Regulated surface potential impacts bioelectrogenic activity, interfacial electron transfer and microbial dynamics in microbial fuel cell

Journal Pre-proof Regulated surface potential impacts bioelectrogenic activity, interfacial electron transfer and microbial dynamics in microbial fuel cell J. Annie Modestra, C. Nagendranatha Reddy, K. Vamshi Krishna, Booki Min, S. Venkata Mohan PII:

S0960-1481(19)31890-7

DOI:

https://doi.org/10.1016/j.renene.2019.12.018

Reference:

RENE 12727

To appear in:

Renewable Energy

Received Date: 25 May 2019 Revised Date:

2 December 2019

Accepted Date: 3 December 2019

Please cite this article as: Modestra JA, Reddy CN, Krishna KV, Min B, Mohan SV, Regulated surface potential impacts bioelectrogenic activity, interfacial electron transfer and microbial dynamics in microbial fuel cell, Renewable Energy (2020), doi: https://doi.org/10.1016/j.renene.2019.12.018. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical Abstract

Synoptic view of electron transfer from bacteria towards electrode and depiction of decreased activation energy (AE) at post potential (PP) phase with positive and negative poised potentials (+100 mV/-100 mV)

1 2 3 4 5 6 7 8 9 10 11 12

Regulated Surface Potential Impacts Bioelectrogenic Activity, Interfacial Electron Transfer and Microbial Dynamics in Microbial Fuel Cell J. Annie Modestra1,2, C. Nagendranatha Reddy1,3, K. Vamshi Krishna1,2, Booki Min3, S. Venkata Mohan*1,2,3 1 Bioengineering and Environmental Sciences Lab, Department of Energy and Environmental Engineering, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, 500 007, India. 2 Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT) campus, Hyderabad, 500 007, India. 3 Department of Environmental Science and Engineering, Kyung Hee University, Seocheon-dong, Yongin-si, Gyeonggi-do 446-701, Republic of Korea.

13 14

*E-mail: [email protected]; [email protected], Tel/Fax: 040-27191765.

15

Abstract

16

Impact of surface anode potential on the performance of microbial fuel cell (MFC) has been

17

evaluated by opting positive and negative poised anode potentials (+100/-100 mV) on two MFCs

18

along with a third MFC operated as control (no applied potential). Variation in physico-chemical

19

factors as well as biocatalytic metabolic efficiency has been observed in terms electron transfer,

20

power density, electro-kinetics and microbiome community. Post potential operation at -100 mV

21

depicted rapid electron transfer, higher redox catalytic currents (-0.44/0.42 mA) and voltage

22

(653±28 mV) in comparison to respective experimental conditions. Disparity in electron carriers

23

is noticed at both the phases with +100 mV (dominantly direct electron transfer)/-100 mV

24

(cytochrome components) potential as well as control (non-specific and multiple carriers) which

25

signify alteration in electron transfer mechanism aligned with respect to change in surface

26

potential. Divergent microbiome community is evidenced, which depicted bacteria belonging to

27

Proteobacteria dominant at -100 mV, while Firmicutes at +100 mV and a mixed bacterial

28

population at control. Electrochemical investigations correlated with biological efficiency of

29

MFC which discerns a way to comprehend the underlying electron transfer process triggered in

30

response to anode potential.

31

Keywords: Microbial electrochemical system; Electrochemically active bacteria; Electro-

32

kinetics; Activation energy; Electron losses; Bioelectrochemical System.

33

1

1

1 Introduction

2

Microbial catalyzed electrochemical systems are being emerged as sustainable technologies that

3

employ microbes as biocatalyst to transform chemical energy stored in organic substrates into

4

electrical energy in an electrode catalyzed environment [1-3]. The total cell potential/voltage

5

contributes to the overall efficiency of a microbial fuel cell (MFC), taking the individual half-cell

6

potentials into account [4]. Surface bio-anodic potential determines the theoretical energy gain

7

for bacteria apart from the metabolic pathway that it undergoes [5, 6]. It is an important factor

8

regulating the electron liberating capabilities of biocatalyst by decreasing the activation energy in

9

association with substrate utilization [7]. The electron transport chain (ETC) of bacteria is

10

dictated by the surface redox potential that enables certain components of ETC to participate in

11

electron transfer based on the potential between electron donor and acceptor [8]. Aelterman et

12

al., 2008 mentioned that bacteria adapts to an electron transferring system to a level just

13

below/near to the existing surface anode potential [9]. It was suggested that low surface potential

14

transfers less energy for the growth and maintenance of bacteria, while a high surface potential

15

enables rapid energy transfer for faster startup and enhanced bacterial growth [10]. However, to

16

attain a steady performance, surface anode redox potential should be maintained optimum where

17

it facilitates the growth and enrichment of stable microbial community to participate in electron

18

transfer for adequate energy gain as well as increased power production [11]. Optimal surface

19

potential depends on various parameters which includes the source of biocatalyst, bacteria

20

enrichment strategies [12], operating microenvironment, pH, electrode materials, reactor

21

configuration etc. [13].

22

As surface potential finds great importance in determining the key performance of MFC, this

23

topic has garnered the attention of researchers and has been studied widely. Several strategies 2

1

can be employed to regulate surface anode potential such as applied potential, applied resistance,

2

disparity in electrode materials as anode and cathode etc. which will have an impact further on

3

bioelectricity generation and microbial community (biofilm). Wagner et al., 2010 [14], Wang et

4

al., 2011 [15] and Nikhil et al., 2018 [16] evaluated the impact of various set anode potentials

5

and external resistance on the performance of MFC. The biofilm matrix is complex and consists

6

of several bacterial species that promote extra-cellular electron transfer (EET) among various

7

redox-active proteins towards electrode [17] generally analyzed through voltammetric techniques

8

[18]. Torres et al., carried out a study on impact of anode potentials on enrichment of anode

9

respiring bacteria (ARB) and determined that acclimation ARB is a key step for promoting EET

10

towards electrogenesis and energy gain [19]. Several studies were performed on using varied set

11

anode potentials on different aspects of a MFC such as biofilm and suspended microbiome [20],

12

microbiome development [21] and composition [22] along with model electrogenic bacteria such

13

as Shewanella sp. [23] and Geobacter sp. [24, 25]. Dennis et al., 2016 carried out a study on

14

evaluating the influence of anode potential on structure and function of microbiomes associated

15

on the electrode surface [26]. Similarly, Yao et al., 2019 carried out a study on using different

16

anode potentials for COD and total nitrogen removal [27].

