Phytoremediation Potential of Sorghum as a Bioenergy Crop in Pb-Amendment Soil

Abstract

Lead contamination is among the most significant threats to the environment. The phytoextraction approach uses plants that can tolerate and accumulate metals in their tissues. Lately, biofuel plants have been recommended to be suitable for remediation and implementation of potentially toxic elements (PTEs)-polluted soil. This research assessed the Pb phytoremediation potential of three Sorghum bicolor [red cultivar (S1), white cultivar (S2) and shahla cultivar (S3)]. A pot experiment with five treatments (0, 100, 200, 400 and 800 mg Pb/kg soil) was carried out to assess the potential possibility of using these cultivars to remediate the soil of Pb. The potential possibility of using these plants to phytoremediate the soil of Pb was also assessed. The results emphasized that all the examined cultivars could attain growth to maturity in high Pb spiked soil. However, Pb influenced morphological and chlorophyll contents, especially in plants grown in soil amended with 800 mg/kg. The S1 cultivar had the most significant reduction in total chlorophyll with an average of 72%, followed by the S2 and S3 cultivars (65% and 58% reduction, respectively). The highest Pb content in root (110.0, 177.6 and 198.9 mg/kg, respectively) and in-plant shoot (83.9, 103.6 and 99.0 mg/kg, respectively) were detected by sorghum (S1, S2 and S3, respectively) grown in soil enriched by 800 mg/kg of Pb. From the calculated results of the contamination indices, contamination factor (CF), translocation factor (TF), plant uptake (UT) and tolerance index (TI), none of the investigated cultivars were considered Pb hyperaccumulators, but all were identified as particularly ideal for phytostabilization.

1. Introduction

Soil is a valuable ecosystem that provides water, energy, nutrients and organic matter, all of which are necessary for the continued existence of a wide variety of organisms [1,2]. Due to unmanaged development, excessive amounts of potentially toxic elements (PTEs) are released into the environment [3]. Metal lead (Pb) is extremely toxic to humans. Inhaling or ingesting even trace amounts of lead is extremely harmful to human health [4,5,6,7]. Anthropogenic activities, such as battery recycling, mining, coal burning, pesticides, Pb-based paints, automobile exhaust and leather tanning, contribute to remarkable levels of Pb in the environment [8,9]. Alloway [10] observed a Pb concentration ranging from 2–300 g/kg in unpolluted soil, with a level of 100 g/kg being a concern to both the environment and humans. Reclamation of Pb-polluted soil is difficult due to its limited mobility, high toxicity, non-biodegradability and persistent nature [11,12,13]. As a result, scientists, policymakers and the general public throughout the world are increasingly concerned about Pb negative impacts on the environment. For the remediation of PTEs-polluted soil, several procedures, such as oxidation, physical separation, isolation and stabilization, are applied [14]. Although these approaches provide effective soil treatment, they are typically time-consuming, expensive, deplete soil fertility and ineffective for treating extremely PTEs-polluted soils [15]. The capability of plants to absorb contaminates, like Pb, could be exploited for substrate phytoremediation [16,17]. These methods include using plants that can absorb PTEs through their roots and then transport and accumulate them into harvestable parts [18,19]. In phytoextraction, plants absorb PTEs and store them in aboveground tissues. In phytostabilization, plants reduce the mobility of PTEs in the soil through root exudates. Few plants have been discovered as Pb hyperaccumulators, the majority of which are Brassicaceae [20,21]. Nonetheless, various studies have been conducted to identify remediation potential, compensating for a reduced Pb level in their tissues by growing fast, producing large biomass and being easily managed and harvested [22]. Through the application of phytoremediation technology, the Robinia pseudoacacia has been utilized successfully for the remediation of PTEs-contained soils and could be used successfully to extract 92.31% Cu and 46.64% Pb [23]. Sorghum (Sorghum bicolor) is a C₄ crop, considered the fifth most important cereal globally. Crops of sorghum can be used for various purposes, and the resulting goods range from syrup and brushes to paper and even building materials. Livestock and people both eat sorghum [24]. This crop can be tolerant and produce high biomass when grown in harsh conditions like drought, heat and pollution [25,26]. Because of its rapid growth and high biomass yield, sorghum crop is regarded as a promising energy plant. Sorghum crop is implanted in a variety of provinces in Saudi Arabia. It is the main cereal crop in the southern area of the Kingdom of Saudi Arabia [27].

Multiple studies demonstrated sorghum’s capacity to tolerate PTEs from a polluted substrate [28,29,30,31,32]. To reduce the potentially disastrous effects of PTEs and their transfer to crops, it is critical to evaluate the impact of soil characteristics on metal availability and crop uptake [33]. Predicting PTEs’ content by crop plants using variables in the soil, such as pH, organic matter (OM) and soil contents, is a useful application of regression equations [34]. According to the authors, no prediction equation is excited for the uptake of Pb metal ions by sorghum crops planted in soil enrichment with Pb. In this regard, only a few studies have been conducted to predict Pb uptake by sorghum plants based on soil characteristics, such as pH and OM. Furthermore, this study is regarded as one of the most recent studies dealing with the application of sorghum crop varieties for the remediation of Pb-contaminated soils in Saudi Arabia.

Consequently, the main objectives of the current study are: (i) to formulate novel prediction equations for predicting the efficiency of three tested cultivars of sorghum crops for uptake of Pb grown in soil amendment with Pb under greenhouse conditions; (ii) to assess and compare the phytoremediation efficiency of three tested cultivars of S. bicolor for removal Pb from contaminated soil; and (iii) to determine the Pb levels in various plant organs and how they affect the tested cultivars’ growth parameters. We hypothesize that using a prediction model to predict Pb levels in contaminated soils based on soil conditions, such as pH and OM, will be beneficial. Furthermore, sorghum crops can be used to remediate Pb-contaminated soils effectively.

