Environmental Science and Pollution Research

[Link]

RESEARCH ARTICLE

Efficient biodegradation of low-density polyethylene by cyanobacteria

isolated from submerged polyethylene surface in domestic
sewage water
Pampi Sarmah 1 & Jayashree Rout 1

Received: 29 January 2018 / Accepted: 27 August 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Two dominant cyanobacterial species, Phormidium lucidum and Oscillatoria subbrevis, isolated from submerged polyethylene
carry bags in domestic sewage water were found to be capable of degrading low-density polyethylene (LDPE) sheets efficiently.
The FT-IR, SEM, NMR, CHN content, thermal, and tensile strength of PE were monitored for structural, morphological, and
chemical changes of PE. The CHN analysis corroborated about 4% carbon utilization by the cyanobacterial species from the PE.
The rapid growth of cyanobacterial species on the PE surface suggested that the microorganisms continued to gain energy from
the PE. The reduction in lamellar thickness, weight, and crystallinity of the cyanobacterial-treated PE pointed to an efficient
biodegradation process without any pro-oxidant additives or pretreatment. Alteration in bond indices computed from FT-IR
spectroscopy revealed changes in functional group and side chain features indicating biodegradation. The enhanced laccase and
manganese peroxidase activity corroborated the biodegradation. The 13C-NMR spectroscopy of the PE is consistent with short
branching providing further evidence of biodegradation. Scanning electron microscopy and optical microscopy exhibited large
grooves on the surface suggesting significant disruption of polyethylene structure.

Keywords Biodegradation . Cyanobacteria . Domestic sewage water . FT-IR . NMR

Abbreviations Introduction
LDPE Low-density polyethylene
FT-IR Fourier transform infrared Polyethylene is a ubiquitous commodity for packaging pur-
SEM Scanning electron microscopy poses throughout the world. Carry bags made of polyethylene
NMR Nuclear magnetic resonance due to its low cost and durability have myriad applications in
CHN Carbon, hydrogen, nitrogen day-to-day life such as their role in packaging, transportation
APHA American Public Health Association of textiles, manufacturing laboratory instruments, and auto-
TGA-DSC Thermogravimetry-differential scanning mobile components (Arutchelvi et al. 2008). The recalcitrant
calorimetry nature of polyethylene due to high molecular weight, complex
ASTM American Society for Testing and Materials three-dimensional structures, and hydrophobic nature causes
these polyethylene bags resistant to natural environment.
They are used as landfills and are usually thrown to natural
water bodies (Shah et al. 2009; Nanda et al. 2010). Consisting
of carbon and hydrogen polymers, polyethylene is remarkably
Responsible editor: Philippe Garrigues resistant to biological decay; it can be degraded to some extent
by sunlight and oxygen resulting brittleness and loss of tensile
* Jayashree Rout strength without proportionate loss of mass, while degradation
routjaya@[Link] by mechanical forces may merely lead to smaller pieces (Potts
Pampi Sarmah 1984). Currently, both marine ecosystem and urban areas are
[Link]@[Link] beset with problems of polyethylene disposal posing severe
1
environmental threats (Caruso 2015). Durability and undesir-
Department of Ecology & Environmental Science, Assam
able accumulation of synthetic polymers in the natural
University, Silchar, Assam 788011, India
 Environ Sci Pollut Res

