Supplementary material for “Journal of Hazardous Materials”
Co-occurrence of microplastics and triclosan inhibited nitrification function and
enriched antibiotic resistance genes in nitrifying sludge
Zhiqi Wang, Jingfeng Gao*, Dingchang Li, Huihui Dai, Yifan Zhao
National Engineering Laboratory for Advanced Municipal Wastewater Treatment
and Reuse Technology, Beijing University of Technology, Beijing 100124, China
*Corresponding author: Dr. Jing-Feng Gao, E-mail: [email protected] or
[email protected], Tel.: 0086-10-67392627; Fax: 0086-10-67391983.
Summary
1. Methods
Method S1 The properties of MPs.
Method S2 Preparation of MPs suspensions.
Method S3 The repeated experiment.
Method S4 TCS mass balance experiments.
2. Tables
Table S1 Primers used in this study.
Table S2 SRCC analysis of the addition of PE, PS, PVC, PA and concentrations of
effluent SS and MLSS (* represents p < 0.05. P values < 0.05 combined with the SRCC
were marked in blue).
Table S3 SRCC analysis of contents of LB-proteins, LB-polysaccharides, LB-EPS,
TB-proteins, TB-polysaccharides, TB-EPS and concentrations of SS in effluent and
1
MLSS (* represents p < 0.05. P values < 0.05 combined with the SRCC were marked in
blue).
Table S4 Summaries of the results of high-throughput sequencing and alpha diversity
of bacterial 16S rRNA gene based on 97% similarity.
3. Figures
Fig. S1 The concentrations of NH4+-N, NO2
--N, NO3--N, the removal rate of NH4
+-N in
R-PVC (a) and R-PA (b).The effluent concentration of SS in R-PVC and R-PA (c). The
MLSS concentration in R-PVC and R-PA at the 0th, 7th, 14th, and 21th day (d).
Fig. S2 SEM photos of four types of fresh MPs. PE (a), PS (b), PVC (c) and PA (d).
Fig. S3 SEM photos of four types of MPs in the SBRs for 28 days. PE (a), PS (b), PVC
(c) and PA (d).
Fig. S4 SEM photos of nitrifying sludge after adding 1 mg/L MPs in four SBRs and
undosing control SBR for 28 days. SBR-CK (a), SBR-PE (b), SBR-PS (c), SBR-PVC
(d) and SBR-PA (e).
Fig. S5 Microbial morphology of nitrifying sludge after adding 1 mg/L MPs in four
SBRs for 28 days. SBR-PE (a), SBR-PS (b), SBR-PVC (c) and SBR-PA (d).
Fig. S6 The adsorption capacity of TCS on PA, PS, PVC and PE.
Fig. S7 Network analysis showing the relationships among environmental variables
and ARGs-MGEs in five SBRs. Solid and dotted lines represent the positive and
negative correlations, respectively.
2
Method S1 The properties of MPs.
In this study, PE, PS, PVC and PA were selected. Among them, PE is used in
pharmaceutical and personal care products (PPCPs) (with TCS), including body and
facial scrubs as well as food packaging films and water bottles (Cheung & Fok, 2017;
Lares et al., 2018; Ziajahromi et al., 2017). Meanwhile, PVC and PS, which have a lot
of usage in daily life, have been reported in the literature as the plastic materials used in
PPCPs (with TCS) (Fendall & Sewell, 2009; Programme, 2015; Zitko & Hanlon, 1991).
In addition, PS is widely used in protective packaging, containers, bottles and lids
(Sussarellu et al., 2016),which could be used as a box for PPCPs (containing TCS). And
PA is all widely used in synthetic clothes (Ziajahromi et al., 2017), which may come
into contact with laundry liquid (containing TCS) when washing clothes. PE, PS, PVC
and PA had frequent contacted with TCS. As manufactured microbeads (primary MPs)
or small plastic fragments derived from the breakdown of plastics (secondary MPs), PE,
PS, PVC and PA were widely used in life and discharged into WWTPs with TCS. Thus,
it was urgent to explore the co-effects of MPs (PE, PS, PVC and PA) and TCS on
nitrifying sludge in WWTPs.
