Accepted Manuscript

Electrically regulating co-fermentation of sewage sludge and food waste to-

wards promoting biomethane production and mass reduction

Zhongxiang Zhi, Yang Pan, Xueqin Lu, Guangyin Zhen, Youcai Zhao, Xuefeng
Zhu, Jianying Xiong, Tianbiao Zhao

PII: S0960-8524(19)30176-2
DOI: https://doi.org/10.1016/j.biortech.2019.01.142
Reference: BITE 21019

To appear in: Bioresource Technology

Received Date: 29 December 2018

Revised Date: 29 January 2019
Accepted Date: 30 January 2019

Please cite this article as: Zhi, Z., Pan, Y., Lu, X., Zhen, G., Zhao, Y., Zhu, X., Xiong, J., Zhao, T., Electrically
regulating co-fermentation of sewage sludge and food waste towards promoting biomethane production and mass
reduction, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.01.142

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 A manuscript submitted to Bioresource Technology

Electrically regulating co-fermentation of sewage sludge and food

waste towards promoting biomethane production and mass reduction

Zhongxiang Zhi a, Yang Pan a, Xueqin Lu a,b, Guangyin Zhen a,c*, Youcai Zhao d,

Xuefeng Zhu e, Jianying Xiong f, Tianbiao Zhao g

a. Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration, School of
Ecological and Environmental Sciences, East China Normal University, Shanghai
200241, PR China
b. Institute of Eco-Chongming (IEC), 3663 N. Zhongshan Rd., Shanghai 200062, PR
China
c. Shanghai Institute of Pollution Control and Ecological Security, 1515 North
Zhongshan Rd. (No. 2), Shanghai 200092, PR China
d. The State Key Laboratory of Pollution Control and Resource Reuse, School of

Environmental Science and Engineering, Tongji University, 200092, Shanghai, PR

China.

e. School of Environmental and Material Engineering, Shanghai Second Polytechnic

University, Shanghai 201209, PR China

f. Shanghai Municipal Engineering Design Institute (Group) Co., Ltd, Shanghai

200092, PR China

g. Shanghai Waterway Engineering Design and Consulting Co., Ltd, Shanghai 200120,

PR China

*Corresponding author: Guangyin Zhen

E-Mail address: [email protected].

7
Abstract

Microbial electrolysis cell (MEC) was integrated into conventional anaerobic

digestion (AD) system (i.e. MEC-AD) to electrochemically regulate the co-

fermentation of food waste (FW) and sewage sludge (SS). Two anaerobic systems (i.e.

MEC-AD, and single AD) were operated in parallel to explore the potential

stimulation of electrical regulation in metabolic behaviors of FW and SS and

subsequent biomethane production. The highest accumulative methane yield was

achieved at an applied voltage of 0.4 V and the FW and SS ratio of 0.2: 0.8,

increasing by 2.8-fold than those in AD. The combined MEC-AD system mitigated

N2O emission and considerably improved ammonia removal and the dewaterability of

digestate, in contrast to AD. Scanning electron microscope (SEM) visualized the

presence of a large number of rod-like and cocci-like electroactive microbes on the

electrode surface. Electrical regulation stimulated the self-growth and proliferation of

typical Methanobacterium and Methanosaeta, accordingly contributing to biomethane

production greatly.

Keywords: Anaerobic digestion; Microbial electrolysis cell; Methane production rate;

Co-digestion; Electron transfer

8
1. Introduction

With the continuous advancement of urbanization in China, the urban population are

increasing quickly and the energy needs more (Luo et al., 2018). However,

superabundant energy consumption and limited fossil fuel storage force researchers to

search for more alternative energy sources, especially renewable energy. According to

IEA’s recent report, the use of renewable energy will account for 60% of the total

energy supply by 2040 all over the world (IEA, 2016). It is assumed that food waste

(FW) yield would be 4.16 billion tons in Asian countries by 2025 (Ren et al., 2018).

The mount of FW in China was about 60 million tons in 2011, and it is increasing by

more than 10% every year with the acceleration of industrial development and

urbanization processes (Zhang et al., 2016). Also, according to China Statistical

Yearbook-2018, about 70.0 billion tons of wastewater was generated in China in

2017, about 1-2% of which was transferred into sewage sludge (SS) during

conventional activated sludge process (Zhen et al., 2017a).

Anaerobic digestion (AD) is a well-studied method for biowastes disposal and

renewable energy recovery, based on the metabolism and interspecies interactions of a

diversity of microorganisms (Huang et al., 2017). The co-digestion cannot only

enhance AD performance of SS and FW but also simplify the downstream processing

of digestate (Xie et al., 2018). Whereas, co-digestion possesses some unfavorable

factors such as excessively long solids retention time (SRT), low organic removal rate

and methane yield, to a certain degree limiting the application of this technology

9
(Chen et al., 2016; Liu et al., 2016b). It is well-accepted that hydrolysis is the key

limiting step for methane production, namely slow conversion of complex organics

into available substrates for Methanogens (Astals et al., 2013; Zhang et al., 2016). To

overcome the thermodynamic limitations, different kinds of pretreatment methods

such as mechanical (e.g. ultrasonic, microwave etc.), thermal, chemical (e.g. alkali,

zonation etc.), and biological options were investigated to enhance the efficiency of

hydrolysis so as to accelerate renewable energy recovery (Zhen et al., 2017a). But the

above pretreatment methods either need high energy input or high chemical cost

(Zhen et al., 2017a), which requests researchers to develop more cost-efficient means

to decompose biowastes.

