Phytorem of CD and Microbial Community Effects
Phytorem of CD and Microbial Community Effects
Phytorem of CD and Microbial Community Effects
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.
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. Ecotoxicology and Environmental Safety 202 (2020) 110958
(Ramakrishna et al., 2019). Among all soil microorganisms, rhizosphere 2. Materials and methods
microorganisms, which are defined as “microorganisms in the zone of
soil surrounding the root which is affected by it”, strongly affect plant 2.1. Experimental reagents and soils
rhizosphere activities due to its direct contact with plant roots
(Sugiyama, 2019). The collective genome of rhizosphere microbial All the reagents used in the experiment were of analytical grade from
communities is also referred to as the plant’s second genome, indicating Sinopharm Chemical Reagent Co., Ltd. Experimental soils with different
they are an essential part of plant rhizosphere (Berendsen et al., 2012). Cd pollution level were collected from Beijing, China and the physico
Actually, each plant possesses its own specific microflora in the rhizo chemical properties of the soil were summarized in Table 1. The soils
sphere, which plays an important role in host plant growth promotion were collected in the same area with different pollution levels. After
and heavy metal detoxification/removal (Lal et al., 2018). As to enhance collected using a stainless-steel shovel at the depth of 0–30 cm, the soil
plant growth, rhizosphere microorganisms secrete IAA (indole acetic was air dried and mixed evenly.
acid), product ACC (1-aminocyclopropane-1-carboxylate) deaminase,
synthetize plant antioxidative enzymes and improve the uptake of 2.2. Plant growth conditions and treatments
essential mineral elements (Etesami and Maheshwari, 2018). Further,
rhizosphere microorganisms help to tolerate and alleviate negative Trifolium repensL. seeds were sterilized with 2% (v/v) sodium hy
impact of abiotic stresses including drought, salinity and pollution of pochlorite for 30 min and then rinsed five times with sterilized water to
heavy metal and antibiotic (Singh et al., 2018). Some of the rhizosphere remove the former solutions. Seeds were pre-germinated at a 90-mm
microorganisms are able to decrease the proportion of phytoavailability plate at 25 � C and kept moist for further treatment. Fifteen seeds were
heavy metals by adsorption, oxido-reduction reactions, extracellular sown in each pot (15 cm in diameter and 15 cm in high) with 750 g soil
matrix and immobilization, and others can improve P nutrition, adjust and ten seedlings were kept after germination. All the pots were placed
osmotic stress under environmental stresses (Nadeem et al., 2014). randomly in the greenhouse with the temperature at 20–25 � C and
During the process of phytoremediation, rhizosphere microorgan watered by deionized water daily.
isms play an important role in plant regulation and this regulation After 16 weeks’ growing, the plants were all carefully harvested from
greatly depends on microbial metabolism and diversity. Generally, the pots. After washed with tap water and deionized water to remove the
metabolic activity and community structure of soil microorganisms are soil and dried the surface with filter paper, plants were measured the
important indicators of soil health, determining soil functionality (Wang growth parameters. For Cd concentration analysis, the plants were
et al., 2019). In soil with heavy metal contamination, the microbial freeze-dried at 50 � C and then grinded to powder with particle size less
community constantly adjusted itself as well as the metabolic capacity to than 0.5 mm (He et al., 2013). The samples were digested by microwave
adapt to different habitats (Zhao, 2019). Meanwhile, the effects of and the cadmium content was analyzed by Inductively Coupled Plas
rhizosphere microorganisms on plant growth and heavy metal enrich ma–Mass Spectrometry (ICP-MS) (SHIMADZU, 2030) in Research Cen
ment also changed, which is considered to be of great significance in ter for Eco-Environmental Science, Chinese Academy of Sciences.
regulating phytoremediation (Zhao, 2019). Microbial communities help
plants resist the toxicity of heavy metal by increasing the abundance of C
2.3. Rhizosphere soil sampling and analysis
and N immobilized microorganisms and the activity of metabolic en
zymes (Das et al., 2017).
