Microbial network for waste activated sludge cascade utilization in an integrated system of microbial electrolysis and anaerobic fermentation
Liu et al. Biotechnol Biofuels
Microbial network for waste activated sludge cascade utilization in an integrated system of microbial electrolysis and anaerobic fermentation
Wenzong Liu 0 3
Zhangwei He 2
Chunxue Yang 2
Aijuan Zhou 1
Zechong Guo 2
Bin Liang 0 3
Cristiano Varrone 4
AiJ‑ie Wang 0 2 3
0 Key Laboratory of Environmental Biotechnology, Research Center for Eco‐Environmental Sciences, Chinese Academy of Sciences , Beijing 100085 , China
1 College of Environmental Science and Engineering, Taiyuan University of Technology , Taiyuan 030024 , China
2 State Key Laboratory of Urban Water Resource and Environment, Har‐ bin Institute of Technology , Harbin 150090 , China
3 Key Laboratory of Environmental Biotechnology, Research Center for Eco‐Environmental Sciences, Chinese Academy of Sciences , Beijing 100085 , China
4 Department of Chemical and Biochemical Engineering, Center for Bio‐ Process Engineering, Technical University of Denmark , Lyngby , Denmark
Background: Bioelectrochemical systems have been considered a promising novel technology that shows an enhanced energy recovery, as well as generation of value‑ added products. A number of recent studies suggested that an enhancement of carbon conversion and biogas production can be achieved in an integrated system of microbial electrolysis cell (MEC) and anaerobic digestion (AD) for waste activated sludge (WAS). Microbial communities in integrated system would build a thorough energetic and metabolic interaction network regarding fermentation communities and electrode respiring communities. The characterization of integrated community structure and community shifts is not well understood, however, it starts to attract interest of scientists and engineers. Results: In the present work, energy recovery and WAS conversion are comprehensively affected by typical pretreated biosolid characteristics. We investigated the interaction of fermentation communities and electrode respiring communities in an integrated system of WAS fermentation and MEC for hydrogen recovery. A high energy recovery was achieved in the MECs feeding WAS fermentation liquid through alkaline pretreatment. Some anaerobes belonging to Firmicutes (Acetoanaerobium, Acetobacterium, and Fusibacter) showed synergistic relationship with exoelectrogens in the degradation of complex organic matter or recycling of MEC products (H2). High protein and polysaccharide but low fatty acid content led to the dominance of Proteiniclasticum and Parabacteroides, which showed a delayed contribution to the extracellular electron transport leading to a slow cascade utilization of WAS. Conclusions: Efficient pretreatment could supply more short‑ chain fatty acids and higher conductivities in the fermentative liquid, which facilitated mass transfer in anodic biofilm. The overall performance of WAS cascade utilization was substantially related to the microbial community structures, which in turn depended on the initial pretreatment to enhance WAS fermentation. It is worth noting that species in AD and MEC communities are able to build complex networks of interaction, which have not been sufficiently studied so far. It is therefore important to understand how choosing operational parameters can influence reactor performances. The current study highlights the interaction of fermentative bacteria and exoelectrogens in the integrated system.
Anaerobic digestion; Microbial electrolysis cell; Biogas; Waste activated sludge
The consideration of wastewater and waste sludge as a
source of renewable energy is growing, together with
the public concerns for future shortage of fossil fuels and
the impact on climate change [
]. Anaerobic wastewater
treatment processes offer the possibility of a net energy
production, rather than aerobic processes that consume
]. Especially, waste activated sludge (WAS), with
its abundance of nutrients, has a great potential as an
alternative resource to extract value-added products [
], even though a long operation time is always required
for anaerobic digestion (AD) to achieve an effective
carbon removal and energy yield [
Recently, a number of studies have suggested that AD
can be enhanced by inserting bioelectrochemical
systems (BESs) [
] in the anaerobic processes, to enhance
acidogenesis , hydrogen production [
methane production [
]. It is well known that
microorganisms capable of extracellular electron transfer have
a major impact on the natural cycling of carbon and
]. Therefore, BESs proved to be highly
versatile in terms of potential application for WAS cascade
utilization, ranging from energy recovery from organic
substrates to product generation and specific
environmental niche creation [
]. Characterization of
microbial community structure in anaerobic digesters has
attracted interest of engineers and researchers, because
understanding microbial behavior and interactions is
essential to improve the fermentation process [
In fact, anaerobic treatment generally requires multiple
groups of microorganisms working together to transform
primary substrates to energy products such as hydrogen
 or methane [
]. However, a thorough work in
relation to the bacterial community shifts, and especially the
impact of fermentation communities to electrode
communities, is lacking.
