Capacitive biocathodes driving electrotrophy towards enhanced CO2 reduction for microbial electrosynthesis of fatty acids

Bioresource Technology 294 (2019) 122181

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Capacitive biocathodes driving electrotrophy towards enhanced CO2 reduction for microbial electrosynthesis of fatty acids J. Annie Modestraa,b, S. Venkata Mohana,b,

T

⁎

a

Bioengineering and Environmental Sciences Lab, Department of Energy and Environmental Engineering, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India b Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT) Campus, Hyderabad, India

GRAPHICAL ABSTRACT

Synoptic view of biocathodes used in MES and elucidating the importance of capacitance linked with electrotrophy for enhanced CO2 reduction towards microbial electrosynthesis of renewable chemicals.

ARTICLE INFO

ABSTRACT

Keywords: Electrotrophs Nyquist plots Energetic efficiency Biobased products Microbial fuel cell (MFC)

Electron transfer towards biocathode is a rate limiting step for CO2 reduction during microbial electrosynthesis (MES). Current study is designed to offer an understanding on electrotrophy using four different electrode materials viz., carbon cloth (CC), stainless-steel mesh (SS), combination of both (CC-SS) and a hybrid material (CC-SS-AC with activated carbon (AC)) as capacitive biocathodes for MES. Non turn-over and turn-over electrochemical investigations revealed electrode properties in terms of electron transfer, capacitance and redox catalytic currents relatively higher with CC-SS-AC and CC-SS. Acetic acid production was higher in CC-SS-AC (4.31 g/l) than CC-SS (4.21 g/l), CC (3.5 g/l) and SS (2.83 g/l) along with noticeable ethanol production with all the biocathodes except SS. Interestingly, long-term operation of all biocathodes witnessed reduction in resistance visualized through Nyquist impedance spectra relatively efficient with CC-SS-AC. Biocompatible property of CCSS-AC with increased surface area was presumed to be a critical factor for enhancing electrotrophy linked with capacitive nature of biocathode towards enhanced bioelectrochemical CO2 reduction.

1. Introduction Incessant rise in CO2 emissions allied with fossil reserves depletion for energy generation developed a quest to search for energy efficient and environmentally benign technologies (Franca and Azapagic, 2015; Venkata Mohan et al., 2016a). In this direction, microbial fuel cells ⁎

(MFC) have been pursued with much interest to generate energy using wastewater as substrate by the catalytic action of bacteria. With the knowledge gained from MFC operation and considering the working principle into account, the applications have been diversified towards treatment of wastes, energy harvesting, resource recovery, desalination, electrosynthesis of renewable chemicals etc. In this perspective,

Corresponding author. E-mail address: [email protected] (S. Venkata Mohan).

https://doi.org/10.1016/j.biortech.2019.122181 Received 30 July 2019; Received in revised form 16 September 2019; Accepted 18 September 2019 Available online 21 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

CC

SS Wastewater +Glucose

VFA, alcohols

CC-SS Treated Wastewater + CO2

CO2+e-+H+

CC-SS-AC

Anodic biocatalyst Cathodic biocatalyst Direction of electron transfer

Fig. 1. Schematic representation of MES used in the study operated with four diverse electrode materials as biocathodes depicting the electron transfer for bioelectrochemical CO2 reduction.

microbial electrosynthesis systems (MES) are emerging as promising and potential routes for CO2 sequestration with simultaneous production of biobased products in an ecofriendly approach (Rojasa et al., 2018; Venkata Mohan et al., 2016b; Rabaey and Rozendal, 2010). MES concept typically relies on the biocatalyst enriched on electrode surface towards catalyzing the bioelectrochemical reduction of CO2 through regulated electron flux either via applied potential or current (Modestra and Venkata Mohan, 2017; Mohanakrishna et al., 2015; Zhang et al., 2013). Several factors such as biocatalyst, applied current/voltage, pH, electrode materials, reactor design, counter electrode etc. will essentially govern the fate of product synthesis in MES (Ganigue et al., 2015; Arends et al., 2017; Modestra et al., 2015; Schlager et al., 2017; Arunasri et al., 2016). Among these, electrotrophy (electron accepting nature) has a key function in conjunction with electrode materials in MES, as the formation of electrotrophic bacteria as biofilm on the surface of electrodes (biocathodes) regulates entire bio-electrochemical reaction (Lovley, 2011). Any electrode can act either as an electron donor or as an acceptor based on the operational criteria. In MES, electrode at cathode (working electrode) serves as electron donor by withdrawing the electrons from counter electrode (anode) under the regulated voltage/ current (Summers et al., 2013; Lovley et al., 1999). Biofilm on the electrode is of utmost importance as it enables the surface catalyzed CO2 reduction efficiently by accepting the electrons from working electrode aimed towards synthesizing the products (Patil et al., 2015a). Electron holding capacity of electrode more generally described as capacitance is based on the material property as well as the affinity of material to facilitate biofilm adhesion on it (Modestra et al., 2016). In MES, capacitive nature of electrode significantly decides the fate