2.3.1. Rhizosphere soil collection and Cd fractionation analysis
Nevertheless, the information on microbial metabolic activities and
Rhizosphere soil were sampled within 0–15 cm, obtained from soil
community structure variation during heavy metal contaminated soil
adhering to plant roots four times: at the 0th, 4th, 9th and 16th week
phytoremediation is very limited. Therefore, in our research, the mi
after planting the seeds. Then, these samples were divided into two
crobial metabolism characteristics and community structure and
parts. One part was immediately used for measuring the number of
composition in the rhizosphere soil during Cd contaminated soil phy
culturable microorganism, microbial metabolic activity and DNA
toremediation by Trifolium repensL. were studied. Trifolium repensL. is
extraction and 16 S rRNA gene amplicon sequencing; the other part was
one of the most important genera of the Leguminosae family with good
air-dried and used for measuring Cd fractionation of after being ground
adaptability to the environment. Under the appropriate conditions of
and sieved through a 0.1-mm mesh.
sunlight and moisture, it can grow with well-developed root and large
Community Bureau of Reference (BCR) sequential extraction pro
biomass in a wide temperature range and in nearly any soil type (Zhang
cedure was applied to rhizosphere soil samples to analyze the chemical
et al., 2018a, b). Previous study has already reported that Trifolium
fractionation of Cd. Acid soluble: 40 mL 0.11 M CH3COOH (16 h, room
repensL. has strong resistance and good removal ability in phytor
temperature); Reducible: 40 mL 0.5 M NH2OH⋅HCl (16 h, room tem
emediation of contaminated soils (Hazrat Ali, 2012). Our study aimed
perature); Oxidizable: 10 mL 8.8 M H2O2 þ 10 mL 8.8 M H2O2 þ50 mL 1
to: I) acquire cadmium enrichment characteristics of Trifolium repensL.
M NH4OAc (1 h, 85 � C; 1 h, 85 � C; 16 h, room temperature); Residual:
and heavy metal distribution in rhizosphere soil; II) analyze the dynamic
digested with microwave dissolver (Oppler Instruments Co., Ltd) by 10
metabolic activities of rhizosphere microbes; III) study the rhizosphere
mL mixed acid (HCl: HNO3: HF ¼ 2:1:1).
microbial community and abundance; IV) determine the correlation
between substrate utilization and microbial communities.
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
Table 1
The physicochemical properties of experimental soils.
Item pH Available N (mg/kg) Available P (mg/kg) Available K (mg/kg) Organic matter (g/kg) Cd concentration (mg/kg)
ND: No detection.
2
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
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.
3
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
Fig. 2. The results of Cd concentrations of rhizosphere soils using the BCR Fig. 3. Responses of rhizosphere soil culturable microbial populations to Cd
sequential extraction. exposure during phytoremediation. Each bar is the mean of five replicates. Bars
with different letters indicate significant differences between the different
reflected its high Cd tolerance. After phytoremediation by Trifolium treatments based on LSD (P < 0.05).
repensL., the fraction of acid-soluble Cd decreased, revealing that
Trifolium repensL. possessed the ability to extract heavy metals. In the resistance of microbe-plant system to heavy metals was enhanced.
addition, root exudates such as carboxylic acids, amino acids, carbo Abundant microorganisms are thought to be beneficial to the growth of
hydrates, released from plant roots during the process of growth (Lica � plants as they contributed to soil nutrient structure, enhance resistance
et al., 2018). These root exudates have a high impact on heavy metal of plants to heavy metals and secrete plant growth-promoting hormones
fractions in the rhizosphere interface and the enrichment of organic (2018).
matter can promote the reducible fractions, which may result in the
increase of reducible Cd fractions (Rugova et al., 2017). The residual Cd 3.3.2. Microbial metabolic activity and substrate utilization
in Trifolium repensL. rhizosphere soil was observed less than 40% and it The soil microbial metabolic activity was evaluated by the AWCD of
was beneficial for phytoremediation as the bioavailability of heavy Biolog EcoPlate™ and a higher AWCD indicates a higher microbial
metal pollutants to plants was the major limitation of phytoremediation metabolic activity. Among the three groups, the AWCD of CL and CM
process (Kaur et al., 2018). 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
3.3. Characteristics of rhizosphere microbial community 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
3.3.1. Microorganisms counts during phytoremediation values almost remain unchanged, which may due to the lag phase of
The abundances of populations of culturable microbes in rhizosphere microorganisms. Then the microorganisms entered the rapid-growth
soils during 16 weeks of phytoremediation by Trifolium repensL. are stage, and reached stability at about 125 h. Differently, the high con
showed in Fig. 3. The amounts of culturable microbes were significantly centration of heavy metal ion restrained the growth and metabolize of
inhibited in Cd contaminated groups at the 0th, 4th, 9th and 16th week, microorganism, leading to the lag-phase extended at the beginning of
though at the 16th week it was almost restored to the uncontaminated plant growth. The lag phase was shortened with increasing exposure
level. During different periods, the number of microbes in rhizosphere time and the AWCD was promoted by the presence of Cd from the fourth
soil showed great difference in high concentration Cd contaminated soil week compared to CL.
