Effect of the Co-Application of Eucalyptus Wood Biochar and Chemical Fertilizer for the Remediation of Multimetal (Cr, Zn, Ni, and Co) Contaminated Soil

Abstract

Contamination of soil with heavy metals is a worldwide problem, which causes heavy metals to release into the environment. Remediation of such contaminated soil is essential to protect the environment. The aims of this study are: first, to compare the effect of biochar and the joint application of biochar with fertilizer for the phytoremediation of heavy metals-contaminated soil using Acacia auriculiformis; second, to study the effect of the application rate of biochar in improving the physicochemical properties of the soil. The soil samples were collected from an active coal mine dump and assessed for their physicochemical properties and heavy metals toxicity. Initial results indicated that the soil has poor physicochemical properties and was contaminated with the presence of heavy metals such as Zn, Ni, Cu, Cr, and Co. Later, the heavy metals-contaminated soil was mixed with the 400 and 600 °C biochar, as well as the respective biochar–fertilizer combination in varying mixing ratios from 0.5 to 5% (w/w) and subjected to a pot-culture study. The results showed that the application of both varieties of biochar in combination with fertilizer substantially improved the physicochemical properties and reduced the heavy metals toxicity in the soil. The biochar and fertilizer joint application also substantially improved the soil physiochemical properties by increasing the application rate of both varieties of biochar from 0.5 to 5%. The soil fertility index (SFI) of the biochar and biochar–fertilizer amended soil increased by 49.46 and 52.22%, respectively. The plant’s physiological analysis results indicated a substantial increase in the plant’s shoot and root biomass through the application of biochar and biochar–fertilizer compared to the control. On the other hand, it significantly reduced the heavy metals accumulation and, hence, the secretion of proline and glutathione hormones in the plant cells. Therefore, it can be concluded that the joint application of biochar with the application rate varying between 2.5 to 5% (w/w) with the fertilizer significantly improved the physicochemical properties of the soil and reduced the heavy metals toxicity compared to the controlled study.

1. Introduction

Heavy metals toxicity is the presence of toxic inorganic elements in the soil in a quantity in excess of the permissible limit [1]. In the last few decades, heavy metal toxicity in the soil reportedly increased worldwide due to the increase in anthropogenic activities, such as mining activities, refinery operations, and other industrial activities [2]. These anthropogenic activities reportedly release various heavy metals, namely, Cd, Cr, Pb, Cu, and Zn, into the environment [3,4,5,6]. Excessive release of heavy metals into the environment and ecosystem poses a threat to land productivity, ecological balance, and soil microbes. Moreover, the leaching of heavy metals reportedly increases metal toxicity in the plants, which ultimately, through various food chain pathways, can pose a threat to human health [7,8,9,10,11,12,13]. The contaminated soils, which are generated through the mining activities, are characterized by variable pH, low organic matter, poor nutrients, low cation exchange capacity (CEC), and heavy metals toxicity [14,15]. The release of heavy metals from such soils is reportedly reduced or prevented by in situ soil remediation techniques [16,17]. Among these, the revegetation technique using a suitable plant species plays an important role in enhancing the physical, chemical, and biological characteristics of the contaminated soil [18,19]. The metal-tolerant species bioaccumulate the heavy metals in the root and shoot parts, thereby reducing their availability and leaching through the soil [20]. However, there is still a chance that the consumption of the metal-tolerant species by the grazing cattle in the area can lead to the biomagnification of the heavy metals within the food chain. Various studies have suggested the role of phytoremediation as a cost-effective technique for the remediation of contaminated soils using a suitable plant species [21,22,23,24].

However, to sustain the vegetation growth during the in situ remediation process, the use of soil ameliorants for the amendment of the contaminated soils plays a vital role [25,26,27]. The various soil-amending materials reportedly used in the in situ remediation of the contaminated soils comprised manure, bonemeal [Ca₁₀ (PO₄)₆ OH₂], fly ash, and lime [25,27,28,29,30]. However, the above-reported ameliorants have certain limitations; for example, lime can only be used in acidic soils to increase the pH, fly ash application could increase the heavy metals content in the soil [31], and the use of manure or bio-solids may increase the mobility of the heavy metal due to their high dissolved organic matter content [31,32,33,34].

