FUNGAL BIODEGRADATION OF POLYSTYRENE

THROUGH Aspergillus tubingensis

FROM WASTE SITES
K.A. Paras1, H.C. Matias2, and M.M. Rivero3
1, 2, 3
Pasay City National Science High School, 2888 Vergel St., Pasay City 1300, Metro Manila,
Philippines
1
krishnaanne.paras@depedpasay.ph, 2hannahcarissa.matias@depedpasay.ph,
3
maureenmay.rivero@depedpasay.ph

INTRODUCTION (Salvodelli et al. 4 and Wang et al. 1417),

The enormous increase in the
as well as bacterial and fungal
production of plastics has been a huge
biodegradation (Ho et al. 7) as a solution
threat to our environment due to its
to PS wastes. Despite the many studies
improper disposal (North and Halden 1).
conducted regarding the fungal
Plastics such as polystyrene (PS) have a
biodegradation of PS, currently
variety of uses in food packaging,
unexplored organisms still may be
electronics, and as insulation (Xue et al.
capable.
461 and Noguchi et al. 19). However, a
A study conducted by Khan et al.
disadvantage is that it lacks degradability
yielded a positive result of
(Abhijith et al. 2140). Improper disposal
biodegradation of polyester polyurethane
of this polymer can lead to depletion of
(PU) through the fungus Aspergillus
ozone layer (Rasul and Arutla 3),
tubingensis. Aspergillus tubingensis is a
contamination of landfills and
black Aspergillus mostly located in warm
groundwater leaching (Horton et al. 10).
climates and tropical areas. Isolated from
Currently, chemical treatments such as
soil, the fungus may grow in areas with
Xylene are used to degrade PS (Garcia
temperatures ranging from 21 C to 36 C,
et al. 1818). However, frequent
and pH levels ranging from 2 to 7 (Lopez
exposures to this treatment can cause
De Leon et al. 1413). Moreover, a report
several health hazards ranging from mild
by Khan et al. states that A. tubingensis
symptoms to severe health conditions
can thrive on PU, making it its sole
that may even lead to death (Rajan et al.
source of carbon; yet, mainly dependent
1-2). On the other hand, degradation
on the medium's environment (11). The
studies utilizing microorganisms are on
fungus A. tubingensis also secrete
the rise, recent works have established
plastic-degrading enzymes such as
the use of thermal degradation
lipase and esterase (Khan et al. 10) (Kim
et al. 6992). The enzymes secreted can
cause fragment degradation of the PS
and PU (Kim et al. 6992) (Khan et al. 10).
Though there are numerous studies
regarding fungal biodegradation of
polystyrene (Chaudhary and
Vijayakumar, 3 and Yanto et al. 2), the
ability of A. tubingensis in degrading PS
has not yet been reported.
Therefore, this study aims to assess
and explore the biodegradation capability
of Aspergillus tubingensis to polystyrene.
Specifically, characteristics of
Aspergillus tubingensis as an effective
biodegradator will be determined along
with its optimum ratio of the temperature
in degrees Celsius to the pH level of the
medium in which A. tubingensis will be
placed in. Tests will also be run to see
and compare the results from the
different set-ups in the study. The
cultured fungus with different ratios such
as 33:3, 33:7, 33:8, 35:3, 35:7, 35:8,
30:3, 30:7, and 30:8 will be characterized
in terms of surface topography
examination, phylogenetic analysis,
scanning electron microscopy (SEM),
Fourier Transform Infrared Spectroscopy
(FTIR) analysis for determining signs of
biodegradation, and through Gas
Permeation Chromatography to
determine the percentage of weight
difference.
Conducting this study will provide
more efficient and ecological ways on
how to degrade plastic specifically
polystyrene. This type of plastic is made
up by linking together large amounts of
styrene molecules which is considered
as carcinogen by the International
Agency on Research for Cancer (IARC).
Considering that this fungus, Aspergillus
tubingensis, has a potential in degrading
polystyrene because of its enzymatic
activity (Kim et al. 6992; Khan et al. 10;
Chaudhary and Vijayakumar 2), it will
reduce the impact not just to the people
and environment but also to other living
organisms since negative effects such
as, contamination of soil and
groundwater, and entering of toxins to
the food chain will be prevented.
LITERATURE REVIEW
 Biodegradation of Polystyrene
(PS) needs centuries to fully biodegrade
for it has a very slow progress in the
