Environmental Pollution 314 (2022) 120316

Contents lists available at ScienceDirect

Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

The dangerous transporters: A study of microplastic-associated bacteria

passing through municipal wastewater treatment
Antonina Kruglova a, *, Barbara Muñoz-Palazón b, c, Alejandro Gonzalez-Martinez b,
Anna Mikola a, Riku Vahala a, Julia Talvitie d
a
Department of Built Environment, Aalto University, PO Box 15200, FI-00076, AALTO, Finland
b
Department of Microbiology, University of Granada, Campus Universitario de la Cartuja C.P., 18071, Granada, Spain
c
Dipartimento di Ecologia e Biologia, Università degli Studi della Tuscia, Largo Università snc, 01100, Viterbo, Italy
d
Marine Research Center, Finnish Environment Institute (SYKE), P.O.Box 140, FI-00251, Helsinki, Finland

A R T I C L E I N F O A B S T R A C T

Keywords: Microplastics (MPs) provide a stable and protective habitat for diverse wastewater bacteria, including pathogenic
Microplastics and antibiotic-resistant species. Therefore, MPs may potentially transport these bacteria through wastewater
Microplastic biofilms treatment steps to the environment and far distances. This study investigated bacterial communities of MP-
Plastisphere
associated bacteria from different stages of municipal wastewater treatment processes to evaluate the poten­
Wastewater treatment
Activated sludge
tial negative effect of these biofilms on the environment. The results showed a high diversity of bacteria that
Wastewater-derived pathogens were strongly attached to MPs. After all treatment steps, the core bacterial groups remained attached to MPs and
escaped from the wastewater treatment plant with effluent water. Several pathogenic bacteria were identified in
MP samples from all treatment steps, and most of them were found in effluent water. These data provide new
insights into the possible impacts of wastewater-derived MPs on the environment. MP-associated biofilms were
proved to be important sources of pathogens and antibiotic-resistant genes in natural waters.

1. Introduction a variety of species (Gouin, 2020; Wang et al., 2021; Yin et al., 2022),
and although field-based evidence is still relatively scarce, many labo­
During the past decades, plastic production has been continuously ratory studies have demonstrated that, in aquatic environments, MPs
increasing (Plastic Europe, 2021). This increase brings along with it an can be transferred in food webs from lower to higher trophic levels and
accumulation of plastic waste in the environment and an escalation of potentially to humans (Nelms et al., 2018). MPs may also include
environmental pollution by microplastics (MPs), a smaller fraction of organic and inorganic micropollutants, originally present or adsorbed to
plastics (1 μm–5mm in diameter) that are harder to stop from spreading their surfaces, which may cause harmful effects on organisms ingesting
due to their small size and persistence (Thompson et al., 2004; Hart­ them (Rochman et al., 2019). In addition, plastic litter and microplastics
mann et al., 2019; Hale et al., 2020). MPs are entering the environment provide a durable substrate for the growth of microbial biofilms, which
either as primary MPs or as secondary microplastics that are breaking can include non-indigenous and harmful species. The microbial com­
down from larger plastic items due to biological, chemical, or biological munity in the plastics can be very different from the surrounding envi­
processes (GESAMP, 2016). Consequently, MPs are everywhere in our ronments forming a unique ecosystem called the plastisphere (Zettler
environment and society: in soils, aquatic environments, wildlife, food et al., 2013; Amaral-Zettler et al., 2020).
products, drinking water, and in the air we breathe (Xu et al., 2020; Municipal wastewater treatment plants (WWTPs) are one of the main
Zhou et al., 2020; Susanti et al., 2020; Kwon et al., 2020; Zhang et al., recipients of MPs originating from our daily activities like washing
2020). synthetic clothes and using MP-containing personal care products
MPs have been suspected to have detrimental impacts on wildlife and (Browne et al., 2011; Carr et al., 2016; Talvitie et al., 2017a, 2017b; Liu
human health (Lo and Chan, 2018; Xia et al., 2020; Vethaak and Legler, et al., 2021). Although WWTPs act as a barrier for MPs to the aquatic
2021; Ragusa et al., 2021). Due to their tiny size, MPs can be ingested by environment, removing microplastics from wastewater (up to 99%),

* Corresponding author.
E-mail address: [email protected] (A. Kruglova).

