Science of the Total Environment 677 (2019) 299–306

Contents lists available at ScienceDirect

Science of the Total Environment

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

Potential of direct interspecies electron transfer in synergetic

enhancement of methanogenesis and sulfate removal in an up-flow
anaerobic sludge blanket reactor with magnetite
Zhen Jin, Zhiqiang Zhao ⁎, Yaobin Zhang
Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024,
China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Potential DIET with magnetite during

AD of sulfate-rich wastewater was eval-
uated.
• Both methanogenesis and sulfate re-
moval were significantly enhanced.
• Fe(III)-reducing genera capable of re-
duction sulfate were enriched with
magnetite.
• The abundance of Methanothrix and
Methanosarcina species was improved
with magnetite.
• DIET between sulfate/Fe(III)-reducing
genus and methanogens was
established.

a r t i c l e i n f o a b s t r a c t

Article history: Anaerobic digestion (AD) has been widely applied in the treatment of industrial wastewater containing oxidized
Received 19 January 2019 sulfur compounds. However, the production of hydrogen sulfide usually limits the syntrophic metabolism
Received in revised form 24 April 2019 proceeded by interspecies hydrogen transfer (IHT), due to its corrosive and toxic properties. The current study
Accepted 25 April 2019
was in an attempt to establish direct interspecies electron transfer (DIET) to resist the toxic inhibition from hy-
Available online 26 April 2019
drogen sulfide and keep syntrophic metabolism stable. The results showed that, in the presence of magnetite,
Editor: Zhen (Jason) He the methane production was improved about 3–10 folds at each ratio of COD/SO2− 4 , while the enhancement of
methanogenesis had almost no negative effect on sulfate reduction. With magnetite, the sludge conductance in-
Keywords: creased about 3 folds, but the concentration of c-type cytochromes decreased, suggesting that the potential DIET
Sulfate-rich wastewater via both electrically conductive pili and outer surface c-type cytochromes was established. Microbial community
Methanogenesis revealed that, Veillonella species, the Fe(III)-reducing genus capable of reducing sulfate to hydrogen sulfide, were
Direct interspecies electron transfer (DIET) specially enriched with magnetite. Together with the relatively higher abundance of Methanothrix and
Fe(III)-reducing genus Methanosarcina species, the novel DIET between Fe(III)/sulfate-reducing genus and methanogens was inferred
to be responsible for the synergetic enhancement of methanogenesis and sulfate removal.
© 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail address: zhiqiangzhao@dlut.edu.cn (Z. Zhao).

