Accepted Manuscript

Synergetic promotion of syntrophic methane production from anaerobic diges-

tion of complex organic wastes by biochar: performance and associated mech-
anisms

Gaojun Wang, Qian Li, Xin Gao, Xiaochang C. Wang

PII: S0960-8524(17)32113-2
DOI: https://doi.org/10.1016/j.biortech.2017.12.004
Reference: BITE 19259

To appear in: Bioresource Technology

Received Date: 11 November 2017

Revised Date: 29 November 2017
Accepted Date: 2 December 2017

Please cite this article as: Wang, G., Li, Q., Gao, X., Wang, X.C., Synergetic promotion of syntrophic methane
production from anaerobic digestion of complex organic wastes by biochar: performance and associated
mechanisms, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.12.004

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Synergetic promotion of syntrophic methane production from anaerobic

digestion of complex organic wastes by biochar: performance and associated

mechanisms

Gaojun Wang, Qian Li, Xin Gao, Xiaochang C. Wang *

International Science and Technology Cooperation Center for Urban Alternative Water Resources

Development; Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE;

Engineering Technology Research Center for Wastewater Treatment and Reuse, Shaanxi; Key

Laboratory of Environmental Engineering, Shaanxi; Xi’an University of Architecture and

Technology, No. 13 Yanta Road, Xi’an 710055, China

*E-mail: xcwang@xauat.edu.cn

Abstract:

Biochar was added to a mesophilic anaerobic digester to promote syntrophic volatile

fatty acids (VFAs) oxidation and methane production from complex organic wastes.

Compared with conventional operation, biochar addition effectively shortened the lag

time by 27.5-64.4% and increased the maximum methane production rate by 22.4% to

40.3%. With a biochar dosage of 15g/L, the system performed well under an organic

loading rate as high as 3g substrate/g inoculums. Biochar showed a remarkable

buffering capacity to alleviate pH decrease caused by VFAs accumulation. In order to

gain knowledge on associated mechanisms, a specific experiment was conducted

using butyrate as substrate. It was identified that syntrophic degradation of butyrate to

acetate occurred under high H2 partial pressure. By microbial community analysis, it

was further revealed that biochar addition brought about the enrichment of
1
Anaerolineaceae and Methanosaeta, typical microorganisms for direct interspecies

electron transfer.

Key words: Anaerobic digestion, Biochar, Volatile fatty acids, Syntrophic metabolism,

Interspecies electron transfer.

1. Introduction
Anaerobic digestion (AD) is a promising sustainable technology for waste

management and energy production (Mao et al., 2015; McCarty, 2001), during which

many kinds of wastes, such as sewage sludge, food waste, organic municipal solid

waste and industrial waste, can be treated efficiently. Additionally, the chemical

energy stored in these wastes can be used to generate cleaner energy in the form of

biogas by anaerobic microbes (Nishio & Nakashimada, 2007).

To achieve high-efficiency energy production, stable and effective methane

production during AD is expected, especially under a high organic loading rate (OLR)

(Appels et al., 2011). However, high OLR increases the risk of AD system failure due

to the imbalance between acidification and methanation, which may result in severe

accumulation of volatile fatty acids (VFAs) and a sharp decrease in pH. In general, the

degradation of VFAs, such as propionate and butyrate, are slow due to the positive

Gibbs free energy, which is thought to be the rate-limiting step of AD (Müller et al.,

2010). During the syntrophic metabolism of propionate and butyrate, interspecies

electron transfer (IET) is a crucial process between the syntrophic VFA oxidizing

bacteria and methanogenic archaea, which is achieved when H2 or formate acts as the

carrier (Stams et al., 2006). However, VFAs accumulation is often accompanied with

2
a rising H2 partial pressure, which can inhibit the syntrophic degradation of VFAs

(Stams & Plugge, 2009).

Recently, direct interspecies electron transfer (DIET) has been identified as an

alternative pathway for syntrophic electron exchange in defined co-culture of two

Geobacter species. These exoelectrogenic bacteria can establish an electrical

connection and achieve DIET between the syntrophic partner with the help of

electrical conductive pili (e-pili) or membrane cytochromes (Lovley, 2011; Shrestha et

al., 2013). Additionally, DIET has also been confirmed in complex microbial

environments. In an upflow anaerobic sludge blanket (UASB) digester, DIET between

