Water Research 230 (2023) 119583

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Water Research
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Two microbial consortia obtained through purposive acclimatization as

biological additives to relieve ammonia inhibition in anaerobic digestion
Shilei Wang a, 1, Zhi Wang a, 1, Muhammad Usman b, Zehui Zheng c, Xiaoling Zhao a,
Xingyao Meng d, Kai Hu e, Xia Shen f, Xiaofen Wang c, Yafan Cai a, *
a
School of Chemical Engineering, Zhengzhou University, Ke xue Dadao 100, Zhengzhou, 450001, China
b
Department of Civil and Environmental Engineering, University of Alberta, Edmonton, AB T6G 2W2, Canada
c
College of Agronomy and Biotechnology/Biomass Engineering Center, China Agricultural University, Beijing, 100193, China
d
Beijing Technology and Business University, State Environmental Protection Key Laboratory of Food Chain Pollution Control, Beijing, 100048, China
e
Shenzhen Derun Biomass Investment Co., Ltd. Shenzhen, 518066, China
f
Key Laboratory of Agricultural Soil and Water Engineering in Arid and Semiarid Areas, Ministry of Education, Northwest A and F University, Yangling, Shaanxi,
712100, China

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

Keywords: Ammonia inhibition is a challenging issue in the anaerobic digestion (AD) of nitrogen-rich substrates and hinders
Bioaugmentation the energy recovery from organic wastes. Bioaugmentation is promising strategy to stabilize AD systems with
Microbial consortia high ammonia concentration. The composition of microbial consortia often determines their effectiveness in
Ammonia inhibition
bioaugmentation. Up to now, the effect of various microbial consortia as biological additives on the AD systems is
Anaerobic digestion
not fully understood. In this study, two microbial consortia (syntrophic microbial consortium, MC, and hydro­
genotrophic methanogen consortium, SS) were obtained through two domestication methods, and were applied
in a nitrogen-rich AD system. The results showed that the MC and SS treatments could restore AD performance
within 21 days and 83 days, respectively. The recovery of digestion performance depended on the methanogenic
archaea Methanospirillum, Methanothermobacter, and Methanoculleus in the early and later stages. Analysis of the
13
C isotope indicated that both MC and SS enhanced the hydrogenotrophic pathway. The KEGG analysis showed
that the MC not only promoted the key enzyme genes in the hydrogenotrophic pathway but also had a positive
effect on the related enzyme genes of propionate and butyrate degradation, which was affected by the abundant
short-chain fatty acids degrading bacteria, such as Syntrophomonas, Syntrophobacter, and Tissierella in the MC.
After recovery of digestion performance, there was no significant difference (p > 0.05) in methane yield between
the MS and SS treatments. Therefore, the best intervention period for bioaugmentation is when the digestion
performance of the AD system is unstable.

1. Introduction nitrogen stripping, and co-digestion) to alleviate ammonia inhibition,

Anaerobic digestion (AD) is an effective process for obtaining bio-
energy from organic wastes (Karki et al., 2021). However, when the
carbon to nitrogen (C/N) ratio of the substrates is lower than 12, the AD
system is vulnerable to ammonia inhibition (Romero-Guiza et al., 2016;
Zheng et al., 2021). For instance, the total ammonia nitrogen (TAN)
concentration in the AD system was higher than 4.2 g L− 1 and volatile
fatty acids (VFAs) accumulated when chicken manure was the main
substrate (Ajayi-Banji et al., 2022).
Among many strategies (e.g., trace elements supplement, ammonia
bioaugmentation is an emerging and promising method to enhance the
digestion performance of AD systems with high TAN concentrations (Cai
et al., 2023; Yang et al., 2022). A previous study showed that hydro­
genotrophic methanogens, such as Methanoculleus and Mathanosarcina,
had high resistance to ammonia toxicity (Yan et al., 2020). Therefore,
they are often used as biological additives to alleviate the ammonia
inhibition (Lee et al., 2022). For instance, Yan et al. (2020) reported that
supplementation of Methanoculleus enhanced the methane yield of
municipal solid waste by 21% and decreased the VFAs by 10%. Some­
times, single archaea is challenging to colonize and reconstruct the

* Corresponding author:
E-mail address: caiyafan@zzu.edu.cn (Y. Cai).
1
These authors contributed equally to work.

https://doi.org/10.1016/j.watres.2023.119583
Received 21 October 2022; Received in revised form 29 December 2022; Accepted 5 January 2023
Available online 6 January 2023
0043-1354/© 2023 Elsevier Ltd. All rights reserved.
S. Wang et al. Water Research 230 (2023) 119583

microbial community in ammonia-inhibited AD systems (Yang et al.,
2019). While, single archaea as biological additives may pose high risks
and uncertainty in relieving the ammonia inhibition. For example, few
studies reported that Methanoculleus and Mathanosarcina had high
resistance to ammonia nitrogen and gave positive results when applied
to ammonia-inhibited AD systems (Capson-Tojo et al., 2020; Yan et al.,
2020), other studies got contradictory results (Gao et al., 2015; Yang
et al., 2018). For instance, Gao et al. (2015) reported that Mathano­
sarcina was gradually replaced by Methanobacterium when the TAN
concentration increased from 2.2 to 4.5 g L− 1. Therefore, microbial
consortia, rich in various hydrogenotrophic methanogens, may have
more advantages as biological additives in reducing this risk and un­
certainty. It is worth noting that the conversion of the VFAs to H2 and
acetate is thermodynamically unfavorable (ΔG0 > 0), and is generally
′
including methane production, pH, soluble chemical oxygen demand
(sCOD), VFAs concentration, total solid (TS), and volatile solid (VS)
removal rates, were explored. Second, their effects on microbial com­
munity structure in the ammonia-inhibited AD system were studied
from the microbial aspects. Finally, the effect of MC and SS on the
methanogenesis pathways and the gene abundance of key enzymes
related to the methanogenesis pathways were analyzed by 13C isotopic
analysis and KEGG data annotation. This study provided a purposive
acclimatization method for obtaining the microbial consortia and
revealed the potential mechanism of two microbial consortia in assisting
the AD of nitrogen-rich substrates.
2. Materials and methods

