Marine Pollution Bulletin 77 (2013) 100–106

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Marine Pollution Bulletin

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Biodegradation of low-density polyethylene by marine bacteria from

pelagic waters, Arabian Sea, India
Kumar Harshvardhan a,b, Bhavanath Jha a,b,⇑
a
Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, GB Marg, Bhavnagar 364002, Gujarat, India
b
Academy of Scientific and Innovative Research, CSIR, New Delhi, India

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

Keywords: Sixty marine bacteria isolated from pelagic waters were screened for their ability to degrade low-density
Pelagic marine bacteria polyethylene; among them, three were positive and able to grow in a medium containing polythene as
Polyethylene the sole carbon source. The positive isolates were identified as Kocuria palustris M16, Bacillus pumilus
Biodegradation M27 and Bacillus subtilis H1584 based on the 16S rRNA gene sequence homology. The weight loss of poly-
Biomass
ethylene was 1%, 1.5% and 1.75% after 30 days of incubation with the M16, M27 and H1584 isolates,
respectively. The maximum (32%) cell surface hydrophobicity was observed in M16, followed by the
H1584 and M27 isolates. The viability of the isolates growing on the polyethylene surface was confirmed
using a triphenyltetrazolium chloride reduction test. The viability was also correlated with a concomitant
increase in the protein density of the biomass. Polyethylene biodegradation was further confirmed by an
increase in the Keto Carbonyl Bond Index, the Ester Carbonyl Bond Index and the Vinyl Bond Index, which
were calculated from FT-IR spectra.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
The development and use of synthetic plastic has changed the
nature of waste in last 3–4 decades (Sheavly, 2005). Over this per-
iod, it has replaced natural material in various aspects of human
life and become an indispensable part of our society. Although
the durability of plastic is ones of its most beneficial qualities, this
same property is a major problem for our environment (Sivan,
2011). Plastics are chemically synthesized long-chain polymers
(Scott, 1999) and are globally produced on a substantial scale. As
per a recent estimate of the Central Pollution Control Board, New
Delhi, India, 8 million tons of plastic products are consumed every
year in India alone. A study on plastic waste generation in 60 major
Indian cities revealed that approximately 15,340 tons per day of
plastic waste is generated in the country (Central Pollution Control
Board (CPCB) New Delhi, India, 2013). Low-density polyethylene
(mainly used as carry bags) constitutes the major portion of this
waste problem.
In the last two decades, the rate of plastic deposition has in-
creased tremendously, and plastic has intruded into the marine
environment. Plastic is found floating in oceans everywhere from
the polar regions to the equator and has become one of the most
common and persistent pollutants of seas and beaches worldwide
⇑ Corresponding author at: Discipline of Marine Biotechnology and Ecology, CSIR-
Central Salt and Marine Chemicals Research Institute, GB Marg, Bhavnagar 364002,
Gujarat, India. Tel.: +91 278 2567552; fax: +91 278 2570885.
E-mail address: bjha@csmcri.org (B. Jha).
(Frias et al., 2010; Moore, 2008; Teuten et al., 2009). Plastic debris
is one of the largest contaminants of the marine environment.
Polyethylene is the most commonly found non-degradable solid
waste and has recently been recognized as a major threat to mar-
ine life. There are reports that suggest that polyethylene causes
blockages in the intestines of fish, birds and marine mammals. In
addition, entanglement in or ingestion of this waste has endan-
gered hundreds of different species (Teuten et al., 2009; Secchi
and Zarzur, 1999; Spear et al., 1995).
Polyethylene represents up to 64% of the synthetic plastics that
are discarded within a short period after use (Byuntae et al., 1991).
It is highly resistant to acids, alcohols, bases and esters. It is also
biologically inactive and considered a recalcitrant material. Its
inertness is due to the high molecular weight, hydrophobicity
and lack of functional groups recognized by microbial enzymatic
systems (Hamid, 2000). Polyethylene is a concern for waste man-
agement due to its accumulation in landfills and natural habitats
(Thompson et al., 2009). Hence, a suitable method for disposal that
is eco-friendly must be found. Recycling of polyethylene was con-
sidered a solution but has failed to provide safe disposal of these
materials (Sivan, 2011); in this regard, microbial degradation is
one of the best options. Some reports on the biodegradation of
plastics indicate that it could be a viable proposition when suitable
microorganisms are utilized (Singh and Sharma, 2008; Shah et al.,
2008). Studies on polyethylene biodegradation (Albertson, 1980;
Albertsson et al., 1987), including the biotic environment (Shah
et al., 2008), have been reported. However, few studies have been

