Chemosphere 222 (2019) 527e533

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

Biodegradation of micro-polyethylene particles by bacterial

colonization of a mixed microbial consortium isolated from a landfill
site
Seon Yeong Park, Chang Gyun Kim*
Department of Environmental Engineering, INHA University, 100 Inha-ro, Michuhol-gu, Incheon 22212, Republic of Korea

h i g h l i g h t s

The feasibility of biodegradation PE microbeads has been studied in the laboratory.

PE degraders were selectively sorted from microbial isolates of municipal solid waste.

PE degraders biologically reduced the weight and particle size of PE microbeads.

The labile organic materials released from microplastic could be biologically broken down.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, we investigated the decomposition of micro-sized polyethylene (PE) by mesophilic mixed
Received 6 August 2018 bacterial culture isolates obtained from a municipal landfill sediment. Among these, Bacillus sp. and
Received in revised form Paenibacillus sp. were more specifically enriched in the non-carbonaceous nutrient medium (i.e., Basal
17 January 2019
medium) as they were the most dominant species when they were exposed to PE microplastics. They
Accepted 27 January 2019
Available online 28 January 2019
reduced the dry weight of particles (14.7% after 60 d) and the mean particle diameter (22.8% after 60 d;
obtained by field-emission scanning electron microscopy analysis). In the gas chromatography-mass
Handling Editor: Y Liu spectrometer analysis of biologically aged particles, the amount and types of organic contents eluted
from the PE microplastics were far lower in the early decomposition phase; however, they increased in
Keywords: the later phase. Thermal gravimetric analysis showed that the aged particles had higher thermal stability
Microplastics at temperatures greater than 570  C compared to the control, thereby suggesting that microplastics were
Polyethylene degraded by enzymatic chain scission, which could in turn be ascribed to the greater refractory fractions
Bacillus sp. of aged particles remaining at a high combustion temperature. It was further verified that PE particles
Paenibacillus sp.
could be biologically utilized as a sole carbon source and broken down during the test period.
Biodecomposition
© 2019 Elsevier Ltd. All rights reserved.

1. Introduction (i.e., secondary microplastics), or as microbeads (i.e., primary

In the last few years, research on plastics and microplastics as
emerging environmental pollutants has greatly increased, with
investigation of their source, distribution, fate, and environmental
effects (Duis and Coors, 2016; Horton et al., 2017; Li et al., 2016;
Pinto et al., 2016). Microplastics are commonly defined as syn-
thetic organic polymers smaller than 5 mm that originate from a
variety of sources by way of fragmentation of larger plastic debris
into smaller polymer particles due to weathering and degradation
* Corresponding author. Tel: þ82-32-860-7561; fax: þ82-32-876-2351.
E-mail address: cgk@inha.ac.kr (C.G. Kim).
microplastics), which are very small pieces of manufactured poly-
mer materials used as additives in personal care products (e.g.,
exfoliants, facial cleansers, and toothpaste). They are easily intro-
duced into freshwater, terrestrial, or marine environments because
they have high molecular weight and hydrophobicity, thereby
making them highly recalcitrant against biodegradation (Restrepo-
flo
rez et al., 2014). They can be further associated with severe
environmental contamination even in remote locations far from
anthropogenic sources of emissions.
Microplastics pose an ecological threat to aquatic organisms. For
example, the freshwater crustacean Daphnia galeata that ingested
nano-sized polystyrene particles has suffered from reduction in
survival rate and hatching rate (Cui et al., 2017). Additionally, plastic

https://doi.org/10.1016/j.chemosphere.2019.01.159
0045-6535/© 2019 Elsevier Ltd. All rights reserved.
528 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533

