Chemosphere 253 (2020) 126721

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Effects of lead (Pb) in stormwater runoff on the microbial

characteristics and organics removal in bioretention systems
Cheng Liu a, b, *, Jie Lu b, Jiaqi Liu b, Tariq Mehmood b, Wei Chen a, b
a
Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing, 210098, China
b
College of Environment, Hohai University, Nanjing, 210098, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

The Pb accumulation reduced the

microbial biomass.

The Pb accumulation produced
small-quantity Pb-tolerance
microorganisms.

The COD removal in the soil and filler
layer was promoted and inhibited by
Pb respectively.

The Pb affected the ability of micro-
organisms to bind to organisms by
altering their structure.

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

Article history: Bioretention systems have been proved to be a natural approach for effectively reducing stormwater
Received 4 September 2019 runoff pollution loads. However, the effects of heavy metals in stormwater runoff on microbial charac-
Received in revised form teristics and organics removal in bioretention systems are unclear. In this study, two lab scale bio-
9 March 2020
retention columns including the control and lead (Pb) treatment with the soil and filler layer were
Accepted 5 April 2020
established. The changes of organic matter and lead in the effluent water and the soil (or fillers) were
Available online 7 April 2020
monitored during 121 operation days. The soil (or fillers) microbial characteristics were also analyzed.
Handling Editor: Chang-Ping Yu The results showed that most of Pb was intercepted by soil, while a small amount accumulated in fillers
after 121 days. The long-term Pb accumulation in the biorentention system negatively affected the mi-
Keywords: crobial biomass and microbial activity, while positively affected the community diversity. Pb accumu-
Bioretention lation killed some microorganisms, but simultaneously stimulated the growth of some Pb-tolerance
Stormwater runoff microorganisms. The abundance of bacteria with COD degradation function in soil layer decreased, while
Organic matters that in fillers increased, indicating the effect of Pb on the community structure of these two layers was
Heavy metals
different. The COD removal in the soil and filler layer was promoted and inhibited by Pb contamination
Microorganisms
respectively. Moreover, Pb affected the removal of organic matter by chelating organic matters and
changing their composition. The results suggested that the long-term accumulation of heavy metals in
bioretention system would affect microbial degradation function and pollutants removal, causing our
concern for the long-term maintenance of the bioretention system.
© 2020 Published by Elsevier Ltd.

1. Introduction
* Corresponding author. Ministry of Education Key Laboratory of Integrated
Regulation and Resource Development on Shallow Lakes, Hohai University, Xikang With the advancement of urbanization, the patterns of urban
Road, Nanjing, 210098, China.
E-mail address: 107489860@qq.com (C. Liu).
land use have changed, and the area of impervious surfaces has

