Energy Conversion and Management 276 (2023) 116551

Contents lists available at ScienceDirect

Energy Conversion and Management

journal homepage: www.elsevier.com/locate/enconman

A novel approach to enhance CO biomethanation by semi-disaggregation of

anaerobic granular sludge
Bingyi Jiang a, Xiao Hu a, Ulf Söderlind b, Erik Hedenström 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: The syngas produced from biomass gasification is a great potential energy resource, which can well be utilized to
Syngas fermentation produce biomass-based substitute natural gas (BioSNG) via syngas biomethanation. CO biomethanation is one of
CO biomethanation the key issues in the biomethanation process and was studied experimentally in this work with respect to the
Syntrophic associations
effect of anaerobic granular sludge semi-disaggregation. The results show 1.07 times higher averaged CH4
Anaerobic granular sludge
Semi-disaggregation
production rate with the semi-disaggregated granular sludge than the whole granular sludge at 35 ◦ C, and 1.69
times higher at 55 ◦ C. The main mechanisms behind the enhanced CH4 production rate, especially under the
thermophilic condition, are the improvement of microbial interspecific syntrophic association caused by the
higher electron and substrate transfer rate, and more active cell growth and metabolism as reflected in higher
abundance of functional genes and enzymes and less useless extracellular polymeric substances. The CO bio­
methanation enhancement occurs in the conversion of the substrate to the intermediate products. The semi-
disaggregation of anaerobic granular sludge or similar way to strengthen interspecific association is an effec­
tive approach to improve the ability and tolerance of microbial cultures under the CO atmosphere. This tech­
nique can well be applied for the energy conversion from the CO-rich gas substrates into BioSNG via CO
biomethanation under the thermophilic condition, or for the production of intermediates as fuels/chemicals
under the mesophilic condition.

1. Introduction
A large scale of syngas is produced worldwide for the productions of
ammonia, refinery hydrogen, synthetic fuels and chemical such as
methanol, Fischer-Tropsch diesel as well as electricity [1]. Many in­
dustrial waste gases and the syngfrefas produced from biomass gasifi­
cation are usually small scale but dispersed in a great potential energy
resource, which can well be utilized via gas fermentation by a wide
range of microorganisms to produce biomass-based chemicals and fuels
[2]. Traditional anaerobic digestion (AD) is a popular method to treat
biomass organic wastes such as animal manure and food waste, but
limited for undegradable lignocellulose biomass. Alternatively, biomass
can be easily gasified into syngas which then can be used as the substrate
to AD process to produce biogas [3]. Thus, the limitation of huge po­
tential forest and agricultural biomass residues to be used as the AD
substrate can be avoided [4]. Syngas biomethanation for the production
of biomass-based substitute natural gas (BioSNG) is a novel and attrac­
tive alternative to the high temperature catalytic synthesis route [5].
On the other hand, 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 sour­
ces or energy carriers [6]. Power to methane (PtM) can be an effective
method for the energy storage, in which hydrogen is produced from
water electrolysis and combined with CO2/CO to produce methane
through methanation [4]. Methane is a versatile chemical feedstock and
energy carrier with easy transport and storage based on present strong
natural gas industry and network worldwide [3]. PtM can play an
important role for the electricity production flexibility from renewable
energy resources and also carbon capture, utilization and sequestration
(CCUS) [7]. The biogas from organic waste AD is usually upgraded via
water scrubbing and pressure swing adsorption (PSA) etc. to remove
CO2 in the biogas, which are expensive [8]. PtM can be an attractive
alternative technology for in-situ or ex-situ biogas upgrading process by
adding hydrogen from surplus renewable electricity or syngas from
biomass gasification [8].
Compared with the high temperature catalytic methanation,

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

https://doi.org/10.1016/j.enconman.2022.116551
Received 29 August 2022; Received in revised form 29 November 2022; Accepted 1 December 2022
0196-8904/© 2022 Elsevier Ltd. All rights reserved.
B. Jiang et al.
biomethanation of H2/CO2/CO has many advantages such as moderate
reaction conditions, the irreversible character of the biochemical re­
actions and less byproducts [9]. Particularly, it is insensitive to the
impurities and the ratio of C/H in the feedstock gas mixture through an
easy conversion of H2/CO2/CO to methane [9]. The sources of gas
substrate for biomethanation can be the CO/CO2-rich industrial off-gas
in small and medium scales [6,10]. 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.
Biomethanation of gas substrates from various hydrogen and carbon
sources has widespread applications as mentioned above. In the
biochemical process, the CO biomethanation is one of the key issues
since CO (or obtained from CO2 water–gas shift reaction) is a major
energy carrier, an excellent carbon source for the growth of anaerobic
microorganisms, but also toxic to methanogenic bacterial [11].
Due to its inhibitory effect on microorganisms, many studies have
addressed the tolerance of mixed anaerobic culture to CO at different
partial pressures. It was found that the methanogenic activity reached
the maximum at 0.2 atm of CO, but was inhibited completely at 1.0 atm
of CO [11]. Esquivel-Elizondo et al. [12] reported that complete inhi­
bition of methanogens occurred at PCO ≥ 51.4 kPa. Although the value of
PCO cannot be well defined due to different inocula and experiment
conditions, the inhibitory trends are consistent. However, few studies
have been reported about how to improve CO biomethanation capacity
of microorganisms. So far, only simple enrichment or acclimation of the
mixed microbial consortia by short-term or long-term exposure to CO
[12,13] has been carried out to improve the tolerance to CO and shorten
the lag phase of CO biomethanation. Such method is very primitive and
needs to be studied in detail. Therefore, it is necessary to find a novel
idea to guide more efficient CO biomethanation.
Many previous studies have focused on the identification of CO
biomethanation pathways which can be summarized in Fig. 1. It has
been proved that the indirect pathway dominates CO biomethanation,
while the direct pathway can be negligible [5]. To a great extent, CO is
first converted to acetate and subsequently to CH4 under the mesophilic
condition [2,14–16]. While under the thermophilic condition, H2/CO2
become the main intermediate products[13,15,17–19]. No matter how
the specific pathways can be drawn, the CO biomethanation routes are
Fig. 1. Major pathways of CO biomethanation. The blue color indicates the preferable pathways under the mesophilic condition. The red color indicates the
preferable pathways under the thermophilic condition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
2
Energy Conversion and Management 276 (2023) 116551
diverse and must involve the interspecific cooperation such as electron
transfer and substrate diffusion between a variety of microorganisms
[20]. This is similar to the traditional fermentation of organic substrates
whose biomethanation involves a complex metabolic network [21]. In
recent years, higher methanogenic performance has been achieved by
adding conductive materials to enhance the interspecific cooperation
during the traditional AD process [22,23]. This provides a theoretical
basis for enhancing the CO biomethanation capacity of microorganisms
by means of promoting microbial syntrophic relationship instead of
forcing microbes to tolerate CO by coarse exposure and acclimation. It
might provide novel feasibility of more efficient energy conversion from
CO to other value-added products.
In fact, anaerobic granular sludge, as a mixture of various microor­
ganisms, is the embodiment of better microbial syntrophic relationship
[24]. In the granule, compared with the free cells, clustering of microbes
is essential to minimize the diffusion distance of interspecies electron
carriers and to maximize the rate of substrate conversion [25]. Thus, one
of the easiest ways to affect the microbial interspecific relationship for
syntrophic cultures is to change the morphology of sludge granule. It
was reported that the complete disaggregation of sludge granule
weakens the CO biomethanation capacity [2]. Similar to the methano­
genesis of organic substrates, the disaggregation of sludge granule can
plays a positive or negative role [26,27]. But these research works
focused on the changes of reaction rates and microbial species rather
than the characterization of more detailed metabolism and interspecific
cooperation, which leads to the lack of sufficient evidence and in-depth
explanation for their results. Semi-disaggregation and complete disag­
gregation of granular sludge may cause totally different performances in
the CO biomethanation, which has not been paid attention to in litera­
ture. The discussions and conclusions with respect to the granular sludge
disaggregation are often simple and inaccurate.
In the present study, the sludge granules were appropriately broken
into small aggregates to change the microbial interspecific relationship,
but the clustering of microbes was retained. The effect of sludge granule
semi-disaggregation on CO biomethanation and the detail mechanism
behind were evaluated to determine the feasibility of changing the
interspecific relationship in order to enhance the performance of CO
biomethanation. In addition, acetate and H2/CO2 were used as the
B. Jiang et al. Energy Conversion and Management 276 (2023) 116551

