Improvement of Microbial Electrolysis Cell Activity by Using Anode Based on Combined Plasma-Pretreated Carbon Cloth and Stainless Steel

: The anode activity in a microbial electrolysis cell (MEC) is known to be a limiting factor in hydrogen production. In this study, the MEC was constructed using di ﬀ erent anode materials and a platinum-coated carbon-cloth cathode (CC). The anodes were comprised of CC, stainless steel (SS), and a combination of the two (COMB). The CC and SS anodes were also treated with plasma to improve their surface morphology and hydrophilic properties (CCP and SSP, respectively). A combined version of CCP attached to SS was also applied (COMBP). After construction of the MEC using the di ﬀ erent anodes, we conducted electrochemical measurements and examination of bioﬁlm viability. Under an applied voltage of 0.6 V (Ag / AgCl), the currents of a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m − 2 , respectively, which are about three times higher compared to the untreated CC and COMB. A MEC utilizing an untreated SS anode exhibited current of only 0.3712 ± 0.0108 A m − 2 . The highest bioﬁlm viability of 0.92 OD 540 ± 0.07 and hydrogen production rate of 0.0736 ± 0.0022 m 3 d − 1 m − 2 at 0.8 V were obtained in MECs based on the COMBP anode. To our knowledge, this is the ﬁrst study that evaluated the e ﬀ ect of plasma-treated anodes and the use of a combined anode composed of SS and CC for hydrogen evolution in a MEC.


Introduction
A microbial electrolysis cell (MEC) is an electrochemical device with a microbial anode, which operates under applied external potential. It bears some similarities to the microbial fuel cell (MFC), which operates as a galvanic cell. A MEC facilitates hydrogen generation by using a hydrogen evolution cathode instead of the oxygen reduction cathode in a MFC. On the other hand, the bioelectrochemical activity of the bioanode is the same in both devices [1,2], and is promoted by bacteria-inducing current production. The anode properties require a high surface area for biofilm formation, functional groups that will support sustainable attachment of the bacteria to the surface, and high conductivity to support effective electron transfer from the bacteria to the anode material.
There are several anode materials that are currently under research [3,4]. Carbon-based materials are cost-effective and serve as electrodes in many configurations, such as carbon cloth [5][6][7], carbon fibers [8], carbon brush [9], graphite-fiber brush [10], graphite granules [11], and graphite plates [12]. Highly pure graphite is chemically more stable compared to carbon (e.g., carbon cloth or carbon felt), and, despite the higher cost of high-grade graphite, it is widely used as a bioanode [13].

LSV Measurements of the Bioanodes
Low hydrogen production in MECs is mostly related to the bacterial anode activity. The limitations of the bioanode are attributed to the bacterial attachment, biofilm development, and handling of electroactive biofilm for a prolonged period [7]. The anode material should be highly conductive, support the biofilm attachment, be chemically stable and cost-effective. Recently, it was reported that the performance of MFCs which were equipped with cost-effective anodes made of different carbon materials (carbon felt, foam, and cloth) demonstrated that the carbon felt anode was the most efficient whereas the least efficient was the carbon cloth. Performance seems to be in direct relationship with the specific area of the anode materials [38][39][40][41][42]. We examined a new concept of microbial anode made of carbon cloth which differs in two ways: the carbon cloth anode was attached to a rigid conductive stainless-steel material, and the carbon cloth anode was also modified by plasma irradiation.
In this study, SS anode material, which is known as a highly conductive and cost-effective metal, was compared to the common CC anode [43]. The anode materials SS and CC were used separately, and also in a combined anode configuration in which the CC and SS were tightly attached (COMB). In addition, the CC and SS were also treated with cold low-pressure plasma, producing CCP and SSP. Since SSP was found to corrode in MEC under 0.3 V, most of the study was conducted with untreated SS; thus, the combined anode configuration, COMBP, was comprised of untreated SS and plasma-treated CC. The MECs were inoculated with Geobacter and were operated under a chronoamperometry potential of 0.3 V vs. Ag/AgCl. LSV measurement was performed on the 10th day of the MEC operation ( Figure 1).
The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
Energies 2019, 12, 1968 5 of 15 attached (COMB). In addition, the CC and SS were also treated with cold low-pressure plasma, producing CCP and SSP. Since SSP was found to corrode in MEC under 0.3 V, most of the study was conducted with untreated SS; thus, the combined anode configuration, COMBP, was comprised of untreated SS and plasma-treated CC. The MECs were inoculated with Geobacter and were operated under a chronoamperometry potential of 0.3 V vs. Ag/AgCl. LSV measurement was performed on the 10th day of the MEC operation ( Figure 1). The results in Figure 1 show th produced higher currents compare Under an applied voltage of 0.6 V v COMBP were 11.66 ± 0.1331 and 16 carbon cloth (CC and COMB) yiel respectively. In the MEC with a SS a applied voltage of 0.6 V.