Hydrolysis has always been considered the rate limiting
stage in the fermentation of waste solids, therefore most
of the studies focused on accelerating sludge hydrolysis
using efficient pretreatment strategies, including
chemical (such as alkaline [
]) and physical methods (such
as ultrasonic [
] or freeze/thaw pretreatment, typically
in cold areas [
]). Clearly, the characteristics of
different pretreated sludge can also substantially influence
the fermentation efficiency and the cascade utilization
in the coupled BESs [
]. Chemical or physical
pretreatments can directly impact the granular size of WAS,
leading to the production of various organic compounds
]. Consequently, digestate from degradation process
in digesters presents prime importance for microbes
in the recycling of carbon and nutrients [
Moreover, the effective diffusion and the biochemical
characteristics of carbons and metabolic products can have a
significant impact on the electrode biofilm matrix (for
example, faster biocatalytic rates were observed under
fatty acid-fed conditions) [
]. On the other hand, the
efficiency in a BES will also be influenced by the initial
anaerobic fermentation community, which will interact
with the electrode community, after connecting the BES
to a conventional AD system. Therefore, it is very
important to evaluate the synergistic effects between
fermentative microbes and electrode respiring microbes and the
influence of carbon source characteristics. It is also worth
noting that metabolic networks and interactions can
determine the electrode respiring community
It is well known that a higher performance of
various BESs can be achieved using mixed culture rather
than pure culture [
]. Though exoelectrogens like
Shewanella and Geobacter are proved to conduct the
electron generation and transport in the mixed culture
], they would not be absolutely dominant compared
to other functional members (like Anaerolinea,
Bacteroida, and Clostridia) in electrode biofilm. It has been
found that interaction between microbes can improve
system performance and energy recovery efficiency i.e.,
when combining Brevibacillus with Pseudomonas [
hinting that microbes worked together and contributed
to carbon recycling and electron transfer. For
example, Acetoanaerobium sp. and Acetobacterium sp. were
reported to be enriched on the bio-electrode of
microbial electrolysis cells (MECs) and acetogenesis occurred
at a limited degree [
]. Fusibacter sp. represented the
enriched anaerobic fermentation community, able to
utilize carbohydrates and produce acetate and butyrate as
end products [
], which represent a favorable substrate
to exoelectrogens. In a previous study on anode
communities, a complex interaction on carbon degradation was
revealed by functional genes (ranging from labile to
recalcitrant carbon) [
]. However, synergistic and interactive
effects of various communities have been insufficiently
examined. In some cases, an unexpected reduction of
efficiency and targeted products occured when applying
microbial electrolysis to wastewater/sludge treatment
], though a high energy harvest can easily be achieved
from artificial wastewater with pure carbon sources [
The relationship between BES and AD (for instance the
understanding of their mutual benefits, or system
stability enhancement, etc.) has become a debated issue [
but clearly the effects of newly introduced communities
on BES function has to be taken into consideration for
further potential application [
]. So far, there were very
few reports investigating the characteristics of cascade
bioconversion, related to electron recovery, in mixed
carbon sources to reveal microbial community interactions
between digestate and BES biofilm. Thus, the present
study wanted to explore the hypothesis that (i) a proper
pretreatment of waste activated sludge can achieve a
satisfactory efficiency in terms of final carbon utilization
and energy recovery; (ii) sludge cascade utilization can
be achieved for hydrogen production in MECs
connecting anaerobic fermentation; (iii) fermentative
communities formed in AD can have an impact on anode respiring
bacteria in batch operation of integrated systems.
Results and discussion
Characteristic change of WAS fermentation using different pretreatment
The particle size primarily changed through
different pretreatment methods, which substantially
influenced the subsequent release and conversion of various
organic matter in the WAS. Ultrasonic treatment played
the most noticeable effect on sludge structure break and
scatter, leading to an average particle size distribution of
29.5 μm (see Additional file 1: Figure S1). Alkaline
treatment slightly improved the sludge particle scatter, with
an average of 56.3 μm in comparison to 60.8 μm of the
control sludge without pretreatment. On the other hand,
an obvious increase of particle size up to 387.5 μm was
obtained by the freeze/thaw treatment, because flocks
were produced after freezing. Consequently, the lysis
ratio of increased SCOD to TCOD was 21, 6, and 11 %
after alkaline, freeze/thaw, and ultrasonic pretreatment,
respectively (see Additional file 1: Figure S2). The freeze/
thaw pretreatment was not as effective as other
methods on SCOD release, indicating that the flocks of larger
particle size were not broken into smaller fragments in a
short reaction time [
]. In another report on the effect
of sludge pretreatment on sludge characteristics, the
disruption of sludge flocks led to the release of
intracellular and extracellular materials [
]. Moreover, alkaline
(pH 10–12) treatment is known to further enhance the
organic release during the pretreatment [
] and favor
VFA production in the subsequent fermentation [
]. In our study, an increased amount of soluble
organics was released after pretreatment, mainly in the form of
carbohydrates, proteins, and volatile fatty acids (VFAs)
(see Additional file 1: Tables S1, S2). After 3 d
fermentation, SCOD increased from 147 mg/L of the raw sludge
to 452 mg/L of the control, 7690 mg/L of the alkaline
pretreatment, 1760 mg/L of the freeze/thaw pretreatment,
and 3461 mg/L of the ultrasonic pretreatment (see
Additional file 1: Table S1). The VFAs were mostly produced
by the alkaline pretreatment, which accumulated up to
5300 mg COD/L, accounting for 69 % of total SCOD. The
same occurred with proteins, which reached 1749 mg/L,
with a 24-fold increase compared to the control sludge
without any pretreatment.