ecosystem and habitats continue to be major concerns. the present study. For ascertaining degradation, the standards
Though bioremediation is considered a potential tool to re- used were as follows: ASTM D 3593 Test Method (FTIR and
duce the adverse effects, plastic waste recycling has largely NMR), ASTM D 638 Test Method (SEM), ASTM D 638 Test
remained an unsuccessful outcome (Shah et al. 2008; Ojo Method (tensile properties), and ASTM D 5247 Test Method
2007; Bonhomme et al. 2003). (weight loss).
Algae are known to colonize on such polyethylene material
submerged in waste water by mucilaginous secretion of extra-
cellular polymeric substances (EPS) (Suseela and Toppo Materials and methods
2007; Sharma et al. 2014; Sarmah and Rout 2017).
Colonization of microbial communities on polyethylene sur- Materials
faces depends largely on the environmental factors, which
may provide an ideal substratum for the colonization PE strip preparation
(Pathak and Navneet 2017). The polyethylenes thrown into
water bodies being exposed to the sunlight are broken into PE sheets of 20-μ thickness were dried under ambient condi-
small pieces by bacterial and algal attachment (Seneviratne tions and cut into strips (1 cm × 1 cm), washed with 70%
et al. 2006). Direct degradation of PE by microorganisms ethanol followed by distilled water.
utilizing only the polymer as sole carbon source has been
documented (Roy et al. 2008). Biodegradation of polyethyl-
ene is a natural process aided by microorganisms such as Isolation of cyanobacteria
bacteria, fungi, actinomycete, or algae (ASTM 1993; Ghosh
et al. 2013; Qi et al. 2017; Ahmed et al. 2018). The degrada- Domestic sewage water was collected in clean polythene bot-
tion rate of polyethylene depends on the crystallinity, surface tles; samples were transported to the laboratory, stored at 4 °C,
treatment, additives, molecular weight, and surfactants. and analyzed within few hours after arrival in the laboratory.
Further, enzymes, both extracellular and intracellular types, Standard protocols were followed for domestic sewage water
play a crucial role in biological degradation (Gu 2003). analysis (APHA 2005). Algal colonized submerged polyeth-
While polyethylene biodegradation by bacteria, fungi have ylene substrates were collected from sewage water of Silchar
been quite extensively studied; studies related to the potential town in the state of Assam, India (Fig. 1a, b). The samples
of algae have received only meager attention (Suseela and colonized on waste water submerged polyethylene carry bags
Toppo 2007; Kumar et al. 2017). Previous research dealt with were collected and observed under microscope, and two dom-
biodegradation potential of PE by Anabaena spiroides, inant species were screened for monoculture development.
Scenedesmus dimorphus, and Navicula pupula (Kumar et al. The algal samples were centrifuged, pellets were homoge-
2017). Selection of microorganisms for biodegradation of nized in sterilized BG-11(N+) medium, and the suspension
polyethylene is crucial. The microorganisms with enhanced was placed onto agar petri plates by pour plate method. The
capacity to produce oxidative and lignolytic enzymes are plates were incubated for 15 days under continuous illumina-
more efficient in the biodegradation of polyethylene (Nayak tion (2000 lx) at 24 ± 1 °C (Rippka et al. 1979). The colony
and Tiwari 2011). We conjectured that dominant algae grow- developed in the agar media has been maintained in liquid
ing on polyethylene substrata utilizing the polymer carbon in BG-11(N+) medium (Table 1). The algal samples were ob-
oligotrophic conditions could be ideal candidates for test ex- served under microscope and identified by diagnostic mor-
periments. Thus, it was deemed fit to explore the efficacy of phological features (Prescott 1952; Desikachary 1959).
cyanobacteria as polyethylene biodegrading agent.
Accordingly, the present work deals with PE biodegradation Biological treatment of PE
potential of two cyanobacterial species, Phormidium lucidum
and Oscillatoria subbrevis, obtained from algal colonized sub- Based on dominance over the occurrence of other algae on
merged polyethylene substrates in domestic sewage water. submerged polyethylene bags in domestic sewage water of
The mechanism of biodegradation has been expounded by Silchar town, two cyanobacterial species, Phormidium
spectroscopic, morphological, mechanical, and thermal lucidum and Oscillatoria subbrevis, were selected for PE bio-
studies. The ASTM (2000) standards were followed for the degradation assay. The PE strips were sterilized before incu-
present study. The ASTM D 5338-98 has been chosen for the bation. In each conical flask, 400 ml of BG-11(N+) media
tests in determining the aerobic biodegradation of polyethyl- with minimal carbon source and 4 ml of cyanobacterial culture
ene materials under controlled composting conditions. The were added. Ten pieces of pre-weighed PE strips (1 cm ×
aqueous test method, Test Method D 5247 (specific microbe 1 cm) were aseptically transferred to the flasks with mild
test), which uses pure microbial cultures to assess the biode- shaking (Fig. 2). PE strips in BG-11 medium with no
gradability of materials based on weight loss has been used in cyanobacteria added served as an abiotic control and that with
Environ Sci Pollut Res

