Journal of Hazardous Materials 324 (2017) 634–644

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journal homepage: www.elsevier.com/locate/jhazmat

Brazilian Cerrado soil reveals an untapped microbial potential for

unpretreated polyethylene biodegradation
Julianna Peixoto a , Luciano P. Silva b , Ricardo H. Krüger a,∗
a
Laboratory of Enzymology, Cellular Biology Department, Biological Sciences Institute, University of Brasilia, Brasilia, 70910-900, DF, Brazil
b
Laboratory of Nanobiotechnology, Embrapa Genetic Resources and Biotechnology, Brasilia, 70770-917, DF, Brazil

h i g h l i g h t s g r a p h i c a l a b s t r a c t

• 9 novel PE-degrading bacteria were

isolated from Cerrado soil.
• PE-degrading bacteria are
from Comamonas, Delftia and
Stenotrophomonas genera.
• Strains are capable of degrading PE
of 191.000 without additives or pre-
treatments.
• Nitrogen metabolism is involved in
the chemical modification of PE.

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

Article history: Discarded PE-based products pose a social and environmental threat because of their recalcitrance
Received 6 May 2016 to degradation, a consequence of the unique set of PE’s physicochemical properties. In this study
Received in revised form 7 October 2016 we isolated nine novel PE-degrading bacteria from plastic debris found in soil of the savanna-like
Accepted 13 November 2016
Brazilian Cerrado. These bacterial strains from the genera Comamonas, Delftia, and Stenotrophomonas
Available online 14 November 2016
showed metabolic activity and cellular viability after a 90-day incubation with PE as the sole car-
bon source. ATR/FTIR indicated that biodegraded PE undergone oxidation, vinylene formation, chain
Keywords:
scission, among other chemical changes. Considerable nanoroughness shifts and vast damages to
Biodegradation
Polyethylene
the micrometric surface were confirmed by AFM and SEM. Further, phase imaging revealed a 46.7%
Comamonas decrease in the viscous area of biodegraded PE whereas Raman spectroscopy confirmed a loss in
Stenotrophomonas its crystalline content, suggesting the assimilation of smaller fragments. Intriguingly, biodegraded PE
Delftia chemical fingerprint suggests that these strains use novel biochemical strategies in the biodegradation

∗ Corresponding author at: Laboratório de Enzimologia, Instituto de Ciências Biológicas, Bloco K, Sala K-10/4, Campus Darcy Ribeiro, Universidade de Brasília, Brasília
70910-900, DF, Brazil.
E-mail addresses: [email protected] (J. Peixoto), [email protected] (L.P. Silva), [email protected] (R.H. Krüger).

http://dx.doi.org/10.1016/j.jhazmat.2016.11.037
0304-3894/© 2016 Elsevier B.V. All rights reserved.
 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644 635

process. Our results indicate that these microbes are capable of degrading unpretreated PE of very high
molecular weight (191,000 g mol−1 ) and survive for long periods under this condition, suggesting not
only practical applications in waste management and environmental decontamination, but also future
directions to understand the unraveled metabolism of synthetic polymers.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction containing microorganisms that actively regulate almost every bio-