In our view, WAS pretreatment initially changed the
particle characteristics, which played a major role in the
studied process, influencing organic release and
fermentative communities during pretreatment, and subsequently
affecting the fermentation and VFAs production. Although
all sludge pretreatments successfully improved hydrolysis
and organic release, the subsequently generated
shortchain fatty acids differed in terms of content and
concentration, which are known to be important factors affecting
the conversion rate and efficiency in bioelectrochemical
]. Clearly, soluble organics play an important
role, reducing the accessibility of substrates to bacterial
disintegration, or stated differently, the initial particle size
can affect the contact surface area, for subsequent
bacterial action [
]. Our results showed that ultrasound led to
the smallest particle size, followed by a higher acetate
production (>80 % of total fermentative products). Alkaline
pretreatment could increase the total production of
shortchain fatty acids with a high conductivity fermentative
liquid. Therefore, it seems to be one of the substantial factors
to interact with electrode biofilm communities.
Furthermore, organics and conductivity of WAS
fermentative liquid were the two key factors to the
bioelectrochemical communities. To evaluate the influence of
pretreatment on COD contribution in different sludge
structures, COD was divided into four parts:
soluble SCOD, loosely bound extracellular polymeric
substances (LB-EPS), tightly bound extracellular polymeric
substances (TB-EPS), and residual particles (Fig. 1).
The alkaline pretreatment effectively released ~25 %
particle organics (compared to the control) into SCOD
(~21 %). A small part of particles (4 %) with reduced
TB-EPS (~3 %) were converted into LB COD (~7 %).
However, COD contribution was reduced to less than
half of SCOD (6–11 %) from particles, when using the
freeze/thaw or ultrasonic pretreatment. The SCOD of
VFAs reached the peak accumulation during
fermentation before methane production started, under the
conditions of this study [
]. The release of soluble
matter also increased conductivity of fermentation
solution. Even during fermentation without any
pretreatment, there was a slight increase from 1.2 to 1.4 mS/
cm in sludge fermentation liquid (SFL) (see Additional
file 1: Table S1). Conductivity was further increased to
1.96–2.63 mS/cm by the freeze/thaw and ultrasonic
treatment respectively, which matched the increasing
trend of SCOD and inorganic ion release. The alkaline
addition, using NaOH, highly enhanced the
conductivity, reaching up to 6.23 mS/cm, which was almost close
to 50 mM PBS (Phosphate buffer solution, pH 7.0) used
for MEC reactor setup . A high conductivity is to
be considered potentially beneficial to electron
transport in the following bioelectrochemical process [
Besides the additional alkaline contribution, organics
and ion release from WAS improves during the
pretreatment and is further enhanced during the fermentation.
A previous study showed that the limiting factors, at the
anodic biofilm, change from potential limitations at low
conductivity, to dual potential and carbon source
transfer limitations at a moderate conductivity, and to only
mass transfer limitations at high conductivity [
]. A low
conductivity (<1 mS/cm) was observed in common AD
effluent after organic removal and biological treatment,
moreover, a higher external voltage was required when
connecting BES after AD to achieve biofuels [
]. In this
respect, pretreatment is an important and flexible tool
to regulate the performance of BES and AD integrated
process, which would determine the total efficiency on
waste treatment and biofuel recovery.
Pretreated SFL utilization and hydrogen production in MECs
The setup performance of the 15 MEC replicate
reactors, before fueling SFL (see Additional file 1: Figure S3).
The average coulombic efficiency was steadily around
92.2 ± 6.5 % in all replicates, with an average peak
current of 3.75 ± 0.22 mA. The COD removal efficiency
of acetate reached 86.8 ± 2.1 %. The 15 MEC reactors
showed similar conversion efficiencies to hydrogen, with
3.3 ± 0.5 mol H2/mol acetate and a hydrogen production
rate of 1.36 ± 0.26 mL/mg COD (1.46 ± 0.28 mL H2/mL
reactor/d). Twelve reactors were randomly divided into
four groups (three replicates each), to be fed with SFL
obtained from the different pretreatment methods.
The pretreated sludge properties determined the
subsequent fermentation process, leading to various levels of
acidification, organic contents, and production rates (see
Additional file 1: Figures S4, S5). The highest amount of
VFAs was produced during the 3rd day fermentation of
alkaline pretreated WAS, containing 2225.81 mgCOD/L
and accounting for 42 % in total VFAs (see Additional
file 1: Figure S4). There was 1077.25 mgCOD/L
acetate produced in ultrasonic pretreated WAS, while still
accounting for 41 % of total VFAs. The lowest amount
of VFAs was observed with the freeze/thaw-pretreated
WAS, though still showing a 3.8-fold increase compared
to WAS without pretreatment. In all pretreated SFL,
more VFAs were firstly utilized in MECs, showing a
similar removal of around 70 % (see Additional file 1: Figure
S5). Over 95 % of acetate and butyrate were utilized in
alkaline SFL and ultrasonic SFL, while only ~85 % acetate
and butyrate were removed in freeze/thaw SFL.