considered to be the rate-limiting step (Tang et al., 2015; Usman et al.,
2021). If biological additives are rich in bacteria, such as propionic acid
and butyric acid degrading bacteria and hydrogen methanogens that
degrade VFAs, a complex relationship between different members may
be formed, which helps to reduce H2 partial pressure and, thus, promote
conversion of VFAs to methane (Li et al., 2022). The complex syntrophic
relationship may be beneficial to relieving the ammonia inhibition. For
example, Yang et al. (2019) combined Methanobrevibacter and syntro­
phic acetate oxidizing bacteria (SAOB) (Syntrophaceticu schinkii) as a
microbial consortium and methane yield was improved by 71%. Based
on the above background, the current study focuses on two potential
microbial consortia, namely, syntrophic microbial consortium (MC) and
hydrogenotrophic methanogen consortium (SS).
Previously, microbial consortia were often constructed artificially.
The main problem with this method is that the criteria for selecting the
microorganisms to combine into a microbial consortium are often based
on the correlation of the functions of microorganisms. There is high
uncertainty about whether these selected microorganisms are reliable or
can fully play their role in complex AD systems. For instance, West­
erholm et al. (2012) combined some SAOBs (Clostridium ultunense sp,
Tepidanaerobacter acetatoxydans, and Syntrophaceticus schinkii) with a
hydrogenotrophic methanogen (Methanoculleus) to construct a micro­
bial consortium as a biological additive but the digestion performance
was not improved after adding the microbial consortium. Similarly,
Yang et al (2019) artificially synthesized a microbial consortium con­
taining Syntrophaceticus schinkii and Methanoculleus bourgensis but no
substantial effect was observed in the ammonia-inhibited AD system.
Interestingly, the microbial consortia obtained by purposive acclimati­
zation have a closer relationship to but a more robust adaptability than
the artificial microbial consortia. Therefore, they may have a better ef­
fect on promoting the digestion performance of the AD systems with
high TAN concentration (Li et al., 2022; Zhao et al., 2022). The potential
mechanism of the acclimatized microbial consortium may be more
complex in mitigating ammonia inhibition than that of single archaea or
artificial combined microbial consortium.
Currently, there is still a knowledge gap on the effect and potential
mechanism of the microbial consortiums obtained through purposive
acclimatization as a biological additive to alleviate the ammonia inhi­
bition. Specifically, the roles of the various members in a microbial
consortium in alleviating the ammonia inhibition are not clear. In this
study, two microbial consortia (MC and SS) were acclimatized by
2.1. Substrates and inoculum
Chicken manure was taken from a laying hen farm. It was grinded
using a pulverizer (FW100, Taisite, Tianjin, China). Then, large particles
(feather, eggshell, and fiber) were removed with a sieve of 1 mm particle
size. In order to create the different TAN concentrations (from low to
high), the ammonia nitrogen (mainly free ammonia nitrogen) in the
chicken manure was partially removed at 50◦ C for two hours. Conse­
quently, the denitrogenated chicken manure was obtained. Denitrified
chicken manure was stored at -20◦ C and thawed at room temperature
before use. The inoculum was obtained from a continuous stirred tank
reactor (CSTR) with a 50 L working volume. The substrates of the 50 L
reactor were chicken manure, corn straw, and cow manure (about 1:1:1
based VS), and its digestion temperature (37±1◦ C) was controlled in a
temperature-controlled room. The inoculum reactor has been running
stably (VFAs/TIC=0.13-0.17 and methane yield=260-325 mL g− 1 VS)
for more than one year when the inoculum was taken. The properties of
chicken manure, denitrified chicken manure, and inoculum are shown in
Table 1.
2.2. Acclimatization and enrichment of MC and SS and the preparation of
additives for experimental treatments
2.2.1. Acclimatization and enrichment of MC and SS
The MC and SS were domesticated and enriched from the inoculum
(see section 2.1). For the acclimatization and enrichment of MC, a
reactor with a 10 L effective volume was used to perform the process. At
the beginning of acclimatization and enrichment, 10 L inoculum was
filled with the reactor. Every day, 410 mL medium and 90 g residue
(centrifuged inoculum, 3214-8228 g, 5-10 mins) was fed into the
reactor, and 500 mL biogas slurry was discharged. To ensure that MC
was rich in POB, BOB, SAOB, and HM, propionate and butyrate were
Table 1
The basic characteristics of experimental substrates and inoculum
Parameters Chicken Chicken Inoculum
manure 1 manure 2*
Total solid (TS) (%) a 34.9±0.2 76.8±0.1 4.5±0.1
Volatile solid (VS) (%) b 68.4±2.0 67.4±1.7 72.8±1.2
Total ammonia nitrogen (TAN) 8.5±0.3 4.3±0.1 3.36±0.18
(g kg− 1) a c

restrictive culture. VFAs (propionic and butyric acids) were used as C/N 6.55 9.65 -
substrates for the enrichment of MC whereas H2/CO2 were used as the pH 8.70±0.0 8.0±0.1 -
main substrate for the enrichment of SS. The two microbial consortia C (g kg− 1) a 125.7±3.4 268.3±4.3 -
H (g kg− 1) a 17.3±0.6 37.5±1.2 -
had different members. The MS was rich in butyric acid degrading
N (g kg− 1) a 19.2±0.5 27.8±0.4 -
bacteria, propionic acid degrading bacteria, SAOB, and HM. The SS was O (g kg− 1) a 96.8±1.8 198.8±2.2 -
mainly composed of HM. These two microbial consortia were applied to S (g kg− 1) a 3.4±0.18 7.45±0.25 -
recover and enhance the digestion performance of an AD system *
Chicken manure is dried at 50 ◦ C to volatilize free ammonia nitrogen fully
impacted by high ammonia nitrogen. The effects of these two microbial a
based on the fresh matter
consortia on the AD system were explored at three levels: digestion b
based on total solid
performance, microorganism, and metabolic pathway. First, from the c
mg L− 1
macro-level, the effects of MC and SS on digestion performance, All tests were carried out three times, and the values in the table are average.

