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PSL 2018 Special Issue 01-12 Dela Torre Et Al PDF
PSL 2018 Special Issue 01-12 Dela Torre Et Al PDF
Biodegradation of low-density
polyethylene by bacteria isolated
from serpentinization-driven alkaline
spring
Denisse Yans Z. Dela Torre, Lee A. Delos Santos, Mari Louise C. Reyes and
Ronan Q. Baculi*
ow-density polyethylene (LDPE), a commonly-used Reduction in percent crystallinity of the films incubated with
Figure 1: Phylogenetic tree showing the relationship among 16S rRNA gene sequences of the bacterial isolates with reference sequences
obtained through BLAST analysis. The sequences alignment was performed using Clustal W program and the tree was constructed using
Neighborhood-Joining with Kimura 2 parameter distances in MEGA 6 software. Bootstrap values (1000) replicates are shown at the nodes. Sequences
designated as PB were obtained from the present study. Methanococcus voltae was used as outgroup.
(http://www.technelysium.com.au/) was used to manually generated have been deposited in the GenBank database under
evaluate the sequences and to remove low quality regions accession numbers MF407325-MF407328.
usually at the start and end of the fragment. DNA sequences
were analyzed using the BLAST tool at the National Centre for Polyethylene film biodegradation assay
Biotechnology Information (NCBI) server Each isolate collected was tested for LDPE-degrading
(http://blast.ncbi.nlm.nih.gov). The sequences were submitted efficiency. Flasks containing 300 mL of SM broth were added
for multiple alignments with reference sequences from the with pre-weighed LDPE strips (1.5 cm x 1.5 cm size) that had
GenBank database using Clustal W. Phylogenetic trees were been dried overnight at 60°C, weighed, disinfected for 30
constructed with the Maximum Likelihood algorithm of MEGA minutes in 70% ethanol, air-dried for 15 minutes in laminar
6 software (Tamura et al. 2011) with evolutionary distances airflow chamber, and treated with mineral oil (0.05%) to allow
calculated according to Kimura’s two-parameter correction bacterial adhesion (Das and Kumar 2014). One mL of a 24- hour
method. The phylogenetic trees were evaluated through bacterial culture maintained at a turbidity equivalent to 0.5
bootstrap analysis from 1000 bootstrap replicates. All sequences McFarland standards was added to the suspension. Flasks
containing non-inoculated SM medium supplemented with
Isolate Cultural characteristics Cell morphology Gram Reaction Catalase Test Oxidase Test
(color, colony form, margin,
surface, elevation
A B
Protein concentration mg/ml
Protein concentration mg/ml
0.6 0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1 0.1
0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14
Incubation Period (Days) Incubation Period (Days)
C D
Protein concentration (mg/ml)
Protein concentration mg/ml
1.2 0.25
1
0.2
0.8
0.15
0.6
0.4 0.1
0.2 0.05
0 0
2 4 6 8 10 12 14 2 4 6 8 10 12 14
Incubation Period (Days) Incubation Period (Days)
Figure 2: Protein content of attached bacterial isolates (A) Bacillus krulwichiae PB1 (B) Bacillus pseudofirmus PB5 (C) Prolinoborus
fasciculus PB8 and (D) Bacillus sp. PB14 on the surface of low-density polyethylene film.
polyethylene served as the control. All flasks were incubated at loss = [(Initial weight - Final weight)/ Initial weight] x 100
37°C for 90 days (Gajendiran et al. 2016). (Konduri et al. 2010).
Determination of dry weight of residual LDPE Quantitative estimation of bacterial population on LDPE
To facilitate accurate measurement of the dry weight of residual films
LDPE, recovered LDPE films were washed with 2% (v/v) Indirect estimation of the bacterial biomass was done by
aqueous sodium dodecyl sulfate (SDS) solution for 4 hours and determining the protein concentration on the LDPE films
then with distilled water (Gilan et al.2004). The mineral oil utilizing the procedure of Hadad et al. (2005) with
added on the LDPE films was removed using chloroform and modifications. Each bacterial isolate was inoculated into 14
then SDS wash. The washed LDPE films were dried overnight flasks each containing SM broth supplemented with 30 mg
on a filter paper at 60°C before weighing. The weight loss was LDPE strips (1.5 cm x 1.5 cm size). The flasks were incubatedat
calculated using the following equation: Percentage of weight 37°C from 1 to 14 days. The protein concentration was checked
Table 4: Carbonyl index obtained from FTIR spectra of low density polyethylene (LDPE) incubated for 90 days with bacterial isolates vs non-
inoculated polyethylene
Carbonyl Index
Isolate 30 days 60 days 90 days
Non-inoculated (Control) 0.009 0.011 0.006
PB1 0.009 0.005 0.008
PB5 0.003 0.004 0.005
PB8 0.014 0.009 0.007
PB14 0.012 0.015 0.007
every day by boiling the films with 5 mL of 0.5M of NaOH incubation. The samples were washed with 2% SDS and distilled
solution for 30 minutes. The solution was then centrifuged for 1 water and finally flushed with 70% ethanol to remove surface-
minute at 5000rpm. The supernatants were combined and adhered organisms. The films were air-dried overnight, gold-
subjected to UV-Vis spectrophotometer with the NaOH solution coated, and visualized using scanning electron microscope
serving as blank solution. Finally, the concentration of proteins (JEOL, Model JSM-6390LV).
