Science of the Total Environment 717 (2020) 137064

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Science of the Total Environment

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

Periphytic biofilm: An innovative approach for biodegradation

of microplastics
Sadaf Shabbir a, Muhammad Faheem b, Naeem Ali c, Philip G. Kerr d, Long-Fei Wang a,
Sathishkumar Kuppusamy a, Yi Li a,⁎
a
Ministry of Education Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, College of Environment, Hohai University, Nanjing 210098, PR China
b
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences, University of Chinese Academy of Sciences, 71, East Beijing Road, Nanjing 210008, Jiangsu, PR China
c
Department of Microbiology, Quaid-i-Azam University, 3rd Avenue, 45320 Islamabad, Pakistan
d
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia

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

• Immobilized periphytic biofilms were

implemented for the biodegradation of
MPs.
• Glucose proved to be the most effective
C-source for effectual MPs' biodegrada-
tion.
• SEM and FTIR confirm biodegradation
showing morphological and structural
changes.
• MiSeq sequencing shows that C-sources
affects microbial community structure.
• Periphytic biofilms are eco-friendly en-
tities effectual for MP biodegradation.

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

Article history: Microplastics (MPs) have been gaining the attention of environmental researchers since the 1960s anecdotal re-
Received 24 November 2019 ports of plastic entanglement and ingestion by marine creatures. Due to their increasing accretion in aquatic en-
Received in revised form 30 January 2020 vironments, as well as resistance towards degradation, marine litter research has focused on microplastics more
Accepted 31 January 2020
recently. In the present study, a relatively new method of biodegradation was implemented for the biodegrada-
Available online 01 February 2020
tion of three structurally different MPs i.e. polypropylene (PP), polyethylene (PE) and polyethylene terephthalate
Editor: Damia Barcelo (PET). Periphytic biofilm was used for this purpose in various backgrounds of carbon sources (glucose, peptone,
and glucose and peptone). Biodegradation of MPs was estimated in terms of weight loss. It was observed that the
Keywords: addition of glucose enhanced the biodegradation of MPs by periphyton biofilm for all MPs (from 9.52%–18.02%,
Microplastics 5.95%–14.02% and 13.24–19.72% for PP, PE and PET respectively) after 60 days compared to natural biofilm alone.
Glucose To the contrary, peptone, and glucose and peptone together, were inhibitory. Biodegradation was further con-
Periphytic biofilms firmed by morphological changes observed using SEM, FTIR spectra and GPC lent further support to the results
Environment whereby new peaks appeared along with reduction in old peaks and decrease in peak intensities. MiSeq sequenc-
Biodegradation of microplastics
ing shows that Deinococcus-thermus N Proteobacteria N Cyanobacteria are the dominant phyla in natural biofilms,
and their relative abundances increase after the addition of glucose. However, the abundances shifted to
Deinococcus-thermus N Cyanobacteria N Firmicutes N Bacteroidetes, when the biofilms were treated with either
peptone alone, or with glucose and peptone together. Therefore, the change in biodegradation capability might

⁎ Corresponding author at: College of Environment, Hohai University, 1 Xikang Road,

Nanjing 210098, PR China.
E-mail address: envly@hhu.edu.cn (Y. Li).

