A marine bacterial community that degrades poly(ethylene terephthalate) and polyethylene

TL;DR: This study establishes a stable and effective marine bacterial community for PET and PE degradation and sheds light on the degradation pathways and associated mechanistic processes, which paves a way to develop a microbial inoculant against plastic wastes.

Abstract: Plastic wastes have become the most common form of marine debris and present a growing global pollution problem. Recently, microorganisms-mediated degradation has become a most promising way to accomplish the eventual bioremediation of plastic wastes due to their prominent degradation potentials. Here, a marine bacterial community which could efficiently colonize and degrade both poly (ethylene terephthalate) (PET) and polyethylene (PE) was discovered through a screening with hundreds of plastic waste associated samples. Using absolute quantitative 16S rRNA sequencing and cultivation methods, we obtained the abundances and pure cultures of three bacteria mediating plastic degradation. We further reconstituted a tailored bacterial community containing above three bacteria and demonstrated its efficient degradation of PET and PE through various techniques. The released products from PET and PE degraded by the reconstituted bacterial community were determined by the liquid chromatography-mass spectrometry. Finally, the plastic degradation process and potential mechanisms mediated by the reconstituted bacterial community were elucidated through transcriptomic methods. Overall, this study establishes a stable and effective marine bacterial community for PET and PE degradation and sheds light on the degradation pathways and associated mechanistic processes, which paves a way to develop a microbial inoculant against plastic wastes.

Summary

Introduction

• Both PET and PE have properties such as lower density, long hydrocarbon chain, high molecular weight and tensile strength, low permeability to gases, durability to physical and chemical degradation, non-biodegradable compound [16] [17] [18] .
• As an environmentally friendly alternative to conventional plastic waste management methods, microorganisms-mediated degradation is the most promising way to accomplish an eventual bioremediation of plastic wastes.
• As compared to the extensive studies about PETdegrading bacteria and enzymes 14, 18, 19, [24] [25] [26] , the researches about PE degradation mediated by microorganisms lag well behind and the degradation efficiency using a single strain or enzyme is still too low to meet the industrial applications requirements.

PE.

• To obtain potential marine bacteria degrading PET or PE, the authors collected about 300 sediment samples contaminated by plastic debris from different locations of a bay of China.
• The copyright holder for this this version posted November 8, 2020.
• Of note, these colonizers formed an obvious biofilm layer and closely interacted each other with filament-like structures on PET (Supplementary Fig. 1g ) and PE films (Supplementary Fig. 1j ).
• Consistently, both PET and PE films were evidently degraded after 4 weeks treatment by the consortium even observed by eyes (Figs. 1b, 1d ), and the four corners of both films lost sharp morphology as that shown in the control (Figs. 1a, 1c ).
• Similar to additive-containing plastics, the consortium prefers to degrade pure PE than PET.

Isolation of the key degraders and reconstitution of the functional community capable of plastic degradation.

• To figure out the composition and dynamics of the above microbial community during the course of plastics degradation, the authors performed an absolute quantitative analysis of 16S rRNA sequences on this microbial flora.
• The copyright holder for this this version posted November 8, 2020.
• Obvious cracks in the films were observed after 7 days incubation (Supplementary Fig. 3f ), indicating the plastics had been degraded in this stage.
• As expected, bacterial strains belonging to above five genera were obtained.
• Notably, SEM observations indicated that the mixture of three above bacteria had a greater degradation efficiency on both PET (Fig. 2h ) and PE (Fig. 2i ), suggesting that the bacterial community had a better capability than single isolate for plastics degradation.

Verification of the degrading effects of the reconstituted bacterial community on

• To further verify the degradation effects of the reconstituted bacterial community on PET and PE, the authors performed multiple techniques to clarify the degradation efficiency and products led by this bacterial community.
• The copyright holder for this this version posted November 8, 2020.
• Consistently, according to a peak-differentiating and imitating calculation analyzed by X-Ray Diffraction (XRD), the relative value of crystallinity degree reduced from 92.55% to 89.85% for treated PET for four weeks (Fig. 4d ), and decreased from 49.10% to 29.50% for treated PE (Fig. 4h ) for four weeks.
• Together, in combination of the results of SEM observation, FTIR, GPC and XRD analyses, the authors conclude that the reconstituted bacterial community indeed possesses a strong capability of degrading both PET and PE.