was computed using the equation described by Groves et al.
(1968): Protein concentration (mg/mL) = A280 (1.55) – A260 (.76). Statistical analysis
Experimental results were expressed as the mean + standard
Fourier transform infrared (FTIR) spectroscopy analysis of deviation of three independent replicates. Test of significance of
polyethylene the differences among mean values was determined by one-way
After 90 days of incubation, the LDPE films were washed with analysis of variance (ANOVA) followed by Tukey’s test at the
2% SDS followed by sterile distilled water and dried overnight 5% level of significance (P < 0.05) using IBM Statistical
at 50°C prior to submission for FTIR analysis. Changes in the Package for the Social Sciences (SPSS) version 20.0 (IBM
structure of polyethylene samples incubated with the bacterial Corporation, Armonk, NY, USA.
strains and non-inoculated control were analyzed by Fourier
transform infrared (FTIR) spectroscopy (Shimadzu Prestige,
Pike Technologies, USA). FTIR scan per sample was taken at RESULTS AND DISCUSSION
spectrum ranging from 400 cm-1 to 4000 cm-1 at ambient room
temperature. Characteristic peaks of the spectra were determined Isolation and identification of LDPE-degrading bacteria
using Essential FTIR Software (Operant LLC, Madison, U.S.A). Bacterial isolates were obtained from Poon Bato Spring, a
The relative absorbance intensities of the ester carbonyl bond natural alkaline environment in the Zambales ophiolite complex
(1740 cm-1), the keto carbonyl bond (1715 cm-1), the terminal of the Philippines. The measured pH was 11 and the temperature
double bond (vinyl) bond (1650 cm-1) and the internal double was 28˚C. Chemical analysis of the water samples revealed the
bond (908 cm-1) to that of the methylene bond (1465 cm-1) were presence of calcium, magnesium, sulfate, chloride, and iron.
evaluated using the following equation (Albertsson et al. 1997): Calcium had the highest amount at 50.3 mg/L (Table 1). The
keto carbonyl bond index (KCBI) = I1715/I1465; ester carbonyl high concentration of calcium detected in the site confirmed that
bond index (ECBI) = I1740/ I1465; vinyl bond index (VBI) = the spring is typical Ca 2+ -OH – type water generated by active
I1650/I1465; and internal double bond index (IDBI) = I908/I1465. The serpentinization process (Cardace et al. 2015). Initially, nine
crystallinity percentage of the polymer was calculated using the isolates were obtained by enrichment culture supplemented with
following equation: % crystallinity = 100 – [{1 – (Ia/1.233Ib) / LDPE as the sole carbon source. However, only four strains
1 + (Ia/Ib)} × 100]: where Ia is the absorbance at 1473 and Ib is have significantly reduced the weight of the polymer after 90
absorbance at 1463. Carbonyl index was computed using the days of incubation. The four isolates were found to be Gram-
following equation: CI = absorption at 1740 cm-1 / absorption at positive except PB8, catalase-positive, and oxidase-positive
1460 cm-1 (Kyaw et al. 2012). Samples incubated with bacterial (Table 2). Analysis of the 16S rRNA gene sequences revealed
isolates were subjected to FTIR analysis after every 30 days of that the isolates belong to two major groups of bacterial phyla,
incubation. phylum Firmicutes and phylum Proteobacteria. Specifically,
three of the isolates were affiliated with phylum Firmicutes,
Scanning electron microscopy of polyethylene which contains low G+C Gram-positive bacteria, represented by
To examine the morphological changes on the surface of the genus Bacillus while the remaining isolate was grouped
polyethylene incubated with bacterial isolates, polyethylene under phylum Proteobacteria, a diverse group of Gram-negative
films were recovered from the SM media after 90 days of bacteria, represented by genus Prolinoborus. All of the
Figure 4: Fourier transform infrared spectra of LDPE after 90 days of incubation with bacterial isolates (A) Bacillus krulwichiae PB1, (B)
Bacillus pseudofirmus PB5, (C) Prolinoborus fasciculus PB8, and (D) Bacillus sp. PB14. The colors indicate the length of incubation period when
the measurement was taken: green = 30 days of incubation, blue = 60 days of incubation, pink = 90 days of incubation).