From the LSV analysis, it ca COMBP) led to higher currents w addition, both the combined anode anode materials alone (CC or SS, a observed on SS (further displayed a mainly served as an electron-curren attachment. However, the electro depended on the contact between th (low resistance) was typically attain the fibers, a common practice in oth cells [44,45]). Typically, in a MEC, t press the anode′s carbon materia conductivity, a highly rigid SS electr assumed that this contact between formation on the CC, while the SS s the electron flow toward the anode.

DPV Measurements of the Bioanod
The MEC based on the SS ano currents obtained by CC and COM under applied voltage of 0.3 V. Thi and COMBP anodes. Each one o connected to one cathode (i.e., fou constant applied potential of 0.3 V v at a time, by connecting the examine OCP mode. The DPV method prov relationship. On the 14th day of ME 14 potentials (between −0.5 V-0.8 V The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode′s carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving the electron flow toward the anode.

DPV Measurements of the Bioanodes
The MEC based on the SS anode led to very low currents of about only 16% and 11% of the currents obtained by CC and COMB, respectively ( Figure 1). In addition, the SSP was corroded under applied voltage of 0.3 V. This study therefore continued by using only the CC, CCP, COMB and COMBP anodes. Each one of four single-cell MECs contained the four different anodes connected to one cathode (i.e., four replicates of each anode). The MECs were operated under a constant applied potential of 0.3 V vs. Ag/AgCl. DPV was measured separately for each anode, one at a time, by connecting the examined anode to the cathode while the other anodes were operated in OCP mode. The DPV method provides a steady-state potentiostatic view of the current potential relationship. On the 14th day of MEC operation, a steady-state polarization curve emerged in a set of 14 potentials (between −0.5 V-0.8 V vs. Ag/AgCl) that were measured by the DPV method ( Figure 2). The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode′s carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving the electron flow toward the anode.

DPV Measurements of the Bioanodes
The MEC based on the SS anode led to very low currents of about only 16% and 11% of the currents obtained by CC and COMB, respectively ( Figure 1). In addition, the SSP was corroded under applied voltage of 0.3 V. This study therefore continued by using only the CC, CCP, COMB and COMBP anodes. Each one of four single-cell MECs contained the four different anodes connected to one cathode (i.e., four replicates of each anode). The MECs were operated under a constant applied potential of 0.3 V vs. Ag/AgCl. DPV was measured separately for each anode, one at a time, by connecting the examined anode to the cathode while the other anodes were operated in OCP mode. The DPV method provides a steady-state potentiostatic view of the current potential relationship. On the 14th day of MEC operation, a steady-state polarization curve emerged in a set of 14 potentials (between −0.5 V-0.8 V vs. Ag/AgCl) that were measured by the DPV method ( Figure 2). The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode′s carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving the electron flow toward the anode.

DPV Measurements of the Bioanodes
The MEC based on the SS anode led to very low currents of about only 16% and 11% of the currents obtained by CC and COMB, respectively ( Figure 1). In addition, the SSP was corroded under applied voltage of 0.3 V. This study therefore continued by using only the CC, CCP, COMB and COMBP anodes. Each one of four single-cell MECs contained the four different anodes connected to one cathode (i.e., four replicates of each anode). The MECs were operated under a constant applied potential of 0.3 V vs. Ag/AgCl. DPV was measured separately for each anode, one at a time, by connecting the examined anode to the cathode while the other anodes were operated in OCP mode. The DPV method provides a steady-state potentiostatic view of the current potential relationship. On the 14th day of MEC operation, a steady-state polarization curve emerged in a set of 14 potentials (between −0.5 V-0.8 V vs. Ag/AgCl) that were measured by the DPV method (Figure 2). The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode′s carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving the electron flow toward the anode.

DPV Measurements of the Bioanodes
The MEC based on the SS anode led to very low currents of about only 16% and 11% of the currents obtained by CC and COMB, respectively ( Figure 1). In addition, the SSP was corroded under applied voltage of 0.3 V. This study therefore continued by using only the CC, CCP, COMB and COMBP anodes. Each one of four single-cell MECs contained the four different anodes connected to one cathode (i.e., four replicates of each anode). The MECs were operated under a constant applied potential of 0.3 V vs. Ag/AgCl. DPV was measured separately for each anode, one at a time, by connecting the examined anode to the cathode while the other anodes were operated in OCP mode. The DPV method provides a steady-state potentiostatic view of the current potential relationship. On the 14th day of MEC operation, a steady-state polarization curve emerged in a set of 14 potentials (between −0.5 V-0.8 V vs. Ag/AgCl) that were measured by the DPV method ( Figure 2). From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode's carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving the electron flow toward the anode.

