake water displayed a significant positive luminescence-inducing activity to Sal94, indicating the presence of strong genotoxic agents in the lake water. Considering that the water samples were collected at algae bloom period, such a result was not surprising [25]. However, the genotoxicity almost completely disappeared at the end of the MFC

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treatment. This result agrees well with the EEM data. The aromatic rings or conjugated bonds mostly possess genotoxic potential, and these genotoxic substances were substantially degraded in the MFC operation, leading to decreased genotoxicity of the lake water. This study demonstrates the feasibility of using a MFC to remediate for polluted lake water. Efficient removal of TOC and genotoxic agents in polluted lake water with concurrent electricity generation was achieved in MFCs. The compositions of DOM were changed in the MFC operation, and biodegradation behaviors of different DOM types varied significantly. Specifically, protein-like substances were found to be biologically removable, while the majority of humic substances were highly refractory to biodegradation. Although the DOM removal efficiency by the MFC treatment is insufficient to meet the discharge criteria yet, the DOM could be altered in its physicochemical nature and genotoxicity. Thus, MFC can be used as an energy-efficient pretreatment step to preliminarily remove most of the DOMs, so that a more convenient and effective polishing of the treated lake water can be favored in subsequent steps. For example, the fouling of membranes, if any, shall be significantly alleviated due to substantial removal of proteins, which are known as a main membrane foulant. Therefore, the MFC system may offer a new and potential water remediation technology for the DOM removal in an energy-efficient way, but more extensive studies are required to further improve its performance in terms of DOM removal and electricity generation. Furthermore, it is yet to be explored that how the degradation of various DOMs in lake water and the electricity generation are related with the different microbial species in the anodic biofilm of MFCs. 5. Conclusions • MFC can be used as an efficient pretreatment method to restore the polluted lake water. The DOM concentration of the lake water decreased significantly, and a maximum power density of 164 mW/m3 was obtained in the MFC. • The DOMs composition was changed substantially after the MFC treatment, and the protein-like substances in the lake water were utilized as a main substrate for MFC. • The genotoxic agents in the polluted lake water were almost completely removed after MFC treatment. • The information obtained in this study is beneficial to develop a MFC-based pretreatment technology for sustainable remediation of polluted water. Acknowledgements The authors wish to thank the NSFC (20907050) and NSFC-JST (21021140001) for the partial support of this study. References [1] H. Cheng, Y. Hu, J. Zhao, Meeting China’s water shortage crisis: current practices and challenges, Environ. Sci. Technol. 43 (2009) 240–244. [2] China EPA, China Water Resources Bulletin, China Water Resources and Hydropower Press, Beijing, 2010.

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