Materials Science for Energy Technologies 2 (2019) 377–384

Contents lists available at ScienceDirect

Materials Science for Energy Technologies

CHINESE ROOTS
GLOBAL IMPACT
journal homepage: www.keaipublishing.com/en/journals/materials-science-for-energy-technologies

Magnetite doped granular activated carbon as an additive

for high-performance anaerobic digestion
Sajib Barua, Basem S. Zakaria, Long Lin, Bipro Ranjan Dhar ⇑
Department of Civil and Environmental Engineering, University of Alberta, 9211-116 Street NW, Edmonton, AB T6G 1H9, Canada

a r t i c l e i n f o a b s t r a c t

Article history: In recent years, granular activated carbon (GAC) has been extensively investigated as an additive for pro-
Received 24 February 2019 moting electroactive microbiome to enhance anaerobic digestion performance. This study first demon-
Revised 1 April 2019 strates that GAC particles doped with magnetite can significantly improve the syntrophic degradation
Accepted 2 April 2019
of propionate, a substrate that is not readily degraded by microbial communities in the anaerobic diges-
Available online 11 April 2019
tion process. The bioreactor amended with magnetite doped GAC achieved the highest methane produc-
tion (220 mL/g COD) and COD removal (70%) in fed-batch tests, which were 1.5 and 1.2 times of those in
Keywords:
control and GAC amended bioreactors, respectively. The acetate accumulation was found to be correlated
Anaerobic digestion
Granular activated carbon
with methane production. Quantitative comparison of microbial communities suggested that the addi-
Magnetite-doped granular activated carbon tion of GAC or doped GAC didn’t achieve higher microbial biomass compared to control. Instead, multiple
Magnetite known electroactive bacterial genera (Shewanella, Pseudomonas, Geobacter, and Desulfuromonas) were
Propionate selectively enriched along with higher levels of heme-binding proteins in the bioreactors amended with
magnetite doped GAC (1.1
105 cells/mL) and GAC (8.4
104 cells/mL) in comparison to control
(2.5
104 cells/mL). Thus, our results suggest that tailoring GAC particles though doping with mag-
netite can provide an efficient method to achieve high-performance anaerobic digestion.
Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

1. Introduction
Anaerobic digestion is a popular bioprocess for biowaste diver-
sion from landfills. Biogas produced from digesters can be used for
heat and energy production on site [1]; CO2 captured from the bio-
gas can be utilized for algae production or microbial electrosynthe-
sis of value-added chemicals [2,3]. Fundamentally, anaerobic
digestion is a complex bioprocess where fermentative bacteria
convert complex organics into various simple metabolites, such
as acetate, H2, formate, and methanol that are utilized by methano-
gens [4,5]. Thus, fermentative bacteria and methanogens syntroph-
ically exchange electrons in the form of these metabolites.
However, this syntropy can be interrupted due to the accumulation
of liquid metabolites [4,6]. For instance, elevated concentration of
⇑ Corresponding author.
E-mail address: bipro@ualberta.ca (B.R. Dhar).
Peer review under responsibility of KeAi Communications Co., Ltd.
Production and hosting by Elsevier
short-chain fatty acids (SCFAs), such as acetate, butyrate, and pro-
pionate can acidify the digester which leads to the inhibition of
methanogenic microbiome [7,8]. In particular, propionate has
often been noticed as the major SCFA in the acidified digester,
and high hydrogen partial pressure (PH2) is usually assumed to
be one of the main reasons for propionate accumulation [6,9,10].
Propionate fermentation requires low PH2 (<104 atm) under stan-
dard condition; hence, interspecies H2 transfer between fermenta-
tive bacteria and methanogens is crucial for maintaining low PH2 in
digesters [11–13].
In recent years, enhancing the methanogenic conversion of var-
ious SCFAs with the addition of conductive materials have been
demonstrated to be an effective approach [4,14–16]. The addition
of various conductive additives, such as granular activated carbon
(GAC), carbon cloth, biochar, carbon fibers, iron nanoparticles in
anaerobic digesters could enable microbiome to directly exchange
electrons instead of metabolites (e.g., H2/formate), which could
ultimately enhance the methanogenic conversion of various SCFAs
[4,14,16,17]. This unique syntrophy is called direct interspecies
electron transfer (DIET). In the literature, bacteria and archaea cap-
able of promoting DIET have been referred to as electroactive bac-
teria and electrotrophic methanogens, respectively [4,18]. In the
absence of conductive additives, electroactive bacteria can estab-

https://doi.org/10.1016/j.mset.2019.04.002
2589-2991/Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
378 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
lish DIET by producing biogenic conductive nanowires, while
biosynthesis of such microbial nanowires requires substantial
energy investment by microbes [4,5]. Hence, conductive additive
like GAC could provide energetically favorable condition for syn-
trophic microbiome to promote DIET.
Among various conductive additives, GAC has been extensively
investigated for promoting DIET in methanogenic bioreactors
[4,14,16,19]. GAC has been frequently used as an adsorbent for
the removal of various pollutants from air and water [20]. GAC
has also been used as an electrode material in various microbial
electrochemical systems, such as microbial fuel cells [21,22]. Sev-
eral studies have shown that surface modification of GAC with
other functionalized materials, such as magnetite and titanium
dioxide, can further increase the effectiveness of GAC for such
applications [23–27]. Most importantly, a few recent studies have
shown that surface modification of carbon-based anode electrodes
with conductive/semi-conductive iron-oxide particles (e.g., mag-
netite, ferrous sulfide) can preferably stimulate growth and
enhancement of electroactive bacterial biomass on their surface
due to reduced charge transfer resistance, which could ultimately
improve performance of microbial electrochemical systems
[24,25]. Thus, we hypothesized that doping of GAC with magnetite
might provide a better additive to promote the growth of elec-
troactive biomass for further enhancement of anaerobic digestion
performance. In general, the development of advanced materials
has received significant attention for improving the efficiency of
various microbially-driven bioenergy systems, such as microbial
fuel cells, enzymatic biofuel cells, microbial desalination cells,
etc. [28–30]. However, to date, no studies have investigated the
impact of the addition of doped carbon-based conductive additives
on anaerobic digestion performance.
