Bulletin of Environmental Contamination and Toxicology (2024) 112:5

https://doi.org/10.1007/s00128-023-03828-z

Revealing How Polyvinyl Chloride Microplastic Physicochemically

Affect the Anaerobic Digestion of Waste Activated Sludge
Yi Cheng1 · Xiaoqing Hu2,3 · Yuxuan Gao2,3 · Lei Wang4 · Guoxiang Wang2,3

Received: 10 May 2023 / Accepted: 30 October 2023

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Recent studies have shown that the microplastics in waste activated sludge (WAS) can directly reduce the microbial activity
and influence the performance of anaerobic digestion. Unfortunately, few studies paid attention on the interactions between
WAS and MPs, since MPs could impact the contact between sludge flocs and microorganisms. We found that PVC-MP
changed the interfacial energy properties of the WAS surface and affected methane production. Low concentration (40 mg/L)
of PVC-MP changed the water affinity and greatly reduced the energy barrier of interfacial reaction. Simultaneously, WAS
surface charge characteristics changed with increasing MPs concentration, which made the sludge difficult to contact with
microorganisms. The change process of WAS surface functional groups also indicated that PVC-MP first cover the sludge
surface to prevent from being utilized by microorganisms, and then affect the surface protein structure before toxic substances
leaching. Our study provides new insights into how MPs affect anaerobic digestion.

Keywords Anaerobic digestion · Interfacial thermodynamics · Polyvinyl chloride microplastic · Surface structure · Waste
activated sludge

Globally, the recycling of waste activated sludge (WAS) has
attracted much attention, among which anaerobic digestion
(AD) has been proved to be a cost-effective way (Yu et al.
2022). Due to the development of chemical industry and
the advancement of detection technology, more and more
studies have found that urban sewage treatment plants have
become an important sink for microplastics (MPs) (Mahon
et al. 2017; Pittura et al. 2021). Microplastics could enter
WAS and stay in the sewage system with traditional bio-
logical treatment processes (Zhang et al. 2020). Studies on
the effects of MPs on the anaerobic digestion process have
drawn controversial conclusions. Some types and concentra-
tions of MPs could promote methane production from AD
* Guoxiang Wang
wangguoxiang@njnu.edu.cn
1
Department of Environmental Science and Engineering,
Fudan University, Shanghai 200082, China
2
School of Environment, Nanjing Normal University,
Nanjing 210023, China
3
Jiangsu Engineering Lab of Water and Soil Eco‑remediation,
Nanjing Normal University, Nanjing 210023, China
4
College of Resources and Environment, Yunnan Agricultural
University, Kunming 650201, China
(Chen et al. 2021), but most studies showed an inhibitory
effect (Mohammad Mirsoleimani Azizi et al. 2021; Zhang
et al. 2020; Wei et al. 2022). In general, the existence of
MPs in WAS showed different effects on hydrolysis, VFAs
conversion and methane production in AD process.
The known effects of MPs on AD process mainly include:
(1) leaching toxic chemicals or additives to affect microbial
activity or key enzyme activities (Wei et al. 2019b, 2020);
(2) MPs catalyze the generation of reactive oxygen species
(ROS) to change the microenvironment for microbial growth
(Wei et al. 2019a, 2021); (3) MPs Directly damage microbial
cell structure and inhibit metabolic function (Fu et al. 2018).
The above mechanisms mainly focus on the effects of micro-
plastics on the activity of anaerobic microorganisms, but
the microbial activity in the process of anaerobic digestion
largely depend on the decomposition of sludge flocs (Bezir-
giannidis et al. 2020). In addition to the direct effects on the
activity of microorganisms, MPs may change the surface
structure of WAS and the interfacial energy between sludge
flocs and water, thus affecting the contact characteristics of
microorganisms and sludge. Finally, it affects the anaero-
bic digestion process and methanogenesis performance (Xu
et al. 2020b).

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Polyvinyl chloride microplastic (PVC-MP) is considered
to be the most toxic in the aquatic environment, and their
inhibitory effect on the anaerobic digestion process is also
unanimously recognized (Mohammad Mirsoleimani Azizi
et al. 2021). Therefore, this study explored the effect of dif-
ferent concentrations of PVC-MP on WAS anaerobic diges-
tion, but uniquely explained its mechanism from the per-
spective of the solid-liquid interface energy of sludge flocs.
