Journal of Hazardous Materials 420 (2021) 126662

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Journal of Hazardous Materials

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

Research Paper

Microplastics as carbon-nutrient sources and shaper for microbial

communities in stagnant water
Xiao Chen a, Yi Wang a, *, Sheng Chen b, Yiran Sun b, Qiaowen Tan b, Zhibin Ding a, *, Yaofeng Lu a,
Yingjun Yu a
a
College of Defense Engineering, The Army Engineering University of PLA, Nanjing 210007, China
b
College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China

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

Editor: Dr. R. Debora Microplastics (MPs) are emerging pollutants as vectors for microbial colonization, but their role as nutrients
sources for microbial communities has rarely been reported. This study explored the impact of six types of MPs
Keywords: on assimilable organic carbon (AOC) and microbial communities over eight weeks. The following were the
Microplastics primary conclusions: (1) MPs contributed to AOC increment and subsequently increased bacterial regrowth
Plastic-deprived nutrients
potential. The maximum AOC reached 722.03 μg/L. The increase in AOC formation corresponded to AOC NOX,
Bacterial growth
except in PVC samples where AOC P17 primarily increased. (2) The MPs accelerated bacterial growth and
Stagnant water
changed the bacterial distribution between the biofilm and water phases. A high MP surface-area-to-volume ratio
or low MPs density contributed to bacterial accumulation and biofilm formation around the plastisphere, thereby
decreasing the relative microbial proportion in the water phase. (3) High-throughput sequencing and scanning
electron microscope revealed that different MPs shaped various microbial communities temporally and spatially.
(4) Biofilm formatting and formatted models were established and simulated to explain the kinetic interaction
between the AOC and bacteria inhabiting the plastisphere. Finally, the challenges that plastic-deprived AOC
represent in terms of anti-bacterial measures and chemical safety are discussed.

1. Introduction
Microplastics (MPs) are plastic particles or debris with diameters <5
mm (Thompson et al., 2004). MPs—as emerging pollutants—have
become the second most important burning issue in the field of envi­
ronmental and ecological research, capturing attentions globally
(Toussaint et al., 2019; Wang et al., 2019). MPs are ubiquitously present
in drinking water supply systems, from source water to point of use
(Koelmans et al., 2019; J.Y. Li et al., 2018). Plastic membranes, pipes,
tanks, cover-plates, bottles, and caps at drinking water consumption
points represent potential sources of MPs for humans (Ding et al., 2020;
Shruti et al., 2020; Wu et al., 2021). It is reported that the estimated
daily intake of MPs for adults and children has arrived at 40.1 g/kg/day
(Zuccarello et al., 2019). MPs have been found in blood system,
lymphatic system, the liver and the guts of human beings, which can
produce hazard to health (Wang et al., 2019). The adverse effects of MPs
on water quality and human health are gradually being recognized.
MPs are emerging pollutants that act as vectors or reservoirs for
microbial communities and other pollutants (Lu et al., 2019, Ateia et al.,
2020). They release toxic materials into water, including phthalates,
polycyclic aromatic hydrocarbons, bisphenol A, and heavy metals
(Cheng et al., 2020; Wagner et al., 2018). Furthermore, MPs can adsorb
and transport microbe due to theirs high surface-area-to-volume ratio
(SAV) (Lambert and Wagner, 2016). MPs selectively enrich opportu­
nistic pathogens (OPs), antibiotic resistance genes (ARGs), and metal
resistance genes (MRGs) (J. Wang et al., 2020; S.S. Wang et al., 2020;
Wu et al., 2019; Yang et al., 2019). Microbial communities inhabiting
the plastisphere are significantly different from those inhabiting other
materials or water environments, with Alpha-proteobacteria and Beta-­
proteobacteria representing the dominant phyla (Ogonowski et al.,
2018). In addition, dissolved organic carbon (DOM) leached from MPs
aggravates chemical risks (Lee et al., 2020). For example, plastic-derived
DOM is correlated with an increase in disinfection by-products (DBPs) in
water (Ateia et al., 2020). An increase in carbon-chlorine (C-Cl) bonds
was detected in chlorinated MPs, thereby aggravating toxicity (Kelkar
et al., 2019). These adverse effects motivate additional studies on the

* Corresponding authors.
E-mail addresses: wyxqh97@126.com (Y. Wang), njwaterdzb@qq.coom (Z. Ding).