compare to others samples. The number of culturable microbes was In order to further analyze the utilization of different carbon sources
gradually increased at the first 9 weeks and maximum at the 9th week. by rhizosphere soil microorganisms, the carbon sources were classified
In general, the count of CL group did not fluctuate greatly and was stable according to functional groups. In CL, the fractional content of amino
at around 200 � 106 CFU g 1. acids and carboxylic acids increased gradually while esters decreased
In our study, the microbial populations in CM and CH were lower sharply from 0.23 to 0.17. Besides, as one of the most familiar carbon
than in CL due to the biotoxicity of heavy metals. Similarly, the results of resources, the fractional content of carbohydrate always kept at the
Adília Oliveira and Jing Pan’s research also showed that the addition of forefront in CL. However, in CM and CH, the percentage of carbohy
toxic substances could reduce the number of microorganisms (Oliveira drates was felled, which may cause by the increase of total utilization of
and Pampulha, 2006; Pan and Yu, 2011). Nevertheless, the microbial carbon sources. In contrast, the fractional content of both alcohols and
populations in Cd contaminated soil were rapidly increased at the first 9 amines was always maintained below 0.1 in the CL, but in CM and CH,
weeks, which can be by the fact that the acid-soluble Cd fractions were the utilization of microorganisms was different and microbial utilization
decreased after phytoremediation (Fig. 2). Previous studies have re of alcohols and amines obviously stronger than in CL. With regard to CH
ported that long-term exposure to heavy metal pollution can make some samples, alcohols became the most preferred carbon source at the 9th
microorganisms adapted to heavy metal stress and obtained heavy metal week. In addition, polymers, and esters, which were the major carbon
resistance (Chen et al., 2018). These results indicated that the soil sources utilized in the CL, have been decreased in CM and CH. The
ecosystem could recover the reduction of microbial abundance in the change of the carboxylic acid fraction content was consistent among all
rhizosphere soil caused by toxic chemicals (Du et al., 2018). In addition, groups that the fractional content was increased with the time went on,
our results also demonstrated that, under the effect of phytoremediation, but the increase in CM and CH was more remarkable.
4
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 initial phase (before week 4), the AWCD of both Cd treatment grown
croorganisms to utilize different carbon sources so that it becomes an slowly due to the lower microbial population (Fig. 2). At the 0th week,
important indicator of microbial functional diversity (Zhu et al., 2017). the lowest AWCD was observed in CH, because the biological activity of
Previous studies have demonstrated that the presence of heavy metals some microorganisms was inhibited in high-cadmium environment. In
can stimulate microbial metabolic activities (2018). Further, both the the following weeks, the lag-phase in Cd treatment obviously decreased
bacteria quantity and diversity of microbial community can cause the because of the increase of microbial number. The microbial carbon
dynamic changes of microbial metabolic activity (Wu et al., 2017; Zhao source utilization ability in CH increased moving toward to even exceed
et al., 2016). The lower carbon conversion and the reduction of soil uncontaminated group, indicating that Trifolium repensL. planting
microbial diversity would lead to the decrease of AWCD. During the increased the population and heavy metal resistance of rhizosphere soil
5
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
microorganisms, as well as metabolic capacities. to protect itself and host plants from heavy metal oxidation (Janar
The utilization of carbon sources was largely affected by microbial thanan et al., 2016). It is found to be related to the nutrients of the soil as
species and soil physical and chemical characteristics. In present study, it exhibits the ability to secrete acid phosphatases, fix CO2 and it is
this difference may be caused by the Cd contamination, and with the correlated with ammoxidation (Lian et al., 2017; Liu et al., 2016; Yu
concentration of Cd increased, a growing number of microorganisms lost et al., 2016). In summary, the increase of Kaistobacter and Flavisolibacter
their metabolic activity. But soon at 4th week, the biotoxicity of heavy could accelerate the nutrient cycling in rhizosphere soil, which may
metals decreased due to Trifolium repensL. planting, and the life activities promote plant growth.
