Crosslinked inter penetrating network of sulfonated styrene and sulfonated PVdF-co-HFP as electrolytic membrane in a single chamber microbial fuel cell

Abstract

In the present study, semi-IPN membranes of sulfonated styrene (SS) and sulfonated PVdF-co-HFP membranes have been analyzed as a polymer electrolyte membrane in single chamber microbial fuel cells (MFCs). 5%, 10%, 20% and 30% sulfonated styrene (SS) with varying concentrations of divinyl benzene (DVB) have been polymerized in the presence of sulfonated PVdF-co-HFP to prepare SPS-0 (0% SS), SPS-5 (5% SS), SPS-10 (10% SS), SPS-20 (20% SS) and SPS-30 (30% SS) membranes, respectively. Progressive improvements in membrane properties were observed with increasing SS and DVB concentrations, where excess DVB (<0.8 wt% of SS) resulted in increased crosslinks within the membrane structure. This eventually impeded the membrane properties and altered their rigidity. The membranes were characterized for their ion exchange capacity (IEC) and proton conductivity; IEC value of 0.39 meq g⁻¹, 0.42 meq g⁻¹, 0.47 meq g⁻¹, 0.54 meq g⁻¹ and 0.63 meq g⁻¹ and proton conductivity of 3.23 × 10⁻³, 1.06 × 10⁻², 1.87 × 10⁻², 2.47 × 10⁻² and 1.61 × 10⁻² S cm⁻¹ were observed for SPS-0 (0% SS), SPS-5 (5% SS), SPS-10 (10% SS), SPS-20 (20% SS) and SPS-30 (30% SS) membranes. The membranes were sandwiched as membrane electrode assemblies (MEA) and employed in single chamber MFCs with open air cathode to analyze their overall performance outputs. It was observed that amongst these membranes, the MFC with SPS-20 membrane showed the maximum power and current density of 447.42 ± 22 mW m⁻² and 1729.63 ± 87 mA m⁻² with an overall ∼91.27% COD removal in 28 days of operation, using electrogenic mixed firmicutes as biocatalysts. Overall, this study reveals the relevance of semi-IPN membranes of sulfonated styrene (SS) and sulfonated PVdF-co-HFP in MFC applications for harvesting bio-energy.

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1. Introduction

The vast majority of the advances made in MFCs have occurred in the last 5 to 10 years. Bennetto et al. were one of the first groups which consistently pursued MFC research in the 1980s and 1990s.^1,2 In addition several studies on alternative polymer electrolyte membranes (PEMs) and their optimization have been conducted. A suitable replacement for the high cost perfluorinated membranes as PEM has always been a requisite in fuel cell technology. Some polymeric materials like polystyrene, polyether ether ketone (PEEK), poly (arylene ether sulfone), phenylated poly sulfone, polyphosphazenes, polyimides, poly(4-phenoxybenzoyl-1,4-phenylene), polybenzimidazole (PBI), low density polyethylene (LDPE), high density polyethylene (HDPE) and polypropylene (PP) have been widely studied.^3–6 Fundamentally, any ion permeable material can function as a barrier and serve as a PEM in fuel cells; for comparison, Kim et al. equated cation-exchange, anion exchange, and ultra filtration (molecular cut-offs of 0.5, 1, and 3 kilodaltons) membranes to determine their effects in MFC performance.^7–9 Similarly, other PEMs using different catholytes e.g.; ferricyanides have been analyzed in dual chamber MFCs.^10–12 Recently, a group led by S. Ayyaru developed a sulfonated polystyrene–ethylene–butylene–polystyrene (SPSEBS) membrane as an alternative of Nafion membrane.¹³ SPSEBS membranes were found to be butylene–polystyrene (SPSEBS) membrane as an alternative of Nafion membrane.¹³ SPSEBS membranes were found to be producing 106.9% higher power density and lower internal resistance in comparison to Nafion 117 in a single chamber MFC (SCMFC). S. Ayyaru and S. Dharmalingam also carried out the sulfonation of polyether ether ketone (PEEK) and used it as a proton exchange membrane (PEM) in a single chamber MFC (SCMFC).¹⁴ MFC with the SPEEK membrane were found to produce approximately 55.2% higher power density than Nafion-117. In another study, the performance of an anion exchange membrane (AEM) in two-chambered microbial fuel cells (MFCs) were analyzed in batch mode using Shewanella putrefaciens by S. Pandit et al., where a maximum voltage of 0.676 V and 0.729 V with a power density of 39.2–7.39 mW m⁻² and 57.8–5.509 mW m⁻² were obtained using Nafion and Ralex membranes, respectively.¹⁵ Another group led by Gangrekar, also evaluated the performance using earthen pot as a proton exchange material of microbial fuel cells (MFCs).¹⁶ Various studies on enhancing membranes properties have been conducted; for instance, surface modification of polyolefins by sulfonation, photosulfonation, plasma treatment, and radiation grafting have been shown to improve hydrophilicity, adhesion, and other properties within membrane.^17–21 Recently, polyvinylidene fluoride (PVdF) and its blends have provided a promising prospective as PEM in fuel cell technology.^22–28 Although, being hydrophobic, various studies on its surface modification have shown increments in water retention capability within the membranes.^29–31 In another study, a semi-interpenetrating network (semi-IPN) of cross-linked sulfonated polystyrene within Nafion showed marked improvement in water uptake properties of the membranes. This significantly improved the overall performance of the system.³²

