Waste and Biomass Valorization (2021) 12:3913–3925

https://doi.org/10.1007/s12649-020-01285-x

ORIGINAL PAPER

CO Biomethanation with Different Anaerobic Granular Sludges

Zhenwen Zhang1,2 · Chao Ding1,2 · Luyang Wang1,2 · Bingyi Jiang1,2 · Ulf Söderlind2 · Wennan Zhang2 · Chunjiang Yu1

Received: 25 May 2020 / Accepted: 26 October 2020 / Published online: 1 November 2020
© Springer Nature B.V. 2020

Abstract 
Biomethanation of the syngas from biomass gasification provides an alternative method for production of biofuel and
chemicals. CO in syngas plays a key role in biomethanation of syngas, as it is both a substrate and an inhibitor of certain
methanogenesis processes. In this study, CO biomethanation by using a mixture of ­N2 and CO as the gas substrate, was
investigated with the help of 5 adapted anaerobic granular sludges under thermophilic and mesophilic conditions. The results
show that CO biomethanation by the adapted inocula is omnipresent. The sludge from the juice plant has a methane yield
more than 80% of the theoretical value both at 37 °C and 55 °C. Increasing the temperature from 37 °C to 55 °C has a slight
effect on the final methane production, but can significantly increase the CO consumption rate and shorten the time for CO
biomethanation.
Graphic Abstract

Keywords  Methane · Carbon monoxide · Biomethanation · Anaerobic granular sludge

Statement of Novelty
Previous studies on CO biomethanation reported in literature
either used nearly 100% pure CO with non-adapted sludge
or a single type of anaerobic granular sludge in CO biom-
ethanation. The present work is directed to the application of
CO biomethanation to the syngas treatment and utilization to
provides an alternative method for the production of biofuels
and chemicals from biomass gasification. The content of CO
in the syngas from biomass gasification is below 50%. In
order to obtain high CO biomethanation rate and conversion
* Chunjiang Yu
chunjiang@zju.edu.cn
1
State Key Laboratory of Clean Energy Utilization, Zhejiang
University, 38 Zheda Road, Hangzhou 310027, Zhejiang,
China
2
Department of Chemical Engineering, Mid Sweden
University, 85170 Sundsvall, Sweden
efficiency for such syngas, in this work, inoculum acclima-
tion is applied, different anaerobic granular sludges from
various industrial wastewater treatment systems are tested,
and thermophilic and mesophilic conditions are compared
with each other.
Introduction
Biogas is a renewable and environmentally sustainable fuel
that is used worldwide [1]. It can replace conventional fuel
for the production of heat and power to reduce carbon emis-
sion and mitigate the greenhouse effect. Upgraded biogas
can substitute natural gas as a vehicle fuel or for chemicals
production. Biogas is usually defined as the gas produced
from anaerobic digestion (AD) of organic substrates by
means of anaerobic microorganisms [2], which ends up with
­CH4 and ­CO2 as the main final products.

