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1 s2.0 S0048969723001067 Main
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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
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.
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X. Zhu et al. Science of the Total Environment 867 (2023) 161491
as a dewatering conditioner which was in liquid form and with a mass measured using a fluorescence spectrophotometer (Hitachi F-2700FL,
concentration of 38 %. Japan) under the voltage of 700 V (Detailed in Text S8). Parallel factor
analysis (PARAFAC), which is more sensitive and accurate, has been used
2.2. Sludge thickening and enhanced dewatering to analyze the full-fluorescence EEMs by decomposing the fluorescent
EEM into different independent components. The analysis was performed
As the schematic diagram of different dewatering operations shown in using R with the staRdom package (Arshad et al., 2021).
Fig. 1, two thickening methods and two enhanced dewatering methods
were performed. Gravity thickening was carried out in 2 L cylinders. 2.4. Microbial community analysis
After settling for 30 min, the gravity thickened sludge (gWAS) was
collected. Centrifuge thickening was processed by centrifugation at 2.4.1. DNA extraction, PCR amplification, and Illumina HiSeq sequencing
12000 rpm for 5 min to obtain centrifuge thickened sludge (cWAS). The Sludge samples (0.50 g) were collected for DNA extraction using Fast
thickened sludge was conditioned by FeCl3 at the dosage of 55 mg/g dry DNA spin Kit for Soil (MP Biomedicals LLC, USA). The quality was checked
matter (DM) and further dewatered by press filtration (gWASfd, cWASfd) by running an aliquot on 1 % agarose gels and the concentration of the
or centrifugation (gWAScd, cWAScd), respectively (Detailed in Text S1, extracted DNA was determined through NanoDrop ND-1000 (Thermo
Text S2, and Fig. S1). Samples used for microbial community structure anal- Scientific, USA). Finally, all the DNA samples were preserved at −80 °C
ysis were stored at −80 °C, otherwise stored at 4 °C for chemical analysis. for further analysis.
A set of bacterial and archaeal primers 515F/806R was used to amplify
2.3. Chemical analysis the hypervariable V4-V5 regions of 16S rRNA genes. Amplicons were then
sequenced using an Illumina HiSeq platform, which was performed by
2.3.1. Characterization of sludge chemical quality Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).
The DM content, volatile matter (VM, in % DM) content, pH values, Fe
content, and Al content of each sample were measured in triplicate, and 2.4.2. Processing of sequencing data
details of the measurement are given in Text S3. In this study, operational taxonomic units (OTUs) were clustered with a
97 % similarity cutoff and α-diversity indices were calculated to evaluate
2.3.2. MVOC analysis the biodiversity of microbial communities in different sludge samples. Prin-
The MVOC analysis was performed using the electronic nose (eNose), cipal Coordinate Analysis (PCoA) and Unweighted Pair Group Method with
headspace gas chromatography-coupled ion mobility spectrometry (HS-GC- Arithmetic mean (UPGMA) clustering were applied based on weighted
IMS), and headspace solid-phase microextraction gas chromatography– UniFrac to present the distance among different samples. Linear discrimi-
mass spectrometry (HS-SPME-GC–MS). Detailed information about the nant analysis effect size (LEfSe) was employed to identify distinguishing
operating conditions is shown in Text S4, Text S5, and Text S6. genera among the groups obtained by PCoA, with p value threshold of
Based on the volatiles identified by eNose, MANOVA (Greenhouse- the Wilcoxon test set to 0.05 and linear discriminant analysis (LDA) log-
Geisser) and clustering analysis (CA) were used as statistical approaches score threshold set to 2.5. To gain insight into the metabolic mechanism
to compare the differences between samples. Principal component analysis of odor production, functional pathway analysis was performed using
(PCA) and CA were applied to evaluate the similarity and differences based PICRUSt2, based on the Kyoto Encyclopedia of Genes and Genomes
on the data set of GC-IMS and GC–MS results. The correlations between (KEGG) database, with distinguishing bacterial genera. Redundancy analy-
physicochemical variables and key odorants were determined by sis (RDA) was carried out to measure the influence of physicochemical
Spearman's rank correlation. parameters on microbial community structure.