of CO2 reduction as well as product synthesis. Barring few studies, mostly research has been confined towards acetate production from CO2, the primarily and easily synthesized product (Nevin et al., 2010; Bajracharya et al., 2017; Jourdin et al., 2015; Marshall et al., 2013). The fate of product synthesis generally relies on the electron flux which is regulated by the applied voltage/current on the well enriched biofilm (Molenaar et al., 2017; Chen et al., 2017; Yu et al., 2017). The concept of electrotrophy and capacitance is linked well in MES regulating the electron flux as well as its acceptance on the electrode surface. However, this phenomenon is governed by several properties of electrode viz., material, biocompatibility, composition, mechanical strength, porosity, impurities, anti-corrosive nature, increased surface area, metal catalyzed surface etc. which promotes the surface catalyzed bioelectrochemical reduction (Modestra et al., 2016; Chen et al., 2017; Su et al., 2019). Understanding electrotrophy interlinked with capacitive nature of electrode in the context of MES and CO2 reduction is limited. Considering the importance of electrode materials, present study is designed by opting carbon cloth (CC), stainless steel mesh (SS), combination of CC and SS (CC-SS) and a hybrid material (CC-SS-AC) composed of CC, SS and activated carbon (AC) to function as biocathodes during MES. Key functioning abilities of electrode materials as biocathodes in terms of electrotrophy, catalytic currents, electrochemical impedance spectra for resistance, reductive behavior etc. were critically analyzed and discussed. Biochemical and electrochemical parameters were comparatively analyzed for four different biocathodes (varied electrode materials and combinations) to evaluate the optimum biocathode electrode material for higher product formation during MES operation. 2

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

2. Experimental details

quantitatively and qualitatively estimate carboxylic acids and alcohols, which was further characterized based on the standard concentration. Gas production/consumption was monitored by Gas chromatography (GC; NUCON 5765). Reduction in bicarbonate concentration was estimated according to standard methods (APHA, 1998). Reductive catalytic currents generation in response to applied voltage was studied using chronoamperometry (CA) technique. Redox catalytic currents and electron discharge phenomenon was studied by performing cyclic voltammetry (CV) at a scan rate of 10 mV/s. The measurements of working electrode electrochemical impedance spectroscopy (EIS) were registered at the designated experimental conditions and within the frequency range from 1 MHz to 1 mHz with a sinusoidal excitation amplitude signal of 10 mV. Nyquist plots were constructed from the resulting EIS data for all the four biocathodes to comparatively assess the resistance to electron transfer/charge transfer resistance. Drop in carboxylic acids concentration and reductive catalytic currents were used as indicators for feed change. Experiment was carried out in fed batch mode with each cycle operated for a retention time of 72 h.

2.1. MES system design Four double chambered MES were designed and fabricated using Schott-Duran glass bottles with a total/working volume of 2.5/2.0 l (Modestra and Venkata Mohan, 2017). MES consisted of a bioanode and a biocathode chamber separated by a Cation exchange membrane (CEM). Plain graphite plate was used as counter electrode to catalyze bioelectrochemical reactions at anode in MES. While in the case of cathode, four different electrode materials viz., carbon cloth (CC), stainless steel (SS-mesh), combination of CC-SS and hybrid combination of CC-SS along with activated carbon (CC-SS-AC) were used as electrode materials for enabling surface catalyzed reduction reactions and the schematic representation of MES has been presented in Fig. 1. Carbon cloth was commercially procured and was used as working electrode with a surface area of 110 cm2, and similarly SS-mesh was commercially procured and was used as working electrode with a surface area of 110 cm2 in two MES reactors respectively. On the other hand, CC-SS electrode was prepared by intertwining SS mesh over CC manually to be used as working electrode in another MES with a surface area of 210 cm2. The hybrid electrode CC-SS-AC was prepared by taking an activated carbon block commercially procured (4 X 2 X 1 cm dimensions) to be used as inner core material. Further, this was wrapped by CC-SS prepared similarly as described above with a total surface area accounting to 290 cm2. Electrical contacts for all the biocathodes was established using stainless steel wire and proper leak proof sealing was done to ensure anaerobic environment at both the chambers. Provisions were made in the design to have inlet and outlet ports, sampling ports and wire input points. All the MES were operated in fed-batch (up flow) mode under anaerobic microenvironment.

3. Results and discussion 3.1. Bio-electrochemistry 3.1.1. Turnover and non-turnover voltammograms To understand the bioelectrochemical and capacitive nature of biocathodes, turn over and non-turn over cyclic voltammetry analysis was carried out. A scan rate of 10 mv/s was employed on the working electrode with respect to Ag/AgCl (s) as reference electrode and graphite as counter electrode. Fig. 2a demonstrates the non-turn over cyclic voltammograms which showed varied current generation in accordance with the nature of diverse electrode materials. During nonturnover voltammetry analysis, maximum redox catalytic currents were displayed by CC-SS-AC (−1 mA; 1 mA) followed by CC-SS (−0.5 mA; 0.4 mA), CC (−0.2 mA; 0.1 mA) and SS (−0.05 mA; 0.02 mA). This depicts the nature of electrode material in the absence of biocatalyst that can hold the electrical charge and display as currents in the vicinity of applied scan potential and window. The hybrid electrode material (CC-SS-AC) displayed higher capacitive nature followed by CC-SS, CC and SS. In general, capacitance is defined as the ability to hold electrical charge on its surface. In the present study, which applies a potential of −0.8 V on the working electrode is a critical parameter which decides the ability of electrode to hold the charge and efficiently transfer the electrons for bioelectrochemical reduction of CO2 towards the targeted product. Maximum redox currents displayed by CC-SS-AC depicts efficient electron holding capabilities in comparison to other electrode materials used as biocathodes. On the other hand, turn-over cyclic voltammograms also showed a definite pattern of current generation with each of the electrode material studied (Fig. 2b). CC-SS-AC displayed bio-electro redox catalytic currents of −12 mA; 9 mA, while CC-SS produced −11 mA; 11 mA followed by CC (−4 mA; 7 mA) and SS (−4 mA; 3 mA). Variation in turn-over voltammograms illustrates the critical intervention of electrode materials favorable for biofilm formation on the surface of electrodes. Although redox catalytic currents seemed to appear slightly similar for CC-SS-AC and CC-SS, capacitive nature was obviously well displayed by CC-SS-AC. Architecture and structure of electrode material greatly contributes for biofilm formation which in turn impacts the process efficiency (Modestra et al., 2016). Porous nature of carbon cloth and activated carbon presents well amenable environment for biocatalyst to form as a biofilm on its surface. Also, surface area will be greatly enhanced through scaffolding of hybrid material which allows higher electron exchange on the surface that enhances and regulates bioelectrochemical CO2 reduction efficiency (Chen et al., 2017). Besides this, redox mediators appeared to participate in electron transfer in CC-SS-AC and CC biocathodes majorly in comparison to other biocathodes studied.