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The AD process is highly efficient to treat biodegrad-
able biomass such as animal manure and food waste, but
is difficult to treat non-biodegradable biomass such as lig-
nocellulosic biomasses [3–5]. The limiting step is biomass
hydrolysis which is difficult to proceed and requires a costly
and complex pretreatment process to enhance the access of
the microorganisms to the raw materials [6–8].
On the other hand, lignocellulosic biomass can be easily
gasified into syngas. As an important intermediate platform
in biorefinery concept, the syngas can be synthesized into
chemicals and high-quality fuels, such as methane, metha-
nol, DME, diesel and gasoline etc. via metal catalysts [9, 10].
However, before downstream syntheses, the syngas must go
through a gas cleaning step to meet the strict standards on
low impurities in the syngas, such as particulate matter, tars,
sulfur compounds, nitrogen compounds, alkali metals and
hydrogen chloride [11]. The key issue for biomass to liquid
(BTL) commercialization is the syngas cleaning which may
account for up to 38% of the total production cost [12].
To avoid the stringent syngas cleaning, the syngas can be
converted into C ­ H4 by means of microbial catalysts which
can tolerate contaminants of syngas in a certain concentra-
tion range [13]. Compared with metal-catalyzed methana-
tion, biomethanation also has milder reaction conditions and
more specific end products [14]. Therefore, integration of
biomass gasification with syngas biomethanation seems a
promising conversion technology of biomass to biofuels in
the future. At present, biomass gasification has been well
commercialized somehow for heat and power production.
Fig. 1  Pathways of CO biomethanation. The standard Gibbs free energy change (ΔG°) was calculated according to Thauer et al. [20]
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Waste and Biomass Valorization (2021) 12:3913–3925
On the other hand, syngas biomethanation is fairly new and
in the earlier stage of research. In particular, the biomethana-
tion of CO as a substrate from syngas is the key issue due to
its inhibitory effect on microorganisms.
Several microbes have been found to be able to produce
methane from CO as the sole substrate, namely Methanobac-
terium thermoautotrophicum, Methanosarcina acetivorans,
Methanosarcina. barkeri. However, methanogenic growth on
CO is fairly slow. Thermophilic Methanothermobacter ther-
moautotrophicus growth on CO is at a rate only about 1% of
its growth rate on H­ 2/CO2 under the pressure of 50 kPa [15].
The mixed microbial consortium seems to be more suitable
for CO biomethanation. The anaerobic granular sludge can
mitigate the inhibitory effect of CO on methanogens dur-
ing the biomethanation since most of the sensitive metha-
nogens are distributed inside the granules to avoid direct
contact with CO [16–18]. This makes anaerobic granular
sludge promising for large-scale syngas biomethanation. The
anaerobic granular sludge contains multiple microorganisms
such as acetogens, hydrogenogens and methanogens. These
microorganisms cooperate to form a metabolic network to
complete the conversion of CO to C ­ H4 by direct or indirect
pathway as presented in Fig. 1. Many studies have shown
that the indirect pathway dominates in CO conversion, and
the direct pathway is negligible [13, 19].
Sipma et al. [21] tested the CO conversion by differ-
ent anaerobic granular sludges from different wastewa-
ter treatment reactors and found the CO biomethanation
performance is different for different anaerobic granular
Waste and Biomass Valorization (2021) 12:3913–3925 3915
sludges. The anaerobic granular sludges from wastewa-
ter treatment of various origins have a great impact on
microbial populations composition and distribution [22,
23]. Identification of sufficiently good anaerobic granular
sludge for CO biomethanation is an important research
work to be done. Navarro et al. [24] used disaggregated
granular sludge to acclimate at 1 atm of CO for 45 days
and found that the methane yield increased from 8 to 90%.
This shows that adapting the sludge to CO beforehand is
crucial for CO biomethanation.
In addition to inocula, other factors such as CO partial
pressure, gas-liquid mass transfer efficiency, and tempera-
ture also have effects on the CO biomethanation. Previous
efforts to evaluate the effect of CO partial pressure for CO
biomethanation suggest that increasing P ­ CO would enhance