2
S. Wang et al.
used as carbon sources of the medium. Specifically, the composition of
the medium was as follows (1 L system): 0.6 g CH3CH2COONa, 0.6 g
CH3CH2CH2COONa, 400 mg NH4Cl, 250 mg MgSO4•6H2O, 400 mg KCl,
120 mg CaCl2•2H2O, 80 mg (NH4)2HPO4, 55 mg FeCl3•6H2O, and 5000
mg NaCO3 (Jiang et al., 2020). Further, in order to make sure that MC
with high tolerance to ammonia nitrogen, urea was used to gradually
adjust the TAN concentration from 4 to 8-9 g L− 1 during acclimatization
and enrichment. When the TAN concentration reached to 8-9 g L− 1, the
reactor was continuously operated under the high TAN concentration
for 40 days to obtain the MC.
For the SS, according to the reaction formula of hydrogenotrophic
methanogen for methane production: 4H2+CO2→CH4+2H2O, the sub­
strate configuration was carried out at the volume ratio of H2: CO2=4:1.
To ensure the anaerobic environment of the system, a portion of N2 was
added to mixed the gas. The configured mixed gas was injected into the
wet gas storage tank, and then the gas in the gas storage tank was cycled
into the reactor using the gas circulation pump. Later, the gas in the
anaerobic reactor was again circulated to the gas storage tank. With the
consumption of H2 and CO2, the suspension hood of the gas storage tank
gradually decreased. When the remaining gas was 1/3 of the original
volume, the gas was extracted, and the mixed gas was reconfigured for
circulation. During the whole cycle, the urea treated with urease (EC
3.5.1.5) was used as the nitrogen source to gradually adjust the TAN
concentration to 8-9 g L− 1. Then, the reactor was continuously operated
for 40 days at the high TAN concentration. After that, the acclimatiza­
tion and enrichment of SS were completed. The mixture in the reactor
was centrifuged (3214-8228 g for 5-10 mins) to obtain the SS. Table 3
showed the main members of bacteria and archaea (genus level) in the
inoculum before domestication, MC, and SS.
2.2.2. Preparation of additives for MC treatment, SS treatment, S-MC
treatment, and S-SS treatment
In section 2.2.1, MC and SS were obtained by centrifugation. MC and
SS were the biological additives of MC and SS treatments, respectively.
Since, MC and SS also contain a certain amount of ash and other sub­
stances, which may interfere with the experiment. Therefore, MC and SS
were sterilized at 121◦ C for 15 min, and later they were used as the
additives for S-MC treatment and S-SS treatment, respectively. To avoid
Fig. 1. Experiment design and flow chart.
3
Water Research 230 (2023) 119583
the decline of microbial activity caused by repeated freezing and
thawing, all the additives (MC, sterilized MC, SS, and sterilized SS) were
aliquoted into 2 mL centrifuge tubes. At the end, they were stored at
− 80◦ C and defrosted for four hours at room temperature before use.
2.3. Set-up and sampling of the semi-continuous experiment
In the semi-continuous experiment, four treatments (MC treatment,
S-MC treatment, SS treatment, and S-SS treatment) and one control were
included. For all treatments, the reactors of 4 L working volume were
used. The agitation speed of the reactors was kept at 120 rpm, and the
digestion temperature (37±1◦ C) was controlled with the circulation of
hot water. At the beginning of the experiment, 4 L inoculum was added
in every reactor, and the OLR was about 3.5 g VS L− 1 d− 1. The reactors
were fed daily, and the substrate was prepared in a beaker. Each time,
about 200 mL of biogas slurry was taken. Consequently, 27 g of deni­
trified chicken manure was added into a beaker, and makeup to 200 g
with distilled water. The substrate was mixed uniformly and then
poured into the reactor to ensure the water level line was at the 4 L mark.
Fig. 1 shows the flow of the whole experiment, which consists of four
phases based on different digestion settings. In the first phase (0-40
days), all the reactors were operated under consistent conditions to
create the same digestion performance, and bioaugmentation and the
regulation of TAN concentration were not performed. Therefore, it was
named as a start-up phase. In the second phase (41-80 days), 4 g urea
was added to each reactor per day around 41-47 days to quickly improve
the TAN concentration from 3-4 g L− 1 to 8-9 g L− 1. During 48-80 days,
the urea loading was adjusted from 4 to 0.8 g d− 1 to maintain the high
TAN concentration in the reactors. Therefore, the second phase was
named as the ammonia-shocking phase. Further, in the second phase,
the reactors were operated under different conditions, and they were
named as control, MC treatment, S-MC treatment, SS treatment, and S-
SS treatment. Table 2 shows the specific meaning and settings of these
treatments. In the third (81-120 days) and fourth (121-200 days) phases,
the reactors continued to maintain at high TAN concentration (8-9 g
L− 1), and the reactors performance started to recover and stabilize.
Therefore, they were named recovery and stable phases, respectively.
The sampling frequency was every five days to determine the
S. Wang et al. Water Research 230 (2023) 119583