2.2. Biocatalyst Indigenous anaerobic bacteria obtained from full scale anaerobic reactor treating wastewater was used as the parent inoculum. Bacteria used as biocatalyst is different at anode and cathode chambers. Anode chamber of MES was inoculated with anaerobic and untreated parent inoculum enriched in synthetic wastewater [NH4Cl-0.5 g/l, KH2PO40.25 g/l, K2HPO4-0.25 g/l, MgCl2-0.3 g/l, CoCl2-25 g/l, ZnCl2-11.5 mg/ l, CuCl2-10.5 mg/l, CaCl2-5 mg/l, MnCl2-15 mg/l] containing glucose (1.5 g/l) as carbon source. While in the case of cathode chamber, parent inoculum was subjected to acid pretreatment (adjusted to pH 3 with HCl) to enrich acidogenic bacteria and to eliminate non-spore forming hydrogen consuming methanogenic bacteria (Goud et al., 2017). The resulting culture was further subjected to a headspace gas mixture of H2 and CO2 (80:20) to selectively enrich homoacetogenic bacteria (Modestra et al., 2015). This selectively enriched homoacetogenic bacteria is ultimately used as biocatalyst at cathode chamber. 2.3. Experimental execution Prior to startup, MES systems were inoculated with untreated bacteria at anode and selectively enriched bacteria at cathode. Four MES systems were operated with four different electrode materials viz., CC, SS, CC-SS and CC-SS-AC respectively at cathode with a polarized potential of −0.8 V vs Ag/AgCl (S) chronoamperometrically using a potentiostat-galvanostat system (EC-Lab). Proper mixing of feed with the biocatalyst was ensured by a magnetic stirrer to avoid concentration gradient. 2.4. MES assessment Performance of MES was assessed in terms of carboxylic acids production, alcohols synthesis, bicarbonate reduction and change in pH. High performance liquid chromatography (HPLC) was used to 3

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

Fig. 2. Evaluation of bioelectrochemical catalytic currents through (a) non-turn over and (b) turn over Cyclic Voltammograms recorded at a scan rate of 10 mV/s for all the varied electrode materials used as biocathodes and evaluation of reduction behavior through (c) non-turn over and (d) turn over Linear Sweep Voltammograms for all the biocathodes and potentiostatic control of MES expressed as Chronoamperometric current density profiles of MES operated with (e) CC (f) SS (g) CC-SS (h) CC-SS-AC as capacitive biocathodes.

Electron transfer necessary for bioelectrochemical CO2 reduction might have taken via two ways i.e., direct as well as mediated through electron carriers. A quasi reversible peak appeared on voltammetric signature on all the biocathodes positioning at 0.35 V, which might be ascribed to the presence of cytochrome components. Also, a peak appeared on CC and SS positioning at −0.64 V which might be attributed to the presence of electron carriers responsible for hydrogen production, and a peak was identified at −0.8 V on CC-SS and CC-SS-AC voltammogram corresponding to the occurrence of acetate to acetaldehyde conversion respectively. These mediators are thought to have participated in electron transfer necessary for bioelectrochemical synthesis of products through CO2 reduction. The identified peak ‘acetate to acetaldehyde’ conversion evidents alcohols synthesis in hybrid biocathodes as observed during bioelectrochemical CO2 reduction. An additional peak was observed on CC-SS-AC positioning at −0.4 V which might be ascribed to ‘acetaldehyde to ethanol’ conversion, which is an evident of acetate reduction to ethanol. The obtained results are quite supportive with the products synthesized during biocathode mediated electrochemical CO2 reduction.

voltammograms depicts the prominence of capacitive biocathodes aiding in electron transfer necessary for CO2 reduction towards value added products. 3.1.3. Responsive reductive current generation Reductive catalytic current generation was observed to vary with each of the biocathode operated bioelectrochemically for CO2 reduction towards value added chemicals production. Relatively higher reductive currents (−6 mA) were displayed with hybrid biocathode (CC-SS-AC) followed by CC-SS (−2.3 mA), CC (−2 mA) and SS (−0.45 mA) (Fig. 2 (e–h)). These reduction currents well illustrate the microbial electrosynthesis reactions upon CO2 reduction (Saheb-Alam et al., 2018). Also, electrotrophy nature can be clearly depicted through CA analysis which defines the electron flow (current generation) over a time period. CCSS-AC displayed a continuous increment in reduction catalytic currents operated for an extended time period. This is in congruence with higher product titre (acetic acid, butyric acids, ethanol, etc.) synthesized upon bioelectrochemical reduction. The porous and well amenable nature for biofilm adherence on this electrode material aided higher current generation necessary for microbial electrosynthesis (Liu et al., 2018). Since the biocatalyst used in each of the biocathode is same, any variation in MES performance is attributed to the nature of electrode material and its biocompatibility to catalyze reduction reactions (Guo et al., 2015). MES operated with CCSS-AC as biocathode depicted a continuous increment in reduction catalytic currents (−3 to −8 mA) with each additional cycle of operation for longer time intervals. While in the case of CC-SS, initially currents were −4 mA which were stable over a time period and slightly decreased with time (−3 mA), which again got stabilized (−2 mA) till the end of operation. The biocathode CC also showed a decrement (−2.5 mA) from initial value (−3 mA) and got stabilized (−2 mA) throughout the operation. On the contrary, although SS biocathode depicted low reduction currents (−0.4 mA), an increased pattern of currents were observed until an extended period (−0.45 mA) and got decreased by the end of operation (−0.35 mA). The displayed current

