Water Research 105 (2016) 209e217

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Water Research
journal homepage: www.elsevier.com/locate/watres

Effect of nonylphenol on volatile fatty acids accumulation during

anaerobic fermentation of waste activated sludge
Xu Duan a, Xiao Wang a, Jing Xie a, Leiyu Feng a, b, *, Yuanyuan Yan a, b, **, Qi Zhou a
a
State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road,
Shanghai 200092, China
b
Research & Service Center for Environmental Industry, Yancheng 224051, Jiangsu Province, China

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

Article history: Most of the reported studies on anaerobic fermentation of sludge focused on the inuences of operating
Received 21 May 2016 conditions, pretreatment methods, and its characteristics, and little attention was paid to those of
Received in revised form persistent organic pollutants (POPs) which widespreadly appeared in sludge. In this study, the effect of
13 August 2016
nonylphenol, a typical POPs in waste activated sludge (WAS), on anaerobic fermentation for volatile fatty
Accepted 29 August 2016
Available online 30 August 2016
acids (VFAs) accumulation was investigated. The concentration of VFAs during WAS anaerobic fermen-
tation was found to be affected positively from 2856 mg COD/L in the control (without NP) to 5620 mg
COD/L with NP of 200 mg/kg dry sludge. Mechanism exploration exhibited that the main reason for the
Keywords:
Waste activated sludge
enhanced VFAs accumulation in the presence of NP was that more acetic acid was generated during the
Nonylphenol acidication of WAS, which was increased by almost three times (3790 versus 1310 mg COD/L). In WAS
Anaerobic fermentation fermentation systems, the abundance of anaerobic functional microorganisms was advantageous to the
Acetic acid accumulation of acetic acid. Further investigation by the pure acetogen revealed that both the viability
Acetogen and activity of Proteiniphilum acetatigenes were improved by NP during anaerobic fermentation, resulting
in more production of acetic acid and showing good agreement with that in the real WAS fermentation
systems.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
In the recent years, the amount of waste activated sludge (WAS)
from wastewater treatment plants (WWTPs) in China has been
growing rapidly and its treatment and disposal to avoid the second
pollutant to the environment is becoming a hot topic. Due to the
presence of high concentration of carbon and nutrient in sludge,
anaerobic fermentation, which is characterized by the production
of valuable bioproducts, such as methane, hydrogen, volatile fatty
acids (VFAs), etc., and the synchronous amount reduction, has been
widely recommended as a preferred method for WAS treatment,
considering its cost-effectiveness and energy recovery. Among
those bioproducts, the fermentative VFAs from WAS has drawn
* Corresponding author. State Key Laboratory of Pollution Control and Resources
Reuse, School of Environmental Science and Engineering, Tongji University, 1239
Siping Road, Shanghai 200092, China.
** Corresponding author. Research & Service Center for Environmental Industry,
Yancheng 224051, Jiangsu Province, China.
E-mail addresses: leiyufeng@tongji.edu.cn (L. Feng), yanyuanyuan@tongji.edu.cn
(Y. Yan).
more and more attention because of the fact that it can act as not
only the external carbon source employed in the biological nitrogen
and phosphorus removal in WWTPs (Liu et al., 2015), but also the
key intermediates involved in the production of methane during
anaerobic fermentation (Schievano et al., 2012).
Meanwhile, in China, due to the widespread application of
combined wastewater collection systems a large proportion of in-
dustrial wastewater entered WWTPs (approximately 35%), bringing
in huge amounts of persistent organic pollutants (POPs), such as
nonylphenol (NP), polycyclic aromatic hydrocarbons (PAHs), etc.
which were nally absorbed in WAS after wastewater treatment
(Feng et al., 2015). For example, the content of NP in WAS from
WWTPs of China has been reported to be in the range of
64.1e200 mg/kg dry sludge (Ma et al., 2002; Hao et al., 2007; Qiao
et al., 2007). Therefore, it can be deduced that the complexity of
WAS caused by a variety of obnoxious POPs would make the bio-
production of VFAs full of uncertainties during anaerobic fermen-
tation, which is a well-known biological process and controlled by
lots of microorganisms, such as acetogen, hydrogen and metha-
nogen. To our best knowledge, however, no information is currently
available on how the POPs present in WAS inuenced anaerobic

