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An overview on biodegradation of polystyrene and

modified polystyrene: the microbial approach

Ba Thanh Ho, Timothy K. Roberts & Steven Lucas

To cite this article: Ba Thanh Ho, Timothy K. Roberts & Steven Lucas (2018) An overview on
biodegradation of polystyrene and modified polystyrene: the microbial approach, Critical Reviews in
Biotechnology, 38:2, 308-320, DOI: 10.1080/07388551.2017.1355293

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 CRITICAL REVIEWS IN BIOTECHNOLOGY, 2018
VOL. 38, NO. 2, 308–320
https://doi.org/10.1080/07388551.2017.1355293

REVIEW ARTICLE

An overview on biodegradation of polystyrene and modified polystyrene:

the microbial approach
Ba Thanh Hoa, Timothy K. Robertsb and Steven Lucasb
a
Faculty of Environment and Natural Resources, Nong Lam University, Ho Chi Minh City, Vietnam; bThe Tom Farrell Institute for The
Environment, University of Newcastle, Newcastle, NSW, Australia

ABSTRACT ARTICLE HISTORY

Polystyrene is a widely used plastic in many aspects of human life and in industries due to its Received 3 May 2016
useful characteristics of low cost, light weight, ease of manufacture, versatility, thermal efficiency, Revised 20 March 2017
durability, and moisture resistance. However, polystyrene is very stable and extremely hard to Accepted 28 May 2017
degrade in the environment after disposal. Polystyrene can be used as a carbon source for micro-
organisms similar to many other hydrocarbons. The ability of microorganisms to use polystyrene KEYWORDS
as a carbon source has been recently established. However, the high molecular weight of poly- Polystyrene; biodegradation;
Downloaded by [49.180.98.150] at 02:21 27 December 2017

styrene limits its use as a substrate for enzymatic reactions to take place. In this paper, we review microbial
studies on biodegradation of polystyrene to give an overview and direction for future studies.

Introduction United States has risen to 28% in 2010 from around

With developments in science and technology, and the
increase in the global population, the demand for plas-
tics is ever increasing. Plastic materials have been
widely used in every aspect of human life and industries
such as packaging, building, furniture, housewares, elec-
trical, electronic, transport, and agriculture. The global
plastics production is about 322 million tons per year
[1]. Plastics are estimated to make up approximately
20%–30% volume of municipal solid waste in landfill
sites such as in the United States, Germany, Australia
[2,3].
Like other plastics, polystyrene (PS) is widely used
because of its good mechanical properties and rela-
tively low cost. PS is widely used in construction materi-
als (insulation), packaging foam, food containers,
disposable cups, plates, cutleries, cassette boxes, and
compact disks. There are about 21 million tons of PS
produced in the world in 2013 [4].
As a result of such wide use, plastics including PS
have accumulated in the environment, causing environ-
mental pollution, human health problems, and ecosys-
tem changes due to their toxicity and recalcitrant
compounds. PS materials can be recycled; however,
most PS foam ends in landfill. Since PS foam is light-
weight and bulky, transportation costs are a major com-
ponent of its recycling. Recycling rate for PS foam in the
20% in 2008 [5].
In order to reduce environmental pollution caused
by PS, some solutions have been applied such as reduc-
ing the use of PS products and using modified PS mate-
rials that can be biodegraded in the environment. PS
foam has been banned from sale and use in some pla-
ces such as San Francisco, Washington DC, Paris,
Toronto, and New York because of the huge problem it
causes in waterways [6,7]. There have been some stud-
ies to find microbes that can digest PS in the environ-
ment such as soil, landfill, and activated sludge.
This paper will review studies on biodegradation of
PS and the analytical procedures for monitoring
biodegradation.
PS and its applications
PS is a synthetic aromatic polymer with high molecular
weight (formula (C8H8)n) made from the monomer styr-
ene (Figure 1). PS can be solid or foamed while the
monomer styrene is liquid. General purpose polystyrene
(GPPS) is clear, hard, and rather brittle. It is an inexpen-
sive resin per unit weight. PS is a rather poor barrier to
oxygen and water vapor and has a relatively low melt-
ing point.
PS has been mainly used in four types of product:
GPPS, high impact polystyrene (HIPS), PS foam, and

CONTACT Ba Thanh Ho thanhbamt@hcmuaf.edu.vn Faculty of Environment and Natural Resources, Nong Lam University, Ho Chi Minh City,
Vietnam
ß 2017 Informa UK Limited, trading as Taylor & Francis Group