https://doi.org/10.1016/j.scitotenv.2020.137064
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
also be due to the change in the microbial community structures after addition of the C-sources. These experi-
ments provide an innovative approach towards effective biodegradation of MPs using a relatively new
environment-friendly method.
1. Introduction
The increasing amount of microplastics (MPs) in marine environ-
ments has been regarded as an inevitable global problem over the last
few decades. The production of plastics is constantly increasing due to
their high demands (annual production from 0.5 million tons in 1950
to 350 million tons in 2017) (PlasticsEurope, 2018). According to an es-
timate, about 8 million metric tons of plastic wastes are dumped to
oceans each year (Viršek et al., 2017), constituting almost 100% of float-
ing wastes (Galgani et al., 2015) in aquatic environments.
Generally, plastics are resistant to degradation in marine environ-
ments, and their slow degradation results in the formation of small frag-
ments known as MPs. Wave action, hydrolysis, photodegradation,
sedimentation, and migration are the main factors that carry out the
natural degradation of plastics in marine environments (Gewert et al.,
2015; Wang et al., 2016; Wright et al., 2013), resulting in the formation
of MPs. Although the term ‘microplastic’ was first introduced in 2004 to
refer to tiny plastic fragments (approx. 50 μm) in marine environments,
in the newer context, this terminology represents a heterogeneous mix-
ture of particles with a variety of shapes (spherical to elongated fibers)
and with a range of sizes (from a few microns to several mm)
(Thompson, 2015).
The presence of MPs has been reported in virtually all types of
aquatic habitats including: beaches, surface waters, the water column,
sea ice, sea floor and subtidal sediments (Alomar et al., 2016; Isobe
et al., 2017; Lusher et al., 2015; Van Cauwenberghe et al., 2013). They
constitute over 90% of the total plastic count in oceans (Eriksen et al.,
2014) and are considered to be harmful primarily because they are mis-
taken as food by fishes and other marine creatures (Steer et al., 2017).
Moreover, the increasing concern about MPs, especially those com-
posed of polyethylene, is due to the fact they contain organic xenobi-
otics, viz. the plasticizers, and are, therefore, the carriers of many
potential toxicants to marine creatures, especially fish, and humans, in-
cluding pathogenic bacteria (Keswani et al., 2016; Viršek et al., 2017).
Hence, it is recommended that these small plastic entities ought to be
removed from marine environments in such a way as to reduce poten-
tial toxicants.
The MP components used in the following study, i.e. polypropylene
(PP), polyethylene (PE) and polyethylene terephthalate (PET) are
among the most extensively worldwide produced and used plastics.
Along with polystyrene (PS) and polyvinyl chloride (PVC), they consti-
tute about 90% of the plastics produced (Andrady and Neal, 2009). Most
MPs made of polypropylene (PP) and polyethylene (PE) and expanded
polystyrene (EPS) have lower densities than sea water (0.85 to
N1.4 kg L−1), therefore they tend to float on the surface of water
(Ryan et al., 2009). Whereas, plastics, such as polyethylene terephthal-
ate (PET), with higher densities, usually end up in the benthic zone of
aquatic environments (Wright et al., 2013).
The properties of the three MPs used in the present study are given in
Table S1. Even though the presence of MPs and their abundance have
been widely reported in the last few decades, there is a dearth of studies
regarding their degradation and/or biodegradation. Due to their pure car-
bon back bones, these MPs are resistant towards degradation by conven-
tional treatment systems (Debroas et al., 2017) and ultimately find their
way into open water environments. In last couple of years, some
physico-chemical methods has been reported to degrade different
microplastics including visible light photocatalysis (Tofa et al., 2019),
functionalized C-nanosprings (Kang et al., 2019) and green photocatalysis
(Ariza-Tarazona et al., 2019) and thermal degradation (Dümichen et al.,
© 2020 Elsevier B.V. All rights reserved.
2017), however, compared to biodegradation, these techniques have cer-
tain shortcomings such as higher operational costs, consumption of
chemicals and polymer specificity. Recently, a few studies on the biodeg-
radation of a few MPs have been reported (Auta et al., 2017; Auta et al.,
2018; Paço et al., 2017). However, these studies are limited in scope
since they have described biodegradation of MPs as the sole source of car-
bon. Previously, it was reported that the addition of an external C-source
can enhance the removal rate of relatively resistant pollutants in aqueous