It is hypothesized that PVC-MP first affected the contact
characteristics of WAS flocs and microorganisms, changing
the WAS surface energy and functional group characteris-
tics, before leaching out toxic chemicals, thereby affecting
the subsequent anaerobic digestion process.
The main purpose of this study is to (1) investigate the
kinetic effects of different concentrations of PVC-MP on the
WAS anaerobic digestion process; (2) elucidate the under-
lying mechanism of PVC-MP affecting the WAS anaero-
bic digestion from the perspective of surface structure and
interfacial thermodynamics. The results of this study provide
new perspectives on the effects of MPs on the AD process.
Materials and Methods
The WAS used in the present study was collected from a
local municipal WWTP in Nanjing, China. The sludge was
concentrated to 40% (V/V) by free settling, stored in a refrig-
erator at 4°C, and used in experiments within 7 days. The
inoculum was taken from a stable anaerobic fermentation
reactor unit in our laboratory. The main characteristics of
the WAS and inoculum are listed in Table S1.
Polyvinyl chloride powder used for experiments were
purchased from Huachuang Plastic Chemical Co., Ltd.
(Dongguan, China), according to our previous study (Wang
et al. 2021). The size of microplastics was confirmed using
Malvern Mastersizer 3000 (Malvern Panalytical) (Fig. S1),
showing a ­Dv(50) of 166 μm. Surface morphology was exam-
ined using Apreo 2 scanning electron microscope (Thermo
Fisher) (Fig. S2). A typical FTIR spectrum is shown in Fig.
S3 using Nicolet iS20 Fourier Transform Infrared Spectrom-
eter (Thermo Fisher).
In this study, 320 mL of the WAS and inoculum with
a 15/1 volume ratio was added into each 600 mL fermen-
tation bottle. To explore the effects of PVC-MP on the
performance of WAS anaerobic digestion, we set up six
PVC-MP treatments (i.e., 0, 40, 80, 120, 160 and 200 mg
­L−1) based on previous researches (Corradini et al. 2019;
Zhang and Chen 2020). The raw WAS substrate (i.e., 0 mg
­L−1) was used as a control. Before the experiment, all bot-
tles were flushed with nitrogen gas for 5 min to maintain
an anaerobic atmosphere. Batch AD was performed in an
automatic methane potential analysis analyzer (MultiTal-
ent 203, Nova Skantek) equipped with a mechanical stirrer
13
(2024) 112:5
­ in−1, which ensured the homogenous suspension
(140 r m
of MPs) and a thermostat water bath (37°C) (Holliger et al.
2016). All the 6 treatments were performed in duplicate
and operated for 25 days. The supernatant was aspirated
using a syringe on days 2, 4, 6, 8, 10, and 12 of the AD
process, respectively, and the resulting volatile fatty acids
(FVAs) were analyzed using meteorological chromatogra-
phy (GC7890A, Agilent, USA).
We used the modified Gompertz equation to calculate
the key parameters of methane production in each treat-
ment (Gu et al. 2020), including lag period, rate, and accu-
mulation (Eq. 1).
{ [ ]}
Rmax × e
Mt = P ⋅ exp − exp × (𝜆 − t) + 1 , (1)
P
where, ­Mt is the accumulated methane production (mL g­ −1
VS) at fermented time (day). P is the potential of methane
production (mL ­g−1 VS). ­Rmax refers to the maximum meth-
ane production rate (mL ­g−1 VS·d). λ is the lag phase time
(day). e refers to the Euler’s number (2.71828). The detailed
calculation can be referred to our previous studies (Cheng
et al. 2021; Cheng et al. 2022).
The thermodynamic parameters of the solid-liquid
interface were calculated based on the result of contact
angle measurement (JY-82B Kruss DSA100, Dataphysics).
Specifically, 5 mL of digestate was filtered by 0.45 μm
membrane filters. The collected filter cake was placed on
a 1% agar plate for 3 h to balance solid-liquid interface.
Before measurement, the filter cake was naturally air-
dried for 10 min, and then swiftly added different liquid
media (water, formamide and 1-brominated naphthalene)
to measure the contact angles via the sessile drop tech-
nique. The detailed calculation was referred to (Xu et al.
2020b). Zetasizer (Nano ZSE, Malvern Panalytical) was
used to test zeta potential for calculating interaction free
energy, and the calculation method is referred to Xu et al.
(Xu et al. 2020a).