Differently, a much higher percentage of propionate (removal
amount was really low) was removed in freeze/thaw
SFL than others at the same time. As a result, hydrogen
production rate differed in MEC reactors, based on acid
types and concentrations that were produced [
Previous results showed that pretreatment methods are very
important to release organics and enhance degradation of
various carbon sources from WAS [
]. Probably, the
cascade utilization of SFL could be regulated according to
composition in VFAs, proteins, and polysaccharide [
], while the energy recovery changed when the suitable
organic compounds were degraded. It is therefore likely
that the alkaline treatment performed best energy gains
(Fig. 2) thanks to the high conductivity (increased from
2.1 ± 0.2 mS/cm for raw sludge to 3.5 ± 0.3 mS/cm for
alkaline pretreatment) [
] and SCOD, as well as high
The current generation varied among different
pretreated and fermented sludge (see Additional file 1:
Figure S6). The highest peak current reached ~3.7 mA,
when feeding with the alkaline pretreated SFL, which
showed the highest acetate production of 2200 mg
COD/L, as well as an enhanced conductivity. The peak
current dropped to 2.5 mA for ultrasonic and 1.8 mA
for freeze/thaw condition. The lowest current was only
1.0 mA, using SFL produced from raw WAS without
pretreatment. The SCOD removal was slightly different in
different SFL, with 61 ± 2 % for alkaline, 66 ± 5 % for
freeze/thaw, and 69 ± 3 % for ultrasonic pretreatment in
MECs (Fig. 2). However, the hydrogen production rate
was quite different. The alkaline pretreated SFL achieved
the highest hydrogen production of 1.22 ± 0.03 mL H2/
mL reactor/d (compared to 1.46 ± 0.28 mL H2/mL
SCOD removal Hydrogen production rate
Control A F U
Fig. 2 SCOD removal and hydrogen production rate in MECs fed
with different sludge fermentative liquids. Control sludge without
treatment, A Alkaline, F freeze/thaw, U ultrasonic pretreatment
reactor/d before fueling with SFL). The ultrasonic
pretreated SFL was converted into hydrogen with a rate of
0.60 ± 0.15 mL H2/mL reactor/d. The freeze/thaw
pretreatment, instead, was not able to effectively improve
the cascade utilization of WAS, compared to raw sludge.
Clearly, organic products were the result of
metabolic activities of the microbial community, which was
characterized by different composition and structure
during fermentation. Previous studies showed that
with the proper enrichment of microbial
communities, anaerobic processes can be improved and perform
more efficiently [
]. In this study, the solid granular
sludge changed based on different pretreatment
methods. Although SCOD was increased and consequently
converted to more hydrogen in MECs, the hydrogen yield
was reduced by ~16 % when influent was changed from
artificial wastewater (acetate, ~1140 mg COD/L) to SFL
(acetate, ~2225 mg COD/L accounting for 29 % of SCOD
in the alkaline pretreatment) with fermentative
communities. Moreover, hydrogen yield was reduced by ~59 %
when feeding the ultrasonic pretreatment SFL
(acetate, ~1080 mg COD/L accounting for 31 % of SCOD).
It has been pointed out that further increases in organic
loading do not vary hydrogen production significantly
]. Therefore, it is likely that MEC performances
changed in relation to anodic community structure,
which interacted with dominant fermentative
communities and organic compounds produced .
Methane production and archaea community change in integrated system
Methane production was detected in all MEC reactors
after 2 weeks feeding SFL, however, the methane
production rate was fluctuating, not being comparable among
different pretreatments over all batch operations (data
not shown). Although methane production was not
substantially increased over 1 month (as we evaluated
]), the MECs feeding SFL without pretreatment
presented the highest methane production over all other
conditions, together with the highest amount of
acetotrophic methanogens (Methanosaeta), both in control
SFL and MEC biofilms fed with control SFL (Fig. 3). It
was interesting that the lowest amount of archaea were
detected under ultrasonic pretreatment, leading to the
lowest growth in MECs as a result. Compared to
methanogens in initial biofilms fed with acetate (MEC sample),
it seem that acetotrophic methanogens were
substantially enriched to anode biofilm in all conditions. But
hydrogenotrophic methanogens were further enriched
with higher amounts than in SFL feeding, as shown
in freeze/thaw and ultrasonic pretreatment,
including Methanocorpusculum, Methanosphaerula,
Methanoregula, Methanospirillum, Methanobacterium, and
Methanobrevibacter. The extra hydrogen generation
from MECs can favor hydrogenotrophic methanogens
in anaerobic condition [
]. The H2 produced in a
single chamber MEC can be lost through methanogenesis,
which causes energy loss in the system [
22, 51, 52
Microbial community structure and anodic biofilm community shift in integrated process
A total of 244,761 raw sequences were analyzed over all
community samples (Additional file 1: Table S2).
Operational taxonomic units (OTUs) at 3 % distance were the
most detected ones in raw WAS (5002), with the
highest diversity (Shannon index 6.96), while being the least
detected in the startup anode biofilm using acetate
(2341), showing a reduced diversity (Shannon index 5.39)
(see Additional file 1: Figure S7). Similar results were
observed from ACE (abundance-based coverage
estimator) and Chao1 indices (see Additional file 1: Table S3).
Interestingly, microbial community diversity in the SFL
decreased, indicating that specific fermentation
bacteria were enriched and became dominant. On the other
hand, an increase of diversity was detected in anodic
biofilm communities, after initial MECs were connected to
sludge fermentation (with or without pretreatment) for
several days, showing an interactive effect of fermentative
communities on initial anodic communities, thus leading
to subsequent changes in MEC reactor performances.
After fermentation, the unpretreated sludge showed
similar community structure to raw sludge (see
Additional file 1: Figure S8). The most abundant phylum in
SFL was Proteobacteria, accounting for 36.7 % in the
control, 40.0 % in the alkaline pretreatment, 28.7 % in the
freeze/thaw pretreatment, and 54.8 % in the ultrasonic
pretreatment, over all microbial communities. Seemingly,
sludge fermentation after ultrasonic pretreatment mostly
increased Gammaproteobacteria. In comparison to the
control sludge, Firmicutes (Bacilli sp. and Clostridia sp.)
were all increased in SFL of the pretreated sludge.