A new option called microbial electrolytic cells (MEC) has been intergraded into

the AD process (i.e. so-called MEC-AD or electro-digestion) for boosting biogas

production (Kadier et al., 2016). The electrochemical controlling made by fixed

external voltage via current circle can serve as electron sinks or sources for redox

reactions in AD process (Moscoviz et al., 2016). Adaptive microorganisms from mixed

cultures would be selected by electrical regulation or stimulation, which would enhance

not only the interactions among microorganisms but the extracellular electron transfer

between microorganisms and the surface of electrodes via cellular mechanisms

(Moscoviz et al., 2016; Zhen et al., 2017b). The application of this technology could

promote the production of biogas (e.g. methane, hydrogen) (Bakonyi et al., 2018;

Beegle and Borole, 2017; Kumar et al., 2017; Rozsenberszki et al., 2017; Zhou et al.,

10
2017). In one of our previous researches, a single-chamber MEC has been continuously

run for around 150 days to provoke the degradation of Egeria densa and bioenergy

recovery, and the electrochemical process indeed made the Egeria densa fermentation

stabilized and improved biomethane production effectively, e.g. the average methane

yield of approximately 248.2 ± 21.0 mL/L/d at 1.0 V (Zhen et al., 2016). Similarly, Liu

et al. (2016a) used raw sludge as substrate for MEC reactor for methane production and

its rate was 1.3 times higher than AD. Not only that, various kinds biowastes have been

employed in MEC-AD system such as Fischer-Tropsch wastewater (Wang et al., 2017),

soybean edible oil refinery wastewater (Yu et al., 2017), beer wastewater (Guo et al.,

2016), table olive brine processing wastewater (Marone et al., 2016), pig slurry

(Cerrillo et al., 2016), fresh incineration leachate (Gao et al., 2017), agricultural wastes

(Zhou et al., 2016), etc. However, there were few reports about the electro-co-digestion

of FW and SS in MEC-AD reactor.

In this study, two continuous anaerobic systems (i.e. MEC-AD, and single AD)

were operated in parallel to evaluate the effect of electrical stimulation on co-digestion

behaviors of FW and SS. The combined MEC-AD aimed at omitting pretreatment

process and achieving in-site enhancement of hydrolysis and methane evolution in

consideration of high cost for biowastes pretreatment. The N2O emissions during the

entire process was detected and the dewaterability of digested solids was determined to

minimize the cost for digestate dewatering and final disposal. Scanning electron

microscope (SEM) and 16S rRNA gene pyrosequencing were conducted to reveal the

11
molecular mechanisms involved within the bioelectrochemically enhanced anaerobic

co-digestion of FW and SS.

2. Material and methods

2.1 MEC-AD and AD system configuration

A single chamber MEC-AD reactor was set up in this research with the size of 6.8 cm

× 11.35 cm (diameter × height), and the working and total volume of 180 and 310

mL, respectively. The port was equipped at the bottom of the reactor for substrate

injecting and sampling. The anode was carbon brush (φ 2.2 cm × 8 cm); the cathode

was a Ti/RuO2 (2 cm × 2.5 cm), which was physically covered with a piece of

graphite felt (0.8 cm × 5.0 cm × 7.2 cm). The distance between two electrodes was set

as 2 cm. Two electrodes were connected to a direct current (DC) power supply

(Lodestar, China) via wire to provide the external voltage. A 1-L gas bag with two

valves was used for biogas collection. For investigating the contribution of the

electrode biofilm on methane production, a single AD reactor with the same

configuration was set up without the use of external power.

FW used in this study was sampled from a canton in East China Normal

University and SS was collected from a Wastewater Treatment Plant (WWTP) located

in Shanghai, China. FW included rice, steamed bun, vegetables, meat etc. These two

substrates were reserved at 4 °C and the FW homogenized in a blender with some tap

water joining. The hybrid co-digestion substrate was still placed in refrigerator at 4 °C

12
and prepared once a week to prevent metamorphism. Table 1 lists the basic

characteristics of FW, SS and co-substrates with different FW: SS ratios.

Table 1.

2.2 Long-term operation

Before the start-up, two reactors were purged with high-purify N2 gas for 5 min to

ensure the anaerobic environment (Choi et al., 2017). The MEC-AD reactor and

single AD reactor (Control) were operated at 37 °C provided by electric heating

thermostatic water bath (YCX-6, Moer, China) for 95 d. The whole operational period

(95 d) was divided into seven periods (i.e. I, II, III, IV, V, VI and VII). The period I

from day 1 to day 14, and period II, III, IV, V, VI and VII were day 15-28, day 29-43,

day 56-69, day 76-85 and day 86-95, respectively. SRT was maintained at 15 d once

the reactor was started up successfully (i.e. SRT 20 d in start-up phase). Throughout

the entire process, the imposed voltage was gradually elevated from 0 V, to 0.2, 0.4,

0.8 and then to 1.2 V. Due to the occurrence of anode damage caused by over-high

voltage (1.2 V), the voltage was re-set to 0.8 V shortly. During the first five periods,

the ratio of FW: SS was fixed at 0.2: 0.8 based on volatile solids (VS), and it was

further increased to 0.4: 0.6 (VI), and then to 0.5: 0.5 (VII) at 0.8 V to explore the

influence of FW: SS ratio on methane production. The voltage was measured over a

10 Ω resistance twice a day inserted in the circuit, using a digital multimeter

(PC7000/PC720M, Sanwa Electric Instrument, Tokyo, Japan), and the current was

calculated via Ohm’s law.

