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Membrane-less MFC based biosensor for monitoring wastewater quality
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Membrane-less MFC based biosensor for monitoring wastewater quality Pinanong Tanikkul a, Nipon Pisutpaisal a,b,c,d,* a
The Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand b Department of Agro-Industrial, Food and Environmental Technology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, 10800, Thailand c The Research and Technology Center for Renewable Products and Energy, King Mongkut's University of Technology North Bangkok, 10800, Thailand d The Biosensor and Bioelectronics Technology Centre, King Mongkut's University of Technology North Bangkok, 10800, Thailand
article info
abstract
Article history:
Microbial fuel cell (MFC), a bioelectrochemical device, can be used to produce bioelectric
Received 31 March 2017
signals from hydrogen carriers, particularly organic compounds in wastewaters. The solid
Received in revised form
correlation between the signals and the hydrogen carrier concentration can be exploited in
9 October 2017
a biosensor for measuring wastewater quality. A small volume of the membrane-less
Accepted 12 October 2017
SCMFCs were operated with various wastewater concentrations to investigate the rela-
Available online xxx
tionship between the concentration of substrates with the current outputs and the performance of the SCMFCs. The results demonstrated that the detection times of current
Keywords:
outputs from low to high peak were significantly short when using a low synthetic
Microbial fuel cell
wastewater (SW) concentration of 25e1000 mg COD.L1. The correlation between the SW
Membrane-less SCMFC
concentration and the current outputs was obtained up to 250 mg COD.L1 (R2 ¼ 0.96).
MFC biosensor
When the SCMFCs were fed with distillery wastewater (DW) from low to high concentra-
Microbial biosensor
tion (50e2000 mg COD.L1), it showed a detection times of the current as short. SCMFCs had a good correlation between the concentration of DW and the current outputs obtained up to 1200 mg COD.L1 (R2 ¼ 0.97). Maximum substrate reduction was found more than 90% when the initial SW concentration was in the range of 25e1000 mg COD.L1. While substrate reduction was more than 60% for the DW concentration in the range of 50e2000 mg COD, L1 was operated. In other words, this membrane-less SCMFCs are able to be a long starvation (5 days) and a high repeatability of the current output in both wastewaters. Indications proved that the detection time of current and substrate degradation were dependant on concentrations, types of substrate, and types of MFC. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand. E-mail address: [email protected] (N. Pisutpaisal). https://doi.org/10.1016/j.ijhydene.2017.10.065 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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Introduction MFC is a bioelectrochemical device that can biologically convert hydrogen carriers, especially renewable organic compounds, to electricity under anaerobic condition [1,2]. Microorganisms in the anode chamber oxidize energy stored in organic matters to produce protons (biohydrogen) and electrons [1e3]. These electrons are transferred to the anode surface and then flow through an external circuit to the cathode chamber, while protons diffuse via a membrane into the cathode chamber and combine with electrons to form water. The voltage difference between two separate chambers that generates bioelectricity directly [1e5]. The MFC technology is more suitable for biohydrogen production from renewable substrates, in term of producing electricity couple with wastewater treatment. It is difficult to compare this technology with other treatment processes, due to the difference of a substrate types, a microbial source, and operating conditions. However, there are some key advantageous of MFCs, namely; (a) MFCs can be operated at ambient temperature; (b) Aeration processes are not required during the operation of MFCs and (c) MFCs provide high energy conversion efficiency to electric current and high treatment efficiency [6,7]. MFCs can generate electricity from various renewable substrates (simple or complex and wastewaters) [8e10]. MFCs are intriguing technology for various work applications such as biohydrogen production, bioremediation, pollutant biosensor and effectively in wastewater treatment, especially the MFC based microbial biosensor [11e13]. In general, the MFC microbial based biosensors have mainly focused on twochamber MFC and a membrane has been used. The twochamber MFC biosensor based on the dissolved oxygen (DO) consumption using a specific aerobic microbial such as Bacillus subtillis, Arxula ardeninivorans [14], and Serratia marcescens [15] have been firstly reported. Nevertheless, these specific single microorganisms have very short-term stability and these are unstable to various substrates [16]. Recently, using the mixed culture has been suitable for a wide range of substrates in the MFCs, where it is possible to use the MFCs as a biosensor. A study of the mixed culture MFC based biosensors showed a direct proportion between the current generated with the organic matter concentrations and high treatment efficiency [17]. Little research has been done to investigate the MFCs, as a biosensor, for detecting the pollutants and determining the concentration in wastes or wastewaters. Twochamber MFC based on biosensor was operated by using starch processing wastewater was investigated by Kim et al. (2003) [17]. The authors found that the coulombs were generated proportionally to the starch concentration up to the concentration of 206 mg COD.L1 and a good linearity (R2 ¼ 0.99). Kumlanghan et al. (2007) [18] reported that the sensor response of the air-cathode SCMFC using glucose solutions were linear against the concentration up to 2500 mg COD.L1. Feng et al. (2008) [19] found that the current generation increased from 29 to 205 mW cm2 (55e1512 mg BOD.L1) is proportional to the brewery wastewater concentration (R2 ¼ 0.996) from the air-cathode SCMFC with an exchange membrane. Lorenzo et al. (2009) [16] reported that the