2. Materials and Methods

2.1. Plant Materials

Seeds of different cultivars of Sorghum bicolor [red cultivar (S1), white cultivar (S2) and shahla cultivar (S3)] were identified and obtained from the Ministry of Environment Water and Agriculture, KSA.

2.2. Experimental Design

At Umm Al-Qura University in Saudi Arabia (21°18′56.3″ N, 39°56′47.8″ E), a pot experiment was conducted between October and December 2020. The temperatures and humidity levels in the greenhouse were controlled to maintain a range from 20 °C to 28 °C and 50 ± 4%, respectively. Several types of sorghum, grown for their bioenergy potential on Pb-contaminated soil, were put through an experiment to measure the amount the PTEs could absorb. The soil was collected from nearby farmland and sands at a ratio of 2:1; then it was air-dried, sieved and blended until it was uniform in texture (loamy sand). The electrical conductivity of this soil was 0.44 dS/cm (EC), and the pH was 7.56. The total background level of Pb was 8.18 ± 2.1 mg/kg, while the DTPA-Pb (available) background level was 2.10 ± 0.05 mg/kg. Lead was added to the soil as lead nitrate [Pb (NO₃)₂] at five different concentrations: 0, 100, 200, 400 and 800 mg Pb/kg dry soil. Based on the ALINORM [35] phytotoxicity threshold, the Pb levels of 400 and 800 mg/kg were chosen. After letting the treated soil equilibrate for around 30 days, a total of 90 individual pots were employed in a completely randomized design (3 cultivars, 5 treatments and 6 replicate). Five seeds of each cultivar were put into 4 kg of soil in pots measuring 21 cm in diameter and 23 cm in height, and the plants were then grown in a greenhouse for 80 days under natural day/light conditions. After 15 days of seeding, we manually weeded the pots and irrigated them twice a week with a drip system, reducing the number of plants per pot from 5 to 3 to remove weak plants and maintain healthy ones.

2.3. Growth Measurements

The crop plants under study were harvested 80 days after planting and dissected into roots and shoots. Each plant’s root and shoot lengths were recorded. Additionally, the fresh and dry weights (g/plant) were calculated by weighing the various components of each plant before and after oven-drying at 65 °C till constant weight.

2.4. Plant and Soil Analysis

For each treatment, we took three samples of the collected plant material, dried them in the oven (at 65 °C), ground them in a metal-free plastic mill until they were uniform in texture and sieved them through a 2-mm mesh sieve. The photosynthetic pigments were extracted from the fresh leaves, and their concentrations were determined colorimetrically. The content of chlorophyll a, b and carotenoids was calculated as described by Arnon [36].

Chlorophyll a (g/L) = 9.784(O.D.at 662) − 9.90(O.D.644)

(1)

Chlorophyll b (g/L) = 21.426(O.D.at 644) − 4.65(O.D.662)

(2)

Carotenoids (g/L) = 4.695(O.D.at 440.5) − 0.268(chlorophyll a + b)

(3)

Pb content was measured using an atomic absorption spectrophotometer (Shimadzu AA-6300; Shimadzu Co. Ltd., Japan) after a 1 g ground sample was digested with 20 mL of a tri-acid mixture consisting of HNO₃:H₂SO₄:HClO₄ (5:1:1, v/v/v) [37].

Three samples of soil were collected before planting crops, allowed to air dry and then sieved through a 2-mm mesh. Soil water extracts of 1/5 (w/v) were made and tested for pH and EC using a pH meter (Model 9107 BN, ORION type), and a conductivity meter (60 Sensor Operating Instruction Corning), respectively. Plant analytical methods were also used to assess the total Pb content of soil samples taken before cultivation. DTPA (diethylene triamine pentaacetate) extract was used to quantify the accessible Pb content in the soil before and after harvesting for each sorghum cultivar across treatments, as described by Lindsay and Norvell [38]. Ten gm of air-dried soil was extracted with 20 mL of DTPA extraction solution. After 2 h of shaking, suspensions were gravity filtered with Whatman 42 filter paper. Filtrates were analyzed for Pb content and measured using an atomic absorption spectrophotometer.

2.5. Data Analysis

Based on growth data, we used the method to determine the tolerance index (TI) of the three sorghum cultivars evaluated for Pb removal [39]. To predict PTEs’ absorption by plant roots from soil, the bioaccumulation factor (BAF_root) is a useful tool. In contrast, PTEs’ translocation from roots to shoots is measured by the translocation factor (TF). They were determined to find out which of the sorghum cultivars used in the study had the best capacity to take up Pb from the soil and move it along to their leaves [40]. Galal et al. [41] provided the following method for measurements:

BAF_root = C_root/C_soil

(4)

TF = C_shoot/C_root

(5)

where, C_soil, C_root and C_shoot are the Pb concentrations (mg/kg) in the soil, root and shoot, respectively.

Also, Pb uptake (μg/pot) was calculated according to Utmazian et al. [42].

TU (µg/pot) = concentration of Pb in roots or shoots dwt × dwt of roots or shoots g/pot

(6)

The simple linear correlation coefficient was used to analyze the association between plant-PTEs and soil factors (r). To validate the data, we chose eighteen shoot and root observations from the entire dataset. To estimate the concentration of Pb in three tested cultivars of sorghum tissues, based on soil pH, OM content and soil Pb as independent variables, we ran a regression approach utilizing 18 additional data for the same tissues. The quality of the models was determined by calculating the following metrics [43]: coefficient of determination (R²), model efficiency (ME), its strength (mean normalized average error, MNAE) and mean normalized bias (MNB).