https://doi.org/10.1016/j.envpol.2022.120316
Received 28 June 2022; Received in revised form 12 September 2022; Accepted 27 September 2022
Available online 30 September 2022
0269-7491/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A. Kruglova et al. Environmental Pollution 314 (2022) 120316

studies show that no wastewater treatment technique leads to complete <0.5% of the incoming microplastics are left in the final effluent and
retention of microplastics, and hence WWTPs are viewed as pathways discharged into the Baltic Sea (Talvitie et al., 2017a). The most common
for microplastics to the aquatic environment (Liu et al., 2021). There­ microplastics in the wastewater at the Viikinmäki WWTP consist of
fore, despite their high reduction ability, conventional WWTPs are a still polyester fibers, and polyethylene (PE) and polypropylene fragments
significant source of MPs in the aquatic environment due to the large (Talvitie et al., 2017a, 2017b).
volumes of discharged effluents (Murphy et al., 2016; Talvitie et al.,
2017a, 2017b; Lares et al., 2018; Salmi et al., 2021). 2.2. Sample collection and processing
The majority of municipal WWTPs include biological treatment
based on the activated sludge (AS) process. Wastewater entering WWTPs The irregular-shaped PE fragments from personal care products
contains a wide range of human microbiota, including pathogenic bac­ commonly detected MPs in the wastewater of Viikinmäki WWTP (Tal­
teria. In the biological treatment step, these bacteria meet with AS mi­ vitie et al., 2017a, 2017b), were chosen to represent wastewater-based
croorganisms (Saunders et al., 2016). In the AS step, the treated water is MPs in our study (hereafter PE). In addition to that, we collected a
separated via sedimentation, and most of the bacteria are returned to the mixture of different kinds of secondary microplastics (hereafter MIX),
process. However, due to their durability and small size associated with including polyethylene and polypropylene fragments, to see the selec­
low density, some of the MPs pass through these wastewater treatment tivity of the MP surface. Polyester fibers were not included in the ex­
steps while being colonized by dense bacterial biofilm (McCormick amination, although they are a very common MP type in wastewater, as
et al., 2014; Auta et al., 2017; Talvitie et al., 2017a, 2017b). it is not possible to distinguish synthetic fibers from natural fibers prior
Few studies have evaluated the role of wastewater-derived MPs as to material analyses. To compare synthetic and natural (organic) mi­
transporters of harmful or pathogenic microbiota to the aquatic envi­ croparticles as selective surfaces for bacterial colonization, the micro­
ronment and how wastewater treatments, particularly disinfection, may scopic (<5 mm) film-like organic microparticles (hereafter OP) were
modify the bacterial community in the plastisphere. Boni et al., 2021 also collected from the effluent.
and Shen et al. (2021) showed in their laboratory studies how MPs can The samples were collected from four sampling points along the
act as a protective habitat for pathogenic bacteria in wastewater, wastewater treatment process: influent, after pre-treatment, after AS,
allowing them to survive disinfection treatments (UV, chlorine disin­ and final effluent (Table 1). Two or three replicates were taken from
fection, and disinfection by peracetic acid), which is usually effective for each sampling site except influent, where the collection of MPs was
free-living bacteria. A recent study of MP biofilms at two different challenging due to the large amount of suspended solids and large
WWTPs demonstrated an increase in bacterial species richness and an variation in microplastic concentration. Sampling was performed with a
abundance of taxa during the wastewater treatment processes, sug­ simultaneous pump and filtering technique, where the wastewater is
gesting that WWTPs can have a significant role in modifying the plas­ pumped from the surface of the wastewater stream into the filter device
tisphere (Kelly et al., 2021). Additionally, studies of MP biofilms and with an electric pump (Biltema art.17–953). The filter device consists of
free-living bacteria before and after the disinfection treatment (ozona­ transparent acryl tubes (60 mm in diameter) and connectors attaching
tion) at a municipal WWTP demonstrated no effect of the treatment on the tubes. The 300 μm mesh-sized filter was placed between the con­
the total bacterial communities, the composition of the potentially nectors (Talvitie et al., 2015). The sample volumes varied from ~1 L
pathogenic bacteria, or the antibiotic resistance gene (ARG) abundances (influent) to >1 m3 (effluent). After the sampling, the filters were
in MP biofilms (Galafassi et al., 2021). This is especially concerning collected in a Petri dish with forceps and kept moist at a temperature of
since Imran et al. (2018) showed that MPs can act not only as trans­ ~4 ◦ C and transported to laboratory facilities for further processing.
porters but also as hotspots for the spread of antibiotic resistance be­ MPs were picked from the filters with sterile forceps under a ste­
tween bacteria, including phylogenetically distinct species. reomicroscope (Fiberoptic – Heim LQ 1100) rinsed with sterile saline
Our study aimed to evaluate the possible effects of wastewater- solution (0.9%) and collected in Eppendorf tube filled also with sterile
derived MP biofilms on the performance of current municipal waste­ saline solution (0.9%). To compare the fate of wastewater-derived MP-
water treatment in terms of hygienic risks and environmental water associated bacteria for free-living bacteria, influent water and activated
quality. For this, we followed the MP bacterial biofilm persistence and sludge bacteria were also collected. The samples were centrifuged in 50
dynamics through the main treatment steps of a large, advanced WWTP, ml Falcon tubes for 10 min at 5000 rpm, and pelleted biomass was
comparing plastispheres of influent, before and after biological treat­ collected. The PE, MIX, natural biofilm microparticles as well as influent
ment and in the effluent released into the aquatic environment. and activated sludge free-living bacteria were collected in separate
Eppendorf tubes, immediately frozen, and kept at − 20 ◦ C before the
2. Materials and methods DNA extraction.

