International Biodeterioration & Biodegradation 143 (2019) 104710

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International Biodeterioration & Biodegradation

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Influence of operation conditions on methane production from swine T

wastewater treated by a self-agitation anaerobic reactor
Mengmeng Jianga,b, Wei Qiaoa,b,∗, Zhengran Rena,b, Ahmed Mahdya,c, Simon M. Wanderaa,b,
Yuyou Lid, Renjie Donga,b
a
College of Engineering, China Agricultural University, Beijing, 100083, China
b
State R&D Center for Efficient Production and Comprehensive Utilization of Biobased Gaseous Fuels, Energy Authority, National Development, and Reform Committee,
Beijing, 100083, China
c
Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, 44511, Zagazig, Egypt
d
Department of Civil and Environmental Engineering, Tohoku University, Sendai, 980-8579, Japan

ARTICLE INFO ABSTRACT

Keywords: This study aimed to evaluate both the feasibility of using ambient temperatures (15, 20 and 25 °C) in a self-
Self-agitation anaerobic baffled reactor agitated anaerobic baffled reactor (SaABR) fed with swine wastewater, and the impact of decreasing the hy-
Swine wastewater draulic retention time (HRT) down to 3 days. The highest specific methane yield (0.61 ± 0.14 L/gVSin) and
Hydraulic retention time organics conversation efficiency (66% on TCOD basis) were attained by elevating temperature from 15 to 25 °C
Ambient temperature
under an HRT of 10 days. Even though the highest volumetric methane yield (0.85 ± 0.17 L/L/day) was re-
Methanogenic activity
gistered during a 3-day HRT, the specific methane yield was lower as a result of volatile fatty acids accumu-
lations (up to 790 ± 39 mg/L). The dominant bacterium was Clostridium sensu stricto while Methanosaeta was
the dominant archaea throughout all the conditions, indicating strong acetoclastic methanogenic activity under
ambient temperatures. These results demonstrate that using ambient temperatures and short HRTs is a promising
strategy to successfully treat wastewater with decreasing energetics entries. This can, thereby, increase the
treatment capacity and enhance the methane yield during the anaerobic digestion process.

1. Introduction
Wastewater from the pig industry can cause serious water and en-
vironmental pollution if not properly managed. A number of methods
are currently used to treat wastewater from piggeries; however, anae-
robic digestion (AD) is preferred because it can both treat and retrieve
energy in the form of methane. AD is a traditional technology that is
widely used to treat animal manure and wastewater (Yuan et al., 2017;
Huynh et al., 2019). Furthermore, the digestate from the process can
also be served as a kind of organic compost for soil conditioner (Meng
et al., 2018).
Various reactor configurations, such as a continuously stirred tank
reactor (CSTR) and an up-flow anaerobic sludge blanket (UASB) are
available in the marketplace. However, different reactor configurations
should be specifically designed for the different feedstock fed into the
anaerobic digester. In this context, the anaerobic baffled reactor (ABR)
is one of the preferred methods for treating swine wastewater due to its
robustness, better performance, and non-requirement of power for
mixing (Plumb et al., 2001; Peng et al., 2013). However, it has been
reported that the ABR has experienced problems due to unsatisfactory
mixing in the reactor as an adequate mixing is vital for improving mass
transfer and enhancing methane production (Li et al., 2007; Karim
et al., 2005). Modified from the normal ABR, a self-agitation anaerobic
bioreactor (SaABR) system, consisting of a container, partition plates
and a U-tube, was developed and used for treating artificial food waste
(Qi et al., 2015). The SaABR is designed to make the liquid to be self-
agitated without an external energy through the function of a U-tube.
While there are many factors influencing the process of AD, the key
among them are: feedstock type, organic loading rate (OLR) (Moestedt
et al., 2014; Park et al., 2016; Bayrakdar et al., 2017), the temperature
and HRT (Cho et al., 2017; Liu et al., 2018). Previous studies have been
conducted under mesophilic and thermophilic conditions, but main-
taining the high temperature (35–55 °C) consumes a considerable
amount of energy during heating process (Naegele et al., 2012), as well
as increases the risk of ammonia inhibition (Deng et al., 2014). Some
reports demonstrated the possibility of operating AD process success-
fully at 15–24 °C (Deng et al., 2014; Wei et al., 2014). Nevertheless, the
morphology of granular sludge became irregular and complex when

∗
Corresponding author. College of Engineering, China Agricultural University, Beijing, 100083, China.
E-mail address: qiaowei@cau.edu.cn (W. Qiao).