geochemical cycle [17,18]. Thus, we infer that the Cerrado soil
Among the various commercially available synthetic polymers, microbiome provides a wide range of metabolic pathways that can
polyethylene (PE) is unquestionably the most utilized. It is fre- contribute to the PE degradation process. In this context, the aim of
quently found in modern products and has a worldwide production this study is to isolate and identify PE-degrading microbial strains
of about 150 million tons per year [1–3]. The wide range of applica- colonizing plastic debris from this biome, as well as to characterize
tions for this material is due to its versatility, durability, resistance, their degradation effects.
and low cost—necessary features to meet the high demands of mod-
ern society [4,5]. Despite the broad applicability of this material,
almost 40% of the total production is destined for single-use pack- 2. Materials and methods
aging [1], highlighting the importance of plastic disposal as a global
problem. The lack of effective strategies to manage post-consume 2.1. Microbial isolation and culture conditions
plastics results in the accumulation of these degradation-resistant
and long-lasting synthetic materials in natural environments, with Plastic debris with strong evidence of degradation, such as
negative impacts on both humans and wildlife [2,3]. cracks, holes, and changes in color and texture, were collected
Although biodegradable plastics have been developed to replace from Cerrado soil (Fig. S1), which is a moderately drained and deep
PE or at least minimize its production [4,6], their rates of degrada- oxisol with a color range from dark red to yellow, a main gran-
tion in natural environments are not well studied, and some have ulometric content of clay (15–80%) and sand, a medium to high
been shown to persist for many years [1,7]. Even if these new mate- carbon content, an acidic pH ranging between 4.3 and 4.9 and a
rials are proved to be a viable solution, strategies are still needed high aluminum saturation [17,19]. The plastic-colonizing microor-
to degrade the inert plastics produced over the last century and to ganisms were suspended in 0.9% NaCl solution (saline buffer),
minimize their dramatic effects on the environment. One promis- which was used to inoculate culture plates containing Nutrient
ing approach is biodegradation, which is the metabolism of these Broth, R2A Broth, Czapek-Dox Broth, Middlebrook Broth, or Marine
compounds by microbes to yield energy for cellular machinery, Broth (Difco, Netherlands) with 15 g/L agar (Acumedia, USA). Sin-
essential carbon compounds, and secondary metabolites that may gle colonies were subcultured overnight in their respective liquid
be useful in biotechnology and commercial applications [8]. media on a rotary shaker (150 rpm). The bacterial isolates were then
However, PE biodegradation is not a trivial process, since this stored at −80 ◦ C with 15% glycerol (Sigma). All bacterial cultures
polymer is extremely resistant to microbial attack [9] due to its were maintained at 28 ◦ C during the study.
molecular structure and chemical composition [10]. PE consists of
very long chain alkanes with high molecular weight, exceeding the
reported limit of 500 g mol−1 for microbial assimilation of alkanes 2.2. Mineral oil degradation assay
[11]. In addition, its highly hydrophobic nature together with the
hydrophilicity of enzymes synthesized by microorganisms inhibits The isolated bacterial strains were first tested for their abil-
formation of the enzyme-substrate complex. Its long hydrophobic ity to degrade mineral oil, a mixture of alkanes of low molecular
carbon chain triggers chain folding, resulting in crystalline, inter- weight. The bacterial isolates were grown in 5 mL of Nutrient Broth
phase and amorphous regions [12]. The interphase regions are medium for 24 h, pelleted by centrifugation at 7000 × g for 5 min,
known to host the beginning of PE degradation [13]. In contrast, and washed 3× with saline buffer for complete removal of culture
crystalline structures are particularly stable and closely packed medium. The bacterial strains were then cultivated in a carbon-
and therefore not readily accessed by enzymes, whereas amor- free mineral synthetic 1.5% agar medium as described by Pridham
phous regions are constituted by inflexible long molecules of higher & Gottlieb [20]. The medium was supplemented with 0.1% mineral
molecular weight that are resistant to enzyme modification. Sev- oil (Sigma Aldrich, USA) as the sole carbon source [21], the cul-
eral studies have reported that PE biodegradation is possible after tures were incubated for 7 days and colonies were selected based
abiotic pretreatment (chemical, thermal, and/or photo-oxidation), on clear-zone formation.
which decreases its hydrophobicity and molecular weight to facil-
itate biotic degradation. However, evidence for the biodegradation
of untreated PE still remains very limited, since up to date only 2.3. Screening of PE-degrading bacterial strains
two bacterial strains, Enterobacter asburiae YT1 and Bacillus sp. YP1,
were shown to degrade untreated PE with an average molecular Mineral oil-degrading bacteria were cultured in glass tubes con-
weight (Mw) of 88,200 [14]. taining 3 mL minimal synthetic media supplemented with 0.1%
Microorganisms comprise more than 60% of the earth’s biomass ultra-high molecular weight PE powder (Sigma Aldrich, USA) in
[15], suggesting the possibility of additional microorganisms capa- three replicates, and maintained in a rotary shaker (150 rpm) for
ble of utilizing PE as a carbon source. Indeed, an increasing number 12 days. RNA was isolated using TRIzol Reagent (Invitrogen, USA)
of microorganisms have been found to be related to this process according to the manufacturer’s instructions. Purified RNA was
[16]. Our previous study demonstrated that the microbial com- resuspended in deionized formamide (Sigma Aldrich, USA), ana-
munity from the soil of the Brazilian biome Cerrado, a neotropical lyzed by denaturing formaldehyde agarose gel electrophoresis and
savanna located in central Brazil, is exceptionally rich and diverse, quantified with the Qubit fluorometer (Invitrogen, USA) using the
RNA Quant-it assay kit (Invitrogen, USA) [22].
636 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644

