Journal of Cleaner Production 279 (2021) 123482

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Journal of Cleaner Production

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

Medium chain fatty acids production from anaerobic fermentation of

waste activated sludge
Shu-Lin Wu a, Gang Luo b, c, Jing Sun a, c, Wei Wei a, **, Lan Song d, e, Bing-Jie Ni a, c, *
a
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, PR
China
b
Shanghai Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan
University, 200433, Shanghai, China
c
Shanghai Institute of Pollution Control and Ecological Security, Shanghai, 200092, PR China
d
School of Environmental Science and Engineering, Southern University of Science and Technology, Shenzhen, 518055, PR China
e
Shenzhen Institute of Sustainable Development, Shenzhen, 518055, PR China

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

Article history: The utilization of waste activated sludge (WAS) to recover energy in the form of methane or short-chain
Received 11 May 2020 fatty acids (SCFAs) is generally restricted by the low energy density of products and poor degradability of
Received in revised form WAS. Herein, this study reported a novel alternative WAS fermentation technology to produce high-
23 June 2020
energy medium-chain fatty acids (MCFAs) from WAS in a one-stage anaerobic fermentation system
Accepted 27 July 2020
using ethanol as electron donor. The MCFAs production and WAS degradation at different ethanol levels
^ as de
Handling editor: Cecilia Maria Villas Bo were investigated. The increased ethanol levels resulted in increasing MCFAs production (from 1875 to
Almeida 6115 mg chemical oxygen demand (COD)/L) and selectivity (from 30.3 to 56.2%). The main MCFAs
products were n-caproate and n-caprylate at lower level of ethanol, while n-caproate was the sole MCFA
Keywords: product at higher level of ethanol with longer chain alcohol (i.e., n-hexanol) produced as well. The
Medium-chain fatty acid ethanol markedly increased WAS degradation, with the greatest degradation (0.72 g COD/g volatile solids
Waste activated sludge (VS)) being 1.9 times of that without ethanol (0.38 mg COD/mg VS, at 0 mmol/L), which was ascribed to
Anaerobic fermentation the advancement of sludge solubilization, hydrolysis and acidification. Microbial community revealed
Chain elongation
that the ethanol participation induced the community shift to the favorable direction for hydrolysis-
Degradation
acidification and chain elongation in anaerobic WAS fermentation.
Ethanol
© 2020 Elsevier Ltd. All rights reserved.

1. Introduction sludge treatment, by which the high levels of organic matters in

Wastewater treatment plants (WWTPs) are increasing rapidly to
serve the requirements of the growing population and the devel-
oping industry (Foladori, 2010). Biological treatment of wastewater
is a highly sustainable treatment process but resulting in large
amount of waste activated sludge (WAS) (Wei et al., 2018; Li et al.,
2018; Wu et al., 2019). Increased sludge production could incur
substantial disposal costs, thus reducing sludge amounts has a
strong economic motivation.
Anaerobic fermentation is the most widely adopted method for
* Corresponding author. State Key Laboratory of Pollution Control and Resources
Reuse, College of Environmental Science and Engineering, Tongji University,
Shanghai, 200092, PR China.
** Corresponding author.
E-mail addresses: hitvivi@126.com (W. Wei), bjni@tongji.edu.cn (B.-J. Ni).
sludge could be bio-converted into value-added products, such as
methane or/and short-chain fatty acids (SCFAs, those with
5
carbon chains), thereby realizing the energy recovery and sludge
reduction/stabilization (Appels et al., 2011; Chen et al., 2014).
However, the sum of energy recovered through WAS anaerobic
fermentation is typically only 5e7% of the total available energy
owing to the low energy density of methane and SCFAs (Steinbusch
et al., 2011; Wei et al., 2017a). Besides, SCFAs feature high hydro-
philicity due to the short carbon chain, thus the operation of sep-
aration and purification of SCFAs from the fermentation liquor is
complicated and energy intensive, which largely limits the eco-
nomic viability of WAS anaerobic fermentation (Wu et al., 2019).
More importantly, the low hydrolysis rates of WAS generally
restrained WAS anaerobic fermentation, resulting in less fermen-
tation products and unfavorable sludge reduction (Wang et al.,
2013).

