Effect of wastewater containing multi-walled carbon nanotubes on dual-chamber microbial fuel cell performance

Abstract

The use of engineered nanomaterials is continuously increasing in commercial products and industrial applications, and a significant portion of these materials may enter domestic and industrial wastewater streams and subsequently, wastewater treatment plants. Microbial fuel cells (MFCs) represent a new emerging technology for simultaneously generating bioenergy and treating wastewater. In this work, the performance of a MFC with wastewater containing multi-walled carbon nanotubes (MWCNTs) was evaluated. No significant negative effect on power generation was observed for MWCNT concentrations from 10 mg L⁻¹ to 200 mg L⁻¹. In fact, there was a stimulating effect due to the increased conductivity resulting from the MWCNTs, therefore slightly enhancing voltage generation (linked to enhanced electron transfer rate). The maximum voltage generation was increased from 0.61 V to 0.68 V (at 1000 Ω external resistance). Low lactate dehydrogenase release at all concentrations of MWCNTs showed that no adverse cell piercing took place and the wrapping of cells by MWCNTs most likely occurred. Chemical oxygen demand (COD) removal efficiency was also enhanced from 74.2% to 84.7%. The experimental results demonstrated that wastewater containing MWCNTs can be applied to MFCs for generating bioelectricity and treating wastewater without any significant adverse effect on performance.

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1. Introduction

Nanoparticles, which have excellent magnetic, electrical, and optical properties, are now being extensively used in commercial products and for industrial purposes.¹ Recently, engineered nanomaterials have been applied to transportation, energy, catalysts, cosmetics, antibiotics, electronics, agriculture, and fillers for plastics and rubber.^2,3 Carbon nanotubes (CNTs) are among the most promising nanomaterials for various applications and their use is increasing at large. This increase in CNT manufacturing and application in nanotechnology will ultimately lead to their arrival at several environmental sites.^4,5 Wastewater treatment plants are the key receivers of all sorts of waste streams, therefore they are among the most probable CNT recipients.⁶

Microbial fuel cells (MFCs) are an alternative emerging and promising technology to current biological wastewater treatment processes, owing to their potential to convert energy-consuming processes into energy-saving processes.⁷ Briefly, MFCs use microbes (mainly electricigens) as inexpensive catalysts to degrade organic material in wastewater and directly transform chemical energy into electrical energy (and/or value-added products). Meanwhile, they yield treated effluent and a low environmental impact, owing to the effective combination of biological and electrochemical processes.^8,9 Therefore, domestic and industrial wastewater (containing organic material) has been tested in MFCs to determine their potential to generate bioenergy.¹⁰ The substrate used largely influences the outcome of MFCs in terms of their treatment and power generation. Hence, the composition of wastewater (including presence/absence of CNTs) can significantly affect MFC performance. In earlier studies, anode has been modified with different type of CNTs to improve and optimize the MFCs performance; however, CNTs were not studied as part of wastewater for positive or negative effects on MFCs performance. CNTs based anodes attribute to highly efficient electrodes that gives very large surface area, enhanced electrochemical activity, and low ohmic loss, as well as offer new designs for next-generation electrodes that can be vastly applicable for highly efficient bioelectrochemical cells.^11–14

In most studies which are related to microbial systems, the toxicity of CNTs in microbes has been established, using single microbial strains.^15,16 It has been proposed that synergistic effects of oxidative stress and cell membrane perturbation are the fundamental molecular mechanisms involved in cell damage. Nevertheless, the toxicity of CNTs on pure microbial strains is not a fair indicator of toxicity to complex microbial communities. To date, only a limited number of studies have examined the effects of CNTs on microbial communities in wastewater treatment systems.^6,17 Moreover, there are contradicting reports on the effect of CNTs on wastewater treatment system performance. Yin et al.¹⁷ found that the presence of single-walled CNTs (SWCNTs) in an activated sludge reactor did not negatively influence the performance of a continuous reactor; in fact, sludge settleability and dewaterability were improved, and chemical oxygen demand (COD) in the effluent was also reduced as a result of absorption by SWCNTs. Luongo et al.¹⁸ found that multi-walled carbon nanotubes (MWCNTs) have a more negative effect on sheared mixed liquor than unsheared mixed liquor in activated sludge processes. Notably, Li et al.¹⁹ found that sludge exposed to CNTs was more electrically conductive, inferring that CNT exposure enhances direct interspecies electron transfer between anaerobic bacteria. This finding can have a significant effect on MFC operation and it is required to investigate that how the CNTs addition effect the wastewater treatment efficiency and power generation.

