Fuel Processing Technology 238 (2022) 107478

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Fuel Processing Technology

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Identification of the biomethanation pathways during biological CO2

fixation with exogenous H2 addition
Bingyi Jiang a, Xiao Hu a, Ulf Söderlind b, Kristina Göransson b, Wennan Zhang b,
Chunjiang Yu a, *
a
State Key Laboratory of Clean Energy Utilization, Zhejiang University, 38 Zheda Road, Hangzhou 310027, Zhejiang, China
b
Department of Chemical Engineering, Mid Sweden University, 85170 Sundsvall, Sweden

A R T I C L E I N F O A B S T R A C T

Keywords: Power-to-gas allows conversion of surplus electricity to methane when CO2 is available, which becomes an
Power-to-gas important technology for carbon capture, utilization and sequestration, as well as for increasing the flexibility of
H2/CO2 biomethanation electricity production from renewable energy resources such as wind and solar energy. H2/CO2 biomethanation
Mixed microbial consortia
is a potentially promising alternative to the conversion of H2/CO2 to methane without limitation of variable
Methanation routes
hydrogen production. To identify mixed culture-based metabolic pathways of H2/CO2 under the mesophilic
Microbial community relationship
(35 ◦ C) and thermophilic (55 ◦ C) conditions, two specific inhibitors, 2-bromoethane sulfonate (BES) and van­
comycin were employed in this experimental study. The combination of hydrogenotrophic and
homoacetogenesis-acetoclastic methanogenesis makes up the pathway for the mesophilic cultivated microbial
consortia. 16S rRNA gene analysis indicates that abundant Bacteria, Methanobacterium and Methanosaeta play
important role in the conversion. Further analysis shows close collaboration between microorganisms by the
formation of microbial clustering and the production of humic acids. The detailed metabolic mechanisms further
confirm a diverse biomethanation network under the mesophilic condition. While under the thermophilic con­
dition, the H2/CO2 biomethanation is fully dominated by the direct hydrogenotrophic methanogenesis mainly
with Methanothermobacter, which is straightforward but more efficient.

1. Introduction
Strong development in solar and wind power industries worldwide in
the recent years leads to large-scale variable electricity production
which must be balanced from other energy sources or energy carriers
[1]. Power-to-gas (P2G) allows conversion of surplus electricity to
hydrogen or preferably to methane when CO2 is available, which be­
comes an important R&D and commercialization technology for carbon
capture, utilization and sequestration (CCUS), as well as for increasing
the flexibility of electricity production from renewable energy resources
such as wind and solar energy [2,3].
Hydrogen produced from water electrolysis can be combined with
CO2 to produce methane through methanation [4]. Compared with
hydrogen, methane has few barriers for commercial implementation and
is a versatile chemical feedstock and energy carrier with easy transport
and storage based on present strong natural gas industry and network
worldwide [3]. Methanation of H2 and CO2 is of a great significance to
help further development of wind and solar power industries and also to
mitigate GHG emission from various sources such as coal, natural gas
and biomass combustion plants [5].
Conventional methanation technology is high temperature catalytic
synthesis. On the other hand, the methanation of H2 and CO2 can also be
realized at low temperature with biological process [3]. Compared with
the catalytic methanation, biomethanation of H2/CO2 has many ad­
vantages such as moderate reaction conditions, the irreversible char­
acter of the biochemical reactions, less byproducts and insensitive to the
impurities and the ratio of C/H in the feedstock gas mixture through an
easy conversion of H2/CO2 to methane without limitation of variable
hydrogen production from wind and solar energy [6,7]. Moreover, the
sources of gas substrate for biomethanation can be the CO/CO2-rich
industrial off-gas and syngas after biomass gasification in small and
medium scalls [8]. The treatment of a wide range of sources not only
provides a great potential for energy recovery from waste streams but
also benefits the environmental protection.

* Corresponding author.
E-mail address: chunjiang@zju.edu.cn (C. Yu).

