Chemosphere 309 (2022) 136669

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Microplastics in sewage sludge destined to anaerobic digestion: The

potential role of thermal pretreatment
Alessandra Cesaro a, *, Francesco Pirozzi a, Antigoni Zafırakou b, Akrivi Alexandraki b
a
Department of Civil, Architectural and Environmental Engineering, University of Napoli Federico II, Via Claudio 21, 80125, Napoli, Italy
b
Division of Hydraulics and Environmental Engineering, Department of Civil Engineering, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece

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

• Coarser microplastics (MPs) tend to

accumulate in municipal sewage sludge
• Sludge anaerobic digestion may be
differently affected by MP presence
• MP fate during the anaerobic digestion
of thermally pretreated sludge was
studied
• Biodegradable compostable (BIO) MPs
led to a methane increase up to 60%
• Sludge pretreatment at 120 ◦ C for 30
min did not affect non-biodegradable
PET-MPs

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

Keywords: Microplastics (MPs) are plastic particles with size smaller than 5 mm: their removal in municipal wastewater
Biomethane treatment plants mostly results in the accumulation of the coarser fraction into the sludge. The common
Circular economy application of the treated sludge as soil amendment raises the issue of the uncontrolled release of MPs into the
Energy
environment, which depicts the need to identify suitable counteraction strategies. This work briefly reviews the
Pollution
Plastic polymers
most recent studies that focus on the fate of MPs during conventional sludge treatments, and, based on the results
Recovery of this analysis, proposes the thermal pretreatment (120 ◦ C, 30 min) of waste activated sludge (WAS) containing
different kinds of MPs, in order to investigate its effect on the anaerobic biodegradability as well as on the
abundance and physical features of MPs. Experimental results show that high temperatures did not alter poly­
ethylene terephthalate (PET) MPs but the biodegradable-compostable ones (BIO-MPs), complying with the UNI
EN 13432 standards. The profile of methane generation from thermally pretreated samples containing PET-MPs
do not indicate any inhibition of the anaerobic process, which was positively influenced by the BIO-MPs in WAS:
a 100% and 25% methane increase was observed over the control samples with and without the thermal pre­
treatment, respectively. Further studies are needed to better understand the mechanisms underlying biode­
gradable MPs behavior as well as to investigate the influence of high temperature treatments on smaller size MPs
during anaerobic processes.

* Corresponding author. Department of Civil, Architectural and Environmental Engineering, University of Napoli Federico II, via Claudio 21, 80125, Napoli, Italy.
E-mail address: alessandra.cesaro@unina.it (A. Cesaro).