ME = 1− {∑ (C_model − C_measured)²/(∑ (C_measured − C_mean)²}

(7)

MNAE = {∑ (|C_model − C_measured|/C_measured)}/n

(8)

MNB = ∑ (C_model − C_measured)/∑ C_measured

(9)

In this equation, C_model represents the projected PTEs concentration; C_measured represents the measured PTEs concentration; C_mean represents the mean of the measured PTEs concentration; and n represents the number of observations. PTEs concentrations in the validation dataset (n = 18 for each root and shoot) were estimated using the derived regression equations. Student’s t-test was used to analyze significant discrepancies between predicted and actual Pb levels in the same tissue [44].

2.6. Statistical Analysis

Data on plant characteristics and Pb-treatment parameters were log-transformed as necessary to ensure normality of distribution and homogeneity of variance before analysis of variance (ANOVA) was performed. Using a two-way analysis of variance (ANOVA), we compared the significance of differences in Pb-concentration between the various tissues of the three sorghum cultivars used in this study. The simple linear correlation coefficient (r) was computed to evaluate the connection between the Pb-tolerance index of the plant growth data and the bioaccumulation criterion, which included the Pb concentration and the bioaccumulation factor. All statistical tests were performed with SPSS [44].

3. Results and Discussion

3.1. Lead Levels in Soil

The soil in the experiment was enhanced with various amounts of Pb (0, 100, 200, 400 and 800 mg/kg), resulting in soil DTPA-Pb values of 2.10, 43.22, 87.55, 108.19 and 135.78 mg/kg (

Table 1. Available Pb content (mean ± SD) of the soil before and after cultivation of the different cultivars of Sorghum bicolor crop plants for 80 days.

Treatments (mg/kg)	Before Harvest	After Harvest
		S. bicolor S1	S. bicolor S2	S. bicolor S3
Control	2.10 ± 0.05	1.79 ± 0.13 *	1.66 ± 0.15 *	1.49 ± 0.06 *
100	43.22 ± 5.84	32.40 ± 4.28 *	31.73 ± 3.51 *	37.01 ± 5.83 *
200	87.55 ± 7.12	55.70 ± 6.97 *	54.00 ± 5.06 *	54.77 ± 6.59 *
400	108.19 ± 8.56	69.32 ± 5.56 *	65.26 ± 6.05 *	69.84 ± 8.81 *
800	135.78 ± 7.12	73.82 ± 8.94 *	75.80 ± 6.60 *	76.51 ± 9.93 *

*: The mean soil value after harvesting was significantly different (p ≤ 0.05) from its corresponding value before cultivation.

). It was found that the content of the soluble fraction of Pb in the soil after 80 days of plantation was significantly (p ≤ 0.05) lower compared to the initial values in the soil before cultivation, as compared to the initial values in the soil before plantation. This was true across all study conditions and all three sorghum cultivars. Such reduction varied from one cultivar to another; for example, the Pb reduction value in soil cultivated with S1, S2 and S3 plants grown in soil amendment with 800 mg/kg reached 45.63, 44.17 and 43.65%, respectively, as compared to soil before cultivation (Table 1).

PTEs are found in soils in various chemical forms. They typically display a range of physical and chemical behaviors regarding their potential toxicity, mobility, biological availability and chemical interactions. Lead, which may form stable complexes with organic material, iron and manganese oxides and clays, is a major ecotoxic element that pollutes both terrestrial and aquatic habitats. Soil carbonates, hydroxides and phosphates are also possible precipitates [45,46]. Smith et al. [47] reported a range of 10–40 mg/kg dry soil for the soil Pb content they utilized in their studies, with a median value of about 20 mg/kg dry soil for the soil Pb content used in the present study. Thus, before additions, the soil utilized in the research was Pb-free, but afterward, it became Pb-polluted. To add extra complexity, the overall Pb content, pH, concentrations of other PTEs and macro-nutrients all impact the amount of Pb that is available to plants [48]. Prior to the experiment, the slightly alkaline (pH = 7.56) soil in the present investigation had an available Pb concentration of between 2.10 and 135.78 mg/kg at the control and 800 mg/kg Pb-treatment, respectively. Soil with a slightly alkaline pH may have reduced metal availability and solubility, as stated by Al-Sodany et al. [49].

3.2. Effect of Pb-Treatments on the Growth Criteria

The high biomass yield of the tested plant for the phytoremediation approach was selected to ensure a high removal rate of different PTEs from a substrate. Sorghum cultivars were chosen in this work based on their capability to uptake metals from a contaminated substrate, high biomass and fast growth rate. In this work, there were significant variations of different tested growth criteria of different cultivars grown on soil enriched with varying levels of Pb (

Table 2. Morphological measurements and tolerance index (TI%) of the different cultivars of Sorghum bicolor crop plants grown under different Pb treatments for 80 days.