Transcriptomic profiling of the plastic degradation process and mechanism led

• To explore the plastic degradation process and potential mechanisms mediated by the reconstituted bacterial community, the authors performed a macro transcriptome analysis of this flora in the presence of PET or preprint (which was not certified by peer review) is the author/funder.
• The copyright holder for this this version posted November 8, 2020.
• The bacteria could obtain energy for growth and reproduction through degradation and preprint (which was not certified by peer review) is the author/funder.

Discussion

• Plastics have become a global concern as the accumulation in the world's oceans and their impacts on marine organisms and human health [2] [3] [4].
• Therefore, their results answer the question that whether oceanic bacteria are capable to degrade plastics, and clearly show that plastic waste associated bacteria in the marine environment have great potentials to develop plastic degradation bio-products.
• The copyright holder for this this version posted November 8, 2020.
• In contrast, mixed flora has stronger environmental adaptability, higher degradation efficiency and more ample scope for the greater use of biotechnologies in biodegradation compared with single pure bacterium due to the synergistic effect of different microorganisms among them 28 .
• PE biodegradation has been observed with an extreme long incubation time (up to couples of months), given appropriate conditions.

Methods

• Screening of microbial community degrading PET and PE.
• About 300 sediment samples contaminated by plastic debris from different locations of Huiquan Bay preprint (which was not certified by peer review) is the author/funder.
• The copyright holder for this this version posted November 8, 2020.
• Three kinds of PET plastic were used for degradation assays in the present study, including plastic drink bottle, type ES301450 (0.25 mm in thickness) and type ES301005 (0.0005 mm in thickness), the latter two were purchased from the Good Fellow Company (UK).
• None additives were contained in the films purchased from the Good Fellow Company according to the manufacturer's standard (Q/SH3180014).

Absolute quantification of individual bacterial abundance in the community.

• The original bacterial community cultivated in the minimal medium without the supplement of any plastics was set as a control group.
• The copyright holder for this this version posted November 8, 2020.
• The spike-in sequences contained conserved regions identical to those of natural 16S rRNA genes and artificial variable regions were distinct from those found in nucleotide sequences in public databases.
• The PET and PE films treated with or without bacterial community were washed in ultrasonic cleaner with 1% SDS, distilled water, and then ethanol 19 .

X-Ray Diffraction (XRD) analysis. XRD was carried out by using the Bruker D8

• Advance instrument with a wavelength of 1.5406 angstrom of CuKα ray.
• The XRD tube current was set as 40 mA, and the tube voltage was set as 40 kV.

High-performance liquid chromatography-Mass spectrometry (HPLC-MS)

• The mobile phase was methanol at a flow rate of 0.5 mL/min, and the effluent was monitored at a wavelength of 240 nm.
• The reaction mixture supernatant was diluted with the mobile phase toward to the calibration range, acidified with concentrated HCl (37%) and centrifuged to remove any precipitation 19, 49 .
• Preprint (which was not certified by peer review) is the author/funder.

Isolation and genome sequencing of three bacteria leading plastics degradation.

• To isolate the bacteria in the community, the biofilm attached to the films were collected and plated on the 2216E solid medium (containing 5 g/L peptone, 1 g/L yeast extract, 1 L filtered seawater, 15 g agar, pH adjusted to 7.4-7.6).
• The purity of bacterial strains was confirmed by repeated partial sequencing of the 16S rRNA gene.
• Sequencing libraries were generated using NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina ® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample.
• Clean data were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data.
• GO enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected.