17

The present study differs from previously carried research in opting lower anode potentials of

18

positive and negative voltage of 100 mV to evaluate the impact on several key functions of

19

MFC. An additional specific feature of this study lies in evaluating at both the phases i.e., during

20

potential (DP) and post potential (PP) which has not been studied earlier. Poising external

21

potential on anode will not only have a critical impact on power production, but also on several

22

parameters of MFC that determines its efficiency. Studies carried earlier majorly focused on

23

either microbiome community or bioelectrogenesis or single strain behavior. However, this study 3

1

will focus on several aspects of MFC such as electron transfer, energy gain, microbiome

2

community, electro-kinetics etc. Keeping in view about various changes associated with respect

3

to surface anode potential, present study is designed to evaluate the impact of positive and

4

negative poised potential (+100/-100 mV) on power generation, change in electron transfer

5

pathways, microbial dynamics and bacterial energy gain at two phases viz., DP and PP. Besides,

6

various effects on poising positive and negative anode potentials were studied and reported

7

which delivered information on disparity in bacterial community structure, electron losses, redox

8

currents, electron carriers, activation energy etc. at PP and DP phases. Several techniques and

9

analysis were employed to elucidate the clear differences with positive and negative anodic

10

external potential on the aforementioned aspects.

11

2 Experimental Details

12

2.1 Biocatalyst

13

Anaerobic consortia obtained from wastewater treatment plant was used as parent culture for

14

MFC operation. Parent culture was washed thrice in saline buffer (5000 rpm, 30°C) and enriched

15

in designed synthetic wastewater (DSW) [Dextrose-3 g/L; NH4Cl-0.5 g/L, KH2PO4-0.25 g/L,

16

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

17

mg/L, MnCl2-15 mg/L] under anaerobic microenvironment to be used for experimental

18

operation.

19

2.2 Microbial Fuel Cell

20

Three single chambered MFCs were designed and fabricated in the laboratory using ‘Perspex’

21

material with a total/working volume of 0.50/0.45 l. Graphite plates (5cmX5cm; 10 mm thick;

22

surface area 70 cm2) were used as electrodes. NAFION-117 obtained from Sigma-Aldrich was 4

1

used as proton exchange membrane (PEM) which was sequentially pretreated in boiling

2

deionized water (80-90oC) followed by 30% H2O2, deionized water (80-90oC), 0.5 M H2SO4 and

3

deionized water (80-90oC) with each step being carried for a period of 1 h to increase porosity

4

and to wash off any impurities if present. Pretreated PEM was sandwiched between the

5

electrodes. Top portion of the cathode was air exposed while bottom portion was suspended in

6

wastewater fixed to PEM. Anode was completely submerged in wastewater while top portion

7

was fixed to PEM-cathode assembly over the liquid layer. Copper wires were used to provide

8

connection after sealing with an epoxy sealant. Provisions were made in the design to have inlet

9

and outlet ports, sampling ports and wire input points. MFCs were operated in fed-batch (up

10

flow) mode under anaerobic microenvironment.

11

2.3 Experimental execution

12

Prior to startup, all the three MFCs were inoculated with enriched anaerobic mixed bacteria

13

containing glucose as carbon source at an organic loading rate of 3 g/l. DSW was fed through

14

inlet provided at the bottom of MFC to facilitate the flow in upward direction passing through

15

anode towards cathode. Proper circulation of feed with biocatalyst was ensured by using a

16

peristaltic pump to maintain uniform distribution of carbon source and to avoid concentration

17

gradient in MFC. Before every feeding event, the inoculum was allowed to settle down (30 min;

18

settling) and the exhausted feed was decanted (15 min; decanting) under anaerobic conditions.

19

The inoculum settled at the bottom was used for subsequent operations. After every feeding

20

event, reactor was sparged with oxygen free N2 gas for 2 min to maintain anaerobic

21

microenvironment. Biocatalyst was initially allowed to acclimatize in MFC for few cycles with

22

each cycle comprising a hydraulic retention time (HRT) of 48h and was operated at room

23

temperature (29 ± 2oC). Constant voltage outputs and substrate (COD) removal efficiency were 5

1

considered as indicators to assess the stabilized performance of MFC. Study was performed with

2

a set surface anode potential of 100 mV (positive and negative) vs Ag/AgCl (S) by using a DC-

3

potentiostat which was evaluated at both ‘during potential (DP) application’ and ‘post potential

4

(PP) application’ phases. The DP phase is followed by PP phase in which the MFCs were

5

operated and monitored after discontinuing the potential application and the performance was

6

comparatively assessed with respect to control. Figure 1 represents the experimental

7

investigation procedure in the form of a flow chart that defines the operation of MFC and the

8

analysis carried. Fig. 1

9

10

2.4 Microbiome identification

11

Microbiome enriched at various experimental conditions along with control was identified using

12

PCR-DGGE technique. Inoculum samples were collected from control, +100 mV and -100 mV

13

at DP and PP conditions. Genomic DNA was extracted using Nucleo Spin soil DNA isolation kit

14

method (Macherey-Nagel GmbH & Co., Germany) following the manufacturer’s protocol. PCR

15

was setup using 100 ng of gDNA and universal primers which are specific for V3 region of 16s

16

rRNA coding gene [341F- CCTACGGGAGGCAGCAG; 517R- ATTACCGCGGCTGCTGG;

17

341

18

CGGGAGGCAGCAG]. A 40 base pair length GC-clamp was added to the 5’ end of the forward

19

primer. PCR was performed according to the protocol described in Sravan et al., 2019 [28]. The

20

amplified PCR product was checked on 1% agarose gel and purified using PCR purification kit

21

(Qiagen, USA). For DGGE analysis, 6% polyacrylamide gel with 40 to 60% denaturant gradient

22

was used. 100 ng of amplified DNA was used for denaturing gradient gel analysis. Gel run was

FGC-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTA

6

1

done at a constant voltage of 90 V for 11 h at 60 °C. The gel images of amplified DNA were

2

captured using a Molecular Imager G: BOX EF System (Syngene International Ltd.) and the

3

bands from all samples were excised with a sterile gel cutter [29]. The excised gel bands were

4

incubated overnight at 4 °C in 50 µl sterile DNase free milli-Q water. The eluted DNA from

5

overnight incubated samples was used as a template for PCR amplification using 341F and 517R

6

primers, and the amplified PCR product was purified and sequenced at Bioserve sequencing

7

facility at Hyderabad. To identify the nearest taxa, all the 16S rRNA gene coding partial

8

sequences were subjected to BLASTN. These sequences were further aligned with closest

9

matches found in the GenBank database (http://www.ncbi.nlm.nih.gov/) using ClustalW.