https://doi.org/10.1016/j.ibiod.2019.05.027
Received 18 March 2019; Received in revised form 15 May 2019; Accepted 25 May 2019
Available online 04 June 2019
0964-8305/ © 2019 Elsevier Ltd. All rights reserved.
M. Jiang, et al. International Biodeterioration & Biodegradation 143 (2019) 104710

operated at 15 °C (Banik et al., 1997). On the other hand, shortening the
HRT can obviously improve the volumetric biogas production yield
(Wandera et al., 2018); and meanwhile, the microbial community may
be considerably influenced (Dennehy et al., 2017; Win et al., 2015; Kim
et al., 2013). It was reported that the relative abundance of Methano-
sarcina decreased while Methanoculleus kept stable as the HRT de-
creased from 21 days to 10.5 days in a digester treating pig manure and
food waste (Dennehy et al., 2017). In addition, it has been reported by
Banik et al. (1997) that a reduction in the specific methanogenic ac-
tivity (SMA) from 1.91 to 1.62 g-COD/g-VSS/d at a HRT of 1 day was
taken place when the temperature was decreased from 25 to 5 °C during
the treatment of swine wastewater with ASBR. Assessing these para-
meters (ambient temperature and HRT) for the SaABR is necessary in
order to study their impact on process performance and stability. In
fact, due to the special structure of such reactors, the hydrolysis, acet-
ogenesis and methanogenesis stages may take place in different com-
partments (Li et al., 2007). This fact is further supported by Li et al.
(2013) who found a dominance of hydrogenotrophic methanogens in-
cluding Methanoregula and Methanosphaerula in the first two compart-
ments, while acetotrophic methanogen Methanosaeta was dominant in
the last two.
The current investigation aimed to evaluate the operation of a
SaABR at a range of ambient temperatures (15, 20 and 25 °C) with the
same HRT (10 days) in order to explore the impact of temperature on
the AD process. To achieve this, the performance of the self-agitated
reactors was monitored, and organic matter removal, VFAs level, and
volumetric/specific methane yield were determined. The reactor cap-
ability was further studied to explore the impact of shortening the HRT
on process performance and stability. An additional aim was to reveal
the effect of these parameters on AD microbiome activity and abun-
dance using maximum consumption rates of different substrates and
16S rRNA gene sequencing, respectively.
2.1. Operation of the SaABR
The SaABR used in this study is depicted in Fig. 1. The SaABR was
functioned at three different temperatures (15, 20 and 25 °C). The
SaABR had a 16 L and 10 L total and working volume, respectively. It
consisted of four compartments, with a U-tube settled between the 1st
and 2nd compartments. The upper part, between the 1st and the 2nd
compartment, was completely sealed, and the biogas produced by the
AD process was stored in the 1st compartment. The liquid level of the
first compartment was lowered by gas pressure. Once the liquid level
was lower than the bottom of U-tube, a stirring occurred but this was
completed rapidly. The second, third and fourth compartments were
connected to each other, and the biogas produced could be directly
discharged and collected by the air bag. A timer and peristaltic pump
(Shenchen BT100N, China) were used to automatically feed it every
day. The digesters were kept under different temperature conditions by
circulating water through water jacket. Feedstock was kept under 4 °C
conditions in a substrate tank connected to a cooling water bath (HX-
105). Effluent from sampling port (SP) 4 was periodically analyzed
every 3–5 days. The design of the experiment is shown in Table S1,
supplementary material.
2.2. Analysis methods
2.2.1. Chemical analysis
Total (TS), volatile (VS), suspended (SS), volatile suspended (VSS)
solids, total chemical oxygen demand (TCOD), soluble COD (SCOD),
ammonium-nitrogen, total alkalinity and bicarbonate alkalinity were
measured in accordance with the standard methods (APHA, 2005). pH
fluctuation was measured by the Mettler-Toledo pH meter. The CH4,
CO2 and VFAs were measured by a GC Shimadzu-GC 8A (Japan)
(Algapani et al., 2019).

