Conductive magnetite nanoparticles trigger syntrophic methane production in single chamber microbial electrochemical systems

Journal Pre-proofs Conductive magnetite nanoparticles trigger syntrophic methane production in single chamber microbial electrochemical systems Mung Thi Vu, Md Tabish Noori, Booki Min PII: DOI: Reference:

S0960-8524(19)31495-6 https://doi.org/10.1016/j.biortech.2019.122265 BITE 122265

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Bioresource Technology

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16 August 2019 10 October 2019 12 October 2019

Please cite this article as: Thi Vu, M., Tabish Noori, M., Min, B., Conductive magnetite nanoparticles trigger syntrophic methane production in single chamber microbial electrochemical systems, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122265

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1

Conductive magnetite nanoparticles trigger syntrophic methane

2

production in single chamber microbial electrochemical systems Mung Thi Vu1, Md Tabish Noori1, Booki Min1*

3 4 5

1Department

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dong, Yongin-si, Gyonggi-do 446-701, Republic of Korea.

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*Corresponding author: Phone: +82-31-201-2463; Fax: +82-31-202-8854; Email:

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[email protected]

of Environmental Science and Engineering, Kyung Hee University, Seocheon-

9 10 11 12 13 14 15 16 17 1

1

Abstract

2

Performance of methane-producing microbial electrochemical systems (MESs) is highly

3

reliant on electron transfer efficiency from electrode to microorganisms and vice versa. In

4

this study, magnetite nanoparticles were used as electron carriers to enhance extracellular

5

electron transfer in single chamber MESs. The MES with magnetite exhibited the highest

6

methane yield and current generation of 0.37 ± 0.009 LCH4/gCOD and 9.6 mA, respectively

7

among the tested reactors. The experimental data was observed to be highly consistent with

8

modified Gompertz model results (R2 >0.99), which also showed 74.2 % and 22.1 %

9

enhanced methane production rate in MES with magnetite as compared to control AD and

10

MES without magnetite, respectively. Cyclic voltammetry and electrochemical impedance

11

spectroscopy analysis confirmed that magnetite enhanced catalytic activity of biofilm and

12

lowered both solution and charge transfer resistance. Therefore, supplementing magnetite

13

in MESs could be a strategy to develop an efficient syntrophic biomethanation in field scale

14

applications.

15

Keywords: Electromethanogenesis; Magnetite nanoparticles; Methane yield; Microbial

16

electrochemical system

17

2

1

1. Introduction

2

Biogas generated from anaerobic fermentation of organic matters mainly contains CH4 (50-

3

70 %), CO2 (50-30 %) and a trace quantities of H2, H2S and NH3 (Kelleher et al., 2002). A

4

mole of CH4 releases 810 kJ of heat combustion energy, making it precious as an off-grid

5

source of fuel (MacDonald, 1990). Therefore, numerous efforts have been developed to

6

maximize the economic value of biogas via increasing CH4 amount and percentage from

7

anaerobic digestion (AD) (Angenent et al., 2018; Aryal et al., 2018). Among the existing

8

upgrade techniques, the utilization of microbial electrochemical systems (MESs) is an

9

attractive and promising method (Cheng and Kaksonen, 2016; Venkata Mohan et al., 2014).

10

For example, in a MES treating waste activated sludge, Liu et al., 2016 observed methane

11

production rate of 0.128 L/L-d, which was about 3 times higher than the value of 0.043

12

L/L-d in control AD. In another test about the potential effects of acidic condition to

13

methane fermentation process, Moreno et al., 2018 used high concentration of glucose as

14

substrate and found that MES exhibited an excellent VFAs decomposition capacity, that

15

lead to about 4 times enhanced CH4 production compared to AD process. Similarly, Park et

16

al., 2018 reported a significant increase of methane yield up to 0.33 LCH4/gCOD when

17

electrodes were employed in the system due to either the increase of VFAs removal or the

18

enrichment of microbial communities that possible for extracellular electron transfer (EET).

19

These reports strongly suggest that bioelectrochemcial system is beneficial for aiding

20

methane production, complex substrate degradation and VFAs consumption, which are

21

prime keys indicating the efficiency and stability of methane fermentation system.

22

Previous studies have investigated that methane upgrading in MESs highly depends on the

23

activity of microorganisms responsible for extracellular electron transfer (exoelectrogens) 3

1

(Aryal et al., 2017; Guo et al., 2015). Recently, adding conductive materials in MESs

2

suspension was found to improve electroactive biofilm activity, thus significant enhanced

3

methane production. For example, Kim et al., 2017 examined the performance of granular

4

activated carbon (GAC) in the electrolyte of a single chamber MES, leading to the

5

improvement of methane accumulation by 52.4% as compared to that of GAC-free MES.

6

Similarly, LaBarge et al., 2017 used GAC to enrich electrotrophic methanogenic

7

communities, which decreased the start-up time and increased methane production rate of

8

MESs by 84.6 % (0.493 L/L-d) as compared to that of the control without GAC

9

supplementation (0.291 L/L-d). These findings proved that conductive materials which

10

were already well-known for accelerating electron transfer in AD process can be applied in

11

MES-AD to further strengthen methane generation and improve the wastewater treatment

12

efficiency.