https://doi.org/10.1016/j.chemosphere.2022.136669
Received 27 April 2022; Received in revised form 23 August 2022; Accepted 27 September 2022
Available online 3 October 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
A. Cesaro et al.
1. Introduction
Plastics are synthetic organic polymers, whose great versatility has
accounted for their large use over the past decades (Derraik, 2002) for a
wide range of consumer and industrial applications. Prior to 1950, the
global plastic production ranged between 4 and 8 million metric tons; in
1950, it reached an average of 2 Mt/year and for two decades later it
kept growing at a 15% rate (Geyer et al., 2020).Plastic waste generation
was observed to develop according to the plastic production pathway,
but with a time delay caused by the use phase (Geyer et al., 2020). Geyer
et al., estimated that between 1950 and 2015, only 9% of the produced
plastic waste was properly recycled, and that approximately 4900 Mt,
corresponding to 60% of all plastics ever produced, were discarded
either in landfills or in natural environment. Nevertheless, the envi­
ronmental threat has been overlooked for long, whereas both inade­
quate or accidental disposal are the main causes for plastic to enter the
marine environment at an ever-increasing rate (Steer and Thompson,
2020). In the absence of appropriate counteractions, global mismanaged
plastic waste could increase from 60 to 100 Mt/year in 2015 to 155–265
Mt/year by 2060 (Steer and Thompson, 2020).
Plastic pollution in natural environments is, therefore, a pressing
issue of our times, especially when considering that plastic debris is
persistent, mobile, and ubiquitous in terrestrial and aquatic environ­
ments, including urban, rural, and remote locations (Hartmann et al.,
2019). The greatest concern is for the so called “microplastics” (MPs).
This term, introduced by Thompson et al. (2004) to describe the accu­
mulation of the smaller plastic particles in marine sediments, now
generally refers to those “plastic particles smaller than 5 mm”. Later, the
definition of MPs was further refined and a categorization based on the
average size was proposed by Barnes et al. (2009) to distinguish the
following classes: mega-debris (>100 mm), macro-debris (>20 mm),
meso-debris (5–20 mm) and micro-debris (<5 mm). The shape is an
additional common classification criteria, allowing the distinction
among beads, fragments, fibers, films, sponges, microspheres.
The MPs which are directly released into the environment are usually
referred to as primary MPs and distinguished from the secondary MPs,
resulting from the deterioration and fragmentation of coarser plastic
items (Frias and Nash, 2019) undergoing physical, chemical, and bio­
logical actions. Most plastic litter occurs as secondary MPs: for instance,
the UN records for 2017 claimed that 15–30% of the ocean plastic litter
consists of primary MPs, while the biggest portion (69–81%) is made of
secondary MPs.
The deterioration of plastic items into MPs may be due to mechanical
forces, heat, ultraviolet (UV) light, oxidation, and biodegradation, and it
is strongly related to the constituting plastic polymer, including chas­
polyethylene (PE), polypropylene (PP), polystyrene (PS), poly­
ethyleneterephthalate (PET), low density polyethylene (LDPE),
polyether urethane, polyamide, acrylamide, polyacrylates, alkyd resin,
polyvinylchloride (PVC), polyvinyl alcohol (PVA), polyether sulfone
(PES) (Bui et al., 2020).
Barnes et al. (2009) mentioned the fragmentation of plastic items
resulting from photo-degradation, due to the prolonged exposure to UV
light, as well as that determined by physical friction, like the one caused
by wave-shore interaction at the coastline. Most of the synthetic poly­
mers are susceptible to degradation initiated by UV and visible light;
however, the most damaging UV wavelength depends on the bonds
present in the plastic; consequently, the maximum degradation usually
occurs at different wavelengths for different types of polymers (Singh
and Sharma, 2008). The photo-chemical and thermal degradation are
very similar and they are both classified as oxidative degradation;
nonetheless, they differ in the sequence of initiation steps leading to the
auto-oxidation cycle and by the fact that thermal reactions occur
throughout the bulk of the polymer, whereas photo-chemical reactions
occur only on the surface (Singh and Sharma, 2008).
Fairbrother et al. (2019) exposed high-density polyethylene (HDPE)
under controlled laboratory conditions to temperatures ranging between
2
Chemosphere 309 (2022) 136669
30 ◦ C and 50 ◦ C and UV light intensities up to 153 W/m2, to find that the
rates of change in HDPE mechanical properties tended to increase under
more extreme exposure conditions. Similarly, Therias et al. (2021)
studied the influence of irradiance and temperature on the
photo-oxidation kinetics of polyethylene. They observed that the kinetic
curves obtained from exposing under the same temperature the samples
to 90 and 300 W/m2 did not change, whereas, in the lower irradiance
range 1.1–41.5 W/m2, the rate of oxidation enhanced with increasing
irradiance. Additionally, the temperature raising from 51 to 65 ◦ C
resulted in a marked increase in the rate of oxidation. More recently,
Ding et al. (2022) investigated the photo-degradation of both PVC and
PET MPs in a photoreactor equipped with 500 W mercury lamp at 25 ◦ C,
as well as in the presence of clay minerals, such as kaolinite. This was
found to significantly advance the aging of MPs by facilitating the pro­
duction of hydroxyl radicals, which were primarily responsible for the
photo-degradation effects.