2.1. Description of the WWTP and removal of microplastics 2.3. Scanning Electron Microscopy (SEM)

The sampling of MPs took place during the winter and spring of The visualization of biomass attached to MPs was done by Scanning
2017. Samples were taken from the Viikinmäki WWTP, a large munic­ Electron Microscopy (SEM). For SEM, samples of MPs were fixated in the
ipal WWTP in Helsinki, Finland, (~300,000 m3 d− 1, population equiv­ glutaraldehyde solution according to Gonzalez-Martinez et al. (2017).
alent to ~800,000). The wastewater treatment process at the Viikinmäki After that, samples were transported to the Center of Scientific Instru­
WWTP consists of pre-treatment (including screening, grit removal, pre- mentation at the University of Granada for visualization by a Carl Zeiss
aeration, and primary sedimentation), conventional AS process (sec­ LEO 906E scanning electron microscope. The detailed protocol of sam­
ondary treatment), secondary sedimentation, and a tertiary biologically ple preparation is described in Gonzalez-Martinez et al. (2017).
active filtration (BAF) for denitrification. The treated effluents are dis­
charged into an open sea area ~4 km away from the shoreline in the Gulf 2.4. FTIR analyses
of Finland, Baltic Sea. The Principle diagram of the WWTP process with
sampling points is presented in Fig. S1 in the Supplementary materials. To ensure that the PE fragments and OP microparticles were what
According to previous studies, Viikinmäki WWTP efficiently removes they were suspected to be, part of those particle types were selected for
MP, 20 μm–5mm in size (Talvitie et al., 2017a, 2017b). Most of the chemical identification with an imaging Fourier Transform Infrared
influent MP (~98%) is already removed during the pre-treatment (from Spectrometer (FTIRi) (Spectrum Spotlight 300, PerkinElmer, Waltham,
686.7 to 10.9 MP L− 1). The AS process further removes about 88% of the Massachusetts, USA). The particles chosen for material analyses were
microplastics from the primary effluent (from 10.9 to 1.3 MP L− 1). Only picked from the filters, rinsed with distilled water and placed onto ZnSe

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Table 1
The types of samples successfully obtained on the six sampling attempts.

windows, and left to dry for approximately 1 h, after which each window Shannon-Wiener, Pielou’s evenness, and Berger Parker indices. The
was photographed and analyzed with the FTIRi. The FTIR spectra were β-diversity was calculated for a pair of samples using Whitaker and
recorded in transmittance mode, in the wavelength region of 700–4000 Williams indices. The diversity indices were calculated whenever
cm− 1 at a resolution of 4 cm− 1 with 15 scans. The Thermo Scientific™ possible according to success in obtaining the replicates for the sample.
Hummel Polymer and Additives FT-IR Spectral Library were used to The principal coordinated analysis (PCoA) was calculated using the
compare the microbead spectra to reference spectra from plastics of Bray-Curtis algorithm under 999 bootstrap using PAST software.
known composition. Similarity Percentages analysis (SIMPER) was used to observe the
contribution of dominant bacterial taxa (OTUs with >1.5% of total
2.5. Microbial analyses relative abundance) to dissimilarities between samples in pairs. This was
done to estimate which taxa in the community contributed to MPs’
For bacterial analyses, MPs were first rinsed with a sterile saline colonization the most and how they affected communities of other MP
solution, then the total DNA was extracted from the surfaces of the MPs types (PE or Mix) at all stages of the treatment (Influent, Before AS, After
using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, Ohio, USA). AS, and Effluent).
A high-throughput sequencing procedure of DNA samples was con­ The compositional statistics captured all the diversity of the biolog­
ducted at Research & Testing Laboratory (Lubbock, Texas, USA) using ical samples, including rare phylotypes. Thus, the OTU table was cor­
Illumina MiSeq equipment and the Illumina MiSeq Reagent Kit v3. The rected to avoid zero values and the log-ratio was transformed using R-
taxonomical affiliation was done using the 16S rRNA gene taxonomy project and CoDaPack software, respectively. The OTU distributions
from the NCBI database, as described by Kruglova et al. (2017). were employed to calculate the expected effect size (EES).