Based on the above observations, we have prepared semi-IPN of sulfonated styrene (SS) and sulfonated PVdF-co-HFP in different proportions, where their properties in terms of water uptake, ion exchange capacity (IEC) and proton conductivity have been analyzed. It should be noted in this respect that the PVdF-co-HFP copolymer utilized in this study has been widely reported as a low-cost membrane material compared to others e.g.; Nafion.^22,30,33,34 Owing to the overall cost reduction, we intuited that the prepared membrane could exhibit significant effect in harnessing energy from MFC. Being a low cost novel material, we have examined the efficiency of these membranes in single chambered open air MFCs for bio-energy generation using mixed firmicutes as biocatalysts.

2. Materials and method

2.1 General conditions

All chemicals used were of analytical and biochemical grade. PVdF-co-HFP (M_w: 455 000), sodium salt of styrene, were brought from Sigma Aldrich. NMP (1-methyl-2-pyrrolidone), dimethyl formamide (DMF) and divinyl benzene (DVB) were bought from Merck Millipore India. All chemicals were used as received. All microbial experiments were performed under strict sterile conditions.

2.2 Membrane preparation

Sulfonation of PVdF-co-HFP polymer was done following the previously described method in which it was treated with 20 mL chlorosulfonic acid at 60 °C for 7 h.³¹ Sulfonated PVdF-co-HFP (SPVdF-co-HFP) copolymer was further re-dissolved in NMP at 60 °C. The obtained solution was transferred into a three-necked round bottom flask containing sodium salt of styrene. Further, initiator AIBN and cross-linker DVB was added in the solution. The resultant mixture was left under stirring condition at 110 °C to allow the SS to polymerize in situ.³⁵ Finally, the viscous solution was casted and kept in the oven at 80 °C for 24 h (Fig. 1). In order to obtain a proper protonation, the prepared membranes were first treated with a mixture of water and H₂SO₄ (50 : 50) for 3 h and later, rinsed with de-ionized (DI) water until a neutral pH was obtained. The resulting membranes were named SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 as per the incorporated SS percentage (0, 5, 10, 20 and 30) respectively. Further, these membranes were subjected to FT-IR and FESEM analysis for structural characterization. The constituent details of the samples prepared are represented in Table 1.

Fig. 1 Constituents of semi-IPN membrane.

Table 1 Chemical composition of the membranes

Samples	S-PVdF-HFP (wt%)	SS (wt%)	DVB (wt% of SS)	AIBN (wt% of SS)
SPS-0	100	0	0	0
SPS-5	95	5	0.4	0.2
SPS-10	90	10	0.6	0.3
SPS-20	80	20	0.8	0.4
SPS-30	70	30	1.2	0.6

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2.3 Solvent extraction

The degree of crosslinking in membranes was characterized by solvent extraction method. Small piece of the membranes were wrapped in filter paper and kept in NMP at 90 °C. The solvent was replaced in every 15 h until no further solubility was observed in the polymer. The remaining part of the samples was dried and weighed. The gel fraction W(gel) of the membranes were calculated from eqn (1):

W(gel) = W₁/W₀ × 100 (1)

where, W₀ is the original weight of the dried membrane, and W₁ is the mass weight of the dried membrane after complete solvent extraction.

2.4 Water uptake and swelling ratios

Small piece of membranes were kept overnight in deionized water. The dry and wet weights of membranes were utilized for water uptake calculation using the following equation:

Water uptake (%) = (W_wet − W_dry)(100)/W_dry (2)

where, W_wet represents the weight of wet membranes obtained after soaking in DI water for 24 h, and W_dry, the weight of the respective dry membranes.