https://doi.org/10.1016/j.scitotenv.2019.04.372
0048-9697/© 2019 Elsevier B.V. All rights reserved.
300 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306
1. Introduction
Conversion of organic wastes to methane via anaerobic digestion
(AD) has been widely applied in the treatment of industrial wastewater
containing oxidized sulfur compounds, such as metal-laden wastewater
(Sun et al., 2018), textile wastewater (Zeng et al., 2017) and pharma-
ceutical wastewater (Li et al., 2015). It is well known that anaerobic sul-
fate reduction is carried out by the sulfate-reducing bacteria (SRB) that
use sulfate as terminal electron acceptor for the oxidation of organic
compounds or hydrogen, resulting in the production of hydrogen sul-
fide (Hao et al., 2014; Muyzer and Stams, 2008; Zeng et al., 2018). How-
ever, hydrogen sulfide has a great inhibition to the metabolism of
environmentally sensitive methanogens, due to its corrosive and toxic
properties, especially for hydrogen-utilizing methanogens known to
have a heavy duty of maintaining the hydrogen partial pressure low
enough and proceeding the decomposition of organic substrates feasi-
ble in thermodynamics via interspecies hydrogen transfer (IHT)
(Demirel and Scherer, 2008; Muyzer and Stams, 2008; Sela-Adler
et al., 2017). In the presence of high-concentration sulfate, the increased
rates of production of hydrogen sulfide can easily break the balance of
syntrophic metabolism via IHT, resulting in the rise of hydrogen partial
pressure as well as stagnation of methanogenesis.
Direct interspecies electron transfer (DIET) as an alternative to IHT
has been studied in defined methanogenic co-cultures of Geobacter
and Methanothrix (Rotaru et al., 2014b) or Methanosarcina species
(Rotaru et al., 2014a) known as the acetate-utilizing methanogens as
well as in some mixed cultures (Lei et al., 2018; Wang et al., 2018;
Zhao et al., 2017b), in which Methanothrix or Methanosarcina species
were dominant in the communities. Compared with IHT, the advantages
of DIET are obvious. It has been reported that, DIET can effectively main-
tain the methanogenic systems stable under some stressed conditions,
such as high organic loading rates (OLRs) (Zhao et al., 2017a), high hy-
drogen partial pressure and acidic impacts (Zhao et al., 2017b). This is
primarily because that, DIET proceeding via electrically conductive pili
or outer surface c-type cytochromes provides the rapider electron trans-
fer (44.9 × 103 e−cp−1 s−1 vs 5.24 × 103 e−cp−1 s−1) (Storck et al.,
2016) and more energy to support the growth of syntrophic partners
than IHT (Cruz Viggi et al., 2014; Lin et al., 2017). Therefore, enhancing
syntrophic metabolism via DIET during AD of high-concentration
sulfate-rich wastewater is expected to promote better methanogenesis.
Conductive materials including metal-based and carbon-based ma-
terials can promote DIET and improve the rate of syntrophic metabo-
lism (Lin et al., 2017; Lovley, 2017; Martins et al., 2018). The primary
mechanism is that, conductive materials serve as electron conduits,
and DIET-based syntrophic partners directly attach to the surface of
conductive materials for the long-range interspecies electron exchange
due to their high electrical conductance (Martins et al., 2018). Based on
this advantage, Li et al. (2017) used the conductive stainless steel to es-
tablish the DIET-based syntrophic metabolism between anaerobic fer-
mentative bacteria and methanogens in an acidogenic digester,
finding that conductive stainless steel effectively relieved the toxic inhi-
bition from hydrogen sulfide and improved methane production about
7.5–24.6%. However, the performances involved in the removal of
sulfate significantly declined (Li et al., 2017). One possible reason was
that, in the presence of conductive stainless steel, the DIET-based
syntrophic metabolism strongly competed electron donors with SRB,
resulting that SRB could not obtain the sufficient energy to support
their growth and metabolism. As a result, the residual high-
concentration sulfate still had a great threat to methanogens in the sub-
sequent methanogenic digester.
A potential strategy for the synergetic enhancement of
methanogenesis and sulfate removal during AD of high-concentration
sulfate-rich wastewater is to establish the DIET-based syntrophic me-
tabolism between SRB and methanogens. In other words, SRB metabo-
lize the organic substrates to acetate and then directly transfer
electrons to methanogens for the reduction of carbon dioxide to
methane. In this case, SRB serving as the DIET-based electron donators
can obtain sufficient energy to support their growth and sulfate-
reducing metabolism. Remarkably, up to now, there is no evidence indi-
cating that SRB can proceed DIET with methanogens in defined co-
cultures, and the DIET-based syntrophic metabolism between the typi-
cal SRB, such as Desulfovibrio or Desulfotomaculum species, and
methanogens is still not established in the traditional methanogenic di-
gesters, even if in the presence of conductive carbon-based materials
(Chen et al., 2014). However, the studies on anaerobic methane oxida-
tion in the mixed cultures comprised of a methane-oxidizing archaea,
ANME-1, and its sulfate-reducing syntrophic partner, Desulfofervidus
auxilii, observed that D. auxilii downregulated the expression of genes
for multiheme cytochromes known as the important biological electri-
cal components, suggesting that D. auxilii known as the special enrich-
ments of SRB were capable of participating in DIET (Krukenberg et al.,
2016; Wegener et al., 2015). Therefore, enriching the novel species of
SRB capable of participating in DIET is necessary.
Magnetite, a common mineral in modern soils and sediments, has
been recently applied in the establishment of DIET-based syntrophic
metabolism in the paddy soils and sediments (Kato et al., 2012;
Rotaru et al., 2018; Xiao et al., 2018). Apart from magnetite serving as
the electron conduits, it has been demonstrated that magnetite can sub-
stitute for the pilus-associated cytochrome OmcS of Geobacter
sulfurreducens (Liu et al., 2015). More importantly, some studies with
magnetite reported that the dissimilatory iron reduction could enrich
some Fe(III)-reducing bacteria capable of extracellular electron transfer
to Fe(III) oxides or electrode (Lovley et al., 2011; Shi et al., 2016). It
should be pointed out that, the reduction of elemental sulfur or sulfate
is very commonly linked to the ability to extracellular transfer electrons
to Fe(III) oxides or electrode (Lovley et al., 2011). Therefore, it is reason-
ably suggested that, magnetite supplemented into the digester treating
sulfate-rich wastewater can enrich the novel species of sulfate/Fe(III)-
reducing bacteria that proceed DIET with methanogens. Based on
these considerations, the study presented here was for the first time
to investigate the potential of DIET between sulfate/Fe(III)-reducing
bacteria and methanogens to the synergetic enhancement of
methanogenesis and sulfate reduction in the presence of magnetite.
2. Material and methods
2.1. Inoculum sludge and magnetite synthesis
Inoculum sludge used in this study was collected from an anaerobic
digester of a municipal sludge treatment plant in Dalian (China) and an-
aerobically stored at 4 °C before using. The initial TSS of the sludge was
17.08 ± 0.16 g/L and ratio of VSS and TSS was 0.68.
The synthetic method of magnetite particles was described in a pre-
viously protocol (Kang et al., 1996). In brief, 0.016 mol FeCl2 and
0.032 mol FeCl3 were dissolved into 0.4 M HCl solution. The mixed solu-
tion was added dropwise into a 1.5 M NaOH solution with vigorous stir-
ring and then generated black precipitate (Fe3O4). The precipitate was
washed three times with deionized water and one time with 0.1 M
HCl. The average size of the magnetite (1.2 ± 0.2 μm) was measured
by a dynamic light scattering (DLS) with a Malvern Nano ZS90 (Malvern
Instruments, UK), and the crystalline structure of these magnetite parti-
cles were evaluated by an X-ray powder diffractometer (XRD) (Empy-
rean, Panalytical, Netherlands) with Cu-K-α-radiation from 5 °C to 80
°C at 45 kV and 200 mA (see Fig. 1B).
2.2. Experimental setup
The continuous-flow experiments were conducted in two parallel
up-flow anaerobic sludge blanket (UASB) reactors (internal diameter
of 70 mm and height of 300 mm) each of which had a working volume
of 1000 mL. At the top of each reactor, a 3 L gas sampling bag was con-
nected with the gas outlet of the three-phase separator (Fig. 1C). Each of
 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306
Fig. 1. Synthesis of magnetite (A), XRD diffraction patterns of the synthesis of magnetite and standard magnetite (B) and schematic diagram of the UASB setup (C).
the UASB reactors received a 400 mL of inoculum sludge before experi-
ments, and was operated at a temperature of 37 ± 1 °C for 80 days.
After 20-day experiments, the synthesized magnetite particles were
added into the suspended sludge of one of both UASB reactors (referred
as RM) with a final concentration of Fe atom at 20 mM. Another UASB
reactor without synthesized magnetite particles supplemented was
served as a control (referred as RC).
The main components of the artificial high-concentration sulfate-
rich wastewater were as follows (per liter): C6H12O6, 5.16 g; NH4Cl,
0.48 g; KH2PO4, 0.11 g; trace element solution, 1 mL; vitamin solution,
1 mL and NaHCO3 was used to adjust pH at approximately 8.20. The
trace element solution contained the following components (per liter)
(Bao and Li, 2017): FeSO4·7H2O, 2.1 g; CoCl2·6H2O, 0.19 g;
ZnSO4·7H2O, 0.144 g; MnCl2·4H2O, 0.1 g; Na2MoO4·2H2O, 0.036 g;
H3BO3, 0.03 g; NiCl·6H2O, 0.024 g; CuCl2·2H2O, 0.002 g. The vitamin so-
lution contained the following components (per liter) (Bao and Li,
2017): pyridoxine dihydrochloride, 0.15 g; calcium D-pantothenate,
0.05 g; 4-aminobenzoic acid, 0.04 g; D-biotin, 0.01 g; nicotinic acid,
0.01 g. Throughout the operational period, the influent OLR was main-
tained at 13.86 kg COD m−3 d−1 with a hydraulic retention time
(HRT) of 8.66 h. The sulfate source was Na2SO4 and the ratio of COD/
SO2−
4 varied from 5.00, 3.75 to 2.50 by gradually increasing the influent
sulfate concentration.
2.3. Analytical methods
Total suspended solid (TSS) and volatile suspended solid (VSS) were
evaluated according to the Standard Methods (Walter, 1998). The pH
was calculated using a pH analyzer (Denver Instrument; UB-10; Den-
ver). Sulfate was determined by ion chromatography (Shimadzu HIC-
VP Super, Japan) with a conductivity detector (Shimadzu SCL-10ASP,
Japan) at a retention time of approximately 25 min. Chemical oxygen
301
demand (COD) was measured with Hach's method 8000 (Hach, DR/
890, USA). VFAs were analyzed by a gas chromatograph, equipped
with a flame ionization detector (FID) (Tianmei, GC-7900P/FID,
China). The biogas volume in the gas sampling bag was measured
with a 100 mL plastic syringe every day. The content of the biogas was
analyzed by another gas chromatograph with a thermal conductivity
detector (TCD) (Tianmei, GC-7900P/TCD, China). The Fe2+ and total Fe
concentration were analyzed by ortho-phenanthroline spectrophotom-
etry at 510 nm (Techcomp, UV-2301, China).
2.4. Sludge conductance
After 80-day experiments, the sludge conductance in both UASB re-
actors was measured as described in previous research (Zhao et al.,
2016). The schematic diagram of sludge conductance measurement
was shown in Fig. S1. Briefly, the sludge samples were washed by
0.1 M NaCl three times and subsequently were placed on the two‑gold
electrodes with a non-conductive gap. Then an electrochemical work-
station (Zhenhua, CHI1030C, China) was used to provide voltages varied
from −0.3 V to 0.3 V with steps of 0.025 V. The time-averaged currents
for different voltages were obtained and used to create the current-
voltage curve. The conductivity of the sludge was calculated by the fol-
lowing formula Eq. (1):
L
σ¼ ð1Þ
RS
where σ is the conductivity (S/m), L is the width of the gap with values
of 0.5 × 10−3 m, R is the reciprocal value of slope obtained by the
current–voltage curve, and S is the cross- sectional area with values of
2.54 × 10−6 m2.
302 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306