environment (Qin et al., 2017; Shen et al., 2015; Zhao et al., 2018). To date,
there is no information regarding the influence of additional C-sources on
the biodegradation of MPs by any kind of microorganisms. Furthermore,
suitable C-sources may enhance the growth of appropriate microorgan-
isms that can elevate the rate of biodegradation of pollutant. However,
studies regarding this aspect are also scarce.
Periphyton biofilms are comprised of ubiquitous microbial entities
that are entangled together by extracellular polymeric substances
(EPS). In the last decade, there are studies that have focused on the bio-
degradation capability of these biofilms and had found them very effec-
tual towards the removal of dyes and different nutrients (Levy et al.,
2017; Shabbir et al., 2017a; Shabbir et al., 2017b; Shabbir et al., 2018;
Sutherland and Craggs, 2017). However, until now there are no studies
that have used these valuable natural entities for the biodegradation of
MPs.
Our previous studies reveal that periphytic biofilms are compara-
tively more resilient in the presence of complex contaminants, and
cope well with changing physico-chemical and biological disparities
(Shabbir et al., 2017a; Shabbir et al., 2017b; Shabbir et al., 2018) in the
surrounding environments. Therefore, periphytic biofilms could also
be an environmentally friendly and cost-effective way for the bioreme-
diation of MPs.
The specific goals of the current study were 1) to evaluate the bio-
degradation of MPs (PE, PP, and PET) by immobilized periphytic
biofilms, and 2) to examine the effects, if any, on the periphytic biofilm
biodegradation efficiencies of MPs as a sole source of carbon, and when
accompanied by other C-sources such as glucose and peptone alone, and
a glucose/peptone mix. Furthermore, an important novelty of this work
is to identify any changes in periphytic community structure as a result
of biodegradation of these MPs either alone or in the presence of addi-
tional C-sources.
2. Material and methods
2.1. Microplastics
Three MPs were used in the study; MP1, polyethylene, MP2, poly-
propylene and MP3 were polyethylene terephthalate. These were ac-
quired from Sigma-Aldrich (USA), and supplied as spheroid pellets,
1–4 mm in diameter, which were mechanically cut to obtain MP pellets
of dimensions b1000 μm (structure and properties are given in
Table S1).
2.2. Periphyton isolation and growth
Periphytic biofilm was collected from Xuan Wu Lake, Nanjing, to ob-
tain a natural microbial entity of complex structure and diverse commu-
nity to facilitate biodegradation of MPs. The biofilm was grown in the
laboratory as already described in our previous work (Shabbir et al.,
2017a; Shabbir et al., 2018) and the average lake conditions were also
determined at the time of sampling. According to our previous findings,
 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
the attached biomass had proved to be more effectual towards the re-
moval of contaminants, therefore the biofilm was grown on artificial
aquatic mats (AAM), diameter 1 cm and length 9 cm). The growth con-
ditions were similar to those already described in our previous work.
After 90 days of incubation, the biofilms were stabilized on the surface
of artificial mats and no further increase was observed in the thickness
of biofilm. After stability was achieved, these mats along with periphy-
ton biofilm were used to determine the biodegradation capability of pe-
riphyton biofilm.
2.3. Screening and biodegradation experiments
Biodegradation studies of MPs were conducted in batch reactors
(pre-sterilized 250 mL flasks) containing 150 mL of deionized water
with one pellet of each of MP in individual flasks and each of those
were inoculated with almost 4 g (wet weight) of immobilized peri-
phytic biofilms. For these experiments, four replicates were made for
each MP. The batch experiment was conducted in the dark at 30 °C
and pH 7 (optimum for periphyton growth). MP samples were collected
at regular intervals (10, 20, 30, 40, 50 and 60 days) for 60 weeks and
control experiments were also conducted with MPs in the absence of
periphytic biofilm.
The biodegradation capability of periphytic biofilms was evaluated
in four experiments: (i) MPs as a sole carbon source, (ii) glucose as an
additional C-source, (iii) peptone as an additional C-source and (iv) glu-
cose and peptone together (detailed experimental design, Table S2).
2.4. Determination of weight loss of MPs
After 60 days of incubation, and biodegradation by periphytic biofilms,
the MPs were taken out of the reactor flask and washed with ethanol,
followed by deionized water to remove biofilm. The pre-weighed MPs
(4 replicates for each of the treatment) were weighed using a 4-decimal
place analytical balance (±0.00005 g). The percentage weight loss (%)
for all treatments was calculated by the following formula:
 