Similarly, swelling ratios of the prepared membranes were calculated from the following eqn (3):

Swelling ratio (%) = (T_wet − T_dry)(100)/T_dry (3)

where, T_wet represents the respective thicknesses of wet membranes soaked in DI water for 24 h, and T_dry is the respective thicknesses of dry membranes.

2.5 Ion exchange capacity (IEC)

Ion exchange capacities (IECs) of the membranes were determined using the conventional titration method. Small square pieces of membranes were kept overnight in 1 M H₂SO₄ solution. After washing with deionized water, excess H₂SO₄ were removed, and the samples were kept overnight in 50 mL (1 M NaCl solution), to replace the protons with sodium ions. The remaining solution was titrated with 0.01 N NaOH solution, using phenolphthalein as indicator. The IEC values (in meq g⁻¹) were calculated using the following equation:

IEC = (V_NaOH)(S_NaOH)/W_dry (4)

where, V_NaOH is the volume of NaOH used in the titration, and W_dry is the dry weight of the membrane in g. S_NaOH is the strength of NaOH used in the experiment for the determination of IEC.

2.6 Proton conductivity

Proton conductivity measurements were done through AC impedance spectroscopy, using a potentiostat (Gamry Potentiostat-600) over a frequency range of 1 Hz to 10⁵ Hz, at an applied potential of 10 mV. The conductivities of the samples (σ) in the transverse direction (Fig. 2) were calculated from the respective impedance data, using the following equation:

σ = T/RA (5)

where, T is the thickness of the sample, A is the cross-sectional area of the sample, R is the resistance derived from the intercept of the high frequency semi-circle on a complex impedance plane with the real (Z) axis.

Fig. 2 A schematic illustration of the setup utilized for analyzing the proton conductivities of the membranes.

2.7 MEA preparation: pre-treatment of the membranes and electrodes

All the membranes were pre-treated in a mixture of water and 3 M H₂SO₄ (7 : 3) for 6 h. The treated membranes were washed with deionized water to maintain their neutrality.

Carbon cloths (Zoltek Pvt. Ltd, USA) of fixed dimension (6 cm²) were used as electrodes (anode and cathode), which prior to use, were kept overnight in de-ionized water to neutralize any unwanted interfacial ionic particles. This also helps in maintaining electrode's total surface positivity for rigorous microbial attachment.

2.8 Preparation and deposition of catalyst ink

A catalyst mixture consisting of 10 : 90 wt% of platinum (Pt/C) was mixed with 5% Nafion solution. It was further sonicated for about 30 min, until a fine ink was obtained. This obtained ink was paint coated on the cathode side of the carbon cloth. A total of 3 mg cm⁻² of supported metal catalyst was loaded on the cathode side facing.

2.9 Fabrication of MEA

Five MEA sets consisting sandwiched membranes (SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30) were assembled between carbon cloths (anodes and cathodes) and hot pressed at 120–140 °C for 20–30 s at 6.89 MPa pressure. This optimum combination of time and temperature allowed perfect membrane assembling, as above it, the carbon cloth turns brittle and loses its texture when subjected to higher temperature and pressure.

2.10 MFC configuration and fabrication

In total, five identical single chambered MFC units namely, MFC-A to E of 150 mL liquid volume (anode chamber) were constructed. MEAs containing SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 membranes were fitted in MFC-A, B, C, D and E respectively, where cathode side (doped with catalysts) facing outward were left open for direct air-exposure. Electrical connections with copper wires of ∼1 ohm were made and requisite fabrications e.g. inlet/outlet sealing, electrode fixing etc. were done properly to avoid leakage (Fig. 3).

Fig. 3 Membrane electrode assembly and single chambered MFCs.

2.11 DNA isolation and PCR amplification

Genomic DNA isolation of pure culture isolates was done using standard phenol–chloroform method.³⁶ Microbial genomic DNA was isolated and treated with RNase A, where its concentration was estimated by taking absorbance at 260 nm (UV spectrophotometer 2700, Thermo Scientific). 16S rRNA gene amplifications were done using PCR (Applied biosystem, US) with universal primers Y1Forward (40th) 5′-TGGCTC AGAACGAACGCGGCGGC-3′ and Y2Reverse (337th) 5′-CCCACTGCTGCCTCC CGTAGGAGT-3′. The amplified regions were sequenced and BLAST tools were employed for bacterial species identification. The strains were allotted accession numbers.