10

Microbial diversity was analyzed through the information obtained from GenBank and was

11

comparatively represented for each experimental condition. Phylogenetic trees were constructed

12

by aligning the obtained sequences (ClustalW) through neighbor-joining method (bootstrap no of

13

500) using MEGA 7.0 software and the evolutionary relationship was studied comparatively.

14

2.5 Instrumentation for analysis

15

MFCs performance was assessed by considering bioelectrogenesis and wastewater treatment as

16

key parameters. Bioelectricity generation through MFCs was evaluated in terms of open circuit

17

voltage (OCV) and current generation patterns. Voltage across external resistor was measured at

18

regular intervals by using a digital multimeter. Anode potential measured with respect to

19

standard reference electrode was calculated and recorded manually at regular time intervals

20

along with the voltage readings. Fuel cell behavior in terms of cell design point (CDP) was

21

calculated by constructing a polarization curve by plotting current density vs power density and

22

voltage across the varying resistance ranges from 30 KΩ to 100Ω. The relative decrease in anode

23

potential (RDAP) was used to evaluate maximum sustainable power. Wastewater parameters 7

1

were determined according to the standard methods [30]. COD removal efficiency (CODR, %)

2

and substrate degradation rate (SDR, kg COD/m3-day) were regularly assessed [31]. Electron

3

transfer and bio-electrochemical behavior of biocatalyst was evaluated through cyclic

4

voltammetry (CV) and linear sweep voltammetry (LSV) employed using a potentiostat-

5

galvanostat system (PGSTAT12, Ecochemie). CV and LSV were performed by applying a

6

potential ramp to the working electrode (anode), at a scan rate of 30 mV/s over a scan window of

7

+0.5 to -0.5 V. Electro-kinetic behavior was analyzed through Tafel analysis obtained from

8

voltammetric profiles using GPES (version 4.0) software, and conclusions were drawn in terms

9

of Tafel slopes and polarization resistance. In order to ensure the validation of results,

10

experiments were carried out in triplicates.

11

3 Results and Discussion

12

3.1 Surface potentials effect on bioelectrogenesis

13

Three MFCs were initially operated without applied surface anode potential and monitored for

14

several cycles to evaluate the consistency in power production. Voltage, current and anode

15

potential was found to be 270±20 mV, 1.2±0.3 mA and -245±20 mV respectively, which is more

16

or less similar for all the three MFCs. This is considered to be control operation (no applied

17

potential), which implies the in situ capacity of the biocatalyst. To the existing anode potentials,

18

a positive surface anodic poised potential of +100 mV and a negative surface anodic poised

19

potential of -100 mV was applied on anodes of two MFCs respectively, leaving the third MFC as

20

control. Performance of MFCs was evaluated at two phases viz., DP and PP. Figure 2 illustrates

21

the significant differences in voltage, current and anode potential was noticed between +100 mV

22

[360± 12 mV; 1.33±0.2 mA; -340 mV] and -100 mV [465±10 mV; 2.3±0.2 mA; -395 mV] at DP

8

1

phase, in which -100 mV yielded more bioelectricity in comparison to +100 mV and control.

2

Increased bioelectrogenic activity observed with this experimental condition implies the critical

3

influence of negative potential on the fuel cell. As the negative charge and surface anode

4

potential increases, electron donation from the electrode might take place during the negative

5

poised potential conditions [14]. An interesting observation can be seen from figure 2, that PP

6

documented relatively higher bioelectrogenesis than DP at both -100 mV poised surface potential

7

[653± 28 mV; 3.5±1.3 mA; -495 mV] and +100 mV poised surface potential [425±12 mV;

8

1.54±0.13 mA; -385 mV]. The observed higher voltage and current at PP phase is ascribed to the

9

prolonged acclimatized and augmented tendency of the biocatalyst in discharging the reducing

10

equivalents into fuel cell environment in response to the attained surface potential. Besides this,

11

electron transferring system of microbes might have been adapted to the level of surface

12

potential, which is high at PP phase, that might enable the membrane components or other

13

compounds as electron carriers to participate in electron transfer [32]. This is also dependent on

14

the redox potential of several compounds that act as electron carriers in between bacteria and

15

electrode. Increase/change in surface anode potential brings a change in electron transfer system

16

of microbes that facilitate the expression of several compounds in transferring electrons towards

17

electrode as electron acceptor [33]. There might also be the possibility of direct electron transfer

18

(DET) [34] or direct interspecies electron transfer (DIET) [35, 36] in biofilm matrix formed on

19

the electrode surface that enables the increase in electron transfer rate towards electrode

20

discussed in detail in the sections below.

21

Fig. 2

22

9

1

3.2 Microbiome constitution

2

Microbiome analysis depicted a total number of 17 operational taxonomic units (OTUs) for all

3

the experimental conditions. A total of 8 OTUs were obtained with control while DP-100mV,

4

DP+100mV, PP-100mV and PP+100mV yielded 5, 8, 10 and 6 OTUs respectively (S Table-1). Figure 3

5

describes about PP-100mV which attained more number of OTUs (10) constituting 3 unique OTUs,

6

whereas DP+100mV and DP-100mV depicted only one unique OTU among the total number of OTUs

7

obtained. Higher number of OTUs represents maximum diversity which might have been

8

enriched at the poised potential conditions. Dominating phyla at each condition consisted of

9

Proteobacteria, Firmicutes and Bacteroidetes. As observed from figure 3, control operated

10

without any poised anode potential depicted the presence of bacteria related to Proteobacteria,

11

Firmicutes and Bacteroidetes in varying fractions. This mixed bacterial population aided for

12

higher substrate utilization as well as power production. While the MFCs poised with positive

13

anodic potential (+100 mV) illustrated variation in percentage abundance of bacterial community

14

at both the phases viz., during potential (DP+100 mV) and post potential (PP+100 mV). At DP+100 mV,

15

higher fraction of Firmicutes followed by Proteobacteria was found to be present. Among the

16

phylum Proteobacteria, maximum of Pseudomonas sp. and Magnetococcus sp. were found to be

17

enriched during +100 mV. These enriched bacteria belong to Gram negative category and are

18

recognized as electrogenic bacteria in several MFC studies. The observed power output from

19

MFCs as well as increased surface potential at DP+100 mV might be attributed to the presence of

20

these enriched electrogenic bacteria that enhances the rate of electron transfer. Most of the

21

Pseudomonas sp. has the ability to secrete redox mediators as well as biofilm formation. Redox

22

mediators that are observed during voltammetric analysis (discussed at below sections) at DP+100

23

mV,

confirms the increased electron transfer in correlation to enriched population. At PP+100 mV, 10

1

members belonging to phylum Firmicutes were present in relatively higher fraction than

2

Proteobacteria and Bacteroidetes. Bacterial groups that might have enriched during DP+100 mV,

3

might not be able to survive at PP+100mV, and only certain population capable of aligning their

4

electron transfer system to this condition might have been evolved during the course of operation

5

time. At PP+100 mV Clostridia sp. were found to be dominant among Firmicutes. These Clostridia

6

sp. are Gram positive bacteria and have more tendency of biofilm formation due to secretion of

7

exo-cellular polymeric substances that aids for adherence to substratum [7]. Electrode that acts as

8

electron acceptor in MFC for facilitating electron transfer can function rapidly according to the

9

biofilm matrix as well as electron transfer system [37]. These enriched bacteria at PP+100

mV,

10

might have formed as biofilm on electrode surface catalyzing higher electron transfer which has

11

been correlated with the enhanced redox catalytic currents as well as decreased electron losses.