Fig. 1 a, b Algal colony on PE

surface. Photomicrographs of c
Phormidium lucidum and d
Oscillatoria subbrevis. e, f Algae
colonizing on test PE strip

only cyanobacteria, and no PE strips were set up in parallel as Model: JSM 6390 LV). The Pt-coated PE strips were placed
a biotic control. on the sample holder and scanned at magnifications of × 1000,
× 3000, × 5000, and × 10,000.
Biodegradation studies
FT-IR spectroscopy
Optical microscopy
The structural changes in PE surface were analyzed by a
The changes on the surface of PE were monitored at × 80 Nicolet (USA) equipment (Model: Impact 410) at a resolution
magnification with an optical reflection microscope of 1 cm−1, in the frequency range of 4000–400 cm−1. Relative
(StereoZoom Leica S8 APO). absorbance intensities (I) of the ester carbonyl bond at
1740 cm−1, keto carbonyl bond at 1715 cm−1, terminal double
SEM analysis bond (vinyl) bond at 1650 cm−1, and internal double bond at
908 cm−1 to that of the methylene bond at 1465 cm−1 were
The surface morphology of the PE strip was observed through evaluated (Albertsson et al. 1987): keto carbonyl bond index
scanning electron microscopy (JEOL equipment, Japan, (KCBI) = I1715/I1465, ester carbonyl bond index (ECBI) =
I1740/I1465, vinyl bond index (VBI) = I1650/I1465, internal dou-
Table 1 The composition of the BG-11 medium per 1000 ml of distilled ble bond index (IDBI) = I908/I1465. The crystallinity of the PE
water strips was measured using the formula: crystallinity (%) =
100 − [(1 − (Ia / 1.233Ib) / 1 + (Ia/Ib)) × 100]; Ia and Ib are the
Ingredients Amount (g)
intensities of absorption bands at 1474 and 1464 cm−1, respec-
Sodium nitrate 1.500 tively (Zerbi et al. 1989). The constant 1.233 corresponds to
Dipotassium hydrogen phosphate 0.0314 the relations of intensity bands of fully crystalline PE.
Magnesium sulfate 0.036
Calcium chloride dihydrate 0.0367
Sodium carbonate 0.020 CHN analysis
Disodium magnesium EDTA 0.001
Citric acid 0.0056
The percentage of carbon, hydrogen, and nitrogen was
Ferric ammonium citrate 0.006
measured using 0.1 g of dried, ground, and homogenized
PE sample using a CHN Analyzer (Perkin Elmer, USA
Final pH after sterilization (at 25 °C): 7.1 Model: 2400 Series 2).
 Environ Sci Pollut Res

Fig. 2 Biological treatment of PE

strips with a Phormidium lucidum
and b Oscillatoria subbrevis

TGA-DSC analysis strips of both control and treated were dissolved in 1,2,4-
trichlorobenzene in 10-mm tubes at 120 °C and a few drops
Melting point (Tm) of the 0.1-g PE strips and enthalpy changes of dimethyl sulfoxide were added as an internal lock.
were analyzed by a Shimadzu Thermal Analyzer (TGA-DSC) Hexamethyldisiloxane was used as chemical shift reference.
(Model: TGA-50 & DSC-60). The lamellar thickness, Lc Spectra were recorded in complete decoupling mode under the
(Lc = 2σe/Δh (Tm0/Tm0-Tm), was estimated from the melting following conditions: pulse interval, 1 s; pulse delay, 5 s; spec-
point following a modified procedure (Hoffman et al. 1976). tral width, 500 Hz; number of accumulations, 1500–2000;
number of data points per spectrum, 8000.
Tensile property
Enzyme activity
The tensile strength, percentage of elongation at break and
modulus of elasticity, and the extension of the material under A 1-ml culture from both cyanobacterial treatments was added
load were measured by a universal testing machine (Model: separately to 1 ml of 2 mM guaiacol and 3 ml of 10 mM
KUT-100 (E) UTNSRL). sodium acetate buffer (pH 4.6). The reaction mixture contain-
ing guaiacol and sodium acetate was incubated at 30 °C for
Cyanobacterial growth and PE degradation 15 min, and the absorptions were recorded in a UV-Vis spec-
trophotometer at 450 nm. One unit of laccase activity was
The growth of cyanobacteria in BG-11 only and on PE strip defined as amount of enzyme required to hydrolyze guaiacol
was measured separately by estimating the chlorophyll a on during incubation period. For manganese peroxidase, a similar
each alternate day for a period of 6 weeks (Kobayasi 1961). procedure was followed with 1 ml of H2O2 added (Papinutti
The growth curves and specific growth rates (μ h−1) were and Martinez 2006).
calculated in log period as per Myers and Kratz (1955). The
total carbohydrate (Spiro 1966) and protein (Herbert et al. Data availability All data generated or analyzed during this
1971) content were analyzed. The extracellular polymeric study are included in this published article.
substances of the cyanobacterial species were estimated by
the method of Underwood et al. (1995). The weight changes
of PE strips were determined using an electronic balance Results and discussion
(Mettler Toledo RS 231C).
Domestic sewage water quality and cyanobacteria
NMR spectroscopy
The temperature of natural water bodies is an important pa-
The PE strips were analyzed using 13C-NMR spectroscopy on rameter for cyanobacterial growth. The water temperature was
a JEOL equipment (Japan Model: ECS-400). For analysis, PE recorded to be 32–34 °C during the summer when algal
Environ Sci Pollut Res