process of degrading naturally (Wikes
and Aistilde 1-2). At the same time, due
to a huge increase production of different
materials related to PS and also to its
improper disposal, the environment is
facing a big threat of plastic pollution
(Wikes and Aistilde 1-2; Devi et al. 1;
Amobonye et al. 3). Since harmful
chemical substances such as Xylene
have been used to degrade PS (Garcia
et al. 1818), current studies have been
conducted about biodegradation,
specifically fungal biodegradation, of the
said polymer (Chaudhary and
Vijayakumar 2). However, there is still the
need to explore other microbes that
might be an efficient variable for
biodegradation (Bahl et al. 4). Hence
further research should be done with
other species of fungi with biodegrading
capabilities.
I. FUNGAL BIODEGRADATION OF POLYMERS
Plastic polymers or commonly
known as plastics have been considered
a global environmental threat due to their
property of being non-biodegradable and
also for their continuous production,
usage, and improper disposal (Pathak
and Naveneet 1). Current methods in
degrading plastics have been proven to
be costly, unsustainable, and do have
negative effects on humans and the
environment (Amobonye et al. 3). On the
other hand, biodegradation using
microorganisms, such as fungi, has been
used for degrading polymers; the
microorganisms used in biodegrading
have many ways on how to degrade the
polymer, two of them are mineralization
due to colonization of the
microorganisms, and hydrolysis due to
the enzymatic activities of the
microorganisms; both are applicable on
Fungal Biodegradation (Bahl et al. 2).
In regards to biodegrading
through colonization on the polymer,
during the process of biodegradation,
colonization of the fungi will lead later on
to the reduction of molecular weight of
the plastic and first, the polymer is turned
into monomers, then the monomers will
be collapsed to carbon dioxide, water,
and methane known as mineralization
(Bahl et al. 2). Meanwhile, for the
degradation activity due to enzymatic
activities, fungi secrete plastic-degrading
enzymes, such as lipase and esterase,
which is then absorbed by the plastic
thus attacking on its polymer substrate Kim et al. 6992). Currently, there has
following hydrolytic division been no reported study about the
(Yogalakshmi and Singh 114; Kim et al. capabilities of the said fungi.
6992; Bahl et al. 2). During hydrolysis,
enzymes that are produced by the fungi
II. ASPERGILLUS TUBINGENSIS AND ITS
will then degrade the polymer into
BIODEGRADING PROPERTIES
smaller molecules; oligomers, dimers,
Aspergillus tubingensis has been
and to monomers (Bahl et al. 2).
recently discovered to degrade polyester
Recently, numerous studies have shown
polyurethane (Khan et al. 11). In addition
positive results of fungal degradation on
to this, it has been proven that external
different types of polymers such as low-
factors such as surfactants, pH,
density polyethylene (LDPE), high-
temperature, and carbon source
density polyethylene (HDPE),
availability can affect the colonization of
polyurethane (PU), polyolefins, polyvinyl
the said fungus (Khan et al. 8).
chloride, poly(vinyl alcohol), and
polyhydroxyalkanoates (Yogalakshmi &
Several studies record the
Singh 114; Khan et al 11; Devi et al. 8;
degrading ability of the genus Aspergillus
Zahra et al. 401). The genera Penicillium,
such as A. japonicus and A. niger on
Aspergillus, and Phanerochaete are the
polythene (Raaman et al. 316), phenol
most investigated fungi for plastic
biodegradation by A. fumigatus
degradation (Yogalakshmi and Singh
(Gerginova et al. 389), and HDPE
114).
biodegradation through A. flavus (Devi et
al.7). Moreover, a report by Kuzikova et
In connection to this study,
al. suggests that A. tubingensis has a
Aspergillus tubingensis, a specie from
possible biotechnological application in
the genera Aspergillus, will be used to
wastewater treatment as it can degrade
degrade PS under different
alkylphenol that resulted in nontoxic
environments. It would colonize on the
products (14).
PS and secrete it's plastic-degrading
enzymes that will help in the process of
Concerning our study, A.
degrading the polymer (Khan et al. 11;
tubingensis was found to secrete
enzymes, mainly esterase and lipase that IV. APPLICATION OF ASPERGILLUS
aids in the biodegradation of polyester TUBINGENSIS IN THE FUNGAL