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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 Sensors S2 and S6 respond to a broad range of compounds, while sensors
removal of VM was also higher for cWAS than gWAS. The DM content of S7, S8, and S9 are selective for sulfur-containing compounds, oxygen-
enhanced dewatered sludge was 7–30 times higher than their corresponding containing organics, and aromatic compounds (Table S1), which usually
thickened sludge. The better dewatering performance of enhanced dewatering have low odor threshold concentrations (OTCs) in the range of
treatment than thickening was attributed to the removal of physically bound 0.0001–0.010 mg/kg (Gemert, 2011; Yang et al., 2019). The MANOVA
water in the sludge flocs (Wang et al., 2017). A higher fraction of organic mat- and CA results in Fig. 2e and Fig. 2f indicated that the gravity and centrifuge
ter was also removed along with water by enhanced dewatering. In addition, thickening showed an insignificant effect on the volatile emission of sludge
the thickening methods showed certain effects on the DM and VM contents of (p > 0.05), although cWAS had slightly higher responses at S6 and S8
enhanced dewatered sludge. For gravity thickened sludge, press filtration con- (Fig. 2b). The two enhanced dewatering treatment methods showed differ-
tributed to a higher moisture removal and lower organic matter removal than ent effects on the volatile emission. The response intensity of sensors S2, S7,
centrifuge dewatering. However, centrifuge dewatered sludge from the centri- and S9 increased slightly and reduced at sensors S6 and S8 for centrifuge
fuge thickening process sequence (cWAScd) had slightly higher DM and VM dewatered sludge (Fig. 2d) (p > 0.05). The intensity of sensors S7 and S9
contents than cWASfd. In addition, the centrifugation-treated sludges had increased prominently for gWASfd and cWASfd (Fig. 2c), indicating distinct
lower pHs. Compared to gravity thickening dewatered sludge (gWASfd and variations of volatile formation and emission after press filtration
gWAScd), the dewatered sludge with centrifuge thickening pre-dehydration dewatering (p < 0.05). The CA results confirmed that the emission from
(cWASfd and cWAScd) had a lower Fe/Al ratio. press filtration dewatered sludge samples (gWASfd and cWASfd) were
significantly different from the others (Fig. 2e, Fig. 2f). Since the limitation
3.2. Chemical profiling of MVOCs emitted from sludge with different dewatering in the identification accuracy, the eNose results only provided a rough
operations profile of the volatile composition. Still, it could be concluded that without
inappropriate anaerobic conditions, the enhanced dewatering treatment
The eNose responses of 10 sensors in Fig. 2a-d show the differences in had a greater effect on the volatile emission than the thickening process,
the volatile emission between sludges with different dewatering opera- and the effect of press filtration and centrifuge dewatering operations on
tions. Generally, sensors S2, S6, S7, S8, and S9 gave strong responses. 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.
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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 and PCA analysis (Fig. S2). Based on the composition and intensity of
discriminating VOC emission of sludges. The topographic plots in Fig. 3a volatiles, the CA and PCA results indicated that the enhanced dewatered
and Fig. 3b show that most of the signals appeared in the Rt of 100–700 s sludge clustered in one group and WAS, gWAS, and cWAS grouped
and Dt of 1.0–1.5 ms. >110 signal peaks were detected and only part of together, nevertheless, cWAS was relatively distant from WAS and gWAS
the signals could be qualitatively identified, such as alcohols, aldehydes, (Fig. 3c, Fig. 3d). Consistent with eNose results, centrifuge thickening
esters, ketones, and nitrogen-containing compounds (Table S2). All the resulted in a higher volatile emission and formation of new compounds,
detected signals were used to develop the fingerprint plot for further CA 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.