debris can adsorb and transport persistent organic pollutants, such

as polychlorinated biphenyls, phenanthrene, or dichloro-diphenyl-
trichloroethane (Bakir et al., 2014), which could strengthen their
bioaccumulation and bioamplification within the ecosystem (Pinto
et al., 2016).
Microbial degradation of polymer materials (e.g., polyethylene,
polypropylene, and polystyrene) has been actively investigated for
the isolation of microorganisms (i.e., bacteria and fungi) from
natural environments that are capable of degrading polymers. For
instance, Bacillus sp. (Auta et al., 2018; Harshvardhan and Jha, 2013;
Roy et al., 2008), Rhodococcus sp. (Auta et al., 2018), Pseudomonas
aeruginosa (Jeon and Kim, 2015), Zalerion maritimum (Paço et al.,
2017), and Aspergillus clavatus (Gajendiran et al., 2016) can utilize
polymer materials as the sole carbon source in minimal nutrient
media, consequently contributing to a reduction in the dry weight
of polymers and induce physicochemical changes, including
changes in surface morphological structure and chemical structure.
Among them, Bacillus licheniformis and Lysinibacillus fusiformis
destroyed PE waste, subsequently forming cracks and rough sur-
faces and producing loosened chemical bonding structures, such as Fig. 1. Locality maps for waste sampling from the landfill site in Incheon, Korea.
carbonyl groups, ketones, or aldehydes, and crystallinity Sampling was conducted at sites A and B at different depths (2 and 5 m) in June of
2017.
(Mukherjee et al., 2015). It was also demonstrated that these mi-
croorganisms could decompose the polymers in two stages, where
they first colonized by adhering to the surface of the polymer (Site A 37 570 99.4900 N, 126 61017.1400 E; Site B 37 570 95.9100 N,
particles (Auta et al., 2018), and then enzymatically dissimilated to 126 61024.3100 E), where plastic wastes had previously been buried.
excrete extracellular enzymes. They subsequently cause a chain They were taken from different depths at 2 and 5 m below the
cleavage of the polymer into a monomer, which can be metabolized landfill cover soil using a boring rig (DESCO 4500 SD Drill Rig,
by microorganisms (Lau et al., 2009). DESCO), from which 1 kg of sediment sample was then contained in
This study was conducted to evaluate the biological decompo- amber jars without headspace and transferred to a laboratory in
sition of micro-sized PE with a size range of 40 mme600 mm after ice-packed storage. Prior to drilling, hollow stem augers were
they were amended with mesophilic bacterial consortium isolated sterilized with 70% (v/v) ethyl alcohol and then rinsed with distilled
from a municipal solid waste disposal site in Incheon, Korea where water three times. The bacterial cultures were prepared by mixing