Table 2 2.5. Carbon isotope analysis

The meaning of different treatments in this experiment.
Treatments The meaning of different treatments Generally, the dominant methanogenesis pathway could be roughly
inferred from the proportions of methanogenic archaea. However, some
Control No MC and SS were supplied.
MC treatment 1.2 g TS MC was added to the reactor every day. methanogenic archaea might not be able to perform their functions at
S-MC treatment 1.2 g sterilized MC was added to the reactor every day. high ammonia nitrogen concentrations, such as Methanosaeta (Cap­
SS treatment 1.2 g TS SS was added to the reactor every day. son-Tojo et al., 2020). Therefore, this study used the isotopic tracer
S-SS treatment 1.2 g sterilized SS was added to the reactor every day. technique to explore the change in the methanogenesis pathway during
different phases. The biogas samples were taken in the middle of the
phase II, III and IV, which were further used for the carbon isotope
Table 3 analysis. 13C signature of δ 13CH4 and δ 13CO2 in biogas samples were
The main members of bacteria and archaea (genus level) in the inoculum before measured using a GC (Trace Ultra GC, CA, USA) equipped with a stable
domestication, MC, and SS. isotope ratio mass spectrometer (Delta V Advantage, CA, USA). δ 13CH4
Taxon % and δ 13CO2 were calculated using V-PDB as the reference. Carbon
Inoculum before MC SS isotope fractionation (αc) was calculated based on equation (2) (Whiti­
domestication
car et al., 1986):
Bacteria Proteiniphilum 23.09 7.3 - (
(Genus) Ignatzschineria 9.67 1.3 - αc = δ13 CO2 +1000)/(δ13 CH4 + 1000) (2)
Bacteroides 7.41 4.1 -
Erysipelothrix 4.55 1.5 - The contribution of different methanogenesis pathways in digestion
Tepidanaerobacter 1.57 3.6 - is referenced to the αc. The hydrogenotrophic pathway was the domi­
Clostridium 1.1 4.6 - nant methanogenesis pathway when the αc was higher than 1.065. While
Syntrophomonas 0.6 16.6 -
the acetoclastic pathway would be dominant when αc was lower than
Pseudomonas 0.29 3.4 -
Syntrophobacter 0.8 18.2 - 1.055 (Conrad et al., 2005).
Tissierella 0.1 6.3 -
Archaea Methanosaeta 77.3 1.4 0 2.6. Microbial analysis and enzyme gene prediction
(Genus) Mathanosarcina 4.9 12.0 16.7
Methanobrevibacter 6.5 5.6 0.6
DNA extraction Kit (Omega Bio-Tek, Norcross, GA, USA) was used to
Methanoculleus 3.7 45.7 71.2
Methanospirillum 1.4 4.9 5.2 extract the DNA of the samples. Then, V3-V4 regions of DNA were
Methanothermobacter 1.9 21.4 6.2 amplified using specific primers. For the bacteria and archaea, they were
Methanobacterium 3.0 4.7 0.1 338F 5′ -ACTCCTACGGGAGGCAGCAG-3′ and 806R 5′ -GGACTACH
Methanimicrococcus 3.5 4.9 0
VGGGTWTCTAAT-3′ , and 524F10extF 5′ -TGYCAGCCGCCGCGGTAA-3′
Note: Only bacteria or archaea with a proportion greater than 3% will be and Arch958RmodR 5′ - YCCGGCGTTGAVTCCAATT-3′ ), respectively.
displayed. The amplification program was performed in a PCR system (GeneAmp
9700, ABI, CA, USA) and further detail can be found in a previous study
physicochemical parameters (TAN concentration, sCOD, VFAs concen­ (Zheng et al., 2021). Sequencing was performed on an Illumina MiSeq
tration, alkalinity, and pH). 10 mL biogas slurry was taken in the middle platform (Illumina, San Diego, USA), and this part of the work was
of the phase II, III and IV for high-throughput sequencing of bacteria and performed by Majorbio Technology. According to the sequencing re­
archaea. These samples were stored at -20◦ C for further analysis. sults, the key enzyme gene was annotated in the module of "functional
predictive analysis" in the cloud platform developed by Majorbio
2.4. Physicochemical parameters analysis Technology. The enzyme was marked in the "Energy Metabolism"
module in the Kyoto Encyclopedia of Genes and Genomes (KEGG)
A gas counter (MGC-1 V3.1, Ritter GmbH, Germany) was used to (https://www.kegg.jp/).
measure the biogas volume. The contents of CH4, CO2, and H2 were
measured using a biogas analyzer (Biogas 5000, Geotech, Germany). TS, 2.7. Statistical analysis
VS, alkalinity, total carbon (TC), and total nitrogen (TN) of the substrate,
inoculum, and biogas slurry were determined based on the standard Statistical analysis was performed among different treatments for
method (APHA, 2005). pH was measured by a pH meter (SX610, Sanxi, some important digestion parameters, such as the pH, alkalinity, VFAs/
China). The VFAs concentration was determined using high- TIC, 13C apparent fractionation factor (αc), TAN, and FAN concentration.
performance liquid chromatography (HPLC) based on the previously The data was significantly different when the p was less than 0.05.
described method by (Zhao et al., 2016). The elements of C, H, N, S, and Bacteria, archaea, and other key parameters including organic acids
O were measured using an elemental analyzer (Vario micro cube, Ele­ concentration, H2 content, sCOD, alkalinity, methane content and yield,
mentar, Germany). The samples were fully combusted and converted TAN, and TS removal rate were used for Redundancy analysis (RDA),
into gases with the help of oxygen. These gases were further separated and the additional detail could be found in the supplementary materials.
by the separation chromatographic column, and then were tested by a The arrow length represents the strength of the correlation between the
thermal conductivity detector and infrared detector. A flow analyzer environmental variables and the microbes. The longer the arrow length,
(AA3, SEAL, Germany) was used to test the TAN concentrations. The free the stronger the correlation. The perpendicular distance between mi­
ammonia nitrogen (FAN) concentration was calculated based on equa­ crobes and environmental variable axes in the plot is presented their
tion (1): correlation. The smaller the distance, the stronger the correlation.
( )− 1
Canoco 4.5 package for Windows was used to perform the RDA. The
process was analyzed by IBM SPSS Statistics 25.0 (IBM Co., Armonk, NY,
pH
10−
[FAN] = [TAN] × 1 + ( ) (1)
− 0.09018+2729.92
T(k)
USA), and AutoCAD was used to draw the diagram of the reactors.
10
Where T(K) is the kelvin temperature.