http://dx.doi.org/10.1016/j.watres.2016.08.062
0043-1354/ 2016 Elsevier Ltd. All rights reserved.
210 X. Duan et al. / Water Research 105 (2016) 209e217
fermentation for the accumulation of fermentative products,
especially VFAs which are the vital intermediates during anaerobic
conversion of sludge organics. Till now, most of the reported
studies on WAS anaerobic fermentation focused on the inuence of
operating conditions, such as pH, temperature, etc (Yuan et al.,
2006; Li et al., 2014)., pretreatment methods, such as thermal, ul-
trasonic, microwave treatments, etc (Eskicioglu et al., 2006; Wilson
and Novak, 2009; Yan et al., 2010; Koupaie and Eskicioglu, 2015).,
and the characteristics of WAS, such as the contents of protein,
carbohydrate even intracellular polyhydroxyalkanoates (PHA)
(Dimock and Morgenroth, 2006; Feng et al., 2009; Wang et al.,
2013).
The main objective of this study was therefore to investigate the
effect of NP, a typical POPs, on the accumulation of VFAs, especially
acetic acid, during WAS anaerobic fermentation. As is well known,
the production of methane and hydrogen is closely related with the
accumulation of VFAs, among which acetic acid plays the most
important role in generating these fermentative biogas. Key stages
of anaerobic fermentation resulted in the changes of VFAs in the
presence of NP was rstly investigated. Then, the diversities of
anaerobic microorganisms responsible for VFAs production when
NP occurred in the anaerobic fermentation systems were exam-
ined. Finally, the viability and activity of a pure acetogen, i.e., Pro-
teiniphilum acetatigenes, in the presence of NP during anaerobic
fermentation were studied to deeply disclose the mechanism for
NP effect on acetic acid production. This work opened a mysterious
veil on the effect of POPs, especially NP on anaerobic fermentation
from macro and micro point of view, and might be helpful for the
control of POPs during WAS anaerobic fermentation.
2. Materials and methods
2.1. NP-containing WAS
The raw WAS was obtained from the secondary sedimentation
tank of a WWTP operated with a traditional biological nutrient
removal process in Shanghai, China. Before anaerobic fermentation,
the sludge was concentrated by settling at 4
C for 24 h, and ltered
with 2 mm  2 mm sieve, and its main characteristics were as
follows: pH 6.9 0.1, total suspended solid (TSS) 15,755 475 mg/L,
volatile suspended solids (VSS) 10,485 307 mg/L, total chemical
oxygen demand (TCOD) 13,660 190 mg/L, soluble chemical oxy-
gen demand (SCOD) 130 10 mg/L, total protein 8311 453 mg
COD/L, total carbohydrate 1240 56 mg COD/L, lipid and oil
170 15 mg COD/L. Protein and carbohydrate were the predomi-
nant organic matters. It is noted here that non NP-existing WAS was
used for anaerobic fermentation because the evaluation of NP effect
on VFAs accumulation from WAS can be carried out more conve-
niently by adding the external NP.
2.2. Experiments of NP affecting the production of VFAs
Batch anaerobic fermentation experiments were conducted in a
series of semi-continuous-ow reactors using glass serum bottles
containing 240 mL stock sludge in the presence of NP at a stirring
speed of 150 revolutions per minute (rpm). The content of NP in the
stock sludge was set at about respectively 0, 20, 50, 100 and
200 mg/kg dry sludge by adding different amount of NP. Sludge
retention time (SRT) in all reactors was controlled at 8 d by with-
drawing 30 mL fermentative mixture and then adding the same
volume of fresh sludge containing the corresponding concentration
of NP each day. The fermentation pH was controlled at 10, which
has been reported to be the most suitable pH for VFAs production
from WAS (Yuan et al., 2006), by adding 4 M sodium hydroxide
(NaOH) or hydrochloric acid (HCl) and the temperature was set at
35 1
C. When WAS fermentation reactors went stable, the sludge
samples were collected to measure the concentration of VFAs. After
operation for 3 months, the analysis of microbial community in the
control and NP fermentation reactors was conducted. All the
fermentation tests were carried out in triplicate, and one way
analysis of variance (ANOVA) at 0.05 level was used to analyze the
data.
In the current study, the mechanism investigations were
focused on the effect of NP on each process involved in WAS
anaerobic fermentation and the activity and viability of anaerobic
microorganism responsible for VFAs accumulation. As it has been
documented that the activity of methanogens was inhibited
completely under the condition of pH 10, only the solubilization,
hydrolysis, acidication and homoacetogesis in the presence of NP
were considered, and the methanogensis was excluded. The effect
of NP on WAS solubilization under the alkaline condition in the
presence of NP was explored by determining the apparent con-
centration of soluble protein and carbohydrate in the fermentation
liquid.
NP inuence on the hydrolysis of WAS organics was examined