https://doi.org/10.1016/j.jhazmat.2021.126662
Received 11 May 2021; Received in revised form 25 June 2021; Accepted 14 July 2021
Available online 17 July 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.
X. Chen et al.
impacts of MPs on the microbiome and drinking water quality.
Despite numerous studies on MPs, the role of MPs as nutrients source
for bacterial growth has rarely been studied. MPs produce persist im­
pacts on water quality over residence time due to their durable nature.
Water stagnation is common in drinking water system, especially in
water tanks, distal pipes in buildings, and distributed small-scale
drinking water purifiers (Chen et al., 2020; Ling et al., 2018; Shruti
et al., 2020). Residual nutrients in stagnant drinking water promote
bacterial regrowth, and assimilable organic carbon (AOC) was regarded
as the main nutrient, which can be absorbed by bacteria and assimilated
into parts of bacteria (Prest et al., 2016). Up to now, impacts of MPs on
AOC concentration and subsequent impacts on microbial activities in
drinking water are still unclear.
The purpose of this study is to explore the role of MPs as nutrients for
microbial growth and microbial community formation in drinking
water. Different MPs were mixed into drinking water to monitor
biochemical indices and microbial communities, with the support of
microbiology, sequencing technology, and numerical simulation anal­
ysis. The research contents consist of three main parts: (1) to evaluate
AOC diversities induced by MPs; (2) to explore impacts of MPs on bac­
terial growth in stagnant water; and (3) to confirm the role of MPs in
shaping microbial communities and related biological risks.
The study was identified as being of importance to evaluate impact of
MPs on drinking water safety with time-delay effect.
2. Materials and methods
2.1. Pretreatment of microplastics
The present study focused on six types of pure MPs that are ubiqui­
tously present in drinking water supply systems, and these include
polyethylene (PE), polypropylene (PP), polyethylene terephthalate
(PET), polyvinyl chloride (PVC), polyethylene random copolymer
(PPR), high-density polyethylene (HDPE), supplied by Dongguan Qimei
Plasticizing Co., Ltd. (Guangdong, China). The diameters of the MP
particles were in the range of 1–3 mm. The MPs particles were first
immersed in 75% ethanol for 1 h, washed with deionized water, and
dried at 50–55 ℃, as previously described by Feng et al. (2020). Pre­
treated MPs (10 g) were sub-packaged in burlap sack (6 × 8 cm, auto­
claved and dried) to facilitate sampling. The three-dimensional sizes of
the MPs were measured, and their volume (V) and surface area (S) were
calculated using the integral formulas of ellipsoids, cylinders, and
elliptic cylinder, following Eqs.(1–3), as detailed in Table 1. These
experimental operations were conducted on a super clean bench.
( / / )
(S/V)Cylinder = 2 1 h + 1 r (1)
/ / /
(S/V)Ellipsoid = 1 a + 1 b + 1 c
/ √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
/ /
(S/V)Ellipticcylinder ≈ 2 h + 2 a2 + 2 b2
2.2. Detection methods in water and biofilm
Physico-chemical water parameters, including pH, oxidation
Table 1
Basic dimensional parameters of the MPs used in the present study.
MPs Shape Size (cm)
PPR Cylinder r = 0.2, h = 0.2
HDPE Cylinder r = 0.2, h = 0.2
PVC Cylinder r = 0.2, h = 0.3
PE Ellipsoid a = 0.2, b = 0.15, c = 0.1
PET Elliptic cylinder a = 0.15, b = 0.1, h = 0.2
PP Elliptic cylinder a = 0.2, b = 0.1, h = 0.2
Journal of Hazardous Materials 420 (2021) 126662
reduction potential (ORP), and ion-specific electrode (ISE) were
analyzed using an ORION 4-Star instrument (ThermoFisher, USA).
UV254 and OD600 were detected using a TU1810 ultraviolet-visible
spectrophotometer (PERSEE, China) with quartz cuvettes, to assess the
level of organic matter and bacterial absorbance, respectively.
Values of heterotrophic plate counts in water (HPCWR) and MP-
biofilm (HPCBM) were analyzed on R2A agar, in accordance with the
procedures shown in Fig. 1b. Measurements of HPCBM were as follows:
five plastic particles were randomly collected, and then were vortexed in
phosphate buffered solution (PBS), to shift bacteria from the plasti­
sphere to the PBS and to obtain the counts of bacteria on the plasti­
sphere. The bacterial counts were then converted into HPC values
(counted as CFU/mL or CFU/cm2) in the biofilm phase on the sizes of
MPs in Table 1.
AOC was measured following a 11-day incubation period on LLA
agar as per the method proposed by Hammes et al. (2006). Briefly,
40 mL water samples were collected in 50 mL glass bottles and then
were pasteurized at 70 ℃ for 0.5 h. Pseudomonas fluorescens (P17) was
incubated into water samples at the concertation of 10,000 CFU/mL,
and was cultured at 25 ℃ in dark environment for 3 days, and then
100 µL sample were cultured on LLA agar for 3 days to count the colony
number. Subsequently, the 40 mL water samples were pasteurized again
to kill P17, then Spirillum (NOX) were incubated and cultured for
another 4 days. Colony number of P17 and NOX were converted into
AOC value as µg/L acetate-carbon.
Bacterial regrowth potential (BRP) was measured following a 5-day
incubation period, as per the protocol proposed by Sathasivan and
Ohgaki (1999). Similarly, 1 mL indigenous inoculum produced from the
tap water were incubated in water samples and cultured for 5 days.
Colony number of indigenous inoculum on R2A agar was marked as BRP
value.
Sequencing analysis was conducted on the Illumina platform. Water
samples were collected to enrich the DNA, and plastic particles were
mixed with PBS and the mixed solution were vortexed to enrich the DNA
on plastisphere. The extracted DNA was amplified with the following
bacterial specific forward 338F and reverse 806R (Read1 – ACTCC­
TACGGGAGGCAGCAG; Read2 – GGACTACHVGGGTWTCTAAT). The
amplified products were purified using a PCR clean-up system (QIAGEN,
Germany). Then DNA concentration of each sample was then measured,
and sequencing was performed.
The morphology of MPs was evaluated using an Inspect F50 scanning
electron microscope (SEM) (FEI, USA). Briefly, impurities attached to
the microplastics were washed with sterilized ultrapure water for 3
times. Then, the microplastics were immersed in 2.5% glutaraldehyde
solution to fix MP-BM at temperature of 4 ℃ for 12 h. The immersed
microplastics were washed with 0.1 mol/L PBS for 3 times, and were
dehydrated using 20%, 40%, 60%, 80% and 99.99% ethanol for 15 min
(2) one after another. Finally, the dehydrated microplastics are dried using
freeze drier and were scanned with SEM.
(3)
2.3. Pilot assay
The pilot test design is depicted in Fig. 1a. A brown glass bottle was
used to store the water and plastic particles. Tap water was collected
from drinking water distribution faucet and was dispensed into 5-L
Density（g/cm3） Volume (V)（cm3） Surface area (S)（cm2） S/V（cm− 1）
0.91–0.92 0.008π 0.16π 20
0.94–0.96 0.008π 0.16π 20
1.16–1.58 0.012π 0.2π 16.67
0.92–0.97 0.004π 0.087π 21.67
1.37–1.45 0.003π 0.13π 43.33
0.9–0.91 0.004π 0.2π 50
2
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