https://doi.org/10.1016/j.jclepro.2020.123482
0959-6526/© 2020 Elsevier Ltd. All rights reserved.
2 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482
Recently, the medium chain fatty acids (MCFAs, those contain
6e12 carbon chains) have been considered (Angenent et al., 2016)
as more profitable anaerobic products. Due to their higher carbon/
oxygen ratio, MCFAs have the higher energy densities and eco-
nomic value than the traditional methane and SCFAs. MCFAs are
superior precursor for biofuels or industrial chemicals process
(Agler et al., 2012a; Shu et al., 2011). Besides, MCFAs have excellent
separation property due to their relatively low solubilities, which
simplify the separation procedure from the fermentation liquor
with much less energy input than SCFAs (Angenent et al., 2016). The
in-line product extraction also prevents the toxic effect of accu-
mulated acids on microorganisms and ensures the stable MCFAs
production (Ge et al., 2015). Chain elongation (CE) (Chen et al.,
2017; Ge et al., 2015; Spirito et al., 2014) is an emerging biotech-
nology to transform SCFAs into MCFAs via the reverse b-oxidization
pathway.
The pathway of chain elongation is a cyclic process and utilizes
an acetyl-CoA molecule each cycle, which comes from an electron
donor (ED), coupling with a SCFA (as the electron acceptor (EA))
and extending its carbon chain length with two carbons (C2)
(Angenent et al., 2016; Wu et al., 2018). Several electron donors that
have been utilized to elongate carboxylates are ethanol (Coma et al.,
2016; Gonza lez et al., 2013; Liu et al., 2017; Steinbusch et al., 2011),
lactate (Kucek et al., 2016a; Zhu et al., 2015), methanol (Genthner
et al., 1981), n-propanol (Kenealy and Waselefsky, 1985), D-galac-
titol (Angenent et al., 2016) and amino acids in peptides (Wallace
et al., 2004). During the use with lactate as ED, it was reported
that lactate would split to the conversion to propionate via acrylate
pathway, reducing the lactate-carbon flow to MCFA-producing via
chain elongation pathway (Wu et al., 2018). Proofs of other electron
donors were only made with pure bacterial cultures (Angenent
et al., 2016) or suggested less production of MCFAs. Therefore,
ethanol is considered as the most suitable electron donor for MCFAs
production with the function of microbiome via chain elongation
(Wu et al., 2018). Many substrates were used for the production of
MCFAs, including glucose, gaseous products and organic wastes
(Levy et al., 1981; Thauer et al., 1968; Kucek et al., 2016c; Xu et al.,
2017; Weimer et al., 2015; Lonkar et al., 2016), which can be con-
verted to different types of SCFAs as starting molecules via anaer-
obic fermentation. MCFAs production performances depended on
the properties of substrates. For example, Weimer et al. (2015)
utilized the cellulosic biomass (alfalfa stems or switchgrass herb-
age) as substrates and the produced MCFA was only caproate with
little amount. In contrast, a large amount of caproate, heptanoate,
caprylate and nonanoic acid were produced by acid whey as sub-
strate (Xu et al., 2017). Different from other organic matters, WAS
has unique property, which is composed of a mass of organic
substances (mainly carbohydrate and protein, occupying 40e80%)
and microbial cells (Wei et al., 2017b). However, the MCFAs pro-
duction from anaerobic fermentation of WAS has never been
documented.
Herein, this study aimed to demonstrate the MCFAs production
from WAS as a feedstock substrate with enhanced WAS degradation
in a one-stage anaerobic fermentation system with ethanol as
electron donor. The MCFAs production, products distribution and
WAS degradation at different ethanol levels were investigated and
compared. The mechanisms of enhanced MCFAs production and
WAS degradation by ethanol were studied from the aspect of the
conversions of metabolic intermediates. Moreover, the shift of
microbial communities was analyzed and linked to the apparent
performance of CE. This work is expected to provide a novel insight
into the sludge reduction and energy recovery.
2. Material and methods
2.1. Substrate and inoculum
The utilized WAS was collected from the secondary sedimen-
tation tank of a municipal WWTP located in Shanghai, China
treating domestic wastewater. After concentrated for 1d by gravity,
the WAS was reserved at 4  C for later use. The primary charac-
teristics of the concentrated sludge were listed in Table S1 in Sup-
porting Information (SI). The inoculum (anaerobically digested
sludge, ADS) was the acclimatized sludge harvested from a semi-
continuous mesophilic anaerobic fermenter (hydraulic retention
time: 15 d) fed with ethanol and acetic acid for MCFAs production.
The semi-continuous reactor was controlled at 37 ± 1  C with a
sludge retention time of 20 d.
2.2. Experiments of MCFAs production from anaerobic WAS
fermentation
In this study, MCFAs production from the WAS with a wide
range of ethanol levels (i.e., 0, 42.5, 85 and 170 mmol/L) was
assessed using four series of test serum bottles (120 mL working
volume) to demonstrate the feasibility of MCFAs production and
the involving mechanisms of ethanol. Within each serum bottle, it
contained 4.8 mL of inoculum and 4.8 mL of WAS with 37.5 mL of
growth medium, whose component was detailed in the SI. After-
wards, 0, 42.5, 85 and 170 mmol/L of ethanol was added into the
four test bottles, respectively. Then, the pH value was set as 5.5 ± 0.1
with 3 mol/L NaOH and HCl solution, which was the optimum pH
for chain elongation (Ge et al., 2015). Finally, after flushed with
nitrogen gas (with high purity) for 5 min, all vials were sealed with
rubber stoppers and aluminum caps, and then stirred in a shaker-
incubator (37 ± 1  C, 170 rpm). Correspondingly, four blanks were
also established to evaluate the effects of the inoculation sludge on
MCFAs production with the studied ethanol levels (i.e., 0, 42.5, 85
and 170 mmol/L). Each blank was identical to the corresponding
test vial except that WAS was replaced by the same volume of tap
water. The fermentation time lasted for over 30 days without
further MCFAs accumulation. Each test was performed in triplicate.
Every 2e3 days, the liquid samples (2 mL) were collected,
centrifuged (12000 rpm, 5 min) and filtered with a 0.45 mm filter.
The products concentration from WAS were calculated by sub-
tracting the concentration in the blank bottle (i.e., ADS with
ethanol) from that in the corresponding test bottle (i.e., WAS and
ADS with ethanol).
2.3. Role of ethanol in solubilization, hydrolysis and acidification in
WAS degradation
Usually, the WAS degradation during anaerobic fermentation
contains three steps, i.e., solubilization, hydrolysis and acidification
(Wei et al., 2019), which were detailed in the SI. These processes
accompany with the production of SCFAs, which were served as the
substrates for the CE process and elongated into MCFAs. It is thus
necessary to explore the influence of ethanol on these processes in
order to explain the different performance with the ethanol
participation. The experiments of effects of ethanol (0 mmol/L as
control and 170 mmol/L as test) on these three processes were
conducted with the model substrates (Mu and Chen, 2011) as
follows.
Solubilization tests: the experiment was carried out under the
same operation as the above MCFAs production experiment. After
