Science of the Total Environment 867 (2023) 161491

Contents lists available at ScienceDirect

Science of the Total Environment

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

Malodorous volatile organic compounds (MVOCs) formation after

dewatering of wastewater sludge: Correlation with the extracellular
polymeric substances (EPS) and microbial communities
⁎
Xinmeng Zhu a,b,c, Xiaofang Yang a,c, , Wei Gao a,b,c, Shan Zhao d, Weijun Zhang e,
Pingfeng Yu f, Dongsheng Wang c,e,f
a
State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Yangtze River Delta Research Center for Eco-Environmental Sciences, Yiwu 322000, China
d
Research and Development Center, Beijing Drainage Group Co., Ltd, Beijing 100124, China
e
Hubei Key Laboratory of Yangtze Catchment Environmental Aquatic Science, School of Environmental Studies, China University of Geosciences, Wuhan 430074, Hubei, China
f
Department of Environment Engineering, Zhejiang University, Hangzhou 310058, 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

• Greater effects of enhanced dewatering on

volatile production than thickening.
• Centrifugation promoted volatile emis-
sions associated with EPS redistribution.
• Tryptophan-like substances in bound EPS
were better correlated with key odorants.
• Desulfobulbus, Gordonia, and
Hyphomicrobium were related to key
odorant production.

A R T I C L E I N F O A B S T R A C T

Editor: Anastasia Paschalidou Malodorous volatile organic compounds (MVOCs) are often the key odorants in determining sludge odor character and
odor impact. However, the emission characterization and generation mechanisms of MVOCs from various dewatered
Keywords: sludge have not been sufficiently understood partly due to the diverse and complex composition and low concentration
Sludge odor of odorants. In this study, waste activated sludge (WAS) was collected to examine the variation of MVOC emission from
HS-GC-IMS
sludge after different dewatering treatment in lab-scale trials. The MVOCs were measured using the electronic nose
HS-SPME-GC–MS
Centrifugation dewatering
(eNose), headspace gas chromatography-coupled ion mobility spectrometry (HS-GC-IMS), and headspace solid-
16s rRNA sequencing phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC–MS). The results showed that centrifu-
gation treatment promoted the generation of various odorous volatiles. The identified key odorants included dimethyl

Abbreviations: MVOCs, malodorous volatile organic compounds; WAS, waste activated sludge; WWTPs, wastewater treatment plants; DM, dry matter; VM, volatile matter; eNose, electronic
nose; HS-GC-IMS, headspace gas chromatography-coupled ion mobility spectrometry; HS-SPME-GC–MS, headspace solid-phase microextraction gas chromatography–mass spectrometry; OAV,
odor activity value; OTC, odor threshold concentration; MeSH, methyl mercaptan; DMS, dimethyl sulfide; DMDS, dimethyl disulfide; DMTS, dimethyl trisulfide; OVAC, organic volatile aromatic
compound; VOSC, volatile organic sulfur compound; PCA, principal component analysis; CA, clustering analysis; EPS, extracellular polymeric substances; TB-EPS, tightly bound EPS; LB-EPS,
loosely bound EPS; SEPS, soluble EPS;; PARAFAC, parallel factor analysis; DOC, dissolved organic carbon; PN, proteins; PS, polysaccharides; OTU, operational taxonomic unit; PCoA, principal co-
ordinate analysis; UPGMA, Unweighted Pair Group Method with Arithmetic mean; LEfSe, Linear discriminant analysis effect size; LDA, linear discriminant analysis; KEGG, Kyoto Encyclopedia of
Genes and Genomes; RDA, redundancy analysis.
⁎ Corresponding author at: State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
E-mail address: xfyang@rcees.ac.cn (X. Yang).

http://dx.doi.org/10.1016/j.scitotenv.2023.161491
Received 31 October 2022; Received in revised form 29 December 2022; Accepted 5 January 2023
Available online 10 January 2023
0048-9697/© 2023 Elsevier B.V. All rights reserved.
X. Zhu et al. Science of the Total Environment 867 (2023) 161491

sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), geosmin, and p-cresol according to their odor
activity values (OAVs). The effects of the enhanced dewatering on volatile production were greater than thickening,
however, the key odorants of dewatered sludge using gravity thickening varied more greatly than sludges from
centrifuge thickening. The distribution of extracellular polymeric substances (EPS) and variation of microbial commu-
nity showed correlations with the production of key odorants. Tryptophan-like substances in the inner layer of EPS
(LB-EPS and TB-EPS) were better correlated with the key odorants. The bound EPS released by centrifugation may
play the role of precursor for odorous microbial volatiles. According to the predicted functions of differential microbial
genera, Desulfobulbus (Desulfobacterota), Gordonia (Actinobacteriota), and Hyphomicrobium (Proteobacteria)
were associated with the production of DMS, DMDS, and DMTS, while Gordonia and Hyphomicrobium were related
to p-cresol production.