2.2.2. Specific hydrolytic, acidogenic activity and acetotrophic

2. Materials and methods methanogenic activity test
Specific hydrolytic, acidogenic, and methanogenic activities test
The swine manure, served as a raw substrate in the current study, were performed on batch assays as described in a previous study
was obtained from a pig farm in Beijing, China. The swine manure was (Hutňan et al., 1999). Batch assays with 120 mL glass serum bottles
then diluted to get swine wastewater, and screened by using a 40-mesh with employed volume of 100 mL (90 mL of basic anaerobic medium
sieve to remove undesired particles. The wastewater was then stored at and 10 g inoculum sludge collected from the SaABR) were operated.
4 °C until used as substrate in the SaABR. The inoculum, which was The basic anaerobic medium composition can be found in a previous
used for the startup of the SaABR at 25 °C, was collected from a me- study (Yin et al., 2018). The 10g of sludge used in the activity test were
sophilic digester (35 °C) treating swine wastewater with a SaABR in our taken at the SP1 and SP3 because of high concentration of micro-
laboratory. The inoculum was cooled down immediately to 25 °C and organisms in these SPs. In addition, substrates used for activity test
then lasted for two weeks without feeding in order to be ready to op- were: 0.5 g starch, 0.5 g glucose, and 0.278 g sodium acetate (formed
erate with an HRT of 10 days. The inoculum for the SaABRs operating 2 g/L acetate concentration) for hydrolytic, acidogenic and methano-
at 20 and 15 °C was collected from the previously mentioned 25 °C genic activities, respectively. Anaerobic conditions were provided ac-
SaABR and then cooled down to use as inoculum. Basic characteristics cording to a previous methodology (Yin et al., 2018). After that, the
of swine wastewater and inoculum (after two weeks of treatment), batch assays were placed in water bath to provide desired temperature.
which were used for the SaABR at 25 °C, are provided in Table 1. The bottles were opened to test SCOD, VFA, and methane production
every 1–4 day(s). They were then discarded and not reused. Calcula-
Table 1 tions and activity expressions are presented with the formula below
Characteristics of substrate and inoculum. according to a previous study (Hutňan et al., 1999).
Parameter Unit Swine wastewater Inoculum 1 d (CSCOD Vr + VCH4/ f )
H= ×
Average Standard Average Standard n
CVSS Vr dt (1)
value deviation value deviation
1 d (CSCOD Vr + VCH4/ f )
A= ×
TS g/L 11.8 1.9 50.0 1.0 3 CVSS Vr dt (2)
VS g/L 7.7 1.7 24.7 1.7 3
SS g/L 6.8 1.6 18.3 1.3 3 1 dVCH4
VSS g/L 5.6 1.3 15.5 0.9 3 SMA =
TCOD g/L 29.4 7.2 / / /
CVSS Vr f dt (3)
SCOD g/L 8.6 2.8 / / /
Where H is hydrolysis; A is acidogenesis; SMA is specific metha-
TVFA mg/L as 3610 530 27 1.9 3
acetate nogenesis activity; CVSS refers to VSS in the reactor, g/L; Vr refers to
sludge volume in serum bottles, L; VCH4 stands for cumulative methane
Note: “/” means no detected. volume, mL; CSCOD is SCOD in serum bottles, g/L; CSCOD(VFA) is VFA as

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M. Jiang, et al. International Biodeterioration & Biodegradation 143 (2019) 104710

Fig. 1. Set-up of SaABR. (Note: “1, 2, 3, 4” are sample ports).

COD, g/L; VCH₄ stands for cumulative methane production, mL; f is a

methane convert factor, 350 mL/g-COD; t is a period which has max-
imum methane production rate, d. The specific hydrolytic, acidogenic
activity and SMA was determined by the maximum consumption rate of
starch, glucose, and sodium acetate rate, respectively.