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X. Zhu et al. Science of the Total Environment 867 (2023) 161491
This result confirmed once again that centrifuge thickening had a greater 3.3. Identification of key odorants by HS-SPME-GC–MS
impact on the formation and emission of volatile compounds than gravity
thickening. The differences in volatiles between centrifuge dewatered sludge 3.3.1. Emission concentration and composition of MVOCs
and thickening pre-dehydrated samples were better revealed by GC-IMS A total of forty compounds were quantitatively detected using HS-
results. >30 new volatiles were detected in gWAScd and cWAScd, such as SPME-GC–MS for all the sludge samples, including nine VOSCs, one terpe-
cyclohexanone and leaf alcohol (Fig. S2, Table S2). noid, ten organic volatile aromatic compounds (OVACs), twelve oxygen-
The type and intensity of volatile compounds emitted from sludge ated (i.e., ketones, esters, alcohols, and furans), three halogenated, three
changed significantly after enhanced dewatering. For instance, the intensi- terpene, and two cyclic alkane compounds (Fig. 4, Fig. S4). According to
ties of acetone and 2-butanone of enhanced dewatered sludge were much the MVOCs identified by GC–MS, WAS and gWAS were clustered in one
higher, and new volatiles appeared along with the disappearance of some group, cWAS and gWAScd were clustered, and the others grouped together
signals, which meant that many volatile substances were raising or forming (Fig. 4a), confirming the differences in composition and concentration of
after enhanced dewatering treatment (Fig. S2, Fig. 3). Moreover, the sludge MVOCs upon dewatering treatment. Based on the composition char-
volatile compounds of gWASfd and gWAScd were greatly different acteristics of MVOCs, the dewatered sludges from the centrifuge thickening
from each other, while cWASfd and cWAScd were more similar in their treatment sequence (cWASfd and cWAScd) were more closely gathered
volatile composition. Compared with gWAS, most aldehydes decreased than from the gravity thickening process sequence (gWASfd and gWAScd).
for gWASfd, particularly pentanal, heptanal, and benzaldehyde, while As shown in Fig. 4a, compared with WAS (91.5 μg/kg), the total VOC
increased for gWAScd, indicating that centrifuge dewatering increased emission capacity of the unit amount of wet sludge increased greatly for
the aldehyde emission (Fig. S3). Moreover, cyclohexanone, leaf alcohol, cWAS (2231.7 μg/kg) while decreased for gWAS (21.3 μg/kg). The VOC
furfural, and propyl acetate were formed in cWASfd and cWAScd concentrations increased for dewatered sludge, reaching 2670.2 μg/kg
(Table S2). The newly formed volatile compounds after dewatering were and 4236.1 μg/kg for gWAScd and cWAScd, respectively, followed by
more likely to be microbial volatiles than blocked compounds in the flocs. gWASfd (2197.3 μg/kg) and cWASfd (3306.6 μg/kg), confirming that
The release of substrates (such as PN and PS) and increase of microbial centrifuge dewatering greatly increased the VOC emission compared with
activity resulting from centrifuge shearing stress were attributed to the press filtration dewatering. However, it was interesting to note that the
increased odor production for dewatered digested sludge (Chen et al., concentration of VOCs (μg/kg) emitted from the unit amount of dry matter
2011). The Fe-bound organics were proposed to show better bioavailability of sludge was the highest for cWAS, due to its high level of VOSCs and
than Al-bound organics in sludge (Higgins et al., 2004; Novak et al., 2007). moisture content (Fig. S4a).
Thus, the dewatering methods affect the release of organic substances from In this study, VOSCs dominated all sludge samples, accounting for
the sludge flocs and the bioavailability of the organics in dewatered sludge, 43.2 %–97.4 % of total MVOC concentrations (Fig. S4b). Nine VOSCs,
which are important influencing factors for the volatile emission. In short, including DMS, DMDS, DMTS, 2,4-dithiapentane, allyl methyl sulfide,
the impacts of the enhanced dewatering on volatiles were greater than methyl allyl disulfide, methyl propyl sulfide, isopropyl methyl sulfide,
thickening, while thickening methods have a certain effect on the volatile and 1-pentanethiol, were detected (Fig. S4c), which were produced largely
emission of downstream dewatered sludge. from the decomposition of sulfur-containing proteins driven by anaerobic
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.