Methanosaeta and Geobacter species aggregates promoted syntrophic ethanol

degradation and methane production (Morita et al., 2011). In order to enhance

electron transfer, some (semi)conductive materials, such as minerals and abiotic

carbon, were assessed for their ability to promote DIET. Conductive mineral

magnetite particles were considered to act as the electron conduits between the

propionate-oxidizing bacteria and methanogenic archaea to enhance propionate

degradation (Viggi et al., 2014). Furthermore, the addition of conductive carbon cloth

was beneficial in the aspects of methane production, volatile solids removal and COD

removal efficiencies for treating the leachate or organic fraction of municipal solids

wastes (Dang et al., 2017; Lei et al., 2016), and helpful to resist heavy acidic impacts

condition via DIET (Zhao et al., 2017). Granular activated carbon (GAC) was shown

to increase methane production potential and waste activated sludge reduction rates

by enriching species like hydrogen-utilizing methanogens and Geobacter, which are

3
capable of DIET (Liu et al., 2012). Moreover, GAC addition stimulated efficient VFA

degradation under high H2 partial pressure (Zhao et al., 2016). It was reported that

single-walled carbon nanotubes could induce faster substrate utilization and methane

production rates, which suggested that DIET among anaerobic fermentative bacteria

and methanogens was enhanced (Li et al., 2015). The addition of 1.0g/L graphene also

resulted in a 25.0% enhancement in methane yield and a 19.5% increase in production

rate compared with the control (Lin et al., 2017).

Multiple lines of evidence suggest that electrical conductive materials stimulate

DIET in anaerobic syntrophic metabolism. However, the production costs of some

commercial materials, such as carbon felt, carbon nanotubes, are prohibitive, and may

not be suitable for field-scale application. Moreover, the environmental risk of

graphene should be assessed in regard to the post disposal of anaerobic digestate

(Clemente et al., 2017; Hansen, 2016). Thus, from the viewpoint of waste

management and sustainable development, adding some conductive material

produced with waste, such as biochar (a carbon rich material produced from waste

pyrolysis) (Lehmann & Joseph, 2015) might be a practical strategy for achieving

DIET in AD systems.

Recently, the effect of biochar addition on AD was studied. It has been reported

that biochar addition can promote IET in co-culture systems (Chen et al., 2014).

Moreover, biochar addition also has some other benefits for AD, such as mitigating

mild ammonia inhibition, supporting the formation of archaea microorganism (Lu et

al., 2016; Mumme et al., 2014), shortening the lag time for methane production (Luo

4
et al., 2015) and increasing the biogas production rate (Sunyoto et al., 2016). However,

to the best of the authors’ knowledge, the effects of biochar addition on different

organic loading rate conditions and the results of biochar dosage on complex organic

wastes have not been reported.

In this study, the effect of biochar dosage on AD was investigated, and the

performance of biochar addition to AD systems was explored under different OLR

conditions. Additionally, butyrate, which was the main VFA that accumulated in the

complex organic wastes AD process, was used as a substrate for degradation in a

biochar amended environment to determine if biochar could promote the syntrophic

degradation of VFAs via DIET.

2. Materials and methods

2.1 Biochar preparation and characterization
The raw feedstock for biochar production was sawdust, which was collected

from a local timber mill. Before pyrolysis, the sawdust was air-dried at 80℃ for 24h

in an oven. The dried feedstock was placed into ceramic crucibles covered with fitted

lids and pyrolyzed under oxygen-limited conditions in a muffle furnace (Shanghai

Jing Hong Laboratory Instrument Co., Shanghai, China). After heating at a rate of

10℃min-1, the pyrolysis temperature reached 500℃, and this was maintained for 1.5h.

After the pyrolysis process, the biochar sample was cooled to room temperature and

then ground and sieved to uniform size fractions of 0.25-1mm. The

Brunauer-Emmett-Teller (BET) surface area of the biochar was measured using a

V-Sorb X800 surface area analyzer (Gold APP Instrument Co., Beijing, China).

5
Organic functional groups of the biochar were measured with Fourier transform

infrared spectroscopy (FT-IR, ThermoFisher, USA) under an attenuated total

reflectance (ATR) model (supplementary materials).

2.2 Substrates and seed sludge

The complex organic wastes used in this study were a mixture of dewatered

activated sludge (DAS) and food waste (FW). The DAS was collected from

dewatered sludge units at Xi’an No.5 wastewater treatment plant, Shaanxi Province,

China. The FW was synthetic and based on the characteristics of FW in China (Li et

al., 2017). The mixture ratio of FW to DAS was 4:1based on wet weights according to

the previous study (Li et al., 2017). The substrate was preserved in a 10L tank at a

temperature of 4℃ before use. The properties of the complex organic wastes were

shown in supplementary materials.