13

Recently, magnetite nanoparticles (NPs) have received extensive attention from researchers

14

towards its positive effects on promoting direct interspecies electron transfer in mix-culture

15

AD systems. As a conductive mineral and a common product of bacterial iron reduction, it

16

is widespread in nature, non-toxic to microorganism and can be artificially synthesized

17

using simple chemical precipitation method (Kang et al., 1996). In a mixed VFAs-utilizing

18

AD, the support of 20 mM magnetite NPs in the sludge suspension accelerated methane

19

yield and methane production rate by 2.7 and 3.4 times, respectively (Yang et al., 2016). In

20

another lab scale study, Cruz Viggi et al., 2014 reported 33 % enhancement in methane

21

accumulation from an AD using 6.3 mM magnetite NPs and propionate as substrate.

22

Similarly, methane production rate was found to increase approximately 30 % from 3.7 to

23

4.8 L/L-d in the addition of 0.13 mM magnetite NPs to the AD treated fresh leachate (Lei et 4

1

al., 2018). In this line, Zhang and Lu, 2016 highlighted the addition of 10 mM magnetite

2

NPs increased the maximum methane production rate of AD by almost 50 % as relative to

3

the control operation. In bioelectrochemical systems, magnetite NPs were noted to make a

4

strong electrically conductive bridge between the anode and electroactive microorganisms,

5

thus supported direct electron transfer (DET) by reducing the charge transfer resistance by

6

32 % and enhanced the electricity generation of the microbial fuel cell (MFC) by 47 %

7

(Liu et al., 2018). While variety methane enhancement levels were reported for ADs using

8

different magnetite NPs concentration ranging from 0.64 mM to 320 mM, no negative

9

effect of magnetite on methane fermentation process has been observed so far. In addition,

10

magnetite NPs did not lose the structural properties even after recycling it for more than

11

250 days in AD system, which make the process could be economically viable (Baek et al.,

12

2017).

13

Despite having excellent properties on improving DET in AD and MFC systems, the

14

application of magnetite for enhancing methane generation in AD-MES is niched as a

15

research lacuna. Therefore, we examined for the first time the effect of suspended

16

magnetite NPs on the methane generation, current production, and substrate removal in

17

single chamber MES integrated with AD system. The hypothesis that magnetite promotes

18

methane production by enhancing DET and reducing electrochemical resistance during the

19

electron transport process was investigated using electrochemical methods including cyclic

20

voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Additionally, the

21

interaction between electroactive microbes and magnetite NPs was observed by scanning

22

electron microscope (SEM) for further understanding of the electrochemical behaviors of

23

magnetite NPs in MES systems. 5

1

2. Materials and methods

2

2.1 Magnetite nanoparticles preparation

3

Magnetite NPs were synthesized following the co-precipitation method with a molar ratio

4

of Fe(II)/ Fe(III) salts of 0.5 under alkaline pH of around 11-12 (Kang et al., 1996). All the

5

steps were conducted under strict oxygen-free environment. Briefly, the required amount of

6

analytical grade FeCl3 and FeCl2.4H2O (Daejung, Korea) was dissolved in 0.4 M HCl to

7

make a homogenous solution of 0.8 M Fe(III) and 0.4 M Fe(II), respectively. The above

8

solution was then added drop-by-drop to a 1.5 M NaOH solution under vigorous mixing

9

condition. This resulted in the rapid precipitation of black colored magnetite NPs. The

10

precipitate was recovered using strong magnetic field created by Neodymium magnets.

11

Recovered magnetite NPs were further washed repeatedly with deionized water and ethyl

12

alcohol to remove impurities followed by drying in a vacuum oven at 50 oC overnight and

13

then the semi-fluid type loose magnetite NPs were stored in air-tight containers for further

14

use.

15

2.2 Inoculum and growth medium

16

Anaerobic mix-consortium enriched inoculum was collected from fermentation tank under

17

operation in Suwon wastewater treatment plant (Suwon, South Korea). Collected sludge

18

was completely purged with pure nitrogen gas for 15 min to maintain anaerobic condition,

19

and then stored at 4 oC in fridge for further utilization. The general chemical composition of

20

as-collected sludge was quantified using standard method (APHA, 1998). The characteristic

21

is as follows: pH 7.21 ± 0.01; total chemical oxygen demand (TCOD) 35,463 ± 1182 mg/L;

22

soluble COD (SCOD) 761 ± 28 mg/L; total suspended solids (TSS) 22,250 ± 1150 mg/L; 6

1

volatile suspended solids (VSS): 17,083 ± 444 mg/L. Before being used in MESs, raw

2

sludge was incubated at 35 oC for 6 hours to activate the microorganisms (Lee et al., 2018).

3

Synthetic nutrient medium required for growth and development of microorganisms had

4

following recipe: 4 g/L glucose; MgCl2.2H2O 0.1 g/L, CaCl2.2H2O 0.08 g/L, NH4Cl 0.53

5

g/L, 1 ml/L trace metals solution and 50 mM phosphate buffer (NaH2PO4 and Na2HPO4)

6

(Kakarla and Min, 2014). Trace metals solution was prepared as described elsewhere (Choi

7

et al., 2017).

8

2.3 Reactor setup and operation

9

The single chamber MESs were fabricated using transparent acrylic cylinders having a

10

working volume of 330 ml. Few sampling and gas collection ports were placed on the top

11

lid of the MESs. Carbon fiber brush with 2.5 cm diameter and 12 cm height (Kemsung

12

brush, Korea) was used as both anode and cathode electrode. An Ag/AgCl reference

13

electrode (+196 mV vs. SHE, Basi Inc., USA) was utilized for measuring cathode potential

14

during the MESs operation. A fixed external voltage of 0.8 V, which was recorded as the

15

optimum applied voltage for the MESs (Ding et al., 2015), was supplied to the MESs using

16

a DC power supply (Array 3645A, Circuit specialists Ln., USA). The negative lead of the

17

power supply was connected to the cathode; whereas the anode was connected with the

18

positive terminal. An external resistance of 10 Ω was loaded between two electrodes and

19

the cell voltage was recorded continuously in every 5 min by using an automatic digital

20

multimeter (National instrument 9205, USA).