https://doi.org/10.1016/j.fuproc.2022.107478
Received 8 June 2022; Received in revised form 24 August 2022; Accepted 25 August 2022
Available online 5 September 2022
0378-3820/© 2022 Elsevier B.V. All rights reserved.
B. Jiang et al.
Different from traditional anaerobic digestion (AD) of organic sub­
strates, H2/CO2 biomethanation is supposed to be a common and direct
process, making previous studies focus more on its applications. H2/CO2
is usually preferred to be an exogenous substrate into the digestion
reactor for better biomethanation performance [1,9–11]. However, H2/
CO2 biomethanation might not be so straightforward and its conversion
routes should be investigated in detail. The main reactions involved in
H2/CO2 biomethanation can be expressed below [12,13]:
Syntrophic acetate oxidation:
CH3 COO− + H+ + 2H2 O→4H2 + 2CO2 (ΔGo = + 94.92 kJ/mol) (1)
Homoacetogenesis:
4H2 + 2CO2 →CH3COO− + H+ + 2H2 O (ΔGo = − 94.92 kJ/mol) (2)
Acetoclastic methanation:
CH3 COO− + 4H2 O→CH4 + HCO3 − (ΔGo = − 31.02 kJ/mol) (3)
Hydrogenotrophic methanation:
CO2 + 4H2 →CH4 + 2H2 O (ΔGo = − 130.75 kJ/mol) (4)
So far, it is not clear how the above reactions compete or collaborate
with each other to establish a biomethanation pathway and which
metabolic interactions may take place in the mixed microbial consortia
at different temperatures. Grimalt-Alemany et al. suggested that aceto­
clastic methanogenesis plays the important role at 37 ◦ C and hydro­
genotrophic methanogens is dominant at 55 ◦ C based on the analysis of
thermodynamic competition [14]. Pan et al. studied the biomethanation
kinetics and proposed an order of high to low methanation rate for
typical methanogenic precursors as H2/CO2 > acetate > formate in
sewage sludge at 37 ◦ C [15]. Kovalovszki et al. also demonstrated the
influence of temperature on microbial growth and by-product genera­
tion through model simulations [16]. In summary, the above studies
suggest that the H2/CO2 biomethanation is indirect. However, the main
pathways of H2/CO2 biomethanation is unclear, especially for mixed
microbial consortia and other key issues such as the cross-feeding re­
lationships and metabolic interactions under both the mesophilic and
thermophilic conditions. The identification of H2/CO2 biomethanation
pathways is valuable for the subsequent industrial application with
respect to the operational temperature, intermediate products and mi­
crobial species.
The objective of this study is to identify the pathways of H2/CO2
biomethanation by enriched anaerobic mixed cultures (anaerobic
granular sludge) under the mesophilic (35 ◦ C) and thermophilic (55 ◦ C)
conditions. H2 + CO2 gas mixture (4:1) and acetate were served as the
substrate with the addition of inhibitors of 2-bromoethane sulfonate
(BES) or vancomycin. The content of each product and reactant was
recorded. High-throughput 16S rRNA gene sequencing analysis was
used to reveal the effects of temperature on the mixed community.
Fluorescence in situ hybridization was used to evaluate the micro-
distribution of microbes. Cyclic voltammetry experiments and excita­
tion emission matrix were conducted to characterize the cellular meta­
bolic capacity.
2. Material and methods
2.1. Growth medium and inoculum
The growth medium used in all test runs of the experiment was a
modified basal anaerobic (BA) medium [17] composed by 4 stock so­
lutions presented in Table S1. The medium was prepared by adding 10
mL A, 2 mL B, 1 mL C, 1 mL D and distilled water to 1 L.
Anaerobic granular sludge for this study was collected from an in­
ternal circulation (IC) bioreactor (Shuierli Environmental Protection
Technology CO. Ltd., Jiangxi, China), which was originally operated
under mesophilic conditions (35–38 ◦ C) for treating industrial
2
Fuel Processing Technology 238 (2022) 107478
wastewater from juice plants. It has been proved to have better perfor­
mance of syngas biomethanation [18]. The total solids (TS) and volatile
solids (VS) contents of the anaerobic granular sludges were 49.46 ±
0.64 g/L and 37.44 ± 0.69 g/L, respectively. Before it was used as an
inoculum, a batch process under H2/CO2 (4:1) was applied for enrich­
ment as well as for removing the original organic matter contained in the
sludges. The anaerobic granular sludges were divided in two series and
incubated under the mesophilic (35 ◦ C) conditions, respectively. The
enrichment details are similar to the previous study [18].
2.2. Method to identify the biomethanation metabolic routes
The experiment was carried out in a bioreactor of pressure-resistant
glass with the working volume of 1.9 L. A shock gas-proof manometer
(YNZ-60, Wenzhou, China) is installed through the reactor cover with
two valves for gas and liquid sampling, respectively. The reactor is
completely isolated through strict pressure and air tightness test.
To identify the possible routes for H2/CO2 conversion to methane,
two different metabolic inhibitors were used: 50 mM BES (sodium salt,
98% purity, Macklin, China) or 0.07 mM vancomycin (hydrochloride
hydrate, USP, Macklin, China), which was added to the bioreactor prior
to inoculation respectively in the experiment. As shown in Fig. 1, BES is
used as the methanogenic inhibitor to block the direct methanation
pathways of H2/CO2 or acetate. Vancomycin is used as an inhibitor of
gram-positive bacteria generally including acetogenic bacteria, to block
the syntrophic acetate oxidation and the homoacetogenesis. The con­
centration, effect and stability of inhibitors have been described previ­
ously [19,20]. After the enrichment, 70 mL of sludge was transferred
into the reactor, and 230 mL of fresh BA medium and 0.12 g of cysteine
hydrochloride were added to bring the liquid volume up to 300 mL,
leaving the headspace of 1600 mL. H2/CO2 (4:1) or acetate (1000 mg/L)
were selected as metabolic substrates. After that, the initial pH was
adjusted to 6.95–7.10 by 4 M HCl or 4 M NaOH according to the real-
time pH measurement. The liquid and headspace were flushed with
the required gas through the valves at a rate of 600 mL/min for more
than 10 min. Finally, the headspace was filled with the required gas (H2/
CO2/N2 = 40%/10%/50% or N2 = 100%) and the initial total pressure
was adjusted to 1.2 atm, so that the final vacuum degree after the re­
action would not be too high. The reactors were divided in two series
and incubated in the shaker-bed at 80 rpm under the mesophilic and the
thermophilic conditions, respectively. Detailed conditions of each
experimental test are shown in Table 1. During the experiment, 4 mL
liquid was extracted every two days to analyze the composition of the
liquid phase and to measure the pH. At the same time, 4 mL fresh BA
medium was added and the pH was adjusted to 6.75–7.30 by adding
about 0.02 mL 4 M NaOH and HCl every time if needed. 2 mL gas was
extracted for analysis of the gas phase composition every day.
2.3. Analytical methods
The composition of H2, CO2, CO and CH4 in the headspace of the
reactor was analyzed by gas chromatography (Shimadzu GC-2014,
Tokyo, Japan) equipped with a thermal conductivity detector (TCD)
and a packed column (TDX-01, 2 m × 3 mm, Stainless Steel) as described
previously [18]. The absolute content of each gas component was
calculated by measuring the gas temperature, total pressure, the volume
of headspace and concentration of each component according to ideal
gas law. The solubilities of H2, CO2, CO and CH4 were calculated using
data from Lide [18] to correct the gas composition.
Volatile fatty acids (VFA) including acetate, propionate, butyrate,
isobutyrate were measured by means of an Agilent 7820A (Wilmington,
Delaware, USA) equipped with a flame ionization detector (FID) and DB-
FFAP column (30 m × 0.25 mm × 0.25 μm). Nitrogen was used as the
carrier gas at flow rate of 40 cm/s. The temperature of inlet and detector
were set up to be 250 ◦ C and 300 ◦ C, respectively. The GC oven was
programmed to begin at 100 ◦ C, held for 5 min, then increased at a rate
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

˄1˅ ˄1˅ ˄1˅

Acetate H2/CO2 Acetate H2/CO2 Acetate H2/CO2
˄2˅ ˄2˅ ˄2˅
˄3˅ ˄4˅ ˄3˅ ˄4˅ ˄3˅ ˄4˅
CH4 CH4 CH4
a) no inhibitor b) with vancomycin c) with BES

Fig. 1. Possible catabolic routes from H2/CO2 to methane: a) without inhibitors, b) with vancomycin and c) with BES. (The solid arrows indicate the existing
pathways; the dotted arrows indicate pathways blocked in the presence of inhibitor, totally or partially. Pathways: (1) syntrophic acetate oxidation; (2) homo­
acetogenesis; (3) acetoclastic methanogenesis; (4) hydrogenotrophic methanogenesis.)