acetogenesis while inhibiting methanogenesis [21, 25–27].
Navarro et al. [24] reported that a maximum methanogenic
activity was achieved at 0.2 atm of CO, and the biometh-
antion rate decreased with the CO partial pressure. When
the partial pressure reached 1.0 atm, the activity of metha-
nogen was completely inhibited. However, Esquivel-Eli-
zondo et al. [28] reported that complete inhibition of meth-
anogens occurred when P ­ CO was ≥ 51.4 kPa (0.48 mM).
Despite differences in conclusions, these studies show that
CO has a strong inhibitory effect on methane production.
Therefore, for syngas from biomass gasification, it is nec-
essary to further evaluate the methanogenic potential of
the adapted anaerobic granular sludges. Guiot et al. [25]
reported that under mesophilic and thermophilic condi-
tions, the conversion of CO appears to proceed through
the formation of ­H2 and hydrogenotrophic methanogensis.
However, other studies indicate that under mesophilic con-
ditions, the conversion of CO is mainly via acetogenesis
and acetoclastic methanogenesis [19, 21, 24]. Therefore,
it would be necessary to further test the conversion of CO
at different temperatures with respect to the integration of
syngas biomethanation with biomass gasification which
takes place at high temperature.
Previous studies either used nearly 100% pure CO with
non-adapted sludge or a single type of anaerobic granular
sludge in CO biomethanation experiments. However, The
content of CO in biomass syngas is typically below 50%
[29]. For the biomethanation of syngas produced by biomass
gasification, it is necessary to evaluate the inoculated granu-
lar sludges with CO concentration below 50%.
So far, the research on syngas biomethanation is fairly
new, and very little has been reported in literature such as the
evaluation of anaerobic granular sludges, sludge acclimation
with CO, which will be studied in this work. Furthermore,
the CO biomethanation rate and the lag phase is paid atten-
tion to in the experimental investigation, and the mesophilic
(37℃) and thermophilic conditions (55℃) are compared and
identified for optimal biomethanation of syngas.
Materials and Methods
Inocula
Anaerobic granular sludges were obtained from Jiangxi
Shuierli Environmental Protection Technology CO. Ltd.
5 different types of sludges originate from the mesophilic
(30–40  ℃) bioreactors that treat industrial wastewater:
alcohol plant wastewater, citric acid wastewater, paper mill
wastewater, starch mill wastewater, and juice plant wastewa-
ter as listed in Table 1. Only starch wastewater sludge was
obtained from the upflow anaerobic sludge blanket (UASB)
reactor, and other sludges were obtained from the internal
circulation (IC) reactors. The cattle manure was obtained
from free-range grazing cattle in the Dabie Mountains. The
characteristics of the inocula are shown in Table 1.
Experimental Facility
The biomethanation reactor used in this study is a 320 mL
serum bottle which is connected to a 100 mL gasbag. The
bottle and gasbag are connected through a 5 mm diameter
silicone hose. The gasbag has a certain degree of flexibility
in volume depending on the gas in the bottle. Thus, the pres-
sure in the bottle can be kept the same level as the outside
atmospheric pressure due to the change in gasbag volume.
To facilitate liquid sampling, a long needle is fixed below the
liquid surface through the bottle cap, and a two-way valve
is installed on the top of the long needle. Besides, another
two-way valve is fixed to the bottle cap for gas sampling. All
interfaces are sealed with high temperature resistant hot glue
to ensure the airtightness of the reactor.
The serum bottle was autoclaved before the start of the
test at a temperature of 121 °C and sterilized for 20 min.
After cooling, 15 mL of the inoculum was inoculated into
the serum bottle, then 60 mL of the basal anaerobic (BA)
medium and 0.03 g of cysteine hydrochloride were added
to the serum bottle [30]. The basal medium is added as a
growth media and as well as a nutrient base. The composi-
tion of the BA medium is shown in Table 2. The reagents in
BA medium are categorized into A, B, C, D and E. The BA
Table 1  The characteristics of inocula
Abbreviation Source of inocula TS (g/L) VS (g/L)
AGS Alcohol plant wastewater 62 50
CGS Citric acid wastewater 96 56
JGS Juice plant wastewater 62 54
PGS Paper mill wastewater 330 74
SGS Starch mill wastewater 48 38
CM Cattle manure 44 42
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3916 Waste and Biomass Valorization (2021) 12:3913–3925