The understanding of the deterioration and degradation mechanisms
that may occur in natural environment is particularly important, since
the size of MPs, along with their shape and constituting polymers, are
pivotal to assess their impact on the biota (Montes-Burgos et al., 2010).
The degradation mechanisms may determine the release of toxic plastic
constituting chemicals, and may produce tiny particles offering a large
surface for the sorption of a wide variety of pollutants (Luo et al., 2022).
In aquatic ecosystems, the presence of MPs has been found to affect both
the water and the sediments (Peng et al., 2020; Song et al., 2019; Zheng
et al., 2020, 2019) as well as a wide variety of living organisms (Celi­
s-Hernández et al., 2021; Ding et al., 2021), from which MPs could also
enter the food chain up to higher tropical levels, exterting their toxic
effects in human bodies (Bhatt et al., 2021).
Identifying the MP sources is, therefore, fundamental to address
possible remediation strategies. Wastewater effluents have been claimed
among the top causes for the environmental release of MPs. Neverthe­
less, wastewater treatment is acknowledged to be effective in the
removal of larger particles, which are mostly transferred and stored into
the sludge produced during the wastewater treatment process (Edo
et al., 2020; Liu et al., 2019), being present in concentrations varying
from to 2.4 ⋅ 104 MPs/kgDW (Mintenig et al., 2017) up to 22.7 ± 12.1 ⋅
103 MPs/kgDW (Li et al., 2018). In this regard, the sludge treatment is of
strategic importance to limit the spread of the MPs retained from
wastewater, especially when it is used in soil enrichment or agriculture.
To date, although the physical, chemical and biological stressors in
sludge treatment have been claimed to influence the fate of MPs, only
few studies have provided a deep understanding (Hatinoğlu and Sanin,
2021). Moreover, the same stressors have not been considered yet as the
driver of MP degradation in sludge. Aims of this work are: i) to provide a
brief review of the scientific reports dealing with the fate of MPs during
conventional sludge treatments; ii) to carry out an experimental activity,
based on the main findings of the literature analysis, to investigate a
possible strategy to reduce MP concentration in sludge; iii) to suggest the
research areas needing more insightful studies. In particular, the liter­
ature analysis suggested the possible use of high temperature to promote
MP degradation during sludge anaerobic digestion and such strategy
was investigated for the first time to highlight its potential and discuss
future research needs.
2. The fate of microplastics during conventional sludge
treatments
In wastewater treatment plants, the sludge produced from both the
primary and secondary settling tanks undergoes separate treatments,
aiming at its biological stabilization and moisture reduction.
The former is usually pursued via anaerobic digestion to produce a
methane-rich gas that can be used for energy purposes; however, in
small plants, the need for easier operation addresses the choice towards
aerobic biostabilization processes. The reduction of sludge water con­
tent relies on thickening and dewatering processes.
A. Cesaro et al.
Alavian Petroody et al. (2021) investigated the effect of sludge
treatment processes on the abundance and characteristics of MPs in
sludge. Sludge samples were collected after thickening, after aerobic
digestion, and after a mechanical dewatering unit and MPs were
extracted following a procedure including sludge purification, density
separation, and filtration. Experimental results showed that thickening
resulted in 6% MP reduction, which was explained by the fact that some
light particles, with a density lower than water, are separated from the
sludge fraction and enter the water phase (supernatant), being recircu­
lated into the wastewater treatment line (Alavian Alavian Petroody
et al., 2021). A relevant decrease in the number of MPs was observed in
the samples of mechanical dewatered sludge. As the dewatering fol­
lowed the aerobic digestion, the authors inferred that such a reduction
should be attributed to the destruction of the biological flocs that likely
determined the release of attached MPs, which, in turn, entered the
reject-water. Li et al. (2018) also showed that the dewatering unit may
play a role in determining MP concentration in solid sludge samples,
with the plate-frame group providing the lower values over
filter-pressure, centrifuge, and belt-type systems. However, such result
may be affected by the presence of lime in the analysed samples that may
have interfered with the MP extraction and quantification procedure.
More studies have been carried out to investigate sludge anaerobic
digestion in the presence of MPs. Although they tend to remain mostly
stable during the process, their presence may influence the bio­
stabilization yields and affect methane production (Zhang and Chen,
2020), depending on the kind of constituting polymer, the particle
concentration and the average size.
Wei et al. (2019b) focused on the effects of polyvinyl chloride (PVC)
MPs (1 mm, 10–60 particles/gTS) on the methane potential of waste
activated sludge (WAS) and found that the presence of 10 particles/gTS
significantly increased methane production by 5.9%. However, higher
concentrations reduced the generation of methane as well as the hy­
drolysis coefficient of WAS, likely due to the adverse effects of the
bisphenol-A leached from PVC MPs. It is worth highlighting that the
leaching of the polymer constituents may also enhance the generation of