air-cathode SCMFC had a linear relationship with the BOD concentration up to 350 mg L1 of synthetic wastewater and had very high reproducibility of signal current. Several types of wastewaters such as food processing wastewater, starch wastewater, brewery wastewater, domestic wastewater, swine manure slurry and paper mill effluent [8e10] have been successfully used as substrates for the electricity generation coupled with the pollution reduction in the MFCs. The distillery wastewater is a by-product of ethanol manufacture using sugarcane molasses materials. The distillery wastewater, high strength with strong odor and brownish color, contains chemical oxygen demand (COD) (134,000e136,000 mg L1) and biochemical oxygen demand (BOD) (69,000e71,000 mg L1) and low pH (4.35). Rich organic content makes it a good candidate feedstock for the bioenergy production [8,9]. This study is focused on the membrane-less SCMFCs based microbial biosensor, which has the ability to convert a specific pollutant in the substrate to the current directly. The relationship between the current generated with the concentration of pollutants can apply the MFC as a biosensor [20e22]. The MFC based biosensors have advantages including shorter response time of the current, high reproducibility, real-time monitoring and long-term stability than the other types of the biosensor [17e22]. The success of the MFC based biosensor depends on many factors such as the MFC configurations, types of microorganisms, types of substrate and the detection time [20e23]. The previous MFCs studies have been illustrated mostly on the electricity generation and the wastewater treatment. Some studies showed unpromising performance toward the membrane-less SCMFCs when applied as a microbial biosensor. In this study, the effectiveness of the current outputs and the biodegradability of the SCMFCs was investigated. The synthetic and distillery wastewaters were used to test the ability of the SCMFCs based biosensor. The relationship between the COD of wastewaters concentrations and the current outputs detection was discussed and the pollutant reduction in both wastewaters were determined.
Materials and methods Membrane-less microbial fuel cells (SCMFCs) The construction of the SCMFCs without an exchange membrane were made of the acrylic cylindrical chamber with a length of 4 cm and a diameter of 3 cm as previously described which has a void volume of 28 mL [24]. The anode electrode was made of a carbon cloth with pre-treated Teflon (without wet proofing; E-Tek, USA). And the cathode electrode was made of a carbon cloth without pre-treated Teflon (30% wet proofed; ETek, USA). In this study, the cathode electrode was coated by a gas diffusion layer on the air contact side [25,26]. Similarly, the platinum catalyst layer loading of 0.5 mg cm2 was coated on the wastewater contact side before use. The titanium wire was used to connect the anode and the cathode electrode while an external resistance (Rext) loading from the resistor box was used to connect the electrodes for complete the circuit.
Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e7
Microorganisms Anaerobic sludge was obtained from a full-scale up-flow anaerobic sludge blanket (UASB) starch-processing wastewater treatment plant of Eiamburapa Co., Ltd (Sakaw, Thailand), operating at the mesophilic condition. At the beginning of the experiment, granular seed with diameter >0.5 mm were washed with tap water to remove sludge matters and cultured with a synthetic wastewater containing a cassava starch (5 g L1) in 10 L of the continuous stirred-tank reactor (CSTR) at 37 C until biogas production was stable. This microbial seed was heat pre-treated at 100 C for 60 min to inhibit the methane producing bacteria prior to use [27,28], which had a concentration of seed as 40 g L1.
Wastewaters Synthetic wastewater (SW) was used for the inoculation and operation. Glucose was used as a carbon source consisted of a nutrient medium with the following (per liter): FeSO4$6H2O, 10.69 mg; MnSO4, 0.589 mg; ZnSO4$7H2O, 0.106 mg; H3BO4, 0.106 mg; CuSO4, 0.003 mg and NH4Cl 8.5 g per g COD [29]. The final pH of a SW was adjusted to 7.0 by 50 mM phosphate buffer solution containing (per liter): NaH2PO4$H2O, 5.62 g; Na2HPO4$H2O, 4.61 g and KCl, 0.13 g [29]. SW was autoclaved at 121 C for 15 min. This wastewater was used to inoculate the microbe biofilm and tested the SCMFCs start-up. Raw distillery wastewater (DW) was collected from the primary clarifier effluent of ethanol production (Sura Bangyikhan Co., Ltd, Pathum Thani, Thailand), which sugar cane molasses were used as the raw material. This wastewater was used to test the ability of the SCMFCs in terms of the relationship among the current generation, the substrate concentration and efficient treatment. DW has high levels of COD as well as BOD and a high temperature (70e80 C) from source. DW was stored in a refrigerator at 4 C until it was used and diluted with deionized water to the given concentration without additions of any other nutrient or trace mineral. The pH of the wastewater dilution was adjusted to 7.0 by phosphate buffer solution of 50 mM. Two types of substrate were flushed with nitrogen gas for 3 min before feeding into the SCMFCs.