21

Prior to the operation, the nutrient medium was mixed with sludge at ratio of 1:1 (based on

22

working volume), and then the whole growth solution was flushed with pure nitrogen for 7

1

20 min to remove the oxygen inside the reactors. After 6 days of operation when the cell

2

voltage decreased down to 1 mV and COD of the solution decreased considerably, the

3

nutrient medium with sludge (1:1) was refreshed. The medium inside the MESs were

4

continuously stirred at 250 rpm by using magnetic stirrer (ATL-4200, Anytech Co., Korea)

5

to avoid any mass transfer limitations. MESs were operated under mesophilic condition (35

6

± 2 °C) maintained by an incubator (Vision, Korea).

7

Magnetite NPs were added initially in the electrolyte suspension at a concentration of 1.55

8

g/L as Fe3O4 (or 20 mM as Fe atom). After each batch cycle, total iron and ferrous

9

concentration in the effluent were examined by using Fe (total) and Fe (ferrous)

10

measurement kits (HUMAS, Korea). Samples were diluted to an approximate range of 0-5

11

mg/L to match the kit recommended range. Because the Fe (ferrous) was detected in trace

12

amount in the medium at the last day of operations, Fe (total) concentration was used to

13

calculate a certain compensation amount of magnetite NPs in the new cycle in order to

14

maintain 20 mM of magnetite inside the reactors.

15

Along with magnetite-amended MES reactors (named MES-M), three different control sets

16

of reactors were constructed and run at similar conditions for performance comparison. The

17

first control reactors (named MES-C) were identical to MES-M, except for the absence of

18

magnetite NPs. The second control reactors (named AD-M) were anaerobic digesters with

19

the same 20 mM magnetite supplementation as compared to MES-M. The last control

20

reactors (named AD-C) were conventional anaerobic digesters without either electrodes or

21

magnetite supplementation. Each experimental treatment was conducted in duplicated and

22

the standard deviation was shown as error bars in the figures.

8

1

2.4 Analysis and calculation methods

2

Biogas was collected using gas bags and the composition of gas was measured using gas

3

chromatography (GC6100 series, Younglin, Korea) equipped with a thermal conductivity

4

detector (TCD) and Carboxen-1000 (Supelco, USA) column of mesh size 60/80. Nitrogen

5

gas of purity 99.99 % was used as carrier gas. The volume of gas reported over time was

6

converted to the value at standard temperature and pressure condition (STP, T=273 K, P=1

7

atm).

8

The modified Gompertz model was used to describe and evaluate methanation process in

9

term of biogas production kinetics (Anthony et al., 2016). The maximum methane

10

accumulation and methane production rate in MES-M and three control systems were

11

obtained by fitting the experimental data with the modified Gompertz function as shown in

12

Eq. (1) (Zwietering et al., 1990).

13

𝑦(𝑡) = 𝑦𝑚.exp ― exp

14

where 𝑦(𝑡) = the cumulative methane at a digestion time t in days (mL); 𝑦𝑚 = the methane

15

production potential (mL); µm = the maximum methane production rate (mL/day) ; λ = lag

16

phase period or minimum time to produce methane (days); t = cumulative time for methane

17

production (days); and e = mathematical constant (2.718282). The best values for kinetic

18

constants ym, µm, λ were obtained via nonlinear regression with “Solver” command in

19

Microsoft Excel by minimizing the sum of squares of the differences between the estimated

20

and the measured values (Anthony et al., 2016).

{

[

𝜇𝑚𝑒

𝑦𝑚 (λ

]}

(1)

― 1) + 1

9

1

Effluent from the MESs was filtered with 0.25 µm pore diameter membrane (Minisart RC,

2

Sartorious) for measurement of SCOD concentration using a medium range COD kit

3

(HUMAS, Korea). Samples were diluted to permissive range of 50-1500 mg/l before the

4

analysis. The pH of the sample was analyzed by PHM 210 pH meter (Radiometer); whereas

5

the volatile fatty acids (VFAs) composition was monitored by ion chromatography (Basic

6

792, Metrohm, Swiss) using organic acids column (Metrosep Organic Acids column) with

7

H2SO4 as a mobile phase.

8

Current (I) was calculated according to Ohm’s law (I=V/R). Coulombic efficiency (CE)

9

represents the amount of electrons transferred through the circuit which were converted into

10

reduced products. Theoretically, CE (%) was obtained by calculating the theoretical

11

coulombs consumed for methane formation over the total coulombs generated by the

12

substrate (based on COD removal) as shown in Eq.(2) (Zhen et al., 2015).