2.4. Progress of bacterial adhesion and viability on PE films 2.8. Scanning electron microscopy (SEM) of PE

Strains that showed metabolic activity after cultivation with PE To characterize surface topography, the disinfected PE samples
as the sole carbon source were grown overnight in 10 mL Nutri- were platinum coated under high vacuum conditions and imaged
ent Broth medium in three replicates for 24 h. The grown cells in using a field emission scanning electron microscope (JSM-7001F;
late log phase were then washed three times with saline buffer and Jeol, Japan) with an acceleration voltage of 5 kV and magnification
inoculated into flasks containing 30 mL mineral synthetic medium of 4500 X. The entire surfaces of the PE films were scanned in order
and a piece of approximately 0.5 cm2 of low-density (0.92 g cm−3 , to obtain representative images.
ISO 1183-A) untreated PE film (Ipethene 111, 100 ␮m thickness,
Carmel Olefins, Israel) without additives with an average molecular 2.9. Atomic force microscopy (AFM) of PE
weight of 191,000 (GPC provided by the manufacturer), melt flow
rate (190 ◦ C/2.16 kg, ISO 1133) of 0.7 g 10 min−1 and a peak melting The disinfected PE film samples were analyzed by AFM (SPM
temperature by DSC of 109 ◦ C (ISO 11357-3) [23]. Cultures were 9600; Shimadzu, Japan) in air at room temperature, using rectangu-
maintained in a rotary shaker for 12, 15, 20, or 90 days. PE films lar silicon cantilevers (tip radius <10 nm), nominal spring constant
containing adherent microbes were immersed into 1 mL of saline of 42 N/m, and resonance frequency of ∼260 kHz. Images were
phosphate buffer then stained using the Live/Dead BacLight Bac- obtained in dynamic-phase mode with a scanner displaying a max-
terial Viability Kit (Invitrogen, USA) and analyzed by fluorescence imum of 125 ␮m in X-Y directions and 7 ␮m in the Z direction.
microscopy. Negative controls for each isolate were incubated SPM-9600 offline software was used to process the AFM images
under the same culture conditions, except by the addition of a glass with only horizontal leveling and plane fitting of the surfaces. For
sheet instead of a PE film. For Gram-positive and Gram-negative each PE film, 10 replicates were performed. Surface analyses of the
strains, respectively, Bacillus subtilis and Escherichia coli were also images were then performed, and nanoroughness parameters were
incubated with PE film as controls to estimate the influence of determined. Subsequently, the AFM images were rendered using
adhesion mechanisms and electrostatic interactions between cells the same software for qualitative evaluation [26]. Finally, phase
surfaces and PE. The entire PE films and glass sheets surfaces were images were also analyzed, and the proportion of viscous areas (%)
analyzed under a 40 X magnification to fully assess the bacterial was calculated using ImageJ software.
viability of each culture.
2.10. Identification of PE-degrading bacterial strains

2.5. Disinfection of PE films The PE-degrading bacterial strains were identified by 16S rRNA
sequencing. After growing bacterial cultures for 24 h in Nutrient
After evaluating the adherent microorganisms, the PE films were Broth (Difco), cell pellets were incubated with 200 ␮L lysozyme
disinfected with 2% (w/v) sodium dodecyl sulfate for 4 h followed (1.82 mg mL−1 ) at 37 ◦ C for 1 h. Lysis buffer was added, and cells
by 70% (v/v) ethanol for 4 h, and then rinsed with distilled water were homogenized for 40 s at 6 m s−1 using the Fast Prep sam-
following an adapted widely used protocol [23,24]. This procedure ple preparation instrument (MP Biomedicals). Bacterial genomic
enables the complete detachment of microbial cells and debris for DNA was then purified with the Wizard SV Genomic DNA Purifi-
investigation of degradation traces in PE films [23]. The untreated cation System Quick Protocol kit (Promega), and 16S rRNA was
PE films were also subjected to this disinfection procedure prior the amplified with bacterial universal primers [27]. The 16S rRNA
analyses. sequences were aligned using the ClustalW algorithm [28] and
manually edited with Bioedit. The sequences were aligned with
2.6. Fourier transform infrared (FTIR) spectroscopy of PE similar sequences from NCBI for taxonomic classification using the
Blastn and Mothur tools [29]. A phylogenetic tree was constructed
The chemical fingerprint of the disinfected PE films was assessed with MEGA6 software [30] using the maximum-likelihood method
by attenuated total reflectance (ATR)-FTIR spectroscopy using the with 1000 bootstrap replicates.
Nicolet iS10 FT-IR Spectrometer (Thermo Scientific, USA). To inves-
tigate the chemical modifications of PE induced by the treatments, 2.11. Statistical analysis
we compared the IR spectra of PE that was biotically treated for
90 days (incubated with bacteria) with PE abiotically treated for All quantitative data were statistically analyzed considering
90 days (exposed to weathering conditions; positive control) and replicates and negative controls. Statistical significance was based
untreated PE (negative control). For these analyses, 10 randomly in confidence levels of less than 0.05.
chosen spots were assayed in each of the three replicates of treated
and untreated PE films. 3. Results and discussion