In recent years, biochar has gained much attention due to its beneficial soil properties, porous structure, presence of functional groups, and ability to immobilize the heavy metals within the soil matrix, thereby possible limiting the bioaccumulation of the heavy metals within the plant’s tissue [25,35]. Biochar is a carbon material derived through the pyrolysis of biomass waste [36,37]. Biochar’s physicochemical properties reportedly vary with the change in pyrolysis parameters, such as pyrolysis temperature and feedstock [38,39,40]. Several studies reported the beneficial effect of biochar on the soil’s physicochemical and biological properties, as well as on the growth of the plants [41,42,43]. Abdelhafez [41] reported the advantageous effect of biochar on the soil physicochemical properties and remediation of Pb contaminated soil. Medyńska-Juraszek [44] reported the effect of biochar in reducing the mobility of the Cu-, Pb-, Zn-, and Cd-contaminated soil. In another study, Lu [45] reported that the application of rice straw biochar at a rate of 5% (w/w) reduced the pool of Cd, Cu, Pb, and Zn by 11, 17, 34, and 6%, respectively, in the soil. However, biochar has certain limitations to act as a fertilizer in the soil due to having limited availability of nitrogen and phosphate sources [46]. In this regard, the application of biochar in combination with nitrogen and phosphorous-enriched material could enhance its heavy metals sorption capacity and agronomic benefits in the soil. For example, Liang [47] reported that the co-application of biochar and compost in the contaminated wetland soil reportedly reduced the exchangeable fractions of Cd and Zn by 67 and 37.5%, respectively. Karami [48] reported that the application of biochar in combination with the green compost significantly reduced the Pb levels in plant shoot part by 63% compared to the single application of biochar. Zeng [49] reported that the application of biochar in combination with compost reduced the availability of Cd by 13.3% in the soil compared to the single application of the biochar. From the above-reported results, it was implied that the joint application of biochar in combination with the compost or other biofertilizer has effectively reduced the availability of heavy metals in soil.

Until now, various studies also reported the beneficial effect of the single application of biochar and the joint effect of the combined biochar and compost in the metal-contaminated soil [50,51,52,53]. Additionally, very few studies have reported the effect of different temperatures of biochar on the physicochemical characteristics of the heavy metals-contaminated soil [54,55,56]. However, none of the studies comprehensively reported the effect of joint application of different pyrolytic temperature Eucalyptus wood biochar with the NPK fertilizer for the remediation of multimetal-contaminated soil using plant species, namely Acacia auriculiformis.

Briefly, this study reports the effect of the application of low (400 °C) and high-temperature (600 °C) biochar and the joint application of respective biochar with fertilizer on the physicochemical properties of the soil based on a pot-culture study, variations in the soil enzymatic activities, and changes in the physiological and biochemical properties of A. auriculiformis. The reason behind the combined application of biochar with fertilizer lies in the fact that the biochar lacks a sufficient intrinsic pool of nitrogen and phosphorous. Hence, the joint application with fertilizer could enhance biochar’s nutrient retention capacity in the soil [40], which would be otherwise lost in the form of leachate. Additionally, the presence of nitrogen, potassium, and phosphorous ions promotes the adsorption of heavy metals within the soil through the precipitation and ion exchange processes [57]. This study also investigated the effect of biochar and the combined effect of the biochar–fertilizer mixture in reducing the availability of the heavy metals in the plant tissues and changes in the plant’s biochemical hormones (indicators of abiotic and toxicity stress). Finally, this study calculated the soil fertility index (SFI) as the pointer toward the increase in the soil fertility level after the co-application of biochar and fertilizer.

2. Materials and Methods

2.1. Study Area and Soil Sample Collection

The study area is located at the geographical coordinates of longitude 86°25′49.41″ E and latitude 23°45′41.82″ N in Bastacolla area, Dhanbad district, Jharkhand, India is known as Bera opencast project as shown in Figure S1. The rest of the details of the study area is given in the Supplementary Material. The soil samples were collected using a randomized design covering the entire area of the dump to represent the aggregate sampling. The samples were collected from a depth of 0–30 cm by forming a regular square grid of 5 × 5 feet, followed by the coning and quartering method to reduce sample volume. Subsequently, the soil samples were stored in airtight polystyrene bags for further use in the laboratory.