some microbial species even were stimulated, result in the changes of At the cluster level, the distance matrix analysis showed that Cd
substrates utilization profiles. Previous study has demonstrated that the contaminated samples shared a relatively high similarity in community
total nitrogen content in soil was negatively correlated with heavy composition, indicating that they had a similar bacterial structural ho
metals and the deficiency of nitrogen content may lead to the mology, and the uncontaminated group was distinct from them. This
enhancement of utilization of nitrogen-containing substrates (amines observation was also supported by the result of PCA analysis (Fig. 5 D),
and amino acids) by microorganisms (Chen et al., 2018). As we know, in which the longest space distance (maximum community dissimilarity)
organic acids and amino acids account for a large proportion and di emerged between CL-1 and CH-1. When the soil environment changed,
versity of root exudates (Haichar et al., 2014). Besides, these root exu microorganisms would start self-regulation function to adjust commu
dates were believed as an important factor affecting the formation of nity structure and composition, and affect the diversity and evenness of
rhizosphere community (Sasse et al., 2018). Therefore, during the pro microorganisms (Zhao, 2019). In our study, heavy metal altered soil
cess of phytoremediation, the capability of microorganisms to utilize microbial communities while phytoremediation influenced more on the
carboxylic acids and amino acids gradually increased, which was re composition and abundance of microbial species. Beat Frey’s study has
flected in the enhanced utilization of these substrates. Heavy metals proposed that heavy metal exposure greatly influenced the microbial
cause soil nutrient deficiency but also promote the utilization of carbon activity and community composition in the soil (Frey et al., 2006). When
sources by rhizosphere microorganisms (Fig. 4) (Huang et al., 2018). In exposed to heavy metals contamination, both the biological activity and
general, rhizosphere microorganisms play a regulatory role in plant the population of sensitive microorganisms was greatly decreased,
growth, while the diversified carbon source utilization capacity con leading to the reduction of their proportion in the community, while
tributes to the formation of diverse rhizosphere community microor other heavy metal resistant species could maintain and even be stimu
ganisms helps with the survival and colonization for microbes in harsh lated vital life processes, becoming the dominant strains (Xu et al.,
environment, which may promote the remediation efficiency of heavy 2018). Such acclimation could decrease the negative effect of heavy
metals contaminated soils (Wang et al., 2019). metals toxicity and stimulate the formation of new microbial pop
ulations with heavy metal resistance, which may explain the equilibrium
3.3.3. Microbial community structure of microbial species in various samples (Demanou et al., 2006). Phy
The Shannon index and Simpson index were used to evaluate the toremediation plays an important role in improving the richness and
Alpha diversity of rhizosphere soil before and after phytoremediation diversity of microbial communities in polluted soil. On the one hand,
(Fig. 5A and B). Species richness was reflected by Shannon index while plants affect microbial communities by protecting from biotic and
species uniformity was embodied by Simpson index. After phytor abiotic stressors, providing habitats, and producing root exudates to
emediation, the value of Shannon index increased while Simpson index regulate metabolism (Feng et al., 2017). On the other hand, plants
decreased among the three groups, indicating both the species richness regulate the characteristics and diversity of microorganisms through the
and species uniformity were increased by Trifolium repensL. planting. To changes of heavy metal bioavailability and soil physiochemical prop
better understand the microbial community composition in rhizosphere erties (Luo et al., 2018). Plant root exudates could relieve the effective
soil, a heatmap showed the relative microbial abundance of each genus toxicity of heavy metals to plants and sustain higher microbial activities
is showed in Fig. 5C. As shown, the composition of microorganisms is and microbial densities as well (Chen et al., 2014; Smalla et al., 2001).
mainly concentrated on few species of bacteria prior to phytor
emediation, but the distribution was more uniform after phytor 3.3.4. Correlation between substrate utilization and rhizosphere microbes
emediation according to the color change of the heatmap. Genus To further grasp the characteristics of microbial community, the
composition in different treatments rhizosphere soil differed markedly correlation of metabolic capacity and structural composition of micro
before and after phytoremediation by Trifolium repensL, indicating that bial community were formed based on Pearson’s correlation coefficients
the Cd contamination and plant growing greatly affected the microbial (Fig. 6). Results showed that different microorganisms contributed
composition. Specifically, Kaistobacter and Flavisolibacter were obviously differently to the substrate utilization. The variables with r >
increased while Bacillus, Adhaeribacter and Pontibacter decreased. 0:9 or r < 0:9 were considered to be significantly correlated.
