Ecotoxicology and Environmental Safety 202 (2020) 110958

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Ecotoxicology and Environmental Safety

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

Responses of microbial communities and metabolic activities in the

rhizosphere during phytoremediation of Cd-contaminated soil
Chenjing Liu a, b, Hai Lin a, b, *, Bing Li a, b, Yingbo Dong a, b, **, Tingting Yin a, b
a
School of Energy and Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, China
b
Beijing Key Laboratory on Resource-Oriented Treatment of Industrial Pollutants, Beijing, 100083, China

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

Keywords: Phytoremediation is an effective way to repair heavy metal contaminated soil and rhizosphere microorganisms
Cadmium contaminated soil play an important role in plant regulation. Nevertheless, little information is known about the variation of mi­
Rhizosphere soil Cd fractions crobial metabolic activities and community structure in rhizosphere during phytoremediation. In this study, the
High-throughput sequencing
rhizosphere soil microbial metabolic activities and community structure of Trifolium repensL. during Cd-
EcoPlate™ substrate utilization
Microbial community composition and
contaminated soil phytoremediation, were analyzed by Biolog EcoPlate™ and high-throughput sequencing.
diversity The uptake in the roots of Trifolium repensL. grown in 5.68 and 24.23 mg/kg Cd contaminated soil was 33.51 and
84.69 mg/kg respectively, causing the acid-soluble Cd fractions decreased 7.3% and 5.4%. Phytoremediation
significantly influenced microbial community and Trifolium repensL. planting significantly increased the rhizo­
sphere microbial population, diversity, the relative abundance of plant growth promoting bacteria (Kaistobacter
and Flavisolibacter), and the utilization of difficultly metabolized compounds. The correlation analysis among
substrate utilization and microbial communities revealed that the relative abundance increased microorganisms
possessed stronger carbon utilization capacity, which was beneficial to regulate the stability of plant-microbial
system. Collectively, the results of this study provide fundamental insights into the microbial metabolic activities
and community structure during heavy metal contaminated soil phytoremediation, which may aid in the bio­
regulation of phytoremediation.

1. Introduction apparently is hazards to humans, which includes diarrhea, damage of

With the development of industry, heavy metal pollution in soil has
increased sharply, which has become one of the most significant envi­
ronmental problems worldwide (Huang et al., 2019). Among all heavy
metal contaminants in soil, cadmium, which is defined as one of the
most dangerous soft metals on the earth, is widely distributed in both
agricultural and industrial soils, and seriously exceeds the standard (Liu
et al., 2019). According to the National Soil Pollution Survey Bulletin
issued in 2014, 16.1% of the soils are polluted by inorganic pollutants, of
which cadmium pollution is the most serious, and its exceeding standard
rate is more than 7%. Cd can interfere with many biochemical and
physiological processes of plants, including plant growth, photosyn­
thesis, nutrient uptake and enzyme activity even at trace concentration.
More important, cadmium absorbed by plants from soil is easily trans­
ferred to animals and humans through the food chain because of its high
mobility and accumulation. Previous study has reported that Cd
bone marrow, kidney failure and loss of sense (Singh et al., 2014).
Consequently, it is urgent to remediate cadmium pollution in soil.
Aiming at the pollution characteristics of heavy metals, several
physico-chemical methods such as soil replacement, soil isolation,
vitrification, electrokinetic remediation, encapsulation, soil washing
and immobilization have been used for the removal of Cd (II) from soil
(Khalid et al., 2017). However, traditional physical and chemical
methods spend a lot of manpower and material resources and even
destroy soil structure, affect soil fertility and introduce new pollutants,
limiting the application in large field (Odoh et al., 2019). In situ phy­
toremediation, which decreases the cost, adds an aesthetic value and has
practical applications on the field which are long-term, is considered
environmentally friendly and economical (Kumar Yadav et al., 2018
Cited Pages).
Soil microorganisms are an important part of soil ecosystem, which
can regulate soil fertility, nutrient cycle and maintain biodiversity

* Corresponding author. University of Science and Technology Beijing, 100083, China.