 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482
fermenting for 2 days, determining the soluble protein and carbo-
hydrate. Besides, the lactate dehydrogenase (LDH) and DNA in
fermentation liquid were measured to assess the cell membrane
integrity. Each fermentation liquid was characterized by excitation
emission matrix (EEM) fluorescence spectroscopy to further indi-
cate the level of WAS degradation.
Hydrolysis tests: bovine serum albumin (BSA, average molecular
weight Mw 67000), and dextran (average molecular weight
Mw~23800) were chosen as model protein and polysaccharide
compounds, respectively (Mu and Chen, 2011). The hydrolysis test
was operated with the identical method depicted in solubilization
test in addition to that the WAS was replaced with the same volume
of synthetic wastewater, whose component were shown in SI. Then,
adding 4 g/L of BSA and 1 g/L dextran in the synthetic wastewater.
By determining the BSA and dextran degradation after the
fermentation for 2 days, the effect of ethanol on WAS hydrolysis
was obtained.
Acidification tests: the same operations were conducted with
the above approach except that selecting 4 g/L of L-glutamate and
1 g/L of glucose as model amino acid and monosaccharide com-
pound. The concentrations of VFAs were measured after the
fermentation for 4 days.
2.4. Microbial community analysis
After the four test groups reached stable performance for MCFAs
production, three samples were withdrawn from each group and
mixed. The microbial communities of four mixed samples from the
four test groups and raw WAS as well as the inoculum were
analyzed using high-throughput 16S rRNA gene sequencing. Briefly,
10 ml of fully mixed and homogeneous sludge mixture was with-
drawn and centrifuged at 10000 rpm for 5min. The microbial
genomic DNA were extracted with the E.Z.N.A. soil DNA kit (Omega
Biotek, Norcross, GA, U.S.). The V4 hypervariable regions of the
bacteria 16s rRNA gene were amplified with primers 515F (50 -
GTGCCAGCMGCCGCGG-30 ) and 806R (50 -GGACTACH
0
VGGGTWTCTAAT-3 ). Purified amplicons were pooled in equimolar
and paired-end sequenced (2  300) on Illumina MiSeq platform
(Illumina, San Diego, USA). The sequence data were processed at
Majorbo Bio-Pharm Technology Co. Ltd. (Shanghai, China).
2.5. Analytical procedures
The measurement including TS, VS, TCOD, SCOD were per-
formed with the same approach as reported in literature (Wei et al.,
2019). The gas composition (i.e., hydrogen and methane) was
analyzed by gas chromatography (LUNAN, SP-6890, China) equip-
ped with a thermal conductivity detector. The liquid component
(i.e., alcohols, SCFAs and MCFAs) were measured by a Gas Chro-
matography (SHIMADAU GC, 2010 Plus, Japan) equipped with a
flame ionization detector. Protein, carbohydrate, LDH/DNA and
EEM fluorescence spectra were measured according to the methods
detailed in SI.
3. Results
3.1. MCFAs production from anaerobic fermentation of WAS
Fig. 1 shows the ethanol utilization and MCFAs production from
WAS (i.e., subtracting the production in the blank vial for ADS with
ethanol solely) over the whole fermentation period. During the
anaerobic fermentation of WAS without the ethanol addition, a
continuous accumulation of total SCFAs (i.e., acetate and n-buty-
rate) from WAS was observed (shown in Fig. 1a). Apparently, MCFAs
was hardly synthesized with single SCFAs, demonstrating that the
3
existence of ED was the prerequisite of feasible CE (Wu et al., 2018).
Expectably, the MCFAs were produced with the ethanol participa-
tion (42.5e170 mmol/L). At ethanol of 42.5 mmol/L (shown in
Fig. 1b), ethanol decreased rapidly and depleted during the
fermentation for 9 days, whereas the n-caproate and n-caprylate as
the main MCFAs components emerged on Day 7, and their pro-
ductions gradually increased and peaked on Day 24 (n-caproate:
710 ± 28 mg COD/L, n-caprylate: 1293 ± 32 mg COD/L). Afterwards,
the concentrations of MCFAs remained stable, which was likely
attribute to the exhaustion of ED. When the ethanol dosage
increased to 85 mmol/L (shown in Fig. 1c), the ethanol was nearly
drained on Day 17. Although the 85 mmol/L of ethanol dosage
extended the lag phase (14 days) in the CE reactions compared to
that (7 days) of the 42.5 mmol/L of ethanol dosage, more MCFAs
from WAS were produced through the 30 days’ CE process. The
maximal n-caproate and n-caprylate concentrations were
1335 ± 64 mg COD/L and 945 ± 21 mg COD/L, respectively. During
the 30 days’ CE process, the highest MCFAs production from WAS
occurred at the 170 mmol/L of ethanol dosage (shown in Fig. 1d). At
the starting point of CE, ethanol was consumed fast to 63.2 mmol/L
while n-caproate as the sole component of MCFAs appeared on day
7 and reached a maximum value of 6320 ± 156 mg COD/L on Day
24. Interesting, 805 ± 92 mg COD/L of n-hexanol was also synthe-
sized. Although the residual ethanol and n-butyrate was observed,
the cumulative MCFAs concentration kept constant. It is likely that
the undissociated caproate exerted toxic effects in the microor-
ganism related to CE process. Vasudevan et al. (2014) reported that
the inhibition of MCFAs production occurred once the concentra-
tion of undissociated caproate reaches ~0.2 g/L. The concentration
of undissociated caproate at the case with 170 mmol/L of ethanol
was determined as ~0.53 g/L.
Overall, the MCFAs production from WAS was successfully
achieved with the addition of ethanol as ED and the increased
ethanol led to higher MCFAs production. The main MCFAs products
were n-caproate and n-caprylate under the ethanol dosage of 42.5
and 85 mmol/L, while n-caproate was the sole MCFA product at the
ethanol dosage of 170 mmol/L, with the production of longer chain
alcohol (i.e., n-heptanol) as well.
3.2. Products distribution and MCFAs selectivity
Fig. 2 summarizes the product distribution based on the con-
verted SCOD from WAS from the four tests. At ethanol concentra-
tion of 0, 42.5, 85, 170 mmol/L, the converted SCOD from WAS
which deduct from ethanol conversion were 0.04, 0.23, 0.42, 0.51 g
COD, respectively. The methane in each case was not detected.
Without the ethanol participation, SCFAs (i.e., acetate and n-buty-
rate) occupied the maximum proportions of 83.9 ± 6.6%, which
could not be further elongated into MCFAs due to the lack of ED. The
5.2 ± 0.7% SCOD existed in and the remaining 10.9 ± 1.8% of SCOD
might be the intermediate metabolites that were not completely
hydrolyzed and acidified. The SCFAs proportion decreased to
25.7 ± 5.2% with the addition of 42.5 mmol/L of ethanol, but the n-
caproate and n-caprylate appeared, accounting for 12.1% and 18.2%,
respectively. Besides, a remaining 42.8 ± 1.2% of the converted
SCOD was not further converted to SCFAs or MCFAs. Increasing
ethanol dosage to 85 mmol/L, the SCFAs proportion further
decreased to 16.8 ± 0.2%, accompanying the increment of MCFAs
(i.e., n-caproate and n-caprylate). The unaccounted SCOD decreased
to 31.9 ± 1.4%. In contrast, n-caproate as the main product of the
fermentation occupied 56.2 ± 1.2% at the highest ethanol level of
170 mmol/L. Interestingly, a longer-chain alcohol, n-hexanol, was
produced with 7.4 ± 0.8% proportion instead of n-caprylate. Only
11.4% of unaccounted SCOD was remained in the fermentation li-
quor, suggesting that ethanol could enhance the SCOD to be further
4 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482

