Journal of Hazardous Materials 423 (2022) 127167

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Journal of Hazardous Materials

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

Research paper

Profiling microbial removal of micropollutants in sand filters:

Biotransformation pathways and associated bacteria
Jie Zhou a, b, 1, Donglin Wang a, b, 1, Feng Ju c, d, Wanchao Hu a, Jinsong Liang e, Yaohui Bai a, *,
Huijuan Liu f, Jiuhui Qu a
a
Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
University of Chinese Academy of Sciences, Beijing 100049, China
c
Key Laboratory of Coastal Environment and Resources of Zhejiang Province, School of Engineering, Westlake University, 18 Shilongshan Road, Hangzhou 310024,
China
d
Institute of Advanced Technology, Westlake Institute for Advanced Study, 18 Shilongshan Road, Hangzhou 310024, China
e
School of Civil and Environmental Engineering, Harbin Institute of Technology, Shenzhen 518055, China
f
Center for Water and Ecology, State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing
100084, China

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

Editor: Dr. R. Maria Sonia Although there is growing evidence that micropollutants can be microbially converted in rapid sand filters of
drinking water treatment plants (DWTPs), little is known about the biotransformation pathways and associated
Keywords: microbial strains in this process. Here, we constructed sand filter columns filled with manganese or quartz sand
Sand filter obtained from full-scale DWTPs to explore the biotransformation of eight micropollutants. Under seven different
Micropollutants
empty bed contact times (EBCTs), the column experiments showed that caffeine and atenolol were easily
Biotransformation
removed (up to 92.1% and 97.6%, respectively) with adsorption and microbial biotransformation of the filters. In
Metagenomic-assembled genome
Oxygenase gene contrast, the removal of other six micropollutants (i.e., naproxen, carbamazepine, atrazine, trimethoprim, sul­
famethoxazole, and sulfadiazine) in the filters were less than 27.1% at shorter EBCTs, but significantly increased
at EBCT = 4 h, indicating the dominant role of microbial biotransformation in these micropollutants removal.
Integrated analysis of metagenomic reads and transformation products of micropollutants showed a shift in
caffeine oxidation and demethylation pathways at different EBCTs, simultaneous occurrence of atrazine hy­
drolysis and oxidation pathways, and sulfadiazine and sulfamethoxazole oxidation in the filters. Furthermore,
using genome-centric analysis, we observed previously unidentified degrading strains, e.g., Piscinibacter,
Hydrogenophaga, and Rubrivivax for caffeine transformation, and Methylophilus and Methyloversatilis for atenolol
transformation.

1. Introduction microbes is mainly attributed to oxidation, including direct oxidation by

Quartz or manganese sand filters, including rapid and slow filters
based on retention time, are commonly used in drinking water treat­
ment. Sand filters were initially designed to remove suspended solids
and reduce turbidity in source water based on the physical retention and
adsorption of filter material. However, many microbes also attach to the
filter sand and develop a thick biofilm, which may contribute to the
removal of various pollutants, even in rapid sand filters (RSFs) (Gryta
et al., 2014; Hedegaard et al., 2018, 2019). In addition to direct utili­
zation of pollutants for microbial growth, the removal of pollutants by
microbial oxidase and indirect oxidation by biogenic oxides (e.g.,
biogenic manganese oxide). For example, monooxygenase, an oxidore­
ductase that catalyzes electron transfer, is reported to play an important
role in pollutant oxidization due to the wide range of substrates present
on filter material (Ricken et al., 2013, 2017; Wojcieszynska et al., 2016);
with the action of multicopper oxidases and superoxide, Mn(II) can be
oxidized to Mn(III&IV) (Liang et al., 2017; Tebo et al., 2004), which can
oxidize heavy metals and organic pollutants in sand filters (Bai et al.,
2016; Zhou and Fu, 2020).
The detection and increasing popularity of micropollutants (e.g.,