** Corresponding author. University of Science and Technology Beijing, 100083, China.
E-mail addresses: linhai@ces.ustb.edu.cn (H. Lin), ybdong@ustb.edu.cn (Y. Dong).

https://doi.org/10.1016/j.ecoenv.2020.110958
Received 7 May 2020; Received in revised form 27 June 2020; Accepted 28 June 2020
Available online 11 July 2020
0147-6513/© 2020 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
C. Liu et al.
(Ramakrishna et al., 2019). Among all soil microorganisms, rhizosphere
microorganisms, which are defined as “microorganisms in the zone of
soil surrounding the root which is affected by it”, strongly affect plant
rhizosphere activities due to its direct contact with plant roots
(Sugiyama, 2019). The collective genome of rhizosphere microbial
communities is also referred to as the plant’s second genome, indicating
they are an essential part of plant rhizosphere (Berendsen et al., 2012).
Actually, each plant possesses its own specific microflora in the rhizo­
sphere, which plays an important role in host plant growth promotion
and heavy metal detoxification/removal (Lal et al., 2018). As to enhance
plant growth, rhizosphere microorganisms secrete IAA (indole acetic
acid), product ACC (1-aminocyclopropane-1-carboxylate) deaminase,
synthetize plant antioxidative enzymes and improve the uptake of
essential mineral elements (Etesami and Maheshwari, 2018). Further,
rhizosphere microorganisms help to tolerate and alleviate negative
impact of abiotic stresses including drought, salinity and pollution of
heavy metal and antibiotic (Singh et al., 2018). Some of the rhizosphere
microorganisms are able to decrease the proportion of phytoavailability
heavy metals by adsorption, oxido-reduction reactions, extracellular
matrix and immobilization, and others can improve P nutrition, adjust
osmotic stress under environmental stresses (Nadeem et al., 2014).
During the process of phytoremediation, rhizosphere microorgan­
isms play an important role in plant regulation and this regulation
greatly depends on microbial metabolism and diversity. Generally,
metabolic activity and community structure of soil microorganisms are
important indicators of soil health, determining soil functionality (Wang
et al., 2019). In soil with heavy metal contamination, the microbial
community constantly adjusted itself as well as the metabolic capacity to
adapt to different habitats (Zhao, 2019). Meanwhile, the effects of
rhizosphere microorganisms on plant growth and heavy metal enrich­
ment also changed, which is considered to be of great significance in
regulating phytoremediation (Zhao, 2019). Microbial communities help
plants resist the toxicity of heavy metal by increasing the abundance of C
and N immobilized microorganisms and the activity of metabolic en­
zymes (Das et al., 2017).
Nevertheless, the information on microbial metabolic activities and
community structure variation during heavy metal contaminated soil
phytoremediation is very limited. Therefore, in our research, the mi­
crobial metabolism characteristics and community structure and
composition in the rhizosphere soil during Cd contaminated soil phy­
toremediation by Trifolium repensL. were studied. Trifolium repensL. is
one of the most important genera of the Leguminosae family with good
adaptability to the environment. Under the appropriate conditions of
sunlight and moisture, it can grow with well-developed root and large
biomass in a wide temperature range and in nearly any soil type (Zhang
et al., 2018a, b). Previous study has already reported that Trifolium
repensL. has strong resistance and good removal ability in phytor­
emediation of contaminated soils (Hazrat Ali, 2012). Our study aimed
to: I) acquire cadmium enrichment characteristics of Trifolium repensL.
and heavy metal distribution in rhizosphere soil; II) analyze the dynamic
metabolic activities of rhizosphere microbes; III) study the rhizosphere
microbial community and abundance; IV) determine the correlation
between substrate utilization and microbial communities.
Table 1
The physicochemical properties of experimental soils.
Item pH Available N (mg/kg) Available P (mg/kg)
CL 7.40 36.52 49.60
CM 7.62 37.09 37.42
CH 7.08 32.35 29.61
ND: No detection.
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Ecotoxicology and Environmental Safety 202 (2020) 110958
2. Materials and methods
2.1. Experimental reagents and soils
All the reagents used in the experiment were of analytical grade from
Sinopharm Chemical Reagent Co., Ltd. Experimental soils with different
Cd pollution level were collected from Beijing, China and the physico­
chemical properties of the soil were summarized in Table 1. The soils
were collected in the same area with different pollution levels. After
collected using a stainless-steel shovel at the depth of 0–30 cm, the soil
was air dried and mixed evenly.
2.2. Plant growth conditions and treatments
Trifolium repensL. seeds were sterilized with 2% (v/v) sodium hy­