4
S. Wang et al.
3. Results and discussion
3.1. Effect of MC and SS on the digestion performance of AD system with
high TAN concentration
As shown in Fig. 2a, the digestion performance was stable for all
treatments, and the methane yield was approximately of 320 mL g− 1 VS
during phase 1 (start-up phase). By adding the urea in all reactors, the
TAN concentration was promptly increased to 8-9 g L− 1 during phase 2
(shocking phase), which resulted in the rapid deterioration of the
digestion system. In addition, it was found that the control, S-SS, and S-
MC treatments completely lost their ability to produce the methane at
the end of phase 2. While the MS treatment produced the methane yield
as low as 211 mL g− 1 VS resulted of digestion performance, which fully
recovered in about 20 days. On the other hand, 184 mL g− 1 VS of
methane was obtained from SS treatment (at the end of phase), resulting
in slow recovery of digestion performance, and the methanogenesis
performance completely recovered after the 80th day. Further, it was
also observed that the methane content had a similar trend with the
methane yield (see Fig. 2(a, b)). In addition, the methane content of MC
treatment and SS treatment was in the range of 47%-68%. While the
methane content of S-SS treatment, S-MC treatment, and control reached
about 20% at the end of phase 2. H2 is the product of the hydrolysis and
acidification of the substrate, and its trend was opposite to that of
methane yield. Under the shock of high TAN concentrations, the H2
content raised rapidly. For the control, S-SS treatment, and S-MC
treatment, the H2 content finally reached up to 1400 ppm, which might
be related to the activity of hydrogenotrophic methanogens (discussed
in section 3.4 in detail). Since, H2 was also the substrate for methane
production, the elevated H2 concentration also reflected that hydro­
genotrophic methanogen didn’t fully convert the H2 to methane.
Furthermore, organic acids were the product of the acidification of
substrates. During phase 2, organic acids accumulated rapidly, showing
that methanogenic archaea could not effectively utilize organic acids in
Fig. 2. The methane yield (a), CH4 content (b), H2 concentration (c), and organic acids concentration (d) in control, MC treatment, S-MC treatment, SS treatment,
and S-SS treatment.
5
Water Research 230 (2023) 119583
phase 2. For the control, S-SS treatment, and S-MC treatment, the total
organic acid concentration was about 8 g L− 1 when the digestion system
deteriorated. On the other hand, there was only a brief increase in the
total organic acid concentration in MS treatment and SS treatment. It
was also observed that the organic acids concentrations were become
lower than 3 g L− 1 during the phase 4 (see Fig. 2d). It indicated that the
digestion system become vulnerable to the interference of ammonia
nitrogen when the TAN concentration fluctuated or increased suddenly
(Cai et al., 2021a). Therefore, the effect of ammonia nitrogen on the
digestion system was not negligible at the start-up stage of the
large-scale biogas project when nitrogen rich-substrate was used. Based
on the results of digestion performance after bioaugmentation using MC
and SS, it was recommended to use microbial consortiums containing
both bacteria and archaea with syntrophic relationships to assist the AD
systems with high ammonia nitrogen.
3.2. Effect of MC and SS on the stability of AD system with high TAN
concentrations
The stability of the AD system is important for high digestion per­
formance (Ajayi-Banji et al., 2022). The key indicators such as alka­
linity, pH, TAN concentration, and VFA/TIC were reflecting the stability
of the AD system (Bah et al., 2014). As shown in Fig. 3 (a-d) and Fig. S2,
these indicators were similar among all treatments and control at phase
1 due to the same digestion condition. In phase 2, the stability of
different treatments began to differ. During phase 2, the alkalinity and
pH of MC treatment and SS treatment were higher than those of S-SS
treatment, while S-MC treatment and control had an opposite trend for
VFA/TIC (see Fig. 3). pH is a vital indicator influencing on the microbial
growth. It was reported that methanogenic archaea are extremely sen­
sitive to pH, and the optimal pH of methanogenic archaea is in the range
of 6.5–8.5 (Cai et al., 2021a). As shown in Fig. 3a, the lowest pH values
for the control, S-SS, and S-MC treatments were 5.8 during phase 2 due
to the accumulation of VFAs. At this condition, the methanogenic
S. Wang et al.
Fig. 3. The alkalinity (a), pH (b), ammonia concentration (c), and the ratio of VFAs/TIC (d) in control treatment (square), MC treatment (circular), S-MC treatment
(plus), SS treatment (cross), and S-SS treatment (triangle).
archaea were almost incapable of producing methane. For MC treatment
and SS treatment, the pH was between 6.5 and 7.8, which was in the
optimal pH range for methane production. Compared with the SS
treatment, the pH of the MC treatment was significantly (p < 0.01)
higher, which might be due to the higher organic acids accumulation in
the SS treatment in phase 2. The digestion system had a high buffer
capacity when the VFA/TIC ratio become lower than 0.4. As shown in
Fig. 3d, the MC and SS treatments were suppressed to a certain extent
during phase 2, the VFA/TIC ratio was still less than 0.4 (see Fig. 3d). For
the Control, S-SS treatment, and S-MC treatment, the VFA/TIC ratio rose
rapidly to higher side (> 0.4), accompanied by deterioration the per­
formance of digestion system. As shown in Fig. S2, there was no sig­
nificant (p > 0.05) difference in TAN concentration among all groups.
This meant that it was reasonable to use urea as the regulator of TAN
concentration, and all digestion systems could effectively hydrolyze the
urea to produce ammonia nitrogen. During phase 3, the pH, alkalinity,
and VFA/TIC of the MC treatment were higher than that of the SS
treatment (see Fig. S2) because ammonia inhibition was higher at the
end of phase 2 in MC treatment. During phase 4 (see Fig. 2a), the
digestion performance of MS treatment and SS treatment recovered. By
considering these four indicators (alkalinity, pH, TAN concentration,
and VFA/TIC), there was no significant difference (p > 0.05) between
MS treatment and SS treatment.
3.3. Effect of MC and SS on the concentration of VFAs and sCOD
Propionic, butyric, acetic, formic, and lactic acids were the most
common organic acids in the AD system Wei et al., 2022). Fig. 4 showed
the composition and change of these organic acids. Acetic acid was the
direct substrate for acetoclastic methanogen. Propionic acid and butyric
acid could be converted to acetate, CO2 and H2 by propionate and
6
Water Research 230 (2023) 119583
butyrate-degrading bacteria (Wu et al., 2022). In phase 1, the total VFA
concentration was lower than 1 g L− 1. During phase 2, the VFAs con­
centration increased rapidly, especially for the control, S-MC treatment
and S-SS treatment. Butyric acid and propionic acid had the fastest
accumulation speed, which might associate with the thermodynamic
characteristics of propionic and butyric acids due to the positive Gibbs
free energy change of conversion from butyric and propionic acids to
acetic acid (Pan et al., 2021) (see Eq 6-(9).
(6)
′
CH3 CH2 COO− +3H2 O=CH3 COO− +HCO−3 +H+ +3H2 ,ΔGo =+76.2kJ
(7)
′
CH3 CH2 CH2 COO − + 2H2 O = 2CH3 COO− +2H2 , ΔGo = +48.4kJ
(8)
′
CH3 COO− + H2 O = CH4 +HCO−3 + H+ , ΔGo = − 31.0kJ
4H2 + CO2 = CH4 + 2H2 O, ΔGo’ = − 135.0kJ (9)
At the end of phase 2, the concentration of the butyric and propionic
acids in control reached to 4.10 and 1.98 g L− 1, respectively. Interest­
ingly, the accumulation of propionic acid and butyric acid was slightly
lower in the S-SS and S-MC treatments compared to control. This might
be due to some mineral elements or other substances that existed in the
sterilized MC and SS promoted the degradation of organic acids
(Sugiarto et al., 2021). Both MC and SS supplementation could effec­
tively promote the conversion of propionic acid and butyric acid, and
the promotional efficiency of MC was better than that of SS. For
instance, the highest butyric and propionic acid concentrations were of
2.6 and 1.09 g L− 1 in the SS treatment. While the highest concentration
of butyric acid and propionic acid were of 2.1 and 0.81, respectively, in
the MC treatment, which was significantly (p < 0.05) lower than that of
the SS treatment. The similar phenomenon was also observed in phase 3.
The difference might be related to the propionic acid and butyric acid
S. Wang et al.
Fig. 4. The composition of VFAs in Control (a), MC treatment (b), S-MC treatment (c), SS treatment (d), and S-SS treatment (e).
oxidizing bacteria that existed in MC. In phase 4, there was no signifi­
cant (p > 0.05) difference in the composition and concentration of VFAs
in MC and SS treatments. The change of sCOD had a similar trend with
the change of VFAs. Simple molecules, such as glucose monomers,
amino acids, and VFAs (acetic acid, propionic acid, butyric acid, and
formic acid) are the main components of sCOD (Yadav et al., 2022). This
study showed a positive correlation between sCOD and total VFA con­
centration. In the S-SS treatment and S-MC treatment, the sCOD reached
to 16000 mg L− 1, while the sCOD of the MC treatment and SS treatment
was only gained to 9000 mg L− 1.
3.4. Effect of MC and SS on the microbial structure and methanogenic
pathway
Bacteria are the major players in the hydrolysis, acidogenic, and
acetogenic stages of AD system. Fig. 5 shows the composition and
abundance of bacteria and archaea in the control and all experimental
treatments in phases 1 to 4. Proteniphilum (23.1%), Ignatzschineria
(9.7%), Bacteroides (7.4%), and Erysipelothrix (4.6%) were the dominant
bacteria in phase I. In this study, the substrate was chicken manure,
containing contained high crude protein content (Kafle et al., 2016).
7
Water Research 230 (2023) 119583
Therefore, Proteniphilum, a protein-decomposing bacteria (Ao et al.,
2020), was dominant in all treatments. Ignatzschineria, Bacteroides, and
Erysipelothrix could decompose the macromolecules into organic acids
(Yang et al., 2017). Although these bacteria had high abundance (Pro­
teniphilum 10.1%–23.1%; Ignatzschineria 4.2%–9.7%; Bacteroides 3.4%–
8.4%; Erysipelothrix 3.7%–5.7%) in the whole digestion process, their
abundance was markedly different among the treatments. For instance,
relative to the control, the abundance of Proteniphilum, Ignatzschineria,
and Bacteroides in the MC treatment decreased by 28.4%, 26.3%, and
50.0%, respectively, in phase 2. Interestingly, some other microorgan­
isms in the MC replaced the original bacteria, such as Tissierella, which
could break down protein (Chen et al., 2018). Cai et al. (2017) reported
that a variety of bacteria have similar functions implying that the
functions of bacteria are redundant. Therefore, in the MC treatment, a
new bacterial community was established due to the supplementation of
MC. Among these rich bacteria in MC, the colonization of Syntropho­
monas, Syntrophobacter, Tepidanaerobacter, and Tissierella was apparent.
Relative to the control, the abundance of Syntrophomonas, Syntropho­
bacter, Tepidanaerobacter, and Tissierella in the MC treatment increased
by 11.7, 10.3, 1.7, and 38.0 times in the phase 2. It was reported that
these four bacteria have ability to degrade VFAs (Wang et al., 2021;
S. Wang et al. Water Research 230 (2023) 119583