Bacteroidetes was the third most abundant community in the
When MECs were connected to the fermentation
process, anodic biofilm composition obviously changed,
compared to the original communities established using
acetate (Fig. 4). Desulfovibrio [
] and Geobacter [
(Deltaproteobacteria), responsible for electron
transfer between bacteria and electrode, represented the key
functional community. Geobacter was the most detected
genus of the anode biofilm, in the case of reactors fed
with acetate (startup MECs), and further increased after
feeding with alkaline pretreated SFL, as well as the
ultrasonic pretreated SFL (with a corresponding high energy
conversion achieved in these reactors). The MEC fed
with the freeze/thaw-pretreated SFL, instead, showed
low abundances of Desulfovibrio and Geobacter, which
was similar to the control SFL. On the other hand,
compared to other treatments, freezing-thaw SFL led to an
increased abundance of Pseudomonas in the anodic
community. Moreover, large particles of organics in SFL led
to enrichment of fermentative communities in the anode
biofilm, including Anaerolinea (Levilinea and Longlinea),
Bacteroida (Paludibacter and Parabacteroides), and
Clostridia (Proteinilclasticum, Proteocatella, and
Sedimentibacter). Compared to the original anode biofilm,
four genera of the class Clostridia (namely
Acetoanaerobium, Acetobacterium, Anaerovorax, and Fusibacter)
decreased in all SFL-fed MECs.
Moreover, hierarchical cluster analysis clearly showed
that SFL communities varied, depending on the different
treatment method (Fig. 5, F-samples). The bacterial
community structure of the control SFL (F-control) without
pretreatment changed less, compared to the raw sludge
(Raw), while the ultrasonic pretreatment led to the
greatest difference in community structure in the SFL (F-f ).
Figure 5 (M-samples) showed how various SFL
communities had different impacts on the change of the anodic
biofilm communities, after combining fermentation and
microbial electrolysis process. Interestingly, anodic
biofilm communities were similarly grouped among the
original MEC (Mec), MECs fed with the alkaline
pretreated SFL (M-a), as well as MECs fed with ultrasonic
pretreated SFL (M-u). Regarding the gas production, they
performed much higher hydrogen yield than the SFL-fed
control or the freeze/thaw pretreatment, which had a low
hydrogen conversion, below 0.2 mL/mg COD, and peak
current below 1.0 mA.
Bioelectrochemical communities were highly enriched
in dominant functional groups related to
Proteobacteria (63 %) and Firmicutes (25 %) when feeding acetate
during reactor setup, inoculated with activated sludge
(See figure on next page.)
Fig. 4 Taxonomic classification of 16S rRNA gene sequences of bacterial communities of anode biofilm at the phylum (a), class (b), and genus (c)
levels. Relative abundance was defined as the number of sequences per sample. Mec MEC initially fed with acetate, M-control SFL‑fed MEC without
treatment (control treatment), M-a MEC fed with alkaline treated SFL, M-f MEC fed with freeze/thaw treated SFL, M-u MEC fed with ultrasonic treated
a Bacterial Phyla
-a -u ec -f -co
M M M M M
(Fig. 6). They played the primary function of electron
transfer and substrate degradation, with great potential
on complex carbon utilization, as already suggested from
a functional genes’ perspective [
]. The most abundant
genera for extracellular electron transfer were Geobacter,
Desulfovibrio, and Acinetobacter, belonging to
Proteobacteria. Geobacter species are considered as the most
efficient exoelectrogens in bioelectrochemical systems [
Desulfovibrio and Acinetobacter species are dissimilatory
metal-reducing bacteria involved in contaminant
degradation and metal reduction, outside the cell membrane
]. In Firmicutes, three genera of Clostridia were
detected, namely Acetoanaerobium (5.9 %),
Acetobacterium (8.5 %), and Fusibacter (5.0 %). They are supposed
to play an important role in carbon recycling for anode
respiring bacteria, as previous studies have shown that
some Firmicutes may closely live with anode respiring
bacteria, when fed with fermentative substrates .
Microbial community network on exoelectrogenic and fermentative communities
Based on the discussion above, a network
representing the community change and linkage was constructed
(Fig. 7), taking alkaline treatment samples as example.
MEC biofilm was inoculated from raw sludge (Raw), then
raw sludge was pretreated to produce fermentation liquid
as feedstock for the MECs. During the cascade process,
MEC biofilm interacted with fermentative communities
to form a new MEC biofilm. The SFL primarily led to an
increase in abundance of Bacteroidetes and Chloroflexi
in anodic communities, thus reducing the abundance of
Proteobacteria. Even though the phylum Bacteroidetes
was commonly detected in bioelectrochemical system
communities, few studies pointed out their negative
impacts on electron transfer efficiency [
]. On the other
hand, Bacteroidetes can be further enriched (over
Proteobacteria) in an open-circuit BES to convert substrates,
thus competing with anode respiring bacteria for power
]. Lately, it was highlighted that Bacteroidetes
can be easily enriched in BES when supplemented with
other electron acceptors (NO3−) [
], thus potentially
enhancing an electron flow that is separated from the
energy yield in MECs. Chloroflexi also represented an
enriched phylum in the open circuit, and it is usually
predominant in anaerobic digester sludge. Bacteroidetes
increased to 25.9 %, compared to 5.6 % in the initial setup
MECs, with high coulombic efficiency and H2 yield.