plastic wastes were once buried. To the best of our knowledge, this 5 g of sediment samples in an isotonic saline solution for 2 h fol-
is the first study that deals with biologically aging the spherical lowed by settling for 30 min, after which the resultant supernatant
type of PE particles not filmed, which will address if primary plastic was inoculated in LB broth (containing 10 g L1 of tryptone, 5 g L1
released into the environment can be decomposed. To do this, of yeast extract, and 10 g L1 of NaCl in distilled water with a pH of
analytical instruments, such as a field-emission scanning electron 7.0). Next, they were cultivated at 30  C in a rotating incubator (VS-
microscope (FE-SEM), Fourier transform infrared (FTIR) spectrom- 8480SF, Vision Scientific Co., Ltd., Korea) for 2 d. Additionally, we
eter, gas chromatography-mass spectrometer (GC-MS), and ther- analyzed the soluble chemical oxygen demand (SCOD) to deter-
mogravimetric analyzer, were comparably used for the mine the degree of biodecomposition for samples. To measure the
measurement of deformed PE microplastics and controls. SCOD, 5 g of sediment sample was suspended in 20 mL of distilled
Morphological or physicochemical changes according to the bio- water, and then filtered with glass fiber filter paper (1.2 mm,
decomposition of concerned particles versus the controls were Whatman®, UK). Subsequently, 5 mL of filtrate were heated at
then addressed. 150  C in an oven in the presence of 7 mL of sulfuric acid reagent
(95% purity) and 3 mL of potassium dichromate solution
2. Materials and methods (0.01667 M). After heating for 2 h, they were cooled down and
ferroin indicator solution was added. They were titrated with
2.1. Microplastics ferrous ammonium sulfate solution (FAS, 0.02 M) until the color
turned to reddish brown. From this, COD was calculated using the
PE microplastic granules (white and amorphous) of CAS number following Eq. (1):
9002-88-4 with a density of 0.94 g mL1 at 25  C were purchased
ðA  BÞ  M  8; 000
from Aldrich Chemical Co. (USA). Prior to the degradation experi- COD mg O2 L1 ¼ (1)
ments, they were sterilized by soaking in ethyl alcohol (anhydrous, V
99.9%, Daejung Chemicals, Korea) in a glass Petri dish (Steriplan®
Where A and B are the titration volume of FAS consumed for the
Petri dish, Duran Group, Czech Republic), dried overnight in an
blank and sample, respectively, M is the molarity of FAS, 8000 is the
oven at 50  C oven (VS-1202D2, Vision Scientific Co., Ltd., Korea),
milliequivalent weight of oxygen, and V is the volume of sample
and stored in a vacuum desiccator at room temperature for further
used for COD analysis.
use. Their size distributions were measured using a FE-SEM (S-
4300SE, Hitachi High Technologies Co., Japan).
2.3. Identification of bacterial strains