Figure 1. Chemical formula of polystyrene.
O
1 2
SMO SOI
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Figure 2. Degradation pathway for styrene [79,80]. 1-styrene, 2-styrene oxide, 3-phenyl acetaldehyde, 4-phenylacetic acid, 5-phe-
nylacetyl coenzyme A SMO: styrene monooxygenase, SOI: styrene oxide isomerase, PAALDH: phenylacetaldehyde dehydrogenase,
PACoA ligase: phenylacetyl coenzyme A ligase.
Table 1. Summary of polystyrene application [8].
Types of polystyrene
General purpose polystyrene (GPPS)/oriented polystyrene (OPS) Produce baskets, pie containers, cookie trays, deli hinged take-out containers, bakery
High impact polystyrene (HIPS)
Polystyrene foam
Expanded polystyrene (EPS) foam
expanded polystyrene (EPS) foam. Their applications are
summarized in Table 1.
Why is PS so hard to degrade in the
environment?
PS is a durable thermoplastic that is generally believed
to be nonbiodegradable. Actually, biodegradation of PS
does occur but at a very slow rate in natural environ-
ments and therefore PS persists for long periods of time
as solid waste. Kaplan et al. stated that in cultivated
soils containing a wide range of fungi, microbes, and
invertebrates, degradation of PS is less than 1% after 90
days with no significant increase in degradation rate
after this time [9]. However, Otake et al. reported that a
sheet of PS buried in soil for 32 years had no sign of
degradation [10].
The hydrophobic character of most thermoplastics
makes them resistant to hydrolysis. The molecular
CRITICAL REVIEWS IN BIOTECHNOLOGY 309
O O O
H OH O SCoA
3 4 5 TCA cycle
PAALDH PACoA
Ligase
Application
cake domes, cutlery (disposable service ware) plates, bowls, platters (disposable service
ware), cups (disposable service ware).
Yogurt containers, creamers, cold drink cups, lids, single-service condiment containers,
plates (single-service and reusable), stirrers
Meat/poultry trays–(prepackaged and store packaged), cold drink cups, hot drink cups,
single-service plates/bowls, hinged take-out containers, school lunch and other food
service trays, other foam sheet (i.e. egg cartons and fruit and vegetable trays)
Cups and containers, coolers (grape and fish boxes), insulated panel systems
composition of plastics affects the hydrophobicity of
the polymer surface, which in turn affects how easily
microorganisms can attach themselves [11].
Thermoplastics have high molecular weights and
their general lack of water solubility prevents microor-
ganisms from transporting them into their cells for
metabolism [12]. Biological processes can start outside
the microbial cell by the secretion of extracellular
enzymes. However, these enzymes are too large to
penetrate deep into the polymer, so they act on the
surface by cleaving the polymer chain via hydrolytic
mechanisms [13]. Biological processes are further
enhanced by the formation of functional groups in the
polymer chain [14,15].
The use of antioxidants, flame retardants, processing
lubricants, and stabilizers in the manufacturing process
also keeps thermoplastics from oxidation and biodeg-
radation, contributing to the quality, life and usefulness
of the resin. Bisphenol A, for example, is widely used as
 310 B. T. HO ET AL.

Table 2. Typical commercial additives using for polystyrene material.

Additives Dose Functions References
2-(20 -hydroxy-50 -methylphenyl)benzotriazole (Tinuvin P) 0.2%–0.3% UV stabilizer [18]
Acrawax 200 ppm Processing lubricant [18]
Alicyclic bromine 4% Antioxidant [19]
bis (2, 2, 6, 6-tetramethyl-4-piperidyl)sebacate (Tinuvin 770) 0.2%–0.5% UV stabilizer [18]
Decabromodiphenyl ethane (S-8010) 1%–12% Flame retardant [19]
Decabromodiphenyl oxide 1%–12% Flame retardant [19,20]
Dibromoethyldibromocyclohexane (Saytex BCL-462) <1% Flame retardant [19]
Ethylene bistetrabromonorbornane dicarboximide (Saytex BT-93) 13% UV resistant [18,19]
Hexabromocyclododecane <1% Flame retardant [19,20]
Mineral oil <3% Processing lubricant [18]
Octadecyl 3,5-di-tert-butyl-4-hydroxycinnamate (Irganox 1076) 0.1%–0.2% Antioxidant [18]
Pentabromochlorocyclohexane <1% Flame retardant [19]
Stearic acid 1000–2500 ppm Processing lubricant [18]
Tetrabromobisphenol A 10%–20% Flame retardant [20]
Tris Nonyl Phenyl Phosphite (Wytox) 0.2% Antioxidant [18]
Zinc stearate 1000–1800 ppm Processing lubricant [18]

an antioxidant and stabilizing material for polymer and PS/PLA/organically modified montmorillonite
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products [16]. Other additives include antimicrobial
agents (used in food packaging to preserve shelf life),
and dyes and pigments (often used to improve esthetic
properties of the material) [17]. Some typical, commer-
cial additives are summarized in Table 2. Recently, silver
nanoparticles have been utilized as an antimicrobial
agent in plastic food packaging materials [21].
Nanosilver damages bacterial cells by weakening cell
membranes and destroying enzymes that transport cell
nutrients, therefore prolonging the shelf life of food
stuffs [22]. Stabilizer technology has the aim to extend
the service life of plastics used in outdoor environ-
ments, especially in regions of the world that have high
temperatures and long summer seasons [23]. UV stabil-
izers, or light absorbers, for example, act to protect the
plastic against UV or sunlight damage such as discolor-
ation, cracking, brittleness, or other loss of desirable
physical properties. Some typical UV stabilizers are ben-
zophenones, hindered amines, and benzotriazole.
Studies on biodegradation of PS and modified
PS
Biodegradability of PS materials has been investigated
in previous studies. PS materials used in these studies
can be pure PS or modified PS, PS blended with other
polymers. These researches are summarized in Table 3.
The biodegradation is complex and not fully under-
stood. Research to clarify the biodegradability of PS
material has two different strategies. In the first
approach, degradation studies have been carried out
using pure microbial strains able to degrade PS materials.
Shimpi et al. [32] recently reported the biodegradation of
modified PS by using a pure strain of Pseudomonas aeru-
ginosa. The modified PSs were PS/polylactic acid (PLA),
(OMMT) composites (a very soft phyllosilicate group of
minerals that typically form in microscopic crystals,
forming clays). Maximum degradation efficiency was at
10% PS/PLA and 2 parts per hundred parts of resin PS/
PLA/OMMT nanocomposite. Pure strains of Rhodococcus
ruber were used to degrade three forms of PS including
pure standard PS flakes, PS powder, and ELISA 96-well
microtiter plates manufactured from pure PS [38]. Most
of the bacterial cells adhered to the PS surface within
few hours, forming a biofilm and a small reduction in
the PS weight (0.8% of gravimetric weight loss) was
found after 8 weeks incubation. Ward et al. used
Pseudomonas putida CA-3 (NCIMB 41162) to convert
styrene oil after pyrolysis to polyhydroxyalkanoate
(PHA) as the sole source of carbon and energy [39]. One
gram of styrene oil was converted to 62.5 mg of PHA
and 250 mg of bacterial biomass in shake flasks. Motta
et al. used Curvularia species to investigate degradation
of atactic PS [40]. The results showed hyphae adhering
to and penetrating the polymeric surface and forming
spores in all the treated samples after 9 weeks.
Therefore, these data showed that biodegradation of PS
material with selected microbial strains might become a
viable solution.
The great advantage of this approach, using pure
strains of microorganism, is that it is a convenient way
to investigate metabolic pathways and to evaluate the
effect of different environmental conditions on the deg-
radation. However, a disadvantage of this approach is
that it ignores the possibility that biodegradability of PS
materials can be the result of a cooperative process of
microbial consortium which acts like a food chains or a
food web. In the first approach, selected microbial
strains are often grown in optimal environmental condi-
tions. Moreover, biodegradation rate might be affected