0025-326X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.marpolbul.2013.10.025
 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106
conducted on non-amended polyethylene (Balasubramanian et al.,
2010; Hadad et al., 2005).
In the present study, we screened sixty marine bacterial isolates
cultured from the pelagic waters of the Arabian Sea, India, for their
polyethylene degradation ability and found three potential strains.
In addition, we characterized the cell-surface hydrophobicity and
growth kinetics of these bacteria and the biodegradation of
polyethylene.
2. Materials and methods
2.1. Screening of bacterial isolates for polyethylene degradation
Sea water samples were collected from different locations along
the Arabian Sea coast, India. The map of different sampling loca-
tions and geographical coordinates is shown in Fig. 1. The bacterial
cultures were isolated on Zobell marine agar 2216 (Himedia) plates
using a dilution technique at room temperature (25 °C). All mor-
phologically distinct colonies were purified and preserved in min-
eral oil.
Sixty bacterial isolates were assayed for their ability to utilize
polyethylene as the sole source of carbon and energy. Each individ-
ual isolate was grown in Bushnell–Haas medium (1.0 g NH4NO3,
0.2 g MgSO47H2O, 1.0 g K2HPO4, 0.1 g CaCl22H2O and 0.15 g KCl
per liter of distilled water) supplemented with 3.5% NaCl. Then,
20 ll of 1% TTC solution was added to 10 ml of medium as an indi-
cator of viability (Alef and Nannipieri, 1999).
2.2. Identification of the bacteria
The genomic DNA was extracted from the bacterial culture
using a standard phenol chloroform extraction procedure.
Amplification of the 16S rRNA gene was conducted using universal
primers (Weisburg and Barns, 1991): 27F (50 -GAGAGTTTGATCCT
GGCTCAG-30 ) and 1495R (50 -CTACGGCTACCTTGTTACGA-30 ). The
Fig. 1. A geographical map showing the sampling sites along the coastline and their locations.
101
reaction mixture (50 ll) contained 0.5 ll of each primer, 400 lM
of dNTPs, 2.5 U of Taq polymerase, 1.5 mM of MgCl2, 50 ng of
DNA template and 5 ll of 10  PCR buffer. The PCR was conducted
at an initial denaturation temperature of 94 °C, 8 min; followed by
28 cycles of 94 °C, 1 min; 58 °C, 1 min; 72 °C, 2 min; and a final
extension at 72 °C for 10 min. The PCR product was purified, ana-
lyzed and sequenced (M/s Microgen Inc., Seoul, South Korea). The
similarity search was conducted in-silico using the BLAST database
of NCBI. The phylogenetic and molecular evolutionary analyses
were conducted using MEGA version 5 (Tamura et al., 2011). Boot-
strap analysis with 100 replicates was performed to estimate the
confidence of the tree topologies.
2.3. Polyethylene film biodegradation assay
The biodegradation tests were performed on samples of low-
density polyethylene film (i.e., pieces of polyethylene bags) that
had been dried overnight at 60 °C, weighed, disinfected (auto-
claved at 105 °C for 1 h) and added to each flask (approximately
50.0 mg of polyethylene film per flask) containing 50 ml of BH
medium. The flasks were inoculated with 2 ml of a mid-exponen-
tial phase culture that were maintained in Zobell marine broth
(ZMB). Before inoculation, the culture was washed with Bush-
nell–Haas (BH) medium to remove medium and cellular soluble
debris. The cell densities of the inoculums were adjusted to
1.5  106 colony-forming units (CFU) per ml. The flasks containing
non-inoculated BH medium supplemented with polyethylene film
served as the control.
2.4. Determination of the dry weight of residual polyethylene
To facilitate accurate measurement of the residual polyethylene
weight, the bacterial film colonizing the polyethylene surface was
removed by supplementing the cultures with a 2% (v/v) aqueous
sodium dodecyl sulfate (SDS) solution. The flasks were then
102 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106
incubated for 4 h at 50 °C and further washed with warm distilled
water (Sivan et al., 2006). The polyethylene samples were collected
on filter paper, rinsed with distilled water and then dried overnight
at 60 °C before they were finally weighed. The initial weights of the
pre-incubated polyethylene samples were measured following the