2.2. Isolation and identification of mixed bacterial culture Twenty single colonies were obtained from four bacterial cul-
tures (A2, A5, B2, and B5 indicating the sampling site by letter and
A number of sediment samples were collected from a decom- depth in meters from the ground surface) by streaking into LB agar
missioned landfill site, as shown in Fig. 1, located in Incheon, Korea plates (containing 1.5% agarose). Extraction of the genomic DNA
 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533
from each of the single colonies was conducted using a HiGene™
Genomic DNA Prep Kit (BIOFACT, Korea). The bacterial 16S rDNA
was amplified using the primer sets of 27F (50 -AGA GTT TGA TCM
TGG CTC AG-30 ) and 1492 R (50 -TAC GGY TAC CTT GTT ACG ACT T-30 ).
Amplifications were performed using a thermal cycler instrument
(Tprofessional Thermocycler, Biometra, Germany) in a final volume
of 25 mL, with each containing 2  Taq Reaction Buffer, 1 mM of
dNTPs, 1 mM of each primer, 1.5 U of Taq polymerase (BIOFACT™
TaqBasic DNA Polymerase, BIOFACT, Koreas), and 3 mL of template
DNA. The PCR conditions were 1 cycle (95  C for 15 min) for initial
denaturation, 30 cycles (95  C for 20 s, 50  C for 40 s, and 72  C for
5 min) for denaturation, annealing, and extension, and 1 cycle
(72  C for 5 min) for the final extension of the amplified DNA. Next,
the PCR products were purified and sequenced with a primer set of
518F (50 -CCA GCA GCC GCG GTA ATA C-30 ) and 805 R (50 -GAC TAC
CAG GGT ATC TAA TC-30 ) using a BigDye® Terminator v3.1 Cycle
Sequencing Kit through an automatic sequencer (ABI 3730XL DNA
Analyzer, Applied Biosystems, USA). A similarity search was con-
ducted using the BLAST database of the National Center for
Biotechnology Information, from which more than 95% matched
bacterial strains were selected. Finally, a phylogenetic tree was
constructed using the neighbor-joining method supplied by MEGA
7.0 software (https://www.megasoftware.net).
2.4. Screening of bacterial strains for PE degradation
The bacterial isolates were screened out according to their
ability to make use of PE microplastics as a sole carbon source. For
this, a mixed bacterial culture was grown on Basal medium con-
taining 2.34 g of K2HPO4, 1.33 g of KH2PO4, 1.0 g of (NH4)2SO4, 0.5 g
of NaCl, 0.2 g of MgSO4$7H2O, and 1 mL of trace element solution
(21.8 mg L1 CoCl2$6H2O, 21.6 mg L1 NiCl2$6H2O, 24.6 mg L1
CuSO4$5H2O, 1.62 g L1 FeCl3$6H2O, 0.78 g L1 CaCl2, and
14.7 mg L1 MnCl2$4H2O) per liter of distilled water (Jeon and Kim,
2015), which was amended with 1% (w/v) sterilized PE micro-
plastics. The microorganisms were incubated for a period of 20 d at
30  C under aerobic conditions. Subsequently, the incubated cul-
tures were cultivated in the LB agar plate for 24 h at 30  C. In the
following, a single colony was each incubated in the same condition
as for the previous and then they were identified as described in
Section 2.3. From this, the number of colonies have been counted to
coincidently match to its identification so that the dominant spe-
cies should be verified.
2.5. Microbial inoculum preparation and assay for PE microplastic
degradation
The bacterial strains previously screened as the microplastic
degraders were inoculated into fresh LB broth and allowed to grow
in a rotating incubator at 30  C until their population density
attained log phase in terms of absorbance intensity of 1.00 ± 0
0.05 at 600 nm. Ten percent of the strains were inoculated into
Erlenmeyer flasks containing 100 mL of basal medium in the
presence of 100 mg of sterilized PE microplastics. For the negative
control test, an uninoculated basal medium supplemented with PE
microplastics was prepared in the same manner as that of the test
with the addition of 1 mL of 1% (w/v) sodium azide (NaN3, 99%,
Samchun Chemicals) to prevent any possible growth from the
medium. All the assays were performed in triplicate.
2.6. Analytical procedures
2.6.1. Determination of dry weight of residual PE microplastics
After the incubation period ended, the PE microplastics in the
529
broth were filtered out using a 0.45 mm cellulose acetate membrane
filter (ADVANTEC®, Tokyo Roshi Kaisha Ltd., Japan). Additionally,
the filtrate was collected to measure SCOD using the same pro-
cedure as that described in Section 2.2 to monitor temporal varia-
tion in organic contents that possibly originated from the break-up
of the mother compound after the incubation progressed. At the
end of the test, the bacterial films that colonized around the surface
of the plastic particles were washed with 2% (w/v) sodium dodecyl
sulfate (SDS) solution for 4 h and dried in an oven at 50  C over-
night. The extent of the amount degraded during the test period
was determined by the amount of dried polymer weight compared
before and after the test using an analytical balance (HR-200, AND,
Korea) with a readability of 0.0001 g. From this, the percentage
weight loss of the PE microplastics due to degradation was deter-
mined using Eq. (2):
 