Fig. 1. Cumulative anaerobic fermentation products from the WAS anaerobic fermentation and the ethanol concentrations with (a) 0 mmol/L ethanol; (b) 42.5 mmol/L ethanol; (c)
85 mmol/L ethanol; and (d) 170 mmol/L ethanol WAS. Error bar represent standard errors of triplicate tests.

converted into SCFAs or MCFAs.
MCFAs selectivity represents the proportion of MCFAs in all
fermentation products. As seen in Fig. 2, MCFAs selectivity was on
the rise as increased ethanol dosage. At the ethanol dosage of 0,
42.5, 85 and 170 mmol/L, the MCFAs selectivity was 0%, 30.3%, 33.0%
and 56.2%, respectively. This revealed that ethanol participation as
ED is effective in enhancing both MCFAs production and selectivity
from WAS fermentation via CE process.
3.3. Enhanced WAS degradation by ethanol addition
ethanol levels. In general, the addition of ethanol as ED led to a
significant increase of SCOD yield and increased ethanol level gave
rise to an enhanced release of SCOD. For the WAS without the
ethanol addition, the SCOD yield was only 0.38 g COD/g VS. In
contrast, SCOD content increased from 0.45 to 0.61 g COD/g VS over
the same period with the increase of ethanol dosage from 42.5 to
85 mmol/L. The SCOD content from WAS further increased to 0.72 g
COD/g VS at the greatest ethanol level tested (170 mmol/L), indi-
cating that the WAS via CE process with 170 mmol/L of ethanol as
EDs was degraded 1.9 times (0.72 versus 0.38 g COD/g VS) that
without ethanol participation (0 mmol/L).