Treatments (mg/kg)	Root Length (cm)	Shoot Length (cm)	Root FW (g/plant)	Root DW (g/plant)	Shoot FW (g/plant)	Shoot DW (g/plant)	TI Root (%)	TI Shoot (%)
Sorghum S1
Control	13.50 ± 2.50 a	106.83 ± 5.01 a	0.82 ± 0.09 a	0.24 ± 0.06 a	6.17 ± 0.99 a	0.97 ± 0.20 a
100	9.50 ± 1.45 b	91.67 ± 7.88 b	0.58 ± 0.06 b	0.20 ± 0.04 a	4.21 ± 0.82 b	0.73 ± 0.06 b	83 ± 5.5 a	75 ± 7.8 b
200	7.13 ± 2.32 c	66.00 ± 8.11 c	0.54 ± 0.06 b	0.12 ± 0.01 b	3.24 ± 0.93 b	0.62 ± 0.05 c	60 ± 4.1 b	85 ± 9.4 a
400	5.00 ± 1.05 d	54.67 ± 9.43 d	0.38 ± 0.02 c	0.06 ± 0.01 b	1.85 ± 0.31 c	0.16 ± 0.04 d	50 ± 4.7 c	16 ± 2.7 c
800	4.23 ± 0.76 d	44.00 ±6.18 e	0.18 ± 0.01 d	0.06 ± 0.01 b	0.98 ± 0.12 e	0.10 ± 0.02 c	25 ± 3.9 d	10 ± 1.9 d
Sorghum S2
Control	12.00 ± 2.00 a	85.67 ± 8.21 a	1.11 ± 0.16 a	0.32 ± 0.07 a	12.78 ± 2.80 a	2.17 ± 0.88 a
100	9.00 ± 1.52 b	69.33 ± 6.21 b	0.81 ± 0.16 b	0.26 ± 0.04 b	9.31 ± 3.07 b	1.85 ± 0.31 b	81 ± 7.2 b	83 ± 6.8 a
200	7.50 ± 1.45 c	62.00 ± 6.50 bc	0.70 ± 0.09 b	0.25 ± 0.06 b	8.74 ± 0.06 b	1.39 ± 0.29 c	96 ± 8.1 a	77 ± 7.2 b
400	5.67 ± 0.76 d	56.07 ± 5.79 cd	0.43 ± 0.06 c	0.16 ± 0.02 c	7.50 ± 0.62 bc	1.10 ± 0.24 d	64 ± 6.5 c	79 ± 5.8 b
800	4.80 ± 0.98 d	49.00 ± 5.09 d	0.26 ± 0.08 d	0.07 ± 0.02 d	5.05 ± 0.51 c	0.76 ± 0.19 e	44 ± 4.2 d	69 ± 5.1 c
Sorghum S3
Control	13.33 ± 3.04 a	115.50 ± 8.91 a	1.29 ± 0.22 a	0.38 ± 0.04 a	9.96 ± 1.01 a	1.96 ± 0.27 a
100	10.17 ± 1.76 b	94.33 ± 7.86 b	1.14 ± 0.11 ab	0.32 ± 0.01 b	8.53 ± 1.63 b	1.77 ± 0.32 a	84 ± 5.5 a	90 ± 7.8 a
200	8.50 ± 1.20 c	80.67 ± 8.21 c	0.96 ± 0.21 b	0.27 ± 0.07 c	8.33 ± 1.45 b	1.35 ± 0.19 b	84 ± 4.9 a	76 ± 6.9 b
400	6.75 ± 0.95 d	68.75 ± 6.25 d	0.68 ± 0.12 c	0.22 ± 0.06 c	6.78 ± 1.10 c	1.27 ± 0.16 b	81 ± 5.7 b	74 ± 5.6 b
800	5.15 ± 0.65 e	47.50 ± 7.50 e	0.51 ± 0.09 c	0.11 ± 0.02 d	5.33 ± 0.69 d	0.79 ± 0.18 c	50 ± 4.8 c	62 ± 6.2 c

Means with different letters are significant at p ≤ 0.05.

). The reduction in the plant length and biomass of both roots and shoots of the tested cultivars was progressive and gradual with the increase of Pb concentration in soil. This is due to the cumulative effect of the bioaccumulated amount of Pb in the different tissues. For example, such reduction may reach 75, 89, 78, 65, 71 and 60%, respectively, in the dry weight of root and shoot in different cultivars (S1, S2 and S3) treated with 800 Pb compared with control treatment (Table 2).

It was found that different cultivars of sorghum (S1, S2 and S3) had their maximum root and shoot length and biomass production (13.5, 115.5 cm, 1.29, 0.38, 12.78, and 2.17 g/plant, respectively) at control, while the lowest (4.2, 44.0 cm, 0.18, 0.06, 0.98 and 0.10 g/plant), respectively was recorded at 800 mg/kg Pb-treatment (Table 2). The significant decline in biomass production in both roots and shoots as the impact of PTEs was outlined in a cultivar of species [50,51,52,53].

High concentrations of PTEs in plants cause significant damage to many physiological activities, ultimately leading to plant death. When plants are exposed to excessive levels of metals, physiologically active enzymes are inhibited [54] and destroy mineral metabolism [55].

The root system is in direct contact with Pb and acts as the main entrance for metal ions [56]. According to Sharma and Dubey [57], plant roots quickly respond to absorbed Pb by changing the branching pattern and reducing root growth. According to several works, root growth is inhibited under Pb stress due to Pb-induced inhibition of cell division in root tips [57,58].

As previously described, a high removal rate of hazardous PTEs requires a high biomass of plants specifically chosen for phytoextraction technology. Three sorghum cultivars were selected for their high biomass, fast growth rate and potential to uptake Pb from polluted soils [59]. Three different cultivars of sorghum showed altered growth and metabolic rates when subjected to PTEs stress, such as Pb. Effects on growth and biomass production were noticeable at high Pb concentrations, and they were similar to those reported by previous researchers in other plant species [53,60,61,62,63]. The cumulative effect of damaged or inhibited physiological function under Pb stress conditions is responsible for the considerable suppression of morphological features in Pb-treated plants compared to control, a finding consistent with the work of Ramana et al. [64] and Ryser and Sauder [65]. Tolerance index (TI) is considered an important parameter that affects selecting the plant with phytoremediation potential. TI was calculated in the root and shoot of three tested sorghum cultivars at various Pb concentrations (Table 2). TI was measured at various Pb concentrations by dividing the dry weight of the plant exposed to different metal concentrations by the dry weight estimated during control growth [66]. Generally, sorghum plants displayed a good TI to Pb (on average, 68%). According to mean TI values at different Pb concentrations, the tested sorghum cultivars can be arranged in the following descending order: S3 (78%), followed by S2 (74%), then 51% for S1. In this regard, Hira et al. [67] found that both tested plants, guar and sesame, had different tolerance indexes (TIs) under Pb stress, and their value decreased by increasing Pb doses. Also, Srinivasan et al. [68] reported that growth inhibition is a common response to PTEs stress and is also one of the most important agricultural indices of PTEs tolerance.