* Corresponding author.
E-mail address: yhbai@rcees.ac.cn (Y. Bai).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jhazmat.2021.127167
Received 18 May 2021; Received in revised form 13 August 2021; Accepted 5 September 2021
Available online 10 September 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.
J. Zhou et al.
pharmaceuticals and personal care products and pesticides) in natural
water sources (Huerta-Fontela et al., 2011; Li et al., 2020; Schwarzen­
bach et al., 2006) have promoted the investigation of their microbial
removal in sand filters. Increasing evidence suggest that some trace
organic micropollutants can be removed in RSFs (Carpenter and Hel­
bling, 2017; Casas and Bester, 2015; Di Marcantonio et al., 2020; Zearley
and Summers, 2012), which is at least partly attributed to microbial
activity (Wang et al., 2020). For example, in a groundwater-fed RSF, the
herbicide bentazone can be co-metabolically transformed into 4-hydrox­
y-bentazone by methanotrophic enrichment, with 92% removal under
microbiological oxidation and demethylation by methanotrophic and
heterotrophic bacteria (Hedegaard et al., 2018, 2019). Nevertheless, a
comprehensive global picture on microbially-mediated micropollutants
metabolism, especially exploration of the biodegradation and biotrans­
formation pathways in RSFs, is still lacking. Addressing this issue is
critical for answering the hanging question “how (and which) microbes
in sand filters are involved in micropollutant removal”, and further
benefits for us to design and manipulate sand filters in drinking water
treatment.
In our previous study on the microbiome of 12 RSFs treating
groundwater or surface water, we observed (i) significant removal of
dissolved organic carbon in RSFs; (ii) the prevalence of carbohydrate-
and xenobiotic-metabolizing genes in the RSFs, especially those treating
the surface water; (iii) the different microorganism community
composition and function between the surface water RSFs (filled with
quartz sands) and groundwater RSFs (filled with manganese sands) (Hu
et al., 2020). These preliminary results strongly imply that certain mi­
croorganisms residing in the RSFs have the metabolic potentials to
biodegrade micropollutants in the source water. However, neither these
recently unraveled genomic potentials in micropollutants biodegrada­
tion have been experimentally validated, nor the key influential factors
and metabolic pathways underlying the differentiation in micro­
pollutants biotransformation between RSFs treating the surface water
versus ground water are known. In the current study, to resolve these
issues, here we established two types of lab-scale sand filters filled with
manganese sands and quartz sands obtained from full-scale water
treatment plants treating groundwater and surface water, respectively.
We selected eight micropollutants that are frequently detected in natural
water sources, i.e., six pharmaceuticals (naproxen, carbamazepine,
atenolol, trimethoprim, sulfamethoxazole, and sulfadiazine), one pesti­
cide (atrazine), and one stimulant (caffeine) (Huerta-Fontela et al.,
2011; Jiang et al., 2011; Zhu et al., 2013), as influent substrates. We
hypothesized that with an increase in empty bed contact time (EBCT),
the contact between the micropollutants and biofilm on the filter ma­
terial would increase and promote micropollutants biodegradation
(Carpenter and Helbling, 2017). Accordingly, we aimed to compara­
tively examine (i) the microbial removal performances of the eight
micropollutants in the two types of sand filters under different EBCTs
and (ii) the metabolic pathways of micropollutants and associated mi­
croorganisms via metagenomic sequencing and transformation products
analysis. The results of this study give a deep understanding of micro­
pollutant biodegradation in sand filters.
2. Materials and methods
2.1. Micropollutants and feedwater preparation
The eight micropollutants in feedwater were prepared with analytic
grade atrazine, naproxen, carbamazepine, atenolol, trimethoprim, sul­
famethoxazole, sulfadiazine, and caffeine. The selected micropollutants
were first diluted in 1% wt. methanol solution, and then diluted with
Milli-Q water to prepare the 50 mg/L stock solution. The standards and
isotope internal standards of corresponding micropollutants as well as
the standards of transformation products were supplied by Tianjin Alta
Technology Co., Ltd. (China) and then stored at − 20 ◦ C.
The feedwater was prepared by adding sodium acetate (equal to 3
2
Journal of Hazardous Materials 423 (2022) 127167
mg/L C), ammonium chloride, potassium nitrate (0.5 mg/L NH4+-N and
2 mg/L NO3--N), dipotassium hydrogen phosphate (equal to 0.15 mg/L
P), and a mixture of the eight micropollutants (approximately 10 µg/L
for each) to dechlorinated tap water. The dechlorinated tap water was
prepared by adding sodium metabisulfite (2 mg/L) to tap water.
2.2. Column experiment startup and operation
A self-made reactor with 12 parallel columns (height, 30 cm; inner
diameter, 4 cm; polymethyl methacrylate) was established to mimic the
sand filters (Fig. 1). We used raw filter sand from two actual RSFs
(operating for more than 10 years) as filter material in the columns. One
RSF used manganese sand (0.6–2.0 mm) as the filter material to treat
groundwater (treatment plant in Tongliao City, Inner Mongolia, China),
and the other RSF used quartz sand (0.5–1.2 mm) as the filter material to
treat surface water (treatment plant in Xuzhou City, Jiangsu Province,
China). The sand filter materials, which consisted of biofilms and asso­
ciated particulates, were collected at depths of 5–15 cm from the two