colonized polyethylenes were collected. The color of the

waste water was black. The values of BOD, DO, and COD
were 600 ± 0.34, 1.2 ± 0.04, and 1502 ± 0.32 mg/l, respective-
ly. The total alkalinity was 10 ± 1.4 mg/l, free CO2 was 64 ±
0.13 mg/l, and TDS was found to be 560 ± 0.18 mg/l. The
suspended solid was 50 ± 0.54 mg/l. The chloride and calcium
contents were found to be 168 ± 0.67 mg/l and 68 ± 0.13 mg/l,
respectively. The sulfate, nitrate, and magnesium concentra-
tions were 214 ± 1.6 mg/l, 12 ± 1.5 mg/l, and 30 ± 0.23 mg/l,
respectively. The ammonia and phosphate contents were 30 ±
0.45 mg/l and 70 ± 0.45 mg/l, respectively. The physicochem-
ical characteristics of the domestic sewage water of Silchar
town were rather similar to those reported recently (Sarmah
and Rout 2017).
The colonization of Phormidium lucidum and Oscillatoria
subbrevis on PE surface and photomicrographs of isolated
cyanobacteria are given in Fig. 1a–d. The species,
Phormidium lucidum and Oscillatoria subbrevis, were found
to be profusely distributed in submerged polyethylene surface
of domestic sewage water with the latter recorded as the largest
genus forming mat-like colonization in submerged polyethyl-
ene bags. The genera, Phormidium and Oscillatoria, were also
found to occur on PE surfaces elsewhere. In a recent field-
based study conducted in oligotrophic water bodies of
Lucknow, Uttar Pradesh, 15 algal genera, viz., Chaetophora,
Coleochaete scutata, Coleochaete soluta, Aphanochaete,
Gloeotaenium, Oedogonium, Oocystis, Oscillatoria,
Phormidium, Chroococcus, Aphanothece, Fragilaria, Fig. 3 Surface morphology of PE strip. a Abiotic control. b Phormidium
Cocconis, Navicula, and Cymbella, were found to be colonized lucidum-treated PE. c Oscillatoria subbrevis-treated PE strip after
6 weeks
on the surface of polyethylene (Suseela and Toppo 2007).
Several species of algae, Phormidium tenue, Oscillatoria
tenuis, Navicula cuspidata, Monoraphidium contortum, growing on polyethylene surface were found to be higher
Microcystis aeruginosa, Closterium costatum, Chlorella relative to the biotic control. Accordingly, the specific growth
vulgaris, and Amphora ovalis, were found to colonize on waste rate (doubling time) of the cyanobacteria on polyethylene sur-
polyethylene materials in various ponds, lakes, and water bod- faces was also found to be higher than that of the biotic con-
ies of Kota City in Rajasthan (Sharma et al. 2014). trol. This provided clear evidence of PE carbon utilization by
the cyanobacteria. Studies on degradation of polypropylene
Optical microscopy and bioriented polypropylene in the field condition have
shown that microorganisms adhered the surface of the samples
The algae growing on PE surface in the field were observed to initiate biodegradation (Longo et al. 2011). The species
under the microscope to check the attachment of microorgan- belonging to the families Chlorophyceae, Cyanophyceae,
isms to the polyethylene surface (Arutchelvi et al. 2008). The and Bacillariophyceae are the common occurrence on the sur-
colonization of Phormidium lucidum and Oscillatoria face of the polyethylene bags (Kumar et al. 2017).
subbrevis on the experimental PE (Figs. 1e, f and 3) started
after 1 week of inoculation which is attributed to initial deg- SEM analysis
radation leading to insertion of hydrophilic groups to the PE
strips as microorganisms prefer hydrophilic surface (Vasile The adhesion of algae to the PE surface is a fundamental
1993).The cyanobacterial species growing on the polyethyl- prerequisite for biodegradation. High surface hydrophobicity
ene surfaces were firmly attached on the PE surfaces and are of polyethylene surface limits the alga colonization process.
not removable by water jet. It is presumed that the Surface erosion, formation of pits, and cavities on the surface
cyanobacterial colonization on the PE surface occurred utiliz- of the treated PE were observed after 6 weeks of treatment
ing the polymer as carbon source (Kumar et al. 2017). The (Fig. 4a1, a2, b1, b2, c1, c2). The surface of the strips treated by
cellular contents (carbohydrates and protein) of the species Oscillatoria subbrevis registered relatively higher damage
 Environ Sci Pollut Res