Membrane-less SCMFCs start-up The SCMFCs were inoculated with 2.5% by the volume of the heat pre-treated seed in the separate reactor. The SCMFCs were operated with a high SW concentration of 1500 mg COD.L1 in fed batch under open circuit voltage (OCV) at 37 C to allow the microbe attraction on the anode electrode. The wastewater was replaced when the voltage dropped until stability of the voltage output was successfully achieved for the microbe adhesion. Liquid samples were collected for analysis of pH, ORP, COD and sugar content. After the batch inoculation, the SCMFCs were acclimated with a SW in fed batch mode under high external resistance of 1000 U at 37 ± 1 C. The wastewater was replaced when the current was dropped until a steady current output allowed the microbe to be covered as biofilm on the anode electrode. During the acclimation, liquid samples were collected for analysis of pH,
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ORP, COD, and sugar content at the end of two batch cycles. The cyclic voltammetry technique was used to measure the characteristic of the anode biofilm compared with a new anode electrode without biofilm. The polarization curves were measured to determine the characteristic of the SCMFCs using the synthetic wastewater concentration as a function of an external resistance load.
Membrane-less SCMFCs testing as a biosensor The SCMFCs acclimated for more than two months were operated in semi-batch mode under low external resistance of 50 U at 37 C by using both types of wastewater. The SCMFCs based on biosensor performances such as current output, response time of current, reproducibility and the substrate degradation were investigated. Both types of wastewater were twice replaced while the current dropped before changing new concentration. The voltage outputs were automatically recorded and the liquid samples were collected for analysis of pH, COD and sugar content. After that, the SCMFCs were starved before being fed with both types of wastewater again and the performance of the SCMFCs including reproducibility of the current together with substrate degradation were investigated.
Analyses and calculations The performances of the SCMFCs biosensor were measured the voltage (V) and used to calculate the current to estimate the detection time. The voltage (V) outputs were automatically recorded by a multimeter every 10 min (Keithley 2700, USA). The current was calculated by applying the Ohms law;I ¼ ðV=RÞ according to the coulombic efficiency (CE) calculated based on the current generation over time and the change in substrate concentration during the SCMFCs operation [29]. In addition, liquid samples in the SCMFC were collected at the end of each batch cycle to analyses the pH, oxidation-reduction potential (ORP), sugar content, total COD and soluble COD. The pH was measured by pH probe using a pH meter. Likewise, the ORP was measured by an Ag/AgCl probe using the pH meter (Mettler Toledo, Germany). Sugar content was measured by the DNS method [30]. The COD was analyzed using the closed reflux method according to the Standard Methods [31], which the liquid samples were centrifuged to determine soluble COD.
Cyclic voltammetry analyses The characteristics of the oxidation and reduction reactions on the anode surface at the start-up compared with the end of batch acclimation were determined by the cyclic voltammetry (CV) technique (potentiostat, Autolab PGSTAT 204, Switzerland), which is connected to a computer. The characteristic of the anode biofilm was measured compared with the new anode electrode without a biofilm. CV was performed by applying a potential ramp from 500 to 1000 mV at a scan rate of 20 mV s1 on the anode surface only. The working electrode as the anode electrode and the counter electrode as the cathode electrode were connected by a titanium wire. The Ag/ AgCl prop was used as the reference electrode and the prop was inserted near the anode electrode. After all, the
Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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wastewater in the SCMFCs was removed and the new wastewater was changed before measuring.
Results and discussion Membrane-less SCMFCs enrichment and power curves The SCMFCs were inoculated with the heat pre-treated seed and fed with the SW concentration of 1500 mg COD.L1 during the start-up stage. The semi-batch open circuit mode was operated. The wastewater was replaced every day (24 h). And a concentration was not changed until the stable open circuit voltage (OCV) was approximately 300 mV within 10 days. The results described that the microbes biodegraded substrate and generated electrons were deposited on the anode in the SCMFCs but were not able to form biofilm on the anode surface [32]. Therefore, the SCMFCs were shifted to close circuit operation with an 1000 U external resistance connected between the anode and cathode. This purpose is to allow the microbes which had been grown as a catalytic biofilm covering the anode electrode surface [30]. Besides, the results showed a stable current peak approximately of 16 mA was obtained after 2 month acclimation (Fig. 1). It was assumed that a stable biofilm was formed at the anode surface of the SCMFCs. And the performance of the SCMFCs include pH of the effluent decreased varying during 6.87e6.95, while the ORP rapidly decreased in all concentration (10 to 226 mV). This acclimation proved the microbes were able to degrade a substrate in the case of soluble COD removal more than 93% (Fig. 1) and sugar content removal to reach 99% everyday (data not shown). When a stable current generation and efficient COD removal are presented, the electrochemical activities of the microbes on the anode surface were determined by CV. The voltammograms showed relatively higher current output in the forward scan while comparing with a new anode (Fig. 2A). Maximum current was 0.038 mA for the forward scan (1000 mV), the potential was 500 mV for the reverse scan, and the highest current was 0.31 mA. While a new anode surface had no redox peaks, demonstrating the anode surface with no microbes activities were obvious. Consequently, results indicated that the microbes were able to utilize substrate for cells
Fig. 2 e Cyclic voltammograms of the anode surface with and without biofilm at a scan rate of 20 mV s¡1 (A), and the voltage as a function of current density and the polarization curve with varied external resistance from 10 to 1000 U external resistance after two months acclimation (B).
growth on the anode surface as a biofilm and the current could also be generated by the microbes activities in the SCMFCs [3]. The characteristic of the SCMFCs were analyzed by using the polarization curves to confirm that SCMFCs were ready to generate electricity and utilize wastewaters as shown in Fig. 2B. The polarization curve was measured with the function of current density and the power density by varying an external resistance from 10 to 1000 U. This curve shows the voltage was rapidly dropped from 629 to 245 mV from the open circuit. It indicated the occurrence of activation loss. Meanwhile, the subsequent slope of the voltage was 652 mA m2. A maximum power density of 88 mW m2 and current density of 408 mA m2 were achieved at an external resistance load of 750 U external resistance.