The seed sludge used in this study was collected from an AD plant of a brewery

factory, located in Xi’an, Shaanxi province, China. The AD plant was operated under

steady mesophilic condition. Seed sludge was preserved in anaerobic conditions

before use. The total solids (TS) and volatile solids (VS) of the seed sludge were 6.82%

and 4.65%, respectively, and the pH of the seed sludge was 7.62.

2.3 Batch experiments

2.3.1 Complex organic wastes AD experiments
To evaluate the effects of different dosages of biochar on AD, addition ratios of 0,

2, 6, 10 and 15g/L biochar were established. A total of 5mL seed sludge and 4mL

substrate were added into a serum bottle to maintain the VS ratio of substrate to

6
inoculums (S/I) at 1.5. The different experimental groups were named BC0, BC2, B6,

BC10 and BC15 according to the biochar dosage.

In the experiment that aimed to evaluate the effects of biochar addition on

different OLR conditions, four groups were designed to simulate the OLR from low to

extremely high conditions. Specifically, 5mL of seed sludge with different substrate

doses (2, 4, 6 and 8mL) was added into the bottles to maintain the S/I at 0.75, 1.5,

2.25 and 3 (based on VS), respectively. For each group, a biochar amended bottle was

set up at 15g/L biochar addition and another without biochar addition was operated as

control. The bottles were referred to as BCL, CTL, BCM, CTM, BCH, CTH, BCEH

and CTEH, with “BC” and “CT” representing the biochar amended or control bottle,

respectively, and “L”, “M”, “H”, and “EM” representing the low, medium, high and

extremely high OLR groups, respectively. Considering the abundant nutrient content

of the synthetic FW (supplementary materials), tap water was added into the bottles to

keep the working volume at 90mL.

All of the bottles were purged with high purity nitrogen gas for 5min to maintain

the anaerobic conditions in the reactors and then sealed with rubber stoppers and

pressed using aluminum caps. The bottles were then placed in a water bath and stirred

at 150 rpm under mesophilic (35℃) conditions. All of these experiments were

conducted in duplicate.

2.3.2 Specific experiment of butyrate degradation

To explore the mechanism underlying the effects to AD, butyrate, which was the

main VFA accumulated in the degradation process of complex organic wastes, was

7
selected as the substrate for further study. Three batch experiment groups, namely BC

(biochar amended), Control A and Control B were designed. The experiments lasted

for two periods.

For the 1st period, 5mL seed sludge, 5mL n-butyrate sodium solution, and 50mL

nutrient medium were added into each serum bottle with a working volume of 90mL

to maintain the concentration of 1300mg/L n-butyrate ions. The pH of the mixture

was adjusted with 6M NaOH and 6M HCl to approximately 7.2. The nutrient solution

(0.5g/L NH4Cl, 0.1g/L MgCl2·6H2O, 0.4g/L K2HPO4 and 0.05g/L CaCl2·2H2O) was

added to the bottles to keep the total working volume at 90mL. To identify the pH

buffering capacity of the biochar, chemical pH buffering reagent (NH4HCO3) was

added to the Control A group to keep the pH stable during reaction process, while the

BC and Control B groups did not receive pH adjustment.

Once the bottles had completely converted the butyrate to methane, the BC and

Control A groups were selected to conduct the 2nd period. Butyrate sodium was added

to these two groups to keep a final butyrate concentration of 700mg/L and a total

working volume of 90mL. All bottles were operated the same as described above to

maintain the anaerobic conditions for the reaction. To explore the effects of high H2

partial pressure on butyrate degradation, 20mL H2 (corresponding to 0.66atm) was

spiked into headspace of each serum bottle. All of these experiments were conducted

in duplicate.

2.4 Analytical methods

The pH values were monitored using a potable pH meter (Horiba, Kyoto, Japan).
8
Hydrogen, methane, and carbon dioxide concentrations of the biogas were measured

using a gas chromatograph (GC) (GC7900, Tianmei, China) equipped with a

molecular sieve packed stainless-steel column (TDX-01, length × diameter of 2.0m ×

3.0mm, Shanghai Xingyi Chrome, China) and a thermal conductivity detector (TCD).

The VFAs were detected with a GC (PANNO, China) equipped with a DB-FFAP

column (φ 0.32mm × 50m; Agilent, USA) and a flame ionization detector (FID).