same procedure mentioned above.
2.5. Viability of the bacteria on the polyethylene surface
The viability and metabolic activity of the surface-attached bac-
teria were measured with the redox probe 2,3,5-triphenyltetrazo-
lium chloride (TTC), which facilitates direct monitoring of
actively respiring bacteria. The colorless TTC is readily reduced
by the bacterial electron transport system (ETS) to red-colored
insoluble triphenylformazan (TPF). The respiration was monitored
by measuring the level of TPF (Alef and Nannipieri, 1999). Surface-
adhered cells were monitored for their viability. Cell pellets were
washed twice with 50 mM phosphate buffer at a pH of 7.6 and cen-
trifuged at 5000 rpm for 10 min. Then, the pellets were re-sus-
pended in 4.5 ml of buffer solution, and 0.5 ml of TTC (0.1 g/l)
was added. The mixture was incubated at 37 °C for 15 min in a
water bath. Then, 5 ml of 96% cold methanol was added to stop
the reaction and to begin the extraction of TPF. The dehydrogenase
activity was quantified at the absorption spectra of 480 nm in the
spectrophotometer. Ice-cold methanol and a phosphate buffer
solution at a 1:1 ratio served as a blank.
2.6. Estimation of the protein content of the surface-attached bacteria
The total protein content of the surface-attached bacterial bio-
mass was determined after alkaline hydrolysis, as follows. The
polyethylene pieces were sampled from flasks containing BH cul-
tures of bacterial isolates, washed gently with water to remove
medium debris and boiled for 30 min in 4.0 ml of 0.5 M NaOH.
The extracts were centrifuged to precipitate cell-debris fragments.
The supernatants were collected, and the pellets were subjected to
the same procedure to minimize the experimental error. The
Fig. 2. A phylogenetic dendrogram showing the relationship between the 16S rRNA gene sequences retrieved from GenBank. The evolutionary relationship was inferred using
the Neighbor-Joining method. The evolutionary distances were computed using the Maximum Composite Likelihood method and are presented in the units of the number of
base substitutions per site. The analysis involved 22 nucleotide sequences, and all ambiguous positions were removed for each sequence pair. All evolutionary analyses were
conducted in MEGA5.
collected supernatants were combined, and the protein content
in the extract was determined spectrophotometrically at 280 nm.
2.7. Evaluation of the bacterial cell surface hydrophobicity
The hydrophobicity of the bacterial cell surface was determined
using the bacterial adhesion to hydrocarbon (BATH) test (Rosen-
berg et al., 1980). For this assay, the bacteria were cultured in
ZMB medium until the mid-logarithmic phase, centrifuged, and
washed (twice) with PUM (phosphate urea magnesium) buffer
containing (per liter): 17 g K2HPO4, 7.26 g KH2PO4, 1.8 g urea and
0.2 g MgSO47H2O. The washed cells were resuspended in PUM
buffer to an optical density at 400 nm (OD400) value of 1.0–1.2. Ali-
quots (1.2 ml each) of this suspension were transferred to a set of
test tubes, to which were added increasing volumes (range 0–
0.2 ml) of hexadecane. The test tubes were shaken for 10 min
and allowed to stand for 2 min to facilitate phase separation. The
OD400 of the aqueous suspension was then measured. A cell-free
buffer served as the reference blank.
2.8. Scanning electron microscopy (SEM) of polyethylene
The polyethylene films were removed from the culture medium
at prescheduled times to observe the bacterial colonization of the
polyethylene film and the surface erosion. The samples were
washed for 2 min in 0.01 M phosphate buffer (pH 7.2) to release
excess medium. In contrast, in the procedure for the examination
of surface erosion, polyethylene samples were washed with a 2%
SDS followed by warm distilled water to completely remove sur-
face-adhered cells. Both types of polyethylene samples were fixed
in 2% glutaraldehyde for 2 h, washed twice (30 min each) in 50%
ethanol, incubated overnight in 70% ethanol, and finally washed
again (30 min  3) in 100% ethanol. After fixation, the samples
were dried in a vacuum, coated with gold and scanned in a LEO-
1430 VP SEM.
 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106 103