substrates to simulate the intermediate products of CO methanogenesis Table 1

to determine how the metabolic pathway is changed after semi- Experimental conditions of the test runs. (W - the whole granular sludge, D - the
disaggregation of sludge granule. Finally, the degree of granular semi-disaggregated granular sludge, M - at the mesophilic temperature of 35 ◦ C,
sludge disaggregation and possible mechanism of CO biomethanation T - at the thermophilic temperature of 55 ◦ C. The subscripts represent the sub­
are discussed in the paper. strates used. The initial total pressure is 1.2 atm, and the rotation speed 80 rpm).
Test run Initial gas composition (v %) Liquid substrate
2. Material and methods
WMCO CO/N2 = 50/50 /
DMCO CO/N2 = 50/50 /
2.1. Growth medium and inoculum WTCO CO/N2 = 50/50 /
DTCO CO/N2 = 50/50 /
WMH2 H2/CO2/N2 = 50/12.5/37.5 /
The growth medium used in this study was a modified basal anaer­
DMH2 H2/CO2/N2 = 50/12.5/37.5 /
obic (BA) medium [28] composed by 4 stock solutions presented in WTH2 H2/CO2/N2 = 50/12.5/37.5 /
Table S1. The medium was prepared by adding 10 mL A (macroele­ DTH2 H2/CO2/N2 = 50/12.5/37.5 /
ment), 2 mL B (microelement), 1 mL C (pH buffer solution), 1 mL D WMAc N2 = 100 12 mM of acetate
(vitamin) and distilled water to 1L. DMAc N2 = 100 12 mM of acetate
WTAc N2 = 100 12 mM of acetate
Anaerobic granular sludge was collected from an internal circulation
DTAc N2 = 100 12 mM of acetate
bioreactor owned by Shuierli Environmental Protection Technology ltd.,
Jiangxi, China, which was originally operated under the mesophilic
condition (35–38 ◦ C) to treat industrial wastewater from juice plants. It The enrichment details are similar to the previous study [15].
has been proved to have better performance of CO biomethanation [15].
The total solids (TS) and volatile solids (VS) contents of the anaerobic
2.2. Experimental tests
granular sludges were 49.46 ± 0.64 g/L and 37.44 ± 0.69 g/L, respec­
tively. Modified from [26], the granular sludge was ground manually to
The experiment was carried out in a 1.9L bioreactor of pressure-
obtain the semi-disaggregated granular sludge. In an anaerobic envi­
resistant glass as shown in Fig. S1. A shock gas-proof manometer
ronment, whole granular sludges were put into the reactor, and an
(YNZ-60, Wenzhou, China) and two valves for gas/liquid sampling were
appropriate amount of BA medium was added to submerge the sludges
installed through the reactor cover. The reactor is completely isolated
fully. Then, a cylinder with flat bottom was used to grind the whole
through strict pressure and air tightness test. The details were exhibited
sludge granules until they were cracked or crushed into small aggre­
in the previous study [15].
gates. In the case that a part of the microorganisms and the extracellular
To study the effect of semi-disaggregation of anaerobic granular
polymeric substances (EPS) were separated from the granules in the
sludge on the CO biomethanation, 50 % CO plus 50 % N2 was used as the
grinding process, the BA medium can prevent their loss which might
substrate for conversion into CH4 by the whole granular sludge and
affect the experiment accuracy. As shown in Fig. 2, the granules after
semi-disaggregated granular sludge respectively under the mesophilic
grinding no longer have smooth surfaces and complete ellipsoid struc­
and thermophilic conditions. In order to identify the biomethanation
tures, but are crushed into small clusters, achieving the purpose of semi-
pathways that might be changed by the semi-disaggregation of granular
disaggregation of granules without completely dispersing microorgan­
sludge, acetate and H2/CO2 were used as the substrates to simulate the
isms. Before sludge was used as the inoculum, a batch process in the
main intermediate products for methanogenic tests. Detailed conditions
atmosphere of H2/CO2 (4:1) was applied for enrichment as well as
of each experimental test are shown in Table 1.
removing the original organic digestible matter contained in the sludges.
70 mL sludge and 230 mL fresh BA medium made the liquid volume

Fig. 2. Photographs of the inoculum: a) semi-disaggregated granular sludge and b) the whole granular sludge.