2.5 Statistical analysis

The experimental data of the batch experiments was simulated using the

modified Gompertz equation:

where P is methane production (mL), P0 is methane production potential (mL), Rmax is

the maximum methane production rate (mL/d), t0 is the lag time (days), and

e=2.718281828. Origin 8.0 software (OriginLab Corporation, USA) was used to

simulate the methane production curve and to coverage the results.

2.6 Microbial community analysis

To identify the effects of biochar addition on the microbial community, samples

of SD0 and SD15 were collected as the methane production ended. The sludge

samples were first centrifuged at 13,000 rpm for 10min, and then the pellets were

rinsed with phosphate-buffered saline twice via resuspension and centrifugation at

room temperature. The supernatant was then discarded, and the samples were dried in

an oven at 55℃ for 10min. A Mag-Bind Soil DNA Kit (Omega Bio-Tek, USA) was

used to extract DNA according to the manufacturer’s protocol. The microbial

9
communities of the sludge were analyzed via high-throughput sequencing on an

Illumina platform (Illumina Miseq PE250, Sangon Biotech, Shanghai, China). For the

bacteria, primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R

(5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the V4 region of the

16S rRNA gene by PCR. For the archaea, the V3-V4 region of 16S rRNA was

amplified by nested PCR. 340F (5’-CCCTAYGGGGYGCASCAG-3’) and 1000R

(5’-GGCCATGCACYWCYTCTC-3’) were the primers for the first cycle, and 349F

(5’-GYGCASCAGKCGMGAAW-3’) and 806R

(5’-GGACTACHVGGGTWTCTAAT-3’) were the primers for the second cycle.

After amplification, the PCR products were checked via agarose gel

electrophoresis to determine the quality of the amplification. After purification with

Agencourt AMPure XP magnetic beads (Backman Coulter, USA) according to the

manufacture’s protocol, the PCR products were quantified using a Qubit 2.0 DNA

detection kit (Sangon Biotech, Shanghai, China).

3. Results and discussion

3.1 Effects of biochar dosage on AD of complex organic
wastes
Cumulative methane production under different biochar dosage conditions was

shown in Figure 1(a). The fitted parameters of the modified Gompertz equation were

listed in Table 1. The lag time of the BC0 group was 21.2 days, while that of the BC2,

BC6, BC10 and BC15 groups were 15.3, 12.1, 10.2, and 7.8 days, respectively. It was

clear that biochar addition significantly shortened the lag time by 27.5% to 64.4%.

10
Moreover, Rmax of the BC0 group was 6.7mL/d, and this increased to 8.2-9.4mL/d

after addition of biochar. Thus, significant positive effects of biochar on both

shortening the lag time and increasing Rmax were confirmed.

The variation in pH was monitored in each group until it recovered to a suitable

range for methane production (Figure 1(b)). Apparently, as the anaerobic process

started, the pH of each group went through a rapid decline over one day due to the

rapid acidification of the carbohydrates contained in the FW (Kawai et al., 2014). In

BC0 group, the pH dropped from 6.7 to 5.0, while in biochar added groups, the drop

in pH were diminished (above 5.1) due to the buffering capacity derived from the

ash-inorganic alkalis and organic alkalis functional groups in biochar (Yuan et al.,

2011). A previous study showed that the vermicompost derived biochar had a

significant effect on improving acidification during AD (Wang et al., 2017). In this

current study, the pH buffering capacity introduced by biochar was confirmed but not

sufficient to completely eliminate the acidification. Considering a slight pH decline in

the AD system would decrease the biogas production efficiency, the addition of

biochar could be an ideal strategy, in practice, to stabilize the pH.

The variation in VFAs during this process was shown in Figure 2. For all groups,

the VFAs rapidly accumulated over the 2nd day after addition of the substrate. The

VFA concentration remained at a high level until the lag time ended. For the BC0

group, the total maximum accumulated VFA concentration was 2296.3mg/L on Day

13. For the BC2, BC6, and BC10 groups, the total maximum accumulated VFA

concentrations were decreased from 2192.6 to 1947.9mg/L on day 11 and that of the

11
BC15 group was 1830.3mg/L on day 9. It appeared that biochar addition was

beneficial for alleviating VFA accumulation and for shortening the duration of

accumulation.

The main accumulated VFAs during this process were n-butyrate and acetate.

For every group, these two VFAs accounted for more than 90% of the total VFAs.