displayed a number of absorptions reflecting the complex na-

ture of the PE (Fig. 5a). Variations were noted in the intensity
of bands in different regions when incubated with
Phormidium lucidum and Oscillatoria subbrevis (Fig. 5b, c).
Significant changes were noted for both the cyanobacterial
species. The intensity of some peaks (2364 cm−1) reduced
more in Oscillatoria subbrevis whereas those at 2326 and
2850 cm−1 became sharper in the treated sample than those
in the control. The peak observed at 2919 cm −1 in
Phormidium lucidum-treated strips attributed to free OH com-
bination while a new peak at 1633 cm−1 corresponding to
C=O in Oscillatoria subbrevis adduced support to depolymer-
ization activity of the cyanobacterial isolates. A peak at
1369 cm−1 in the control PE strip usually observed for
1000C short branching is found missing in the treated PE
strips presumably owing to cyanobacterial degradation (Blitz
and McFaddin 1994). The FT-IR spectrum of Phormidium
lucidum-treated PE showed peak at 3422 cm−1 assigned to
υOH mode indicating the occurrence of alcohol.
The peak at 1629 cm−1 for υCO mode originated from the
presence of carboxylic acid group. For Oscillatoria subbrevis-
treated PE, peaks at 667 and 468 cm−1 confirmed the presence
of nitrogen-containing bio-ligands. The υ CO peak at
1633 cm−1 is concordant with the presence of carboxylic
group. The FT-IR signatures of ester, keto, vinyl, and internal
Fig. 4 Scanning electron micrographs of the surface of abiotic control PE double bond were quite diagnostic, and the corresponding
strip (a1, × 3000) (a2, × 10,000) and PE strip after incubation with indices validated PE biodegradation (Albertsson et al. 1987;
Phormidium lucidum (b1, × 3000) (b2, × 10,000) and Oscillatoria
Gardette 2006). The KCB, ECB, VB, and IDB indices were
subbrevis (c1, × 3000) (c2, × 10,000) (treatment with BG-11 media and
2% SDS) all found to be higher in the treated Phormidium lucidum case.
A relatively higher value of the KCB and EBC of the
cyanobacterial-incubated PE strips has been attributed to en-
than that of Phormidium lucidum-treated strips. The
zymatic activity of the organisms (Albertsson et al. 1994) (Fig.
Anabaena spiroides and Navicula pupula colonization on
6). However, when incubated with Oscillatoria subbrevis, the
polyethylene surface exhibited rather similar surface features
bond indices KCB, ECB, and VB though registered an in-
(Kumar et al. 2017). The SEM images provided evidence for
crease, the IDB got reduced. In a previous study involving
the breakdown of the PE into its monomeric components
bacteria, Arthrobacter sp., an increase in the bond indices
(Sanin et al. 2003). The grooves and cracks confirmed the
was noted while that with Pseudomonas sp., the IDB index
fragility of the PE strip on treatment with the cyanobacterial
was reported to be lower (Balasubramanian et al. 2010). The
strains. Comparison with the control sheet clearly attested
keto and ester carbonyls have been reported as major degraded
degradation. The grooves on the surface testify carbon utiliza-
products in the presence of enzyme oxidoreductase (Karlsson
tion from the surface of polymer which upset the uniform
and Albertsson 1998). The formation of double bonds in the
branching of polymer matrix (Manzur et al. 2004). The corro-
treated PE strip may have been effected by Norrish type II
sion of PE surface by cyanobacteria was scattered, not uni-
photochemical reaction (Albertsson et al. 1987; Chiellini
form, indicating that the amorphous region of the polymer was
et al. 2003). The crystallinity of PE strips was found to de-
more susceptible to cyanobacterial adhesion and degradation.
crease up to 2% and 62% after incubation with Phormidium
It is generally believed that cyanobacterial attack is mainly
lucidum and Oscillatoria subbrevis, respectively. The free rad-
limited to surface or near-surface accessible particles
ical driven chain scission may have disrupted the crystalline
(Albertsson et al. 1994).
order (Restrepo-Flórez et al. 2014). The cyanobacteria, in the
present case, are believed to access the amorphous regions of
FT-IR spectroscopy the polymer most (Roy et al. 2008; Khabbaz et al. 1999;
Patani and Sorrentino 2013). Cyanobacterial interaction may
FT-IR spectroscopy has been used to assess the biodegrada- enhance the surface hydrophilicity of the PE by the formation
tion of polyethylene strips. Spectra of control PE strip of additional groups such as Bcarbonyl^ that can be utilized by
Environ Sci Pollut Res