2.5. C-type cytochromes

Sludge samples (1 mL) collected from the end of experiments were

resuspended in 0.01 M PBS solution (9 mL) by manual homogenization
in a glass homogenizer about 8 min. Then the mixtures were centri-
fuged at 3000 rpm within 15 min to extract supernatants. Subsequently
the concentration of c-type cytochromes was determined by utilizing
the Cytochrome c ELISA kit (Shanghai Lanpai Biotechnology Co., Ltd.,
China). The O.D. value was measured by a microplate reader at
450 nm (Bio-Rad iMark, USA).

2.6. High-throughput sequencing

Sludge samples (10 mL) were collected from RM and RC, respec-
tively and immediately frozen and stored at −80 °C after sampling to
analyze microbial communities and functional genes via high-
throughput sequencing. The E.Z.N.A™ Mag-Bind Soil DNA kit was used
to extract DNA from these samples in accordance with the manufactur-
er's protocols. Bacterial and archaeal V3-V4 region of the 16S rRNA
genes were amplified by PCR. PCR amplification was carried out under
the following thermal cycler: 94 °C for 3 min, followed by 5 cycles of
94 °C for 30 s, 45 °C for 20 s and 65 °C for 30s, and 20 cycles of 94 °C
for 20 s, 55 °C for 20 s and 72 °C for 30s after which a final extension
at 72 °C for 5 min. According to DNA concentrations and molecular
weight, multiple samples were pooled together in the same proportions
and were purified using calibrated Agencourt Ampure XP beads. Then
Qubit3.0 DNA was used to accurate quantification of DNA and sequence.
Similarity searches for the assembled sequences were analyzed using
the NCBI Blast search program (http://www.ncbi.nlm.nih.gov/blast/).
STAMP was also used to analyze differences between RC and RM of
functional genes. The raw sequence files were submitted to the NCBI Se-
quence Read Archive database under accession No. SRP186846.

2.7. Calculation

The methane conversion efficiency (MCE, %) was calculated by the

following formula Eq. (2):

CODmethane
MCE ¼  100% ð2Þ
CODinf −CODeff

where CODmethane is the methane concentration (gCOD/L), CODinf is the

influent COD concentration (gCOD/L), and CODeff is effluent COD con-
centration (gCOD/L).

3. Results Fig. 2. Methane production rate (A), COD removal efficiency (B) and methane conversion
efficiency (MCE) (C) in both UASB reactors during the 80-day experiments. Error bars
represent standard deviations of three-group parallel measurements.
3.1. Effects of magnetite on methanogenesis

The performances in both UASB reactors with equivalent (P N 0.05)
methane production (Fig. 2A) and organic removal (Fig. 2B) diverged
after magnetite were added into one of both UASB reactors during the
initial 20-day experiments at a COD/SO2−
4 ratio of 5.00. When magnetite
were supplemented into RM, the methane production rate gradually in-
creased to about 157 mL/d at day 40, while the methane production rate
in RC without magnetite supplemented presented an initially increased
but then decreased trend. Finally, the methane production rate in RC
stabilized at about 48 mL/d, which was about 1/3 of that in RM at the
same time. After 40-day experiments, the COD/SO2− 4 ratio was gradually
decreased from 5.00 to 2.50 via increasing the influent concentration of
sulfate over the next 40 days. The gap of methane production rate be-
tween RM and RC increased further (Fig. 2A). At each ratio of COD/
SO2−
4 , the average methane production rate with magnetite supple-
mented was 3–10 folds higher than that without magnetite
supplemented.
As expected from the methane production data (Fig. 2A), the COD
removal efficiency was also higher in the presence of magnetite at a rel-
atively lower COD/SO2− 4 ratio (Fig. 2B). Specially, as the ratio of COD/
SO2−
4 was decreased from 5.00 to 2.50, the COD removal efficiency in
RM increased from 38.4% to 46.7%, while that of RC stabilized at about
36.0%. As showed in Fig. S2, the total VFA concentrations in RC at COD/
SO2−
4 ratios of 5.00, 3.75 and 2.50 were higher than in RM. It was also
noticed that, after magnetite supplemented into RM, a significant de-
crease in the concentration of acetate was observed.
A gradual improvement in the reduction of sulfate led to a quite
lower MCE in RC (Fig. 2C). Namely, the sulfate reduction might contrib-
ute more organic removal than methanogenesis. Besides, the further de-
crease of the ratio of COD/SO2−
4 to 2.50 immediately disrupted the MCE,
which was nearly declined to 0 (Fig. 2C), and the performances of
methanogenesis in RC declined soon thereafter. Conversely, the MCE
in RM with magnetite supplemented gradually increased (Fig. 2C). At
 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306 303