Table 3. Summary of studies on biodegradability of polystyrene and modified polystyrene.
Materials
Blank polystyrene (PS), Blend of PS,
and starch-irradiated PS /10 wt%
starch
High impact polystyrene (HIPS)
with decabromodiphenyl oxide
and antimony trioxide as sole
carbon source
High impact polystyrene (HIPS)
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PS
(disposable plate)
Styrofoam containing PS >98%
with Mn of 40,430 and Mw of
124,200
PS films 0.02 mm thick were syn-
thesized in lab condition
PS -graft-starch copolymers
PS/CaSO4 nanocomposites
Methods
Samples were buried for 6 months in soil
including agricultural and desert soils.
Fourier transform infrared (FTIR), swelling
behavior, mechanical properties, thermogra-
vimetric analysis (TGA), and scanning elec-
tron microscopy (SEM) were used to
measure biodegradability.
Enrichment medium containing the test sam-
ples was used to isolate microbial cultures.
16 S rRNA sequencing was used to identify
isolated bacteria. FTIR, TGA, nuclear mag-
netic resonance (NMR), and SEM were used
to measure biodegradability.
Bacillus spp. and Pseudomonas spp. were iso-
lated from soil with HIPS as a sole carbon
and identified by 16SrRNA sequencing.
These techniques of high-pressure liquid
chromatography (HPLC), NMR, FTIR, TGA,
and weight loss analysis were used to con-
firm biodegradation.
PS-degrading microorganisms were isolated
from five different soil samples collected at
five different locations.
The degradation rate of five strains of micro-
organism including Pseudomonas aeruginosa,
Bacillus subtilis, Staphylococcus aureus,
Streptococcus pyogenes, and Aspergillus niger
on plastic samples was observed separately
by calculating percentage weight loss separ-
ately by calculating percentage weight loss.
Mealworms (the larvae of Tenebrio molitor
Linnaeus) were fed with Styrofoam as a sole
diet.
Gel permeation chromatography (GPC), solid-
state 13C cross-polarization/magic angle spin-
ning NMR (CP/MAS NMR) spectroscopy, and
thermogravimetric Fourier transform infrared
(TG  FTIR) spectroscopy were used to ana-
lyze fecula egested from Styrofoam-feeding
larvae.
Mealworms (the larvae of Tenebrio molitor
Linnaeus) were fed with the test samples.
Exiguobacterium sp. strain YT2 from guts of the
mealworms that ate the PS film was isolated
and identified by 16 S rDNA sequencing.
PS biodegradation by the isolated was charac-
terized by weight loss and molecular weight
shifts after 60 days of incubation. Water con-
tact angle, gas chromatography/mass spec-
trometry (GC/MS), X-ray photoelectron
spectroscopy, and SEM were used to confirm
the biodegradation.
The disc-shaped samples with radius of 3 cm
and thickness of 0.5 cm were buried at 6 cm
depth in the vessel volume of 1 L in three
different types of commercially available
soils for 6 months.
Monitor mass degree. The mass of samples was
measured every 15 days.
FTIR spectroscopy and SEM were used as meth-
ods for characterization of grafted copoly-
mers of PS and starch.
50 mg of PS/CaSO4 nanocomposites films in
50 ml of mineral salts basal were inoculated
with 2 ml of a Gram-positive bacterium,
Rhodococcus pyridinivorans NT2 (1.5  106
CFU/ml).
CRITICAL REVIEWS IN BIOTECHNOLOGY 311
Results References
The PS is generally found to be more resistant [24]
to the biodegradation in the two types of
soil. The degradation of irradiated PS/10 wt%
starch bio-blend at a dose of 5 kGy in agri-
cultural soil is slightly higher than that in
desert soil. Irradiated PSty/10 wt% starch
bio-blend at a dose of 5 kGy can be used as
a potential candidate for packaging material
due to the improvement in its mechanical
and thermal properties.
Four bacterial strains were isolated and identi- [25]
fied as Enterobacter sp., Citrobacter sedlakii,
Alcaligenes sp. and Brevundimonas diminuta.
12.4% (w/w) of the test sample lost within
30 days using an isolate, Enterobacter sp. PS
intermediates were detected in the degrad-
ation medium.
Degradation with Bacillus spp. showed a weight [26]
loss of 23% (w/w) of HIPS film in 30 days.
Reduction in turbidity in 4 days incubation
of HIPS emulsion with Bacillus spp. and
Pseudomonas spp. was 94% and 97%,
respectively.
The maximum percentage of biodegradation of [27]
PS was by Gram-negative cocci (in single)
isolated from garbage soil after four months
of incubation period.
The percentage loss in weight of PS was high-
est by Bacillus subtilis
Within a 16 day test period: 47.7% of the [28]
ingested Styrofoam carbon was converted
into CO2.
49.2% was egested as fecula with a limited
fraction incorporated into biomass.
The 13C-labeled PS was mineralized to 13CO2
and incorporated into lipids.
Exiguobacterium sp. could form biofilm on PS [28]
film over 28 days of incubation and made
obvious pits and cavities (0.2  0.3 mm in
width) on PS film surfaces. This strain was
able to degrade 7.4 ± 0.4% of the PS pieces
(2500 mg/L) over 60 days of incubation in
suspension culture.
During biodegradation process in PS-graft- [29]
starch copolymers, only starch was
degraded, while PS remains intact.
The highest degradation rate was achieved in
soil for cactus growing (81.30%).
There is a steep increase in protein content [30]
over 3 weeks of incubation.
A linear positive correlation was observed
between the biomass attached on the poly-
mer surfaces and weight loss over the whole
incubation period studied.
(continued)
 312 B. T. HO ET AL.
Table 3. Continued
Materials
Grafted copolymers of corn starch
and PS and corn starch and pol-
y(methacrylic acid)
PS:PLA and PS:PLA:organically
modified montmorillonite
(OMMT) composites.
Loose-fill foams contain corn starch
and PS at ratios of 70:30 and
80:20
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PS and expanded polystyrene (EPS)
solution (2%) in chloroform was