Consequently, this study first demonstrated that the effective-
ness of GAC as a conductive additive in anaerobic digestion could
be further enhanced through doping with magnetite particles.
The impact of magnetite doped GAC particles on methanogenic
degradation of propionate was assessed in consecutive fed-batch
cycles. We compared the performance of bioreactors amended
with GAC, magnetite-doped GAC, and unamended control in terms
of methane productivity, accumulation of SCFAs, qualitative and
quantitative differences in microbial communities, and concentra-
tions of heme-binding proteins in extracellular polymeric sub-
stances (EPSs) extracted from biomass samples.
2. Materials and methods
2.1. Preparation of magnetite-doped GAC particles
The doping of GAC with magnetite was performed according to a
method previously described in the literature after slight modifica-
tion [24]. Before doping, GAC particles (mesh size: 8–20, Sigma
Aldrich, Canada) were washed thoroughly with deionized water
and dried at 105 ± 5 °C. Then, 6.5 g of magnetite particles (95% pure,
powder size < 5 mm; Sigma Aldrich, Canada) was suspended into
150 mL of deoxygenated water. After that, 100 g of GAC particles
were added into the suspension and gently mixed for 12 h at
300 rpm. Then, the liquid was vacuum dried at 120 °C. After the
completion of this process, we thoroughly washed the doped parti-
cles and analysed them for element mapping and composition with
scanning electron microscopy (SEM) and energy dispersive spec-
trometry (EDS) to confirm the doping of particles. As discussed later
(Section 3.1), higher iron (Fe) content on the GAC surface confirmed
the effectiveness of doping procedure used in this study. Here, GAC
particles doped with magnetite are referred to as ‘magnetite doped
GAC’, and GAC particles received the same treatment as described
above except magnetite doping, are referred to as ‘GAC’.
2.2. Propionate biodegradation experiments
Nine glass anaerobic bioreactors (working volume of 0.8 L)
equipped with mechanical mixers and reactor lids along with gas
and liquid sampling ports were used in this study. Bioreactors
supplemented with 22.5 g/L of GAC, and magnetite doped GAC are
referred to as ‘GAC’ and ‘MDGAC’, respectively. Bioreactors without
any GAC or doped GAC particles served as the control. Triplicate
bioreactors were operated for each condition. The bioreactors were
inoculated with a mixture of anaerobic digester sludge and effluent
from a lab-scale mother microbial electrolysis cell (MEC), as previ-
ously described in the literature [6,31]. The mother MEC was oper-
ated with 25 mM sodium acetate medium for over one year. The
anaerobic digester sludge (TSS: 12.4 ± 0.05 g/L, VSS: 6.4 ± 0.03 g/L,
TCOD: 9.6 ± 0.06 g/L, SCOD: 1.5 ± 0.09 g/L) was collected from a
full-scale anaerobic digestion facility at the Gold Bar Wastewater
Treatment Plant (Edmonton, AB, Canada). The volume ratio of
digested sludge to MEC effluent was 8:1, and 400 mL of inoculum
and 400 mL of substrate medium were added into each reactor. Ini-
tially, ethanol (TCOD: 4860 ± 410 mg/L) was used as the substrate to
stimulate enrichment of electroactive methanogenic communities
as previously suggested in the literature [6,9,32]. Then, we switched
from ethanol to a mixture of ethanol and propionate, and then
finally switched to propionate as a sole substrate to gradually adopt
the microbial communities. The substrate medium was always sup-
plemented with NaHCO3 (3.5 g/L) and trace element solution (5 mL/L)
as described elsewhere [6]. The performance of bioreactors was
assessed in consecutive fed-batch cycles operation with propionate
medium (TCOD: 13280 ± 90 mg/L). The duration of each cycle was
approximately 90 h. After the completion of each cycle, mixing was
stopped for three hours, and 150 mL of supernatant was replaced with
fresh propionate medium, corresponding to a hydraulic residence
time of 20 days. During this process, a gas bag filled with ultra-pure
nitrogen gas was used to alleviate oxygen diffusion into bioreactors.
The bioreactors’ temperature was maintained at 37 ± 1 °C using a
water bath and liquid medium was stirred at 300 rpm.
2.3. Analytical methods
COD concentration was measured with HACH COD reagent kit
(High Range, 20–1500 mg/L; HACH Co., Loveland, CO, USA). The
concentrations of various SCFAs were analysed with an ion chro-
matograph (Dionex ICS-2100, Dionex, Sunnyvale, CA) equipped
with an electrochemical detector (ECD) and microbore AS19 col-
umn. The concentration of heme-binding proteins was measured
using the Heme Assay Kit (Heme Assay Kit, Sigma-Aldrich, USA)
according to the manufacturer’s instructions. The samples were
prepared according to the method described in the literature after
some minor modification [33,34]. Biomass samples from different
reactors were collected in 50 mL centrifuges tube and suspended
in 10 mL phosphate buffer (2 mM Na3PO4
12 H2O, 4 mM
NaH2PO4
1 H2O, 9 mM NaCl, 1 mM KCl, pH 7.0). Then, 2 gm of
cation exchange resin (CER) (DowexÒ MarathonÒ C sodium form,
Sigma-Aldrich, USA) washed with phosphate buffer (15 min;
10 mL g1 Dowex) was added followed by shaking at maximum
capacity on shaker (Vortex Mixer, Fisher Scientific, USA) for 20
mins, then centrifuged at 20,000g for 20 min at 4 °C. Then,
supernatant was moved into a new centrifuge tube (50 mL); the
centrifuge step was repeated twice, followed by filtration using
0.20 mm Nylon filter. The samples were analysed according to the
manufacturer’s instructions. For directly monitoring methane gas
production from different bioreactors, the gas sampling port of
each bioreactor was connected to a CO2 sequestration bottle
(80 mL; 3 M NaOH with thymolphthalein indicator) followed by a
gas bag [6]; the volume of methane was intermittently measured
with a syringe.