each ratio of COD/SO2− 4 , the MCE with magnetite supplemented was

6–13% higher than that without magnetite supplemented, suggesting
that an effective interspecies electron transfer pathway capable of
resisting the toxic inhibition from hydrogen sulfide was established to
proceed syntrophic metabolism between syntrophs and methanogens.

3.2. Effects of magnetite on sulfate removal

When magnetite was added into RM, the sulfate removal efficiency
was only slightly higher than that of RC (Fig. 3A) at a COD/SO2−4 ratio
of 5.00. Then the influent concentration of sulfate was gradually in-
creased over the next 40 days, and the gap of sulfate removal efficiency
between RM and RC increased further (Fig. 3A), suggesting that magne-
tite supplemented into the UASB reactor also had a positive effect on
sulfate reduction. Commonly, the effective syntrophic methanogenic
metabolism between syntrophs and methanogens that resulted in the
significant increase of MCE (Fig. 2C) might have a great advantage to
compete organic substrates with sulfate reduction. The hydrogen sul-
fide production rates and total soluble sulfide in RM were slightly higher
than in RC (Fig. 3B, Table S1), when the ratio of COD/SO2− 4 was de-
creased from 5.00 to 2.50, suggesting that the sulfate reduction was
also enhanced with magnetite. To evaluate the relationship between
methanogenesis and sulfate reduction, mass balance based on COD re-
moval was calculated (Fig. 3C). Surprisingly, with magnetite supple-
mented, the contribution of sulfate reduction to COD removal also
gradually increased as the decrease of the ratio of COD/SO2− 4 , similar
to the trend of MCE, suggesting that effective syntrophic methanogenic
metabolism established by magnetite did not limit the sulfate-reducing
metabolism. In addition, from Fig. 3C when the COD/SO2− 4 ratio de-
creased from 5.00 to 2.50, the percentage of effluent COD accounting
for total influent COD decreased by about 8% (from 66.9% to 59.4%) in
RM reactor, while the percentage of COD-sulfate accounting for total in-
fluent COD was increased by about 8% (from 13.8% to 21.5%). Therefore,
it seemed like that, the improved COD removal might be primarily as-
cribed to the improved sulfate reduction.