2.2. Biochar Production and Characterization

Biochar was produced through the pyrolysis of Eucalyptus wood waste at 400 °C and 600 °C for 150 min using a slow pyrolysis unit reported in the author’s previous study [58] and denoted as 400 and 600 EB. Eucalyptus wood biochar (EB) was crushed and sieved through 1 mm and stored for further characterization. The details of the biochar characterization techniques are given in the Supplementary Material.

2.3. Pot Experiment Using Biochar and Fertilizer as Amending Materials

A pot experiment (Figure S2) was done in a poly-house to reckon the efficacy of biochar and the co-application of biochar and fertilizer for the remediation of the multimetal-contaminated soil planted with the A. auriculiformis seeds [59,60]. The details of the pot study are given in Supplementary Materials.

The design of the experiment for the pot-culture study was divided into four sets: soil with biochar, soil with biochar and fertilizer, soil with fertilizer (control 1), and only soil (control 2). A third control (control 3) representing the field sample before the pot-culture study was also included in the study. Moreover, the application of biochar with the soil was further divided into two sets, i.e., soil with 400 °C EB and 600 °C EB, to determine the effect of the pyrolysis temperature on the remediation potential of the biochar. Moreover, the 400 °C EB-fertilizer and 600 °C EB-fertilizer mixture were incorporated into the soil to estimate the co-effect of both biochar and fertilizer for the remediation of the soil. The 400 and 600 °C EB were mixed with 5 kg of the soil in the pots in a weight percentage of 0.5, 1, 2, and 5% to represent the 6, 12, 24, and 60 tonnes per hectare application of biochar in the top layer (up to 20 cm) of the soil layer. The fertilizer (containing nitrogen:phosphorous:potassium in a weight ratio of 10:8:10) was mixed with the 5 kg of the soil in the pot along with the biochar in a dose an equivalent to 75 kg of N ha⁻¹, 30 kg of P ha⁻¹, and 90 kg of K ha⁻¹, equivalent to the dose of fertilizer recommended for the reclamation of sandy soil [61,62].

2.4. Characterization of Soil

Pre- and Post-Soil Physicochemical Characterization

The soil was characterized for the physicochemical properties before and after the pot culture study to evaluate the effect of biochar and joint effect of biochar–fertilizer amendments on the properties of the soil. The pH of the soil was determined using the method described in earlier studies [63,64]. The soil organic carbon was determined using the Walkley–Black method, as reported in detail in the earlier studies [65,66]. The soil organic matter was determined using the loss-on-ignition method [67,68]. The plant-available nutrients in the soil (Na, K, Ca, and Mg) were determined by extracting the soil samples with 1 M ammonium acetate with a weight to volume ratio of 1:5, followed by N analysis on ion chromatography (IC) (Model: Dionex ICS 2100, Thermo Scientific, Sunnyvale, CA, USA). The plant-available phosphorous (H₂PO₄) in the soil was determined using Bray’s method as reported in earlier studies [63,69]. The plant-available nitrogen (NH₄-N) in the soil was determined using the alkaline permanganate method on the Kjeldahl apparatus [63,70]. Soil cation exchange capacity was determined using the sodium acetate (buffered at pH 8.2) and ammonium acetate (buffered at 7.0) method [63,71]. Acid extractable heavy metals in the mine soil were determined using a method reported in an earlier study [72]. Briefly, 0.5 g of soil sample was digested using the acids HNO₃ (concentrated 69%) and HCl (strength 37%) in a 3:1 (v/v) ratio in a microwave digester (Model No. SK 10/HPR-GE-12, Milestone Technologies, Fremont, CA, USA) at 200 °C. The diethylenetriaminepentaacetic acid (DTPA) extractable heavy metals in the mine soil were determined by the method described in an earlier study [73]. Soil catalase activity was determined using the method reported in an earlier study [74,75]. The β-glucosidase in the soil was determined using the colorimetric method for the estimation of p-nitrophenol (pNP) formed by the hydrolysis of p-nitrophenyl-β-D-glucopyranoside [75,76]. Soil urease activity in the soil was determined using the method described in an earlier study [77]. The soil fertility index for pre- and the post-pot culture study was calculated using Equation (1) [78,79], to determine the effect of biochar and co-application of biochar–fertilizer on the fertility level of the soil.