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X. Zhu et al. Science of the Total Environment 867 (2023) 161491
microorganisms (Bak et al., 1992; Drotar et al., 1987; Higgins et al., 2006). cWASfd and cWAScd. 2-hexanone and methyl acrylate were also high in
DMS, DMDS, and DMTS had higher concentrations in enhanced dewatered cWASfd and cWAScd with concentrations of 6.3–11.8 μg/kg (Fig. S4f).
sludge. The increased production of reduced sulfur compounds corresponded According to Rappert and Muller (2005), incomplete aerobic degradation
to the decreased oxidation/reduction potential with increased dry matter processes result in the emission of oxygenated compounds. Compared
content of sludge (Kim et al., 2002). Moreover, the VOSC concentration of with the thickened sludge, the higher concentration of oxygenated com-
cWAS was 100 times higher than gWAS, and the enhanced dewatered sludge pounds in the enhanced dewatered sludge was likely due to the alteration
by centrifugation (gWAScd and cWAScd) emitted higher VOSCs compared of the micro-environment, especially for centrifuge dewatering. Although
with press filtration dewatered sludge (gWASfd and cWASfd). The increase high concentrations of oxygenated compounds were emitted, the olfactory
of MVOC concentration after enhanced dewatering of gWAS and cWAS was system of humans seems to be more tolerant of them as evidenced by their
not simply consistent with the residual organic matter content in sludge. It higher OTCs.
has been reported that the activity of microorganisms in VOSC generation The origin of cyclic alkane, terpene, and halogenated compounds may
can be induced and promoted upon centrifugation due to the release of nutri- derive directly from industrial or municipal influent to the sewer catchment
ent substrates, such as available protein/amino acids (Chen et al., 2011). (Fisher et al., 2017). In the present work, the concentrations of these
Geosmin was detected though it was less frequently reported in waste- compounds were relatively low (< 7 μg/kg) (Fig. S4g) and were not key
water sludge. Geosmin exhibits a strong earthy/musty odor with a low odorants though they were frequently detected.
OTC (8 × 10−7 mg/kg) and is mainly produced by members of
Cyanobacteria, Actinomycetes, and Proteobacteria (Gemert, 2011; Gerber 3.3.2. SOAV and key odorants
and Lechevalier, 1965; Suurnakki et al., 2015). Among the samples, the Since the chemical concentration of MVOCs did not represent its odor
highest concentration of geosmin occurred in WAS (4.0 μg/kg). After impact, the OAV was calculated for each compound. The SOAV of each
thickening, the concentrations of geosmin in gWAS and cWAS were 2.6 sludge sample is shown in Fig. 4b. The SOAV was in the order of cWAScd
and 1.4 μg/kg, while the filtrate of gWAS also had a higher concentration (22956) > cWASfd (20339) > cWAS (19967) > gWAScd > (14506) >
than cWAS filtrate (result not shown) and geosmin in the filtrate of cWAS gWASfd (9009) WAS (5509) > gWAS (3312).
was not even detected. The geosmin concentration in gWASfd (2.6 μg/kg) Table S3 gives 13 compounds with OAV > 1 in at least one sample. The
was higher than gWAScd (1.9 μg/kg) and the filtrate of gWASfd also had key odorants are identified as DMS, DMDS, DMTS, geosmin, and p-cresol,
a higher concentration than gWAScd filtrate (result not shown). Interest- which contributed to >99.8 % of SOAV in each sample (Fig. 4c). Geosmin
ingly, after enhanced dewatering of cWAS, geosmin was not detected in was the principal odorant in WAS and gWAS, accounting for 90.0 % and
the dewatered sludge (Fig. S4d) and their filtrate. In contrast to VOSC 98.1 %, respectively. As to cWAS, DMTS, DMS, and geosmin contributed
emission, the operation of the centrifugation treatment reduced the concen- to 78.7 %, 11.0 %, and 8.8 % of the SOAV. The major SOAV contributors
tration of geosmin. This finding indicated that the microbial generation were different for gWASfd and gWAScd. gWASfd was dominated by
mechanism or generation conditions of geosmin were distinct from DMTS and geosmin (57.8 % and 36.1 %, respectively), however, gWAScd
VOSCs. Geosmin is often a metabolism product of Actinomycetes and was dominated by DMS (50.3 %), DMTS (30.3 %), and geosmin (16.4 %).
