DOI: 10.3303/CET2293003
Paper Received: 8 February 2022; Revised: 19 March 2022; Accepted: 26 April 2022
Please cite this article as: Puello-Mendez J., Marin-Batista J.D., Fusaro C., Lambis-Miranda H.A., Ramírez-Cuadro N.E., Teherán-Romero J.A.,
2022, Biodegradation Potential of White Rot Fungus in Waste Tire Rubber, Chemical Engineering Transactions, 93, 13-18
DOI:10.3303/CET2293003
CHEMICAL ENGINEERING TRANSACTIONS
VOL. 93, 2022
A publication of
The Italian Association
of Chemical Engineering
Online at www.cetjournal.it
Guest Editors: Marco Bravi, Alberto Brucato, Antonio Marzocchella
Copyright © 2022, AIDIC Servizi S.r.l.
ISBN 978-88-95608-91-4; ISSN 2283-9216
Biodegradation Potential of White Rot Fungus in Waste Tire
Rubber
Juliana Puello-Mendeza,*, Jose D. Marin-Batistaa, Carmine Fusaroa, Henry A.
Lambis-Mirandab, Nestor E. Ramirez-Cuadroa, Jefferson A. Teheran-Romeroa
aGICI Research Group, Chemical Engineering Department, Universidad de San Buenaventura Cartagena, Diag. 32 # 30-
966, Cartagena, Colombia
bProcesses and Systems Program, CIPTEC Research Group, Fundación Universitaria Tecnológico Comfenalco, Cartagena,
Colombia
jpuello@usbctg.edu.co
Vehicle tires are high demand inputs in the transportation sector, however, at the end of their useful life they
become a technical, economic, environmental and public health problem, due to their complex structure made
up of various materials, especially vulcanized rubber. One possible way to mitigate the environmental impact
of waste tires is to biodegrade rubber through the enzymatic action of fungi. The present study evaluated the
biodegradation potential of waste rubber tires by enzymatic action of a white rot fungus (Pleurotus Ostreatus).
Initially, the effect of the enzymatic action of the fungus on the mechanical structural and chemical properties
of the rubber was evaluated. Sterilized tire rubber cuts of 1 cm2 were innoculated with the fungus. After 16 and
40 days, surface changes were evaluated by environmental scanning electron microscopy (ESEM). It was
detected that the enzymatic action on the molecular bonds caused material erosion; changes in chemical
structure by Fourier transform infrared spectroscopy (FTIR) were evaluated after 40 days. Also, potential of
the scrap tire material to produce fungi biomass was analyzed, by preparing different concentrations of
pulverized waste tire material in Agar Sabourad, and innoculating them with a suspension of the fungus
mycelia. After 21 days, the germinated biomass at each substrate concentration was gravimetrically analyzed
and modeled. Results showed a proportional dependence of the biomass growth as a function of the substrate
concentration, and a potential of the white rot fungus to help biodegradation of waste tires.
1. Introduction
Natural rubber production worldwide in 2020 amounted to almost 13 million metric tons, showing an important
increase since 2000, when a total of around 6.8 million metric tons of natural rubber was produced globally
(Tiseo, 2021a). In the case of synthetic rubber around 10.9 million metric tons were produced in 2000, while in
2020 this amount reached over 14.4 million metric tons (Tiseo, 2021b). A large part of the rubber production is
destined to the manufacture of tires for airplanes, heavy and light vehicles (Ruibal Villaseñor et al., 2017).
Worldwide, millions of tires are discarded annually in landfills, generating problems of both environmental
pollution at the time of burning them, which disintegrates the rubber, releasing not only carbon monoxide and
dioxide, but also sulfur dioxide, which in the atmosphere can be converted into acid rain. The accumulation of
waste tires is also associated with public health problems when they are buried, stored or destroyed by
incineration, due to the fire hazards (Mohamed et al., 2017). Bioremediation has attracted the attention of
researchers for its minimal energy consumption, efficient abatement and reduced production of toxic by-
products (Srivastava et al., 2015). Fungal bioremediation represents a promising alternative within the
biological treatment of tires due to the implementation of dry media for fermentation. Dry media lead to
bioreactor designs with low operation volume and economical, as they do not require water consumption,
generate a low volume of effluent. The biodegradation of tires by fungi has been studied for Aspergillus sp and
Penicillium sp, showing a 40% reduction in the weight and mechanical properties of rubber due to surface
erosion. Additionally, fungi have a high potential for the generation of enzymes, which, when recovered, would
represent an added value for bioremediation (Shah et al., 2013).