3
B. Jiang et al.
to be 300 mL, in which 0.12 g cysteine hydrochloride was added. The
headspace and the bioreactor were flushed with the gas to be used in the
test run at a rate of 500 mL/min for more than 10 min through the
valves. The bioreactors were incubated in the shaker-bed at 80 rpm
under the mesophilic and thermophilic conditions, respectively. During
each test run, 4 mL liquid was extracted every day to measure the pH and
analyze the composition of the liquid phase. Then, 4 mL fresh BA me­
dium was added and the pH was adjusted to 6.6–7.5 with 4 M NaOH and
HCl if needed. 2 mL gas was extracted for analysis of the gas phase
composition every day.
2.3. Measurements of chemical components in the gas and liquid phases
The composition of H2, CO, CH4 and CO2 in the headspace of the
bioreactor was measured 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 [15]. The conversion of gas volumes in liter to moles was
calculated according to ideal gas law at 1 bar and 293 K after measuring
the gas temperature, total pressure, the headspace volume and con­
centration of each component. The solubilities of H2, CO, CO2 and CH4
were calculated to correct the gas composition using data from Lide
[29].
Volatile fatty acids (VFA) including acetate, propionate, butyrate and
isobutyrate were analyzed by Agilent 8860 (Wilmington, Delaware,
USA) equipped with a flame ionization detector (FID) and DB-WAX
column (30 m × 0.53 mm × 1.0 μm). Nitrogen was used as the carrier
gas at a flow rate of 40 cm/s. The temperatures of inlet and detector
were set up to be 250 ◦ C. The GC oven was programmed to begin at
50 ◦ C, held for 1 min, then increased at a rate of 20 ◦ C/min to 250 ◦ C,
and finally held at 250 ◦ C for 6 mins.
The total solids (TS) and volatile solids (VS) contents of the anaerobic
granular sludge were measured according to standard methods [6]. The
pH was measured by a pH meter (pH-100, Shanghai, China).
2.4. Morphological analysis of the microbial communities
The microorganism morphology of cultured sludge was observed by
a scanning electron microscope (SEM). The sludge samples were fixed,
dehydrated, coated with gold–palladium [30] and finally observed in
SEM (SU-8010, Hitachi, ltd., Japan) in Bio-ultrastructure Analysis Lab.
of Analysis Center of Agrobiology and Environmental Sciences, Zhejiang
University (Hangzhou, China).
The spatial distribution of Archaea and Bacteria inside the sludge
granules was marked by fluorescence in situ hybridization (FISH).
Frozen section was taken in order to obtain the 10 μm central slices of
sludge granule. Then, the slices were taken to perform FISH analysis.
Oligonucleotide probes specific for Bacteria (EUB338 probe 5′ –FAM-
GCTGCCTCCCGTAGGAGT-3′ ) and Archaea (ARC915 probe 5′ –CY3-
GTGCTCCCCCGCCAATTCCT-3′ ) domains were used [25,31]. Samples
after FISH were photographyed by confocal laser scanning microscopy
(NIKON Eclipse Ti, Nikon ltd., Japan). The hybridized bacterial cells
were excited with the 488 nm line of an Ar laser (excitation) and
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 in the red channel of 590 nm (emission).
2.5. Electrochemical analysis of anaerobic digestion residues
To calculate the electron transfer during anaerobic process, cyclic
voltammetry (CV) measurements were conducted using a single-
chamber three-electrode electrochemical cell. The electrochemical cell
was made of two graphite electrodes (a working electrode and a counter
electrode, each with a 95 mm length and 5 mm diameter) and a refer­
ence electrode (Ag/AgCl) [32]. 20 mL digestion residue containing 5 mL
sludges and 15 mL medium was added into the electrochemical cell, and
4
Energy Conversion and Management 276 (2023) 116551
the cell was then purged with pure N2 for 10 min to establish an
anaerobic environment. CVs were measured using an electrochemical
analyzer (Bio-Logic, Claix, France). The scanning voltage of the working
electrode was ranged from − 1.0 ~ 1.0 V with scanning rates of 20 ~
100 mV/s.
According to the Laviron equation, the electron transfer rate constant
kapp in the system was calculated [32]. First, α was calculated according
to Eqs. (1) and (2) as follow:
( ) [ ]
RT αnF νc
Epc = Ec0 − ln (1)
αnF RTkapp
( ) [ ]
RT (1 − α)nF νa
Epa = Ea0 − ln (2)
(1 − α)nF RTkapp
where α is the electron transfer coefficient, Epc the reduction peak po­
tential, Epa the oxidation peak potential, ν the scanning rate, R 8.314 J
mol− 1 K− 1, T 298 K and F 96,483C mol− 1. The straight slopes of Ep-E0
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. (3)
as follows:
( )
RT nFΔE
lgkapp = αlg(1 − α) + (1 − α)lgα − lg − (1 − α)α (3)
nF ν 2.303RT
where ΔE indicates the potential difference between the reduction and
oxidation peaks.
2.6. Excitation mission matrix fluorescence analysis
The extracellular polymeric substances (EPS) after the experiments
were tested by a Fluorescence Spectrophotometer (F7000, Hitachi,
Japan). EPS were separated with heat extraction methods [33]. The
excitation 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 at 5 nm and the scanning speed was set up at 2400 nm/min.
2.7. Gene sequencing and functional analysis
Biomass samples were collected at the end of the experiments to
analyze the microbial community. Total genomic DNA was extracted
using the DNeasy PowerSoil DNA extraction kit (Qiagen, Hilden, Ger­
many). The 16S rRNA gene V4 ~ V5 hypervariable region was amplified
using the universal forward 515F (5′ -GTGCCAGCMGCCGCGG-3′ ) and
the reverse 907R (5′ -CCGTCAATTCMTTTRAGTTT-3′ ) primers for Bac­
teria, and using the universal forward 519F (5′ -CAGCCGCCGCCGTAA-
3′ ) and the reverse 915R (5′ -GTGCTCCCCCGCCAATTCCT-3′ ) primers
for Archaea [34]. The PCR products were verified using 1.5 % agarose
gel electrophoresis, and the DNA bands were excised and purified fol­
lowed by using NanoDrop 2000 (Theromo Fisher Scientific, Waltham,
USA) to determine the concentration and quality of DNA. High-
throughput sequencing of 16S rRNA was conducted using the Ion Gen­
eStudio S5 (Thermo Fisher Scientific, Waltham, USA) at Analysis Center
of Agrobiology and Environmental Sciences of Zhejiang University
(Hangzhou, China). QIIME package was used to handle the original
sequence. The detailed steps were based on the published methods [35].
Alpha diversity metrics were calculated using Chao1. The Beta diversity
was estimated using the phylum/genus-level abundance [36]. For
functional profiles, taxonomy annotation and enzymes of KEGG
Ontology (KO) were made relying on Kyoto Encyclopedia of Genes and
Genomes (KEGG) Database [37].
3. Results and discussion
3.1. CO biomethanation
In the CO biomethanation experimental test, 50 % CO plus the rest of
B. Jiang et al. Energy Conversion and Management 276 (2023) 116551

N2 was fed as the initial substrate, and the whole granular sludge and Table 2
semi-disaggregated granular sludge were used respectively under the The performance of CO biomethanation in the experimental tests.
mesophilic and thermophilic conditions to identify the influence of the Averaged Averaged End- Product yieldb
granular sludge semi-disaggregation on the AD process. The consump­ consumption production product
tion of CO, the productions of CH4 and CO2, as well as the intermediate rate rate
products of H2 and acetate are registered against the biomethanation tmaxa CO (mmol/g CH4 (mmol/ CH4 CH4 CO2
time as shown in Fig. 3. The digestion time to reach the maximum (d) VSS/d) g VSS/d) (mmol)
product, tmax, the amount of end–product and the product yield are also WMCO 12 1.21 0.29 8.65 90.89 68.75
DMCO 11 1.36 0.31 9.26 94.12 68.11
summarized in Table 2. In general, the CO biomethanation is faster and WTCO 7 2.12 0.52 10.00 102.00 72.07
the CH4 yield is higher under the thermophilic condition than that under DTCO 4 3.78 0.88 10.15 101.92 70.04
the mesophilic condition, in agreement with the conclusion made from a
Time to reach maximum amount of the product.
previous study [15]. It is also noticed in Fig. 3 that the lag phase for CO b
stoichiometric yield: 1/4 mol CH4 and 3/4 mol CO2 per mol CO.
biomethanation under the thermophilic condition is shorter, about 1 and
2 days in the cases of DTCO and WTCO, in comparison to about 4 days
granular sludge at 35 ◦ C, and 1.69 times higher at 55 ◦ C as calculated
under the mesophilic condition as seen in Fig. 3a, 3b and 3c. A certain
from Table 2, whereas the amount of CH4 end-product and the product
amount of the intermediate product H2 is produced under the thermo­
yield are almost unchanged. As a conclusion, granular sludge semi-
philic condition, which cannot be seen under the mesophilic condition
disaggregation changes almost nothing more than to speed up the CO
as shown in Fig. 3d, suggesting different CO biomethanation pathways
biomethanation significantly under the thermophilic condition.
[15,16].
Comparing the test runs of the semi-disaggregated granular sludge
with the test runs of the whole granular sludge, it can be seen that the CO 3.2. Microbial community
consumption rate is slightly increased under the mesophilic condition,
but significantly increased under the thermophilic condition as seen in The species richness is characterized by Chao1 index [13] as seen in
Fig. 3a. The same trends are true for the productions of CH4 and CO2 Table 3. After the enrichment under the CO atmosphere, species richness
shown in Fig. 3b and 3c in response to the CO consumption. The lag of both archaea and bacteria increases. Comparing the test runs of WMCO
phase for CH4 to start to be produced is slightly shorten under the with DMCO, and WTCO with DTCO respectively, it can be seen that the
mesophilic condition, but clearly shorten from 4 days to 1 day under the species richness of archaea in semi-disaggregated granular sludge is
thermophilic condition with the help of granular sludge semi- lower than that in the whole granular sludge, but the species richness of
disaggregation. The averaged CH4 production rate with the semi- bacteria does not exhibit any obvious change. This indicates that the
disaggregated granular sludge is 1.07 times higher than the whole disaggregation of granular sludge makes the microorganisms inside the
sludge to be more direct exposure to CO, thus part of them are

Fig. 3. Consumption and production of the gas in the bioreactor headspace, a) CO, b) CH4, c) CO2, and d) intermediate H2 and acetate in the liquid, in the CO
biomethanation with the whole and semi-disaggregated granular sludge at 35 ◦ C and 55 ◦ C, respectively.