2.2.3. 16S rRNA gene sequencing

In this study, one sample was used for microbial analysis at each
HRT. To ensure the sample was representative, the withdrawn sludge
from the digester was feedback for three times. After that, the wanted
sludge was immediately sampled for genetic analysis. The extraction of
total DNA was carried out by using CTAB/SDS method (Chen et al.,
2010). PCR amplifications using universal primers 338F/806R and
524F/958R for bacterial and archaeal sequences, respectively, were
implemented. Thermal cycling was carried out in accordance with our
previous study (Wandera et al., 2018). PCR products were purified with
GeneJET Gel Extraction Kit (Thermo Scientific). Sequencing libraries
were generated using NEB Next® Ultra™ DNA Library Prep Kit for Il-
lumina (NEB, USA) following manufacturer's recommendations. The
library quality was assessed using the Qubit 2.0 Fluorometer (Thermo
Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library
was sequenced on an Illumina HiSeq platform to generate paired-end
reads of 250 bp. The paired-end reads were merged according to Magoč
and Salzberg (2011) and analyzed based on a previous study (Caporaso
et al., 2011), and were filtered by QIIME quality filters. Sequences were
then clustered into operational taxonomic units (OTUs) defined at 97%
similarity. Typical sequences were carefully chosen for each OTU.
Taxonomic classification was performed with 16S rRNA reference
(RDP) database.
Fig. 2. Performance of SaABR at HRT 10 days under 15, 20 and 25 °C.
3. Results and discussion
enhancement occurred when other reactors were fed with swine was-
3.1. SaABR performance under 15 to 20 and 25 °C tewater at different temperatures (Deng et al., 2014). A 7.3-fold en-
hancement in methane production was demonstrated with elevating
Methane yield and TVFAs profiles in the three digesters at different temperature from 15 to 35 °C. This behavior is similar with the finding
temperatures (15, 20 and 25 °C) are displayed in Fig. 2, and the overall by Luo et al. (2015) who concluded that temperature played an im-
reactors performances in a stable state are summarized in Table S2 portant role in reconfiguring the profile of the microbial community in
(supplementary material). The fluctuations in the methane yield at the terms of its composition and activity in biogas reactors. However, in
beginning were attributed to the methane production from the organics order to elucidate this behavior, a closer look into organic matter ki-
in the inoculum. Alternatively, the microbial communities may also netics and common AD inhibitors (VFAs) is required.
have needed time for metabolic adaption to the new temperatures. The accumulation of VFA in a SaABR operated at lower tempera-
After approximately a 30-day operation, volumetric methane yield for tures (15 and 20 °C, up to 769 mg/L) at the beginning, indicating that
all the reactors at the three temperatures became relatively stable the acidogenesis rate was higher than the methanogenesis rate.
(Fig. 2a). In comparison to the SaABRs which were operated at 15 and Nevertheless, the VFAs concentrations were far away from significant
20 °C and exhibited volumetric methane yield (0.18 ± 0.05 and process inhibition. In contrast, the VFA concentration of the reactor
0.23 ± 0.07 L/g-VSin), the methane yield of SaABR that operated at operated at 25 °C was low (44 ± 10 mg/L), confirming that the activity
25 °C was increased by 3.4 and 2.7-fold, respectively (Fig. 2a and b). of acidogens and methanogens was balanced. The aforementioned
These findings were similar to a previous research in which