3.3. Sludge conductance and C-type cytochromes

Electrical aggregates have been considered as an important evidence

to support the DIET-based syntrophic metabolism established in the
methanogenic communities (Wang et al., 2019; Zhao et al., 2016).
Therefore, to investigate the potential establishment of DIET-based
syntrophic metabolism via electrically conductive pili in the presence
of magnetite, the conductivity of suspended sludge in both UASB reac-
tors was measured. The conductivity of sludge in RM was 25.6 ± 1.5
μS/cm, about 3 folds higher than that of in RC (8.7 ± 0.5 μS/cm)
(Fig. 4A).
Apart from electrically conductive pili, the c-type cytochromes is an-
other crucial biological electrical component for nanowire which played
a significant role in improving long-range extracellular electron transfer
(Wang et al., 2018). As shown in Fig. 4B, the concentration of c-type cy-
tochromes in RM was 113.54 nmol/L, which was about half of that in RC,
suggesting the potential interspecies electron transfer via magnetite
that substitutes for the pilus-associated c-type cytochromes.
3.4. Microbial community composition
The predominant methanogenic genus in RC belonged to
Methanobacterium species known as the typical hydrogen-utilizing
methanogens, accounting for N90% of the methanogenic communities
(Fig. 5A). In iron corrosion environment, a novel Methanobacterium-
like archaea could produce methane faster than hydrogen-using
methanogens, indicating that it could get electrons through a direct ac-
cess than hydrogen consumption (Dinh et al., 2004). In addition, in the
first study of ‘electromethanogenesis’, Cheng et al. (2009) observed that
the dominant methanogenic species in the cathodic biofilm belonged to
Fig. 3. Sulfate removal efficiency (A), hydrogen sulfide production rate (B) and mass
balance (based on COD removal) (C) in both UASB reactors during the 80-day
experiments. Error bars represent standard deviations of three-group parallel
measurements.
Methanobacterium palustre. Together with no molecular hydrogen de-
tected, it was suggested that Methanobacterium palustre might partici-
pate in the direct conversion of electric current into methane.
However, there was no evidence demonstrated that Methanobacterium
species could directly accept electrons from Geobacter species via DIET
in defined co-cultures. In RM, however, Methanobacterium species
remained abundant, but less so. Their decline was accompanied by the
significant increase of Methanothrix species known as the actetate-
utilizing methanogens, which accounted for 23.4% of the methanogenic
communities and was about 20% higher than that in RC (Fig. 5A). The
most closest relative of Methanothrix species detected (Table S2),
Methanothrix concilii (100%, similarity), were reported to have a heavy
duty of accepting electron via DIET for the reduction of carbon dioxide
to methane in methanogenic digester treating brewery wastes (Morita
et al., 2011). Another acetate-utilizing methanogens, Methanosarcina
species, were almost not detected in RC, but accounted for 5.5% of the
304 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306
Fig. 4. Conductivity (A) and c-type cytochromes (B) of the suspended sludge in both UASB reactors after the 80-day experiments. Error bars represent standard deviations of three-group
parallel measurements.
methanogenic communities in RM (Fig. 5A). The closest relative of
Methanothrix species detected (Table S2), Methanosarcina barkeri
(98%) species were capable of accepting electrons from Geobacter spe-
cies for the reduction of carbon dioxide to methane in defined methan-
ogenic co-cultures (Rotaru et al., 2014a).
In RC, the dominant bacteria belonged to the typical sulfate-reducing
genus, Desulfovibrio species, accounting for 24.4% of the bacterial com-
munities (Fig. 5B). The second dominant bacteria, Veillonella species ca-
pable of dissimilatory reduction of radionuclides and iron, only
accounted for 6.7% of the bacterial communities (Fig. 5B) (Woolfolk
and Whiteley, 1962; Mohapatra et al., 2010). In the presence of magne-
tite, the abundance of Desulfovibrio species significantly declined by
about 8%, but Veillonella species became the dominant sulfate-
reducing genus with a relative abundance of 23.3% (Fig. 5B). It should
be noted that, Veillonella species also have the ability to reduce elemen-
tal sulfur or sulfate to hydrogen sulfide (Dash et al., 2012), and it was
suggested that magnetite could support the growth of Veillonella species
to participate in sulfate-reducing metabolism. The microbial commu-
nity analysis also detected some anaerobic fermentation genus capable
of converting complex organics to short-chain fatty acids and alcohols,
such as Lactococcus and Aminobacterium species, but the differences in
the abundance of these genuses between RC and RM were not signifi-
cant (Fig. 5B).
4. Discussion
The results present here demonstrated that, magnetite could effec-
tively enhance syntrophic metabolism to resist the toxic inhibition
from hydrogen sulfide (Fig. 3) and improve methanogenesis (Fig. 2)
during AD of high-concentration sulfate-rich wastewater. The effluent
concentration of Fe2+ was 1.46 mg/L and 5.81 mg/L in RC and RM, re-
spectively (Fig. S3), and the nutritional effect of Fe2+ on the improve-
ment of methanogenesis was negligible since its concentration was
significantly lower than 10 mg/L (Kato et al., 2012; Wang et al., 2018).
However, the Fe2+ and total Fe concentration in RM were slightly
higher than RC. Unlike the carbon-based materials, such as graphene
did not function as electron shuttles, Fe(III) release from magnetite
might aid in shuttling electrons for EET via redox cycling of Fe(II)/Fe
(III) (Baek et al., 2016; Tian et al., 2017). Therefore, magnetite
proceeded the dissimilatory iron reduction to provide the energy for
the growth of these Fe(III)-reducing genera, which was supported by
the high expression of functional genes involved in the inorganic ion
transport and metabolism (Fig. S4) (Baek et al., 2016; Zhao et al.,
2017a). Generally, Fe2+ produced by dissimilatory iron reduction
might precipitate FeS to reduce the inhibition of hydrogen sulfide to
methanongens. However, the results in our study were not support
this assumption. On the one hand, the hydrogen sulfide concentration
in RM was higher than in RC. On the other hand, the XRD of the
suspended sludge after 80-day experiments (Fig. S5) showed that, the
main mineral was quartz in both RC and RM, and no FeS was detected
in RM.
Therefore, the primary reason involved in the synergetic enhance-