polyurethane (Khan et al. 6-10). These BIODEGRADATION OF POLYSTYRENE

enzymes are involved in a series of Polystyrene atoms are strongly
reactions that includes the bonded to one another making it very
depolymerization of the polymer, turning stable and extremely hard to degrade
it into styrene (Amobonye et al.16). after disposal (Ho et al. 2). However,
fungi have a great potential for degrading
petroleum-based plastics due to their
III. ISOLATION OF ASPERGILLUS
characteristics such as their enzymatic
TUBINGENSIS FROM WASTE SITES
system with the capacity for pollutant
It is usually important to isolate
detoxification and unspecificity of
and culture fungi from its sources before
substrates. Their ability to produce
identifying and utilizing them for tests
hydrophobin for surface coating to attach
(Rand 6). It has been shown that shorter
hyphae to hydrophobic substrates,
periods of incubation are enough for the
makes it possible for them to degrade
isolation of most fungal pathogens,
polystyrene. (Sánchez 8)
however, it still depends on the type of
Although there is no study yet that
specimen and the organism being sought
proves Aspergillus tubingensis can
(Murray 213).
degrade polystyrene, it is discovered that
A. tubingensis is capable of degrading
To isolate A. tubingensis, it is
polyester polyurethane (PU). The first
needed to collect soil samples from the
indication in PU degradation is the
waste sites first. As per the study of Khan
attachment or adhesion of fungal spores
et al., plastic films are buried at least 4
to the surface of PU showing a
inches vertically in the soil and are then
hydrophobic interaction is responsible for
sterilized after a one-month time interval.
the adhesion of microorganisms to the
It was then re-cultured at a separate plate
surface of buried PU which could be
and will then be incubated at a
influenced by environmental conditions.
temperature of 37°C for 20 days. The
Second indication is the growth of
fungi which can colonize the film surface
mycelia on the surface of PU and
is then observed and identified (2).
secretion of different types of enzymes Commonly used methods in
allowing the PU to degrade and lastly the testing the biodegrading capability of a
most important step in biodegradation is microorganism are scanning electron
the amount of enzymes produced by the microscopy (SEM) and Fourier
microbe that are responsible for the Transform Infrared Spectroscopy (FTIR)
degradation of PU films depending on Analysis. Microscopic techniques such
different as SEM are often used in assessing
factors such as pH of the medium, visible changes on the surface structure
carbon source availability, nature of of the degraded polymer and the
substrate, the temperature of the morphological and topographical
environment, humidity etc. (Khan et al. 8- characteristics of the surface (Rydz et al.
10). 1). Other techniques might also be used,
With regards to polystyrene, just namely, photonic microscopy,
like other microorganisms, PS can be polarization microscopy, electronic
used as a carbon source. However, its microscopy, atomic force microscopy
high molecular weight limits its use as a (AFM), and scanning force microscopy
substrate and for enzymatic reactions to (Ho et al. 7).
take place (Ho et al. 2). A study by Tahir Moreover, the changes in the
et al. recorded the presence of the chemical properties of the polymer
enzyme esterase in Lentinus tigrinus and including the formation or disappearance
is able to effectively degrade polystyrene of functional groups is determined
(1). Therefore, polymers such as through Fourier transform infrared
polystyrene, have different ways in which spectroscopy (FTIR) (Alshehrei 9). The
they’ll biodegrade and that its abiotic and appearance or disappearance of the
biotic environment also affects the rate of bands in certain functional groups,
degradation. indicates a sign of biodegradation (Khan
. et al. 4).
Weight loss percentage will be
determined through Gel Permeation
Chromatography (GPC). A study
V. TESTING THE BIODEGRADING
conducted by Fu et al. utilized GPC in
CAPABILITY OF ASPERGILLUS TUBINGENSIS
characterizing molecular weight and
element state, in which they can analyze
the molecular structure of their
component. Quantitative data will be
obtained from this test. The
polydispersity index of the samples will
be calculated using the molecular weight
data (Chaudhary and Vijayakumar 4).