Proteobacteria, on the contrary, decreased to 17.8 % in SFL
control MECs, although they increased to 26.8 % by
feeding with freeze/thaw-pretreated SFL. More specifically,
Gammaproteobacteria and Deltaproteobacteria were
partially decreased, while Betaproteobacteria partially
increased, thus leading, as a result, to a lower efficiency
of electron transfer and hydrogen recovery.
MECs fed with alkaline pretreated SFL showed the best
energy yield and shared the largest community of
Geobacter, Desulfovibrio, Pseudomonas, and Clostridium,
with initial MECs (Fig. 7). Simultaneously, Firmicutes
substantially decreased, including Acetoanaerobium
(1.1 %), Acetobacterium (0.2 %), and Fusibacter (0.3 %).
Some of the anaerobes (>20 %), i.e., the class Bacilli
(Pasteuria and Lactococcus) and Clostridia
(Fusibacter, Anaerovorax, and Proteiniclasticum), were clearly
enriched on electrode biofilm by SFL feeding,
suggesting a synergistic effect with exoelectrogens to degrade
complex organic matter [
] (like Lactococcus
producing soluble electron shuttles to promote electron transfer
between cells and the electrode surface [
genera belonging to Clostridia (Acetobacterium and
Acetoanaerobium) (<10 %) were probably enriched in MECs by
the availability of products such as hydrogen (electron).
Initial ARB shared in
Evolutive ARB shared
with AD communities
However, their function on carbon/electron recycling
seems to be very limited in various BES systems [
which would suggest only a limited hydrogen loss (or
over 100 % coulombic efficiency) in MECs fed with
], carbohydrate [
], or fermentation liquid [
It is worth noting that a high protein or polysaccharide
but low fatty acid content would lead to the dominance
of Proteiniclasticum and Parabacteroides (increased
by >10 %), which are able to produce VFAs as end
products from fermentation [
]. Thus, part of the
microbial communities did not function on extracellular
electron transfer; however, they were maintained in the
fermentation niche of electrode biofilm, where they could
provide labile products for electrode respiring bacteria. A
substantial reduction in current and hydrogen recovery
(Alkaline vs. Ultrasonic) was observed when
introducing SFL with an increased abundance in
Proteiniclasticum (Alkaline vs. Ultrasonic genus level in Additional
file 1: Figure S8). Proteiniclasticum reached 11.4 % in the
ultrasonic pretreated SFL, but only 0.4 % in the alkaline
pretreated SFL. In fact, MECs presented a similar COD
removal (61 and 69 %) but coulombs were reduced by
31 % and hydrogen production by 50 %.
Alkaline pretreatment could provide more short-chain
fatty acids and higher conductivities than other
pretreatments. These two aspects are known to favor mass
transfer in anodic biofilm [
]. Geobacter increased in both,
alkaline and ultrasonic pretreated SFL, in which
accumulated VFAs were higher than 2500 mgCOD/L (with
acetate reaching >1000 mgCOD/L). It appeared that some
species, belonging to Parabacteroides, Clostridium, and
Pseudomonas, were potentially enriched more in
alkaline SFL with higher SCOD and conductivity, compared
to ultrasonic SFL. But a delayed fermentation process
on raw WAS, as well as freeze/thaw-pretreated WAS,
substantially led to different fermentative communities
in the anode biofilm (such as Parabacteroides and
Proteinilclasticum), which produced little VFAs for anode
respiring bacteria. In this situation, long time would be
required to WAS cascade utilization, with a slow
fermentation and inefficient electron generation in BES.
WAS, rich in organic carbon, can be used as an
alternative bioresource, and energy recovery can be potentially
improved when combining proper pretreatment method
and novel AD technology. During the cascade utilization
of WAS, organic removal could be improved by various
pretreatment methods, while energy recovery of MEC
was impacted by fermentation liquid and microbial
community composition. A smaller particle size of WAS
after pretreatment substantially favored the
fermentation process and formation of suitable byproducts for
BES. Alkaline pretreatment gave the highest VFAs
production, achieving the most effective energy recovery in
the MECs with abundance of Geobacter >10 %. Energy
yield of electron transfer in integrated system was
influenced, on one side, by the initially formed
exoelectrogen community, but also by the newly formed one, after
interacting with fermentative communities. A high
protein or polysaccharide (but low fatty acids) content led
to the dominance of Proteiniclasticum and
Parabacteroides, which had a delayed contribution to extracellular
Pretreatment of WAS and short fermentation process
The WAS was collected from the secondary
sedimentation tank of Wenchang municipal WWTP in Harbin,
China. The sludge was concentrated by settling for 24 h
and moving the water layer away, then stored in the
refrigerator at 4 °C. The large particles were separated
through the 40 mesh sieve. In order to facilitate
comparison among different treatments, the volatile suspended
solid (VSS) content of the WAS used in this study was
adjusted to 14 g/L. Three kinds of pretreatment methods
were used, prior to fermentation: (a) alkaline treatment,
in which to the pH was adjusted to 10 (by NaOH) and
the sludge reacted for 20 min at 81 °C [
]; (b) freeze/
thaw treatment, by freezing the sludge at −20 °C for 72 h
and then thawing it at room temperature, before use [
(c) ultrasonic treatment, in which the sludge was treated
for 10 min with 0.5 kW/L energy density, using 40 kHz
bi-frequency ultrasound; (d) the control, without any
treatment of the raw sludge [
After pretreatment, the particle size distribution was
analyzed with a particle analyzer (Mastersizer 2000
Malvern) with a Hydro 2000MU dispersing unit and detected
by means of laser diffractometry (within approximately
30 s). The following parameters were set: refractive index
(RI) values for particles and basis solution (water, 20 °C)
were 1.52 and 1.33, respectively and the measurement
cycle is 10 s. The analysis was repeated three times, and
the average readings were obtained by Mastersizer 2000
software. The output of the measurements is depicted in
a graph of volume (%) against particle size (μm) within a
range from 0.02 to 2000 μm. The stirrer and pump speed
were kept at 600 rpm, which is the minimum
pump/stirrer speed of the instrument, to eliminate the potential
damage to the sludge flocks [
]. The WAS
fermentation took place in sealed glass bottles (effective volume
of 500 mL, Sichuan Shubo CO., China). Each bottle
contained 350 mL WAS. All bottles were flushed with
nitrogen gas for 10 min to remove oxygen. The bottles were
stirred in an air-bath shaker at 35 ± 2 °C for 3 days to
achieve a good accumulation of VFAs [
supernatant was taken from SFL after settling overnight (12 h),
and used as carbon source for MECs.