methane, as found in the presence of polyamide 6 (PA6). As a monomer
for PA6 synthesis, the caprolactam leached from PA6 MPs promoted the
activity of key enzymes (protease, α-glucosidase, acetate kinase, buty­
rate kinase, and F420), thus improving acidification and methano­
genesis during WAS anaerobic digestion: in the presence of 10
particles/gTS, methane production enhanced by almost 40% (Chen et al.,
2021).
Different consideration emerged for polyethylene (PE) MPs: their
presence in concentration up to 10 particles/gTS did not affect methane
generation that was, however, significantly inhibited when the PE levels
increased to 100 and 200 particles/gTS during biomethane potential
tests. Consistently with these results, continuous trials showed that at
200 particles/gTS, the stable state methane production was 101 ± 8 mL/
gVS, almost 30% lower than the control. This decrease was partly
attributed to the Reactive Oxygen Species (ROS) induced by PE-MPs,
which caused significant loss of cell viability, as further indicated by
the decreased abundances of microbes relevant to these processes (e.g.,
Proteobacteria) observed in continuous WAS anaerobic digesters (Wei
et al., 2019a). ROS were also identified as the main cause of the
decreased production of hydrogen from the alkaline fermentation of
WAS in the presence of polyethylene terephthalate (PET) MPs (Wei
et al., 2019c). The reduced methane production potential during the
anaerobic digestion of WAS containing up to 200,000 particles/gTS was
attributed to the incomplete digestion induced by the MP presence, since
any significant difference in the microbial community was observed (Li
et al., 2020). However, the type of polymer is not the only variable
affecting methane production during anaerobic digestion, since the size
of the plastic particles can also play a role.
The study of the impact of polystyrene (PS) nano-plastic showed that
the reactor start-up was prolonged and, at a concentration of 0.2 g/L,
both methane yield and maximum daily methane yield decreased by
3
Chemosphere 309 (2022) 136669
14.4% and 40.7%, respectively. This result was attributed to the
attachment of the particles to the cell membrane of Acetobacteroides
hydrogenigenes, which inhibited its growth and metabolism (Fu et al.,
2018). Zhang et al. (2020) found that, at a concentration of 0.25 g/L, the
lower was the nanoparticle PS size, the higher was the induced metha­
nization inhibition. Nevertheless, the possible damage of archaeal cells
by nano-plastic has been recently identified as a pressing research issue
(Mohammad Mirsoleimani Azizi et al., 2021).
In contrast with the studies claiming the inhibition of anaerobic
digestion from MPs, Mahon et al. (2017) inferred that the role of
degradation by microorganisms during anaerobic digestion should be
further investigated. These authors characterized MPs in sludge samples
from seven WWTPs, using anaerobic digestion, thermal drying or lime
stabilization and found that the lower abundance of smaller size MPs
was achieved in anaerobic digestion samples. As previous studies had
provided evidence of microbial breakdown of polymers through the
activity of exo-enzymes and mineralization, they were keen to consider
that the lower MP abundance in anaerobic digestion samples should be
attributed to this kind of mechanisms. In this regard, both microbial and
enzymatic degradation processes have been proposed as a remediation
strategy for a wide range of MPs, but still a more accurate demonstration
of the degradation itself is needed (Jaiswal et al., 2022).
The biodegradation of MPs appears as a promising approach and any
method to enhance the biodegradation yield will be of the utmost
importance. In this regard, Chen et al. (2020) obtained the highest value
ever reported for MP biodegradation after 45 days of hyperthermophilic
composting: in this study, the authors stated that high temperatures
promoted the growth of bacteria particularly effective at attacking and
oxidizing plastic structures, leading to effective bio-oxidation and
biodegradation.
Therefore, assessing the fate of MPs during the anaerobic digestion of
thermally pretreated sludge seemed worth investigating to contribute to
the discussion of possible research directions to promote the degrada­
tion of MPs during sludge treatment.
3. Materials and methods
3.1. Sample preparation
For the experimental purposes, waste activated sludge (WAS) sam­
ples were collected from the biological oxidation tanks of the municipal
wastewater treatment plant of Cuma (Italy). The plant is based on a
conventional activated sludge system and serves approximately 1.2
million people equivalent, treating an average daily flow rate of 2.7 m3/
s. After sampling, WAS was thickened for 24 h by settling before use.
MPs were prepared from daily-use items, made of either non-
biodegradable polyethylene terephthalate (PET) or biodegradable
compostable (BIO) polymer following the requirements of UNI EN
13432. These were manually cut to form 5 mm tokens and added to WAS
samples in concentration of 1 g/L, based on the analysis of scientific
literature (Fu et al., 2018) as well as on analytical determination needs.
Sludge samples, either containing MPs or not, were characterized by
their main chemical and physical parameters (Table 1) and thermally
pretreated at 120 ◦ C for 30 min (Kumar Kumar Biswal et al., 2020).
Table 1
Main physico-chemical characteristics of WAS samples.
Parameter Value
pH [− ] 7.3 ± 0.1
TS [%] 2.35 ± 0.24
VS [%TS] 73.03 ± 0.43
COD [mg/L] 11,900 ± 1899
sCOD [mg/L] 1688 ± 141
sCarbohydrates [mg/L] 21 ± 1
sProteins [mg/L] 263 ± 10
A. Cesaro et al. Chemosphere 309 (2022) 136669