Fig. 5. The composition of microorganisms (bacteria and archaea) in control, MC treatment, S-MC treatment, SS treatment, and S-SS treatment at the genus level in
the whole experiment.

Ziels et al., 2017). The low concentration of VFAs in the MC treatment lower than 1.055 and did not have a significant difference (p > 0.05) in
indicated that their colonization and growth were enhanced in the AD the first phase since all reactors were operated under the same condi­
system. Tepidanaerobacter and Tissierella were typical SAOBs (Solli et al., tions. Therefore, the dominant methanogenesis pathway in the first
2018). Syntrophomonas and Syntrophobacter efficiently degraded the phase was the acetoclastic pathway. This is consistent with the finding of
propionic acid and butyric acid. In the third and fourth phases, the previous studies. For instance, Pan et al. (2021) showed that 67% of
abundance of Syntrophomonas and Syntrophobacter in the MC treatment methane production was contributed by the acetoclastic methano­
and SS treatment reached to 7% and 9%, respectively, and the abun­ genesis pathway when the AD system was not stressed by toxic sub­
dance of Tepidanaerobacter and Tissierella was higher than 2%. Inter­ stances. A previous study reported that Methanosaeta could hardly
estingly, although the SS didn’t initially contain Tepidanaerobacter and survive and function properly when the TAN concentration was higher
Tissierella, their abundance also increased in the SS treatment along with than 8 g L− 1 (Capson-Tojo et al., 2020; Cai et al., 2021b). In phase 2, the
the adaptation of the reactor to high TAN concentration and the re­ TAN concentration was close to 9 g L− 1. Capson-Tojo et al. (2020)
covery of digestion performance. This indicated that the archaea showed that hydrogenotrophic methanogens had higher ammonia ni­
occurring in the SS provided a suitable environment or promoted the trogen tolerance (Capson-Tojo et al., 2020). In phase 2, the abundance of
growth of these bacteria. Additionally, these findings showed that the Methanosaeta decreased whereas that of Methanothermobacter, Matha­
archaea in the SS promoted the formation of the syntrophic relationship nosarcina, and Methanoculleus increased. Interestingly, the most abun­
between archaea and these bacteria. Moreover, the change in abundance dant archaea, Methanoculleus, in SS (71.2%) and MC (45.7%) did not
of some microorganism was not directly related to bioaugmentation. For grow rapidly in both treatments. It was found that the abundance of
instance, the abundance of Clostridium increased nearly 10 times in all Methanospirillum (1.9x increase) and Methanothermobacter (11.7x in­
treatments and the control in phase 2. One possible cause was that crease) rose substantially with the rapid increase in the TAN concen­
Clostridium tended to form spores to resist the extreme environment tration in phase 2. This meant that Methanothermobacter might have
(Canada et al., 1964). In phase 2, the TAN concentration increased played the most important role in methane recovery in phase 2. Meth­
rapidly, which might also be the main reason for the rapid increase in anoculleus was considered the archaea with the highest tolerance to
the abundance of Clostridium. ammonia nitrogen (Capson-Tojo et al., 2020). In this study, Meth­
Archaea are responsible for converting organic acids and CO2/H2 to anoculleus could not rapidly grow to became the dominant archaea at the
methane(Cai et al., 2022). Eight methanogenic archaea were identified beginning of ammonia inhibition (phase 2). This showed an advantages
at the genus level (Fig. 5). Six of them were hydrogenotrophic metha­
nogens (Methanobacterium, Methanobrevibacter, Methanothermobacter,
Methanoculleus, Methanospirillum, and Methanimicrococcus). Meth­ Table 4
anosaeta was a typical acetoclastic methanogen. Mathanosarcina could The 13C apparent fractionation factor (αc) in the four phases for all treatments.
produce methane through three methanogenic pathways. In this study, Phase I Phase II Phase III Phase IV
the carbon isotope technology was used to analyze changes to the
CK 1.051 1.154 - -
methanogenic pathway caused by bioaugmentation. It is commonly MC 1.052 1.274 1.282 1.281
believed that the dominant methanogenesis pathways were hydro­ S-MC 1.051 1.153 - -
genotrophic methanogenesis and acetoclastic methanogenesis, respec­ SS 1.052 1.219 1.249 1.278
tively, when αc > 1.065 and < 1.055 (Yang et al., 2019). In phase 1, S-SS 1.052 1.149 - -
Methanosaeta had the highest abundance, accounting for more than 40% Note: “-” means undetermined. The 13
C apparent fractionation factor (αC) was
of the microbial consortium. In addition, as shown in Table 4, αc was calculated using Eq. (2).