Fig. 5 FT-IR spectra of PE. a

Abiotic control. b Phormidium
lucidum treated. c Oscillatoria
subbrevis treated

the microorganisms (Albertsson 1980; Ibiene et al. 2013). The treated PE strip, a key marker for biodegradation. It is perti-
carbonyl index increased considerably for both the algal- nent to mention herein that 2% SDS buffer solution has been

Fig. 6 Carbonyl and double bond

indices as determined from FT-IR
data of pre- and post-treated
(6 weeks) PE
 Environ Sci Pollut Res

used for washing the PE strips before recording the FT-IR melting points and may be explained by invoking the
spectra to remove any extraneous substances such as micro- cyanobacteria’s quest for energy with concurrent cleaving
bial metabolites or culture media from the strips (Gu 2017). and rearrangement of chemical bonds.

CHN analysis Tensile property

Carbon analysis after 6 weeks of incubation revealed the per- The changes in the tensile strength and elongation at break due
centage of carbon in the control PE strips to be 84%. The to cleavage of the polymer chain by the cyanobacterial species
extent of carbon utilized by Phormidium lucidum from the were monitored. The cyanobacterial-treated PE strips expect-
PE strip was 3% whereas Oscillatoria subbrevis utilized 4%. edly registered a reduction in tensile strength to ~ 7 from
For polyethylene treated with bacteria, Achromobacter 11.8 MPa. The elongation break of Phormidium lucidum-
denitrificans have shown 2% of carbon being utilized from and Oscillatoria subbrevis-treated PE strips was observed at
polymer by the bacteria after long exposure (Devi et al. 2015). 254 and 243 compared to 299% of extension of control strip.
The hydrogen and nitrogen in control PE strip were 14.50% A reduction of 42.5% tensile strength of PE, 31.5% reduction
and 0.12%, respectively. The treated ones showed hydrogen in elongation, and 28.8% reduction in modulus of elasticity
and nitrogen to be about 15% and 0.1%, respectively. Low for polyethylene were noted earlier following biodegradation
nitrogen availability (0.12%) is a limiting factor for microbial (Devi et al. 2015).
growth. The adhesion mechanism of the cyanobacteria to PE
surface may be attributed to the low carbon availability in the Growth study and PE degradation
medium and confirms its ability to use the PE as a carbon and
energy source (Awasthi et al. 2017a). Cyanobacterial growth on test PE got initiated from the first
week of treatment itself. Cyanobacterial growth in PE strips
TGA-DSC analysis was studied with reference to the biotic control (without PE
strips, Fig. 8b). The growth of cyanobacterial species on PE
Melting points for the treated PE strips were found to be was found to be higher in terms of chl a by about 3.5 μg/ml
slightly lower than those for the control (Fig. 7a). For from that of the respective biotic control at the end of 42 days.
Phormidium lucidum, onset temperature was 121.56 °C (heat The fast growing Phormidium lucidum showed specific
enthalpy − 42.17 mJ) (Fig. 7b). For Oscillatoria subbrevis, the growth rate (μ) in biotic control and on PE strips to be 0.134
onset temperature was 121.37 °C (heat enthalpy − 74.41 mJ ± 0.12 (generation time, G = 178.25 ± 0.45 h) and 0.123 ± 0.12
(Fig. 7c).The results from the present study demonstrated that (G = 182.21 ± 0.23 h), respectively. The fast growing
heat release was relatively much higher in Oscillatoria Oscillatoria subbrevis showed specific growth rate (μ) in biot-
subbrevis. For biodegradation of polyethylene by the bacteria ic control and on PE strips to be 0.158 ± 0.23 (generation time,
Aspergillus sp., a reduction in melting point by 1.2 °C has G = 151.34 ± 0.67) and 0.143 ± 0.23 (G = 156.24 ± 0.37), re-
been noted (Raaman et al. 2012). The melting and crystalliza- spectively. This can be attributed to the acclimatization of
tion temperature of PE treated by Phormidium lucidum is cyanobacteria in the PE strips with the degraded carbon source
found to be higher than that treated by Oscillatoria subbrevis from the polyethylene (Arutchelvi et al. 2008). The algal
in the present experiment. Formation of high molecular growth increased with time and after a period of 6 weeks found
weight substances in Phormidium lucidum-treated case is be- to level off. In the present case, the BG-11 medium used had
lieved to be one primary reason for this (Farukkawa et al. citric acid, ferric ammonium citrate, EDTA, and Na2CO3 as
2006). The TGA traces (Fig. 8a) of cyanobacterial-treated sources of minimal carbon, and hence to annul the effect of
PE strips showed that initial decomposition temperature of such chemicals on the polyethylene substratum, control exper-
the control PE strip decreased from 88 to 20 °C iments were simultaneously performed with the BG-11 medi-
(Phormidium lucidum) and to 18 °C (Oscillatoria subbrevis), um alone (Gu 2017). The enhancement of cellular contents
respectively. The weight loss versus temperature curve (carbohydrates and protein) of the species growing on PE sur-
(Fig. 8a) showed a marked decrease in thermal stability of face was also observed. The carbohydrate content on PE and
the treated PE strips. Similar decrease in the onset tempera- biotic control for Phormidium lucidum were 245 ± 0.26 μg/ml
tures and corresponding weight loss were also observed in and 240 ± 0.56 μg/ml, respectively. The carbohydrate content
fungus-treated polyethylene films, though, in the presence of on PE and biotic control for Oscillatoria subbrevis were 377 ±
pro-oxidant (Corti et al. 2010). The lamellar thickness (Lc ~ 0.26 μg/ml and 370 ± 0.56 μg/ml, respectively. The protein
110 nm) for control PE strip following cyanobacterial expo- content of Phormidium lucidum on PE and biotic control were
sure got reduced to 97.78 nm and 54.45 nm for Phormidium 214 ± 0.12 μg/ml and 210 ± 0.56 μg/ml, respectively. The pro-
lucidum and Oscillatoria subbrevis treatments, respectively. tein content of Oscillatoria subbrevis on PE and biotic control
The results are congruent with the observed trends in the were 240 ± 0.12 μg/ml and 230 ± 0.56 μg/ml, respectively.
Environ Sci Pollut Res