1. Introduction The deteriorative odor from centrifuge dewatering of anaerobic

Large quantities of waste activated sludge (WAS) are produced in mod-
ern sewage treatment. To minimize the amount of WAS, different thicken-
ing and enhanced dewatering treatment processes have been developed
(Cao et al., 2021). The consequent inevitable odor emission from sludge
treatment facilities have become an annoying problem (Jiang et al.,
2017). Compared to other processing sites, dewatered sludge caused a
high level of VOC emission and sustained emission in the subsequent stor-
age duration (Fisher et al., 2018b; Fisher et al., 2017). Thus, the unpleasant
odors emitted from the dewatering processes and dewatered sludge have
been of great concern to wastewater treatment plants (WWTPs).
Rather than H2S and NH3, the odor character of wastewater sludge was
often determined by various malodorous volatile organic compounds
(MVOCs) (Chen et al., 2006; Decottignies et al., 2010; Fisher et al., 2018a;
Gao et al., 2022; Novak et al., 2012). Based on the frequency of detection
and odor active value (OAV), methyl mercaptan (MeSH), dimethyl sulfide
(DMS), diethyl sulfide, ethanethiol, p-cresol, and butanoic acid were often
identified as key odorants from the dewatered sludge (Bylinski et al.,
2019; Fisher et al., 2018b; Fisher et al., 2017). Among various MVOCs, vol-
atile organic sulfur compounds (VOSCs) were the most frequently detected
and concerned (Han et al., 2021; Ras et al., 2008; Zarra et al., 2009). The
emissions of MVOCs should be seriously concerned in the evaluation and
control of sludge odor. In addition to the offensive odor impact, the high
emission of VOCs can be harmful to the health of WWTP employees and sur-
rounding residents and may also contribute to the formation of ground-level
ozone via photochemical reactions (Sillman, 1999; Xue et al., 2022).
The emissions of MVOCs from dewatered sludge varied significantly in
concentration and composition between batches and sites, resulting in dif-
ferent odor impacts. Such differences are largely related to the sludge prop-
erties depending on the physic-chemical and biological processes that the
sludge had gone through (Fisher et al., 2018b; Fisher et al., 2017). For
instance, inappropriate aeration in wastewater secondary treatment
resulted in deteriorated sulfur/cabbage/garlic and rancid/putrid odors of
WAS (Fisher et al., 2018a). The long-term stagnation of sludge during grav-
ity thickening also results in odor-forming by anaerobic fermentation of
WAS. The sludge organic matter, such as protein, carbohydrate, lipid, and
starch, was used as the substrate for the microbial generation of MVOCs.
The anaerobic degradation of sulfur-containing proteins, such as cysteine
and methionine, was attributed to the release of MeSH, DMS, and dimethyl
disulfide (DMDS) (Higgins et al., 2006). p-Cresol and indole were produced
from microbial degradation of tyrosine and tryptophan, respectively (Chen
et al., 2006; Rappert and Muller, 2005). Moreover, the variation of sludge
quality, such as moisture, organic matter, and oxygen content could largely
influence the microbial diversity and activities that eventually affect the
odor emission from sludge. Chen et al. (2011) associated the increased
microbial activity with shear and oxygen availability during sludge
dewatering, which resulted in increased VOSCs production. In short, the
odor production of sludge was greatly related to the metabolism behaviors
of sludge microbial communities which were responsive to the variation of
substrates and ambient conditions caused by sludge treatment. However,
the succession of the microbial community and characteristics of metabo-
lisms in dewatered sludge related to odor have not been fully understood.
digested sludge was attributed to the promoted microbial activity by the re-
lease of more bioavailable organic matter (Chen et al., 2011), which should
be mainly derived from extracellular polymeric substances (EPS). EPS is a
mixture of bio-macromolecular polymers produced by microorganisms
and accounts for 60 %–80 % of WAS biomass, containing abundant proteins
(PN) and polysaccharides (PS) (Liu and Fang, 2003). EPS could be absorbed
on the cell surfaces and distributed in the surroundings, forming three EPS
layers, namely tightly bound EPS (TB-EPS), loosely bound EPS (LB-EPS),
and soluble EPS (SEPS) (Li and Yang, 2007; Poxon and Darby, 1997). The
relationship between EPS and sludge dewaterability has been studied for
years. EPS, especially the protein components, are important influencing
factors of sludge dewaterability (Cao et al., 2019; Yu et al., 2008; Zhang
et al., 2014). It was found that the variations of MVOC emissions from
sludge upon chemical conditioning were related to the damage or floccula-
tion of EPS in conditioning processes (Gao et al., 2017). However, it was
still not clear how the dewatering methods would influence odor produc-
tion and its correlation with EPS. Vega et al. (2015) found that SEPS
might have a greater contribution (r2 = 0.65) to the volatile sulfur com-
pound emission than bound EPS (LB-EPS and TB-EPS) for the thermally
dried sludge. Higgins et al. (2006) proposed that the degradation of weakly
bound PN and PS was the main source of VOSCs and odors of dewatered
biosolids. However, the relevance between EPS and MVOC formation has
not been specifically investigated, especially for the sludge without or
before stabilization treatment. Nevertheless, it could be hypothesized that
EPS works as substrates for MVOC generation considering the important
role of EPS in the determination of sludge properties and metabolism of mi-
croorganisms. Thus, in the present work, we investigated the relationship
between the MVOC emission characteristics and the corresponding EPS and
sludge quality changes and the microbial community structure of dewatered
WAS to gain new insights into the mechanism of odor production.
To reveal the characteristics and variations in MVOC generation by
sludge dewatering, four different dewatering process sequences were
performed on fresh WAS in the laboratory. The aims of the study were to
(1) evaluate the effects of different dewatering treatment processes on
MVOC emission behavior by identifying key odorants and comparing the
profile of MVOC emissions; (2) investigate the correlation between the gen-
eration of key odorants and the variation of sludge quality characteristics
and the evolution of microbial community induced by sludge dewatering.
These results will provide a better understanding of the underlying mecha-
nisms responsible for the variation in odor emissions by sludge dewatering.
2. Materials and methods
2.1. Materials
The WAS used in this study was without stabilization treatment and col-
lected from the secondary sedimentation tank of a WWTP in Beijing, China.
The WWTP utilizes the anaerobic/anoxic/oxic process to treat domestic
sewage with a treatment capacity of 1 million m3/d. Approximately 150 L
of WAS sample was collected and immediately transported to the labora-
tory on ice within 24 h. The sludge sample was stored at 4 °C before exper-
iments and used for analysis and dewatering within 7 days. FeCl3 was used

2
X. Zhu et al.
as a dewatering conditioner which was in liquid form and with a mass
concentration of 38 %.
2.2. Sludge thickening and enhanced dewatering
As the schematic diagram of different dewatering operations shown in
Fig. 1, two thickening methods and two enhanced dewatering methods
were performed. Gravity thickening was carried out in 2 L cylinders.
After settling for 30 min, the gravity thickened sludge (gWAS) was
collected. Centrifuge thickening was processed by centrifugation at
12000 rpm for 5 min to obtain centrifuge thickened sludge (cWAS). The
thickened sludge was conditioned by FeCl3 at the dosage of 55 mg/g dry
matter (DM) and further dewatered by press filtration (gWASfd, cWASfd)
or centrifugation (gWAScd, cWAScd), respectively (Detailed in Text S1,
Text S2, and Fig. S1). Samples used for microbial community structure anal-
ysis were stored at −80 °C, otherwise stored at 4 °C for chemical analysis.
2.3. Chemical analysis
2.3.1. Characterization of sludge chemical quality
The DM content, volatile matter (VM, in % DM) content, pH values, Fe
content, and Al content of each sample were measured in triplicate, and
details of the measurement are given in Text S3.
2.3.2. MVOC analysis
The MVOC analysis was performed using the electronic nose (eNose),
headspace gas chromatography-coupled ion mobility spectrometry (HS-GC-
IMS), and headspace solid-phase microextraction gas chromatography–
mass spectrometry (HS-SPME-GC–MS). Detailed information about the
operating conditions is shown in Text S4, Text S5, and Text S6.
Based on the volatiles identified by eNose, MANOVA (Greenhouse-
Geisser) and clustering analysis (CA) were used as statistical approaches
to compare the differences between samples. Principal component analysis
(PCA) and CA were applied to evaluate the similarity and differences based
on the data set of GC-IMS and GC–MS results. The correlations between
physicochemical variables and key odorants were determined by
Spearman's rank correlation.
2.3.3. Sludge EPS characterization
The EPS extraction method is given in Text S7. The dissolved organic
carbon (DOC) of different EPS fractions was measured with a TOC/TN-
VCSH analyzer (Shimadzu, Japan). PN content in extracted EPS was
quantified by the modified Lowry-Folin method using bovine serum
albumin as a standard (Le et al., 2016). PS content was determined using
the anthrone method with a glucose standard. Fluorescence EEMs were
Fig. 1. Schematic diagram of different dewatering trials.
3
Science of the Total Environment 867 (2023) 161491
measured using a fluorescence spectrophotometer (Hitachi F-2700FL,
Japan) under the voltage of 700 V (Detailed in Text S8). Parallel factor
analysis (PARAFAC), which is more sensitive and accurate, has been used
to analyze the full-fluorescence EEMs by decomposing the fluorescent
EEM into different independent components. The analysis was performed
using R with the staRdom package (Arshad et al., 2021).
2.4. Microbial community analysis
2.4.1. DNA extraction, PCR amplification, and Illumina HiSeq sequencing
Sludge samples (0.50 g) were collected for DNA extraction using Fast
DNA spin Kit for Soil (MP Biomedicals LLC, USA). The quality was checked
by running an aliquot on 1 % agarose gels and the concentration of the
extracted DNA was determined through NanoDrop ND-1000 (Thermo
Scientific, USA). Finally, all the DNA samples were preserved at −80 °C
for further analysis.
A set of bacterial and archaeal primers 515F/806R was used to amplify
the hypervariable V4-V5 regions of 16S rRNA genes. Amplicons were then
sequenced using an Illumina HiSeq platform, which was performed by
Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).
2.4.2. Processing of sequencing data
In this study, operational taxonomic units (OTUs) were clustered with a
97 % similarity cutoff and α-diversity indices were calculated to evaluate
the biodiversity of microbial communities in different sludge samples. Prin-
cipal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with
Arithmetic mean (UPGMA) clustering were applied based on weighted
UniFrac to present the distance among different samples. Linear discrimi-
nant analysis effect size (LEfSe) was employed to identify distinguishing
genera among the groups obtained by PCoA, with p value threshold of
the Wilcoxon test set to 0.05 and linear discriminant analysis (LDA) log-
score threshold set to 2.5. To gain insight into the metabolic mechanism
of odor production, functional pathway analysis was performed using
PICRUSt2, based on the Kyoto Encyclopedia of Genes and Genomes
(KEGG) database, with distinguishing bacterial genera. Redundancy analy-
sis (RDA) was carried out to measure the influence of physicochemical
parameters on microbial community structure.
3. Results and discussion
3.1. Chemical characterization of the sludge
The chemical parameters of sludge samples are given in Table 1. Differ-
ent dewatering methods had effects on sludge chemical properties. The DM
content of gWAS and cWAS was close to 1 % and 2 %, respectively,
X. Zhu et al. Science of the Total Environment 867 (2023) 161491