3.1.2. Reductive nature evaluation In order to further analyze the reductive nature of biocathodes which is an imperative feature for bioelectrochemical synthesis, linear sweep voltammetric analysis (LSV) was carried out in a scan window of −1.2 V to 1.0 V at a scan rate of 10 mV/s. Turn over as well as non-turn over LSV sweeps were performed for all the biocathodes which depicted a definite pattern of reductive capacity (Fig. 2 (c&d)). During non-turn over conditions, hybrid biocathode (CC-SS-AC) depicted maximum catalytic currents (−1.1 mA; 1 mA) in comparison to CC-SS (−0.4 mA; 0.2 mA), CC (−0.3 mA; 0.6 mA) and SS (−0.1 mA; 0.1 mA). While in the case of turn over LSV sweeps, redox catalytic currents were relatively higher than non-turn over conditions. CC-SS-AC displayed higher reduction current of −45 mA followed by CC-SS (−20 mA), CC (−18 mA) and SS (−10 mA). Extensively higher redox catalytic currents generation as observed during biocatalyst mediated turn over 4

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

Fig. 2. (continued)

generation pattern is in synchrony with bioelectrochemical reduction of CO2 for products synthesized accordingly for each of the biocathode studied. On the other hand, charge profiles were drawn along with reduction currents during amperometric analysis for each of the biocathode. Charge is usually defined as the number of electrons or the electric property of the material. Charge expressed in milli coulombs is found to be higher with CC-SS-AC (−380 mC) followed by CC-SS (−330 mC), CC (−220 mC) and SS (−140 mC). This variation observed might be due to the difference in biocatalyst capabilities on respective electrode materials used as biocathodes. Electrotrophy is a key phenomenon during microbial electrosynthesis as it enables surface catalyzed bioelectrochemical CO2 reduction through the enriched electrotrophic bacteria as biofilm on the electrode. Charge of a system can be well related with the capacitance in adjunction with the applied voltage. The correlation between MES system parameters such as charge and applied voltage to deduce capacitance can be obtained from Eq. (1) in which Q is charge in milli coulombs (mC), C is the capacitance in milli Farads (mF) and V is the applied voltage in volts (V).

Q= CV

‘energy stored’ is also derived which will deliver information on the potential energy stored within the system in correlation with the applied voltage and derived charge. Energy stored is represented by Eq. (2), where W is the energy stored in mJ, Q is the charge in mC and V is the voltage in mV.

W=

1 QV 2

(2)

W is found to be significantly higher (152 mJ) with hybrid electrode (CC-SS-AC) as biocathode followed by CC-SS (132 mJ) and relatively low with CC (88 mJ) and SS (56 mJ). Similarly, the number of electrons (n) that were stored in MES during the bioelectrochemical synthesis as derived through CA analysis was found to be extensively higher with CC-SS-AC (−2.4E+21) followed by CC-SS (−2.1E+21) and comparatively lower with CC (−1.4E+21) and SS (−8.8E+20). This has been deduced through the charge in coulombs Q(C) equivalent to the charge in electron charge Q(e) multiplied by 1.60217646*10−19 represented as Eq. (3).

n= Q/1.6 E

(1)

19

(3)

Although the actual number of electrons that are stochiometrically required for acetate synthesis from CO2 is 8, the obtained values depict the total accumulated electrons that are stored and utilized for product synthesis.

CC-SS-AC (475 mF) resulted in higher capacitance followed by CCSS (412.5 mF) and relatively lower capacitance was noticed with CC (275 mF) and SS (175 mF). This capacitive nature of various biocathodes is attributed to several factors such as the property of electrode material which lists the specific features of biocompatibility, porous nature, amenable for biofilm formation etc. along with the applied voltage and operating conditions. Since the applied voltage and operational parameters lies same for MES systems, any variation in the responsive currents as well as product yield is linked to the varied electrode material that acts as the backbone for biofilm formation. Electrotrophic bacteria act as key players for enhanced microbial electrosynthesis wherein the capacitive nature of biocathode well supports their adaptability and existence for longer duration that enhances the overall performance. Bio-electrochemical analysis in particular chrono amperometry and voltammetry are in congruence with the capacitive nature of biocathodes that in turn alleviated the MES performance in terms of product formation. In addition to capacitance which signifies the electron holding or electron storage capacity of MES in the current study, the parameter