Fig. 1. Pilot test and methods for investigating the impact of MPs on AOC and bacteria in simulated stagnant drinking water. (a) Pilot test, (b) Method for measuring
microbial counts onto plastisphere; and (c) Method for measuring unculturable bacteria using scanning electron microscopy and agar.

brown glass bottle as samples. Then, pretreated MPs (10 g, marked as
PP, PE, PVC, PPR, PET, and HDPE) were added into a burlap sack, and
the burlap sack was fastened with a string and placed in individual water
samples. No MPs were added to the control group (marked as Control).
All samples were stored in an air-conditioned room at a temperature of
25 ℃. Water samples were then collected on the1st, 3rd, 5th, 7th, 14th,
28th, and 56th days. pH, ORP, UV254, TDS, ISE, EC, BRP, AOC, HPC were
monitored in the water samples. To detect microbial changes in MP-
biofilm (MP-BM) and water, DNA extracted from water samples and
MPs particles were sequenced at the time of 14th, 28th, and 56th day,
and DNA extracted from initial tap water was tested as the control
group.
2.4. Data processing
Time-delay differential equations were calculated and simulated
using Maple 2020. Matrix analysis were conducted using Matlab 2020.
Correlations among parameter were analyzed using R version 3.6.2. The
significance tests were conducted using SPSS version 22. Data visuali­
zation were operated using Origin 2019, R version 3.6.2 and Adobe
Illustrator 2019.

3
X. Chen et al.
3. Results
3.1. MPs altered AOC formation and resulted in an increase in BRP
Briefly, AOCP17 showed a descending trend with stagnation time in
most samples with MPs, while AOCNOX showed an ascending trend, and
the total AOC showed an ascending trend, as shown in Fig. 2a–d. The
detailed changes in AOC are as follows:
AOC concentration ranged from 3.63 to 110 μg/L in the control
samples, whereas the AOC concentration ranged from 3.89 to
722.03 μg/L in the MPs samples. Peak values of AOC followed the
following order: PET > PVC > PPR > HDPE > PP > PE > Control. In
addition, the average AOC content in the MP samples was higher than
that in the control samples.
The AOC levels in all MPs samples were higher than those in the
control samples during the first four weeks. Interestingly, the AOC
content increased again in the 8th week in the control sample, in which
it was higher than that in the MP samples, the exception being PET and
PVC. The second increase in AOC content in the control sample was
attributed to the release of highly bioavailable intracellular organic
matter (Wen et al., 2017), along with a decrease in the abundance of
intact bacteria. Another explanation for this phenomenon is that the
nutrient and bacteria levels fluctuated periodically one after another,
with a time-delay effect (Al Marzooq et al., 2018; Chi and Zhao, 2018).
The formation of AOC varied in the different MPs samples. AOCNOX
concentration in the MPs samples rapidly increased during the first two
weeks, then stabilized from the 2nd week to the 4th week, and decreased
from the 4th week to the 8th week. A pronounced increase of AOCNOX
was observed in most MPs samples (as shown in Fig. 2d), while the in­
crease in AOCP17 was the maximum in the PVC samples. AOCP17 is
characterized by amino acids, carboxylic acids, ethanol, carbohydrates,
etc., which account for more than 60% in natural water. In contrast,
AOCNOX is characterized by organic acids, aldehydes, and ketones
(Hammes et al., 2007a, 2007b). The absorbance at different wavelength
revealed that the main changes occurred in the range from 220 to
400 nm in near-ultraviolet bands (see Fig. S1), a finding that is consis­
tent with a recent study by Lee et al. (2020).
The increased AOC caused biological instability in the water. The
average values of AOC in the MPs samples were greater than 135 μg/L
and subsequently contributed to bacterial growth, which could poten­
tially lead to biological instability in drinking water (Zhang et al., 2016).
Thus, a higher dose of disinfectants would be required to balance the
increased AOC for maintaining biological stability. In addition,
plastic-derived organic matters—along with increased amount of dis­
infectants—would aggravate formation of DBPs, resulting in increased
chemical risks (Ateia et al., 2020).
The BRP increased with exposure time in the MPs samples, whereas
the BRP decreased with stagnation time in the control samples (see
Fig. 2e). BRP levels were positively correlated with the AOC level in the
first four weeks (R2 = 0.230), with a coefficient of R2 = 0.744 in the 2nd
week and R2 = 0.174 in the 4th week. During the 8th week, the corre­
lation weakened (R2 = 0.002). A possible reason for the weaker corre­
lation with time is that parts of indigenous bacteria used to detect BRP
cannot be counted using colony method. Some bacteria may either enter
the viable but not culturable (VBNC) state or the unculturable bacteria
change (see Fig. S3), when nutrients are limited (Wu et al., 2016).
Another reason may be that the yield coefficient of P17 or NOX is more
stable than that of endogenous bacteria, due to a shift in the microbial
community or VBNC bacteria (S. Chen et al., 2018; X. Li et al., 2018;
Zhang et al., 2016).
3.2. MPs promote high microbial counts and rapid growth rate
Overall, the HPCWR values ranged from 7 to 93 CFU/mL in the initial
tap water (Control_0). HPCWR ranged from 6 to 9.27 × 104 CFU/mL and
HPCBM ranged from 5 to 7.21 × 105 CFU/mL, as shown in Fig. 2f and h.
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Journal of Hazardous Materials 420 (2021) 126662
Three further observations related bacterial growth are detailed below:
(1) The HPCWR level was higher in water samples with MPs than that
in the control group (no MPs added). The presence of MPs was
positively correlated with the abundance of bacteria in water
samples. One possible reason may be that MPs release organic
matters that includes nutrients available for bacterial growth (Lee
et al., 2020). The proportion of unculturable bacteria ranged
from 97% to 99.5%, as shown in Fig. S2.
(2) The HPCBM values were higher than the HPCWR values. The
reason for this may be that MPs have a high void rate and a high
SAV ratio, and are thus able to absorb abundant microbial com­
munities. Furthermore, the plastisphere represents the core area
of the nutrient source, therefore bacterial concentration was
higher near to MPs.
(3) The MPs accelerated bacterial growth rate and affected the bac­
terial growth phases. It has been reported that bacteria grew
faster on solid culture medium than that on liquid culture me­
dium (Schoenborn et al., 2004), which explains the higher bac­
terial growth rate of HPCBM during the exponential phase. The
stationary phases of bacterial growth were much longer in the
MP-BM (from the 5th day to the 28th day) than those in the water
environment (from the 7th day to the 14th day), and the time
difference was almost two weeks. A previous study had reported
that bacterial growth would be deregulated when the bacterial
population reached the dead phase if no additional nutrients were
added (X. Chen et al., 2018). It can be deduced that nutrients
level increased due to the presence of MPs, according to the time
difference of the stationary phase.
3.3. MPs shaped different microbial communities in water and
plastisphere
Microbial diversity was analyzed spatially and temporally through
morphological assessment using SEM and high-throughput sequencing.
3.3.1. Changes in morphological features of plastisphere
SEM (see Fig. 3), revealed significant differences in the morphology
of the different MPs, primarily in two aspects:
(1) The initially rough surface of the plastic became dense, and the
extent of the denseness increased with time. The formation of
multi-layered cells is regarded a significant sign of biofilm for­
mation (Liu et al., 2016). The surface of PET was the last to form a
biofilm among all plastic samples, which is contrary to the bio­
film formation results observed upon using seawater (Feng et al.,
2020). A previous study had reported that specific microbial
communities have the potential to degrade polyesters, such as
commercial PET from beverage bottles (Mueller, 2006), which
may explain the slow biofilm formation on the PET surface.
(2) Abundant rod-shaped bacteria and micrococci were observed on
the MPs. On the PPR surface—which was similar to the
morphology of the PP surface—Bacilli initially represented the
dominant community, and in later stages, Cocci represented the
dominant community. On the HDPE surface, scattered Phages and
Cocci were observed. On the PE surface, Cocci initially repre­
sented the dominant community, after which the proportion of
Bacilli then gradually increased. Streptobacilli and Bacilli were
found to be densely distributed around the PVC surfaces, and
Bacilli represented the dominant community on the PET surface.
3.3.2. Changes in microbial diversities
Additionally, high-throughput analysis on operational taxonomic
units (OTU) level revealed different spatial and temporal changes onto
different plastic materials. Three differences were observed:
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