 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
The elemental composition of GAC and magnetite-doped GAC
particles were examined with SEM (Zeiss Sigma 300 VP-FESEM,
Carl Zeiss, Cambridge, UK) and EDS. The Brunauer–Emmett–Teller
(BET) surface areas of GAC and doped GAC particles were measured
with gas adsorption method [35].
2.4. Microbial community analyses
Microbial communities of biomass samples collected from the
control, GAC and MDGAC bioreactors were characterized by high
throughput 16S rRNA gene sequencing. The metagenomic DNA of
the biomass samples were extracted using the PowerSoilÒ DNA
Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to
the manufacturer’s protocol. The properties of the extracted DNA
samples (e.g., concentration, quality, and integrity) were deter-
mined using a spectrophotometer (NanoDrop 2000C, Thermo
Fisher Scientific, Waltham, MA, USA). The extracted DNA samples
were stored immediately at 70 °C until Illumina Miseq sequenc-
ing was performed at the Research and Testing Laboratory (Lub-
bock, TX, USA) using the specific primer set 341F:
CCTACGGGNGGCWGCAG and 805R: GACTACHVGGGTATCTAATCC
to target the V3-V4 region of 16S rRNA gene. The software Quanti-
tative Insights Into Microbial Ecology (QIIME v2) was used to anal-
yse the demultiplexed sequencing data [36]. The sequences were
first processed to remove and/or correct noisy reads, remove chi-
meric sequences and singletons, and join denoised paired-end
reads (DADA2 method) [37]. The denoised sequences were
assigned to species-equivalent operational taxonomic units (OTUs)
at a 97% sequence similarity level using the open-reference OTU
picking method (vsearch method against 2013–08 Greengenes
database) [38]. The distribution of major bacterial and archaea
genera (each represented by >0.1% of their population) was further
analysed using heatmap with a double hierarchical dendrogram
[39]. Bacterial and archaeal quantity in the DNA samples were
evaluated by real-time quantitative polymerase chain reaction
(qPCR) analysis.
2.5. Calculation of hydrogen partial pressure (PH2)
The hydrogen partial pressure (PH2) that allows propionate fer-
mentation (C3H5O – – +
2 + 3H2O ? CH3COO + HCO3 + H + 3H2) to pro-
ceed was computed using the measured concentrations of
acetate and propionate according to Eq. (1).
0
00  113
DG
@ e RT
½HPr A
P H2 ¼ ð1Þ
½HAc
½HCO3  
where, DGo’ is the standard Gibbs free energy for propionate fer-
mentation (+76.1 kJ/mole) under standard biochemical conditions
(i.e., substrates and products at 1 M or 1 atm, pH 7, and 298 K), R
is the ideal gas constant (8.314 J/mol-K), T is the temperature (K),
[HPr] is the propionate concentration (M), [HAc] is the acetate con-
centration (M), and [HCO–3] is the concentration of bicarbonate (M),
which was assumed to be 3.5 g/L (0.04 M) for the computations.
2.6. Statistical analysis
All the statistical analyses were performed with JMP 11 (SAS
Institute Inc., NC, USA). The Analysis of Variance (ANOVA) and stu-
dent’s t-test at a 95% confidence level was conducted and p < 0.05
was considered statistically significant. A summary of the Student’s
t-test results is provided in the Supplementary Information.
379
3. Results
3.1. Physicochemical properties of doped GAC
Fig. 1 shows SEM images of GAC and magnetite doped-GAC.
Both GAC and doped GAC exhibited rough and porous surfaces.
Magnetite particles were tightly attached on the surface of GAC
particles (Fig. 1B). However, the distribution of magnetite particles
on the GAC surface was not uniform (Fig. 1D). The elemental anal-
ysis with SEM-EDS also confirmed the presence of higher iron con-
tent on the surface of magnetite-doped GAC (16.8–27.4%) than that
of GAC (0.76–1.48%) (Table 1). The EDS spectrums were provided in
the Supplementary Information. Both GAC and doped GAC particles
contained traces of other elements such as calcium, magnesium,
and aluminum, which is consistent with the previous literature
[40,41]. On the other hand, the BET surface area of doped GAC par-
ticle decreased by 12% (Table 1), which was due to the impregna-
tion of magnetite particles on the GAC surface [23].
3.2. Methane productivity from propionate
Fig. 2A illustrates methane productivities from different biore-
actors in four consecutive fed-batch cycles; the time course cumu-
lative methane production for each condition is provided in the
Supplementary Information. The addition of GAC and doped GAC
led to significantly higher methane productivities over the control.
The MDGAC showed the highest methane production among all
reactors, which was 1.52- and 1.21-times of that in the control
and GAC bioreactors (p < 0.05), respectively. On the other hand,
GAC showed 25% higher methane production than that in control
during four consecutive fed-batch cycles. There was no significant
difference in the ultimate methane production from MDGAC in
four consecutive cycles (p > 0.05), while methane production from
both control and GAC gradually decreased (p < 0.05) after each fed-
batch cycle.
As shown in Fig. 2B, MDGAC also exhibited 50% and 20% higher
COD removal efficiencies than that of control and GAC, respec-
Fig. 1. SEM image for (A) GAC and (B) magnetite doped GAC; SEM-EDS image of
elemental mapping for (C) GAC and (D) magnetite doped GAC.
Table 1
Physicochemical properties of GAC and magnetite doped GAC particles.
Fe content (%) BET surface area (m2/g)
GAC 0.76–1.48 654
Magnetite doped GAC 16.8–27.4 576
380
A 250 Control
200
Specific CH 4 Production
(mL/ g CODinitial)
150
100
50
0
80
B
COD Removal Efficiency (%)
60
40
20
0
Cycle #1 Cycle #2
Fig. 2. (A) Average specific methane production and (B) COD removal efficiencies in
four consecutive fed-batch cycles.
tively. However, GAC also provided 30% higher COD removal effi-
ciency over the control. Analogous to methane production, COD
removal efficiencies from MDGAC remained almost constant dur-
ing all cycles. Also, COD removal efficiencies in the GAC and control
decreased from Cycle #1 to 4 (GAC: 62 ± 1% vs. 47 ± 8% and control:
50 ± 8% vs. 36 ± 2%). Nonetheless, based on the methane productiv-
ities and COD removal efficiencies, the following ranking could be
established: MDGAC > GAC > control.