Total solids (TS), volatile solids (VS) and COD of
all samples were determined according to the standard
method (APHA 2005), and pH was measured by online
pH meter.
A 3D-EEM (excitation-emission matrix, F-380, Thermo
Fisher) fluorescence was employed to investigate the
effects of PVC-MP on the generation of dissolved organic
matters (Wang et al. 2018). In brief, Excitation (Ex) wave-
lengths were stepwise increased from 200 to 450 nm at
an interval of 5 nm, and the emission (Em) spectra were
detected incrementally from 350 to 550 nm at an interval
of 5 nm. The scanning speed was 1200 nm/min. The par-
allel factor analysis method (PARAFAC) was applied to
classify and analyze the composition of fermented prod-
ucts based on the EEM scan results. Raman Units (RU)
Bulletin of Environmental Contamination and Toxicology (2024) 112:5
was used as the unit of fluorescence intensity and each
component was quantified by the maximum fluorescence
intensities ­(Fmax). The percentage of each region in all the
EEM spectra was calculated by cc (FRI) technique.
Attenuated total reflection-Fourier transform infra-
red spectroscopy (ATR-FTIR) spectrometer (VERTEX
70, Bruker AXS) was used to analyze the distribution of
functional groups of WAS after dosing PVC-MP (Xu et al.
2017). The wavenumber of transmission was at the range of
400–4000 ­cm−1 with a 4 ­cm−1 resolution. ATR-FTIR data
were analyzed using 2D-COS with the methods of Hur et al.
2011 and Noda and Ozaki 2005.
All sample analysis was conducted in triplicate. Data
were presented in average values with error bars. The sig-
nificance of differences among different treatment groups
were tested by one-way analysis of variance (ANOVA) using
IBM SPSS Statistics 22 software.
Results and Discussion
The effects of different concentrations of PVC-MP on the
performance of WAS digestion were significantly different
(Fig. 1a, P < 0.05). The final methane production in the 40
mg ­L−1 PVC-MP treatment was slightly higher than that
in the control group, whereas the other groups were lower
than the control group, which were similar to the previous
study (Wei et al. 2019b). The maximum methane yield was
reached at 401.94 mL/ g VS with 40 mg/L of PVC-MP
addition which exceeded 1.64% of the control and highest
concentration of PVC-MP (200 mg/L) reduced methane pro-
duction by 14.49%. The reaction kinetic parameters show
that a certain range of PVC-MP dosing will increase the
methane production rate (Table S2), and the methanogenesis
process will not be completely inhibited until the concentra-
tion threshold is reached, only low concentration PVC-MP
Fig. 1  Effects of PVC-MP on the performance of WAS digestion. A methane production and kinetic fitting curve of each treatment; B maximum
fluorescence intensity (­ Fmax) of soluble digested product components
Page 3 of 7 5
dosing can keep the methane production rate always higher
than that of the control group (Wei et al. 2019b; Zhang and
Chen 2020), but the higher the PVC microplastics concentra-
tion, the sooner the methanogenesis process is approaching
the end, which means that PVC microplastics reduces the
biochemical methanogenesis potential of WAS.
Independent component signals were identified and
semi-quantified based on the results of 3DEEM analysis via
parallel factor analysis (PARAFAC). The results showed
that the three fluorescent components model was suitable
for these samples (Fig. 1b) and each component was deter-
mined using the OpenFluor online database. Specially, C1
(Ex/Em: 340−360/425−445 nm) was considered as the
aromatic compounds, C2 (Ex/Em: 385−415/465−490 nm)
were considered as the humic acid-like organics and C3 (Ex/
Em: 285−295/370−405 nm) was categorized as microbial
metabolites such as tryptophan-like protein produced by
protein hydrolysis. As is shown in Fig. 1b, the C2 produc-
tion was hardly affected by the concentration of PVC-MP
addition while both C1 and C3 were affected in different
ways. C1 increased slightly when the PVC-MP concentra-
tion was 40 mg/L, but C1 gradually decreased when the
PVC-MP concentration further increased. This means that
a small number of PVC-MP can promote the degradation of
aromatic compounds in WAS during AD process, which may
be the main reason for its increased methane production. In
contrast, C3 decreases with the increase of PVC-MP con-
centration, which means that the presence of PVC-MP has
an inhibitory effect on the decomposition of proteinaceous in
WAS. From the change of VFAs in the process of methano-
genesis (Fig. S4), it can be found that the addition of PVC-
MP inhibits the production of fatty acids and weakens the
conversion of long-chain fatty acids to short chains, which
means that the rate-limiting step of the methanogenesis pro-
cess, the hydrolysis process, is inhibited. It is speculated that
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 5 Page 4 of 7
Fig. 2  Changes of the interfacial interaction energies( between
) sludge
flocs and water. A Electrostatic interfacial energy ΔGEI SLS ; B Lif-
( AB )free energy ΔGSL ; C Lewis
shitz–van der LW BL Waals interaction
( LW )
acid-base interaction free energy ΔGSL
PVC-MP may be caused by changing the interaction free
energy of sludge and affecting functional groups.