Influence of wastewater concentrations on biosensor performance
Fig. 1 e Current generation and soluble COD removal (%) during the SCMFCs acclimation with the synthetic wastewater.
The SCMFCs achieved approximately two months acclimation were tested the relationship between organic loading and the current outputs by varying COD glucose concentrations in SW at a low external resistance of 50 U. The experiment showed
Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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the detection time of the current which was too short to reach the highest current output with the changes in the SW concentration of 25e1000 mg COD.L1 being around 1.7e8.4 h. However, this result illustrated a longer detection time (1.7e8.4 h) than single and two-chamber MFCs that were previously reported [17,20]. The correlation between the concentration of glucose and the coulombs were observed in the range of 25e250 mg COD.L1 (R2 ¼ 0.96) (Fig. 3A). Nevertheless, the SW concentration was higher than 400 mg COD.L1 that no increase of the current output was observed. Different DW concentrations were used to determine the effect of the real wastewater on the current outputs, and tested to identify the relationship between the current outputs with the concentrations of wastewater. The DW was diluted to obtain the COD concentration at the range of 50e2000 mg COD.L1 and was operated in semi-batch mode under 50 U external resistance. The current outputs with the changes in the concentrations of DW were shown in Fig. 4A. The detection time of the current was approximately detected of 2.5e0.9.3 h from low to high concentration. The relationships between the concentrations of DW and the coulombs were observed in the range of 50e1200 mg COD.L1 (R2 ¼ 0.97) (Fig. 4B). While the concentration of DW lower than 50 mg COD.L1, it could be detect the current output. The current increased quickly, but it dropped rapidly especially when a high DW concentration up to 1200 mg COD.L1. Moreover, the high DW concentration up to 1200 mg COD.L1 required a long time to oxidize the high
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Fig. 4 e Maximum current outputs with different DW concentrations of 50e2000 mg COD.L¡1 under 50 U external resistance (A). Inset figures represent the correlation between maximum current with DW concentrations. Total COD removal and efficiency (B).
organic matter concentration in wastewater to complete the batch cycle (>3 days). Although, the result showed longer detection time and low current outputs from these SCMFCs, this type of MFC leading to high reproducibility of the current was conducted with the same initial concentration of both wastewaters (data not shown). Nevertheless, the performance of the system was still potentially high in the case of linear correlation and high substrate reduction.
Substrate reduction
Fig. 3 e Maximum current outputs with different SW concentrations of 25e1000 mg COD.L¡1 under 50 U external resistance (A). Inset figure represent the correlation between maximum current with SW concentrations. Total COD removal and efficiency (B).
The COD removal efficiency in both of wastewaters experiments displayed a similar pattern. High substrate reduction efficiency was observed for the SW more than 97% (1000 mg COD.L1) (Fig. 3B), which was similar to the sugar content removal (>98%). While the DW has the highest substrate reduced efficiency that was 66.5% (2000 mg COD.L1) (Fig. 4B), there was slightly increased in accordance with the increased concentration. The sugar contained in the DW could be reduced more than 95% in all concentration tests. It indicated that the microbes were firstly utilized the preferable reducing sugar due to easier degraded than the other organic content. In addition, the pH of the effluent of the two types of wastewater slightly increased (7.01e7.08) from the initial indicating the microbes which controlled metabolisms activity during the current generation.
Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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that the SCMFCs can not only potentially generate electrical current but can also be applied to measure the organic substrate values as a biosensor and used to treat wastewater.
Acknowledgements The authors are grateful to Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0322/ 2552) and the Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi (Grant No. JGSEE/THESIS/226) for scholarship. The authors also would like to express their gratitude to King Mongkut's University of Technology North Bangkok (grant no. KMUTNB-60-ART-080) for the financial support.
references
Fig. 5 e Current regeneration after the microbes starvation in the SCMFCs fed with SW (A) and DW (B). Number in the figures represent the concentration of wastewater (mg COD.L¡1).
The starvation of the SCMFCs on biosensor The SCMFCs were starved before both of wastewaters were fed again to determine the current regeneration as shown in Fig. 5A and B. The results demonstrated the current could be rapidly regenerated to reach the highest peak of current after 5 days of the starvation with both of wastewaters. The current regeneration was similar to the original value current output before the starvation. All in all, this work suggested that the SCMFCs are good in practice for applying as a microbial biosensor due to rapid regeneration of the current when the system is in starvation.
