Chemical Engineering Journal 477 (2023) 147307

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Chemical Engineering Journal

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Exogenous hydrogen supply improves in-situ biogas upgrading of sewage

sludge: Performance and mechanisms
Zhong-Fang Sun a, Lei Zhao a, *, Jie-Ting Wu b, Zi-Han Wang a, Kai-Kai Wu a, Chuan Chen a,
De-Feng Xing a, Dong-Mei Liu a, Shan-Shan Yang a, Ai-jie Wang c, d, Nan-Qi Ren a
a
School of Environment, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
b
School of Environment, Liaoning University, Shenyang 110000, China
c
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
d
School of Civil and Environmental Engineering, State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology Shenzhen, Shenzhen
518055, China

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

Keywords: In-situ biogas upgrading is a promising approach for enhancing carbon dioxide capture and efficient resource use.
In-situ biogas upgrading Recent investigations have highlighted that exogenous hydrogen can be used to purify biogas. However, the
Anaerobic digestion effects of exogenous hydrogen supply on in-situ biogas upgrading remain poorly understood. This study inves­
Hydrogen
tigated the effects of direct and indirect hydrogen supply on in-situ biogas upgrading and elucidated potential
Zero valent iron
Sewage sludge
underlying mechanisms. Significant enhancements were observed in methane production efficiency and biogas
upgrading using an exogenous hydrogen supply. Batch tests showed that adding 10 g/L coarse zero valent iron
(cZVI) resulted in a high cumulative methane yield of 286.4 mL/g-volatile solids (VS) and methane content of
95.5%, representing a 2.2-fold and 1.5-fold increase, respectively, compared to results in the absence of cZVI. In
semi-continuous tests, the use of hollow fibers to enhance hydrogen diffusion led to an increased methane
production rate (52.0 mL/g-VS⋅d), while less hydrogen was more favorable for biogas purification (92.0%
methane). Further analysis revealed that an exogenous hydrogen supply enhances cooperative interactions be­
tween hydrolysis microbes, acetogenesis microbes, and methanogens to promote methane production and biogas
upgrading. These findings provide valuable insights into the impact of exogenous hydrogen supply on in-situ
biogas upgrading during anaerobic digestion of sewage sludge, informing future development of strategies for
enhancing methane production and biogas upgrading from waste sources.

1. Introduction
Sewage sludge is a by-product of wastewater treatment that contains
a variety of pollutants (organic substances, microorganisms, harmful
substances, heavy metals, and inorganic particles). Conventional
disposal methods, such as landfilling and incineration, are challenging
due to the complexity of sewage sludge and its environmental threats.
Anaerobic digestion is considered an efficient resource recovery tech­
nology for sewage sludge treatment [1]. However, raw biogas contains
CO2 (30–50%) and other trace gases (1–10%), including H2, H2S, CO,
N2, and water vapor, which reduce its calorific value from 50.2 to 17.7
MJ/m3 [2], limiting its range of applications [3]. Biogas with over 95%
methane content can be used for domestic and industrial electricity and
heat generation, directly injected into the natural gas grid, or used as a
vehicle fuel, substantially increasing its value [4]. Therefore, upgrading
biogas to increase its methane content is a crucial step in the reuse of
biogas derived from anaerobic digestion.
Biological biogas upgrading is an attractive technology that has
garnered significant interest due to its mild operational conditions and
low chemical and energy requirements [5,6]. Hydrogen-assisted biogas
upgrading is one of the most promising methods for biogas upgrading, in
which hydrogen is used as an electron donor by hydrogenotrophic
methanogens during anaerobic digestion to facilitate conversion of CO2
into methane (i.e., in-situ biogas upgrading) [7]. For instance, Mulat [8]
achieved an increase in methane content in biogas to 89% by injecting
hydrogen into an anaerobic digestion reactor in pulse mode. To improve
the gas–liquid mass transfer rate of hydrogen, Thapa [9] explored the
feasibility of hydrogen addition for in-situ biogas upgrading using a

* Corresponding author.
E-mail address: lzhao@hit.edu.cn (L. Zhao).