Hainan Lu et al. have also found that the abundance of Kaistobacter could For instance, D,L-α-Glycerol phosphate were closely related with Meth
be changed in plant treatments (Lu et al., 2019). Kaistobacter is ylophaga, Lactobacillus, Cellvibrio and Bacteroides. Moreover, Glycogen
frequently detected in terrestrial environments and it is associated with was negatively correlated to three microbiological species, including
disease suppression and able to degrade some toxic refractory organics, Balneimonas, Streptomyces and Rhodocytophaga. Kaistobacter, Methyl
such as phenanthrene (Kumar et al., 2018; Li et al., 2019; Suzuki et al., ophaga, Devosia, Lactobacillus, Rhodoplanes, Cellvibrio, Methyloversatilis,
2018; Zhang et al., 2018a, b). It is also a kind of microorganism that can Bacteroides and Pseudomonas were positively correlated to most of the
carry out photosynthesis and contribute to the stability of soil ecosystem substrates, indicating the strong carbon source utilization capacity.
especially in the harsh environment of hypoxia and lack of light. Coincidently, the relative abundance of those kinds of genes were all
(Thomas et al., 2017; Zhong et al., 2018). Previous study reported that increased after phytoremediation (Fig. 5 C).
some toxic chemicals can stimulate the abundance of Flavisolibacter, The differences in the utilization of 31 common carbon sources by
which supported our findings. It was reported that the relative abun soil microorganisms were caused by the diverse functional groups of
dance of Flavisolibacter is much higher in Cd contaminated soil before organic compounds(Wang et al., 2019). In general, among all substrates,
phytoremediation (Lin et al., 2016). However, after phytoremediation, benzene-containing compounds, polymers and partial carboxylic acids
Flavisolibacter significantly increased in CL group, which may cause by were difficult to utilize, but in our study, phytoremediation enhanced
the variation of soil properties and plant root exudates. Flavisolibacter is the utilization of difficultly metabolized compounds. An increase in the
positive with the organic acids and the root exudates may be responsible utilization of substrates by soil microbes may cause by the changes of
for its increase after phytoremediation (Lin et al., 2016). Flavisolibacter, microbial community composition and diversity during the phytor
belonging to Bacteroidetes, is functional of catalyzing hydrogen peroxide emediation process by Trifolium repensL. The growth of Trifolium repensL.
6
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
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 regulate the stability of plant-microbial system and the stability of
pounds in rhizosphere, attracting a large number of microorganisms that plant-microbial system played an important role in maintaining soil
can use these organic compounds as carbon source (Johnston-Monje and ecological function (Müller et al., 2002). More importantly, Wang et al.
Raizada, 2011). Such microorganisms accumulated and reproduced in have already proposed that the metabolism ability and activity of soil
the rhizosphere, and then gradually became the dominant bacteria. As microorganisms were critical in environmental pollution control (Wang
one of the main driving factors for shaping the structure of soil microbial and Oyaizu, 2011).
community, the composition of plant root exudates is largely depended
on plant developmental stages, which is also an reason for the differ 4. Conclusion
ences of microbial community and carbon source utilization during
phytoremediation (Li et al., 2017). In this study, microorganisms with A pot experiment was designed to study the microbial metabolic
increased relative abundance adapted to the changes of soil organic activities and community structure in rhizosphere during heavy metal
matter composition quickly rather than being inhibited and could utilize contaminated soil phytoremediation by Trifolium repensL. The results
a wider range of carbon sources (Fig.4; Fig.5). These genera were able to showed that heavy metal stress decreased plant growth but enhanced
8
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
metal uptake both in roots and shoots, which may explain the decrease He, H., et al., 2013. Characterization of endophytic Rahnella sp. JN6 from Polygonum
pubescens and its potential in promoting growth and Cd, Pb, Zn uptake by Brassica
of acid-soluble Cd fractions. High concentration of heavy metal inhibi
napus. Chemosphere 90, 1960–1965.