5
B. Jiang et al. Energy Conversion and Management 276 (2023) 116551

Table 3
Parameters related to microbial community richness and diversity.
Sample Chao1 Sample Chao1
Archaea Bacteria
Initial 4726.418 Initial 3576.103
WMCO 8343.784 WMCO 7803.888
DMCO 6527.119 DMCO 7849.523
WTCO 7335.356 WTCO 6359.618
DTCO 6152.174 DTCO 7103.904
eliminated because of the toxic effect of CO. Compared with bacteria,
many methanogens are less able to tolerate pure CO under high pressure
[2] so that the effect on archaea is more significant. However, such
exposure to CO does not mean a negative phenomenon. It makes a faster
adaption to CO and shortens the lag phase of methanogenesis, which is
one of the reasons for the enhancement of CO biomethenation [17].
As seen in Fig. 4, after the exposure to CO, the dominant archaea
have been changed completely, while the relative abundance of bacteria
is similar to that in the initial sludge before inoculation at the phylum
level. This further confirms that methanogens are less tolerant to CO
than bacteria. The bacterial community is relatively conventional,
which is dominated by Bacteroidetes, and Firmicutes and Proteobacteria.
These bacteria are common in anaerobic mixed cultures and usually
responsible for hydrolysis and acidogenesis [38,39] in traditional
anaerobic digestion, as well as required in CO biomethanation. As for
Archaea, the methanogens only occupy about 10 % of the relative
abundance in 4 experimental tests, which are mainly consist of Meth­
anosaeta, Methanolinea, Methanothermobacter and Methanobacterium. It
suggests that a large number of methanogens are inhibited by CO. The
dominant archaeal class of these tests is Thaumarchaeota, whose relative
abundance ranges from 25.5 % to 45.1 %. They are ammonia-oxidizing
archaea (AOA) and play a role in nitrification, during which ammonium
(NH+ 4 ) is oxidized to nitrite (NO2) or nitrate (NO3) [40]. The absence of
– –
methanogens and the presence of NH+ 4 in the medium might lead the
dominant growth of AOA. It should be noted that the CO2 yield is just
around 70 % as given in Table 2, indicating that part of carbon is utilized
in the form of CO2 or other carbon-containing products by microor­
ganisms for their cell growth [41] and the production of extracellular
polymeric substances [42]. But then the system lacks a supply of elec­
trons to match the process. The reasonable explanation is that anaerobic
ammonium oxidation (Anammox) carried out by AOA releases
electrons. Some studies also have reported the potential autotrophs
function of metabolizing bicarbonate of AOA [43], suggesting that CO2
reduction is possible. However, there is lack of the information about
those Archaea, making it difficult to determine how they participate in
the methanogenesis process and whether there is a possible link between
ammonium removal and CO2 removal. Because high ammonium con­
centration will inhibit methane production [44] and too much CO2 in
the product gas will also reduce the quality of biogas.
In conclusion, the relative abundance of microbial community shows
that whether the sludge is broken or not will not fundamentally affect
the shift of microorganisms under CO atmosphere at a high concentra­
tion. Although the semi-disaggregation accelerates the microorganism
adaptation to CO, it is not sufficient to explain the enhanced CO
biomethanation.
3.3. Microbial interspecific syntrophic associations
The 16S rRNA gene analysis above and previous study [42] show
that the sludge structure has no significant effect on microbial com­
munity. But the destruction of granular structure might change the
syntrophic associations between the microflora and give rise to an
impact on the methanogenesis process. The formation of granular sludge
is partly forced by the signaling and collective action in microbial
community [24] and contributes to the mutualistic interactions among
different members of a mixed microbial consortium in the sludge [5]. In
such syntrophic cultures, clustering of Bacteria and Archaea maximizes
the rate of substrate conversion as well as the interspecies electron
transfer [25]. The transfer of substrates and electrons between various
functional microorganisms plays an essential role during the process of
CO biomethanation [20]. Thus, the semi-disaggregation of granular
sludge possibly enhances the interspecific symbiosis and speeds up the
CO biomethanation process.
In order to evaluate the interspecific symbiosis in detail, SEM of
sludge and CV experiment of the residues after CO biomethanation were
made. It can be seen in Fig. 5 that the granular sludge is broken up into
small aggregates, while the cluster link among microorganisms, extra­
cellular polymeric substances and other inert substances is reserved.
Although the surface of the whole granular sludge is more compact and
smoother, the spatial structure required for the interspecific link in the
semi-disaggregated granular sludge is not completely destroyed.
To better understand the effects of disaggregation, the wide range of

Fig. 4. Relative abundance of a) archaea and b) bacteria communities using V4-V5 region primer set.