13

𝐶𝐸 (%) = 𝐹𝑉𝑚𝛥𝑆𝐶𝑂𝐷 × 100

14

where ∫𝐼𝑑𝑡 = total coulombs recovered as current I; 8 = the number of electrons generated

15

during the oxidation of 1g of COD; F = the Faraday constant (96,485.33 C/mol), Vm = the

16

working volume of reactor (L); and 𝛥SCOD = consumed SCOD (gCOD/L)

17

2.5 Electrochemical tests

18

The cyclic voltammetry (CV) was carried for MES-M and MES-C at day 0 and the last day

19

of cycle 5th using a potentiostat (Versastat 3, USA) with a conventional three electrodes

20

system consisting of cathode, anode and Ag/AgCl (+196 mV vs. SHE, Basi Inc., USA) as

21

working, counter, and reference electrodes, respectively. The CV measurements were

8∫𝐼𝑑𝑡

(2)

10

1

conducted with cathodic potential ranging from -1 V to 1 V (vs. Ag/AgCl) and a constant

2

scan rate of 10 mV/s (Noori et al., 2017). The electrochemical impedance spectroscopy

3

(EIS) analyses were pursued within a frequency range from 100 kHz to 100 mHz, and a

4

sinusoidal perturbation of 10 mV. The EIS spectra was simulated using Randles equivalent

5

circuit from Versa Studio software for the evaluation of charge transfer resistance (Rct) and

6

solution resistance (Rs).

7

2.6 Scanning electron microscopy and X-ray diffraction analysis

8

The morphology of magnetite NPs was observed using a field emission scanning electron

9

microscope (FESEM, Zeiss SUPRA, Germany). The samples were prepared following

10

previous protocol (Noori et al., 2018). Along with SEM, energy dispersive X-ray analysis

11

(EDX, Oxford, UK) was used to identify the elemental composition of the NPs. X-ray

12

diffraction analysis (XRD) was conducted using a D-max diffractometer (Rigaku

13

Corporation, Japan) with a Cu Kα radiation source (λ = 1.45 Å). The results of EDX and

14

XRD spectra was compared with the pattern of pure magnetite to further confirm the

15

successful synthesis of magnetite NPs.

16

SEM integrated with EDX analysis was used to gain insight on the interaction between

17

cells, magnetite, and electrode surface. For SEM observation, several pieces of carbon

18

fibers were cut off from the biocathode of MES-M and MES-C before and after the

19

operation. The samples were prepared following reported study (Miloslav et al., 2008).

20

Briefly, biofilm on cathode fibers was immobilized in a buffer fixative solution containing

21

3 % glutaraldehyde for 24 hours. Subsequently, fixed specimens were washed several times

11

1

with buffer, dehydrated with graded water-ethanol series, then dry and coated with

2

platinum before taking images.

3

3. Results and discussion

4

3.1 Current generation

5

The collected current profiles under an applied voltage of 0.8 V were depicted over the

6

operation time of a single chamber MES with (MES-M) and without (MES-C) magnetite

7

addition (Fig. 1). Both MESs exhibited current growth after 2 days of inoculation, but

8

MES-M was more efficient in achieving 5.4 mA at the first loading, which was about 2

9

times higher than that of MES-C (2.6 mA). In consecutive cycles of operation, the

10

continuous intensification in current generation was observed, confirming the successful

11

formation and stabilization of electroactive biofilm on the electrodes during this startup

12

period (Choi et al., 2017). At steady state, the highest achievable current value of MES-M

13

was 9.6 mA corresponding to the maximum normalized current density of 35.56 A/m3,

14

which was about 1.2 times greater as compared to that of 8.6 mA and 31.85 A/m3 obtained

15

from MES-C, respectively. The current density of MESs in this study was comparable to

16

the literature values of 18.89 A/m3 (Choi et al., 2017) and 14.2 A/m3 (Dou et al., 2018)

17

obtained from other AD-MES, possibly due to the differences in the reactor operating

18

conditions. It is proved that quantitative current generation is a prime indicator for the

19

relative electron transport efficiency from exoelectrogens to electrode and vice versa for the

20

electrochemical conversion of carbon dioxide to methane (Zhen et al., 2015). Therefore,

21

the higher current response in MES-M might indicate toward a better biofilm electrogenic

22

activity, suggesting that magnetite NPs possibly facilitated the electron transfer process by

12

1

supporting biofilm conductivity or/and increasing the biofilm attachment on electrodes. The

2

average coulombs transferred via external circuit during stable performance state of MES-

3

M (2029 ± 201 C) was also improved approximately 1.5 fold as compared to MES-C (1379

4

± 104 C). Similarly, the average columbic efficiency (CE) was enhanced over 1.5 fold,

5

from 19 ± 1.5 % in MES-C to 29 ± 2 % in MES-M. This CE value of MES-M was also

6

found to be higher than that of other MES performing electromethanogenesis at 23.1 %

7

(Eerten-Jansen et al., 2012) and 20.2 % (Zhen et al., 2015). The comparative analysis of CE

8

value obtained in MES-M with MES-C and other reported values in literatures clearly

9

demonstrates the enhancement of extracellular electron transfer in the addition of magnetite

10

NPs, which eventually promoted electrochemical methanogenesis by recovering additional

11

coulombs into methane (Noori et al., 2016).

12

3.2 Methane production

13

In order to assess the effects of magnetite nanoparticles on the methane generation, the

14

biogas potential of MES-M and three control reactors was evaluated by measuring methane

15

production (volume) as a function of time (Fig .2). The maximum methane accumulative

16

volume was obtained with MES-M (215 mL), followed by MES-C (191 mL), AD-M (181

17

mL), and conventional AD-C (144 mL). It can be seen that MES-C increased methane

18

volume by 34 % as compared with AD-C, whereas MES-M with magnetite NPs further

19

upgraded methane volume by about 54.1 %. The addition of magnetite NPs to AD-M also

20

improved methane production by about 26 % compared to AD-C, that agrees with

21

previously reported enhancement of AD ranging from around 10 % to 30 % with the

22

addition of magnetite NPs (Yin et al., 2018).