3.3. Accumulation of SCFAs and estimation of PH2
The accumulation of SCFAs in the effluent was monitored dur-
ing four consecutive fed-batch cycles (Fig. 3). The detailed initial
and final SCFAs concentrations are provided in the supplementary
information. The syntrophic propionate degradation in anaerobic
digester could lead to a different spectrum of metabolites or by-
products, while propionate conversion to acetate and H2 is consid-
ered to be the most predominant pathway. As evident from the
Fig. 3A, all bioreactors showed minor accumulation of propionate
(<250 mg COD/L), indicating microbial consortia efficiently fer-
mented propionate. In contrast, an accumulation of acetate
(>1000 mg COD/L) was observed in all reactors (Fig. 3B). However,
MDGAC successfully maintained a significantly lower acetate con-
centration (1020–1290 mg COD/L) in all cycles than GAC and con-
trol (p < 0.05). Acetate concentration in both control and GAC
gradually increased in consecutive cycles; however, GAC was able
to maintain significantly lower acetate accumulation over control
(p < 0.05). The trend of the bioreactors in maintaining low acetate
concentration in the effluent was MDGAC > GAC > control.
Fig. 4 shows the time course degradation profiles of SCFAs in
different reactors in Cycle#5. Initial acetate and propionate con-
centration in control was noticeably higher as compared to GAC
and MDGAC reactors which was due to the accumulation of these
SCFAs over four consecutive fed-batch cycles. Nonetheless, compa-
S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
GAC MDGAC
Cycle #3 Cycle #4
Fig. 3. Average effluent (A) acetate and (B) propionate concentrations during four
consecutive fed-batch cycles.
rable trends in changes in acetate and propionate concentrations
were observed in all bioreactors regardless of the differences in ini-
tial concentrations; acetate concentration quickly reached a pla-
teau. Similar to the first four cycles, MDGAC exhibited the lowest
accumulation of acetate and propionate concentration among all
reactors. The PH2 required for the propionate fermentation to pro-
ceed was decreased with time from 1.2
104 atm to 6
105 atm
for all three bioreactors because propionate decreased while acet-
ate increased with time.
3.4. Microbial community
3.4.1. Quantitative analysis of microbial population
The quantitative analysis of variations in microbial cell biomass
was performed to attain further insights into the differences in
methane productivities from different reactors (Table 2). The qPCR
results showed that the amounts of both bacterial and archaeal cells
were substantially higher in control (bacteria: 3.50
106 cells/mL,
archaea: 1.65
105 cells/mL) compared to the two amended
bioreactors. Furthermore, MDGAC (bacteria: 8.85
105 cells/mL,
archaea: 7.74
104 cells/mL) showed relatively higher abundances
of both bacteria and archaea than those in GAC reactor (bacteria:
7.64
105 cells/mL, archaea: 3.78
104 cells/mL). Thus, the addition
of conductive materials did not increase total microbial cell numbers
in this study.
3.4.2. Bacterial community
Fig. 5A shows the relative abundance of bacterial communities
at the phylum level, which shows apparent changes in bacterial
populations in GAC and MDGAC reactors compared to the control.
The two most predominant phyla were Proteobacteria and Chlo-
roflexi in all reactors, both accounting for  60% of the bacterial
 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384 381

Fig. 5. Relative abundance of (A) bacterial community at phylum level and (B)
archaeal community at genus level. Note: sequences that accounted for less than 2%
of their population were grouped into ‘‘Others”.

groups. Group I contains three genera that prevailed in all three

bioreactors, including Syntrophobacter, candidate genus T78, and
HA73. Syntrophobacter is well-known for syntrophic propionate
oxidation to acetate and H2. The candidate genus T78 belongs to
the family Anaerolinaceae (phylum Chloroflexi). Notably, group IIa
contains genera that were almost undetected in control, while
increased to 2–6 orders of magnitude higher in GAC and MDGAC
reactors. Most of these genera are known as electroactive bacteria,
including Shewanella, Pseudomonas, Geobacter, and Desulfuromonas,
Fig. 4. Degradation profiles of SCFAs and critical partial pressure of hydrogen gas
which all belong to Proteobacteria [42]. The total relative abun-
(PH2) in (A) control, (B) GAC, and (C) MDGAC bioreactors from cycle #5.
dances of these possible electroactive genera were substantially
higher in GAC, and MDGAC amended reactors (11–12%) than that
in control (0.7%). Another genus Pelotomaculum (phylum Firmi-
cutes) in Group IIa contains well-defined syntrophic propionate-
Table 2
Total microbial counts and heme-binding protein concentrations from three biore- oxidizing bacteria (e.g., P. propionicicum) [44]. Group IIb contains
actors. Note: the microbial quantity was determined by analysis of qPCR targeted very diverse bacteria; most of them were present in all reactors
bacterial and archaeal 16S rRNA gene. with similar abundances, such as Syntrophomonas and Acetobac-
Bacteria (cells/mL) Archaea (cells/mL) Heme-binding terium, while some of them were considerably lower in GAC and
protein (mg/L) MDGAC compared to control bioreactor, such as Clostridium. The
Control 3.50
106 1.65
105 621 genera Clostridium and Syntrophomonas contain various well-
GAC 7.64
105 3.78
104 1740 defined syntrophic hydrogen-producing bacteria [45]. Acetobac-
MDGAC 8.85
105 7.74
104 2362 terium is recognized as homoacetogen (usually convert H2/CO2 to
acetate) [46].