granule. Then the slices were performed on FISH analysis. Oligonucle­

Table 1
otide probes specific for Bacteria (EUB338 probe 5′ –FAM-
Experiment conditions in the tests to identify catabolic routes. (G - the substrate
GCTGCCTCCCGTAGGAGT-3′ ) and Archaea (ARC915 probe 5′ –CY3-
is gas H2/CO2, A - the substrate is acetate, B - the inhibitor is BES, V - the in­
hibitor is vancomycin, M - at the mesophilic temperature of 35 ◦ C, T - at the GTGCTCCCCCGCCAATTCCT-3′ ) domains were used [24,25]. Samples
thermophilic temperature of 55 ◦ C. The initial total pressure is 1.2 atm, and the after FISH were photographed by confocal laser scanning microscopy
rotation speed 80 rpm). (NIKON Eclipse Ti, Nikon Ltd., Japan). The hybridized bacterial cells
were excited with the 488 nm line of an Ar laser (excitation) and
Test Initial gas composition (v %/v %/v Substrate in the Inhibitor
%) liquid observed in the green channel of 550 nm (emission). Archaea cells were
excited with the 560 nm line of a He–Ne laser (excitation) and observed
GM H2/CO2/N2 = 40/10/50 / /
GBM H2/CO2/N2 = 40/10/50 / BES in the red channel of 590 nm (emission).
GVM H2/CO2/N2 = 40/10/50 / Vancomycin
AM N2 = 100 Acetate / 2.6. Excitation mission matrix (EEM) fluorescence analysis
ABM N2 = 100 Acetate BES
AVM N2 = 100 Acetate Vancomycin
The metabolites after the anaerobic process were tested by a Fluo­
GT H2/CO2/N2 = 40/10/50 / /
GBT H2/CO2/N2 = 40/10/50 / BES rescence Spectrophotometer (F7000, Hitachi, Japan). The tested me­
GVT H2/CO2/N2 = 40/10/50 / Vancomycin tabolites were loosely bound extracellular polymeric substances (LB-
AT N2 = 100 Acetate / EPS) and tightly bound extracellular polymeric substances (TB-EPS).
ABT N2 = 100 Acetate BES
EPS was separated with heat extraction methods [26]. The excitation
AVT N2 = 100 Acetate Vancomycin
wavelength (EX) was set up at 200–450 nm and the emission wavelength
(EM) at 250–550 nm. The excitation and emission slits were maintained
of 10 ◦ C/min to 250 ◦ C, and finally held at 250 ◦ C for 12 min. at 5 nm and the scanning speed was set up at 2400 nm/min.
The pH was measured by a pH meter (pH -100, Shanghai, China).
The total solids (TS) and volatile solids (VS) contents of the anaerobic 2.7. Electrochemical measurements of anaerobic digestion residues
granular sludges were measured according to standard methods [1].
To calculate the electron transfer during anaerobic process, cyclic
voltammetry (CV) measurements were conducted using a single-
2.4. High-throughput 16S rRNA gene sequencing and analysis
chamber three-electrode electrochemical cell. The electrochemical cell
was made of two graphite electrodes (a working electrode and a counter
High-throughput sequencing of the 16S rRNA gene (V4-V5 hyper­
electrode, each with a 95 mm length and 5 mm diameter) and a refer­
variable region) of bacteria and archaea was conducted to analyze the
ence electrode (Ag/AgCl) [27]. 20 mL of digestion residue (containing 5
differences in microbial community compositions between the inocula
mL of sludges and 15 mL of medium) was added into the electrochemical
at two different temperatures of 35 and 55 ◦ C. Total genomic DNA was
cell and the cell was then purged with pure N2 for 10 min to establish an
extracted using the DNeasy PowerSoil DNA extraction kit (Qiagen,
anaerobic environment. CVs were measured using an electrochemical
Hilden, Germany). 16S rRNA amplification and quantification were
analyzer (Bio-Logic, Claix, France). The scanning voltage of the working
conducted using the SmartChip Real-time PCR system (Wafergen Inc.
electrode was ranged from − 1.0– 1.0 V with scanning rates of 20– 100
USA). The 16S rRNA gene V4 ~ V5 hypervariable region was amplified
mV/s.
using the universal forward 515F (5′ -GTGCCAGCMGCCGCGG-3′ ) and
According to the Laviron equation, the electron transfer rate constant
the reverse 907R (5′ -CCGTCAATTCMTTTRAGTTT-3′ ) primers for bac­
kapp in the system was calculated [27]. First, α was calculated according
teria, and using the universal forward 519F (5′ -CAGCCGCCGCCGTAA-
to Eqs. (5) and (6) as follow:
3′ ) and the reverse 915R (5′ -GTGCTCCCCCGCCAATTCCT-3′ ) primers
( ) [ ]
for archaea [21]. High-throughput sequencing of 16S rRNA was con­ RT αnFνc
Epc = E0c − ln (5)
ducted using the Ion GeneStudio S5 (Thermo Fisher Scientific, Waltham, αnF RTkapp
USA) at Analysis Center of Agrobiology and Environmental Sciences of
( ) [ ]
Zhejiang University (Hangzhou, China). QIIME package was used to RT (1 − α)nFνa
Epa = E0a − ln (6)
handle the original sequence. The detailed steps were based on the (1 − α)nF RTkapp
published methods [22]. Alpha diversity metrics were calculated using
chao1 and shannon. The Beta diversity was estimated using the phylum/ where α is the electron transfer coefficient, Epc the reduction peak po­
genus - level abundance [23]. The major genera of archaea community tential, Epa the oxidation peak potential, ν the scanning rate, R 8.314 J
and the major phyla of bacterial community discussed in this study are mol− 1 K− 1, T 298 K and F 96,483C mol− 1. The straight slopes of Ep-E0
referred to those with a relative abundance no less than 3% at least. and ln ν with the reduction and oxidation peaks are RT/αnF and RT/(1-
α)nF, respectively. Following this, kapp was obtained according to Eq. (7)
as follows:
2.5. Fluorescence in situ hybridization (FISH) of the sludge granules
( )
RT nFΔE
lgkapp = αlg(1 − α) + (1 − α)lgα − lg − (1 − α)α (7)
The spatial location of Archaea and Bacteria inside the sludge gran­ nF ν 2.303RT
ules was marked by fluorescence in situ hybridization (FISH). Frozen
section was taken in order to obtain the 10 μm of central slices of sludge where ΔE indicates the potential difference between the reduction and

3
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

oxidation peaks.
3. Results and discussions
3.1. H2/CO2 biomethanation routes at 35 ◦ C and 55 ◦ C
In the case of H2/CO2 used as the initial substrate, the consumption
of H2 and CO2, the production of CH4 and acetate are registered against
the biomethanation time as shown in Fig. 2. The digestion time to reach
the maximum product, tmax, the amount of end–product as well as the
product stoichiometic yield are also given in Table 2. Comparing the test
runs of GM and GVM with GT and GVT, it can be seen that the H2/CO2
biomethanation rate under the thermophilic condition is about twice of
that under the mesophilic condition, so that the tmax is shorten from 6 to
7 days to 3 days. However, very little has been changed for the amount
of the end-product, CH4, and the product stoichiometric yield. Vanco­
mycin inhibits the conversion of H2/CO2 to acetate and then to CH4, but
not hydrogenotrophic methanogenesis which completes the conversion
of H2/CO2 to CH4 in the experiment. The inhibition of the bio­
methanation route via acetate affects the CH4 production rate, but
hardly change the final CH4 production [19]. As for the intermediate
product, VFAs with acetate as the dominant component, the content in
the liquid phase were almost undetected as seen in Fig. 2d. In the H2/
CO2 biomethanation process, a very small amount of acetate is produced
since acetate is immediately converted to the end-product CH4 as soon as
produced in the process.
At 35 ◦ C, when vancomycin is added to inhibit the H2/CO2 conver­
sion to acetate, both the consumption of H2 and CO2 and the methane
production delay one day later than the H2/CO2 biomethanation
without vancomycin addition as seen in Fig. 2a, b and c from compar­
ison of the test run GVM with GM. The methane production rate de­
creases from 0.53 mmol/g VSS/d to 0.45 mmol/g VSS/d as given in
Table 2, which suggests that H2/CO2 - Acetate - CH4 is one of the con­
version routes for H2/CO2 biomethanation. With the inhibition of ace­
tate formation, the CH4 production through the route of
homoacetogenesis and aceticlastic methanogenesis might be partly
stopped and partly replaced by hydrogenotrophic methanogenesis. On
the other hand, at 55 ◦ C, with the addition of vancomycin, H2 and CO2
metabolism curves and CH4 production curve of GT and GVT are iden­
tical, indicating that blocking acetate production did not affect H2/CO2

35
a) 12
b)
GM
GM
30
CO2 content in the headspace (mmol) GVM
GVM
H2 content in the headspace (mmol)

10
GBM
GBM
GT
25 GT
8 GVT
GVT
GBT
20 GBT
6
15
4
10

2
5

0
0
0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)
c) d)

GM 1600
CH4 production in the headspace (mmol)

8
Acetate production in the liquid (mg/L)

GVM
GBM 1400
GT
6 1200
GVT
GBT 1000

4 800
GM
600 GVM
GBM
2 400 GT
GVT
200
GBT
0
0
0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)
Fig. 2. Consumption and production of the gas in the bioreactor headspace, a) H2 and b) CO2 and c) CH4, and d) acetate concentration in the liquid phase for the
substrate H2/CO2/N2 = 40%/10%/50% to be digested at 35 ◦ C and 55 ◦ C respectively.