12

Comparatively higher MFC performance observed at PP+100

13

ascribed to the change in microbial community structure that in turn influences the electron

14

transfer mechanism that aids towards bio-electrogenic activity.

15

On the contrary, operation of MFC at negative anode potential resulted in a difference in

16

bacterial community structure at DP-100 mV and PP-100

17

abundance of bacteria belonging to phylum Proteobacteria followed by Firmicutes and

18

Bacteroidetes were present. A significant shift in percentage abundance of bacteria has been

19

noticed with respect to control and positive potential (DP+100

20

phylum Proteobacteria is found to be dominant which comprised of bacteria belonging to

21

species Shewanella, Pseudomonas, Rhodospirillum etc. in higher fractions. Shewanella is an

22

exo-electrogen and one of the widely studied model organisms in MFC. Shewanella sp., a potent

23

exo-electrogen has the capability of forming nanowires that aids in facilitating electron transfer 11

mV.

mV

than at DP+100

mV,

might be

At DP-100 mV, a maximum percentage

mV

and PP+100

mV).

At DP-100

mV,

1

towards electrode at a higher rate with minimal electron losses in comparison to other modes of

2

electron transfer. Although nanowires were not identified or analyzed in the study, abundance of

3

these bacteria might have contributed for higher bio-electrogenic performance of MFC at DP-

4

100mV.

5

community as a corner stone responsible for overall MFC performance. Similarly, the microbial

6

community evaluated at PP-100

7

phylum Proteobacteria in higher fraction followed by Firmicutes and Bacteroidetes. Almost

8

two-fold increment in the abundance of Proteobacteria has been observed at PP-100 mV than at

9

DP-100

Higher redox catalytic currents depict the involvement of exo-electrogenic bacterial

mV,

mV,

depicted the percentage abundance of bacteria belonging to

with enriched bacteria majorly comprising of Geobacter sp., Shewanella sp.,

10

Pseudomonas sp., etc. in higher fraction. Geobacter sp. a notable and model electrogen has been

11

found to be enriched in higher fraction which might have been responsible for overall increase in

12

power production in comparison to other experimental conditions [38]. At PP-100

13

higher power output has been observed, redox mediators were less noticeable in comparison to

14

other experimental conditions, which might be ascribed to the occurrence of direct electron

15

transfer either through biofilm matrix or nanowires, a characteristic feature of enriched bacteria

16

as observed at PP-100

17

phylum Bacteroidetes and PP-100

18

higher fraction than at DP-100 mV, depicting the dynamics associated with the redox potential of

19

MFC consisting of surface anode potential, membrane potential etc. Adaptable tendency of

20

bacteria and its electron transferring system to the anode potential generated as a result of

21

negative poised potential contributed to the shifts in microbial community structure that is

22

further correlated with enhanced MFC performance.

23

Fig. 3

mV

mV,

although

[39]. Flavobacteria sp. has been found in higher fraction among the mV

also revealed bacteria belonging to Firmicutes in slightly

12

1

3.3 Bioelectro-catalytic activity

2

Influence of positive and negative applied anode potentials along with control on electron

3

discharge and energy generation pattern was evaluated by employing cyclic voltammetry which

4

visualized a clear variation during both the phases viz., DP and PP. Figure 4a illustrates lower

5

redox catalytic currents (OC: 0.39 mA; RC:-0.22 mA) documented by control operation

6

compared to positive and negative poised anode potentials. Extensively higher redox catalytic

7

currents were noticed with -100 mV (DP; OC: 0.42 mA, RC: -0.43 mA; PP; OC: 0.42 mA, RC: -

8

0.44 mA) in comparison to +100 mV (DP; OC: 0.32 mA, RC: -0.22 mA; PP; OC: 0.41 mA, RC:

9

-0.37 mA). Higher currents recorded during -100 mV is attributed to the enhanced electron

10

discharge capabilities by the biocatalyst in response to attained surface potential. Abundance of

11

Gram negative electrogenic bacteria closely related to Geobacter and Shewanella sp., at -100

12

mV supports increased electron transfer rates as observed through voltammetric analysis. It was

13

reported that lower and negative potentials drive the electron transfer at a higher rate and

14

enhance the startup of MFC contributing towards increased power output. This can be further

15

correlated with higher substrate (COD) removal during negative poised potential conditions,

16

which might have been occurred due to the large potential gradient between anode surface and

17

bacterial cell that would enhance the metabolic capabilities of the biocatalyst towards increased

18

electron transfer thereby contributing towards an increment in the redox currents [40]. Also,

19

positive potential has influenced the electron transfer in accordance with surface potential which

20

depicted higher redox catalytic currents in comparison to control.

21

3.3.1 Electron carriers

22

Electron transfer from bacterial membrane towards electrode takes place through a series of

23

electron carriers/mediators [41] observed as peaks during the voltammetric analysis [42]. A 13

1

perfect redox catalytic wave in the form of a peak, positioning at -100 mV was observed on

2

voltammograms during negative poised potential (both DP and PP) which might be attributed to

3

the presence of Fe-S proteins participating in the electron transfer, while the same peak was not

4

observed in control and positive poised potential (DP and PP) conditions. Besides this, the peak

5

height was observed to increase and was found to be highest at PP-100 mV, depicting the enhanced

6

activity of this electron transfer chain (ETC) component in electron transfer towards electrode

7

aiding for higher bioelectrogenic activity. Although predominant peaks were appeared to be less

8

during negative poised conditions, higher bio-electrogenic activity was observed which might be

9

accredited to the occurrence of direct electron transfer (DET), congruence with the observed

10

bacterial species (Shewanella sp.) closely related to phylum Proteobacteria encompassing a

11

group of electrogenic bacteria. Since bacteria belonging to model electricigens were observed,

12

there might also have been nanowires formation contributing for long range electron transfer

13

which is not analyzed during the study. While in positive anodic poised potential conditions,

14

redox catalytic wave positioning at 80 mV (DP+100 mV), 100 mV (PP+100V) and 375 mV (DP and

15

PP) were observed, which might be ascribed to the involvement of cytochrome-C, Ubiquinone

16

and cytochrome-aa3 respectively as electron carriers. These components are believed to be

17

involved in bacterial ET, in which few species facilitate oxidation and few facilitating reduction

18

reactions [43]. Positive poised anode potential illustrated Gram positive bacterial species, most

19

of which are confined under the category of ‘weak electricigens’. These are reported to perform

20

electron transfer under specified conditions which enable either membrane bound proteins as

21

carriers or soluble shuttlers. In control operation, two peaks were appeared positioning at 110

22

mV and 410 mV during voltammetric analysis, which might be attributed to the presence of

23

diverse bacteria as well as occurrence of DIET.