pochlorite for 30 min and then rinsed five times with sterilized water to
remove the former solutions. Seeds were pre-germinated at a 90-mm
plate at 25 � C and kept moist for further treatment. Fifteen seeds were
sown in each pot (15 cm in diameter and 15 cm in high) with 750 g soil
and ten seedlings were kept after germination. All the pots were placed
randomly in the greenhouse with the temperature at 20–25 � C and
watered by deionized water daily.
After 16 weeks’ growing, the plants were all carefully harvested from
the pots. After washed with tap water and deionized water to remove the
soil and dried the surface with filter paper, plants were measured the
growth parameters. For Cd concentration analysis, the plants were
freeze-dried at 50 � C and then grinded to powder with particle size less
than 0.5 mm (He et al., 2013). The samples were digested by microwave
and the cadmium content was analyzed by Inductively Coupled Plas­
ma–Mass Spectrometry (ICP-MS) (SHIMADZU, 2030) in Research Cen­
ter for Eco-Environmental Science, Chinese Academy of Sciences.
2.3. Rhizosphere soil sampling and analysis
2.3.1. Rhizosphere soil collection and Cd fractionation analysis
Rhizosphere soil were sampled within 0–15 cm, obtained from soil
adhering to plant roots four times: at the 0th, 4th, 9th and 16th week
after planting the seeds. Then, these samples were divided into two
parts. One part was immediately used for measuring the number of
culturable microorganism, microbial metabolic activity and DNA
extraction and 16 S rRNA gene amplicon sequencing; the other part was
air-dried and used for measuring Cd fractionation of after being ground
and sieved through a 0.1-mm mesh.
Community Bureau of Reference (BCR) sequential extraction pro­
cedure was applied to rhizosphere soil samples to analyze the chemical
fractionation of Cd. Acid soluble: 40 mL 0.11 M CH3COOH (16 h, room
temperature); Reducible: 40 mL 0.5 M NH2OH⋅HCl (16 h, room tem­
perature); Oxidizable: 10 mL 8.8 M H2O2 þ 10 mL 8.8 M H2O2 þ50 mL 1
M NH4OAc (1 h, 85 � C; 1 h, 85 � C; 16 h, room temperature); Residual:
digested with microwave dissolver (Oppler Instruments Co., Ltd) by 10
mL mixed acid (HCl: HNO3: HF ¼ 2:1:1).
2.3.2. Numbers of culturable microorganism
To determine the number of culturable microorganism, viable counts
of rhizosphere microbes were conducted using the dilution plate-count
technique (Hiroki, 1992). Ten grams fresh soils were homogenized in
90 mL sterilized normal saline for 30 min in a constant temperature
Available K (mg/kg) Organic matter (g/kg) Cd concentration (mg/kg)
115.36 15.85 ND
112.71 14.06 5.68
97.43 12.58 24.23
C. Liu et al.
shaker. Then the soil samples were serially diluted by mixing 1 mL of the
previous dilution with 9 mL of 0.1% (w/v) sterile normal saline and
incubated on LB solid medium. After 5 days’ incubation at 28 � C, the
solid medium with 30–300 Colony-Forming Unit (CFU) were counted.
All the determination was carried out in triplicates and the results were
expressed as CFU/g.
2.3.3. Microbial metabolic activity
Metabolic characteristics of microbial communities were accom­
plished with Biolog EcoPlate™ (Biolog, USA). The plate is composed of
96 wells, which contains 31 carbon sources (4 carbohydrates, 6 amino
acids, 3 alcohols, 3 amines, 4 polymers, 4 esters and 7 carboxylic acids)
and one water blank. A multichannel repeated pipette was used to
dispense the rhizosphere soil diluent into 96 wells (150 μ l per hole) of
the microplate under aseptic conditions. Plates were incubated at 28 � C
for 10 days in the dark and the optical density at both 590 nm and 750
nm was read automatically every 12 h by the computerized Micro­
Station™ system. AWCD (Average Well Color Development) was used to
evaluate the microbial metabolic activity. AWCD was calculated as
followed (Kong et al., 2013):
1 X31
AWCD ¼
31 i¼1
ðRit R0t Þ
where Rit is OD in each carbon source well at time t and R0t OD of the
control at time t.
2.3.4. Microbial community analysis
The microbial community diversity and structure of all the rhizo­
sphere soil samples were characterized by Beijing Zhongke Jingyun
Technology Co., Ltd, Institute of Computing Technology, Chinese
Academy of Sciences, using 16 S rRNA high throughput sequencing
technique. Briefly, the V3–V4 hypervariable regions of bacteria 16 S
rRNA gene were amplified with primers 515 F (50-
GTGCCAGCMGCCGCGGTAA-30) and 806 R (50-GGAC­
TACHVGGGTWTCTAAT-30). PCR was performed using the following
program: 3 min of denaturation at 95 � C, 27 cycles of 30 s at 95 � C, 30s
for annealing at 55 � C, and 45s for elongation at 72 � C, and a final
extension at 72 � C for 10 min. UPARSE(version 7.1 http://drive5.com/
uparse/) was used to cluster operational taxonomic units (OTUs) with
97% similarity.
2.4. Statistical analysis
All the statistical analyses of data were performed using Statistical
Package for Social Sciences (SPSS) version 19.0 software. PCA analyses
were performed to assess the possible correlations among soil types and