Although biochar addition had no obvious effect on the accumulated VFAs types, it

was interesting that the ratio of acetate to n-butyrate was significantly altered after

biochar addition. The syntrophic degradation of butyrate to acetate (Equation (1)) was

energetically unfavorable and only occurred together with the hydrogen-consuming

methanogenic process (Equation (2)). In this study, due to the low pH value, almost

no methane was produced during the lag time. However, as the biochar dosage was

increased, the concentration (mg/L) ratio of acetate to n-butyrate demonstrated a

significant increasing trend (Figure 3). The average ratio of acetate to n-butyrate for

the BC0 group was 2.22, while that of the BC2, BC6, BC10 and BC15 groups were

3.19, 3.88, 5.35 and 10.18, respectively, which suggested that biochar addition

stimulated the conversion from butyrate to acetate by promoting DIET in the absence

of metabolism by hydrogen-consuming methanogen. In a previous study, it was

reported that biochar could act as an ideal additive to promote IET (Chen et al., 2014).

CH3CH2CH2COO- +2H2O + 2CO2 2CH3COO- + H+ + 2H2 (1)

G0’=+48.3kJ/mol

4H2 + CO2 CH4 + 2H2O (2)

G0’= -131.7kJ/mol

12
The authors attributed this phenomenon to the electrical conductivity of biochar,

though its conductivity was 1000-fold less than that of granular activated carbon

(Huggins et al., 2014). Noticeably, one important property of biochar for

environmental management was the redox active property, due to the richness of

organic functional groups of biochar (supplementary materials)(Klüpfel et al., 2014;

Yuan et al., 2017). Thus, another explanation for the occurrence of DIET was likely

that the butyrate oxidizing bacteria oxidized butyrate to acetate using biochar as the

temporary electron acceptor in the inactive metabolism of methanogenic archaea.

However, in the control group, syntrophic butyrate degradation could not occur due to

the lack of electron acceptors, which resulted in butyrate accumulation.

The experimental results of this section revealed that biochar alleviated VFA

accumulation and stimulated methane production in AD. From the aspects of relieving

VFA accumulation and avoiding acidification, which were two main complications

often responsible for the failure of AD systems in engineering applications, addition

of 15g/L biochar was demonstrated as the optimal dosage for promoting methane

production.

3.2 Effects of biochar addition on AD of complex organic

wastes under different OLRs
To explore whether biochar addition could promote methane production under

high OLR, four OLR conditions were selected and the results of cumulative methane

production was shown in Figure 4. For the L and M groups, methane production was

achieved independent of whether biochar was added or not. However, biochar

13
addition both shortened the lag time and increased Rmax. The lag time of the BCL and

BCM groups was 6.5 days and 9.1 days, respectively, which were reduced by 39.2%

and 52.8% compared with that of the BCL and CTM groups. Correspondingly,

compared with the Rmax of the CTL and CTM groups, those of the BCL and SDM

groups were also increased. For the H and EH groups, the lag times of the BCH and

BCEH groups were 14.9 days and 19.5 days, respectively, which were prolonged

compared to those of the SDL and SDM groups due to the high VFA accumulation.

The Rmax of the BCH and BCEH groups were 13.5mL/d and 14.3 mL/d, respectively,

which were quite higher than that of the SDL and SDM groups. It was noticeable that

no methane production was detected in the CTH and CTEH groups until the time

point at which the methane production process had ended in the BCH and BCEH

groups. In the CTH and CTEH groups, the activity of methanogenic archaea was

completely inhibited due to the heavy VFA accumulation (data not shown). The pH of

the CTH and CTEH groups was also maintained at lower than 5.5 over this duration.

These results revealed the significance of adding biochar to the AD system, especially

under high OLR conditions. Additionally, the results of this study illustrated that the

metabolic activity of methanogenic archaea could be recovered after biochar addition

even in the presence of high VFA accumulation. Thus, biochar addition could also be

an efficient and practical strategy for the rapid metabolic recovery of acidified AD

reactors. Considering the batch operation mode of this study, the continuous run mode

should be studied in the future to explore the long-term response of the AD system

performance to biochar addition.

14
3.3 Effect of biochar addition on butyrate degradation
As the above results showed, n-butyrate and acetate were the main accumulated

VFAs in the AD of complex organic wastes. Considering the fact that acetate could

immediately be utilized by methanogenic archaea, n-butyrate was used as a substrate

to study the effects of biochar addition on the VFAs degradation further.

During the 1st period, all three groups started to produce methane accompanied

by butyrate degradation after a 6-day lag time (Figure 5(a)). However, the control B

group ceased methane production at day 12. The cumulative methane production of

the Control B group was just less than 20% that of the other two groups. The main

reason for this was the decrease in pH, which was caused by the accumulation of

acetate (Figure 5(c), supplementary materials). Apparently, the pH buffering capacity

was important for the AD system to maintain stable and efficient methane production.