Figures

Figures 630

Full text

1
A marine bacterial community that degrades poly(ethylene
1
terephthalate) and polyethylene
2
Rongrong Gao
1,2,3,4
, Chaomin Sun
1,2,4*
3
1
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese
4
Academy of Sciences, Qingdao, China
5
2
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory
6
for Marine Science and Technology, Qingdao, China
7
3
College of Earth Science, University of Chinese Academy of Sciences, Beijing,
8
China
9
4
Center of Ocean Mega-Science, Chinese Academy of Sciences, Qingdao, China
10
11
*
Corresponding author
12
Chaomin Sun Tel.: +86 532 82898857; fax: +86 532 82898857.
13
E-mail address: sunchaomin@qdio.ac.cn
14
15
Keywords: ocean, plastics, bacterial community reconstitution, degradation,
16
pollution
17
Running title: A marine bacterial community degrades PET and PE
18
19
20
21
22
23
24
25
26
27
28
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted November 8, 2020. ; https://doi.org/10.1101/2020.11.07.372490doi: bioRxiv preprint

2
Abstract
29
Plastic wastes have become the most common form of marine debris and present a
30
growing global pollution problem. Recently, microorganisms-mediated degradation
31
has become a most promising way to accomplish the eventual bioremediation of
32
plastic wastes due to their prominent degradation potentials. Here, a marine bacterial
33
community which could efficiently colonize and degrade both poly (ethylene
34
terephthalate) (PET) and polyethylene (PE) was discovered through a screening with
35
hundreds of plastic waste associated samples. Using absolute quantitative 16S rRNA
36
sequencing and cultivation methods, we obtained the abundances and pure cultures
37
of three bacteria mediating plastic degradation. We further reconstituted a tailored
38
bacterial community containing above three bacteria and demonstrated its efficient
39
degradation of PET and PE through various techniques. The released products from
40
PET and PE degraded by the reconstituted bacterial community were determined by
41
the liquid chromatography-mass spectrometry. Finally, the plastic degradation process
42
and potential mechanisms mediated by the reconstituted bacterial community were
43
elucidated through transcriptomic methods. Overall, this study establishes a stable and
44
effective marine bacterial community for PET and PE degradation and sheds light on
45
the degradation pathways and associated mechanistic processes, which paves a way to
46
develop a microbial inoculant against plastic wastes.
47
48
49
50
51
52
53
54
55
56
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted November 8, 2020. ; https://doi.org/10.1101/2020.11.07.372490doi: bioRxiv preprint

3
Introduction
57
Plastics have been found widespread in the world’s oceans
1-4
. It has been reported that
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about 4.8 to 12.7 million tons of plastic debris per year enter the ocean
5
. Plastics in the
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marine environment are of increasing concern because of their persistence and effects
60
on the oceans
6
, wildlife
7
, and, potentially, humans
3
. An estimated one million birds
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and ten thousand marine animals die each year as a result of ingestion of or trapping
62
by plastics in the oceans
8,9
. Moreover, after weathering, mechanical wear and
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ultraviolet radiation, the large plastic may be broken into fragmentation, when it is
64
smaller than 5 mm in diameter, it was commonly defined as microplastics
10
. Of note,
65
microplastics also negatively impact upon marine biota and can be ingested and
66
accumulated along trophic webs until top predators
11,12
.
67
Among various types of plastic wastes, poly (ethylene terephthalate) (PET) and
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polyethylene (PE) constitute the major 46.5% portion of the tremendous amount of
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plastic pollution debris
13
. PET is a type of semi-aromatic thermoplastic co-polymer
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resin from polyester family, which has aromatic groups heteroatoms in the main
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chain
14
. PE has a carbon-carbon backbone which is solely built of carbon atoms and
72
has high resistance against various degradation processes, due to non-hydrolyzable
73
covalent bonds
15
. Both PET and PE have properties such as lower density, long
74
hydrocarbon chain, high molecular weight and tensile strength, low permeability to
75
gases, durability to physical and chemical degradation, non-biodegradable
76
compound
16-18
.
77
Landfilling, incineration, recycling and biodegradation are the principal strategies
78
to solve the plastic waste problem
8
. As an environmentally friendly alternative to
79
conventional plastic waste management methods, microorganisms-mediated
80
degradation is the most promising way to accomplish an eventual bioremediation of
81
plastic wastes. Microbial degradation of plastics is usually an enzymatic activity that
82
catalyzes the cleavage of polymer bond into monomer entity
19,20
. Thus far, PET
83
hydrolyzing activity has been reported for members of the cutinase, lipase and
84
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted November 8, 2020. ; https://doi.org/10.1101/2020.11.07.372490doi: bioRxiv preprint