6
B. Jiang et al.
Fig. 5. SEM images of the whole sludge granule (a and b) and the semi-disaggregated granular granule (c and d).
electron transfer processes that occur during the CO biomethanation
should be analyzed. Although it is difficult to determine the association
between the specific redox reaction and the redox peaks [33], CV
experiment has been used in many previous studies to quantify extra­
cellular electron transfer capacity of biological experiments [32], so
does in this study. As shown in Fig. S2a, c, e and g, the peak currents
increase linearly with increasing scan rates for Peak 1 and 2. Fig. S2b, d,
f and h show the dependence of peak potential on the scan rate by
plotting Epeak versus ln ν. Based on Laviron theory. The values of the
electron transfer rate kapp in the 4 test runs are calculated from the two
redox peaks.
The kapp values of the whole granular sludge and the semi-
disaggregated granular sludge are 0.00276 s− 1 and 0.00738 s− 1
respectively under the mesophilic condition, and 0.00775 s− 1 and
0.01302 s− 1 respectively under the thermophilic condition. The results
show that during the process of CO biomethanation, the electron
transfer capacity between microorganisms in the sludge under the
thermophilic is stronger than that under the mesophilic condition. The
electron transfer capacity of the semi-disaggregated granular sludge is
stronger than that of the whole granular sludge. This is consistent with
the CO methanogenesis performance of different sludge structures under
the mesophilic and thermophilic conditions, indicating that the electron
transfer capacity changed by the granular sludge semi-disaggregation is
an important reason to explain the enhanced CO biomethanation reac­
tion rate. It should be noted that the electron transfer rate of semi-
disaggregated granular sludge is 2.67 times higher than that of the
whole granular sludge under the mesophilic condition, while 1.68 times
under the thermophilic condition. They are not the same as the
enhanced multiples of the methanogenic rates since biological reactions
are often caused by multiple factors and electron transfer capacity
cannot completely represent the actual CH4 production rate. In the
whole granular sludge, due to the limitation of mass transport in the
inner layer of the granule, only part of the granule will contribute to the
conversion process [27]. Once the whole granule is crushed to small
aggregates, the diffusion resistance will be weakened and microbes will
be released. At the same time, those aggregates are still able to perform
the function for interspecific collaboration, offering more opportunities
for microbial consortia to participate in the syntrophic CO fermentation.
Such enhanced interspecific syntrophic associations are characterized
by the higher electron transfer capacity, which is one of the important
reasons to explain the improved CO utilization capacity of semi-
disaggregation. It is consistent with many studies that the strength­
ening of interspecific syntrophic association will significantly promote
biological reaction [25,32,45].
Many previous studies [46,47] have shown that the internal mass
transfer resistance of granular sludge plays a more important role than
the external mass transfer resistance with respect to the substrate mass
transfer, which causes a lower substrate concentration in the core of the
sludge granules. The substrate concentration decreases as the granule
diameter is increased. Thus, the semi-disaggregation brings a dual effect.
On one hand it enhances the interspecific syntrophic associations,
making more microbes join in the methanogenesis. On the other hand, it
leads a higher mass transfer rate [26].
7
Energy Conversion and Management 276 (2023) 116551
3.4. Cell growth and metabolism
To further explain the semi-disaggregation effect on metabolic pro­
cess, EEM was used to evaluate the characteristic of extracellular poly­
meric substances (EPS). As shown in Fig. 6, four main peaks are
recorded. According to the location of emission/emission (Ex/Em)
fluorescence peaks of various dissolved organic matters [48], Peak A and
B are observed at an Ex/Em = 275–280/305–340 nm and assigned to
simple aromatic protein-like substances; Peak C and D are observed at an
Ex/Em = 225–230/305–340 nm and assigned to soluble microbial
byproduct-like materials such as tryptophan and tyrosine protein-like
substances. Organic matters in other regions such as humic acid-like
and heme-like substances are almost absent. Comparing the test runs
of WMCO with DMCO, and WTCO with DTCO, respectively, it can be seen
that the whole granular sludge has higher content of protein-like EPS
than the semi-disaggregated granular sludge. This agrees with the
observation of more extracellular metabolites on the surface of the
whole granular sludge in Fig. 5. Although protein-like substances usu­
ally participate in electron transfer as electron shuttles [45]. But in this
experiment, the electron transfer rate of the whole granular sludge is
lower, which is contrary to the higher EPS content. Therefore, these
protein-like EPS do not play the major role of electron shuttles [19].
Instead, they contribute more to the structural stability of sludge granule
and the defense against the external selection pressure [49]. This can
also be demonstrated by the different EPS content at 35 ◦ C and 55 ◦ C in
Fig. 6. The thermophilic condition is not suitable for many microor­
ganisms, making formation of more proteins such as heat-resistant en­
dospores [50], so that WTCO and DTCO have more EPS. These protein-
like EPS did not help accelerate interspecific association, but resulted
in a compact and stable structure in Fig. 5 which might increase the
limitation of diffusion of substrate and electrons, resulting in the
decrease of CO biomethanation rate.
Previous studies has proved that the disaggregated sludge has a
higher cell specific growth rate and a more active biomass [26], which is
partly related to the less production of EPS [42], so that more power will
be used for more efficient CO metabolism. Therefore, a further meta­
genomic analysis was conducted to reveal the effect of semi-
disaggregation on functional profiling using KEGG database (Fig. 7).
Metabolism, genetic information processing, environmental information
processing and cellular processes are identified as the four primary
pathways on KEGG Level 1. The dominant KEGG pathway level is
metabolism, including amino acid metabolism, carbohydrate meta­
bolism and energy metabolism, which normally exist in AD system with
large quantities [37]. In addition, membrane transport, replication and
repair and translation are dominant pathways of genetic information
processing and environmental information processing, respectively,
which shows that translation regulation mechanism plays an important
role in the normal operation of physiological function [37]. As shown in
Fig. 7, most of the functional gene clusters of semi-disaggregated gran­
ular sludge have higher abundance than those of the whole granular
sludge under both the mesophilic and thermophilic conditions. This
indicates that semi-disaggregation effectively stimulates the cell growth,
collaboration and metabolic functions related to methane production.
Thus, it can be concluded that enhanced CO biomethenation rate by
B. Jiang et al.
Fig. 6. 3-D EEM fluorescence spectra for EPS of sludge after CO biomethanation in the test runs of a) WMCO, b) DMCO, c) WTCO and d) DTCO.
semi-disaggregated granular sludge is a result of increased substrate and
electron transport rates (analyzed in 3.3) and improvement in cell
growth, collaboration and metabolism.
3.5. Possible enhanced pathways
Regarding the CO biomethanation pathway, it has be concluded that
acetate is the main intermediate product under the mesophilic condition
and H2 under the thermophilic condition, while carboxydotrophic
methanogenic activity is negligible, although the interconversion of
intermediate products as well as by-products may occur [2,14,17].
Therefore, in this experimental work, acetate and H2/CO2 were used to
simulate the main intermediate products for methanogenic tests to
determine which pathway or process is promoted by the granular sludge
semi-disaggregation.
As seen in Fig. 8a, 8b and 8c, the curves of acetate consumption and
CH4 and CO2 productions in the cases of WMAc and DMAc are almost
identical, suggesting that crushing granular sludge does not affect the
route of acetoclastic methanogenesis under the mesophilic condition.
Under the thermophilic condition, the rate of acetate conversion to CH4
is higher when the granular sludge is crushed. However, this pathway
can be neglected as acetate is not the main intermediate product at
55 ◦ C. As seen in Fig. 8d, 8e and 8f, the trend of H2/CO2 consumption
and CH4 production during H2/CO2 biomethanation is not affected by
8
Energy Conversion and Management 276 (2023) 116551
whether the sludge is semi-disaggregated or not. In conclusion, the
granular sludge semi-disaggregation does not affect the pathways of
acetoclastic methanogenesis and hydrogenotrophic methanogenesis. It
can be inferred that the enhancement of CO biomethanation process
should be attributed to the impact on the processes before the digestion
of intermediate products, which is the pathway of carboxydotrophic
acetogenesis under the mesophilic condition and water–gas shift reac­
tion under the thermophilic condition, or the transfer process of sub­
strates and electrons between donors and receivers after the formation
of intermediate products. Most hydrogenogens and acetogens belong to
Bacteria, dominating the production of H2/CO2 and acetate from CO
[20]. On one hand, because of the higher tolerance of Bacteria to CO
compared with methanogens, the higher activity of bacteria provides a
higher potential for enhancement of the former methanogenesis process.
On the other hand, because the Archaea species involved in the latter
process are simpler, while the Bacteria species involved in the former
process are richer, the closer collaborations among microorganism make
the strengthening effect of the former process more obvious.
In order to further explore the effects on gene functions and meta­
bolic pathways, the key functional enzymes associated with the meth­
anogenic pathways (based on the KEGG database) are analyzed in Fig. 9
[20,21,37]. It is noted in Fig. 9b that the semi-disaggregation of granular
sludge has different impacts on the abundance of different enzymes.
After semi-disaggregation, most enzymes including CODH/ACS,
B. Jiang et al. Energy Conversion and Management 276 (2023) 116551

Fig. 7. Metabolic pathways abundance on KEGG categories (level 2).

Fig. 8. Consumption of a) acetate in the liquid phase and production of b) CO2 and c) CH4 in the bioreactor headspace during anaerobic digestion of acetate as the
substrate, and consumption of d) H2 and e) CO2, and production of f) CH4 in the bioreactor headspace during the biomethanation of H2/CO2/N2 = 50 %/12.5 %/37.5
% as the substrate.

formylmethanofuran dehydrogenase (EC: 1.2.99.5), methenyltetrahy­
dromethanopterin cyclohydrolase (EC: 3.5.4.27), 5,10-methylenetetra-
hydromethanopterin reductase (EC: 1.5.98.2) and tetrahy­
dromethanopterin S-methyltransferase (EC: 2.1.1.86) have higher
abundance, while the abundance of enzymes including coenzyme F420
hydrogenase (EC: 1.12.98.1) and phosphate acetyltransferase (EC:
2.3.1.8) decreases.
According to the methane metabolism pathways based on KEGG, the
enzymes are divided into four modules in Fig. 9a: CO oxidation [20,51],
acetate decarboxylation, CO2 reduction and methanogenesis pathways
[21,37]. The semi-disaggregation of granular sludge reduces the abso­
lute abundance of enzymes in acetate decarboxylation and CO2 reduc­
tion pathways, but increases in the common methanogenesis pathway,
which results in an unnoticeable change in enzyme abundance in the
complete process from intermediates (H2/CO2 or acetate) to methane.
However, the abundance of CODH/ACS, the key enzyme for CO con­
version to intermediate products, increases significantly by 67.46 %
under the mesophilic condition and 185.14 % under the thermophilic