RSFs after evacuating the supernatant water. We then loaded the man­
ganese sand material into three columns and quartz sand material into
three columns, with graded gravel as the support layer. The graded
gravel layer and sand (2–5 mm particle size) fill layer were 5 cm and 15
cm high, respectively. Pumps and flowmeters were installed at both
inflow and outflow sites to control the flow rates.
All six sand columns were loaded with prepared feedwater using a
pump for 15 d for microbial adaptation inside the filters. In total, 1 L of
influent and effluent water were collected every 24 h for in-situ physi­
cochemical measurements. At the end of each EBCT, ~40 mL of water
was collected and stored in a refrigerator (4 ◦ C) to measure the con­
centrations of micropollutants and transformation products, and ~10 g
of sand was collected from the surface (~1 cm) of each filter and stored
at − 80 ◦ C for DNA extraction. The entire column experiment was per­
formed at room temperature (22 ± 3 ◦ C) and was covered with
aluminum foil to avoid photodegradation of trace contaminants.
To verify the microbial role in the removal of micropollutants, the
filter material, i.e., quartz and manganese sand, was treated with sodium
azide and used to fill the remaining six parallel columns (three for quartz
sand and three for manganese sand) as control groups. The control
groups were run with the same feedwater but only under EBCTs of 4 and
0.5 h. Micropollutants biodegradation in the sand filters was roughly
estimated as: micropollutants removal in non-treated groups (attributed
to filter material adsorption and microbial metabolism)
− micropollutants removal in control groups (mainly attributed to filter
material adsorption).
2.3. In-situ physicochemical measurements of water samples
Temperature, conductivity (COND), oxidation-reduction potential
(ORP), total dissolved solids (TDS), and pH were measured in the
influent and effluent water samples using a multi-parameter water
quality detector (Myron Co., USA). Dissolved oxygen (DO) and turbidity
(NTU) in water samples were measured using a dissolved oxygen probe
detector (Hach Co., USA) and turbidimeter (WGZ-2000, Shanghai
Shengguang, China), respectively.
2.4. Analysis of micropollutants in water samples
Micropollutants analysis was performed by liquid chromatography-
mass spectrometry. For low concentration detection, online solid-
phase extraction (SPE) was applied, with structure and settings similar
to Huntscha et al. (2012) using an Atlantis T3 column (3.0 (I.D.)
× 150 mm, 3 µm particle size; Waters, BadenDättwil, Switzerland). The
online SPE apparatus was connected to a high-performance liquid
chromatograph (Ultimate 3000 HPLC, Thermo Fisher Scientific, Wal­
tham, USA). A binary gradient system was applied, consisting of mobile
phase A, 0.1% formic acid in nanopore water, and mobile phase B,
J. Zhou et al.
Fig. 1. Experimental design of sand filter columns. (a) Schematic of sand filter columns; (b) experimental procedure. Control: sterile group, i.e., quartz and man­
ganese sand were sterilized using sodium azide.
acetonitrile (HPLC analysis grade), as separate analytes in a Hypersil
GOLD column (2.1 mm × 100 mm, particle size 1.8 µm, Thermo Sci­
entific, Bellefonte, USA). The solvent gradient was 5% B to 95% B in
7 min, then maintained at 95% B to 15 min. The HPLC was connected to
an electrospray ionization probe (ESI) of a TSQ Altis Quantum Ultra
triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA,
USA) operated under unit resolution in the selected reaction monitoring
(SRM) mode. Details on the mass spectrometry precursor and secondary
ions are listed in Supporting Material Table S1. Nitrogen was used as the
sheath gas (60 arbitrary units) and auxiliary gas (15 arbitrary units) and
argon was used as the collision gas (1.5 m Torr). Analyses were per­
formed at a spray voltage of + 3 800 V (positive mode) and capillary
temperature of 350 ◦ C.
For quantification, the internal standard method was used. Isotope-
labeled internal standards were available for all analytes. The concen­
tration of each product ion in a sample was calculated by comparing the
peak area ratio of the analyte and its assigned internal standard to the
corresponding ratio in the calibration standard curve. Calibration curves
were obtained by weighted (1/x) linear least square regression of seven
standards spiked in nanopure water between 1 and 12 µg/L. Within each
point of the calibration curves, the relative standard deviation (RSD) of
the internal standard was controlled under the limitation of 15%.
Quality control was checked within each experiment using batch sam­
ples picked randomly by spike recovery experiments (spiking at 5 μg/L),
which showed a maximum RSD range within 15%. The limit of quan­
titation (LOQ) of micropollutants was 0.2 μg/L and LOQ of their trans­
form products was 0.1 μg/L.
2.5. Active biomass and metabolic characteristics of filter material
samples
Adenosine triphosphate (ATP) bioluminescence was adopted to es­
timate the living microbial biomass attached to the sand filter material.
ATP was measured using a BacTiter-Glo Microbial Cell Viability Assay
Kit (Promega Corporation, Switzerland) and a GloMax 20/20 Lumin­
ometer (Turner BioSystems, Sunnyvale, CA, USA). The specific experi­
mental steps followed previous study (Hammes et al., 2010).
Biolog™ EcoPlates (Biolog Inc., Hayward, CA, USA) were used to
evaluate the physiological metabolic characteristics of the microbial
community in the sand filters. By measuring and analyzing optical
density (OD) in the wells, the metabolic pattern of 31 carbon substrates