2.6. Bioinformatics pipeline 3. Results

The raw data treatment from massive parallel sequencing was done During our six sampling campaigns, ten successful (with at least 20
using the software mothur v.1.45.3. First, the forward and reverse microparticles found in the sample) PE samples and 8 MIX samples were
paired ends were merged into contigs. The contigs were subjected to collected and analyzed from four sampling points at the WWTP. AS was
quality analysis in order to remove any ambiguous bases and sequences sampled to see the effect of the biological process step on MP biofilm.
with more than eight polymers. The unique sequences were identified One sample of the influent microbial community and one sample of
and aligned against the SiLVA SEED using Needleman alignment con­ biofilm attached to OP were also included in the study as references.
ditions. The aligned sequences that were not in the right forward and However, due to the lack of replicates, these samples were excluded
reverse positions were eliminated. Then the chimerical sequences were from statistical analyses.
removed using the VSEARCH algorithm as a template. Then any
sequence belonging to any lineage other than Bacteria was removed. The 3.1. Microplastic colonization
remaining sequences were clustered into OTUs with a similarity up to
97% and a cut-off of 0.03. The cluster was calculated employing the Examples of PE and MIX samples are shown in Fig. 1 (A). SEM pic­
algorithm based on an abundance-based greedy algorithm to construct tures of bacteria attached to effluent MPs after rinsing are shown in
the operational taxonomic unit (OTUs). The sequences were classified Fig. 1 (B), demonstrating a persistent bacterial biofilm attached to MPs.
using the k-nearest-neighbor algorithm with the k-mer search method
using a k-mer size of 8 bp with the SiLVA SEED v132. Finally, the 3.2. Statistical comparison: α-diversity, principle component analysis, and
singleton OTUs were removed, and the remaining sequences were used β-diversity analysis
to create a taxonomic consensus.
The Simpson, Shannon, and Chao indices varied considerably among
2.7. Statistical analyses the samples as well as the replicates (Table S1), showing great differ­
ences in communities. These differences could be driven by the
Heat maps of the samples were represented using the most dominant morphology of the MPs such as size, roughness, form, etc. (Yang et al.,
OTUs, with more than 1% of total relative abundance in at least one 2020). There were no conclusive results about differences in species
sample. diversity between the PE and Mix, although previously, some authors
Estimates for α-diversity and β-diversity were calculated using Past described materials such as polystyrene and PE as having lower species
v3.14 software. The α-diversity calculation for species richness, di­ richness than micropieces of natural materials (Kettner et al., 2019),
versity, and evenness was estimated through the Chao index, Simpson, while in general, the species richness is greater than in free-living

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Fig. 1. (A) Examples of PE (polyethylene) and MIX (mixture) samples of MPs. (B) SEM images showing biofilm bacteria on the effluent microplastic surface.

bacterial niches (Dussud et al., 2018). β-diversity analyses (Fig. S2 in the Supplementary material) have
In the evenness study, the results similarly presented great variances. demonstrated remarkable differences between the free-living bacterial
The highest evenness was observed in influent MIX and effluent MIX populations found in influent and AS compared to all MP biofilms. For
samples, showing the selective growth of bacteria on MP passing MP biofilms, the larger differences among pairs of samples were found
through wastewater treatment compared to influent MPs and especially before and after the AS for both types of MPs but to a greater extent for
on PE. PE, which is consistent with other analyses. In contrast, the samples
The Bray-Curtis PCoA plot (presented in Fig. 2) demonstrated a clear taken after the biological treatment (after AS and from effluent) were
clustering of AS communities, influent MP biofilm, and MP biofilms highly similar to each other, demonstrating no effect of the tertiary
before and after the AS process (secondary treatment). In addition, MIX treatment. However, it is noteworthy to mention that the Whittaker
communities were separated from PE communities, especially after the index revealed a great standard deviation in the data. Altogether, the
AS process. analyses showed that the composition of AS influenced biofilm coloni­
These results suggest that the AS bacteria, despite their high diversity zation. But it is important to mention that the nature of MPs also re­
and population density, have only a limited effect on the MP biofilm flected differences in the dominant OTUs (Section 3.4), as described by
bacteria of PE. However, the AS step noticeably altered the MIX biofilm Kelly et al. (2021).
communities. This shows the selective properties of PE for bacterial
attachment compared to the variety of surface materials in the MIX
samples.

Fig. 2. Principal Coordinate Analysis (PCoA) analysis of the studied samples based on Bray-Curtis dissimilarity.