3.2. Batch anaerobic digestion tests

Anaerobic digestion tests were carried out in batch, using both

thermally pretreated and untreated samples, either containing MPs or
not, under mesophilic conditions (35 ± 1 ◦ C). The inoculum was pre­
pared by digesting WAS under the same thermal regime selected for the
anaerobic digestion tests.
Schott glass bottles (Duran, Germany) were used as reactors, where
both samples and inoculum were added to ensure a substrate to inoc­
ulum ratio of 1 gVS/gVS (Kumar Kumar Biswal et al., 2020). Each reactor
was sealed by a screw cap with a sampling port for daily quantification
of methane, which was performed by water displacement method, using
a two-column system: the first column was filled with a 15% NaOH
solution to allow CO2 trapping and it was connected at the top to a
second column filled with tap water.
Anaerobic digestion tests were run in triplicate and monitored for 55
days.
Fig. 1. Soluble COD variation after the thermal treatment (120 ◦ C, 30 min) of
3.3 Analytical set up.
WAS samples, either containing MPs or not.
The fate of MPs during anaerobic digestion of both untreated and
thermally pretreated sludge was assessed in terms of MP abundance, by
extracting (Bretas Alvim et al., 2020), weighing and counting plastic
tokens before and after the biological trials. Additionally, the variation
in shape was assessed by visual observation and a gauge monitored the
average size.
The soluble fraction of WAS samples, obtained after centrifugation
and filtration at 0,45 μm, was analysed by the COD, carbohydrate as well
as protein content. The former was carried out according to the Standard
Method APHA 5220B, which was followed to determine total COD as
well. Soluble carbohydrates were obtained by the Dubois method and
soluble proteins were assessed by Lowry method.
Both total (TS) and volatile (VS) solids were analysed according to
the Standard Method (APHA, 2005). pH was monitored using a Hanna
pH meter (Limena, Italy).

