Innovative Biotic Symbiosis for Plastic Biodegradation to Solve their
End-of-Life Challenges in the Agriculture and Food Industries
PATRIZIA CINELLI1, NICCOLETTA BARBANI1, SARA FILIPPI1, GIOVANNA STRANGIS1,
MARCO SANDRONI1, ANTONIO PRATELLI1, MARIA J LOPEZ2, PABLO BARRANCO2,
TOMAS CABELLO2, PATRICIA CASTILLO2, MARIE ALINE PIERRARD3,
MAURIZIA SEGGIANI1
1Department of Civil and Industrial Engineering, University of Pisa,
Largo Lucio Lazzarino 2, 56122,
ITALY
2Unit of Microbiology, Department of Biology and Geology, CIAIMBITAL, ceiA3,
University of Almeria, Almeria, 04120,
SPAIN
3IDELUX Environment, Drève de l’arc-en-ciel 98, 6700 Arlon,
BELGIUM
Abstract: - At present just about 30% of the waste plastic collected is efficiently recycled, while the rest is
incinerated, disposed in landfills, or can end up in compost and be released in the environment, inducing a very
negative effect on safety and health of flora and fauna. Sustainable management of hardly recyclable plastic
waste generated by light weight single use packaging and agricultural films can be improved by applying
biotechnological approaches, combining microorganisms, new enzymes, earthworms, and insects to work
collaboratively, not only to promote the degradation of these plastics but also to obtain, by-products of the
biodegradation process to be valorized as fertilizers, functional polysaccharides, etc.
In order to develop a feasible process, mapping and characterization of the most diffused agri-food waste
plastic were conducted isolating the main types of plastic involved. Plastic waste in agriculture is mainly
constituted by polyethylene (PE) both linear low density (LLDPE) and high density (HDPE), polypropylene
(PP) and polystyrene (PS), whereas in food packaging polyethylene is still present together with a large
presence of polypropylene, polystyrene and polyethylene terephthalate (PET). Combining plastic presence and
availability of organisms for their degradability, representative samples of plastics (PE, PET, PS) were selected
for analysis of deterioration and potential subsequent biodegradation by enzymes and organisms. To monitor
the plastic degradability by enzymes, and larvae, methods for the plastic analysis were set, outlining some
differences in virgin and post consumer plastic in particular after use in agriculture, assessing the possibility to
monitor the degradability of plastic with time and different treatments, in particular, some evidence of
polyethylene degradability from larvae of Tenebrio molitor was observed.
Key-Words: - Agri-food waste plastic, enzymes, biodegradation, larvae, biomass, end of life.
Received: May 13, 2022. Revised: October 15, 2022. Accepted: November 16, 2022. Published: December 30, 2022.
1 Introduction
Plastics are important in our society, providing a
range of benefits for human health and for the
environment. For example, plastic packaging
protects food and goods from getting wasted and/or
contaminated, thereby saving resources. The light
weight of plastic packaging compared to other
materials, such as glass, saves fuel and decreases
emissions during transportation, similarly low-
density plastic materials, used as replacements for
metals or ceramics in cars and aircraft, save fuel and
decrease emissions. However, such diverse
properties lead to a diverse waste stream, [1].
In 2018 over 360 million tons of plastic were
produced worldwide, of which up to 40% and 3.5%
are consumed by the packaging and agricultural
industries, respectively, [2].
During their use for application, such as those in
food packaging and in agriculture, plastic materials
are contaminated by different impurities such as
food, soil and agrochemical particles difficult to be
removed, washed away, from the post consume
plastic. These plastics end-of-life is generally
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DOI: 10.37394/232015.2022.18.120
Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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represented by incineration or landfilling when they
are properly collected. Highly degraded or
contaminated plastics that cannot be mechanically
recycled can be successfully used as an alternative
fuel in power plants or in cement factories.
Unfortunately, abandonment and burning are
practices still frequently used, although they are
against the law in several European Countries, [3].
To encourage plastic reuse, the EU has adopted a
recycling target of 55% by 2030 for household
plastic packaging waste, complemented by
voluntary commitments from the European plastics
industry to recycle 70% (plastic packaging) and
50% (all plastic waste) by 2040. However, there is a
lack of knowledge about possible tangible solutions
to achieve these ambitions. The challenge is to
increase recycling rates and change the unfavorable
structure of plastic waste reuse. Currently, the
energy recovery rate (41.6%) is even higher than the
recycling rate (31.1%), and the recycling rate only
slightly exceeds that of the landfill rate (27.3%), [4].
Poor segregation of waste at source, multilayers in
plastic items and inefficient collection of recyclable
materials are among the barriers to achieving a
higher recycling rate.