4
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

Table 2
H2, CO2, acetate average consumption rates, CH4, acetate average pruduction rates and end-products yields in the experiments to identify the biomethanation catabolic
routes.
Consumption rate Production rate End-products Product
stoichiometric
yieldb

tmaxa H2 (mmol/g CO2 (mmol/g Acetate (mg/L/g Acetate (mg/L/g CH4 (mmol/g CH4 Acetate (mg/ CH4 Acetate
(d) VSS/d) VSS/d) VSS/d) VSS/d) VSS/d) (mmol) L)

GM 6 1.92 0.50 0.53 8.14 102.98

GVM 7 1.79 0.47 0.45 8.65 100.39
GBM 8 1.13 0.45 80.50 1636.33 122.68
GT 3 3.97 0.87 0.89 7.79 105.44
GVT 3 4.07 0.97 1.00 8.44 107.39
GBT 18 0.33 0.11 30.22 1208.77 142.63
AM 4 100.63 0.37 4.40 118.47
AVM 6 65.60 0.24 4.15 113.39
ABM / / / / / / / / / /
AT 16 24.52 0.10 4.40 119.37
AVT 28 13.62 0.06 4.35 123.09
ABT / / / / / / / / / /
a
Time to reach maximum amount of product.
b
Stoichiometric yield: 1 mol of CH4 per mol CO2, 1/2 mol of acetate per mol CO2, and 1 mol of CH4 per mol acetate.

biomethanation. This confirms that anaerobic granular sludges metab­
olize H2/CO2 fully through hydrogenotrophic methanogenesis under the
thermophilic condition, in agreement with the conclusion made from
previous study [14].
When BES is added to inhibit the H2/CO2 conversion to CH4, acetate
becomes the dominant end product, suggesting the central role that
homoacetogenesis plays in the H2/CO2 digestion reaction network. The
rates of H2/CO2 consumption and acetate production under the meso­
philic condition are about 3 times higher than those under the ther­
mophilic condition, and the final acetate yield is also higher as seen in
Table 2. The difference between the CH4 and acetate productions at two
temperatures indicates that homoacetogenesis does not dominate H2/
CO2 biomethanation at 55 ◦ C, but plays an important role at 35 ◦ C. The
CO2 consumption rate decreases only slightly from 0.50 mmol/g VSS/
d to 0.45 mmol/g VSS/d as seen in Table 2, indicating that the anaerobic
microbial consortia under the mesophilic condition has a stronger ca­
pacity to convert H2/CO2 into acetate. However, the microbal commu­
nity at 55 ◦ C is more difficult to metabolize H2/CO2 into acetate and then
to produce methane.
In order to know more about the metabolism of the intermediate
product, acetate was used as the sole carbon source in the bio­
methanation experiment. As shown in Table 2 and Figs. 3 AM and 3AT,
the rate of metabolizing acetate to methane at 35 ◦ C is about four times
faster than at 55 ◦ C. This suggests that acetate, as a intermediate product
of H2/CO2 biomethanation, is much easier to be metabolized to methane
under the mesophilic condition than the thermophilic condition.
In the case of acetate as the substrate used in the biomethanation
experiment, H2 and CO2 can also be found in the bioreactor headspace
more and less as shown in Fig. 3, but the content varies irregularly and is
extremely low, one order of the magenitude lower than the case of H2/
CO2 used as the substrate. H2 is accumulated in the headspace for the
test runs AM and AT, but disappears when vancomycin is added in the
test runs AVM and AVT. In the same time, the rates of acetate con­
sumption and methane formation are reduced as seen in Table 2. Acetate
decomposes fast and the concentration levels off after 5 days bio­
methanation under the mesophilic condition, while acetate decomposes
slowly and the concentration levels off after 10 days under the ther­
mophilic condition. These indicates that besides aceticlastic methano­
genesis, acetate can be transformed into H2/CO2 to produce CH4 both
under the mesophilic and the thermophilic condition, although the ac­
etate metabolism is weak at 55 ◦ C. It can be concluded that acetoclastic
methanation and syntrophic aceteate oxidation (SAO) are two more
active routes at 35 ◦ C. They are also present at 55 ◦ C but the pathways
are fairly week.
In the case of BES addition as presented in Figs 3ABM and 3ABT, the
gas composition in the headspace and the acetate concentration in the
liquid remian almost unchanged. It can be explained that the reaction
syntrophic aceteate oxidation (SAO) is nonspontaneous thermodynam­
ically, as Gibbs energy is 104 kJ/mol [18]. After inhibiting the formation
of methane as the end-product, H2 and CO2 are difficult to be consumed,
which further causes the negative feedback for the reaction SAO and
makes the whole system in a state of equilibrium basically. Because the
reaction is entropy-increasing and thermodynamically favorable, the H2
concentration is a little higher at 55 ◦ C. The acetate concentration in the
test runs ABM and ABT remains fairly constant or decreases first and
returns back to its initial value at the end. In fact, the choices of meta­
bolic routes for H2/CO2 biomethanation can be partly explained by
thermodynamics as described above. Due to the decrease of entropy in
homoacetogenesis, the acetogenic process are more limited under the
thermophilic. Of course, biological reaction is more complex, and the
kinetics and microbial metabolic habits should be considered
comprehensively.
Based on all the experimental results presented above, the pathways
of H2/CO2 biomethanation can be described in Fig. 4. Under the mes­
ophilic condition, the pathways of H2/CO2 biomethanation are complex
and both hydrogenotrophic methanogenesis and homoacetogenesis-
acetoclastic methanogenesis co-exist, in agreement with the previous
work [14]. The anaerobic granular sludges have strong capacity of
producing and metabolizing acetate. After acetate formation from H2/
CO2, a part of acetate is directly digested into methane, while the other is
converted into H2/CO2 again to produce methane via hydrogenotrophic
methanogenesis. Previous study has shown that both H2/CO2 and ace­
tate are typical methanogenic precursors [15]. Under the thermophilic
condition, H2/CO2 biomethanation by means of anaerobic granular
sludges completely depend on the direct and more efficient route of
hydrogenotrophic methanogenesis. Although the microbial community
has a weak ability to reconvert acetate into H2/CO2, the conversion will
not take place during H2/CO2 biomethanation because of the absence of
homoacetogenesis.
3.2. Analysis of the microbial communities
The species richness is characterized by Chao1 index as seen in
Table 3. The archaea richness under the thermophilic condition is lower
than that under the mesophilic condition. The Shannon index is used to
represent the microbial community diversity as well as the evenness of
the species [28]. It shows a decreasing trend of species diversity and
evenness under the thermophilic condition, which is consistent with the

5
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

AM Acetate H2 CO2 CH4 AT Acetate H2 CO2 CH4

5 5

Acetate concentration in the liquid (mg/L)

Acetate concentration in the liquid (mg/L)

1000 1000
Headspace composition (mmol)

Headspace composition (mmol)

4 4
800 800

3 3
600 600

2 400 2 400

1 200 1 200

0 0
0 0
0 5 10 0 5 10 15 20
Time (days) Time (days)

AVM Acetate H2 CO2 CH4 AVT Acetate H2 CO2 CH4

5 5

Acetate concentration in the liquid (mg/L)

Acetate concentration in the liquid (mg/L)

1000 1000
Headspace composition (mmol)

Headspace composition (mmol)

4 4
800 800

3 3
600 600

2 400 2 400

1 200 1 200

0 0
0 0
0 5 10 0 5 10 15 20 25 30
Time (days) Time (days)

ABM Acetate H2 CO2 CH4 ABT Acetate H2 CO2 CH4

5 6
Acetate concentration in the liquid (mg/L)

Acetate concentration in the liquid (mg/L)

1000 1000
Headspace composition (mmol)

Headspace composition (mmol)

5
4
800 800
4
3
600 600
3

2 400 400
2

1 200 200
1

0 0
0 0
0 5 10 15 0 5 10 15
Time (days) Time (days)

Fig. 3. The amounts of H2, CO2, CH4 in the bioreactor headspace and acetate concentration in the liquid phase when the substrate was 1000 mg/L acetate under
different experimental conditions of AM, AVM, ABM, AT, AVT, ABT described in Table 1.