https://doi.org/10.1016/j.chemosphere.2020.126721
0045-6535/© 2020 Published by Elsevier Ltd.
2 C. Liu et al. / Chemosphere 253 (2020) 126721
increased enormously, leading to the increase of runoff volume and
peak flow rates. Due to the scouring of stormwater runoff, many
pollutants such as heavy metals, suspended solids, nutrients and
some organics become a part of water-bodies without specific
treatment (Li and Davis, 2016; Park et al., 2019; Pumo et al., 2017).
The pollution in stormwater runoff can cause urban waterlogging
and the deterioration of aquatic environment, which has attracted
more and more attention in recent years. Li et al. (2014) reported
that the EMC (Event Mean Concentration) values for TSS, TP and Pb
in road runoff were respectively 364.99e1208.07 mg L1 and
0.23e1.13 mg L1, and 25.89e171.07 mg L1 by monitoring the
runoff quality from different surfaces in Shenyang. Leroy et al.
(2016) collected runoff of a moderately busy road (<2500 veh
d1) in a commercial area, and the two-year studies observed that
COD and BOD mean values were 120 and 8 mg L1, respectively.
Park et al. (2019) pointed out that the TN and TP concentrations in
the stormwater runoff exceeded 4.0 and 0.3 mg L1, respectively.
Hereby, such pollutants like heavy metals and polycyclic aromatic
hydrocarbons (PAHs) in road runoff have received increasing
concern. Heavy metals such as lead, copper, zinc can pose a serious
threat to plants and aquatic animals because of their toxicity and
low biodegradability. Tyre wear and suspended soil dust contribute
to Pb and Zn contamination in the initial rain (Men et al., 2018). The
Pb and Cr concentrations in road runoff were very high, exceeding
the class V surface water standard (100 mg L1), the recommended
guideline for agricultural and general landscape water (CEPA,
2002). With the bioaccumulation of heavy metals in the food
chain, there is a potential health risk to human beings. It is reported
that at present, the anthropometric release of potentially toxic el-
ements has exceeded the heavy metal inputs in the global weath-
ering process (Touceda-Gonza
lez et al., 2017). Therefore, it is
imperative and urgent to reduce the contamination in the storm-
water runoff.
In order to reduce the occurrence of urban flooding disasters
and alleviate the pollution of urban stormwater, some new urban
stormwater management methods, such as USA’s low impact
development (LID), Britain’s sustainable discharge systems (SUDs)
and China’s sponge city (Beza et al., 2019; Li et al., 2019), have been
put forward in recent years. One of the most efficient and common
measures is bioretention. Bioretention can effectively purify water
quality by taking advantage of the interaction in the matrix-
microbes-plant systems, which includes sorption, biodegradation,
plant uptake (Wang et al., 2018). In recent years, there have been
many studies showing the removal efficiency of bioretention and
analyzing the microbial role and some parameters that affect effi-
ciency. Zhang et al. (2019) compared up-flow and mixed-flow
bioretention systems with the conventional bioretention methods
for their pollutant removal. The results reflected that the TN
removal efficiencies of both up-flow and mixed-flow bioretention
improved when compared with the conventional bioretention,
increasing from 17% (conventional) to 41% (up-flow) and 31%
(mixed-flow), while only the mixed-flow bioretention increased
the COD removal efficiencies by 10%. Shrestha et al. (2018)
addressed that the nutrients from excessive compost in soil me-
dia, rainfall intensity and peak inflow rate negatively affected
nutrient removal. Fan et al. (2019) adopted 15N isotope tracer
method to continuously monitor the dynamic changes of nitrogen
in the bioretention systems within 60 h after rainfall, which
established nitrogen balance model and improved the nitrogen
removal efficiency. Wang et al. (2017) constructed 18 stepped bio-
retention systems to treat the urban stormwater runoff in the
mountainous cities. Results showed that the use of peat soil would
result in heavy nitrogen release, and the high removal efficiencies
of TN and NO3eN were achieved without additional carbon, which
is very valuable for the field application of bioretention systems in
the mountainous cities. Zuo et al. (2019) analyzed the diversity of
microorganisms in the used bioretention impacts of microbial
metabolism on nutrient removal and concluded that the
enhancement of dominant bacteria, such as Chloroflexi and Nitro-
spirae, could strengthen nitrogen removal.
Concerning the heavy metals, many researchers mostly have
focused on the accumulative characteristics of heavy metals from
the rainwater in the bioretention systems. Most heavy metals were
intercepted on the surface of the soil, while a few heavy metals
were absorbed by plants (Zhao et al., 2007). Li found that 88%e97%
of Cu, Zn and Cd in stormwater were intercepted by the fillers of the
bioretention filter columns and 0.5%e3.3% were absorbed by plants
(Li et al., 2018). The column sorption experiments by Gülbaz et al.
(2015) showed that the higher sorption capacity of Pb and Cu
was attributed to their higher cation exchange capacity, potential
precipitation and filtration.
However, most of the studies have neglected the impact of
heavy metals accumulation on the bioretention system. With the
passage of time, an excessive amount of heavy metals usually
accumulate in bioretention system. The heavy metals can cause
toxicity to microbes, affect the biodegradation of organic matter
and finally make bioretention system inefficient. Therefore, it is
necessary to study the effects of heavy metals on microbial char-
acteristics and organics removal in bioretention systems.
In this study, two bioretention columns including the control
and the Pb treatment were established to compare the removal of
organics and investigate the effects of Pb accumulation on the
microbial communities and their biological activities. This study
aimed to observe the variations of microbial characteristics and
organic matters under the Pb influence with the increasing of the
operation day. The specific objectives were (1) to quantify the
accumulation of Pb in the soils and the fillers respectively of the Pb
treatment column; (2) to know the potential toxic risks of Pb
accumulation from the bioavailability of Pb fractions in the bio-
retention soil; (3) to explore the effects of Pb on the microorgan-
isms; (4) to compare the organics removal by the Pb treatment
column with that by the control column; (5) to analyze the influ-
ence mechanisms of the long-term Pb treatment on the organics
removal.
2. Materials and methods
2.1. Materials
The soil was obtained from the upper 0e20 cm layer of a garden
in Hohai University, Nanjing, China. Before use, the soil was air-
dried, crushed and sieved through a 3 mm mesh to remove
stones or roots. Zeolite and ceramsite were used as the filler layer
media of the bioretention columns because of their low-price, easy
availability and strong sorption capacity. The particle size of the