communities. In control, Chloroflexi (34%) was dominant, whereas
Proteobacteria (37–40%) prevailed in GAC and MDGAC. Moreover,
Firmicutes (7% vs. 16–18%) was lower, while Bacteroidetes (14%
vs. 5–7%) was higher in control compared to the two amended
bioreactors. Most of the well-known electroactive bacteria
(i.e., Geobacter, Shewanella) reported in the literature were from
Proteobacteria [42]. In contrast, Firmicutes contains various fermen-
tative bacteria, such as Clostridium and Syntrophomonas [43].
Nonetheless, the bacterial communities were quite similar in
GAC and MDGAC reactors regarding bacterial phylum structure.
The distribution of major bacterial genera was further analysed
using heatmap (Fig. 6). Clustering of bacteria resulted in two
3.4.3. Archaeal community
Fig. 5B shows the relative abundance of methanogens at the
genus level. Methanosaeta was the most dominant genus in all
bioreactors, and its proportion was the highest in MDGAC (85%),
followed by GAC (49%) and control (43%) reactors. Methanosarcina
was also present at low relative abundance in all reactors and was
slightly higher in control and GAC (3–4%) than that in MDGAC (1%).
In contrast, known hydrogenotrophic methanogens including
Methanobacterium and Methanoculleus accounted for 46% in con-
trol, 44% in GAC, only 13% in MDGAC. Other H2 utilizing methano-
gens such as Methanolinea were present at low proportions in all
reactors and decreased in relative abundance with the addition
382 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
Fig. 6. Double hierarchical dendrogram based on log cell number of bacterial genera that were assigned a genus name (each represented >0.1% of their population).
of GAC and MDGAC. Overall, compared to MDGAC, various hydro-
genotrophic methanogens were higher in relative abundance in
control and GAC reactors.
3.5. Heme-binding protein concentration
The concentrations of heme-binding proteins in EPSs in biomass
samples collected from different bioreactors were also measured
(Table 2). The biomass EPS from MDGAC reactor showed a signifi-
cantly higher level of heme-binding proteins (2362 ± 23 mg/L)
compared to the GAC (1740 ± 1 mg/L) and control (621 ± 9 mg/L)
bioreactors (p < 0.05). The qPCR quantification of bacterial cells
Fig. 7. Correlation between concentrations of heme-binding protein and bacterial
cells belonging to four known electroactive genera (Shewanella, Pseudomonas,
Geobacter, and Desulfuromonas).
belonging to four potential electroactive genera (Shewanella, Pseu-
domonas, Geobacter, and Desulfuromonas) in different bioreactors
was positively correlated with the concentrations of heme-
binding protein (R2 = 0.9967) (Fig. 7).
4. Discussion
To date, various conductive materials have demonstrated a pos-
itive impact on anaerobic methanogenesis [4,5,47]. However, to
our knowledge, iron-doped GAC particles have not yet been inves-
tigated for improving anaerobic digestion. Thus, the results of this
study first demonstrated that doping of GAC particles with mag-
netite could offer further improvement of methanogenesis from
propionate, a substrate that is not readily degraded by methano-
genic microbiome. Propionate conversion to methane may include
multiple biochemical reactions, while the most common being
propionate conversion to acetate and H2 and their subsequent con-
version to methane through acetoclastic and hydrogenotrophic
methanogenesis pathways, respectively [6,48]. On the other hand,
DIET-based pathway may involve propionate conversion to acetate
and electrons. Electrons can be directly utilized by electrotrophic
methanogens via reduction of CO2, while acetoclastic methanogens
can consume acetate [6,48]. The pathways involved in IHT and
DIET-based methanogenesis were summarized in the Supplemen-
tary Information. It appeared that acetate accumulation was the
primary factor for the inferior performance of control and GAC
bioreactors. Generally, it is believed that high PH2 (104 atm)
can establish a thermodynamically unfavorable condition for ace-
toclastic methanogenesis [10,42,43]. However, estimated PH2 val-
ues for different bioreactors were comparable or lower than the
threshold value of 104 atm. Thus, it seems unlikely that high
PH2 would promote acetate accumulation in control and GAC
reactors.
 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
Multiple known electroactive bacterial genera (Shewanella,
Pseudomonas, Geobacter, and Desulfuromonas) were selectively
enriched in the GAC and MDGAC reactors in comparison to control
(Control: 2.5
104 cells/mL, GAC: 8.4
104 cells/mL, MDGAC:
1.1
105 cells/mL). Given the significantly higher methane produc-
tivities from GAC and MDGAC reactors compared to control, it is
possible that they might be involved in the DIET to methanogens.
A few studies previously suggested that some electroactive bacte-
ria could degrade propionate to acetate and electrons; subse-
quently, acetate and electrons could be utilized by acetoclastic
and electrotrophic methanogens, respectively [6,9,48]. Addition-
ally, electroactive bacteria could oxidize acetate and transfer elec-
trons to electrotrophic methanogens [6,9,31]. Since no apparent
electroactive bacteria were detected in control, it is possible that
the methanogenesis in control bioreactor was primarily accom-
plished through traditional methanogenesis pathways (i.e., aceto-
clastic and hydrogenotrophic).
Interestingly, both the addition of GAC or magnetite doped GAC
did not bring any positive influence on the total amount of bacteria
and archaea, suggesting that the conductive materials did not con-
tribute to the increase of total microbial biomass. A possible reason
might be the collision of granular particles, leading to reduced bio-
mass retention [18]. Nonetheless, our results suggested that bio-
mass retention was not the reason for high performance from
GAC or MDGAC reactors over the control. Instead, enrichment of
known electroactive bacteria could be considered as a decisive fac-
tor in improving the performance of these reactors. Notably, the
total amount of bacteria and archaeal cell biomass was higher in
MDGAC in comparison with GAC. Thus, the amount of electroactive
bacterial biomass would also be higher in MDGAC, which was con-
sistent with higher levels of heme-binding protein in biomass EPS
collected from MDGAC. Heme-binding proteins are vital redox
component facilitating extracellular electron transport in various
electroactive bacteria, such as Shewanella and Pseudomonas [49].
Thus, MDGAC appears to be more advantageous in enriching elec-
troactive bacteria.