3.1.4. Resistance evaluation-Nyquist plots In order to assess the electron transfer efficiency and relative resistivity of each of the electrode material used as biocathode, potentioelectrochemical impedance spectroscopy (PEIS) was carried out within the frequency range of 1 MHz to 1 mHz at an amplitude of 10 mV. PEIS was carried out chronologically during MES operation to determine the impact of biofilm on decrease in resistance towards increased electron transfer (Fig. 3). A blank PEIS was initially carried out prior to biocatalyst inoculation to evaluate the potential of electrode materials to be used as biocathodes. Hybrid electrode material (CC-SS-AC) depicted relatively less resistance for electron transfer followed by CC-SS, plain carbon cloth (CC) and SS. The resistivity range is comparatively lower for hybrid electrode materials in comparison to plain electrode materials which depict the innate property of material to act as a source of electron conduction either as an acceptor or as a donor. 5

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

(a)

(b)

800

400

-Z" (Ohm)

500

-Z" (Ohm)

1000

600

400

300

CC

CC-SS-AC

CC-SS

SS

200

CC SS

200

100

CC-SS CC-SS-AC

0

0 0

(c)

2

4

6

8

10

12

14

16

0

18

Z' (Ohm)

5

10

Z' (Ohm)

15

20

(d)

(e)

(f)

Fig. 3. Nyquist impedance spectral plots recorded for (a) uninoculated and (b) inoculated MES operated with all the varied electrode materials used as working electrode and (c-f) Impedance spectral profiles for long term operated MES.

MES operation with biocathodes. Generally impedance spectra consists of two major elements such as Ohmic and charge transfer resistances often described in Nyquist plots. The substantial decrease in resistance as a result of biocathodic operation is an indicative of augmented electron transfer efficiency which might be ascribed to accelerated interaction of biofilm on electrode surface. Although biocathodic operation resulted in distorted semicircular plots, an indication of solution/MES electrolyte resistance and system resistances were noticed. The primary semicircular plot appeared to be less for hybrid materials, and the plot continued to be more like a sweeping parabola. This is attributed to the presence of Ohmic resistance of electrode material and the minimal charge transfer resistance (Yellappa et al., 2019). On the contrary, SS displayed two semicircular plots that display information on the existence of both Ohmic as well as charge transfer resistance. Occurrence of charge transfer resistance on SS might be due to poor biofilm formation in comparison to respective electrode materials studied. To validate the stability and efficiency of biocathodes in the long

After inoculating the biocatalyst and operating MES for more than 20 cycles until stabilized performance, PEIS was again carried out. Interesting observations were noticed post inoculation which displayed relatively less resistance in comparison to blank PEIS with all the electrode materials studied. This elucidates the impact of biofilm in contributing towards efficient electron transfer. In further, hybrid electrode materials (CC-SS-AC and CC-SS) showed less resistance followed by plain electrode materials (CC and SS). This is in congruence with the capacitive nature of hybrid electrode materials as biocathodes in comparison to plain electrodes. The intertwined structure of hybrid materials might be more favorable for biofilm formation as well as increased surface area for rapid electron transfer rather than the plain material, thus favouring electrotrophy (Guo et al., 2015). 3.1.4.1. Long term operation. The pattern of Nyquist impedance spectra were significantly different during blank and biocathodic operation which delivers information on less resistance for electron transfer as well as the adaptability of biofilm on electrode material for efficient 6

Bioresource Technology 294 (2019) 122181

J. Annie Modestra and S. Venkata Mohan

term, PEIS was carried out after operating MES for six months. Less resistance is observed in long term operated biocathodes in comparison to blank as well as post inoculated MES as appeared from Nyquist plots. Interesting results appeared on Nyquist plots which showed a circular pattern with each of the biocathode studied. Similar pattern of Nyquist plots appeared in a study earlier by Yang et al., which assessed long term operated graphene and graphite electrodes in MFC (Yang et al., 2016). Appearance of circular loop demonstrates the absence of Ohmic resistance which generally implies to electrode and the electrolyte in its vicinity. A very low frequency loop in fourth quadrant was displayed in impedance spectra for all the biocathodes operated, a feature described as pseudo inductive behaviour (Yang et al., 2016). This was suggested as surface relaxation phenomena of adsorbed intermediates such as CO from platinum materials. In the present study, we speculate the electron acceptance on the surface of biocathodes by redox mediators towards CO2 bioelectrochemical reduction or adsorption of dissociated components from CO2/electrolyte particulates resulted from feed. Although scanty literature is available on such pattern of impedance spectra, it can be hypothesized that biofilm formation was well supported by the electrode materials (hybrid materials richer than plain materials) as appeared through significantly decreased resistance range in impedance spectra. In addition, circular loop also suggested a speculation about adsorption of intermediates from CO2 dissociation as well as electrotrophy. Negligible charge transfer resistance and Ohmic resistances were found during impedance spectra with long term operated biocathodes.