zeolite and the ceramsite was 2e4 mm. The particle sizes of quartz
sand and gravel used as drainage filters were respectively 2e4 mm
and 4e8 mm. All the above bioretention media were received from
Wuxi city in Jiangsu province, China.
All other chemicals employed in this research were at least of
analytical grade and purchased from Sinopharm Chemical Reagent
Co. (Shanghai, China).
2.2. Device setup
Two plexiglass bioretention columns were designed and
established at an outdoor field in the campus of Hohai University,
Nanjing, China. The columns were rectangular with size (15 cm
 C. Liu et al. / Chemosphere 253 (2020) 126721
long by 15 cm wide by 50 cm height). Considering the convenience
of the operation, the total height of the bioretention columns were
50 cm referring to Ding et al. (2019). One port equipped with a valve
was installed at the bottom to allow for water outflow and water
samples collection. Another port was placed at the top ends of each
column for water overflow. At one side of the bioretention column,
two ports were set up horizontally for collecting both the water
samples and the fillers samples of different depths. The locations of
the two ports were sequentially 15 cm and 50 cm from the top. The
structural configuration in each bioretention column was planting
layer (5 cm), soil layer (10 cm), filler layer (30 cm), filtration layer
(3 cm), drainage layer (2 cm) from top to bottom. A permeable
geotextile was set between the soil layer and the filler layer to
separate the two layers and prevent the soil erosion. Ryegrass,
which is common in the park or roadside green space and has
excellent moisture resistance, was used as the bioretention plant in
this study. At the beginning of the experiment, the plant height of
ryegrass was 1e2 cm, and the top space was for the growth of
ryegrass. The soil layer consisted of a mixture of soil and sand (2:3).
Zeolite and ceramsite were mixed in the ratio of 1:1 and then added
to the filler layer. The filtration layer and the drainage layer were
composed of quartz sand and gravel, respectively. Outside of each
column was attached to black shade paper to simulate the dark
underground environment. The waterproof board was used to
cover the bioretention columns in case of rain.
2.3. Environmental design
The experiment began on March 17, 2019. The temperature
during the operation ranged from 5 to 35  C. Precipitation was
calculated based on the formula of rainfall intensity in Nanjing. The
64:300þ53:800lgP
formula was i ¼ , where i denoted rainfall intensity
ðtþ32:900Þ1:011
1
(mm min ), P meant recurrence interval of rainfall (a) and t meant
rainfall duration (min). The flow rate of the influent was calculated
as Q ¼ aiA, where a is a comprehensive runoff coefficient, i is
rainfall intensity (mm min1) and A is catchment area (mm2).
In this study, we hypothesized that P and t were 1 a and 10 min,
respectively. It was calculated that the rainfall intensity was
1.438 mm min1. In general, the comprehensive runoff coefficient is
between 0.6 and 0.9. We set a to 0.85 in this study and the catch-
ment area was 0.0225 m2 (0.15 m  0.15 m). Ultimately, the flow
rate of the influent was calculated to be 165.01 mL min1. The
simulated rainfall experiment was conducted every four days for a
total of thirty times. The time of each influent process was 30 min
and the experimental water for each column was 5 L. After the
experimental water began to enter the column, the port next to the
bottom of the column started to flow out with an interval of about
2 min. The amount of effluent collected from the measuring glass
was approximately 1.8 L. The duration of each simulated rainfall
experiment lasted 40 min.
The influent concentrations were mainly simulated according to
actual stormwater runoff, which depended on the investigation and
monitoring of stormwater runoff quality in Nanjing. The chemicals
Table 1
Influent concentrations of simulated stormwater runoff and reagents.
Pollutant Influent concentration (mg L1)
pH 6.8e7.2
COD 220
NHþ4 -N 2 Ammonium chloride (NH4Cl)
NO3 -N 4 Potassium nitrate (KNO3)
TP 0.5
Pb 0.4
3
added to the water applied as the simulated influent to the bio-
retention columns are shown in Table 1. Suspended solids (SS) were
not included in the experimental water, because between the inlet
and the bioretention tank is usually set up a pre-treatment tank
which can remove the majority of suspended solids. The bio-
retention columns were considered to be placed after the pre-
treatment tank in this study. The experimental water was pum-
ped into the bioretention columns by a peristaltic pump.
2.4. Samples collection and analysis
Water samples of two depths (15 cm and 50 cm) of each bio-
retention column were collected 1 h after the end of the inflow into
150 mL acid-washed glass bottle, and then stored in a refrigerator at
4  C until they were analyzed. The water samples were immedi-
ately analyzed for determination of chemical oxygen demand
(COD), UV254, total organic carbon (TOC), the concentration of Pb
and some other physicochemical parameters including water
temperature, pH value and dissolved oxygen (DO). A pH meter was
used to test the pH value, while potassium dichromate titration
standard method was used for COD analysis. The handy dissolved
oxygen analyzer (Mettler Toledo, China) was used to measure the
DO and water temperature. For the measurement of UV254, a UV-
VIS spectrophotometer (Shimadzu, Japan) and a quartz cuvette
with a 1 cm path length (Orient, China) were used to measure
absorbance at the wavelength of 254 nm. TOC was measured by a
TOC analyzer (Shimadzu, Japan). The concentration of Pb was
measured by an inductively coupled plasma optical emission
spectrometer (ICP-OES) (PerkinElmer, USA). Also, a molecular
fluorescence spectrophotometer (Hitachi, Japan) was utilized to
analyze the species of organic matters that existed in water
samples.
For each bioretention column, soil samples were collected into
plastic bags by hand using soil sampler from the surface (0e2 cm)
of the sandy loam soil layer. Soil samples were air-dried, sieved
through a 2 mm mesh to remove roots and significant organic
residues, and then store at 4  C before analyses. It was found that
after the experiment, the thickness of soil layer was reduced by
about 0.8 cm, which could be considered to have little effect on the
experiment. The soil pH was measured with a ratio of 1:2.5 (soil:
water) by using a pH meter. Soil organic matter content was
determined with the potassium dichromate digestion - ammonium
ferrous sulfate titration method. The concentrations of Pb in the
soils were determined by an inductively coupled plasma optical
emission spectrometer (ICP-OES) (PerkinElmer, USA) after micro-
wave digestion with concentrated HNO3 and HF. The partitioning of
Pb in the soils was determined by Tessier’s five-step sequential
extraction procedure (Tessier et al., 1979). The speciation of Pb was
divided into five different kinds of binding forms: exchangeable
(F1), carbonate bound (F2), iron/manganese oxides bound (F3),