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3.3. Contribution to dissimilarities was the most challenging and that only one sample was collected, this
may have affected this result. Similarly, for OP biofilm, more sample
The contribution of OTUs to dissimilarities between samples was replicates would be needed to support these results.
calculated by SIMPER analysis, which showed that 52 OTUs contribute The relative abundance of bacterial phyla and bacterial classes is
to dissimilarities with more than 1.00% in at least one sample (Fig. S3 in presented in Fig. 4. Consistent with most of the published MP biofilm
Supplementary materials). The most striking contribution to dissimi­ data (Yang et al., 2020), all studied MPs were mostly colonized by
larities between sampling points was given by Blastocatella (OTU007). Proteobacteria (up to 60% of the community). Additionally, Firmicutes,
Being dominant in AS and almost absent in all other samples, Blastoca­ Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, and Fusobacteria
tella contributed more than 20% of the relative abundance to differen­ were among the dominant phyla with a great variety between the
tiate the bacterial communities found in the AS compared to other samples and even the replicates.
samples. Next, Corynebacterium (OTU004) had the heaviest impact on In contrast to MPs, influent free-living bacteria were mostly repre­
dissimilarities of MP biofilm samples after AS since its phylotypes sented by Firmicutes (30%) and Fusobacteria (17%), followed by Proteo­
proliferated after the biological treatment and almost completely dis­ bacteria (only 9%). Among AS free-living bacteria, the most abundant
appeared in the biofilm of effluent, regardless of the nature of the MP dominant phylum was Acidobacteria (up to 50%), mostly represented by
pieces. Furthermore, Methylotenera (OTU001) and Aquabacterium the family Microtrichaceae, followed by Proteobacteria (up to 27%). OP
(OTU002) contributed to wide dissimilarities between MP biofilm biofilm had a very different structure of the community, with four
samples before AS and the rest, with values of dissimilarity promotion dominant phyla: Proteobacteria (35%), Firmicutes (20%), Bacteroidetes
ranging from 5 to 10% of relative abundance. The same pattern in the (12%), and Actinobacteria (7%).
OTU001 and OTU002 SIMPER analyses was shown between effluent and Within the phylum Proteobacteria, the dominant bacteria of all PE
the rest of the samples, which demonstrates selectivity for colonization biofilms were from the class Betaproteobacteria (up to 55% of the total
of these two phylotypes to the MPs during the wastewater treatment community), followed by Gammaproteobacteria (up to 18%) and Epsi­
steps. lonproteobacteria (up to 6%).
The expected effect size analyses determined the greatest number of By contrast, MIX biofilm in influent was colonized mostly by Gam­
OTUs differences between the influent MIX and effluent MIX samples maproteobacteria (29% of the total community), followed by Alphapro­
(Fig. S4 E in Supplementary materials) as well as before and after AS teobacteria (6.5%). However, in the next treatment steps,
(Fig. S4 F in Supplementary materials). In contrast, the PE samples had Betaproteobacteria become the most abundant class in MIX biofilm (up to
minor effects on the OTU populations observed between influent and 51% of the total community after primary treatment and up to 29% in
effluent, and even lower before and after AS (Fig. S3 C in Supplementary the effluent). This change could be explained by the different compo­
materials). Furthermore, the dominant OTUs in MIX had a stronger ef­ sition of MIX microparticles due to the effect of the primary wastewater
fect on the dissimilarities, while the dominant and rare phylotypes of PE treatment.
bacteria had similar importance. Finally, for all types of MPs, the effect Altogether, these results demonstrated that MP-associated bacterial
of spatial disturbance in the number of OTUs showed statistically more communities have a different structure and dominant groups than free-
significant differences before and after AS compared to influent and living or OP-associated bacteria. In addition, during secondary waste­
effluent MP biofilms. water treatment (AS process), some of the AS free-living bacteria
attached to MPs develop a new bacterial composition of MP biofilms.
3.4. Bacterial community composition Both wastewater-derived and AS-derived bacterial populations were
detected in effluent MPs and OPs.
The results showed a high diversity of bacteria attached to MPs of
both PE and MIX sample types in all of the successfully processed 3.5. Microplastic biofilm dynamics through the wastewater treatment
samples. The taxonomic richness of bacterial communities (presented in process
Fig. 3) was similar between all MP biofilm samples as well as free-living
bacteria of influent and AS samples. MP biofilm analysis on a genera level revealed that bacterial com­
Noticeably lower taxonomic richness was observed in MIX biofilm munities shifted greatly after primary, secondary, and tertiary treat­
from influent and OP biofilm from effluent. Given that influent sampling ment. The most abundant genera of MP biofilms at different steps of the

Fig. 3. Number of operational taxonomic units observed within the studied bacterial communities. Bars represent the mean values. The error bars represent the
standard deviation.

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Fig. 4. Relative abundance of bacteria at the class level associated with mixture (MIX) microplastics, polyethylene microbeads (PE), and free-living bacteria at
different steps in the municipal wastewater treatment process. Others are classes with less than 2.5% abundance.

municipal wastewater treatment process are presented in Fig. 5. colonization. Additionally, both genera presumably include species with
Influent PE biofilm was mostly represented by Aquabacterium (up to plastic-degrading capabilities (Kelly et al., 2021; McCormick et al.,
32%) and Pseudomonas (up to 18%) genera, followed by unclassified 2016).
Betaproteobacteria (up to 17%). These taxa continued to dominate the PE Similarly to the results obtained by Kelly et al. (2021), in this study,
biofilm communities through the whole wastewater treatment process. the abundance of Arcobacter significantly decreased during the treat­
Both Aquabacterium and Pseudomonas were previously reported as ment steps (from 2% to 6% before secondary treatment to below 0.2% in
dominant bacterial genera of wastewater MP, and our data support the effluent). Previously, Kristensen et al. (2020) reported a high abundance
suggestions on the selective advantage of these bacterial groups for PE of Arcobacter in many wastewater treatment plant effluents in Denmark.