Comparable to this finding, previous studies also reported the
dominance of Methanosaeta in digesters amended with conducive
additives [6,50]. Several studies confirmed the electrotrophic
activity of Methanosaeta, suggesting its potential to be involved
in DIET. Considering the fact that quantitatively Methanosaeta
population was more abundant in the control reactor (Control:
7.1
104 cells/mL, GAC: 1.7
104 cells/mL, MDGAC: 6.6
104
cells/mL), it does not provide conclusive support on their role in
improving methane productivity from propionate. Moreover,
recent studies have confirmed the DIET capability of diverse
methanogens [8,17,48]. Hence, further studies would be needed
to identify electrotrophic methanogens more explicitly.
It must be asserted that both GAC and magnetite have found to
be effective in improving methanogenic activity in digesters in pre-
vious studies [4,14,16]. Although increased GAC loading in diges-
ters can boost digester performance [51], it is expected that
increased GAC loading will inevitably lead to increased digester
footprint as GAC occupies a working volume of the digester. Con-
versely, magnetite particles may easily wash out during continu-
ous digester operation [52]. Thus, economic and energy benefits
of engineering DIET with these materials will be traded off or be
negative. Hence, tailoring GAC particles though doping with mag-
netite can provide a practical solution to these challenges.
5. Conclusions
This study first demonstrates that doping of GAC particles with
magnetite could offer further improvement of methanogenesis
from propionate as a sole substrate. Magnetite doped GAC signifi-
383
cantly enhanced syntrophic propionate degradation over undoped
GAC. Our results suggested that biomass retention was not the rea-
son for the improvement of methane productivities in GAC or
MDGAC reactors over the control. The enrichment of various elec-
troactive bacteria was found as a pivotal factor in enhancing
methanogenesis. Thus, our results suggest that the deployment
of tailored conductive materials could assist in shaping efficient
methanogenic microbiome with electroactivity for high-
performance anaerobic digestion.
Acknowledgements
This work was supported by the Faculty of Engineering Start-up
Grant (University of Alberta), Natural Sciences and Engineering
Research Council of Canada Discovery Grant (#2017-05608), and
Future Energy Systems-Canada First Research Excellence Fund for
Early Career Researcher (FES-T01-Q01).
Conflict of interest
The authors declare that there is no conflict of interest regard-
ing the publication of this research manuscript.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.mset.2019.04.002.
References
[1] Rajesh K. Srivastava, Materials science for energy technologies, Mater. Sci.
Energy Technol. 2 (2019) 08–318, https://doi.org/10.1016/j.mset.2018.12.007.
[2] C. Yan, L. Zhu, Y. Wang, Photosynthetic CO2 uptake by microalgae for biogas
upgrading and simultaneously biogas slurry decontamination by using of
microalgae photobioreactor under various light wavelengths, light intensities,
and photoperiods, Appl. Energy. (2016), https://doi.org/10.1016/j.
apenergy.2016.06.012.
[3] L. Jourdin, S. Freguia, V. Flexer, J. Keller, Bringing high-rate, CO2-based
microbial electrosynthesis closer to practical implementation through
improved electrode design and operating conditions, Environ. Sci. Technol.
(2016), https://doi.org/10.1021/acs.est.5b04431.
[4] S. Barua, B.R. Dhar, Advances towards understanding and engineering direct
interspecies electron transfer in anaerobic digestion, Bioresour. Technol.
(2017), https://doi.org/10.1016/j.biortech.2017.08.023.
[5] Q. Cheng, D.F. Call, Hardwiring microbes via direct interspecies electron
transfer: mechanisms and applications, Environ. Sci. Process. Impacts. 18
(2016) 968–980, https://doi.org/10.1039/C6EM00219F.
[6] S. Barua, B.S. Zakaria, B.R. Dhar, Enhanced methanogenic co-degradation of
propionate and butyrate by anaerobic microbiome enriched on conductive
carbon fibers, Bioresour. Technol. (2018), https://doi.org/10.1016/j.
biortech.2018.06.053.
[7] Z. Zhao, Y. Zhang, Y. Li, Y. Dang, T. Zhu, X. Quan, Potentially shifting from
interspecies hydrogen transfer to direct interspecies electron transfer for
syntrophic metabolism to resist acidic impact with conductive carbon cloth,
Chem. Eng. J. 313 (2017) 10–18, https://doi.org/10.1016/J.CEJ.2016.11.149.
[8] Y. Dang, D. Sun, T.L. Woodard, L. Wang, K.P. Nevin, D.E. Holmes, Stimulation of
the anaerobic digestion of the dry organic fraction of municipal solid waste
(OFMSW) with carbon-based conductive materials, Bioresour. Technol. 238
(2017) 30–38, https://doi.org/10.1016/j.biortech.2017.04.021.
[9] Z. Zhao, Y. Zhang, Q. Yu, Y. Dang, Y. Li, X. Quan, Communities stimulated with
ethanol to perform direct interspecies electron transfer for syntrophic
metabolism of propionate and butyrate, Water Res. 102 (2016) 475–484,
https://doi.org/10.1016/j.watres.2016.07.005.
[10] H. Xu, C. Wang, K. Yan, J. Wu, J. Zuo, K. Wang, Anaerobic granule-based
biofilms formation reduces propionate accumulation under high H2 partial
pressure using conductive carbon felt particles, Bioresour. Technol. (2016),
https://doi.org/10.1016/j.biortech.2016.06.010.
[11] W. Gujer, A.J.B. Zehnder, Conversion processes in anaerobic digestion, Water
Sci. Technol. (1983).
[12] A.J.M. Stams, C.M. Plugge, Electron transfer in syntrophic communities of
anaerobic bacteria and archaea, Nat. Rev. Microbiol. (2009), https://doi.org/
10.1038/nrmicro2166.
[13] L. Shen, Q. Zhao, X. Wu, X. Li, Q. Li, Y. Wang, Interspecies electron transfer in
syntrophic methanogenic consortia: From cultures to bioreactors, Renew.
Sustain. Energy Rev. (2016), https://doi.org/10.1016/j.rser.2015.10.102.