13
2.3 Analytical methods

2.3.1 General parameters detected methods

Total solids (TS), volatile solids (VS), total chemical oxygen demand (TCOD),

soluble chemical oxygen demand (SCOD), total protein (TPN), soluble protein (SPN),

total polysaccharose (TPS), soluble polysaccharose (SPS), water content, ammonia

nitrogen (NH4+-N), alkalinity and pH were determined on the basis of the Standard

Methods (APHA, 1998). Volatile fatty acids (VFAs) were analyzed by a gas

chromatograph (Agilent, 7890GC-5975MS, USA) with a capillary column equipped

with a FID. Before GC analysis, the VFAs samples were centrifuged at 8000 rpm/min

and filtered through 0.45 mm membrane filters. The equipped gas collection bag

volume was 1 L, and the produced biogas was measured by a class syringe. The

content of methane (CH4), carbon dioxide (CO2) and nitrous oxide (N2O) were

analyzed by a gas chromatograph (Agilent, 7890A, USA), with a packed column and

a TCD detector. The digestate dewaterability was measured via a capillary suction

timer (Type 304M, England) equipped with a 0.535-cm inner diameter funnel and

Triton CST paper (7 × 9 cm, Electronics Ltd., England). The Hitachi S-4800 field

emission SEM was used to visualize the microstructural morphology of electrode

surface, and the SEM samples obtained from anode and cathode materials were

pretreated in accordance with previously reported method (Zhen et al., 2014).

14
2.3.2 DNA extraction and 16S rRNA gene pyrosequencing for microbial community

analysis

The microbial community analysis was based on a high-throughput sequencing

platform. The sequencing of 16S rRNA regions can determine the species diversity

and composition of microbial community in samples. Biomass samples were taken

from anode carbon brush, cathode graphite felt and digested liquid using sterile

scissors and then were kept in an ultra-low temperature refrigerator at -80 °C before

DNA extraction, PCR amplification and pyrosequencing. Before DNA extraction, the

solid samples were slightly rinsed with deionized water to remove the adsorbed

digestate. The liquid samples were centrifuged at 8000 r/min to remove supernatant.

According to the manufacturer's instructions, DNA was extracted using a rapid soil

DNA isolation Kit (12888-100, MOBIO Laboratories, Carlsbad, CA, USA) and was

quantified for PCR amplification (Lee et al., 2017). The primers were used to target

the V4 region of the 16S rRNA gene, with the extracted DNA as a template and the

used primers were 515F (5’-GTGCCAGCMGCCGCGG-3’) and 806R (5’-

GGACTACHVGGGTWTCTAAT-3’). Both PCR amplicons and PCR products were

confirmed via 1% agarose gel electrophoresis and visualized utilizing a Gel Doc

system. Then the quantified PCR products were send to pool for sequencing, which

was carried out by MAGIGENE Inc., Shanghai, China.

2.3.3 The calculation of energy conversion balance

The energy conversion efficiency was calculated based on the input electric energy

and output chemical energy. The input electric energy is referred to the electric energy
15
consumed in the MEC-AD system. The output energy is equal to the thermal energy

from the complete burning of net methane production. The net methane recovery was

calculated via subtracting the methane production in single AD process from the

methane production of the combined MEC-AD system (Eqs. (1-2)).

𝑡
̅𝐼 ̅
𝑊𝐸 = ∫0 𝑈 (1)

∆𝐻𝑠 （𝑉𝑀𝐸𝐶−𝐴𝐷 −𝑉𝐴𝐷 ）

𝑊𝐶𝐻4 = (2)
𝑉𝑚

where, WE is electric energy (J), t is operational time (s),U is average voltage

(V) andI is mean current (A), WCH4 is the output energy (J), ΔHs is complete

combustion heat of methane (890.31 kJ/mol-CH4), VMEC-AD is the methane production

of combined system (mL), VAD is the methane production of single AD process (mL)

and Vm is gas molar volume (22.4 L/mol).

3. Results and discussion

3.1 Current generation and methane production

The current variation with the applied external voltage was shown Fig. 1a. As can be

seen, the current showed a gradual rise with the applied voltage. The increase in current

reflected the successful development of electroactive biofilms and the increase in

extents of electrochemical reactions occurring on both electrodes. Fig. 1b-c showed

methane production behaviors throughout the whole process. The data of methane

production during the initial period (0-14 d) was not shown because of leakage of

equipment. In phase II, the accumulative methane production in both MEC-AD system

and single AD process showed the obvious difference (44.3 mL/L-reactor and 72.0 mL/L-

16
reactor), presumably also caused by leakage of equipment. From the phase III, a small

voltage of 0.2 V was poised in MEC-AD system, which induced an accumulative

methane production of around 58.9 mL/L-reactor, in sharp contrast to 49.6 mL/L-reactor in

single AD process. This result suggested that MEC-AD has been started up successfully.

During this phase, electroactive fermentative microorganisms were selected and

enriched with the help of electrical regulation, which thus motivated the solubilization

of particulate organics and the subsequent hydrolysis and methanogenesis processes

(Liu et al., 2016b). Whereas, the methane yield was still unstable and irregular during

the first 40 days (period I-III). When the external voltage was increased to 0.4 V at FW:

SS ratio of 0.2: 0.8 (i.e. phase IV), the accumulative methane production in MEC-AD

system was 2.8-times higher than the single AD process, and it was 1.9-times at 0.8 V

(phase V), comparably higher than the previous researches. For example, Park et al.