organic and sulfide bound (F4) and residual (F5) fractions. As for the
assessment of the soil microorganisms, microbial biomass, micro-
bial activity, and microbial community structure of soil samples are
three aspects considered. Soil microbial biomass C (SMBC) was
Reagent
Hydrogen chloride (HCl) or Sodium hydroxide (NaOH)
Glucose (C6H12O6)
Potassium dihydrogen phosphate (KH2PO4)
Lead nitrate (Pb(NO3)2)
4 C. Liu et al. / Chemosphere 253 (2020) 126721
determined by the chloroform fumigation extraction method
(Vance et al., 1987). Soil enzymes regarding urease, invertase and
dehydrogenase were measured to assist in the description of mi-
crobial activity. Urease was determined by sodium phenate-sodium
hypochlorite colorimetry at 578 nm. Invertase was measured
through colorimetry at 508 nm using 3,5-dinitrosalicylic acid. The
measurement of dehydrogenase adopted was triphenyl tetrazolium
chloride (TTC) reduction method (Mersi and Schinner, 1991). The
structure of soil (fillers) microbial community was characterized by
high throughput sequencing after 120 operation days. Primers for
16S RNA gene in the V3eV4 region were designed to amplify spe-
cific areas. The alpha diversity and taxonomy of the samples were
studied by bioinformatics analysis, and sequences were clustered
into operational taxonomic units (OTUs) at 97% sequence identity.
The community diversity was determined by Shannon and Simp-
son diversity indexes. The community richness was determined by
Ace and Chao indexes. The distribution bar plot and the heatmap
graph of the community structure components were used to
evaluate the number of microbes sequences in the samples, that is,
the relative abundance of each microorganism, which intuitively
reveals the difference in the ratio of relative richness among
different samples. The Venn diagram was used to count the number
of common and unique OTUs in the samples, which can intuitively
show the similarity and overlap of the composition of the microbial
community in the samples at different classification levels.
With regard to the determination of the characteristics of the
mixed fillers, the measurement of the Pb concentration and the
microbial characteristics were referred to the detection methods of
the soil samples above-mentioned. The determination of the mi-
crobial biomass of the fillers adopted the lipid-phosphorus method.
2.5. Data analyses
All experiments were conducted in three replicates. All data
were analyzed using Origin 9.0 (OriginLab, USA) and SPSS 20.0
software (IBM Corporation, USA). The values are expressed as
mean ± standard deviation (SD) and all data were checked for
normality. Comparisons between control and treated columns were
made by statistical analysis of variance. The value of p < 0.05 rep-
resents a significant difference.
3. Results and discussion
3.1. Accumulation of Pb in the bioretention media
As is shown in Fig. 1a, the removal rates of Pb were above 78% in
the Pb treatment column, and the discrepancy of the average
removal rate between the 50 cm depth and the 15 cm depth was
1.72%. The accumulation of Pb in the soil was 37.234 mg kg1, while
that in the filler layer was very little with the content of 1.4 mg kg1
after 121 days (Fig. 1b). These make clear that most of Pb was
intercepted in the upper 10 cm soil, which was similar to some
previous studies (Al-Ameri et al., 2018; Hatt et al., 2008). These
studies reported that the heavy metals usually accumulated in the
top 2e10 cm of the filter media followed by a rapid decrease in
concentration with increasing depth. The exogenous Pb was nor-
mally fixed on the surface soil layer after it entered the soil. Since
most Pb salt minerals, such as Pb(OH)2, PbCO3, PbSO4, were insol-
uble in water, it was tough for Pb to move both horizontally and
vertically in the soil (Guanxing et al., 2011). With the advance of the
experimental process, the removal rates of Pb decreased slowly,
from 99.04% to 79.62%, but on the 85th day increased slightly. Buffle
(1988) mentioned that the humic substances, a major component
of soil organic matter, which contain a large number of binding
sites, hence they behave as a natural multiligand complexed
system. The increasing soil organic matter due to the continuous
input of exogenous organic matter provided more binding sites for
lead. The Pb removal might increase shortly, however finally
decreased caused by the weakening of the adsorption efficiency. As
is shown in Fig. 1b, the increasing rate of Pb accumulation was
0.35 mg kg1 d1 from 1st day to 81st day, but decreased to
0.2 mg kg1 d1 from 81st day to 121th day. This might attributed
to the increased uptake of Pb by the grown-up plants.
3.2. Bioavailability of Pb in the soil
Although the total amount of heavy metals in the soil has been
widely concerned and used in many soil environmental quality
standards in various countries, more and more researchers realized
that the total amount of heavy metals could not accurately reflect
the pollution of heavy metals in the soil. It is the concentration of a
metal in its available form that is important to understand the
metal toxicity (Tu et al., 2011). Sometimes, the microbial effects of
the soil environment with the same heavy metals contents differ
significantly. It is necessary to understand the impact of heavy
metals on microorganisms by utilizing the bioavailability of heavy
metals, which refers to the degrees of absorption, accumulation or
toxicity of heavy metals generated in the microbes or plants. The
extraction of different forms of heavy metals is an excellent way to
evaluate the bioavailability of heavy metals (Tessier et al., 1979).
Speciation distribution of heavy metals in different soil samples of
several periods was characterized, as presented in Fig. 1c.
During the whole operation days, only the organic bound frac-
tion remained stable, which fluctuated between 0.58% and 2.04%,
while the proportion of other forms changed obviously. The
speciation distribution was as follows: F2 (43.15%) > F3
(23.08%) > F1 (20.60%) > F5 (11.13%) > F4 (2.04%) on the first day.
The surface soil intercepted a large amount of water-soluble Pb2þ in
the form of PbCO3 after Pb2þ entered the system. 23.08% of iron/
manganese oxides bound fraction was considered to be that there
were a small amount of iron, manganese, silicon oxides or hydrates
in the soil, which were easy to cause the specific adsorption re-
actions. Nomeda et al. (2008) reported, exchangeable, carbonate
bound and iron/manganese oxides bound fractions were labile
because they were easy to cause biological reactions, while organic
bound and residual fractions were stable because of their insolu-
bility and low bioavailability. Therefore, 86.83% labile fraction of Pb
was more likely to be used by microorganisms and had a potential
threat to the environment. However, low concentration of Pb,
which provided an inverse environment for the microorganisms, in
turn stimulated them growth instead of having a toxic effect. With
the proceeding of the experiment, the proportion of labile fractions
decreased significantly. The speciation distribution on the 81st day