by the hydrolysis efciencies of bovine serum albumin (BSA,
average molecular weight (Mw) 67,000, model protein used in this
study) and dextran (Mw 23,800, model carbohydrate) in the syn-
thetic wastewater, which was synthesized by dissolving the
mixture of BSA (0.72 g) and dextran (0.15 g) (the mass ratio of BSA
and dextran was almost the same as that of protein to carbohydrate
in the raw WAS) into 216 mL tap water and then added 24 mL
withdrawn WAS from the semi-continuous-ow fermentation re-
actors with or without NP (200 mg/kg) as inoculum with a nal
sludge concentration of about 1500 mg/L. The hydrolysis ef-
ciencies of BSA and dextran were expressed by the following
equation.
Hydrolysis efficiency % C0  C=C0  100 (1)
where C0 and C represent the initial and residual concentration of
BSA and dextran in the synthetic wastewater during the fermen-
tation tests. The pH was controlled at 10 by the addition of 4 M
NaOH or HCl. After being ushed with nitrogen gas for 10 min to
remove oxygen, all fermentation reactors were capped with rubber
stoppers, sealed and stirred at 35 1
C and 100 rpm.
The investigation of NP effect on the acidication of hydrolyzed
products was carried out under the same conditions with those
during the hydrolysis process except that the substrates were
replaced by mixing 0.72 g L-glutamate and 0.15 g glucose. Based on
the analysis of VFAs concentration and composition in the
fermentation reactors, the inuence of NP on the acidication
during anaerobic fermentation was revealed.
Homoacetogesis is a biological process during which the
homoacetogenic bacteria utilize hydrogen and carbon dioxide as
the substrates to produce acetic acid. In order to investigate the
contribution of homoacetogenic bacteria to acetic acid production
in the presence of NP, the synthetic gas which included 70% ni-
trogen, 20% hydrogen and 10% carbon dioxide (v/v) was fed into
600 mL serum bottles containing 240 mL sludge from the control
and NP-added (200 mg/kg) semi-continuous-ow reactors. The
fermentation reactors with the pure nitrogen were set as the con-
trol. By monitoring the hydrogen consumption, the effect of NP on
homoacetogesis was examined.
In order to monitor the conversion of main organic substances
and illustrate the effect of NP on their fates during WAS fermen-
tation, carbon balance study was conducted in the current
fermentation systems by observing the carbon contents of protein,
carbohydrate, SCFAs, lactic acid, ethanol, carbon dioxide, methane
et al. The samples were collected from the fermentation reactors,
 X. Duan et al. / Water Research 105 (2016) 209e217
which went stable, to examine the content of carbon-containing
matters, such as protein, carbohydrate, VFAs, carbon dioxide et al.
The carbon content of carbohydrate and protein were calculated
respectively using C6H10O5 and C5H7NO2 as formulas (Maya-
Altamira et al., 2008).
2.3. Experiments of NP affecting the functional microorganism
Based on results derived from the batch tests about the effect of
NP on each step of anaerobic fermentation and microbial com-
munity, Proteiniphilum acetatigenes which is a model acetogen and
known to produce acetic acid from the proteinaceous substrates,
was used to deeply study the impact of NP on WAS anaerobic
fermentation, especially acetic acid production. During the explo-
ration, Proteiniphilum acetatigenes was rstly cultured at 35 1
C
for 24 h in the peptone-yeast extract-glucose medium, which were
composed of (mg/L of tap water): glucose 5000, tryptone 20,000,
NaHCO3 10,000, yeast extract 10,000, L-Cysterine-HCl-H2O 500,
NaCl 80, NaHCO3 400, CaCl2 8, MgSO4 8, K2HPO4 80, Vitamin K1 5
and Chlorohemin 12.5, and then inoculated to the substrates for
acetic acid production. The volume of substrates and inoculum was
respectively 100 and 2 mL, and the mixed fermentation substrates
were discarded and replenished each day to control the SRT at 8 d.
The dosage of NP was 2.6 mg/L which was equal to that in WAS
fermentation system (200 mg/kg), and the pH was also adjusted to
10. All the operation and inoculation were conducted under the
anaerobic condition with nitrogen as the protecting gas. When the
fermentation reactors went stable, the analysis of activity of key
enzyme and its encoding gene closely related with acetic acid
production were carried out. The membrane potential (MP) and
optical density at 600 nm (OD600) of Proteiniphilum acetatigenes
were also detected to reect the effect of NP on the viability of
acetogen.
2.4. Analytic methods
The determinations of VFAs, protein, carbohydrate, COD, TSS,
VSS, lactic acid and ethanol were the same as described in the
previous publications (Feng et al., 2009; Zhao et al., 2010). Carbon
elemental compositions of fermentation substrates were analyzed
by an elemental analyzer (NA 2500, Thermo). NP in WAS was
analyzed according to a method (Hesselsoe et al., 2001) with minor