9
B. Jiang et al.
Fig. 9. a) The main methanogenic pathways and b) Absolute abundance of functional enzyme based on KEGG, and c) Absolute abundance of modules involved in
methanogenesis.
condition. This is consistent with the conclusions analyzed from Fig. 8
that the semi-disaggregation of granular sludge significantly enhances
the process of CO to intermediate products instead of the latter process
from the intermediate products to methane.
However, the improvement of functional genes and enzymes are by
no means the only reason for the enhanced CO biomethanation capacity.
Although the total abundance of enzymes in the acetate decarboxylation
and methanogenesis pathways increases slightly under the thermophilic
10
Energy Conversion and Management 276 (2023) 116551
condition (Fig. 9c), it does not seem to be enough to make the CH4
production rate increase significantly as shown in Fig. 8. Thermophilic
condition is not suitable for the direct digestion of acetate, which is
usually converted into CH4 through acetate-oxidizing reaction [19]. This
process needs syntrophic associations of a variety of bacteria [5]. Similar
to the CO biomethanation, the digestion of acetate under the thermo­
philic condition is also enhanced by crushed sludge. From this point of
view, the pathway or process enhanced by semi-disaggregation usually
B. Jiang et al. Energy Conversion and Management 276 (2023) 116551

Fig. 9. (continued).

involves closer interspecific association. Thus, the enhancement of the
diffusion process of the substrate and the electron after the formation of
the intermediate products cannot be negligible.
The above analysis and discussions once again show the effect of
granular sludge semi-disaggregation caused by a combination of phys­
ical factors such as substrate and the electron transfer and biological
factors such as cell metabolism. In future work, more attention could be
paid to the quantitative analysis of different factors and model
development.
3.6. The degree of granular sludge disaggregation
The disaggregated sludge has been found to be detrimental to the
biological process in many studies [2,26,40]. After a long-time exposure
to CO, the disaggregated sludge showed a negative effect on its meth­
anogenic activity and was less able to tolerate CO at higher CO partial
pressures [2], which is contrary to the conclusion of this study. The
previous theory of granule structure describes methanogens (Archaea)
concentrated in the inner layer and other acetogens and H2 consuming/
producing organisms (Bacteria) forming the exterior layer [52]. Navarro
et al. [2] believe that such structure of granular sludge protects metha­
nogens against the toxicity of CO and provides sludge with a better
conversion of CO into CH4. In order to explain these two contradictory
conclusions and determine the spatial distribution of microorganism
inside the sludge granule, FISH experiment was carried out. The fluo­
rescence micrographs were taken after hybridization with the specific
probe EUB338 for Bacteria and the probe ARC915 for Archaea [25].
As shown in Fig. 10, Archaea (red) and Bacteria (green) are evenly
distributed inside the sludge granule without obvious stratification after
acclimation by 50 % CO gas mixture, as well as in semi-disaggregated
granular sludge (Fig S3). This indicates that the physical barrier of
sludge structure is inadequate for methanogen protection under inten­
sive acclimation by CO, and Bacteria and Archaea in the whole and semi-
disaggregated granular sludge are indiscriminately acclimated by CO.
Eventually, the stratified structure of the sludge in normal anaerobic
digestion [19] disappears, regardless of whether the sludge granule is

Fig. 10. FISH images of the sections of the whole sludge granule after enrichment or acclimation by 50% CO gas mixture: a) distribution of archaea (red) and
bacteria (green) in sludge granule, b) distribution of archaea and c) bacteria. (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)