(Table S4) by microorganisms in the sand filters was determined (Liao
et al., 2018). The color absorbance and turbidity values of each culture
well were measured using a SPARK 10-M multifunctional microplate
reader (Tecan Group Ltd., Männedorf, Switzerland) at 590 nm and
750 nm, respectively. The metabolic rate was calculated based on
average well color development (Liao et al., 2018). Details are described
in Text S1.
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Journal of Hazardous Materials 423 (2022) 127167
2.6. DNA extraction, metagenomic sequencing, and taxonomic
identification
DNA was extracted from the sand using a DNeasy PowerMax Soil Kit
(Qiagen, Germany) following the manufacturer’s instructions. Accord­
ing to micropollutants determination, we selected 16 DNA samples for
metagenomic sequencing, including two parallel samples of the original
manganese and quartz sand obtained from the treatment plants, and two
parallel samples of manganese and quartz sand at EBCTs of 4, 2, and
0.5 h, respectively. Metagenomic sequencing was performed using an
Illumina Hiseq X-Ten system at the Beijing Genomics Institute, China
(Hu et al., 2020). All clean reads were deposited in the NCBI Sequence
Read Archive database under accession number PRJNA517235.
Metagenomic 16S rRNA gene fragments were extracted from the
clean reads and taxonomically classified using GraftM v0.13.1 (Boyd
et al., 2018). The GraftM package was chosen based on Glasl et al.
(2020), with using Silva (release NR99 132) (Quast et al., 2012) as a
reference database after clustering sequences at 97% identity with
cd-hit-est v4.8.1(Fu et al., 2012).
2.7. Construction of reference databases and identification of
micropollutants biotransformation genes in sand filters
Based on the published literatures and enviPath (Wickert et al.,
2016), we collected information on the biotransformation pathways of
the eight micropollutants, as shown in Table S2. We downloaded the
gene sequences involved in caffeine and atrazine biotransformation
from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and con­
structed biotransformation gene databases. We also downloaded the
amino acid sequences of biphenyl dioxygenase (involved in carbamaz­
epine biotransformation), atenolol amidohydrolase (atenolol), and
monooxygenase (naproxen, sulfamethoxazole, and sulfonamide) from
Reviewed Swiss-Prot (https://www.uniprot.org/uniprot/?query=rev
iewed:yes) and constructed their biotransformation enzyme databases.
No reports were found regarding the biotransformation genes/enzymes
of trimethoprim. All customized reference databases are available at htt
ps://github.com/byhfly/open-source.git.
Reads of each sample were aligned to the above customized data­
bases and Greengenes database (16 S rRNA of gg_135.fasta) (DeSantis
et al., 2006) to quantify the biotransformation genes in each sample
using the ublast command in USEARCH (v10.0.240) (Edgar, 2010) with
the parameters (-e value 1e-10 -accel 0.8). For each reference sequence
in each database, the lengths of all aligned reads in each sample were
added and then divided by the length of the reference sequence to obtain
the average copy number of the reference sequence, which was then
normalized based on the 16 S rRNA gene copy number acquired by the
same method.
J. Zhou et al.
2.8. Genome-centric analysis to identify bacterial strains carrying
micropollutants biotransformation genes
The filtered clean reads for each metagenome (4.96 ± 0.09 Gbp)
were assembled into contigs using MEGAHIT (v1.1.3) (Li et al., 2015).
The contigs recovered were clustered into metagenome-assembled ge­
nomes (MAGs) using MetaBAT (Kang et al., 2015), CONCOCT (Alneberg
et al., 2014), and MaxBin (Wu et al., 2014) respectively. The MAGs
generated from single-sample assembly and mixed assembly of the
manganese and quartz sand filters using the above software were
together refined using the bin_refinement module in the MetaWRAP
pipeline (Uritskiy et al., 2018) to retain the best representative bins
(completeness > 50%; contaminant < 10%); dRep (Olm et al., 2017)
was then used to de-replicate (cutoff for filtering: minimum genome
completeness 50%; maximum genome contamination 10%) MAGs from
all samples by identifying groups of highly similar MAGs and choosing
the best representative for each MAG set, finally generating a nonre­
dundant MAG set. To determine the percentage of the total community
captured by the MAGs, the “pipe” and “appraise” functions of SingleM
v0.13.2 were used to search for single-copy marker genes in both the
MAGs and clean reads to assess the fraction of marker genes recovered in
the MAGs (https://github.com/wwood/singlem). Taxonomic classifi­
cation of all bins was performed with a standardized bacterial taxonomy
based on genome phylogeny using the genome taxonomy database
(gtdbtk.r86_v3_data.tar.gz) (Chaumeil et al., 2019). The coverage of
each non-redundant MAG in each sample was calculated using a
mapping-based method (BBMap (v38.43), under slow mode/high
sensitivity) and customized script, as described previously (Hu et al.,
2020). Details are described in Supporting Material Table S3.
2.9. Analysis of transformation products proposed from metagenomes
Approximately 1.0 mL filtered water samples obtained from the ef­
fluents of column experiment were used for the determination of
transformation products, using the HPLC-MS/MS as described above.
External standard method was adopted for each transformation product
(parameters are shown in Tables S5 and S6). The standards of naproxen
and carbamazepine transformation products were not purchased. Stan­