modication. Typically, the NP-containing sludge was rstly freeze-
dried, and then extracted with 25 mL of methanol/acetone (90: 10)
and 20 mL of water. The extracts were centrifuged, and 20 mL of
supernatant was puried using solid phase extraction (SPE) col-
umns. The eluate from SPE columns with hexane/acetone (40: 60)
was concentrated under N2 and measured using a high perfor-
mance liquid chromatogram (HPLC, Agilent 1200) with C18 column
(ODS Hypersil, Thermo). Gas component (CO2, CH4 and H2) was
measured using a gas chromatograph (GC-2010, SHIMADZU)
equipped with a thermal conductivity detector (TCD).
The analysis of microbial community responsible for VFAs pro-
duction in the presence of NP in WAS fermentation systems was
conducted by Illumina MiSeq sequencing technique (Supporting
Information). The barcoded primers used in multiplex pyrose-
quencing for bacteria were 338F (50 -ACTCCTACGGGAGGCAGCAG-
30 ) and 806R (50 -GGACTACHVGGGTWTCTAAT-30 ).
The content of extracellular polymeric substances (EPS) from
the pure acetogen was measured by the method of Wang et al.
(Wang et al., 2009). The content of EPS from the pure acetogen was
rstly extracted by centrifugation to remove bulk solution, and the
remaining pellet was washed and resuspended with saline water.
The mixed liquor was then subjected to heat treatment (100
C and
1 h) and centrifuged again. Finally, the centrifuged supernatant was
211
used to measure protein and carbohydrate.
The MP of acetogen cells was determined by ow cytometry
using the anionic lipophilic potential-sensitive uorescence dye, i.e.
bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)].
Usually, the uorescence signal can be ingnored when DiBAC4(3)
does not enter the cells. As the membrane of cells is damaged, the
dye would react with the cytosol and the uorescence intensities
increase (Supporting Information).
The determination of the activity of acetate kinase (AK) was
carried out according to the previous investigation (Feng et al.,
2009). Normally, the extract from pure acetogen was obtained af-
ter ultrasonication and centrifugation to remove the cell debris. The
assay was conducted at room temperature (25
C), and the mixture
for analysis contained (in mmol/L) tris-HCl buffer (pH 7.4) 81,
adenosine 5-triphosphate (ATP) 4.0, MgCl2 4.0, phosphoenolpyr-
uvic 1.6, acetate 81, NADH 0.4, and (in units) pyruvic kinase 0.4 and
lactic dehydrogenase 0.4. The reaction was started by adding 160 mL
of extract, and a control without acetate was used to correct for the
adenosine diphosphate (ADP) present in the ATP preparations. The
quantication of acetate-related gene, which encodes AK, was
conducted via Real-time quantication PCR (Supporting
Information).
3. Results and discussion
3.1. Effect of NP on VFAs accumulation from WAS during anaerobic
fermentation
The production of VFAs from WAS during anaerobic fermenta-
tion in the presence of NP was shown in Fig. 1(a). It was observed
that the accumulation of VFAs from WAS was positively affected by
NP during anaerobic fermentation. The concentration of VFAs was
increased with NP in the range of 0e200 mg/kg dry sludge. When
the content of NP in WAS was 200 mg/kg dry sludge, the production
of VFAs was 5620 mg COD/L, which was almost 2.0-fold of that in
the control (2856 mg COD/L). The individual VFA produced from
WAS in anaerobic fermentation systems was shown in Fig. 1(b).
Acetic, propionic and iso-valeric acids were three dominant prod-
ucts in all fermentation tests, among which acetic acid occupied
about 50% of the total VFAs. When the NP occurred in the
fermentation systems, the concentrations of propionic, butyric and
valeric acids from WAS were almost the same with those in the
control. However, the production of acetic acid was changed
signicantly in the presence of NP. The yield of acetic acid during
WAS anaerobic fermentation was only 1310 mg COD/L without NP,
whereas with NP of 200 mg/kg dry sludge it was increased to as
high as 3790 mg COD/L which was nearly three times of that in the
control. Further calculation found that the enhancement of acetic
acid production (3790e1310 2480 mg COD/L) accounted for most
of the increased total VFAs (5620e2856 2764 mg COD/L), which
meant that the effect of NP on WAS anaerobic fermentation for
VFAs accumulation was mainly attributed to the variation of acetic
acid production (2480/2764 0.90). Consequently, the mechanism
for VFAs production enhanced by NP during WAS anaerobic
fermentation should be focused on acetic acid.
3.2. Contribution of NP degradation to acetic acid production
In this study, NP in the WAS was observed to be partly bio-
degraded during anaerobic fermentation (Table S4), which might
act as the carbon source for the production of acetic acid and then
improved its concentration. During the fermentation tests, the
degradation efciency of NP in the anaerobic reactor with 200 mg
NP/kg dry sludge was 33.6%, and the corresponding quantity of NP