was F5 (69.00%) > F3 (13.52%) > F2 (9.97%) > F1 (6.60%) > F4
(0.91%), which indicated that the bioavailability of Pb decreased
during the first 80 days. Such phenomenon was also referred to as
natural attenuation of heavy metals (Tom-Petersen et al., 2004;
Vazquez et al., 2011). With the prolongation of time, the greater the
intensity of Pb binding in the soil, the more stable solid phase could
be formed by the water-soluble Pb. The carbonate bound Pb would
be labile under the release of the soil Hþ and came into the trans-
formation to residual fractions. The mechanisms regarding the
natural attenuation of heavy metals included particle diffusion,
precipitation and cluster formation, eventually forming the inner or
outer-sphere complex (Sparks, 2006). Moreover, microorganisms
could secrete SMP to chelate Pb2þ to relieve the toxic environ-
mental pressure of heavy metals, which affected the bioavailability
of Pb. During the last 40 days of the experiment, the labile fractions
increased, while the residual fractions decreased slightly. This may
be due to the reasons that (1) the attenuation rate slowed down
 C. Liu et al. / Chemosphere 253 (2020) 126721
Fig. 1. (a) Removal rate of Pb of different depths, (b) Pb accumulation in soil and filler layer, (c) fractional percent of Pb in soil layer, and (d) organic carbon contents in soil layer with
the operation day.
with the prolongation of time; (2) the utilization of labile Pb by
microorganisms and plants came to a limit; (3) new water-soluble
Pb entered into the system. At the end stage of the experiment, the
total amount of Pb was still increasing and the bioavailability of Pb
was on the rise, which indicated that there were potential threats
and risks to the bioretention system, and the microorganisms and
plants in the system were at risk of toxicity, so further long-term
observation should be strengthened.
3.3. Effects of Pb on microorganisms
Numerous reports have demonstrated that microbial charac-
teristics of soil or fillers change when exposed to heavy metal stress
(Rajkumar and Freitas, 2008; Tang et al., 2019; Zhang et al., 2016).
The microbial biomass, microbial activity and microbial community
structure are major indicators of microbes for evaluating soil
quality (Khan et al., 2007; Renella et al., 2005). Microbial biomass
can reflect the actual quantity of microorganisms and nutrient cycle
and transformation in the media, but does not reveal the changes of
community structure (Hu et al., 2014). Microbial activity plays an
important role in the occurrence and catalysis of many biochemical
5
reactions (Shi and Ma, 2017). Microbial community structure can
affect the ability of community to achieve different functions
(Torsvik and Øvreås, 2002). None of them can be used as a separate
microbiological indicator to diagnose media quality. In order to
reflect soil or filler quality more accurately, it is best to include a
complete set of biological indicators, i.e., microbial biomass, mi-
crobial activity, microbial community.
3.3.1. Microbial biomass
When the operation day increased, the fillers biomass and the
soil microbial biomass carbon tended to increase gradually and
then flattened, which was mainly due to the continuous entry of
influent pollutants into the system and the constant increase of
temperature in Nanjing from March to July, which provided an
opportunity for the growth of microorganisms. As is presented that
in Fig. 2a and b, the fillers biomass and the soil microbial biomass
carbon in the Pb treatment column were 95.54 nmol P g1 and
185.14 mg kg1, which were 7.75% and 15.94% lower than those in
the control column, respectively. The reason was that when the
pollution load of Pb was high, it produced toxicity and inhibited
microbial biomass. Rajkumar and Freitas (2008) suggested that
6 C. Liu et al. / Chemosphere 253 (2020) 126721
Fig. 2. (a) Soil microbial biomass, (b) fillers microbial biomass, (c) COD removal of different depths, and (d) TOC removal of different depths in two bioretention columns with the
operation day.
copper and zinc had little effect on microbial biomass at low con-
centration, and once the level of copper and zinc was inclined to
high, the biomass could decrease by 40%. The microbial biomass in
the soil was much higher than that in the fillers, because the soil
contained more nutrients and organic matters (Fig. 1d). The soil
layer undertook higher metal pollution load, so the effect of Pb
pollution on the soil microbial biomass carbon was more severe.
Although the amount of Pb accumulated in the fillers was only
1.4 mg kg1 which belonged to the low pollution load, the fillers
biomass might also be restrained under the effect of the long-term
increasing accumulation treatment.
3.3.2. Microbial activity
This study determined the activities of three enzymes including
urease, dehydrogenase and invertase to investigate the microbial
activity in the soil and fillers. Urease exists in most of bacteria, fungi
and plants. It reflects the transformation ability of organic nitrogen
to available nitrogen and the supply capacity of inorganic nitrogen.
Dehydrogenase can be used as an index of microbial redox ability
and has attracted considerable attention in the study of biokinetics.
Invertase can hydrolyze sucrose to glucose and fructose which
plays an essential role in increasing soluble nutrients in soil.
As is shown in Fig. 3c, the urease, dehydrogenase and invertase
activities in the soil showed a trend of slowly increasing and finally
stabilized at 0.839 mg NH3 g1 d1, 2.842 mg TF g1 h1, 6.135 mg
glucose g1 d1 respectively, 4.7, 3.9 and 5.2 times higher than that
in the fillers. On the 21st and 81st day, enzyme activities in the Pb
treatment were higher than those in the control. This was because
that low concentration of exogenous Pb2þ became the prosthetic
group of the protein, and also could promote the binding of the
enzyme with the substrate in the media, thus changing the equi-
librium state of the reaction process and the charged state of the
enzyme. At the end stage of the experiment, the activity of the
enzymes, especially urease, was depressed by the Pb contamina-
tion. Soil urease in the Pb treatment column was 19.41% lower than
that in the control column on the 81st day. There are two reasons
for the inhibition of enzyme activity by heavy metals. The first is
that the pollution of heavy metals has a direct inhibitory effect on
the enzymes, which makes the enzyme genes change; the second is
the indirect inhibition, which affects the growth of microbes to
reduce the enzyme activity (Shi and Ma, 2017). Therefore, the
inhibitory effect of Pb on urease may be due to (1) as the accu-
mulation of Pb in the media increased, Pb bound to urease activity
sites, competing with substrate, to form stable complexes; (2) Pb-
organics complexes inhibited the growth and reproduction of
media microorganisms and reduced the synthesis and secretion of
endogenous enzymes, which led to the decrease of urease activity
in the media.
 C. Liu et al. / Chemosphere 253 (2020) 126721 7