3.3. Effect of Pb-Treatment on Pigment Contents

Pigment contents are a significant reflection of plant response to different stress; it could be due to total chlorophyll, chl. a, and b contents under these conditions were declined. Increasing the level of Pb in the soil led to a decline in the growth of three tested cultivars of sorghum, which led to significant (p ≤ 0.05) gradual inhibition of total chlorophyll, chl. a and b contents (

Table 3. Chlorophyll contents of the different cultivars of Sorghum bicolor crop plants grown under different Pb treatments for 80 days.

Treatments (mg/kg)	Chl. a	Chl. b	Total Chl.	A/B ratio	Carotenoids
Sorghum S1
Control	1.75 ± 0.20 a	1.12 ± 0.12 a	2.87 ± 0.20 a	1.56 ± 0.18 b	2.51 ± 0.48 a
100	1.33 ± 0.17 b	1.15 ± 0.14 a	2.48 ± 0.29 b	1.16 ± 0.28 c	2.38 ± 0.46 ab
200	1.10 ± 0.14 c	0.93 ± 0.12 b	2.03 ± 0.26 c	1.19 ± 0.13 c	1.90 ± 0.22 bc
400	0.91 ± 0.12 d	0.62 ± 0.09 c	1.53 ± 0.21 d	1.47 ± 0.18 b	1.52 ± 0.15 c
800	0.53 ± 0.08 e	0.27 ± 0.05 d	0.79 ± 0.09 e	1.97 ± 0.27 a	0.69 ± 0.06 d
Sorghum S2
Control	1.98 ± 0.39 a	1.37 ± 0.11 a	3.35 ± 0.54 a	1.47 ± 0.51 ab	2.86 ± 0.24 a
100	1.33 ± 0.21 b	1.00 ± 0.08 b	2.33 ± 0.26 b	1.33 ± 0.32 b	2.35 ± 0.28 b
200	0.99 ± 0.14 bc	0.87 ± 0.09 c	1.86 ± 0.36 c	1.14 ± 0.22 b	1.84 ± 0.25 c
400	0.89 ± 0.13 bc	0.58 ± 0.07 d	1.47 ± 0.35 cd	1.55 ± 0.27 ab	1.18 ± 0.24 c
800	0.76 ± 0.12 c	0.41 ± 0.08 e	1.17 ± 0.32 d	1.84 ± 0.34 a	0.82 ± 0.15 d
Sorghum S3
Control	2.05 ± 0.31 a	1.52 ± 0.24 a	3.57 ± 0.24 a	1.37 ± 0.22 a	2.88 ± 0.29 a
100	1.58 ± 0.26 b	1.09 ± 0.17 b	2.67 ± 0.30 b	1.44 ± 0.21 a	2.28 ± 0.24 b
200	1.12 ± 0.21 b	0.94 ± 0.14 b	2.06 ± 0.04 c	1.20 ± 0.16 ab	1.85 ± 0.10 c
400	1.07 ± 0.11 c	0.86 ± 0.18 bc	1.94 ± 0.39 c	1.25 ± 0.10 ab	1.34 ± 0.21 d
800	0.82 ± 0.13 d	0.67 ± 0.07 c	1.49 ± 0.20 d	1.21 ± 0.09 ab	0.72 ± 0.11 e

Means with different letters are significant at p ≤ 0.05.

). This is due to the threatening impact of excessive Pb contents, which cause plants’ weakness and vital metabolic activities. Chlorophyll a and b, total chl. and carotenoids were lowered by (mean from three tested cultivars) 64.5, 67.3, 65.8 and 73.1%, respectively, with 800 mg/kg Pb contents as compared to untreated plants (Table 3). Several authors detected the same results in different plants [51,69,70,71].

The plant subject to PTEs’ stress, especially Pb, leads to inhibition of chlorophyll contents. This may be associated with the high ability of these plants to accumulate metals that destroy the chloroplasts and/or inhibit chlorophyll synthesis [72,73] or directly destroy the photosynthetic apparatus and decrease the photosynthetic production due to enhanced Reactive Oxygen Species (ROS) [74].

3.4. Lead Content in Plant Tissue and Phytoremediation Efficiency

A close positive correlation was observed between the concentrations of Pb in the soil and its concentrations in the root and shoot of the tested sorghum cultivars (

Table 4. Pb-concentration (mean ± SE, n = 3) in the roots and shoots, translocation factor (TF), bioaccumulation factor (BAF_root), root, shoot and total plant uptake of different cultivars of Sorghum bicolor crop plants grown under different Pb-treatments for 80 days.