was 0.93 mg. It can be calculated that the theoretical production of
212
7000
(a) control
100 mg/kg
Concentration of total VFAs (mg COD/L)
6000
5000
4000
3000
2000
20 28 36
acetic acid from the degraded NP was 2.8 mg COD/L, accounting for
only about 0.1% of the enhancement of acetic acid production
(2480 mg COD/L). Therefore, in the current study the contribution
of NP biodegradation to acetic acid could be negligible, and the
enhancement of VFAs, especially acetic acid, came from WAS itself.
3.3. Carbon mass balance of WAS fermentation systems
Usually, the carbon mass balance is used to monitor the con-
version of carbon-containing organic substances and illustrate their
fates during WAS anaerobic fermentation. In this study various
carbon-containing matters were involved in sludge fermentation,
such as protein, carbohydrate, VFAs, CO2 and other organic carbons
(such as lactic acid, ethanol et al.). It was observed in Fig. 2 that the
main carbon substrates in the initial WAS were protein and car-
bohydrate, which were respectively 706.1 and 123.6 mg (account-
ing for about 61.8% of the total carbon). When WAS was fermented
under the anaerobic condition, the contents of protein and carbo-
hydrate were signicantly decreased. The carbon from residual
protein and carbohydrate were respectively 546.7 and 59.8 mg in
the fermentation reactor without NP. The corresponding carbon
CO2
Acetic acid
1500
Carbon (mg)
1000
500
0 directly related with the content of hydrolyzed organic matters for
Initial 0 20
NP content (mg/kg dry sludge)
Fig. 2. Carbon balance analysis in WAS fermentation systems (The other VFAs included
propionic, butyric and valeric acids, and the others mainly referred to lipid, lactic acid,
ethanol and some unknown organic carbons. Methane was not detected in this study.
The data was the average values after the reactors went stable.).
X. Duan et al. / Water Research 105 (2016) 209e217
6000
20 mg/kg 50 mg/kg (b) n-valeric acid iso-valeric acid
200 mg/kg n-butyric acid iso-butyric acid
4800 propionic acid acetic acid
Individual VFA (mg COD/L)
3600
2400
1200
0
44 52 60 68 76 84 92 100 0 20 50 100 200
Operation time (d) NP content (mg/kg dry sludge)
Fig. 1. Concentration of total VFAs and individual VFA from WAS in the presence of NP.
contents were further decreased in the NP-added fermentation
reactor. As the concentration of NP was 200 mg/kg dry sludge in the
fermentation reactors, the carbon contents of residual protein and
carbohydrate were 333.0 and 24.0 mg, much lower than those in
the control. As a result, the carbon content of VFAs, especially acetic
acid, was found to be much greater in the fermentation reactor with
NP (200 mg/kg dry sludge). In the control reactor, the carbon from
acetic acid was 117.7 mg, whereas it was increased to 340.0 mg in
the presence of NP. Obviously, a larger amount of acetic acid was
produced from WAS when NP occurred in the fermentation re-
actors. Based on the results of carbon variations in the WAS
fermentation reactors, it can be concluded that in WAS fermenta-
tion systems the enhanced accumulation of acetic acid in the
presence of NP compared with that in the control was mainly
attributed to more bioconversion of protein and carbohydrate.
3.4. Effect of NP on each stage of WAS anaerobic fermentation
Based on the experimental results, the inuence of NP on VFAs
production from WAS could be concentrated on the change of
acetic acid, which is the most important intermediate during
anaerobic fermentation of organic matters. It is well known that the
solubilization, hydrolysis, acidication and homoacetogenesis are
Others Other VFAs involved during WAS anaerobic fermentation when the methano-
Total carbohydrate Total protein genesis of acidied products was inhibited under the strong alka-
line condition. The effect of NP on each step associated with the
accumulation of acetic acid during sludge anaerobic fermentation
was highly desirable.
During the solubilization of WAS anaerobic fermentation, the
particulate substrates are rstly changed into the soluble ones. In
this study, the concentration of soluble protein and carbohydrate in
the fermentation reactors with 200 mg/kg dry sludge was 935.7
and 224.6 mg/L, which was almost the same with that in the control
(932.2 and 237.6 mg/L) (Fig. 3(a)), indicating that NP gave no in-
uence on WAS solubilization when it occurred in the fermentation
systems. Usually, during anaerobic fermentation the hydrolysis of
soluble substrates is considered as the rate-limiting step and
50 100 200
VFAs production. In this investigation, the hydrolysis efciencies of
model soluble protein and carbohydrate showed no signicant
difference in the fermentation reactors with and without NP (15.5%
versus 15.9% for protein, and 62.3% versus 65.0% for carbohydrate)
(Fig. 3(b)), which demonstrated that the hydrolysis of WAS was also
not affected by NP.
 X. Duan et al. / Water Research 105 (2016) 209e217 213