Fig. 2. Changes in biological stability values in the samples over 8 weeks. (a–d) Variations in AOC formation with time, (e) Changes in BRP with time, and (f–g)
Changes in HPC with time.

5
X. Chen et al.
Fig. 3. Morphology differences in biofilm over a period of 8 weeks. Remark: The figure of PET (5 µm) in the 2nd week was not available and a figure of PET (10 µm)
was inserted.
(1) The OTU number increased with stagnation time, and the OTU
number followed the following descending order: biofilm phase
> water phase > control, as shown in Fig. 4a, indicating that
plastic particles contributed to an increase in microbial richness.
The OTU abundance was much higher than that in the sample
with no stagnation (Control_0, with 107 OTU types), and the
bacterial abundance increased with stagnation time, ranging
6
Journal of Hazardous Materials 420 (2021) 126662
from 338 to 699 OTU types. Moreover, there were 44 core OTUs,
and the special OTU increased with time, especially during the
8th week, up to 96 and 100 OTUs. A possible reason for this
phenomenon is that the oligotrophic environment in the water
contributed to the microbial richness. Microbial communities in
biofilm onto PPR particles hold the most abundant OTU value.
X. Chen et al.
Fig. 4. Changes in microbial diversities. (a) OTU number, (b) Shannon values, (c) Hierarchical clustering tree based on OTU; (d) PCoA on OTU level.
(2) Shannon index primarily reflects changes in microbial evenness,
as shown in Fig. 4b. Shannon indexes increased with stagnation
time, and the level followed the order: biofilm > water > control.
In the 8th week, the value decreased in biofilm phase while
increased in water phase, in sample with PE particles inserted.
The possible reason was that microbial communities shifted from
biofilm to water.
(3) Beta diversities on the OTU level were more significant in the
spatial dimension compared to beta diversities in the temporal
dimension, as shown in Fig. 4c and d. The differences between the
control and biofilm groups were much more significant than
those between the control and water groups. Beta diversities
analysis revealed that the group distance induced by spatial dif­
ferences was longer than that induced by stagnation time. The
impacts of the plastisphere-biofilm on microbial diversities in the
surrounding water decreased with stagnation time, and the dif­
ferences subsequently become more pronounced, which indi­
cated that interactions between mature biofilm and the
surrounding water were weakened. This represents indirect proof
that the levels of plastic-deprived nutrients decrease with mature
biofilm development, which is consistent with the previously-
described AOC and SEM.
3.3.3. Changes in microbial community formation
According to impacting factors including stagnation time, spatial
phases, and plastic materials, the average levels of microbial community
formation (on levels of phyla and classes) were analyzed by group:
7
Journal of Hazardous Materials 420 (2021) 126662
(1) The coupling impacts of stagnation time and spatial phases
showed obvious differences, as shown in Fig. 5a. Proteobacteria
was the only one dominant phylum that shared in all samples.
The changes in microbial community formation were insignifi­
cant with stagnation time in the control group, compared to those
in samples with MPs added, both in water phase and biofilm
phase. The proportions of Firicutes, Actinobacteriota, Planctomy­
cetota had a significant increase due to the presence of MPs. The
primary increased classes included γ-Proteobacteria, Actinobacter,
Phycisphaerae, and Bailli. Spatial differences had a more signifi­
cant impact compared to temporal differences, which was
consistent with the beta-diversities analysis based on OTU data.
(2) The changes in microbial community formation induced by the
specific plastic particles were shown in Fig. 5b. The primary
phyla were Proteobacteria, Bacteroidota, Firmicutes and Plancto­
mycetota, and the primary classes were α-Proteobacteria, γ-Pro­
teobacteria, Actinobacter, and Bacteroidia. The proportion of
α-Proteobacteria was higher than that of γ-Proteobacteria in bio­
film, except biofilm onto PVC particles. There was a significant
increase in Clostirdia in biofilm phase onto PP, PET, PPR, PVC.
Biofilm onto PET particles had the most abundant microbial
community formation. The increment might be helpful to explore
microbial communities for polymer biodegradation.
3.4. MPs altered chemical indices in water with time
The changes in chemical indices, including pH, ORP, turbidity, ESI,
EC and TDS, are shown in Fig. S3. The pH values ranged from 7.53 to
9.02, the values first increased and then slowly decreased in the MP
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