Conclusion Simultaneous bioelectricity generation and degradation of organic hydrogen carriers were achieved by using the SCMFCs. The SCMFCs were successfully operated, which clearly showed a linear correlation between the low concentrations of wastewaters and the current outputs. The detection time of current was short and the current reached its highest quickly after changing to a new concentration of wastewater. The results demonstrated a high reproducibility of the current after the SCMFCs were starved for a long period. This is an important part of the MFC based biosensor. On the whole, these suggest
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Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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ScienceDirect journal homepage: www.elsevier.com/locate/he
Membrane-less MFC based biosensor for monitoring wastewater quality Pinanong Tanikkul a, Nipon Pisutpaisal a,b,c,d,* a
The Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand b Department of Agro-Industrial, Food and Environmental Technology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, 10800, Thailand c The Research and Technology Center for Renewable Products and Energy, King Mongkut's University of Technology North Bangkok, 10800, Thailand d The Biosensor and Bioelectronics Technology Centre, King Mongkut's University of Technology North Bangkok, 10800, Thailand
article info
abstract
Article history:
Microbial fuel cell (MFC), a bioelectrochemical device, can be used to produce bioelectric
Received 31 March 2017
signals from hydrogen carriers, particularly organic compounds in wastewaters. The solid
Received in revised form
correlation between the signals and the hydrogen carrier concentration can be exploited in
9 October 2017
a biosensor for measuring wastewater quality. A small volume of the membrane-less
Accepted 12 October 2017
SCMFCs were operated with various wastewater concentrations to investigate the rela-
Available online xxx
tionship between the concentration of substrates with the current outputs and the performance of the SCMFCs. The results demonstrated that the detection times of current
Keywords:
outputs from low to high peak were significantly short when using a low synthetic
Microbial fuel cell
wastewater (SW) concentration of 25e1000 mg COD.L1. The correlation between the SW
Membrane-less SCMFC
concentration and the current outputs was obtained up to 250 mg COD.L1 (R2 ¼ 0.96).
MFC biosensor
When the SCMFCs were fed with distillery wastewater (DW) from low to high concentra-
Microbial biosensor
tion (50e2000 mg COD.L1), it showed a detection times of the current as short. SCMFCs had a good correlation between the concentration of DW and the current outputs obtained up to 1200 mg COD.L1 (R2 ¼ 0.97). Maximum substrate reduction was found more than 90% when the initial SW concentration was in the range of 25e1000 mg COD.L1. While substrate reduction was more than 60% for the DW concentration in the range of 50e2000 mg COD, L1 was operated. In other words, this membrane-less SCMFCs are able to be a long starvation (5 days) and a high repeatability of the current output in both wastewaters. Indications proved that the detection time of current and substrate degradation were dependant on concentrations, types of substrate, and types of MFC. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. The Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand. E-mail address: [email protected] (N. Pisutpaisal). https://doi.org/10.1016/j.ijhydene.2017.10.065 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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Introduction MFC is a bioelectrochemical device that can biologically convert hydrogen carriers, especially renewable organic compounds, to electricity under anaerobic condition [1,2]. Microorganisms in the anode chamber oxidize energy stored in organic matters to produce protons (biohydrogen) and electrons [1e3]. These electrons are transferred to the anode surface and then flow through an external circuit to the cathode chamber, while protons diffuse via a membrane into the cathode chamber and combine with electrons to form water. The voltage difference between two separate chambers that generates bioelectricity directly [1e5]. The MFC technology is more suitable for biohydrogen production from renewable substrates, in term of producing electricity couple with wastewater treatment. It is difficult to compare this technology with other treatment processes, due to the difference of a substrate types, a microbial source, and operating conditions. However, there are some key advantageous of MFCs, namely; (a) MFCs can be operated at ambient temperature; (b) Aeration processes are not required during the operation of MFCs and (c) MFCs provide high energy conversion efficiency to electric current and high treatment efficiency [6,7]. MFCs can generate electricity from various renewable substrates (simple or complex and wastewaters) [8e10]. MFCs are intriguing technology for various work applications such as biohydrogen production, bioremediation, pollutant biosensor and effectively in wastewater treatment, especially the MFC based microbial biosensor [11e13]. In general, the MFC microbial based biosensors have mainly focused on twochamber MFC and a membrane has been used. The twochamber MFC biosensor based on the dissolved oxygen (DO) consumption using a specific aerobic microbial such as Bacillus subtillis, Arxula ardeninivorans [14], and Serratia marcescens [15] have been firstly reported. Nevertheless, these specific single microorganisms have very short-term stability and these are unstable to various substrates [16]. Recently, using the mixed culture has been suitable for a wide range of substrates in the MFCs, where it is possible to use the MFCs as a biosensor. A study of the mixed culture MFC based biosensors showed a direct proportion between the current generated with the organic matter concentrations and high treatment efficiency [17]. Little research has been done to investigate the MFCs, as a biosensor, for detecting the pollutants and determining the concentration in wastes or wastewaters. Twochamber MFC based on biosensor was operated by using starch processing wastewater was investigated by Kim et al. (2003) [17]. The authors found that the coulombs were generated proportionally to the starch concentration up to the concentration of 206 mg COD.L1 and a good linearity (R2 ¼ 0.99). Kumlanghan et al. (2007) [18] reported that the sensor response of the air-cathode SCMFC using glucose solutions were linear against the concentration up to 2500 mg COD.L1. Feng et al. (2008) [19] found that the current generation increased from 29 to 205 mW cm2 (55e1512 mg BOD.L1) is proportional to the brewery wastewater concentration (R2 ¼ 0.996) from the air-cathode SCMFC with an exchange membrane. Lorenzo et al. (2009) [16] reported that the