Table 1
Chemical parameters of the sludge samples.
Samples DM % VM (% DM) pH Fe mg/g (DM) Al mg/g (DM) Fe/Al

WAS 0.435 ± 0.25 46.8 ± 0.00 7.04 ± 0.02 20.7 43.2 0.479
gWAS 1.02 ± 0.05 46.2 ± 0.49 6.72 ± 0.01 20.2 45.2 0.448
gWASfd 30.3 ± 0.54 41.6 ± 0.21 6.77 ± 0.04 29.0 40.6 0.713
gWAScd 7.32 ± 0.43 41.5 ± 0.10 6.01 ± 0.01 37.9 48.8 0.776
cWAS 1.87 ± 0.03 44.6 ± 0.29 6.80 ± 0.04 22.9 47.9 0.478
cWASfd 19.2 ± 0.62 40.2 ± 0.27 6.39 ± 0.11 31.8 48.4 0.656
cWAScd 21.6 ± 0.96 40.9 ± 0.67 6.18 ± 0.04 32.5 49.4 0.657

DM: dry matter; VM: volatile matter; VM, Fe, and Al mentioned above are based on dry matter.

indicating a higher dehydration of centrifuge thickening. Accordingly, the
removal of VM was also higher for cWAS than gWAS. The DM content of
enhanced dewatered sludge was 7–30 times higher than their corresponding
thickened sludge. The better dewatering performance of enhanced dewatering
treatment than thickening was attributed to the removal of physically bound
water in the sludge flocs (Wang et al., 2017). A higher fraction of organic mat-
ter was also removed along with water by enhanced dewatering. In addition,
the thickening methods showed certain effects on the DM and VM contents of
enhanced dewatered sludge. For gravity thickened sludge, press filtration con-
tributed to a higher moisture removal and lower organic matter removal than
centrifuge dewatering. However, centrifuge dewatered sludge from the centri-
fuge thickening process sequence (cWAScd) had slightly higher DM and VM
contents than cWASfd. In addition, the centrifugation-treated sludges had
lower pHs. Compared to gravity thickening dewatered sludge (gWASfd and
gWAScd), the dewatered sludge with centrifuge thickening pre-dehydration
(cWASfd and cWAScd) had a lower Fe/Al ratio.
3.2. Chemical profiling of MVOCs emitted from sludge with different dewatering
operations
The eNose responses of 10 sensors in Fig. 2a-d show the differences in
the volatile emission between sludges with different dewatering opera-
tions. Generally, sensors S2, S6, S7, S8, and S9 gave strong responses.
Sensors S2 and S6 respond to a broad range of compounds, while sensors
S7, S8, and S9 are selective for sulfur-containing compounds, oxygen-
containing organics, and aromatic compounds (Table S1), which usually
have low odor threshold concentrations (OTCs) in the range of
0.0001–0.010 mg/kg (Gemert, 2011; Yang et al., 2019). The MANOVA
and CA results in Fig. 2e and Fig. 2f indicated that the gravity and centrifuge
thickening showed an insignificant effect on the volatile emission of sludge
(p > 0.05), although cWAS had slightly higher responses at S6 and S8
(Fig. 2b). The two enhanced dewatering treatment methods showed differ-
ent effects on the volatile emission. The response intensity of sensors S2, S7,
and S9 increased slightly and reduced at sensors S6 and S8 for centrifuge
dewatered sludge (Fig. 2d) (p > 0.05). The intensity of sensors S7 and S9
increased prominently for gWASfd and cWASfd (Fig. 2c), indicating distinct
variations of volatile formation and emission after press filtration
dewatering (p < 0.05). The CA results confirmed that the emission from
press filtration dewatered sludge samples (gWASfd and cWASfd) were
significantly different from the others (Fig. 2e, Fig. 2f). Since the limitation
in the identification accuracy, the eNose results only provided a rough
profile of the volatile composition. Still, it could be concluded that without
inappropriate anaerobic conditions, the enhanced dewatering treatment
had a greater effect on the volatile emission than the thickening process,
and the effect of press filtration and centrifuge dewatering operations on
the volatile emission characteristics of sludge was different.

Fig. 2. Polar plots of the responses of the sensor array to the sludge samples. (a) WAS; (b) gWAS and cWAS; (c) gWASfd and cWASfd; (d) gWAScd and cWAScd; (e) and
(f) Cluster analysis of sludge from different dewatering process sequences.

4
X. Zhu et al. Science of the Total Environment 867 (2023) 161491

The GC-IMS analysis was used as a non-targeted analytical method for
discriminating VOC emission of sludges. The topographic plots in Fig. 3a
and Fig. 3b show that most of the signals appeared in the Rt of 100–700 s
and Dt of 1.0–1.5 ms. >110 signal peaks were detected and only part of
the signals could be qualitatively identified, such as alcohols, aldehydes,
esters, ketones, and nitrogen-containing compounds (Table S2). All the
detected signals were used to develop the fingerprint plot for further CA
and PCA analysis (Fig. S2). Based on the composition and intensity of
volatiles, the CA and PCA results indicated that the enhanced dewatered
sludge clustered in one group and WAS, gWAS, and cWAS grouped
together, nevertheless, cWAS was relatively distant from WAS and gWAS
(Fig. 3c, Fig. 3d). Consistent with eNose results, centrifuge thickening
resulted in a higher volatile emission and formation of new compounds,
such as triethylamine, ethyl heptanoate, and alpha-phellandrene (Table S2).

Fig. 3. (a) and (b) Topographic plots of sludge samples from different dewatering process sequences; (c) Cluster analysis, and principal component analysis score plot of
(d) PC1-PC2 and (e) PC2-PC3 with 95 % confidence ellipses for assessing the variance of MVOC emissions according to HS-GC-IMS.