Soil fertility index (SFI) = pH + Organic matter (%) + available P (mg kg⁻¹) +
exchangeable K (cmol kg⁻¹) + exchangeable Ca (cmol kg⁻¹) + exchangeable
Mg (cmol kg⁻¹) − exchangeable Al (cmol kg⁻¹)

(1)

2.5. Plant Analysis

The plant biomass from the shoot and root parts was collected from the top, bottom, and middle parts to represent the whole plant and cleaned with distilled water to remove dust. Subsequently, the cleaned shoot and root biomass were subjected to the ultrasonic bath using an ultrasonicator (Phoenix instrument) to remove metals, if any, present in the leaf and root tissues that might have loosely bound within the pores through the dust particles deposited on them. The cleaning of the shoot and root parts through ultrasonication is essential, as otherwise, it may lead to variations in the concentration of heavy metals during analysis. Furthermore, the cleaned shoot and root biomass were oven-dried at 70 °C, until constant weight was obtained, and the shoot and root biomass was recorded. The lengths of the plant’s shoot and root in all the pots were measured using a digital measuring tape to ascertain the plant growth above and below the soil layer.

Total glutathione content in freshly collected plant leaves was determined using the method described in an earlier study [80]. Proline content in the plant leaves was determined using the acid-ninhydrin method [81]. The chlorophyll a, chlorophyll b, and total carotene in the fresh plant leaves were determined using the method described in an earlier study [82]. The content of chlorophyll a, chlorophyll b, and total carotene was calculated using the Equations (2)–(4), respectively [83]:

C_a = 10.05A₆₆₂ − 0.766A₆₄₄

(2)

C_b = 16.37A₆₄₄ − 3.140A₆₆₂

(3)

C_T = 1000A₄₇₀ − 1.280C_a − 56.7C_b/230

(4)

where C_a is chlorophyll a, C_b is chlorophyll b, C_T is the total carotene, A₆₆₂ is the absorbance at 662 nm, A₆₄₄ is the absorbance at 644 nm, and A₄₇₀ is the absorbance at 470 nm.

The heavy metals content in the plant’s shoot and root parts was determined using the method reported in an earlier study [84]. The digested shoot and root samples were diluted and analyzed for heavy metals (Zn, Ni, Co, Cr, Fe, Pb, and Mn) using an atomic absorption spectrophotometer (AAS ICE 3000 series, Thermo Scientific, Waltham, MA, USA). The bioaccumulation factor (BAF) in shoot and root parts was calculated using Equations (5) and (6), respectively [85]. The translocation factor in plants was calculated using Equation (7) [85,86]:

$${\mathrm{BAF}}_{\mathrm{shoot}}=\frac{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{the} \mathrm{shoot}\right)}{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{soil}\right)}$$

(5)

$${\mathrm{BAF}}_{\mathrm{root}}=\frac{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{root}\right)}{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{soil}\right)}$$

(6)

$$\mathrm{TF}=\frac{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{the} \mathrm{shoot}\right)}{\left(\mathrm{heavy} \mathrm{metals} \mathrm{concentration} \mathrm{in} \mathrm{root}\right)}$$

(7)

where BF_shoot is the bioaccumulation factor for the shoot, BF_root is the bioaccumulation factor for root, and TF is the translocation factor.

2.6. Statistical Analysis

The pot culture study in the polyhouse using biochar and biochar–fertilizer mixture as the soil-amending material was conducted in a completely randomized design. All the data generated from the soil and plant analysis were tested for normal distribution using SPSS 20 for the calculation of Skewness Z-score and Shapiro-Wilks p-value as shown in Table S7 before being analyzed using one-way ANOVA statistical. It can be observed from Table S7 that all the soil data have shown normal distribution due to having a Skewness z-score between ±1.96 and the Shapiro-Wilks test p-value was greater than 0.05, which showed the acceptance of the null hypothesis that the data are normally distributed. Hence, all the data were further subjected to the one-way ANOVA analysis at a 95% confidence interval using Origin pro-2020 software (Copyright, Origin Lab, Northampton, MA, USA) followed by a post-hoc Tukey test to find the significant mean difference among the cases (biochar and biochar–fertilizer mixture amendment) and controls (control 1, control 2, and control 3). The correlation plots among the various groups of data were plotted using the corrplot function in the R stats (version 3.6.3) statistical package.