casted on petri plates to get
thin films (0.3–0.5 mm)
PS /cornstarch blends (films were
2 mm in thickness)
PS and TPS were mixed in different
ratios 0.9:0.1, 0.7:0.3, 0.5:0.5,
and 0.3:0.7 TPS was obtained by
mixing starch powder, water,
and glycerol or buriti oil in
50:15:35 (mass/vol/vol) ratios
Atactic PS Homopolymer samples
(Mw) of 270,000 g mol-1 (with-
out prooxidant additive) and
286,000 g mol-1 (with prooxi-
dant additive with a cobalt-
based salt additive; and with a
manganese-based salt additive.)
Three forms of PS were used: pure
standard PS flakes; PS powder;
ELISA 96-well microtiter plates
manufactured from pure PS.
Methods
The biodegradation was assessed by the quanti-
tative estimation of bacterial biomass of bio-
film, weight loss study, FTIR, and Raman
spectroscopy, GPC, contact angle measure-
ments, GC-MS analysis, and CO2 release.
The synthesized copolymers and products of
degradation were characterized by FTIR
spectroscopy and SEM. Biodegradation was
monitored by mass decrease and the num-
ber of microorganisms by the Koch method.
Confirmation of surface modification using FTIR.
Put these polymers in broth medium containing
pure Pseudomonas aeruginosa in shaking
incubator in 28 days at room temperature.
Determination of biomass, protein, and degrad-
ation. SEM was used to view film surfaces.
The structures and biodegradability of loose-fill
foams were evaluated using a laboratory
composting system, five 6 L chambers.
Biodegradability was expressed as the percent-
age of CO2 in the exhaust gas eluted from
the individual chambers. The concentrations
of CO2 in the exhaust gas from the cham-
bers were recorded using a gas chromato-
graph and the net CO2 production (from the
foams) was calculated by subtracting the
CO2 production in the control (chamber)
SEM was used to view the microstructures of
foams.
The films remained buried in garden soil for
eight months.
Bacteria were isolated and identified molecular
characterization.
FTIR spectroscopy was employed to study sur-
face changes of PS films.
Biodegradation products were analyzed by high
pressure liquid chromatography.
The samples were evaluated by soil burial test
under laboratory conditions for a period of
60 days.
Weight loss of the specimens with time was
used to evaluate degradation.
The morphology of the samples was observed
with a SEM.
PS/TPS blends were buried in a perforated box
to allow the samples to be attacked by the
microorganisms and moisture. The box was
buried at a depth of 7 ± 9 inches beneath
the soil surface.
Thermogravimetry was used to determine the
mass loss and decomposition temperature
(Td) of the blends.
The samples were subjected to degradation by
ultraviolet radiation and heat over three dif-
ferent time periods. The oxidized surface res-
idues detached from the samples were
incubated in a stabilized compost of urban
waste (58  C) or in an aqueous mineral
medium (25  C), the latter being inoculated
with urban waste compost and also with a
microbial consortium. Microbial growth was
assessed through optical density at 600 nm.
Respirometry tests in compost were measure
by CO2 value.
Cultivate strain of Rhodococcus ruber on the
experimental samples.
Estimating bacterial biomass of the biofilm.
Estimation of the protein content of biofilms
by a spectrophotometer at 280 nm.
Biofilm respiration measurements. SEM for bio-
film analysis.
Results References
The starch-graft-poly(methacrylic acid) copoly- [31]
mers had completely degraded after 21
days, the starch-graft- PS had partially
degraded (45.8%–93.1% mass loss) after 27
days.
All composition supported to the degradation [32]
nature properly.
The bacterial growth and extracellular protein
concentration varies with various compos-
ition. 10% PS: PLA and 2 phr (parts per hun-
dred parts of resin) PS: PLA: OMMT
nanocomposite showed maximum degrad-
ation efficiency.
The CO2 generation peaked after about 15 days [33]
of composting, and then decreased. The rate
and amount of CO2 eluted depended on the
starch content in the foams.
At the end of the composting tests, the remain-
ing foam material had fibrous and crumbly
textures, presumably consisting primarily of
PS. FTIR and NMR spectra of the foams,
taken after 39 days of composting, did not
reveal the spectral features of starch,
thereby confirming the decomposition of the
starch.
The bacterial isolated strains were identified as [34]
Microbacterium sp. NA23, Paenibacillus urina-
lis NA26, Bacillus sp. NB6, Pseudomonas aeru-
ginosa NB26. They were able to extract
some carbon from the complex molecules of
PS but the process is very slow and causes
no significant chemical changes on the
surface.
The microbial activity inside the specimens was [35]
accelerated in the first 15 days of evaluation.
There were significant differences in film images
after 30 days of incubation in soil.
Fractured surfaces covered with a heteroge-
neous microorganism community.
After 6 months, PS/TPS blends with buriti oil [36]
presented only one thermal degradation
stage with a significant increase in mass
loss.
These samples underwent biodegradation and [37]
gave mineralization values of 2%–5% over
2–3 months of incubation in compost and
perlite or in mineral aqueous medium.
Biodegradation of the residues from the
samples not containing prooxidant additives
was also observed, but at levels which were
lower than those obtained for oxo-bio-
degradable samples.
This study shows the colonization, biofilm for- [38]
mation and, presumably, partial biodegrad-
ation of PS by R. ruber (C208).
Prolonged incubation (of up to 8 weeks) of
C208 with PS resulted in a dense biofilm on
the PS surface which may have led to a par-
tial degradation (about 0.8% weight loss) of
(continued)
 CRITICAL REVIEWS IN BIOTECHNOLOGY 313