https://doi.org/10.1016/j.cej.2023.147307
Received 9 September 2023; Received in revised form 22 October 2023; Accepted 11 November 2023
Available online 11 November 2023
1385-8947/© 2023 Elsevier B.V. All rights reserved.
Z.-F. Sun et al.
trickling filter bed reactor, demonstrating that hydrogen injection
resulted in a 66.5% increase in methane content and a 67.4% reduction
in CO2 content. Similarly, Khan [10] confirmed that adding hydrogen
can increase the methane content to 98.9%. Multiple studies have
confirmed that addition of hydrogen can promote biogas upgrading, but
the low solubility and mass transfer efficiency of hydrogen need to be
addressed [11]. Moreover, exceeding the inhibition partial pressure (10-
5
atm for volatile fatty acids degradation) can dilute the purity of
methane in biogas [12] and inhibit microbial activity. Excess hydrogen
can also reduce the buffering capacity of anaerobic digestion and
damage its stability. Wahid [12] confirmed that when the ratio of
hydrogen to CO2 was greater than 4:1, the pH increased to > 8, leading
to an imbalance in the anaerobic digestion and a decrease in methane
production. Additionally, hydrogen itself is an energy source and an
unnecessarily high dose wastes resource. Therefore, studying the most
efficient and effective approach to indirect supply of hydrogen con­
tributes to development of hydrogen-assisted in-situ biogas upgrading.
Previously, studies have shown that addition of ZVI can improve
methane production in anaerobic digestion [13]. For example, Zhang
[14] added 99.9% pure commercial nanoscale zero valent iron (nZVI) to
tetracycline wastewater, resulting in an increase of 21.2% in methane
production and 26.9% in methane content. Charalambous and Vyrides
[15] used ZVI scraps collected from lathe machines to digest cheese
whey (CW), resulting in a methane yield of 17.9 mL CH4/mL CW and
97.7% methane content. In addition to its direct effect on methane
production by facilitating electron transfer and enhancing microbial
activity [13], ZVI indirectly contributes to hydrogen supply via anaer­
obic corrosion, in which ZVI releases hydrogen through reactions such
as equations (1) and (2) [13].
Fe + 2H2O → Fe(OH)2 + H2 (1)
3Fe(OH)2 → Fe3O4 + H2 + 2H2O (2)
Tang [16] showed that nZVI corrosion-generated hydrogen can in­
crease methane content to 90.5%. However, with excess ZVI concen­
tration, methane production is inhibited. Dong [17] reported that when
ZVI was added at a concentration of 12.5 g/L, CO2 was completely
converted and the methane production rate was 3.93 μmol/h. At the
same time, the relative abundance of hydrogenotrophic methanogens
increased, strengthening the conversion of H2 and CO2 into CH4. How­
ever, when the concentration of ZVI was > 25 g/L, methane generation
was significantly inhibited and acidogenic bacteria dominated, leading
to the accumulation of acetic acid. Therefore, the concentration of ZVI is
critical for orderly bioconversion of CO2 into CH4. However, it is not yet
clear whether ZVI can promote biogas upgrading by releasing hydrogen
or how to achieve this effect. Residual ZVI could have negative envi­
ronmental impacts by increasing the recycling workload. Therefore,
finding an appropriate exogenous hydrogen supply method and con­
trolling the amount of ZVI to regulate hydrogen supply are important to
increase efficiency. In addition, there is currently no research on the
effectiveness of simultaneously directly and indirectly adding hydrogen.
The aim of this study was to address the limitations of exogenous
hydrogen supply in in-situ biogas upgrading to expand the scope of its
application. To achieve this, the effects of various methods of exogenous
hydrogen addition were evaluated through batch tests, including direct
hydrogen injection, indirect hydrogen addition using ZVI with different
particle sizes and concentrations, and a combination of both. Subse­
quently, bubble-free hollow fiber membranes were used to provide
hydrogen and enhance hydrogen gas–liquid mass transfer efficiency.
Semi-continuous experiments were conducted concurrently to verify the
beneficial effects of exogenous hydrogen. To gain a better understanding
of how exogenous hydrogen can improve the efficiency of in-situ biogas
upgrading, the impacts of hydrogen and ZVI on each stage of anaerobic
digestion and corresponding changes in the microbiome were evaluated.
The results have significant implications for biogas upgrading technol­
ogy advancement and contribute to reducing carbon emissions.
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Chemical Engineering Journal 477 (2023) 147307
2. Materials and methods
2.1. Sewage sludge sources
The study used sewage sludge from the secondary sedimentation
tank at the Wenchang Wastewater Treatment Plant in Harbin, China.
The sewage sludge was settled for 24 h, the supernatant was removed,
and the remaining sludge was stored under anaerobic conditions.
Anaerobic digested sewage sludge was used as an inoculum, and sewage
sludge that had not undergone anaerobic digestion was heat-treated at
90 for 1 h as substrate [18]. To prevent accumulation of volatile fatty
acids (VFAs), an inoculum to substrate ratio of 1.3 on a dry VS basis was
maintained. The inoculum and substrate volumes were adjusted based
on their primary characteristics (Table S1).
2.2. Batch tests for in-situ biogas upgrading using various exogenous
hydrogen supply methods
The impacts of in-situ biogas upgrading using different exogenous
hydrogen supply methods were assessed via batch tests. The tests were
conducted in 150 mL serum bottles, with a working volume of 100 mL
consisting of 65 mL inoculum and 35 mL substrate. Three different
hydrogen supply groups, namely direct hydrogen injection, indirect
hydrogen addition, and mixed hydrogen supply, were used as described
below. All tests were performed in triplicate, with a control group
comprising only inoculum and substrate. Gas and liquid samples were
collected every 1–3 days to assess biogas upgrading performance.
(1) Direct hydrogen injection. The serum bottles were flushed with
nitrogen gas for at least 10 min to ensure an anaerobic condition,
after which they were sealed with butyl rubber stoppers and fixed
with aluminum crimp caps. Subsequently, the serum bottles were
placed in an air-bath shaker at 35 and 150 rpm. During anaerobic
digestion, based on the yield of biogas produced, hydrogen
(99.9%) was injected into the serum bottle daily at a rate of
26.7–80.0 mL/L⋅d using a syringe to ensure a 4:1 H2/CO2 ratio.
(2) Indirect hydrogen injection. Various particle sizes of ZVI were
added, including cZVI (250–380 μm), medium ZVI (mZVI;
75–180 μm), fine ZVI (fZVI; 25–48 μm), and nZVI (100 nm), at a
concentration of 5 g/L along with the inoculum and substrate.
The experimental method was as described above. After identi­
fying the most effective ZVI particle size, the impacts of cZVI
concentrations (5, 10, 15, and 20 g/L) and nZVI concentrations
(2, 6, 10, and 14 g/L) on in-situ biogas upgrading were further
evaluated as described previously [19,20].
(3) Mixed hydrogen supply. Three different amounts of hydrogen
(26.7, 53.3, and 80.0 mL/L⋅d) were added daily to three series of
serum bottles containing 10 g/L cZVI. Batch tests were conducted
over a period of 25 d or until biogas production was negligible.
2.3. Semi-continuous tests for in-situ biogas upgrading with exogenous
hydrogen supply
For this study, two 400-mL anaerobic reactors were used, each
consisting of 260 mL inoculum and 140 mL substrate. The two reactors
had the same basic structure, but reactor F1 had an additional hollow
fiber membrane module (482 fibers, Teijin Limited, Japan) for direct
hydrogen supply. The membrane is made of polypropylene material and
the inner and outer diameters of the membrane fibers are 90 μm and 200
μm. Reactor F2 indirectly supplied hydrogen by introducing 10 g/L
cZVI. Both reactors operated for 57 days at 35℃ and a rotation speed of