(2018) created a MEC reactor and the substrate was FW. The methane production was

1.7 times higher than AD at 0.3 V and 35 ℃. In addition, in the research of Chen et al.

(2016), the application of MEC improved methane production 76.2% than AD at o.6 V.

Its substrate was SS and the temperature kept at 35 ± 2℃. In the following phase, the

external voltage was elevated to 1.2 V. The anode showed the serious damage because

of the high external voltage (1.2 V), which leaded to short circuit of MEC-AD reactor

and the deteriorated methane production (period VI). After repairing anode and

adjusting the ratio of FW and SS (period VII), MEC-AD reactor was recovered.

Methane production became to increase, but was obviously lower compared with before.

17
 Nonetheless, the average methane production rate of MEC-AD in period VII was

13.6 mL/L-reactor while nearly no methane was detected in AD reactor. In terms of

accumulative methane production in each phase, an obvious ever-decreasing trend

was observed, remarkable decrease in methane production for both reactors in period

VII might be presumably attributed to high grease, salinity, and low pH caused by

high proportion of FW added (Park et al., 2018). In spite of this, this result

demonstrated that the use of electrochemical stimulation can indeed improve the

performance of AD process. In-situ electrochemical stimulation enriched putative

exoelectrogens at the anode biofilm and hydrogenotrophic methanogens at the

cathode biofilm, thereby effectively promoted methane production and process

stability (Dou et al., 2018; Liu et al., 2016b). As a result, the combined MEC-AD

system produced higher methane than the single AD process. Based on the above

observations, the optimal operational conditions for the co-fermentation of FW and

SS in MEC-AD system can be SRT15 d, voltage 0.4 V and FW: SS 0.2: 0.8.

Fig. 1. and Table 2

3.2 Greenhouse gas (N2O) emissions

N2O is an undesirable intermediate in denitrification and it can strongly destroy the

ozone sphere (Liu et al., 2018). Nitric oxide reduction caused by microorganisms is

the main source for N2O, whose ozone-depleting capability is 310 times potential than

carbon dioxide (Saraiva et al., 2004). Thus, a process with low N2O emissions will be

more preferred. As can be seen in Fig. 2, low N2O emissions and high ammonia

18
removal were observed during the steady-state condition (after day 40). MEC-AD

system had higher average N2O yield rate (2.4 × 10-4 mL/L-reactor/d) during period III

compared with 8.9 × 10-5 mL/L-reactor/d in single AD process, but it showed a clear

decline (i.e. 1.2 × 10-4 mL/L-reactor/d) during period IV. On contrary, the average N2O

yield in single AD profoundly increased to 5.3 × 10-4 mL/L-reactor/d. In period V, VI

and VII, N2O production rate of combined MEC-AD system still kept much lower

than that of single AD process, revealing that the external voltage was able to mitigate

the emissions of N2O effectively. As for ammonia nitrogen, the average concentration

in single AD process in period III and IV, was 1.3 and 1.2 times higher than MEC-AD

system, respectively. The similar phenomenon was also observed during the period V,

VI and VII, strongly reflecting that combined MEC-AD system could reduce

greenhouse gas emission and improve ammonia removal efficiencies. The microbial

transformation of nitrogen compounds consists of 14 redox reactions, involving 8 key

inorganic nitrogen species of different oxidation states. Ammonia nitrogen is

converted into N2 mainly through the nitrification and the denitrification or the

anaerobic ammonium oxidation (Kuypers et al., 2018). In nitrification, a molecule of

ammonia is oxidized to nitrite, which is then oxidized to nitrate. As for denitrification,

the whole process can be expressed as NO3- → NO2- → NO → N2O → N2. The N2O

reduction to N2 is manly dependent on nitrous oxide reductase (NOS). The decrease in

N2O content reflected that the use of external voltage might have promoted the

secretion of NOS and the conversion of N2O to N2, thereby mediating the N2O

19
emissions. A small amount of N2O was still detected, indicating the incomplete

reduction. The possible reason for such phenomenon might be the inhibition of NOS

in complete denitrifies caused by environment (e.g. oxygen, pH, sulfide, etc.);

besides, the presence of incomplete denitrifies could also lead to the imbalance

between N2O production and reduction (Kuypers et al., 2018). Nonetheless, more

works are still urgently required to disclose the underlying mechanisms for N-

metabolic behaviors in combined MEC-AD system.

Fig.2.

3.3 Alkalinity/pH variations and solids removal

The pH and alkalinity are important parameters indicating the buffering capacity of

anaerobic digestion to warn instability of the process (Wang et al., 2018). Throughout

the whole process, there were no obvious fluctuations in both pH and alkalinity in

both reactors (Fig.3 a-b). Overall, the pH fell within a range of 6.9-7.7, suitable for

the growth of anaerobic microorganisms (fermentative bacteria: 4.0-8.5 and

methanogens: 6.5-7.2 (Zhang et al., 2014)). Total alkalinity maintained at 1000-1500

mg-CaCO3/L, indicating that both reactors run well.