DPV Measurements of the Bioanodes
The MEC based on the SS anode led to very low currents of about only 16% and 11% of the currents obtained by CC and COMB, respectively ( Figure 1). In addition, the SSP was corroded under applied voltage of 0.3 V. This study therefore continued by using only the CC, CCP, COMB and COMBP anodes. Each one of four single-cell MECs contained the four different anodes connected to one cathode (i.e., four replicates of each anode). The MECs were operated under a constant applied potential of 0.3 V vs. Ag/AgCl. DPV was measured separately for each anode, one at a time, by connecting the examined anode to the cathode while the other anodes were operated in OCP mode. The DPV method provides a steady-state potentiostatic view of the current potential relationship. On the 14th day of MEC operation, a steady-state polarization curve emerged in a set of 14 potentials (between −0.5 V-0.8 V vs. Ag/AgCl) that were measured by the DPV method ( Figure 2).  It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an  It is very difficult to compare results from other studies regarding MEC/MFC, since there are many parameters differentiating between the reported devices (for example: electrode materials, distance between electrodes, MEC/MFC configuration, reactor volume, carbon source, and bacterial community). Several MEC/MFC studies reported using SS-anode material whose surface was treated with a method other than plasma. A tubular MEC based on a pleated SS-felt anode and a platinum cathode was reported to have low internal resistance and high conductivity of the electrodes. The MEC reached a maximum current density of 1.09 ± 0.04 mA cm −2 at 1 V vs. Ag/AgCl [46]. In that report, the SS that served as an anode in the MEC was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. At an applied voltage of 0.2 V, their reported MEC based on the clean SS showed a current density of around 0.7 A m −2 and 2.4 A m −2 with 5 mM and 10 mM acetate, respectively [16].
A MFC based on a 25 cm 2 area of projected SS grids, which were cleaned by immersion in a 2% HF/0.5 M HNO 3 solution, led to an efficient microbial anode that provided a current density of 8 A m −2 at −100 mV (SCE) [47]. Enhancement of biocompatibility and anode electroactivity was observed in a MFC utilizing flame oxidation of SS felt coated by iron-oxide nanoparticles (IONPs). This SS felt produced a maximum current density of 27.42 mA cm −3 and 1.92 mA cm −2 at −0.2 V, which was 16.5 and 4.8 times higher than the untreated SS felt and carbon felt, respectively [48].
The most commonly used anode material in MECs is CC. It was reported that in a single-cell MEC utilizing a carbon-felt anode and a Pt gas-diffusion cathode, where a J-cloth was separating the adjacent electrodes, the obtained current was 6 A m −2 under an applied voltage of 1 V [49]. A multi-electrode MEC stack design with increasing electrode pairs (up to 10 electrode pairs) in which activated carbon cloth (pretreated with surfactant) and SS mesh served as anode and cathode, respectively, was compared for electric current and COD reduction. A maximum current density of 520 A m −3 at an applied voltage of 1.1 V was observed in the 10-pair MEC compared to the single-pair MEC with a current density of 45 A m −3 . These results showed that increasing the number of electrodes in MECs can magnify the current production [50]. Single-chamber MECs based on a type-A commercial carbon-cloth anode (without a wet-proofing coating) and a type-B carbon-cloth cathode (30% wet-proofing), separated by J-cloth, were inoculated by a mixed culture. Under an applied voltage of 0.6 V, the MEC achieved a current density of 9.3 A m −2 at pH 7 and 14 A m −2 at pH 5.8 [51].
In summary, the reported MECs/MFCs utilizing SS anodes had high current performance. However, in our study the MEC based on SS led to a negligible current density of 0.3712 ± 0.3711 A m −2 at an applied voltage of 0.6 V. SS that was plasma-treated to increase hydrophilicity was corroded under an applied potential of 0.3 V. However, a combined anode of SS and CCP (COMBP) demonstrated a high current density of 16.36 ± 0.3172 A m −2 . The current contributed by each of the different active elements of the MEC to the overall obtained current was determined at the end of the MEC operation period. The oxidation currents generated by the full MEC construction were measured on the 21st day. The biofilm anode was transferred to a sterile MEC to examine currents contributed by the biofilm (the "biofilm anode"). A sterile anode was inserted into the MEC with the planktonic bacteria to measure the contribution of the planktonic bacteria to the obtained current of the full MEC (the "planktonic bacteria"). In addition, at the beginning of the MEC operation, the currents were measured before inoculation (the "abiotic anode"). The currents were measured when the MECs were operated under selected voltages of between 0.2 V to 0.8 V (Table 1). The MECs based on the different anodes were operated for 14 days. Increasing the voltage from 0.2 V to 0.8 V vs. Ag/AgCl led to higher currents in the four MECs (based on COMBP, COMB, CC, CCP anodes) as well as in each element (biofilm anode, planktonic bacteria, and abiotic electrode). The currents obtained under an applied voltage of 0.6 V from the MEC based on the COMBP anode were the highest (5.593 mA ± 0.067), while the currents obtained from the MEC based on COMB, CC, and CCP were 4.948 0.152, 3.817 ± 0.209, and 4.724 ± 0.054 mA, respectively. In all cases, the biofilm anode contributed higher currents than the planktonic bacteria. For example, in the MEC based on the COMBP anode under an applied voltage of 0.6 V, the biofilm anode led to a current of 2.931 mA ± 0.056, while the planktonic bacteria produced only 0.450 ± 0.005 mA. In the MEC based on the COMB anode, the biofilm anode led to a current of 1.154 ± 0.035 mA, while the planktonic bacteria led to only 0.426 ± 0.013 mA. The sum total of the currents obtained from the planktonic bacteria and from the biofilm anode did not equal the total current exhibited in the full constructed MEC. We ascribe this phenomenon to damage occurring to the bacterial anode while moving it to a sterile MEC.