5
X. Zhu et al.
This result confirmed once again that centrifuge thickening had a greater
impact on the formation and emission of volatile compounds than gravity
thickening. The differences in volatiles between centrifuge dewatered sludge
and thickening pre-dehydrated samples were better revealed by GC-IMS
results. >30 new volatiles were detected in gWAScd and cWAScd, such as
cyclohexanone and leaf alcohol (Fig. S2, Table S2).
The type and intensity of volatile compounds emitted from sludge
changed significantly after enhanced dewatering. For instance, the intensi-
ties of acetone and 2-butanone of enhanced dewatered sludge were much
higher, and new volatiles appeared along with the disappearance of some
signals, which meant that many volatile substances were raising or forming
after enhanced dewatering treatment (Fig. S2, Fig. 3). Moreover, the
volatile compounds of gWASfd and gWAScd were greatly different
from each other, while cWASfd and cWAScd were more similar in their
volatile composition. Compared with gWAS, most aldehydes decreased
for gWASfd, particularly pentanal, heptanal, and benzaldehyde, while
increased for gWAScd, indicating that centrifuge dewatering increased
the aldehyde emission (Fig. S3). Moreover, cyclohexanone, leaf alcohol,
furfural, and propyl acetate were formed in cWASfd and cWAScd
(Table S2). The newly formed volatile compounds after dewatering were
more likely to be microbial volatiles than blocked compounds in the flocs.
The release of substrates (such as PN and PS) and increase of microbial
activity resulting from centrifuge shearing stress were attributed to the
increased odor production for dewatered digested sludge (Chen et al.,
2011). The Fe-bound organics were proposed to show better bioavailability
than Al-bound organics in sludge (Higgins et al., 2004; Novak et al., 2007).
Thus, the dewatering methods affect the release of organic substances from
the sludge flocs and the bioavailability of the organics in dewatered sludge,
which are important influencing factors for the volatile emission. In short,
the impacts of the enhanced dewatering on volatiles were greater than
thickening, while thickening methods have a certain effect on the volatile
emission of downstream dewatered sludge.
Fig. 4. (a) Variation of the VOCs concentration emitted from sludge samples; (b) SOAV of seven MVOC families in different sludge samples; (c) SOAV contribution of key
odorants.
6
Science of the Total Environment 867 (2023) 161491
3.3. Identification of key odorants by HS-SPME-GC–MS
3.3.1. Emission concentration and composition of MVOCs
A total of forty compounds were quantitatively detected using HS-
SPME-GC–MS for all the sludge samples, including nine VOSCs, one terpe-
noid, ten organic volatile aromatic compounds (OVACs), twelve oxygen-
ated (i.e., ketones, esters, alcohols, and furans), three halogenated, three
terpene, and two cyclic alkane compounds (Fig. 4, Fig. S4). According to
the MVOCs identified by GC–MS, WAS and gWAS were clustered in one
group, cWAS and gWAScd were clustered, and the others grouped together
(Fig. 4a), confirming the differences in composition and concentration of
sludge MVOCs upon dewatering treatment. Based on the composition char-
acteristics of MVOCs, the dewatered sludges from the centrifuge thickening
treatment sequence (cWASfd and cWAScd) were more closely gathered
than from the gravity thickening process sequence (gWASfd and gWAScd).
As shown in Fig. 4a, compared with WAS (91.5 μg/kg), the total VOC
emission capacity of the unit amount of wet sludge increased greatly for
cWAS (2231.7 μg/kg) while decreased for gWAS (21.3 μg/kg). The VOC
concentrations increased for dewatered sludge, reaching 2670.2 μg/kg
and 4236.1 μg/kg for gWAScd and cWAScd, respectively, followed by
gWASfd (2197.3 μg/kg) and cWASfd (3306.6 μg/kg), confirming that
centrifuge dewatering greatly increased the VOC emission compared with
press filtration dewatering. However, it was interesting to note that the
concentration of VOCs (μg/kg) emitted from the unit amount of dry matter
of sludge was the highest for cWAS, due to its high level of VOSCs and
moisture content (Fig. S4a).
In this study, VOSCs dominated all sludge samples, accounting for
43.2 %–97.4 % of total MVOC concentrations (Fig. S4b). Nine VOSCs,
including DMS, DMDS, DMTS, 2,4-dithiapentane, allyl methyl sulfide,
methyl allyl disulfide, methyl propyl sulfide, isopropyl methyl sulfide,
and 1-pentanethiol, were detected (Fig. S4c), which were produced largely
from the decomposition of sulfur-containing proteins driven by anaerobic
X. Zhu et al.
microorganisms (Bak et al., 1992; Drotar et al., 1987; Higgins et al., 2006).
DMS, DMDS, and DMTS had higher concentrations in enhanced dewatered
sludge. The increased production of reduced sulfur compounds corresponded
to the decreased oxidation/reduction potential with increased dry matter
content of sludge (Kim et al., 2002). Moreover, the VOSC concentration of
cWAS was 100 times higher than gWAS, and the enhanced dewatered sludge
by centrifugation (gWAScd and cWAScd) emitted higher VOSCs compared
with press filtration dewatered sludge (gWASfd and cWASfd). The increase
of MVOC concentration after enhanced dewatering of gWAS and cWAS was
not simply consistent with the residual organic matter content in sludge. It
has been reported that the activity of microorganisms in VOSC generation
can be induced and promoted upon centrifugation due to the release of nutri-
ent substrates, such as available protein/amino acids (Chen et al., 2011).
Geosmin was detected though it was less frequently reported in waste-
water sludge. Geosmin exhibits a strong earthy/musty odor with a low
OTC (8 × 10−7 mg/kg) and is mainly produced by members of
Cyanobacteria, Actinomycetes, and Proteobacteria (Gemert, 2011; Gerber
and Lechevalier, 1965; Suurnakki et al., 2015). Among the samples, the
highest concentration of geosmin occurred in WAS (4.0 μg/kg). After
thickening, the concentrations of geosmin in gWAS and cWAS were 2.6
and 1.4 μg/kg, while the filtrate of gWAS also had a higher concentration
than cWAS filtrate (result not shown) and geosmin in the filtrate of cWAS
was not even detected. The geosmin concentration in gWASfd (2.6 μg/kg)
was higher than gWAScd (1.9 μg/kg) and the filtrate of gWASfd also had
a higher concentration than gWAScd filtrate (result not shown). Interest-
ingly, after enhanced dewatering of cWAS, geosmin was not detected in
the dewatered sludge (Fig. S4d) and their filtrate. In contrast to VOSC
emission, the operation of the centrifugation treatment reduced the concen-
tration of geosmin. This finding indicated that the microbial generation
mechanism or generation conditions of geosmin were distinct from
VOSCs. Geosmin is often a metabolism product of Actinomycetes and
Proteobacteria in soil and water systems under aerobic conditions (Abd El-
Hack et al., 2022; Clercin and Druschel, 2019; Dionigi and Ingram, 1994).
Without centrifugation treatment, the emission of geosmin barely changed
for gWAS and gWASfd, although the emission per dry matter content
increased. It implied that if centrifugation was employed, geosmin may
not be greatly accumulated in dewatered WAS like VOSCs.
There was no significant difference in the OVAC concentration between
WAS and thickened sludge, and their concentrations were relatively low
(0.51–0.86 μg/kg). Compared with press filtration dewatered sludge, the
concentration of OVACs was higher in gWAScd and cWAScd, reaching
551.2 and 74.3 μg/kg, respectively (Fig. S4e). p-Cresol was dominant in
gWAScd and cWAScd with concentrations of 458.0 and 69.4 μg/kg, respec-
tively. In addition to p-cresol, the concentration of indole was also high in
gWAScd (81.4 μg/kg), while it was not detectable in cWAScd. p-Cresol
and indole have been reported as the key odorants in emissions from
other WWTPs (Fisher et al., 2017; Gruchlik et al., 2013). The concentration
of OVACs in gWAScd and cWAScd was about 1000 and 80 times higher
than their corresponding thickened sludge, respectively, and p-cresol and
indole were not even detected in thickened sludge. Rappert and Muller
(2005) and Chen et al. (2006) reported that the presence of p-cresol and
indole was likely due to the ongoing degradation of tyrosine and trypto-
phan after centrifuge dewatering. Novak et al. (2012) also reported that
p-cresol and indole were associated with shear release in the dewatering
process similar to VOSCs.
A total of twelve oxygenated compounds including eight ketones, two
alcohols, one ester, and one furan were quantified, while no oxygenated
compounds were detected in WAS and gWAS. 3-methyl-1-butanol was the
main oxygenated compound in cWAS with a concentration of 54.1 μg/kg
accounting for 2.5 % of the VOC emission concentration (Fig. S4b). Large
differences between gWASfd and gWAScd were observed. 2-Pentanone,
3-pentanone, 2-hexanone, methyl isobutyl ketone, and methyl acrylate
were dominant in gWASfd with concentrations of 7.5–893.9 μg/kg, while
3-methyl-1-butanol was the main oxygenated compound in gWAScd with
the concentration of 473.7 μg/kg. cWASfd and cWAScd had relatively
close concentrations of 513.7–897.5 μg/kg. 2-pentanone dominated
7
Science of the Total Environment 867 (2023) 161491
cWASfd and cWAScd. 2-hexanone and methyl acrylate were also high in