In order to distinguish the differences in the performances between two reactors,

the average TS, VS, COD removal efficiencies in each period were calculated (Table

2). The effluent TS and VS in MEC-AD system showed much lower levels, even such

enhancement was not statistically significant. In period IV, the TS removal efficiency

in MEC-AD system and single AD process were 20.7 ± 28.8% and 5.2 ± 6.5%,

20
respectively. Similarly, the VS removal were 46.6 ± 25.0% and 35.3 ± 11.6%,

respectively. In period IV, both TS and VS removal showed high efficiency, as

observed in period V. In period VI, the effluent TS and VS removal efficiency of

MEC-AD were 15.2 ± 17.8% and 49.8 ± 26.6%, respectively, which were somewhat

better than that of single AD process (21.1 ± 10.8% and 47.7 ± 20.8%, respectively);

but after repairing, they gradually surpassed the single AD in the last period.

Therefore, the destruction of anode might reduce the rate of organics decomposition.

The considerable enhancement in solids removal might be closely related the

inoculation and enrichment of exoelectrogens and electrotrophs on both electrodes of

the MEC-AD system during the in-situ electrical stimulation. Those electroactive

microorganisms own more robust biological degradation capacity in complex organic

matters, which subsequently accelerated the decomposition and degradation of

organic solids, and supported methane production. Low solids left in the effluent will

be very important from the view of engineering, considering the cost for final disposal

and the potential environmental impact. Similar results were also observed in protein

and carbohydrate removal.

Table 3. and Fig. 3.

3.4 Organics solubilization and biodegradation

As can be seen in Fig.4 a-f, the concentration of COD, protein and polysaccharide in

the combined MEC-AD system were lower than those in the single AD process. The

average TCOD removal efficiency of combined MEC-AD system in period III and

21
period IV were 1.83 times and 1.28 times higher than those of single AD process,

respectively. Similarly, TPN removal efficiency of MEC-AD and AD in period III

were 26.4% and 5.6%, respectively, and in period IV, the efficiency was 28.0% and

12.8%, respectively. In all periods, the TPS removal efficiency of both reactors were

more than 50%. Obviously, PS was easy to be metabolized by microorganisms. FW

contains a high content of PS, thus the co-digestion of SS and FW were able to offset

the shortage of low biodegradability of SS. Specially, the bioelectrocatalysis promoted

organics conversion, which was more conducive to the uptake and metabolism by

electroactive bacteria for methane generation.

VFAs concentration are given in Fig. 4g-h. The addition of FW led to the

increase in VFAs production. In period I, VFAs content of the two reactors were

almost the same. Acetic acid was 60.0 mg/L and 69.4 mg/L, and propionic acid was

55.1 mg/L and 69.5 mg/L, respectively. At this moment, the acetotrophic

methanogens translated such simple acids into methane, which caused the rapid

decline of acetic acid. As operational time went on, the VFAs were gradually

produced and consumed. Whereas, the VFAs content kept stable initially and then

slightly increased. After day 40, the VFAs content in MEC-AD were slightly lower

than that of AD. The average acetic acid content of MEC-AD and AD were 7.0 ± 1.5

mg/L and 7.5 ± 4.0 mg/L, respectively. Meanwhile, the average propionic acid content

were 6.9 ± 3.2 mg/L and 7.6 ± 3.5 mg/L, and the average n-butyric acid content were

12.2 ± 5.2 mg/L and 13.1 ± 7.4 mg/L, respectively. This indicated that electrical

22
stimulation could facilitate the solubilization of particulate organics, favoring

subsequent acidification and methanogenesis, in line with the biomethane production

tendency. It was probably because that the external voltage enhanced the extracellular

electrons transport between electrodes surface and microorganisms and the direct

interspecies electrons transport.

Fig. 4.

3.5 Characterizations of microbial biofilm growth on the bioelectrodes

The SEM analysis was conducted to visualize the microbial biofilm growth on the

bioelectrodes. The graphite felt fiber and carbon brush had lumpy-looking surface and

plenty of carbon fiber silk on the surface. The great superficial area provided a

sufficient space for electroactive microorganisms to grow and adhere. The intimate

contact enhanced the electron transfer between microorganisms and electrodes. It is

crucial for MEC-AD system to choose high-surface electrode materials with good

conductivity, otherwise the growth of functional microorganisms would be impacted

(Zhen et al., 2018).

Cocci-shaped, rod-shaped microbial cells were obviously identified. Meanwhile,

some gel-like components were also observed, possibly belonging to the

extracellularly metabolic products of the microorganisms. Similarly, plenty of rod-

shaped microorganisms and zoogloea were also prevailing on the cathode graphite felt

fiber. The microorganisms attached to each other closely on the electrode surface,

either enhancing the electrons exchange between electrode and microorganisms or

23
being beneficial to the direct interspecies electron transfer. The observations showed

that microbial biofilm grew and cooperated well on the electrodes surface. Note that

the electrode materials surface is easy to be contaminated by some impurities or

inorganic particles. Shell-like structure was formed on the surface of anode carbon

brush where functional electroactive bacteria live and work. Thus, how to

avoid/eliminate the contamination of electrode surface in long-term operation is a new

research direction in future works.