3
M. Jiang, et al.
results could be explained by the faster adaptation of acidogenic bac-
teria compared to methanogens as they generally grow more slowly and
have a narrower temperature range (Yu and Fang, 2003). When the
microbes adapted to the new temperature (after 30 days), the TVFAs
concentrations were similar for all three reactors. The TVFAs con-
centrations were 82 ± 4, 59 ± 11 and 44 ± 10 mg/L for the reactors
operated at 15, 20, 25 °C, respectively (Fig. 2c). Similar behavior was
observed by Ho and Sung (2010) who reported a higher TVFA con-
centration in AnMBR reactors fed with synthetic municipal wastewater
when the reactors were operated at 15 °C compared to those operated
initially at 25 °C. They attributed this fact to a microbial adaption phase
during the first month in the reactor. Acetate was the main VFA, as seen
in Fig. S1a (supplementary material). Acetate concentrations were
(73 ± 7), (45 ± 7) and (39 ± 3) mg/L in the reactors operated at 15,
20 and 25 °C, respectively, which were in agreement with the con-
centration of TVFA. Therefore, TVFA and acetate concentrations were
negligible, indicating that organic overload did not occur (Fig. S1), and
consequently, pHs remained close to neutrality throughout the experi-
mental time. In particular, pH levels were between 7.3 and 8.0
throughout the experiment and remained inside the permissible range
(6.5–8.4) for continuous AD (Yi et al., 2014).
The effluent SCOD of the reactors which were operated at 20 and
25 °C were similar, and this coincided with the SS values (Fig. S1b).
They were also slightly higher than those attained with a lower tem-
perature. However, remarkable differences between the three tem-
peratures in volumetric methane yield (Fig. 2) were achieved, implying
that the process rate was not limited by the hydrolysis stage.
3.2. Performance of SaABR under 25 °C and HRT (3–10) days
The volumetric methane production rate in this study increased
from 0.51 to 0.77 then 0.85 L/L/d with HRT reduction from 10, 5 to 3
days, as depicted in Fig. 3a. However, specific methane yield decreased
from 0.61 to 0.56 then 0.33 L/g-VSin, respectively. The specific me-
thane yield obtained in current study was comparably higher than
Fig. 3. Performance of SaABR at 25 °C under HRT of 10, 5 and 3 days, respectively.
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International Biodeterioration & Biodegradation 143 (2019) 104710
reported yield with swine manure and other substrates. For instance,
Adl et al. (2012) obtained a methane yield of 0.20 L/g-VSin in a plug-
flow reactor treating swine wastewater at an OLR of 2.4 g-VS/(L∙d). A
higher specific methane yield of 0.36 L/g-VSin at a 12-day HRT and an
OLR of 7.4 g-VS/(L∙d) was also reported for treating pig manure and
dried pellet of exhausted sugar beet wastes by Aboudi et al. (2015).
The VFAs are the appropriate indicator for anaerobic process sta-
bility. When the methanogenesis rate is lower than acidogenesis, VFA
accumulation takes place. A short HRT is the main cause of VFA ac-
cumulation due to organic overloading, leading to a decrease in di-
gester pH, which ultimately results in instability of the anaerobic di-
gestion process. As shown in Fig. 3b, VFA concentration in SaABR
effluents was < 100 mg/L at HRT of 10 and 5 days, implying that the
activity of acidogens, acetogens and methanogens was balanced.
However, immediately after reducing the HRT to 3 days, the VFAs
concentration sharply accumulated to 790 ± 39 mg/L. This accumu-
lation was concomitant with a slightly reduction in pH level from 7.9 to
8.0 to 7.3. Given the concentration of acetate (375 ± 194, Table S2), it
would seem that the methanogenic substrates were available, but the
methanogens were inhibited from converting this acetate to biogas,
resulting in a reduction in the methane yield by half, which is com-
parable to the methane yield achieved at a HRT of 10 days (Fig. 3a).
Likewise, Wandera et al. (2019) found that the acetate amplified from
38 to 376 mg/L after decreasing the HRT from 10 to 3 days in CSTR
reactors fed with sewage sludge. The most likely cause of the observed
suboptimal methane yield was the relatively low growth rate of me-
thanogens. The different growth rates could have affected the microbial
population which is abundant in anaerobic digestion. It has been found
that acetoclastic methanogens (the most abundant methanogens in this
current study as will be discussed later) need 1–4 days to double their
numbers (Lee et al., 2011), making them vulnerable to washing-out
effects during a short HRT. In fact, many studies have highlighted the
effect of HRT as a key parameter for methanogenic community devel-
opment (Demirel and Scherer, 2008; Liu et al., 2018), and have at-
tributed this fact to the imbalance of VFA producers and consumers.
M. Jiang, et al. International Biodeterioration & Biodegradation 143 (2019) 104710

Fig. 4. Genus level of bacteria under different temperature and HRTs.