150 rpm. During Phase I (Days 0–10), the reactors operated until biogas
production stabilized. In Phase II (Days 11–27), hydrogen and ZVI were
supplied to reactors F1 and F2, respectively, to enhance in-situ biogas
upgrading. In Phase III (Days 27–57), the sludge retention time (SRT)
was set to 10 days; 40 mL of sludge was replaced daily with an equal
Z.-F. Sun et al.
volume of substrate. Gas and liquid samples were collected from the
anaerobic reactors every 1–2 days to monitor biogas upgrading
performance.
2.4. Effects of exogenous hydrogen supply on each stage of anaerobic
digestion
The study used 150-mL serum bottles with a 100-mL working vol­
ume, including 20 mL inoculum and 80 mL synthetic medium, to
investigate each step of anaerobic digestion. Exogenous hydrogen was
supplied directly or indirectly by adding 80.0 mL/L⋅d H2 and 10 g/L
cZVI to the serum bottles. Synthetic medium consisting of bovine serum
albumin (BSA), glucose, sodium propionate, sodium acetate, and syngas
(80% H2 and 20% CO2) was used to examine the impact of hydrogen on
the various steps of anaerobic digestion, including hydrolysis, acido­
genesis, acetogenesis, acetoclastic methanogenesis, and hydro­
genotrophic methanogenesis [21,22]. A blank was set up by replacing
the synthetic medium with distilled water to evaluate the impact of the
inoculum on the results. The experiments were conducted in triplicate.
The degradation rate of BSA, glucose, sodium propionate, and sodium
acetate, as well as the hydrogen consumption rate, were measured by
collecting liquid and gas samples daily.
2.5. DNA extraction and high-throughput 16S rRNA gene sequencing
Samples of microorganisms were obtained from the inoculum on Day
0, as well as during Phase I on Day 10, Phase II on Day 27, and Phase III
on Day 33, from both the F1 and F2 reactors. According to the manu­
facturer’s protocol, genomic DNA was extracted using the TIANamp Soil
DNA Kit (Beijing, China). Bacterial DNA amplification was carried out
using primers 341 F and 805 R, while archaea DNA amplification was
done through nested PCR using two sets of primers: 340 F and 1000 R for
the first round and 349 F and 806 R for the second round [16]. The
amplified DNA was sequenced using the Illumina MiseqTM platform
(Sangon Biotech, Shanghai, China) to analyze changes in the micro­
biome composition during the different phases. The 16 S rRNA
sequencing data were obtained from the NCBI Sequence Read Archive
database (submission ID: SUB12958288 and SUB12962988).
2.6. Analytical methods
Liquid samples were passed through disposable filters with a pore
size of 0.22 μm prior to being analyzed for VFAs using an Agilent 7890A
gas chromatograph with a flame ionization detector, as described by
Zhang [23]. A DB-FFAP gas chromatography capillary column (Agilent,
USA) was used, with high-purity air and nitrogen as the carrier gas. The
column box temperature was 50℃ and the injection port and detector
temperatures were 200℃. Total solids (TS), VS, total chemical oxygen
demand (TCOD), soluble chemical oxygen demand (SCOD), proteins,
polysaccharides, and glucose concentrations were determined following
the protocols outlined in Wang [24]. Biogas produced was collected in
gas bags and its composition was analyzed, including H2, CH4, and CO2
using a gas chromatograph (GC, 7890A, Agilent, USA) with a thermal
conductivity detector [25]. The carrier gas was high-purity nitrogen
with a column box temperature of 35℃, a sample inlet temperature of
250℃, and a detector temperature of 200℃. The absorption peaks of the
three gases were able to be completely separated. From 0 to 100 %, the
regression coefficients (R2) of the standard curve determined by external
standards were all > 0.99, indicating good repeatability. X-ray photo­
electron spectroscopy (XPS) was used to detect the final valence of ZVI
after in-situ biogas upgrading. Based on the obtained full spectrum and
narrow spectrum data for elemental Fe, a fine spectrum of elemental Fe
was constructed and the positions of the different valence states were
compared using an XPS electron binding energy comparison table to
calculate the peak areas of different valence states. The obtained peak
area ratio was used to determine the proportion of Fe in different
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Chemical Engineering Journal 477 (2023) 147307
valence states, thus determining the theoretical amount of hydrogen
evolution from the ZVI.
The modified Gompertz equation [26] was used to provide reason­
able estimates of methane yield, methane production rate, and lag time
(Equation (4):
{ [ ]}
Rm × e × (λ − t)
y(t) = Pm × exp − exp +1 (4)
Pm
where y (t) is the cumulative methane yield (mL/g-VS), Pm is the
methane production potential (mL/g-VS), Rm is the maximum methane
production rate (mL/g-VS⋅d), λ is the lag phase period before producing
methane (d), t is time (d), and e is the mathematical constant
(2.718282).
3. Results
3.1. Effects of direct exogenous hydrogen supply on in-situ biogas
upgrading
The appropriate range for hydrogen addition was determined to be
26.7–80 mL/L⋅d based on the amount of CO2 produced during anaerobic
digestion. Methane yield after direct hydrogen injection is shown in
Fig. 1a. Cumulative methane yield reached a maximum of 255.8 mL/g-
VS after 25 d of anaerobic digestion, 132.5% higher than in the control
(110.0 mL/g-VS). A maximum methane content of 71.5% was also
observed, which is 13.7% higher than that obtained without hydrogen
(62.9%) (Fig. 1b). These findings suggest that direct hydrogen supply
can enhance both methane production and content. Additionally, con­
centrations of SCOD and VFAs exhibited a decreasing trend, with values
higher than the control at the end of anaerobic digestion (Fig. S1). This
may be attributed to the promotion effect of hydrogen on organic matter
dissolution and transfer in the sludge [27].
Previous studies have investigated the potential of direct hydroge­
nation as a solution to upgrading biogas. These studies have reported
biogas production levels ranging from 125.0 to 897.0 mL/g-VS, with
methane content of 70–100% [28]. Our results fall within this range,
which can be attributed to the absence of high organic content or easily
degradable substrates, such as pig manure [29], waste activated sludge
[30], or glucose [31], as well as the use of smaller hydrogenation vol­
umes compared to previous studies.
3.2. Effects of indirect exogenous hydrogen supply on in-situ biogas
upgrading
The cumulative methane yield obtained by adding 5 g/L ZVI with
different particle sizes is presented in Fig. 2a. After 25 days of anaerobic
digestion, cZVI achieved the highest methane yield of 259.6 mL/g-VS.
Addition of nZVI resulted in the highest methane content of 77.1%,
followed by cZVI (75.4%), mZVI (74.9%), and fZVI (70.2%), 22.6%,
19.9%, 19.1%, and 11.6% higher than that without ZVI, respectively
(Fig. 2b). Variations in SCOD and VFAs concentrations with different
ZVI particle sizes are illustrated in Fig. S2a and S3. SCOD concentrations
decreased during the first 15 d of anaerobic digestion but increased
somewhat at the end. This phenomenon could be due to the promotion
effect of ZVI on organic matter transformation. At the beginning of
anaerobic digestion, higher microbial activity allowed for timely utili­
zation of organic matter. However, as microbial activity decreased to­
wards the end of anaerobic digestion, organic matter accumulated,
leading to an increase in SCOD. In the test with cZVI, SCOD and VFA
were completely consumed earlier than in the other tests, suggesting
that cZVI enhances substrate consumption at a faster rate than does
other particle sizes of ZVI. Overall, the findings indicated that addition
of cZVI resulted in an increased methane yield, whereas addition of nZVI
resulted in a higher methane content. However, the maximum methane
yield and methane content attained in this study for various particle
Z.-F. Sun et al. Chemical Engineering Journal 477 (2023) 147307