Table 2  Stock solution of basal anaerobic medium Table 3  Experimental conditions

Component Reagent Concentration Unit Test Sludge Gas composition (%/%) Tempera- Rotating
ture (°C) speed
A NH4Cl 100 g/L (rpm)
NaCl 10
1 AGS1 CO/N2 = 50/50 37 80
MgCl2·6H2O 10
CGS1
CaCl2·2H2O 5
JGS1
B K2HPO4·3H2O 200 g/L
PGS1
C Resazurin 0.5 g/L
SGS1
D FeCl2·4H2O 2 g/L
CM1
H3BO3 0.05
2 AGS2 CO/N2 = 50/50 55 80
ZnCl2 0.05
JGS2
CuCl2·2H2O 0.038
PGS2
MnCl2·4H2O 0.05
SGS2
(NH4)6Mo7O24·4H2O 0.05
CM2
AlCl3 0.05
CoCl2·6H2O 0.05
NiCl2·6H2O 0.092
EDTA 0.5 was measured once every three days and a small amount of
Na2SeO3·5H2O 0.1 liquid consumed by the measurement was replenished with
Concentrated HCl 1 mL the fresh BA medium. The biomethanation reactor was cul-
E Biotin 2 mg/L tured in shaker-bed at 80 rpm and 2 different temperatures
Folic acid 2 of 37 °C and 55 °C in the dark.
Pyridoxal HCl 10
Thiamine HCl 5
Riboflavin 5 Experimental Tests
Nicotnic acid 5
Ca-d-Pantotheinate 5 The inocula used in this study contains five types of sludge
Cyanocobalamin 0.1 and one type of cattle manure, as shown in Table 1. Prior to
P-aminobenzoic acid 5 the start of the tests, all the inocula were cultured under CO/
Thioctic acid 5 N2 = 50/50 environment and at two different temperatures
until the gas composition did not change. There are two rea-
sons for that: (1) to adapt the inoculums to the temperature
medium are made from 10 mL A, 2 mL B, 1 mL C, 1 mL D and gas composition used in the experiment, (2) to consume
and 1 mL E in addition to distilled water for a total volume the original digestible in the inoculums. This process lasted
of 1 L, similar to reference [31]. 31 days. Then the reactor was filled with the BA medium,
The gas substrate used in this study is a mixture of CO sealed, flushed and filled again before the test starts. The
and ­N2 with a ratio of 1:1 in volume, which was obtained experiment is organized with 2 series of tests for mesophilic
from Hangzhou Jingong Special Gas Co. Ltd. Before the and thermophilic conditions respectively as seen in Table 3.
start of the tests, the gas was used to flush the biomethana-
tion reactor at a rate of 300 mL/min for 15 min to exhaust the
air in the reactor. After flushing, the entire reactor was filled Analytical Methods
with the gas at 1 atm. Then a syringe was used to adjust the
gasbag volume to 100 mL through the gas sampling valve. The TS and VS were determined by heating the samples
Therefore, the volume of the headspace gas of the bottle is in an oven at 105 ℃ overnight until the quality remained
245 mL and the volume of the gas in the gasbag is 100 mL, unchanged, and then in a furnace at 600 °C for 2 h [32].
leading to the total volume of the gas substrate equal to The values of TS and VS are calculated from the sample
345 mL. The total volume of the liquid is 75 mL. During amount remained at different times. Use the data from Lide
the tests, a syringe was used to siphon out 2 mL of gas from [33] to calculate the solubility of H
­ 2, CO, C
­ O2 and C ­ H 4,
the reactor every day to measure its gas composition. The taking into account the amount of gas phase dissolved into
liquid in the reactor was siphoned out by syringe from the the liquid phase.
long needle to measure the pH which was then adjusted to Lower calorific values (LCV) of the initial CO and the
6.5–7.5 by 4 M HCl or 4 M NaOH if necessary. Liquid pH final gas were determined according to Yeqing Li et al. [34]:

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Waste and Biomass Valorization (2021) 12:3913–3925 3917
LCV(MJ∕Nm3 ) = (30.0 × CO + 25.5 × H2 + 85.4 × CH4 ) × 0.42.
where CO, H ­ 2 and C
­ H4 represent the corresponding gas
concentrations (%) of the gas.
The content of CO, ­CO2, ­CH4, ­H2 in the headspace of the
biomethanation reactor was determined by gas chromatog-
raphy (Shimadzu GC-2014, Kyoto, Japan) equipped with a
thermal conductivity detector and a packed column (TDX-
01, 2 m × 3 mm, Stainless Steel). The carrier gas is argon
and the flow rate is 30 mL/min. The injector and detector
temperatures are 110 °C and 160 °C, respectively. The col-
umn temperature was first retained at 70 °C for 3 min, then
increased to 100 °C at a rate of 3 °C/min, and finally retained
for 0.5 min.
The concentrations of acetate, butyrate, and propionate
were determined by liquid chromatography (UltiMate 3000,
USA) equipped with a differential refractive Index detector
and Aminex HPX-87H Column (300 × 7.8 mm). The column
and detector temperatures are 60 °C and 40 °C, respectively.
The flow phase is 5 mM sulfuric acid at a rate of 0.6 mL/
min. The sample injection volume is 5 µL and the analysis
time is 31 min.
Results and Discussion
Methanogenesis of CO at 37 ℃
The measurement results for the gas substrate of CO and
­N2 mixture under mesophilic conditions of 37 ℃ with help
of 6 different inocula are presented in Fig. 2. As seen in the
figure, AGS and JGS are the best inocula for fast CO biom-
ethanation. After 31 days’ adaptation, AGS and JGS are able
to give a certain amount of methane production in a very
short lag phase after gas substrate introduction, less than 2
days. The methane content gradually increases and remains
constant around 12 days in response to the CO consumption
rapidly at the beginning of the tests and leveling-off after
12 days as expected. The CO consumption rate gradually
decreases with time. ­H2 (< 50 µmol) were detected anyway,
but at an extremely low level as seen in Fig. 2 for all different
inoculum cases. The final methane production of AGS and
JGS is fairly high, 1.38 mmol and 1.51 mmol, respectively,
as summarized in Table 4.
Different from AGS and JGS, CGS, PGS, PGS and CM
give a much lower level of methane production in the early
stage as seen in Fig. 2. The lag phase is longer, approxi-
mately 8 days. After the end of the lag phase, the CO con-
sumption rate increases significantly, while methane begins
to accumulate. Similarly, only traces of H ­ 2 were detected
during the tests. For CM, the CO consumption rate is sig-
nificantly lower and only a small amount of ­CH4 is gener-
ated after 16 days even a week longer as observed in the
experiment, while the gas composition of CM is hardly
changed. This may be related to the microbial structure in
cattle manure. These microbial consortiums may need longer
acclimation time to adapt to the CO atmosphere under meso-
philic condition of 37 ℃.
As shown in Fig. 2, after inoculum acclimation to CO for
31 days, AGS and JGS can quickly consume CO at 0.5 atm
partial pressure of CO under the mesophilic condition. For
SGS and PGS, when CO consumption rate is increased sig-
nificantly, the partial pressure of CO is reduced down to
0.27 atm, meanwhile methane starts to be produced signifi-
cantly. Although all the inocula were acclimated for 31 days
under the same condition, the anaerobic granular sludges
from different sources still have different CO consumption
capacity and start to produce methane at different levels of
CO partial pressure.
Sipma et al. [21] reported that acetate seems to be the
main intermediate under the mesophilic condition and it
was completely converted into methane at the end of experi-
ments. On the other hand, the results of Esquivel-Elizondo
et al. [28] showed that acetate was the main end product
when CO is used as a single carbon source and energy
source. The present work shows that, following CO con-
sumption, acetate, butyrate, and propionate start to appear
and accumulate corresponding to the changes of pH as seen
in Fig. 3.
The main volatile fatty acid (VFA) obtained during the
CO biomethanation process is acetate. At the end of the
experiment, acetate is reduced to an extremely low level
while methane becomes the main end product for AGS,
CGS, JGS and PGS. For SGS and CM, however, Acetate
is kept at a high level in the content. At the beginning of
the experiment, with the formation of VFA, the pH value
dropped significantly as seen in Fig. 3. In order to ensure
a better CO methanation, when the pH drops too much,
the pH would be adjusted back to around 7 in time. As the
experiment progressed, the pH value gradually stabilized.
This can be explained by that the CO was converted into
VFA firstly under the mesophilic condition, and resulted in
a dramatic drop in pH. Then the formation and conversion
of VFA gradually reached a certain balance.
The concentration of VFA in the case of J­ GS1 is very low
over the whole experimental period of time, in response to a
high concentration of ­CH4 in the headspace as seen in Fig. 2.
This can be explained by fast and complete conversion of
VFA to C ­ H4 under the mesophilic condition with the JGS
inoculum. For PGS and SGS, however, the concentrations of
VFA are high in response to low ­CH4 concentrations in the
headspace. Anyway, the accumulated acetate in the reactor
liquid phase is just an intermediate product for PSG in the
test period of time, which is consumed mostly at the end
of the experiment, resulting in a higher methane produc-
tion comparing to SGS. As a result, whether VFA is a main
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3918 Waste and Biomass Valorization (2021) 12:3913–3925