Treatments (mg/kg)	Pb Concentration in Plant Tissues µg/g		TF	BAFroot	Pb Uptake (µg/pot)
	Root	Shoot			Root	Shoot	Total
Sorghum S1
Control	0.16 ± 0.01 e	0.11 ± 0.03 e	0.69 ± 0.08 ab	0.09 ± 0.01 e	0.06 ± 0.02 c	0.33 ± 0.06 d	0.39 ± 0.05 d
100	15.39 ± 3.35 d	5.28 ± 0.95 d	0.35 ± 0.07 c	0.48 ± 0.07 d	4.71 ± 0.83 b	11.46 ± 2.44 c	16.17 ± 2.51 c
200	62.36 ± 6.32 c	39.46 ± 1.73 c	0.64 ± 0.04 b	1.12 ± 0.17 c	11.01 ± 1.74 a	73.44 ± 5.18 a	84.42 ± 8.58 a
400	88.12 ± 5.24 b	60.63 ± 5.97 b	0.69 ± 0.06 ab	1.28 ± 0.13 b	8.37 ± 1.26 ab	28.47 ± 3.08 b	36.84 ± 4.05 b
800	110.00 ± 8.47 a	83.93 ± 6.81 a	0.76 ± 0.05 a	1.49 ± 0.14 a	10.95 ± 2.56 a	25.62 ± 2.09 b	36.99 ± 3.74 b
Sorghum S2
Control	0.18 ± 0.02 e	0.13 ± 0.01 e	0.71 ± 0.10 ab	0.11 ± 0.01 e	0.18 ± 0.05 d	0.81 ± 0.09 d	0.99 ± 0.08 d
100	16.50 ± 2.88 d	8.03 ± 1.08 d	0.49 ± 0.06 c	0.52 ± 0.03 d	12.99 ± 2.83 c	44.43 ± 5.27 c	57.42 ± 6.67 c
200	66.70 ± 5.90 c	45.56 ± 4.63 c	0.69 ± 0.07 ab	1.24 ± 0.13 c	50.88 ± 7.65 a	189.54 ± 7.01 b	240.42 ± 9.67 b
400	100.52 ± 8.38 b	74.16 ± 6.12 b	0.74 ± 0.10 a	1.55 ± 0.15 b	39.20 ± 4.54 b	234.77 ± 10.42 a	273.97 ± 10.91 ab
800	177.64 ± 13.37 a	103.56 ± 10.15 a	0.59 ± 0.08 bc	2.35 ± 0.23 a	39.33 ± 7.42 b	238.05 ± 13.70 a	277.38 ± 10.28 a
Sorghum S3
Control	0.15 ± 0.01 e	0.11 ± 0.01 e	0.74 ± 0.11 b	0.10 ± 0.01 d	0.18 ± 0.04 d	0.63 ± 0.08 c	0.81 ± 0.09 d
100	21.62 ± 2.61 d	8.54 ± 1.66 d	0.40 ± 0.05 d	0.59 ± 0.10 c	20.67 ± 3.76 c	45.60 ± 4.73 b	66.30 ± 5.24 c
200	61.83 ± 5.21 c	50.17 ± 4.14 c	0.81 ± 0.13 a	1.13 ± 0.21 b	50.34 ± 6.74 b	203.37 ± 9.55 a	253.71 ± 10.81 b
400	79.30 ± 7.45 b	58.70 ± 6.70 b	0.74 ± 0.11 b	1.14 ± 0.14 b	52.95 ± 4.05 b	222.90 ± 14.54 a	275.82 ± 11.60 ab
800	198.91 ± 12.58 a	99.03 ± 8.31 a	0.50 ± 0.09 c	2.60 ± 0.30 a	65.7 ± 5.82 a	233.73 ± 9.84 a	299.81 ± 13.53 a
Safe limit *	Up to 10
Excessive or toxic limit **	30–300

* ALINORM [35]; ** Kabata-Pendias [75]. Means with different letters are significant at p ≤ 0.05.

). From the tabulated data, it is clear that increasing Pb contents in the soil led to a significant (p ≤ 0.05) increase in Pb content in different parts of the tested cultivars of sorghum (Table 4 and

Table 5. Two-way analysis of variance (ANOVA) indicates the variations in the Pb contents in root and shoots, translocation factor (TF), bioaccumulation factor (BAF), root, shoot and plant uptake of the three tested cultivars of Sorghum bicolor plants grown under different Pb treatments.

Variable	F-Value
	Treatment	Plant	Intercept
Root Pb	1609.10 ***	63.77 ***	8682.65 ***
Shoot Pb	2477.50 ***	47.33 ***	14,119.01 ***
TF	51.08 ***	0.416	5880.73 ***
BAF	572.10 ***	29.95 ***	5128.61 ***
Root Uptake	140.51 ***	195.83 ***	1426.41 ***
Shoot Uptake	234.01 ***	263.96 ***	1966.09 ***
Plant Uptake	293.65 ***	342.07	2561.82 ***

***: p < 0.001.

). As expected, the highest concentration of Pb was found in plants grown in soil amended with 800 mg/kg of Pb for all tested cultivars of sorghum. In contrast, the minimum Pb contents were detected in sorghum cultivars grown in untreated soil. The highest Pb content in root (110.0, 177.6 and 198.9 mg/kg, respectively) and in-plant shoot (83.9, 103.6 and 99.0 mg/kg, respectively) was detected by sorghum (S1, S2 and S3, respectively) grown in soil enriched by 800 mg/kg of Pb.

Regarding the distribution of Pb among the different parts of all tested cultivars of sorghum under the different levels of soil enrichment with Pb, the data revealed a tendency to accumulate Pb ions in roots rather than in shoots (Table 4). This could be attributed to PTEs being absorbed by roots from soil solution and then translocated to leaves (via xylem tissues), where they are stored in vacuoles [76]. Since it prevents metal translocation to shoots, Pb accumulation in roots is thought to increase plant tolerance to Pb toxicity [77].