14

1

3.3.2 Electron transfer - Interfacial

2

In order to elucidate whether the identified peaks are surface absorbed or diffusive, CV’s were

3

performed at varied scan rates and the linear correlation was considered as a determining factor

4

[44]. Surface confined/absorption controlled phenomena of redox species refers to the migration

5

of molecules/redox species from bulk aqueous phase to interface, while the diffusion controlled

6

phenomena refers to the migration of molecules from bulk aqueous phase to subsurface and

7

further towards interface [45]. The peaks were identified to be diffusive in both the poised

8

potential conditions, since linearity has been observed. However, the electrode material used in

9

the study is graphite with confined porous surface, and the identified linear correlation might not

10

be affirmative to the diffusion controlled phenomenon of redox species on voltammogram

11

surface. The increase in peak current with corresponding increase in scan rate implies the active

12

electron transfer process that is well illustrated in terms of either adsorption or diffusion

13

controlled phenomenon of redox species. On the contrary, control operation depicted surface

14

controlled phenomena (adsorption) of redox species. Salient observation drawn during

15

voltammetric analysis is the generation of higher redox catalytic currents during negative poised

16

potential conditions, which in further is at post potential conditions. As the surface potential

17

changed with respect to varied applied potentials at both DP and PP phases, the electron transfer

18

behavior also varied accordingly, which enabled specific ETC components at each experimental

19

variation to participate in electron transfer as visualized through voltammetric analysis.

20

3.3.3. Electron transfer - Redox mediators

21

The maxima of first derivative of CV (DCV) correspond to the inflection points of the catalytic

22

waves and helps in identifying the undetected redox active species (RS) involved during electron

15

1

transport [42]. Figure 4b depicts the presence of distinct peaks elucidating the involvement of RS

2

during DCV analysis for both the surface anodic poised potential conditions (+100 mV/-100

3

mV) along with control operation. Two quasi reversible peaks were detected with peak potentials

4

of -0.151 V and 0.272 V corresponding to the involvement of Fe-S proteins and cytochrome-C

5

respectively at -100 mV. While +100 mV documented the involvement of cytochrome aa3,

6

cytochrome bc1 exclusive of cytochrome complex, whereas -100 mV documented the

7

involvement of flavoproteins, cytochromes, cytochrome aa3, Fe (CN)63-/Fe (CN)64+ and

8

NAD/NADH couple. Fe (CN)63-/Fe (CN)64+are impermeable to the plasma membrane and is an

9

extracellular electron receptor during the redox reactions in cells. Also, the presence of Fe

10

(CN)63-/Fe (CN)64+can be attributed to secretions of reductants or "Trans Plasma Membrane

11

Electron Transport" (TPMET) activity. Several RS observed during CV and DCV infers the role

12

of ETC components in electron transfer aligned with respect to change in anode potential. Fig. 4

13

14

3.3.4 Feasible current generation

15

Linear sweep voltammetry, a potentio-dynamic polarization technique was employed to

16

understand maximum possible current generation. Figure 5 infers the maximum feasible current

17

generation to be higher at PP phase (-100 mV: 48 mA; +100 mV: 37 mA) followed by DP phase

18

(-100 mV: 48mA; +100 mV: 40 mA) and control (30 mA). This is in correlation with the bio-

19

electrogenic activity as well as phylogenetic analysis which delivers information on higher

20

metabolic activity, less electron losses and maximum currents at post potential phase.

21

Fig. 5

22 16

1

3.5. Energy gain

2

From a thermodynamic perspective, surface anode potential is one of the determining elements

3

for bacterial energy gain [46]. Bacteria tend to gain more energy based on the potential of

4

terminal electron acceptor, which is anode surface in the case of a MFC [47]. An optimal surface

5

potential would aid bacteria in gaining more energy as well as to facilitate the electron transfer.

6

Relative decrement in anode potential (RDAP) with the function of applied external resistance

7

was used to evaluate the maximum sustainable power generation ability of MFC operated at

8

various phases [48]. Figure 6 illustrates the sustainable resistance to be 2.5 kΩ for PP-100

9

followed by 3 kΩ for DP-100 mV, 6 kΩ for PP+100 mV, 6.5 kΩ for DP+100 mV and 7 kΩ for control

10

[39]. Lower oxidation potential at anode surface provides less energy for the growth and

11

maintenance of the biocatalyst, while higher oxidation potential supports early start up of the

12

electron discharge and higher current generation [49]. Higher oxidation potential at anode

13

observed during PP phase is attributed to the prolonged acclimatized capabilities of the bacteria

14

acquired during the poised potential conditions that results in rapid electron discharge

15

contributing for higher power generation in addition to acquiring energy. Besides this, electron

16

transferring system of the microbes might have aligned to a level in accordance with the surface

17

anode potential generated during poised potential conditions. These results also reveal the

18

findings on more energy gain by bacteria at lower anode surface potentials (-100 mV) in

19

comparison to higher anode surface potentials (+100 mV), which has been depicted through the

20

observed resistance contributing for sustainable and increased power production. Maximum

21

sustainable power generation in a system against an external load is an indicator of the fuel cell

22

efficiency [31]. Also, higher sustainable resistance indicates that the fuel cell can be operated at

23

steady state even at higher external loads depicting its efficiency subsiding the activation losses. 17

mV,

Fig. 6

1

2

3.6 Electro-kinetics

3

Bacteria require certain energy to cross the activation energy barrier for the redox reactions to

4

take place [50]. During this, there are many possibilities of electron losses which can be

5

described as activation losses, Ohmic losses and concentration losses. Electro-kinetic behavior of

6

biocatalyst at various phases along with control was studied through polarization profile as well

7

as Tafel slope analysis.