8
S. Wang et al.
of microbial consortia as biological additives over single archaea, is that
they reduce the uncertainty, which was mentioned above. During phase
2, the change in the archaea community structure and the abundance of
Methanosaeta, Methanothermobacter, Mathanosarcina, and Meth­
anoculleus also influenced the nature of succession of methanogenic
pathway. As shown in Table 4, during the second phase, αc was higher
than 1.065. Therefore, the hydrogenotrophic methanogenesis pathway
was the dominant pathway. The αc in the MC treatment (αc=1.274) and
SS treatment (αc=1.219) was higher than that in the control (αc=1.154),
S-SS (αc=1.149), and S-MC (αc=1.153) treatments, indicating that the
contribution of the hydrogenotrophic pathway was higher in the MC and
SS treatments. A previous study reported that Methanoculleus still pro­
duced methane efficiently even when the TAN concentration was higher
than 25 g L− 1 (Poirier et al., 2016). In this study, Methanoculleus showed
unique growth ability in phases 3 and 4. Mathanosarcina, which had a
high abundance in SS treatment and MC treatment, did not proliferate in
phases 3 and 4. In short, the recovery of digestion performance depen­
ded on Methanospirillum and Methanothermobacter at the early stage of
the ammonia inhibition and Methanoculleus at the later stage. We could
speculate from these finding that which archaea in the microbial con­
sortia played a major role was closely related to the reactor environ­
ment, such as ammonia nitrogen conditions. In phase 3, the αc continued
to increase from 1.219-1.274 to 1.249-1.282, which meant that the
contribution of the hydrogenotrophic pathway rose. In phase 4, there
was no significant difference (p > 0.05) in αc of MC treatment and SS
treatment, indicating that the hydrogenotrophic pathway contributed
equally to the both treatments.
3.5. The relationship between critical parameters and microorganisms
Fig. 6 shows the redundancy analysis (RDA) between microorgan­
isms (bacteria (a) and archaea (b)) and key digestion parameters. As
Fig. 6. Redundancy analysis (RDA) between critical parameters and bacteria
(a) and archaea(b). The numbers behind every treatment refer to the diges­
tion phase.
9
Water Research 230 (2023) 119583
shown in Fig. 6a, the samples were divided into three clusters, the first
cluster between MC-2, MC-3, and MC-4; the second cluster among SS-2,
SS-3, and SS-4; and the third cluster between C-2, S-SS-2, and S-MC-2.
This meant that bioaugmentation affected the bacterial composition of
the digestive system, and the effect was not limited by digestion phase.
As shown in Fig. 6a, the abundances of Clostridium, Proteiniphilum,
Ignatzschineria, Bacteroides, and Alkaliphilus were positively correlated
with VFA (except for acetic acid) concentration, H2 content, and sCOD.
Previous studies reported that these bacteria were mainly involved in
hydrolysis, acidogenesis, and acetogenesis stages (Ao et al., 2020; Yang
et al., 2017; Awasthi et al., 2022). Except for Clostridium, all other SAOBs
were negatively correlated with acetic acid concentration, and Tissierella
had the highest correlation with acetic acid concentration. This indi­
cated that the function of Clostridium to oxidize the acetic acid could be
inhibited by high TAN concentration (Zhang et al., 2022). Syntropho­
monas (butyrate-oxidizing bacteria) and Syntrophobacter (propiona­
te-oxidizing bacteria) showed a clear negative correlation with butyrate
and propionate concentrations, respectively. Therefore, the high abun­
dance of Syntrophomonas and Syntrophobacter might be majorly
contributing to alleviating the accumulation of propionic acid and
butyric acid (Li et al., 2022). Four of the five bacteria (Tepidanaerobacter,
Clostridium, Syntrophomonas, Syntrophobacter, and Tissierella) were rich
in MC, Tepidanaerobacter, Syntrophomonas, Syntrophobacter, and Tissier­
ella were negatively correlated with the concentration of VFAs and
positively correlated methane yield. This meant that these bacteria
abundant in MC played a positive role in reconstructing the bacterial
community in the ammonia-inhibited AD system.
The regularity of the correlations between methanogenic archaea
and digestion parameters differed from that of bacteria (Fig. 6b).
Although bioaugmentation also changed with the community structure
of archaea, the difference in the effects of adding SS and MC was
negligible during the period of ammonia nitrogen rapid shock (phase 2)
which was due to the SS-2 and MC-2 closest to each other. Additionally,
the composition of archaea in phase 2 differed from that in the phases 3
and 4, which indicated that the change of TAN concentration affected
the growth of methanogenic archaea and TAN concentration was the key
factor affecting the results of bioaugmentation. The most positive and
negative correlation with TAN concentration was Methanoculleus and
Methanosaeta, respectively, which was consistent with findings of a
previous study. For instance, Capson-Tojo et al. (2020) found that
Methanosaeta hardly survived when the FAN concentration was higher
than 300 mg L− 1 and Methanoculleus could still have high methanogenic
activity when the concentration of FAN was higher than 500 mg L− 1.
The abundance of Methanoculleus was highest in both MC (45.7%) and
SS (71.2%). Methanoculleus had the highest positive correlations with
methane yield and TS removal and it was negatively correlated with
VFAs, H2, and sCOD. This meant that the archaea Methanoculleus in MC
and SS was the major archaea in restoring and maintaining the high
reactor performance (Yan et al., 2020). The role of some other archaea,
such as Methanospirillum, with strong ammonia nitrogen resistance and a
positive correlation with TAN concentration could not be ignored.
Additionally, Mathanosarcina accounted for 12.0% and 16.7% in MC and
SS, respectively. However, this study found no correlation between
Mathanosarcina and TAN concentration, which was inconsistent with
finding of previous studies. For instance, Capson-Tojo et al. (2020) re­
ported that the abundance of Mathanosarcina would increase along with
the rise of TAN concentration. The discrepancy might be due to the di­
versity of Mathanosarcina’s functions. Cai et al. (2021b) reported that
the members of Mathanosarcina could produce methane by three path­
ways, namely acetoclastic pathway, hydrogenotrophic, and methylo­
trophic pathways. When the concentration of ammonia nitrogen
changed, Mathanosarcina resisted high TAN concentration by changing
the methanogenic pathway. Therefore, the abundance of Mathano­
sarcina showed inertia to the change of ammonia nitrogen concentration
in the current study.
S. Wang et al.
3.6. Effect of MC and SS on the gene abundance of key enzymes
Fig. 7(a-b) showed the enzyme genes and their change in the abun­
dances of the propionic acid degradation pathway, butyric acid degra­
dation pathway, hydrogenotrophic pathway, and acetoclastic pathway.
Fig. 7c showed the total abundance of enzyme genes associated with the
four metabolic pathways. As shown in Fig. 7(a-b), a total of eight,
thirteen, six, and three enzyme genes were selected of the propionic acid
degradation pathway, butyric acid degradation pathway, hydro­
genotrophic pathway, and acetoclastic pathway, respectively, due to the