During the WAS anaerobic fermentation, the cells and/or par-

ticulate organic substrates in WAS is degraded with the release of
soluble organic substrates. The total SCOD from WAS were 0.04,
0.23, 0.42, 0.51 g COD at ethanol concentration of 0, 42.5, 85 and
170 mmol/L, respectively. Fig. 2 presents the SCOD distribution
from WAS during the entire CE period (i.e., 30 days) at different
3.4. Mechanisms of enhanced WAS degradation and MCFAs
productions
From the above results, the ethanol participation is effective in
enhancing both WAS degradation and MCFAs production with WAS
 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482
Fig. 2. The products distribution in SCOD at different ethanol levels: (a) 0 mmol/L; (b) 42.5 mmol/L; (c) 85 mmol/L; and (d) 170 mmol/L.
as substrate. During WAS anaerobic fermentation, WAS degrada-
tion usually undergoes several key steps, such as the solubilization,
hydrolysis and acidification processes, accompanying the pro-
ductions of SCFAs, which were subsequently elongated into MCFAs.
It is thus required to investigate the influence of ethanol on these
processes in order to explain the potential mechanism. These
processes usually co-occur in the real anaerobic fermenters. In this
work, consequently, a succession of model substrates was used to
indicate the role of ethanol in these processes.
Protein and carbohydrate generally are the dominant constitu-
ents of WAS (Mu and Chen, 2011), which can be released from
sludge cells and/or converted from sludge particulate organic
matters during the solubilization process. Fig. 3a presents the
protein and carbohydrate contents after 2 days fermentation with
and without the ethanol participation. As seen from Fig. 3a, the
ethanol significantly (P ¼ 0.03) increased the release of protein,
while the polysaccharide content did not show a change (P > 0.05)
compared to that without the ethanol addition. Besides, the LDH
and DNA, as cell membrane integrity markers (Wang et al., 2018),
which could be used to reveal the effect of ethanol on disruption of
WAS cells. Fig. 3b showed the measurements of LDH and DNA re-
leases after the fermentation for 2 days with and without the
ethanol participation. Aligning to the results of protein and car-
bohydrate contents, the 170 mmol/L of ethanol significantly
increased the LDH and DNA releases by 24.5 ± 0.71% and 25 ± 1.41%,
respectively, suggesting that the addition of ethanol did incur more
leakage of intracellular substrates. The organics released in
fermentation liquid were further characterize by the three-
dimension EEM fluorescence spectroscopy. The images with and
without the ethanol participation are shown in Fig. 3c and d. One
major peak at the Ex/Em of 270e280/360e370 nm presented in
EEM fluorescence spectra of two fermentation liquids. This location
of EEM peak is generally related to tryptophan & Protein-like of
Region IV (Wen et al., 2015). The ethanol participation clearly
enhanced the fluorescence intensity of this peak, implying that the
5
ethanol promoted more protein substances be released in the
fermentation liquid.
The impact of ethanol on the hydrolysis of WAS solubilized
products (i.e., soluble protein and carbohydrate) is shown in Fig. 3e.
The ethanol enhanced the degradation of the BSA (model protein)
rather than the dextran (model carbohydrate). Without the ethanol
addition, the BSA degradation was 31.0 ± 4.2% after the anaerobic
fermentation for 2 days. In comparison, the BSA degradation with
the 170 mmol/L of ethanol over the same period was 50.5 ± 2.1%,
indicating a significant (P ¼ 0.03) increase of 19.5%. The dextran
degradation efficiency was relatively high and the impact of
ethanol on the dextran degradation was insignificant (P > 0.05). The
effect of ethanol on the accumulation of SCFAs is shown in Fig. 3f.
The produced SCFAs in each case mainly contained acetate and n-
butyrate. The total SCFA concentrations after the fermentation for 4
days with 0 and 170 mmol/L of ethanol were 2428 ± 34 and
3092 ± 62 mg COD/L, respectively. Obviously, the ethanol had a
positive effect on acidification process.
Based on these results, the ethanol participation improved WAS
degradation by enhancing the solubilization, hydrolysis and acidi-
fication processes, thereby increasing the SCFAs production. As the
substrates of MCFAs, the more SCFAs were subsequently elongated
into more MCFAs via CE process.
3.5. Changes of microbial community for MCFAs production
To figure out the mechanisms of MCFAs production using WAS
as substrate deeply, the microbial community was characterized
based on the four samples (R0, R1, R2 and R3) from the bottles with
the studied dosage of ethanol (i.e., 0, 42.5, 85 and 170 mmol/L) after
reached stable performance for MCFA production. In addition, two
samples from the inoculum and the WAS were also included. For all
samples, 28,933 high-quality sequence reads were obtained after
normalization. The overall number of operational taxonomic units
(OTUs) was 748, with 252 shared by the six samples (Fig. S1). The
6 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482