8

3.6.1 Electron losses

9

Electron delivering nature of biocatalyst in MFC with respect to applied anode surface potential

10

was studied by performing polarization across a wide range of resistances (30 KΩ to 100 Ω). A

11

typical polarization curve illustrates the fuel cell behavior in terms of CDP (Cell Design Point),

12

power density and current density against voltage along with electron losses [16]. Figure 7

13

depicts the variation in CDP at each experimental phase studied, in which higher CDP was

14

observed during PP-AP-100mV (320 Ω) followed by DP-AP-100mV (200 Ω), PP-AP+100 mV (195 Ω),

15

DP-AP+100 mV (195 Ω) and control operation (120 Ω). There is no significant difference in CDP at

16

+100 mV and DP-100mV, which reflects the ability of the biocatalyst in ED more or less similar at

17

the above phases studied. Also, higher CDP observed with PP-AP-100mV indicates the enhanced

18

capabilities of the biocatalyst in rapid electron discharge against the external load. A maximum

19

of 320 Ω resistance could be sustained by the fuel cell during PP-AP-100mV. Concentration losses

20

as well as activation losses are found to be dominant during the fuel cell operation at PP-

21

AP+100mV, DP-AP+100mV and DP-AP-100mV, whereas, Ohmic losses were found to be predominant

22

during the operation at PP-AP-100mV. In the case of control operation, electron losses were 18

1

relatively higher (concentration losses) indicating the poor ability of biocatalyst in ED against

2

the external load. Involvement of several electron carries in electron transfer as observed through

3

voltammetric analysis is also in congruence with less electron losses at PP phase. Fig. 7

4

5

3.6.2 Tafel Plots and associated kinetics

6

Tafel analysis is used to derive active kinetic parameters in the form of redox Tafel slopes as

7

well as Polarization resistance (Rp). Oxidation slopes (ba) were significantly lower in comparison

8

to reduction slopes (bc) at all the experimental conditions studied. This is related to bacterial

9

energy gain where the oxidizing ability facilitates bacteria to gain more energy for its growth and

10

cell maintenance. Figure 8 illustrates the redox slopes to be higher with control operation (ba:

11

0.087 V/dec; bc: 1.031 V/dec) followed by +100 mV [(DP: ba: 0.075 V/dec; bc: 0.809 V/dec);

12

(PP: ba: 0.062 V/dec; bc: 0.681 V/dec) and -100 mV (DP: ba: 0.09 V/dec; bc: 0.681 V/dec); (PP:

13

ba: 0.08 V/dec; bc: 0.798 V/dec). This is in direct correlation to the surface poised potential in

14

enhancing the electron discharge capacity of biocatalyst as well as the oxidation reactions at a

15

higher rate than its native (control) state (Table-1). Less reduction capabilities in comparison to

16

oxidation reactions in MFC is attributed to concentration as well as activation losses.

17

Concentration losses appeared to be predominant as the electrons and protons liberated during

18

oxidation are high, but are not effectively utilized towards reduction reactions. However, total

19

electron losses are found to be lower during PP phase due to rapid electron transfer capabilities,

20

as well as the altered electron transfer pathway which is depicted through change in voltage and

21

current density plot. All the electron losses at various zones viz., Ohmic (ZOL), concentration

22

(ZCL) and activation (ZAL) appeared to be relatively less at PP phase in positive and negative

19

1

poised surface potential conditions. This is in concurrence with higher redox currents as well as

2

the electrogenic activity in terms of OCV and higher CDP (320 Ω) at PP phase in comparison to

3

DP phase and control. Shifts in Tafel plots provide a clear and visual understanding of the

4

behavior of MFC with respect to poised surface anode potentials. Control operation was found at

5

-0.19V (reduction phenomena) whereas the positive potential conditions exhibited more shift

6

towards reduction (DP: -0.32 V; PP: -0.27 V). On the contrary, negative potential conditions

7

exhibited behavioral shift towards midpoint potential (0 V)/oxidation (DP: -0.1 V; PP: -0.06 V).

8

The positive tendency of the negative potential towards midpoint/near oxidation is a good sign of

9

its electrogenically higher activity over positive potential in liberating more number of reducing

10

equivalents (e- and H+).

11

3.6.3 Electron transfer resistance

12

Polarization resistance (Rp) is the resistance for electron transfer either from the biocatalyst

13

towards anode surface or from the anode surface to the cathode. Figure 8 portrays Rp higher with

14

control operation (1.94 Ω) followed by positive potential (DP: 1.08 Ω; PP: 0.82 Ω) and negative

15

potential (DP: 0.65 Ω; PP: 0.49 Ω). Lower resistance observed at PP phase is ascribed to the

16

efficient and higher electron delivering capabilities acquired by the biocatalyst during the DP

17

phase of anodic surface poised potential conditions. Also, the specifically enriched bacteria at the

18

poised potentials will be electrochemically active in delivering the electrons effectively at a

19

higher rate [51]. Electro-kinetic analysis illustrated enhanced electron delivery capabilities

20

acquired by the bacteria that are specifically enriched at poised potential conditions over a period

21

of time. Lower redox Tafel slopes and Rp supports the positive influence of negative poised

22

potential at a higher rate on the electron transfer and losses minimization than the positive poised

20

1

potentials [16]. A detailed and consolidated electrochemical performance of MFC is presented in

2

Table-1. Fig. 8; Table-1

3

4

3.7 Substrate degradation

5

The degradation of substrate was found to vary at each experimental condition studied. Substrate

6

degradation capabilities of the bacteria depend on the microenvironment, external factors,

7

metabolic activities etc. As potential was poised to the system during the fuel cell operation, the

8

behavior of the biocatalyst would definitely get influenced in terms of various parameters viz.,

9

electrogenesis, substrate degradation. Irrespective of the experimental variation, a gradual

10

depletion in substrate was observed in the fuel cell which was calculated at regular (for every 6h)

11

time intervals. During the control operation, substrate degradation in terms of COD removal was

12

found to be 52.23%. Figure 9a depicts a marked improvement in COD removal during the poised

13

potential of +100 mV (DP: 63.27%; PP: 67.1%), along with considerable increment in COD

14

removal was observed during -100 mV potential (DP: 68.23%; PP: 72.2%). The increased COD

15

removal efficiency at each condition is attributed to the function of poised potential in enhancing

16

the metabolic activities of the biocatalyst that enhance substrate degradation with respect to

17

anodic potential.

18

3.8 Redox microenvironment

19

The redox microenvironment of MFC was assessed by monitoring the system pH and volatile

20

fatty acids (VFA) generation. Figure 9b depicts that poised potential has a marked influence on

21

the redox microenvironment during the operation. pH and VFA express the intrinsic acid–base

22

conditions as well as the balance between the diverse bacteria groups. MFC showed a distinct 21

1

trend towards acidification during the initial period of the cycle operation especially up to 30 h.