W˳−W t
%weight loss ¼  100
W˳
where W˳ is the initial weight of the MPs, and Wt is the weight of MPs
after biodegradation.
2.5. Polymer reduction rate
The first-order kinetic model based on initial and final weights of
three kinds of MPs was used to determine the rate constants of MPs re-
duction for specific intervals (10 days). The following equation was
used to determine the rate constants:
 
1 W˳
k¼ ln
t Wt
where t is the time in days and k is the first-order rate constant per day
uptake of MPs. This model was useful to obtain a constant fraction per
unit time present/removed within the MPs.
Following the generation of the rate constant for the reduction of
MPs, the half-life (t1/2) of all the MPs in different treatments was deter-
mined by using the following equation:
ln2
t 1=2 ¼
k
2.6. Analytical techniques
The changes in the structure of the three MP polymers, after degra-
dation by periphytic biofilms, were evaluated by attenuated total
3
reflectance-Fourier transform infrared spectroscopy (FTIR-ATR) (Nico-
let 8700, Thermo Electron Co., USA) analysis at a 4 cm−1 resolution
within the 4000–550 cm−1 range. The analysis was carried out for all
the MPs including the control (un-inoculated) and biodegraded sam-
ples. To observe degradation, and to visualize the growth of biofilm on
the surface of all the three MPs, the samples were individually placed
on carbon tape, gold plated and observed by scanning electron micros-
copy (SEM) (EVO 18, Zeiss, Germany). Additionally, Gel permeation
chromatography (GPC) was carried out to evaluate the changes in mo-
lecular weight of polymers after all the treatments using PL-GPC PL50
(Varian, Inc.) system, with three Mixed-B columns and an RI detector.
Briefly, PP and PE (16–20 mg) samples were dissolved in 1,2,4-
tricholobenzene (TCB) + 0.15% butylated hydroxytoluene (BHT) at
160 °C whereas PET (16–20 mg) samples were dissolved in 4-
chlophenol at 100 °C in capped amber jars. The dissolved MP samples
were then filtered through 0.5 μm polytetrafluoroethylene (PTFE) filter
to remove the solid particles and were immediately placed in the
autosamplers of GPC at their respective dissolution temperatures. The
flow rate of respective eluents (TCB + 0.15% BHT and 4-chlorophenol)
was 1 mL min−1 for each polymer (Abrusci et al., 2013; Imel et al.,
2015) and duplicate samples were run for each treatment and control.
Agilent Chemstation software was used to determine the average mo-
lecular weights (Mw and Mn) and polydispersity index (Mw/Mn). The
calibration curves were made by using polystyrene standards.
2.7. Isolation and characterization of periphytic biofilms
In order to determine the variability in structure and composi-
tion of biofilms, they were peeled off the mats before and after
the treatment and observed under SEM (EVO 18, Zeiss, Germany).
The microbial activity in different environments was estimated
using the Biolog™ plate technique. Briefly, the microbial diversity
of periphytic biofilms was determined by peeling 2 g of biofilm
from the surface of mats which was inoculated into plate wells,
which were incubated at 25 °C for seven days, and average well
color development (AWCD) was evaluated using a Biolog Micro-
ð1Þ
plate Reader (590 nm) after every 24 h during the experiment
(Shabbir et al., 2018).
2.8. DNA extraction and MiSeq sequencing
DNA was extracted from all the samples by using a FastDNA
SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) following the
manufacturer's manual. The quality of extracted genomic DNA
was evaluated by using 2% agarose gel electrophoresis. PCR ampli-
fication was carried out in triplicate using forward primer 341F (5′-
CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTA
ð2Þ AT-3′) (Mori et al., 2014). ABI GeneAmp® 9700 (ABI, USA) in a total
volume of 20 μL containing 4 μL × 5 FastPfu Buffer, 2 μL dNTPs
(2.5 mM), 0.8 μL forward primer (5 μM), 0.4 μL reverse primer
(5 μM), 0.4 μL FastPfu Polymerase, 10 ng template DNA and 20 μL
ddH 2 O. The quality of PCR products was estimated by agarose gel
(2%) detection after amplification.
All the samples were stored at −80 °C before further processing. V3-
V4 regions of 16S rRNA were targeted by PCR using an ABI GeneAmp®
9700 DNA thermal cycler. Illumina HiSeq 4000 Sequencing platform
was used to further analyze the PCR products at Shanghai BIOZERON
ð3Þ Biotechnology Co., Ltd. (Shanghai, China).
After processing the raw data of sequencing, the paired-end reads
were overlapped and separated followed by removing mismatched
reads. The remaining 16S rRNA gene sequences were paired into oper-
ational taxonomic units (OTUs) at a similarity of 97% (or 3% cutoff).
The filtered sequences were classified by using the SILVA reference da-
tabase (Release119) (Quast et al., 2012).
4 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064

Fig. 1. (a). Percentage weight loss of the three MPs during 60 days under different treatment conditions, (b). pH variations during the treatment of MPs by periphytic biofilms. (MP1 =
polyethylene, MP2 = polypropylene, MP3 = polyethylene terephthalate, P = periphytic biofilm, G = glucose, Pep = peptone).