Chao1 results. For bacterial community, the species richness and di­ [14].
versity of microbial consortia under the thermophilic condition fall off a The main phyla of archaea community for all the samples are Eur­
cliff than those under the mesophilic condition. This illustrates that the yarchaeota and Crenarchaeota as seen in Fig. 5a. The relative abundance
higher temperature eliminates a large number of inviable bacteria. of Euryarchaeota ranges from 53.17% to 73.93% while Crenarchaeota
Fewer microbial species under the thermophilic condition can be one of ranges from 14.31% to 23.13%. Analysis at the genus level for the
the reasons for the single H2/CO2 biomethanation pathway shown in archaea communities was further carried out. As the initial sludge was
Fig. 4. Under the mesophilic condition, more species are involved in H2/ collected in the reactor under the mesophilic, it has high similarity in
CO2 metabolism, making the biomethanation network more complex community abundance with that in GM. The archaea community of

6
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

Acetate H2/CO2 Acetate
CH4 CH4
Mesophilic Thermophilic
Fig. 4. The pathways suggested from the experiment for conversion of CO2/H2
into CH4 by a mixed anaerobic sludge. (The thickness of the arrow represents
the relative importance of the pathway. The dotted lines indicate the theoretic
pathways but do not happen in actual reaction.)
Table 3
Parameters related to microbial community richness and diversity.
Sample Chao1 Shannon Sample
Archaea Bacteria
Initial 4360.093 6.490 Initial
GM 4297.259 6.655 GM
GT 3860.382 5.563 GT
Initial: Anaerobic granular sludges before inoculating.
those is dominated by Methanobacterium and Methanosaeta. Under
continuous enrichment, it is noted that the relative abundance of
Methanobacterium increased from 10.69% (Initial) to 24.6% (GM),
indicating that Methanobacterium is one of the dominant archaea genus
converting H2/CO2 to produce methane under the mesophilic condition.
It belongs to Methanobacteriales, mediating hydrogenotrophic meth­
anogenesis [28]. Another dominant genus, Methanosaeta in GM
(20.08%) is significantly higher than that in GT (9.86%). It is a strict
acetoclastic methanogen genus [28] and its relative abundance is very
small in hydrogenotrophic mixed cultures [29], indicating that aceto­
clastic methanogenesis exists under the mesophilic condition. In the case
of GM, a part of H2/CO2 is converted to acetate firstly, then converted to
CH4 by Methanosaeta, which is consistent with the analysis in section
3.2. The existence of hydrogenotrophic and acetoclastic methanogen
proves that H2/CO2 biomethanation involves the pathways of homo­
acetogenesis, hydrogenotrophic and acetoclastic methanogenesis.
Under the thermophilic condition, the archaea communities are domi­
nated by Methanothermobacter, which increased from 0.15% (Initial) to
38.38% (GT). Methanothermobacter, hydrogenotrophic methanogen
H2/CO2 genus [29], is thermophilic methanogens active at a wide temperature
range of 45–80 ◦ C with the optimum temperature of 65 ◦ C [30,31]. It can
be concluded from the above discussion and also from the fact of almost
missing Methanosaeta that H2/CO2 biomethanation is strictly completed
through hydrogenotrophic methanogenesis under the thermophilic
condition.
The analysis of bacteria at the phylum level in Fig. 5b shows that the
initial sludge has high similarity in community abundance in the case of
GM. They are dominated by Bacteroidetes, Chloroflexi and Proteobacteria.
The initial sludge was used to digest organic matters in sewage treat­
ment, and the similar and abundant bacterial community abundance
makes the sludge in GM also capable of hydrolysis and acidogenesis
[32,33], providing more potential pathways in H2/CO2 biomethanation
such as homoacetogenesis and syntrophic aceteate oxidation. Under the
Chao1 Shannon
thermophilic condition, it is doubtful whether the remaining bacteria
play any role in biomethanation, because H2/CO2 biomethanation at
7440.400 9.431 55 ◦ C relies on the direct hydrogenotrophic methanogenesis of Meth­
7449.588 9.909
4230.644 6.073
anothermobacter. The left bacteria are just because they are more
tolerant to extreme environments. The low species richness reduces the
possibility of mutual conversion between H2/CO2 and acetate and
weakens the interaction between microorganisms.
The fluorescence micrographs were taken after hybridization with
the specific probe EUB338 for Bacteria and the probe ARC915 for
Archaea. The former is mainly representative of the hydrogenogen and
acetogen and the latter is representative of the methanogenic fraction of
the biomass [25]. The spatial distribution of Bacteria (green) and
Archaea (red) identified by FISH help to give more evidence about the
dominant pathways during H2/CO2 biomethanation process.
As shown in Fig. 6a, the initial sludge granule is compact. Bacteria
and Archaea are evenly distributed in the sludge. After cultivation under
the mesophilic and thermophilic conditions, the internal voids of
sludges gradually enlarge, as shown in Fig. 6b and c. This can be related
to the elimination and selection of microorganisms during the enrich­
ment and domestication. The largest voids inside sludge granules at
55 ◦ C further confirm the above analysis of community abundance that
the high temperature can be the limit for some microflora to survive.
From Fig. 6, it can also be seen that the spatial distribution of Archaea
and Bacteria are changed after cultivation under different temperatures.
Under the mesophilic condition, the main colour of the granule exterior
layer after fluorescence is green, while the red area increases gradually

100 100
p: [Parvarchaeota]; o: [Micrarchaeles] Thermotogae
p: Euryarchaeota; f: [Methanomassiliicoccaceae] Spirochaetes
p: Euryarchaeota; g: Methanosaeta Proteobacteria
p: Euryarchaeota; g: Methanolinea Planctomycetes
80 80
Relative Abundance (%)

p: Euryarchaeota; g: Methanocorpusculum OP8

Relative Abundance (%)

p: Euryarchaeota; o: Methanobacteriales; f: WSA2 Firmicutes

p: Euryarchaeota; g: Methanothermobacter Chloroflexi
p: Euryarchaeota; g: Methanobacterium Chlorobi
p: Crenarchaeota; c: Thermoprotei Bacteroidetes
60 60
p: Crenarchaeota; c: MCG; o: pGrfC26 Armatimonadetes
Other Actinobacteria
Other

40 40

20 20

0 0
Initial GM GT Initial GM GT

a) Archaea-Genus Level b) Bacteria-Phylum Level

Fig. 5. Relative abundance of a) archaea and b) bacteria communities using V4-V5 region primer set. (Other represents the sum of all genera/ phyla with the relative
abundance below 3% in the individual samples.)

7
B. Jiang et al. Fuel Processing Technology 238 (2022) 107478

Fig. 6. FISH images of the sections of sludge granules a) before enrichment and cultivated with H2/CO2 under the b) mesophilic and c) thermophilic conditions. (The
communities in the sludge granule were stained to reveal the archaea in red and bacteria in green.) (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

Fig. 7. Cyclic voltammetry (CV) curves of anaerobic sludges containing mixed cultures at different scan rates of 20, 40, 60, 80 and 100 mV/s (from the inside out)
under the a) mesophilic and c) thermophilic conditions. (Insets) Peak currents are presented as a function of scan rates. Peak potentials are presented as a function of
the logarithm of the scan rates under the b) mesophilic and d) thermophilic conditions.