Fig. 2  Gas composition in the headspace of bioreactor with the initial gas composition of CO/N2 = 50%/50% at 37 ºC for different inocula: AGS,
CGS, JGS, PGS, SGS and CM

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Waste and Biomass Valorization (2021) 12:3913–3925 3919

Table 4  CO average Inoculum CO consumption rate CH4 production at the CH4 yield stoichio- Energy conver-
consumption rate, C­ H4 (mmol/gVS d) end (mmol) metric %a sion efficiency %b
production, ­CH4 yield
stoichiometric and energy 37 °C 55 °C 37 °C 55 °C 37 °C 55 °C 37 °C 55 °C
conversion efficiency at 37 and
55 ℃ respectively AGS 0.58 0.75 1.39 1.18 76.6 65.2 73.2 60.8
CGS 0.52 – 1.61 – 88.8 – 77.8 –
JGS 0.51 0.73 1.51 1.55 83.6 85.6 77.1 69.6
PGS 0.39 0.49 1.20 1.31 66.3 72.2 60.8 70.0
SGS 0.78 0.96 0.99 1.21 54.5 66.7 47.3 63.5
CM 0.25 0.53 0.23 0.83 12.4 45.6 77.4 57.4
a
 CH4 yield stoichiometric: 1/4 mol of ­CH4 per mol CO (4CO + 2H2O → ­CH4 + 3CO2)
b
 Energy conversion efficiency = (LCV (the product gas)/LCV (the initial CO) × 100%)