Fig. 5. Changes in microbial community formation induced by different impacting factors. (a) Impacts of stagnation time and phases; (b) Impacts of different
plastic particles.

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X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

samples, while the pH slowly increased from 8.0 to 8.5 in the control
group. The PPR and HDPE samples were at the risk of exceeding the pH
standard recommended by the World Health Organization (WHO,
2017). Besides, acidic and basic pH levels would contributed to the
deprivation of MPs pieces, compared to neutral pH levels, and acidic pH
levels are more aggressive (Mortula et al., 2021). The ORP levels first
decreased and then increased in MPs samples, ranging from − 40.7 to
35.3 mV, which was much higher than that in the control group at the
end of the pilot test. The turbidity ranged from 0.22 to 2.63 NTU. Large
municipal supplies should consistently produce water with no visible
turbidity (and should be able to achieve 0.5 NTU before disinfection at
all times and average 0.2 NTU or less) (WHO, 2017). Turbidity levels
were much higher than the standard of WHO due to MPs, especially
during the first weeks. The biggest increase in turbidity was as much as
10.95 times. Turbidity levels first increased sharply then slowly
decreased, and turbidity always showed a decreasing trend in the con­
trol samples. The changes in EC and TDS were not significant. Therefore,
changes in pH and turbidity induced by MPs should be paid attentions
to.
4. Discussions
4.1. Correlations among biostability indices and impacts of MP properties
4.1.1. Correlations among biostability indices
Biostability indices, including AOC, BRP, and HPC, are often used to

Fig. 6. Correlations among the biostability indices, microbial evolution distance, and MPs’ properties. (a) Biostability indices; (b) impacts of MPs’ properties; (c)
Normalized Euclidian distance from Control_0 sample based on OTU; and (d) Weights of impacting factors on evolution distance.