Most plastics are non-biodegradable and when
subjected to degradative environments slowly break
down into smaller fragments, first as microplastics,
then further as nano-plastics, [5].
Biotechnological approaches, combining
microorganisms, enzymes, earthworms, and insects
working collaboratively may promote sustainable
management of hardly recyclable plastic waste
generated by light weight single use packaging and
agricultural films.
In the present paper, we report a mapping and
characterization of the most diffused agri-food
waste plastic, which brought to the selection of
polyethylene (PE), polyethylene terephthalate
(PET), and polystyrene (PS), as plastic substrate to
be investigated for biodegradability, and a brief
overview of recent biotic symbiosis approaches for
plastic assimilation as well as the setting of
analytical tools to monitor the possible degradation,
in selected plastic samples by environment and by
the action of larvae. The use of enzymes and
microorganisms for plastic degradation and
valorisation, [6], [7], is widely investigated as well
the action of some worms and insects on plastic
degradation has been proved and is under further
investigation to be promoted on a real scale system.
Thus, the degradation of usually recalcitrant plastic
in the environment, and the possible approach to
improve it, is a topic of growing interest, [8].
Indeed, slow polyethylene (PE) degradation was
recorded after 4 to 7 months of exposure to the
bacterium Nocardia asteroides, [9], and by bacterial
strains [10], [11], from the guts of plastic-eating
waxworms.
Bacteria are also reported to degrade PET, [12].
Thus, by screening natural microbial communities
exposed to PET in the environment, a novel
bacterium, Ideonella sakaiensis 201-F6, was
isolated that is able to use PET as its major energy
and carbon source. When grown on PET, this strain
produces two enzymes capable of hydrolysing PET
and the reaction intermediate, mono(2-
hydroxyethyl) terephthalic acid. Both enzymes are
required to enzymatically convert PET efficiently
into its two environmentally benign monomers,
terephthalic acid and ethylene glycol.
As well several papers report evidence of the
degradation of PS, for example, mealworms (the
larvae of Tenebrio molitor) from different sources
chew and eat Styrofoam, a common PS product,
[13], [14].
2 Biotic Symbiosis Approach
In order to conduct a systematic and specific study
to promote the degradability of most representative
plastic samples, generated by the food packaging
and agricultural sectors, in the present study an
inventory collection of plastic waste derived from
food packaging and agriculture sectors was
conducted. On the selected plastic types some
representative samples of pre and post consumer
plastic were analysed for morphology and structure,
in order to estimate degradation undergone during
use and to set a study on the possible degradability
of these plastics by the action of microorganisms
and insects.
Thus, for example, recently researchers in Spain
and England claimed that the larvae of the greater
wax moth can efficiently degrade polyethylene,
which accounts for 40% of plastics waste, [11]. In
this work macro-organisms such as yellow flour
worms (Tenebrio molitor) (Figure 1) were tested to
confirm their ability to chew and digest the LDPE,
[13].
In our running experiments selected
representative samples of plastic wastes will be pre-
treated (chemical, enzymes, etc) to improve
degradability and will undergo the action of
microorganisms and insects to promote their
biodegradation.
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DOI: 10.37394/232015.2022.18.120
Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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Fig. 1: Tenebrio molitor.
2.1 Selection of Plastic Samples
Analysis of mixed municipal plastic waste was done
on samples collected by IDELUX Environnement, a
municipal waste treatment plant located in the south
of Belgium.
The analysis was conducted by selecting and
characterizing the mixed plastic waste (Figure 2).
Fig. 2: Municipal solid waste selection at Idelux
facilities.
Post consume samples were washed carefully with
water and detergent, then rinsed with water and
mechanical action. Selected samples were dried 48
hours at 80 °C, and analysed for weight distribution.
The combined analysis of chemical structure (FTIR)
and thermal properties by thermal gravimetric
analysis (TGA) and Differential scanning
calorimetry (DSC) allowed identifying the
polymeric samples and drawing a distribution of the
most present plastic types.
Linear low-density polyethylene (LLDPE), resulted
to be one of the main polymers present in food
packaging, and was selected for testing the effect of
different abiotic treatments on the polymer structure
and using it as a diet for insect breeding.
Thermal/thermomechanical, photo-oxidation and
chemical/thermochemical treatments were applied
to representative LLDPE samples.
2.2 Mealworms and Test Materials
Tenebrio molitor larvae (length: approximately 2
cm) were utilised to test LDPE ingestion and
biodegradation. Larvae were fed with LDPE in four
batches of 100 mass larvae with 100% plastic and
50 individualized larvae with doses of 90% and
100% of LDPE.