3. Results and Discussion

3.1. Soil and Biochar Physicochemical Characterization

3.1.1. Biochar Characterization Results

The details of the biochar characterization results are given in the Supplementary Material in the results and discussion Section 3.1.1.

3.1.2. Pre-Soil Characterization

Initial evaluation of the physicochemical properties of the soil samples collected from the two-year-old overburden dump was done to ascertain the current status of the soil physicochemical properties before being implemented in the pot-culture study. The initial characterization results showed that the soil has a sandy loam texture (

Table 1. Pre-pot culture soil physicochemical properties (mean ± S.D, n = 3).

Soil Parameter	Values
Particle size	Sand	49.50%	Sandy loam texture
	Silt	46.79%
	Clay	3.80%
Water holding capacity, WHC (%)	13.18 ± 1.53
pH	5.86 ± 0.17
Total organic carbon (%)	0.516 ± 0.12
Organic Matter (%)	1.03 ± 0.18
Exchangeable Na (mg/kg)	131.45 ± 10.58
Exchangeable K (mg/kg)	95.88 ± 7.11
Exchangeable Ca (mg/kg)	146.68 ± 6.08
Exchangeable Mg (mg/kg)	16.21 ± 3.27
Available P (mg/kg)	0.70 ± 0.11
Available N (mg/kg)	194.05 ± 8.37
Cation exchange capacity, CEC (cmol/kg)	6.16 ± 0.74
Nickel, Ni (mg/kg)	40.03 ± 9.25
Copper, Cu (mg/kg)	34.60 ± 5.56
Zinc, Zn (mg/kg)	52.62 ± 11.16
Cobalt, Co (mg/kg)	63.60 ± 11.03
Chromium, Cr (mg/kg)	139.66 ± 13.04
DTPA-extractable Ni (mg/kg)	11.25 ± 1.38
DTPA-extractable Cu (mg/kg)	9.31 ± 1.50
DTPA-extractable Zn (mg/kg)	8.55 ± 2.78
DTPA-extractable Co (mg/kg)	12.33 ± 1.45
DTPA-extractable Cr (mg/kg)	17.89 ± 2.81
Soil catalase (0.1 mol KMnO4 g-1 of soil)	0.59 ± 0.05
β-glucosidase (mol PNF g-1 h-1)	0.76 ± 0.08
Urease (µg N-NH4 kg-1 h-1)	0.30 ± 0.04

) having acidic pH (5.86 ± 0.178). The soil was low in both organic carbon (<0.52%) and organic matter content (<1.03%) along with having meagre availability of the nutrients, which makes it imperative to categorize the entire area of the dump as a degraded land [87]. The degraded lands reportedly require a very long time (>30 years) to reach the complete reclamation stage [88]. The CEC of the soil were also very low (Table 1), hence making it difficult to hold the applied nutrients within the soil matrix. Apart from having degraded characteristics, the soil was also contaminated with the presence of multiple heavy metals (Ni, Zn, Co, and Cr) (Table 1). It can be observed that the total concentration of Ni (60.03 mg/kg), Co (63.60 mg/kg), Zn (52.62 mg/kg), and Cr (139.66 mg/kg) were higher than their respective permissible limit of (as per ecological and health risk guidelines) in the soil [1,89]. Moreover, the plant-available concentration of heavy metals, namely, Cr (17.89 mg/kg), Cu (9.31 mg/kg), and Ni (11.25 mg/kg), were also higher than their respective permissible limits in the soil [1]. The microbial activities comprising soil catalase, β–glucosidase activity, and urease activities were very low in the soil, which might be limited due to the absence of organic carbon, organic matter, nutrients, acidic pH, and toxicity of heavy metals in the soil. Such conditions of the soil can be improved using the remediation process, which utilizes the soil amendment and phytoremediation technique [90].

