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Dehdezi, et al.

to identify the key bacteria that are crucial for the 2.2. Plant sampling and analysis
remediation process. 2.2.1. Leaves and roots dry weight measurement
Leaf and root samples were dried at 70°C for 48 h and
2. Materials and methods subsequently weighed.

2.1. Soil preparation and plant material 2.2.2. HM leaching and analytical process
Soil samples were taken from Chitgar Forest Park, After harvesting, the leaves and roots of V. zizanioides
Tehran, Iran, and were transferred to the laboratory. were carefully washed with deionized water to remove
According to the soil taxonomy, the samples were any surface contaminants. The washed plant material
classified as typic haplocambids. The samples were was then dried at 70°C for 48 h to ensure complete
air-dried at room temperature; a 2 mm mesh screen moisture removal. Once dried, the plant tissues were
was used to sieve the samples. In addition, the soil finely ground to a homogeneous powder to facilitate
texture type was defined using a hydrometer test, and efficient digestion and analysis. For metal analysis, the
soil electrical conductivity, cation-exchange capacity, samples were digested in a mixture of concentrated
pH, total organic carbon, CaCO3, and elements were nitric acid (HNO3) and hydrogen peroxide (H2O2) in
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measured according to standard methods (Table 1). order to break down the plant matrix and release the
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Cd(NO ) solution was applied as a single dose, metals into solution. This step was crucial to ensure
3 2
at concentrations of 20 and 60 mg/kg soil, with the that the metals of interest were fully extracted from the
control group (0 mg/kg) consisting of soil without plant material. The digestates were then filtered and
metal addition. Table 2 presents the treatment plan for diluted with deionized water as needed. Finally, metal
various soil samples. All experiments involving these concentrations in the resulting solutions were determined
treatments were replicated 3 times. All soil samples were using (furnace atomic absorption spectrometry; model
maintained under field capacity moisture conditions for 603 Perkin-Elmer, PerkinElmer, Inc., USA). This
3 weeks. Each pot contained 2 kg of soil. Eight seeds technique was chosen for its sensitivity and precision in
were planted in each pot. After 10 days, five unhealthy measuring trace metal concentrations.
seedlings were eliminated. The pots were weighed
and irrigated at intervals of 24 – 48 h. The plants were 2.3. Soil microbial community analysis
harvested once the growing season was completed. 2.3.1. DNA extraction
Microbial DNA from the total soil samples (Table 1)
®
Table 1. Chemical and physical characteristics of was extracted using the NucleoSpin Microbial DNA
substrates used in the study Isolation Kit (Macherey-Nagel, Germany) following the
Sand Silt N EC pH P K Zn Mn Fe manufacturer’s instructions. The quality of the extracted
DNA was assessed using Nano-drop ultraviolet-Vis’s
(%) (Ds/m) (mg/kg) spectrophotometer (Thermo Fisher Scientific, USA) as
27 53 0.09 62.3 7.8 18.9 940 0.63 5.43 3.53 previously reported by Yang et al. Then, agarose gel
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electrophoresis (1%) was applied to study the structural
Table 2. Experimental treatments integrity of the DNA molecules. DNA from the soil
Sample Treatment name samples was utilized to analyze bacterial diversity
abbreviation through 16S rRNA amplicon sequencing.
B Clean soil, no Cd, and no V. zizanioides
plants (negative control) 2.3.2. Illumina sequencing data analysis
V Clean soil in the presence of V. zizanioides In the present study, the impact of Cd-phytoremediation
plants (positive control) by V. zizanioides grass on the soil microbial diversity
Cd20 Soil with 20 mg/kg Cd+ was investigated using NGS. The 16S protocol was
V. zizanioides plants designed to amplify both bacteria and archaea using
Cd60 Soil with 60 mg/kg Cd+ paired-end 16S community sequencing on the Illumina
V. zizanioides plants platform. PCR amplification was performed following
Abbreviation: V. zizanioides: Vetiveria zizanioides; B: Clean soil standard procedures, using primers with barcodes:
without Vetiveria zizanioides; V: Clean soil in the presence of 515F (GTGCCAGCMGCCGCGGTAA) and 806R
V. zizanioides; Cd20: Cd-contaminated soil (20 mg/kg); (GGACTACHVGGGTWTCTAAT), which target the
Cd60: Cd-contaminated soil (60 mg/kg). V4 region of the 16S rRNA. Subsequently, sequencing

Volume 22 Issue 2 (2025) 34 doi: 10.36922/AJWEP025040021