intermediate product or end product depends on experimen-
tal conditions such as inoculum used and incubation time.
It is worth emphasizing that in this experiment, it cannot
be distinguished whether the acetate was converted directly
from CO through carboxydotrophic acetogenesis or con-
verted indirectly from ­H2 + CO2 through water-gas shift
reaction and homoacetogenesis. Although the H ­ 2 concen-
tration is always very low but ­H2 can be expected to appear
and then converted into other products after it is formed[35].
Methanogenesis of CO at 55 °C
For CO biomethanation at 55 °C, the gas composition in
the headspace as function of time is shown in Fig. 4. As
seen in the figure, the time-dependent CO consumption
rate under the thermophilic condition is generally higher.
With the exception of ­CM2, CO is rapidly consumed with
almost no lag phase or extremely short lag phase, less than
2 days. After the short lag phase, methane began to form,
and methane production reaches its maximum after about a
week. These indicate that under the thermophilic condition,
the time taken for the biomethanation process of CO/N2 is
significantly shorter than the mesophilic condition. Guiot
et al. [25] also observed a faster CO conversion potential
during CO biomethanation with anaerobic granular sludge
under the thermophilic condition.
As shown in Fig. 4, a certain amount of ­H2 appears in
the headspace, and its maximum value reaches 1.08 mmol,
which is completely consumed after a few days. Based on
the maximum value of ­H2, it can be calculated that at least
44% of CO was converted into ­CO2 + H2.
As shown in Fig. 4 CM2, under the thermophilic condi-
tion, adapted cattle manure still requires a long lag phase
of 16 days to consume CO. Afterwards, the CO consump-
tion rate starts to increase, and C
­ H4 as well as H
­ 2 start to
accumulate. The final methane production reaches a much
higher level comparing to the mesophilic condition shown
in Fig. 2CM1. The long lag phase required might be attrib-
uted to CO inhibition effect. The microbial community in
the CM is dissociated state in the liquid phase, so that CO
can directly be in contact with the methanogens to inhibit
its methanogenic activity. Navarro et al. [24] observed that
as ­PCO increasing from 0.5 to 1 atm, aceticlastic methano-
genesis was replaced by hydrogenotrophic methanogen-
esis. Hydrogenotrophic methanogens are less sensitive to
CO inhibition in comparison to acetoclastic methanogens.
Therefore, under the thermophilic condition, methane pro-
duction proceeds mainly through the synergy of hydrog-
enogens and hydrogenotrophic methanogens after a longer
phase lag. This further confirms that anaerobic granular
sludge has a unique advantage in CO biomethanation due to
the structure of granular sludge.
Under the thermophilic condition, the accumulation of
VFA was also detected, as shown in Fig. 5. It can be seen
that the VFA accumulated during the CO biomethanation
is significantly lower than the mesophilic condition. Mean-
while, the amount of hydrogen has increased significantly as
mentioned above. This confirms that temperature can affect
the intermediate process of CO conversion to ­CH4. The drop
in pH is also slightly lower than the mesophilic condition.
This can be explained by the decrease in the amount of VFA.
Comparison of CO Biomethanations at 55 ℃
and at 37 ℃
Comparing the two cases of thermophilic and mesophilic
conditions, the intermediate products vary from each other
to a great degree, in spite of similar amount of end product
of methane. In the case of the thermophilic condition, C ­ O2
is rapidly produced in the beginning after 2 or 3 days in
response to H­ 2 production for all inocula except for C­ M 2.
The production of ­H2 is significantly higher than that under
the mesophilic condition. Similar to previous studies[21],
under the thermophilic condition, ­H2 is the main intermedi-
ate in the CO biomethanation, which is subsequently con-
verted to C
­ H4 by hydrogenotrophic methanogenesis at pres-
ence of abundant thermophilic hydrogenogens in the sludge.

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Fig. 3  VFA accumulation and pH in the bioreactors with the initial gas composition of CO/N2 = 50%/50% at 37 ºC for different inocula: AGS,
CGS, JGS, PGS, SGS and CM. (The dotted line represents the pH adjustment from outside)

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Waste and Biomass Valorization (2021) 12:3913–3925 3921

Fig. 4  Gas composition in the headspace of bioreactor with the initial gas composition of CO/N2 = 50%/50% at 55 ºC for different inocula: AGS,
JGS, PGS, SGS and CM

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Fig. 5  VFA accumulation and pH in the bioreactors with the initial gas composition of CO/N2 = 50%/50% at 55 ºC for different inocula: AGS,
JGS, PGS, SGS and CM. (The dotted line represents the pH adjustment from outside)