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White-rot fungi (WRF), belonging to the Basidiomycetes group, are considered suitable microorganisms for
the production of enzymes through solid state fermentation, due to the similarity between their natural
environment and the conditions of the fermentation process (Ellouze and Sayadi, 2016). This fungus has an
enzymatic system made up of the enzymes Lignin peroxidase, Manganese peroxidase and laccase, capable
of decomposing ligninolytic compounds. Additionally, WRF are known for their feasibility in the degradation of
pesticides, aromatic hydrocarbons and some colorants. Taking into account the versatility of WRF to
biodegrade polluting organic compounds, it enables the study window towards the search for new applications
in bioremediation. The fungus used in this research was Pleurotus Ostreatus. This type of mushroom is
characterized by growing in climates with tropical and subtropical forests, and can also be cultivated artificially
(Andler et al., 2021). According to the bibliographic review, no bioremediation studies are reported that
evaluate the biodegradation feasibility of tires by enzymatic action of Pleurotus Ostreatus. The main
component of tires is poly-cis-1,4-isoprene (2-methyl-1,3-butadiene) which, being an organic molecule,
represents a carbon source available for obtaining energy (Andler, 2020). Poly-cis-1,4-isoprene is naturally
extracted from the bark of trees during logging processes for rubber production (Singh et al., 2017). Given its
lignocellulosic origin, poly-cis-1,4-isopropene has characteristics similar to lignin, making its polymeric
structure and molecular bonds susceptible to decomposition by the WRF enzymatic complex. The intrinsic
ability of rubber to be transformed into a simpler chemical structure by microbial means defines the
biodegradation potential (Stevenson et al., 2008). However, the measurement of the biodegradative potential
of a vulcanized rubber is a non-standard parameter since the metabolic route in the decomposition of poly-cis-
1,4-isoprene has not been fully identified; this makes it difficult to determine a quantitative value that
expresses the potential of the white rot fungus to transform poly-cis-1,4-isopropene to reusable metabolic
products. The biodegradative potentials of solid substrates can be measured indirectly from the changes in the
physicochemical, mechanical and structural properties of the material. Likewise, the mathematical models
allow to carry out kinetic characterizations of the bioprocess from the identification of biodegradative
parameters, cell growth, yields in the formation of products and consumption of substrate. In this way,
biodigesters can be designed that lead to studies under controlled operating conditions, substrate formulation,
inhibition control, optimization of nutritional requirements and process scaling.
2. Methods and materials
The waste tire rubber sample was obtained from an industrial waste dump, this tire rubber was washed with
distilled water and dried in an oven at 105 °C. Subsequently, the tire fraction was crushed until obtaining a fine
rubber powder with a particle size of 2-4 mm in diameter. The elemental composition of the tire was
determined by Energy Dispersive Spectroscopy (EDS) at 25°C and 1 atm pressure, in order to evaluate the
amount of carbon (C) that is potentially available as a nutrient for the fungus.
2.1 Preparation of the inoculum
A white rot fungus (Pleurotus ostreatus) sample was recovered from a rotting wooden log under local
meteorological conditions. The fungus sample was cut into 1 cm2 pieces, and surface cultured in 20 ml of solid
Sabouraud Agar using 9 cm diameter Petri dishes. The culture was kept at a constant temperature of 37±2 °C
for 10 days in an incubator.
The mycelia resulting from the culture were transferred to a previously sterilized mixer, together with 60 ml of
sterilized water to obtain a complete homogenized spore suspension.
2.2 Fungus germination kinetics
Six concentrations of tire powder (2.5, 5, 7.5, 10, 12.5 mg/L) were prepared, each one in 20 ml of Sabouraud
Agar in Petri dishes of 9 cm in diameter. Each preparation was inoculated with 2 ml of the spore suspension
described in section 2.1) and kept at 37 ± 2 °C in a Memmert incubator, Model 600. The mycelia obtained
after 21 days of incubation were collected and dried at 105°C in an oven and weighed on an analytical
balance. A blank test consisting of 20 ml of Agar and 2 ml of inoculum was performed in triplicate to discount
the weight of germinated biomass by the consumption of nutrients provided by the Agar.