Fig. 5. Relative abundance of the most abundant bacterial genera associated with polyethylene microbeads (PE) and mixture (MIX) microplastics at different steps in
the municipal wastewater treatment process.

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The suggested reason for the low removal of these bacteria was their detected on PE, but up to 2.1% of the effluent MIX community still
loose attachment to flocs, which could also be a reason for them to be belonged to this genus. Furthermore, members of the previously
washed out from MP biofilms. In contrast, all other dominant bacterial mentioned abundant genus Arcobacter include several dangerous path­
taxa showed no decrease in abundance after primary, secondary, and ogens for humans and animals (Collado and Figueras, 2011).
tertiary treatment. Lachnospiraceae are common human and animal enteric bacteria
The composition of MIX biofilms showed similarities to MP biofilms (McLellan et al., 2013). Though they were abundant on influent MIX
previously reported by Galafassi et al. (2021), mostly after secondary biofilm (18% of the total community), these bacteria seem to be quickly
treatment. In particular, similarly to this study, MIX MPs after AS had removed from MPs and the total wastewater bacteria composition
high an abundance of Flavobacteriaceae (~50% after AS), Comamona­ already after the primary treatment steps. Similarly, the genus Lacto­
daceae (up to 13%), and Rhodocyclaceae (up to ~5%). Additionally, bacillus is a known group of human enteric bacteria that appeared to be
Microtrichaceae had a clear increase in abundance after secondary present on PE biofilm (about 7% of the bacterial community) but have
treatment (up to 23% in effluent MIX biofilm) due to the effect of the not been detected in any MP samples after secondary treatment.
dominant bacteria from AS. Methylophilaceae abundance is also most In addition to the groups presented in Fig. 6, two important gen­
likely related to the effect of wastewater treatment that is typical in a era—Diezia and the previously discussed Pseudomonas—were observed
bacteria community (Hultman et al., 2018). in high abundances on MP biofilms. Bacteria from the Diezia genus
represented up to 2.5% of MP biofilm after secondary treatment. Species
3.6. Human-associated and potentially pathogenic bacteria colonizing of these genera may have various habitats and also include human
microplastics pathogens (Gharibzahedi et al., 2014).
Almost 45% of one PE biofilm sample was represented by the Neis­
Almost 35% of influent free-living bacteria and up to 48% of influent seriaceae family. In other effluent PE samples, it was present in 0.1%, and
MP biofilm bacteria could be associated with the human gastrointestinal in MIX samples, up to 4.1%. Although no Neisseriaceae were found in the
system and potentially pathogenic species. The abundances of identified influent, after pre-treatment, it represented 0.1% of PE biofilm and up to
families and genera of enteric bacteria are presented in Fig. 6. 0.5% of MIX biofilm. Interestingly, after secondary treatment, a higher
Leptotrichiaceae represent 16% of influent free-living organisms and abundance was indicated (up to 1% in both types of MP biofilm
from 0.5 to 1% of MP communities in both MIX and PE in all the samples).
treatment steps, including 0.1% of PE biofilm and 0.4% of MIX biofilm in Finally, Nocardiaceae were detected in AS (up to 1%) after secondary
the effluent. Leptotrichiaceae are human-associated taxa that include treatment in MIX (up to 1.7%) and in effluent samples in both PE and
dangerous pathogens (Eisenberg et al., 2016). Another dominant group MIX (~2%). Nocardiaceae include multiple species commonly detected
of influent bacteria was represented by the Streptococcus genus, which in wastewater, some of which are the clinically significant and patho­
was about 15% of free-living bacteria and up to 1.2% of MP biofilm genic species Nocardia spp. (Bafghi and Yousefi, 2016; Jia and Zhang,
bacteria in influent. After the secondary treatment, no Streptococcus was 2020).

Fig. 6. Abundance of human-associated and potentially pathogenic bacteria among studied microbial communities.