9
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

evaluate biostability in water. Correlations among these indices were growth in biofilm. Moreover, the growth rate on solid media was faster
illustrated in Figs. 6a and S4. The correlation between AOC and AOCNOX than that on liquid media (Owen et al., 2006; Schoenborn et al., 2004),
was the most significant (R2 = 0.904), as AOCNOX was the main which can explain the contribution effect of particles with high RSV on
component responsible for the increase in AOC formation in the MPs numerical changes. However, the impacts on microbial evolution di­
samples, and subsequently led to an increase in BRP (R2 = 0.317). rection is still unclear now.
HPCWR was positively correlated with AOCP17 (R2 = 0.465), but the
env = [ t p v s sv ρ ] (4)
correlation between HPCWR with AOCNOX was less significant (R2
= 0.290). [
t p v s sv ρ
]
A= (5)
|t| |p| |v| |s| |sv| |ρ|
4.1.2. Impacts of MPs’ properties on biostability
The impacts of the MPs on biostability indices were analyzed in ( )− 1
AX = d⇒X = AT A AT d
respect to properties including surface area, volume, surface area to (6)
X = [ 0.067 0.193 0.318 0.337 0.34 − 0.125 ]T
volume ratio (S/V), density, and number of MPs, as depicted in Fig. 6b.
Density had a more significant impact on the AOC and BRP levels in the Second, we tried to use the differences in indices instead of indices
water than the other four parameters. The AOC in water increased with themselves to conduct matrix analysis, for exploring the impacts of MPs
MP’s density. Similarly, HPCWR increased with MP density, whereas nature on evolution direction:
HPCBM decreased with MP density. Correlations with density mean that
the upper layer is more suitable for bacterial growth than the bottom (1) Building matrixes and equation. A matrix M was introduced to
layer so that MPs with low density have high performance of biofilm define the differences in MPs. If two samples had the same MPs,
formation. More mature biofilm resulted in the development of a more the value was marked as 0 or else was marked as 1, as shown in
compact plastisphere. Hence, nutrients release from mature MP-BM Eq. (7). Matrixes, including T, V, S, R, and ρ, were introduced and
become more difficult (Fish et al., 2017). Mature biofilm formation separately the differences in stagnation time, volume, superficial
may therefore block the release of nutrients. As a result, AOC decay rates area, RSV and density. The definition of these matrixes followed
were higher in samples with low-density MPs. the regulation in Eq. (8). Essentially, the microbial evolution
Furthermore, the properties of the MPs impacted the distribution of differences are the comprehensive results of differences in time,
bacteria between the biofilm and water phases. Although HPCWR was MPs type, density, size and other indices. Therefore, we can re­
positively correlated with HPCBM (R2 = 0.372), the correlations of these gard that the linear combination of these differences decided the
indices with MP properties revealed converse trends, especially the differences in standard Euclidean distance, as shown in Eq. (9).
correlations with density. This trend mainly resulted from the absor­ All MP-BM samples were selected to form the matrix sets, as
bance performance of the MPs. The average absorbance performance shown in Fig. S7.
was positively correlated with the SAV value. A higher is SAV ratio was (2) Solving equations and ranking impacting factors. All matrix ei­
associated with a stronger absorbance performance. The ratios were genvalues were calculated and formed a new linear equation.
positively correlated with the reciprocal of the MPs’ size and the Since all matrixes were linearly correlated, so were their eigen­
gradient decreased most rapidly in the smallest dimension. This in­ values, as shown in Eq. (10). The equation was solved in Matlab
dicates that small-sized MPs are associated with high potential risks, software, and the results were shown in Eq. (11). Besides, as
which is consistent with the results of a previous study by Lambert and shown in Fig. 4d, impacts of spatial differences were more sig­
Wagner (2016). Similarly, a large number of MPs contributes to the total nificant than that of temporal differences. Therefore, as for mi­
absorbance performance of the MPs. MPs with a high absorbance per­ crobial community differences concerned, the impacting factors
formance can easily absorb bacteria onto the plastisphere where the followed the order: phase position > stagnation time > density >
nutrient level is high, thereby promoting rapid biofilm formation. Pre­ MPs difference > S > V > RSV.
vious studies have shown that continuous MPs fragmentation would {
increase the SAV ratio and subsequently increase the absorbance Mij =
1, samplei, jaredifferentMPs
(7)
0, samplei, jaresameMPs
performance.
[ ⃒⃒ ⃒ ]
ti − tj ⃒
4.1.3. Weights of impacting factors Tij = ⃒ ⃒ (8)
Microbial community evolution of each sample can be regarded as max⃒ti − tj ⃒
one vector (the Euclidean distance from the Control_0 is r, and the
evolution direction is θ), as shown in Fig. S6. Levels of r indicated the x1 ⋅T + x2 ⋅V + x3 ⋅S + x4 ⋅R + x5 ⋅M + x6 ⋅ρ + o(x)⋅U = D
(9)
⇒x1 ⋅T + x2 ⋅V + x3 ⋅S + x4 ⋅R + x5 ⋅M + x6 ⋅ρ ≈ D
microbial evolution distance, while θ levels of indicated the evolution
direction. To quantitively rank the effects of the impacting factors
in which, o(x) is infinitely small quantity, and U is the matrix of
(marked as matrix env, including stagnation time, temporal phases,
unknown impacting factors.
volume, surface area, SAV, and density) on differences in microbial
⎡ ⎤ ⎡ ⎤
community evolution, matrix analysis was conducted in two aspects: 1 1
λ λ
First, impacts on evolution distance and evolution speed were ⎢ k
⎢
⎥
⎥
⎢ D
⎢
⎥
⎥
analyzed. The Normalized Euclidian distance from Control_0 sample ∑6 ⎢
⎢ λ2k ⎥
⎥
⎢
⎢ λ2D ⎥
⎥
based on OTU was calculated, marked as d (see Fig. 6c). The matrix env xk ⋅ ⎢
⎢
⎥
⎥ = ⎢
⎢
⎥
⎥
⎢ ⋱ ⎥ ⎢ ⋱ ⎥
was processed with method of vector unitization, as shown in Eq.5.
k=1
⎢ ⎥ ⎢ ⎥
⎣ ⎦ ⎣ ⎦
Since all physical and chemical characteristics cannot be defined with a 15
λk 15
λD
simple value in the matrix, the impacts of MPs nature were not taken ⎡ ⎤
15×15
⎡ ⎤
15×15
(10)
into accounted in this part temporally. The impacting weights of env 1
λT λV ⋯ λρ 1 1
⎡ ⎤ 1
λ
were calculated following the matrix transformation as shown in Eq. (6) ⎢
⎢
⎥
⎥
x1 ⎢ D ⎥
⎢ ⎥
⎢ λ2 λ2 ⋯ λ1 ⎥ ⎢ ⎥ ⎢ λ2 ⎥
and were depicted in Fig. 6d. The impacting weights on evolution dis­ ⎢ T V ρ ⎥ ⎢ x 2 ⎥ ⎢ D ⎥
⇒⎢ ⎢ ⎥ ⎢ ⎥
⋅⎢ ⎥ = ⎢ ⎢ ⎥⇒A′ X ′ = d′
tance followed the following order: SAV > surface area > volume > ⎢⋮
⎥
⎥ ⎣⋮ ⎦ ⎢⋮ ⎥
⎥
⋮ ⋱ ⋮
temporal phases > density > stagnation time. High levels of SAV ⎢
⎣
⎥
⎦
⎢
⎣
⎥
⎦
x6
contributed to bacterial attachment and nutrient absorption onto plas­ λ15
T λ 15
V ⋯ λ1
ρ λ15
D
tisphere (Lambert and Wagner, 2016), which accelerated bacterial 15×6