Proteobacteria in soil and water systems under aerobic conditions (Abd El- DMS and DMTS were also the main SOAV contributors for cWASfd and
Hack et al., 2022; Clercin and Druschel, 2019; Dionigi and Ingram, 1994). cWAScd, accounting for 96.6 % and 96.4 %. These findings indicated that
Without centrifugation treatment, the emission of geosmin barely changed the two dewatering methods had significantly different effects on odor
for gWAS and gWASfd, although the emission per dry matter content formation, while thickening methods also had certain effects, possibly
increased. It implied that if centrifugation was employed, geosmin may owing to the differences in the breakage of sludge flocs and changes in
not be greatly accumulated in dewatered WAS like VOSCs. the micro-environment of microorganisms. Centrifugation treatment
There was no significant difference in the OVAC concentration between caused a higher level of odor emission from sludge and alteration of volatile
WAS and thickened sludge, and their concentrations were relatively low composition. The key odorants of dewatered sludge from the gravity
(0.51–0.86 μg/kg). Compared with press filtration dewatered sludge, the thickening process sequence varied more greatly than from centrifuge
concentration of OVACs was higher in gWAScd and cWAScd, reaching thickening.
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- 3.4. Effect of dewatering on sludge EPS and microbial community structure
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 3.4.1. Variation of EPS distribution and composition upon sludge dewatering
and indole have been reported as the key odorants in emissions from To understand the relevance of EPS as the hypothetical MVOC precur-
other WWTPs (Fisher et al., 2017; Gruchlik et al., 2013). The concentration sor, the sludge EPS was compared. Fig. 5a-c shows the concentration
of OVACs in gWAScd and cWAScd was about 1000 and 80 times higher changes of DOC, PN, and PS in all three extracted fractions of sludge EPS.
than their corresponding thickened sludge, respectively, and p-cresol and The variations in PN and PS concentration are similar to that of the total
indole were not even detected in thickened sludge. Rappert and Muller concentration of EPS. After thickening, the bound EPS (LB-EPS and TB-
(2005) and Chen et al. (2006) reported that the presence of p-cresol and EPS) concentration of gWAS and cWAS was lower than WAS, but the oppo-
indole was likely due to the ongoing degradation of tyrosine and trypto- site pattern was observed for SEPS. The changes in the distribution of EPS
phan after centrifuge dewatering. Novak et al. (2012) also reported that indicated the release of bound EPS to out layer by thickening treatment.
p-cresol and indole were associated with shear release in the dewatering Moreover, bound EPS in gWAS and cWAS decreased by 43.2 % and
process similar to VOSCs. 67.8 % compared with WAS, indicating a greater effect of centrifuge
A total of twelve oxygenated compounds including eight ketones, two thickening on the redistribution of EPS than gravity thickening. The greater
alcohols, one ester, and one furan were quantified, while no oxygenated disturbance on the surrounding structure of EPS resulting from centrifuge
compounds were detected in WAS and gWAS. 3-methyl-1-butanol was the thickening was due to the deformation of the sludge flocs, destruction of
main oxygenated compound in cWAS with a concentration of 54.1 μg/kg cells, and even the liberation of intercellular materials into the aqueous
accounting for 2.5 % of the VOC emission concentration (Fig. S4b). Large phase by the shear force. Subsequently, the bound EPS and total EPS
differences between gWASfd and gWAScd were observed. 2-Pentanone, content of cWASfd and cWAScd were rather similar, suggesting a limited ef-
3-pentanone, 2-hexanone, methyl isobutyl ketone, and methyl acrylate fect of press filtration and centrifuge dewatering on cWAS EPS. The similar
were dominant in gWASfd with concentrations of 7.5–893.9 μg/kg, while composition of EPS was consistent with the similar VOC emission charac-
3-methyl-1-butanol was the main oxygenated compound in gWAScd with teristics of cWASfd and cWAScd. However, the gWAS EPS was frail and
the concentration of 473.7 μg/kg. cWASfd and cWAScd had relatively responded differently to the following dewatering treatment in terms of
close concentrations of 513.7–897.5 μg/kg. 2-pentanone dominated SEPS removal and bound EPS increase. The gWASfd had the lowest SEPS
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X. Zhu et al. Science of the Total Environment 867 (2023) 161491
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 like substances in SEPS of gWAS decreased after press filtration dewatering,
cWASfd and cWAScd. However, for gWAScd, the SEPS removal was limited while its content in cWASfd SEPS was higher than cWAS, which indicated
and the bound EPS increased markedly (6.8 mg/g (DM)), leading to a certain effects of thickening methods on press filtration. However, unlike
higher content of total EPS, SEPS, PN, and PS in gWAScd than other the EPS, PN, and PS, tyrosine-like and tryptophan-like substances in SEPS
dewatered sludge samples. The less efficient removal of EPS by the for centrifuge dewatered sludge were higher than their thickened sludge,
combined application of gravity thickening and centrifuge dewatering which also suggested the great difference between press filtration and
treatment was consistent with its bad performance in dehydration. centrifuge dewatering on tyrosine-like and tryptophan-like contents.