2.9. Fourier Transform Infrared analysis of polyethylene
Two types of polyethylene samples were analyzed: (i) incu-
bated with the bacterial strain and (ii) an un-inoculated control.
Changes in the polyethylene structure with subsequent bacterial
incubation were analyzed by Fourier Transform Infrared (FTIR)
(Perkin Elmer, Spectrum EX). Spectra in the frequency range of
4000–400 cm-1 were used at a resolution of 1 cm-1. The relative
absorbance intensities of the ester carbonyl bond at 1740 cm-1,
the keto carbonyl bond at 1715 cm-1, the terminal double bond (vi-
nyl) bond at 1650 cm-1 and the internal double bond at 908 cm-1 to
that of the methylene bond at 1465 cm-1 were evaluated using the
following formula (Albertsson et al., 1987): Keto Carbonyl Bond In-
dex (KCBI) = I1715/I1465; Ester Carbonyl Bond Index (ECBI) = I1740/
I1465; Vinyl Bond Index (VBI) = I1650/I1465; and Internal Double Bond
Index (IDBI) = I908/I1465. The crystallinity percentage of the polymer
showed only a 10% reduction in turbidity at the lowest concentra-
tion and an approximately 15% reduction at the maximum concen-
tration (Fig. 3b). The logarithmic cells were more hydrophobic than
those of the stationary phase. The greater affinity to hydrocarbon
(hydrophobicity) suggests a higher colonization interaction of
these isolates with the polyethylene surface. The viable coloniza-
tion of the bacteria was confirmed by an increased respiration level
and protein content on the polyethylene surface. It has previously
been reported that a polyethylene-degrading bacterium, Brevibacil-
lus borstelensis, resulted in a 10% maximum reduction in turbidity
(a) 105
Log phase
100
Stationary Phase
95