For the Control A group, to avoiding a decreasing pH, a chemical buffering reagent

(1g/L NH4HCO3) was added twice (at Day 0 and Day 10). Although the pH was

maintained within a suitable range during the methane production process, a stagnant

period occurred between Day 10 and Day 15. In the BC group, no chemical reagent

was added for controlling the pH, as an appropriate pH value was maintained during

the whole process due to the buffering capacity introduced by the organic functional

groups of biochar (Yuan et al., 2011). The potential of biochar as an ideal substitution

for chemical reagents for controlling pH was highlighted in this experiment.

During the 2nd period, to study syntrophic butyrate degradation under the

conditions of a high H2 partial pressure, 20mL H2 was added to the BC and Control A

15
groups. In the BC group, methane production and butyrate degradation began without

a lag time, indicating that a high H2 partial pressure did not inhibit syntrophic butyrate

degradation in this group. However, for the Control A group, although 4.61 mL

cumulative methane was produced over the first two days, butyrate degradation was

completely inhibited. Analysis of the biogas components revealed that H2 was

consumed by the hydrogenophilic methanogens over first two days. After this, the

butyrate began to degrade at Day 3. This result suggested that different metabolism

types of syntrophic butyrate degradation likely existed between the Control A and BC

groups. Due to the positive Gibbs free energy (Equation (1)), one critical requirement

of this process is that the H2 concentration should be keep extremely low (typically

below 10-5 atm). This was consistent with the results of the Control A group, which

started butyrate degradation after the H2 was completely consumed, and was also

consistent with a previous study (Viggi et al., 2014). Nevertheless, in the BC group,

butyrate degradation began under a high H2 partial pressure condition. It was

suggested that DIET could be occurring in the BC group. The biochar added here was

considered as a temporary electron acceptor for promoting butyrate oxidation, and

methanogens accepted these electrons from the reductive biochar to reduce the carbon

dioxide to methane. In this manner, the high H2 partial pressure could not inhibited

butyrate oxidation.

3.4 Response of the microbial community to biochar

addition
To identify the response of the microbial community structure to biochar

16
addition, the microbial mixtures of BC15 and BC0 were used for microbial

community analysis. As shown in Table 2, for the bacteria community after biochar

addition, the Shannon index decreased from 4.73 to 4.01 and the Simpson index

increased from 0.03 to 0.06. Meanwhile, for the archaea community, the Shannon

index decreased from 1.90 to 1.58 and the Simpson index increased from 0.25 to 0.38.

These results revealed that biochar addition decreased the diversity of both the

bacteria and archaea communities. However, as presented above, VFA degradation

and methane production of the BC15 group were significantly promoted compared

with that of the BC0 group. Thus, it was suggested that biochar addition stimulated

the growth of some predominant microbes, which were crucial for promoting VFA

degradation and methane production.

Analysis of 16S rRNA gene sequences showed that biochar addition caused

changes in the bacteria community structure (Figure 6(a, b)). For example, the relative

abundance of Anaerolineaceae in the BC15 group was 30.08%, while in the BC0

group it was only 5.47%. It appeared that biochar addition stimulated the growth of

Anaerolineaceae. Multiple lines of evidence suggested that Anaerolineaceae (phylum

Chloroflexi) had the capacity of DIET. First, high transcription of the pilA gene was

detected in some genus of Anaerolineaceae, which was prerequisite for the formation

of the electrical conductive nanowires for DIET (Yu et al., 2016). Previous reports

also showed that Anaerolineaceae played key roles in the syntrophy metabolism of

AD and could transfer electrons to ferric iron (Kawaichi et al., 2013; Narihiro et al.,

2015). In an anaerobic system with added fulvic acids, Anaerolineaceae became the

17
dominant microbe as the dosage of fulvic acids was increased, which suggested that

Anaerolineaceae were able to transfer electrons to fulvic acids for syntrophic

metabolism (Dang et al., 2016). Moreover, Anaerolineaceae were considered as one

of predominant population in the anode of plant-based sediments Microbial Fuel Cell

(MFC) system, and their relative abundance (17%) were comparable with that of

Geobacter (19%), the well-known exoelectrogenic bacteria (Cabezas et al., 2015).

Figure 6(a) illustrated the similar microbial community shifts occurring at the phylum

level. In the BC0 Group, Firmicutes (43.81%) were the predominant bacteria, while

Chloroflexi increased from 5.52% to 30.19% and Firmicutes decreased to 12.21%

after addition of biochar. These results were also consistent with (Dang et al., 2016).