2

pH drop was relatively higher at DP (DP+100: 4.1±0.1; DP-100: 4.3±0.2) in comparison to PP

3

(PP+100: 4.7±0.3; PP-100: 4.2±0.1) and control operation (4.2±0.2). pH also influences the

4

efficiency of substrate metabolism, biofilm structure, electron transfer, protein synthesis,

5

synthesis of storage material and metabolic by-product release. VFA synthesis co-exists with the

6

anaerobic metabolic pathway as a byproduct due to the function of anaerobic or facultative

7

bacteria in the process of substrate degradation. VFA generation was observed maximum at

8

during potential (DP) conditions (DP+100: 1300±100 mg/l; DP-100: 1700±200 mg/l) in comparison

9

to negative poised anode (PP+100: 1200±100 mg/l; PP-100: 1100±200 mg/l) and control (1600±100

10

mg/l). This is in accordance with pH and substrate degradation which incurs electron distribution

11

partly to bioelectricity generation and organic intermediate metabolites synthesis. Fig. 9

12

13

4. Conclusions

14

Study investigated the regulatory influence of both positive and negative surface potentials on

15

the performance of a single chamber MFC at two phases i.e., during potential (DP) and post

16

potential (PP). Significant variation in electron transfer, bioelectrogenic activity and microbiome

17

community composition has been observed among all the experimental variations.

18

Electrochemical investigations performed through cyclic and linear sweep voltammetry,

19

polarization curves and Tafel analysis revealed MFC efficiency interms of redox catalytic

20

currents, electron transfer phenomenon, electron carriers involved, power density and electro-

21

kinetics to be relatively higher at post potential phase with negative poised potential in

22

comparison to other variations.

22

1

2

The conclusions obtained from the study are depicted below: •

Negative poised surface potential resulted in higher bioelectrogenic activity (653± 28

3

mV) associated with minimal electron losses in comparison to positive poised surface

4

potential (425±12 mV) and control operation (270±20 mV).

5

•

Interestingly, post potential operation illustrated increased redox catalytic currents as well

6

as sustainable power generation in comparison to during potential condition at both

7

positive as well as negative poised anodic surface potential.

8

•

potential conditions is found to be associated with change in electron transferring system,

9

which is correlated with voltammetric profiles as well as bioelectrogenic activity.

10 11

Dynamics in microbial community structure with respect to poised anode surface

•

Members belonging to phylum Proteobacteria consisting of Gram negative bacteria were

12

abundant at negative potential while Firmicutes consisting of Gram positive bacteria

13

appeared in higher fraction at positive potential.

14

•

Possibility of occurrence of overlapping electron transfer mechanisms were identified

15

i.e., direct electron transfer (DET), direct interspecies electron transfer (DIET) and

16

mediated electron transfer (MET) with the experimental conditions studied, wherein

17

DET might have been predominant in negative poised surface potential followed by MET

18

in control and positive poised surface potential.

19

•

Current study provided new insights in to the adaptability of electron transfer mechanism

20

of bacteria with respect to surface potentials (positive and negative) that depicted

21

augmented electrogenic activity specifically at post potential operation inferring the

22

triggered response attained at DP phase sustained till PP phase.

23 23

1

Acknowledgements

2

JAM and KVK acknowledges CSIR for providing research fellowship. CNR and BM would like

3

to acknowledge the research grants from National Research Foundation of Korea

4

(2018R1A2B6001507) and Korea-India S&T Cooperation Program (2016K1A3A1A19945953).

5

The authors wish to thank the Director, CSIR-IICT (manuscript No. IICT/Pubs./2018/196) for

6

supporting the research.

7

8

9

10

11

12

13

14

15

16

17

18

24

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8

microbial fuel cell performance with the function of open and closed circuitry, Biores.

9

Technol. 101 (2010) 5337-5344.

10 11

50. B.E. Logan, J.M. Regan, Microbial fuel cells-Challenges and applications, Env Sci and Technol. 40 (2006) 5172-5180.

12

51. C.I. Torres, A.K. Marcus, H.S. Lee, P. Parameswaran, R. Krajmalnik- Brown, B.E.

13

Rittmann, A kinetic perspective on extracellular electron transfer by anode-respiring

14

bacteria, FEMS Microbiol Rev. 34 (2009b) 3-17.

15

31

Table 1: Consolidated electro-chemical performance of MFCs operated at various conditions

Experimental variation

ba (V/dec)

bc (V/dec)

Rp (Ω)

I (mA)

Control

1.031

0.087

1.94E+00

1.42

DP-AP+100

0.809

0.075

1.08E+00

1.33

PP-AP+100

0.681

0.062

8.28E-01

1.64

DP-AP-100

0.88

0.088

6.57E-01

2.42

PP-AP-100

0.798

0.09

4.91E-01

4.48

Figure Captions Fig 1 Flow chart depicting the experimental investigation carried with three independent MFCs as control, positive poised surface potential (+100 mV) and negative poised surface potential (-100 mV) respectively at both during potential (DP) and post potential (PP) phases Fig 2 Variation in total cell emf, power density and anode potential at all the experimental conditions with respect to time Fig 3 (a) Microbial diversity expressed in percentage abundance of bacterial phyla for all the experimental conditions (b) Venn diagram representing common and unique OTUs identified at each experimental condition Fig 4 (a) Representative turnover cyclic voltammograms and (b) Derivatives of cyclic voltammograms depicting the presence of redox mediators in the form of peaks obtained in a scan window of -0.5 V to +0.5 V with anode as working electrode, cathode as counter electrode and Ag/AgCl (S) as reference electrode Fig 5 Potentio-dynamic polarization recorded at a scan rate of 20 mV/s in a scan window of -0.5 V to +0.5 V for all the experimental variations represented with respect to Ag/AgCl (S) as reference electrode Fig 6 Relative decrement in anode potential with respect to varied external resistance for all the experimental conditions Fig 7 Cell polarization depicting voltage and power density with respect to current density recorded at an external resistance range of 30 KΩ to 50 Ω for all the experimental variations Fig 8 Bio-electrochemical performance of MFCs in terms of (a) charge, capacitance, energy and number of electrons and Electro-kinetics represented in the form of (b) Tafel plots (c) Tafel Slopes and (d) Electron losses representing the zone of activation (ZAL), ohmic (ZOL) and concentration losses (ZCL) Fig 9 Substrate degradation represented as (a) COD concentration and substrate degradation rate (SDR) and (b) Redox metabolism expressed as change in pH and volatile fatty acid (VFA) generation for all the experimental variations