Fig. 3. (a) Venn diagram, (b) distribution bar plot of the relative abundance of bacteria community in phylum, (c) enzyme activity (urease, dehydrogenase and invertase), and (d)
heatmap graph of community structure components in genus of soil and filler samples in two bioretention columns. The shift of the bacterial community compositions in heatmap
graph was depicted by the color intensity ranged from 0 to 3. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this
article.)

3.3.3. Microbial community structure
As shown in Table 2, the observed OTU number varied signifi-
cantly among the four media samples. There was little difference in
the OTU number between the control and the Pb treatment, but a
big difference between the soil and fillers. Though Pb had the
apparent effects on the microbial diversity and richness, it was very
slight compared with the impact of the media properties. It can also
be seen from Fig. 3a that high similarity only existed between the
two soil samples. The Shannon and the Simpson indexes showed
that long-term Pb accumulation could positively affect the com-
munity diversity. The community richness in the Pb treatment
media increased compared with the control, seen from the Chao
and the Ace indexes. This meant that the toxicity of Pb on many
microorganisms did reduce the microbial biomass by killing some
microorganisms, such as Truepera and Soehngenia, meanwhile, it
increased the abundance of some microorganisms, like Thauera,
Shinella and Rhodobacter (Fig. 3b and d), which might be in a small
quantity but could tolerate Pb and survive.
As shown in Fig. 3b, the most abundant phylum in soil was
Preteobacteria, which constituted 29.01%e40.04% of the bacteria,
followed by Chloroflexi and Bacteroidetes. In fillers, three domi-
nating bacteria phyla were Preteobacteria, Firmicutes, and Bacter-
oidetes. The structure and characteristics of bacterial communities
depend on media pH mainly. Chodak et al. (2013) reported that