Consequently, it can be suggested that a longer HRT (> 3 days) could also done based on at least pilot scale study to make the result more
help to decrease VFA concentration and thus increase the methane applicable.
yield. In addition, negligible variation was observed between different
HRTs in biogas composition as the results showed similarities, with 3.3. Microbial communities in SaABR
76–79% and 18–21% for methane and CO2 contents, respectively
(Fig. 3c). The differences in operational conditions, such as the HRT and
Variations in the organic removal efficiencies represented by TS, VS temperature, affect both behavior and abundance of the microbial
and TCOD removal efficiencies are shown in Fig. 3d. With the HRT community. It was therefore crucial to study the microbial community
shortening, these removal efficiencies apparently decreased. In com- structures with regard to the variations in these operational conditions
parison, the SaABR operated at a shorter HRT, and when it operated at in order to elucidate the differences in methane yields.
a 10-day HRT, the organic matter removal increased by 9–14%,
10–29% and 7–14%, which is represented by TS, VS and TCOD, re-
3.3.1. Bacterial community
spectively. These values are in agreement with the values attained for
The proportion of bacteria at phylum level in Table S3 (supple-
methane production. The efficiency removal achieved in the current
mentary material), shows that Firmicutes (> 70%) was the most
study was similar to the values obtained for other reactors fed with
dominant bacteria during all the temperature levels regardless SPs,
swine manure (Lopes Pereira et al., 2010), and exceeding that attained
followed by Bacteroidetes (8–20%) and Proteobacteria (1–13%). This
by Wandera et al. (2019). Lopes Pereira et al. (2010) reported a 65%
result was in agreement with a number of previous studies which
removal efficiency of TCOD when treating swine wastewater with ABR
treated swine wastewater (Liu et al., 2018; Da Silva et al., 2015; Ros
at a HRT of 15 h and a temperature of 24 °C (it was 69% in current
et al., 2017). However, Firmicutes showed no clear changes in relative
study, Fig. 3d) while the efficiency obtained by Wandera et al. (2019)
abundance at different HRTs. The analysis of the bacteria community in
was only 47% in a CSTR during a 10-day HRT. Inadequate retention
SP1 showed that the relative abundance of Firmicutes at 25 °C was 8%
time for organics biodegradability could be the reason for organic
and 17% higher than that attained at 20 and 15 °C, respectively. Certain
matter efficiency reductions with the shortening of the HRT and, thus, a
bacteria belonging to Firmicutes are capable of diverse metabolic reac-
longer HRT, of 10 or 5 days for example, is required if higher removal
tions including the break-down of complex organic material in the
efficiency is wanted.
hydrolysis and acidification stages (Wirth et al., 2012; Lin et al., 2016),
If economic considerations are factored in, it is worth mentioning
as well as the oxidation of fatty acids in combination with hydro-
that the anaerobic processes in the current investigation were con-
genotrophic methanogens (Song et al., 2010). Another noteworthy re-
ducted with self-agitation. Application of the anaerobic digestion pro-
sult was that phylum Proteobacteira decreased from 6-11% to 1–2%
cesses with external-agitation, for example by using gas recirculation,
with the reduction of the HRT from 10 to 3 days. Like Firmicutes, both
mechanical pumping or stirring (Karim et al., 2005), could have been
Proteobacteria and Bacteroidetes have the ability to degrade complex
highly efficient. However, more energy for mixing would have been
substrate such as the organics found in swine manure (Fitamo et al.,
required and, thus, the overall costs would have compromised their
2017).
feasibility. In addition, the abundance of methanogens and syntrophic
With regard to genus level, as illustrated in Fig. 4, the dominant
bacteria may be negatively affected in the agitated digester. Tian et al.
bacterium was Clostridium sensu stricto, which is always found in pig
(2014) attribute this fact to the supreme sensitivity of obligate syn-
guts and is associated with dietary protein (Fan et al., 2017). This was
trophy to H2. This is due to the presence of bacterial genera Acet-
consistent with previous reports treating swine waste in which the
anaerobacterium and Ruminococcus which are known as H2 producer
dominant bacterium was also Clostridium sensu stricto (Meng et al.,
through sugar fermentation with a relatively high abundance in an
2018). Furthermore, the influence of both HRT and temperature on the
agitated digester. In the case of self-agitation used in this study, the
relative abundance of this bacterium was obvious. At 25 °C and a 10-
results have shown that the conversion efficiency and process stability
day HRT in SP1, the Clostridium sensu stricto accounted for 46%, which
were not diminished. However, further research on mass balance and
was 9–12% higher than another HRTs, and 11–21% higher than the two
stoichiometry is highly required to further elucidate the responsible
other low temperatures. However, the proportion of Clostridium sensu
biochemical processes. Further studies for economic analysis should be
stricto in the SP3 at 25 °C was the lowest at 38%, which was 3–9% less