Fig. 1. Cumulative methane yield (a) and methane content (b) with direct hydrogen supply.

Fig. 2. Cumulative methane yield (a) and methane content (b) with indirect hydrogen supply at different ZVI particle sizes.

sizes were lower than the values obtained by Tang [16], who achieved a
methane yield of 388.5 mL/g-VS and a methane content of 90.5% by
adding 10 g/L nZVI. It was hypothesized that the concentration of ZVI
has an impact on biogas upgrading performance. Therefore, the influ­
ence of different concentrations of cZVI and nZVI on biogas upgrading
was subsequently examined.
In-situ biogas upgrading performance at various concentrations of
cZVI (5, 10, 15, and 20 g/L) is illustrated in Fig. 3a, c. At 10 g/L cZVI, the
maximum methane yield of 286.4 mL/g-VS was observed, representing
a 124.5% increase compared to the control (Fig. 3a). The corresponding
maximum methane content was 95.5%, a 51.8% increase over the
control. Among the tested nZVI concentrations (2, 6, 10, and 14 g/L), the
highest cumulative methane yield of 192.8 mL/g-VS was achieved at 6
g/L nZVI, a 51.1% increase compared to the control (Fig. 3b). Similarly,
the highest methane content was 80.8%, 28.5% higher than that without
nZVI (Fig. 3d). Excess ZVI concentrations, regardless of their type, can

Fig. 3. Cumulative methane yield (a: cZVI; b: nZVI) and methane content (c: cZVI; d: nZVI) with indirect hydrogen supply at different concentrations.