Hydrogen Formation in the MECs
In the following experiments, we examined the effect of the anode materials and surface plasma-pretreatment of the carbon cloth to improve the hydrogen evolution reaction (HER) activity. For these measurements (Figure 3), the MECs were connected to the potentiostat in a configuration of two electrodes (the working electrode was connected to the platinized carbon cloth while the reference and counter electrodes were connected to the bacterial anode). and CCP were 4.948 0.152, 3.817 ± 0.209, and 4.724 ± 0.054 mA, respectively. In all cases, the biofilm anode contributed higher currents than the planktonic bacteria. For example, in the MEC based on the COMBP anode under an applied voltage of 0.6 V, the biofilm anode led to a current of 2.931 mA ± 0.056, while the planktonic bacteria produced only 0.450 ± 0.005 mA. In the MEC based on the COMB anode, the biofilm anode led to a current of 1.154 ± 0.035 mA, while the planktonic bacteria led to only 0.426 ± 0.013 mA. The sum total of the currents obtained from the planktonic bacteria and from the biofilm anode did not equal the total current exhibited in the full constructed MEC. We ascribe this phenomenon to damage occurring to the bacterial anode while moving it to a sterile MEC.

Hydrogen Formation in the MECs
In the following experiments, we examined the effect of the anode materials and surface plasma-pretreatment of the carbon cloth to improve the hydrogen evolution reaction (HER) activity. For these measurements (Figure 3), the MECs were connected to the potentiostat in a configuration of two electrodes (the working electrode was connected to the platinized carbon cloth while the reference and counter electrodes were connected to the bacterial anode). The results depicted in Figure 3 show that the highest hydrogen reduction current was obtained in the MEC applying the combined anode, COMBP. At the maximal applied cell voltage (0.8 V) the reduction current obtained in the MEC based on a COMBP anode was 6.08 ± 0.182 A m −2 , while in the MEC based on a COMB anode the current was only 3.7 ± 0.131 A m −2 (an improvement of 64%). Similar results were obtained in the MEC based on plasma-treated CCP, where the reduction current was 59% higher than the current in the MEC based on the untreated anode (CC).
Calculation of hydrogen evolution rates under applied constant potentials ranging from 0.2 to 0.8 V were performed according to Equations (1)-(3) and results are presented in Table 2. Under an applied voltage of 0.6 V, the current densities obtained in the MECs based on CC, COMB, CCP or COMBP anodes were 2.386 ± 0.074, 2.590 ± 0.084, 4.232 ± 0.104, and 5.186 ± 0.163 A m −2 , respectively. In these MECs, the hydrogen evolution rates per cubic meter of the anodic medium were 0.0902 ± 0.0028, 0.0980 ± 0.0032, 0.1600 ± 0.004, and 0.1961 ± 0.006 m 3 d −1 m −3 , respectively. The hydrogen evolution rates per square meter of anode were 0.0289 ± 0.0009, 0.0313 ± 0.0010, 0.0512 ± 0.0013, and 0.0627 ± 0.002 m 3 d −1 m −2 , respectively. These results show that under applied voltages of 0.6 V, the MEC based on the combined anode (COMBP) led to a higher hydrogen evolution rate of 0.0627 ±  The results in Figure 1 show that MEC produced higher currents compared to M Under an applied voltage of 0.6 V vs. Ag/A COMBP were 11.66 ± 0.1331 and 16.36 ± 0. carbon cloth (CC and COMB) yielded cu respectively. In the MEC with a SS anode, applied voltage of 0.6 V.