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Interestingly, OP biofilm in effluent had almost none of the discussed Pathogenic microorganisms are considered to be efficiently removed
bacterial taxa except Neisseriaceae in 0.1% of the community and a ten during the municipal wastewater treatment process. However, this study
times lower abundance of Streptococcus. shows that while some pathogenic groups of bacteria are effectively
removed, for other species, microplastics can provide a protective
4. Discussion habitat. Not only do these bacteria survive all wastewater treatment
steps, including water-sludge separation, but the retention of micro­
Overall, in this study, several factors showed a strong effect on the plastics in sludge could increase their abundance. Selective growth of
MP biofilm structure. First, the diversity of MP materials increased the these bacteria on MPs forced by wastewater treatment operational
diversity of bacteria attached to MP, showing that the polymer structure conditions could be one possible explanation for these results. Most of
can be selective for attaching bacteria. Secondly, despite the abundance MPs together with bacteria are returned to the AS process after sec­
and density of AS bacterial populations and different water processing ondary sedimentation (Fig. S1 of Supplementary material). Therefore,
steps, MP biofilms were able to keep a similar abundance of human gut these MPs may spend a long time returning to the AS before they reach
bacteria. Once the MP particle was colonized by certain groups of bac­ effluent, while process conditions could favor the growth of certain
teria, the porous surface structure of MPs with pore sizes similar to bacterial groups. For instance, in our study, a noticeable increase in
bacterial sizes (Fig. 1B) could accommodate bacterial cells as well as act Neisseriaceae was observed on MPs between primary and secondary and
as a shield against different environmental factors. Finally, AS bacteria after tertiary treatment, suggesting preferable growth conditions for this
colonized MP biofilms, increasing the diversity of particle-attached group of bacteria. Earlier, Hultman et al. (2018) reported Neisseriaceae
bacterial communities. AS is a diverse population of environmental among the dominant bacteria of effluents from two Finnish WWTPs,
bacteria mixed with wastewaters. In wastewater, activated sludge is including Viikinmäki WWTP hosting tetracycline resistant genes (Hult­
constantly receiving different types of human-associated bacteria as well man et al., 2018). Additionally, antibiotic resistance genes associated
as trace amounts of multiple antibiotics. Close and long-term contact with Neisseriaceae were found in effluents of several WWTPs by Chu Binh
with environmental and human gut bacteria may potentially increase et al. (2018). Therefore, the presence of certain pathogenic bacteria in
the spread of antibiotic resistance among environmental bacterial spe­ WWTP effluents could be region-specific and attributed to a combina­
cies through interspecies horizontal gene transfer. tion of local factors such as wastewater sources, wastewater composi­
The above-mentioned factors could also be attributed to OP biofilm tion, climate, and operational conditions. Furthermore, the diversity of
formation. However, according to our results, biofilm from organic region-specific human-associated bacteria released into natural waters
particles accommodated much less human gut bacteria, which can be can be linked to the spread of certain antibiotic resistance genes in this
attributed to the different origins of these microparticles. Additionally, region.
the effect of OP on the environment would generally be secondary, Additionally, presence of Streptococcus, Pseudomonas, Lactobacillus
considering the rather short surface material lifetime due to faster and Acinetobacter on MP biofilms in our study raises concerns about the
decomposition and higher biodegradability compared to plastic possibility for transferring antibiotic resistance. For decades, antibiotic
polymers. treatment of Streptococcus diseases has been challenged due to the in­
Earlier, Basili et al., 2020 demonstrated on marine plastic biofilm crease in resistance to penicillin and non-β-lactam antibiotics in its
that a microbial community depends on both environmental features species worldwide. The same resistance mechanisms have emerged in
and the biofilm formation process. In the study by Martínez-Campos other gram-positive pathogens (Ambrose Karita et al., 2005). The strains
et al. (2021) on wastewater MP biofilm, a bacteria community was of Pseudomonas, Lactobacillus, and Acinetobacter have well-known mul­
mostly defined by the environment rather than the polymer structure. tidrug-resistant isolates and are responsible for growing
Yang et al. (2020) reported that colonization and distribution of mi­ healthcare-related infections (Anisimova and Yarullina, 2019; Santajit
croorganisms in MPs biofilm depend on the interaction between exo­ and Indrawattana, 2016). Also, Pseudomonas was previously reported
polysaccharides segregated by bacteria with factors not yet known among major antibiotic resistance carriers at WWTPs (Sun et al., 2016).
(Amaral-Zettler et al., 2020). If the biofilm formation process could be The high abundance of the above-mentioned bacteria on effluent MPs
controlled by operational conditions at a WWTP, this knowledge would demonstrates the potential risks of antibiotic resistance spread through
help limit the amount of dangerous MP-associated bacteria escaping the wastewater-derived MPs and the need to optimize the wastewater
process. Therefore, more studies are needed on the effect of engineering treatment process for better MP removal.
design and conditions of WWTPs on the microbial communities’ According to our results, MPs provide a durable substrate for biofilm-
development and bacterial metabolic functions in MPs biofilms. forming micro-organisms in municipal wastewater, potentially
The analyses on the diversity and richness of bacterial-associated including pathogens and antibiotic resistance genes. WWTPs can effi­
biofilms have indicated no effect of treatment processes and waste­ ciently remove MPs from the wastewater, but due to their small size and
water treatment process conditions on multiple bacterial groups, light weight, part of the MPs escape the wastewater treatment processes
including potentially pathogenic and antibiotic-resistant bacteria. and end up in the aquatic environments with attached biofilm. To un­
Therefore, the source and amount of microplastics passing through derstand what kinds of harm, if any, can wastewater-derived biofilms
WWTPs are important to control in order to secure environmental water cause in aquatic environments, more information is needed on the effect
quality. Similarities between the samples before and after tertiary of MP-associated biofilm on the spread of antibiotic-resistant genes,
treatment demonstrated the importance of additional advanced polish­ particularly among AS bacteria. In addition, the effect of advanced
ing steps, although in this case, the purpose of the tertiary treatment was treatment technologies such as advanced oxidation and membrane
only to denitrify solids (not remove them). Previous studies reported technologies on biofilm formation and MP removal should be examined.
that effluent MP biofilms have higher microbial diversity compared to Finally, studies on the fate of microplastic-associated bacteria in envi­
effluent free-living bacteria (Martínez-Campos et al., 2021; McCormick ronmental water near WWTP discharge are of high importance.
et al., 2014). This could be explained by the beneficial conditions of the
MP surface for bacterial survival and growth. It also suggests the 5. Conclusions
importance of MP-associated biofilm analysis in evaluating water safety.
The microbiological safety of wastewater effluents is traditionally MPs provide a long-lasting favorable and protective habitat for the
evaluated through coliform indication methods, which give very limited high diversity of bacteria, including several known human gastrointes­
information compared to high-throughput sequencing (Lu et al., 2015). tinal and pathogenic species. MPs’ surface composition affects bacterial
Our data suggest that targeted cultivation tests may not reflect the di­ colonization, selecting certain groups of bacteria, after which environ­
versity of dangerous bacteria escaping WWTPs hidden in MPs. mental factors have a smaller effect on the bacterial community. Both,