3.1.3. Post-Soil Characterization

Effect of Biochar and Fertilizer Amendment on the Physicochemical Properties of the Soil

Here, the post-soil is represented by the soil collected after the completion of the pot-culture study. Representative post-soil characterization was done to evaluate the effectiveness of the biochar and the joint biochar–fertilizer amendments on its physicochemical properties. It can be observed from the initial characterization of the soil (Table 1) that the soil has acidic pH and insufficiency of nutrients and organic matter to support revegetation. The results of the post-soil analysis showed that the pH of the soil that was amended with 400 and 600 EB and the biochar–fertilizer mixture was significantly (p < 0.05) improved (Figure 1a) compared to the control 1, control 2, and control 3. Additionally, it can be concluded from Figure 1a that increasing its application rate from 0.5 to 5% significantly increased the pH by 0.8 units.

The joint application of 400 EB with fertilizer did not show much of an effect on the pH of the soil as compared to the sole biochar, except at the application rates of 2 and 5%. However, the joint application of 600 EB with fertilizer substantially (p < 0.05) improved the pH of the soil at the application rate of 1 and 2% by 0.44 and 0.33 units, respectively. A similar observation of the increase in the soil pH after the application of biochar and biochar with compost was reported in earlier studies [91,92]. An increase in the pH of the soil after the completion of the pot-culture study might be related to the liming effect of the biochar (Table S2) and the release of carbonates into the soil. Additionally, the joint application of biochar with fertilizer (pH∼7.80) may have increased the liming effect, and hence, showed a considerable increase in the soil pH [92].

The total organic carbon (TOC), organic matter (OM), available nutrients (NH₄-N, K, Ca, Mg, P), and cation exchange capacity (CEC) of the soil were significantly increased (p < 0.05, Figure 1 and Figure 2) compared to the controls (C1, C2, and C3) at the end of the pot-culture study. The TOC and OM contents in the soil amended with 400 EB were significantly increased (p < 0.05, Figure 1d) by 29.77 and 27.96%, respectively, as the biochar’s application rate was increased up to 5% (w/w). Likewise, the TOC and OM content in the soil amended with 600 EB was increased by 20.95 and 21.18%, respectively. It can be observed that the increase in the TOC and OM in the soil amended with 400 EB was relatively higher compared to 600 EB. This can be attributed to the increase in the presence of non-liable carbon in the biochar with the rise in the temperature [93,94]. The joint application of biochar with the fertilizer at a low application rate (up to 2%) has shown a significant change in TOC and OM in the soil. A similar increase in the soil organic carbon and organic matter after the application of biochar and biochar with compost or cow dung were reported in the earlier studies [95,96,97,98,99]. The increase in the soil’s organic carbon and organic matter can be correlated with the change in the soil’s physicochemical properties, (Figure 1 and Figure 2a). The improvement in the soil physicochemical properties is positively correlated with the soil microbial activities (r² > 0.90, Figure S5), which might have increased the humification process in the soil. A similar mechanism was reported in a study by Jien and Wang [97], which stated that the soil environment could accelerate the self-humification of biochar and release of complex organic carbons into the soil. Another study by Liang et al. [98] reported that biochar is more prone to oxidation of carbon rings on its surface than on the inner core surface, which might have made it possible to break the complex organic carbon into simpler forms to be utilized by the microbes, and hence, had increased the soil organic carbon.