ted the AWCD and substrate utilization at the initial time, while during Hiroki, M., 1992. Effects of heavy metal contamination on soil microbial population. Soil
phytoremediation, it was gradually recovered and even was stimulated Sci. Plant Nutr. 38, 141–147.
at harvest. Heavy metal altered the soil microbial communities while Huang, L., et al., 2018. Shift of soil bacterial community and decrease of metals
bioavailability after immobilization of a multi-metal contaminated acidic soil by
phytoremediation influenced more on the composition and abundance inorganic-organic mixed amendments: a field study. Appl. Soil Ecol. 130, 104–119.
of microbial species. Trifolium repensL. planting significantly increased Huang, B., et al., 2019. Loss characteristics of Cd in soil aggregates under simulated
the rhizosphere microbial population, diversity, the relative abundance rainfall conditions. Sci. Total Environ. 650, 313–320.
2018 Investigation on microbial community in remediation of lead-contaminated soil
of plant growth promoting bacteria, and the utilization of difficultly by Trifolium repensL. Ecotoxicol. Environ. Saf., 2018 52–60.
metabolized compounds. The correlation analysis among substrate uti Islam, M.N., et al., 2016. Chemical speciation and quantitative evaluation of heavy metal
lization and microbial communities revealed that the relative abun pollution hazards in two army shooting range backstop soils. Bull. Environ. Contam.
Toxicol. 96, 179–185.
dance increased microorganisms possessed stronger carbon utilization Janarthanan, O.M., et al., 2016. Fluxes in PHA-storing microbial communities during
capacity, which was beneficial to regulate the stability of plant- enrichment and biopolymer accumulation processes. N. Biotech. 33, 61–72.
microbial system. The improving of microbial diversity and metabolic Johnston-Monje, D., Raizada, M.N., 2011. Conservation and diversity of seed associated
endophytes in Zea across boundaries of evolution, ethnography and ecology. PloS
activity was related to plant adaptation to heavy metal stress. Further One 6, e20396.
studies are needed to determine the influence of exogenous plant Kaur, P., et al., 2018. Role of earthworms in phytoremediation of cadmium (Cd) by
beneficial bacteria inoculation on phytoremediation effects. modulating the antioxidative potential of Brassica juncea L. Appl. Soil Ecol. 124,
306–316.
Khalid, S., et al., 2017. A comparison of technologies for remediation of heavy metal
Declaration of competing interest contaminated soils. J. Geochem. Explor. 182, 247–268.
Kong, X., et al., 2013. Analysis of microbial metabolic characteristics in mesophilic and
The authors declare that they have no known competing financial thermophilic biofilters using Biolog plate technique. Chem. Eng. J. 230, 415–421.
Kumar, U., et al., 2018. Continuous application of inorganic and organic fertilizers over
interests or personal relationships that could have appeared to influence 47 years in paddy soil alters the bacterial community structure and its influence on
the work reported in this paper. rice production. Agric. Ecosyst. Environ. 262, 65–75.
Kumar Yadav, K., et al., 2018. Mechanistic understanding and holistic approach of
phytoremediation: a review on application and future prospects. Ecol. Eng. 120,
CRediT authorship contribution statement 274–298.
Lal, S., et al., 2018. Biosurfactant and exopolysaccharide-assisted rhizobacterial
Chenjing Liu: Conceptualization, Writing - original draft, Investi technique for the remediation of heavy metal contaminated soil: an advancement in
metal phytoremediation technology. Environ. Technol. Innov. 10, 243–263.
gation, Formal analysis, Data curation. Hai Lin: Conceptualization, Li, Y., et al., 2017. Characterizing rhizosphere microbial communities in long-term
Supervision, Validation, Resources, Writing - review & editing, Funding monoculture tea orchards by fatty acid profiles and substrate utilization. Eur. J. Soil
acquisition, Project administration. Bing Li: Writing - original draft, Biol. 81, 48–54.
Li, J., et al., 2019. Diversity of the active phenanthrene degraders in PAH-polluted soil is
Data curation. Yingbo Dong: Conceptualization, Supervision, Writing - shaped by ryegrass rhizosphere and root exudates. Soil Biol. Biochem. 128, 100–110.
review & editing. Tingting Yin: Investigation, Formal analysis. Lian, T., et al., 2017. Bacterial communities incorporating plant-derived carbon in the
soybean rhizosphere in Mollisols that differ in soil organic carbon content. Appl. Soil
Ecol. 119, 375–383.