developed on the polyethylene. This observation confirms the Fourier transform infrared (FTIR) spectroscopy analysis
results of Hadad et al. (2005) on biodegradation of polyethylene Fourier transform infrared analysis enables the determination of
by the thermophilic bacterium, Brevibacillus borstelensis. In degradation products, chemical moieties incorporated into the
their study, bacterial colonization of the polyethylene measured polymer such as branches, co-monomers, unsaturation, and
as extractable protein by B. bortelensis initially increased presence of additives such as antioxidants (Sudhakar et al.
followed by a gradual decrease leading to the absence of 2008). Analyses were conducted to monitor the formation or
extractable protein until the 20th day of incubation. A slow and disappearance of acids (1715 cm-1), ketones (1740 cm-1), and
constantly proliferating biofilm eventually developed on the double bonds (1640 and 908 cm-1) to explain the mechanisms of
polyethylene film following the decline in the protein content of biodegradation process. The control spectra of the polyethylene
the fast-proliferating cells. Similarly, Gilan et al. (2004) reported film not treated with any of the isolates displayed distinctive
the development of low-density biofilm of Rhodococcus ruber bands that characterize a typical LDPE (Fig. 3). The
using LDPE film as carbon source after the utilization of mineral characteristic absorption bands were assigned at 2920 cm-1 (CH2
oil. asymmetric stretching), 2850 cm-1 (CH2 symmetric stretching),
94
92
90
88
% Crystallinity
86
84
82
80
78
76
74
30 DAYS 60 DAYS 90 DAYS
Days
1473, 1462, and 1437 cm-1 (C=C bending deformation), and 727 of the films to these isolates led to a decrease in carbonyl index
and 723 cm-1 (C-H bend mono) (Gulmine 2002). presumably due to the utilization of low molecular weight
oxidation products. Similar activity was reported in the
The intensity of peaks varied in different regions after 30, 60 and polyethylene degradation studies by Gilan et al. (2004),
90 days of incubation of the test samples with the bacterial Sudhakar et al. (2008), Hadad et al. (2005), and Volke-
isolates. Generally, there was a decrease in the intensity of peaks Sepulveda et al. (2001). On the other hand, the increase in
that corresponded to C=C bending deformation (1473, 1462, and carbonyl index was evident throughout the 90-day incubation of
1437 cm-1), and C-H bend mono (727 and 723 cm-1) on the films the polyethylene films with isolate PB5 which can be attributed
incubated with the isolates (Fig. 4). Moreover, strong absorption to biological activities of the microorganisms that lead to the
peaks at 1462 cm-1 and 1437cm-1 became less prominent after formation of ketone or aldehyde C=O groups (Esmaeili 2013)
microbial treatment. The intensity of these peaks was further which was confirmed by peak formations. Moreover, this may
reduced in the case of isolates PB1 and PB8. also be due to microbial oxidation of short chains of alkanes
forming carboxylic acids (Manzur et al.2004). This increase in
Carbonyl residues at the typical carbonyl peak (1712 cm-1) were carbonyl index as a result of biodegradation is similar to the
reduced upon incubation with bacterial isolates PB1, PB8, and results of Balasubramanian et al. (2010).