From the LSV analysis, it can be d COMBP) led to higher currents when co addition, both the combined anode COMB anode materials alone (CC or SS, as well observed on SS (further displayed and disc mainly served as an electron-current collec attachment. However, the electron con depended on the contact between the carb (low resistance) was typically attained by the fibers, a common practice in other fuel cells [44,45]). Typically, in a MEC, the ano press the anode′s carbon material. In o conductivity, a highly rigid SS electrode m assumed that this contact between the two formation on the CC, while the SS support the electron flow toward the anode. The results in Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) produced higher currents compared to MECs based on untreated anodes (SS, CC and COMB). Under an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and COMBP were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated carbon cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , respectively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an applied voltage of 0.6 V.
From the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and COMBP) led to higher currents when compared to the untreated anodes (CC and COMB). In addition, both the combined anode COMB as well as COMBP exhibit higher currents than any of the anode materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability observed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode mainly served as an electron-current collector, rather than a material capable of supporting biofilm attachment. However, the electron conduction through the carbon-cloth electrode strongly depended on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity (low resistance) was typically attained by applying external pressure to force good contact among the fibers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel cells [44,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to press the anode′s carbon material. In our study, instead of pressing the anode to increase conductivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We assumed that this contact between the two anode materials provided better conditions for biofilm formation on the CC, while the SS supported better current collection through the CC by improving  Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) ed higher currents compared to MECs based on untreated anodes (SS, CC and COMB). an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , ively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an voltage of 0.6 V. m the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and ) led to higher currents when compared to the untreated anodes (CC and COMB). In n, both the combined anode COMB as well as COMBP exhibit higher currents than any of the aterials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability d on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode served as an electron-current collector, rather than a material capable of supporting biofilm ent. However, the electron conduction through the carbon-cloth electrode strongly ed on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity sistance) was typically attained by applying external pressure to force good contact among rs, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel ,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to he anode′s carbon material. In our study, instead of pressing the anode to increase tivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We d that this contact between the two anode materials provided better conditions for biofilm on on the CC, while the SS supported better current collection through the CC by improving  Figure 1 show that MEC utilizing plasma-pretreated anodes (CCP and COMBP) ed higher currents compared to MECs based on untreated anodes (SS, CC and COMB). an applied voltage of 0.6 V vs. Ag/AgCl, the currents obtained in a MEC based on CCP and P were 11.66 ± 0.1331 and 16.36 ± 0.3172 A m −2 , respectively; while MEC based on untreated cloth (CC and COMB) yielded currents of only 3.272 ± 0.1792 and 5.541 ± 0.1705 A m −2 , tively. In the MEC with a SS anode, the observed current was only 0.3712 ± 0.0108 A m −2 at an voltage of 0.6 V. om the LSV analysis, it can be demonstrated that plasma-pretreated anodes (CCP and P) led to higher currents when compared to the untreated anodes (CC and COMB). In n, both the combined anode COMB as well as COMBP exhibit higher currents than any of the materials alone (CC or SS, as well as CCP or SS, respectively). The poor biofilm viability ed on SS (further displayed and discussed in Figure 4) led us to conclude that the SS electrode served as an electron-current collector, rather than a material capable of supporting biofilm ent. However, the electron conduction through the carbon-cloth electrode strongly ed on the contact between the carbon fibers (woven or nonwoven). Thus, high conductivity sistance) was typically attained by applying external pressure to force good contact among ers, a common practice in other fuel cells (e.g., hydrogen polymer electrolyte membrane fuel 4,45]). Typically, in a MEC, the anode is immersed in the medium without a mechanism to the anode′s carbon material. In our study, instead of pressing the anode to increase ctivity, a highly rigid SS electrode mesh was attached to the CC to form the COMB anode. We ed that this contact between the two anode materials provided better conditions for biofilm ion on the CC, while the SS supported better current collection through the CC by improving ) in a single-cell MEC containing G. sulfurreducens in Geobacter medium and 0.1 M PB, pH 7. Scan rate was 5 mV s −1 .
The results depicted in Figure 3 show that the highest hydrogen reduction current was obtained in the MEC applying the combined anode, COMBP. At the maximal applied cell voltage (0.8 V) the reduction current obtained in the MEC based on a COMBP anode was 6.08 ± 0.182 A m −2 , while in the MEC based on a COMB anode the current was only 3.7 ± 0.131 A m −2 (an improvement of 64%). Similar results were obtained in the MEC based on plasma-treated CCP, where the reduction current was 59% higher than the current in the MEC based on the untreated anode (CC).