Acknowledgements Lic�a, I.C.L., et al., 2018. Biological properties and pharmacological potential of plant
exudates. Food Res. Int. 105, 1039–1053.
This work was financially supported by Open Fund of National En Lin, H., et al., 2016. A compositional shift in the soil microbiome induced by tetracycline,
sulfamonomethoxine and ciprofloxacin entering a plant-soil system. Environ. Pollut.
gineering Laboratory for Site Remediation Technologies, China (No. 212, 440–448.
NEL-SRT201706), the Fundamental Research Funds for the Central Liu, L., et al., 2016. Relationships of decomposability and C/N ratio in different types of
Universities (FRF-TP-19-019 A) and National Key R&D Program of organic matter with suppression of Fusarium oxysporum and microbial communities
during reductive soil disinfestation. Biol. Contr. 101, 103–113.
China (2018YFC0604604). Liu, S., et al., 2019. A newly discovered Cd-hyperaccumulator Lantana camara L.
J. Hazard Mater. 371, 233–242.
References Lu, H., et al., 2019. Mixed-surfactant-enhanced phytoremediation of PAHs in soil:
bioavailability of PAHs and responses of microbial community structure. Sci. Total
Environ. 653, 658–666.
Berendsen, R.L., et al., 2012. The rhizosphere microbiome and plant health. Trends Plant
Luo, Y., et al., 2018. Bacterial community structure and diversity responses to the direct
Sci. 17, 478–486.
revegetation of an artisanal zinc smelting slag after 5 years. Environ. Sci. Pollut.
Chen, B., et al., 2014. Improvement of cadmium uptake and accumulation in Sedum
Control Ser. 25, 14773–14788.
alfredii by endophytic bacteria Sphingomonas SaMR12 : effects on plant growth and
Müller, A.K., et al., 2002. The diversity and function of soil microbial communities
root exudates. Chemosphere 117, 367–373.
exposed to different disturbances. Microb. Ecol. 44, 49–58.
Chen, L., et al., 2016. Sex-specific responses of Populus deltoides to Glomus intraradices
Nadeem, S.M., et al., 2014. The role of mycorrhizae and plant growth promoting
colonization and Cd pollution. Chemosphere 155, 196–206.
rhizobacteria (PGPR) in improving crop productivity under stressful environments.
Chen, Y., et al., 2018. Long-term and high-concentration heavy-metal contamination
Biotechnol. Adv. 32, 429–448.
strongly influences the microbiome and functional genes in Yellow River sediments.
Odoh, C.K., et al., 2019. Status, progress and challenges of phytoremediation - an African
Sci. Total Environ. 637–638, 1400–1412.
scenario. J. Environ. Manag. 237, 365–378.
Das, S., et al., 2017. Arsenic-enrichment enhanced root exudates and altered rhizosphere
Oliveira, A., Pampulha, M.E., 2006. Effects of long-term heavy metal contamination on
microbial communities and activities in hyperaccumulator Pteris vittata. J. Hazard
soil microbial characteristics. J. Biosci. Bioeng. 102, 157–161.
Mater. 325, 279–287.
Pan, J., Yu, L., 2011. Effects of Cd or/and Pb on soil enzyme activities and microbial
Demanou, J., et al., 2006. SchloterShifts in microbial community functions and nitrifying
community structure. Ecol. Eng. 37, 1889–1894.
communities as a result of combined application of copper and mefenoxam. FEMS
Ramakrishna, W., et al., 2019. Plant growth promoting bacteria in agriculture: two sides
Microbiol. Lett. 260, 55–62.
of a coin. Appl. Soil Ecol. 138, 10–18.
Du, Z., et al., 2018. Effects of the herbicide mesotrione on soil enzyme activity and
Rugova, A., et al., 2017. Elucidating rhizosphere processes by mass spectrometry – a
microbial communities. Ecotoxicol. Environ. Saf. 164, 571–578.
review. Anal. Chim. Acta 956, 1–13.
Etesami, H., Maheshwari, D.K., 2018. Use of plant growth promoting rhizobacteria
Sarwar, N., et al., 2017. Phytoremediation strategies for soils contaminated with heavy
(PGPRs) with multiple plant growth promoting traits in stress agriculture: action
metals: modifications and future perspectives. Chemosphere 171, 710–721.
mechanisms and future prospects. Ecotoxicol. Environ. Saf. 156, 225–246.