Fig. 3. Effects of ethanol on waste activated sludge anaerobic digestion: (a) the concentrations of protein and carbohydrate after sludge solubilization for 2 days; (b) the release of
LDH and DNA after sludge solubilization for 2 days; (c) the EEM profiles of liquid after sludge solubilization for 2 days with 0 mmol/L ethanol addition and (d) with 170 mmol/L of
ethanol. (e) the degradation of solubilized products (BSA and dextran) after 2 days reaction; and (f) the concentration of acidification products (SCFAs) after 4 days reaction. Error
bars represent standard deviations.

rarefaction curves (Fig. S2) of all samples nearly reached apexes and
smoothness, indicating that the sequencing data of each sample
had a high quality and was reliable to cover the whole microbial
diversity.
Principal component analysis (PCA) based on the community
diversity of the whole microbiomes shown in Fig. 4a reflected the
divergence 16S rRNA sequences of different CE sludge samples. The
PC1 and PC2 occupied for 75.63% of the total community variations.
According to the distribution distance, microbiomes of “R1” “R2”
and “R3” with the ethanol clustered together, while the micro-
biomes of “R0” “WAS” and “inoculum” remained dispersed, sug-
gesting clear shifts of microbial communities after the addition of
ethanol. Fig. 4b exhibits the phylum-level distributions of the mi-
crobial abundances of the six samples. With the ethanol partici-
pation, Aminicenantes, Bacteroidetes, Firmicutes and Proteobacteria
were the four dominant phyla, occupying for ~59%, 64% and 62% in
 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482 7

Fig. 4. Distribution of microbial populations at the phylum level: (a) Principal component analysis (PCA) of the sequencing data in a 2D graph of PC1 and PC2; and (b) Relative
abundances of microorganisms of different sludge samples.