2.9. Statistical and network analysis
The Vegan package in R 3.4.3 (https://www.reproject.org/) was
used to calculate the α-diversity indices (OTU richness, Chao,
Shannon index, Simpson index, and Evenness index). Principal
component analysis (PCA) was carried out to determine the simi-
larity between the samples.
Spearman's correlation analysis was applied to determine the co-oc-
curring networks in the microbial communities. Specifically,
Spearman's rank correlation coefficients (ρ) between all 97% cutoff
OTUs, with occurrence in at least 25% of samples and at least 30 counts
in each cluster, were calculated pairwise using the R package. Network
visualization and modular analyses were conducted using Gephi
(http://gephi.github.io/). The topological properties of the networks,
including average degree, betweenness centrality, modularity, and av-
erage path length were also calculated using Gephi. The size of each
node is proportional to the relative abundance of the bacterial commu-
nity (log (n + 1)).
The functional potential of the biofilm was evaluated by using the
Phylogenetic Investigation of Communities by Reconstruction of Unob-
served States (PICRUSt) version 1.0.0 (Langille et al., 2013). A similarity
threshold of 0.97 of OTUs was used in QIIME for the prediction of
PICRUSt and the Greengenes database (13_5_release) was used as a
clustering reference and the metagenome functional profiles were pre-
dicted using the predict_metagenomes.py script.
The statistical analysis of data for MPs was conducted using analysis
of variance with the p b 0.05 indicated statistical significance (SPSS
21.0).
 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
(i)
(ii)
(iii)
Fig. 2. SEM micrographs of three MPs. {(i) PE, (ii) PP and (iii) PET, (A = control, B = MP as a sole source of carbon, C = glucose as an additional C-source, D = peptone as an additional C-
source, and E = glucose + peptone was added as additional C-sources).
3. Results and discussion
3.1. Changes in weight of MPs during biodegradation
The three MP polymers used in this study can be classified into two
groups based on their backbone structure; (i) PP and PE have a C-C
backbone structure and (ii) PET, with ester linkages, has heteroatoms
in the backbone structure. The determination of weight loss of MPs
after treatment with periphytic biofilms is a direct quantitative method
to determine the extent of biodegradation. The MP samples were ex-
posed to periphytic biofilms for 60 days, and showed marked variation
in their recovery weights (Fig. 1a). Additionally, the effect of substrate
competition on biodegradation of MPs was also examined by using dif-
ferent C-sources. Previous reports indicate that during the biodegrada-
tion of benzo(a)pyrene and 4-chlorophenol, the addition of different
C-sources such as glucose, starch, sucrose and peptone, have a profound
effect on the biodegradation capability of microbes, changing the micro-
bial community structure (Qin et al., 2017; Zhao et al., 2018). Therefore,
the aim was to check the effect of the additional C-sources on the bio-
degradation capability for MPs.
We observed that polypropylene was degraded most in the presence
of glucose as additional C-source (18.02%), next, in the absence of any
additional C-source (13.27%), followed by degradation in the presence
of peptone (10.45%), and finally in the presence of glucose and peptone
together (9.62%). Similar trends were noted for both polyethylene and
PET, where the highest degradation was observed in the presence of
5
glucose, 14.02% and 19.72%, respectively. The addition of peptone
alone, or glucose and peptone together, appeared to inhibit the degra-
dation of MPs. Moreover, the uninoculated control samples did not
show any weight loss, implying that the periphytic biofilms were re-
sponsible for the weight loss due to the degradation of the MPs.
The additional C-sources were added in the treatments to evaluate
the inhibitory or facilitatory effect on the removal of MPs. According
to previous studies, the addition of simple and easily assimilated carbon
sources is an effective amendment to the bioremediation of recalcitrant
compounds such as benzo(a)pyrene and 4-chlorophenol (Qin et al.,
2017; Shen et al., 2015; Zhao et al., 2018).
We obtained similar results, where glucose enhanced the biodegra-
dation of all the three MPs. Periphytic biofilms have the natural capabil-
ity to attach to the surface and grow on a given medium. Additionally,
the community composition changes in response to additional C-
source, that might have enhanced the secretion of intra- and extracellu-
lar enzymes that results in higher biodegradation.
3.2. pH variations during biodegradation
The physiological and biochemical processes of microorganisms are
influenced by pH, which plays a key role in the growth of periphytic
biofilms as well as the removal of contaminants (Shabbir et al.,
2017a). Therefore, the initial pH of the experiment was set at 6.8, an en-
vironmentally relevant condition, suitable for growth and function of
periphytic biofilms. As the growth of periphytic biofilm, followed by
6 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
MP biodegradation, proceeded, the pH of the medium was found to
have slightly increased after 60 days (Fig. 1b). The pH changes were
greater in the samples with glucose i.e. 8.78–9.08, as an additional C-
source followed by the samples without any C-source i.e. 8.54–8.72.
The pH changes in the other treatments were lower than these two
treatments.
We have reported in our previous studies that periphyton has the
biodegradation capability over a wide range of pH values (3−11)
(Shabbir et al., 2017a; Shabbir et al., 2018). Auta et al. (2018) have re-
ported that the change in pH (alkaline) during the biodegradation of
MPs is due to; (i) the production of alkaline aromatic compounds and
(ii) the excretion of metabolites by microorganisms. These changes
might also have disturbed backbone structure of MPs, thus facilitating
periphytic biofilm for further biodegradation.
3.3. Reduction rate and half-life of MPs
The removal constant (k) for the biodegradation of MPs was deter-
mined based on first-order kinetics, along with the half-life supporting
the biodegradation of MPs. After 60 days of biodegradation, the uptake
rate of PP by periphytic biofilm was 0.0024 day−1, in the presence of
glucose, 0.033 day−1, in the presence of peptone, 0.0018 day−1 and in
the presence of both glucose + peptone, 0.0016 day−1.
The shortest half-life was for the glucose treated PP whereas the one
with peptone + glucose treatment was the longest, with similar trends
being followed by PE and PET (Table S3). The shortest half-life of PP
(209.29 days), PE (275.27 days) and PET (189.31 days) was observed
in the treatments with glucose as additional C-source, and the corre-
sponding removal rate constants were 0.0033 day−1, 0.0025 day−1
Fig. 3. FTIR of MPs (a) PE, (b) PP and (c) PET. {A = MP as a sole source of carbon, B = glucose as an additional C-source, C = peptone as an additional C-source, and D = glucose + peptone
were added as additional C-sources}.
and 0.0036 day−1, respectively. The higher removal rate by microbial
communities shows their capability to degrade the MPs (Auta et al.,
2017). Moreover, compared to previous studies, periphytic biofilms re-