2.3 Experimental Methods
Differential Scanning Calorimetry (DSC)
measurements were performed with a Pyris (Perkin
Elmer Instrument, Waltham, MA, USA) equipped
with a Perkin Elmer IntraCooler 1 as a refrigerating
system. Dry nitrogen was used as purge gas at a rate
of 25 mL/min. Samples of about 10 mg were
analysed from room temperature to a temperature
subsequent to the melting point at a heating rate of
10 °C/min in two runs alternated by cooling at the
same speed, with an aluminium empty pan as
reference.
Thermogravimetric analysis (TGA) was
performed using a TA Q-500 (TA Instruments,
Waters LLC, New Castle, DE, USA). About 15 mg
of sample were placed into a platinum pan and
heated from room temperature to 700 °C at 10
°C/min under nitrogen atmosphere.
FTIR technique has been used to obtain
information on the functional groups that
characterise the polymeric structure of the material
allowing its identification. Infrared spectroscopic
analysis was performed using an FTIR ATR
Spectrum 400 Perkin-Elmer (germanium crystal)
spectral sensitivity 4 cm-1, in the frequency range of
4000-650 cm-1.
Morphological and FTIR analysis of sample
surfaces was also performed using the optical
microscope combined with a FTIR Spotlight
Chemical Imaging Perkin Elmer instrument (300X
magnification).
3 Results and Discussion
Based on the results of the analysis of the mixed
municipal plastic waste, and of the agri-waste
provided by European farmers to Idelux was
conducted a pre-selection of samples of agri-food
packaging waste types, including in each case virgin
plastics and post consume counterparts picked
individually from Municipal solid waste (MSW) or
agricultural films.
In food packaging waste, the main plastic types
present in the non-recyclable fraction were multi-
layers (20%), different polymers, and multi-
materials including layers with aluminium (30%).
Among raw polymers, polyethylene, both low
density (LDPE) and linear low-density polyethylene
(LLDPE), (17%) were the most present, followed by
polypropylene (PP) (15%), polystyrene (PS) (8%),
polyethylene terephthalate (PET) (5%), and high-
density polyethylene (HDPE) (5%).
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Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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Concerning agricultural plastics, the most common
polymers retrieved were PE, PP and PS in particular
expanded PS.
PE and more specifically LDPE and LLDPE,
PET, and PS were considered most representative
and suitable for this study for both presence in
MSW and the availability of enzymes,
microorganisms and insects promising for their
biodegradation.
The analysis of plastic multi layers outlined that
most of them were based on PE and PET confirming
the large presence of these polymers in plastic
packaging waste. It is very common to associate the
good moisture barrier properties of PE with oxygen
barrier properties of PET, for food packaging film
production. Figure 3 reported the DSC analysis of a
multi-layer film that was composed of PE and PET.
While figure 4 reports the FTIR of the upper and
lower side of the film, proving it is made with PE
and PET layers.
Fig. 3: Differential scanning calorimetry analysis of
a PE/PET multi-layer in food packaging waste.
In the DSC graph, we can see the melting peak of
PET at about 250 °C, and the melting peak of PE at
about 110-120 °C, while in the cooling scan we can
see a peak for crystallization of PET at 210°C, and
one for PE at 105 °C, [15], [16].
Fig. 4: FTIR of PET/PE bi-layer films, respectively
side a) PET and side b) PE.
The FTIR spectra of Figure 4, confirm the presence
of typical functional chemical groups of respectively
PET, and PE outlining the composition of the film
as a bi-layer of a PE film and a PET film.
Considering the large amount of PE and PET
present in both food packaging and agriculture
plastic waste, these two polymers were selected as
main ones to be studied for the degradation with
enzymes and larvae. Considering that the films
which will undergo degradation will be post-
consumed plastic, we conducted an analysis to
compare the post-consumed films with the raw ones,
in order to estimate the degradation induced by the
stress during use. Analysis of LDPE films coming
from post-consumption in agriculture outlined some
differences when compared to same LDPE films
pre-use “virgin” material.
In a comparison between the spectra of samples
LDPE films used in horticultural mulching in Spain,
and same film as virgin material, main variations are
due to the presence, in the used sample, of a
widened band with a maximum of 1028 cm-1
(Figure 5) attributable to soil contamination as
highlighted optical image (Figure 6).
Fig. 5: FTIR of LDPE mulching film a) virgin and
b) post consume.
Fig. 6: Surface morphology of respectively LDPE
virgin and LDPE used.
The ratio between the intensity of the band relative
to the amorphous phase at 717 cm-1 and that relative
to the crystalline phase at 730 cm-1 was measured,
using the instrument software and was noted an
increase in the superficial amorphous phase in the
used sample compared to the virgin sample.
Similarly, for LDPE films we can observe some
differences in thermal stability (Figure 7) between
the virgin and the used samples.