Calculation of hydrogen evolution rates under applied constant potentials ranging from 0.2 to 0.8 V were performed according to Equations (1)-(3) and results are presented in Table 2. Under an applied voltage of 0.6 V, the current densities obtained in the MECs based on CC, COMB, CCP or COMBP anodes were 2.386 ± 0.074, 2.590 ± 0.084, 4.232 ± 0.104, and 5.186 ± 0.163 A m −2 , respectively. In these MECs, the hydrogen evolution rates per cubic meter of the anodic medium were 0.0902 ± 0.0028, 0.0980 ± 0.0032, 0.1600 ± 0.004, and 0.1961 ± 0.006 m 3 d −1 m −3 , respectively. The hydrogen evolution rates per square meter of anode were 0.0289 ± 0.0009, 0.0313 ± 0.0010, 0.0512 ± 0.0013, and 0.0627 ± 0.002 m 3 d −1 m −2 , respectively. These results show that under applied voltages of 0.6 V, the MEC based on the combined anode (COMBP) led to a higher hydrogen evolution Energies 2019, 12, 1968 9 of 15 rate of 0.0627 ± 0.002 m 3 d −1 m −2 , which is a two-fold increase over the rates of HER in the MECs based on CC and COMB. But it was only a 1.23-fold increase compared to CCP.
where Q(V r ) H 2 -HER production rate per cubic meter of the anodic medium, V H 2 -Hydrogen production volume (m 3 s −1 , calculated from Equation (1)), t-time in seconds normalized to 24 h, and V r -reactor volume normalized to cubic meter (m 3 ).
where Q(A e ) H 2 -HER production rate per square meter of electrode, V H 2 -Hydrogen production volume (m 3 s −1 , calculated from Equation (1)), t-time in seconds normalized to 24 h, and A e -electrode geometric area normalized to square meter (m 2 ). The results presented in Table 2 show that increasing the applied voltage from 0.2 V to 0.8 V resulted in a 10-fold enhancement of the reduction current density in MECs based on plasma-treated anodes (CCP and COMBP), while in MECs based on untreated anodes (CC and COMB) the reduction current density was increased by 28 and 16-fold, respectively. The highest hydrogen production rate (0.0736 ± 0.0022 m 3 d −1 m −2 at 0.8 V) was obtained in the MEC based on the COMBP anode, which was 40%, 18% and 49% higher than the MEC based on the COMB, CCP and CC anodes, respectively.
A single-chamber MEC made from narrow-mouth media bottles (total volume of 320 mL, which includes a liquid volume of 100 mL) was inoculated with a mixed culture. The anode and cathode were made of carbon cloth, and the cathode was Pt-coated. This MEC was operated under an applied voltage of 1.0 V and reached a hydrogen production rate of 3.0 L L −1 D −1 [52]. A MEC made from wide-mouth glass bottles (500 mL) was inoculated with a mixed culture. The anode and Pt-coated cathode were made of carbon cloth and separated by a J-cloth. Under an applied voltage of 0.6 V, the MEC reached a hydrogen production rate of 0.53 m 3 day −1 m 3 (0.11 m 3 d −1 m 2 ) at pH 7, and a higher rate of 0.69 m 3 d −1 m −3 (0.15 m 3 d −1 m −2 ) at pH 5.8 [51]. A cubic-shaped single-chamber MEC (liquid volume of 90 mL) based on a graphite-fiber brush anode and a carbon-cloth cathode was operated at 4 • C and 9 • C, which led to a hydrogen production rate varying from 0.23 ± 0.03 to 0.53 ± 0.04 m 3 d −1 m −3 at an applied voltage of 0.6 V [53]. A membrane-free MEC (100 mL working volume) consisting of anode and cathode (3 × 3 cm 2 E-Tek carbon cloth without wet-proofing), where the cathode was coated with 0.5 mg Pt/cm 2 and the anode was enriched with exoelectrogens from anaerobic sludge, reached a hydrogen production rate of 0.82 ± 0.606 m 3 d −1 m −3 at 1 V. In this MEC, the anode surface area/electrolyte volume ratio was 1850 m 2 m −3 [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 • C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).  [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b) As shown in Figure 4a, a higher biofilm viability of about 4-fold was developed on CC (0.42 OD ± 0.02) compared to the SS (0.093 OD540 ± 0.017). The biofilm viability of CCP was 0.64 OD540 ± 0.034, compared to 0.42 OD540 ± 0.02 on the untreated CC. Higher biofilm viability was also observed in the combined anode where the carbon cloth was treated by plasma (COMBP), compared to the   [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b)  [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b)  [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b)  [51]. A cubic-shaped single-chamber MEC (liquid volume of 90 mL) based on a graphite-fiber brush anode and a carbon-cloth cathode was operated at 4 °C and 9 °C, which led to a hydrogen production rate varying from 0.23 ± 0.03 to 0.53 ± 0.04 m 3 d −1 m −3 at an applied voltage of 0.6 V [53]. A membrane-free MEC (100 mL working volume) consisting of anode and cathode (3 × 3 cm 2 E-Tek carbon cloth without wet-proofing), where the cathode was coated with 0.5 mg Pt/cm 2 and the anode was enriched with exoelectrogens from anaerobic sludge, reached a hydrogen production rate of 0.82 ± 0.606 m 3 d −1 m −3 at 1 V. In this MEC, the anode surface area/electrolyte volume ratio was 1850 m 2 m −3 [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b) As shown in Figure 4a, a higher biofilm viability of about 4-fold was developed on CC (0.42 OD ± 0.02) compared to the SS (0.093 OD540 ± 0.017). The biofilm viability of CCP was 0.64 OD540 ± 0.034, compared to 0.42 OD540 ± 0.02 on the untreated CC. Higher biofilm viability was also observed in the combined anode where the carbon cloth was treated by plasma (COMBP), compared to the  [51]. A cubic-shaped single-chamber MEC (liquid volume of 90 mL) based on a graphite-fiber brush anode and a carbon-cloth cathode was operated at 4 °C and 9 °C, which led to a hydrogen production rate varying from 0.23 ± 0.03 to 0.53 ± 0.04 m 3 d −1 m −3 at an applied voltage of 0.6 V [53]. A membrane-free MEC (100 mL working volume) consisting of anode and cathode (3 × 3 cm 2 E-Tek carbon cloth without wet-proofing), where the cathode was coated with 0.5 mg Pt/cm 2 and the anode was enriched with exoelectrogens from anaerobic sludge, reached a hydrogen production rate of 0.82 ± 0.606 m 3 d −1 m −3 at 1 V. In this MEC, the anode surface area/electrolyte volume ratio was 1850 m 2 m −3 [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b) As shown in Figure 4a, a higher biofilm viability of about 4-fold was developed on CC (0.42 OD ± 0.02) compared to the SS (0.093 OD540 ± 0.017). The biofilm viability of CCP was 0.64 OD540 ± 0.034, compared to 0.42 OD540 ± 0.02 on the untreated CC. Higher biofilm viability was also observed in the combined anode where the carbon cloth was treated by plasma (COMBP), compared to the  [51]. A cubic-shaped single-chamber MEC (liquid volume of 90 mL) based on a graphite-fiber brush anode and a carbon-cloth cathode was operated at 4 °C and 9 °C, which led to a hydrogen production rate varying from 0.23 ± 0.03 to 0.53 ± 0.04 m 3 d −1 m −3 at an applied voltage of 0.6 V [53]. A membrane-free MEC (100 mL working volume) consisting of anode and cathode (3 × 3 cm 2 E-Tek carbon cloth without wet-proofing), where the cathode was coated with 0.5 mg Pt/cm 2 and the anode was enriched with exoelectrogens from anaerobic sludge, reached a hydrogen production rate of 0.82 ± 0.606 m 3 d −1 m −3 at 1 V. In this MEC, the anode surface area/electrolyte volume ratio was 1850 m 2 m −3 [54]. A tubular single-cell MEC (28 mL) comprised of a carbon-cloth anode (3 cm in diameter, type A without wet-proofing) and a Pt cathode (2 × 2 cm 2 ) was operated under an applied voltage of 0.6 V, which led to a hydrogen production rate of 2.3 m 3 d −1 m −3 [55].

Biofilm Viability Depending on Anode Materials and Plasma Treatment before MEC Construction and after MEC Operation for 30 Days
Examination of G. sulfurreducens ability to produce biofilm on the different anodes was performed before constructing the MEC. The biofilm viability on the different anode material, where the carbon cloth was plasma-treated or untreated, was examined in Geobacter medium and in the presence of a soluble organic electron collector (fumarate). The anodes were incubated for 10 days at 35 °C, followed by MTT analysis, a colorimetric reaction based on the activity of the bacterial oxygenase (Figure 4a). The viability of the bioanodes was also examined at the end of MEC operation (30 days) (Figure 4b).