11
B. Jiang et al.
intact, semi-disaggregated, or totally disaggregated. This means that the
conclusion that the granule structure protects the internal methanogens,
making the CO biomethanation capacity of the whole sludge granule
stronger than that of the crushed sludge is incorrect. This is consistent
with the results of 16S rRNA analysis. In fact, temperature should have
been a significant acclimation factor for microorganisms [53,54], but no
obvious difference of relative abundance under different temperatures is
found in Fig. 4. It can be concluded that the toxicity of CO is the key
factor to influence the microbial community composition and distribu­
tion, which overrides the influence of the other factors.
The contradictory conclusions on CO biomethanation with respect to
the granular sludge disaggregation might be associated to the disag­
gregation degree of sludge granule and the reasons should be explained
in terms of the change of interspecific relationship. In this study, the
granule is crushed to small aggregates, referred to as semi-
disaggregation. The granular sludge semi-aggregation can increase the
mass transfer between the substrate and microorganisms [46,47], and
shorten the acclimation time for the microorganisms to be adapted to
the CO atmosphere [11]. So that, the biomethanation rate can be
increased.
Once the granule is totally disaggregated, the microorganisms in the
sludge are completely separated and the interspecific association will be
thoroughly destroyed, resulting in the reduction of the tolerance level of
CO. This phenomenon can also be verified in other studies. For example,
semi-disaggregated granular sludge leads an increase in the degradation
activity of acetate and butyrate [27]. The fully disaggregated sludge
reduces the amount of methane converted from the intermediate H2
[26], which might be related to disruption of substrate and electron
diffusion. In addition, sludge structure has different effects on different
substrates. Sludge, even at the same degree of disaggregation, is good to
degrade acetate and butyrate, but bad to degrade propionate [27]. Thus,
it is important to maintain the syntrophic associations in the process of
methanogenesis. In the biomethanation of syngas consisting mainly of
CO, sludge structure and granule size will largely determine the bio­
methanation rate. In industrial applications, properly selection of sludge
granule size should be carried out according to the specific substrates
and target products to achieve the best performance. The granular
sludge disaggregation has the potential to enhance biological reaction,
but the adaptability and stability of microorganisms to the environment
can be deteriorated [40]. Therefore, the enhancement of CO and acetate
biomethanation by semi-disaggregated granular sludge under thermo­
philic condition could be more significant than that under the meso­
philic condition. The conclusion of the enhanced process indicates that
the biomethanation enhancement mainly occurs before the digestion of
intermediates.
Although sludge crushing is a simple physical means, the mechanism
to influence microbial metabolism can guide the practical application in
order to speed up the biomethanation process. As a recommendation of
this work, to enhance the ability and tolerance of microbial cultures
under the CO atmosphere, the granular sludge semi-disaggregation
approach can be employed under the thermophilic condition or for the
production of intermediates. In essence, with the guidance of strength­
ening the interspecific syntrophic association between microorganisms,
more ways might be found to enhance the CO biomethanation in future
work.
4. Conclusion
The anaerobic granular sludge semi-disaggregation can enhance the
CO biomethanation by slightly increasing the reaction rate under the
mesophilic condition, but significantly increasing the reaction rate
under the thermophilic condition. The main reasons are the improve­
ment of microbial interspecific syntrophic association and more active
cell growth and metabolism. The possible enhancement occurs in the
conversion of the substrate to the intermediate products, i.e. the
pathway of carboxydotrophic acetogenesis under the mesophilic
12
Energy Conversion and Management 276 (2023) 116551
condition and water–gas shift reaction under the thermophilic condi­
tion, or the transfer process of substrates and electrons between donors
and receivers in the formation of intermediate products. The mecha­
nisms about strengthening interspecific association can be applied for
the energy conversion of the CO-rich gas substrates into BioSNG or other
liquid fuels and chemicals via biochemical process.
CRediT authorship contribution statement
Bingyi Jiang: Conceptualization, Methodology, Investigation,
Visualization, Writing – original draft. Xiao Hu: Investigation. Ulf
Söderlind: Methodology. Erik Hedenström: Writing - review & edit­
ing. Wennan Zhang: Funding acquisition, Project administration,
Writing - review & editing. Chunjiang Yu: Conceptualization, Re­
sources, Supervision, Funding acquisition, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This work is supported by EU Regional Development Program
[ERUF20353185] and “Bao Yu Gang” Foundation for Foreign Guest
Professor Program at Zhejiang University.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.enconman.2022.116551.
References
[1] Figueras J, Benbelkacem H, Dumas C, Buffiere P. Biomethanation of syngas by
enriched mixed anaerobic consortium in pressurized agitated column. Bioresour
Technol 2021:338. https://doi.org/10.1016/j.biortech.2021.125548.
[2] Navarro SS, Cimpoia R, Bruant G, Guiot SR. Biomethanation of syngas using
anaerobic sludge: Shift in the catabolic routes with the CO partial pressure
increase. Front Microbiol 2016:7. https://doi.org/10.3389/fmicb.2016.01188.
[3] Andreides D, Bautista Quispe JI, Bartackova J, Pokorna D, Zabranska J. A novel
two-stage process for biological conversion of syngas to biomethane. Bioresour
Technol 2021;327:124811. https://doi.org/10.1016/j.biortech.2021.124811.
[4] Achinas S, Achinas V, Euverink GJW. A technological overview of biogas
production from biowaste. Engineering 2017;3:299–307. https://doi.org/10.1016/
J.ENG.2017.03.002.
[5] Grimalt-Alemany A, Skiadas IV, Gavala HN. Syngas biomethanation: state-of-the-
art review and perspectives. Biofuels, Bioprod Biorefining 2018;12:139–58.
https://doi.org/10.1002/bbb.1826.
[6] Kougias PG, Tsapekos P, Treu L, Kostoula M, Campanaro S, Lyberatos G, et al.
Biological CO2 fixation in up-flow reactors via exogenous H2 addition. J Biotechnol
2020;319:1–7.
[7] Fang Z, Zhou J, Zhou X, Koffas MAG. Abiotic-biotic hybrid for CO2
biomethanation: From electrochemical to photochemical process. Sci Total Environ
2021;791:148288. https://doi.org/10.1016/j.scitotenv.2021.148288.
[8] Xia A, Cheng J, Murphy JD. Innovation in biological production and upgrading of
methane and hydrogen for use as gaseous transport biofuel. Biotechnol Adv 2016;
34:451–72. https://doi.org/10.1016/j.biotechadv.2015.12.009.
[9] Rönsch S, Schneider J, Matthischke S, Schlüter M, Götz M, Lefebvre J, et al. Review
on methanation - From fundamentals to current projects. Fuel 2016;166:276–96.
[10] Asimakopoulos K, Grimalt-Alemany A, Lundholm-Høffner C, Gavala HN,
Skiadas IV. Carbon sequestration through syngas biomethanation coupled with H2
supply for a clean production of natural gas grade biomethane. Waste Biomass
Valoriz 2021;12:6005–19. https://doi.org/10.1007/s12649-021-01393-2.
[11] Li C, Zhu X, Angelidaki I. Syngas biomethanation: effect of biomass-gas ratio,
syngas composition and pH buffer. Bioresour Technol 2021;342:125997. https://
doi.org/10.1016/j.biortech.2021.125997.
B. Jiang et al.
[12] Esquivel-Elizondo S, Delgado AG, Krajmalnik-Brown R. Evolution of microbial
communities growing with carbon monoxide, hydrogen, and carbon dioxide. FEMS
Microbiol Ecol 2017;93:1–12. https://doi.org/10.1093/femsec/fix076.
[13] Luo G, Wang W, Angelidaki I. Anaerobic digestion for simultaneous sewage sludge
treatment and CO biomethanation: Process performance and microbial ecology.
Environ Sci Technol 2013;47:10685–93. https://doi.org/10.1021/es401018d.
[14] Navarro SS, Cimpoia R, Bruant G, Guiot SR. Specific inhibitors for identifying
pathways for methane production from carbon monoxide by a nonadapted
anaerobic mixed culture. Can J Microbiol 2014;60:407–15. https://doi.org/
10.1139/cjm-2013-0843.
[15] Zhang Z, Ding C, Wang L, Jiang B, Söderlind U, Zhang W, et al. CO biomethanation
with different anaerobic granular sludges. Waste Biomass Valoriz 2021;12(7):
3913–25.
[16] Luo G, Jing Y, Lin Y, Zhang S, An D. A novel concept for syngas biomethanation by
two-stage process: Focusing on the selective conversion of syngas to acetate. Sci
Total Environ 2018;645:1194–200. https://doi.org/10.1016/j.
scitotenv.2018.07.263.
[17] Bu F, Dong N, Kumar Khanal S, Xie L, Zhou Q. Effects of CO on hydrogenotrophic