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damage the cell membrane structure, leading to partial cell dissolution
and release of cell components [32], which inhibits anaerobic digestion.
For example, in the presence of 14 g/L nZVI in this study, the concen­
trations of SCOD and VFAs significantly increased (Fig. S2c and S5d),
which inhibited methane production and resulted in only 16.4 mL/g-VS
of methane being produced. Similarly, Charalambous and Vyrides [15]
found that higher concentrations of powdered ZVI (100 g/L) can inhibit
acetoclastic methanogenesis and disrupt the symbiosis between bacteria
and methanogens during anaerobic digestion by facilitating rapid
hydrogen production and high hydrogen partial pressure, resulting in
accumulation of VFAs and a reduction in methane production. Addi­
tionally, high concentrations of ZVI can form stable complexes with
phosphate, sequestering key nutrients (phosphorous) that can inhibit
methanogens. While excess concentrations of ZVI can lead to lower
hydrogen production rates (Table 1), thereby affecting the performance
of in-situ biogas upgrading. According to Fig. S2b, 10 g/L cZVI acceler­
ates hydrolysis and consumption of organic matter, while also providing
optimal oxidation–reduction potential for methanogens [33]. Thus, in
this study, addition of 10 g/L cZVI to sewage sludge significantly
improved biogas upgrading performance.
experimental results of adding hydrogen or ZVI (R2 > 0.99). The kinetic
model parameters estimated by the Gompertz model are shown in
Table S2. After adding hydrogen or ZVI, Pm and Rm followed the actual
cumulative methane yield, while the lag time was longer than in the
control without hydrogen or ZVI. The same phenomenon was also re­
ported by Zhou [34], Zhang [35], and Dai [36]. However, these reports
did not address why the addition of hydrogen or ZVI prolonged the λ. We
suggest two possible reasons: first, hydrogen or ZVI may contribute more
to the hydrolysis and acetogenesis stages early in anaerobic digestion,
and methane production occurs after sufficient energy reserves are
available. Second, the methanogens are relatively sensitive, so after
adding hydrogen or ZVI, the hydrolysis-acidogenesis microbes quickly
adapt and become enriched, after which methanogens begin to produce
methane. Collectively, the experimental results for methane yield and
the well-fitted model indicated stable performance of the anaerobic
digestion, confirming the enhancement of in-situ biogas upgrading with
exogenous hydrogen supply.
3.4. Effects of the exogenous hydrogen supply on semi-continuous in-situ
biogas upgrading

3.3. Effects of the mixed exogenous hydrogen supply on in-situ biogas To improve the efficiency of hydrogen mass transfer in the liquid–gas
upgrading phase, semi-continuous in-situ biogas upgrading was conducted using a
hollow fiber membrane in reactor F1, while 10 g/L cZVI was directly
The effect of the mixed exogenous hydrogen supply for biogas added to reactor F2 as a comparison. In the first phase, both reactors
upgrading was investigated by injecting different volumes of hydrogen underwent anaerobic digestion. On Day 10, the methane production
(26.7, 53.3, and 80.0 mL/L⋅d) in addition to 10 g/L cZVI. The maximum rates and methane contents in F1 and F2 were 14.3 mL/g-VS⋅d and
methane yield of 242.8 mL/g-VS was obtained with 80.0 mL/L⋅d 51.3%, and 20.6 mL/g-VS⋅d and 63.1%, respectively. Subsequently,
hydrogen and 10 g/L cZVI. The highest methane content of 77.9% was hydrogen partial pressure of 1.025 atm and 10 g/L cZVI were introduced
achieved with 53.3 mL/L⋅d hydrogen and 10 g/L cZVI (Fig. 4). Thus, into F1 and F2 to promote biogas upgrading. On Day 27, the methane
higher volumes of added hydrogen resulted in higher cumulative production rates and contents increased to 31.3 mL/g-VS⋅d and 73.2% in
methane yield. However, excess hydrogen addition reduced the biogas F1, and 23.4 mL/g-VS⋅d and 90.0% in F2. The two reactors were then
upgrading performance. Based on SCOD content, organic matter release operated semi-continuously to ensure stable in-situ biogas upgrading. In
increased with increasing hydrogen dose at a constant cZVI concentra­ F1, the methane production rate reached a maximum of 52.0 mL/g-VS⋅d
tion (Fig. S2d). Additionally, increasing the hydrogen dose led to greater and a consistent methane content of 88.1 % was maintained after 3 SRTs
conversion of organics into VFAs (Fig. S6). of semi-continuous operation (Fig. 5a). In F2, addition of 10 g/L cZVI as
Currently, there has been little research on application of a mixed a hydrogen source resulted in a maximum methane production rate of
exogenous hydrogen supply for in-situ biogas upgrading. Contrary to 32.2 mL/g-VS⋅d and a methane content of 92.0% (Fig. 5b). Toward the
expectations, adding a mixture of hydrogen and ZVI resulted in lower end of anaerobic digestion, VFAs increased in F1, while no significant
methane content and yield compared to a hydrogen supply alone or changes in VFAs were observed in F2 (Fig. S8). Furthermore, approxi­
addition of ZVI separately. One possible explanation is that the com­ mately 10% hydrogen was detected in F1 at this point, possibly due to
bined addition of hydrogen and ZVI promotes dissolution of organic accumulation of hydrogen, leading to excessive conversion of dissolved
matter, resulting in an increase in organic load [15]. Furthermore, organic matter to acid and accumulation of VFAs.
introduction of both hydrogen and ZVI increases the hydrogen dose,
which elevates hydrogen partial pressure and can impede anaerobic (1) Impact of exogenous hydrogen on each stage of anaerobic
digestion, thereby decreasing both the methane volume and content digestion
[29].
Additionally, the cumulative methane yield was fitted using the To further explore the mechanisms involved in promoting anaerobic
modified Gompertz model during the batch experiment (Fig. S7). The digestion of sewage sludge and achieving enhanced biogas upgrading
fitted methane yield curve for the model was in good agreement with the through exogenous hydrogen supply, we examined the effects of direct
and indirect hydrogen supply on each stage of sewage sludge anaerobic
Table 1 digestion, including hydrolysis, acidogenesis, acetogenesis, aceticlastic
Theoretical hydrogen evolution amount and methane production effects of ZVI methanogenesis, and hydrogenotrophic methanogenesis. The relative
with different particle size and concentrations. activities of these stages were determined by measuring the degradation
ZVI Theoretical hydrogen Cumulative Methane rates of BSA, glucose, sodium propionate, and sodium acetate, as well as
evolution amount methane yield content (%) the rate of hydrogen consumption. Without hydrogen addition, the
(mL/L) (mL/g-VS) degradation rate of BSA was 192.8 ± 4.4 mg/g-VS⋅d (Table 2), which is
Particle Coarse 1020.3 259.6 ± 8.3 75.4 ± 0.1 considered the baseline activity of the hydrolytic microorganisms [37].
size Nano 953.1 229.7 ± 7.0 77.1 ± 2.5 When 80.0 mL/L⋅d hydrogen was added, the degradation rate of BSA
cZVI (g/ 5 1077.0 200.9 ± 5.9 89.7 ± 0.2 increased significantly to 247.6 ± 2.1 mg/g-VS⋅d, a 28.4% improvement
L) 10 2219.4 286.4 ± 4.9 95.5 ± 2.1
in the relative activity of the hydrolytic microbes. Similarly, when 10 g/
15 3598.8 258.4 ± 5.0 93.1 ± 1.2
20 4087.6 175.2 ± 6.1 71.6 ± 3.1 L cZVI was added, the relative activity of the hydrolytic microbes
nZVI (g/ 2 390.1 187.9 ± 6.6 79.3 ± 6.0 improved by 24.1%. Addition of hydrogen and ZVI increased degrada­
L) 6 1187.7 192.8 ± 6.4 80.8 ± 5.5 tion of propionate and consumption of hydrogen by 31.6% and 48.2%,
10 2016.6 123.3 ± 13.6 78.1 ± 0.0 respectively. These results suggest that the relative activity of microbes
14 2691.0 16.4 ± 8.3 42.8 ± 0.0
responsible for hydrogenotrophic methanogenesis and acetogenesis