Three fluorescent components of EPS, hereafter referred to as compo-
nents C1, C2, and C3, were identified by PARAFAC analysis (Fig. S5, 3.4.2. Variation of microbial community structure and functions upon sludge
Fig. S6). C1 had two excitation maxima (220 nm and 260 nm) and one dewatering
emission maximum at 290 nm. C2 displayed two excitation maxima Table S5 displays the α-diversity indices of the microbial community in
(225 nm and 270 nm) that corresponded to a single emission maximum different sludge samples. The Good's coverage of each sample was beyond
(315 nm). C1 and C2, resembled Peak “B” and Peak “T”, were identified 98 %, indicating that the sequence libraries constructed could cover the
as tyrosine-like and tryptophan-like substances originating from biological diversity of the microbial community. The richness was evaluated by the
activities (DeFrancesco and Guéguen, 2021; Gao and Guéguen, 2018; number of OTUs and ACE, and Shannon and Invsimpson indices were
Romero et al., 2017; Wang et al., 2020). C3 displayed two excitation calculated for diversity evaluation. Press filtration dewatered sludge had
maxima (220 nm and 280 nm) and an emission maximum at 380 nm, lower richness and higher diversity than the corresponding thickened
which was similar to a mixture of polycyclic aromatic hydrocarbons and sludge. However, the microbial community richness and diversity of
protein-like components associated with anthropogenic sources (Amaral gWAScd were both higher than gWAS, and cWAScd had lower richness
et al., 2021). The spectral Ex/Em maxima of these components are summa- and diversity than cWAS. Although the press filtration of gWAS achieved
rized in Table S4. The fluorescence intensity of the three components in the best dewatering performance, centrifuge thickening and dewatering
each extracted fraction of EPS is shown in Fig. 5d-f. The tyrosine-like facilitated the increase of microbial diversity due to the larger effects on
and tryptophan-like substances were the major fluorescence proportions the sludge physical-chemical properties, such as EPS composition and pH
in EPS, accounting for an average of 60.6 % and 26.5 % of the total value. However, twice centrifugation may decrease the diversity.