MECs reactor setup and operational conditions
Fifteen single chamber MEC reactors were set up (see
Additional file 1: Figure S9). The reactors were made of
polycarbonate. The effective volume was 40 mL,
including a 28 mL chamber (3 cm diameter × 4 cm length) and
a 10 mL injection syringe as a gas collection tube (valid
volume 12 mL). The anode was a graphite brush (1.6 cm
diameter × 8 cm; 0.22 m2 surface area). The cathode was
made of carbon cloth (YW-50, YiBang Technology Co.,
Ltd., China), covered with a Pt catalyst layer (0.5 mg Pt/
cm2 in one side) [
The reactors were firstly inoculated with the sludge from
the Wenchang municipal WWTP and were fed with
acetate (1500 mg/L) as carbon source, in 50 mM phosphate
buffer solution (PBS, containing NH4Cl 0.31 g/L, KCl
0.13 g/L, NaH2PO4·2H2O 5.618 g/L, Na2HPO4·12H2O
6.155 g/L, pH 7.0 ± 0.5, 7.3 ± 0.3 mS/cm) [
external voltage was 0.80 ± 0.01 V. All the reactors kept
running stably, in 24 h batch operation, for at least 10
cycles till all replicates performed similar current, COD
removal, and gas production. Twelve reactors were
picked out of the 15, and then every three reactors were
set as replicates, which were randomly divided into four
groups for the test of different sludge fermentative liquid,
obtained from different treatment, as described above.
The SFL for MECs was discharged and refilled at the end
of every 3 d batch cycle over 1 month. A flow schematic
representation of experimental methodology and reactor
setup is shown in (see Additional file 1: Figure S10).
is 4 mol-e−/mol; COD is measured from influence and
effluence of SFL.
Analysis and calculation methods
The currents were automatically monitored (Acquisition
system; Keithley Instrument, US) through a 10 Ω resister.
The gas was collected in a gas bag (50 mL; Cali5-Bond;
Calibrated Instrument Inc, US). Gas composition was
analyzed by a gas chromatograph (Fuli, GC9790;
Zhenjiang Instrument Inc, China), with a packed column [
(TDX-01; 2 m length) and equipped with a TCD detector.
The volume of gas was measured by a glass syringe.
LB-EPSs and TB-EPSs were extracted according to
previous studies [
], and were modified
appropriately. The specific method was: firstly, 10 mL sample was
centrifuged at 4000g for 10 min, the supernatant was
filtered with 0.45 μm cellulose nitrate membrane filters,
and the filtrate was serviced as dissolved organic
matters (DOMs). Secondly, the residue in centrifuge tube
was treated according to the EPS extraction method for
LB-EPSs . The filtrate was processed as the LB-EPSs.
Finally, after LB-EPSs extraction, the residue in
centrifuge tube was treated according to the EPSs extraction
method for TB-EPSs and filtrate was regarded as the
The short-chain fatty acids (SCFAs) were recorded as
the sum of acetic (HAc), propionic (HPr), n-butyric
(nHBu), iso-butyric (iso-HBu), n-valeric (n-HVa), and
isovaleric acids (iso-HVa). SCFAs were analyzed by a gas
chromatograph (Agilent 4890; J&W Scientific, USA)
equipped with a FID detector and a capillary column
(19095N-123HP-INNOWAX; 30 × 0.530 mm × 1.00 μm;
J&W Scientific, USA) [
]. The samples were centrifuged
at 10,000 rpm and filtered through a 0.45 μm membrane
filter, before analysis. Soluble carbohydrate and protein of
filtrate samples were analyzed immediately.
The energy and coulombic efficiency were
calculated to characterize the performance of MEC
reactor. Columbic efficiency indicated the electron recovery
from substrates, which was defined by the ratio of
coulomb recovery to the total coulombs in the substrate.
The coulomb recovery can be calculated by the equation
Q = ∫i·∆t, where i is the current of the external circuit.