nature as well as enlarged surface area might have supported the development of biofilm that majorly constitute chemolitho-electrotrophic bacteria among mixed bacterial community (Zhang et al., 2013). These electrotrophic bacteria on biocathodes might have aided in yielding higher surface bioelectrochemical reduction of CO2 with hybrid biocathodes than the plain electrode based cathodes. Several studies were carried out using different electrode materials such as carbon felt [9.8 g/l/d] (Jourdin et al., 2018), carbon felt [7.8 g/l] (Song et al., 2019), composite carbon felt [149 mg/l/d] (Bajracharya et al., 2013), carbon cloth [56 mM/m2/d], CNT’s modified on carbon cloth [122 mM/m2/d], graphene nano sheets [GN: 213 mM/m2/d], modified GN with CNTs [278 mM/m2/d] (Han et al., 2019), activated carbon based VITO CoRE [142.2 mg/l/d] (Mohanakrishna et al., 2013), graphite [2.1 g/l, 3.01 g/l] (Modestra et al., 2015, 2017), multi walled carbon nano tubes [1330 g/m2/d] (Jourdin et al., 2016), graphite granules [1.04 g/l/d] (Marshall et al. 2013) etc. and attained considerably good acetate production rates. However, this again depends on various factors such as initial concentration of substrate, the applied voltage, surface area, biocatalyst (pure/mixed) etc. The hybrid combination of CC-SS-AC has not been evaluated in previous studies, and relatively good acetic acid production has been attained in batch mode MES operation. Despite the addition of AC to CC-SS-AC, the plain hybrid electrode i.e CC-SS displayed marginally similar acetic acid production rates in the present study. This might be ascribed to the fact that AC has been functioned as a supporting substratum for efficient biofilm formation thereby promoting electrotrophy. On the contrary, if AC would have been deposited on the outer surface of CC-SS, the results might have been varied which has already been studied in several studies that employed surface modifications with granular activated carbon (GAC), MWCNTs, graphene nano sheets (GNs) etc. However, the observation that was noted in the present study is the imperative role of AC as favourable substratum for CC-SS hybrid material contributing towards capacitance thereby enhancing microbial electrosynthesis.

3.2. Biobased products from CO2 reduction 3.2.1. Acetic acid Acid treated and selectively enriched bacterial culture was inoculated in to four MES reactors operated with varied electrode materials as biocathodes. MES operation was carried out by applying a potential of −0.8 V vs Ag/AgCl (S) on biocathode considering it as working electrode against anode as counter electrode and Ag/AgCl (S) as reference electrode through a potentiostat-galvanostat system. Bioelectrochemical CO2 reduction towards acetic acid production was quantified using HPLC which depicted variation in production with respect to each biocathode studied. CC-SS-AC yielded higher product titer (4.31 g/l) followed by CC-SS (4.21 g/l), CC (3.5 g/l) and SS (2.83 g/l). Fig. 4a & b represents acetic acid production for a single batch cycle and the production rates of best five cycles is expressed among 20 cycles operated. It is observed from a batch cycle data plotted with respect to cycle length that the production of acetic acid appeared to increase until 60 h of operation followed by a slight decrement thereafter. This decrease might be ascribed to the utilization of acetic acid towards synthesis of other value added products such as ethanol or other carboxylic acids (discussed in sections below). This variation in yield with different electrode materials might be attributed to the enrichment of bacterial biofilm that will have characteristic feature of electrotrophy to enable surface catalyzed bio-electrochemical reactions. Although study depicted relatively higher yield of acetic acid with hybrid biocathode (CC-SS-AC), the other hybrid material without AC i.e CC-SS also displayed marginally similar yield, and hence statistical significance of the results obtained for acetic acid production were analyzed through T-test performed using Minitab® software (version 19.0) for both the biocathodes which exhibited marginal significance. However, in terms of catalytic current generation and electrotrophy properties which are responsible for enhancing bio-electrocatalytic CO2 reduction, CC-SS-AC exhibited higher significance in comparison to other biocathodes studied. Comparatively, lower production observed with SS might be attributed to the electrolysis at electrode surface that is in support with the rise in pH (represented in below sections) that generates hydroxide ions (Guo et al., 2014, Hou et al., 2014). The biocompatible property of hybrid materials such as being porous, intertwined skeleton, coarse

3.2.2. Total carboxylic acids Besides the production of acetic acid in major fraction, MES also depicted the bio-electrochemical synthesis of a mixture of carboxylic acids. Higher carboxylic acids production is observed in hybrid electrode combination (CC-SS-AC; 4.7 g/l) followed by CC-SS (4.5 g/l), CC (3.72 g/l) and SS (3.36 g/l) (Fig. 4c&4d). Carboxylic acids were bioelectrochemically synthesized by utilizing bicarbonate as sole carbon source along with an applied potential as driving force for directing the reducing equivalents. Among the mixture of carboxylic acids synthesized, acetic acid (C2) concentration was high followed by butyric acid (C4) (Fig. 4e–g). Synthesis of higher chain carbon compounds is a value addition and is of high economical value offering multiple benefits besides CO2 reduction (Jourdin et al., 2019). A gradual increment in concentration of carboxylic acids was noticed till 60 h followed by a slight decrement at 72 h. Hybrid material displayed higher titre of carboxylic acids which might be due to the favourable positioning sites for bacteria to form as biofilm that well exhibits electrotrophy (Jourdin et al., 2014). The decrement in carboxylic acids concentration might be due to the reduction of C2-C4 compounds towards alcohols synthesis. 3.2.2.1. Alcohols. Besides the production of carboxylic acids, ethanol was also detected in small quantities in MES operated with hybrid electrode combination [CC-SS-AC (0.29 g/l), CC-SS (0.26 g/l)] and CC (0.2 g/l) along with acetic and butyric acids. It is speculated that ethanol production might have been initiated due to the reduction of synthesized carboxylic acids (Srikanth et al., 2018). This is also in agreement with the observed pH profiles, where alcohol synthesis was initiated due to the accumulation of carboxylic acids. Generally ethanol synthesis was reported to take place upon reduction of acetic acid with lower pH as favourable conditions (Sarkar et al., 2017). It is also to be noted in the present study that biocathodes based on CC alone or as a component in hybrid material combination displayed ethanol synthesis. 7

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(a)

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Fig. 4. Acetic acid production expressed for (a) a batch cycle with a HRT of 72 h (b) five consecutive cycles and total carboxylic acids production expressed for (c) a batch cycle and for (d) five consecutive cycles along with compositional variation of renewable chemicals expressed as (e) other carboxylic acids (f) butyric acid (g) ethanol in MES operated with varied biocathodes and (h) acetic acid production for 20 cycles.