13

1

The modified Gompertz model is frequently used to describe the methane generation in a

2

close relationship with the growth of methane-producing microorganisms inside the

3

reactors. The key parameters obtained from modified Gompertz model (Table 1) revealed

4

the significant differences in kinetic biogas production of the digesters with and without

5

magnetite NPs addition. As shown in Fig .2, all the curves which represent the estimated

6

methane accumulation gave excellent fittings with the experimental data points with

7

correlation coefficient R2 ≥ 0.99, suggesting that the modified Gompertz model can be used

8

to predict the methane production rate and methane accumulation in the scaled-up studies.

9

According to the model results, the maximum methane accumulation (𝑦𝑚) and production

10

rate (µm) in MES-M (217.0 mL and 0.391 L/L-d, respectively) was observed to be highest

11

among four systems. The second corresponding lower methane accumulation was found to

12

be MES-C at 192.2 mL, followed by AD-M at 181.3 mL and AD-C at 143.4 mL,

13

respectively. Interestingly, the methane production rate in AD-M (0.341 L/L-d) was

14

estimated to be slightly higher than that of MES-C (0.320 L/L-d). The model predicted the

15

lag time of 0.59 d for AD-M and from 0.06 to 0.08 d for other digesters, which is

16

significant lower than that of other studies using same model, for example 6.5 d in

17

methane-producing MES (Yang et al., 2011) and 8.2 d in conventional AD (Choi and Lee,

18

2019). This is possibly due to the use of inoculum from AD fermentation tank in which the

19

microbial community was already developed. In addition, the inoculum was placed inside

20

the incubator for 6 h before using to activate the microbial activity (please refer to the

21

material and method section).

22

Regarding to methane yield (Fig. 3A), the highest value was obtained in MES-M at 0.37 ±

23

0.009 LCH4/gCOD, which was about 1.42 times higher than that of AD-C (0.26 ± 0.01 14

1

LCH4/gCOD). The methane yield of AD-M (0.31 ± 0.007 LCH4/gCOD) and MES-C (0.33 ±

2

0.004 LCH4/gCOD) also exhibited about 1.2 and 1.3 times higher than that of AD-C,

3

respectively. Taking the theoretical methane yield of AD process (0.35 LCH4/gCOD) into

4

consideration, the superior yield of MES-M (0.37 ± 0.009 LCH4/gCOD) clearly clarified the

5

huge contribution of electro-methanogenesis on overall methane enhancement. That value

6

was also higher than the achievable methane yield ranging from 0.31 to 0.36 LCH4/gCOD in

7

other MES without magnetite NPs utilization (Choi and Lee, 2019; Dou et al., 2018).

8

Comparing the methane yield of MES-M and AD-M, the presence of electrode system

9

significant enhanced the yield value by 0.06 LCH4/gCOD, which clearly pointed out the vivid

10

contribution of electrochemical reactions on the overall methane extension in MES-M. At

11

the end of each cycle, around 5.2 ± 0.4 mM of magnetite was found in the bulk solution,

12

indicating that most of nanoparticles were present around the electrode. Therefore, to

13

evaluate the origin of extra methane from bulk solution reactions, this 5.2 mM of

14

compensated magnetite was not added in cycle 7th, leading to the slightly decrease of

15

methane yield to 0.36 LCH4/gCOD. This value, however, was still considerable higher than

16

that of AD-M and MES-C, which confirms the main role of magnetite NPs associated with

17

electrode on facilitating electrochemical reactions for methane generation.

18

Along with methane accumulation, CO2 and H2 variation were simultaneously measured for

19

further investigation of methanogenic pathway. The CO2 levels in MES-M and MES-C

20

were always higher than that of AD-M and AD-C (Fig. 3B), which might result from the

21

more efficient acidogenesis and acetogenesis processes in bioelectrochemcial systems (Lee

22

et al., 2018). At these last days of the cycle, the CO2 levels decreased more rapidly in MES-

23

M compared to MES, suggesting more efficient cathodic CO2 reduction with the presence 15

1

of magnetite NPs. Recent studies about the route of extra methane in AD with magnetite

2

addition have proposed the ability of magnetite NPs in promoting direct interspecies

3

electron transfer (DIET) between exoelectrogens (Zhang and Lu, 2016). Although

4

magnetite NPs have not been tried in AD-MES to improve the methane production, several

5

studies performed in MFC system advocated that DET in the presence of magnetite was

6

enhanced during anodic oxidation processes (Liu et al., 2018; Peng et al., 2013). They have

7

found that magnetite NPs promoted the charge transfer process among exoelectrogens by

8

enhancing the electrode-cell-magnetite-cell networks as solid mediators. Considering the

9

excellency of magnetite NPs on electroactive biofilms in earlier reports, it can be postulated

10

here that the magnetite might play as electrical conduit among electron donor and electron

11

acceptor bacterial cells, which eventually enhanced electron transfer efficiency during the

12

electrochemical methanation process.

13

H2 was not detected in the off-gas of all tests throughout the operations possibly suggesting

14

that the facilitation of hydrogen interspecies electron transfer was limited in this study.