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Z.-F. Sun et al. Chemical Engineering Journal 477 (2023) 147307

Fig. 4. Cumulative methane yield (a) and methane content (b) with mixed exogenous hydrogen supply.

Fig. 5. Methane production rate (a) and methane content (b) in different reactors. F1: the hydrogen-adding reactor; F2: the ZVI-adding reactor.

Table 2
Effect of exogenous hydrogen supply on each stage of anaerobic digestion.
Anaerobic Hydrolysis (mg/g- Acidogenesis (mg/g- Acetogenesis (mg/g- Acetoclastic methanogenesis (mg/ Hydrogenotrophic methanogenesis (mL/
digestion VS⋅d) VS⋅d) VS⋅d) g-VS⋅d) g-VS⋅d)

Control 192.8 ± 4.4 395.4 ± 1.3 38.6 ± 0.6 116.6 ± 0.6 64.1 ± 2.7
Hydrogen 247.6 ± 2.1 396.0 ± 0.2 50.8 ± 6.9 103.0 ± 2.3 139.8 ± 4.8
10 g/L cZVI 239.3 ± 4.9 394.3 ± 0.3 57.2 ± 6.1 116.3 ± 1.8 99.7 ± 1.9

increased in the presence of hydrogen and ZVI, while there was no
significant effect on acidogenesis microbes and acetoclastic metha­
nogens. Overall, both hydrogen and ZVI achieved in-situ biogas
upgrading by promoting hydrolysis, acetogenesis, and hydro­
genotrophic methanogenesis. Promotion of hydrogenotrophic meth­
anogenesis was more efficient with addition of hydrogen, while
acetogenesis was more effectively promoted by an indirect exogenous
hydrogen supply. Thus, direct exogenous hydrogen supply accelerated
the methane production rate and indirect hydrogen supply increased the
methane content.
(2) Change in microbial communities in response to different exog­
enous hydrogen supply methods
The microbial community in the semi-continuous reactors was
analyzed through high-throughput 16S rRNA sequencing to compare
community structure and function. Microbial changes in different pha­
ses of in-situ biogas upgrading are illustrated in Fig. 6 and S9. At the
phylum level, six archaeal phyla were identified, with Euryarchaeota
and Nanoarchaeaeota being dominant. Following addition of hydrogen
or ZVI, the abundance of Euryarchaeota responsible for methane pro­
duction reached 100%. Proteobacteria, Bacteroidetes, Firmicutes, and
Chloroflexi were identified as dominant bacterial phyla. The total
abundance of these four bacterial phyla before and after addition of
hydrogen (or ZVI) was 80.7% and 84.7% (or 80.7% and 92.3%). Thus,
addition of exogenous hydrogen only affected the abundance of micro­
organisms, but did not significantly affect the microbial community
structure at the phylum level.
At the genus level, the addition of hydrogen in F1 (Phase II) signif­
icantly increased the relative abundance of methanogens, reaching
78.6%. In Phase III, the relative abundance of methanogens reached
98.2% of the total archaea (Fig. 6a), indicating that hydrogen supple­
mentation can maintain stable methane production via enrichment of
methanogens. Specifically, the total reads of hydrogenotrophic metha­
nogens (Methanoregula, Methanobacterium, Methanospirillum, Meth­
anolinea, Methanomassiliicoccus, Methanosphaerula, and
Methanobrevibacter) increased from 37.2% in Phase II to 59.5% in Phase
III, accompanied by a decrease in the reads of aceticlastic methanogens
(Methanosaeta and Methanosarcina) to 38.7%, suggesting a transition
from acetoclastic methanogenesis to hydrogenotrophic methanogenesis
[38]. After addition of hydrogen, Tangfeifania and Longilinea dominated
the community of hydrolysis microbes, with relative abundances of
7.0% and 3.4%, respectively. In Phase III, the combined abundance of
Pseudomonas, Treponema, and Gelria increased to 42.9%. These bacteria
have been identified as key participants in hydrolysis-acidification
[9,39]. Genera affiliated with Gelria can also participate in direct
interspecific electron transfer and promote methane production in
cooperation with methanogens [40]. These findings suggest that the
observed increase in VFA concentration (Fig. S8a) may be attributed to
an increase in these bacterial genera.