This particular feature observed with CC based biocathodes might be ascribed to the development of similar sort of biofilm community that would constitute both carboxylic acid and alcohol producers. Consolidated electrochemical and biochemical performance of MES operated with four biocathodes is represented in Table 1.

3.3. Electron recovery and energetic efficiency Applied potential or current acts as the driving force for MES in directing the bioelectrochemical reduction of CO2 towards product formation. The electrical energy that has been supplemented accounts for input costs, and if the overall electron recovery efficiency is high, the process is said to be economically favourable and efficient taking 8

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Table 1 Comparative performance of four biocathodes operated with varied electrode materials in MES.

a

Biocathode

Capacitance (mF)

Maximum reduction current (mA)

Maximum drop in pH

Acetic acid production (g/l)

Total carboxylic acids (g/l)

Other carboxylic acids (g/l)

Ethanol (g/l)

Bicarbonate reduction (%)

CC SS CC-SS CC-SS-AC

275 ± 4.32 175 ± 5.78 412.5 ± 6.32 475 ± 5.87

−2.0 ± 0.45 −0.450.02 −2.3 ± 0.99 −7 ± 1.13

5.7 ± 0.39 7 ± 0.12 5.4 ± 0.27 5.21 ± 0.18

3.5 ± 0.25 2.83 ± 0.19 4.21 ± 0.23 4.31 ± 0.20

3.72 ± 0.07 3.36 ± 0.18 4.5 ± 0.23 4.7 ± 0.15

0.8 ± 0.10 0.71 ± 0.17 0.45 ± 0.13 0.49 ± 0.17

0.2 ± 0.08 ND 0.26 ± 0.10 0.29 ± 0.09

66 59 72 75

± ± ± ±

5.76 3.96 4.43 4.45

Results are expressed here as ± standard deviation.

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Fig. 5. Reduction in substrate concentration expressed as bicarbonate removal for (a) batch cycle operation and (b) five consecutive cycles and variation in redox behavior expressed as pH for (c) a batch cycle operation and (d) five consecutive cycles of MES operated with varied biocathodes.

the process parameters into consideration. Since MES involves biocathodic operation dominantly, the CE is equal to the percentage of electrons recovered in the product i.e acetic acid in the present study (Patil et al., 2015b). This CE is often termed as electron recovery efficiency which determines the process efficiency considering the bioelectrochemical CO2 reduction with simultaneous product formation. CE has been calculated for all the four biocathodes as per Patil et al., 2015b and was found to be higher for CC-SS-AC (47%) followed by CC-SS (44%), CC (40%) and SS (37%). This CE has been obtained considering only acetate as the product, and the remaining electrons might have been recovered/utilized for ethanol and butyric acid production respectively as determined besides being utilized for biomass growth. SS as biocathode depicted comparatively good CE than the product yield observed, which might be ascribed to the fact that less butyric acid and no ethanol formations were noticed. Similarly, energetic efficiency (EE) has been calculated for all the biocathodes considering only the cathodic process. Higher EE was depicted by CC-SS-AC (6.3%) followed by CC-SS (5.6%), CC (4.3%) and SS (3.2%) which is in accordance with the carboxylic acids profiles. The observed efficiency values are in correlation with the product profiles obtained for each of the

biocathode studied which illustrates the efficacy as well as electrotrophy responsible for reducing CO2 bioelectrochemically. 3.4. CO2/Bicarbonate reduction MES electrolyte (cathodic) samples were collected at regular time intervals for each cycle to monitor the changes in carbon concentration. Ideally, one mole of bicarbonate is equivalent to one mole of CO2, and the decrease in bicarbonate concentration equivalent to CO2 has been expressed in percentage reduction. Considering the molar mass (80.0066 g/mol), one gram of sodium bicarbonate is equal to 0.0125 mol. This can be again represented in terms of CO2, by considering the molar mass of CO2 (44.01 g/mol), which is equivalent to 0.0125 mol of sodium bicarbonate. While depicting the concentration of CO2 in grams, this can be expressed by considering molar mass of CO2 multiplied by 0.0125 mol of sodium carbonate. Hence, 1 g of sodium bicarbonate can yield 0.550 g of CO2. The liquid samples were periodically collected from MES and have been analyzed for reduction in bicarbonate concentration as per the standard methods (APHA, 1998). The carbon source (bicarbonate/CO2) was observed to decrease 9

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with the increase in time for each cycle operation (Fig. 5a & 5b). Maximum carbon reduction was depicted in MES operated with hybrid combination CC-SS-AC (75%) followed by CC-SS (72%), CC (66%) and SS (59%). This depicts the utilization of inorganic carbon substrate by the enriched chemolithoautotrophic bacteria. The carbon reduction profiles of various biocathodes are in congruence with carboxylic acids production, which yielded the products in accordance with carbon utilization. Carbon reduction appeared to be considerably good with all the biocathode materials studied except SS, which in turn is reflected in the production of carboxylic acids. Prevalence of alkaline range of pH might also have favoured dissolution of bicarbonate in catholyte that aided in the bioelectrochemical CO2 reduction by the biocatalyst towards products synthesis.