3.1. Isolation and screening of PE-degrading bacteria

2.7. Raman spectroscopy of PE
Brazilian Cerrado soil microbiome is a rich and diverse envi-
The crystallinity of the disinfected PE films was inferred by ronment that hosts a remarkable variety of metabolic reactions,
Raman spectroscopy using a confocal Raman microscope (WITec suggesting the presence of microbes capable of degrading a wide
Alpha 300 RA), charge-coupled device detector, and XTRA II single- range of substrates [17,31]. After cultivating the plastic-colonizing
frequency diode laser at 785 nm (power <400 mW). Raman spectra microbes from Cerrado soil in different agar media, we successfully
were acquired in spectroscopy mode using a high numerical aper- isolated and cultured a total of 647 bacterial strains. These microor-
ture Zeiss EC Epiplan-Neofluar 100× air objective (NA = 0.9 DIC). ganisms were first screened for the ability to degrade mineral oil, a
The integration time was 0.2 s, and 100 acquisitions were per- low molecular weight substrate chemically similar to PE, in which
formed using 10 replicates for each PE film. The mass fractions of 54 mineral oil-degrading microorganisms were identified. We then
the orthorhombic crystalline (␣c ), isotropic amorphous (␣a ), and evaluated the metabolic activity of the oil-degrading bacteria after
anisotropic disordered (␣b ) phases were determined following the incubation with PE as the sole carbon and energy source. Results of
Strobl and Hagedorn method [25]. total RNA analysis revealed 28 metabolically active bacterial strains
 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644 637

and 16 strains were selected for further analysis based on total RNA are successfully breaking down PE and, subsequently, assimilating
concentrations (data not shown). its products as carbon sources to support biosynthesis and energy
production. On the other hand, biofilms that consisted primarily
3.2. Taxonomic analysis and identification of PE-degrading of dead cells were detected over the entire surface of the glass
bacteria sheets (negative controls). Furthermore, cells incubated without PE
were mainly attached to each other rather than to the glass surface.
Results of 16S rRNA sequencing grouped the 16 isolates that Closer observation revealed that live cells in the negative control
showed signs of the ability to degrade PE into three genera: cultures were above/surrounded by cellular debris, suggesting the
Comamonas, Delftia, and Stenotrophomonas (Fig. S2). Further charac- use of dead cells as the carbon and energy source. Based on the
terization is necessary for complete taxonomic classification and to contrasts between the PE-containing cultures and their negative
determine whether they represent novel species. No previous stud- controls observed over the 20-day period, we perceived a positive
ies have reported Comamonas species able to degrade PE, despite relationship between the presence of PE and the viability of the
their known ability to degrade mineral oil [32]. Similarly, Delftia PE-degrading bacteria. We then assessed the long-term viability of
and Stenotrophomonas species have been reported to biodegrade these potential PE-degrading bacterial strains cultured with PE film
other synthetic polymers [33,34], suggesting the actual potential as the sole carbon source. Fig. 2 shows PE films densely colonized
of these strains to degrade PE. by viable cells after 90 days, with very few dead cells observed. In
contrast to the usual average incubation of 60 days, the strong via-
3.3. Adhesion and viability of PE-degrading bacteria bility of Comamonas sp., Delftia sp. and Stenotrophomonas sp. after
90 days shows these strains can remain viable after even longer
Cellular adhesion and viability of the bacteria cultured with periods under this condition [3].
PE film were investigated by fluorescence microscopy using the To evaluate the influence of (1) electrostatic interactions
well-established live/dead assay with the LIVE/DEAD baclight bac- between the outer membrane and the surface of the PE film and (2)
terial viability kit [24]. Since non-soluble PE was the sole carbon adhesion cellular machinery, non-PE-degrading synthetic strains of
and energy source provided, cells would mainly adhere to the B. subtilis and E. coli were also incubated with PE film or a glass sheet
polymer surface, secrete extracellular enzymes and trigger the to serve as negative controls for Gram-positive and Gram-negative
molecular modifications required for assimilation of those hydro- bacteria, respectively (Figs. S6 and S7). In contrast to the cultures of
carbons, such as bond scission, chemical transformation, structural potential PE-degrading strains, the results showed no relationship
changes, and formation of new functional groups [4,23]. Because between the presence of PE and cellular viability, as sparse biofilms
of its extremely high molecular weight, this polymer is not readily and an increasing number of dead cells were observed over the
assimilated and demands to be broken down [9]. Microbial assim- 20-day incubation period in both cultures.
ilation is mediated by catalytic membrane proteins or secreted
extracellular enzymes that induce PE’s chain cleavage to the for- 3.4. Characterization of PE biodegradation using physicochemical
mation of smaller compounds, the first step of the biodegradation and imaging methods
process. Depolymerization is the crucial step and some oxidore-
ductases have been described to have this ability [35]. In contrast to Since hydrophobicity and high molecular weight of PE impede
planktonic cells, attached cells can secrete degrading enzymes and biodegradation by preventing the bacterial adhesion and the assim-
auxiliary molecules near PE’s surface, maximizing their concentra- ilation of the alkane, oxidation and/or chain scission emerge as
tion around the polymer and increasing the probability of substrate the limiting steps [4,11]. Indeed, with an increasing variety of PE-
modification. In addition, adherence ensures that starved cells are degrading enzymes reported in the literature [16,35], it has been
close enough to assimilate degradation products efficiently enough shown that the microbial ability to consume this polymer is intrin-
to meet their carbon and energy demand. sically related to its prior oxidation or molecular weight reduction.
Although dead cells may also provide a consistent carbon and Subsequently, the smaller and oxidized products can be assimi-
energy source, bacteria would need to be adjacent to dead cells in lated by microbes, which mineralize them mainly through ␻ or
order to maintain their viability and, still, few viable cells would ␤-oxidation [11].
remain after 20 days if the bacterial strain was not capable of The first step of the general PE degradation mechanism is typ-
degrading and utilizing PE. This scenario is represented by the nega- ically auto-oxidation through (i) the initial formation of radicals
tive control that consists in cells cultivated with a glass sheet, which R• and H• , which involves the activation of PE by a loss of hydro-
reflects the metabolic state of the bacterial strains grown without gen after energy input or formation of charge transfer complexes
added carbon sources. This would also allow for inferences about [36–42]. A complex “domino-effect” is triggered by this macro-
a possible utilization of stored energy sources, which would aid radical formation (R• ), leading to (ii) the formation of cross-linked
the cellular viability during carbon starvation. Fig. 1 shows repre- structures within the polymer or (ii’) the oxidation (e.g. via atmo-
sentative bacterial strains from each genus incubated with PE film spheric O2 , water) of its carbon chain. This oxidation produces (iii)
or glass sheets for 12, 15, or 20 days (see Figs. S3–S5 for further other reactive molecules that can either (iv) result in the formation
details). of other radicals to restart the process or (iv’) stabilize in the form
Since the only differing parameter between the cultures of each of a hydroxyl, carbonyl, or nitro group and often result in double
isolate was the presence/absence of PE, it is possible to access the bond formation (formation of a vinylene group) [11,37,43,44].
impact of the polymer over the maintenance of bacterial viabil- The unique spectroscopic fingerprints of the treated PE are
ity. Although the amount of adherent cells qualitatively differed contrasted with that of untreated PE in Fig. 3a, thus allowing to
between cultures incubated with PE film and negative controls target the chemical modifications induced by either bacteria or
(Table S1), all cultures had a similar proportion of viable cells after weather. The highlighted absorption peaks represent the major
12 days. However, differences between cultures incubated with chemical modifications induced by the treatments, such as chain
PE and negative controls emerged after 15 days, and these dif- scission (915–905 cm−1 ; H2 C C ) and formation of alkoxy groups
ferences were even more obvious after 20 days. At this moment, (1026 cm−1 , 1090 cm−1 ; C O), acyl groups (1220 cm−1 ; C O),
the PE films were densely colonized by the viable PE-degrading nitro groups (1500–1600 cm−1 ; N O), double bonds (1640 cm−1 ;
strains, with very few dead cells observed. The strong predomi- C C ), and carbonyl groups (1715 cm−1 ; C O), revealing clear
nance of viable cells on PE-containing cultures indicates that they differences between biotic and abiotic degradations.
638 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644