In this work, the response of MWCNT dosage on MFCs with simulated wastewater was evaluated. The power generation and substrate utilization was monitored with increasing MWCNT concentrations. The effect of MWCNTs on voltage, maximum power density, internal resistance of the cell, coulombic efficiency, and lactate dehydrogenase (LDH) release was also monitored. These results provide insights into the response of exoelectrogens to MWCNTs in bioelectrochemical systems.

2. Materials and methods

2.1. Bioelectrochemical cell assembly and operation

A rectangular dual-chamber MFC was employed for this study, as previously reported.²⁰ A schematic of the MFC is shown in Fig. 1. Each chamber had a volume capacity of 200 mL. The anode electrode (5 cm × 5 cm) was made of porous carbon felt with a thickness of 3.18 mm (Alfa Aesar, Haverhill, USA), while the cathode electrode (5 cm × 5 cm) was 1 mg cm⁻², 20% (wt) platinum-coated carbon cloth (Fuel Cell Earth, Wakefield, USA). Both chambers and electrodes were separated by a proton exchange membrane (PEM) (Nafion 117, Dupont Co., USA). Nafion has been used broadly as a proton exchange substance in MFC research, owing to its very high selective ionic conductivity and permeability. In this work, a minimum distance between electrodes was also ensured to reduce the internal resistance of the cell. PEMs were pre-treated by boiling in H₂O₂ (3%, v/v) and deionized water, followed by treatment with 0.5 M H₂SO₄ and then deionized water, each for 1 h. Electrodes were connected via an external circuit by a resistor of 1000 Ω. The MFC was operated under fed batches in a temperature-controlled environment at 30 ± 1.0 °C.

Fig. 1 A schematic diagram for voltage generation by MFCs using wastewater containing MWCNTs.

2.2. Anolyte and catholyte

Inoculum source used was mixed anaerobic culture obtained from the lab MFC system, already in operation for more than six months with lactate as the main carbon source. Originally, anaerobic sludge was obtained from the local domestic wastewater treatment plant in Daegu, South Korea. The simulated anode nutrient medium was comprised of the following: K₂HPO₄, 0.50 g L⁻¹; NH₄Cl, 1.0 g L⁻¹; FeSO₄·7H₂O, 0.10 g L⁻¹; MgSO₄·7H₂O, 0.06 g L⁻¹; L-ascorbic acid, 0.1 g L⁻¹; along with 1.0 mL L⁻¹ of trace elements and vitamins. Sodium lactate was used as the main carbon source for the growth of exoelectrogens. MWCNTs were purchased from Carbon Nano-material Technology Co. Ltd, Korea. Properties of the MWCNTs are shown in Table 1. MWCNTs (10–200 mg L⁻¹) without any pretreatment were added to the medium and mixed with ultrasonication using a Vibra-Cell probe sonicator (VCX-750, Sonics, USA). This anolyte medium was then deoxygenated with ultra-high pure nitrogen gas. The medium was continuously stirred in the anodic chamber with a magnetic stirrer. Since the major focus of this study was on the anodic process, the cathodic process was only utilized as an electron sink, hence a strong and stable cathodic reaction was ensured. The cathode electron acceptor used for this purpose was aerated potassium ferricyanide (50 mM) complemented with 50 mM phosphate buffer (pH, 7.0). The cathode reaction was mainly reduction of ferricyanide as:

Fe(CN)₆³⁻ + e⁻ = Fe(CN)₆⁴⁻

Table 1 Properties of multi-walled carbon nanotubes used in this study

Parameter	Value
Diameter	∼20 nm
Length	∼10 μm
Aspect ratio	>500
Purity	>85%
Specific surface area	100–700 m^2 g^−1
Bulk density	0.08–0.1 g cm^−3

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The use of potassium ferricyanide is considered non-sustainable in comparison to oxygen (air), but it is very useful for this kind of study, as it provides a constant and easily replenished electron sink that does not interfere with the anodic processes.²¹

2.3. Measurements and calculations

The voltage outputs of MFCs were automatically monitored by a digital multimeter (model 2700; Keithley Instruments, Cleveland, OH, USA) along with logging by a personal computer through a data acquisition system (EXCELINK, Keithley Instruments). The current and power values were calculated as previously described.²² Polarization curves were drawn to determine the maximum power density by varying the external resistance from 10 Ω to 10 kΩ. Power density and current density were normalized to the anode single-side projected area. Coulombic efficiency (CE) was calculated as described previously.²³ Internal resistance of cell was measured by linear polarization curve, the relationship between the external voltage (E) and current (I) can be described as follows:

E = E_b − R_int × I

where R_int is the total internal resistance of an MFC (which can be easily obtained from the slope of the linear curve) and E_b is the linear extrapolation open circuit voltage.²⁴

LDH is a glycolytic enzyme that is mainly found in the cell cytoplasm. Under normal circumstances, LDH is rarely found extracellularly because it is sequestered by the cell membrane. However, when the cell membrane is damaged, LDH leaks into solution. Consequently, LDH can be used as an indicator for cell rupture and membrane integrity. The bacterial cell suspensions, after interaction with MWCNTs during 48 h batch experiments, were centrifuged at 12 000 × g for 5 min. LDH in the supernatant was quantified following a standard protocol.²⁵ Briefly, in 100 μL of supernatant, an equal volume of 30 mM sodium pyruvate was added along with 2.8 mL of 200 mM Tris HCl. The change in absorbance at 340 nm was measured with respect to the control, using a UV-Vis Spectrophotometer (DR/4000U, Hach, USA). Soluble COD was measured in a Hach COD reactor (DRB200, Hach Co., Loveland, CO), using Hach vials and a spectrophotometer (1412V, Optizen, Mecasys Co., Korea). Total organic carbon (TOC) was measured using a TOC analyzer (TOC-V_CPH, Shimadzu, Kyoto, Japan). For Field Emission-Scanning Electron Microscopy (FE-SEM) analysis, microbial samples were collected from the anode by cutting the anode electrode in 1 cm × 1 cm sections from different locations. Cells were fixed on the electrode in a 4% glutaraldehyde solution for 4 h at 4 °C and then exposed to increasing concentrations of ethanol (20%, 50%, 80%, and 100%). The biofilms were washed with phosphate-buffered saline between every fixation step. The anode pieces were dried at 35 °C, and finally coated with platinum by sputtering. FE-SEM (S-4800, Hitachi, Japan) was performed to observe the morphology and surface structure of the MWCNTs and carbon felt with biofilm attached.