fluorescence intensity. The fluorescence intensities of tyrosine-like and As shown in Fig. 6a, a total of 56 phyla were identified. Proteobacteria
tryptophan-like substances (C1 and C2) were substantially strengthened was one of the most abundant phyla in all samples, accounting for
in LB-EPS and TB-EPS of cWAS than gWAS, and the intensity of 20.2 %–33.9 % (averaging at 25.0 %) of total effective sequences. This
tryptophan-like substances in bound EPS of cWAS was also higher than result was similar to the previous study reported by Zhang et al. (2012),
WAS, confirming the release of protein and amino acid by the break of who observed that Proteobacteria was the dominant phylum and accounted
flocs and redistribution of EPS via centrifuge thickening. For dewatered for 36 %–65 % of total sequences in active sludge or biosolids. Other dom-
sludge, tyrosine-like and tryptophan-like substances in bound EPS or TB- inant phyla were Chloroflexi (13.7 %–26.5 %, averaging 21.9 %),
EPS were higher than their thickened sludge, which confirmed the break Bacteroidota (15.7 %–25.2 %, averaging 18.8 %), Actinobacteriota (5.4 %–
of flocs and even destruction of cells during enhanced dewatering. For 11.3 %, averaging 7.5 %), Acidobacteriota (1.8 %–11.4 %, averaging
press filtration dewatered sludge (gWASfd and cWASfd), tryptophan-like 6.9 %), and Planctomycetota (2.0 %–7.8 %, averaging 5.6 %). The relative
substances in SEPS were removed. It was interesting to note that tyrosine- abundances of the bacterial genus showed distinct differences among
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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, and amino acid metabolism occupied the highest relative abundance
norank_f__Saprospiraceae, norank_f__Caldilineaceae, norank_f__A4b, and (Fig. S9b). Most of the pathways were common for the different dewatered
unclassified_f__Comamonadaceae. sludge samples, however, some metabolic pathways exhibited differences
Both UPGMA and PCoA showed that the sludge samples could be clus- in abundance. The functional microbial community related to amino acid
tered into five groups according to their bacterial communities, namely metabolism and lipid metabolism was enriched in cWAScd, while energy
WAS and gWAS in one group; press filtration dewatered sludge samples metabolism was abundant in gWAScd.
(gWASfd and cWASfd) in one group; cWAS, gWAScd, and cWAScd were
distant from one another (Fig. 6). Apparently, centrifuge thickening and 3.5. Relationships between key odorants and sludge characteristics
enhanced dewatering treatment had a great effect on sludge microbial
community structure, which was consistent with the PCA result of GC- 3.5.1. Relationship between key odorants and physicochemical characteristics
IMS, suggesting a relationship between microbial community structure To test the relationship between sludge characteristics and key
and VOC emission. The LEfSe analysis with LDA > 2.5 was conducted to odorants, Spearman's rank correlation was performed. As shown in
examine the differential genus with statistical significance that was affected Fig. 7a, several key odorants had significant correlations with sludge phys-
by dewatering methods (Fig. S8). At the genus level, the abundance icochemical variables. C2 was positively correlated with DMS (r = 0.86,
of Hyphomicrobium (Proteobacteria), Gordonia (Actinobacteriota), and p < 0.05), p-cresol (r = 0.91, p < 0.01), and 3-methylbutanal concentra-
Desulfobulbus (Desulfobacterota) were significantly enriched in cWAScd, tions (r = 0.89, p < 0.01). Heptanal was positively correlated with EPS
while norank_f__Rhizobiales_Incertae_Sedis (Proteobacteria) were more abun- (r = 0.79, p < 0.05) and PN (r = 0.82, p < 0.05), and VM had a positive
dant in the group including gWASfd and cWASfd. The significant difference correlation with geosmin (r = 0.81, p < 0.01). Moreover, PN and
in the genus might be attributed to the variation of sludge properties. tryptophan-like substances in the inner layer of EPS (LB-EPS and TB-EPS)
The functional characteristics of microbes in sludge samples were pre- were more positively correlated with key odorants than the EPS compo-
dicted by the PICRUSt2 based on the differential genera. As shown in nents in the outer layer (SEPS) (Fig. S10). This might be due to the greater
Fig. S9a, the most active metabolic pathway (level 1) was metabolism involvement of the inner layer EPS in the secondary metabolism of
(77.7 %–78.4 %), followed by genetic information processing (7.0 %– microbes. In addition, DM also showed a strong correlation with DMDS
7.6 %), environmental information processing (4.8–5.1 %), cellular (r = 0.82, p < 0.05) and acetal (r = 0.93, p < 0.01), implying a relatively
processes (4.1 %–4.4 %), human diseases (3.3 %–3.8 %), and organismal low dependence of generation of DMDS and acetal on the moisture content
systems (1.8–1.9 %). The distribution of metabolism in level 2 showed of the sludge. Similar to C2, Fe had strong positive correlations with DMS
that functions of global and overview maps, carbohydrate metabolism, (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. Science of the Total Environment 867 (2023) 161491
(r = 0.89, p < 0.01). Hence, it was suspected that Fe had a significant role in odorants than the SEPS. The functional characteristics were predicted
the generation of odorous microbial VOCs via regulating protein content, based on the differential genera. Functional profiles involving global
particularly tryptophan-like substances. In addition, Al was also positively and overview maps, carbohydrate metabolism, amino acid metabolism,
correlated with p-cresol (r = 0.87, p < 0.05). Similar trends have been pre- and energy metabolism predominated microbial metabolic activity.