8
B. Jiang et al.
as it gets closer to the center of the granule. It indicates that Bacteria are
mostly distributed in the exterior layer of the granule, while Archaea are
distributed in the inner layer. This is similar to the previous theory [34]
of granule composition that methanogen is in the inner layer and other
acidogens and H2 consuming/producing organisms form the exterior
layer. In syntrophic cultures, such clustering is essential to minimize the
diffusion distance of interspecies electron carriers between a producing
and a consuming organism to improve the rate of substrate conversion
[25]. Such a structure makes Bacteria in the exterior layer firstly convert
a part of H2/CO2 into intermediate products, mainly acetate, which will
be later utilized by the internal Archaea. Thus, the pathways of H2/CO2
methanogenesis under the mesophilic condition become more diverse
and involve more collaboration between species.
Under the thermophilic condition, most of the areas in the sludge are
occupied by Archaea. It can be concluded from above discussion that H2/
CO2 methanogenesis at 55 ◦ C is mainly carried out by hydrotrophic
methanogen archaea directly, causing the simpler metabolic pathway
and the weaker interspecific relationship between microorganisms.
In summary, more abundant bacteria and archaea species and their
cooperation under the mesophilic condition provide various H2/CO2
biomethanation pathways. On the other hand, Methanothermobacter
shifted by high temperature makes H2/CO2 biomethanation simpler but
faster.
3.3. Comparison of metabolism between different biomethanation
pathways
After H2/CO2 biomethanation, the residues were tested via CV
experiment to analyze the electron transfer capacity. Fig. 7 displays the
CVs of H2/CO2 biomethanation under the mesophilic and the thermo­
philic conditions at different scan rates, similar to typical anaerobic
digestion processes [35]. Two distinct redox peaks with potentials of
0.05 (Peak 1) and − 0.40 V (Peak 2) (versus Ag/AgCl) are observed in
the CV curves. Another peak is found at 0.6 V (Peak 3) when the tem­
perature is 55 ◦ C. Peak 2 might be associated with methane formation
from carbon dioxide [27], but the identity of the Peak 1 and Peak 3 are
not certain. Although it is difficult to determine the association between
the specific redox reaction and the redox peaks, the CV experiment can
also be used to quantitatively determine the capacity of extracellular
electron transfer between the biomass and electrode [26]. The peak
currents increase linearly with increasing scan rates, as seen in Fig. 7a
and c. Fig. 7b and d show the dependence of peak potential on the scan
rate by plotting Epeak versus ln ν. Based on Laviron theory, values of kapp
calculated from Peaks 1 and 2 under the mesophilic and the thermo­
philic conditions are 0.0223 s− 1 and 0.0068 s− 1, respectively. The
greater value of kapp suggests a faster extracellular electron transfer rate
under the mesophilic condition, which is contrary to the conclusion that
the rate of methane production is slower under the mesophilic condi­
tion. It can be concluded that the indirect extracellular electron transfer
is not the key factor in the process of H2/CO2 biomethanation under the
thermophilic condition. Due to the direct and simple biomethanation
pathway under the thermophilic condition, H2 is the main electron
donor for CO2 methanogenesis [32] in the direct electron transfer with
few bacteria involved. However, under the mesophilic condition, the
interspecific electron transfer between the acetogen, hydrolytic bacte­
rium and methanogen is more active in the processes of hydro­
genotrophic methanogenesis and homoacetogenesis, strengthening the
electron transfer capacity. The electron transfer capacity cannot be used
to represent the biomethanation rate but indicate the differences of H2/
CO2 methanogenic pathways under the mesophilic and the thermophilic
conditions.
To further explain metabolic process, EEM was utilized to evaluate
the characteristic of dissolved organic matter in the EPS. As shown in
Fig. 8, the EEM spectrum is classified to region I-V according to the
previous study [36–38] and eight main peaks are recorded. Region I and
II represent simple aromatic protein-like substances, and Peak C and D
9
Fuel Processing Technology 238 (2022) 107478
are assigned to aromatic protein-like and tyrosine aromatic protein-like
substances, respectively. Region III represents fulvic acid-like and heme-
like substances, and Peak F belonged to this region. Region IV represents
soluble microbial byproduct-like material, and Peak A and B are
assigned to tryptophan and tyrosine protein-like substances. Region V
represents humic acid-like organics, and Peak E, G and H belong to this
region. Peak fluorescence intensities are listed in the Table S2. On the
whole, the substances can be classified to protein of Peak A, B, C and D
and humic acid of Peak E, F, G and H.
The EPS are believed to be associated with the defense and the
growth of living organisms [38]. It was reported that the protein-like
substances usually participate in electron transfer as electron shuttles
[37]. Meanwhile, protein-like EPS contribute more to the structural
stability of sludge granules, encourage microbial aggregation, and help
to defend against the external selection pressure [39]. Humic acids are
typical electron shuttles responsible for the indirect extracellular elec­
tron transfer between the electron acceptors and donors [40]. Fulvic
acids have smaller molecular weights and higher redox abilities than
other humic acids [41]. Heme groups are also found to be involved in the
electron transport [40].
As shown in Fig. 8 and Table S2, the anaerobic granular sludges
under the mesophilic condition have less protein-like EPS, but the
content of humic acids is larger compared to those under the thermo­
philic condition. The cultures should have the higher electron transport
capacity under the thermophilic condition according to the larger total
content of EPS, but the fact is the opposite. It is speculated that humic
acids play a major role in extracellular electron transport during H2/CO2
methanation. More interactions between bacteria and archaea through
higher content of humic acids lead to the stronger electron transport
capacity under the mesophilic condition. Since high temperature is not
suitable for many microorganisms, proteins are used more often to
enhance the structure stability of the microbial communities and to
defend against the adverse external environment, such as the formation
of protein-like heat-resistant endospores [42], but these proteins do not
play the role of electron shuttles. The production of more humic acids
also indicates that microbes interact more intensively under the meso­
philic condition, which confirms more abundant biomethanation
pathways.
From CVs and EEMs, it is further proved that H2/CO2 methano­
genesis pathways are more complex under the mesophilic condition
involving more collaboration between microorganisms. But under the
thermophilic condition, there is lack of collaboration between micro­
organisms, which becomes one of the reasons for the single pathway of
H2/CO2 biomethanation.
4. Conclusions
Under the mesophilic condition, methanogenesis of H2/CO2 by
anaerobic mixed cultures is completed through the pathways of both
hydrogenotrophic methanogenesis and homoacetogenesis-acetoclastic
methanogenesis, resulting from the close cooperation of various bacte­
ria and archaea. The higher richness of microbial communaties, specific
microbial spatial distribution, higher electron transport capacity and
content of humic acid-like substances contribute to the diversity and
complexity of the pathways. Under the thermophilic condition, H2/CO2
biomethanation fully depends on hydrogenotrophic methanogenesis
and Methanothermobacter dominates the archaeal population. The pro­
cess lacks the interspecific collaboration, is simple and direct but more
efficient in comparison to the case under the mesophilic condition.
CRediT authorship contribution statement
Bingyi Jiang: Conceptualization, Methodology, Investigation,
Visualization, Writing – original draft. Xiao Hu: Investigation. Ulf
Söderlind: Funding acquisition. Kristina Göransson: Investigation.
Wennan Zhang: Funding acquisition, Writing – review & editing.
 B. Jiang et al.
450 450 2800

a) GM-LB-EPS Peak H 2600

b) GT-LB-EPS
2400
Excitation Wavelength (nm) 400 400 2200

Excitation Wavelength (nm)

2000

Peak G 1800
350 Peak G 350
1600

1400
IV V 1200
300 300 Peak B
Peak A 1000
Peak E
800.0

600.0
250 Peak F
250
Peak C Peak F
400.0

I II III Peak D 200.0

200 200 0.000

250 300 350 400 450 500 550 250 300 350 400 450 500 550
Emission Wavelength (nm) Emission Wavelength (nm)
450 450
10