3.6 Microbial community analysis and speculated mechanism of MEC-AD system

The prominent bacteria communities were analyzed on the phylum and family level in

combined MEC-AD system and AD planktonic cells (Fig. 5). On the anode biofilm,

four dominated bacteria communities were detected at the phylum level. They were

Proteobacteria, Bacteroidetes, Chloroflexi, and Firmicutes, and the abundance were

24.8%, 14.6%, 11.0% and 7.3%, respectively. Proteobacteria is a facultative

anaerobe, which plays a very important role in the degradation of cellulose and

biofilm formation in anaerobic conditions. Chloroflexi has the ability to degrade

various sugars such as starch into acetate and other short chain fatty acids, and

Bacteroidetes is able to decompose high molecular weight (HMW) compounds (Lu et

al., 2018). On family level, the abundance of Spirochaetaceae and Anaerolineaceae

were 5.6% and 6.0%, respectively. In addition, as for Archaea, the abundance of

Methanobacteriaceae, Methanosaetaceae and Methanospirillaceae were 7.2%, 5.3%

and 0.1%, respectively. Especially, Geobacter was detected on the genus level,

24
accounting for 3.8% of total genus. Geobacter are the typical microbial community

for extracellular electron transport on the biofilm of anode in bioelectrochemical

system (Liu et al., 2016a). These microorganisms lived together and cooperated for

organics solubilization/degradation, electron transport and methane production.

On the cathode biofilm, Proteobacteria, Chloroflexi, Actinobacteria,

Bacteroidetes and Firmicutes were identified as the dominating bacteria communities

with the abundance of 19.8%, 19.6%, 17.2%, 7.7% and 6.2%, respectively. On family

level, Anaerolineaceae had a high abundance (16.0%), and Methanobacteriaceae and

Methanosaetceae accounted for 10.9% and 4.2%, respectively. On genus level, the

percentage of Geobacter was just 0.7%. Methanobacterium (hydrogenotrophic

methanogen) and Methanosaeta (acetotrophic methanogen) were observed as the

dominant communities, and the corresponding abundance were 15.6% and 6.0%.

Methanobacterium relies on H2/CO2 and formate as carbon source and belongs to the

class Methanobacteria (Demirel and Scherer, 2008). In addition, Methanolinea, a

unique Archaea, accounted for just 0.6% in total genus detected. The abundance of

Methanospirillum, a kind of hydrogenotrophic methanogen, was just 0.8%. These data

implied that hydrogenotrophic methanogenenesis played an important role in the

enhanced methane evolution (Wang et al., 2009).

In the suspensions of MEC-AD, the abundance of Proteobacteria, Bacteroidetes,

Chloroflexi, Firmicutes and Actinobacteria were 19.8%, 18.7%, 10.9%, 8.9% and

9.4%, respectively. The abundance of Methanobacteriaceae and Methanosaetceae on

25
the family level were 13.1% and 13.1%, respectively. Besides, Anaerolineaceae,

Saprospiraceae and Porphyromonadaceae, were also identified, with the

corresponding abundance of 5.9%, 4.4% and 4.0%, respectively. Methanobacterium

accounted for 17.2% in total genus detected and Methanosaeta was 16.8%, thus the

suspensions might be also a key space for methane production. In the suspensions of

single AD process, Methanosaeta was the only genus detected in raw sludge (Liu et

al., 2016b), and the abundance of Methanosaeta in AD (19.1%, obviously higher than

that in MEC-AD) verified that Methanosaeta is the main methanogen in conventional

AD process. The other prominent microbial communities were Methanosaetaceae,

Methanobacterium and Anaerolineaceae, about 13.1%, 8.8% and 7.5% of total

family. This indicated that the combined MEC-AD system enriched functional

microbial communities, thereby increasing methane production yield considerably.

Based on the above observations, the possible internal mechanisms were proposed

(Fig. 6). MEC-AD mostly relied on the syntrophic interactions between exoelectrogens

and fermentative partners. The two electrodes provided the abundant space for the

growth and attachment of electroactive microorganisms. The electrical stimulation

could further promote the selection/enrichment of such functional microbes. As

illustrated in the figure, fermenters in anode could convert the complex substrates to

metabolites accessible to electroactive microorganisms, which made the overall

fermentation thermodynamically favorable thus increasing the methane recovery (Zhen

et al., 2017a). Acetotrophic methanogens utilized acetate to produce methane (i.e.

26
CH3COOH + H2O → CH4 + H2CO3, ΔGo’ = -31 kJ/mol). On the one hand, the residual

acetate can be further decomposed into H+ and CO2 via anode oxidation, while releasing

some electrons which were subsequently transferred to cathode via the external circuit.

The electroactive microorganisms on the cathode biofilm could take the electrons to

convert CO2 to methane via direct (CO2 + 8e-+ 8H+ → CH4 + 2H2O, ΔGo’ = -136

kJ/mol) or indirect electron transport pathway (2H+ +8e- → H2 E = -0.421 V vs. SHE;

CO2 + 4H2 → CH4 + 2H2O, △Go’ = -131 kJ/mol) (Rotaru et al., 2014, Morita et al.,

2011), thus further upgrading biogas. Besides, some metal cations and other ions (e.g.

PO43-, NH4+) can be moved towards electrodes under electric field. This induced the

precipitation of non-conductive crystals on the biocathode surface, e.g. struvite crystals

(MgNH4PO4·6H2O), an essential phosphorous-rich nutrient, accordingly

accomplishing the simultaneous nitrogen and phosphorous recovery from digestate.

Fig. 5. and Fig. 6.