5
M. Jiang, et al.
than the other HRTs and temperatures. The obvious decrease in relative
abundance of Clostridium sensu stricto at lower temperature and shorter
HRT support the results of the decreased abundance of Firmicutes and
was most probably caused by finite digestion time and/or unsuitable
temperature. Furthermore, given the importance of highly variable
metabolism of Clostridium sensu stricto which are able to metabolize
various organic matters such as carbohydrate amino-acids, alcohols and
purines (Alou et al., 2018), it is likely that the suboptimal organic re-
moval efficiency attained at lower temperature and shorter HRT may be
attributed to the reduction in relative abundance of such bacteria
(Figs. 2 and 3). Regarding the second abundant bacteria, Terrispor-
obacter (7–10%), it seems that no significant changes were found be-
tween different reactors. Terrisporobacter can use various sugars, or-
ganic acids, and proteins to produce acetate in anaerobic processes
(Deng et al., 2015). Other genera in SaABRs, such as Sedimentibanter,
Smithella, Syntrophomonas and Acinetobacter, occupied a relatively
lower proportion. Among these genera, Sedimentibanter (1–7%) and
Acinetobacter (0–5%) have been recognized as being responsible for the
degradation of the complex organic compounds (Ros et al., 2017).
Alongside this, Smithella (0–6%) has been reported as being able to
convert short-chain fatty acids, such as propionate and butyric acid (Liu
et al., 2018). Syntrophomonas (1–3%) is also capable of butyrate and
propionate degradation to produce acetate for methanogens (Li et al.,
2017; Jiang et al., 2019). In addition, Acinetobacter was found to be
responsible for hydrolysis and acidification in AD of food waste and pig
manure (Jiang et al., 2019). All of these bacteria contribute to the AD
process and, in particular, the hydrolysis and acidification stage, re-
sulting in a better performance in SaABR.
3.3.2. Archaea community
The influence of temperature on the archaea community is shown in
Fig. 5a. The dominant archaea were Methanosaeta (27–80%) and Me-
thanosarcina (10–35%) in both SP1 and SP3 at three temperatures.
Methanosaeta was found to be more capable of adapting at low-tem-
peratures than other methanogens (Liu et al., 2018). In this study, the
dominance of Methanosaeta did not have significant response when
temperature decreased. However, slightly higher abundance of
Fig. 5. Phylum level of archaea under different temperatures and HRTs.
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International Biodeterioration & Biodegradation 143 (2019) 104710
Methanosaeta in the SP3 than in the SP1 was observed. The similar
distribution of Methanosaeta in a 4 chambers ABR treating sugar re-
finery wastewater was also found in a previous study (Li et al., 2013).
Six main methanogens were detected at 25 °C during different HRTs
(Fig. 5b). The archaea community was dominated by the obligate
acetotrophic methanogen Methanosaeta (35–84%) and mixotrophic
methanogen Methanosarcina (1–35%), both of which can degrade
acetate to produce methane (Vrieze et al., 2012). In addition, the Me-
thanosarcina in SP1 was a little abundant than in SP3. A small pro-
portion of hydrogenotrophic methanogens such as Methanocorpusculum
(4–29%), Methanobrevibacter (0–15%), Methanosphaera (0–7%) and
Methanobacterium (0–1%) were attained. With the shortening of the
HRT from 10 days to 5 and 3 days, Methanosaeta steadily increased
from 35% to 53% and 62% in SP1, and from 44% to 66% and 84% in
SP3, respectively. An opposite trend was observed with Methanosarcina,
which decreased from 35% to 23% then 1% in SP1 and from 20% to
16% and 1% in SP3. This reduction in the relative abundance of Me-
thanosarcina may be explained by two hypotheses. Firstly, it has been
concluded that the pH shock of Methanosarcina is 0.8–1.0 (Vrieze et al.,
2012), and as a result, the significant decrease in its proportion at a 5-
day HRT and a 3-day HRT may be due to pH disturbance caused by HRT
shorten (7.95, 7.87 and 7.26 at HRT 10, 5 and 3 days, respectively, Fig.
S1). The second reason that could explain this reduction is the ability of
some Methanosarcina species to shift their metabolic pathway between
acetotrophic and hydrogenotrophic methanogenesis under different
conditions (Demirel and Scherer, 2008) and thus, there is the possibility
that, to a minor extent, some of the Methanosarcina species were
minimizing the hydrogenotrophic pathway. Conclusively, in this study
the acetoclastic methanogenesis can be presumed to be the pre-
dominant pathway in the SaABR at the three ambient temperatures.
In this study, Methanosaeta dominated at different conditions, which
tended to increase under HRT shortening conditions. The methano-
genesis was supposed to be progressed via the acetoclastic pathway.
The methanogenesis was supposed to be through the acetoclastic
pathway. The dominance of Methanosaeta thus may determine the low
concentration of acetate and thus lay the foundation for process stabi-
lity.
M. Jiang, et al.
Table 2
Summary of hydrolytic, acidogenic and methanogenic activities (g-COD g-
VSS−1 d−1).
Activity Sampling port 15 °C 20 °C 25 °C