methanogenesis under thermophilic and extreme-thermophilic conditions:
Microbial community and biomethanation pathways. Bioresour Technol 2018;266:
364–73. https://doi.org/10.1016/j.biortech.2018.03.092.
[18] Grimalt-Alemany A, Łężyk M, Kennes-Veiga DM, Skiadas IV, Gavala HN.
Enrichment of mesophilic and thermophilic mixed microbial consortia for syngas
biomethanation: The role of kinetic and thermodynamic competition. Waste
Biomass Valoriz 2020;11:465–81. https://doi.org/10.1007/s12649-019-00595-z.
[19] Jiang B, Hu X, Söderlind U, Göransson K, Zhang W, Yu C. Identification of the
biomethanation pathways during biological CO2 fixation with exogenous H2
addition. Fuel Process Technol 2022:238. https://doi.org/10.1016/j.
fuproc.2022.107478.
[20] Oelgeschläger E, Rother M. Carbon monoxide-dependent energy metabolism in
anaerobic bacteria and archaea. Arch Microbiol 2008;190:257–69. https://doi.
org/10.1007/s00203-008-0382-6.
[21] Yang Z, Sun H, Zhou L, Arhin SG, Papadakis VG, Goula MA, et al. Bioaugmentation
with well-constructed consortia can effectively alleviate ammonia inhibition of
practical manure anaerobic digestion. Water Res 2022;215:118244.
[22] Zhang Z, Guo L, Wang Yi, Zhao Y, She Z, Gao M, et al. Application of iron oxide
(Fe3O4) nanoparticles during the two-stage anaerobic digestion with waste sludge:
Impact on the biogas production and the substrate metabolism. Renew Energy
2020;146:2724–35.
[23] Cheng J, Li H, Ding L, Zhou J, Song W, Li Y-Y, et al. Improving hydrogen and
methane co-generation in cascading dark fermentation and anaerobic digestion:
The effect of magnetite nanoparticles on microbial electron transfer and
syntrophism. Chem Eng J 2020;397:125394.
[24] Liu Y, Lou XH, Yang SF, Tay JH. Mechanisms and models for anaerobic granulation
in upflow anaerobic sludge blanket reactor. Water Res 2003;37:661–73. https://
doi.org/10.1016/S0043-1354(02)00351-2.
[25] Cruz Viggi C, Rossetti S, Fazi S, Paiano P, Majone M, Aulenta F. Magnetite particles
triggering a faster and more robust syntrophic pathway of methanogenic
propionate degradation. Environ Sci Technol 2014;48:7536–43. https://doi.org/
10.1021/es5016789.
[26] Carrillo-Reyes J, Celis LB, Alatriste-Mondragón F, Razo-Flores E. Different start-up
strategies to enhance biohydrogen production from cheese whey in UASB reactors.
Int J Hydrogen Energy 2012;37:5591–601. https://doi.org/10.1016/j.
ijhydene.2012.01.004.
[27] van Lier JB, Martin JLS, Lettinga G. Effect of temperature on the anaerobic
thermophilic conversion of volatile fatty acids by dispersed and granular slugde.
Water Res 1996;30:199–207.
[28] Asimakopoulos K, Gavala HN, Skiadas IV. Biomethanation of syngas by enriched
mixed anaerobic consortia in trickle bed reactors. Waste Biomass Valoriz 2020;11:
495–512. https://doi.org/10.1007/s12649-019-00649-2.
[29] P D. CRC Handbook of Chemistry and Physics. J Mol Struct 1992;268:320. https://
doi.org/10.1016/0022-2860(92)85083-s.
[30] Cheng J, Sun J, Huang Y, Feng J, Zhou J, Cen K. Dynamic microstructures and
fractal characterization of cell wall disruption for microwave irradiation-assisted
lipid extraction from wet microalgae. Bioresour Technol 2013;150:67–72. https://
doi.org/10.1016/j.biortech.2013.09.126.
[31] Crocetti G, Murto M, Björnsson L. An update and optimisation of oligonucleotide
probes targeting methanogenic Archaea for use in fluorescence in situ
hybridisation (FISH). J Microbiol Methods 2006;65:194–201. https://doi.org/
10.1016/j.mimet.2005.07.007.
[32] Lee JY, Lee SH, Park HD. Enrichment of specific electro-active microorganisms and
enhancement of methane production by adding granular activated carbon in
anaerobic reactors. Bioresour Technol 2016;205:205–12. https://doi.org/10.1016/
j.biortech.2016.01.054.
13
Energy Conversion and Management 276 (2023) 116551
[33] Yue L, Cheng J, Zhang H, Yuan L, Hua J, Dong H, et al. Inhibition of N-
Vanillylnonanamide in anaerobic digestion of lipids in food waste: Microorganisms
damage and blocked electron transfer. J Hazard Mater 2020;399:123098.
[34] Cui E, Wu Y, Zuo Y, Chen H. Effect of different biochars on antibiotic resistance
genes and bacterial community during chicken manure composting. Bioresour
Technol 2016;203:11–7. https://doi.org/10.1016/j.biortech.2015.12.030.
[35] Huang L, Wang Q, Jiang L, Zhou P, Quan X, Logan BE. Adaptively evolving
bacterial communities for complete and selective reduction of Cr(VI), Cu(II), and
Cd(II) in biocathode bioelectrochemical systems. Environ Sci Technol 2015;49:
9914–24. https://doi.org/10.1021/acs.est.5b00191.
[36] Lozupone C, Knight R. UniFrac: A new phylogenetic method for comparing
microbial communities. Appl Environ Microbiol 2005;71:8228–35. https://doi.
org/10.1128/AEM.71.12.8228-8235.2005.
[37] Wang X, Wang P, Meng X, Ren L. Performance and metagenomics analysis of
anaerobic digestion of food waste with adding biochar supported nano zero-valent
iron under mesophilic and thermophilic condition. Sci Total Environ 2022;820:
153244. https://doi.org/10.1016/j.scitotenv.2022.153244.
[38] Jaenicke S, Ander C, Bekel T, Bisdorf R, Dröge M, Gartemann K-H, et al.
Comparative and joint analysis of two metagenomic datasets from a biogas
fermenter obtained by 454-pyrosequencing. PLoS One 2011;6(1):e14519.
[39] Lu B, Jiang C, Chen Z, Li A, Wang W, Zhang S, et al. Fate of polylactic acid
microplastics during anaerobic digestion of kitchen waste: Insights on property
changes, released dissolved organic matters, and biofilm formation. Sci Total
Environ 2022;834:155108.
[40] Akizuki S, Natori N, Cuevas-Rodríguez G, Toda T. Application of nitrifying granular
sludge for stable ammonium oxidation under intensive light. Biochem Eng J 2020;
160:107631. https://doi.org/10.1016/j.bej.2020.107631.
[41] Hu X, Jiang B, Yu C, Söderlind U, Göransson K, Zhang W. Product gas
biomethanation with inoculum enrichment and grinding. Biomass Convers
Biorefinery 2022. https://doi.org/10.1007/s13399-022-03490-1.
[42] Vital-Jacome M, Buitrón G, Moreno-Andrade I, Garcia-Rea V, Thalasso F.
Microrespirometric determination of the effectiveness factor and biodegradation
kinetics of aerobic granules degrading 4-chlorophenol as the sole carbon source.
J Hazard Mater 2016;313:112–21. https://doi.org/10.1016/j.
jhazmat.2016.02.077.
[43] Jia Z, Conrad R. Bacteria rather than Archaea dominate microbial ammonia
oxidation in an agricultural soil. Environ Microbiol 2009;11:1658–71. https://doi.
org/10.1111/j.1462-2920.2009.01891.x.
[44] Gangagni Rao A, Sasi Kanth Reddy T, Surya Prakash S, Vanajakshi J, Joseph J,
Jetty A, et al. Biomethanation of poultry litter leachate in UASB reactor coupled
with ammonia stripper for enhancement of overall performance. Bioresour Technol
2008;99(18):8679–84.
[45] Shi Y, Liu T, Chen S, Quan X. Accelerating anaerobic hydrolysis acidification of
dairy wastewater in integrated floating-film and activated sludge (IFFAS) by using
zero-valent iron (ZVI) composite carriers. Biochem Eng J 2022;177:108226.
https://doi.org/10.1016/j.bej.2021.108226.
[46] Gonzalez-Gil G, Seghezzo L, Lettinga G, Kleerebezem R. Kinetics and mass-transfer
phenomena in anaerobic granular sludge. Biotechnol Bioeng 2001;73(2):125–34.
[47] Ting WH, Huang JS. Influence of mass transfer resistance on overall nitrate
removal rate in upflow sludge bed reactors. Chemosphere 2006;65:148–58.
https://doi.org/10.1016/j.chemosphere.2006.02.013.
[48] Qian J, Li K, Wang P, Wang C, Shen M, Liu J, et al. Toxic effects of three crystalline
phases of TiO2 nanoparticles on extracellular polymeric substances in freshwater
biofilms. Bioresour Technol 2017;241:276–83.
[49] Liang Z, Tu Q, Su X, Yang X, Chen J, Chen Yi, et al. Formation, extracellular
polymeric substances and microbial community of aerobic granules enhanced by
microbial flocculant compared with poly-aluminum chloride. J Clean Prod 2019;
220:544–52.
[50] Tu Z, Setlow P, Brul S, Kramer G. Molecular physiological characterization of a
high heat resistant spore forming bacillus subtilis food isolate. Microorganisms
2021:9. https://doi.org/10.3390/microorganisms9030667.
[51] Sipma J, Henstra AM, Parshina SM, Lens PN, Lettinga G, Stams AJM. Microbial CO
conversions with applications in synthesis gas purification and bio-desulfurization.
Crit Rev Biotechnol 2006;26:41–65. https://doi.org/10.1080/
07388550500513974.
[52] Hulshoff Pol LW, De Castro Lopes SI, Lettinga G, Lens PNL. Anaerobic sludge
granulation. Water Res 2004;38:1376–89. https://doi.org/10.1016/j.
watres.2003.12.002.
[53] Jiang H, Wu F, Wang Y, Feng L, Zhou H, Li Y. Characteristics of in-situ hydrogen
biomethanation at mesophilic and thermophilic temperatures. Bioresour Technol
2021;337:125455. https://doi.org/10.1016/j.biortech.2021.125455.
[54] Li Y, Jing Z, Pan J, Luo G, Feng Lu, Jiang H, et al. Multi-omics joint analysis of the
effect of temperature on microbial communities, metabolism, and genetics in full-
scale biogas reactors with food waste. Renew Sustain Energy Rev 2022;160:
112261.