Together, these previous studies suggested that Anaerolineaceae were capable of

extracellular electron transfer to electron acceptors.

For the archaea community, as shown in Figure 6(c), Methanosaeta

(Methanothrix), Methanobacterium and Methanolinea were the three predominant

genera in the methane production system regardless of the addition of biochar. These

three genera accounted for more than 85% of the total archaea OTUs. Noticeably,

with the addition of biochar, Methanosaeta was further enriched by 43.8% (from

40.77% in the BC0 Group to 58.64% in the BC15 Group). Traditionally,

Methanosaeta were considered as the methanogens producing methane by using

acetate. However, in the presence of some electron donors, the Methanosaeta genus

could also reduce carbon dioxide to methane via DIET (Morita et al., 2011; Rotaru et

al., 2014). Furthermore, it was also found that Anaerolineaceae and Methanosaeta

18
were enriched in the conductive carbon felt particles used for enhancing the

degradation of propionate, which suggested syntrophic metabolism between these two

microbes was achieved via DIET (Xu et al., 2016). All the evidence suggested that the

addition of biochar could achieve the enrichment of the electro-active

Anaerolineaceae and Methanosaeta, and further indicated the advantage of biochar

assisted AD over conventional process.

4. Conclusions
Syntrophic VFAs degradation and methane production was promoted by biochar

addition in this study. Biochar showed a remarkable buffering capacity to alleviate the

pH decrease caused by VFAs accumulation. By using butyrate as a substrate in a

specific experiment, it was identified that syntrophic degradation of butyrate to

acetate occurred under high H2 partial pressure, evidently supporting the suggestion

that biochar would have assisted in DIET in AD process. Biochar addition also

resulted in enrichment of electro-active Anaerolineaceae and Methanosaeta, which

further indicated the advantage of biochar assisted AD over conventional process.

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

Acknowledgements:
This work was supported by National Natural Science Foundation of China (Grant No.

51608430), the Natural Science Foundation for Young Scientists of Xi’an University

of Architecture and Technology, China (Grant No. QN1615), and the Scientific

Research Program Funded by Shaanxi Provincial Education Department (Grant No.

19
17JS077).

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Figure and Table captions

Figure 1. Cumulative methane production (a) and pH variation (b) under

different biochar dosages condition.

Figure 2. Volatile fatty acids variation under different biochar dosages

conditions.

Figure 3.Acetate to n-butyrate mass concentration ratio variation under

different biochar dosage conditions.

Figure 4. Cumulative methane production and pH variation under

different organic loading rates condition

Figure 5. Cumulative methane production and VFAs concentration

variation in 1st (a, c) and 2nd periods (b, d)

Figure 6. Relative abundance of bacterial communities under phylum (a)

and genus (b) level and archaeal communities under genus level (c) at the

end of complex organic wastes anaerobic digestion.

Table 1. The fitting results of methane production on different biochar

dosage addition by modified Gompertz equation.

Table 2. Microbial community diversity analysis of bacteria and archaea

of biochar amended (BC) and Control (CT) groups.

26
 Cumulative methane production (mL)
120
(a)
100

80

60
BC2
BC6
40
BC10

20 BC15
BC0
0
0 5 10 15 20 25 30 35 40 45 50 55
Time (day)

7.3
(b)
6.8

6.3
pH

BC2
5.8
BC6
BC10
5.3
BC15
BC0
4.8
0 4 8 12 16 20 24 28 32 36
Time (day)

Figure 1. Cumulative methane production (a) and pH variation (b) under

different biochar dosages condition.

27
 2400 2400
BC0-acetate BC2-acetate
2100 BC0-propionate BC2-propionate
2000 BC2-n-butyrate
BC0-n-butyrate
Cumulative volatile fatty acids

1800 BC2-iso-valerate

Cumulative volatile fatty acids

BC0-iso-valerate
concentration (mg/L)

1600

concentration (mg/L)
1500

1200 1200

900
800
600

400
300

0
0
0 1 3 5 7 9 11 13 15 17 21 25 28 33 36 39 0 1 3 5 7 9 11 13 15 17 21 25
Time (day) Time (day)

2400 2400
BC6-acetate BC10-acetate
BC6-propionate 2000 BC10-propionate
2000

Cumulative volatile fatty acids

BC6-n-butyrate
Cumulative volatile fatty acids

BC10-n-butyrate
BC6-iso-valerate
concentration (mg/L)
concentration (mg/L)

1600 1600 BC10-iso-valerate

1200 1200

800 800

400 400

0 0
0 1 3 5 7 9 11 13 15 17 21 25 0 1 3 5 7 9 11 13 15 17 21 25
Time (day) Time (day)

2400
BC15-acetate
Cumulative volatile fatty acids concentration

BC15-propionate
2000
BC15-n-butyrate
BC15-iso-valerate
1600
(mg/L)

1200

800

400

0
0 1 3 5 7 9 11 13 15 17 21 25
Time (day)

Figure 2. Volatile fatty acids variation under different biochar dosages

conditions.