384 S. Barua et al. / Materials Science for Energy Technologies 2 (2019) 377–384
[14] J.-H. Park, H.-J. Kang, K.-H. Park, H.-D. Park, Direct interspecies electron
transfer via conductive materials: A perspective for anaerobic digestion
applications, Bioresour. Technol. 254 (2018) 300–311, https://doi.org/
10.1016/J.BIORTECH.2018.01.095.
[15] D.R. Lovley, The microbe electric: conversion of organic matter to electricity,
Curr. Opin. Biotechnol. 19 (2008) 564–571, https://doi.org/10.1016/
j.copbio.2008.10.005.
[16] Q. Cheng, D.F. Call, Hardwiring microbes: Via direct interspecies electron
transfer: Mechanisms and applications, Environ. Sci. Process. Impacts. (2016),
https://doi.org/10.1039/c6em00219f.
[17] D.R. Lovley, Syntrophy goes electric: direct interspecies electron transfer,
Annu. Rev. Microbiol. 71 (2017) 643–664, https://doi.org/10.1146/annurev-
micro-030117-020420.
[18] S. Zhang, J. Chang, C. Lin, Y. Pan, K. Cui, X. Zhang, P. Liang, X. Huang,
Enhancement of methanogenesis via direct interspecies electron transfer
between Geobacteraceae and Methanosaetaceae conducted by granular
activated carbon, Bioresour. Technol. (2017), https://doi.org/10.1016/j.
biortech.2017.08.111.
[19] D.R. Lovley, Syntrophy goes electric: direct interspecies electron transfer,
Annu. Rev. Microbiol. (2017), https://doi.org/10.1146/annurev-micro-030117-
020420.
[20] C. Brasquet, P. Le Cloirec, Adsorption onto activated carbon fibers: Application
to water and air treatments, Carbon N. Y. (1997), https://doi.org/10.1016/
S0008-6223(97)00079-1.
[21] S. Kalathil, J. Lee, M.H. Cho, Granular activated carbon based microbial fuel cell
for simultaneous decolorization of real dye wastewater and electricity
generation, N. Biotechnol. (2011), https://doi.org/10.1016/j.nbt.2011.04.014.
[22] D. Jiang, B. Li, Granular activated carbon single-chamber microbial fuel cells
(GAC-SCMFCs): A design suitable for large-scale wastewater treatment
processes, Biochem. Eng. J. (2009), https://doi.org/10.1016/j.bej.2009.06.013.
[23] L.C.A. Oliveira, R.V.R.A. Rios, J.D. Fabris, V. Garg, K. Sapag, R.M. Lago, Activated
carbon/iron oxide magnetic composites for the adsorption of contaminants in
water, Carbon N. Y. (2002), https://doi.org/10.1016/S0008-6223(02)00076-3.
[24] N.G. Yasri, G. Nakhla, Electrochemical behavior of anode-respiring bacteria on
doped carbon electrodes, ACS Appl. Mater. Interfaces 8 (2016) 35150–35162,
https://doi.org/10.1021/acsami.6b09907.
[25] N.G. Yasri, G. Nakhla, The performance of 3-D graphite doped anodes in
microbial electrolysis cells, J. Power Sources (2017), https://doi.org/10.1016/j.
jpowsour.2016.12.081.
[26] J.H. Xu, N.Y. Gao, Y. Deng, S.Q. Xia, Nanoscale iron hydroxide-doped granular
activated carbon (Fe-GAC) as a sorbent for perchlorate in water, Chem. Eng. J.
(2013), https://doi.org/10.1016/j.cej.2012.07.141.
[27] Q. Chang, W. Lin, W. chi Ying, Preparation of iron-impregnated granular
activated carbon for arsenic removal from drinking water, J. Hazard. Mater.
(2010), https://doi.org/10.1016/j.jhazmat.2010.08.066.
[28] G. Anusha, M.T. Noori, M.M. Ghangrekar, Application of silver-tin dioxide
composite cathode catalyst for enhancing performance of microbial
desalination cell, Mater. Sci. Energy Technol. (2018), https://doi.org/10.1016/
j.mset.2018.08.002.
[29] A. Kumar, S. Sharma, L.M. Pandey, P. Chandra, Nanoengineered material based
biosensing electrodes for enzymatic biofuel cells applications, Mater. Sci.
Energy Technol. (2018), https://doi.org/10.1016/j.mset.2018.04.001.
[30] M.I. Ahamed, K.A. AlAmry, A.M. Asiri Beenish, Inamuddin, Biocompatible
mediated bioanode prepared by using poly(3,4-ethylene dioxythiophene) poly
(styrene sulfonate) (PEDOT:PSS) and sulfonated graphene oxide integrated
enzyme for biofuel cells applications, Mater. Sci. Energy Technol. (2018),
https://doi.org/10.1016/j.mset.2018.03.003.
[31] S. Barua, B.S. Zakaria, L. Lin, B.R. Dhar, Shaping microbial communities with
conductive carbon fibers to enhance methane productivity and kinetics,
Bioresour. Technol. Reports (2018), https://doi.org/10.1016/j.
biteb.2018.11.008.
[32] C. Wang, W. Qiao, H. Chen, X. Xu, L. Zhu, A short-term stimulation of ethanol
enhances the effect of magnetite on anaerobic digestion, Appl. Microbiol.
Biotechnol. (2018), https://doi.org/10.1007/s00253-018-9531-2.
[33] S. Jachlewski, W.D. Jachlewski, U. Linne, C. Brasen, J. Wingender, B. Siebers,
Isolation of extracellular polymeric substances from biofilms of the
thermoacidophilic archaeon Sulfolobus acidocaldarius, Front. Bioeng.
Biotechnol. (2015), https://doi.org/10.3389/fbioe.2015.00123.
[34] X.M. Liu, G.P. Sheng, H.W. Luo, F. Zhang, S.J. Yuan, J. Xu, R.J. Zeng, J.G. Wu, H.Q.
Yu, Contribution of extracellular polymeric substances (EPS) to the sludge
aggregation, Environ. Sci. Technol. (2010), https://doi.org/10.1021/es9016766.