1200 400 80

(a) protein carbohydrate (b) BSA Dextran

1000
300 60

Soluble carbohydrate (mg/L)

Degradation efficiency (%)
Soluble protein (mg/L)

800

600 200 40

400
100 20
200

0 0 0
0 200 0 200
NP content (mg/kg dry sludge) NP content (mg/kg dry sludge)

1200 20
(c) control
(d) control
200 mg/kg 200 mg/kg
Concentration of acetic acid (mg/L)

900

Hydrogen percentage (%) 15

600

300
10

0
0 1 2 3 0 1 2
Operation time (d) Operation time (d)

Fig. 3. Effect of NP on the solubilization (a), hydrolysis (b), acidication (c) and homoacetogenic (d) processes during WAS anaerobic fermentation. The concentration of soluble
protein and carbohydrate was measured as the fermentation reactors went stable after being operated for more than 20 d. The operation time for BSA and dextran degradation was
3 d.

It is well known that during WAS anaerobic fermentation the WAS fermentation systems. The increased accumulation of acetic
production of acetic acid from organic matters is mainly derived acid from WAS with NP was attributed to the improvement of
from two pathways, etc., utilizing the hydrolyzed substrates, such bioconversion of hydrolyzed substrates.
as amino acids and monosaccharides, by acidogenic bacteria and
consuming hydrogen and carbon dioxide by homoacetogenic bac-
3.5. Effect of NP on microbial community during WAS anaerobic
teria. In this study, the effect of NP on sludge acidication during
fermentation
anaerobic fermentation was therefore rstly investigated by fer-
menting the synthetic wastewater with L-glutamate and glucose
Normally, anaerobic microorganisms play a vital role in WAS
(model amino acid and monosaccharide), and determined by the
fermentation systems for VFAs production. In order to further
yield of acetic acid. It was observed that within operation time of
investigate the reasons for the effect of NP on the production of
3 d the concentration of acetic acid produced from hydrolyzed
acetic acid from WAS, the communities of anaerobic bacteria rele-
substrates with NP was 944.2 mg COD/L, whereas it was 360.5 mg
vant to acetic acid accumulation were analyzed. It was found in
COD/L in the control (Fig. 3(c)), which exhibited that the occurrence
Fig. 4 that anaerobic microorganisms in all fermentation reactors
of NP beneted the accumulation of acetic acid during WAS
were mainly grouped into three phyla, i.e. Firmicutes, Bacteroidetes
acidication.
and Proteobacteria, which have all been reported to have the ability
The exploration on the homoacetogenic contribution to the
of degrading a wide range of organic matters, including protein and
enhancement of acetic acid production in the presence of NP was
carbohydrate, and closely related with the accumulation of acetic
also necessary, which was studied by examining the hydrogen
acid (Ariesyady et al., 2007; Leven et al., 2007; Lu et al., 2012; Wong
consumption via providing sufcient hydrogen and carbon dioxide
et al., 2013; Zheng et al., 2013). However, the abundance of these
for homoacetogenic bacteria. Experimental data showed that the
anaerobes varied between the control and NP fermentation re-
consumption efciency of hydrogen with NP was 48.2%, exhibiting
actors. The abundance of Firmicutes, which was the predominant
no difference with that in the control (48.0%) (Fig. 3(d)). The
bacteria in the present fermentation systems, and possessed the
contribution of homoacetogenic bacteria to the enhancement of
ability of consuming acetic acid (Jang et al., 2014), was reduced due
acetic acid production could be neglected when NP appeared in
to the presence of NP. The decrease of Firmicutes abundance in the
214 X. Duan et al. / Water Research 105 (2016) 209e217
Others
Verrucomicrobia
Chloroflexi
Chlorobi
Actinobacteria
Acidobacteria 1%
30%
Proteobacteria 60%
Bacteroidetes
Firmicutes
control 200 mg/kg
Relative abundance (%)
Fig. 4. Phylum level distribution of bacterial populations in WAS fermentation
reactors.
NP fermentation reactors (58.5% in the control reactor versus 50.8%
in the NP reactor) was obviously advantageous to the accumulation
of acetic acid. Meanwhile, in the WAS fermentation reactor with
200 mg NP/kg dry sludge the abundance of Bacteroides, which has
been documented to produce acetic acid during anaerobic degra-
dation of organic matters (Riviere et al., 2009; Jang et al., 2014), was
nearly 2-fold of that in the control, revealing that more acetic acid
could be generated in the presence of NP. Obviously, the microbial
community in the fermentation reactor with NP beneted the
accumulation of acetic acid, which was consistent with the higher
concentration of acetic acid from WAS. Further exploration at genus
level found that the abundances of microbes, i.e. Guggenheimella,
Comamonadaceae, Erysipelothrix, Proteiniclasticum, Rhizobiales,
Anaerovorax, Phyllobacteriaceae, related with degradation of com-
plex organic matters and acetic acid production (Satoh et al., 2002;
Jin et al., 2016; Yuan et al., 2016; Liu et al., 2016) were all increased
remarkably in the NP reactors (Seen in Table S5), beneting the
accumulation of VFAs, especially acetic acid.
3.6. Evaluation of NP effect on acetic acid production by pure
acetogen during anaerobic fermentation
On the basis of above ndings that the proportion of acetic acid-
forming bacteria was changed remarkablely due to the presence of