Table 3. Continued
Materials
PS
Three synthetic 14C-labeled poly-
mers: poly (methyl methacryl-
ate), phenol formaldehyde, and
PS.
Methods
Pyrolysis of PS to styrene oil, followed by the
bacterial conversion of the styrene oil to pol-
yhydroxyalkanoate (PHA) by Pseudomonas
putida CA-3 (NCIMB 41162) as the sole
source of carbon and energy.
Radioactivity was measured in a Beckman LS
100C liquid scintillation counter for baseline
rates of 14CO2 release.
Results References
the polymer the formation of biofilms on
hydrophobic polymers, such as PS, may be
promoted by carbon starvation.
Styrene oil (1 g) was converted to 62.5 mg of [39]
PHA and 250 mg of bacterial biomass in
shake flasks.
All three polymers are highly recalcitrant to [9]
biological decay. The addition of cellulose
and minerals failed to increase decompos-
ition rates significantly.

when microbes live in the environment without carbon
starvation. In an environment where PS materials are as
a sole carbon source, selected microbial strains can use
this carbon source for their growth. However, if these
strains live in an environment having many carbon
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supercritical CO2 from the shell and the pulp of ripe
fruits. The obtained results showed that only one ther-
mal degradation stage with a significant increase in
mass loss on PS/TPS blends with buriti oil. The degrad-
ability characteristics correlated with the amount of

sources including PS materials, the rate of PS biodeg-
radation may be significantly decreased. Therefore, a
second approach has been used to overcome this
disadvantage.
In the second approach, PS materials were placed in
different environmental conditions such as marine, soil,
water, sludge or compost. The purpose of this approach
was to find capacity of PS biodegradation and/or to
find microbes that digest PS. Nikolic et al. [29] buried
samples of PS-graft-starch copolymer at 6 cm depth in
1 L vessels containing three different types of commer-
cially available soils, for 6 months, in order to examine
the biodegradation rate. Biodegradation was monitored
by mass decrease, and the highest degradation rate
was achieved in soil with cactus growing (81.30%).
Pushpadass et al. [33] investigated biodegradation
of loose-fill foams that contain corn starch and PS at
ratios of 70:30 and 80:20. The samples were put in 6 L
chambers as a laboratory composting system.
Biodegradability was determined by the percentage of
CO2 in the exhaust gas eluted from individual cham-
bers. The CO2 generation peaked after about 15 days
composting, and subsequently decreased. The starch
content in the foams correlated with the rate and
amount of CO2 produced. Schlemmer et al. [36] used PS
and thermoplastic starch (TPS) which were mixed in dif-
ferent ratios of mass (0.9:0.1, 0.7:0.3, 0.5:0.5, and 0.3:0.7)
by solvent casting techniques and then buried in a per-
forated box to allow the samples to be attacked by
microorganisms and moisture. The box was buried at a
depth of 7–9 inches beneath the soil surface to be later
analyzed by thermogravimetry and solid state 13C NMR
spectroscopy. The TPS was obtained by mixing starch
powder, water, and glycerol or buriti oil in 50:15:35
(mass/vol/vol) ratios. Buriti oil was extracted with
starch. PS’s degradability can be improved when TPS
plasticized with buriti oil is added to it.
PS biodegradation in water was investigated by a
study of grafted copolymers of corn starch and PS in
model river water. Biodegradation was determined by
mass decrease and the number of microorganisms by
the Koch method. The obtained results showed that
biodegradation of copolymers advanced with time. The
starch-graft-PS had partially degraded (45.8%–93.1%
mass loss) after 27 days [31]. In an environment of acti-
vated sludge, Naz et al. [41] monitored the growth and
physiological activities of biofilm during succession on
PS (1 inch thickness in cubical pieces) under aerobic and
anaerobic conditions. After 9 weeks incubation with
activated sludge, forming biofilm, mainly Bacillus cereus,
was monitored by gravimetric weight analysis, spectro-
photometric absorbance, and scanning electron micros-
copy (SEM). Biofilm development proved to be involved
in the biochemical transformation of the PS medium as
indicated by Fourier transform infrared spectroscopy.
A similar study investigated the degradation of HPS
with starch (16wt%, 24wt%, or 32 wt%) and without
starch in concentrated activated sludge. The samples
remained for 12 weeks in the 3 L vessel containing acti-
vated sludge in aerobic conditions maintained by aer-
ation. The results showed that samples with starch had
biodegradation rates higher than the samples without
starch and the effectiveness of starch inclusion as a filler
accelerated the structural molecular changes [42].
Therefore, the approach of using real environments
such as soil, river water, and activated sludge for assess-
ment of biodegradation of PS material also yields inter-
esting results. In general, the biodegradation is often
low and the rate of the biodegradation much depends
on the additive such as starch. Another drawback of
 314 B. T. HO ET AL.
these studies is that microorganisms responsible are
not characterized. Moreover, it is hard to determine the
rate of PS biodegradation in these environments. Most
reported studies terminated at early stage of degrad-
ation. This leaves the vexing questions whether PS
materials are completely degraded in a real environ-
ment such as soil, landfill, and how long such complete
biodegradation will take.
To increase biodegradability of PS in the environ-
ment, besides modifying PS by grafting or blending
with copolymers, using the addition of prooxidants
such as metal salt (iron, cobalt, and manganese) was
also investigated. It is stated that trace amounts of met-
als such as Co, Mn, Fe, Cu, and Ni, considerably increase
the rate of oxidative degradation [43]. Prooxidants are
used to promote oxidative degradation of PS, in order
to break the molecules into smaller fragments contain-
ing hydrophilic oxygenated groups that can be biode-
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graded by microorganism in the environment [44].
Ojeda et al. [37] used Co and Mn based prooxidant
additives to increase biodegradability of foamed PS.
The results showed that the molar masses of the eroded
materials from the prooxidant activated samples were
significantly lower than the initial sample molar masses
without the prooxidant additive. These samples under-
went biodegradation and gave mineralization values of
2%–5% over 2–3 months of incubation in the compost
or in mineral aqueous medium. Biodegradation of the
residues from the samples containing prooxidant addi-
tives was higher than samples not containing prooxi-
dant additives.
Mechanism of PS biodegradation
Biodegradation of plastics is the process in which micro-
organisms (fungi, bacteria, and archaea) degrade them
by their extracellular or intracellular enzymes and use
the plastics as a substrate for growth [3,45,46]. PS bio-
degradation starts when microorganisms begin growing
on the surface of PS and secrete their enzymes to
degrade the polymer into smaller molecular fragments
called oligomer and maybe monomeric units. Styrene
itself is able to be used as a carbon source for growth
by some microorganisms. Rhodococcus ruber has been
shown to form biofilms on PS and partially degrade it
[38]. A biofilter consisting of Brevibacillus sp. has been
shown to remove 3 kg of styrene in a day. The biodeg-
radation rate depends on the thickness and the molecu-
lar weight of the plastic [47].
In fact, a large number of microorganisms can bring
about styrene biodegradation [48–55]. There are several
ways of styrene catabolism; however, a predominant
pathway involves the oxidation of styrene to
phenylacetate, which is then converted via the TCA
cycle. This pathway is shown in Figure 2.
Can we find microbes that can digest PS?
Although relatively few, biodegradation of PS has been
reported in some previous studies. In the literature, few
reports describe the microbial utilization of PS as a car-
bon source [9,38,40,56]. However, there are few reports
of microbes degrading PS in the real environment such
as landfill, soil, etc.
Oikawa et al. [57] isolated and identified
Pseudomonas sp. and Bacillus sp. for styrene degrad-
ation; also Xanthomonas sp. and Sphingobacterium sp.
for PS decomposition by 16 S ribosomal DNA analysis
from soil. Four microbial strains have been isolated
from garden soil after 8-month buried samples of PS
and EPS solution (2%) in chloroform. They were identi-
fied as Microbacterium sp. NA23, Paenibacillus urinalis
NA26, Bacillus sp. NB6, and Pseudomonas aeruginosa
NB26. They were able to extract some carbon from the
complex molecules of PS but the process was very slow
and caused no significant chemical changes on the sur-
face [34]. Biodegradation of PS involved Gram-positive
coccobacillus, Gram-negative cocci, Gram-negative rod-
shaped bacillus, Gram-positive cocci (in clusters) in
Garden soil, and Gram-negative cocci (in singles) in gar-
bage soil with weight loss up to 30% [27].
Microbial studies on biodegradation of PS materials
are summarized in Table 4. Although PS material can be
degraded by microbes, there have been few studies
reporting the identification of microorganisms or what
enzymes are used in the process of biodegradation.
Analytical procedures for monitoring PS
biodegradation
Visual observations
The visual observation to evaluate the macroscopic
changes on the surface structure of the biodegraded
polymer can be used for an initial estimation of biodeg-
radation. These changes include roughening or scab-
rous of the surface, creation of holes and cracks,
changes in color, formation of microbial colonies over
the surface, etc. Visual changes can be used as a first
indication of any microbial attack. Microbial colonies
can be seen by the naked eye and their quantity of
microorganisms on Petri dishes can be estimated by
several normalized tests [29,61,62]. However, to obtain
information about the degradation mechanism, more
sophisticated observations can be employed such as
SEM, photonic microscopy, polarization microscopy,
 CRITICAL REVIEWS IN BIOTECHNOLOGY 315