2.3 Inspection by electronic microscope
The structural changes during the biodegradation of the rubber by the enzymatic action of the fungus were
evaluated in a Quanta 650 FEG scanning electron microscopy (SEM). For this, 1 cm2 tire cuts were
impregnated with the mycelia of the fungus and incubated at a constant temperature of 37 ± 2 °C. The
samples were analyzed in their initial state (without impregnation), and after 16 and 40 days of impregnation.
The samples were covered in gold and morphologically analyzed by Environmental Scanning Electron
Microscope (ESEM), in a FEG Quanta 650 equipment at 15 KV.
14
2.4 Fourier Transform Infrared Spectroscopy (FTIR)
To evaluate molecular changes, an uninoculated tire sample was analyzed together with tire samples obtained
after 45 days of inoculation, by Fourier Transform infrared analysis. The analysis was performed using a
Perkin Elmer Infrared equipment, Spectrum 100 model, DTGS detector. A KBr pellet was made from each
sample in an approximate ratio of 100:1mg KBr-Sample. Subsequently, the pellet-shaped samples were
analyzed in the spectrometer at 22°C, resolution of 4 cm-1, number of scans of 16 and a wavenumber range of
400-4500 cm-1.
3. Results
3.1 Tire composition
Table 1 shows the elemental composition detected by EDS. The waste tire rubber sample is composed of
around 88.13% w/w carbon; due to the organic nature of carbon in vulcanized rubber, it becomes a
biodegradable substrate for the fungus (Tsuchii and Tokiwa, 2001).
Table 1: Waste tire rubber composition (% w/w)
Element C O Na S
% w/w 88.13 6.50 2.50 2.79
On the other hand, the sodium content detected was 2.50% w/w. Sodium concentrations greater than 3%
affect transport through the membrane, causing significant losses in the metabolic performance of the fungus.
However, the substrate used presented favorable nutritional conditions for the growth of microorganisms, as
can be seen in results showed in section 3.4.
3.2 SEM inspection of the biodegradation process
Figure 1 shows the changes in the surface of the scrap tire before and after 16 and 40 days of enzymatic
biodegradation with the white-rot fungus.
(a) (b) (c)
Figure 1: Electron microscopy analysis for the rubber surface (a) before biodegradation and after (b) 16 and
(c) 40 days of enzymatic action of the fungus.
In Figure 1a, a previous deterioration of the material surface caused by the metabolic activity of native rubber
bacteria is observed. Figure 1b shows the tire after 16 days of continuous biodegradation. Figure 1b shows
that the fungus was fixed to the surface of the tire, generating erosion of the material, which is probably due to
the enzymatic action of the fungus that broke the sulfur bridges in poly-cis-1,4-isopropene. Therefore,
structural damages similar to those presented in wood by fracture of the ductile material were generated,
creating cavities and fissures. After 40 days of biodegradation, the fissure of the material was visually greater,
indicating that the erosion is progressive as the material is exposed to the enzymatic action of the fungus.
Therefore, the superficial inspection of the material showed that the white rot fungus has biodegradative
activity and affinity for the hydrocarbons that make up the polymeric material of the tire (Rodrigues da Luz et
al., 2019).
15
3.3 Inspection by FTIR analysis
Figure 2 shows the transmittance vs wave number spectrum for the waste tire rubber sample without
inoculation (black spectrum) and the wave spectrum of the sample after being exposed to 45 days of
biodegradation (blue spectrum).
Figure 2: FTIR spectra of rubber samples before inoculation with fungi (black spectrum) and after 45 days of
biodegradation (blue spectrum)
Figure 2 shows a decrease in the peaks of the spectrum in the region from 400 cm-1 to 1200 cm-1, which
indicates breakage of some functional groups in the polymeric chain of poly-cis-1,4-isopropene such as C=C,
carbonyl, methyl, and ester bonds. Changes in the stretch band between the 1400 cm-1 and 1200 cm-1 peaks
indicate the formation of alkaloids. Additionally, there was a decrease in the stretching band between the 3200
cm-1 peak and the 2800 cm-1 peak, lengths where the vibration of the CH2 radical occurs. However, the
presence of biological activity by the white rot fungus on the surface of the used tire is confirmed by changes
in the chemical structure of the rubber surface. These results are consistent with the studies conducted by
Shah et al. (2013), who reported variations in the 834 cm-1 peak, where there is stretching of the cis-1.4
double bond. As well as the appearance of ketones and alkaloids as metabolites of the enzymatic
decomposition process of rubber. Based on the changes mentioned above, it is suggested that the
biodegradation mechanism is oxidative.