6
Z.-F. Sun et al. Chemical Engineering Journal 477 (2023) 147307

Fig. 6. Taxonomic profiles of microbial communities in the inoculum and the Phase I-III at genus level: (a) relative abundance of bacteria in F1; (b) relative
abundance of bacteria in F2; (c) relative abundance of archaea in F1; (d) relative abundance of archaea in F2. Minor genera accounting for < 2 % of the total
sequence are presented as “others”.

The abundance of methanogens increased from 82.9% to 98.2%
following ZVI addition, whereas it decreased to 79.1% in Phase III,
possibly due to formation of siderite, which hindered the hydrogen
evolution of ZVI. Unlike with direct hydrogen supply, the proportion of
hydrogenotrophic methanogens (Methanoregula, Methanobacterium, and
Methanospirillum) was reduced from 61.4% (Phase II) to 16.3% (Phase
III). Conversely, the relative abundance of aceticlastic methanogens
(Methanosaeta and Methanosarcina) increased from 36.8% to 62.8%,
indicating a transition from hydrogenotrophic methanogenesis to ace­
toclastic methanogenesis. This coincided with a decline in the total
methanogen abundance (98.2% to 79.1%), which may account for the
lower methane production rate. Similarly, ZVI addition caused a sig­
nificant change in the bacterial community. The total proportion of the
20 genera responsible for hydrolysis-acidification increased from 22.5%
in Phase I to 63.1% after adding ZVI (Fig. 6). The primary genera
involved were Petromonas, Parabolides, Tangfeifania, Levitinea, and Ace­
toanerobium, as previously identified [41,42]. In Phase III, this propor­
tion of these genera decreased to 28.8%, combined with an increase in
the abundance of acetoclastic methanogenesis, which may explain the
lack of VFAs accumulation in F2. Notably, the relative abundance of
bacteria involved in hydrolysis-acidification exhibited a similar trend to
that of hydrogenotrophic methanogenesis, suggesting a potential