Table 4. Microbial studies on biodegradation of polystyrene (PS) materials.

Microorganism Results References
Basidiomycete, P.chrysosporium, Trametes versicolor, Styrene (1-phenylethene) graft copolymers of lignin to make testing sample. The [58]
and Pleurotus ostreatus fungi secreted lignin degrading oxidative enzymes and also degraded polystyrene
component of the copolymer. FTIR spectroscopy showed the loss of functional
groups after incubation with fungi. Nuclear magnetic resonance (NMR) spectra
also demonstrated reduced resonances of aromatic region.
Bacillus coagulans Graft copolymers of Cassava starch and PS made PS sheet. The destroyed areas of [59]
starch in composite PS sheets that made holes under SEM indicated that bac-
teria promote the biodegradation of PS plastics.
Serratia marcescens, To increase the rate of degradation of functionalized PS, maleic anhydride (14% [60]
Pseudomonas sp. by weight) was used to functionalize PS and anchored small quantity of differ-
Bacillus sp. ent monomeric sugars like glucose, lactose, or sucrose on it. Pure cultures of
these soil bacteria were used to study their growth pattern on these polymers.
Infrared (IR) spectroscopy analysis, weight loss data, and gel permeation chro-
matography data illustrated that polymers were degraded by the bacteria.
Pseudomonas sp. Pseudomonas sp. and Bacillus sp. can degrade styrene while Xanthomonas sp. and [57]
Bacillus sp. Sphingobacterium sp. can degrade PS. Bacillus sp. STR-Y-O strain decomposed
Xanthomonas sp. both styrene and PS. They were reduced 40% (9 g) and 56% (34 g) of initial
Sphingobacterium sp. concentrations (quantity), respectively, in 8 days by strain STR-Y-O.
Bacillus sp. STR-Y-O
Pseudomonas putida CA- Pyrolysis of PS to styrene oil, followed by the bacterial conversion of the styrene [39]
3 (NCIMB 41162) oil to polyhydroxyalkanoate (PHA) as the sole source of carbon and energy.
Styrene oil (1 g) was converted to 62.5 mg of PHA and 250 mg of bacterial biomass
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in shake flasks.
Rhodococcus ruber C208 The strain produced colonies on synthetic medium agar plates containing PS pow- [38]
der. 0.8% weight loss was observed within 8 weeks of treatment. The study
reported that R. ruber C208 was able of partial biodegradation, biofilm forma-
tion, and colonization of PS.
Curvularia The fungus colonized the oxidized PS in Sabouraud plates within 9 weeks. Hyphae [40]
adhering to and penetrating the polymer’s surface were observed in micro-
scopic examinations. The fungus utilized PS by co-metabolism.
Microbacterium sp. These bacteria were isolated from soil buried expanded PS films showing adher- [34]
NA23 ence and growth with the PS as a sole carbon source. Bacterial isolates NA26,
Paenibacillus urinalis NB6, NB26 were able to extract some carbon from the complex molecules of
NA26 PS but the process is very slow and causes no significant chemical changes on
Bacillus sp. NB6 the surface after 4 weeks of incubation with PS films.
Pseudomonas
aeruginosa NB26
Pseudomonas aeruginosa After 1 month at room temperature in nutrient broth and Bushnell Hass broth, [27]
Bacillus subtilis maximum weight loss of PS was by B. subtilis with 20% and 58.8%, respect-
Staphylococcus aureus ively. The percentage of weight loss was followed by S. aureus and S. pyogenes
Streptococcus pyogenes in Bushnell Hass broth with 37.5% and 11.1%, respectively. In nutrient broth, it
was 4.7% and 8.3% of weight loss done by S. aureus and S. pyogenes. The low-
est biodegradation rate causing by P. aeruginosa was 5% in nutrient broth and
0% in Bushnell Hass broth.
Exiguobacterium sp. This strain was isolated from the guts of the mealworms (the larvae of Tenebrio [28]
strain YT2 molitor Linnaeus). Over a 28 day incubation period on PS film, this strain could
form biofilm and made obvious pits and cavities (0.2–0.3 mm in width) on the
film surfaces associated with decreases in hydrophobicity and the formation of
C–O polar groups. Over a 60 day incubation period in a suspension culture of
the strain (108 cells/mL), PS pieces (2500 mg/L) was able to degrade
7.4% ± 0.4%, and molecular weight of the residual PS pieces was lower.
Bacillus spp. Degradation of HIPS film with Bacillus spp. showed a weight loss of 23% (w/w) in [26]
Pseudomonas spp. 30 days. Reduction in turbidity in four days incubation of HIPS emulsion with
Bacillus spp. and Pseudomonas spp.
Enterobacter sp. 12.4% (w/w) of the high impact polystyrene (HIPS) with decabromodiphenyl oxide [25]
Citrobacter sedlakii and antimony trioxide (synthesized in lab) as sole carbon source lost within
Alcaligenes sp. 30 days using an isolate, Enterobacter sp.
Brevundimonas diminuta
Strains TM1 and ZM1 were isolated from guts of TM1 and ZM1 could utilize PS as their carbon sources. [61]
Tenebrio molitor and Zophobas morio. TM1 and ZM1 with yeast extract had more degrading activities than which that
without yeast extract.

electronic microscopy, atomic force microscopy, and

scanning force microscopy. This method is simple,
cheap, and quick, but its results are qualitative because
the microbial colonies may be utilizing additives within
the polymer and not the polymer itself. Furthermore,