2400
c) GM-TB-EPS d) GT-TB-EPS Peak H 2200
Peak H
400 400 2000
Excitation Wavelength (nm)

Excitation Wavelength (nm) 1800

Peak G 1600
350 350
Peak G 1400

1200
Peak A
300 300 Peak B 1000

Fuel Processing Technology 238 (2022) 107478

800.0
Peak E
600.0
250 250 Peak F
Peak F 400.0

Peak C Peak D 200.0

200 200 0.000

250 300 350 400 450 500 550 250 300 350 400 450 500 550
Emission Wavelength (nm) Emission Wavelength (nm)
Fig. 8. 3-D EEM fluorescence spectra for the LB-EPS and TB-EPS of anaerobic sludge granules under the mesophilic (a and c) and thermophilic conditions (b and d).
B. Jiang et al.
Chunjiang Yu: Conceptualization, Resources, Supervision, Writing –
review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no competing financial interest
associated with this work.
Data availability
Data will be made available on request.
Acknowledgements
This work is supported by EU Regional Development Program
[ERUF20201371] and the “Bao Yu Gang” Foundation for Foreign Guest
Professor Program.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fuproc.2022.107478.
References
[1] P.G. Kougias, P. Tsapekos, L. Treu, M. Kostoula, S. Campanaro, G. Lyberatos,
I. Angelidaki, Biological CO2 fixation in up-flow reactors via exogenous H2
addition, J. Biotechnol. 319 (2020) 1–7, https://doi.org/10.1016/j.
jbiotec.2020.05.012.
[2] A. Maroufmashat, M. Fowler, Transition of future energy system infrastructure;
through power-to-gas pathways, Energies. 10 (2017), https://doi.org/10.3390/
en10081089.
[3] A. Lewandowska-Bernat, U. Desideri, Opportunities of power-to-gas technology in
different energy systems architectures, Appl. Energy 228 (2018) 57–67, https://
doi.org/10.1016/j.apenergy.2018.06.001.
[4] C.J. Quarton, S. Samsatli, Power-to-gas for injection into the gas grid: what can we
learn from real-life projects, economic assessments and systems modelling? Renew.
Sust. Energ. Rev. 98 (2018) 302–316, https://doi.org/10.1016/j.rser.2018.09.007.
[5] J. Browne, A.S. Nizami, T. Thamsiriroj, J.D. Murphy, Assessing the cost of biofuel
production with increasing penetration of the transport fuel market: a case study of
gaseous biomethane in Ireland, Renew. Sust. Energ. Rev. 15 (2011) 4537–4547,
https://doi.org/10.1016/j.rser.2011.07.098.
[6] H. Li, E. Larsson, E. Thorin, E. Dahlquist, X. Yu, Feasibility study on combining
anaerobic digestion and biomass gasification to increase the production of
biomethane, Energy Convers. Manag. 100 (2015) 212–219, https://doi.org/
10.1016/j.enconman.2015.05.007.
[7] S. Rönsch, J. Schneider, S. Matthischke, M. Schlüter, M. Götz, J. Lefebvre,
P. Prabhakaran, S. Bajohr, Review on methanation - from fundamentals to current
projects, Fuel. 166 (2016) 276–296, https://doi.org/10.1016/j.fuel.2015.10.111.
[8] A. Xia, J. Cheng, J.D. Murphy, Innovation in biological production and upgrading
of methane and hydrogen for use as gaseous transport biofuel, Biotechnol. Adv. 34
(2016) 451–472, https://doi.org/10.1016/j.biotechadv.2015.12.009.
[9] I. Díaz, F. Fdz-Polanco, B. Mutsvene, M. Fdz-Polanco, Effect of operating pressure
on direct biomethane production from carbon dioxide and exogenous hydrogen in
the anaerobic digestion of sewage sludge, Appl. Energy 280 (2020), 115915,
https://doi.org/10.1016/j.apenergy.2020.115915.
[10] D. Andreides, J.I. Bautista Quispe, J. Bartackova, D. Pokorna, J. Zabranska, A novel
two-stage process for biological conversion of syngas to biomethane, Bioresour.
Technol. 327 (2021), 124811, https://doi.org/10.1016/j.biortech.2021.124811.
[11] K. Asimakopoulos, A. Grimalt-Alemany, C. Lundholm-Høffner, H.N. Gavala, I.
V. Skiadas, Carbon sequestration through syngas biomethanation coupled with H2
supply for a clean production of natural gas grade biomethane, Waste and Biomass
Valorization. 12 (2021) 6005–6019, https://doi.org/10.1007/s12649-021-01393-
2.
[12] S.S. Navarro, R. Cimpoia, G. Bruant, S.R. Guiot, Biomethanation of syngas using
anaerobic sludge: Shift in the catabolic routes with the CO partial pressure
increase, Front. Microbiol. 7 (2016), https://doi.org/10.3389/fmicb.2016.01188.
[13] C. Li, X. Zhu, I. Angelidaki, Syngas biomethanation: effect of biomass-gas ratio,
syngas composition and pH buffer, Bioresour. Technol. 342 (2021), 125997,
https://doi.org/10.1016/j.biortech.2021.125997.
[14] A. Grimalt-Alemany, M. Łężyk, D.M. Kennes-Veiga, I.V. Skiadas, H.N. Gavala,
Enrichment of mesophilic and thermophilic mixed microbial consortia for syngas
biomethanation: the role of kinetic and thermodynamic competition, Waste and
Biomass Valorization. 11 (2020) 465–481, https://doi.org/10.1007/s12649-019-
00595-z.
[15] X. Pan, I. Angelidaki, M. Alvarado-Morales, H. Liu, Y. Liu, X. Huang, G. Zhu,
Methane production from formate, acetate and H2/CO2; focusing on kinetics and
11
Fuel Processing Technology 238 (2022) 107478
microbial characterization, Bioresour. Technol. 218 (2016) 796–806, https://doi.
org/10.1016/j.biortech.2016.07.032.
[16] A. Kovalovszki, L. Treu, L. Ellegaard, G. Luo, I. Angelidaki, Modeling temperature
response in bioenergy production: Novel solution to a common challenge of
anaerobic digestion, Appl. Energy 263 (2020), 114646, https://doi.org/10.1016/j.
apenergy.2020.114646.
[17] K. Asimakopoulos, H.N. Gavala, I.V. Skiadas, Biomethanation of syngas by
enriched mixed anaerobic consortia in trickle bed reactors, Waste and Biomass
Valorization. 11 (2020) 495–512, https://doi.org/10.1007/s12649-019-00649-2.
[18] Z. Zhang, C. Ding, L. Wang, B. Jiang, U. Söderlind, W. Zhang, C. Yu, CO
biomethanation with different anaerobic granular sludges, Waste and Biomass
Valorization. 12 (2021) 3913–3925, https://doi.org/10.1007/s12649-020-01285-
x.
[19] S.S. Navarro, R. Cimpoia, G. Bruant, S.R. Guiot, Specific inhibitors for identifying
pathways for methane production from carbon monoxide by a nonadapted
anaerobic mixed culture, Can. J. Microbiol. 60 (2014) 407–415, https://doi.org/
10.1139/cjm-2013-0843.
[20] J. Figueras, H. Benbelkacem, C. Dumas, P. Buffiere, Biomethanation of syngas by
enriched mixed anaerobic consortium in pressurized agitated column, Bioresour.
Technol. 338 (2021), https://doi.org/10.1016/j.biortech.2021.125548.