ment of methanogenesis and sulfate removal might be ascribed that,
magnetite served as electron conduits and substituted for the c-type cy-
tochromes to establish the DIET-based syntrophic metabolism between
syntrophs and methanogens. The most direct evidence to support this
was that, with magnetite, the sludge conductance (Fig. 4A) was signifi-
cantly increased, but the concentration of c-type cytochromes (Fig. 4B)
was declined to half of that without magnetite supplemented. The
mechanism involved in the increase of sludge conductance was that
electrical aggregates which was DIET-based syntrophs, such as
Geobacter species, facilitated DIET via their electrically conductive pili
to form the electrical networks (Morita et al., 2011). Furthermore, Liu
et al. (2015) demonstrated that, magnetite could substitute for the
pilus-associated cytochrome OmcS in defined co-cultures of two
Geobacter species. In addition, the concentration of c-type cytochromes
was also decreased with magnetite, suggesting that magnetite im-
proved establishment of DIET by alleviating the need for pili and c-
type cytochromes (Wang et al., 2018; Wang et al., 2019).
In the presence of magnetite, the relative abundance of both
Methanothrix and Methanosarcina species significantly increased
(Fig. 5A), suggesting that the DIET-based electron acceptors for the re-
duction of carbon dioxide to methane were most likely them. However,
Li et al. (2018) recently reported that the conductive carbon cloth might
promote acetoclastic methanogenesis (mainly Methanosarcina species)
instead of the reduction of carbon dioxide in wetland soil. It should be
pointed out that, the metabolism rates of methanogenesis between
the natural conditions, such as soils and sediments, and high-OLR di-
gesters are quite different, even if Methanothrix or Methanosarcina spe-
cies are similarly dominant. In the high-OLR methanogenic digesters,
 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306
Fig. 5. Archaeal (A) and bacterial (B) communities in the suspended sludge of both UASB
reactors after 80-day experiments.
Methanothrix or Methanosarcina species are required to obtain more en-
ergy to support their growth and metabolize, but their conversion of
acetate to methane via traditional acetoclastic pathway only yields little
energy (Dang et al., 2016). Similar to this, our previous study (Zhao
et al., 2018) observed the significant accumulation of acetate
due to the slow acetoclastic methanogenesis in an anaerobic digester
treating waste activated sludge (WAS), which resulted in the decreased
abundance of Methanothrix species. However, Methanothrix and
Methanosarcina species may benefit from accepting electrons via DIET
from conductive materials, since DIET provides the rapid electron trans-
fer (Storck et al., 2016) and more energy (Cruz Viggi et al., 2014) to sup-
port the growth of Methanothrix and Methanosarcina species.
Furthermore, Methanosarcina species are more favorable for utilizing
the high-concentration acetate to produce methane than Methanothrix
species, and the high-concentration acetate may hereby stimulate
the methanogenic communities to enrich Methanosarcina species.
However, the gradual increase in the effluent concentration of acetate
(Fig. S2) suggested a lowered ability of acetate consumption,
which was not in accordance with the increase in abundance of
Methanosarcina species.
No Geobacter species were detected in this study, even if magnetite
were supplemented into the reactor (Fig. 5B), suggesting the DIET-
based electron donators might be not the familiar syntrophs. However,
305
in the presence of magnetite, some Fe(III)-reducing genera, such as
Veillonella and Enterobacter species (De Castro and Ehrlich, 1970;
Emde et al., 1992; Mohapatra et al., 2010), were significantly enriched
(Fig. 5B). The mechanisms involved in extracellular electron transfer
to Fe(III) oxides are very commonly linked to that of electron transfer
to methanogens via DIET (Kato et al., 2012; Shi et al., 2016). Therefore,
it seemed likely that, Veillonella or Enterobacter species might partici-
pate in DIET to proceed the syntrophic metabolism. Also, DIET
established by magnetite provided the rapider electron transfer and
more energy to support the growth of the DIET-based electron donators
(Baek et al., 2016; Wang et al., 2018). As a result, the abundance of
Veillonella and Enterobacter species significantly increased.
Remarkably, Veillonella species are also capable of participating in
sulfate-reducing metabolism. Veillonella species were found in direct
agar shake dilutions of sediment with L-lactate as the only energy source
which can fermentation of lactate to propionate, acetate and carbon di-
oxide (Delwiche, 1985). Furthermore, Dash et al. (2012) reported that
an isolate strain (WS-1) which was collected from sewage water was
closely related to Veillonella species and had ability to reduce sulfate to
sulfide. In the presence of magnetite, special enrichment of Veillonella
species not only proceeded the DIET with methanogens, but were re-
sponsible for reducing the sulfate to hydrogen sulfide. This might be
the reason that, even though DIET-based methanogenic metabolism
strongly competed with the electron donors, the sulfate reduction was
still facilitated (Fig. 3). However, the abundance of the typical SRB,
Desulfovibrio species, significantly declined (Fig. 5B), suggesting that
Desulfovibrio species were not capable of participating in DIET. Actually,
it was reported that, the conductive carbon-based materials could not
accelerate the syntrophic metabolism of a co-culture of Desulfovibrio
vulgaris and Geobacter sulfurreducens (Chen et al., 2014). One possible
reason was that, Desulfovibrio species grew syntrophically primarily
via producing hydrogen or formate as interspecies electron carriers
(Matias et al., 2005) (Fig. S6).
5. Conclusions
The study presented here demonstrated that, magnetite supple-
mented into the anaerobic digester treating high-concentration
sulfate-rich wastewater could enrich some Fe(III)-reducing bacteria ca-
pable of participating in sulfate reduction, such as Veillonella species.
These special sulfate/Fe(III)-reducing bacteria might proceed DIET via
magnetite serving as electron conduits or compensating outer surface
c-type cytochromes with Methanothrix and Methanosarcina species to
resist the toxic inhibition from hydrogen sulfide, maintain methano-
genic metabolism stable, and more importantly provide the more en-