15

Yang et al., 2016 testified the promotion of hydrogen interspecies electron transfer in an

16

AD using 20 mM magnetite for improving the syntrophic ethanol oxidation. They found

17

that the dominance of hydrogenotrophic methanogenesis species (Fe(III)-reducers and

18

Methanobacterium) resulted in a significant increased amount of H2 in gas phase and Fe(II)

19

concentration in the medium. However, in several mix-culture utilizing AD and MES

20

systems, the absence of H2 in effluent gas was also reported due to the efficient H2

21

consumption of hydrogenotrophic methanogens (Cheng et al., 2009; Dou et al., 2018;

22

Flores-Rodriguez et al., 2019). In addition, the utilization of single chamber MESs in this

16

1

study made it difficult to quantify the portion of methane generated from hydrogen-

2

consuming pathway.

3

3.3 Substrate removal and VFAs composition

4

The maximum COD removal efficiency was observed in MES-M at 90.4 %, followed by

5

MES-C (90 %), AD-M (89.2 %) and AD-C (82.5 %), respectively. Previously, the

6

combined MES-AD was reported to assist the rapid substrate oxidation due to the

7

enrichment of exoelectrogens that capable for dependent respiration without electron

8

acceptors (Flores-Rodriguez et al., 2019). Interestingly, AD-M reached similar degradation

9

efficiency as MES-C, which showed about 6.7 % enhanced consumption rate as compared

10

to AD-C. This observation is in agreement with other studies using magnetite in AD tests.

11

For example, Lei et al., 2018 found that addition of magnetite in an AD treating municipal

12

solid waste incineration leachate at high organic loading, COD removal efficiency was

13

improved from 78.8 % to 89.0 %, mostly attributed from the increase of biofilm clusters

14

containing acetogenic and mixed acid fermentative bacteria. In this line, Yin et al (Yin et

15

al., 2018) also reported the enrichment of both acidogens (Proteiniclasticum and

16

Proteiniclasticum) in the conductive magnetite-sludge aggregates that answered for the

17

faster hydrolysis and acidification of protein-based synthetic water in the addition of 10 g/L

18

magnetite NPs. In this study, the addition of magnetite in MES system showed further

19

increase of COD removal by 7.9 % as compared to AD-M, which possibly correlated with

20

the quantitative increase of electroactive microorganisms on electrode site. In comparison

21

with MES-C, however, MES-M performed just slightly higher COD removal efficiency

22

about 0.4 %. Considering the much higher methane yield of MES-M as compared to MES-

23

C (0.37 vs 0.33 LCH4/gCOD ), the similar COD removal between two operations might 17

1

suggest that magnetite NPs presumably facilitated electrochemical reduction of CO2 for

2

methane generation rather than strengthening the decomposition of recalcitrant substrate.

3

The VFAs concentrations were determined as a function of operation time for all reactors

4

(Fig. 5). In all tests, the acetate was formed as the main intermediate byproduct, and other

5

VFAs including butyrate, propionate, and valerate were also observed. The maximum

6

concentration of total VFAs was recorded in AD-C after the first day of operation (around

7

1264 mgCOD/L), then it was gradually decreased until the end of all operations. The addition

8

of magnetite nanoparticles in AD-M or MES-M further decreased the highest total VFAs in

9

the early stage of the operation to 1103 and 1086 mgCOD/L, respectively. The presence of

10

magnetite contributed the degradation of VFAs, especially longer chain acids such as

11

propionate and butyrate (Jing et al., 2017). Recent studies suggested that adding magnetite

12

to AD possibly facilitate the methanation of propionic acid by promoting syntrophic

13

interaction of propionate metabolize species (Cruz Viggi et al., 2014). In this study,

14

magnetite addition in MES-M and AD-M both reduced the propionate concentration more

15

quickly as compared to the MES-C and AD-C, respectively. Additionally, MES-M with the

16

addition of electrodes systems represented the better propionate conversion rate possibly

17

due to the rapid utilization of H for the electrochemical methanogenic reactions on the

18

cathode side (Baek et al., 2017)

19

3.4 Electrochemical characteristic of MES in the presence of magnetite

20

CV analysis was performed to investigate the catalytic activity of cathodic biofilm and

21

further clarify the role of magnetite in improving the electrochemical behaviors of active

22

microbes in MESs. In the agreement with previous observation of Choi et al., the current

23

response of both MESs were negligible before the biofilm formation and then increased 18

1

significantly after the establishment of biofilm on the electrodes (Fig. 6A). This illustrates

2

the vital role of biofilm on the electrochemical reactions for methane production in MES

3

system. The CV profiles of MES-M and MES-C with biofilm exhibited clear redox peaks,

4

showing current exchange during Faradic processes at a scan rate of 10 mV/s (Noori et al.,

5

2017). The CV plot of MES-M depicted one pair of reduction current peak (2.23 mA) at

6

around -500 mV (vs Ag/AgCl) and the complementary oxidation current peak (2.89 mA) at

7

around -60 mV. Similarly pattern of CV profile was obtained for MES-C having formal

8

redox potentials around -420 mV and 4 mV and current response of 1.05 mA and 2.03 mA,

9

respectively. Based on the position of redox peaks appeared in this study, two redox

10

couples of MES-M and MES-C were possibly coherent with two different types of c-type

11

cytochromes OmcZ and OmcB electron carriers, that responsible for the electron transfer

12

between cells membrane and electrodes, and vice versa (Katuri et al., 2010). Therefore, it

13

can be postulated that the biofilm on the cathode of both the MESs had electrochemical

14

activity, which indicates towards (bio)electro-methanogenesis. It is also worthy to note that

15

voltammetry behavior can quantitatively determine efficiency of DET (Yin et al., 2018).