PB14 indicating carbonyl utilization (Fig. 4). The initial abiotic
step in degradation of polyethylene involves polymer chain The KCBI, ECBI, and VBI of the polyethylene increased when
oxidation which forms carbonyl groups that subsequently forms incubated with most of the isolates (Fig. 5). Although KCBI and
carboxylic groups that eventually undergo β-oxidation. VBI were lower than the control when using isolate PB5, a
Complete degradation happens via citric acid cycle resulting in consistent increase in KCBI and VBI values was observed with
the formation of CO2 and H2O (Albertsson et al. 1997). The this isolate throughout the 90-day incubation time presumably
carbonyl index is the ratio between the absorbance peaks of due to the emergence of keto and ester carbonyl as the primary
carbonyl group to CH2 at 1462-1463cm-1. Initially, carbonyl products of enzyme activity particularly involving
index increased relative to the control for strains PB1, PB8, and oxidoreductase (Karlssonand Albertsson 1998). Furthermore,
PB14 after 30 days of incubation (Table 4). Prolonged exposure
IDBI increased when the polyethylene was incubated with more resistant to enzymatic attack.The subsequent decrease in
isolates PB1 and PB8. crystallinity at the end of the 90-day incubation for both strains
was possibly caused by the microbial attack on smaller crystal
Low-density polyethylene is insoluble in nature due to their structures which were formed during the initial attack on
crystalline structure (Pierre and Chiellini 1986). In this study, amorphous fractions (Manzur et al. 2004). The results obtained
there was an observed reduction in the crystallinity of the are similar to the findings of Manzur et al.(2004) on the
polyethylene after 90 days incubation with isolates PB5 and PB1 biodegradation of physicochemically- treated LDPE by a
(Fig. 6). The decrease in crystallinity supports the conversion of consortium of filamentous fungi and of Volke-Sepúlveda et
crystalline structures of the LDPE films into an amorphous al.(2001) on biodegradation of LDPE.
structure as a consequence of degradation. On the other hand,
abiotic degradation results in the remaining polymer becoming Scanning electron microscopy (SEM) analysis
more organized after release of the degradation products The changes in the surface morphology of the LDPE films were
(Albertsson et al.1995) as indicated by the increase in percent investigated by SEM after 90 days of incubation. The control
crystallinity of the control. Furthermore, the observed increase sample, incubated without any of the bacterial isolates, had a
in crystallinity in the film incubated with PB1 and PB8 after 60 smooth surface with no pits, cracks or any particles attached to
days of incubation can be attributed to microbial attack on the surface. In contrast, LDPE films treated with the bacterial
amorphous fractions rather than crystals because the latter are isolates had surface alterations characterized by pits, cracks,
The present study on in-vitro biodegradation clearly showed that Albertsson AC, Karlsson S. The influence of biotic and abiotic
bacteria isolated from a hyperalkaline spring can degrade low- environments on the degradation of polyethylene. Prog
density polyethylene, a polymer that is resistant to natural Polym Sci 1990; 15 ;177–192.
process of degradation. Bacterial isolates, phylogenetically
related to Bacillus krulwichiae, Bacillus pseudofirmus, Albertsson AC, Andersson SO, Karlsson S. The mechanism of
Prolinoborus fasciculus, and Bacillus sp., were able to colonize biodegradation of polyethylene. Polym Degrad Stab1997;
and degrade LDPE films based on weight reduction, appearance 18:73–87.
and disappearance of various functional groups as revealed by
FT-IR analysis, and structural changes on the film by SEM Andrady AL. Microplastics in the marine environment. Marine
analysis. Moreover, the actively increasing metabolism of the Pollution Bulletin 2011; 62:1596–1605.
isolates as revealed by protein quantification supported their
ability to utilize LDPE as carbon source. The degradation Baculi R, Lantican NB, delos Reyes III FL, Raymundo AK.
capability of the bacterial strains could be due to the occurrence Prokaryotic community analysis of a hyperalkaline spring in
of certain enzymatic activities that lead to a chain cleavage and the Philippines using 16S rRNA gene clone library
assimilation of the hydrocarbon backbone of the polymer. This construction. Phil J Sci 2015:144 (1):1-12.
study demonstrated the ability of the isolates to degrade
polyethylene even in the absence of prior oxidation treatments. Balasubramanian V, Natarajan K, Hemambika B, Ramesh N,
The results showed that selected microorganisms exhibited great Sumathi CS, Kottaimuthu R, Rajesh Kanan V. High-density
potential for LDPE biodegradation, a discovery which can be polyethylene (HDPE)-degrading potential bacteria from
used in reducing solid waste currently accumulating in natural marine ecosystem of Gulf of Mannar, India. Lett Appl
environments. Determination of bacterial protein concentrations Microbiol 2010;51, 205-211.
using samples incubated at longer periods combined with cell
surface hydrophobicity analysis will lead to improvements in Bhatia M, Girdhar A, Tiwari A, Nayarisseri A. Implications of
understanding the colonization and biofilm formation on the a novel Pseudomonas species on low density polyethylene
film. Microbial consortia can be formulated and evaluated for biodegradation: an in vitro to in silico approach. Springer
their efficacy in enhancing the rate of LDPE biodegradation. Plus 2014; 3:497.