sulted in greater weight loss and shorter half-lives of MPs, which dem-
onstrates their potential towards implementation in wastewater
treatment plants.
3.4. SEM of biodegraded MPs
SEM of 60-day samples clearly exhibits the growth and colonization
of periphytic biofilms on the surface of all three microbially treated MPs
(PP, PE and PET) compared to the smooth surfaces of un-inoculated
MPs. The surface of microbially treated MPs shows various grooves,
pores and cracks, thus corroborating the capability of periphytic
biofilms in the biodegradation of MPs (Fig. 2). These results verify the
previous studies, that microbial communities have the potential to
cover the polymer (MP) surface leading to its surface erosion (Auta
et al., 2018; Paço et al., 2017). SEM results show, that in all the three
MPs investigated, the best results are obtained by using glucose as an
additional C-source, followed by treatment where the microplastics
themselves are the sole source of carbon.
It was also observed that peptone as an additional C-source shows
comparatively less biodegradation, followed by periphyton treatment
in the presence of glucose and peptone. As the periphytic biofilm is
composed of a variety of microorganisms, it is likely that they excrete
EPS and extracellular enzymes after attachment to the surface of MPs.
These extracellular enzymes oxidatively attack the C-C back bone (for
PP and PE), or hydrolytically cleave the ester backbone (PET) structure
of the MP polymers resulting in their degradation.
 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
Fig. 3 (continued).
3.5. FTIR-ATR analysis of MPs
FTIR-ATR was performed to evaluate the structural changes in all
three MPs (PP, PE, and PET) after 60 days exposure to periphytic
biofilms, and to confirm biodegradation in terms of changes in struc-
tures of these polymers. The FTIR-ATR spectra of MPs are shown in
Fig. 3(a–c), the spectra show a comparison of the control un-
inoculated MPs and different type of MPs after inoculation with peri-
phytic biofilms. Evident changes had been observed in the spectra of
MPs treated with periphytic biofilms. Rather than shift, most of the
peaks had shown the reduction in their intensity.
3.5.1. PE
In PE, 3394.10 cm−1 would represent the O\\H bond of the oxidized
methylenic carbons (CH2) of the partially degraded polymer. The peak
at 1463.22 cm−1 is for sp2 C_C stretch of aromatic moieties that form
during the oxidative degradation of PE. The peak at 1463 cm−1 may
also be showing the presence of a chemically complex intrinsic constit-
uent of bacteria, of protein level, and the amino and neutral polysaccha-
rides (Ma et al., 2014). The peaks of PE occurring at, 2914 cm−1,
2684.6 cm−1, 1463 cm−1 and 728 cm−1 indicate –CH bond stretches
and bends remained at their place in all the treated samples whereas
the peak at 1376 cm−1 disappeared after treatment, this peak
7
represents aliphatic C\\H bending in structure (Fig. 3a). In sample,
treated with peptone and glucose together, 1645 cm−1 had also disap-
peared which is also an indicator of double bond. In sample 4B, three
new peaks were formed at 1577 cm−1, 1540 cm−1 and 1061 cm−1, in-
dicating the formation of new double bond structures. Additionally,
peak at 3184 cm−1 has also disappeared in all the samples, which rep-
resents the O\\H group formation. In sample C, a new peak was ap-
peared at 1082.35 cm−1, indicating the formation of tertiary alcohol.
In sample D, 3184 cm−1, 1463 cm−1 and 728.96 cm−1 disappeared
whereas the peak at 1463 cm−1 was shifted to 1470 cm−1.
3.5.2. PP
In PP, new peaks have appeared at 3391.69–3392.17 cm−1 in sam-
ples B, C and D, showing the presence of O\\H bonds, indicating the for-
mation of tertiary alcohol functions via oxidation of the methylene
groups of the polymer backbone. The reduction in the peaks at
972 cm−1 and 992 cm−1 in samples B-D may be attributed to C\\C
stretching (Fig. 3b).
Appearance of a new peak at 1747.67 cm−1 in C indicates the forma-
tion of carbonyl compound and the one appeared in B, i.e. 873.11 cm−1
shows the C\\H alkyl bend. The peaks 1453 cm−1 indicating the C\\H
bend of methylene group (CH2) and the one at 1375 cm−1 indicating
C\\H bend of the methyl group (CH3) were present in treated samples
8 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
Fig. 3 (continued).
as well. The peaks at 1645 cm−1 for primary amine and peaks from 1358
indicates CH2 bend whereas the band at 1202 cm−1 and the one at
1102 cm−1 are due to C\\O stretching. A new peak had also appeared
at 668.70 cm−1 in sample B, C and D that stands for an alkyne C\\H
bend.
3.5.3. PET
The native peaks of PET were observed in un-inoculated and inocu-
lated samples, these peaks include 1712 cm−1 (C_O stretch),
1240 cm−1 (sp2 C\\O), 1092 cm−1 (sp3 C\\O stretches of the ester moi-
eties) (Guo et al., 2018) and 871 cm−1 (aromatic CH bending) (Fig. 3c).
However, evident reduction in the intensity of all peaks was found in all
the samples of PET treated with periphytic biofilm compared to the in-
oculated control sample.
A new peak at 1849.31 cm−1 appeared in samples A, B and C, addi-
tionally, a band also appeared at 1646 cm−1 in sample A indicates a pri-
mary amide stretch; at 846.11 cm−1 indicates para di-substitution of an
aromatic ring, in all samples and 668.7 cm−1 in sample A, B and C show-
ing a C\\H bending vibration of a new alkyne bond. Moreover, the peaks
in the control sample at 1578–1579 cm−that stands for 1 secondary
amid H-bending and the peak at 970.5 cm−1 showing trans\\CH out-
of-plan bend of an alkene along with the peak at 791–792 cm−1 indicat-
ing aromatic\\CH out-of-plan bend were disappeared and were no lon-
ger distinctive in the spectra.
Auta et al. (2017) described the biodegradation of MPs as being due
to oxidation and reduction reactions that result in the production of
new carbonyl, hydroxyl, alcohol and phenolic functional groups. This re-
sults in an increase in hydrophilicity of the MPs that will in turn enhance
the adherence of microbes to the surface of MPs, thus enhancing the
process of biodegradation. Previously, similar results had been reported
that the microbial biofilms can change the structure of MPs and results
in the biodegradation of MPs (Auta et al., 2017; Auta et al., 2018; Paço
et al., 2017) as observed by ATR-FTIR.
3.6. GPC analysis of MPs
GPC was used to evaluate the biodegradation of three kinds of MPs
by periphytic biofilm with and without different C-sources. The analysis
showed that the results obtained by this experiment re highly coherent
as the data obtained by analyzing duplicate samples of each MP was co-
inciding with each other (data not shown here). GPC analysis results of
samples after 0 and 60 days of treatments are shown in Table 2. The
values of number-average molar mass (Mn) and weight- average
molar masses (Mw) were decreased after 60 days compared to control
sample (Table 2), indicating MPs has been degraded by biofilm. The
polydispersity (Mw/Mn) was also reduced which implies the reduction
of overall molecular weight distribution. The most significant changes
in Mw and Mn was found in the samples with glucose as an additional
C-source. For PP, the decrease in Mw and Mn was 25% and 14%,
 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064 9