The total coulombs can be calculated by the equation
Qt = (CODin − CODeff ) · V · F · b/MO2, where F
represents the Faraday constant, 96,485 C/mol; MO2 is the
molar mass of oxygen, 32 g/mol; b is the complete
oxidation requirement of electron per mole oxygen and b
DNA extraction and Illumina sequencing
Microbial community samples were collected from
fermentation liquid on the 3rd day of fermentation and
anode biofilm at the end of MEC operations.
PowerSoil DNA Isolation Kit (Mo Bio Laboratories,
Carlsbad, CA, US) was used to extract the genomic DNA of
the samples, according to the manufacturer’s
instructions. The quantity and quality of the extracted DNA
were checked by measuring its absorbance at 260 and
280 nm, using a spectrophotometer. Amplicon was
constructed for Illumina sequencing, using bacterial fused
primers 341F (5′-CCTACACGACGCTCTTCCGATCTN
(barcode) CCTACGG-GNGGCWGCAG-3′) and 805R
(barcode) GACTACHVGGGTATCTAATCC-3′) for
the V3–V4 region of the 16S rRNA gene [
were performed in a total volume of 50 μL,
containing 1 × PCR buffer, 1 mM dNTPs, 5 μM each primer, 1
U Plantium Taq, and 10 ng of template DNA. The PCR
amplification program contained an initial denaturation
at 94 °C for 3 min, followed by 5 denaturing cycles at
94 °C for 30 s, annealing at 45 °C for 20 s, and extension
at 65 °C for 30 s, then followed by 20 cycles of denaturing
at 94 °C for 20 s, annealing at 55 °C for 20 s, and
extension at 72 °C for 30 s, finally followed by a final extension
at 72 °C for 5 min.
Before sequencing, PCR products of different
samples were normalized in equimolar amounts in the final
mixture, which was used to construct the PCR
amplicon libraries. Sequencing was carried out on an Illumina
HiSeq 2000, and the raw sequences have been deposited
in the NCBI Short Read Archive (SRA) database, under
the accession number SRR1532554. With similarity set
at 97 % and a confidence threshold of 95 %, the obtained
sequences were phylogenetically allocated down to the
phylum, class, and genus level with the MOTHUR
program (http://www.mothur.org/wiki/Main_Page). To define
the relative abundance of a given phylogenetic group, the
number of sequences affiliated to that group were divided
by the total number of obtained sequences. The results
were used for the analysis and comparison of microbial
community structure differences. The Cytoscape network
layout used in this work was made by Cytoscape 3.2.1,
using detected OUTs as node connectivity to illustrate
groups and inter-group relationships [
Additional file 1. Table S1. The main contents of sludge fermentative
liquid on the 3rd day, after different pretreatments. Table S2. The main
characteristics of raw waste activated sludge. Table S3. The detected
number of sequences, OTUs and diversity. Fig. S1. Particle size distribu‑
tion in sludge, obtained through different pretreatments. Control: sludge
without treatment; A: Alkaline; F: Freeze/thaw; U: Ultrasonic treatment.
Fig. S2. Lysis ratio of increased SCOD to TCOD in sludge, after different
pretreatments. Fig. S3. MEC reactor setup: COD removal and Coulombic
efficiency obtained by feeding artificial wastewater. Fig. S4. VFAs content
in different sludge fermentative liquids. Fig. S5. Main contents of COD
removal (Polysaccharide, protein, VFAs) in MECs fed with different sludge
fermentative liquids. Fig. S6. Current change in the last three batch
MECs fed with different sludge fermentative liquids. Fig. S7. Rarefaction
curves (a) and Shannon diversity (b) base on pyrosequencing of bacterial
communities. The OTUs were defined by 3 % distance. Fig. S8. Taxonomic
classification of bacterial communities of sludge fermentative liquid at the
phylum (a), class (b) and genus (c) levels. Relative abundance was defined
as the number of sequences per sample. Fig. S9. The setup of single
chamber microbial electrolysis cell (MEC). Fig. S10. A flow schematic
representation of experimental methodology and reactor setup.
MEC: microbial electrolysis cell; AD: anaerobic digestion; WAS: waste activated
sludge; BESs: bioelectrochemical systems; COD: chemical oxygen demand;
SCOD: soluble chemical oxygen demand; TCOD: total chemical oxygen
demand; VFAs: volatile fatty acids; LB‑EPS: loosely bound extracellular poly‑
meric substances; TB‑EPS: tightly bound extracellular polymeric substances;
SFL: sludge fermentation liquid; PBS: phosphate buffer solution; OTUs:
operational taxonomic units; ACE: abundance‑based coverage estimator; VSS:
volatile suspended solids; DOMs: dissolved organic matters.
WL and ZH designed and carried out the experiments, performed the data
analysis, and drafted the manuscript. CY, AZ, and ZG carried out the pretreat‑
ment method, participated in the setup operation and maintenance of the
bioreactors. BL and CV participated in data analysis for pyrosequencing results
and revised the manuscript. WL and AW conceived of the study, put forward
the hypothesis, and gave the final approval of publication. All authors read
and approved the final manuscript.
This research was supported by National Science Foundation for Distin‑
guished Young Scholars (Grant No. 51225802), by National Natural Science
Foundation of China (No. 51578534), by “Hundred Talents Program” of the
Chinese Academy of Sciences, and by Project 135 of Chinese Academy of
Sciences (No. YSW2013B06), and by Sino‑EU International S&T cooperation
program (No. S2015GR1012).
The authors declare that they have no competing interests.
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