4. Results and discussion

4.1. Effects of the thermal pretreatment on sludge and MPs

Over the last decades, sludge pretreatment methods have been a key
topic in a number of scientific studies, aiming at overcoming the limits
posed by the hydrolysis stage, releasing simpler compounds and
improving anaerobic digestion yields. Sludge pretreatment acts towards
the disruption of cell wall and the degradation of the extracellular
polymeric substances, so that the organic substances are released as
soluble compounds, more readily available to anaerobic bacteria. For
this reason, the increased solubilisation of organic compounds is a
common indicator of sludge pretreatment effects.
Fig. 1 plots the soluble COD (sCOD) after the thermal pretreatment at
120 ◦ C for 30 min of sludge samples either containing MPs or not.
Notwithstanding the presence of plastic items, sCOD enhanced
approximately three times after the thermal treatment. As expected, the
particulate polymers and microbial cells were thermally degraded into
soluble monomers and brought into the liquid phase, leading to the Fig. 2. Variation in soluble carbohydrates (a.) and soluble proteins (b.) after
sCOD increase (Xue et al., 2015). the thermal treatment (120 ◦ C, 30 min) of WAS samples, either containing MPs
The sCOD fraction mainly consists of soluble proteins and carbohy­ or not.
drates; a minor fraction, usually lower than 10% is composed of volatile
fatty acids (VFAs) originating from the thermal hydrolysis reactions its exposure at a temperature of 120 ◦ C for 30 min. Any significant
(Han et al., 2017). Consistently with sCOD composition, its increase difference could be attributed to the presence of MPs, which seemed to
came along with that of soluble carbohydrates and soluble proteins, as not influence the solubilisation effect of the thermal process. This effect
shown in Fig. 2. was predominantly attributed to the sludge organic matter: carbohy­
After the thermal treatment, soluble carbohydrates enhanced five drates are, indeed, the main constituents of extracellular polymeric
times, moving from an average 21 ± 1 mg/L in WAS to 132 ± 2 mg/L, substances (EPS), whereas proteins are present inside the cell. The
127 ± 35 mg/L and 105 ± 30 mg/L in thermally pretreated WAS degradation of these WAS components led to the increase in sCOD, as
without MPs, with PET-MPs and with BD-MPs, respectively. The in­ well as in soluble carbohydrates and proteins.
crease in soluble proteins was even more relevant, as the WAS content of The negligible effect of the high temperature on MPs was confirmed
263 ± 10 mg/L reached a value as high as 2342 ± 127 mg/L following by both visual observation and mass variation of the tokens extracted