some structural differences may be due to physical/
chemical degradation rather than biodegradation [63].
Changes in mechanical properties and molar mass
Stress-strain tests (tensile strength, elongation at break,
modulus, and yield stress) are used to measure mechan-
ical changes during degradation. A disadvantage of
using mechanical properties, or any other property that
relies on the macromolecular nature of the substrate for
 316 B. T. HO ET AL.
the estimation of biodegradability, is that these proper-
ties can only address the early stages of the biodegrad-
ation process. Mechanical properties are usually used to
support the results of other tests.
A decrease in the average molecular weight, and the
broadening of the molecular weight distribution, pro-
vide initial evidence of the degradation of a polymer. A
change in molecular weight is a measure of bulk deteri-
oration, whereas biodegradation occurs initially on the
polymer’s surface. Therefore, no degradation may be
observed from molecular weight measurement even
when there has been a significant amount of weight
loss. However, the method can be used to indicate
where cleavage occurs in the polymer chain during bio-
degradation. Change in molecular weight is an easy
measurement of biodegradation, and when used with
other methods, it can be a useful indicator of the
degree of biodegradability [63]. The molecular weight
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of a biodegradable polymer can be measured by gel
permeation chromatography [28,30].
Weight loss measurements: determination of
residual polymer
Measurement of weight loss for the estimation of bio-
degradation is a frequently used method when the
polymer is exposed to a mixture of selected microbes
in culture media with the polymer as the sole source of
carbon. This method is standardized for in-field and
simulation biodegradability tests [64]. Problems can
arise with the correct cleaning of the sample or if the
material disintegrates excessively [65]. Measurement of
the weight loss of samples is not really representative
of material biodegradability, since this loss of weight
can be due to the vanishing of volatile and soluble
impurities [62]. Furthermore, the method only
addresses the early stages of the biodegradation pro-
cess, but gives no information on the extent of mineral-
ization [66].
Determination of biogas (CO2/CH4) evolution
Evolved CO2 or CH4 from biodegradation are used as
analytical parameters to determine the ultimate bio-
degradability of polymers. Under aerobic conditions,
microbes use oxygen to oxidize carbon compounds
and form CO2 as one of the major metabolic prod-
ucts. The amount of CO2 evolved is a measure for the
extent of biodegradation achieved. It is expressed as
a percentage of the theoretically expected value for
total carbon conversion to CO2. A value of 60% car-
bon conversion to CO2 achieved within 28 days for
the resins made from single polymer is generally
taken to indicate ready degradability. A respirometer
is used to measure CO2 production. CO2 evolution is
the method most often used to measure biodegrad-
ation [30,33].
A number of well-known tests have been standar-
dized for aerobic biodegradation, such as the modified
Sturm test and the laboratory-controlled composting
test discussed in [67–70]. Anaerobic tests generally fol-
low biodegradation by measuring the increase in pres-
sure and/or volume due to methane evolution, usually
in combination with gas chromatographic analysis of
the gas phase.
A number of well-known tests have been standar-
dized for anaerobic biodegradation of polymers, such
as the anaerobic sludge test and the anaerobic diges-
tion test as in ISO 13975:2012, ASTM 5526–12, and
ASTM D5511–12 [71–73].
Oxygen consumption
The amount of oxygen consumed during biodegrad-
ation is also an indicator of biodegradation. It can be
measured by comparing the biological oxygen demand
to the chemical oxygen demand. A respirometer is used
to measure the oxygen consumption [63]. The method
for the determination of oxygen consumption is based
on the so-called MITI (Ministry of International Trade
and Industry, Japan) test and is standardized in stander
organization as ISO 14851:1999, ISO 17556:2012, EN
14048:2002 [69,74,75].
Clear-zone formation
This is an agar plate test in which the polymer is dis-
persed as very fine particles within the synthetic
medium agar. This results in the agar having an opaque
appearance. After inoculation with microorganisms, the
formation of a clear halo around the colony. This indi-
cates that the microorganisms are at least able to
depolymerize the polymer, which is the first step of bio-
degradation. The method is usually applied to screen
microorganisms that can degrade a certain polymer,
but it can also be used to obtain semiquantitative
results by analyzing the growth of clear zone [62].
Radiolabelling
Radiolabelling is a nondestructive technique for meas-
uring biodegradation. Using this technique for poly-
meric materials to investigate biodegradability in
different microbial environments shows a high degree
of precision and consistency. In this technique, the
 CRITICAL REVIEWS IN BIOTECHNOLOGY 317