2.12 Anolyte preparation

Isolated microbial strains were found to be firmicutes class lysinibacillus species (EMBL accession no. HE648059, HE648060, HF548664), which when tested for viability in gas pack jar were observed as facultative anaerobes. The COD composition of the feed wastewater was 2100 ± 300 mg L⁻¹ (total nitrogen: 117 ± 30 mg L⁻¹, PO₄–P: 36 ± 3 mg L⁻¹, MgSO₄: 50 mg L⁻¹). These mixed strains were suspended in 50 mM phosphate buffer (50 mL volume) and subsequently transferred to 100 mL synthetic wastewater (COD = 150 mg L⁻¹, pH = 7.0). In total, a final volume of 150 mL anolyte (microbial enriched) was prepared and transferred in each MFC unit.

2.13 MFC startup, electrical parameters and measurements

Prior to operation, the assembled MFC units were stored with sterilized de-ionized water overnight, and later replaced by pumping anolyte using peristaltic pump. All MFCs were continuously monitored (24 h intervals) using a multimeter (Keithley Instruments, Cleveland, OH, USA), and a potentiometer (G600; Gamry Instrument Inc., Warminster, PA, USA) connected to a personal computer. Fuel cells were operated continuously for 28 days, where current (I) and potential (V) measurements were recorded after allowing the circuit to stabilize for 2–5 min. Power (W) was calculated using relation P = IV, where I and V represents current and voltage, respectively. Power and current densities were calculated by dividing the obtained power and current by projected surface area of the anode.

Substrate removal was analyzed by measuring chemical oxygen demand (COD) periodically at 420 nm (Anatech Labs India Pvt. Ltd, India). The measurements were done in triplicate. The Columbic efficiency (CE) of the fed-batch mode MFCs were calculated by applying the following formula:

where ‘M’ represents the molecular weight of oxygen (M = 32), ‘F’ is Faraday's constant, ‘b’ denotes the number of electrons ex-changed per mole of oxygen (b = 4), ‘V_an’ is the liquid volume in anode, and ΔCOD is the change in the chemical oxygen demand over time t.

2.14 Electrochemical impedance spectroscopy (EIS)

For internal resistance assessment, potentiostatic EIS were performed for all MFCs at a frequency range of 10³ kHz to 1 mHz at 10 mV. The small AC response (lower amplitude of the applied potential), stimulates current response in the system without affecting the performance of the MFC. The obtained Nyquist graphs were plotted and the internal resistances of all MFCs (R_in) were determined.³⁷

2.15 Cyclic voltammetry (CV)

Microbial electrochemical activity was analyzed using CV at a scan rate of 2 mV s⁻¹ (G600; Gamry Instrument Inc., Warminster, PA, USA). The setup contained anode as the working, cathode as the counter, with an Ag/AgCl reference electrode, placed near the anode (for minimal iR drop). To ensure microbial activity, control experiments (without microbes) were conducted separately.

2.16 Scanning electron microscopy (SEM)

For confirming the microbial adhesion at electrode's surface, the biofilm containing electrode material was subjected for SEM analysis. The biofilm developed on the electrode was fixed with 2.5% glutaraldehyde and 0.1 M phosphate buffer solution, and then dehydrated by using a continuously higher (from 30 to 100%) concentration of ethanol.³⁸ The samples were dried, followed by sputter-coating with a thin layer of gold under vacuum to neutralize the charging effects. The morphology was observed under a scanning electron microscope (Carl Zeiss EVO® 18 electron microscope), using an acceleration voltage of 15 kV.