cWASfd and cWAScd with concentrations of 6.3–11.8 μg/kg (Fig. S4f).
According to Rappert and Muller (2005), incomplete aerobic degradation
processes result in the emission of oxygenated compounds. Compared
with the thickened sludge, the higher concentration of oxygenated com-
pounds in the enhanced dewatered sludge was likely due to the alteration
of the micro-environment, especially for centrifuge dewatering. Although
high concentrations of oxygenated compounds were emitted, the olfactory
system of humans seems to be more tolerant of them as evidenced by their
higher OTCs.
The origin of cyclic alkane, terpene, and halogenated compounds may
derive directly from industrial or municipal influent to the sewer catchment
(Fisher et al., 2017). In the present work, the concentrations of these
compounds were relatively low (< 7 μg/kg) (Fig. S4g) and were not key
odorants though they were frequently detected.
3.3.2. SOAV and key odorants
Since the chemical concentration of MVOCs did not represent its odor
impact, the OAV was calculated for each compound. The SOAV of each
sludge sample is shown in Fig. 4b. The SOAV was in the order of cWAScd
(22956) > cWASfd (20339) > cWAS (19967) > gWAScd > (14506) >
gWASfd (9009) WAS (5509) > gWAS (3312).
Table S3 gives 13 compounds with OAV > 1 in at least one sample. The
key odorants are identified as DMS, DMDS, DMTS, geosmin, and p-cresol,
which contributed to >99.8 % of SOAV in each sample (Fig. 4c). Geosmin
was the principal odorant in WAS and gWAS, accounting for 90.0 % and
98.1 %, respectively. As to cWAS, DMTS, DMS, and geosmin contributed
to 78.7 %, 11.0 %, and 8.8 % of the SOAV. The major SOAV contributors
were different for gWASfd and gWAScd. gWASfd was dominated by
DMTS and geosmin (57.8 % and 36.1 %, respectively), however, gWAScd
was dominated by DMS (50.3 %), DMTS (30.3 %), and geosmin (16.4 %).
DMS and DMTS were also the main SOAV contributors for cWASfd and
cWAScd, accounting for 96.6 % and 96.4 %. These findings indicated that
the two dewatering methods had significantly different effects on odor
formation, while thickening methods also had certain effects, possibly
owing to the differences in the breakage of sludge flocs and changes in
the micro-environment of microorganisms. Centrifugation treatment
caused a higher level of odor emission from sludge and alteration of volatile
composition. The key odorants of dewatered sludge from the gravity
thickening process sequence varied more greatly than from centrifuge
thickening.
3.4. Effect of dewatering on sludge EPS and microbial community structure
3.4.1. Variation of EPS distribution and composition upon sludge dewatering
To understand the relevance of EPS as the hypothetical MVOC precur-
sor, the sludge EPS was compared. Fig. 5a-c shows the concentration
changes of DOC, PN, and PS in all three extracted fractions of sludge EPS.
The variations in PN and PS concentration are similar to that of the total
concentration of EPS. After thickening, the bound EPS (LB-EPS and TB-
EPS) concentration of gWAS and cWAS was lower than WAS, but the oppo-
site pattern was observed for SEPS. The changes in the distribution of EPS
indicated the release of bound EPS to out layer by thickening treatment.
Moreover, bound EPS in gWAS and cWAS decreased by 43.2 % and
67.8 % compared with WAS, indicating a greater effect of centrifuge
thickening on the redistribution of EPS than gravity thickening. The greater
disturbance on the surrounding structure of EPS resulting from centrifuge
thickening was due to the deformation of the sludge flocs, destruction of
cells, and even the liberation of intercellular materials into the aqueous
phase by the shear force. Subsequently, the bound EPS and total EPS
content of cWASfd and cWAScd were rather similar, suggesting a limited ef-
fect of press filtration and centrifuge dewatering on cWAS EPS. The similar
composition of EPS was consistent with the similar VOC emission charac-
teristics of cWASfd and cWAScd. However, the gWAS EPS was frail and
responded differently to the following dewatering treatment in terms of
SEPS removal and bound EPS increase. The gWASfd had the lowest SEPS
X. Zhu et al.
Fig. 5. (a) The EPS distribution (SEPS, LB-EPS and TB-EPS); Variations of (b) PN and (c) PS in EPS matrix; (d)-(f) Fluorescence intensity of three PARAFAC-derived
components in EPS of different sludge samples.
content among all the samples and similar bound EPS composition as
cWASfd and cWAScd. However, for gWAScd, the SEPS removal was limited
and the bound EPS increased markedly (6.8 mg/g (DM)), leading to a
higher content of total EPS, SEPS, PN, and PS in gWAScd than other
dewatered sludge samples. The less efficient removal of EPS by the
combined application of gravity thickening and centrifuge dewatering
treatment was consistent with its bad performance in dehydration.
Three fluorescent components of EPS, hereafter referred to as compo-
nents C1, C2, and C3, were identified by PARAFAC analysis (Fig. S5,
Fig. S6). C1 had two excitation maxima (220 nm and 260 nm) and one
emission maximum at 290 nm. C2 displayed two excitation maxima
(225 nm and 270 nm) that corresponded to a single emission maximum
(315 nm). C1 and C2, resembled Peak “B” and Peak “T”, were identified
as tyrosine-like and tryptophan-like substances originating from biological
activities (DeFrancesco and Guéguen, 2021; Gao and Guéguen, 2018;
Romero et al., 2017; Wang et al., 2020). C3 displayed two excitation
maxima (220 nm and 280 nm) and an emission maximum at 380 nm,
which was similar to a mixture of polycyclic aromatic hydrocarbons and
protein-like components associated with anthropogenic sources (Amaral
et al., 2021). The spectral Ex/Em maxima of these components are summa-
rized in Table S4. The fluorescence intensity of the three components in
each extracted fraction of EPS is shown in Fig. 5d-f. The tyrosine-like
and tryptophan-like substances were the major fluorescence proportions
in EPS, accounting for an average of 60.6 % and 26.5 % of the total
fluorescence intensity. The fluorescence intensities of tyrosine-like and
tryptophan-like substances (C1 and C2) were substantially strengthened
in LB-EPS and TB-EPS of cWAS than gWAS, and the intensity of
tryptophan-like substances in bound EPS of cWAS was also higher than
WAS, confirming the release of protein and amino acid by the break of
flocs and redistribution of EPS via centrifuge thickening. For dewatered
sludge, tyrosine-like and tryptophan-like substances in bound EPS or TB-
EPS were higher than their thickened sludge, which confirmed the break
of flocs and even destruction of cells during enhanced dewatering. For
press filtration dewatered sludge (gWASfd and cWASfd), tryptophan-like
substances in SEPS were removed. It was interesting to note that tyrosine-
8
Science of the Total Environment 867 (2023) 161491
like substances in SEPS of gWAS decreased after press filtration dewatering,
while its content in cWASfd SEPS was higher than cWAS, which indicated
certain effects of thickening methods on press filtration. However, unlike
the EPS, PN, and PS, tyrosine-like and tryptophan-like substances in SEPS
for centrifuge dewatered sludge were higher than their thickened sludge,
which also suggested the great difference between press filtration and
centrifuge dewatering on tyrosine-like and tryptophan-like contents.
3.4.2. Variation of microbial community structure and functions upon sludge
dewatering
Table S5 displays the α-diversity indices of the microbial community in
different sludge samples. The Good's coverage of each sample was beyond
98 %, indicating that the sequence libraries constructed could cover the
diversity of the microbial community. The richness was evaluated by the
number of OTUs and ACE, and Shannon and Invsimpson indices were
calculated for diversity evaluation. Press filtration dewatered sludge had
lower richness and higher diversity than the corresponding thickened
sludge. However, the microbial community richness and diversity of
gWAScd were both higher than gWAS, and cWAScd had lower richness
and diversity than cWAS. Although the press filtration of gWAS achieved
the best dewatering performance, centrifuge thickening and dewatering
facilitated the increase of microbial diversity due to the larger effects on
the sludge physical-chemical properties, such as EPS composition and pH
value. However, twice centrifugation may decrease the diversity.
As shown in Fig. 6a, a total of 56 phyla were identified. Proteobacteria
was one of the most abundant phyla in all samples, accounting for
20.2 %–33.9 % (averaging at 25.0 %) of total effective sequences. This
result was similar to the previous study reported by Zhang et al. (2012),
who observed that Proteobacteria was the dominant phylum and accounted
for 36 %–65 % of total sequences in active sludge or biosolids. Other dom-
inant phyla were Chloroflexi (13.7 %–26.5 %, averaging 21.9 %),
Bacteroidota (15.7 %–25.2 %, averaging 18.8 %), Actinobacteriota (5.4 %–
11.3 %, averaging 7.5 %), Acidobacteriota (1.8 %–11.4 %, averaging
6.9 %), and Planctomycetota (2.0 %–7.8 %, averaging 5.6 %). The relative
abundances of the bacterial genus showed distinct differences among
X. Zhu et al. Science of the Total Environment 867 (2023) 161491