Fig. 7 Melting points of PE. a

Abiotic control. b Phormidium
lucidum treated. c Oscillatoria
subbrevis treated

Studies also revealed that gram-negative bacteria could easily environmental conditions (Garrett et al. 2008). The
adapt to the environment rich in polyethylene and similar poly- cyanobacterial colonization on the polyethylene surface was
mers. Since cyanobacteria are gram-negative, it is reasonable observed to be maximum at the end of log phase.
to conclude that they colonize the PE surface utilizing the Extracellular polysaccharide (EPS) is believed to have aided
polyethylene as partial carbon source (Dey et al. 2012). The in intercellular adhesion of cyanobacteria to the polyethylene
structure of cell wall of gram-negative bacteria comprising surface (Dunne 2002). In the present study, EPS of
either homopolysaccharides or heteropolysaccharides is cyanobacterial species were estimated to be 19.99 and
known to impart mechanical stability enabling adhesion and 22.43 μg/ml for Phormidium lucidum and Oscillatoria
cohesion on the polymeric surface and negotiate dynamic subbrevis, respectively. The rapid growth of cyanobacteria on
 Environ Sci Pollut Res

Fig. 8 a Change in the thermal

behavior of PE due to
cyanobacterial exposure (abiotic
control, (a) Phormidium lucidum,
(b) Oscillatoria subbrevis). b
Growth pattern of algae on PE for
a period of 6 weeks. c Percentage
of weight loss of PE after incuba-
tion with algae, *PL Phormidium
lucidum, PLP Phormidium
lucidum on PE, OS Oscillatoria
subbrevis, OSP Oscillatoria
subbrevis on PE