4
A. Cesaro et al.
from the sludge after the thermal treatment.
All the plastic items collected at the end of the thermal process
appeared in a different shape than the original ones: the flat tokens that
were prepared and added to WAS samples were differently deformed,
depending on the constituting polymer. PET-MPs were slightly wrinkled
(Fig. 3a), likely due to the fact that the pretreatment was carried out at a
temperature higher than 70 ◦ C, which is the glass transition temperature
of PET. Conversely, BIO-MPs were crumbled on themselves (Fig. 3b)
since the constituting polymers is more sensitive to high temperature.
However, no relevant change either in the mass or in the number of
extracted tokens was detected, indicating that the selected thermal
conditions did not alter the structure of the plastic items.
4.2. Methane generation from thermally pretreated sludge in presence of
MPs
Among sludge pretreatment techniques, the thermal ones have
gained great attention: in his review, Pilli et al. (2015) distinguished
between high (>100 ◦ C) and low (<100 ◦ C) temperature pretreatment.
The former is reportedly more efficient and less time consuming:
40–80% biogas increase was found after sludge pretreatment at tem­
peratures ranging between 160 ◦ C and 180 ◦ C and duration from 30 to
60 min, whereas several hours were required to obtain a 20–50% in­
crease in biogas production at 70 ◦ C (Volschan Junior et al., 2021).
Fig. 4 plots the cumulative methane generation from the anaerobic
digestion of both untreated and thermally pretreated (120 ◦ C, 30 min)
Fig. 3. Shape variation in PET-MPs (a.) and BIO-MPs (b.) after the thermal
treatment of WAS samples. In both images, MPs extracted from pretreated WAS
samples (on the right) were compared to the 5 mm original tokens (on the left).
5
Chemosphere 309 (2022) 136669
Fig. 4. Cumulative methane generation from the anaerobic digestion of both
untreated and thermally pretreated (120 ◦ C, 30 min) WAS samples, either
containing MPs or not.
WAS samples, containing either PET- or BIO-MPs.
The presence of PET-MPs in untreated samples did not affect
methane generation: despite the slower start up, the anaerobic degra­
dation of WAS samples with PET-MPs reached similar average values
observed for the control (sole WAS) sample (155.1 ± 19.9 NLCH4/kgVS
and 154.9 ± 1.3 NLCH4/kgVS for WAS and WAS with PET-MPs, respec­
tively). This result was not consistent with previous studies on the
anaerobic degradation of PET nanoplastic (Wei et al., 2019c), confirm­
ing the role of the particle size over that of the concentration, which in
this study was higher than those reported to be inhibiting (Fu et al.,
2018). Conversely, the presence of BIO-MPs seemed to promote
methane generation (Chen et al., 2021): samples containing this kind of
particles exhibited an initial methane production trend close to the
control; from the 11th day of test, this trend diverted from that of the
control samples, reaching values approximately 25% higher than those
registered for the sole WAS.
The thermal pretreatment further increased the generation of
methane: as shown in Fig. 4, all the pretreated samples exhibited a
greater methane production than the respective untreated substrates.
This outcome is consistent with the solubilisation results, since the
greater availability of soluble carbohydrates and proteins of thermally
pretreated samples made the organic material more easily degradable by
anaerobic bacteria during the biological process; this, in turn, led to
higher methane generation. After 55 days mesophilic digestion, the
thermally pretreated WAS produced 40% more methane than the un­
treated one, consistently with previous results (Carrère et al., 2010;
Kumar Kumar Biswal et al., 2020; Volschan Volschan Junior et al.,
2021). After the thermal pretreatment, WAS samples with PET-MPs
were observed to provide a slightly higher methane production than
the one obtained from pretreated WAS, as shown in Fig. 4. However,
when interpreting the variability of results as standard deviation (not
shown in Fig. 4 for clarity reasons), the presence of PET-MPs did not
seem to influence the generation of methane, even after the thermal
pretreatment. Therefore, the exposure of PET-MPs to high temperature
during the pretreatment stage showed not to promote the release of any
inhibiting substance for the anaerobic microbial consortium, nor the
environmental conditions of the biological process addressed any
further deformation of the plastic items.
Both the visual observation and the abundance monitoring resulted
in negligible differences in the shape and weight of the PET items
extracted from the digestate, and the number of items was the same
before and after the digestion tests, despite the pretreatment
application.
Different consideration rose for the pretreated samples containing
BIO-MPs that provided the best performances in terms of methane
A. Cesaro et al.
generation: as highlighted in Fig. 4, the biological process did not show a
lag phase and it proceeded fast, reaching higher methane volumes than
the other samples from the eighth day. At the end of the test, the average
cumulative methane production from the thermally pretreated samples
containing BIO-MPs was 310.5 ± 10.1 NLCH4/kgVS, which was 60%
higher than the respective untreated sample.
Experimental results suggest that the presence of BIO-MPs played a
role in the anaerobic process, which was amplified when the substrate