soil microbial community. All the figures were made using Origin Graph
2016 software and the microbial community parameters were calcu­
lated on website https://www.mothur.org/wiki/Mothur_manual.
3. Results and discussion
3.1. The effect of Cd on plant growth response and Cd translocation
The growth of Trifolium repensL. under Cd stress conditions was
affected, by the decrease of plant length and biomass. Relatively in CL,
the biomass of shoot and root of CH samples were decreased 37.8% and
35.1%, which might be explained that Cd was toxic and characteristi­
cally inhibited plant growth at higher concentrations. As shown in
Fig. 1, uptake was generally higher in soil seriously contaminated by
cadmium, with corresponding values of 34.36 and 84.69 mg/kg in shoot
and root respectively, but only lower at 1.62 and 1.35 mg/kg in CL. This
also indicated a significant correlation between Cd uptake by the plant
in various soils and total soil Cd concentration. By statistical analysis,
the correlation coefficient between soil Cd concentration and root, shoot
3
Ecotoxicology and Environmental Safety 202 (2020) 110958
Fig. 1. Trifolium repensL. growth parameters and Cd content in different
treatments. The bar on the columns is the standard deviation. Bars with
different letters indicate significant differences between the different treat­
ments based on LSD (P < 0.05).
Cd concentration was 0.987 and 0.970, respectively. In addition, the
data demonstrated that more uptake was observed in roots than shoots
in all conditions and it was observed more significantly in heavy metal
enrichment groups.
Cadmium, a non-essential element for plants, is toxic even in trace
concentrations and its contamination in soil could cause anatomical and
physiological changes such as the length and fresh weight of shoot and
root (Chen et al., 2016). Cd exposure leads to alteration of normal
metabolism including the synthesis of chlorophyll and photosynthesis
reduction, nitrogen inhibition and mineral uptake decrease, which are
directly reflected by plant height and biomass (Kaur et al., 2018). In our
study, the root length in CH soil was observed to decrease 30.0% while
shoot length decreased 20.4%. Nevertheless, Trifolium repensL. was able
to survive and propagate under both 5 and 25 mg/kg Cd contaminated
soil. Many studies have suggested heavy metal pollutants are toxic to
plants but some tolerant species possess the potential to absorb, trans­
port and accumulate heavy metal. Like most heavy metal tolerant plants,
Trifolium repensL. is believed to adopt different strategies (like seques­
tration, exocytosis, compartmentalization) to cope with metals toxicity
(Sarwar et al., 2017). More importantly, Trifolium repensL. could accu­
mulate heavy metals from the soil and transport them to the shoots,
indicating Trifolium repensL. possessed excellent potential for
phytoremediation.
3.2. Analysis of Cd fractions during phytoremediation
Residual, oxidable, reducible and acid-soluble Cd were extracted to
research the changes of rhizosphere soil heavy metal fractions during
phytoremediation (Fig. 2). As shown in Fig. 2, the acid-soluble and re­
sidual Cd fractions decreased gradually while the reducible Cd fractions
increased over time. The acid-soluble Cd fractions were decreased 7.3%,
5.4%, and the residual Cd fractions were decreased 4.3%, 2.7%. Only the
reducible Cd fractions were showed an increased trend of 8.8% and
6.1% with a most extent during the study. As for the oxidable Cd frac­
tions, it was stabilized at about 30% among all samples. Overall, the
total proportion of acid-soluble, reducible and oxidable cadmium
accounted for a large part in the contaminated soil, while the residual
cadmium did not exceed 35%.
Observing heavy metal chemical form distribution and content
changes could evaluate the bioavailability of heavy metals. As the
bioavailability is depended on solubility, the bioavailability of metal
increases in the order of residual, oxidable, reducible and acid-soluble
forms. (Islam et al., 2016). In our study, though the first three forms
of Cd were the main ones, Trifolium repensL. could grow, which also
C. Liu et al.
Fig. 2. The results of Cd concentrations of rhizosphere soils using the BCR
sequential extraction.
reflected its high Cd tolerance. After phytoremediation by Trifolium
repensL., the fraction of acid-soluble Cd decreased, revealing that
Trifolium repensL. possessed the ability to extract heavy metals. In
addition, root exudates such as carboxylic acids, amino acids, carbo­
hydrates, released from plant roots during the process of growth (Lica �
et al., 2018). These root exudates have a high impact on heavy metal
fractions in the rhizosphere interface and the enrichment of organic
matter can promote the reducible fractions, which may result in the
increase of reducible Cd fractions (Rugova et al., 2017). The residual Cd
in Trifolium repensL. rhizosphere soil was observed less than 40% and it