dard recovery experiments showed a maximum RSD range within 20%.
As some transformation products of atrazine proposed by meta­
genomes were not detected in the column experiment, we further con­
ducted the cultivation experiment. A series of 150-mL Erlenmeyer flasks
were used and each flask contained 50 mL sterile feeding water used for
the column experiment and 5.0 g quartz- or manganese sand. Flasks
with autoclaved sand (120 ◦ C for 20 min) were used as negative con­
trols. All flasks were sealed with sealfilm and shaken at 30 ◦ C and
170 rpm. Suspensions (~1 mL) were sampled at 2 h, 4 h, 8 h, 18 h, 24 h,
and 48 h, and then were filtered through a 0.45-μm membrane for the
determination of transformation products.
2.10. Statistical analysis
A paired student’s t-test (α = 0.05) was applied to identify differ­
ences in biomass ATP, metabolic rate, and removal of micropollutants
between the quartz and manganese sand filters at each EBCT. The Wil­
coxon paired signed-rank test was used to analyze differences in the
abundance of bacterial genus-encoding micropollutants biotransforma­
tion genes between the manganese and quartz sand filters, using the R
command “wilcox.test”.
3. Results
3.1. Micropollutants removal in sand filters
Manganese and quartz sand filters were operated under seven EBCT
(from 0.5 to 4 h) to examine the removal efficiency of eight
4
Journal of Hazardous Materials 423 (2022) 127167
representative micropollutants for 64 days, during which pH, DO, and
TDS showed no statistically different (p > 0.05) between influent and
effluent (Fig. S1). According to micropollutants determination (Fig. 2),
the removal efficiencies of atenolol and caffeine were comparatively
higher than those of the other six micropollutants in the sand filters.
Atenolol removal in the manganese sand filters gradually increased from
48.0% ± 13.6–83.6% ± 1.1% with increased EBCT. However, the
removal efficiencies of atenolol in the quartz sand filters (93.1% ±
2.1–97.6% ± 0.1%) under all seven EBCTs were similar and were all
significantly higher than that in the manganese sand filters (Student’s t-
test, p < 0.05). The other six micropollutants (atrazine, naproxen, car­
bamazepine, trimethoprim, sulfamethoxazole, and sulfadiazine)
demonstrated the same removal trend, i.e., a sharp increase in removal
at EBCT = 4 h.
We further used sodium azide-treated sand filters as controls to
roughly evaluate the role of microbes in micropollutants removal.
Comparing treated and non-treated sand filters, we calculated the mi­
crobial removal of micropollutants (Table 1). Results demonstrated
significant microbial removal of each micropollutants in the sand filters
at EBCT = 4 h. Compared with the quartz sand filters, the manganese
sand filters showed lower microbial removal capacity for micro­
pollutants at EBCT = 4 h (except for atrazine). At EBCT = 0.5 h, no or
only limited microbial removal was observed in the sand filters (except
for atenolol in the manganese sand filters). Collectively, these results
revealed that (i) micropollutant-degrading microbes already exist in the
sand filters but show weak removal capacity for micropollutants in RSFs
under less contact time, and (ii) prolonging EBCT may improve micro­
bial removal of micropollutants to a certain extent.
3.2. Microbial biomass and carbon metabolism in sand filters
The ATP bioluminescence assay was used to assess the concentration
of active microbial biomass attached to the sand filters. Results revealed
that with the extension of EBCT, active microbial biomass did not in­
crease in the sand filters. However, as shown in Fig. 3a, the microbial
biomass in the quartz sands collected from a surface water treatment
plant (inoculated to quartz sand filters) was more than 12 times than
those in the manganese sands collected from a groundwater treatment
plant (inoculated to manganese sand filters). This result is consistent
with the microbial removal of micropollutants at EBCT = 4 h (Table 1),
with higher removal efficiency exhibited in the quartz sand filters.
We used Biolog™ EcoPlates to determine the utilization of 31 carbon
sources (Table S4) by the sand filter material. Data showed that the
metabolic rate of the microbial community in the quartz sand filters was
slightly higher or not significantly different from that in the manganese
sand filters at different EBCTs (Fig. 3b). Principal coordinate analysis
based on the categories of carbon sources (carbohydrates, amino acids,
carboxylic acids, polymers, miscellaneous, and amines) showed that the
two types of filters had no obvious preference for the utilization of
different carbon substances (Fig. S2).
3.3. Microbial community composition
We sequenced whole-community DNA from 16 biofilm samples ob­
tained from the filters (details in Table S3), with two parallel samples for
each condition. A total of 2 341 operational taxonomic units (OTUs)
were obtained from the metagenomic data. Alpha and beta diversity
analyses showed that the differences in microbial diversity (Figs. S3 and
S4) between the manganese and quartz sand filters decreased markedly
after feeding the micropollutant-containing water, suggesting inputs of
micropollutants led to a more homogenous microbial community in the
two types of sand filter. At the phylum level (Fig. S5), Proteobacteria
(48.7% ± 10.6%) and Acidobacteria (20.6% ± 10.1%) were dominant
in the quartz sand filters, while Proteobacteria (65.0% ± 6.0%) and
Nitrospirae (5.7% ± 3.9%) were dominant in the manganese sand fil­
ters. Genus analysis (Fig. S6) revealed that Nitrospira and
J. Zhou et al. Journal of Hazardous Materials 423 (2022) 127167