R1, R2 and R3, respectively. Among in the above phyla, a lot of stricto_12 sp. surged in the relative abundances. Moreover, Oscil-
bacteria were reported capable of promoting organic matters (e.g., libacter sp. is generally consider as the predominant bacteria for
proteins and carbohydrates) degradation (Wang et al., 2018). In MCFA production during ethanol-acetate fermentation system (Wu
contrast, these four phyla mentioned above in the R0 (with 0 mmol/ et al., 2018) and their relative abundances in R1, R2 and R3 were
L ethanol) just accounted for 44%. In particular, the addition of higher than that in R0. These results confirmed that the microbial
ethanol caused increase in the abundances of Firmicutes from 5% to community with the ethanol participation was diverted to the
13e18%, which was correlated with CE process (Wu et al., 2018). favorable direction toward hydrolysis, acidification and chain
The microbial communities at genus level (Fig. 5) in the six elongation, which was accord with the higher produce of MCFAs
samples contained of lots of anaerobes related with hydrolysis (e.g., discovered in the experiments.
Bacteroides sp.), acidogenesis (e.g., Proteiniclasticum sp., Escherichia-
Shigella sp., Parabacteroides sp. and Collinsella sp.) as well as chain
4. Discussion
elongation (e.g., Clostridium sp., Oscillibacter sp. and Dechloromonas
sp.) (Kucek et al., 2016b; Wang et al., 2018; Wu et al., 2018). Part of
This research experimentally demonstrated for the first time the
these microbial abundances were influenced by the ethanol. For
MCFAs production from anaerobic fermentation with WAS as a
instance, the relative abundance of Bacteroides sp. in R0 was 0.02%,
feedstock and ethanol as electron donors with enhanced WAS
lower than those (0.2e1.2%) in the vials with the ethanol partici-
degradation in a one-stage anaerobic fermentation system.
pation. The above result indicates that the addition of ethanol had
The addition of ethanol as electron donor was clearly beneficial
positive correlation on the abundance of hydrolytic microorgan-
to the MCFAs production from WAS and the increased ethanol
isms. The relative abundances of Escherichia-Shigella sp. and Para-
levels led to higher MCFAs production. The maximum MCFAs pro-
bacteroides sp. also increased from 0.001% to 0.003% in R0 to
duction from WAS was achieved at 5.37 mg COD/L. This finding was
0.05e0.1% and 0.26e2.5% in R1-R3, respectively, which was in
accordance with previous reports. Liu et al. (2016) found that the
accord with the results of enhanced acidification test with the
higher ratio of ethanol to acid improved the n-caproate production
ethanol addition observed in Fig. 3f. When MCFAs have been pro-
(exceed 3000 mg/L) at the reaction stable phase. Lonkar et al.
duced with the ethanol as EDs, an OTU for Clostridium sensu
(2016) reported that although MCFAs yield almost doubled when
8 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482
Fig. 5. Heatmap showing the difference of the relative abundances of microorganisms (by genus) related to MCFAs production.
the ethanol level was increased to 108 mmol/L, the ethanol con-
centration should be within the appropriate range. The maximum
tolerable concentration of pure-culture and open-culture micro-
organisms to ethanol was 460 mmol/L and 300 mmol/L, respec-
tively (Kucek et al., 2016; Weimer and Stevenson, 2012). Exceeding
this range, MCFAs production would be affected.
Strikingly, the n-caprylate was produced at the lower ethanol
level (e.g., 42.5 and 85 mmol/L) rather than the higher level (e.g.,
170 mmol/L). This was likely attributed to the toxicity of mass-
produced MCFAs affecting the further chain elongation. Lonkar
et al. (2016) also reported that the high concentration of propanol
(i.e., above 15 g/L) as ED even ceased the production of n-hepta-
noate. However, Spirito et al. (2018) applied MCFA extraction
method in the experiment to alleviate the toxicity of undissociated
MCFAs and found that higher ethanol levels result in higher spec-
ificities for n-caprylate production. Besides, a longer chain alcohol
(i.e., n-heptanol) was produced at the higher ethanol level in this
study, which may be attributed to the reduction of carboxylates
(Agler et al., 2011). The carboxylates reduction has been observed in
several fermentation process (Diender et al., 2016; Steinbusch et al.,
2008). Carboxylates are first reduced to aldehydes with ethanol or
other organic substance as electron donor, and then further is
reduced to their corresponding alcohols (Liu et al., 2014; Richter
et al., 2016; Tashiro et al., 2004). Agler et al. (2011) also found
that the produced carboxylates were reduced to alcohols with
hydrogen or ethanol via secondary fermentation. Besides, the
increased ethanol could enhance the converted SCOD from WAS to
be further converted into MCFAs, thereby improving the MCFAs
selectively. Grootscholten et al. (2013a) also reported that
increasing the ethanol load from 13 g L1 d1 to 26 g L1 d 1 led to
MCFA selectivity increased from 78% to 91%. The maximum MCFA
selectivity according to the converted SCOD from WAS and
consumed ethanol in this study was 56.2% (Fig. 2). This selectivity is
lower than that obtained in previous reports with the SCFAs as the
direct substrate (Grootscholten et al., 2013b, 2014; Wu et al., 2018),
which is mainly limited by SCFAs conversion from WAS via hy-
drolysis and acidification. Similarly, Lonkar et al. (2016) also ob-
tained a lower MCFA selectivity of ~23% with office paper and
chicken manure as the substrates and ethanol as ED.
The ethanol increased WAS degradation to up to 1.9 times as
much as that without the ethanol, which was mainly ascribed to the
enhanced sludge solubilization, hydrolysis and acidification with