Table 2
Microbial diversity and richness indexes of soil and filler samples.

Index control þ soil Pb treatment þ soil control þ fillers Pb treatment þ fillers

OTU 1356 1834 440 709

Shannon 5.36 ± 0.02 6.10 ± 0.02 4.66 ± 0.02 5.21 ± 0.02
Simpson 0.019 ± 0.001 0.007 ± 0.001 0.027 ± 0.001 0.017 ± 0.001
Ace 1695 ± 64 2161 ± 58 448 ± 5 718 ± 6
Chao 1708 ± 83 2151 ± 69 455 ± 9 718 ± 5

Notes: Indexes were calculated from OTU relative abundance of each replicate.
8 C. Liu et al. / Chemosphere 253 (2020) 126721
higher pH values favored Firmicutes and Verrucomicrobia, while
Chloroflexi and Gemmatimonadetes were more abundant in acidic
soils. The pH values of soil and filler samples after 121 operation
days were determined to be 6.89 (control þ soil), 6.85 (Pb
treatment þ soil), 8.04 (control þ fillers) and 7.68 (Pb
treatment þ fillers), respectively. In addition to the influence of pH,
the difference in dissolved oxygen between the soil and fillers also
caused the discrepancy of microbial community structure. Bacter-
oidetes often exist in anoxic environment, which can explain why
the abundance of Bacteroidetes in fillers was higher than that in soil.
Proteobacteria showed slightly increase in tested soil samples in
response to the Pb accumulation, but exhibited an adverse trend in
fillers. This may be due to the fact that the accumulated concen-
tration in the soil was much higher than that in the fillers (Fig. 1b).
The phylum Proteobacteria was found to be abundant in many high
heavy metal content environments, such as heavy metal-polluted
river water (Serkebaeva et al., 2013) and sediments (Yin et al.,
2015), whereas Proteobacteria had opposite trend reported and
showed negative correlation with Pb concentration (Gillan et al.,
2005). The change in response of Proteobacteria to heavy metals
might be attributed to the reason that the phylum had a diverse
nature which helped them to get carbon, nitrogen and energy by
using various forms of organic matters (Bouskill et al., 2010). These
properties made it performed differently towards environmental
change, for example, the different concentrations of heavy metal
environmental pressure. In Fig. 3d, the changes of microbial com-
munity structure in genus level could be found more clearly and
concretely. These changes might be explained by the selection of
tolerant groups, while sensitive ones were clearly reduced (Singh
et al., 2014). Pb contamination led to more variability and insta-
bility of microorganisms, therefore the microorganisms adapted to
Pb accumulation by changing microbial community structure.
Moreover, the variations in interactions between the microorgan-
isms in the bioretention systems may be a way for microorganisms
to adapt to heavy metal pollution.
3.4. Organics removal by the bioretention columns
The changes of microbial characteristics caused by the Pb
contamination affected the removal of organic matters. As shown in
Fig. 2c, the COD removal at different depths exhibited a trend of
increasing and then stabilized gradually. Similar to the COD
removal, the TOC removal increased rapidly at first, and began to
rise slowly on the 17th day and stabilized gradually on the 61st day
(Fig. 2d). The increasing rate of TOC removal was slower than that of
COD. This was supposed to be due to the fact that some organic
matters, which did not change in the short-term of the soil utili-
zation, were oxidized in TOC determination but not completely
oxidized in COD determination. The removal rates of COD at the
beginning of operation were negative, because the dissolved
organic matters contents of the soil layer were very high initially. At
the beginning, the concentrations of COD decreased considerably
with the decreasing dissolution concentrations of organic matters.
Although the soil had certain physical adsorption capacity for COD,
the adsorption of COD by the soil could not offset the dissolution of
organic matters, so the removal rate of COD at 15 cm depth was still
negative (12.63%) until the 13th day. From day 13 to day 21, the
COD removal at 15 cm depth had a slight decline, which meant the
adsorption was close to the limit and the adsorption effect was
weakened. From day 29 to day 53, the period was just the time
when the temperature in Nanjing rose rapidly (from 19  C to 32  C).
The temperature increase provided a favorable environment for
biological growth in the bioretention media. The mechanisms of
COD removal changed from sorption to biodegradation. The rapid
growth of microorganisms through the degradation of organic
matters led to a rapid increase in the COD removal rate, which
increased significantly from 26.88% to 33.06% at the depth of
15 cm from day 21 to day 53. From day 53 to day 69, the COD
removal rate experienced a short decline, which probably was that
the drop of local temperature had affected the biological activity
during this time. The COD removal tended to be stable with an
average removal rate of 59.91% and 53.72% in the control and Pb
treatment column respectively from the 77th day. It indicated that
the microorganisms began to enter a stable growth period.
Compared with the control, the COD removal rate at 15 cm depth in
the Pb treatment was 6.79% higher from day 9 to day 25, but 10.99%
lower from day 77 to day 117. Similar findings were also found in
TOC removal. This was because that the amount of Pb accumulated
in the two periods determined the positive and negative effects on
microorganisms. However, at 50 cm depth, the COD removal rate in
the Pb treatment was only 6.19% lower than that in the control,
which meant the filler layer (15e50 cm) of the Pb treatment
exhibited more COD removal than that of the control. In other
words, the effects of the soil and the filler layer on the COD removal
under the stress of Pb environment were opposite. The reason will
be explained in Section 3.5.
3.5. Influence mechanisms of Pb on organics removal
As we all know, the removal mechanisms of organic matters by
the bioretention system mainly include abiotic treatment (filtration
and adsorption) and biological treatment. It can be seen from Fig. 2c
that the adsorption of the media was limited, and the long-term
removal of organic matters depended on microbial degradation.
Through the formation of biofilm on the matrix, the metabolism of
biofilm and the hydrolysis of extracellular enzymes are used to
adsorb and degrade the organic matters; mineralizing them to form
small molecular inorganic substances (LeFevre et al., 2012; Wang
et al., 2017).
With the increase of Pb accumulation in the bioretention col-
umn, different microorganisms made different responses to Pb
contamination. Heavy metal pollution can inhibit the metal sensi-
tive species and meanwhile stimulate metal-resistant species (Liu
et al., 2018). Different concentrations of heavy metals may also
have different effects on the same microorganism. Liu et al. (2018)
found that the abundance for Azoarcus decreased under low Pb
concentration but increased under high Pb concentration, which
was similar to the result of this study. In this study, most of Pb was
intercepted in the soil layer, and only a small amount existed in the
filler layer. Therefore, Pb contamination might have different effects
on the microorganisms in the soil and filler layer. The abundances
for both Proteiniphilum and norank_o_SBR1031 in the soil decreased
to different degrees after 121 operation days, while that in the
fillers increased (Fig. 3d). The genera Proteiniphilum and nor-
ank_o_SBR1031 were famous for their capabilities in degradation of
polysaccharide and proteins (Hahnke et al., 2016; Sekiguchi et al.,
2003), indicating they both contributed to the COD removal. Due
to the different accumulation of Pb, the abundances of bacteria with
COD degradation function in soil and fillers were adversely affected.
The COD removal in the soil and filler layer was promoted and
inhibited by Pb respectively.
In addition, the water samples were also analyzed for organic
matter identification. According to an analytical approach named
fluorescence regional integration (FRI) proposed by Chen et al.
(2004), the two-dimensional fluorescence regions formed by
excitation and emission wavelengths are divided into five parts,
which represent five different types of organic compounds,
including aromatic proteins I (Region I) (220e250/280-330), aro-
matic proteins II (Region II) (220e250/330-380), fulvic acid-like
substances (Region III) (220e250/380-500), soluble microbial
 C. Liu et al. / Chemosphere 253 (2020) 126721 9