To further investigate the mechanism of PVC-MP dos-
ing on the interaction between sludge flocs and digestate,
solid–liquid interfacial interaction energies of digested
sludges were explored. It has been confirmed that the inter-
action energy between solid-liquid( systems) includes Lewis
acid-base interaction free energy ΔGAB , van der LW BL
( LW ) SL
Waals interaction free energy ΔGSL , electrostatic interfa-
cial energy ΔGEI
SLS and Brownian movement (BR) interac-
( )
tions free energy (Van Oss 2006), among which the first
three are dominant. In detail, ΔGAB represents the interac-
SL
tion energy between polar particles at the interface of the 2
phases. ΔGAB > 0 suggests that the solid phase is hydrophilic
SL
and ΔGSL < 0 indicates hydrophilic repulsion; ΔGLW
AB
rep-
SL
resents the interaction between non-polar particles between
the interfaces, the larger the absolute value, the stronger the
surface interaction force; different from above, ΔGEI SLS
char-
acterizes the electrostatic interaction energy between two
solid particles in the solid–liquid system, and the larger the
absolute value, the stronger the surface interaction force (Xu
et al. 2020a).
Changes in the interfacial free energy between sludge
particles and water between groups are shown in Fig. 2.
ΔGLWSL
is mainly distributed between − 20 and − 40 mJ/m2
in each group, which is different from previous reports
(Xu et al. 2020b), that may be caused by the difference in
sludge characteristics. Obviously, the introduction of 40
13
Bulletin of Environmental Contamination and Toxicology (2024) 112:5
mg/L PVC-MP increased the absolute value of ΔGLW SL
on
the sludge surface, which means a larger exchange driving
force for surface non-polar particles (Fig. 2a). Moreover,
the addition of PVC-MP did not enhance the spatial dif-
ference in the interfacial free energy of sludge surface,
and even reduced this difference at low concentrations
(40 mg L ­ −1), which may be one of the physical structural
mechanisms for the improved methane-producing perfor-
mance of sludge at low PVC-MP concentrations. Due to
the hydrophobic character of PVC-MP (Liu et al. 2008),
ΔGABSL
became negative for all the dosing groups compared
to control, but the average value of ΔGAB SL
for the small
−1
dose group (40 mg ­L ) fell at a suitable value, similar
to the previous study (Cheng et al. 2021), which means
that low-concentration PVC-MP appropriately reduced the
energy barrier for material exchange between sludge and
the system (Copeland 2000), and it was not too hydro-
phobic to make it difficult for the sludge to be utilized by
microorganisms (Wu et al. 2002). Change of ΔGEI SLS
in each
group reflected the effects of PVC-MP on the charge char-
acteristics of sludge particles, which may be related to the
charge properties of PVC-MP. The change of ΔGEI value
SLS
also confirms that the addition of different PVC-MP con-
centrations affects the methane production performance
(Xu et al. 2017).
Attenuated total reflection-Fourier transform infrared
spectroscopy (ATR-FTIR) is usually used to reflect the
composition and characteristics of functional groups on
the sludge surface. Using 2D-COS can more finely distin-
guish hidden signal peaks and eliminate the interference of
false peaks (Aftab et al. 2018). With the increase of PVC-
MP dosage, the reflected signal intensity of each group
decreased, but also increased locally (2967−2987 c­ m−1
in Fig. 3a). Compared with the peak-finding results of the
2D-COS synchronous map, the functional groups affected
by the addition of PVC-MP mainly include: (1) peaks gen-
erated by the vibration of the –CH bond generated by the
H vibration (2922 ­cm−1) which considered to be methylene
in organics (Akkas et al. 2007; Rodrigues et al. 2019); (2)
peaks resulting from C-H stretching at 2975 ­cm−1 which
considered to be characteristic of PVC-MP (Refaat et al.