NP, a pure strain, etc., Proteiniphilum acetatigenes, which belongs to
the phylogenetic group of Bacteroides and have the ability of fer-
menting protein-containing organic matters (the predominant
organic in WAS) into acetic acid (Zeppilli et al., 2015), was used for
further disclose the interaction between NP and acetic acid accu-
mulation. The production of acetic acid by Proteiniphilum acetati-
genes in the control and fermentation reactor with 200 mg NP/kg
dry sludge was presented in Fig. S1. It was observed that the yield of
acetic acid in both anaerobic reactors was increased with operation
time, and within 6 d it was accumulated to 6037.4 mg/L with
200 mg NP/kg dry sludge, much higher than that in the control
(3658.6 mg/L). Obviously, the effect of NP on the pure acetogen
showed a similar result with that in the real WAS fermentation
tests, etc., that the production of acetic acid was distinctly enhanced
by NP under the anaerobic condition.
3.7. Effect of NP on cell viability of Proteiniphilum acetatigenes
The physiological status of acetogen is closely related with its
ability to produce acetic acid, while the most fundamental physi-
ological characteristic of microbial cells is their viability (Diaz et al.,
2010; Gunnigle et al., 2013; Cangelosi and Meschke, 2014). Only the
viable acetogen could carry out a serious of life events, including
consuming the organic matters and forming acetic acid. In this
study, the OD600 and MP of Proteiniphilum acetatigenes were
selected as two critical parameters to evaluate the microbial cell
viability in the presence of NP.
Usually, OD600 is employed to measure the growth of microbial
cell, which represents its viability to a certain extent. The amount of
cell in the bacterial suspension was in proportion to turbidity and
inversely proportion to transparency in a certain concentration
range, which meant that the concentration of bacteria showed
agreement with optical density. As can be seen from Fig. 5, the
OD600 of acetogen suspension was originally 0.043, and increased
with operation time, indicating that the cells of Proteiniphilum
acetatigenes began to grow when adapting to the fermentation
environment and survived. Despite of the fact that anaerobic ace-
togens in two fermentation reactors were growing, their growth
rates and thus total number of cells were signicantly different.
Within operation time of 3 d, the OD600 of acetogen suspension in
the presence of NP was 0.68, whereas it was only 0.37 in the con-
trol, demonstrating that the growth of Proteiniphilum acetatigenes
was simulated by NP, resulting in more production of acetic acid
during anaerobic fermentation.
Meanwhile, all microbial cells are bounded by the cytoplasmic
membrane, which allows themselves to communicate selectively
with the immediate environment. MP was kept negative inside and
positive outside when the cells are in normal state, which called
polarization. Membrane depolarization, i.e. the increase of MP,
often indicates the structural damage of cell membrane and thus
decreases in cell viability, resulting in the change of physiological
status, such as cell size and cell density, and the loss of cellular
function (Kitada et al., 2006). Usually, MP is determined by ow
cytometry with proper probes, which enter the damaged cell and
emit the uorescence while those cells with intact membranes are
impermeable to molecular probes.
As shown in Fig. 6, the average uorescence intensity of ace-
togen cells in the control (pH 10) was 770.7, which meant that the
cell membranes of acetogens were destructed by the strong alka-
line condition, because that of anaerobic microorganisms under the
neutral condition was much lower (336.9). When the acetogen was
exposed to NP, the negative impacts of pH 10 on cells were
observed to be less severe. The average uorescence intensity of
acetogen cells at pH 10 in the presence of NP was 450.6, declining
remarkably than that in the control and indicating that the ace-
togen cells in the fermentation reactors with NP were more in-
clined to maintain their normal MP, and thus showed better
viability than that at pH 10. The reason for this might be attributed
to the stimulating effect derived from NP during anaerobic
fermentation. It has been reported in the literature that microor-
ganisms can produce extra EPS to protect themselves when being
exposed to the toxic compounds by making the cell wall thicker to
reduce the mass transfer of toxicant into the cells(Aquino and
Stuckey, 2004). In this study, due to the presence of NP the ace-
togen was stimulated to synthesize more EPS to resist the negative
effect of strong alkaline condition on the viability of cells. The
content of EPS (protein plus carbohydrate) from Proteiniphilum
acetatigenes was measured to be enhanced by 28% in the fermen-
tation reactors with NP compared with that in the control
(Fig. 5(b)). Thus, the current acetogen was more tolerant under the
alkaline condition with NP, showing stronger viability with more
production of acetic acid.
3.8. Effect of NP on the activity of Proteiniphilum acetatigenes
After the most fundamental viability assays, the deeper analysis
was concentrated on activities of acetogen in two fermentation
reactors. Acetic acid is the metabolite of acetogen, and its
 X. Duan et al. / Water Research 105 (2016) 209e217 215