3. Result and discussion

3.1 Crosslink degree, water uptake and swelling ratio analysis

Using eqn (1), the gel fraction of different membranes was calculated, where a linear increase in the membrane crosslink degree was observed with a maximum value of 42.3% in SPS-30. Because of the crosslinked network of sulfonated polystyrene, the membranes revealed a crosslink density of 5.6%, 23.7% and 31.5% in SPS-5, SPS-10, and SPS-20 membrane. With increasing DVB concentration, a maximum of 42.3% crosslink density was observed in SPS-20 membrane because of the presence of higher content of crosslinker (1.2% DVB). The corresponding water uptake values of all the samples were calculated using eqn (2). PVDF-co-HFP, being hydrophobic does not account much in terms of water affinity, but with increased degree of sulfonation, subsequent increase in membrane hydrophilicity has been observed.³¹ The water uptake values for the casted SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 membranes were 18.6%, 22.2%, 31.3%, 39.2% and 28.1% respectively (Fig. 4). Increasing SPS percentage showed clear improvement in water uptake with a highest percentage of 39.2% in SPS-20 membrane. The greater hydrophilic nature of SPS entailed strong interactions between the surface –SO₃H groups and water molecules, resulting in the formation of hydrogen bonds and higher water retention within the membranes. As a consequence of these neutral sulfonic acid groups, progressive improvements in water uptake for all SPS membranes were observed. However, in case of SPS-30, despite of having maximum SS (30%) content, lower water uptake values in comparison to SPS-10 and SPS-20 membranes were found. This decrease was attributed to the greater constraints imposed by the higher content of cross-linker DVB that resulted in increased cross-linking in the membrane that eventually allowed fewer –SO₃ groups exposed to water molecules. This additional cross-linking proportionally reduced the available free space in the membrane and constricted the membrane, leading to lower water uptake in comparison to SPS-20 membrane. Nevertheless, improvements in the water uptake capacities in all membranes ensured the added effects of SS within the SPVDF-co-HFP membrane. In order to rationalize the results, swelling behaviours were studied. The corresponding swelling values were calculated from eqn (3). The swelling ratios for SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 membranes were 7.2%, 12.1%, 18.2%, 37.3% and 12.1% respectively. The observed decrease in the swelling ratio of SPS-30 compared to that obtained for SPS-10, SPS-20 membrane was due to the previously mentioned reason.

Fig. 4 Water uptake (WU) and swelling ratio (SR) LDPE membranes.

Excess DVB, increased membrane rigidity and resulted in comparatively lower swelling ratios. In effect, SS incorporation resulted in higher water uptake and swelling ratios within membranes, where excess DVB restricted inter chain mobility and thereby reduced the available free volume within the membrane. Nevertheless, the enhancements in water uptake and swelling ratios provided a promising aspect of these semi-IPN membranes as better PEM.

3.2 FT-IR analysis

Incorporation of sulfonated styrene (SS) within sulfonated PVDF-co-HFP structure on varying divinyl benzene (DVB) concentration resulted in crosslinked semi-IPN membranes. The structural identities of the functional groups were characterized using FT-IR spectroscopy, where major peak differences were observed in sulfonated and SS incorporated SPVDF-co-HFP membranes (Fig. 5). A characteristic sharp peak at 1682 cm⁻¹ was observed in all SS incorporated SPVDF-co-HFP membranes, which was assigned to the symmetrical stretching vibrations of C=C bonds present in the aromatic benzene ring of sulfonated styrene (SS).³² This characteristic peak was absent in case of SPS-0 and with increasing SS concentration its increased prominence was observed within membranes, reaching highest in the case of SPS-30. Alternatively, decrements in C–F and C–H peak intensities at 1397 and 1200 cm⁻¹ were observed with increasing SS incorporation, which were plausibly due to the substitution of host polymer i.e. SPVdF by SPS. Other specific IR peak intensities at 1066 and 1164 cm⁻¹ range were found indicating the symmetric and asymmetric stretching vibrations of the constituent S=O bonds. These were indicative of the presence of –SO₃H groups in the membranes. In overall, the peak differences in the casted membranes revealed successful SS incorporation within SPVDF-co-HFP membranes.

Fig. 5 FTIR of membranes.

3.3 FESEM-EDAX analysis

For elemental qualitative and quantitative analysis, EDAX of the casted membranes (SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30) were obtained with three iterations (Table 2). With increasing SS concentration, respective increments in membrane elemental compositions, including carbon, oxygen and sulphur were observed. A higher C : O : F : S atomic ratio of 56.5 : 10 : 30 : 3.3 was observed in SPS-30, with carbon (50.12 wt%), oxygen (12.42 wt%) and sulfur (4.90 wt%) approximately. This increase was attributed to the incorporated SS in the copolymer structure. The increments in sulfur and oxygen contents were indicative of the increased sulfonic groups within membranes. In comparison, an approximate increment of ∼19.57 wt%, ∼32.12 wt% and ∼48.97 wt% were observed in SPS-30 than SPS-0 with respect to carbon, oxygen and sulfur contents. Evidently, the incorporated SS within SPVDF-co-HFP structure increased the elemental compositions in the respective casted membranes. SEM analysis revealed increased porosity in all membranes with increasing SS incorporation, except SPS-30. In spite of added 30% SS, minimal porosity were found in SPS-30 in comparison to other membranes (SPS-10 and SPS-20). This reduction was due to the increased concentration of DVB and AIBN used for in situ polymerization of SS. Excess DVB resulted in increased cross linking within membrane structure that in turn, reduced the available free volume and porosity in the membrane. This in turn increased the rigidity in SPS-30 membrane. Relatively, in other membranes (SPS-5, SPS-10 and SPS-20) marked porosity was observed in the copolymer structure, which was majorly due to the controlled crosslinking that undertook with lower DVB concentration. In effect, the membrane compactness depended majorly on the cross linker (DVB) concentration, which defined the available free space within semi-IPN membranes.