% of Initial OD600
was measured based on the method suggested by Zerbi et al. 90
(1989) and calculated using the following formula:
85
% of Crystallinity ¼ 100  ½f1  ðIa=1:233IbÞ=1 þ ðIa=IbÞg  100: 80
75
where Ia is the absorbance at 1473 and Ib is absorbance at 1463.
70

3. Results and discussion 65

60
3.1. Screening and identification of the bacterial isolates 0 0.05 0.1 0.15 0.2 0.25
Hexadecane added (ml)
Sixty marine bacterial isolates from pelagic waters were
screened for the potential degradation of low-density polyethyl- (b)105 Log Phase
ene. Three isolates were determined positive and utilized polyeth- Stationary Phase
100
ylene as a sole source of carbon (Supplementary S1). These isolates
95
were identified on the basis of their 16S rRNA gene sequence
% of Initial OD600

homology. The isolates M16 (accession no. KC355367), M27 90

(accession no. KC355368) and H1584 (accession no. KC355366) 85
showed 99% homology with Kocuria palustris, 99% homology with
80
Bacillus pumilus and 100% homology with Bacillus subtilis, respec-
tively. A phylogenetic analysis confirmed their similarity to the 75
respective species (Fig. 2). 70
Polyethylene degradation was initially monitored by the dry
65
weight loss, and after 30 days of incubation, the loss was
1 ± 0.033%, 1.5 ± 0.038% and 1.75 ± .06% (mean dry weight 60
0 0.05 0.1 0.15 0.2 0.25
loss ± SD%) for K. palustris M16, B. pumilus M27 and B. subtilis
Hexadecane added(ml)
H1584, respectively. The degradation was further confirmed based
on hydrophobicity and growth kinetics of bacterial cells and FT-IR
analysis of the degraded polyethylene. (c) 105 Log Phase
100 Stationry Phase
3.2. Bacterial cell surface hydrophobicity 95
% of Initial OD600

The ability of a microorganism to utilize any substrate depends 90

on its growth on and adherence to that substrate. Bacterial adhe- 85
sion to either a hydrophilic or hydrophobic substrate is governed
80
by many factors, including the forces by which the bacterium at-
taches to the surface and the properties of the substrate and micro- 75
organism. Generally, a hydrophobic bacterium prefers a 70
hydrophobic surface for attachment, whereas the opposite is true
65
for bacteria with hydrophilic properties. As the polyethylene sur-
face is hydrophobic in nature, it has been suggested that the more 60
0 0.05 0.1 0.15 0.2 0.25
hydrophobic the bacterial cell surface, the higher the interaction
with polyethylene (Gilan et al., 2004). To determine the bacterial Hexadecane added (ml)
interaction with polyethylene, the bacterial cell surface hydro- Fig. 3. The hydrophobicity of bacterial isolates (a) Kocuria palustris M16, (b) Bacillus
phobicities of the positive isolates, in both log and stationary pumilus M27 and (c) Bacillus subtilis H1584 was determined by the bacterial
phases, were assayed using a BATH test and the results are shown adhesion to hydrocarbon test. Aliquots of logarithmic (Log.) and stationary cell
in Fig. 3a–c. The M16 and H1584 isolates were hydrophobic even at suspensions were supplemented with increasing concentrations of hexadecane. The
transfer of hydrophobic cells from the aqueous phase to hexadecane is reflected as a
the lowest concentration of hexadecane (0.25 ll), resulting in an decrease in the turbidity (optical density at 600 nm, OD600) of the bacterial
approximately 24% reduction in the turbidity (Fig. 3a–c) and a suspension. The data represent the means of three replicates, and the error bars
maximum reduction in turbidity (32%) at 150 ll. The M27 isolate indicate the percent error.
104 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106

at 150 ll of hexadecane (Hadad et al., 2005). A 20% maximum
reduction in turbidity has been reported for Rhodococcus ruber (Gi-
lan et al., 2004). In the present study, we report a significant in-
crease (32%) in the hydrophobicity of two marine bacteria, K.
palustris and B. subtilis.
3.3. Viability and metabolic activity of the isolates
The respiration of the surface-attached bacteria on the polyeth-
ylene films was monitored regularly for 14 days. The reduction of
TTC to TPF, driven by the ETS, was used to determine the respira-
tion level of the surface-attached bacteria. The formation of TPF in-
creased sharply on the first day of incubation, followed by a
constant increase until the fourteenth day (Fig. 4a–c). This observa-
tion indicates a regular increase in the respiration rate and sug-
gests bacterial viability and growth. Dehydrogenases, a group of
intracellular enzymes, are involved in microbial oxidoreductase
metabolism. These enzymes have been frequently used as an index
of microbial activity in soil and waste water (Alef and Nannipieri,
1999; Camina et al., 1998). The activities of these enzymes are
linked to respiratory and energy processes in the cell, which de-
pend on the metabolic state of microorganisms. To determine
dehydrogenase activity, tetrazolium salts such as TTC are used as
artificial electron acceptors. TTC replaces oxygen as the final H+/
e acceptor and is reduced by the aerobic cytochrome system to
water-insoluble, red-colored formazans by microbial activities
(Alef and Nannipieri, 1999; Schimmel and Morrison, 1989).