3.7 Digestate dewaterability and environmental-energy analysis.

Dewaterability of digestate produced at different phases was assessed. Higher CST

indicated the poor dewaterbility and vice versa. With increasing the content of FW in

the co-substrate, the dewaterability of raw co-substrate became worse first (0.4: 0.6)

but then better (0.5: 0.5) compared with the ratio of 0.2: 0.8. Sludge often has high

difficulty in dewatering because the water is bound between microbial flocs or inside

cells. Agreeing well with Zhen’s finding (Zhen et al., 2014), the CST of digestate after

co-digestion in single AD process in the three periods were all increased in contrast to

27
the raw co-substrate especially in the period VII, increasing by about 1.68 times. This

suggested that single AD process attenuated the dewaterability of biowaste. Whereas,

after the co-digestion in the combined MEC-AD system, the CST dropped from 446.4

± 22.2 s to 249.6 ± 52.9 s in period VI and from 338.5 ± 14.1 s to 199.7 ± 0.5 s in

period VII. The dewaterability of digestate was promoted evidently.

The results showed that the combined MEC-AD system elevated the

dewaterability of the co-digestion digestate, by altering physicochemical

characteristics of digestate through electrical stimulation. The combined MEC-AD

system enhanced organics removal and aided the release of bound water, although

colloidal particles and biopolymers released during hydrolysis step could re-absorb

part of the released water (Zhen et al., 2014). In this regard, the dewaterability became

better. The superior dewaterability of digestate showed a great potential for post-

treatment such as mechanical dewatering and volume reduction, and the costs

associated with the transport and final disposal can be saved considerably. Moreover,

less land resources will be demanded for land application or landfill of the dewatered

digestate.

In addition, the energy balance for MEC-AD system was calculated. Total

electricity recovery efficiency for this system was 50.4%, 39.7%, 4.5%, -1% and

4.5% for phase III, IV, V, VI and VII, respectively. The highest electricity recovery

efficiency was still as low as 50.4%, only half of the input energy, suggesting the

serious electron loss. This might be the main factor limiting the wide-spread

28
implementation of MEC-AD. Overall, the electrical regulation in the traditional AD

process cannot only upgrade renewable energy recovery from complex biomass but

also in-situ post-treat digestate. It should be noted that except for DC power, MEC

can also use various kinds of energy (e.g. wind energy, water energy, tidal energy,

geothermal energy, etc.) as external power supply, which allows them to be converted

and fixed into transportable electro-fuels (Park et al., 2018).

4. Conclusions

The MEC-AD have produced 2.8 times higher methane production yield than AD, at

FW: SS 0.2: 0.8, SRT 15 d and 0.4 V. The TS, VS and COD removal all had apparent

advantages over AD, indicating that the combined MEC-AD system has the unique

potential to facilitate the solubilization of organics (e.g. protein and polysaccharides),

which undoubtedly has improved methane production, increased NH4+-N removal and

decreased N2O emissions. Besides, the electrical regulation/stimulation enhanced the

digestate dewaterability, facilitating the post-treatment. Nonetheless, a further work is

still required to maximize the energy recovery efficiency in MEC-AD system.

Acknowledgments

This work was sponsored by the National Natural Science Foundation of China (No.

51808226), the Distinguished Professor in Universities of Shanghai (Oriental Scholar,

No. TP2017041), the Shanghai Pujiang Program (No. 17PJ1402100), and Shanghai

Institute of Pollution Control and Ecological Security.

29
Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version.

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36
Figure Legends

Fig. 1. Applied external voltage and current of MEC-AD (a), variations in methane

production rate (b), accumulative methane yield (c) at different stages in two systems.

Fig. 2. N2O emissions (a) and ammonia nitrogen concentration (b).

Fig. 3. Variations in pH (a), total alkalinity (b), effluent TS (c) and VS (d) at different

stages in two systems.

Fig. 4. Variations in effluent TCOD (a), SCOD (b), TPN (c), SPN (d), TPS (e), SPS (f)

and VFAs (g-h) at different stages in two systems.

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(Bacteria) and genus (Archaea) level.

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38
 SRT=20d SRT=15d
FW:SS=0.2:0.8 0.4:0.6 0.5:0.5
0V 0V 0.2 V 0.4 V 0.8 V 1.2 V0.8 V 0.8 V 0.8 V
1400 6

1200
External voltage (mV)

（ a） 5

Current (mA)
1000
4
800 External voltage
Current 3
600
2
400
200 1

0 0
12 （ b）
(mL-CH 4/L-reactor d)

MEC-AD
10
Methane yield

AD
8
6
4
2
Accumulative CH 4 production

0
（ c）
MEC-AD
(mL-CH4/L-reactor)

60
AD

40

20

0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Operational time (d)

Fig. 1.

39
 SRT=20d SRT=15d
FW:SS=0.2:0.8 0.4:0.6 0.5:0.5

0V 0V 0.2 V 0.4 V 0.8 V 1.2 V0.8 V 0.8 V 0.8 V

0.0008

(mL-N 2O/L-reactor d)
（ a）

N2O emission 0.0006 MEC-AD

AD
0.0004

0.0002

0.0000
Ammonia nitrogen（ mg/L）

350 （ b）
300
250
200
150
100
50
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Operational time (d)

Fig. 2.

40
 SRT=20d SRT=15d
FW:SS=0.2:0.8 0.4:0.6 0.5:0.5

0V 0V 0.2 V 0.4 V 0.8 V 1.2 V0.8 V 0.8 V 0.8 V

8.5

8.0 （ a）
7.5
pH
7.0

6.5 MEC-AD
6.0 AD
Total alkalinity (CaCO3mg/L)

5.5
（ b）

1500

1000

500

0
20
（ c）

16
TS (mg/L)

12

0
10 （ d）

8
VS (mg/L)

0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Operational time (d)

Fig. 3.