air-cathode SCMFC had a linear relationship with the BOD concentration up to 350 mg L1 of synthetic wastewater and had very high reproducibility of signal current. Several types of wastewaters such as food processing wastewater, starch wastewater, brewery wastewater, domestic wastewater, swine manure slurry and paper mill effluent [8e10] have been successfully used as substrates for the electricity generation coupled with the pollution reduction in the MFCs. The distillery wastewater is a by-product of ethanol manufacture using sugarcane molasses materials. The distillery wastewater, high strength with strong odor and brownish color, contains chemical oxygen demand (COD) (134,000e136,000 mg L1) and biochemical oxygen demand (BOD) (69,000e71,000 mg L1) and low pH (4.35). Rich organic content makes it a good candidate feedstock for the bioenergy production [8,9]. This study is focused on the membrane-less SCMFCs based microbial biosensor, which has the ability to convert a specific pollutant in the substrate to the current directly. The relationship between the current generated with the concentration of pollutants can apply the MFC as a biosensor [20e22]. The MFC based biosensors have advantages including shorter response time of the current, high reproducibility, real-time monitoring and long-term stability than the other types of the biosensor [17e22]. The success of the MFC based biosensor depends on many factors such as the MFC configurations, types of microorganisms, types of substrate and the detection time [20e23]. The previous MFCs studies have been illustrated mostly on the electricity generation and the wastewater treatment. Some studies showed unpromising performance toward the membrane-less SCMFCs when applied as a microbial biosensor. In this study, the effectiveness of the current outputs and the biodegradability of the SCMFCs was investigated. The synthetic and distillery wastewaters were used to test the ability of the SCMFCs based biosensor. The relationship between the COD of wastewaters concentrations and the current outputs detection was discussed and the pollutant reduction in both wastewaters were determined.
Materials and methods Membrane-less microbial fuel cells (SCMFCs) The construction of the SCMFCs without an exchange membrane were made of the acrylic cylindrical chamber with a length of 4 cm and a diameter of 3 cm as previously described which has a void volume of 28 mL [24]. The anode electrode was made of a carbon cloth with pre-treated Teflon (without wet proofing; E-Tek, USA). And the cathode electrode was made of a carbon cloth without pre-treated Teflon (30% wet proofed; ETek, USA). In this study, the cathode electrode was coated by a gas diffusion layer on the air contact side [25,26]. Similarly, the platinum catalyst layer loading of 0.5 mg cm2 was coated on the wastewater contact side before use. The titanium wire was used to connect the anode and the cathode electrode while an external resistance (Rext) loading from the resistor box was used to connect the electrodes for complete the circuit.
Please cite this article in press as: Tanikkul P, Pisutpaisal N, Membrane-less MFC based biosensor for monitoring wastewater quality, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/j.ijhydene.2017.10.065
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Microorganisms Anaerobic sludge was obtained from a full-scale up-flow anaerobic sludge blanket (UASB) starch-processing wastewater treatment plant of Eiamburapa Co., Ltd (Sakaw, Thailand), operating at the mesophilic condition. At the beginning of the experiment, granular seed with diameter >0.5 mm were washed with tap water to remove sludge matters and cultured with a synthetic wastewater containing a cassava starch (5 g L1) in 10 L of the continuous stirred-tank reactor (CSTR) at 37 C until biogas production was stable. This microbial seed was heat pre-treated at 100 C for 60 min to inhibit the methane producing bacteria prior to use [27,28], which had a concentration of seed as 40 g L1.
Wastewaters Synthetic wastewater (SW) was used for the inoculation and operation. Glucose was used as a carbon source consisted of a nutrient medium with the following (per liter): FeSO4$6H2O, 10.69 mg; MnSO4, 0.589 mg; ZnSO4$7H2O, 0.106 mg; H3BO4, 0.106 mg; CuSO4, 0.003 mg and NH4Cl 8.5 g per g COD [29]. The final pH of a SW was adjusted to 7.0 by 50 mM phosphate buffer solution containing (per liter): NaH2PO4$H2O, 5.62 g; Na2HPO4$H2O, 4.61 g and KCl, 0.13 g [29]. SW was autoclaved at 121 C for 15 min. This wastewater was used to inoculate the microbe biofilm and tested the SCMFCs start-up. Raw distillery wastewater (DW) was collected from the primary clarifier effluent of ethanol production (Sura Bangyikhan Co., Ltd, Pathum Thani, Thailand), which sugar cane molasses were used as the raw material. This wastewater was used to test the ability of the SCMFCs in terms of the relationship among the current generation, the substrate concentration and efficient treatment. DW has high levels of COD as well as BOD and a high temperature (70e80 C) from source. DW was stored in a refrigerator at 4 C until it was used and diluted with deionized water to the given concentration without additions of any other nutrient or trace mineral. The pH of the wastewater dilution was adjusted to 7.0 by phosphate buffer solution of 50 mM. Two types of substrate were flushed with nitrogen gas for 3 min before feeding into the SCMFCs.