2019); (3) peaks due to intermolecular and intramolecular
hydrogen-bonded –O–H groups at 3230−3550 ­cm−1 (Hint-
erstoisser and Salmén 1999; Shinzawa et al. 2017); (4)
peaks due to C double bonds in olefin functional groups
at 3012 ­cm−1 (Stitt et al. 2012).
According to the Noda rules proposed in the summary
(Noda and Ozaki 2005), a comparative analysis of the
synchronous and the asynchronous maps can lead to the
sequential rather than coincidental change order of the two
observations ­v1 and v­ 2. In short, when the response is both
positive or negative in 2 synchronous and asynchronous
Bulletin of Environmental Contamination and Toxicology (2024) 112:5
Fig. 3  Effect of PVC-MP on
the functional groups on the
surface of WAS. A ATR-FTIR
reflected signal; B Synchronous
and C asynchronous maps from
2D-COS based on the ATR-
FTIR reflected signal
Fig. 4  Schematic (qualitative)
representation indicating that
PVC-MP cover the sludge
surface to prevent from being
utilized by microorganisms
maps, ­v1 occurs before v­ 2, and when the response is one
positive and one negative, ­v1 occurs after ­v2. Comparing
the orthogonal position response values of the above four
key peaks (Fig. 3b and c), the reaction sequence of the
above four functional groups can be obtained: C–H (2975
­cm−1) → –CH (2922 c­ m−1) → –O–H (3230−3550 c­ m−1)
→ C=C (3012 ­cm−1). The interface changes after PVC-MP
addition can be inferred from the above functional group
change sequence. First, PVC-MP adsorbed on the surface
of sludge flocs, which made the C–H bond on the surface
significantly increased. After that, under the influence of
microplastics, the surface structure of the sludge changed
and the interfacial energy mutated (Fig. 2), resulted in the
reduction of surface proteins, and the -CH and –O–H in
organic matters decreased in turn (Zhang and Chen 2020).
With time passed, PVC-MP leached out toxic elements
such as bisphenol A (Wei et al. 2019b), which adhere to
the surface of sludge flocs and significantly increases the
Page 5 of 7 5
C=C bond, and then further hinders the anaerobic diges-
tion process of sludge and poisons microorganisms (Zhang
et al. 2021).
Anaerobic digestion of WAS is generally considered
to be primarily rate-limited by the hydrolysis step (Zhen
et al. 2017), but little attention has been paid to the contact
between microorganisms and WAS before the microorgan-
isms act on WAS (i.e., before the hydrolysis process). The
contact between microorganisms and WAS also significantly
affects the anaerobic digestion performance of WAS, which
has been confirmed in our previous study (Cheng et al. 2021,
2022). Therefore, it is important to pay attention to the effect
of PVC-MP on the contact process before it affects the
anaerobic digestion process.Consistently, our present study
clearly confirmed that PVC-MP usually cover the surface of
the sludge and severely affect the contact between WAS and
microorganisms (Fig. 4). The change of the structure on the
sludge surface has a direct effect on the anaerobic digestion
13
 5 Page 6 of 7 Bulletin of Environmental Contamination and Toxicology
process, and this effect takes precedence over its microbial
toxicity. After covering and affecting the surface structure,
the effect of PVC-MP on WAS significantly changed the
interaction state of particles at the solid–liquid interface of
sludge, and low doses of PVC-MP could benefit from this
change and promote the methanogenesis process.
To the best of our knowledge, this is the first study that
investigated the effects of PVC-MP on methane production
from the perspective of interfacial energy and functional
group changes of sludge surface, and a new mechanism
was proposed to explain the reason why PVC-MP affect the
anaerobic digestion process. In subsequent studies, surface
X-ray photoelectron spectroscopy etching or X-ray absorp-
tion near edge structure can be introduced to further explain
the internal process of structural change.
Supplementary Information The online version contains supplemen-
tary material available at https://​doi.​org/​10.​1007/​s00128-​023-​03828-z.
Acknowledgements This study was financially supported by the
National Natural Science Foundation of China (41971043) and the
Postgraduate Research & Practice Innovation Program of Jiangsu Prov-
ince (KYCX21_1400). We would like to thank Dr. Chen Yun and Lv
Yue for their support and help in this research.
Declarations
Conflict of interest The authors declare that they have no known com-
peting financial interests or personal relationships that could have ap-
peared to influence the work reported in this paper.
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