Table 2 Elemental analysis of membranes (EDAX)

	Carbon (C)	Oxygen (O)	Fluorine (F)	Sulfur (S)
SPS-0 (weight%)	40.31	8.43	48.76	2.50
SP-5 (weight%)	43.67	10.42	45.87	2.78
SP-10 (weight%)	46.02	10.71	40.05	3.22
SP-20 (weight%)	48.92	11.14	35.38	4.56
SPS-30 (weight%)	50.12	12.42	32.56	4.90

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3.4 Analysis of IEC and proton conductivity

Ion-exchange capacity (IEC) of a membrane defines its ability to displace ions initially attached/incorporated in its structure by an oppositely charged ion present in the surrounding solution. After titration, the IEC values of the respective membranes were obtained (Fig. 6). The IEC for the casted SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 membranes were 0.39 meq g⁻¹, 0.42 meq g⁻¹, 0.47 meq g⁻¹, 0.54 meq g⁻¹ and 0.63 meq g⁻¹ respectively. The values clearly signified the effect of SS incorporation on IEC over that obtained for only sulfonated PVdF-co-HFP membrane, SPS-0 (devoid of SS). A direct correlation existed between IEC and the percentage of incorporated SS within membrane structure. The incorporated sulfonic groups (present in the SS structure) resulted in higher IEC upon rational increments in SS concentration. In a similar relation, enhanced proton conductivities were observed in SPS-0, SPS-5, SPS-10, SPS-20 and SPS-30 membranes with a respective value of 3.23 × 10⁻³, 1.06 × 10⁻², 1.87 × 10⁻², 2.47 × 10⁻² and 1.61 × 10⁻² S cm⁻¹. In case of SPS-30, reduction in proton conductivity was observed with respect to SPS-20 and SPS-10. The reason was the used DVB concentration (<0.8 wt% of SS), which due to the formation of additional cross links, limited the available free volume required for ionic mobility in SPS-30 membrane. Proton hopping depends directly on the concentration of free –SO₃ groups and available free space for ionic mobility. DVB concentration overall regulated the SS polymerization and membrane porosity which in turn, affected the overall water uptake and proton conductivities in the membranes. As observed earlier, despite of having increased IEC (due to higher SS concentration), lower water uptake and increased rigidness, restricted transverse proton conduction in SPS-30 membrane. As a consequence, lower proton conductivity was observed in SPS-30 in comparison to other (SPS-10 and SPS-20) membranes. Nevertheless, incorporated SS in SPVDF-co-HFP copolymer with cross linker DVB, exhibited SPS-20 as better PEM with higher water uptake, IEC and increased proton conductivity among other casted membranes.

Fig. 6 Ion exchange capacity (IEC) and proton conductivity of membranes.

3.5 Cyclic voltammetry analysis

In order to enumerate the microbial electrochemical potency, cyclic voltammograms with and without biofilm, were performed in a 15 mL Bob cell (Gamry Instruments, USA) at a scan rate of 2 mV s⁻¹ (Fig. 7). Electrochemical activities were found absent in bacterial devoid medium, as no observable redox peaks were observed in the control experiments (using PBS buffer solution). On the other hand, the presence of biofilms revealed prominent redox activities with an oxidation peak at 889 mV vs. ref. at 10.3 μA, and two distinct reduction peaks at 222 mV and −262 mV vs. ref. at −10.6 μA and −7.9 μA respectively.

Fig. 7 Cyclic voltammograms of biofilm.

The oxidation was indicative of electron transfer from electrode to biofilm, whereas reduction peaks corresponded to the charge transfer from biofilm to electrode. The plausible reason for this redox activity could be attributed to the microbial cell surface proteins that resulted in sustained microbial electrochemical activity on repeated potential cycling. The electrogenic activities of the employed firmicutes evidently ensured their biocatalytic potency for substrate oxidation.