Fig. 2. Micropollutant removal by manganese and quartz sand filters at different empty bed contact time. Data are means ± standard deviation (n = 3). Paired
student’s t-test was used to compare differences in micropollutant removal between manganese and quartz sand filters. Asterisks indicate significance level of
comparisons: * , p < 0.05; * *, p < 0.01; * ** , p < 0.001.

5
J. Zhou et al. Journal of Hazardous Materials 423 (2022) 127167

Table 1 completeness, and contamination rates are provided in the Supporting

Microbial removal of eight micropollutants at EBCT = 0.5 and 4 h. Note: Mi­ Information Excel).
crobial removal was estimated as: micropollutant removal in non-treated groups
− micropollutant removal in sodium azide-treated groups. MS: manganese sand (i) Caffeine and atrazine
filter; QS: quartz sand filter. Read-based metagenomic analysis revealed that the cdhA genes
Micropollutant Removal at EBCT = 0.5 h (%) Removal at EBCT = 4 h (%) involved in caffeine oxidation to 1,3,7-trimethyluric acid were
MS QS MS QS present in all sand samples. The ndmB gene involved in caffeine
demethylation to paraxanthine mainly appeared in the filter
Atenolol 21.5 ± 14.7 3.5 ± 1.6 16.7 ± 6.3 49.4 ± 12.7
Caffeine 3.8 ± 2.6 10.1 ± 4.3 -4.4 ± 3.9 51.5 ± 1.1 samples at EBCT = 0.5 and 2 h (Fig. 4a, left and middle sub­
Atrazine 9.6 ± 3.5 -2.8 ± 1.9 16.6 ± 5.2 19.8 ± 3.8 figures). These results suggest that the dominant transformation
Naproxen 4.1 ± 1.3 0.7 ± 9.4 11.1 ± 7.9 27.7 ± 2.6 pathways of caffeine may change (from caffeine oxidation to
Trimethoprim 4.9 ± 5.2 3.2 ± 7.2 15.3 ± 2.2 45.6 ± 0.4 caffeine oxidation + demethylation) with the reduction of EBCT.
Sulfadiazine -0.9 ± 1.3 4.0 ± 4.1 30.6 ± 8.6 48.0 ± 2.2
Carbamazepine 3.2 ± 0.9 0.7 ± 2.0 10.7 ± 1.6 16.1 ± 3.0
Compared with the manganese sand filters, the quartz sand filters
Sulfamethoxazole 5.4 ± 2.2 5.6 ± 4.3 41.5 ± 10.2 52.3 ± 0.4 contained more abundant cdhA genes (Fig. 4a, left subfigure).
From further analysis of MAGs carrying cdhA and ndmB genes, we
recovered 32 potential caffeine degraders (17 carrying cdhA and
15 carrying ndmB). These MAGs were identified as 14 bacterial
genera or families affiliated with Burkholderiales (58.2%) and
Rhizobiales (24.7%). The most abundant degraders potentially
involved in caffeine oxidase and demethylation were Hyphomi­
crobiaceae and Rubrivivax, respectively. Furthermore, we
observed that most MAGs carrying the cdhA and ndmB genes were
significantly more abundant in the manganese sand filters than in
the quartz sand filters (non-parametric Wilcox signed-rank test,
p < 0.05) (Fig. 4a, right subfigure), except for MAGs affiliated
with Gemmata and Anderseniellaceae.
We also mapped the metagenomic reads against the reference
databases of atrazine biotransformation genes (including
atzABCDEF, trzN, and thcb). We found that the atzA genes
involved in atrazine hydrolytic dechlorination to hydroxya­
trazine and thcb genes involved in atrazine oxidation to deiso­
propylatrazine and deethylatrazine were present in all sand
samples (Fig. 4b, left and middle subfigures). From MAG analysis,
we only identified MAGs carrying atzA genes, with no MAGs
carrying thcb genes found in the filters. The dominant atrazine
degraders were Hyphomicrobiaceae in the manganese sand filters
and Pseudomonas in the quartz sand filters (Fig. 4b, right
subfigure).
(ii) Atenolol, carbamazepine, sulfamethoxazole, sulfadiazine, and
naproxen biotransformation
Fig. 3. Data are means ± standard deviation (n = 3). Paired student’s t-test was
used to compare differences in ATP concentration and metabolic rate between
Analysis of metagenomic reads against the reference databases of
manganese and quartz sand filters. Asterisks indicate significance level of
comparisons: * , p < 0.05; * *, p < 0.01. Microbial biomass ATP determination atenolol amidohydrolase revealed that abundant gene sequences
(a) and metabolic rate of 31 carbon sources (b) in manganese and quartz sand (12.4–27.2 gene copies per 16 S RNA gene) encoding atenolol amido­
filters at different empty bed contact time. hydrolase were present in all metagenomic samples (Fig. 5a, left sub­
figure). Thus, it was suggested that atenolol was potentially hydrolyzed
Hyphomicrobium were dominant in all filter columns, and six genera, to atenolol acid with the contribution of atenolol amidohydrolase
including Nitrosomonas, showed significant differences (Welch’s t-test, (Fig. 5a, middle subfigure) in the sand filters. From MAG analysis, 22
p < 0.05) between the manganese and quartz sand filters (Fig. S7). MAGs were identified to 12 genera or families, and the most abundant
degrader was Methylophilus in both the manganese and quartz sand fil­
ters. (Fig. 5a, right subfigure).
3.4. Potential genes, pathways, and microbes for biotransformation Similarly, we identified the biphenyl dioxygenase gene sequences
(1.8–5.8 gene copies per 16 S RNA gene) that metabolized carbamaze­
To identify micropollutants transformation genes, we first con­ pine to cis-10,11-dihydroxy-10,11-dihydrocarbamazepine and cis-2,3-
structed the reference sequence database of biotransformation genes/ dihydroxy-2,3-dihydrocarbamazepine (Fig. 5b, left and middle sub­
enzymes for selected micropollutants according to the NCBI GenBank figures), as well as nine MAGs carrying biphenyl dioxygenase genes in
database, enviPath, and previous literatures (Table S2). These functional the filters (Fig. 5b, left subfigure). Sphingomonadaceae was the domi­
genes and enzymes included first-step biotransformation genes involved nant carbamazepine degrader in the two types of sand filter (Fig. 5b,
in caffeine and atrazine and first-step biotransformation enzymes right subfigure).
involved in atenolol, carbamazepine, sulfamethoxazole, sulfadiazine, Flavin-dependent monooxygenase are reported to be effective at
and naproxen. No genes or enzymes were found for trimethoprim biodegrading sulfonamide drugs, such as sulfadiazine and sulfameth­
transformation. Then, metagenomic reads were first mapped against the oxazole (Kim et al., 2019; Ricken et al., 2017). Monooxygenase can also
reference databases to obtain the abundances of biotransformation degrade naproxen (Wojcieszynska et al., 2016). Analysis of meta­
genes/enzymes. To determine the occurrence and abundance of specific genomic reads revealed that genes encoding monooxygenase were
MAGs carrying the biotransformation genes/enzymes, we recovered abundant (27.1–51.7 gene copies per 16 S RNA gene) in the filter mi­
draft genomes from the sand filter metagenomes (coverage, croorganisms (Fig. 5c, left subfigure), which may be involved in the