16

Thus, the higher peak current response of MES-M as compared to MES-C might suggest

17

the enhancement of DET for the recovery of electrons to methane. In addition, MES-M also

18

showed a larger background current both in reduction and oxidation scan with a value of

19

12.87 mA and 22.2 mA, respectively, which was noted slightly higher than that of MES-C

20

(11.6 and 18.0 mA, respectively). Background currents are related to the diffusion of

21

electrochemical specifies from electrolyte to electrode and vice versa. Higher diffusion

22

current in MES-M as compared to MES-C possibly indicates towards the mobility of

19

1

magnetite NPs from electrolyte to the electrodes that created a favorable conductive

2

environment to enhance the activity of electroactive microorganisms.

3

EIS analysis was conducted to evaluate the electrochemical resistance during the electron

4

transport process and to understand the activation energy profile of different MESs. The

5

EIS results were fitted in a suitable equivalent circuit to obtain simulated Nyquist plots

6

composing imaginary impedance at Y-axes and real impedance at X-axes. The distance

7

between origin to the appearance of first impedance data in X-axes represents solution

8

resistance (Rs); whereas the largest extent of semicircle quantifies the value of charge

9

transfer resistance (Rct) (Noori et al., 2018). The Rs and Rct values collectively depict the

10

performance of electrochemical reactions occurring at the electrodes. For example, the

11

lower the values of Rs and Rct, the higher the performance of bioelectrochemical system can

12

be expected due to lower hindrance in the mobility of charge from bulk solution to the

13

reaction interface (Noori et al., 2018). The obtained EIS data from MES-C and MES-M

14

was found to be best fitted to a simple Randles’s circuit and the simulated Nyquist plot with

15

the equivalent circuit is shown Fig. 6B. The Nyquist plot for both MESs shows well

16

defined semicircle in the high frequency region and a long tail in low frequency region

17

making a 45o angle with the imaginary component of the impedance. It reveals that both

18

MESs had diffusion controlled reactions supported by electroactive biofilm, which also can

19

be corroborated with the high diffusion current from CV results (Paul et al., 2018).

20

However, the MES with magnetite demonstrated significantly lower Rs and Rct values of

21

4.9 Ω and 0.11 Ω, respectively as compared to that of MES without magnetite (5.2 and 0.17

22

Ω, respectively). The Rs value generally represents the conductivity of the solution. The

23

low Rs value for MES-M as compared to MES-C is an obvious implication of 20

1

supplementation of conductive magnetite NPs in the suspension. The Rct on the other hand

2

demonstrates the catalytic activity of the microorganisms attached with the biofilm, which

3

is directly related to the activation overpotential of the desired electrochemical reaction, for

4

instance biomethanation reaction in this study. It is worth to note here that the EIS results

5

can give us the qualitative explanations of the performance system i.e. lower the Rs and Rct

6

value higher the output from the MES (Noori et al., 2019). Hence, based on the results, it

7

will be difficult to know the exact amount of methane that could be enhanced due to

8

increase in solution conductivity and reduction in charge transfer resistance. However, the

9

lower Rct value in MES-M pointed out the less activation overpotential losses during the

10

biomethanation process, which therefore aided the MES-M to enhance methane generation.

11

3.5 SEM observation

12

SEM analysis was conducted to observe the morphology of cathode of MES-M and MES-

13

C. The carbon fibers showed a smooth surface structure before the microbial inoculation.

14

After 5 repeated cycles in both MES-C and MES-M, diverse cells were observed on the

15

cathodes in rod, spiral and spherical shape co-existed with extracellular polymeric

16

substances (EPS) layers. Although it is hard to determine the exact difference in the

17

biomass amount of electrodes with and without magnetite NPs, the cathode of MES-M

18

seemed to have a richer biofilm with magnetite-like spherical particles covering the carbon

19

fibers. This observation was likely in coherence with the higher current and methane yield

20

observed in MES-M in this study. The SEM image of pure magnetite NPs shows spherical

21

particles with around 10 nm diameter. In biofilm of MES-M cathode, magnetite NPs was

22

found to be attached with EPS layer making a strong interaction with microorganisms. This

23

agrees with EDX energy peaks patterns of different elements attached with biofilm that 21

1

confirmed the presence of magnetite on biocathode. The energy peak of Fe was found to be

2

more significant in MES-M with a mass percentage of 3.97 % as compared to 0.35 % in

3

MES-C. It means that the MES-M biofilm had more Fe compound, in fact due to the

4

external addition of magnetite NPs. This observation further advocated that magnetite NPs

5

attached with the electroactive biofilm and might act as electric conduit to facilitate direct

6

electron transfer during methanogenesis.