4
esterase
20
. PE, as one of the most abundant plastics in the ocean, shows obvious signs
85
of degradation when incubated with specific microorganisms under controlled
86
laboratory conditions
21-23
. However, as compared to the extensive studies about PET-
87
degrading bacteria and enzymes
14,18,19,24-26
, the researches about PE degradation
88
mediated by microorganisms lag well behind and the degradation efficiency using a
89
single strain or enzyme is still too low to meet the industrial applications requirements.
90
Alternatively, using microbial community to degrade PE might be a good choice
91
given their inherent multiple robust function among synergistic effect of different
92
species
27
. Actually, the construction of artificial microbial consortia has opened a new
93
horizon in environment bioremediation in terms of removing hard biodegradable
94
harmful compounds
28
.
95
Herein, a marine bacterial community efficiently degrading both PET and PE
96
was obtained by a large-scale screening. Three bacteria driving plastic degradation
97
were isolated and reconstituted to an artificial bacterial community with a similar
98
degradation capability to that of the original bacterial flora. The degradation effects
99
and possible products of PET and PE treated by this reconstituted bacterial
100
community were further clarified by various techniques. Lastly, the potential
101
degradation process and associated enzymes were disclosed through transcriptomics
102
methods.
103
Results
104
Discovery of a marine bacterial community efficiently degrading both PET and
105
PE. To obtain potential marine bacteria degrading PET or PE, we collected about 300
106
sediment samples contaminated by plastic debris from different locations of a bay of
107
China. Using these samples, we initiated to screen microorganisms that could use
108
plastic drink bottles (whose main component is PET) or commercial PE bags as major
109
carbon sources for growth. With that, a distinct consortium derived from one of the
110
plastic debris samples could efficiently colonize on both PET (Supplementary Fig. 1b)
111
and PE films (Supplementary Fig. 1d). Scanning electronic microscopy (SEM)
112
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted November 8, 2020. ; https://doi.org/10.1101/2020.11.07.372490doi: bioRxiv preprint

5
observation confirmed that the consortium could evidently colonize on PET
113
(Supplementary Fig. 1f) and PE films (Supplementary Fig.1i). Of note, these
114
colonizers formed an obvious biofilm layer and closely interacted each other with
115
filament-like structures on PET (Supplementary Fig. 1g) and PE films
116
(Supplementary Fig.1j). After removing the microbial layer from the films, significant
117
morphological changes in both PET (Supplementary Figs. 2b-2d) and PE films were
118
observed by SEM, especially for PE which showed large amount of heavy cracks and
119
deep holes in the surface and even the inside of the film (Supplementary Figs. 2f-2h).
120
Given the fact that most commercial plastics contain various additives (such as
121
dyes, plasticizer, antistatic agents etc.), it is necessary to make sure that the
122
consortium indeed degraded the plastics rather than the additives. We thus repeated
123
the degradation test by the above consortium with the PET and PE films without any
124
additives. Consistently, both PET and PE films were evidently degraded after 4 weeks
125
treatment by the consortium even observed by eyes (Figs. 1b, 1d), and the four
126
corners of both films lost sharp morphology as that shown in the control (Figs. 1a, 1c).
127
Similar to the results obtained with the additive-containing plastics, SEM observation
128
revealed that the consortium could efficiently colonize on the surface of films (Figs.
129
1f, 1i) and caused obvious degradation by forming pits, cracks or holes in the surface
130
and inside of PET (Fig. 1g) and PE film (Fig. 1j). Similar to additive-containing
131
plastics, the consortium prefers to degrade pure PE than PET. Overall, we conclude
132
that this consortium could efficiently degrade both PET and PE, and is worthy of
133
further study.
134
Isolation of the key degraders and reconstitution of the functional community
135
capable of plastic degradation. To figure out the composition and dynamics of the
136
above microbial community during the course of plastics degradation, we performed
137
an absolute quantitative analysis of 16S rRNA sequences on this microbial flora. The
138
growth curve of this microbial flora showed that it took about 10 h to enter the
139
stationary phase and kept a stable population quantity after 7 d- or even longer
140
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted November 8, 2020. ; https://doi.org/10.1101/2020.11.07.372490doi: bioRxiv preprint