the PE may be attributed to metabolizable compounds from the Oscillatoria subbrevis-treated PE strips, was found to be about
polymers (Koutny et al. 2006a). It is assumed that a significant 30% after 42 days (Fig. 8c). In the abiotic control (without
amount of low molecular weight compounds is released to inoculation), no significant weight loss was observed, thus
aqueous media from the PE strips (Koutny et al. 2006b). ruling out any effect of the nutrient chemicals of the culture
The degradation for PE strips by the cyanobacterial species medium on the polyethylene substratum. Previous study in-
was monitored weekly by weight loss measurements. Weight volving polyethylene degradation by Anabaena spiroides,
loss of polyethylene is an important and well-recognized in- Scenedesmus dimorphus, and Navicula pupula recorded
dicator of biodegradation. The highest reported weight loss so weight loss up to 8% only (Kumar et al. 2017). The reduction
far by any microorganism appears to be ~ 60% after 3 months in weight of cyanobacterial-treated PE strips in the present
(Rajandas et al. 2012). In the present case, the weight loss study vis-a-vis the dual control used in the experiment un-
associated with the cyanobacteria, Phormidium lucidum- and equivocally represents significant biodegradation by the tested
Environ Sci Pollut Res

end product and initiation of short branching due to degrada-

tion. It is quite plausible that degraded polyethylene furnished
an organic soluble fraction implying a carbon uptake process
linked to the metabolic pathway of the cyanobacteria
(Miyazaki et al. 2012; Balasubramanian et al. 2014).
Formation of carboxylic acid and other byproducts of polyeth-
ylene degradation is believed to facilitate carbon assimilation
(Arnaud et al. 1994).

Enzymatic activity

Activity of laccase (0.009 IU/ml) was higher as compared

to manganese peroxidase (0.0075 IU/ml) after 6 weeks
treatment for both the cyanobacteria. High molecular
weight polymer limits the enzymatic reactions leading to
biodegradation. Two key mechanisms are believe to be
operative; one is the reduction of molecular weight and
the other oxidation of molecules (Yoon et al. 2012). The
breakdown of large polyethylene molecules is in fact be-
lieved to have been initiated by enzymatic action accom-
panied by molecular weight reduction and enhancement of
keto carbonyl index in the present case (Santo et al. 2012).
Microorganisms are known to produce necessary oxidative
and degradative enzymes and assimilate the polymeric car-
bon into their biomass (Hadad et al. 2005; Tribedi and Sil
2013). We conclude that cyanobacterial enzymes present in
the liquid phase of the media interact with the macromol-
ecules available at the surface of the PE strips triggering
Fig. 9 13C-NMR of PE. a Abiotic control. b Phormidium lucidum biodegradation (Chinaglia et al. 2018).
treated. c Oscillatoria subbrevis treated

Conclusion
cyanobacteria (Awasthi et al. 2017b). In a recent study, about
81 ± 4% and 38 ± 3% weight loss was observed for LDPE Spectroscopic, enzymatic, thermal, mechanical, and morpho-
strips and pellets, respectively, by Enterobacter sp. and logical studies in relation to dual experimental control clearly
Pantoea sp. bacterial consortia screened from plastic garbage demonstrated efficient polyethylene biodegradation by the
processing area (Skariyachan et al. 2016). cyanobacteria. The fast growing, readily available and easily
isolable cyanobacteria are capable of effectively colonizing on
NMR spectroscopy the PE utilizing carbon without any pro-oxidant additives or
pretreatment. The results are of significance for development
The 13C-NMR spectroscopy (Fig. 9) of the cyanobacterial- of biodegradation protocol for PE using freshwater nontoxic
treated PE vis-à-vis the untreated PE ones (abiotic control) cyanobacteria which are more efficient besides being conve-
revealed microstructural changes in the polyethylene. The ab- nient, easy to handle, and less hazardous as compared to other
sorption peaks centered at 20 ppm in the control and treated PE bacteria or fungi. Under natural conditions, these
strips are believed to have originated from common plastic cyanobacterial species have the potential to degrade polyeth-
additives like phosphoric acid esters. The carbon signals ob- ylene even more efficiently furnishing a tangible alternative
served at 35 (multiplet) and 36.20 ppm in the control PE got solution to polyethylene waste management.
altered slightly both in terms of position and multiplicity in the
Acknowledgements The authors thank Sophisticated Analytical
treated ones (Fig. 9b, c). Multiplet character of the signal at
Instrumentation Centre (SAIC), Tezpur University, Napaam, Assam,
34.5 ppm in the treated polyethylene can be assigned to car- India, for providing some instrumentation facilities. One of the author
bonyl group of the acid moiety (Brandolini and Hills 2000). (PS) acknowledge University Grant Commission (UGC) for fellowship.
This observation may be correlated with formation of ester as
 Environ Sci Pollut Res

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