[21] E. Cui, Y. Wu, Y. Zuo, H. Chen, Effect of different biochars on antibiotic resistance
genes and bacterial community during chicken manure composting, Bioresour.
Technol. 203 (2016) 11–17, https://doi.org/10.1016/j.biortech.2015.12.030.
[22] L. Huang, Q. Wang, L. Jiang, P. Zhou, X. Quan, B.E. Logan, Adaptively evolving
bacterial communities for complete and selective reduction of Cr(VI), Cu(II), and
Cd(II) in biocathode bioelectrochemical systems, Environ. Sci. Technol. 49 (2015)
9914–9924, https://doi.org/10.1021/acs.est.5b00191.
[23] C. Lozupone, R. Knight, UniFrac: a new phylogenetic method for comparing
microbial communities, Appl. Environ. Microbiol. 71 (2005) 8228–8235, https://
doi.org/10.1128/AEM.71.12.8228-8235.2005.
[24] G. Crocetti, M. Murto, L. Björnsson, An update and optimisation of oligonucleotide
probes targeting methanogenic Archaea for use in fluorescence in situ
hybridisation (FISH), J. Microbiol. Methods 65 (2006) 194–201, https://doi.org/
10.1016/j.mimet.2005.07.007.
[25] C. Cruz Viggi, S. Rossetti, S. Fazi, P. Paiano, M. Majone, F. Aulenta, Magnetite
particles triggering a faster and more robust syntrophic pathway of methanogenic
propionate degradation, Environ. Sci. Technol. 48 (2014) 7536–7543, https://doi.
org/10.1021/es5016789.
[26] L. Yue, J. Cheng, H. Zhang, L. Yuan, J. Hua, H. Dong, Y. you Li, J. Zhou, Inhibition
of N-Vanillylnonanamide in anaerobic digestion of lipids in food waste:
Microorganisms damage and blocked electron transfer, J. Hazard. Mater. 399
(2020), 123098, https://doi.org/10.1016/j.jhazmat.2020.123098.
[27] J.Y. Lee, S.H. Lee, H.D. Park, Enrichment of specific electro-active microorganisms
and enhancement of methane production by adding granular activated carbon in
anaerobic reactors, Bioresour. Technol. 205 (2016) 205–212, https://doi.org/
10.1016/j.biortech.2016.01.054.
[28] G. Luo, W. Wang, I. Angelidaki, Anaerobic digestion for simultaneous sewage
sludge treatment and CO biomethanation: Process performance and microbial
ecology, Environ. Sci. Technol. 47 (2013) 10685–10693, https://doi.org/10.1021/
es401018d.
[29] F. Bu, N. Dong, S. Kumar Khanal, L. Xie, Q. Zhou, Effects of CO on
hydrogenotrophic methanogenesis under thermophilic and extreme-thermophilic
conditions: Microbial community and biomethanation pathways, Bioresour.
Technol. 266 (2018) 364–373, https://doi.org/10.1016/j.biortech.2018.03.092.
[30] L. Cheng, L. Dai, X. Li, H. Zhang, Y. Lu, Isolation and characterization of
Methanothermobacter crinale sp. nov., a novel hydrogenotrophic methanogen
from the shengli oil field, Appl. Environ. Microbiol. 77 (2011) 5212–5219, https://
doi.org/10.1128/AEM.00210-11.
[31] A.K. Kaster, M. Goenrich, H. Seedorf, H. Liesegang, A. Wollherr, G. Gottschalk, R.
K. Thauer, More than 200 genes required for methane formation from H2 and CO2
and energy conservation are present in methanothermobacter marburgensis and
methanothermobacter thermautotrophicus, Archaea. 2011 (2011), https://doi.
org/10.1155/2011/973848.
[32] J. Cheng, H. Dong, H. Zhang, L. Yuan, H. Li, L. Yue, J. Hua, J. Zhou, Improving CH4
production and energy conversion from CO2 and H2 feedstock gases with mixed
methanogenic community over Fe nanoparticles, Bioresour. Technol. 314 (2020),
123799, https://doi.org/10.1016/j.biortech.2020.123799.
[33] S. Jaenicke, C. Ander, T. Bekel, R. Bisdorf, M. Dröge, K.H. Gartemann,
S. Jünemann, O. Kaiser, L. Krause, F. Tille, M. Zakrzewski, A. Pühler, A. Schlüter,
A. Goesmann, Comparative and joint analysis of two metagenomic datasets from a
biogas fermenter obtained by 454-pyrosequencing, PLoS One 6 (2011), https://doi.
org/10.1371/journal.pone.0014519.
[34] L.W. Hulshoff Pol, S.I. De Castro Lopes, G. Lettinga, P.N.L., Lens, Anaerobic sludge
granulation, Water Res. 38 (2004) 1376–1389, https://doi.org/10.1016/j.
watres.2003.12.002.
[35] J. Li, L. Xiao, S. Zheng, Y. Zhang, M. Luo, C. Tong, H. Xu, Y. Tan, J. Liu, O. Wang,
F. Liu, A new insight into the strategy for methane production affected by
conductive carbon cloth in wetland soil: Beneficial to acetoclastic methanogenesis
instead of CO2 reduction, Sci. Total Environ. 643 (2018) 1024–1030, https://doi.
org/10.1016/j.scitotenv.2018.06.271.
[36] W. Chen, P. Westerhoff, J.A. Leenheer, K. Booksh, Fluorescence excitation-
emission matrix regional integration to quantify spectra for dissolved organic
matter, Environ. Sci. Technol. 37 (2003) 5701–5710, https://doi.org/10.1021/
es034354c.
[37] Y. Shi, T. Liu, S. Chen, X. Quan, Accelerating anaerobic hydrolysis acidification of
dairy wastewater in integrated floating-film and activated sludge (IFFAS) by using
B. Jiang et al.
zero-valent iron (ZVI) composite carriers, Biochem. Eng. J. 177 (2022), 108226,
https://doi.org/10.1016/j.bej.2021.108226.
[38] J. Qian, K. Li, P. Wang, C. Wang, M. Shen, J. Liu, B. Lu, X. Tian, Toxic effects of
three crystalline phases of TiO2 nanoparticles on extracellular polymeric
substances in freshwater biofilms, Bioresour. Technol. 241 (2017) 276–283,
https://doi.org/10.1016/j.biortech.2017.05.121.
[39] Z. Liang, Q. Tu, X. Su, X. Yang, J. Chen, Y. Chen, C. Liu, H. Li, Q. He, Formation,
extracellular polymeric substances and microbial community of aerobic granules
enhanced by microbial flocculant compared with poly-aluminum chloride,
J. Clean. Prod. 220 (2019) 544–552, https://doi.org/10.1016/j.
jclepro.2019.02.180.
12
Fuel Processing Technology 238 (2022) 107478
[40] X. Liu, L. Shi, J.D. Gu, Microbial electrocatalysis: Redox mediators responsible for
extracellular electron transfer, Biotechnol. Adv. 36 (2018) 1815–1827, https://doi.
org/10.1016/j.biotechadv.2018.07.001.
[41] Y. Dang, Y. Lei, Z. Liu, Y. Xue, D. Sun, L.Y. Wang, D.E. Holmes, Impact of fulvic
acids on bio-methanogenic treatment of municipal solid waste incineration
leachate, Water Res. 106 (2016) 71–78, https://doi.org/10.1016/j.
watres.2016.09.044.
[42] Z. Tu, P. Setlow, S. Brul, G. Kramer, Molecular physiological characterization of a
high heat resistant spore forming bacillus subtilis food isolate, Microorganisms. 9
(2021), https://doi.org/10.3390/microorganisms9030667.