Fig. 1

Control

During Potential (DP) Post Potential (PP)

o E (mV) Cell PD(mW/m )

600

2

300 0

600

Control DP-AP+100 PP-AP+100

300

DP-AP-100 PP-AP-100

EAnode (mV)

0 -300 -600 0

144

288

432

Time (h) Fig. 2

576

720

864

(a) Firmicutes

Proteobacteria

Bacteroidetes

Percentage abundance (%)

100

80

60

40

20

0 Control

DP-AP+100

DP-AP-100

b)

Fig. 3

PP-AP+100

PP-AP-100

(a) 0.05

i/A

0.03

0

Control - -AP+100mV DP PP -AP+100mV

- 0.03

- -AP-100mV DP – -AP -100mV PP - 0.05

-0.8

-0.5

0

-0.3

0.3

0.5

0.8

E/V vs Ag/Agcl (S)

(b)

Control DP-AP+100 PP-AP+100 DP-AP-100 PP-AP-100

di/dt / A/s

0.1

0.0

-0.1 -0.6

-0.4

-0.2

0.0

0.2

E/V vs Ag/AgCl (S)

Fig. 4

0.4

0.6

0.05

i/ A

0.03 0

Control DP-AP +100mV PP-AP +100mV DP-AP -100mV PP-AP -100mV

-0.03 -0.05 -0.8

-0.5

-0.3

0

0.3

E/V vs Ag/Agcl (S) Fig. 5

0.5

0.8

Control DP-AP+100 PP-AP+100 PP-AP-100 DP-AP-100

100

RDAP(%)

80 60 40 20 0 0

5

10

15

20

Resistance (KΩ) Fig. 6

25

30

160 140

2

PD (mW/m )

120 100

PP-AP-100 (AP) AP-100mV DP-AP-100 (DP) AP-100mV PP-AP+100 (AP) AP+100mV DP-AP+100 (DP) AP+100mV Control Control

80 60 40 20

700 0

AP-100mV (AP) AP-100mV (DP) AP+100mV (AP) AP+100mV (DP) Control

600

V (mV)

500 400 300 200 100 0.00

0.05

0.10

0.15

0.20

0.25

0.30 2

CD (mA/m ) Fig. 7

0.35

0.40

0.45

3.50E+01

1.2E+20 Charge(C)

3.00E+01 2.50E+01

1E+20

Capacitance (F) Energy stored (J)

8E+19 No. of electrons (n) 2.00E+01 6E+19 1.50E+01 4E+19 1.00E+01 2E+19

5.00E+00 0.00E+00

0

Control

DP-AP+100 PP-AP+100 DP-AP-100 PP-AP-100

(b)

-16 -17

(ln I)

-18 -19 Control

-20

DP-AP+100 PP-AP+100

-21 -22 -0.6

DP-AP-100 PP-AP-100

-0.4

-0.2

0.0

E/V

0.2

0.4

0.6

No. of electrons (n)

Charge (C), Capacitance (F) and Energy stored (J)

(a)

Tafel Slopes (V/dec) and

∆ (Voltage/Current density) Polarization Resistance(Ω)

(c, d) 2.0 1.6

βa (V/dec) β (V/dec) c

Rp (Ω)

1.2 0.8 0.4 0.0 3.0

ZAL

ZOL

ZCL

2.5 2.0 1.5 1.0 0.5 0.0

DP-AP PP-AP Control DP-AP-100 PP-AP -100 +100 +100

Fig. 8

(a) SDR- Control SDR- PP-AP+100

SDR- DP-AP-100

1.0

SDR- PP-AP-100

2800

0.8

3

COD Concentration (mg/l)

3200

1.2

SDR- DP-AP+100

SDR (Kg COD/m day)

3600

2400 0.6 2000 0.4 1600 0.2

1200 COD- Control

COD- DP-AP+100

COD- PP-AP+100

800

0.0

COD- DP-AP-100

COD- PP-AP-100

400

0

6

12

18

24

30

36

42

48

-0.2

Time (h) (b) Control

During Potential (DP) Post Potential (PP)

6.5

pH

6.0 5.5 5.0 4.5

VFA Conc. (mg/l)

4.0 2500

Control DP-AP+100 PP-AP+100

2000

DP-AP-100 PP-AP-100

1500 1000 500 0

144

288

432

Time (h)

Fig. 9

576

720

864

Highlights •

MFC at -100 mV depicted higher bioelectrogenesis (653± 28 mV) than +100 mV

•

Post-potential phase illustrated less electron losses than during-potential phase at -100 mV

•

Dominantly, Proteobacteria were enriched at -100 mV and Firmicutes at +100 mV

•

Requirement of activation energy was less at post potential phase in -100 mV

•

Varied electron transfer modes viz. direct and mediated appeared for +100/-100 mV and Control

CRediT author statement Article Type: Research Paper Title: Regulated Surface Potential Impacts Bioelectrogenic Activity, Interfacial Electron Transfer and Microbial Dynamics in Microbial Fuel Cell Authors: J. Annie Modestra1,2, C. Nagendranatha Reddy1,3, K. Vamshi Krishna1,2, Booki Min3, S. Venkata Mohan*1,2,3 Corresponding author: Dr. S. Venkata Mohan Corresponding author *E-mail: [email protected]; [email protected], Tel/Fax: 04027191765. Affiliations: 1Bioengineering and Environmental Sciences Lab, Department of Energy and Environmental Engineering, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad, 500 007, India. 2 Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT) campus, Hyderabad, 500 007, India. 3 Department of Environmental Science and Engineering, Kyung Hee University, Seocheondong, Yongin-si, Gyeonggi-do 446-701, Republic of Korea.

Author Contributions:

Dr. S. Venkata Mohan: Conceptualization, methodology and supervision; J. Annie Modestra: Data curation, writing- original draft preparation and data validation; C. Nagendranatha Reddy: Data curation, manuscript preparation and data validation; K. Vamshi Krishna: Microbial community analysis; Dr. Booki Min: Reviewing of manuscript.

Declaration of interests ×The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Article Type: Research Paper Title: Regulated Surface Potential Impacts Bioelectrogenic Activity, Interfacial Electron Transfer and Microbial Dynamics in Microbial Fuel Cell Authors: J. Annie Modestra, C. Nagendranatha Reddy, K. Vamshi Krishna, Booki Min, S. Venkata Mohan* Declaration: All the authors prepared manuscript by abiding with the guidelines of Elsevier’s ethical requirements and mutually agreed for submission of the work in the journal of Renewable Energy. The work reported in the manuscript is the original work of authors and was not submitted elsewhere. Conflict of interest: All the authors of the manuscript have declared no conflict of interest.