was beneficial for phytoremediation as the bioavailability of heavy
metal pollutants to plants was the major limitation of phytoremediation
process (Kaur et al., 2018).
3.3. Characteristics of rhizosphere microbial community
3.3.1. Microorganisms counts during phytoremediation
The abundances of populations of culturable microbes in rhizosphere
soils during 16 weeks of phytoremediation by Trifolium repensL. are
showed in Fig. 3. The amounts of culturable microbes were significantly
inhibited in Cd contaminated groups at the 0th, 4th, 9th and 16th week,
though at the 16th week it was almost restored to the uncontaminated
level. During different periods, the number of microbes in rhizosphere
soil showed great difference in high concentration Cd contaminated soil
compare to others samples. The number of culturable microbes was
gradually increased at the first 9 weeks and maximum at the 9th week.
In general, the count of CL group did not fluctuate greatly and was stable
at around 200 � 106 CFU g 1.
In our study, the microbial populations in CM and CH were lower
than in CL due to the biotoxicity of heavy metals. Similarly, the results of
Adília Oliveira and Jing Pan’s research also showed that the addition of
toxic substances could reduce the number of microorganisms (Oliveira
and Pampulha, 2006; Pan and Yu, 2011). Nevertheless, the microbial
populations in Cd contaminated soil were rapidly increased at the first 9
weeks, which can be by the fact that the acid-soluble Cd fractions were
decreased after phytoremediation (Fig. 2). Previous studies have re­
ported that long-term exposure to heavy metal pollution can make some
microorganisms adapted to heavy metal stress and obtained heavy metal
resistance (Chen et al., 2018). These results indicated that the soil
ecosystem could recover the reduction of microbial abundance in the
rhizosphere soil caused by toxic chemicals (Du et al., 2018). In addition,
our results also demonstrated that, under the effect of phytoremediation,
4
Ecotoxicology and Environmental Safety 202 (2020) 110958
Fig. 3. Responses of rhizosphere soil culturable microbial populations to Cd
exposure during phytoremediation. Each bar is the mean of five replicates. Bars
with different letters indicate significant differences between the different
treatments based on LSD (P < 0.05).
the resistance of microbe-plant system to heavy metals was enhanced.
Abundant microorganisms are thought to be beneficial to the growth of
plants as they contributed to soil nutrient structure, enhance resistance
of plants to heavy metals and secrete plant growth-promoting hormones
(2018).
3.3.2. Microbial metabolic activity and substrate utilization
The soil microbial metabolic activity was evaluated by the AWCD of
Biolog EcoPlate™ and a higher AWCD indicates a higher microbial
metabolic activity. Among the three groups, the AWCD of CL and CM
was stabilized at about 0.7 and 1.0 respectively, while the value of CH
group was gradually increased with the increase of times. Fig. 4 illus­
trates the effects of Cd on variation in AWCD and the AWCD curves of all
samples were essentially the same over time. In the first 25 h, the AWCD
values almost remain unchanged, which may due to the lag phase of
microorganisms. Then the microorganisms entered the rapid-growth
stage, and reached stability at about 125 h. Differently, the high con­
centration of heavy metal ion restrained the growth and metabolize of
microorganism, leading to the lag-phase extended at the beginning of
plant growth. The lag phase was shortened with increasing exposure
time and the AWCD was promoted by the presence of Cd from the fourth
week compared to CL.
In order to further analyze the utilization of different carbon sources
by rhizosphere soil microorganisms, the carbon sources were classified
according to functional groups. In CL, the fractional content of amino
acids and carboxylic acids increased gradually while esters decreased
sharply from 0.23 to 0.17. Besides, as one of the most familiar carbon
resources, the fractional content of carbohydrate always kept at the
forefront in CL. However, in CM and CH, the percentage of carbohy­
drates was felled, which may cause by the increase of total utilization of
carbon sources. In contrast, the fractional content of both alcohols and
amines was always maintained below 0.1 in the CL, but in CM and CH,
the utilization of microorganisms was different and microbial utilization
of alcohols and amines obviously stronger than in CL. With regard to CH
samples, alcohols became the most preferred carbon source at the 9th
week. In addition, polymers, and esters, which were the major carbon
sources utilized in the CL, have been decreased in CM and CH. The
change of the carboxylic acid fraction content was consistent among all
groups that the fractional content was increased with the time went on,
but the increase in CM and CH was more remarkable.
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958