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Waste and Biomass Valorization (2021) 12:3913–3925 3923
The CO average consumption rate, the C ­ H4 production
at the end and the ­CH4 yield from biomethanation of the
gas substrate of CO and ­N2 mixture under the thermophilic
condition and the mesophilic condition with help of 5 differ-
ent inocula are summarized in Table 4. Generally speaking,
in comparison to the mesophilic condition, the CO average
consumption rate under the thermophilic condition is sig-
nificantly higher, and the final ­CH4 yield in most cases is
slightly higher. The thermophilic condition has been shown
to favor higher product yields and lower growth yields for
the microbial community, which could be explained by
the lower free energy yield when increasing the tempera-
ture[36]. The lower energy yield at 55℃ may force them to
consume more CO to obtain the same amount of energy for
microbial growth.
As shown in Table 4, At 37 °C, the energy conversion
efficiency of most inocula exceeds 70%. The energy con-
version efficiency of PGS and SGS is lower because a part
of CO is converted into VFA without further conversion to
­CH4 as shown in Fig. 3. It is worth noting that the methane
production of CM is 0.23 mmol at 37 ℃, much lower than
55 ℃, but the energy conversion efficiency is higher. This is
because most of the CO has not been converted into methane
under the mesophilic condition as shown in Fig. 2.
Although the temperature increasing has a negative effect
on the solubility of the gas, which will weaken the gas-liquid
mass transfer. However, for the thermophilic condition, the
­CH4 production is higher and the biomethanation time is
shorter as described above. This indicates that the limiting
factor of 0.5 atm CO biomethanation process is likely to be
the biochemical reaction. Since syngas is normally generated
at high temperature up to 850 ºC, the thermophilic condi-
tion is expected to be more appropriate for the process con-
figuration of syngas biomethanation with respect to energy
optimization.
As shown in Fig. 1. It can be calculated from thermo-
dynamic analysis that the water-gas shift reaction and the
aceticlastic methanation reaction are entropy-increasing
reactions, the reactions of the hydrogenotrophic metha-
nation, carboxydotrophic acetogenesis and the homoace-
togenesis are entropy-decreasing reactions, the standard
entropy change (ΔS°) was calculated according to Lide [33].
Increasing the temperature from 37 to 55 ℃ makes water-
gas shift reaction and the aceticlastic methanation reaction
more thermodynamically favorable, but the reactions of the
hydrogenotrophic methanation, carboxydotrophic acetogen-
esis and the homoacetogenesis are slightly limited due to
the changes in Gibbs energy. This means that at 55 °C, the
hydrogenogenic process is more thermodynamically favora-
ble while the processes of hydrogen consumption are slightly
limited. This may explain the significant accumulation of ­H2
detected under the thermophilic condition of 55℃. Similarly,
at 55 °C, the acetogenic processes are slightly limited while
the acetoclastic methanogenesis process is more thermody-
namically favorable. This may explain that the accumulation
of acetate at 55 °C is less than 37 °C in most cases. Under
the thermophilic condition of 55 ℃, higher H ­ 2 concentration
can be considered for ­H2 production from CO in application.
Based on the stoichiometry of the reaction of carboxydo-
trophic methanation as shown in Fig. 1, the ­CH4 yields of
the anaerobic granular sludges are calculated to be between
50 and 88%. It is worth noting that, at either 37 °C or 55 °C,
the methane yields of JGS are above 80%. This indicates that
the adapted JGS has better performance in the process of
CO conversion to C ­ H4, the JGS is more appropriate for the
biomethanation of CO from biomass gasification. Navarro
et al.[24] reported that the ­CH4 yield is 24% during the CO
biomethanation by disaggregated anaerobic sludge gran-
ules under 0.5 atm initial CO partial pressure. The results of
Guiot et al. [25] show that the methane yields close to 100%
even more than 100%. It may be attributed to the degradable
organic matter contained in the inoculum used. As shown in
Fig. 4 ­JGS2 and Fig. 2 ­JGS1, there is almost no lag phase in
the consumption of CO, i.e. CO is rapidly consumed as soon
as the experiment starts. In the present case of 0.5 atm CO
partial pressure, JGS has better CO conversion ability and
shorter time of biomethanation. In conclusion, the anaerobic
granular sludge from a juice plant is more suitable as the
original inoculum for CO biomethanation.
Conclusion
In this study, CO biomethanation by using a mixture of ­N2
and CO as the gas substrate, was investigated with the help
of 5 anaerobic granular sludges under thermophilic and
mesophilic conditions. The results show that the inoculum
acclimation to CO is important for CO biomethanation.
The sludge from the juice plant has a methane yield more
than 80% of the theoretical value both at 37 °C and 55 °C.
Increasing the temperature from 37 to 55 °C has a slight
effect on the final methane production, but can significantly
increase the CO consumption rate and shorten the time for
CO biomethanation. This work can provide a good refer-
ence for the biomethanation application to the syngas from
biomass gasification.
Acknowledgements  This work is supported by the National Key
Research and Development Program of China [2018YFB1501405],
EU Regional Development Program [ERUF20201371] and the “Bao
Yu Gang” Foundation for Foreign Guest Professor Program.
Compliance with ethical standards 
Conflict of interest  The authors declare that there is no conflict of in-
terest.
13

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