10
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

in which, k = 1, 2, …6 represent indices of stagnation time, volume, where S represents the AOC concentration, b represents the bacterial
superficial area, RSV, MPs type, and density. concentration, k is the average desorption rate of AOC, μmax is the
maximum growth rate, Km is the AOC concentration when the growth
( )− 1
X ′ = A′ TA′ A′ Td′ rate is half of μmax, δ is the yield coefficient, d is the natural mortality rate
(11)
= [ − 1.8191 0.0098 0.0301 0.005 1.0745 1.6831 ]T of bacteria, and Ae is the proportion of unblock plastisphere.
In the following, we analyze the model system (Eq. 13) using stability
theory of differential equations. For the solutions of model system, the
(3) Interpretation of matrix analysis. Due to the decreased nutrients region of attraction is given by the set in Eq. (15).
in waters, all samples come into a comparative poor-nutrients ⎧
abrk μmax Sb db
with stagnation time (Chen et al., 2020; Liu et al., 2013), so the ⎪
⎪
⎨ k − 3 − δ(Km + S) + δ = 0,
⎪ ( )
impacts of stagnation time were negative. The impacts of density 3 Km d
⇒(b∗ , S∗ ) = (0, 0)or ,
were significant, and previous studies indicated that MP-BM ⎪ ar μmax − d
⎪ μmax Sb − db = 0,
⎪
⎩
development would change the density of the mixture (Wang Km + S
et al., 2019), and the changes of density would again alert mi­ (14)
crobial community formation. This interaction with time de­ { }
serves more attention in future study. Ω= (b, S) : b ≤
3
,S ≥
Km d
(15)
ar μmax − d
Moreover, impacts of MPs nature can also be observed qualitatively. Moreover, with the development of the biofilm, these two phases
It can be noticed that the particles of HDPE and PPR applied in the would transfer. A previous study described the complete biofilm
present study has the similar shape, size and pores, as shown in Table 1. development phases, including attachment, growth, maturation, and
Drawing lessons from the idea of contradiction, we tried to prove the detachment (Zhang et al., 2019).
impacts of MPs themselves excluding size. According to the morpho­ Finally, based on the results of present study—and previous stud­
logical features in Fig. 3, spherical bacteria were distributed on PPR, ies—MPs can be regarded as nutrient producers or nutrient transporters,
while rod-shaped bacteria were distributed on HDPE. Phyla of Cyano­ but the dominant role of MPs strongly depends on the original nutrient
bacteria and Planctomycetota were much more abundant on plastisphere level (Romera-Castillo et al., 2018). For example, MPs mostly absorb
of HDPE, compared to PPR, as shown in Fig. 5b. Besides, even though and transport nutrients in seawater, while MPs slowly release organic
PPR and HDPE have the similar size, yet the proportion of the special matter in drinking water (Lee et al., 2020).
genera ranged from 6.4% to 10.14%, as shown in Fig. S8.
4.2.2. Numerical solution for differential equations
4.2. Kinetic analysis of AOC and bacteria using time-delay differential To simply the process of solving differential equations, we replaced
equations the bacterial growth equation b(t) with three empirical models to
character changes in microbial counts, including Monod model (Eq. 15),
According to integrated analyses of SEM, AOC, and bacterial ana­ Logistic model (Eq. 16), and Exponential model (Eq. 17), as shown in
lyses in the previous sections, we propose two possible MP-BM forma­ Fig. 7c. Monod model had the best simulation result. The numerical
tion phases, as shown in Fig. 7. solution for differential equations were deduced in Maple 2020, ac­
cording to the initial values of differential equations coefficients
4.2.1. Conceptional models for MP-BM formation (Table 2), as illustrated in Fig. 7d. It provides some views for overcoming
During the first phase (named the MP-BM formatting phase), the MPs biological risks in field of storing stand-by drinking water, especially in
were not completely enclosed within the biofilm and could be regarded remote area. As microbial communities have the capability of consume
as consistent AOC sources (see Fig. 7a), in accordance with the con­ AOC rapidly, so the traditional model (harvesting-treating-storing-sec­
centration diffusion model of a point pollution source. ondary treating) might be replaced by a new model (harvesting-storing-
During the second phase (named the MP-BM formatted phase), the treating). This can convert the adverse effect of water stagnation into
MPs were completely enclosed within the biofilm and the AOC release biodegradation.
was blocked (see Fig. 7b), thereby terminating the release of new nu­ ( )
b(t) bmax
trients and antibacterial compounds into water, so the value of D(MP) is ḃ(t) = μmax 1 − b(t)⇒b1 (t) = (16)
bmax 1 + C1 bmax e− μmax t
none, as shown in Eq. (13) (Fish et al., 2017).
The regulation of AOC and bacteria can be summarized as a set of
ḃ(t) = μmax b(t)⇒b2 (t) = C2 e− μmax t
(17)
equations, followed the Monod model, as shown in Eq. (13).
( ) ( )
Ṡ x, y, z, t = D Sxx + Syy + Szz b(t) = b0 (1 + p)t (18)

(12) We discussed impacts of AOC desorption rate (k) on the stability of

∰ Ω S(t)dv
S(t) = ≈ kt the system. When the value of k was 27.5 μg/L/day, the supply and the
∰ Ω dv
consumption of AOC reached a balance (see Fig. 7e) and the microbial
counts would stabilize at a relative high level so that the stationary
phase was expanded, which was harmful to the biosafety of water. The

˙ = D(MP)A k − μmax S(t)b(t) + db(t), ḃ(t) = μmax S(t)b(t) − db(t),

{ S(t) e
δ(Km + S(t)) δ Km + S(t)
/ (13)
4π r2 − 4 3πr3 b(t)a
D(MP) = { 1, immatureMP − BM 0, matureMP − BM, Ae =
4πr2

11
X. Chen et al. Journal of Hazardous Materials 420 (2021) 126662

Fig. 7. Mechanism underlying microplastic biofilm formation and AOC diversities. (a) MP-BM formatting phase, (b) MP-BM formatted phase, (c) Three models for
bacterial growth, (d) Deduced AOC curve based on three models, and (e) Impact of k values on AOC curves.