was exposed to high temperature pretreatment. The biodegradable
items extracted from the digestate exhibited a relevant variation in
shape and size (Fig. 5). Moreover, the average reduction in the mass (of
approximately 10 and 20 mg in both untreated and thermally pretreated
samples, respectively) was consistent with a minor variation in the
abundance recorded after the digestion tests. It is necessary to highlight
that the removal of residual organic matter was particularly difficult on
BIO-MPs extracted from the digested samples, so that the results of the
mass variation are further affected by this issue, which should be
adequately addressed in future studies.
5. Research perspectives
The issue of the presence of MPs in sludge is particularly challenging
and any strategy promoting their degradation during conventional
sludge processes may be worth investigating. The identification of
physical agents, like high temperatures, to which plastic items may be
sensitive can be interesting if they may display positive effects on the
Fig. 5. Shape variation in BIO-MPs extracted from the digestate of both pre­
treated (a.) and thermally pretreated (b.) WAS samples. In both images, MPs
extracted from WAS samples (on the right) were compared to the 5 mm original
tokens (on the left).
6
Chemosphere 309 (2022) 136669
sludge as well, enhancing its solubilisation and anaerobic
biodegradability.
The experiments carried out in this study showed that such a strategy
is particularly interesting for biodegradable MPs: the high temperature
effects on this type of items indicates to promote the generation of
methane. Further studies are necessary to understand the underpinning
mechanisms. The sludge weight loss as well as the monitoring of MP
abundance may be indicative, but not sufficient to address unambiguous
conclusions on the fate of biodegradable MPs within anaerobic pro­
cesses, which may be positively affected by their degradation and/or by
the release of additives promoting the microbial activity. In this regard,
polymers labeled with radioisotopes has been recently proposed as a
mean to monitor bioplastic degradation (Zhang et al., 2021). The
monitoring of target substances that may be reasonably released,
depending on the bioplastic composition, the microbial consortium
variation as well as the enzymatic activity can be also used to better
understand the fate of biodegradable MPs during anaerobic processes.
Experimental outcomes further provided an insight into the behavior
of PET-MPs. In this study, no inhibiting effect on anaerobic digestion
was detected, nor high temperatures addressed the deterioration of
plastic items. This evidence suggests the need for future studies inves­
tigating the combined effects of both particle size and concentration of
MPs, as the threshold concentration for the inhibition of the biological
process would depend on both the type of polymer and the particle size.
It cannot be omitted that the application of high temperature pretreat­
ment on PET-MPs smaller than those used in this study could lead to
different results. In this view, it would be interesting to perform addi­
tional tests to verify this hypothesis, by varying the size and the con­
centration of MPs in sludge as well as the constituting polymer.
6. Conclusions
This study aimed at contributing to the discussion of possible
research directions to promote the degradation of MPs during conven­
tional sludge treatments. Following the analysis of the most recent
works in this field, the fate of MPs during the anaerobic digestion of
thermally pretreated sludge was investigated.
Experimental results showed that high temperature pretreatment
deformed the plastic items in sludge, depending on the sensitivity of the
constituting polymer. However, this affected the anaerobic biodegrad­
ability of WAS only in samples containing BIO-MPs. The cumulative
methane production from these samples after thermal pretreatment was
60% higher than that obtained from the untreated ones, indicating that
the exposure at 120 ◦ C for 30 min of the BIO-MPs contained in WAS
improved their capability of enhancing the biological process yields.
Further studies are, thus, necessary to better understand the underlying
mechanisms, which may be related to the degradation of BIO-MPs and/
or to the release of substances promoting the enzymatic activity of
anaerobic bacteria.
High temperatures, which have been claimed to play a role in pro­
moting MP degradation, were observed not to be effective when used as
pretreatment of WAS containing PET plastic tokens and destined to
anaerobic digestion. The increase in methane generation from thermally
pretreated WAS samples with PET-MPs by 52% over the respective un­
treated samples is likely related to the improved solubilisation of the
sludge organic matter exposed to high temperature. Moreover, both the
size and the concentration of PET-MPs employed in this study were not
found to adversely affect the anaerobic process, indicating that further
studies should investigate the combined influence of these parameters,
both in presence and absence of a thermal pretreatment.
Author statement contribution
Conceptualization: A. Cesaro, F. Pirozzi, A. Zafırakou, A. Alexan­
draki; writing-original draft preparation: A. Cesaro, A. Alexandraki;
writing-review and editing: A. Cesaro, F. Pirozzi, A. Zafırakou, A.
A. Cesaro et al. Chemosphere 309 (2022) 136669

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