ergy to support the DIET-based syntrophic partners. As a result, the
sulfate removal was also kept at a relatively stable efficiency.
Conflict of interest statement
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Acknowledgments
The authors acknowledge the financial support from National Natu-
ral Science Foundation of China (51578105, 51808097).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2019.04.372.
306 Z. Jin et al. / Science of the Total Environment 677 (2019) 299–306
References
Baek, G., Kim, J., Lee, C., 2016. A long-term study on the effect of magnetite supplementa-
tion in continuous anaerobic digestion of dairy effluent - enhancement in process
performance and stability. Bioresour. Technol. 222, 344–354. https://doi.org/
10.1016/j.biortech.2016.10.019.
Bao, P., Li, G., 2017. Sulfur-driven iron reduction coupled to anaerobic ammonium oxida-
tion. Environ. Sci. Technol. 51 (12), 6691–6698. https://doi.org/10.1021/acs.
est.6b05971.
Chen, S., Rotaru, A.E., Liu, F., Philips, J., Woodard, T.L., Nevin, K.P., Lovley, D.R., 2014. Carbon
cloth stimulates direct interspecies electron transfer in syntrophic co-cultures.
Bioresour. Technol. 173, 82–86. https://doi.org/10.1016/j.biortech.2014.09.009.
Cheng, S., Xing, D., Call, D.F., Logan, B.E., 2009. Direct biological conversion of electrical
current into methane by electromethanogenesis. Environ. Sci. Technol. 43 (10),
3953–3958. https://doi.org/10.1021/es803531g.
Cruz Viggi, C., Rossetti, S., Fazi, S., Paiano, P., Majone, M., Aulenta, F., 2014. Magnetite par-
ticles triggering a faster and more robust syntrophic pathway of methanogenic pro-
pionate degradation. Environ. Sci. Technol. 48 (13), 7536–7543. https://doi.org/
10.1021/es5016789.
Dang, Y., Holmes, D.E., Zhao, Z., Woodard, T.L., Zhang, Y., Sun, D., Wang, L., Nevin, K.P.,
Lovley, D.R., 2016. Enhancing anaerobic digestion of complex organic waste with
carbon-based conductive materials. Bioresour. Technol. 220, 516–522. https://doi.
org/10.1016/j.biortech.2016.08.114.
Dash, J., Das, T., Mishra, B.K., Kar, R.N., 2012. Isolation, characterization and growth control
of a sulfate reducing bacteria from sewage water. Appl. Ecol. Env. Res. 10 (3),
233–242. https://doi.org/10.15666/aeer/1003_233242.
De Castro, A.F., Ehrlich, H.L., 1970. Reduction of iron oxide minerals by a marine Bacillus.
Anton. Leeuw. 36 (3), 317–327. https://doi.org/10.1007/BF02069033.
Delwiche, E.A., 1985. The veillonella: Gram-negative cocci with a unique physiology. Annu.
Rev. Microbiol. 39, 175–193. https://doi.org/10.1146/annurev.mi.39.100185.001135.
Demirel, B., Scherer, P., 2008. The roles of acetotrophic and hydrogenotrophic
methanogens during anaerobic conversion of biomass to methane: a review. Rev. En-
viron. Sci. Bio. 7 (2), 173–190. https://doi.org/10.1007/s11157-008-9131-1.
Dinh, H.T., Kuever, J., Mußmann, M., Hassel, A.W., Stratmann, M., Widdel, F., 2004. Iron
corrosion by novel anaerobic microorganisms. Nature 427, 829–832. https://doi.
org/10.1038/nature02321.
Emde, K.M.E., Smith, D.W., Facey, R., 1992. Initial investigation of microbially influenced
corrosion (MIC) in a low-temperature water distribution-system. Water Res. 26
(2), 169–175. https://doi.org/10.1016/0043-1354(92)90216-Q.
Hao, T., Xiang, P., Mackey, H.R., Chi, K., Lu, H., Chui, H., van Loosdrecht, M.C., Chen, G.,
2014. A review of biological sulfate conversions in wastewater treatment. Water
Res. 65, 1–21. https://doi.org/10.1016/j.watres.2014.06.043.
Kang, Y.S., Risbud, S., Rabolt, J.F., Stroeve, P., 1996. Synthesis and characterization of
nanometer-size Fe3O4 and γ-Fe2O3 particles. Chem. Mater. 8, 2209–2211. https://
pubs.acs.org/doi/abs/10.1021/cm960157j.
Kato, S., Hashimoto, K., Watanabe, K., 2012. Methanogenesis facilitated by electric
syntrophy via (semi)conductive iron-oxide minerals. Environ. Microbiol. 14 (7),
1646–1654. https://doi.org/10.1111/j.1462-2920.2011.02611.x.
Krukenberg, V., Harding, K., Richter, M., Glockner, F.O., Gruber-Vodicka, H.R., Adam, B.,
Berg, J.S., Knittel, K., Tegetmeyer, H.E., Boetius, A., Wegener, G., 2016. Candidatus
Desulfofervidus auxilii, a hydrogenotrophic sulfate-reducing bacterium involved in
the thermophilic anaerobic oxidation of methane. Environ. Microbiol. 18 (9),
3073–3091. https://doi.org/10.1111/1462-2920.13283.
Lei, Y., Wei, L., Liu, T., Xiao, Y., Dang, Y., Sun, D., Holmes, D.E., 2018. Magnetite enhances
anaerobic digestion and methanogenesis of fresh leachate from a municipal solid
waste incineration plant. Chem. Eng. J. 348, 992–999. https://doi.org/10.1016/j.
cej.2018.05.060.
Li, J., Xiao, L., Zheng, S., Zhang, Y., Luo, M., Tong, C., Xu, H., Tan, Y., Liu, J., Wang, O., Liu, F.,
2018. A new insight into the strategy for methane production affected by conductive
carbon cloth in wetland soil: beneficial to acetoclastic methanogenesis instead of CO2
reduction. Sci. Total Environ. 643, 1024–1030. https://doi.org/10.1016/j.
scitotenv.2018.06.271.
Li, W., Niu, Q., Zhang, H., Tian, Z., Zhang, Y., Gao, Y., Li, Y., Nishimura, O., Yang, M., 2015.
UASB treatment of chemical synthesis-based pharmaceutical wastewater containing
rich organic sulfur compounds and sulfate and associated microbial characteristics.
Chem. Eng. J. 260, 55–63. https://doi.org/10.1016/j.cej.2014.08.085.
Li, Y., Zhang, Y., Yang, Y., Quan, X., Zhao, Z., 2017. Potentially direct interspecies electron
transfer of methanogenesis for syntrophic metabolism under sulfate reducing condi-
tions with stainless steel. Bioresour. Technol. 234, 303–309. https://doi.org/10.1016/j.
biortech.2017.03.054.
Lin, R., Cheng, J., Zhang, J., Zhou, J., Cen, K., Murphy, J.D., 2017. Boosting biomethane yield
and production rate with graphene: the potential of direct interspecies electron
transfer in anaerobic digestion. Bioresour. Technol. 239, 345–352. https://doi.org/
10.1016/j.biortech.2017.05.017.
Liu, F., Rotaru, A.E., Shrestha, P.M., Malvankar, N.S., Nevin, K.P., Lovley, D.R., 2015. Magne-
tite compensates for the lack of a pilin-associated c-type cytochrome in extracellular
electron exchange. Environ. Microbiol. 17 (3), 648–655. https://doi.org/10.1111/
1462-2920.12485.
Lovley, D.R., 2017. Syntrophy goes electric: direct interspecies electron transfer. Annu.
Rev. Microbiol. 71, 643–664. https://doi.org/10.1146/annurev-micro-030117-
020420.
Lovley, D.R., Ueki, T., Zhang, T., Malvankar, N.S., Shrestha, P.M., Flanagan, K.A., Aklujkar, M.,
Butler, J.E., Giloteaux, L., Rotaru, A.E., Holmes, D.E., Franks, A.E., Orellana, R., Risso, C.,
Nevin, K.P., 2011. Geobacter: the microbe electric's physiology, ecology, and practical