Hydrolytic 1 0.37 ± 0.01 0.40 ± 0.01 1.67
3 0.17 ± 0.03 0.29 ± 0.04 1.23
Acidogenic 1 0.32 ± 0.01 0.35 ± 0.01 0.93
3 0.15 ± 0.03 0.20 ± 0.04 0.49
Methanogenic 1 0.03 ± 0.01 0.06 ± 0.02 0.16
3 0.04 ± 0.01 0.07 ± 0.03 0.13
3.4. Differences of anaerobic activities under different operation
temperatures
The highest hydrolysis activity was observed in the reactors oper-
ated at 25 °C. Under the higher temperature, the hydrolysis efficiency
increased by 4.2-fold in comparison with the reactors operated at 20 °C,
regardless of the SPs. It was also 4.5-and 7-fold higher than in the re-
actors operated at lower temperatures in SP1 and SP3, respectively
(Table 2). More specifically, after the temperature rose from 15 °C to
20, and then to 25 °C, the hydrolytic activity increased from 0.37 to
0.40 and 1.67 g-COD g-VSS−1 d−1 in SP1, and from 0.17 to 0.29 and
1.23 g-COD g-VSS−1 d−1 in SP3. The significant difference between the
hydrolysis activity at different temperatures indicated that the hydro-
lytic bacteria was more sensitive to temperature flocculation and was
the cause of restrictions in anaerobic digestion processing under lower
temperatures (15 and 20 °C).
The same trend was observed with acidogenic activity, although it
was lower than for the hydrolysis. The acidogenic activity increased
sharply from 0.32 to 0.35 to 0.93 g-COD g-VSS−1 d−1 in SP1, and from
0.15 to 0.2 to 0.49 g-COD g-VSS−1 d−1 in SP3 after temperature was
elevated from 15 to 20, then to 25 °C. This trend was expected as the
products of hydrolysis are the substrates for acidogens. In addition, the
hydrolytic and acidogenic activities in SP1 were greater than that of
SP3. These results are in agreement with a previous study which treated
synthetic wastewater with ABR (Hutňan et al., 1999), thus indicating
that the first two stages of anaerobic digestion occurred more easily in
the first two compartments of the SaABR. Furthermore, at the same
temperature, the difference in the ratio between both the hydrolytic
and acidogenic activities in the two compartments was 26–54%.
However, at different temperatures, the ratio of the hydrolytic and
acidogenic activity in the same compartments was 59–86%, which in-
dicated that temperature impact on the hydrolytic and acidogenic ac-
tivities was greater than compartments distribution. The promotion of
hydrolysis and acidogenic activities, corresponding to increased abun-
dance of Firmicutes (specially, Clostridium sensu stricto) and was in ac-
cordance with highest organic matter efficiency attained at 25 °C, re-
sulting in highest methane yield.
In contrast, the specific methanogenic activity in the two compart-
ments did not apparently differ at the same temperature. However,
after temperature changed from 15 °C to 20 and then 25 °C, the SMA
increased from 0.03 to 0.06 to 0.16 g-COD g-VSS−1 d−1 in the SP1 and
from 0.04 to 0.07 to 0.13 g-COD g-VSS−1 d−1 in the SP3, respectively.
A previous study treating synthetic municipal wastewater with AnMBR
showed that the SMA at 15 °C was lower than for the 25 °C conditions
(Ho and Sung, 2010). When concentrated slaughterhouse wastewater
was treated in an ASBR, a higher average methanogenic activity of 0.34
gCH4-COD/g-VSS was obtained at 25 °C; however, this activity was
decreased to 0.12 gCH4-COD/g-VSS at 20 °C (Daniel and Masse, 2001).
These studies together with the current investigation demonstrate that a
low temperature considerably affects the methanogenic activity re-
gardless of the reactor configurations. The highest activities of hydro-
lysis, acidogenesis, and methanogenesis in the reactor operated at 25 °C
were consistent with the highest methane yield obtained at that tem-
perature (Fig. 2). Nevertheless, the lowest methanogenic activity, when
International Biodeterioration & Biodegradation 143 (2019) 104710
compared to the hydrolytic and acidogenic activities at all tempera-
tures, implies that methanogenesis was the rate limiting stage for AD
processing at ambient temperature ranges.
4. Conclusions
± 0.14
± 0.05
The stability of the anaerobic digestion performance obtained in this
± 0.03
± 0.04 study showcases the potential of utilizing ambient temperatures (25 °C)
± 0.04 and short HRTs (10 days for best conversion efficiency or 3 days if plant
± 0.03 treatment capacity is the target) as a promising strategy to decrease
energetic inputs and maximize the methane yield of the anaerobic di-
gestion plant. Under these conditions, the specific methane yield was
increased by 1.8- to 3.4-fold, and acetotrophic methanogens
(Methanosaeta) were the most dominant methanogens. The value of
hydrolytic, acidogenic and methanogenic activities were the highest at
25 °C, demonstrating the considerable influences of temperature and
HRT on microbial abundance and activities.
Acknowledgements
This work was partially supported by the National Key Research and
Development Program of China (2016YFD0501403). Ahmed Mahdy
would like to thank for the financial support from State Administration
of Foreign Experts Affairs, China. (Project No. WQ20180011).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ibiod.2019.05.027.
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