The exchangeable nutrients (nutrients that are available for plant’s uses in the soil through the cation or anion exchange process) in the soil amended with 400 and 600 EB were significantly higher than those of controls (Figure 1). Additionally, the availability of the nutrients was increased (p < 0.05, Figure 1b,c,e) with the increase in the application rate of the EB from 0.5 to 5% in the soil. The joint application of 400 and 600 EB with fertilizer in the soil marked a noticeable increase (p < 0.05, Figure 1c–e) in the availability of nutrients compared to the merely applied biochar. Such an increase in the plant-available nutrient can be linked to the reduction in nutrient leaching and increased availability [40,99,100]. Moreover, at a high application rate (2 and 5%) of biochar, the soil amended with the 600 EB showed higher nutrient content compared to the 400 EB. Such variation can be linked to the higher mineral matter content and CEC in 600 EB compared to the 400 EB [37,93]. The cationic nutrients are usually present in the biochar either in the electrostatically bonded form to the negatively charged surface of the biochar or on its surface through the weak van der Waals force. A similar observation of an increase in the availability of the applied nutrients (NH₄⁺, NO₃⁻, and PO₄³⁻) in the sandy loam soil amended with the rice straw biochar was reported in our earlier study [40]. The availability of nutrients in the soil can also be correlated (r² > 0.80, Figure S5) with the CEC, which was also significantly increased (p < 0.05, Figure 2a) in the soil amended with 400 and 600 EB. The CEC of the soil amended with the biochar–fertilizer mixture (Figure 2a) was significantly higher at the application rate greater than 0.5% (w/w) compared to the soil merely mixed with the 400 and 600 EB. A similar observation of the increase in the CEC of the soil amended with biochar and compost was reported in the earlier studies [91,92]. The increase in the CEC of the soil can be associated with the increase in the organic matter, charge density, and pH of the soil [92]. Moreover, the high specific surface area of the biochar and increase in the surface negative chare due to the surface oxidation of aromatic carbon to form carboxylate groups can also be linked to the increase in the CEC of the soil [92,98,101].

Soil microbial activities, i.e., catalase, β-glucosidase, and urease, are the indicators of an increase in the aerobic microbial activity, microbial activity related to the carbon cycle, and conversion of nitrogen to ammonium form, respectively, in the soil [92]. In the present study, the soil microbial activities were evinced increasing with the application of 400 and 600 EB, as well as biochar–fertilizer mix compared to the controls (C1, C2, and C3). Nevertheless, the rate of application of biochar (from 0.5 to 5%) in the soil also significantly increased (p < 0.05, Figure 2b–d) the microbial activities. The increase in the soil microbial activities can be correlated (r² > 0.75, Figure S5) with the increase in the soil pH, CEC, available nutrients, and organic matter in the soil, which synergistically improved the local soil environment for their growth. The above finding of the association of microbial activities with the soil physicochemical properties is in line with the facts reported in earlier studies, which stated that an increase in the soil pH, CEC, and available nutrients are the key factors for the abundance of microbes in the soil [102,103]. Apart from this, another reason for the significant increase (p < 0.05) in the soil microbial activities could be the reduction in the availability of the toxic heavy metals due to their sorption on the biochar surface [101].

The soil fertility index (SFI), which is an indicator of overall soil fertility level, was substantially increased (Figure 3) for the soil amended with the 400 and 600 EB, as well as biochar and fertilizer compared to the controls (C1, C2, and C3). The SFI of the soil mixed with the biochar, as well as the biochar–fertilizer mixture at a higher mixing ratio, i.e., at 5% (w/w), was comparable to the SFI (29.80, SFI was calculated using the data reported in the paper) of the 7-year-old soil, as reported in an earlier study [104]. The increase in the SFI might be correlated (Figure S5) with the increase in the pH (r² > 0.80), available nutrients (r² > 0.60), OM (r² > 0.70), and CEC (r² > 0.60). Moreover, the SFI also significantly increased (p < 0.05, Figure 3) as the biochar application rate in the soil increased from 0.5 to 5 % (w/w) ratio. The SFI of the soil mixed with 600 EB biochar was significantly higher (p < 0.05) beyond the 2% application rate compared to the 400 EB, which might be due to the significantly higher (p < 0.05, Figure 2 and Table S2) availability of plant-available nutrients, liable organic carbon, organic matter, pH, and large surface area that might have helped in the growth of microbes in the soil. The SFI of the soil amended with the co-application of biochar–fertilizer was significantly higher (p < 0.05) than the sole application of 400 and 600 EB at the application rate of 2% (w/w) and above.