Table 1 Out of 29 phyla detected in the sequencing of the periphytic biofilm

Diversity statistics of periphytic biofilm for the different treatments.
samples, among them, Proteobacteria, Cyanobacteria, Deinococcus-
Treatment Sequences OTU Simpson Shannon Chao1 Thermus and Bacteroidetes were the most abundant phyla (Fig. 4a).
Raw Effective The periphytic communities were dominated by phylum Deinococcus-
Thermus in the control and glucose treated samples (relative abun-
MP1 58,271 56,435 563 0.08 0.91 789.5
MP1 +G 31,660 30,517 467 0.31 0.69 499.8
dance = 28–56%). These were followed by Proteobacteria with a rela-
MP1 + Pep 39,018 30,694 360 0.11 0.88 431 tive abundance of 21–35% and Cyanobacteria in these samples.
MP1 + Pep + G 48,460 46,326 392 0.10 0.89 456.3 However, the relative abundances of these phyla were relatively higher
MP2 25,336 24,590 575 0.08 0.95 802.8 in the treatment where glucose was added as an external source. This
MP2 +G 58,524 57,489 472 0.43 0.72 487
higher abundance might be the reason for the higher biodegradation
MP2 + Pep 41,700 40,577 368 0.13 0.85 408
MP2 + Pep + G 57,527 54,802 398 0.20 0.87 411 of MPs after glucose addition. Microbes from phylum Deinococcus-
MP3 40,548 35,813 553 0.07 0.94 790 Thermus are already known for their resistance to DNA damaging condi-
MP3 +G 48,218 45,502 456 0.39 0.66 567 tions as well as potential in bioremediation processes due to their ther-
MP3 + Pep 31,863 30,862 349 0.14 0.86 583.9 mostable enzymes (Theodorakopoulos et al., 2013).
MP3 + Pep + G 59,813 56,335 396 0.18 0.81 582
Contrarily, the relative abundance of these phyla changed in the
treatments with different C-sources i.e. Peptone and glucose + peptone.
The abundance in these groups shifted to Deinococcus-Thermus and
respectively, for PE, it was approximately 35% and 25% whereas for PET, Cyanobacteria followed by Firmicutes and Bacteroidetes. The relative
it was 28% and 23%, respectively when glucose was used as an external abundance of Cyanobacteria was found to be increased after these treat-
C-source. Other treatments have also shown reduction; however, it was ments. This shows that the phototrophic bacteria had increased after
lower compared to this treatment and followed the same trend as biodegradation of MPs in these treatments. The growths of other
weight loss results (MP + G N MP N MP + Pep N MP + Pep + G). GPC phyla were relatively low in all the treatments (b1%), these include
results signify the fact that periphytic biofilms are effectual towards bio- Firmicutes, Chloroflexi, Ignavibacteriae and Verrucomicrobia (Fig. 4a).
degradation of high molecular weight MPs used in the present study. Similarly, when the community composition was compared at class
Recently, a few studies have reported the role of microorganisms in level, it was observed that the biodegradation process has changed the
the degradation of MPs (Auta et al., 2017; Auta et al., 2018; Paço et al., dominance of different classes. This might be due to one or both of the fol-
2017), however, the present study is the first report that has verified lowing two reasons, (i) the addition of different C-sources in the
the microbial biodegradation of MPs by using GPC. treatment process or (ii) the formation of byproducts after biodegrada-
tion. After biodegradation of MPs, a change in the abundances of different
3.7. Periphytic biofilms classes of microorganisms was found. However, Alphaproteobacteria,
Betaproteobacteria, Cyanobacteria, Gammaproteobacteria and
Periphytic biofilms are the amalgamation of different kinds of micro- Sphingobacteria remained the dominant classes both before and after
organisms including bacteria, algae and protozoa. They are found either biodegradation. The similarities and differences in periphytic biofilms
attached to some surface or suspended and float freely in natural under different treatments were also estimated on the basis of OTUs
aquatic environments. The capability of these biofilm in the biodegrada- (Fig. S2(A–C)). OTUs basically represent the diversity of the biofilm but
tion of different contaminants is already well-established (Basilico et al., it is not related to relative abundance of a microbial community. It was ob-
2016; Miao et al., 2015; Shabbir et al., 2017a; Shabbir et al., 2018). How- served that the number of common and specific OTUs was different for
ever, there have been no studies published regarding the biodegrada- the different treatments that might also be due to the addition of different
tion of microplastics using these biofilms. C-sources during the treatment.
SEM shows the structure of periphytic biofilm as being interwoven Although the dye degradation capability of these biofilms has
clusters with voids, additionally, algae, bacteria and diatoms were already been discussed in our previous work (Shabbir et al.,
clearly visible in the micrographs. The voids and tunnels present in peri- 2017a; Shabbir et al., 2018), the MP degrading capability of
phytic biofilms might assist in the attachment of biofilms to MPs periphytic biofilm have not yet been reported in the literature.
(Fig. S1, A–D). Once they are attached to the surface of MPs, the biofilms There is also a need to evaluate their capability of these biofilms
secrete different enzymes that enable the degradation processes that ef- to degrade MPs in the presence of other important environmental
fectively provide fuel for the periphyton community as a whole. variables.
In the Biolog® experiments, the AWCD is used as an indicator of the
degree of activity of microorganisms. The darker the color of the well,
the higher would be the activity of the microorganisms. The diversity in-
Table 2
dices determined demonstrate higher community richness, community Changes in molecular weights of PP, PE and PET determined by GPC.
dominance, and homogeneity of the microbial community, and species
Microplastic Treatments 60 days
evenness of the periphytic biofilm that were used in our study
(Fig. S1E). For example, the Shannon index as determined by Biolog® Mw Mn Mw/Mn
was 3.4 after 7 days of incubation. That means a higher diversity of mi- Polyethylene (PE) Control 182,366 23,321 7.82
croorganisms in the periphytic biofilm. Without C-source 147,824 19,237 7.68
Glucose 118,475 17,365 6.82
Peptone 160,053 21,214 7.54
3.8. Microbial community structure with different C-sources Glucose + peptone 162,646 21,393 7.60
Polypropylene (PP) Control 274,258 61,364 4.47
On the basis of supplying different C-sources and biodegradation ex- Without C-source 236,293 54,372 4.35
periments of different MPs, the microbial diversity and community dif- Glucose 203,847 52,293 3.90
Peptone 253,887 56,914 4.46
ferences were analyzed and compared between different treatments.
Glucose + peptone 260,183 57,923 4.49
The Illumina MiSeq sequencing of different periphytic biofilm samples Polyethylene terephthalate Control 62,934 25,838 2.44
generated 538,230 high quality reads (24,463–55,700 reads per sam- (PET) Without C-source 48,142 20,747 2.32
ple) after quality trimming and subsampling (Table 1). The taxonomic Glucose 45,238 19,847 2.28
categorization of OTUs at different levels generated 29 phyla, 94 classes, Peptone 52,234 21,786 2.40
Glucose + peptone 52,239 22,187 2.35
188 orders, 354 families and 750 genera.
10 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064

Fig. 4. Relative abundance (%) of periphytic community at (a) phyla and (b) class levels.