W0  W
% Weight loss ¼  100 (2)
W0
Where W0 is the initial weight of the PE microplastic (g) and W is
the residual weight of the PE microplastic (g).
2.6.2. FE-SEM analysis of PE microplastics
The morphology and particle size distribution of the degraded
PE microplastics were compared to those of raw PE microplastics
using a FE-SEM (S-4300SE, Hitachi High Technologies Co., Japan).
For this, they were sputter coated with a platinum layer under an
argon atmosphere followed by visualization using a SEM at a
magnification of 40 (for particle size distribution analysis) and
8000 (for observing surface morphology, including the bacterial
colonization on the microplastic surface). Finally, the particle size
distribution was obtained through the image processing software
Image Pro Plus 6.0 (Media Cybernetics, USA).
2.6.3. FTIR analysis of microplastic polymers
The changes in the surficial structure of the microplastic poly-
mers after incubation were analyzed using FTIR spectroscopy
(VERTEX 80V, Bruker, Germany) in the frequency range of 4000-
400 cm1 with 4 cm1 of scanning mode. It was also conducted for
the uninoculated PE microplastic as the control. For this purpose,
100 mg of KBr and 5 mg of dried PE particles were grinded in an
agate mortar and pelletized using an Atlas™ manual hydraulic
press (SPECAC Inc., USA), which was used for FTIR spectroscopic
analysis. Background scanning of FTIR was conducted under vac-
uum conditions, and it was compared with the FTIR observation of
the sample. It consequently revealed its own spectrum for the given
sample.
2.6.4. GC-MS analysis
To extract organic matters formed around the surface of the PE
particles during the test, a procedure suggested in the literature
(Contat-Rodrigo et al., 2001) was modified and implemented. That
is, a total of 50 mg of sample was mixed with 10 mL of chloroform
(99.5%, Daejung Chemicals, Korea) in a 20 mL glass vial and
ultrasonicated in a Branson 5510 apparatus for 2 h in a hot water
bath held at 55  C. The residual PE particles were removed using
filter paper (Whatman™ Grade 2 Qualitative Cellulose Filter Paper,
8 mm, GE Healthcare Life Science, United Kingdom). The aliquot was
then concentrated up to 1 mL by evaporating chloroform with ni-
trogen gas purged in a TurboVap® II Automated Solvent Evapora-
tion System (Biotage®, Sweden). The extracts were characterized
with a GC-MS (1200 L Single Quadrupole GC-MS system with
3800 GC, Varian Inc., USA) using helium as the carrier gas, which
was equipped with DB-5MS (30 m  0.25 mm ID, film thickness
0.25 mm, Agilent, USA) of non-polarity. The oven temperature was
530 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533
kept at 40  C for 3 min, increased to 280  C at 10  C min1, and then
finally held at 280  C for 4 min (Roy et al., 2008). Unknown com-
pounds that originated from the incubation were identified by
comparing their mass spectra with the NIST/WILEY database.
2.6.5. Thermogravimetric analysis of PE microplastics
Thermogravimetric analyses of PE particles were performed
using TGA Instruments (TG 209 F3 Tarsus®, NETZSCH, Germany).
Approximately 10 mg of decomposed microplastics were subjected
to heating at a rate of 10  C min1, starting from 30  C ramping and
increasing to 600  C under ambient conditions.
3. Results and discussion
3.1. Isolation and identification of bacterial strains
The bacterial strains for screening PE-degrading bacteria were
obtained from four different points (A2, A5, B2, and B5) where
plastic wastes were buried in the landfill site. Each sample had a
different environmental organic content with 4385 mg O2 L1 (A2),
4106 mg O2 L1 (A5), 1914 mg O2 L1 (B2), and 1714 mg O2 L1 (B5)
of SCOD. The varying organic concentration among the samples
was derived from the difference in historical backgrounds
depending on the reclamation period of wastes and relevant de-
gradability. Sampling point A had more recently buried wastes than
sampling point B. Nevertheless, regardless of the location, the
deeper sites showed a lower concentration than that of shallower
sites, thereby indicating that more active decomposition occurred
at deeper sites.
Different historical backgrounds of the organic content and
types of plastic wastes buried at each sampling point and depth
might have directly induced different microbial diversity. From this,
we chose 7 microbial strains among the 20 single colonies grown
on the LB agar plates. These were strain numbers 21, 22, 23, and 25
from A2; 26 from B2; 51 from A5; and 56 from B5; respectively