level of k would decrease with the formation process of MP-BM, then led (2) There were genera that prefer to grow in biofilm environment,
to the shocks of AOC and microbial counts. such as Curvibacter, Enhydrobacter, Enterobacter, Escherichia-
Shigella, Klebsiella and Mycobacterium.
4.3. Challenges in MP-contaminated water and associated pollution (3) Some genera only existed in a certain period, such as Erysipela­
controlling strategies toclostridium, Escherichia-Shigella, Helicobacter, Klebsiella, Limno­
bacter, and Mycobacterium. These genera were detected in the 8th
4.3.1. Changes in opportunistic pathogens and pathways week.
The absolute proportion of OPs in all samples ranged from 0.97% to (4) Some genera only existed in samples with specific MPs. Pseudo­
37.21%, the proportion increased with stagnation time, and MPs monas was correlated with the presence of MPs, as there was
increased levels of OPs, as shown in Fig. S5. Rations of OPs in biofilm almost no Pseudomonas in control samples. High level of Escher­
were higher than those in water during the period of the 2nd week to the ichia-Shigella was only observed onto plastisphere of PVC, PPR,
4th week. To be noticed, rations of OPs in water phase (PP, PPR, PET) and PE, whereas high level of Flavobacterium was observed onto
exceeded those in biofilm phase in the 8th week, which might be plastisphere of PVC, PPR, PE, and HDPE.
induced by biofilm detachment.
There were 20 genera belonged to opportunistic pathogens, and their Moreover, changes in prediction of KEGG pathways were conducted
relative proportion in OPs were shown in Fig. 8. The possible diseases based on Tax4Fun package and were illustrated in Fig. 9. Prediction on
induced by these OPs include human cat scratch disease, colonic dis­ level 1 mainly consisted of six types, including Human diseases, meta­
eases, pneumonia, sepsis, urogenital tract diseases, etc. These genera can bolism, genetic information processing, cellular processing, organismal
be divided into four groups according to their relative proportions: system, and environmental information processing. MPs contributed to
all these functions in water phase and biofilm phase except cellular
(1) There were genera that prefer to grow in water environment, processing and environmental information processing, compared to the
such as Afipia, Burkholderiales, Staphylococcus, and control samples. The average level of human diseases was higher in
Xanthobacteraceae. biofilm phase than that in water phase. Metabolism function of micro­
bial communities in HDPE samples, PVC samples and PP samples were
Table 2 obviously stimulated. The aggregated metabolism was also proved in a
Values of coefficient for numerical solution. previous study due to water stagnation (Zhang et al., 2021). Due to the
low level of nutrients in the control samples, functions of cellular pro­
Coefficient Values Unit Reference
cessing and environmental information processing were enhanced to
k 10 μg/L/day Data from this study
survive in the competition for nutrients.
μmax 2.5 /day (King and Mody, 2011)
δ 50,000 cells/(μg/L) (Hammes et al., 2007a, 2007b)
Km 500 μg/L (Zhang et al., 2016) 4.3.2. Chemical risks in subsequent disinfection
d 0.01 (Misra and Singh, 2012) Currently, disinfection is one of the most effective measures
bmax 35,000 cells/mL (Zhang et al., 2016) employed to ensure microbial safety. Plastic-derived nutrients stimulate
p 0.5 Data from this study
microbial activities, leading to an increase in the minimum disinfectant
b0 100 cells/mL Data from this study
S0 110 μg/L Data from this study requirement (Ohkouchi et al., 2013). The increased disinfectant dose

12
X. Chen et al.
Fig. 8. Relative proportion of opportunistic pathogens in different samples.
may result in further potential risks.
Disinfection methods, including chlorination, ultraviolet treatment,
and ozonation, contribute to the aging of MPs. The aging process is
accompanied by fragmentation, and persistent organic pollutants
release may result in secondary contamination (Cheng et al., 2020;
Wagner et al., 2018). High-dose disinfectants may accelerate the
development of bacterial tolerance to disinfection (Zhang et al., 2018).
Efficient MPs removal would contribute to better control of DBPs in the
subsequent disinfection process and some researchers have recom­
mended methods, such as electro-sorption (Xiong et al., 2020).
Conversely, electrolysis may be a promising strategy for MPs pollution
controlling. Electrolysis can result in potential absorbing of high levels
of MPs onto the electrode, which contributes to the removal of MPs and
to buffering the disinfection stress.
The present study mainly focused on the impact of plastic-deprived
AOC on bacterial growth with stagnation time, and more attentions
will be paid to the impact of different disinfection methods on MP-BM in
our further work.
13
Journal of Hazardous Materials 420 (2021) 126662
5. Conclusion
The present study explored changes in plastics-deprived AOC and the
subsequent impacts on microbial activities in stagnant water. The main
conclusions are as follows:
(1) Plastic-derived nutrients were detected and contributed to bac­
terial growth.
(2) Differences in MPs resulted in variations in AOC formation and
subsequently shaped the various microbial communities.
(3) The properties of MPs, including the density and surface to vol­
ume ratio, affected the distribution of bacteria between water and
biofilm phases.
(4) Two phases, the MP-BM formatting and MP-BM formatted pha­
ses, were proposed to explain the kinetics of bacterial growth and
AOC.
X. Chen et al.
Fig. 9. Prediction of KEGG pathway based on Tax4Fun package.
CRediT authorship contribution statement
Xiao Chen: Methodology, Investigation, Mathematic modeling,
Formal analysis, Writing – original draft. Yi Wang: Conceptualization,
Writing – review & editing, Project administration. Sheng Chen, Yiran
Sun: Methodology, Investigation, Resources, Writing Reviewing.
Qiaowen Tan: Data processing, Mathematic modeling, Formal analysis.
Zhibin Ding: Conceptualization, Methodology, Writing – review &
editing, Supervision. Yaofeng Lu: Investigation, Resources. Yingjun:
Methodology, Resources.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This study was supported by: (1) Logistic Project (No. BY115C002);
and (2) National Key R & D Plan-Resource Utilization Technology of
Major Wastes in Typical Seas of South China Sea (No.
2017YFC0506304). Thanks to professor Wang Zaihua and professor
Zhao Ying for their guidance and inspiration in mathematics modelling
and calculation.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2021.126662.
14
Journal of Hazardous Materials 420 (2021) 126662
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