Fig. 4. Effects of Cd treatment on average well color development on the (A) 0th weeks, (B) 4th week, (C) 9th week, (D) 16th week. Variation in average well color
development (AWCD) over time in ECO plates of (E) CL, (F) CL and (G) CH during Cd-contaminated soil phytoremediation by Trifolium repensL. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of this article.)

The AWCD value in EcoPlate wells represents the ability of soil mi­
croorganisms to utilize different carbon sources so that it becomes an
important indicator of microbial functional diversity (Zhu et al., 2017).
Previous studies have demonstrated that the presence of heavy metals
can stimulate microbial metabolic activities (2018). Further, both the
bacteria quantity and diversity of microbial community can cause the
dynamic changes of microbial metabolic activity (Wu et al., 2017; Zhao
et al., 2016). The lower carbon conversion and the reduction of soil
microbial diversity would lead to the decrease of AWCD. During the
initial phase (before week 4), the AWCD of both Cd treatment grown
slowly due to the lower microbial population (Fig. 2). At the 0th week,
the lowest AWCD was observed in CH, because the biological activity of
some microorganisms was inhibited in high-cadmium environment. In
the following weeks, the lag-phase in Cd treatment obviously decreased
because of the increase of microbial number. The microbial carbon
source utilization ability in CH increased moving toward to even exceed
uncontaminated group, indicating that Trifolium repensL. planting
increased the population and heavy metal resistance of rhizosphere soil