Fig. 6. (a) The relative abundance of microbial communities at the phylum level and UPGMA analysis among different dewatered sludges; (b) Principal coordinate analysis
(PCoA) among different dewatered sludges.

sludge samples (Fig. S7). The dominant genera were Ferruginibacter,
norank_f__Saprospiraceae, norank_f__Caldilineaceae, norank_f__A4b, and
unclassified_f__Comamonadaceae.
Both UPGMA and PCoA showed that the sludge samples could be clus-
tered into five groups according to their bacterial communities, namely
WAS and gWAS in one group; press filtration dewatered sludge samples
(gWASfd and cWASfd) in one group; cWAS, gWAScd, and cWAScd were
distant from one another (Fig. 6). Apparently, centrifuge thickening and
enhanced dewatering treatment had a great effect on sludge microbial
community structure, which was consistent with the PCA result of GC-
IMS, suggesting a relationship between microbial community structure
and VOC emission. The LEfSe analysis with LDA > 2.5 was conducted to
examine the differential genus with statistical significance that was affected
by dewatering methods (Fig. S8). At the genus level, the abundance
of Hyphomicrobium (Proteobacteria), Gordonia (Actinobacteriota), and
Desulfobulbus (Desulfobacterota) were significantly enriched in cWAScd,
while norank_f__Rhizobiales_Incertae_Sedis (Proteobacteria) were more abun-
dant in the group including gWASfd and cWASfd. The significant difference
in the genus might be attributed to the variation of sludge properties.
The functional characteristics of microbes in sludge samples were pre-
dicted by the PICRUSt2 based on the differential genera. As shown in
Fig. S9a, the most active metabolic pathway (level 1) was metabolism
(77.7 %–78.4 %), followed by genetic information processing (7.0 %–
7.6 %), environmental information processing (4.8–5.1 %), cellular
processes (4.1 %–4.4 %), human diseases (3.3 %–3.8 %), and organismal
systems (1.8–1.9 %). The distribution of metabolism in level 2 showed
that functions of global and overview maps, carbohydrate metabolism,
and amino acid metabolism occupied the highest relative abundance
(Fig. S9b). Most of the pathways were common for the different dewatered
sludge samples, however, some metabolic pathways exhibited differences
in abundance. The functional microbial community related to amino acid
metabolism and lipid metabolism was enriched in cWAScd, while energy
metabolism was abundant in gWAScd.
3.5. Relationships between key odorants and sludge characteristics
3.5.1. Relationship between key odorants and physicochemical characteristics
To test the relationship between sludge characteristics and key
odorants, Spearman's rank correlation was performed. As shown in
Fig. 7a, several key odorants had significant correlations with sludge phys-
icochemical variables. C2 was positively correlated with DMS (r = 0.86,
p < 0.05), p-cresol (r = 0.91, p < 0.01), and 3-methylbutanal concentra-
tions (r = 0.89, p < 0.01). Heptanal was positively correlated with EPS
(r = 0.79, p < 0.05) and PN (r = 0.82, p < 0.05), and VM had a positive
correlation with geosmin (r = 0.81, p < 0.01). Moreover, PN and
tryptophan-like substances in the inner layer of EPS (LB-EPS and TB-EPS)
were more positively correlated with key odorants than the EPS compo-
nents in the outer layer (SEPS) (Fig. S10). This might be due to the greater
involvement of the inner layer EPS in the secondary metabolism of
microbes. In addition, DM also showed a strong correlation with DMDS
(r = 0.82, p < 0.05) and acetal (r = 0.93, p < 0.01), implying a relatively
low dependence of generation of DMDS and acetal on the moisture content
of the sludge. Similar to C2, Fe had strong positive correlations with DMS
(r = 0.86, p < 0.05), p-cresol (r = 0.91, p < 0.01), and 3-methylbutanal

Fig. 7. (a) Heat map of Spearman's correlation coefficients between the key odorants and physicochemical variables, and (E)-2-octenal and (E)-2-nonenal were not included
due to their low detection frequencies; (b) RDA revealing the relationship between the microbial communities and physicochemical variables; (c) Bubble plot of abundance
changes of 15 genera in sludge that were more actively involved in the pathways of metabolism (level 3).