(Fig. 2). Based on the 16S rDNA gene sequencing, the bacterial
cultures isolated from the site mainly belonged to four genera of
class Bacilli in terms of Bacillus, Paenibacillus, Fontibacillus, and
Enterococcus together with a genus of order Aeromonadales (Aer-
omonas). They included strains of Bacillus velezensis, Bacillus pseu-
domycoides, Paenibacillus alvei, Paenibacillus motobuensis,
Fontibacillus phaseoli, Enterococcus avium, Aeromonas veronii, and
Fig. 2. Phylogenetic dendrogram of the relationships between 16S rDNA gene se-
quences retrieved from GenBank.
Aeromonas caviae, as shown in Fig. 2. Among these, Aeromonas sp.,
an obligatory anaerobe, was commonly detected at 2 and 5 m for
site B, which had been buried earlier than site A, while Bacillus spp.
were found in the more recently buried site A.
Selectively isolated PE degraders from the sediments were
inoculated in the basal medium containing 1% (w/v) PE micro-
plastics for 20 d under aerobic conditions. It was evident that Ba-
cillus sp. and Paenibacillus sp. were dominantly present at more
than 85% as PE degraders that had been enriched from the A2
bacterial isolates among others, which showed the highest PE
degradation efficiency of approximately 15%. These bacterial strains
are capable of degrading various types of polymer materials,
including low-density polyethylene (LDPE) (Das and Kumar, 2015a),
polyester polyurethane (Shah et al., 2016), and poly(lactic acid)
(Teeraphatpornchai et al., 2003). Particularly, Bacillus amylolique-
faciens contributed to reducing the dry weight of LDPE film by 16%
after 60 d (Das and Kumar, 2015b). In addition, the mixture of Ba-
cillus sp. and Paenibacillus sp. isolated from an extinct volcano
crater could decrease the dry weight of LDPE film (type FGNX23-
D022) by 7.53% and 13.54% after 75 d and 150 d, respectively
(Nowak et al., 2011). Thus, we undertook a subsequent biodegra-
dation experiment by employing the blended PE degraders
composed of Bacillus sp. and Paenibacillus sp. for 60 d to assess the
variation in physicochemical changes in granular PE microplastics.
3.2. Determination of weight loss of PE and variation in SCOD
The apparent degradation efficiency of PE particles was assessed
by comparing the dry weight loss of PE microplastics. To do this,
they were washed with 2% (w/v) SDS solution to remove the cell
and its debris from the collected sample after the microbial
degradation test (Fig. 3 (a)). The weight loss of PE microplastic was
14.7% after 60 d of incubation where the dry weight loss was sig-
nificant in the early phase of incubation. In the negative control
(without inoculation), weight loss of PE microplastic was lower
than 5%. The difference in weight loss might have been due to
microbial metabolism and partial dissolution of PE particles. Recent
studies reported that the rate of degradation of PE polymer mate-
rials widely ranged from 1.5% to 13% according to varying experi-
mental conditions and types of microbes. Out of these, the
degradation efficiency observed in this study was comparable to
that of LDPE film degraded by mixed microbial cultures of Bacillus
sp. and Paenibacillus sp. mentioned in Section 3.1. Additionally,
Fig. 3 (b) shows that the growth patterns of microbes under the
presence of PE microplastic were consistent with its own degra-
dation efficiency compared to that of the control.
The organic matter content was measured as SCOD during the
incubation. For the control, 675 mg O2 L1 of SCOD was observed,
while approximately 60 mg O2 L1 originated from the liquid me-
dium itself. After the inoculation, SCOD decreased to less than
60 mg O2 L1. This clearly verified that the given microbes could
biologically attack the structure of PE particles that were eventually
decomposed.
3.3. SEM observation of treated PE microplastics
Changes in the morphological structure of untreated and bio-
logically aged PE microplastics was observed using a SEM, as shown
in Fig. 4 (a) and (b), respectively. Each polymer particle had very
typical features in their shape, showing generally spherical forms.
In the initial stage of incubation, larger particles were almost all
present regardless of the experimental condition, on which a
number of cracks and pits remained. In the meantime, relatively
small polymer particles tended to attract onto the surface of larger
particles regardless of the experimental conditions and with either
 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533 531