3.6 MFC performance

All five MFCs were monitored under similar operating conditions. For stabilization, initially MFCs were allowed to run without any external load and data were recorded thereafter periodically. The anolyte pH was kept 7.2 and the air facing side of cathode was covered with parafilm to favour an initial anodic start-up condition. Prior to system stabilization, random fluctuations were observed for two days with an average anodes and cathodes potential of −216 mV and +175 mV (with Ag/AgCl ref. electrode) from MFCs (data not shown here). These fluctuations were considered as the system acclimatization period, where gradual OCV (open circuit voltage) increments were observed in all MFCs from day 3. Within 10 days, MFC-D with fitted SPS-20 membrane showed frequent OCV rise, where similar, but lower OCV increments were observed in other units. A respective maximum open circuit potential of 611 ± 20 mV, 629 ± 30 mV, 681 ± 18 mV, and 722 ± 20 mV, and 642 ± 15 mV were observed from MFC-A, B, C, D and E (Fig. 8). Comparatively, MFC-D showed apparent increase in cell efficiencies in terms of current and voltage drop over other units. When measured at multiple resistances, a maximum current of 0.83 mA (0.20 V), 0.87 mA (0.21 V), 0.96 mA (0.24 V), 1.02 mA (0.26 V), 0.91 mA (0.23 V) were observed from MFC-A, B, C, D and E respectively at 250 Ω. Increased current with relatively lower voltage drop in MFC-D, seemingly indicated the efficiency of fitted SPS-20 membrane that served as better PEM than other employed membranes. Here, MEA effect was an inclusive factor in MFCs besides sulfonation and in situ SS polymerization within copolymer structure. SS incorporation resulted in increased ionic conductivity across the membrane, which indicated enhanced performance from MFC-D, in comparison to other units. In case of MFC-B, C and E lower OCV and current outputs were observed, which were mainly because of the relatively hindered water uptake, IEC and proton conductivity of the membrane as stated above. Based on the employed membranes, the variations in the performance primarily distinguished the MFCs. Reduction in electrode spacing due to the used MEA reduced the electrolyte resistance, which in turn aided in increased power production from the units. The electrode materials and spacing in between have been found to produce pronounce effects on fuel cell performances.³⁹ On varying resistors, polarization curves for all MFCs were obtained (Fig. 9). At higher external resistance, higher activation losses were observed with rapid voltage drops, which can be attributed to the energy lost in initiating oxidation–reduction reaction, that is, in electron transfer from cell terminal protein/enzyme to the anode surface.⁴⁰ Voltage drop was found greater at lower resistances which were mainly due to the ease in electron flow within the circuit. A respective maximum power and current density of 287.42 ± 14 mW m⁻² and 1383.63 ± 69 mA m⁻², 311.9 ± 16 mW m⁻² and 1441.37 ± 72 mA m⁻², 382.65 ± 19 mW m⁻² and 1597.36 ± 80 mA m⁻², 447.42 ± 22 mW m⁻² and 1729.63 ± 87 mA m⁻² and 347.65 ± 17 mW m⁻² and 1523.63 ± 76 mA m⁻² were observed from MFC-A, B, C, D and E. The obtained results in terms of power generation have been compared with other relevant previous studies (ESI Table S2†). Approximately 7%, 24% and 17% higher power densities were observed from MFC-B, C and E in comparison to MFC-A. SS incorporation in sulfonated PVDF-co-HFP copolymer showed marked increase in cell efficiencies, where maximum output was observed from MFC-D (SPS-20 fitted MFC). In comparison, the cell efficiencies obtained from MFC-D were approximately 36%, 30%, 14% and 22% higher than MFC-A, B, C, and E respectively. Thus, in effect, the discernible efficacy of SPS-20 with respect to its higher power output indicated its promising potential in MFC over other employed membranes.

Fig. 8 Open circuit potentials of MFCs.

Fig. 9 Polarization and power curves of MFCs.

3.7 Electrochemical impedance spectroscopy (EIS) analysis

Two-electrode geometry was used to measure the internal resistance (R_in) of the whole unit. Anode served as the working electrode whereas both reference and counter electrode were connected to the cathode. R_in is generally categorized in three components: activation (charge transfer) resistance, ohmic resistance (R_m, representing the resistance from electrode materials, solution and membrane) and concentration (diffusion) resistance. Nyquist representations of the electrochemical impedance of the cells as a function of applied frequency were plotted (Fig. S2, ESI†), and the respective cell resistances of the system were segmented in various specific components as described above. The impedance at higher frequency represented the ohmic resistance R_m, whereas the diameter of the semicircle denoted R_p. The polarization resistance, R_p (or charge transfer resistance), in general shows the kinetics of the electrode reaction. It has been observed that the biofilm formation and substrate utilization increases the internal resistance in the system with time, which affects the overall performance of the cell.⁴¹ Nevertheless, the ohmic resistances, R_m as calculated from the equivalent circuit (Fig. S2, ESI†), were ∼9.4 Ω, ∼7.6 Ω, ∼6.2 Ω, ∼4.3 Ω, and ∼7.3 Ω for MFC-A, B, C, D and E respectively. MFC-D with SPS-20 showed minimal ionic resistance as compared to other MFCs. The reason could be the abundant transverse ionic movement in SPS-20 due to the incorporated SS within the membrane, which enhanced its polarity and conduction. In addition, Warburg effect at lower frequencies were observed, indicating limited ionic diffusion in all units. This behaviour showed that the electron transfer at the electrode was significantly reduced at lower frequencies. Nevertheless, differences in the charge transfer evidently ensured the better performance of SPS-20 over other membranes. This was expected, as the increased IEC and proton conductivity of SPS-20 relatively allowed more ionic conduction over other fitted MFC membranes. This in turn, increased the cell efficiency with comparatively lower resistance in MFC-D.