applications. Adv. Microb. Physiol. 59, 1–100. https://doi.org/10.1016/B978-0-12-
387661-4.00004-5.
Martins, G., Salvador, A.F., Pereira, L., Alves, M.M., 2018. Methane production and conduc-
tive materials: a critical review. Environ. Sci. Technol. 52 (18), 10241–10253. https://
doi.org/10.1021/acs.est.8b01913.
Matias, P.M., Pereira, I.A., Soares, C.M., Carrondo, M.A., 2005. Sulphate respiration from hy-
drogen in Desulfovibrio bacteria: a structural biology overview. Prog. Biophys. Mol.
Biol. 89 (3), 292–329. https://doi.org/10.1016/j.pbiomolbio.2004.11.003.
Mohapatra, B.R., Dinardo, O., Gould, W.D., Koren, D.W., 2010. Biochemical and genomic
facets on the dissimilatory reduction of radionuclides by microorganisms – a review.
Miner. Eng. 23 (8), 591–599. https://doi.org/10.1016/j.mineng.2010.03.004.
Morita, M., Malvankar, N.S., Franks, A.E., Summers, Z.M., Giloteaux, L., Rotaru, A.E., Rotaru,
C., Lovley, D.R., 2011. Potential for direct interspecies electron transfer in methano-
genic wastewater digester aggregates. MBio 2 (4), e00159-11. https://doi.org/
10.1128/mBio.00159-11.
Muyzer, G., Stams, A.J., 2008. The ecology and biotechnology of sulphate-reducing bacte-
ria. Nat. Rev. Microbiol. 6 (6), 441–454. https://doi.org/10.1038/nrmicro1892.
Rotaru, A.E., Shrestha, P.M., Liu, F., Markovaite, B., Chen, S., Nevin, K.P., Lovley, D.R., 2014a.
Direct interspecies electron transfer between Geobacter metallireducens and
Methanosarcina barkeri. Appl. Environ. Microbiol. 80 (15), 4599–4605. https://doi.
org/10.1128/AEM.00895-14.
Rotaru, A.E., Shrestha, P.M., Liu, F., Shrestha, M., Shrestha, D., Embree, M., Zengler, K.,
Wardman, C., Nevin, K.P., Lovley, D.R., 2014b. A new model for electron flow during
anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the re-
duction of carbon dioxide to methane. Energy Environ. Sci. 7 (1), 408–415. https://
doi.org/10.1039/C3EE42189A.
Rotaru, A.E., Calabrese, F., Stryhanyuk, H., Musat, F., Shrestha, P.M., Weber, H.S.,
Snoeyenbos-West, O.L.O., Hall, P.O.J., Richnow, H.H., Musat, N., Thamdrup, B., 2018.
Conductive particles enable syntrophic acetate oxidation between Geobacter and
Methanosarcina from coastal sediments. mBio 9 (3), e00226-18. https://doi.org/
10.1128/mBio.00226-18.
Sela-Adler, M., Ronen, Z., Herut, B., Antler, G., Vigderovich, H., Eckert, W., Sivan, O., 2017.
Co-existence of methanogenesis and sulfate reduction with common substrates in
sulfate-rich estuarine sediments. Front. Microbiol. 8, 766. https://doi.org/10.3389/
fmicb.2017.00766.
Shi, L., Dong, H., Reguera, G., Beyenal, H., Lu, A., Liu, J., Yu, H.Q., Fredrickson, J.K., 2016. Ex-
tracellular electron transfer mechanisms between microorganisms and minerals. Nat.
Rev. Microbiol. 14 (10), 651–662. https://doi.org/10.1038/nrmicro.2016.93.
Storck, T., Virdis, B., Batstone, D.J., 2016. Modelling extracellular limitations for mediated
versus direct interspecies electron transfer. ISME J 10 (3), 621–631. https://doi.org/
10.1038/ismej.2015.139.
Sun, R., Zhang, L., Zhang, Z., Chen, G.H., Jiang, F., 2018. Realizing high-rate sulfur reduction
under sulfate-rich conditions in a biological sulfide production system to treat metal-
laden wastewater deficient in organic matter. Water Res. 131, 239–245. https://doi.
org/10.1016/j.watres.2017.12.039.
Tian, T., Qiao, S., Li, X., Zhang, M., Zhou, J., 2017. Nano-graphene induced positive effects
on methanogenesis in anaerobic digestion. Bioresour. Technol. 224, 41–47. https://
doi.org/10.1016/j.biortech.2016.10.058.
Walter, W.G., 1998. APAH standard methods for the examination of water and wastewa-
ter. Am. J. Public Health Nations Health. 56 (3), 387.
Wang, C., Liu, Y., Jin, S., Chen, H., Xu, X., Wang, Z., Xing, B., Zhu, L., 2019. Responsiveness
extracellular electron transfer (EET) enhancement of anaerobic digestion system dur-
ing start-up and starvation recovery stages via magnetite addition. Bioresour.
Technol. 272, 162–170. https://doi.org/10.1016/j.biortech.2018.10.013.
Wang, T., Zhang, D., Dai, L., Dong, B., Dai, X., 2018. Magnetite triggering enhanced direct
interspecies electron transfer: a scavenger for the blockage of electron transfer in an-
aerobic digestion of high-solids sewage sludge. Environ. Sci. Technol. 52 (12),
7160–7169. https://doi.org/10.1021/acs.est.8b00891.
Wegener, G., Krukenberg, V., Riedel, D., Tegetmeyer, H.E., Boetius, A., 2015. Intercellular
wiring enables electron transfer between methanotrophic archaea and bacteria. Na-
ture 526 (7574), 587–590. https://doi.org/10.1038/nature15733.
Woolfolk, C.A., Whiteley, H.R., 1962. Reduction of inorganic compounds with molecular
hydrogen by Micrococcus Lactilyticus I. J. Bacteriol. 84, 647–658.
Xiao, L., Liu, F., Liu, J., Li, J., Zhang, Y., Yu, J., Wang, O., 2018. Nano-Fe3O4 particles acceler-
ating electromethanogenesis on an hour-long timescale in wetland soil. Environ. Sci:
Nano 5, 436–445. https://doi.org/10.1039/c7en00577f.
Zeng, Q., Hao, T., Mackey, H.R., Wei, L., Guo, G., Chen, G., 2017. Alkaline textile wastewater
biotreatment: a sulfate-reducing granular sludge based lab-scale study. J. Hazard.
Mater. 332, 104–111. https://doi.org/10.1016/j.jhazmat.2017.03.005.
Zeng, Q., Hao, T., Mackey, H.R., van Loosdrecht, M.C.M., Chen, G., 2018. Recent advances in
dissimilatory sulfate reduction: from metabolic study to application. Water Res. 150,
162–181. https://doi.org/10.1016/j.watres.2018.11.018.
Zhao, Z., Zhang, Y., Yu, Q., Dang, Y., Li, Y., Quan, X., 2016. Communities stimulated with
ethanol to perform direct interspecies electron transfer for syntrophic metabolism
of propionate and butyrate. Water Res. 102, 475–484. https://doi.org/10.1016/j.
watres.2016.07.005.
Zhao, Z., Li, Y., Quan, X., Zhang, Y., 2017a. Towards engineering application: potential
mechanism for enhancing anaerobic digestion of complex organic waste with differ-
ent types of conductive materials. Water Res. 115, 266–277. https://doi.org/10.1016/
j.watres.2017.02.067.
Zhao, Z., Zhang, Y., Li, Y., Dang, Y., Zhu, T., Quan, X., 2017b. Potentially shifting from inter-
species hydrogen transfer to direct interspecies electron transfer for syntrophic me-
tabolism to resist acidic impact with conductive carbon cloth. Chem. Eng. J. 313,
10–18. https://doi.org/10.1016/j.cej.2016.11.149.
Zhao, Z., Li, Y., He, J., Zhang, Y., 2018. Establishing direct interspecies electron transfer dur-
ing laboratory-scale anaerobic digestion of waste activated sludge via biological
ethanol-type fermentation pretreatment. ACS Sustain. Chem. Eng. 6, 13066–13077.
https://doi.org/10.1021/acssuschemeng.8b02618.