Membrane-less SCMFCs start-up The SCMFCs were inoculated with 2.5% by the volume of the heat pre-treated seed in the separate reactor. The SCMFCs were operated with a high SW concentration of 1500 mg COD.L1 in fed batch under open circuit voltage (OCV) at 37 C to allow the microbe attraction on the anode electrode. The wastewater was replaced when the voltage dropped until stability of the voltage output was successfully achieved for the microbe adhesion. Liquid samples were collected for analysis of pH, ORP, COD and sugar content. After the batch inoculation, the SCMFCs were acclimated with a SW in fed batch mode under high external resistance of 1000 U at 37 ± 1 C. The wastewater was replaced when the current was dropped until a steady current output allowed the microbe to be covered as biofilm on the anode electrode. During the acclimation, liquid samples were collected for analysis of pH,
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ORP, COD, and sugar content at the end of two batch cycles. The cyclic voltammetry technique was used to measure the characteristic of the anode biofilm compared with a new anode electrode without biofilm. The polarization curves were measured to determine the characteristic of the SCMFCs using the synthetic wastewater concentration as a function of an external resistance load.
Membrane-less SCMFCs testing as a biosensor The SCMFCs acclimated for more than two months were operated in semi-batch mode under low external resistance of 50 U at 37 C by using both types of wastewater. The SCMFCs based on biosensor performances such as current output, response time of current, reproducibility and the substrate degradation were investigated. Both types of wastewater were twice replaced while the current dropped before changing new concentration. The voltage outputs were automatically recorded and the liquid samples were collected for analysis of pH, COD and sugar content. After that, the SCMFCs were starved before being fed with both types of wastewater again and the performance of the SCMFCs including reproducibility of the current together with substrate degradation were investigated.
Analyses and calculations The performances of the SCMFCs biosensor were measured the voltage (V) and used to calculate the current to estimate the detection time. The voltage (V) outputs were automatically recorded by a multimeter every 10 min (Keithley 2700, USA). The current was calculated by applying the Ohms law;I ¼ ðV=RÞ according to the coulombic efficiency (CE) calculated based on the current generation over time and the change in substrate concentration during the SCMFCs operation [29]. In addition, liquid samples in the SCMFC were collected at the end of each batch cycle to analyses the pH, oxidation-reduction potential (ORP), sugar content, total COD and soluble COD. The pH was measured by pH probe using a pH meter. Likewise, the ORP was measured by an Ag/AgCl probe using the pH meter (Mettler Toledo, Germany). Sugar content was measured by the DNS method [30]. The COD was analyzed using the closed reflux method according to the Standard Methods [31], which the liquid samples were centrifuged to determine soluble COD.
Cyclic voltammetry analyses The characteristics of the oxidation and reduction reactions on the anode surface at the start-up compared with the end of batch acclimation were determined by the cyclic voltammetry (CV) technique (potentiostat, Autolab PGSTAT 204, Switzerland), which is connected to a computer. The characteristic of the anode biofilm was measured compared with the new anode electrode without a biofilm. CV was performed by applying a potential ramp from 500 to 1000 mV at a scan rate of 20 mV s1 on the anode surface only. The working electrode as the anode electrode and the counter electrode as the cathode electrode were connected by a titanium wire. The Ag/ AgCl prop was used as the reference electrode and the prop was inserted near the anode electrode. After all, the
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wastewater in the SCMFCs was removed and the new wastewater was changed before measuring.
Results and discussion Membrane-less SCMFCs enrichment and power curves The SCMFCs were inoculated with the heat pre-treated seed and fed with the SW concentration of 1500 mg COD.L1 during the start-up stage. The semi-batch open circuit mode was operated. The wastewater was replaced every day (24 h). And a concentration was not changed until the stable open circuit voltage (OCV) was approximately 300 mV within 10 days. The results described that the microbes biodegraded substrate and generated electrons were deposited on the anode in the SCMFCs but were not able to form biofilm on the anode surface [32]. Therefore, the SCMFCs were shifted to close circuit operation with an 1000 U external resistance connected between the anode and cathode. This purpose is to allow the microbes which had been grown as a catalytic biofilm covering the anode electrode surface [30]. Besides, the results showed a stable current peak approximately of 16 mA was obtained after 2 month acclimation (Fig. 1). It was assumed that a stable biofilm was formed at the anode surface of the SCMFCs. And the performance of the SCMFCs include pH of the effluent decreased varying during 6.87e6.95, while the ORP rapidly decreased in all concentration (10 to 226 mV). This acclimation proved the microbes were able to degrade a substrate in the case of soluble COD removal more than 93% (Fig. 1) and sugar content removal to reach 99% everyday (data not shown). When a stable current generation and efficient COD removal are presented, the electrochemical activities of the microbes on the anode surface were determined by CV. The voltammograms showed relatively higher current output in the forward scan while comparing with a new anode (Fig. 2A). Maximum current was 0.038 mA for the forward scan (1000 mV), the potential was 500 mV for the reverse scan, and the highest current was 0.31 mA. While a new anode surface had no redox peaks, demonstrating the anode surface with no microbes activities were obvious. Consequently, results indicated that the microbes were able to utilize substrate for cells
Fig. 2 e Cyclic voltammograms of the anode surface with and without biofilm at a scan rate of 20 mV s¡1 (A), and the voltage as a function of current density and the polarization curve with varied external resistance from 10 to 1000 U external resistance after two months acclimation (B).
growth on the anode surface as a biofilm and the current could also be generated by the microbes activities in the SCMFCs [3]. The characteristic of the SCMFCs were analyzed by using the polarization curves to confirm that SCMFCs were ready to generate electricity and utilize wastewaters as shown in Fig. 2B. The polarization curve was measured with the function of current density and the power density by varying an external resistance from 10 to 1000 U. This curve shows the voltage was rapidly dropped from 629 to 245 mV from the open circuit. It indicated the occurrence of activation loss. Meanwhile, the subsequent slope of the voltage was 652 mA m2. A maximum power density of 88 mW m2 and current density of 408 mA m2 were achieved at an external resistance load of 750 U external resistance.