[35] ISO (International Organisation for Standardisation), Determination of the
specific surface area of solids by gas adsorption - BET method (ISO 9277), 2010.
http://doi.org/10.1007/s11367-011-0297-3.
[36] J.G. Caporaso, J. Kuczynski, J. Stombaugh, K. Bittinger, F.D. Bushman, E.K.
Costello, N. Fierer, A.G. Peña, J.K. Goodrich, J.I. Gordon, G.A. Huttley, S.T. Kelley,
D. Knights, J.E. Koenig, R.E. Ley, C.A. Lozupone, D. McDonald, B.D. Muegge, M.
Pirrung, J. Reeder, J.R. Sevinsky, P.J. Turnbaugh, W.A. Walters, J. Widmann, T.
Yatsunenko, J. Zaneveld, R. Knight, QIIME allows analysis of high-throughput
community sequencing data, Nat. Methods 7 (2010) 335–336, https://doi.org/
10.1038/nmeth.f.303.
[37] B.J. Callahan, P.J. McMurdie, M.J. Rosen, A.W. Han, A.J.A. Johnson, S.P. Holmes,
DADA2: high-resolution sample inference from Illumina amplicon data, Nat.
Methods 13 (2016) 581, https://doi.org/10.1038/nmeth.3869.
[38] J.R. Rideout, Y. He, J.A. Navas-Molina, W.A. Walters, L.K. Ursell, S.M. Gibbons, J.
Chase, D. McDonald, A. Gonzalez, A. Robbins-Pianka, J.C. Clemente, J.A. Gilbert,
S.M. Huse, H.-W. Zhou, R. Knight, J.G. Caporaso, Subsampled open-reference
clustering creates consistent, comprehensive OTU definitions and scales to
billions of sequences, PeerJ 2 (2014) e545.
[39] S. Babicki, D. Arndt, A. Marcu, Y. Liang, J.R. Grant, A. Maciejewski, D.S. Wishart,
Heatmapper: web-enabled heat mapping for all, Nucl. Acids Res. 44 (2016)
W147–W153, https://doi.org/10.1093/nar/gkw419.
[40] V.K. Gupta, M. Sharma, K. Singh, R.K. Vyas, Reactive adsorption of phenol onto
Fe-GAC: Parallel pore batch modeling and experimental studies, J. Taiwan Inst.
Chem. Eng. (2016), https://doi.org/10.1016/j.jtice.2016.02.017.
[41] Z. Liu, H. Meng, H. Zhang, J. Cao, K. Zhou, J. Lian, Highly efficient degradation of
phenol wastewater by microwave induced H2O2-CuOx/GAC catalytic oxidation
process, Sep. Purif. Technol. (2018), https://doi.org/10.1016/j.seppur.2017.11.010.
[42] C. Koch, F. Harnisch, Is there a specific ecological niche for electroactive
microorganisms?, ChemElectroChem (2016), https://doi.org/10.1002/
celc.201600079.
[43] Y.Q. Tang, T. Shigematsu, S. Morimura, K. Kida, Dynamics of the microbial
community during continuous methane fermentation in continuously stirred tank
reactors, J. Biosci. Bioeng. (2015), https://doi.org/10.1016/j.jbiosc.2014.09.014.
[44] H. Imachi, S. Sakai, A. Ohashi, H. Harada, S. Hanada, Y. Kamagata, Y. Sekiguchi,
Pelotomaculum propionicicum sp. nov., an anaerobic, mesophilic, obligately
syntrophic, propionate-oxidizing bacterium, Int. J. Syst. Evol. Microbiol. 57
(2007) 1487–1492, https://doi.org/10.1099/ijs.0.64925-0.
[45] X. Guo, C. Wang, F. Sun, W. Zhu, W. Wu, A comparison of microbial
characteristics between the thermophilic and mesophilic anaerobic digesters
exposed to elevated food waste loadings, Bioresour. Technol. 152 (2014) 420–
428, https://doi.org/10.1016/j.biortech.2013.11.012.
[46] F. Kremp, A. Poehlein, R. Daniel, V. Müller, Methanol metabolism in the
acetogenic bacterium Acetobacterium woodii, Environ. Microbiol. (2018),
https://doi.org/10.1111/1462-2920.14356.
[47] D.R. Lovley, Bug juice: harvesting electricity with microorganisms, Nat. Rev.
Microbiol. 4 (2006) 497–508, https://doi.org/10.1038/nrmicro1442.
[48] Y. Jing, J. Wan, I. Angelidaki, S. Zhang, G. Luo, iTRAQ quantitative proteomic
analysis reveals the pathways for methanation of propionate facilitated by
magnetite, Water Res. 108 (2017) 212–221, https://doi.org/10.1016/j.
watres.2016.10.077.
[49] S.W. Li, G.P. Sheng, Y.Y. Cheng, H.Q. Yu, Redox properties of extracellular
polymeric substances (EPS) from electroactive bacteria, Sci. Rep. (2016),
https://doi.org/10.1038/srep39098.
[50] J.H. Park, J.H. Park, H. Je Seong, W.J. Sul, K.H. Jin, H.D. Park, Metagenomic
insight into methanogenic reactors promoting direct interspecies electron
transfer via granular activated carbon, Bioresour. Technol. 259 (2018) 414–
422, https://doi.org/10.1016/j.biortech.2018.03.050.
[51] Y. Yang, Y. Zhang, Z. Li, Z. Zhao, X. Quan, Z. Zhao, Adding granular activated
carbon into anaerobic sludge digestion to promote methane production and
sludge decomposition, J. Clean. Prod. 149 (2017) 1101–1108, https://doi.org/
10.1016/j.jclepro.2017.02.156.
[52] G. Baek, H. Jung, J. Kim, C. Lee, A long-term study on the effect of magnetite
supplementation in continuous anaerobic digestion of dairy effluent –
Magnetic separation and recycling of magnetite, Bioresour. Technol. 241
(2017) 830–840, https://doi.org/10.1016/J.BIORTECH.2017.06.018.