(a) 12
(a) 60
Concenctration of TPF (µg)

10
50

Protein (µg) cm-2 Polyethylene

8

40
6

4 30

2 20

0
0 2 4 6 8 10 12 14 10

Incubation period (Days)

0
0 2 4 6 8 10 12 14
(b) 14 Incubation Period (Days)
Concentration of TPF (µg)

12
(b) 70
10
Protein (µg) cm-2 Polyethylene

60
8
50
6
40
4
30
2
20
0
0 2 4 6 8 10 12 14 10
Incubation period (Days) 0
(c) 14 0 2 4 6 8 10
Incubation Period (Days)
12 14

(c) 70
Concentration of TPF (µg)

12

10 60
Protein (µg) cm-2 Polyethylene

8 50

6 40

4 30

2 20

0 10
0 2 4 6 8 10 12 14
Incubation period (Days) 0
0 2 4 6 8 10 12 14
Fig. 4. The respiration of the surface-attached marine bacterial culture (a) Kocuria Incubatin Period (Days)
palustris M16, (b) Bacillus pumilus M27 and (c) Bacillus subtilis H1584 in Bushnell–
Haas medium supplemented with polyethylene as a sole source of carbon. The Fig. 5. The protein content of surface-attached marine bacterial culture (a) Kocuria
respiration was measured by the intracellular reduction of triphenyltetrazolium palustris M16, (b) Bacillus pumilus M27 and (c) Bacillus subtilis H1584 in Bushnell–
chloride (TTC) by cytochrome of the electron transport system to triphenylforma- Haas medium supplemented with polyethylene as a sole source of carbon. The data
zan (TPF). The data represent the means of three replicates, and the error bars represent the means of three replicates, and the error bars indicate the percent
indicate the percent error. error.
 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106
0.25 Control
M16
0.2 M27
Absorbance (Cm-1 )
H1584
0.15
0.1
0.05
0 in the presence of enzyme oxidoreductase (Karlsson and Alberts-
KCBI ECBI VBI IDBI
Fig. 6. Fourier Transform Infrared analysis of polyethylene incubated with marine
bacteria Kocuria palustris M16, Bacillus pumilus M27 and Bacillus subtilis H1584 for 1
month (KCBI – Keto Carbonyl Bond Index; ECBI – Ester Carbonyl Bond Index; VBI –
Vinyl Bond Index; IDBI – Internal Double Bond Index). The data represent the means
of three replicates and the error bars indicate the percent error.
3.4. Growth kinetics of surface-attached bacteria
The growth kinetics of bacteria on the polyethylene film was
monitored by quantifying the total protein extracted from the sur-
face of the film. The data depicted in Fig. 5a–c shows a biphasic
pattern of surface attachment on the polyethylene. The first phase
was characterized by a steep increase in protein content over
10 days of incubation and is reflected by an increase in the sur-
face-attached biomass. This phase was followed by a second period
in which the protein content remained constant from the 12th to
14th days. The continuous and slow increase in extractable protein
suggests a regular growth of isolates on the polyethylene. These
data may also suggest that the biomass on the polyethylene is con-
tinuously proliferating. This result agrees with the TTC reduction
analysis, which was used to measure the metabolic activity of
the bacteria.
Fig. 7. A SEM photograph of polyethylene after 30 days, (a) blank (uninoculated with bacteria), (b) inoculated with K. palustris M16, (c) inoculated with B. pumilus M27 and d)
inoculated with B. subtilis H1584. The images show erosion on the polyethylene surface with a reference to the blank.
105
3.5. Effect of biodegradation on the structure of polyethylene
Synthetic polymers are water insoluble, which is due to their
crystallinity (Pierre and Chiellini, 1986). We observed 10.33, 8.6
and 4.6% decreases in crystallinity after 30 days of incubation for
M16, M27 and H1584, respectively. Hydrophobic polymers (poly-
ethylene) are more stable because they have no hydrolysable
bonds (Zheng et al., 2005). In biodegradation, enzymes catalyze a
specific or series of reactions that lead to various types of chemical
conversions, such as oxidation, reduction, hydrolysis, esterification,