Sasse, J., et al., 2018. Feed your friends: do plant exudates shape the root microbiome?
Feng, N., et al., 2017. Efficient phytoremediation of organic contaminants in soils using
Trends Plant Sci. 23, 25–41.
plant–endophyte partnerships. Sci. Total Environ. 583, 352–368.
Singh, D., et al., 2014. Citric acid coated magnetic nanoparticles: synthesis,
Frey, B., et al., 2006. Microbial activity and community structure of a soil after heavy
characterization and application in removal of Cd(II) ions from aqueous solution.
metal contamination in a model forest ecosystem. Soil Biol. Biochem. 38,
J. Water Process Eng. 4, 233–241.
1745–1756.
Singh, V.K., et al., 2018. Interaction of plant growth promoting bacteria with tomato
Haichar, F.E.Z., et al., 2014. Root exudates mediated interactions belowground. Soil Biol.
under abiotic stress: a review. Agric. Ecosyst. Environ. 267, 129–140.
Biochem. 77, 69–80.
Hazrat Ali, M.N.M.A., 2012. Phytoremediation of heavy metals by Trifolium
alexandrinum. Int. J. Environ. Sci. 3, 1459–1469.
9
C. Liu et al. Ecotoxicology and Environmental Safety 202 (2020) 110958
Smalla, K., et al., 2001. Bulk and rhizosphere soil bacterial communities studied by Yu, Z., et al., 2016. Effectiveness of elevated CO2 mediating bacterial communities in the
denaturinggradient gel electrophoresis: plant-dependent enrichment and soybean rhizosphere depends on genotypes. Agric. Ecosyst. Environ. 231, 229–232.
seasonalshifts revealed. Appl. Environ. Microbiol. 67, 4742–4751. Zhang, H., et al., 2018a. Transcriptome analysis reveals potential genes involved in
Sugiyama, A., 2019. The soybean rhizosphere: metabolites, microbes, and beyond—a flower pigmentation in a red-flowered mutant of white clover (Trifolium repens L.).
review. J. Adv. Res. 19, 67–73. Genomics 110, 191–200.
Suzuki, Y., et al., 2018. Effects of bacterial pollution caused by a strong typhoon event Zhang, S., et al., 2018b. Influence of high-carbon basal fertiliser on the structure and
and the restoration of a recreational beach: transitions of fecal bacterial counts and composition of a soil microbial community under tobacco cultivation. Res.
bacterial flora in beach sand. Sci. Total Environ. 640–641, 52–61. Microbiol. 169, 115–126.
Thomas, T.B., et al., 2017. Postmortem microbial communities in burial soil layers of Zhao, Z., et al., 2016. Influences of iron and calcium carbonate on wastewater treatment
skeletonized humans. J. Forensic Legal Med. 49, 43–49. performances of algae based reactors. Bioresour. Technol. 216, 1–11.
Wang, Y., Oyaizu, H., 2011. Enhanced remediation of dioxins-spiked soil by a Zhao, X., et al., 2019. Study on the influence of soil microbial community on the long-
plant–microbe system using a dibenzofuran-degrading Comamonas sp. and Trifolium term heavy metal pollution of different land use types and depth layers in mine.
repens L. Chemosphere 85, 1109–1114. Ecotoxicol. Environ. Saf. 170, 218–226.
Wang, S., et al., 2019. Soil aggregate-associated bacterial metabolic activity and Zhong, Z., et al., 2018. Long-term effects of legume mulching on soil chemical properties
community structure in different aged tea plantations. Sci. Total Environ. 654, and bacterial community composition and structure. Agric. Ecosyst. Environ. 268,
1023–1032. 24–33.
Wu, M., et al., 2017. Bioremediation of hydrocarbon degradation in a petroleum- Zhu, L., et al., 2017. Microbial functional diversity responses to 2 years since biochar
contaminated soil and microbial population and activity determination. application in silt-loam soils on the Loess Plateau. Ecotoxicol. Environ. Saf. 144,
Chemosphere 169, 124–130. 578–584.
Xu, Y., et al., 2018. Biochar modulates heavy metal toxicity and improves microbial
carbon use efficiency in soil. Sci. Total Environ. 621, 148–159.
10