Table 5. Existing techniques for assessment of polystyrene biodegradation.

SN Changes in properties of polymer
1 Physical
Morphology-micro cracks
Density, contact angle, viscosity,
molecular weight distribution
Melting and glass transition
temperature
Residual polymer
2 Chemical properties
3 Molecular weight
4 CO2/CH4 evolution test
5 Others
Type of technique Reference
Filed emission scanning electron microscopy (FESEM), scanning [24,26,29,30,32,34,38,41];
electron microscopy (SEM) Kundu et al., 2014
High temperature gel permeation chromatography (HT-GPC), gel [28,30,77]
permeation chromatography (GPC)
Thermogravimetric analysis (TGA), differential scanning calorimetry [25,26,36]
(DSC)
Weight loss [24,26–29,31,32,38,41,77]
Fourier transformed infrared spectroscopy (FTIR) [25,26,28,29,33,34,77]
Gas chromatography (GC), nuclear magnetic resonance (NMR), gas [25,26,30,33,34,36,77]
chromatography-mass spectrometry (GC-MS), high-pressure
liquid chromatography (HPLC), cross-polarization/magic angle
spinning NMR (CP/MAS NMR) spectroscopy
Respirometer, GC [30,33]
Protein analysis, turbidity assay, plate count [30,31,32,38,41,61]

carbon in the polymer is radiolabeled with carbon iso- Another important area of future research is the
tope 14C and exposed to a microbial environment. identification of additives to enhance the rate of PS
It is possible to determine the duration of exposure by biodegradation. Theoretically, PS can be used as a
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comparing the amount of radioactive 14CO2 or 14CH4 to
the original radioactive of the label product. The
amount of 14CO2 evolved is measured using a scintilla-
tion counter. This method is not subject to interference
by biodegradable impurities or additives in the polymer.
The disadvantages of this method are the difficulty and
cost in preparing radiolabeled polymers (e.g. particular
laboratory, and specific equipment). Licensing (trained
technicians) and waste disposal of the radioactive sam-
ples may also be drawbacks [66,76].
There are several techniques available for checking
PS degradation, which are summarized in Table 5.
Conclusion and perspectives
The demand for plastic is still increasing and the
result is an ever increasing amount of plastic waste.
The scarce landfill space, hazards of waste inciner-
ation, and increasing costs of disposing solid wastes
have resulted in a search for new approaches for
waste management, particularly of plastic waste. Two
strategies of PS degradation, using pure microbial
strains as well as complex microbial communities,
have proved that PS can be biodegradable although
the biodegradation rate is slow. However, research
performed so far is mainly of a descriptive nature. It
is likely that future investigations will focus on the
isolation of microbes and enzymes able to oxidize
and break PS chains and use as a substrate to eluci-
date the mechanisms of PS degradation and the fate
of PS inside microorganisms. Especially, in the anaer-
obic conditions of landfill environment, biodegrad-
ation of PS is useful by saving landfill space as well
as energy recovery from the evolved biogas.
carbon source for microorganisms similar to many
other hydrocarbons. However, a high molecular
weight of PS limits its use as a substrate for
enzymatic reactions to take place. However, the
convenience and increasing demand for these poly-
mers together with the environmental protection
have made requirements to convert them into bio-
degradable materials in significantly shorter time. A
possible solution is to use an additive capable of
accelerating the reaction of the plastic with atmos-
pheric oxygen and incorporating oxygen atoms into
the carbon chains in the first stage of degradation.
The additives that accelerate this process and pro-
mote biodegradation are widely used such as metal
salts (iron, cobalt, and manganese) and copolymer
[37,78]. It has been known that additives are used
to facilitate biodegradation. However, the relation
and interaction between additives and rate of bio-
degradation has been not been clarified. It has sig-
nificance both from an environmental viewpoint of
plastic waste accumulation and conservation of
integrity for infrastructures incorporating this plastic.
It is expected that this review will encourage young
scientists to identify more microbial strains in nature
for the potential biodegradation of PS waste.
Acknowledgments
This work has been financed by the scholarship of University
of Newcastle and Ministry of Education and Training of
Vietnam.
Disclosure statement
No potential conflict of interest was reported by the authors.
 318 B. T. HO ET AL.
Funding
This work has been financed by the scholarship of University
of Newcastle (Grant No. 0650111006935X) and Ministry of
Education and Training of Vietnam (3133/QD-BGDDT).
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