Fig. 1. Cellular adhesion and viability of Comamonas sp., Delftia sp., and Stenotrophomonas sp. after 12, 15, or 20 days cultured in minimal synthetic medium with a PE film
(PE) or a glass plate (Control): live cells (green), dead cells (red), and overlap between live/dead cells (yellow). During the first 12 days of incubation, there was only a discrete
impact of PE to the cellular viability of these strains, with few dead cells in the absence of carbon sources. However, after 15 days, a strong cellular viability could be observed
in the presence of PE as the sole carbon source, contrasting to the vast predominance of dead cells in the carbon-free cultures. Still, by the end of the 20th day of incubation,
almost no dead cells could be found in PE-containing cultures. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)

The IR spectra of weathered PE indicate chain scission ( C H interaction with OH− from water molecules. Alkyl radicals may also
bending, 905 cm−1 ) and carbonyl formation (C O stretching, be converted into vinyl radicals after the abstraction of a hydro-
1715 cm−1 ), suggesting two possible photodegradation mecha- gen from the end CH2 group, resulting in the observed terminal
nisms: Norrish type I and Norrish type II reactions [11,23]. Oxidized double bond at 905 cm−1 . Alternatively, the peaks at 905 cm−1
PE (R (CH O) R) undergoes the Norrish type I reaction to produce and 1715 cm−1 may also indicate the occurrence of an additional
carbonyl and alkyl makro-radicals after the homolytic scission of mechanism: Norrish type II reaction, which results in the simulta-
PE’s carbon skeleton. Since FTIR spectroscopy cannot detect radi- neous formation of carbonyl and vinyl groups from the heterolytic
cals, a peak at 1715 cm−1 related to carbonyl is found. However, scission of PE [11]. Since weathered PE was exposed not only
carbonyl and alkyl makro-radicals may stabilize as carboxylic acid to UV irradiation but also to many other chemical and physical
(1715 cm−1 ) and alcohol (1000–1150 cm−1 ) respectively following factors related to the weathering process (e.g. wide temperature
 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644 639