3.9. Diversity and richness of biofilms
Due to effectual biodegradation of different MPs both individually
and in the presence of different C-sources, the microbial community
structure of the biofilm was analyzed and compared by high throughput
pyrosequencing. The diversity of microbial biofilms can be evaluated by
the evenness and richness, and in the present study, this has been deter-
mined by the Simpson, Shannon and Chao1 indices (Table 1). Approxi-
mately, 349–575 OTUs were detected in all the samples after clustering
them at a 97% similarity level. As shown in Table 1, the samples without
any C-source had the highest OTUs followed by the samples with glu-
cose as an additional C-source. Our results show that biodegradation
without any additional C-source has the highest diversity of anaerobic,
aerobic, facultative and other kinds of bacteria followed by other treat-
ments. The Simpson and Chao1 indices reveal the community diversity,
and the Shannon diversity index shows the community richness (Yang
et al., 2015). The addition of different C-sources reasonably changes the
Simpson, Chao1 and Shannon diversity indices, indicating that these C-
sources have an important influence on the diversity and richness of
periphytic biofilm. Xie et al. (2018) has also evaluated the impact of dif-
ferent C-sources. Briefly, the results show that the additional C-sources
vary the biofilm structure not only at the phylum and class levels but
also at the species level. Additionally, the changes were also evident in
richness and diversity as shown by diversity indices, which is the reason
behind the variation in the biodegradation capability of biofilm for each
of the MP.
 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
Fig. 5. Co-occurrence network of different communities in periphytic biofilm showing strong interspecies correlations.
3.9.1. Co-occurrence network analysis and prognosticate potential of
biofilm
Co-occurrence network analysis of different co-existing communi-
ties is a useful method to give a visual summary of the association be-
tween microorganism in a community. In order to evaluate the
interaction between different communities, network analysis was car-
ried out. Based on Spearman correlation analysis, the microbial network
of periphytic biofilm in all the treatments comprised of 241 nodes and
4988 edges (Table S4), and the average network distance was 2.58 for
all the nodes. In the modules, obtained from respective OTUs, most of
the nodes show strong co-relation with each other in all samples
(Fig. 5). The modularity of all the networks differs from each other,
thus confirming the fact that the earlier work regarding this aspect
(Zhang et al., 2018). However, the strong co-relation between nodes
shows that the microbe-microbe interaction remains strong even with
the addition of external source or by the biodegradation products.
Additionally, the functional attributes of the microbial community in
periphytic biofilm was estimated using PICRUSt, a method used to pre-
dict the functional proficiency of the community at different levels. This
method also validates the presence of genes involved in various pro-
cesses such as environmental information processing, cellular pro-
cesses, genetic information processing, and metabolism (Wu et al.,
2019). The relative abundance of various genes was relatively higher
in the treatments with glucose as C-sources followed by treatments
with no additional C-source, with peptone, and finally, glucose and pep-
tone (p b 0.05), which is an indication of the reduction in functionality of
various genes involved in the processes (Fig. 6). As the following exper-
iment was carried out to evaluate the impact of degradation as well var-
ious C-sources on biofilm structure, this heatmap also shows that the
11
genes involved in the xenobiotic metabolism of biodegradation as well
as those for metabolic reactions, were lower in treatment with peptone
alone as well as in treatment with glucose and peptone together
(P + G). This might be an indication of less functional genes, resulting
in lower biodegradation by the treatments with peptone and P + G. Fur-
thermore, the microorganisms that are more resistant to external
sources as well as biodegradation products, are more efficient towards
the biodegradation of relatively resistant MPs, and so can be directly ap-
plied to the degradation and removal of these pollutants.
Briefly, the present study provides a novel method to effectively bio-
degrade resistant microplastics (PP, PE and PET) by natural periphytic
biofilms. These biofilms have effectively degraded PP, PE and PET
(from 9.52%–18.02%, 5.95%–14.02% and 13.24–19.72%, respectively) as
verified by different tests such as SEM, ATR-FTIR and GPC. It has already
been reported in our previous studies that periphytic biofilms possess a
remarkable capability to resist against various environmental changes
and its structure, function and diversity changes even with a small
change in the surrounding environments (Shabbir et al., 2017a) that
has been also confirmed in the present study. Compared to other micro-
bial methods, periphytic biofilms do not need any specific sterile condi-
tions that make them more advantageous to be used in wastewater
treatments systems. Overall, these biofilms require lower maintenance,
little supervision and are cost-competitive novel biological entities for
the treatment of microplastics.
4. Conclusion
This study highlights the remarkable potential of immobilized peri-
phytic biofilms for the biodegradation of a range of MPs in the presence
12 S. Shabbir et al. / Science of the Total Environment 717 (2020) 137064
Fig. 6. Predicted functions of periphytic community during the treatment processes.
of various C-sources. Perhaps unsurprisingly, glucose was found to be
the best C-source for the effectual biodegradation of MPs compared to
others. Additionally, Illumina Pyrosequencing shows that adding and/
or changing a C-source changes the density and diversity of periphytic
biofilms. This variation is evident at phylum and class levels, and influ-
ences the biodegradation of MPs by periphytic biofilms. The biodegra-
dation has been confirmed by MP weight loss, SEM and FTIR analyses
that shows structural, morphological and chemical changes in all the
MPs. Due to the natural presence and miscellaneous community struc-
tures of periphytic biofilms, they present an environmentally friendly
bioremediation strategy that can be implemented wastewater treat-
ment systems and also in aquatic environments contaminated with
MPs under controlled conditions.
Declaration of competing interest
The manuscript entitled “Periphytic Biofilm: an innovative approach
for biodegradation of Microplastics”, the authors whose names are
listed immediately below certify that they have NO affiliations with or
involvement in any organization or entity with any financial interest
(such as honoraria; educational grants; participation in speakers' bu-
reaus; membership, employment, consultancies, stock ownership, or
other equity interest; and expert testimony or patent-licensing arrange-
ments), or non-financial interest (such as personal or professional
relationships, affiliations, knowledge or beliefs) in the subject matter
or materials discussed in this manuscript.
Acknowledgement
This study was supported by the National Natural Science Founda-
tion of China (No. 51779076); the Foundation for Innovative Research
Groups of the National Natural Science Foundation of China (No.
51421006); the Funds for Key Research and Development Project of Sci-
ence and Technology Department of Jiangsu Province (BE2018738); the
Innovation Program for Ocean Science and Technology of Jiangsu Prov-
ince (HY2018-2); the Six Talent Peaks Project in Jiangsu Province
(2016-JNHB-007); the 333 Talent Project Foundation of Jiangsu Prov-
inceandtheTop-NotchAcademicProgramsProjectofJiangsuHigherEdu-
cation Institutions (TAPP).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2020.137064.
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