Consistent with our findings, different works detected that the underground portions were the principal depot for accumulating Pb in sorghum, followed by the aerial portions [30,31,32,78,79]. In this regard, Blanco et al. [31] performed a study that collected sorghum plants grown in a former battery recycling plant in Córdoba city (Argentina), indicating that sorghum accumulated Pb principally in roots and only a small amount was detected in the aerial portion. Furthermore, Memoli et al. [32] noticed that Pb accumulated primarily in the sorghum’s roots, the leaves and stems and the fruits.Also, the roots accumulate Pb twenty folds than leaves.

Estimates of Pb ion uptake by roots were obtained using the BAF_root. In most cases, elevated soil Pb levels were associated with a rise in BAF_root values (Table 4). Potential accumulation of Pb from the soil to the roots of the three sorghum cultivars was shown by a BAF_root higher than 1 at each Pb-treatment, with the exception of the control and 100 mg/kg treatments for tested cultivars. Treatment with 800 mg/kg Pb produced the greatest BAF_root (2.60) in S3. Furthermore, TF values ranged from 0.35 to 0.81 for the tested cultivars of sorghum, which means that tested cultivars did not transfer Pb from root to shoot with TF less than 1. The maximum values of TF were detected in the case of S3 plants, while the lowest ones were found in the case of S1 plants (Table 4). The tested cultivars exhibited significant variation in the TF values at different Pb contents (Table 4 and Table 5). Goni et al. [80] investigated the uptake and translocation of Fe, Zn, Cr, Pb, Cu, Ni and Cd in various parts of rice plants irrigated with contaminated water and detected that the TF for tested metals was below 1, which indicates that the majority of the metals were stored in the roots. In our case, TF of Pb ranged between 0.35 and 0.81. Similar results were reported by Baldi et al. [81] and Yoon et al. [82] on sorghum and Alaboudi et al. [53] on sunflower.

Concentration of the Pb in the plant and dry matter yield affect the absorption of tested metal (PT). Therefore, a plant species that can tolerate and accumulate moderate levels of Pb would be suitable for remediating a contaminated site by producing high biomass yielding crop. There is higher uptake in the shoot (238.05 g/pot) over the root (65.7 g/pot), and the uptake of the overall plant is (299.81 g/pot) at Pb 800 mg/kg, indicating that the plant’s uptake pattern of Pb does not reflect the trend of its concentration in the tissue (Table 4). It may be because shoots generate more biomass than roots do. The uptake levels varied from cultivar to cultivar based on each plant’s ability to absorb Pb from the soil and its total biomass. Despite ragweed (Ambrosia artemisiifolia) and sunflower (H. annus) having plant Pb concentrations more than two times the values for redtop (Agrostis alba L.) and corn (Zea mays), Cooper et al. [83] reported that the greatest plant Pb uptake occurred in redtop and corn, which generated the greatest biomass.

3.5. Determining Cause and Effect through a Correlation Analysis

The Pb-tolerance index was negatively connected with root Pb contents (r = −0.384, −0.383, −0.555 and −0.366) and shoot Pb contents (r = −0.483, −0.666, −0.362 and −0.787), as shown by the simple linear correlation coefficient (

Table 6. Correlation matrix of the Pb tolerance index (TI) and accumulation characteristics for all tested cultivars of Sorghum biolocor.

Variable	Tolerance Index
	Root Length (cm)	Shoot Length (cm)	No. of Leaves	Root Weight (g/plant)	Shoot Weight (g/plant)
Pb content in root (mg/kg)	−0.384 **	−0.383 **	−0.688 **	−0.555 **	−0.366 **
Pb content in shoot (mg/kg)	−0.753 **	−0.736 **	−0.713 **	−0.571 **	−0.696 **
BAFroot	−0.550 **	−0.542 **	−0.588 **	−0.631 **	−0.440 **
TF	−0.621 **	−0.619 **	−0.625 **	−0.691 **	−0.591 **
Plant Uptake	−0.279 *	−0.278 *	−0.092	−0.320 **	−0.141

*: p < 0.05, **: p < 0.01.

). Root bioaccumulation factor (BAF_root), translocation factor (TF) and plant uptake (UT) were all inversely connected with root and shoot length tolerance index (Table 6).

Three features must be met for a plant to be considered a Pb hyperaccumulator: (a) TF > 1, (b) a concentration of 1000/g dry wt in the shoot and (c) BAF > 1, as stated by (Baker and Walker [84]). The current investigation failed to find evidence that the plant fulfilled all three of the criteria for a Pb hyperaccumulator. That is why it is considered an excludable item. Even in the presence of high concentrations of PTEs, excluders have been observed to tolerate severely polluted soils and to take up low amounts of PTEs [85].

3.6. Regression Models

As shown in

Table 7. Regression models between Pb concentrations in three sorghum cultivars tissues (mg/kg) and soil Pb (mg/kg), pH and organic matter content (OM%).

Equation	R2	ME	MNAE	MNB	Student’s t-test
					t-Value	p
Sorghum S1
Pbshoot = 691.621 + (1.223 × Pbsoil) − (94.162 × pH) + (1.331 × OM)	0.941	0.979	0.185	0.029	0.246	0.811
Pbroot = 694.125 + (1.632 × Pbsoil) − (95.143 × pH) + (2.576 × OM)	0.965	0.988	0.149	0.011	0.142	0.891
Sorghum S2
Pbshoot = −617.666 + (1.335 × Pbsoil) + (81.480 × pH) − (0.659 × OM)	0.909	0.926	0.213	0.046	0.575	0.579
Pbroot = −1556.612 + (2.139 × Pbsoil) + (206.009 × pH) + (0.532 × OM)	0.923	0.950	0.207	0.036	0.574	0.580
Sorghum S3
Pbshoot = −633.742 + (1.231 × Pbsoil) + (81.468 × pH) + (1.260 × OM)	0.899	0.922	0.284	0.046	1.393	0.197
Pbroot = −1717.556 + (2.197 × Pbsoil) + (221.489 × pH) + (4.900 × OM)	0.858	0.865	0.301	0.052	1.979	0.079

R²: coefficient of determination; ME: model efficiency; MNAE: mean normalized average error; MNB: mean normalized bias.