3. Results and discussion

3.1. Impact of MWCNTs on power generation

Bioelectricity was stably generated by MFCs after a successful acclimation period (Fig. 2). A sharp increase in voltage was observed after every addition of fresh batch, due to the presence of carbon sources easily degradable by anodic bacteria. As these degraded substances were consumed, power output began to decrease. The maximum voltage generation state was achieved within 2 h and remained stable for more than 24 h in each cycle. The batch studies showed that a maximum voltage of 610 mV was achieved with lactate as the main carbon source. The main degradation products of lactate were acetate and propionate, which were further degraded with time and electron release. After adding MWCNTs in the anolyte, an increase in voltage up to 678 mV, approximately 11% higher, was observed after increasing the MWCNT concentration to 25 mg L⁻¹. The voltage generated by all batches containing MWCNTs up to 200 mg L⁻¹ were higher than batches containing lactate only (without MWCNTs). Improvements in power generation with CNTs most likely arise because of better interactions between exoelectrogens and electrodes mainly due to improved electrical conductivity and increased surface areas. Furthermore, it was earlier reported that CNTs help in an excellent charge transfer characteristics due to π–π stacking between the carbon rings of CNTs and the pili of microbes. It also helped in lowering ohmic losses.¹¹ The increased generation of bioelectricity when MWCNTs were present compared to that in the presence of lactate only indicated that the addition of MWCNTs in the feed hardly suppressed the activity of exoelectrogens in the anode chamber. Moreover, electrons from the oxidation of organic compounds were transferred to the anode electrode through electricigens for bioelectricity generation, and this process was facilitated by MWCNTs. Previously, Li et al.¹⁹ studied the response of anaerobic granular sludge to SWCNT exposure and found that SWCNT addition largely improved the conductivity of both fresh and incubated sludge. In this work, polarization curves and power outputs showed a similar trend to voltage generation (Fig. 2b). The maximum power (normalized to anode unit area) obtained from power density and a polarization curve was 259 mW m⁻², which corresponds to the current density of 720 mA m⁻² (without the addition of MWCNTs). The maximum power attained was 320 mW m⁻², which corresponds to the current density of 800 mA m⁻² after the addition of MWCNTs (25 mg L⁻¹). It is important to mention here that reason of addition of MWCNTs was not intentionally to attach them to the anode surface. However, MWCNTs can be seen on the anode biofilm and in bulk phase. Moreover, CNTs attachment on anode surface from wastewater is also possible as it has been reported earlier that even CNTs (for anode modification) can be attached to carbon cloth, papers, or textiles by using simple dipping in CNT solutions to increase the electrical conductivity and surface area of electrodes (resulting in 20–150% enhancement in power density).¹¹ It was also previously reported that adding CNTs to activated sludge has a minimal negative impact for several reasons. For instance, the presence of extracellular polymeric substances (EPS) in activated sludge processes mitigated the toxicity of CNTs since they are likely to become embedded in EPS, preventing their direct contact with cell membranes.^18,26 In this study, a decrease in internal resistance (197 Ω to 183 Ω) was observed after the addition of MWCNTs. It is well-known that lowering internal resistance helps in efficient electron transfer in the MFC circuit. The overall results related to power generation by MFCs after adding MWCNTs showed a positive outcome.

Fig. 2 Voltage generation (a) and power density curves (b) from MFCs containing different MWCNT concentrations.

3.2. Impact of MWCNTs on COD/TOC removal

The removal of organic compounds by MFCs is very important, as this is a fundamental objective of wastewater treatment. The MFC anode section performs well as an anaerobic growth chamber and this indicates the great potential of the MFC system for removing organic compounds as an alternative to conventional biological wastewater treatment processes.^27,28 In this study, efficient removal of organic compounds was achieved in all batches. Most importantly, an increase in soluble COD and TOC removal was achieved after the addition of MWCNTs (Fig. 3). The COD removal was increased from 74.2% to 84.7%, while TOC removal was increased from 81.6% to 90.8% when the concentration of MWCNTs was increased from 0 to 200 mg L⁻¹. This increase in removal rate can be explained in two ways. Firstly, it was most likely due to enhanced electron transfer after the addition of MWCNTs, which increased microbial metabolism and led to greater removal of organic compounds. It has been reported in earlier studies that the organics degradation in MFCs as compared to traditional anaerobic treatment was enhanced attributed to the presence of the anode, which increases the metabolic rate of anaerobic bacteria with sufficient anaerobic terminal electron acceptors.²⁹ CNTs addition increased the conductivity and hence increases the electron flow to anode, which could result in faster conversion of the substrate to maintain the microbial metabolism rate. Secondly, soluble organic matter (measured by the soluble COD) was strongly absorbed by MWCNTs and such absorption increased with MWCNT concentration. Yin et al.¹⁷ also reported that 17% of the soluble COD was absorbed by 250 mg L⁻¹ of SWCNTs. Similarly, Li et al.¹⁹ investigated the effects of SWCNT exposure on methanogenic sludge in an anaerobic digestion process. It was reported that SWCNTs had no severe negative impact on CH₄ production. Interestingly, far higher substrate consumption and CH₄ production rates were observed upon SWCNT exposure. It is also important to note that a substantial proportion of the organic matter that was used up by bacterial processes did not produce bioelectricity; the coulombic efficiency was less than 7% in all cases. Various studies are already underway to enhance the percentage of organic matter that can be converted into electricity. However, the organic substances in wastewater are essentially free, therefore as long as COD removal is accomplished by electricity generation or other methods, the goal of wastewater treatment is achieved.