28
 10
BC2

BC6
8
Acetate to n-butyrate mass ratio

BC10

BC15
6
BC0

0
0 5 10 15 20 25 30 35
Time (day)

Figure 3. Acetate to n-butyrate mass concentration ratio variation under

different biochar dosage conditions.

29
 7.4 7.3

Cumulative methane production

120
Cumulative methane prduction
70
(a) (b)
7.2 6.8
60 100
7
50 80 6.3
6.8

(mL)

pH
(mL)

40
60

pH
30 6.6 BCM 5.8
BCL 40
6.4 CTM
20 CTL
BCM pH 5.3
10 BCL pH 6.2 20
CTL pH CTM pH
0 6 0 4.8
0 5 10 15 20 25 0 5 10 15 20 25 30 35 40 45 50 55
Time (day) Time (day)

180 7.5 250 7.5

Cumulative methane production

Cumulative methane production

160 (c) 225 (d)

7 7
140 200
120 6.5 175 6.5
100 150

(mL)
(mL)

BCH 6 125 6

pH
80 pH BCEH
CTH 100
60 5.5 CTEH
BCH pH
75 5.5
BCEH pH
40 CTH pH
5 50 CTEH pH 5
20
25
0 4.5 0 4.5
0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45 50
Time (day) Time (day)
Figure 4. Cumulative methane production and pH variation under different organic loading rates condition.

30
 40
Cumulative methane production BC

Cumulative methane production

50 35 BC
Control A Control A
40 30
Control B
25
(mL)

30

(mL)
20

20 15
10
10
(a) 5
(b)
0 0
0 4 8 12 16 20 24 28 0 1 2 3 4 5 6 7 8 9
Time (day) Time (day)
1800 1000
Bu-BC
Volatile fatty acids concentration

Volatile fatty acids concentration

Bu-BC
1600 Bu-Control A
Bu-Control B Bu-Control A
1400 Ac-BC 800
Ac-Control A Ac-BC
1200 Ac-Control B
Ac-Control A
600
(mg/L)

(mg/L)
1000
800
400
600
400 200
200 (c) (d)
0 0
0 4 8 12 16 20 24 28 0 1 2 3 4 5 6 7 8 9
Time (day) Time (day)
Figure 5. Cumulative methane production and VFAs concentration variation in 1 st (a, c) and 2nd periods (b, d)

31
 (a)

(b)

(c)

Figure 6. Relative abundance of bacterial communities under phylum (a)

and genus (b) level and archaeal communities under genus level (c) at the

end of complex organic wastes anaerobic digestion.

32
 Table 1. The fitting results of methane production on different biochar dosage addition
by modified Gompertz equation.
Group t0 (day) Rmax (mL/day) P0 (mL) R2
BC0 21.2±0.2 6.7±0.1 111.7±1.5 0.997
BC2 15.3±0.1 8.7±0.2 114.6±2.1 0.996
BC6 12.1±0.1 9.4±0.2 116.2±1.7 0.996
BC10 10.2±0.2 8.2±0.2 112.1±2.1 0.993
BC15 7.5±0.2 7.8±0.2 109.5±1.8 0.993

Table 2. Microbial community diversity analysis of bacteria and archaea of biochar

amended (BC) and Control (CT) groups.
Sample OTU number Shannon index Simpson index
Bacteria-BC 1300 4.01 0.06
Bacteria-CT 1618 4.73 0.03
Archaea-BC 506 1.58 0.38
Archaea-CT 537 1.90 0.25

33
Highlights:

Biochar (BC) was added for enhancing anaerobic digestion of complex organic wastes

BC addition shortened lag time and raised maximum CH4 production rate

BC showed good buffering capacity to mitigate pH decrease caused by VFAs

accumulation

BC addition promoted syntrophic oxidation of butyrate under high H 2 partial pressure

Electro-active Anaerolineaceae and Methanosaeta were enriched after BC addition

34