3.4 Effect of substrate concentration on biomass production
Figure 3 shows the growth of the fungus for substrate concentrations in the range of 0 mg/L to 15 mg/L.
Figure 3: Germination of white rot fungus at different concentrations of rubber
16
The substrate concentration of 0 mg/L corresponds to the blank tests, which allowed discounting the amount
of 110 ± 4 mg of dry biomass obtained from the nutrients present in the medium. The highest amount of dry
biomass (619 mg) was obtained at tire concentrations of 15 ± 0.07 mg/L. On the other hand, the lowest
amount of dry biomass of 365 mg was obtained for the tire concentration of 2.5 ± 0.07 mg/L. This result is
consistent with the statements of Monod's law, which describes microbial growth as an exponential function of
substrate concentration (Cavaleiro, 2016).
3.5 Modeling of the germination process
For the determination of the biodegradative potential of the fungus, three germination models were considered
for a kinetic analysis of the process. The kinetic parameters of each model were calculated by using the curve
fitting toolbox of the MATLAB® software. The description of each of these models is found below (Cavaleiro,
2016)
Monod equation
𝑓(𝑥) = 𝑢𝑚𝑎𝑥 (
𝑥
𝑘𝑚 + 𝑥
) (1)
𝑢𝑚𝑎𝑥 = maximum specific growth rate (h-1)
𝑘𝑚 = average saturation coefficient (g/L)
𝑥 = substrate concentration
Haldane equation
𝑓(𝑥) = 𝑢𝑚𝑎𝑥 (
𝑥
𝑘𝑚 + 𝑥 + 𝑘1
) (2)
𝑘1 = Haldane’s growth kinetic inhibition coefficient (g/L)
Hinshelwood equation
𝑓(𝑥) = 𝑢𝑚𝑎𝑥 (
𝑥
𝑘𝑚 + 𝑥
)(1 − 𝑘𝑝𝑃) (3)
𝑘p = Propagation coefficient (g/L)
According to the results, the three models showed a good fit, with R2 values between 0.90 and 0.97 (see
Table 2). The Monod model showed greater predictive capacity with R2 = 0.97, which indicates that this model
is adequate to predict the parameters calculated based on the real data obtained. The main advantage of the
Monod model is its ability to satisfactorily fit the curved forms of fungus germination. Based on these results, it
is possible to affirm that there is an affinity of the fungus towards the tire and, in turn, the growth of the fungus
is due to a greater consumption of substrate.
Table 2: Mathematical model fit values
Model R2 Adjusted R2 RMSE SSE
Monod 0.97 0.83 0.06 0.02
Haldane 0.90 0.88 0.05 0.01
Hinshelwood 0.97 0.72 0.08 0.02
Table 3 presents the coefficients of the models, which have in common the coefficient of 𝜇𝑚𝑎𝑥 defined as the
maximum specific growth rate or maximum speed of biomass generation. The values of the constant 𝜇𝑚𝑎𝑥 are
indicators of the potential of the tire to generate Fungi biomass. Since the saturation coefficient (𝑘𝑚) is lower
than the substrate concentration, this suggests that as the fungus grows, it will consume more substrate.
Table 3: Parameters of the microbial germination models
Model µmax km kp k1 P
Monod 0.66 2.65 - - -
Haldane 0.34 0.03 - - 0.03 -
Hinshelwood 0.82 2.65 0.92 - 0.21
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4. Conclusions
In this research, a qualitative evaluation of the changes in the structural and chemical properties of waste tire
rubber was carried out, evaluating the tolerant and biodegradation potential of the white rot fungus Pleurotus
Ostreatus. It was found that the amount of biomass formed (fungal growth) is proportional to the concentration
of waste tire rubber in the culture medium. According to the SEM and FTIR analyses, it was observed that the
enzymatic action of the fungus caused erosion on the rubber surface. The germination modeling of the fungus
allowed us to conclude that there is an affinity of the fungus towards the tire and that, as the fungus grows
there will be greater consumption of substrate, since the saturation constant (𝑘𝑚) is lower than the substrate
concentrations. The models showed that germination data were predicted with confirmed accuracy at R2 >
94%.
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Biodegradation Potential of White Rot Fungus in Waste Tire Rubber