41
 SRT=20d SRT=15d SRT=20d SRT=15d

FW:SS=0.2:0.8 0.4:0.6 0.5:0.5 FW:SS=0.2:0.8 0.4:0.6 0.5:0.5

1.2 V0.8 V 1.2 V0.8 V
0V 0V 0.2 V 0.4 V 0.8 V 0.8 V 0.8 V 0V 0V 0.2 V 0.4 V 0.8 V 0.8 V 0.8 V
20000

16000 （ a） 2000
（ b）
TCOD (mg/L)

SCOD (mg/L)
12000 1500

8000 1000
MEC-AD
4000 500
AD
0 0
（ c） （ d）

Soluble Polysaccharide Soluble Protein (mg/L)

MEC-AD
Total Protein (mg/L)

5000
AD 60
4000

3000 40

2000
20
1000

0 0
（ e） （ f）
Total Polyccharide

1600 40

1200 30
(mg/L)

(mg/L)
800 20

400 10
Volatile fatty acids (mg/L)

0 0
（ g） MEC-AD （ h） AD
300 Acetic acid
Volatile fatty acids

150 Propionic acid

250 Isobutyric acid
N-butyric acid
(mg/L)

200
100 Isovaleric acid
150 Valeric acid

100
50
50

0 0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95100 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95100
Operational time (d) Operational time (d)

Fig. 4.

42
Fig. 5.

43
Fig. 6.

44
Lists of Tables

Table 1 Characteristics of waste activated sludge, food waste and mixed substrate a.

Table 2 TS, VS, and TCOD removal efficiencies (%).

Table 1

FW SS FW: SS=0.2: FW: FW: SS=0.5:

0.8 SS=0.4:0.6 0.5

pH 4.4 ± 0.1 6.8 ± 0.1 6.9 ± 0.1 6.3 ± 0.1 5.4 ± 0.1

C/N 17.1 ±
6.3 ± 0.1 7.1 ± 0.1 8.2 ± 0.1 9.3 ± 0.1
0.1

TS (g/L) 51.8 ±
45.3 ± 0.1 15.5 ± 0.4 18.5 ± 2.8 20.0 ± 0.2
0.4

VS (g/L) 47.1 ±
22.4 ± 0.1 8.1 ± 0.8 11.4 ± 2.3 14.2 ± 0.3
0.5

TCOD (g/L) 69.7 ±

25.6 ± 0.3 15.1 ± 0.7 15.6 ± 0.5 19.9 ± 0.7
3.8

SCOD (g/L) 45.5 ±

0.32 ± 0.1 1.93 ± 0.1 2.7 ± 0.2 5.4 ± 0.2
0.4

TPN (g/L) 3.1 ± 0.1 1.4 ± 0.1 2.9 ± 0.2 2.8 ± 0.3 3.3 ± 0.4

SPN (g/L) 13.5 ±

0.6 ± 0.1 1.1 ± 0.1 2.1 ± 0.3 4.0 ± 0.5
0.1

TPS (g/L) 0.6 ± 0.1 13.5 ± 0.1 2.1 ± 0.3 2.9 ± 0.1 3.9 ± 0.5

NH4+-N 13.8 ± 143.4 ±

174.6 ± 15.7 59.6 ± 9.1 43.8 ± 4.8
(mg/L) 0.4 22.9
a
TS: total solids; VS: volatile solids; TCOD: total chemical oxygen demand; SCOD:
soluble chemical oxygen demand; TPN: total protein; SPN: soluble protein; TPS: total
polysaccharose; NH4+-N: ammonia nitrogen.

45
Table 2

0V 0V 0.2 V 0.4 V 0.8 V 0.8 V

Reactor Parameters 0.4:
0.2: 0.8 0.5: 0.5
0.6
MEC- 31.0 ± 35.2 ± 29.6 ± 20.7 ± 15.1 ± 15.2 ± 34.0 ±
AD 19.3 5.8 10.5 28.8 15.7 17.8 13.8
TS
42.1 ± 5.8 ± 19.0 ± 5.2 ± 12.3 ± 21.1 ± 24.0 ±
AD
31.5 16.2 16.4 6.5 12.3 10.8 4.7
MEC- 32.2 ± 38.0 ± 52.6 ± 46.6 ± 29.8 ± 49.8 ± 51.3 ±
AD 19.7 11.7 14.0 25.0 20.2 26.6 12.3
VS
41.9 ± 12.6 ± 45.8 ± 35.3 ± 28.0 ± 47.7 ± 44.4 ±
AD
30.0 16.3 22.1 11.6 17.3 20.8 13.3
MEC- 41.1 ± 33.2 ± 35.8 ± 40.3 ± 31.8 ± 45.1 ± 42.5 ±
AD 19.2 9.4 11.9 20.1 12.9 6.7 19.8
TCOD
36.5 ± 34.5 ± 19.6 ± 31.8 ± 39.7 ± 23.9 ± 47.0 ±
AD
27.1 8.8 14.9 12.8 12.2 7.1 20.1

46
Graphical Abstract

47
Research Highlights:

A united microbial electrolysis cells-anaerobic system was designed.

The use of microbial electrolysis cells improved dewaterability of digestate.

Electrical regulation induced 2.8-fold increase in biomethane production.

The united system internal mechanism was speculated.

Syntrophic interaction of microorganisms promoted system performance.

48