6
J. Zhou et al. Journal of Hazardous Materials 423 (2022) 127167

Fig. 4. Potential transformation pathways of caffeine (a) and atrazine (b) and associated bacteria in sand filters. In (a) and (b), left subfigures show abundances of
identified gene(s) involved in first step of caffeine/atrazine transformation in manganese (MS) and quartz sand filters (QS); middle subfigures show first step of
caffeine/atrazine biotransformation pathways according to identified biotransformation gene(s) (see Table S1); right subfigures show abundance of associated
bacteria carrying specific biotransformation gene(s) based on genome-centric analysis. Bacteria carrying cdhA (upper blue) or ndmB (lower red) are present in right
subfigure of (a) and bacteria carrying atzA (blue) are present in right subfigure of (b) as we did not search the thcb gene. Note: In left subfigures of (a) and (b), data are
means ± average deviation (n = 2). In right subfigures of (a) and (b), abundance was calculated by the mean of associated bacterial abundances from original, EBCT
= 0.5, EBCT = 2, and EBCT = 4 in MS or QS. Asterisks indicate differences in abundance of each strain between MS and QS (Wilcoxon’s signed-rank test): * ,
p < 0.05; * *, p < 0.01.

oxidation of sulfadiazine, sulfamethoxazole and naproxen.
3.5. Transformation products analysis
According to the micropollutants pathways proposed from identified
catabolic genes, we further measured the potential biotransformation
products of caffeine, atrazine, atenolol, sulfadiazine, and sulfamethox­
azole in the water samples obtained from the column experiment. As
shown in Table 2, all the transformation products proposed from met­
agenomes were detected in column samples except two transformation
products of atrazine (i.e., hydroxyatrazine and deisopropylatrazine). We
further confirmed the occurrence of these two intermediates in the
cultivation experiment (Fig. S9). In addition, we also observed the in­
crease of 1,3,7-teimethyluric acid along with the decrease of para­
xanthine at different EBCTs (0.5–2.5 h), which further verified a shift in
caffeine oxidation and demethylation pathways, as proposed by meta­
genomes analysis. Although caffeine, atrazine, and sulfamethoxazole
were rapidly degraded at EBCT = 4 h, their transformation products
were not detected in our analysis. It was speculated that they probably
were further degraded into the other products, e.g., paraxanthine and
1,3,7-teimethyluric acid were transformed into 7-methyxanthine
(Summers et al., 2015) and 1,3,7-trimethyl-5-hydroxyisourate
(Mohanty et al., 2012), respectively; hydroxyatrazine was transformed
into N-isopropyl amide (Zhang et al., 2019a).
4. Discussion
In this study, we first demonstrated the significant microbial role in
the selected micropollutants removal, and then further explored the
microbial transformation pathways and micropollutants and associated
microbes. Our study proved metagenomic is a power tool to understand
the mechanism of micropollutants biodegradation in sand filters.
4.1. Microbial removal of micropollutants in sand filters
A previous study on seven micropollutants (diclofenac, propranolol,
iopromide, iohexol, iomeprol, tebuconazole, and propiconazole)
demonstrated single first-order removal in slow sand biofilter reactors in
the EBCT range of 0–60 h (Casas and Bester, 2015). In the present study,
no clear linear relationship between EBCT and micropollutants removal
was observed, which may be due to our comparatively lower EBCT range
(0.5–4 h) (Fig. 2). In this range, microbial removal of micropollutants
was weak (except for atenolol) under short EBCT (0.5 h) and adsorption
dominated the removal process; whereas under a longer EBCT (i.e., 4 h),
both adsorption and microbial biodegradation contributed to micro­
pollutants removal (Table 1).
Atenolol and caffeine were the two most easily removed micro­
pollutants in our study, even at the short EBCT of 0.5 h (Fig. 2).
Compared with previous reports on biofiltration, the removal effi­
ciencies of these two micropollutants show high variability, which is
likely due to the filter materials and water sources. For example, Rattier
et al. (2014) found no significant removal of atenolol but up to 80%
removal of caffeine using an anthracite filter (EBCT = 8 min); Reungoat
et al. (2011) reported weak removal of atenolol and moderate removal
of caffeine under sand biofiltration of wastewater treatment plant
effluent (EBCT = 2 h); and Teerlink et al. (2012) described a >68%
removal of atenolol and caffeine in onsite wastewater silica sand col­
umns (EBCT = 1–30 days).
Previous studies on biofiltration revealed that the removal of atra­
zine, carbamazepine, sulfamethoxazole, naproxen, and trimethoprim is

7
J. Zhou et al. Journal of Hazardous Materials 423 (2022) 127167

Fig. 5. Potential transformation pathways of atenolol (a), carbamazepine (b), sulfamethoxazole, sulfadiazine, and naproxen (c) and associated bacteria in sand
filters. In (a), (b) and (c), left subfigures show abundances of identified genes (ublast against customized enzyme database) involved in first step of micropollutant
transformation in manganese (MS) and quartz sand filters (QS); middle subfigures show first step of micropollutant biotransformation pathways according to
identified biotransformation enzymes (see Table S1); and right subfigures show average abundance of associated bacterial genera carrying specific biotransformation
gene(s) based on genome-centric analysis. Note: In left subfigures of (a), (b), and (c), data are means ± average deviation (n = 2). In right subfigures of (a), (b), and
(c), abundance was calculated by the mean of associated bacterial abundances from original, EBCT = 0.5, EBCT = 2, and EBCT = 4 in MS or QS. Asterisks indicate
differences in abundance of each strain between MS and QS (Wilcoxon’s signed-rank test): * , p < 0.05; * *, p < 0.01.

Table 2
Occurrence of transformation products in the column experiment and cultivation experiment. Measurement at each EBCT was shown in Fig. S8. MS: manganese sand
filter; QS: quartz sand filter; ⊗: detected; ⃝ : undetected; /: not performed.
Micropollutants Genes/enzymes Transformation Products Columns Cultivation

MS QS MS QS

Caffeine cdhA 1,3,7-teimethyluric acid ⊗ ⊗ / /

ndmB Paraxanthine ⊗ ⊗ / /
Atrazine atzA Hydroxyatrazine ⃝ ⃝ ⊗ ⊗
thcb Deethylatrazine ⊗ ⊗ ⊗ ⊗
Deisopropylatrazine ⃝ ⃝ ⊗ ⊗
Atenolol Atenolol amidohydrolase Atenolol acid ⊗ ⊗ / /
Sulfadiazine Monooxygenases 2-aminopyrimidine ⊗ ⊗ / /
Sulfamethoxazole Monooxygenases 3-amino-5-methylisoxazole ⊗ ⊗ / /
4-aminophenol ⊗ ⊗ / /