, regression models were used to predict the levels of Pb in sorghum shoots and roots related to soil Pb contents, pH and OM contents. Correlations were clearly observed between the measured and predicted Pb levels in shoots and roots with a high ME and R² and low mean MNAE. In addition, p-values indicated no significant differences between the actual and estimated Pb levels in either shoots or roots of tested sorghum cultivars. The obtained results demonstrated that the used model fit well for estimating Pb levels in shoots and roots based on their Pb concentrations in soils and pH and OM levels of the tested soils. As shown in (Table 7), a high R² value was associated with a high ME value. Sorghum cultivar S1 had the highest R² and ME values recorded, with averages of 0.94 and 0.98 in shoots and 0.97 and 0.99 in roots. Sorghum cultivar S3 had lower R² and ME values, with averages of 0.90 and 0.92 in shoots for R² and ME, respectively, and 0.86 and 0.87 in roots. It is worth noting that soil pH and OM contents both play major roles in Pb absorption by the growing sorghum cultivars. The previous studies conducted by Eid et al. [86] showed that soil pH has negative correlations with tested PTEs, i.e., Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb and Zn. In addition, they have observed that OM contents ultimate PTEs uptake by the growing plants. In this study, we went almost to similar findings; soil pH and OM contents significantly affected Pb uptake by sorghum cultivars. In this regard, the utilized model is effective for calculating Pb absorption based on soil Pb content, pH and OM content of the tested soil. T-tests for Pb in tissues of tested cultivars found no significant differences (p > 0.05) between the measured and estimated Pb in plant tissues.

The Pearson correlation analysis is shown in Figure 1 and Supplementary Material S1. The solubility of PTEs is controlled by the adsorption and desorption phenomena in soil, which are primarily influenced by soil characteristics, i.e., pH, OM, CEC, Eh and the concentration of specific PTEs ion [33]. The obtained results showed that Pb concentrations in the growing sorghum cultivars were strongly linked to the Pb concentrations in soils where they were grown. These findings imply that Pb can be easily transferred from soils to plants. The previous studies went almost to similar findings [87]. They have mentioned that, as the concentration of PTEs increased in soil, the growing plants’ uptake increased. Sorghum cultivar S1 showed a negative correlation between soil pH and plant Pb absorption. The average pH value of the examined soil was 7.59, and raising soil pH resulted in less Pb ion uptake by the sorghum plant. PTEs solubility and absorption by growing plants are influenced by soil pH, as demonstrated by Abdelhafez et al. [6] and Abdelhafez and Li [3]. Raising soil pH is well-known to limit Pb absorption by forming less soluble forms of Pb metal ions [6,88,89]. S2 and S3 cultivars, on the other hand, showed a different pattern, with average pH values of investigated soils of 7.42 and 7.55, respectively. These were lower than those of the S1 cultivar; as a result, Pb solubility increased under low pH, resulting in elevated Pb absorption by the growing plants.

Organic matter plays an essential role for metal ions speciation in soil [90]. Most studies demonstrated that increasing soil organic matter contents led to increased PTE solubility of PTEs and their uptake by the growing plants [33,91]. However, in this study, OM contents in soils negatively but not significantly affected the absorption of Pb by the tested sorghum cultivars, except for S2 sorghum cultivar.

Figure 2 displays the scatter plot of Pb bioaccumulation factor (BAF) values. The results revealed that the BAF of the investigated cultivars had high BAF values above 1 at soil Pb concentrations > 50 mg/kg. This finding suggests that metal content is the primary factor influencing Pb absorption by growing plants.

4. Conclusions

This study tested the Pb phytoremediation potential of three S. bicolor cultivars grown on different Pb-contaminated soils. Our research showed that sorghum cultivars might be used to clean up Pb-contaminated soil. Some soil properties, including pH, EC, OM, CEC, Eh and Pb ion concentration, may affect the availability of Pb ions in the tested soils. Therefore, it contributes considerably to the efficiency with which sorghum cultivars may extract Pb. Compared to the S1 and S2 cultivars, the Pb removal efficiency of S3 was clearly the highest. Soil Pb levels may be accurately predicted using the evaluated regression model, which showed excellent performance and efficiency.

These plants were also evaluated for their potential to be used in Pb phytoremediation. It was shown that all tested cultivars were capable of growing in soil heavily spiked with Pb. However, Pb affected plant morphology and chlorophyll content, especially in soil treated with 800 mg/kg. Sorghum cultivars (S1, S2 and S3) planted in soil enriched by 800 mg/kg of Pb had the greatest Pb concentration in root (110.0, 177.6 and 198.9 mg/kg, respectively) and in plant shoot (83.9, 103.6 and 99.0 mg/kg, respectively). Despite not being Pb hyperaccumulators, the contamination indices (CT, TF, UT and TI), contamination factor (CF), translocation factor (TF), plant uptake (UT) and tolerance index (TI) revealed that evaluated cultivars were ideal candidates for phytostabilization. Overall, the regression model results and Pb uptake by sorghum cultivars confirmed our hypothesis. More research is needed to study the potential combinations of plant growth promoters and phytoremediation technologies to obtain high Pb removal effeminacy from polluted soils.