3.8 Bio-film growth on electrode surface

Bio-film adhesion is a well known characteristic of microbes, due to the presence of EPS (exo-polysaccharide) on microbial cell-wall. On day 30, small portion of electrode with bio-film was taken for SEM analysis. SEM image, revealed dominant microbial colonies at electrode's surface (Fig. 10). On cloth fibers, these colonized bacterial population acted as bio-catalysts, which engaged electrochemically in the system and eventually led to power production with subsequent substrate utilization.

Fig. 10 SEM images of biofilm on carbon cloth (electrode).

3.9 Wastewater treatment

Substrate depletion is a perpetual phenomenon in MFC and to analyze it, anolytes were monitored every 4^th day. Substrate removal efficiency (γ) in MFC operation was evaluated using the equation

γ = (COD_initial − COD_final)/COD_initial × 100 (7)

where, COD_initial represents the initial COD and COD_final denotes the final COD in the reactor. An overall COD removal of ∼88.66%, ∼90.09%, ∼91.27%, ∼93.21%, and ∼90.45% were observed from MFC-A, B, C, D and E respectively from 150 mg L⁻¹ anolyte in 28 days operation (Fig. 11). The COD removal differences were minimal in MFCs and mainly due to the used membranes.

Fig. 11 Substrate removal from MFCs.

Increased substrate utilization indicates the energy harnessing efficiency of the microbes. It was majorly observed in MFC-D, where the major reason attributed was the employed SPS-20 membrane. Also, in other units, the energy performance correlated with the COD removal that corresponded affirmatively with the respective used membranes.

The increased SS resulted in higher IEC and proton conductivity that allowed more current to be drawn out of the system. This eventually resulted in comparative higher COD removals from SPS-5, 10 and 20 fitted MFCs (MFC-B, C and D). Relatively, in case of SPS-30 MFC, lower current sourced because of its higher impedance with reduced COD removals in comparison to MFC-C and D. The plausible reason could also be attributed to the reduced water uptake, swelling ratio and proton conductivity of SPS-30 membrane. Adverse effects of charge accumulation are well known in MFCs, which majorly hampers anolyte exhaustion and microbial sustenance. Since COD removal solely depends on the employed microbes, multiple enzymatic steps involved in substrate degradation also augment kinetic limitations in the system. In addition, the coulombic efficiencies of the studied systems varied between 1–3%. This lower coulombic efficiency was due to the different factors involved. One major factor was the fermentative or methanogenic reaction that predominates in the system. This, in turn, reduces the microbial metabolism and thus, results in overall lower efficiencies from the units.^42–45 Nevertheless, successive COD removals from all units showed microbial adequacy in substrate utilization, where SPE-20 served as relatively efficient PEM over other employed membranes in MFCs.

4. Conclusion

In summary, different semi-IPN membranes composed of varied concentrations of sulfonated styrene (SS), sulfonated PVdF-co-HFP and crosslinker divinyl benzene (DVB) were compared as MEA in single chambered MFCs with mixed electrogenic firmicutes as biocatalysts. Increasing SS concentration showed improved water uptake, swelling, IEC and proton conductive properties in membranes, where best PEM qualities were observed with 20% incorporated SS (SPS-20) with minimal internal resistance. DVB concentration of ∼0.8 wt% of SS was found optimum for in situ polymerization of sulfonated styrene (SS) within sulfonated PVdF-co-HFP structure that served it as a cost efficient PEM. Being a future technology, MFC needs more profound research in such diversified areas of membrane technology for relevant cost effective practical alternatives.

Acknowledgements

Financial support from Council of Scientific and Industrial Research (CSIR), and Ministry of Environment and Forest, Govt. of India (MOEF) is duly acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03411f

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