Fig. 3 COD and TOC removal efficiency by MFCs containing wastewater and MWCNTs.

3.3. Lactate dehydrogenase activity and SEM

Microbial cell membrane integrity was assessed by measuring the release of LDH in MFCs after exposure to MWCNTs (Fig. 4). LDH release was slightly increased with increasing MWCNT concentrations. After 48 h batch operations, LDH release was determined to be 2.66, 3.14, 3.14, 3.60, 3.86, and 4.1 U L⁻¹ in 0, 10, 25, 50, 100, and 200 mg L⁻¹ of MWCNTs, respectively. Earlier studies suggested that physical contact between carbon-based nanomaterials such as MWCNTs and cells is mainly responsible for the inactivation of bacteria.^16,30 However, the effects of CNTs are largely determined by their surface properties, matrix type, and preparation methods.³¹ In this study, the addition of MWCNTs up to 25 mg L⁻¹ showed increased voltage generation, beyond which there was slight decrease (likely due to inactivation of few microbes), but the overall impact was positive up to 200 mg L⁻¹. Microbes form biofilms on MFC anodes, and biofilms are considered to be more resistant to CNT treatment.³² Therefore, it can be inferred that LDH release was mostly from the microbes in suspension in the anodic chamber rather than from biofilms attached to the anode. Furthermore, the EPS excreted by microbes form a gel-like matrix, which agglomerate microbes within biofilms and thus build a protective barrier against the harsh external environment.³³

Fig. 4 LDH release activity at different MWCNT concentrations.

Fig. 5 shows SEM images of MWCNTs and biofilm formed on the MFC anode electrode surface. After ultrasonication and nitrogen purging, most MWCNTs were evenly spread and dispersed as loose aggregates (Fig. 5a). Moreover, MWCNTs existed in this form when they were added into the MFC reactor. SEM observations also clearly showed that the microbial community formed a densely packed biofilm on the anode (Fig. 5b and c), which most likely prevented the MWCNTs from penetrating into the internal layer, mitigating their toxicity. This prevented MFC performance from deteriorating significantly. Most microbes on the anode were Bacillus sp. (rod-shape) (Fig. 5d), which were most likely wrapped with EPS excretions.

Fig. 5 SEM images of MWCNTs and biofilm attached to the MFC anode-electrode.

4. Conclusions

This study established the possibility of producing bioelectricity in MFCs from wastewater containing MWCNTs. The spontaneous anaerobic oxidation of carbon sources in MFCs, even after the addition of MWCNTs, was observed. The enhanced electron transfer rate upon MWCNT addition resulted in higher power generation and COD/TOC removal efficiency. LDH release was minimal and SEM images showed that MWCNTs did not significantly damage the microbial communities attached to the anode. This work provides a valuable foundation for determining the response of MFC microbial communities to MWCNTs.

Acknowledgements

This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education (ME) and National Research Foundation (NRF) of Korea (NRF-2014H1C1A1066929). This study was also supported by grants (NRF-2009-0093819, NRF-2016R1A2B4010431) through the ME and NRF of Korea. This research was also supported by an NRF grant from the Korean government (MSIP) (NRF-2015M2A7A1000194).

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