Influence of wastewater concentrations on biosensor performance
Fig. 1 e Current generation and soluble COD removal (%) during the SCMFCs acclimation with the synthetic wastewater.
The SCMFCs achieved approximately two months acclimation were tested the relationship between organic loading and the current outputs by varying COD glucose concentrations in SW at a low external resistance of 50 U. The experiment showed
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the detection time of the current which was too short to reach the highest current output with the changes in the SW concentration of 25e1000 mg COD.L1 being around 1.7e8.4 h. However, this result illustrated a longer detection time (1.7e8.4 h) than single and two-chamber MFCs that were previously reported [17,20]. The correlation between the concentration of glucose and the coulombs were observed in the range of 25e250 mg COD.L1 (R2 ¼ 0.96) (Fig. 3A). Nevertheless, the SW concentration was higher than 400 mg COD.L1 that no increase of the current output was observed. Different DW concentrations were used to determine the effect of the real wastewater on the current outputs, and tested to identify the relationship between the current outputs with the concentrations of wastewater. The DW was diluted to obtain the COD concentration at the range of 50e2000 mg COD.L1 and was operated in semi-batch mode under 50 U external resistance. The current outputs with the changes in the concentrations of DW were shown in Fig. 4A. The detection time of the current was approximately detected of 2.5e0.9.3 h from low to high concentration. The relationships between the concentrations of DW and the coulombs were observed in the range of 50e1200 mg COD.L1 (R2 ¼ 0.97) (Fig. 4B). While the concentration of DW lower than 50 mg COD.L1, it could be detect the current output. The current increased quickly, but it dropped rapidly especially when a high DW concentration up to 1200 mg COD.L1. Moreover, the high DW concentration up to 1200 mg COD.L1 required a long time to oxidize the high
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Fig. 4 e Maximum current outputs with different DW concentrations of 50e2000 mg COD.L¡1 under 50 U external resistance (A). Inset figures represent the correlation between maximum current with DW concentrations. Total COD removal and efficiency (B).
organic matter concentration in wastewater to complete the batch cycle (>3 days). Although, the result showed longer detection time and low current outputs from these SCMFCs, this type of MFC leading to high reproducibility of the current was conducted with the same initial concentration of both wastewaters (data not shown). Nevertheless, the performance of the system was still potentially high in the case of linear correlation and high substrate reduction.
Substrate reduction
Fig. 3 e Maximum current outputs with different SW concentrations of 25e1000 mg COD.L¡1 under 50 U external resistance (A). Inset figure represent the correlation between maximum current with SW concentrations. Total COD removal and efficiency (B).
The COD removal efficiency in both of wastewaters experiments displayed a similar pattern. High substrate reduction efficiency was observed for the SW more than 97% (1000 mg COD.L1) (Fig. 3B), which was similar to the sugar content removal (>98%). While the DW has the highest substrate reduced efficiency that was 66.5% (2000 mg COD.L1) (Fig. 4B), there was slightly increased in accordance with the increased concentration. The sugar contained in the DW could be reduced more than 95% in all concentration tests. It indicated that the microbes were firstly utilized the preferable reducing sugar due to easier degraded than the other organic content. In addition, the pH of the effluent of the two types of wastewater slightly increased (7.01e7.08) from the initial indicating the microbes which controlled metabolisms activity during the current generation.
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that the SCMFCs can not only potentially generate electrical current but can also be applied to measure the organic substrate values as a biosensor and used to treat wastewater.
Acknowledgements The authors are grateful to Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0322/ 2552) and the Joint Graduate School of Energy and Environment (JGSEE), King Mongkut's University of Technology Thonburi (Grant No. JGSEE/THESIS/226) for scholarship. The authors also would like to express their gratitude to King Mongkut's University of Technology North Bangkok (grant no. KMUTNB-60-ART-080) for the financial support.
references
Fig. 5 e Current regeneration after the microbes starvation in the SCMFCs fed with SW (A) and DW (B). Number in the figures represent the concentration of wastewater (mg COD.L¡1).
The starvation of the SCMFCs on biosensor The SCMFCs were starved before both of wastewaters were fed again to determine the current regeneration as shown in Fig. 5A and B. The results demonstrated the current could be rapidly regenerated to reach the highest peak of current after 5 days of the starvation with both of wastewaters. The current regeneration was similar to the original value current output before the starvation. All in all, this work suggested that the SCMFCs are good in practice for applying as a microbial biosensor due to rapid regeneration of the current when the system is in starvation.
Conclusion Simultaneous bioelectricity generation and degradation of organic hydrogen carriers were achieved by using the SCMFCs. The SCMFCs were successfully operated, which clearly showed a linear correlation between the low concentrations of wastewaters and the current outputs. The detection time of current was short and the current reached its highest quickly after changing to a new concentration of wastewater. The results demonstrated a high reproducibility of the current after the SCMFCs were starved for a long period. This is an important part of the MFC based biosensor. On the whole, these suggest
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