5
C. Liu et al.
microorganisms, as well as metabolic capacities.
The utilization of carbon sources was largely affected by microbial
species and soil physical and chemical characteristics. In present study,
this difference may be caused by the Cd contamination, and with the
concentration of Cd increased, a growing number of microorganisms lost
their metabolic activity. But soon at 4th week, the biotoxicity of heavy
metals decreased due to Trifolium repensL. planting, and the life activities
some microbial species even were stimulated, result in the changes of
substrates utilization profiles. Previous study has demonstrated that the
total nitrogen content in soil was negatively correlated with heavy
metals and the deficiency of nitrogen content may lead to the
enhancement of utilization of nitrogen-containing substrates (amines
and amino acids) by microorganisms (Chen et al., 2018). As we know,
organic acids and amino acids account for a large proportion and di­
versity of root exudates (Haichar et al., 2014). Besides, these root exu­
dates were believed as an important factor affecting the formation of
rhizosphere community (Sasse et al., 2018). Therefore, during the pro­
cess of phytoremediation, the capability of microorganisms to utilize
carboxylic acids and amino acids gradually increased, which was re­
flected in the enhanced utilization of these substrates. Heavy metals
cause soil nutrient deficiency but also promote the utilization of carbon
sources by rhizosphere microorganisms (Fig. 4) (Huang et al., 2018). In
general, rhizosphere microorganisms play a regulatory role in plant
growth, while the diversified carbon source utilization capacity con­
tributes to the formation of diverse rhizosphere community microor­
ganisms helps with the survival and colonization for microbes in harsh
environment, which may promote the remediation efficiency of heavy
metals contaminated soils (Wang et al., 2019).
3.3.3. Microbial community structure
The Shannon index and Simpson index were used to evaluate the
Alpha diversity of rhizosphere soil before and after phytoremediation
(Fig. 5A and B). Species richness was reflected by Shannon index while
species uniformity was embodied by Simpson index. After phytor­
emediation, the value of Shannon index increased while Simpson index
decreased among the three groups, indicating both the species richness
and species uniformity were increased by Trifolium repensL. planting. To
better understand the microbial community composition in rhizosphere
soil, a heatmap showed the relative microbial abundance of each genus
is showed in Fig. 5C. As shown, the composition of microorganisms is
mainly concentrated on few species of bacteria prior to phytor­
emediation, but the distribution was more uniform after phytor­
emediation according to the color change of the heatmap. Genus
composition in different treatments rhizosphere soil differed markedly
before and after phytoremediation by Trifolium repensL, indicating that
the Cd contamination and plant growing greatly affected the microbial
composition. Specifically, Kaistobacter and Flavisolibacter were obviously
increased while Bacillus, Adhaeribacter and Pontibacter decreased.
Hainan Lu et al. have also found that the abundance of Kaistobacter could
be changed in plant treatments (Lu et al., 2019). Kaistobacter is
frequently detected in terrestrial environments and it is associated with
disease suppression and able to degrade some toxic refractory organics,
such as phenanthrene (Kumar et al., 2018; Li et al., 2019; Suzuki et al.,
2018; Zhang et al., 2018a, b). It is also a kind of microorganism that can
carry out photosynthesis and contribute to the stability of soil ecosystem
especially in the harsh environment of hypoxia and lack of light.
(Thomas et al., 2017; Zhong et al., 2018). Previous study reported that
some toxic chemicals can stimulate the abundance of Flavisolibacter,
which supported our findings. It was reported that the relative abun­
dance of Flavisolibacter is much higher in Cd contaminated soil before
phytoremediation (Lin et al., 2016). However, after phytoremediation,
Flavisolibacter significantly increased in CL group, which may cause by
the variation of soil properties and plant root exudates. Flavisolibacter is
positive with the organic acids and the root exudates may be responsible
for its increase after phytoremediation (Lin et al., 2016). Flavisolibacter,
belonging to Bacteroidetes, is functional of catalyzing hydrogen peroxide
6
Ecotoxicology and Environmental Safety 202 (2020) 110958
to protect itself and host plants from heavy metal oxidation (Janar­
thanan et al., 2016). It is found to be related to the nutrients of the soil as
it exhibits the ability to secrete acid phosphatases, fix CO2 and it is
correlated with ammoxidation (Lian et al., 2017; Liu et al., 2016; Yu
et al., 2016). In summary, the increase of Kaistobacter and Flavisolibacter
could accelerate the nutrient cycling in rhizosphere soil, which may
promote plant growth.
At the cluster level, the distance matrix analysis showed that Cd
contaminated samples shared a relatively high similarity in community
composition, indicating that they had a similar bacterial structural ho­
mology, and the uncontaminated group was distinct from them. This
observation was also supported by the result of PCA analysis (Fig. 5 D),
in which the longest space distance (maximum community dissimilarity)
emerged between CL-1 and CH-1. When the soil environment changed,
microorganisms would start self-regulation function to adjust commu­
nity structure and composition, and affect the diversity and evenness of
microorganisms (Zhao, 2019). In our study, heavy metal altered soil
microbial communities while phytoremediation influenced more on the
composition and abundance of microbial species. Beat Frey’s study has
proposed that heavy metal exposure greatly influenced the microbial
activity and community composition in the soil (Frey et al., 2006). When
exposed to heavy metals contamination, both the biological activity and
the population of sensitive microorganisms was greatly decreased,
leading to the reduction of their proportion in the community, while
other heavy metal resistant species could maintain and even be stimu­
lated vital life processes, becoming the dominant strains (Xu et al.,
2018). Such acclimation could decrease the negative effect of heavy
metals toxicity and stimulate the formation of new microbial pop­
ulations with heavy metal resistance, which may explain the equilibrium
of microbial species in various samples (Demanou et al., 2006). Phy­
toremediation plays an important role in improving the richness and
diversity of microbial communities in polluted soil. On the one hand,
plants affect microbial communities by protecting from biotic and
abiotic stressors, providing habitats, and producing root exudates to
regulate metabolism (Feng et al., 2017). On the other hand, plants
regulate the characteristics and diversity of microorganisms through the
changes of heavy metal bioavailability and soil physiochemical prop­
erties (Luo et al., 2018). Plant root exudates could relieve the effective
toxicity of heavy metals to plants and sustain higher microbial activities
and microbial densities as well (Chen et al., 2014; Smalla et al., 2001).
3.3.4. Correlation between substrate utilization and rhizosphere microbes
To further grasp the characteristics of microbial community, the
correlation of metabolic capacity and structural composition of micro­
bial community were formed based on Pearson’s correlation coefficients
(Fig. 6). Results showed that different microorganisms contributed
differently to the substrate utilization. The variables with r ​ >
​ 0:9 ​ or ​ r ​ < ​ 0:9 were considered to be significantly correlated.
For instance, D,L-α-Glycerol phosphate were closely related with Meth­
ylophaga, Lactobacillus, Cellvibrio and Bacteroides. Moreover, Glycogen
was negatively correlated to three microbiological species, including
Balneimonas, Streptomyces and Rhodocytophaga. Kaistobacter, Methyl­
ophaga, Devosia, Lactobacillus, Rhodoplanes, Cellvibrio, Methyloversatilis,
Bacteroides and Pseudomonas were positively correlated to most of the
substrates, indicating the strong carbon source utilization capacity.
Coincidently, the relative abundance of those kinds of genes were all
increased after phytoremediation (Fig. 5 C).
The differences in the utilization of 31 common carbon sources by
soil microorganisms were caused by the diverse functional groups of
organic compounds(Wang et al., 2019). In general, among all substrates,
benzene-containing compounds, polymers and partial carboxylic acids
were difficult to utilize, but in our study, phytoremediation enhanced
the utilization of difficultly metabolized compounds. An increase in the
utilization of substrates by soil microbes may cause by the changes of
microbial community composition and diversity during the phytor­
emediation process by Trifolium repensL. The growth of Trifolium repensL.
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958