the function of ethanol (Fig. 3). This was also supported by the
changes of relevant microbial community. The ethanol participa-
tion enriched lots of anaerobes related to hydrolysis (e.g., Bacter-
oides sp.) and acidogenesis (e.g., Escherichia-Shigella sp. and
Parabacteroides sp.). The WAS degradation was improved by
ethanol, leading to the higher production of SCFAs, which was the
substrate of chain elongation, thereby enhancing MCFAs produc-
tion. The final pH value in fermentation system was ~5.2, which was
still favorable for microbes relevant to chain elongation. The MCFAs
production was found to be positively related to the abundance of
chain elongation bacteria, such as Clostridium sp. and Oscillibacter
sp. In previous studies with similar inoculum, the positive corre-
lations between the MCFAs production and the relative abundance
of these microbes responsible for CE were also observed (Wu et al.,
2018).
A social and economic evaluation was conducted based on the
results of lab-scale test in this study to estimate the potential social
and economic feasibility of the proposed alternative WAS fermen-
tation technology. With increasing MCFAs production (from 1875 to
6115 mg chemical oxygen demand (COD)/L) from WAS anaerobic
fermentation, the net economic benefit is estimated to be around
487.80 to 1650.40 $/ton TS (Table S2), remarkable higher than that
of control system without MCFAs production. This net benefit arises
from the enhanced MCFAs and extra alcohols production associated
benefit overweighing the additional costs for ethanol and methane
inhibitor. Therefore, this novel WAS fermentation technology for
MCFA production is economically favorable. The social conse-
quence of this technology was also potentially friendly. In WWTPs,
a large amount of WAS are produced during wastewater treatment,
which can serve as substrates for MCFAs production via this novel
and alternative WAS fermentation technology. Meanwhile, the
WAS degradation is improved, reducing the amounts of sludge to
be discarded into the environment. The generated MCFAs could be
readily extracted onsite from anaerobic fermentation liquor using
 S.-L. Wu et al. / Journal of Cleaner Production 279 (2021) 123482
physical method, like a membrane liquid-liquid extraction system,
thereby achieving a viable commercial production and application.
The remaining highly degradable SCFAs-containing fermentation
liquor serves, in turn, as a supplementary carbon source for
improving the biological nutrients removal in WWTPs (Feng et al.,
2009), ensuring WWTPs’ continuous and regular task. In general,
this strategy shifts WWTPs from ‘linear economy’ to ‘circular
economy’.
It should be noted that the aim of this study was to demonstrate
the feasibility of MCFAs production from anaerobic WAS fermen-
tation, thus the possible optimization of the overall process was not
specifically explored. In particular, although a wide levels of ethanol
were investigated aiming to cover a broad picture of the process, it
is not necessary to apply these studied levels for MCFAs production
from WAS. The further efforts on condition optimization should be
carried out to facilitate MCFAs production. While ethanol
enhancing MCFA production from WAS has potential, the addition
of the ethanol might compromise the overall benefit of the system.
As such, the alternative or more sustainable source of ethanol such
as the use of ethanol-containing wastes (e.g., brewing wastewater)
or in-situ ethanol generation from wastewater in a bio-
electrochemical system might be capable of improving the overall
benefit of the process, which require further investigations. It
should also be noted that MCFAs produced by this technology
might be a superior precursor for biofuels or industrial chemicals
process. However, the direct application of the MCFAs produced
from WAS into foods and medications may not yet be applicable at
this stage.
5. Conclusion
In this work, the feasibility of MCFAs production from WAS with
ethanol as electron donor were experimentally demonstrated. The
following conclusions can be summarized:
 MCFAs production and selectivity from WAS increased with the
ethanol levels in the anaerobic WAS fermentation system.
 The solubilization, hydrolysis and acidification processes for
SCFAs production from WAS were enhanced with the ethanol
participation, thereby subsequently promoted the MCFAs pro-
duction via CE process and WAS degradation.
 The microbial community analyses confirmed the increased
abundance of hydrolytic microorganism (i.e., Bacteroides sp.),
acidification microorganisms (i.e., Escherichia-Shigella sp. and
Parabacteroides sp.) and CE microorganisms (i.e., Clostridium
sensu stricto_12 sp. and Oscillibacter sp.) for MCFAs production.
CRediT authorship contribution statement
Shu-Lin Wu: Conceptualization, Investigation, Methodology,
Formal analysis, Data curation, Writing - review & editing. Gang
Luo: Methodology, Software, Investigation. Jing Sun: Conceptuali-
zation, Writing - review & editing. Wei Wei: Conceptualization,
Formal analysis, Writing - review & editing, Supervision. Lan Song:
Methodology, Formal analysis, Data curation. Bing-Jie Ni:
Conceptualization, Formal analysis, Writing - review & editing,
Funding acquisition, Supervision.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
9
Acknowledgement
This work was partially supported by the Recruitment Program
of Global Experts China; the National Natural Science Foundation of
China (51578391 and 51608374); and the State Key Laboratory of
Pollution Control and Resource Reuse Foundation, China (No.
PCRRK18007).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.jclepro.2020.123482.
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