9
X. Zhu et al.
(r = 0.89, p < 0.01). Hence, it was suspected that Fe had a significant role in
the generation of odorous microbial VOCs via regulating protein content,
particularly tryptophan-like substances. In addition, Al was also positively
correlated with p-cresol (r = 0.87, p < 0.05). Similar trends have been pre-
viously reported by Higgins et al. (2006) and Fisher et al. (2018b), which
suggested that Fe and Al can form flocs containing organic matter bound
to the cations. Therefore, higher contents of Fe and Al salts resulted in a
decrease in the amount of protein in the filtrate, and Fe and Al cations
have been shown to sequester soluble protein and deposit it into the sludge
phase. The increased protein content in sludge then became bioavailable
substrate for MVOC production.
3.5.2. Relationship between key odorants and sludge microbial characteristics
The correlation between the microbial community and physicochemi-
cal variables of sludge was investigated by performing RDA (Fig. 7b).
According to the forward selection, the six most important contributors of
microbial communities were DM, VM, Fe, Al, C2, and C3, and these
variables were statistically significant for both the first canonical axis
(p = 0.04, F = 4.8) and all canonical axis (p = 0.008, F = 18.0). As
shown in Fig. 7b, both ordination axes of the biplot combined to explain
82.3 % of the microbial community variations, indicating that these physi-
cochemical variables were major factors shaping the microbial community
dynamics. WAS and gWAS were associated with VM, and cWAS was related
to C3. For enhanced dewatered sludge, the decline of moisture content and
enrichment of Fe, Al, and C2 contents changed the microbial communities
of sludge. Centrifuge dewatered sludge showed a stronger relationship
with these parameters, suggesting the responses of microbial community
structures to the variation of sludge properties.
The association between differential genera and predicted KEGG metab-
olism in level 3 showed that 15 genera were more actively involved in the
metabolic pathways (Fig. S11, Fig. 7c). These bacterial genera possess func-
tions, such as amino acid metabolism, biosynthesis of other secondary
metabolites, and metabolism of terpenoids and polyketides. The biotic
origin analysis of key odorants performed using MetOrigin online platform
(Yu et al., 2022) indicated that Desulfobulbus (Desulfobacterota), Gordonia
(Actinobacteriota), and Hyphomicrobium (Proteobacteria) could be involved
in the production of DMS, DMDS, and DMTS, while Gordonia and
Hyphomicrobium might also contribute to p-cresol generation. However,
Cyanobacteria, Actinomycetes, and Proteobacteria in our study which were
reported as major producers of geosmin didn't match with the genus in
the platform. Meanwhile, further studies are needed to confirm the specific
microorganisms responsible for the production of sludge key odorants.
4. Conclusion
Odorous VOCs emitted from dewatered sludge were studied using
eNose, GC-IMS, and GC–MS analysis. The chemical profiling results
revealed the changes in the composition of VOCs and the formation of
new volatile compounds after sludge dewatering, including various
sulfur-containing compounds, oxygen-containing organics, and aromatic
compounds. Based on the OAV, DMS, DMDS, DMTS, geosmin, and p-
cresol were considered as the key odorants. The total concentration of
quantitatively detected compounds by GC–MS was higher for dewatered
sludge than thickened sludge and WAS, however, the SOAV was in the
order of cWAScd > cWASfd > cWAS >gWAScd > gWASfd > WAS >
gWAS. The composition of characteristic odorants was different for sludge
treated by different dewatering methods, namely geosmin was dominant
for WAS and gWAS, and VOSCs dominated dewatered sludge and cWAS
odor. The promoted production of MVOCs was consistent with the greater
variation in sludge properties and microbial community compositions
induced by dewatering than thickening treatment. Centrifugation
treatment promoted the secretion of microbial VOCs by changing the envi-
ronmental factors of microbes, such as the concentration and composition
of EPS. PN and tryptophan-like substances of EPS were positively correlated
with DMS, p-cresol, 3-methylbutanal, and heptanal production. The inner
layer EPS (LB-EPS and TB-EPS) were better correlated with these key
10
Science of the Total Environment 867 (2023) 161491
odorants than the SEPS. The functional characteristics were predicted
based on the differential genera. Functional profiles involving global
and overview maps, carbohydrate metabolism, amino acid metabolism,
and energy metabolism predominated microbial metabolic activity.
Moreover, Desulfobulbus (Desulfobacterota), Gordonia (Actinobacteriota),
and Hyphomicrobium (Proteobacteria) were involved in the production of
DMS, DMDS, DMTS, and p-cresol. This study provides new insights into
the characteristics and generation mechanism of odors from wastewater
sludge that was essential for better control of sludge odor.
CRediT authorship contribution statement
Xinmeng Zhu: Conceptualization, Performed the experiments, Data
analysis and Visualization, Writing-original draft, Investigation; Xiaofang
Yang: Conceptualization, Resources, Writing-Reviewing and Editing,
Project administration, Funding acquisition; Wei Gao: Data validation
and group discussion; Shan Zhao: Resources; Weijun Zhang: Methodol-
ogy; Pingfeng Yu: Methodology; Dongsheng Wang: Funding acquisition
and supervision.
Data availability
Data will be made available on request.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the
work reported in this paper.
Acknowledgments
This project is supported by the National Natural Science Foundation of
China (Grant No. 52070186, 52030003) and the special fund from the State
Key Joint Laboratory of Environment Simulation and Pollution Control
(Research Center for Eco-environmental Sciences, Chinese Academy of
Sciences) (Project No. 19Z01ESPCR).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2023.161491.
References
Abd El-Hack, M.E., El-Saadony, M.T., Elbestawy, A.R., Ellakany, H.F., Abaza, S.S., Geneedy,
A.M., et al., 2022. Undesirable odour substances (geosmin and 2-methylisoborneol) in
water environment: sources, impacts and removal strategies. Mar. Pollut. Bull. 178.
Amaral, V., Romera-Castillo, C., Forja, J., 2021. Submarine mud volcanoes as a source of chro-
mophoric dissolved organic matter to the deep waters of the Gulf of Cadiz. Sci. Rep. 11
(1), 3200.
Arshad, Z., Maqbool, T., Shin, K.H., Kim, S.H., Hur, J., 2021. Using stable isotope probing and
fluorescence spectroscopy to examine the roles of substrate and soluble microbial prod-
ucts in extracellular polymeric substance formation in activated sludge process. Sci.
Total Environ. 788, 147875.
Bak, F., Finster, K., Rothfuß, F., 1992. Formation of dimethylsulfide and methanethiol from
methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic
bacteria. Arch. Microbiol. 157 (6), 529–534.
Bylinski, H., Barczak, R.J., Gebicki, J., Namiesnik, J., 2019. Monitoring of odors emitted from
stabilized dewatered sludge subjected to aging using proton transfer reaction-mass spec-
trometry. Environ. Sci. Pollut. Res. Int. 26 (6), 5500–5513.
Cao, B., Wang, R., Zhang, W., Wu, H., Wang, D., 2019. Carbon-based materials reinforced
waste activated sludge electro-dewatering for synchronous fuel treatment. Water Res.
149, 533–542.
Cao, B., Zhang, T., Zhang, W., Wang, D., 2021. Enhanced technology based for sewage sludge
deep dewatering: a critical review. Water Res. 189, 116650.
Chen, Y.C., Higgins, M., Murthy, S., Maas, N., Covert, K., Toffey, W., 2006. Production of
odorous indole, skatole, p-cresol, toluene, styrene, and ethylbenzene in biosolids.
J. Residuals Sci. Technol. 3 (4), 193–202.
Chen, Y.C., Higgins, M.J., Beightol, S.M., Murthy, S.N., Toffey, W.E., 2011. Anaerobically
digested biosolids odor generation and pathogen indicator regrowth after dewatering.
Water Res. 45 (8), 2616–2626.
X. Zhu et al.
Clercin, N.A., Druschel, G.K., 2019. Influence of environmental factors on the production of MIB