obvious relative change. In this study, the enzyme gene was considered
significantly up-regulated when the ratio of the abundance of the same
enzyme gene from different phases was higher than 1.2. By comparing
with S-MC treatment, 28 enzyme genes were found up-regulated
(+25–59%) in the MC treatment, except for acetate-CoA transfer (EC:
2.8.2.3) and aldehyde dehydrogenase (NAD+) (EC: 1.2.1.3) (Fig. 7b). As
shown in Fig. 7c, the MC treatment had the highest abundance of
enzyme gene related to methane production. Relative to the S-MC
treatment, the gene abundance of enzymes related to the butyric acid
degradation pathway, the propionic acid degradation pathway
(M00013), the hydrogenotrophic pathway (M00567), and the aceto­
clastic pathway (M00357) increased by 20.8%, 34.1%, 42.4%, and
28.2%, respectively. This showed that bacteria and archaea abundant in
the MC played a positive role in strengthening the four pathways related
to methane production. Interestingly, among these enzyme genes, the
supplement of SS (SS treatment) had the greatest impact on the abun­
dance of enzyme genes related to the hydrogenotrophic pathway. The
abundance of formylmetalofuran tetrahydromethanopterin N-for­
myltransferase (EC: 2.3.1.101) and methenyltetrahydromethanopterin
cyclolase (EC: 3.5.4.27) increased by 40.2% and 89.6%, respectively.
This might be related to the abundant hydrogenotrophic methanogens in
SS, such as Methanothermobacter (6.2%) and Methanoculleus (71.2%)
(Yang et al., 2022). It should be noted that the abundance of these
enzyme genes in MC treatment and SS treatment was higher than that of
Fig. 7. The key enzymes related to propionic acid degradation, butyric acid degradation, hydrogenotrophic and acetoclastic pathway (a); the abundance of essential
enzyme genes in MC treatment, S-MC treatment, SS treatment, S-SS treatment (b); the abundance of total enzyme genes related to propionic acid degradation, butyric
acid degradation, hydrogenotrophic and acetoclastic pathway. Note: M00567, M00357, and M00013 mean hydrogenotrophic pathway, acetoclastic pathway, and
propionic acid degradation pathway in the KEGG database (https://www.kegg.jp).
10
Water Research 230 (2023) 119583
S-MC and S-SS treatments, which meant that the main contribution to
recovering digestion performance of AD systems with high ammonia
nitrogen in MC and SS treatments initiated from the microorganisms
existing in the MC and SS, rather than other substances such as ash in MC
and SS (Li et al., 2018). By comparing with the SS treatment, the MC
treatment showed an increase in the abundance of all enzyme genes
from 13.4% to 35.4% except for aldehyde dehydrogenase (NAD+) (EC:
1.2.1.3) (− 1.6%) and methenyltetrahydrometherobin cyclohydrolase
(EC: 3.5.4.27) (− 11.1%). In the propionic acid degradation pathway, the
gene abundance of malate dehydrogenase (EC: 1.1.1.37), pyruvate de­
hydrogenase (acetyl transferring) (EC: 1.2.4.1), fumarate hydratase (EC:
4.2.1.2), methylalonyl CoA mutase (EC: 5.4.99.2), propionyl CoA
carboxylase (EC: 6.4.1.3), and methylalonyl CoA epimerase (EC:
5.1.99.1) increased by 24.1%, 24.1%, 35.7%, 27.4%, 23.2%, and 25.8%,
respectively. These enzymes are responsible to converting the propionic
acid to acetyl CoA (Liu et al., 2021). Further, the enzyme genes of the
MC treatment related to the butyric acid degradation pathway, such as
3-hydroxybutyryl CoA dehydrogenase (EC: 1.1.1.157) (+27.6%), acet­
aldehyde dehydrogenase (acetylating) (EC: 1.2.1.10) (+30.5%), buty­
rate kinase (EC: 2.7.2.7) (+27.3%), propionate CoA transfer (EC:
2.8.3.1) (+25.2%), enoyl-CoA hydratase (EC: 4.2.1.17) (+28.9%),
3-hydroxybutyryl-CoA epimerase (EC: 5.1.2.3) (+25.1%), acetate-CoA
ligase (ADP-forming) (EC: 6.2.1.13) (+25.0%) were significantly upre­
gulated compared with SS treatment. Among them, the enzyme gene of
EC: 1.2.1.10 was up-regulated by 31%. EC: 1.2.1.10 could catalyze
acetyl CoA to acetaldehyde, which was the precursor of acetic acid. The
increase of abundance of enzyme genes related to the degradation of
propionic acid and butyric acid might be related to the enrichment of
propionic acid and butyric acid degrading bacteria, such as Syntropho­
monas and Syntrophobacter (Li et al., 2022). Relative to the SS treatment,
the abundance of enzyme genes related to acetoclastic pathway in the
MC treatment increased by 13%–26.7%. While, only the gene abun­
dance of phosphate acetyltransferase (EC: 2.3.1.8) (responsible for
converting acetyl phosphate to acetyl CoA) was significantly increased
S. Wang et al.
(+26.7%) among these enzyme genes. In the hydrogenotrophic
pathway, the gene abundance of EC: 2.3.1.101 and formylmethylofuran
dehydrogenase subunit A (EC: 1.2.7.12) only increased by 15–18% and
the gene abundance of methylmethylethanopterin cyclohydrolase (EC:
3.5.4.27) decreased by 11%. Therefore, MC did not show obvious
advantage in promoting the hydrogenotrophic pathway than SS, as MC
was mainly composed of propionic acid and butyric acid degrading
bacteria, which didn’t lead to the absolute quantity of hydrogenotrophic
methanogens. The composition of microorganisms in the microbial
consortia was the main factor determining its effectiveness as biological
additive (Yang et al., 2019).
4. Conclusion
The microbial consortia of MC and SS were obtained through pur­
posive acclimatization. The microbial composition of the microbial
consortia is the key to the effectiveness of bioaugmentation. Both MC
and SS could recover the digestion performance of AD in 20 and 80 d,
respectively, when the AD system was shocked by high TAN concen­
tration (>8 g L− 1). The MC performed better in methane production
recovery, which might be related to the abundant propionic and butyric
acid-degrading bacteria in MC, such as Syntrophomonas and Syntropho­
bacter. The RDA showed a clear negative correlation between the
abundance of these bacteria and VFAs concentrations. Methanospirillum
and Methanothermobacter, and Methanoculleus contributed most to
restoring ammonia in MC and SS treatments at the early and later stages
of the ammonia inhibition, respectively. Based on the results of KEGG
and 13C isotopic analyzes, MC and SS showed different mechanisms for
relieving the ammonia inhibition. The MC improved the conversion of
propionic and butyric acids and methane production whereas SS mainly
enhanced the hydrogenotrophic pathway. Overall, the bioaugmentation
is an effective method to enhance mesophilic AD performance of chicken
manure, and the effect of the syntrophic consortium is better than that of
the hydrogenotrophic consortium.
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 study was supported by the National Natural Science Founda­
tion of China (project number: 52200178), the start-up fund of
Zhengzhou University (project number: 32213091), the Open Research
Fund Program of State Environmental Protection Key Laboratory of
Food Chain Pollution Control (project number: FC2022YB06), and Na­
tional Natural Science Foundation of Chian (project number:
52109101). We are very grateful to Yujie Liu for her help on the reactor
drawing.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.watres.2023.119583.
11
Water Research 230 (2023) 119583
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