Fig. 2. Cellular adhesion and viability of Comamonas sp., Delftia sp., and Stenotrophomonas sp. on the surface of PE film (top) and their growth in nutrient broth (bottom)
after 90 days of culture in minimal synthetic medium with PE as the sole carbon source. Top panel: live cells (green), dead cells (red). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. (a) IR spectra of PE film incubated with Comamonas sp. (blue), Delftia sp. (magenta), or Stenotrophomonas sp. (green) for 90 days. The positive control was PE exposed to
weather for 90 days (light orange), and the negative control was the untreated PE (red). Absorption peaks corresponding to the main chemical modifications are highlighted.
(b) Comparison of carbonyl, double bond, and terminal double bond indices among treated and untreated PE film samples (mean ± SE, n = 3). *No significant difference from
untreated PE (P > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
640 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644

range, humidity shifts, mechanical wear and water), additional specific bacterial strains, especially since this mechanism would
chemical mechanisms are expected to be involved in its degrada- lead to decreased hydrophobicity and molecular weight. Consid-
tion/photodegradation. ering further mechanistic analyses are beyond the scope of this
Biotically treated films showed a distinct absorption pattern as work, additional research to explore this biochemical novelty will
a consequence of the chemical environment created in the culture be presented in future works.
media by the microbial metabolism due to the action of bacterial Additionally, we calculated the carbonyl (1715 cm−1 ), termi-
transmembrane transport, excretion and secretion systems. PE film nal double bond (905 cm−1 ) and internal double bond (1640 cm−1 )
incubated with bacteria showed evidence of alcohol, ether, acid and indices (Fig. 3b and Table S2), which correspond to the concentra-
ester formation, as indicated by the presence of alkoxy and acyl C O tions of each of these chemical species over the polymer [11]. They
stretching bands (Fig. 3a) [45]. The hydroxylation inferred from the also account for inferences and estimations about oxidation, viny-
peak at 1000–1150 cm−1 related to alkoxy group formation (C O) lene formation and chain scission (Table S2) [11]. Fig. 3b shows that
provides evidence for PE hydrolysis. Another possible explanation biodegradation resulted in a remarkable increase in double bond
for C O formation is the stabilization of peroxy radicals. As stated formation of up to 3.4-fold, whereas the smaller increase in car-
by Massey et al., a weak dissociation enthalpy for the hydroper- bonyl and terminal double bond indices indicate the formation and
oxide O O bond (R O OH) suggests that the 1000–1150 cm−1 the subsequent microbial assimilation of the oxidized and lower
peaks do not represent peroxy groups [36]. Although hydroperox- molecular weight fragments. Besides, the substantial vinylene for-
ides are well-described products of the oxidative degradation of PE mation confirms PE chemical modification by bacterial metabolism.
[11,39,40], they decompose, leading to the formation of hydroxyl These data also highlight that biotic and abiotic degradations occur
and carbonyl groups as well as carbonyl radicals, all related to by different mechanisms.
chemical species detected on the IR spectra of treated PE in this Microscale analysis of surface topography revealed disruption of
study. the surface of PE film after microbial treatment (Fig. 4c) contrast-
According to previous reports [11,37,40,46], the intense peak at ing to the smooth and flat surface of untreated PE film (Fig. 4a). As
1640 cm−1 represents C C stretching. It is not clear how microbes emphasized by the micrographs, the surface disruption and deteri-
induce vinylene formation (R C C R) in PE film, but this mod- oration caused by the biotic treatment (Fig. 4c) was far more evident
ification appears to reflect a relevant step of the biodegradation than that caused by abiotic treatment (Fig. 4b). This may be due to
process and indicates the occurrence of chain scission [37]. We the action of transmembrane or extracellular enzymes, which break
speculate that the complex chemical environment created by bac- down PE reducing its molecular weight and modifying its surface. In
terial metabolism along with the reagents from culture media may fact, different hydrolases and oxidases were found in the genomes
induce radical (R• ) formation due to charge-transfer complexes, of these strains (data not shown).
and one of the possible configurations is a vinylene group [44]. Further topographic analysis by AFM also indicates consider-
Instead, vinylene formation may also result from dehydrogenation able modification after biotic and abiotic treatment. Compared with