7

Implications

8

This study firstly demonstrated that the application of 20 mM magnetite NPs in the

9

suspension of single chamber MESs can facilitate methane production, current generation,

10

and VFAs consumption rate, which are prime indicators determining the efficiency and the

11

stable operation of MESs system. In our results, the overall enhancement of MES-M lies on

12

the stimulation of electrochemical reactions on electrode site, which was determined by

13

comparing methane generation of MES-M after stop adding around 5.2 mM compensated

14

magnetite in the medium. In addition, electrochemical methods showed a decrease of

15

overpotential losses during the electron transfer process, together with SEM images

16

showing the connection among magnetite-bacterial cells-electrode that strongly supports

17

the hypothesis that magnetite might play as electrical conduit between electron donors and

18

electron acceptors to facilitate the DET for methane generation. Although the microbial

19

community analysis was not performed in this study, it is reported that the microbial

20

structure extremely reflects the pathway in which methane is produced (Mateos et al.,

21

2020). Hence, further studies on community analysis are needed to clearly understand how

22

the microbial species interact in the presence of magnetite NPs. In addition, the mechanism

23

behind the migration of magnetite NPs from suspension to the biofilm EPS matrix is not 22

1

clearly understood. Also, the dispose of effluent wastewater containing a certain amount of

2

magnetite can be a critical problem in scale-up application, either in cost or environmental

3

aspects. To date, magnetite with its magnetic property was proved to be recycle efficiently

4

by magnet in recent anaerobic digesters, that make it economically viable (Baek et al.,

5

2017). However, developing a new electrode containing magnetite NPs as a catalyst to

6

enhance DET could be another strategy to increase the stability and economic feasibility of

7

the system.

8

Conclusions

9

Supplementation of magnetite NPs in suspension enhanced methane yield of the MESs up

10

to 0.37 ± 0.009 LCH4/gCOD, which was significant higher than the theoretical methane yield

11

of conventional AD (0.35 LCH4/gCOD) and obtained yield of other control operations in this

12

study. MESs with magnetite enhanced cathodic current production with significantly less

13

Rs and Rct value as compared to the MESs without magnetite, which supports that the

14

magnetite presumably played as electrical conduit to promote DET between electrode and

15

electroactive microorganisms, which lowered the activation overpotential losses during the

16

bioconversion of methane.

17

Acknowledgement

18

This study was carried out with a grant from the National Research Foundation of Korea

19

(2018R1A2B6001507).

20

Appendix A. Supplementary

21

E-supplementary data for this work can be found in e-version of this paper online.

22 23

1 2

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assessment of biogas production from co-digestion of horse and cow dung. Res.

3

Agric. Eng. 57, 97–104.

4

47. Zhang, J., Lu, Y., 2016. Conductive Fe3O4nanoparticles accelerate syntrophic

5

methane production from butyrate oxidation in two different lake sediments. Front.

6

Microbiol. 7, 1–9. https://doi.org/10.3389/fmicb.2016.01316

7

48. Zhen, G., Kobayashi, T., Lu, X., Xu, K., 2015. Understanding methane

8

bioelectrosynthesis from carbon dioxide in a two-chamber microbial electrolysis

9

cells (MECs) containing a carbon biocathode. Bioresour. Technol. 186, 141–148.

10 11 12

https://doi.org/10.1016/j.biortech.2015.03.064 49. Zwietering, M.H., Jongenburger, I., Rombouts, F.M., van ’t Riet, K., 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–81.

13 14 15

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LIST OF TABLE

1 2 3

Table 1. Modified Gompertz parameters predicted for the tested operations. Modified Gompertz model parameters Operations

ym (mL)

µm (L/L-d)

λ (days)

R2

MES-M

217.0

0.391

0.08

0.9997

MES-C

192.2

0.320

0.07

0.9982

AD-M

181.3

0.341

0.59

0.9966

AD-C

143.4

0.224

0.06

0.9979

4

32

1

LIST OF FIGURES

2

3 4 5

Fig. 1. Current generation respects with time in MES-M (with magnetite) and control MES-C (without magnetite) operations.

6 7 8 9 10 11 12 13

33

Methane accumulation (ml)

250

200

150

100

MES-M MES-C AD-M AD-C

50

0 0

1

2

3

4

5

6

7

Time (d) Fig. 2. Comparison of experimental data and modified Gompertz model for methane accumulation. Scatters represent experimental data and lines represent nonlinear estimated by modified Gompertz model.

1

34

Fig. 3. Comparison of (A) methane yield and columbic efficiency and (B) dynamics of CO2 in MES-M and three control operations.

1

35

2500

MES-M MES-C AD-M AD-C

SCOD (mg/l)

2000

1500

1000

500

0 0

1

2

3

4

5

Time (d) Fig. 4. SCOD removal with respect to time in MES-M and three control operations. 1 2

36

6

1

2 3 4

Fig. 5. VFAs variation during the operation of (A) control AD-C, (B) control AD-M, (C) control MES-C, and (D) MES-M operation.

5

37

1

2 3Fig. 6. Cyclic Voltammetry profiles (A) and Nyquist plot showing electrochemical impedance 4

data (B) of MES-M and control MES-C operations.

5 6 7 8 9 10

Highlights

11 12



Methanogenesis in MES was significantly enhanced with magnetite nanoparticles

13



Magnetite nanoparticles promoted current generation, substrate and VFAs removal

14



Overpotential losses of MES was decreased with magnetite nanoparticles addition

15



Magnetite may act as electrical conduit for extracellular electron transfer

38

1 2 3 4

GRAPHICAL ABSTRACT

5

6

Conductive magnetite nanoparticles trigger syntrophic methane production in

7

single chamber microbial electrochemical systems

8

Mung Thi Vu1, Md Tabish Noori1, Booki Min1*

9

1Department

of Environmental Science and Engineering, Kyung Hee University, Seocheon-

10

dong, Yongin-si, Gyonggi-do 446-701, Republic of Korea.

11

*Corresponding author: Phone: +82-31-201-2463; Fax: +82-31-202-8854; Email:

12

[email protected]

39

1 2

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