Fig. 5. The microbial diversity index in

rhizosphere soils under different treat­
ment. (A) Shannon index; (B) Simpson
index. (C) Heatmap showing the levels of
relative microbial abundance of each
genus in the two different phytor­
emediation periods. Different color blocks
represent different microbial genera. (D)
PCA of the rhizosphere soil microbial
community based on the OUT, according
to the different treatments. CL-1, CM-1
and CH-1 represent the rhizosphere soil
before phytoremediation while CL-2, CM-
2 and CH-2 represent the rhizosphere soil
after phytoremediation. (For interpreta­
tion of the references to color in this figure
legend, the reader is referred to the Web
version of this article.)

7
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958

Fig. 6. Correlations among substrate utilization and the most abundant genera. The white box corresponds to r > 0.90 while the yellow box corresponds to r <
0.90. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

is accompanied by the release of carboxyl, hydroxyl and amino com­
pounds in rhizosphere, attracting a large number of microorganisms that
can use these organic compounds as carbon source (Johnston-Monje and
Raizada, 2011). Such microorganisms accumulated and reproduced in
the rhizosphere, and then gradually became the dominant bacteria. As
one of the main driving factors for shaping the structure of soil microbial
community, the composition of plant root exudates is largely depended
on plant developmental stages, which is also an reason for the differ­
ences of microbial community and carbon source utilization during
phytoremediation (Li et al., 2017). In this study, microorganisms with
increased relative abundance adapted to the changes of soil organic
matter composition quickly rather than being inhibited and could utilize
a wider range of carbon sources (Fig.4; Fig.5). These genera were able to
regulate the stability of plant-microbial system and the stability of
plant-microbial system played an important role in maintaining soil
ecological function (Müller et al., 2002). More importantly, Wang et al.
have already proposed that the metabolism ability and activity of soil
microorganisms were critical in environmental pollution control (Wang
and Oyaizu, 2011).
4. Conclusion
A pot experiment was designed to study the microbial metabolic
activities and community structure in rhizosphere during heavy metal
contaminated soil phytoremediation by Trifolium repensL. The results
showed that heavy metal stress decreased plant growth but enhanced

8
C. Liu et al.
metal uptake both in roots and shoots, which may explain the decrease
of acid-soluble Cd fractions. High concentration of heavy metal inhibi­
ted the AWCD and substrate utilization at the initial time, while during
phytoremediation, it was gradually recovered and even was stimulated
at harvest. Heavy metal altered the soil microbial communities while
phytoremediation influenced more on the composition and abundance
of microbial species. Trifolium repensL. planting significantly increased
the rhizosphere microbial population, diversity, the relative abundance
of plant growth promoting bacteria, and the utilization of difficultly
metabolized compounds. The correlation analysis among substrate uti­
lization and microbial communities revealed that the relative abun­
dance increased microorganisms possessed stronger carbon utilization
capacity, which was beneficial to regulate the stability of plant-
microbial system. The improving of microbial diversity and metabolic
activity was related to plant adaptation to heavy metal stress. Further
studies are needed to determine the influence of exogenous plant
beneficial bacteria inoculation on phytoremediation effects.
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.
CRediT authorship contribution statement
Chenjing Liu: Conceptualization, Writing - original draft, Investi­
gation, Formal analysis, Data curation. Hai Lin: Conceptualization,
Supervision, Validation, Resources, Writing - review & editing, Funding
acquisition, Project administration. Bing Li: Writing - original draft,
Data curation. Yingbo Dong: Conceptualization, Supervision, Writing -
review & editing. Tingting Yin: Investigation, Formal analysis.
Acknowledgements
This work was financially supported by Open Fund of National En­
gineering Laboratory for Site Remediation Technologies, China (No.
NEL-SRT201706), the Fundamental Research Funds for the Central
Universities (FRF-TP-19-019 A) and National Key R&D Program of
China (2018YFC0604604).
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