1.0 45
(a) 0 mg/kg
(b) Bound EPS-protein
200 mg/kg Bound EPS-carbohydrate
0.8 40

Carbohydrate/protein (mg/L)
0.6 35
OD600

0.4 30

0.2 25

0.0 20
0 3 6 0 200

Operation time (d) NP content (mg/kg dry sludge)

Fig. 5. OD600 and EPS concentration of Proteiniphilum acetatigenes.

Fig. 6. Fluorescence intensities for Proteiniphilum acetatigenes cultured without NP at pH 7 (a and b) and pH 10 (c and d) and with 200 mg/kg NP at pH 10 (e and f).

production is directly related with bacterial activity which is
commonly reected by the corresponding enzymes. During
anaerobic fermentation, AK, which was the most important and
direct enzyme related with the formation of acetic acid, was uti-
lized to catalyze the conversion of acetyl-phosphate to acetic acid.
As can be seen from Fig. 7, when the NP existed in the fermentation
medium, the relative activity of AK was found to be increased by
30%, demonstrating that more acetyl phosphate was catalyzed to
acetic acid. The enhancement of AK activity exhibited that the
acetogen was much more active, which showed good agreement
with the improvement of acetic acid production in the fermenta-
tion reactors with NP.
It is well known that the enzymes produced by microorganisms
were encoded and controlled by the corresponding functional
genes, and their expressions and activities were closely associated
with the quantity of related gene. The enzyme AK involved in acetic
acid production is encoded by its operon, and in this study whether
the AK operon was inuenced due to the presence of NP was very
important. Based on the results investigated by real-time qPCR, it
was observed that the quantity of AK gene from Proteiniphilum
acetatigenes in the fermentation reactor with NP of 200 mg/kg dry
sludge was 3.83  106 copies/mL, which was much greater than
that in the control (2.11  106 copies/mL). The greater quantity of
AK gene from Proteiniphilum acetatigenes indicated that more
acetic-forming related gene participated and regulated the syn-
thesis and expression of corresponding AK in the fermentation
216 X. Duan et al. / Water Research 105 (2016) 209e217
6 140
Quantity
Activity 120
5
Quantity (106 copies/mL)
100
4 Eskicioglu, C., Kennedy, K.J., Droste, R.L., 2006. Characterization of soluble organic
Activity (%)
80
3 Feng, L.Y., Chen, Y.G., Zheng, X., 2009. Enhancement of waste activated sludge
60
2 pH. Environ. Sci. Technol. 43 (12), 4373e4380.
40
1 Gunnigle, E., McCay, P., Fuszard, M., Botting, C.H., Abram, F., O'Flaherty, V., 2013.
20
0 0
0 200
NP content (mg/kg dry sludge)
Fig. 7. Activity of AK and quantity of its encoding gene of Proteiniphilum acetatigenes.
reactors with NP, leading to the enhancement of acetic acid
production.
4. Conclusion
During anaerobic fermentation of waste activated sludge, NP
exerted the positive inuence on the accumulation of VFAs. When
the content of NP in WAS was 200 mg/kg dry sludge, the production
of VFAs was 5620 mg COD/L, which was almost 2.0-fold of that in
the control. The enhancement of VFAs accumulation from WAS in
the presence of NP was mainly attributed to the improvement of
acetic acid, which was increased to 3790 mg COD/L and nearly three
times of that in the control. Mechanism investigation found that
the effect of NP on WAS fermentation focused on the acidication,
instead of the solubilization and hydrolysis. Also, the abundance of
anaerobic functional microorganisms was advantageous to the
accumulation of acetic acid. Further exploration based Proteini-
philum acetatigenes demonstrated that both the viability reected
by the OD600 and MP and activity expressed by key enzyme and its
encoding gene were improved by NP during alkaline fermentation
of organic matters.
Acknowledgement
The work is nancially supported by the National Natural Sci-
ence Foundation of China (Nos. 51108332 and 51208371), National
Major Science and Technology Project of China (No. 2012ZX07302-
002), State Key Laboratory of Pollution Control and Resource Reuse
Foundation (No. PCRRK16003), Cooperative Innovation Fund of
Jiangsu Province (Nos. BY2014114 and BY2014115), Fundamental
Research Funds for the Central Universities and the Research Fund
of Tianjin Key Laboratory of Aquatic Science and Technology (No.
TJKLAST-ZD-2014-05).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.watres.2016.08.062.
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