(a) (b) As shown in Figure 4a, a higher biofilm viability of about 4-fold was developed on CC (0.42 OD ± 0.02) compared to the SS (0.093 OD540 ± 0.017). The biofilm viability of CCP was 0.64 OD540 ± 0.034, compared to 0.42 OD540 ± 0.02 on the untreated CC. Higher biofilm viability was also observed in the combined anode where the carbon cloth was treated by plasma (COMBP), compared to the As shown in Figure 4a, a higher biofilm viability of about 4-fold was developed on CC (0.42 OD ± 0.02) compared to the SS (0.093 OD 540 ± 0.017). The biofilm viability of CCP was 0.64 OD 540 ± 0.034, compared to 0.42 OD 540 ± 0.02 on the untreated CC. Higher biofilm viability was also observed in the combined anode where the carbon cloth was treated by plasma (COMBP), compared to the combined anode where the CC was untreated (COMB): the biofilm viability was 0.91 OD 540 ± 0.09 and 0.61 OD 540 ± 0.1, respectively. The viability of the bioanodes was also examined at the end of the MEC operation. From the results described in Figure 4b, it can be seen that the highest biofilm viability as 0.92 OD 540 ± 0.07 at OD 540 was developed on the COMBP anode, compared to 0.57 OD 540 ± 0.04, 0.7 OD 540 ± 0.09, and 0.39 OD 540 ± 0.06 on the COMB, CCP and CC, respectively. The biofilm viability on the treated anode materials (Figure 4a) and after MEC operation (Figure 4b) were significantly higher (p-value < 0.05) compared to the untreated anodes (CCP/CC; SSP/SS and COMBP/COMB). Improvement in SS bio-compatibility toward electroactive biofilm was observed in a tubular MEC, when a 316 L stainless steel-fiber felt anode was pretreated in a furnace at 600 • C for 5 min under ambient air pressure [46]. High biofilm attachment was observed on a MEC's SS anode that was modified with polyaniline and polypyrrole, compared to an unmodified SS plate [56]. Stainless steel was cleaned with a mixture of ethanol and acetone to dissolve organic materials, and with a solution of fluoridric/nitric acids to dissolve the oxide layer. Using epifluorescence microscopy, it was shown that the biofilm was densely and well distributed all over the electrode surface [16]. In our recent study, a carbon cloth was exposed to cold nitrogen plasma which resulted by changing its hydrophobic properties. The carbon cloth surface hydrophilicity was measured using a goniometer. It was shown that the contact angle of a water droplet that was placed over the pristine carbon cloth surface was of~130 • . This result indicated that the pristine carbon cloth is a hydrophobic surface. When a water contact angle was measured after cold plasma treatment for 2 min, the contact angle changed to 0 • , which is considered as a super-hydrophilic surface. In this case, the improvement of the carbon cloth cathode surface wettability properties improved the mechanical adherence of the catalyst [31]. In our current study, the hydrophilic pattern of the plasma treated carbon cloth anode led to an increasement of the bioanodes biofilm viability and hydrogen evolution rate as shown in Figure 4 and Table 2.
Carbon nanotubes (CNTs) used in MFCs/MECs as anode material [57,58] were treated by plasma and were found to support selective biofilm formation. Enhancement of biofilm formation of Gram-positive bacteria such as S. epidermidis and B. subtilis was displayed on the CNT after plasma treatment. However, the Gram-negative bacteria P. aeruginosa and E. coli responded differently, and their biofilm formation was not affected by the plasma treatment [30]. In another study, a CC anode that was coated by graphene oxide, followed by calcination under an atmospheric-pressure plasma jet, leading to an increase in the surface area as well as in hydrophilicity; consequently, a higher bacterial growth was observed on the treated CC electrode compared to the untreated one [59].
To summarize, examination of plasma treatment on biofilm viability in Geobacter medium containing fumarate as an electron collector showed that on SSP, there was a slight improvement in biofilm viability of 30% (with a significant p-value < 0.05), compared to the untreated SS. It is important to note, however, that plasma treatment led to corrosion of the SS. Contrary to this result, plasma treatment of CC led to a significant increase in biofilm viability, of 50% and 67% in CCP and COMBP, respectively, compared to the untreated anodes (CC and COMB). The phenomenon of higher biofilm viability was also observed at the end of MEC operation. The biofilm viability on CCP and COMBP was 80% and 62% higher, respectively, compared to the untreated anodes.

Conclusions
Our single-chamber MECs were constructed using different anode materials: CC, SS, and a combination of these (COMB); along with a platinum-coated CC cathode. The CC and SS anodes were also treated with cold low-pressure nitrogen plasma to improve their surface hydrophilicity, resulting in CCP and SSP, respectively. Plasma treatment led to higher biofilm viability compared to the untreated Energies 2019, 12, 1968 12 of 15 anodes. However, plasma treatment led to corrosion of SSP under an applied voltage of 0.3 V. Thus, the experimental MECs were based on CC, SS, COMB, CCP and COMBP (a combination of SS and CCP) anodes. LSV measurements showed approximately 3-fold increases in currents in the MECs based on CCP and COMBP, compared to the untreated CC and COMB. An analysis of the contribution of the elements: (biofilm anode, planktonic bacteria, and abiotic electrode) to the overall current obtained in the different MECs showed that the biofilm anode provided the majority of the current density, relative to the other elements. The highest hydrogen production rate (0.0736 ± 0.0022 m 3 d −1 m −2 at 0.8 V) was obtained in MECs based on the COMBP anode, which was 40%, 18% and 49% higher than the MEC based on the COMB, CCP and CC anodes, respectively.