and geosmin metabolites by bacteria in a eutrophic reservoir. WRR 55 (7), 5413–5430.
Decottignies, V., Bruchet, A., Suffet, I., 2010. Dried sludge odours: classification and case
study. Proc. Water Environ. Fed. 2010 (3), 72–82.
DeFrancesco, C., Guéguen, C., 2021. Long-term trends in dissolved organic matter composi-
tion and its relation to sea ice in the Canada Basin, Arctic Ocean (2007–2017).
J. Geophys. Res. Oceans 126 (2).
Dionigi, C.P., Ingram, D.A., 1994. Effects of temperature and oxygen concentration on
geosmin production by streptomyces-tendae and penicillium-expansum. J. Agric. Food
Chem. 42 (1), 143–145.
Drotar, A., Burton, G.A., Tavernier, J.E., Fall, R., 1987. Widespread occurrence of bacterial
thiol methyltransferases and the biogenic emission of methylated sulfur gases. Appl. En-
viron. Microbiol. 53 (7), 1626–1631.
Fisher, R.M., Barczak, R.J., Suffet, I.H., Hayes, J.E., Stuetz, R.M., 2018a. Framework for the
use of odour wheels to manage odours throughout wastewater biosolids processing.
Sci. Total Environ. 634, 214–223.
Fisher, R.M., Le-Minh, N., Alvarez-Gaitan, J.P., Moore, S.J., Stuetz, R.M., 2018b. Emissions of
volatile sulfur compounds (VSCs) throughout wastewater biosolids processing. Sci. Total
Environ. 616–617, 622–631.
Fisher, R.M., Le-Minh, N., Sivret, E.C., Alvarez-Gaitan, J.P., Moore, S.J., Stuetz, R.M., 2017.
Distribution and sensorial relevance of volatile organic compounds emitted throughout
wastewater biosolids processing. Sci. Total Environ. 599, 663–670.
Gao, H., Zhang, W., Song, Z., Yang, X., Yang, L., Cao, M., et al., 2017. Transfer behavior of
odorous pollutants in wastewater sludge system under typical chemical conditioning pro-
cesses for dewaterability enhancement. Sci. Rep. 7 (1), 3417.
Gao, W., Yang, X., Zhu, X., Zhao, S., Yu, J., Wang, D., et al., 2022. The variation of odor char-
acteristics of wastewater sludge treated by advanced anaerobic digestion (AAD) and the
contribution pattern of key odorants. Sci. Total Environ. 840, 156722.
Gao, Z., Guéguen, C., 2018. Distribution of thiol, humic substances and colored dissolved organic
matter during the 2015 Canadian Arctic GEOTRACES cruises. Mar. Chem. 203, 1–9.
Gemert, L.J.V., 2011. Odour Thresholds: Compilations of Odour Threshold Values in Air,
Water and Other Media. Oliemans Punter & Partners BV, Houten, Netherlands.
Gerber, N., Lechevalier, H., 1965. Geosmin, an earthy-smelling substance isolated from acti-
nomycetes. ApMic 13 (6), 935–938.
Gruchlik, Y., Heitz, A., Joll, C., Driessen, H., Fouche, L., Penney, N., et al., 2013. Odour reduc-
tion strategies for biosolids produced from a Western australian wastewater treatment
plant: results from phase I laboratory trials. Water Sci. Technol. 68 (12), 2552–2558.
Han, Z., Li, R., Shen, H., Qi, F., Liu, B., Shen, X., et al., 2021. Emission characteristics and as-
sessment of odors from sludge anaerobic digestion with thermal hydrolysis pretreatment
in a wastewater treatment plant. Environ. Pollut. 274, 116516.
Higgins, M.J., Adams, G., Card, T., Chen, Y.-C., Erdal, Z., Forbes, R.H., 2004. Relationship be-
tween biochemical constituents and production of odor causing compounds from anaer-
obically digested biosolids. Odors and Air Pollutants Conference 2004. Water
Environment Federation, pp. 471–486.
Higgins, M.J., Chen, Y.C., Yarosz, D.P., Murthy, S.N., Maas, N.A., Glindemann, D., et al., 2006.
Cycling of volatile organic sulfur compounds in anaerobically digested biosolids and its
implications for odors. Water Environ. Res. 78 (3), 243–252.
Jiang, G., Melder, D., Keller, J., Yuan, Z., 2017. Odor emissions from domestic wastewater: a
review. Crit. Rev. Environ. Sci. Technol. 47 (17), 1581–1611.
Kim, H., Murthy, S., McConnell, L., Peot, C., Ramirez, M., Strawn, M., 2002. Characterization
of wastewater and solids odors using solid phase microextraction at a large wastewater
treatment plant. Water Sci. Technol. 46 (10), 9–16.
Le, C., Kunacheva, C., Stuckey, D.C., 2016. "Protein" measurement in biological wastewater
treatment systems: a critical evaluation. Environ. Sci. Technol. 50 (6), 3074–3081.
11
Science of the Total Environment 867 (2023) 161491
Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS)
on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41
(5), 1022–1030.
Liu, Y., Fang, H.H.P., 2003. Influences of extracellular polymeric substances (EPS) on floccu-
lation, settling, and dewatering of activated sludge. Crit. Rev. Environ. Sci. Technol. 33
(3), 237–273.
Novak, J.T., Murthy, S., Higgings, M.J., Forbes, B., Erdel, Z., 2012. Ten years of odor research
on biosolids-what have we learned. Proc. Water Environ. Fed. 2012 (3), 527–541.
Novak, J.T., Verma, N., Muller, C.D., 2007. The role of iron and aluminium in digestion and
odor formation. Water Sci. Technol. 56 (9), 59–65.
Poxon, T.L., Darby, J.L., 1997. Extracellular polyanions in digested sludge: measurement and
relationship to sludge dewaterability. Water Res. 31 (4), 749–758.
Rappert, S., Muller, R., 2005. Odor compounds in waste gas emissions from agricultural oper-
ations and food industries. Waste Manag. 25 (9), 887–907.
Ras, M.R., Borrull, F., Marce, R.M., 2008. Determination of volatile organic sulfur compounds
in the air at sewage management areas by thermal desorption and gas chromatography-
mass spectrometry. Talanta 74 (4), 562–569.
Romero, C.M., Engel, R.E., D'Andrilli, J., Chen, C., Zabinski, C., Miller, P.R., et al., 2017. Bulk
optical characterization of dissolved organic matter from semiarid wheat-based cropping
systems. Geoderma 306, 40–49.
Sillman, S., 1999. The relation between ozone, NOx and hydrocarbons in urban and polluted
rural environments. AtmEn 33 (12), 1821–1845.
Suurnakki, S., Gomez-Saez, G.V., Rantala-Ylinen, A., Jokela, J., Fewer, D.P., Sivonen, K.,
2015. Identification of geosmin and 2-methylisoborneol in cyanobacteria and molecular
detection methods for the producers of these compounds. Water Res. 68, 56–66.
Vega, E., Monclus, H., Gonzalez-Olmos, R., Martin, M.J., 2015. Optimizing chemical condi-
tioning for odour removal of undigested sewage sludge in drying processes. J. Environ.
Manag. 150, 111–119.
Wang, H.-F., Wang, H.-J., Hu, H., Zeng, R.J., 2017. Applying rheological analysis to under-
stand the mechanism of polyacrylamide (PAM) conditioning for sewage sludge
dewatering. RSC Adv. 7 (48), 30274–30282.
Wang, S., Matt, M., Murphy, B.L., Perkins, M., Matthews, D.A., Moran, S.D., et al., 2020. Or-
ganic micropollutants in New York Lakes: a statewide citizen science occurrence study.
Environ. Sci. Technol. 54 (21), 13759–13770.
Xue, S., Ding, W., Li, L., Ma, J., Chai, F., Liu, J., 2022. Emission, dispersion, and potential risk
of volatile organic and odorous compounds in the exhaust gas from two sludge thermal
drying processes. Waste Manag. 138, 116–124.
Yang, X., Jiao, R., Zhu, X., Zhao, S., Liao, G., Yu, J., et al., 2019. Profiling and characterization
of odorous volatile compounds from the industrial fermentation of erythromycin. Envi-
ron. Pollut. 255, 113130.
Yu, G., Xu, C., Zhang, D., Ju, F., Ni, Y., 2022. MetOrigin: discriminating the origins of micro-
bial metabolites for integrative analysis of the gut microbiome and metabolome. iMeta 1
(1), e10.
Yu, G.H., He, P.J., Shao, L.M., He, P.P., 2008. Stratification structure of sludge flocs with im-
plications to dewaterability. Environ. Sci. Technol. 42 (21), 7944–7949.
Zarra, T., Naddeo, V., Belgiorno, V., Reiser, M., Kranert, M., 2009. Instrumental characteriza-
tion of odour: a combination of olfactory and analytical methods. Water Sci. Technol. 59
(8), 1603–1609.
Zhang, T., Shao, M.F., Ye, L., 2012. 454 pyrosequencing reveals bacterial diversity of activated
sludge from 14 sewage treatment plants. ISME J. 6 (6), 1137–1147.
Zhang, W., Xiao, P., Liu, Y., Xu, S., Xiao, F., Wang, D., et al., 2014. Understanding the impact
of chemical conditioning with inorganic polymer flocculants on soluble extracellular
polymeric substances in relation to the sludge dewaterability. Sep. Purif. Technol. 132,
430–437.