viously reported by Higgins et al. (2006) and Fisher et al. (2018b), which Moreover, Desulfobulbus (Desulfobacterota), Gordonia (Actinobacteriota),
suggested that Fe and Al can form flocs containing organic matter bound and Hyphomicrobium (Proteobacteria) were involved in the production of
to the cations. Therefore, higher contents of Fe and Al salts resulted in a DMS, DMDS, DMTS, and p-cresol. This study provides new insights into
decrease in the amount of protein in the filtrate, and Fe and Al cations the characteristics and generation mechanism of odors from wastewater
have been shown to sequester soluble protein and deposit it into the sludge sludge that was essential for better control of sludge odor.
phase. The increased protein content in sludge then became bioavailable
substrate for MVOC production. CRediT authorship contribution statement
3.5.2. Relationship between key odorants and sludge microbial characteristics Xinmeng Zhu: Conceptualization, Performed the experiments, Data
The correlation between the microbial community and physicochemi- analysis and Visualization, Writing-original draft, Investigation; Xiaofang
cal variables of sludge was investigated by performing RDA (Fig. 7b). Yang: Conceptualization, Resources, Writing-Reviewing and Editing,
According to the forward selection, the six most important contributors of Project administration, Funding acquisition; Wei Gao: Data validation
microbial communities were DM, VM, Fe, Al, C2, and C3, and these and group discussion; Shan Zhao: Resources; Weijun Zhang: Methodol-
variables were statistically significant for both the first canonical axis ogy; Pingfeng Yu: Methodology; Dongsheng Wang: Funding acquisition
(p = 0.04, F = 4.8) and all canonical axis (p = 0.008, F = 18.0). As and supervision.
shown in Fig. 7b, both ordination axes of the biplot combined to explain
82.3 % of the microbial community variations, indicating that these physi- Data availability
cochemical variables were major factors shaping the microbial community
dynamics. WAS and gWAS were associated with VM, and cWAS was related Data will be made available on request.
to C3. For enhanced dewatered sludge, the decline of moisture content and
enrichment of Fe, Al, and C2 contents changed the microbial communities Declaration of competing interest
of sludge. Centrifuge dewatered sludge showed a stronger relationship
with these parameters, suggesting the responses of microbial community The authors declare that they have no known competing financial inter-
structures to the variation of sludge properties. ests or personal relationships that could have appeared to influence the
The association between differential genera and predicted KEGG metab- work reported in this paper.
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- Acknowledgments
tions, such as amino acid metabolism, biosynthesis of other secondary
metabolites, and metabolism of terpenoids and polyketides. The biotic This project is supported by the National Natural Science Foundation of
origin analysis of key odorants performed using MetOrigin online platform China (Grant No. 52070186, 52030003) and the special fund from the State
(Yu et al., 2022) indicated that Desulfobulbus (Desulfobacterota), Gordonia Key Joint Laboratory of Environment Simulation and Pollution Control
(Actinobacteriota), and Hyphomicrobium (Proteobacteria) could be involved (Research Center for Eco-environmental Sciences, Chinese Academy of
in the production of DMS, DMDS, and DMTS, while Gordonia and Sciences) (Project No. 19Z01ESPCR).
Hyphomicrobium might also contribute to p-cresol generation. However,
Cyanobacteria, Actinomycetes, and Proteobacteria in our study which were Appendix A. Supplementary data
reported as major producers of geosmin didn't match with the genus in
the platform. Meanwhile, further studies are needed to confirm the specific Supplementary data to this article can be found online at https://doi.
microorganisms responsible for the production of sludge key odorants. org/10.1016/j.scitotenv.2023.161491.
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