of PE molecules [45]. the wavy nanometric surface of the untreated PE film (Fig. 5e), PE
Intriguingly, an unprecedented extensive nitro group forma- incubated with bacterial isolates (Fig. 5a–c) or exposed to weather
tion (N O) occurred exclusively in all biodegraded samples, as (Fig. 5d) for 90 days appeared clearly abraded. Nanoscale surface
demonstrated by the presence of high peaks at 1500–1600 cm−1 . analyses suggested the formation of deep cavities after mainly
The same effect was previously described as a result of the abi- the biotic treatments. The nanoroughness parameters (Fig. 5f) of
otic treatment of PE by its exposure to a nitric oxide atmosphere all PE film samples incubated with bacteria differed significantly
[37]. Since the three genera have known nitrification and/or den- compared to the untreated film. Topographic changes suggest-
itrification abilities [47–50], we speculate that nitric oxide (NO) ing biodegradation include substantially higher values for Ra
is synthesized by the microbes and diffuses through the outer (untreated, 21.5 ± 1.4 nm; biotic treatment, ≤39.2 ± 10.1 nm), Rq
membrane to its interface with PE. In the extracellular environ- (untreated, 26.8 ± 1.6 nm; biotic treatment, ≤49.8 ± 11.9 nm), RzJIS
ment, NO is oxidized by atmospheric O2 to the reactive nitrogen (untreated, 82.0 ± 3.9 nm; biotic treatment, ≤203.5 ± 34.2 nm), and
dioxide (2NO + O2 → • NO2 ), which then interacts with activated Rz (untreated, 168.5 ± 8.2 nm; biotic treatment, ≤432.5 ± 69.6). The
PE (R• ), resulting in nitro formation (C O N O) [37]. Indeed, the large peak-to-peak amplitude (Rz) values for both biotic and abi-
genomes of our Comamonas, Delftia and Stenotrophomonas isolates otic treatments indicate the formation of broad and deep cavities
indicate they have denitrification abilities (data not shown). We in treated PE film samples. Besides, Rp and Rv values are comple-
found that all the strains were capable of synthesizing NO2 − dur- mentary to Rz, supporting this result. Microbial fragmentation of
ing incubation with PE, which was quantified after its coupling PE by enzymatic action is a plausible mechanism for topography
with Griess reagent (0.5% sulphanilamide in 2.4 M HCl and 0.3% changes in PE treated with bacteria, whereas in abiotic treatment
naphthy-lethylenediamine dihydrochloride in 1.2 M HCl) (data not these changes are due to cross-linking and embrittlement.
shown). Furthermore, nitrite concentrations in the supernatants Phase imaging of AFM (Fig. 6) allowed the evaluation of the vis-
of PE-containing cultures were approximately four times higher coelastic behavior of treated and untreated PE, which is closely
than the ones from carbon-free cultures. The production of nitrite, related to molecular weight [53]. Polymers behave as both flu-
while ammonia was the sole provided source of nitrogen, sug- ids and solids; thus, investigating physical properties such as
gests these microbes are also capable of heterotrophic nitrification viscoelasticity can provide valuable information about molecu-
– aerobic denitrification. Since NO is a by-product of nitrification lar weight and size changes [54]. In untreated PE, the relative
(conversion of NH4 + to nitrate – NO3 − ), it is plausible that het- contributions of viscous and elastic components were quite sim-
erotrophic nitrification also releases NO, even though the genes and ilar (Fig. 6e). Quantitative viscoelasticity data (Fig. 6f) show that
enzymes related to this process are still unidentified [51,52]. More- viscous regions comprise approximately 59.9 ± 10.4% (mean ± SE)
over, NO is also a product of chemodenitrification, the chemical of the untreated PE film, 39.9 ± 5.9% of the weathered PE film,
decomposition of nitrite (NO2 − ) as a consequence of the presence and up to 31.9 ± 4.9% (mean ± SE) of the biodegraded PE, which
of reduced metals such as ferrous iron (Fe2+ ) [52]. Considering represents a 46.7% decrease after biotic treatment. As reported,
the media composition, it is likely that this mechanism does take degradation changes the average molecular weight of PE [45,53,55].
place in the described cultures. Because of the intensity of these PE biodegradation decreases its viscous fraction area within the
peaks in Fig. 3, the set of reactions that lead to this chemical mod- polymer, which is related to smaller carbon chains, since microbial
ification might represent main degradation mechanisms of these enzymes break PE allowing the assimilation of the oxidized and
 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644 641

Fig. 4. Scanning electron microscopy of (a) untreated PE film, (b) PE film exposed to weather for 90 days, and (c) PE film incubated with bacteria over 90 days. Noticeable
surface disruption can be observed after incubation with bacteria, suggesting biodegradation has occurred.