In addition, the MPs (the size of MPs > 100 μm) in the influent of WWTPs varied
from 1 to 293 particles/L (approximately 0.004 to 1.23 mg/L) (Carr et al., 2016; Dris et
al., 2015). Furthermore, the MPs (the size of MPs > 10 μm) in the influent of WWTPs
varied from 2223 to 18285 particles/L (approximately 0.09-0.84 mg/L) (Simon et al.,
2018). Notably, Ziajahromi et al. (Ziajahromi et al., 2017) quantified MPs in
wastewater, including 3.1 particles/L (approximately 0.012 mg/L) PE, 1.4 particles/L
(approximately 0.006 mg/L) PS, 1.0 particles/L PA (approximately 0.004 mg/L), and a
small number of PVC. PE is one of the most abundant type MPs. Furthermore,
3
Ziajahromi et al. (Ziajahromi et al., 2016) expected the concentration of PE in UK
wastewater is 0.27-1.4 mg/L. Thus, in this study, 1 mg/L MPs, which might be an
environmental concentration of MPs in WWTPs, were selected to study their effect on
activated sludge. Meanwhile, the size of the activated sludge in this study was about 110
m, so the MPs with a size slightly larger than the sludge was selected to study their
impact on sludge.
4
Method S2 Preparation of MPs suspensions.
PE, PS, PVC and PA were dispersed into water by magnetic stirring in four bottles
at a concentration of 100 mg/L, respectively. Before feeding stage, the magnetic
stirrings worked to make the suspensions in bottles sufficiently mixed for 30 min. And
then, the 30 mL MPs dispersions were added into the SBRs during feeding stage.
5
Method S3 The repeated experiment.
In this study, the inhibition on nitrification of “TCS and PVC” or “TCS and PA”
co-loading, were found during 14 days. Thus, the experiment, which co-added TCS and
PVC (named as R-PVC), TCS and PA (named as R-PA), respectively, was repeated by
simulating the same experimental conditions as before. Because the experiment used
domestic sewage as the influent, and formal experiment was completed in summer,
while repeated experiment was conducted in winter, the concentration of NH4+-N and
COD in the sewage in winter is slightly higher than that in summer. The influent
concentration of NH4+-N and COD fluctuated between 58-79 mg/L and 110-150 mg/L,
respectively. Each SBR ran four cycles per day. Each cycle included 0.1 h feed, 3 h
aerobic reaction, 0.15 h settling, 0.15 h decant and 2.6 h idle. The other parameters were
same with before. Among them, 0.5 mg/L TCS and 1 mg/L PVC, 0.5 mg/L TCS and 1
mg/L PA, respectively, were fed to the SBRs. The concentrations of TCS, PVC and PA
were exactly the same as before. Due to the decrease of temperature in winter, the heater
band was used to maintain the temperature in each reactor at 25 ± 2 oC. Meanwhile, the
SBRs were started up with the MLSS concentration of 2000 mg/L, and the seed sludge
was collected from a lab-scale SBR which was dosed with TCS under stable operation.
The repeated experiment ran 24 days.
6
Method S4 TCS mass balance experiments.
The analysis of TCS was made by UV detection at 230 nm, using a Waters X-
bridge C18 Column (250 mm × 4.5 mm, 5 μm). The injected volume was 10 μL with the
flow rate of 1 mL/min. The TCS removal by nitrifying activated sludge may be
contributed by adsorption and degradation (Eq. (1)). The removal TCS mass and
removal efficiency during MPs loading at the14th and 28th day were tracked using the
following Eqs. (1) - (2), where Mlost is the removal mass of TCS (g); Madsorption is the
mass of adsorbed TCS (g); Mdegration is the mass of degraded TCS (g); MTCS is the mass of
total TCS (g); Mresidue is the residual mass of TCS (g); V inf is the volume of influent (L);
Cinf is the total concentration of TCS in influent (g/L); Veff is the volume of effluent (L);
Ceff is the total concentration of TCS in effluent (g/L) (Heidler & Halden, 2007):
(1)
(2)
The adsorbed TCS mass, adsorption efficiency and degradation efficiency are
calculated as Eqs. (3) - (5), respectively, where Vsludge is the volume of sludge in SBR
(L); Csludge, before is the total concentration of TCS in sludge before influent (g/L); Csludge, after
is the total concentration of TCS in sludge after effluent (g/L).
(3)
(4)
(5)
7
Table S1 Primers used in this study.