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comparative genomics of the family Leptotrichiaceae and introduction of a novel
Antonina Kruglova: Conceptualization, Methodology, Investiga­ fingerprinting MLVA for Streptobacillus moniliformis. BMC Genom. 17, 864. https://
doi.org/10.1186/s12864-016-3206-0.
tion, Visualization, Writing- Original draft preparation. Barbara
Galafassi, S., Sabatino, R., Sathicq, M.B., Eckert, E.M., Fontaneto, D., Dalla Fontana, G.,
Muñoz-Palazón: Validation, Software, Visualization, Writing- Review­ Mossotti, R., Corno, G., Volta, P., Di Cesare, A., 2021. Contribution of microplastic
ing and Editing. Alejandro Gonzalez-Martinez: Methodology, Soft­ particles to the spread of resistances and pathogenic bacteria in treated wastewaters.
ware, Project administration. Anna Mikola: Supervision, Project Water Res. 201, 117368 https://doi.org/10.1016/j.watres.2021.117368.
GESAMP, 2016. Sources, fate and effects of microplastics in the marine environment:
administration, Writing- Reviewing and Editing. Riku Vahala: Super­ part two of a global assessment. In: Kershaw, P.J., Rochman, C.M. (Eds.), IMO/FAO/
vision, Funding acquisition. Julia Talvitie: Conceptualization, Meth­ UNESCO-IOC/UNIDO/WMO/IAEA/UN/UNEP/UNDP Joint Group of Experts on the
odology, Investigation, Writing- Reviewing and Editing, Project Scientific Aspects of Marine Environmental Protection, vol. 93. Rep Stud GESAMP,
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Gonzalez-Martinez, A., Muñoz-Palazon, B., Rodriguez-Sanchez, A., Maza-Márquez, P.,
Declaration of competing interest Mikola, A., Gonzalez-Lopez, J., Vahala, R., 2017. Start-up and operation of an
aerobic granular sludge system under low working temperature inoculated with
The authors declare the following financial interests/personal re­ cold-adapted activated sludge from Finland. Bioresour. Technol. 239, 180–189.
Gouin, T., 2020. Toward an improved understanding of the ingestion and trophic transfer
lationships which may be considered as potential competing interests: of microplastic particles: critical review and implications for future research.
Antonina Kruglova reports financial support was provided by Finnish Environ. Toxicol. Chem. 39 (6), 1119–1137.
Water Utilities Association. Hale, R.C., Seeley, M.E., La Guardia, M.J., Mai, L., Zeng, E.Y., 2020. A global perspective
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P., Lusher, A.L., Wagner, M., 2019. Are we speaking the same language?
Recommendations for a definition and categorization framework for plastic debris.
Data will be made available on request.
Environ. Sci. Technol. 53 (3), 1039–1047. https://doi.org/10.1021/acs.est.8b05297.
Hultman, J., Tamminen, M., Pärnänen, K., Cairns, J., Karkman, A., Virta, M., 2018. Host
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Acknowledgments
effluent. FEMS Microbiol. Ecol. 94 https://doi.org/10.1093/femsec/fiy038.
Imran, Md., Das, K.R., Naik, M.M., 2018. Co-selection of multi-antibiotic resistance in
The research was supported by a grant from the Finnish Water bacterial pathogens in metal and microplastic contaminated environments: an
Utilities Association (FIWA). emerging health threat. Chemosphere. https://doi.org/10.1016/j.
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Kelly, J.J., London, M.G., McCormick, A.R., Rojas, M., Scott, J.W., Hoellein, T.J., 2021.
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