Fig. 3. (a) Weight loss of PE microplastic and (b) microbial population density of
attached and suspended cells observed for 20, 40, and 60 d after the mixed bacterial
strains mainly composed of Bacillus sp. and Paenibacillus sp. were incubated.

the biologically aged or the control particles. Unlike this, studies

showed that filmed polymer with an intact smooth surface without
cracks became cracked or pitted after incubation began (Mukherjee
et al., 2015; Tribedi and Sil, 2013). In these experiments, the result
was used to verify that bacterial decomposition clearly occurred on
the polymer surface. However, in this study, the differences in the
surficial change of morphology between the biologically degraded
and control particles were not clearly distinguished by the FE-SEM
analysis. Meanwhile, Fig. 4 (d) shows that the microbes were
strongly attached on the particle surface area where they could
biologically utilize the PE microplastics, comparing to the control
which has no microbes on its surface (Fig, 4 (c)), as demonstrated
by a previous study (Das and Kumar, 2015a).
Additionally, the particle size distribution for the biologically
deformed particles was determined using a SEM analysis, as shown
in Fig. 4 (e). The particle size range of raw PE microplastic was from
40 to 600 mm, and the mean particle size was approximately
231 mm. Upon incubation, the particle size decreased as the incu-
bation time increased from 0 to 60 d. That is, the mean particle size
gradually decreased to 211 mm after 20 d, 210 mm after 40 d, and
176 mm after 60 d. At this time, the control test, which was con-
ducted in the same manner as that for the biodecomposition
experiment without inoculum, decreased the particle size to
approximately 225 mm mean diameter. This clearly showed that the
greatest particle size range that appeared initially was shifted to a
relatively smaller size range from which the mean particle diameter
of 225 mm shown in the beginning was reduced to 176 mm after
Fig. 4. Scanning electron microscope images of clusters of polyethylene (PE) micro-
plastic of (a) control and (b) biologically aged particles. The magnified surface of (c)
control and (d) biologically aged PE microplastic where the microbes colonized after
60 d of incubation. Cumulative particle size distribution analysis of PE microplastic
particles measured by Image Pro software (e).
incubation was completed. As mentioned in Section 3.2, it was
consistent with the weight loss of PE microplastic for the biologi-
cally degraded particles, which was 14.7% for 60 d of incubation,
compared to that of the control, which showed a weight loss of less
than 5%. Additionally, a previous study demonstrated that many
small particles could be deformed by weathering and decomposi-
tion (i.e., exposure to sunlight and oxygen) of plastic materials
(Lambert and Wagner, 2016).
3.4. FTIR analysis of microplastic polymers
The surficial structure changes in the PE microplastic with
bacterial decomposition were determined using FTIR spectroscopy
(VERTEX 80V) in the frequency range from 4000 to 400 cm1. The
FTIR spectra for both raw and biologically deformed particles are
shown in Fig. 5. For the control (Fig. 5 (a)), critical characteristic
absorption bands were assigned at 3447 cm1 (eOeH stretching),
2920 cm1 (eCH3 stretching), 2851 cm1 (eCHO stretching),
532 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533
Fig. 5. Fourier transform infrared spectra of (a) control (after incubating for 60 d in the
medium without inoculum) and (b) biologically treated polyethylene microplastics
incubated for 20 d, (c) 40 d, and (d) 60 d.
1472 cm 1
(C]C double bond stretching), 1377 cm (eOeH 1
stretching), and 719 cm1 (C]C-H stretching). For the deformed
particles after 20 d of incubation (Fig. 5 (b)), the FTIR spectra of each
peak appeared to be at the lowest level, except for the 3447 cm1
band (eOeH stretching), which showed a very similar pattern for
the biologically decomposed and control particles. Extending the
incubation to 40 and 60 d (Fig. 5 (c) and (d)), the spectral peak
increased compared to that after 20 d, thereby indicating that mi-
crobes could be increasingly cultivated to attach to the PE surface.
3.5. GC-MS analysis of eluted contents from degraded PE