SYNTHESIS
This study will be using
Aspergillus tubingensis obtained from
waste sites in order to degrade
polystyrene. Polystyrene, a carbon Fungus isolated from waste soils
source of microorganisms and A. will be used in this study as the
tubingensis a fungus able to secrete independent variable, along with the pH
enzymes that are able to degrade of the media and the set temperature in
polystyrene; its promising degradation incubation. This variable will determine
capability of makes it capable and and is expected to significantly affect the
applicable for degradation of other fungal biodegradation of polystyrene
polymers such as polystyrene. films.To perform this study, a total of 11
Environmental factors of the media are set-ups will be made. With the first set-up
also said to affect the rate of degradation. being the positive control, which is
Then, it will be subjected to different xylene. Second will be the negative
characterization and evaluation tests to control which is set according to the
determine if A. tubingensis is a possible normal conditions of the Sabouraud
microorganism that is able to degrade Dextrose Agar plate without any
polystyrene. modifications. While the remaining set-
ups are ratios of temperature in degrees
MATERIALS AND METHODS
Celsius and the pH level. These set-ups
will undergo characterization tests,
namely, surface topography examination
and phylogenetic analysis. As well as,
evaluation tests, specifically, Scanning
Electron Microscopy (SEM), Fourier
Transform Infrared Spectroscopy (FTIR)
analysis and Gas Permeation
Chromatography. Both qualitative and
quantitative data will be obtained and will
undergo statistical analysis through the
use of two-way ANOVA.
1. GATHERING OF MATERIALS
The main materials in this study
are soil samples from a waste site and
polystyrene films. The two (2) kg of soil
samples will be acquired from Rizal
Provincial Sanitary Landfill; on the other
hand, 500 g of polystyrene films along
with the 2L of deionized water will be
bought from Bambang, Sta. Cruz,
Manila. Two (2) pcs of polyethylene bags
will be purchased from Donewell plastics.
The 6L Distilled water will be obtained
from Puregold, Libertad. Lastly, 100 mL
HCl 1M, 100 mL NaOH 1M, 500 g
Sabouraud Dextrose Agar (SDA) Powder
and 15 mL of Lactophenol Cotton Blue
Stain will be provided by Adamson
University’s Chemistry Laboratory.
2. ISOLATION AND BIODEGRADATION OF
POLYSTYRENE
2.1. Isolation of fungus
In isolating the fungus, vertically
cut polystyrene films will be buried 4-6
inches below the obtained waste soil.
After a one-month time interval the
polystyrene films inoculated with the
fungus will be washed gently with distilled
water and will be then shifted to an SDA
plate in which it will be incubated at a
temperature of 37°C for one (1) week and
then re-cultured on a separate SDA plate
at
37°C for another 20 days (Khan 2).
2.2. Preparation of Culture Media
A modified SDA broth will be
made with regards to the pH level in
which the fungus will be placed in terms
of: 3, 7, and 8. Hydrochloric acid and
sodium hydroxide will be used to modify
the pH levels of the SDA plate.
2.3. Incubation of SDA petri plate 3. EVALUATION OF THE BIODEGRADABILITY
Streak plate method will be used OF ASPERGILLUS TUBINGENSIS

in obtaining the cultures with the

polystyrene film on it. It will be incubated 3.1. Characterization of the Fungal
at set temperatures in degrees Celsius, Strain
namely 30, 33, and 35 for four days. It will The cultured SDA plate in which
be then shifted to a Mineral Salt Medium the fungus is inoculated will undergo
(MSM) plate at 37°C for 24-48 hours. characterization tests such as Surface
Topography Examination and
Phylogenetic Analysis.
Table 1. Set conditions for each set-up

Set-ups Temperature pH level

3.2. Evaluation of the degrading ability
in degrees
Celsius of A. tubingensis
On the other hand, evaluation
A (positive ----- -----
tests for the degrading ability of A.
control)
tubingensis will be conducted through
B (negative 37 5.6
Scanning Electron Microscopy (SEM),
control)
Fourier Transform Infrared Spectroscopy
C 30 3 (FTIR) Analysis, and Gas Permeation

D 30 3 Chromatography.

E 30 3

F 33 7

G 33 7

H 33 7

I 35 8

J 35 8

K 35 8