8
J. Zhou et al.
comparatively lower than that of atenolol and caffeine (Carpenter and
Helbling, 2017; Rattier et al., 2014; Teerlink et al., 2012; Zearley and
Summers, 2012), in agreement with our column experiment (Fig. 2). In
addition to the low adsorption of these micropollutants in the sand fil­
ters, the low removal efficiencies may also be related to the biode­
gradability of micropollutants. Remarkably, in our study, a sharp
increase in the removal of these micropollutants was observed at EBCT
= 4 h. This was mainly attributed to the increase of contact time be­
tween the micropollutants and biofilm on the filter material.
4.2. Biotransformation pathways of micropollutants and associated
microbes
Metagenomic reads analysis can help predict comprehensive meta­
bolic pathways and enzymes to explain the observed micropollutants
biotransformation (Fang et al., 2014; Weigold et al., 2016). Further­
more, genome-centric analysis can accurately identify the biotransfor­
mation gene carriers and thus obtain potential micropollutants
biodegraders, as demonstrated in recent studies (Gaytan et al., 2020;
Hidalgo et al., 2020; Zhang et al., 2019b). Here, we analyzed the
micropollutants biotransformation genes by metagenomic reads and
associated bacteria by genome-centric analysis in the sand filters. Using
transformation products analysis, we further verified the pathways
proposed by metagenomes.
A variety of microorganisms can biodegrade caffeine, including
Pseudomonas (Summers et al., 2012; Woolfolk, 1975; Yu et al., 2008),
Alcaligenes (Mohapatra et al., 2006), Serratia marcescens (Mazzafera
et al., 1996), Klebsiella, and Rhodococcus (Madyastha and Sridhar, 1998).
We recovered 32 potential caffeine degraders (Fig. 4a, right subfigure),
some of which have not been isolated or identified as caffeine degraders
previously (e.g., Piscinibacter, Hydrogenophaga, and Rubrivivax), which
may be due to frequent horizontal gene transfer in the biofilm, leading to
many microorganisms carrying degradation genes and becoming
potentially degrading bacteria (Bhandari and Karn, 2019). For atrazine,
various biodegraders have been isolated previously, including strains of
Pseudomonas (Martinez et al., 2001), Pseudaminobacter (Topp et al.,
2000), Citricoccus (Yang et al., 2018), Ensifer (Ma et al., 2017), Rhodo­
coccus (Shao and Behki, 1996), Klebsiella variicola (Zhang et al., 2019a),
and Rhizobium (Chen et al., 2019). We observed abundant Pseudomonas
and Pseudaminobacter in the sand filters, which could play a primary role
in the process of atrazine hydrolysis and dechlorination.
To date, genetic and enzymatic studies on the degradation of aten­
olol by microorganisms are still under exploration. Previous research
has suggested that the transformation of atenolol mainly occurs via
amide hydrolysis (Helbling et al., 2010); however, but the strains
involved in this process are yet to be confirmed. Our metagenomic
analysis indicated that diverse bacterial populations have the potential
to participate in the hydrolysis of atenolol amide in the sand filters,
including Nitrosomonas, Methylophilus, Methyloversatilis, and Rhizobia­
ceae (Fig. 5a, right subfigure). Notably, these bacteria have not been
isolated as atenolol degraders previously. Although researchers have
identified carbamazepine degraders, such as Acinetobacter (Cui et al.,
2008), Pseudomonas (Li et al., 2013), Streptomyces (Popa et al., 2014),
Starkeya and Rhizobium (Bessa et al., 2017), few studies have explored
the enzymes and genes involved in carbamazepine biodegradation. To
the best of our knowledge, only one study has proposed biphenyl
dioxygenase as a candidate enzyme for carbamazepine metabolism
(Aukema et al., 2017). Based on this enzyme, we identified six potential
carbamazepine biodegraders, with one belonging to Sphingomonada­
ceae, which is reportedly linked to carbamazepine degradation (The­
lusmond et al., 2016).
Various sulfonamide degraders have been isolated in previous
studies, including Arthrobacter (Deng et al., 2016), Pimelobacter (Deng
et al., 2018), Achromobacter denitrificans and Leucobacter (Reis et al.,
2018), and Microbacterium (Ricken et al., 2017). Similarly, Steno­
trophomonas (Wojcieszynska et al., 2014), Planococcus (Wojcieszynska
9
Journal of Hazardous Materials 423 (2022) 127167
et al., 2016), Bacillus thuringiensis (Marchlewicz et al., 2016), and
Amycolatopsis (Alanis-Sanchez et al., 2019) have been isolated as nap­
roxen degraders. Some of these bacteria carry monooxygenase, which
may be involved in sulfonamide biodegradation, for example Achro­
mobacter denitrificans and Leucobacter (Reis et al., 2018), and Micro­
bacterium (Ricken et al., 2017). Stenotrophomonas(Wojcieszynska et al.,
2014) and Planococcus (Wojcieszynska et al., 2016) were involved in
naproxen biodegradation by carry monooxygenase. Here, we identified
34 MAGs affiliated with 16 genera/families that have the potential to
secrete monooxygenase. These microbes may be involved in the
biotransformation of sulfonamides and naproxen as well as other com­
pounds in the sand filters.
5. Conclusions
● Atenolol (97.6% removal) and caffeine (92.1% removal) were easily
removed micropollutants in both the quartz and manganese sand
filters. The removal efficiencies of atrazine, carbamazepine, sulfa­
methoxazole, sulfadiazine, naproxen, and trimethoprim were low at
short EBCTs (0.5, 1, 1.5, 2, 2.5, and 3 h) but increased markedly
when the EBCT was prolonged to 4 h, which could be attributed to
the microbial degradation of these six micropollutants.
● Active microbial biomass did not increase significantly with the
extension of EBCT in the filters, but biomass in the quartz sand filters
was approximately twice that in the manganese sand filters. This was
consistent with the higher microbial removal in the quartz sand fil­
ters than in the manganese sand filters at EBCT 4 h.
● Integrated analysis of metagenomic reads and transformation prod­
ucts revealed that the pathways of caffeine oxidation and demethy­
lation might shift at different EBCTs. Analysis of metagenomic-
assembled genomes revealed 32 bacterial MAGs involved in the
first step of these two pathways and unclassified Hyphomicrobiaceae
and Rubrivivax were the most abundant for caffeine oxidation and
demethylation, respectively. With the same approach, we identified
two biotransformation pathways for atrazine, i.e., hydrolysis and
oxidation. However, we only identified 10 bacterial MAGs carrying
atzA genes, with Pseudomonas and unclassified Hyphomicrobiaceae
dominant in the quartz and manganese sand filters, respectively.
● Atenolol was hydrolyzed by amidohydrolase, and 22 bacterial MAGs
affiliated with 12 genera/families had the potential to participate in
this process; carbamazepine was potentially oxidized by biphenyl
dioxygenase with five previously unidentified genera potential
involved. We identified 34 MAGs affiliated with 16 genera/families
with the potential to secrete monooxygenase that might oxidize
micropollutants in the sand filters.
CRediT authorship contribution statement
Jie Zhou: Investigation, Methodology, Experiment, Formal analysis,
Writing – original draft. Donglin Wang: Methodology, Data pre­
processing, Writing – original draft. Feng Ju: Validation. Wanchao Hu:
Investigation, Methodology, Experiment. Jinsong Liang: Software.
Yaohui Bai: Investigation, Writing – review & editing, Supervision.
Huijuan Liu: Resources, Funding acquisition. Jiuhui Qu:
Conceptualization.
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 work was supported by the National Natural Science Foundation
of China (Funding No. 51778603 and 31700106) and Chinese Academy
J. Zhou et al.
of Sciences (QYZDY-SSW-DQC004). The authors thank the Beijing Ge­
nomics Institute Central China for providing high-throughput
sequencing services.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jhazmat.2021.127167.
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