microplastics
Each chloroform extract of the raw and bio-deformed particles
was characterized using GC-MS analysis (Fig. 6). The unknown
organic compounds derived from the extracts were identified as 2-
dodecanol (C13H28O; at the retention time of 20.0 and 23.9 min),
1,8-nonanediol (C10H22O2; 22.0, 25.5, 27.1, and 28.9 min), and 1-
dencene (C11H22; 28.4 min) in the search of the GC-MS library. They
all had a linear carbon chain structure that originated from the PE
microplastic, which were functionalized into eCHO and eOH, as
was also found from the FTIR analysis. Meanwhile, in the compar-
ison of two types of peak patterns in the raw and deformed
Fig. 6. Gas chromatography-mass spectrometry chromatogram of (a) neat poly-
ethylene (PE) microplastic and (b) biologically treated PE microplastics incubated for
20 d, (c) 40 d, and (d) 60 d.
Fig. 7. Thermal gravimetric analysis of polyethylene microplastics before and after
biodegradation according to different incubation times.
particles, the peaks for the deformed particles in terms of 1 and 2
(Fig. 6) disappeared after 20 d of incubation. This was consistent
with the SCOD for the deformed particles, which significantly
decreased, as described in Section 3.2. The peak heights increased
after 40 d of incubation. In other words, this implied that the carbon
chain in the microplastic composition could be released into the
medium. This was also strongly related to the FTIR observation for
the differences in spectral patterns of peaks between 20 d and after
40 d.
3.6. Thermogravimetric analysis of PE microplastics
Thermogravimetric analysis and thermal stability of PE micro-
plastics were characterized using a TGA, as shown in Fig. 7. For the
deformed particles, T5, or the temperature at which 5% weight loss
of PE particles occurred, was slightly decreased from the starting
temperature of 290  Ce285  C during the analysis, whereas the
control was kept at 290  C to lose the same weight. It was similarly
found that T5 of LDPE was decreased regardless of the type of in-
cubation mode in terms of abiotic (i.e., photocatalytic degradation
on titania nanoparticles) or biotic conditions (Tahir et al., 2016).
Upon increasing the temperature to more than 570  C, the control
completely lost weight, but the deformed PE still maintained its
weight to some extent regardless of incubation time. This demon-
strated that such formation of a higher thermal stability could not
be explained as a consequence of severe biological chain scission
(Tahir et al., 2016). Among them, the deformed particles after 20 d
showed the highest extent of thermal stability with 9.6% of its
weight still residually sustained. Its stability was relatively more
staggered after 40 and 60 d, thereby implying that a more persis-
tent composition of particles after 20 d might be further broken up
as incubation continues.
4. Conclusion
This study showed that a mixed bacterial culture mainly con-
sisting of Bacillus sp. and Paenibacillus sp. isolated from a landfill
site could help accelerate PE microplastic degradation. The bacte-
rial culture was grown in an aqueous medium containing PE
microplastic as the sole carbon source, contributing to 14.7% of
weight loss together with a reduction of 22.8% for mean particle
diameter. The bacterial colonization on the microplastic surface
was observed by SEM, which could lead to the decomposition of PE
microplastic as confirmed by the GC-MS and SCOD analyses in
 S.Y. Park, C.G. Kim / Chemosphere 222 (2019) 527e533
association with the FTIR and TGA. As incubation time increased,
daughter compounds appeared, so their size decreased to conse-
quently lose weight, which could in turn be attributed to keeping
their stability much stronger compared to that of the control.
Nonetheless, the SCOD gradually decreased when incubation time
increased, which meant that relatively more labile organic contents
were clearly decomposed to decrease their SCOD concentration,
even though the types and levels of daughter compounds were
increasingly released into the media.
Acknowledgments
This research was supported by the Basic Science Research
Program through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education (NRF-
2017R1D1A1B03034029).
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