C (1998). Arends, J.B.A., Patil, S.A., Roume, H., Rabaey, K., 2017. Continuous longterm electricitydriven bioproduction of carboxylates and isopropanol from CO2 with a mixed microbial community. J. CO2 Util. 20, 141–149. Arunasri, K., Annie Modestra, J., Dileep Kumar, Y., Vamshi Krishna, K., Venkata Mohan, S., 2016. Polarized potential and electrode materials implication on electro-fermentative di-hydrogen production: Microbial assemblages and hydrogenase gene copy variation. Bioresour. Technol. 200, 691–698. Bajracharya, S., Vanbroekhoven, K., Buisman, C., Strik, D., Deepak, P., 2017. Bioelectrochemical conversion of CO2 to chemicals: CO2 as next generation feedstock for the electricity-driven bioproduction in batch and continuous mode. Faraday Discuss. 202, 433–449. Bajracharya, S., Vanbroekhoven, K., Buisman, C., Strik, D., Pant, D., 2013. Bioelectrochemical Conversion of CO2 to Chemicals: CO2 as Next Generation Feedstock for the Electricity-driven Bioproduction in Batch and Continuous mode. Faraday Discussions 2013 (00), 1–3. Chen, W., Wu, D., Wan, H., Tang, R., Li, C., Wang, G., Feng, C., 2017. Carbon-based cathode as an electron donor driving direct bioelectrochemical denitrification in biofilm-electrode reactors: Role of oxygen functional groups. Carbon. 118, 310–318. Franca, C.R.M., Azapagic, A., 2015. Carbon capture, storage and utilization technologies: a critical analysis and comparison of their life cycle environmental impacts. J. CO2 Util. 9, 82–102. Ganigue, R., Puig, S., Batlle-Vilanova, P., Balaguer, M.D., Colprim, J., 2015. Microbial electrosynthesis of butyrate from carbon dioxide. Chem. Comm. 51, 3235–3238. Goud, R.K., Arunasri, K., Yeruva, D.K., Vamshi Krishna, K., Dahiya, S., Venkata Mohan, S., 2017. Impact of selectively enriched microbial communities on long-term fermentative biohydrogen production. 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Critical biofilm growth throughout unmodified carbon felts allows continuous bioelectrochemical chain elongation from CO2 up to caproate at high current density. J. Mater Chem. A. 2, 13093–13102. Jourdin, L., Freguia, S., Flexer, V., Keller, J., 2016. Bringing high-rate, CO2-based microbial electrosynthesis closer to practical implementation through improved electrode design and operating conditions. Environ. Sci. Technol. 50, 1982–1989. Jourdin, L., Grieger, T., Monetti, J., Flexer, V., Freguia, S., Lu, Y., et al., 2015. High acetic acid production rate obtained by microbial electrosynthesis from carbon dioxide. Environ. Sci. Technol. 49, 13566–13574. Jourdin, L., Raes, S.M.T., Buisman, C.J.N., Strik, D.P.B.T.B., 2018. Critical biofilm growth throughout unmodified carbon felts allows continuous bioelectrochemical chain elongation from CO2 up to caproate at high current density. Front. Energy Res. 6, 7. Jourdin, L., Winkelhorst, M., Rawls, B., Buisman, C.J.N., Strik, P.B.T.B., 2019. 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3.5. Redox change-pH During microbial electrosynthesis, variation in pH is noticed throughout the cycle with respect to carboxylic acids and alcohols production. Initially, all MES systems were operated at a set pH of 8. A drop in pH is noticed from 6 h in all the MES operated with various electrode materials (CC-SS-AC: 7.2; CC-SS: 7.1; CC: 7.1; SS: 7.4) (Fig. 5c & d). Maximum drop in pH was noticed in hybrid combination (CC-SSAC; 5.21) followed by CC-SS (5.4), CC (5.7) and SS (7), which is in congruence with the products synthesized. In the current study, maximum accumulation of carboxylic acids lead to a higher drop in pH in hybrid biocathodes in comparison to other biocathodes studied. This is in accordance with the ethanol production observed in hybrid combination at lower pH conditions. It was speculated that acetic acid reduction might have been favoured at lower pH towards synthesis of ethanol. Also, higher proton availability at lower pH and the incessant availability of electron flux through regulated electro-potential conditions drifted the pathway of acidogenesis towards solventogenesis. Thereafter, a rise in pH is observed in all the MES, which might be ascribed to the utilization of carboxylic acids towards higher carbon chain compounds or as carbon source by other bacterial population. It is also to be noted that MES operated with SS as biocathode depicted less drop in pH which might be due to the formation of hydroxyl ions upon bicarbonate dissociation that causes marginal/no drop in pH, that is in support with bicarbonate reduction profiles. 4. Conclusion Current study demonstrated the efficiency of hybrid electrodes (CCSS-AC and CC-SS) as potential biocathodes with capacitive nature that would support electrotrophy towards enhanced product synthesis. Bioelectrochemical reduction of CO2 resulted in various renewable chemicals viz., carboxylic acids and alcohols. The importance of electrode materials that enable electrotrophic biofilm formation towards augmented and regulated electron transfer for product synthesis was identified. Study unraveled the potential of MES in not only synthesizing multi-carbon biobased products, but also efficient electrotrophy deciphering the role of electrode materials as capacitive biocathodes. Acknowledgements The research work is supported by Department of Biotechnology (DBT), Government of India in the form of project (No. BT/PR20759/ BCE/8/1218/2016). J.A.M wishes to acknowledge CSIR for providing research fellowship. The authors would also like to thank the Director, CSIR-Indian Institute of Chemical Technology (IICT manuscript No. IICT/Pubs./2019/255). References APHA, Standard methods for the examination of water and wastewater. 20th Ed, American Water Works Association & Water Environment. Federation, Washington D.

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