Fig. 5. Nanometric topography of PE film incubated with (a) Comamonas sp., (b) Delftia sp., or (c) Stenotrophomonas sp. for 90 days. (d) PE film exposed to weather for 90 days
served as a positive control, and (e) untreated PE film as a negative control. Sidebars represent amplitude distribution and maximum amplitude (nm) within a 100-␮m2
surface. (f) Roughness parameters (mean ± SE, n = 10) of untreated PE (red), weathered PE (light orange), and PE incubated with Comamonas sp. (blue), Delftia sp. (magenta),
and Stenotrophomonas sp. (green): average roughness (Ra), root mean square roughness (Rq), Japanese industrial standard average roughness (RzJIS), average height (Rz),
maximum peak height (Rp), and maximum valley depth (Rv). *No significant difference from untreated PE (P > 0.05). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
642 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644

Fig. 6. Viscoelastic behavior of PE films incubated with (a) Comamonas sp., (b) Delftia sp., or (c) Stenotrophomonas sp. for 90 days, as determined by phase imaging. (d) PE
film exposed to weather for 90 days served as a positive control, and (e) untreated PE film as a negative control. Elastic areas are light, and viscous areas are dark. Sidebars
indicate the phase angle of probe oscillation. (f) Viscous area (%) of the PE film after microbial treatment (blue, magenta, and green), exposure to weather (light orange), or no
treatment (red). All areas showed significant difference from untreated PE (P < 0.05; each group, n = 10). (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

lower molecular weight fragments. The simultaneous production the integral intensities of its absorption peaks are proportional to
and consumption of these lower molecular weight fragments by the sum of all phases. Based on the mass fractions of the fully
bacteria would ultimately increase the average molecular weight of crystalline (␣c = 1: integral intensity of 1416 cm−1 band = 0.46) and
the material, since smaller fragments are being assimilated, thereby totally amorphous samples (␣a = 1: integral intensity of 1080 cm−1
increasing the proportion of the elastic regions (Fig. 6a–c) com- band = 0.79), whereas taking the internal standard into account, it
pared with untreated PE (Fig. 6e). was possible to calculate ␣a , ␣b , and ␣c .
Although weathering also decreases the molecular weight of The results showed that the amorphous fraction increased in all
polymers, a decrease in the proportion of viscous regions of PE treated PE films, but the interphase fraction was the most strongly
was observed (Fig. 6d). This result is due to crosslinking provoked affected (Fig. S8) [13]. Only a small increase in crystalline con-
by radicals produced during degradation, which increase the aver- tent was observed after weathering in contrast to the great one
age molecular weight and decrease chain flexibility [43,56], thus expected as a consequence to the break down of PE into smaller
enhancing the elastic area in the weathered PE film [57]. In addi- alkanes, which would accumulate within the material. This effect
tion, the phase angle related to the advance or delay of the wave is also a consequence of cross-linking, which compensates for the
phase (Fig. 6a–e) of cantilever oscillation tended to shift from the decrease in molecular weight due to abiotic degradation [43] and
broad bimodal distribution of the untreated PE control to a sharp is consistent with the viscoelasticity findings. PE film incubated
unimodal distribution after both biotic or abiotic treatment, sup- with Comamonas sp. and Delftia sp. showed a decrease in crys-
porting the quantitative viscoelasticity data [56]. tallinity due to the assimilation of the lower molecular weight
Ultimately, Raman spectroscopy was used to estimate propor- fragments, produced by the action of the microbial enzymes, that
tions of the crystalline phase (␣c ), amorphous phase (␣a ), and constitute the crystalline fraction. In contrast, PE film incubated
interphase (␣b ) of the PE samples, since ␣a + ␣b + ␣c = 1 [25]. Phase with Stenotrophomonas sp. showed a considerable increase in the
fractions can be calculated from the integral intensities of spe- crystalline fraction, indicating its potential to break the long alkane
cific absorption peaks in spectral regions related to C C stretching chain. However, it remains unclear whether this strain breaks PE
(1062–1129 cm−1 ) and twisting (1250–1350 cm−1 ) and methylene more efficiently or consumes the smaller alkanes slower. A pro-
bending (1390–1510 cm−1 ) vibrations [13,25]. Peaks at 1416 cm−1 posed summary of biotic and abiotic degradation effects to PE is
and 1080 cm−1 were used to estimate crystalline and amorphous presented in Fig. S9.
fractions, respectively. The twisting region served as an internal Overall, Delftia sp. appears to have the highest degradation
standard, since it does not respond to conformational changes, and capability according to our results, followed by Comamonas sp.
 J. Peixoto et al. / Journal of Hazardous Materials 324 (2017) 634–644 643

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