and molecular inner conversion (Kopecek and Rejmanova, 1983).
Keto and ester carbonyls have been reported as major products
son, 1998). Analyses were conducted to monitor the formation or
disappearance of acids (1715 cm-1), ketones (1740 cm-1) and dou-
ble bonds (1640 and 908 cm-1) to explain the mechanisms of the
biodegradation process. The formation of ester, keto, vinyl and
internal double bonds were clearly shown by FT-IR spectra, which
further confirms polyethylene biodegradation. In this study, the
biodegradation of polyethylene film was confirmed by FT-IR anal-
ysis and showed changes in functional groups and side chain mod-
ifications because of microbial activities. The KCBI, ECBI, VBI and
IDBI were increased because of enzymatic activity (Fig. 6). It has
previously been reported that the biotic environment supports
the formation of terminal double bonds (IR 905-915 cm-1) (Alberts-
son et al., 1987); the same was observed for terminal double bonds
after incubation with all three bacterial strains (Supplementary
S2a, S2b and S2c). The formation of double bonds in the polymer
chain may be due to the Norrish type II reaction proposed earlier
(Albertsson et al., 1987) or through the formation of esters.
Previous reports of polyethylene bio-degradation utilized UV-
irradiated polyethylene films (Gilan et al., 2004; Hadad et al.,
2005). The exposure to UV radiation causes increased carbonyl
and terminal double bond indices and provides functional groups
to bacteria for attachment on the polyethylene surface. In the pres-
ent study, we used non-irradiated polyethylene film and show that
106 K. Harshvardhan, B. Jha / Marine Pollution Bulletin 77 (2013) 100–106
bacteria were able to colonize and grow efficiently by using it as a
carbon source.
For decades, discussions surrounding polyethylene have
determined it to be a non-biodegradable polymer, as long as
high-molecular-weight-olefin-degrading enzymes had not been
identified (Griffin, 1983). After three decades, these enzymes have
still not been completely characterized. However, our study sup-
ports the occurrence of enzymatic activity on polyethylene as
SEM photographs of the polyethylene film showed some localized
degradation around the bacterial cells (Fig. 7a–d). A cell-like
molded pattern on the polyethylene has previously been observed
for biodegradable polymers, e.g., poly-b-hydroxybutyrate (Otake
et al., 1995; Watanabe et al., 2009).
There are four different mechanisms for the study of biodegra-
dation: solubilization, charge formation, hydrolysis and enzyme-
catalyzed degradation (Gilding, 1981; Kronenthal, 1975). Our
results supports the three mechanisms: loss of dry weight supports
solubilization, an increase in carbonyl indexes supports charge for-
mation, and SEM images support enzyme-catalyzed degradation.
4. Conclusion
Polyethylene, which has a wide range of applications, is accu-
mulating in the environment. Its inert properties that resist deteri-
oration and degradation are creating a serious environmental
concern. This in vitro biodegradation study of polyethylene sug-
gests the suitability of three marine bacteria, K. palustris M16, B.
pumilus M27 and B. subtilis H1584. Their actively increasing metab-
olism after 14 days of incubation supports the additional use of
these bacteria for biodegradation. Based on the growth results on
the polyethylene surface, hydrophobicity, metabolic activity and
FT-IR data, we were able to determine that B. subtilis H1584 is
more efficient than the other bacteria.
Acknowledgments
We thankfully acknowledge the financial support of the CSIR,
Govt. of India (W2W, CSC 0120) for making this work possible.
KH gratefully acknowledges the Senior Research Fellowship from
CSIR, New Delhi, India.
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.marpolbul.
2013.10.025.
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