7
Z.-F. Sun et al.
synergistic relationship.
4. Discussion
4.1. Role of exogenous hydrogen in enhanced in-situ biogas upgrading
In recent years, the potential for an exogenous hydrogen supply
(either direct or indirect) to improve the methane yield and content via
in-situ biogas upgrading has attracted increasing attention [27,43].
Despite this, comprehensive investigation of the effects of different
exogenous hydrogen supply methods on in-situ biogas upgrading had not
yet been conducted. The results of this study fill this gap by confirming
that a single hydrogen supply can significantly promote in-situ biogas
upgrading rather than a combination of both. A previous study reported
that nZVI can promote ex-situ biogas upgrading with the assistance of
hydrogen [44]. However, in this study, the combined additional effect of
hydrogen and ZVI is lower than either one alone (Figs. 1–4), which may
be related to the following factors: 1) the synergistic effect of hydrogen
and ZVI addition on hydrolysis acid-producing bacteria resulted in sig­
nificant accumulation of organic matter, leading to an increase in SCOD
and VFAs concentrations, which hindered methane production (Fig. S2d
and S6) and 2) the microbial composition of ex-situ biogas upgrading is
more uniform, avoiding competition with other microorganisms
involved in anaerobic digestion.
In this study, the presence of cZVI resulted in higher methane yield,
while the presence of nZVI led to higher methane content. Based on
these observations, a higher hydrogen concentration may be more
favorable for methane production, while a lower hydrogen concentra­
tion is more suitable for improving methane purity. This hypothesis was
verified by the results obtained from X-ray photoelectron spectroscopy
(XPS) fine spectrum and the assessment of hydrogen evolution (Fig. S10
and Table 1). When mixed hydrogen supply was performed, it was
observed that increased quantities of supplemented hydrogen in the
presence of 10 g/L cZVI led to a higher cumulative methane yield, while
moderate hydrogen addition resulted in the highest methane content.
Furthermore, this finding further supported the conclusion from direct
hydrogen injection that higher hydrogen partial pressure is more
Fig. 7. Microbiological mechanism diagram of in-situ biogas upgrading of sewage sludge with exogenous hydrogen supply.
8
Chemical Engineering Journal 477 (2023) 147307
conducive to methane generation, while lower hydrogen partial pres­
sure is more conducive to conversion of CO2 into methane.
The microbial mechanism underlying exogenous hydrogen supply
for promoting anaerobic digestion for in-situ biogas upgrading is
depicted in Fig. 7. The continuous direct hydrogen supply enriches many
bacteria involved in hydrolysis and acidification, thereby strengthening
these processes after adding hydrogen. This continuous promotion of
hydrolysis and acidification may have caused accumulation of VFAs,
despite the high total abundance of methanogens in the reactor. How­
ever, the increase in the total abundance of methanogens accelerated
methane production. Meanwhile, the continuous supply of hydrogen
diluted the methane in the biogas due to the higher sensitivity of
hydrogenotrophic methanogens to elevated hydrogen/hydrogen partial
pressure compared to other methanogens [45]. As a result, addition of
excessive amount of hydrogen showed a lower methane content. In
contrast, after adding 10 g/L ZVI, the high abundance of microorgan­
isms involved in hydrolysis-acidification and methanogenesis after ZVI
addition synergistically promoted consumption of organic matter, VFAs,
and production of methane. However, the ZVI may have been protected
by siderite, thereby reducing evolution of hydrogen and the abundance
of hydrogenotrophic methanogens and bacteria involved in hydrolysis-
acidification [27]. Consequently, the addition of ZVI had a lower
methane production rate and no accumulation of VFAs.
These results obtained in this study indicate that use of exogenous
hydrogen supply has a strong impact on in-situ biogas upgrading.
Moreover, adding hydrogen, directly or indirectly, to anaerobic diges­
tion promotes hydrolysis, acetogenesis, and hydrogenotrophic meth­
anogenesis. Importantly, the quantity of hydrogen supplied can
substantially influence both methane production and biogas upgrading.
4.2. Implications of the study
Hydrogen-driven biogas upgrading has gained increasing interest
recently, with the potential to convert biological CO2 into a sustainable
energy source and reduce carbon emissions. However, the low solubility
of hydrogen and its negative impact on pH balance, microbial meta­
bolism, and “acidification” have limited its application in biogas
Z.-F. Sun et al.
upgrading. To improve in-situ biogas upgrading, research needs to be
conducted on increasing the gas–liquid mass transfer efficiency of direct
hydrogen supply. Studies have shown that ZVI can release hydrogen
under anaerobic conditions and promote biogas upgrading [15,16,46],
making it a potential substitute for hydrogen. Therefore, the magnitude
of two potential contributions (the H2 released by ZVI and the role of ZVI
itself) of ZVI to promoting biogas upgrading should be further clarified
in future experiments. Moreover, attention also needs to be given to the
protective layer gradually formed after adding ZVI during anaerobic
digestion, which can hinder hydrogen generation and direct effects on
anaerobic digestion. Future research endeavors should investigate
strategies to prevent this phenomenon while also exploring alternative
materials to enhance the efficiency of in-situ biogas upgrading. Addi­
tional studies should delve into the genetic knockout of hydrogenase
genes associated with hydrogenotrophic methanogenesis to verify the
promoting effect on hydrogenotrophic methanogenesis. Simultaneously,
transcriptomic analysis of microbial communities is required to confirm
the electron transfer mode facilitated by exogenous hydrogen supply,
thus promoting methane production.
Furthermore, it’s important to note that this study was conducted on
a lab scale. To facilitate future applications on a larger industrial scale, it
is advisable to consider the utilization of scrap ZVI as a cost-effective
alternative to pure ZVI. Additionally, the production of hydrogen via
the electrolysis of water, using excess electricity generated by wind
power plants or photovoltaic power generation facilities, offers the po­
tential to further reduce operational costs. Expanding to a larger in­
dustrial scale may result in a reduced mass transfer interface per unit
volume, potentially impacting the hydrogen utilization rate. To main­
tain an efficient hydrogen transfer environment, it is essential to main­
tain a constant hydrogen mass transfer efficiency by setting it at the
same level as in small-scale or pilot scale reactors. This can be achieved
by incorporating gas distributors, larger membrane components, or gas
recirculation equipment in the scaled-up reactor. Moreover, as the re­
action scale increases, the dosage of ZVI will also need to be adjusted,
and the ZVI may tend to settle at the bottom of the reactor due to its high
density, potentially affecting its effectiveness. To address this, it is
advisable to introduce a bottom aeration device or stirring device to
ensure even distribution of ZVI. This not only improves the distribution
of ZVI but also addresses fluid distribution changes that may occur when
expanding the reactor. Therefore, suitable reactor configurations should
be selected based on the application scenarios to achieve an optimal
efficiency in future expanded applications.
5. Conclusions
This study aimed to investigate the underlying mechanisms of
exogenous hydrogen supply on in-situ biogas upgrading during sewage
sludge anaerobic digestion. The main findings include the following:
(1) Exogenous hydrogen supply improved the efficiency of in-situ
biogas upgrading. Among them, 10 g/L cZVI resulted in the
highest cumulative methane yield (286.4 mL/g-VS) and methane
content (95.5%). In contrast, the effect of a mixed hydrogen
supply was inferior to direct or indirect hydrogen supply alone.
(2) The theoretical hydrogen evolution of ZVI indicated that the
methane yield and content were correlated with the amount of
hydrogen. Specifically, in semi-continuous experiments, direct
exogenous hydrogen supply achieved a methane production rate
of 52.0 mL/g-VS⋅d, while indirect exogenous hydrogen supply
(10 g/L cZVI) led to a maximum methane content of 92.0%.
(3) Microbial analysis revealed that exogenous hydrogen supply
enriched microorganisms involved in hydrolysis-acidification
and methanogenesis, with the highest relative abundance of
63.1% and 98.2%, respectively. The synergistic interaction
among these microorganisms contributed to enhanced methane
production and biogas upgrading, which aligned with the effects
9
Chemical Engineering Journal 477 (2023) 147307
of exogenous hydrogen supply in each stage of anaerobic
digestion.
(4) The findings of this study deepen our understanding of the po­
tential impact of exogenous hydrogen supply on in-situ biogas
upgrading and will be helpful in developing strategies to enhance
methane production and biogas upgrading from waste sources in
the future.
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.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China [No. U22A20443]; the Fundamental Research Funds for the
Central Universities [No.HIT. OCEF.2023014]; the Heilongjiang Pro­
vincial Natural Science Foundation of Excellent Young Scholars (Grant
No.YQ20123C031); Open Project of Key Laboratory of Environmental
Biotechnology, CAS [Grant No kf2020009]; Heilongjiang Touyan
Innovation Team Program; and the Fundamental Research Funds for the
Central Universities [HIT.DZJJ.2023058].
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cej.2023.147307.
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