In the samples of LDPE post use, there is an
additional step of degradation at 620 °C that might
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Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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be attributed to the soil debris adhered to the film or
to some oxidised form of PE.
Even for samples based on PS, used as
germination trays, a difference in morphology
between the virgin and used samples is reported in
Figure 8. In the used samples, an increase in the
superficial amorphous phase was compared to the
virgin sample, and a rougher surface was also
reported. The FTIR of the used PS samples was
significantly different compared to the virgin PS
(Figure 9).
These most significant differences in the used
samples from plastic batches, in the comparison of
virgin and used plastic in food packaging, are
attributed to the use in agriculture and thus a more
serious exposure to temperature and sunlight than
what happens to polymers used in food packaging.
Fig. 7: Thermal gravimetric analysis on mulch film
sample virgin LDPE and used LDPE.
Fig. 8: Morphology of respectively sample virgin PS
and used PS.
Fig. 9: Morphology of respectively sample virgin PS
and post consume PS.
Selected samples of the virgin and post consumer
plastic were used for feeding larvae of different,
macro-organism. Preliminary tests have already
allowed us to observe promising results in the case
of larvae of Tenebrio molitor, tested for LDPE
ingestion and biodegradation (Figure 10).
Fig. 10: LDPE film in contact with Tenebrio molitor
larvae.
FTIR analysis (Figure 11) evidenced incorporated
oxygen functional groups as can be observed at
3500-3100 cm−1(-OH) and 1800–1500 cm−1(C=O).
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Patrizia Cinelli, Niccoletta Barbani,
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Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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Fig. 11: LDPE film surface after being fed to
Tenebrio molitor larvae.
The presence of spherical particles with a diameter
range of 150 to 300 μm in the larvae feces suggests
the presence of polymer residues (Figure 12).
The FTIR spectra acquired in the selected area in
Fig.12, revealed the presence of bands indicating the
presence of LDPE residues presenting sign of
depolymerisation (Figure 13).
Fig. 12: Tenebrio molitor feces with spherical
particles.
Fig. 13: FTIR of the selected area in Tenebrio
molitor feces.
The appearance of peaks at 1654 cm -1 (C=C stretch)
suggests de-polymerization of LDPE.
4 Conclusion
At present, a low amount of plastic used in the
agricultural and food packaging sector is recycled
and most of it ends up in landfill or goes to thermal
valorisation. An analysis was performed in these
plastic streams evidencing that the main polymers
present were PE, PP, PET and PS. A selection of
polymers was conducted in order to apply biotic
approaches to improve recalcitrant plastic waste
biodegradation, considering both plastic presence in
the waste stream and availability of enzymes and
organisms to attempt their degradation. Considering
all these factors we decided to focus further research
on LDPE, LLDPE, PET and PS. Representative
samples of these polymers, both virgin and used
(recycled stream) were acquired and investigated to
observe the degradative effect due to use. The post-
consume samples presented some evidence of
degradation versus the virgin material due to the
stress of real application, evident in particular in
PET and PS. The polymers were subjected to attack
with enzymes and larvae. In preliminary tests with
Tenebrio molitor larvae the same evidence of
assimilation was observed for LDPE films. On the
basis of these positive results, more tests are
currently running on the degradation of selected
plastic samples with larvae and adults of insects and
worms.
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Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
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Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
Patrizia Cinelli, Maurizia Seggiani conceptualised
plastic analysis and data organisation; Antonio
Pratelli coordinated logistic for plastic management,
Niccoletta Barbani, Sara Filippi, Giovanna Strangis,
Marco Sandroni, performed chemical, thermal,
morphological analysis and samples preparation.
Marie Aline Pierrard coordinated plastic collection
and analysis of waste plastic.
Maria Jose Lopez, Pablo Barranco, Tomas Cabello,
Patricia Castillo performed plastic pre-treatment and
test on larvae feeding with plastic.
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
This project has received funding from the Bio
Based Industries Joint Undertaking (JU) under grant
agreement “GA887648” project RECOVER
“Development of innovative biotic symbiosis for
plastic biodegradation and synthesis to solve their
end-of-life challenges in the agriculture and food
industries” The JU receives support from the
European Union's Horizon 2020 research and
innovation programme and the Bio Based Industries
Consortium.
Creative Commons Attribution License 4.0
(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.en
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WSEAS TRANSACTIONS on ENVIRONMENT and DEVELOPMENT
DOI: 10.37394/232015.2022.18.120
Patrizia Cinelli, Niccoletta Barbani,
Sara Filippi, Giovanna Strangis,
Marco Sandroni, Antonio Pratelli,
Maria J Lopez, Pablo Barranco,
Tomas Cabello, Patricia Castillo,
Marie Aline Pierrard, Maurizia Seggiani
E-ISSN: 2224-3496
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