Gene Name Forward Primer (5’-3’) Reverse Primer (5’-3’) Source
16S rRNA ATGGCTGTCGTCAGCT ACGGGCGGTGTGTAC(Ferris et al.,
1996)
AOA amoA ATAGAGCCTCAAGTAGGAAAGTTCTA CCAAGCGGCCATCCAGCTGTATGTCC(Meinhardt et
al., 2015)
AOB amoA GGGGTTTCTACTGGTGGT CCCCTCKGSAAAGCCTTCTTC(Rotthauwe
et al., 1997)
NOB CCTGCTTTCAGTTGCTACCG GTTTGCAGCGCTTTGTACCG(Liu &
Wang, 2013)
acrA-03 CAGACCCGCATCGCATATT CGACAATTTCGCGCTCATG
(Zhu et al.,
2013)
acrB-01 AGTCGGTGTTCGCCGTTAAC CAAGGAAACGAACGCAATACC
adeA CAGTTCGAGCGCCTATTTCTG CGCCCTGACCGACCAAT
mexF CCGCGAGAAGGCCAAGA TTGAGTTCGGCGGTGATGA
fabI AGGCATAACGCGCTCAGAAC GCACACGCTTGATGGTGACT (Chen et al.,
8
2009)
fabI1 GGCGATTTCCGCTATATCCT CAGCAGATACAAGCCCGAAT(Pycke et al.,
2010)
PafabVACCGAGGTTCATATGATGATCATCAAAC
CGCGCAGGCAAGCTTGTCGCTGAAAACGCGAAC
(Zhu et al.,
2010)
intI1 CGAACGAGTGGCGGAGGGTG TACCCGAGAGCTTGGCACCCA(Gillings et
al., 2015)
intI3 GCCACCACTTGTTTGAGGA GGATGTCTGTGCCTGCTTG(Barraud et
al., 2010)
IS613 AGGTTCGGACTCAATGCAACA TTCAGCACATACCGCCTTGAT(Zhu et al.,
2013)
9
SS MLSS
PESRCC -0.282 0.491
P values 0.498 0.217
PSSRCC -0.282 0.000
P values 0.498 1.000
PVCSRCC 0.773 -0.525
P values 0.039* 0.182
PASRCC 0.800 -0.659
P values 0.017* 0.075
Table S2 SRCC analysis of the addition of PE, PS, PVC, PA and concentrations of
effluent SS and MLSS (* represents p < 0.05. P values < 0.05 combined with the SRCC
were marked in blue).
10
Table S3 SRCC analysis of contents of LB-proteins, LB-polysaccharides, LB-EPS,
TB-proteins, TB-polysaccharides, TB-EPS and concentrations of SS in effluent and
MLSS (* represents p < 0.05. P values < 0.05 combined with the SRCC were marked in
blue).
SS MLSS
LB-proteinsSRCC 0.735 -0.810
P values 0.038* 0.015*
LB-
polysaccharides
SRCC 0.181 -0.500
P values 0.668 0.207
LB-EPSSRCC 0.759 -0.810
P values 0.029* 0.015*
TB-proteinsSRCC 0.566 -0.667
P values 0.143 0.071
TB-
polysaccharides
SRCC 0.458 -0.571
P values 0.254 0.139
TB-EPSSRCC 0.482 -0.619
P values 0.227 0.102
11
Table S4 Summaries of the results of high-throughput sequencing and alpha diversity of bacterial 16S rRNA gene based on 97%
similarity.
SampleEffective
readsobserved_species
Good’s
coveragePD_whole_tree Chao1 Shannon Simpson
Control-14d
35470
1491 0.99 169.92 2095.16 7.38 0.98
PE-14d 1517 0.98 107.27 2189.88 7.44 0.99
PS-14d 1447 0.99 117.84 2063.13 7.29 0.99
PVC-14d 1440 0.98 99.60 2053.21 6.92 0.98
PA-14d 1300 0.99 78.93 1906.00 6.57 0.97
Control-28d 1558 0.99 122.40 2043.03 7.89 0.99
PE-28d 1590 0.98 94.89 2224.28 7.97 0.99
PS-28d 1668 0.98 110.72 2293.29 7.89 0.99
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Fig. S1 The concentrations of NH4+-N, NO2
--N, NO3--N, the removal rate of NH4
+-N in
R-PVC (a) and R-PA (b).The effluent concentration of SS in R-PVC and R-PA (c). The
MLSS concentration in R-PVC and R-PA at the 0th, 7th, 14th and 21th day (d).
13
Fig. S2 SEM photos of four types of fresh MPs. PE (a), PS (b), PVC (c) and PA (d).
14
Fig. S3 SEM photos of four types of MPs in the SBRs for 28 days. PE (a), PS (b), PVC
(c) and PA (d).
15
Fig. S4 SEM photos of nitrifying sludge after adding 1 mg/L MPs in four SBRs and
undosing control SBR for 28 days. SBR-CK (a), SBR-PE (b), SBR-PS (c), SBR-PVC
(d) and SBR-PA (e).
16
Fig. S5 Microbial morphology of nitrifying sludge after adding 1 mg/L MPs in four
SBRs for 28 days. SBR-PE (a), SBR-PS (b), SBR-PVC (c) and SBR-PA (d).
17
Fig. S6 The adsorption capacity of TCS on PE, PS, PVC and PA.
18
Fig. S7 Network analysis showing the relationships among environmental variables
and ARGs-MGEs in five SBRs. Solid and dotted lines represent the positive and
negative correlations, respectively.
19
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