products (SMP) (Region Ⅳ) (250e280/280-380) and humic acid-
like substances (Region Ⅴ) (250e400/380-500). The organic pol-
lutants in the four samples mainly contained fulvic acid, humic acid
and aromatic proteins (Fig. 4 and Table 3), which mostly were the
decomposition and degradation products of plants or microor-
ganisms in soil or water. The fluorescence intensity of Region I
(aromatic proteins) and Region Ⅳ (SMP) in the Pb treatment was
obviously reduced than that in the control (Table 3), indicating that
the metabolism of microorganisms was affected by Pb. A large
number of functional groups such as eOH, eCOOH, eSH containing
in SMP made them able to chelate the heavy metal ions (Kuo and
Parkin, 1996), like Pb2þ. In addition to the adjustment of microbi-
al community structure, the change of organic composition would
also affect the utilization of organic matter by microorganisms.
Therefore, Pb would affect the removal of organic matter by
chelating organic matters and changing their composition. At
present, there is no suitable way to define the accumulation of
heavy metals in bacteria. The complexity of the interaction be-
tween the heavy metals and microorganism make it difficult to
better understand the basic mechanisms. Therefore, it needs
further research to explore the mechanisms.
4. Conclusions
This study used outdoor bioretention columns with stormwater
runoff simulation to study the effects of Pb accumulation on mi-
crobial characteristics and organics removal, and obtained the
following conclusions. Most of Pb was intercepted in the upper
10 cm soil. Pb accumulation in the soil was 37.234 mg kg1, while
that in the filler layer was very little with the content of 1.4 mg kg1
after 121 days. The long-term Pb accumulation in the biorentention
system not only reduced the microbial biomass by killing some
microorganisms, but also increased the community diversity by
stimulating the growth of some Pb-tolerance microorganisms. Due

Fig. 4. Fluorescence EEM Spectra diagrams of (a) controlþ15 cm, (b) Pb treatmentþ15 cm, (c) controlþ50 cm, and (d) Pb treatmentþ50 cm of organics in effluent.
10 C. Liu et al. / Chemosphere 253 (2020) 126721

Table 3
Fluorescence intensity and integral volume in five regions of the fluorescence EEM spectra of organics in effluent.

Sample Index Region I Region II Region III Region Ⅳ Region Ⅴ

controlþ15 cm FI (au) 1080 966.8 1470 2103 1413

F (106 au nm2) 10.71 15.02 19.40 6.51 29.36
Pb treatmentþ15 cm FI (au) 548.4 1020 1538 915 1480
F (106 au nm2) 8.15 16.17 20.55 5.54 29.89
controlþ50 cm FI (au) 752.1 1012 1468 1049 1453
F (106 au nm2) 8.42 15.51 19.91 5.10 29.49
Pb treatmentþ50 cm FI (au) 333 1025 1456 559.8 1436
F (106 au nm2) 4.45 16.47 20.03 4.12 28.55

Notes: FI denotes the maximum fluorescence intensity of peaks in each region of each fluorescence EEM spectra. F denotes the integral volume calculated by the FRI method
referring to the fluorescence intensity in each region of each fluorescence EEM spectra.

to the different Pb accumulation in the soil layer and filler layer, the
effect of Pb on the community structure of these two layers was
different. The abundance of bacteria with COD degradation func-
tion in soil decreased, while that in fillers increased. The COD
removal in the soil and filler layer was promoted and inhibited by
Pb respectively. In addition to the adjustment of microbial com-
munity structure, Pb affected the removal of organic matter by
chelating organic matters and changing their composition. The
long-term accumulation of heavy metals in bioretention system
would affect microbial degradation function and pollutants
removal, causing our concern for the long-term maintenance of the
bioretention system.
Author contribution
Cheng Liu, Conception and design of study, analysis and/or
interpretation of data, drafting the manuscript, revising the
manuscript critically for important intellectual content, approval of
the version of the manuscript to be published. Jie Lu, Conception
and design of study, acquisition of data, analysis and/or interpre-
tation of data, revising the manuscript critically for important in-
tellectual content, approval of the version of the manuscript to be
published. Jiaqi Liu, Conception and design of study, acquisition of
data, analysis and/or interpretation of data, approval of the version
of the manuscript to be published. Tariq Mehmood, Conception and
design of study, analysis and/or interpretation of data, drafting the
manuscript, approval of the version of the manuscript to be pub-
lished. Wei Chen, Conception and design of study, analysis and/or
interpretation of data, approval of the version of the manuscript to
be published.
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 (51378174, 51438006), the Project Funded by
the Priority Academic Program Development of Jiangsu Higher
Education Institutions.
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