Effect of Biochar and Fertilizer Amendment on the Availability of the Heavy Metals in the Soil

It has been discussed in the previous Section 3.1.1. that the soil collected from the overburden dump was found contaminated with multiple heavy metals (Ni, Co, Zn, Cu, and Cr). The joint application of the biochar and fertilizer was made to promote the remediation of the soil using the revegetation process, to reduce the metal toxicity to the plants and prevent the leaching of the metals from the soil. It can be observed from Table S6 that the concentrations of the acid-extractable heavy metals (Ni, Co, Cu, Zn, and Cr) in the soil amended with both 400 and 600 EB, as well as the respective biochar and fertilizer, were significantly reduced (p < 0.05) compared to the controls (C1 and C2). The application rate of the biochar also had a considerable effect on the sorption of the heavy metals in the soil. The content of total heavy metals (acid extractable) in the soil was significantly reduced (p < 0.05, Table S4) by increasing the mixing ratio of the biochar (EB 400 and 600) from 0.5 to 5% (w/w). Such a reduction in the heavy metals in the soil can be linked to the higher adsorption capacity of the EB that might have adsorbed the heavy metals through the formation of metal ion complexes, electrostatic attraction, and precipitation in the soil-biochar matrix. A similar observation was reported in an earlier study, where the acid extractable contents of the Cd, Cu, Zn, and Pb were significantly reduced with the increase in the application rate of bamboo and rice straw-derived biochar from 1 to 5% (w/w) [45]. Importantly, the soil amended with the same varying amount of biochar with the fixed-dose of fertilizer had a significantly (p < 0.05) lower content of acid-extractable heavy metals compared to the soil amended with 400 and 600 EB. Such a reduction in the concentration of acid extractable heavy metals can be linked to the fact that the jointly applied biochar–fertilizer in the soil might have promoted the higher adsorption of the metals within the soil [105]. The evidence of the adsorption of heavy metals onto the EB surface can be evinced from the post-pot culture biochar characterization study through SEM, EDAX mapping (Figure S8a), and XRD analysis (Figure S8b). It can be observed from the SEM image analysis and EDAX mapping (Figure S8a) that the heavy metals successfully adsorbed onto the biochar surface. This fact can further be verified through the post-XRD analysis results of biochar, which indicated an increase in the number of peaks of both 400 and 600 EB after the completion of the pot-culture study compared to the pre-biochar analysis. The change in the peaks can be marked at the 2 θ angles of 13°, 21°, 26.5°, 36°, 42°, 45°, 47°, 50°, 55°, 60°, 64°, 68°, and 79°, respectively (Figure S8b). Heavy metals are reportedly immobilized within the soil matrix by forming complex ionic compounds with the phosphate and carbonates to form precipitates, electrostatically bonding to the surface of the biochar, forming complexes with the deprotonated functional groups due to increasing pH, and reduction by accepting the π electrons from the biochar [105,106]. The detailed mechanism of heavy metals sorption onto the biochar within the soil matrix is diagrammatically shown in Figure 4.

The soil containing biochar–fertilizer had shown higher sorption capacity compared to the soil merely containing only biochar, and hence, has high adsorption efficiency for heavy metals. Furthermore, the soil containing 600 EB at the application rate greater than 1% had significantly lower (p < 0.05, Table S6) metal content compared to the 400 EB. A similar observation was reported in a study by Xiao et al. [107], who reported a high sorption capacity of Pb adsorbed using biochar produced at 600 °C compared to the low-temperature biochar (300 °C).

The DTPA-extractable heavy metals represent the available form of the metals, which are usually present in the soil organically bounded, and can be phytoextracted by the plants in their shoot and root parts [108]. It can be observed from Table S5 that the plant-available forms of heavy metals in the soil amended with both 400 and 600 EB, as well as the biochar–fertilizer, were significantly reduced (p < 0.05) compared to the controls (C1 and C2). Moreover, by increasing the application rate of 400 EB from 0.5 to 5% (w/w) in the soil, the availability of the heavy metals was substantially reduced by 64.48, 39.75, 33.73, 20.47, and 34.15%, for Ni, Cu, Zn, Co, and Cr, respectively (Table S3). Similarly, for the 600 EB the availability of the heavy metals was reduced by 59.89, 44.85, 36.2, 30, and 37.57%, respectively. The 600 EB showed a better reduction in the availability of heavy metals compared to the 400 EB. This might be due to the effective adsorption of heavy metals by 600 EB, owing to having high CEC, mineral matter content, and surface area [38]. The joint application of 400 and 600 EB with the fertilizer reduced the availability of the heavy metals at an application rate greater than 1% (w/w) (Table S5). It can be concluded from the above results that the co-application of both 400 and 600 EB up to 5% (application rate (w/w) along with the fertilizer further reduced the availability of Zn, Co, and Cr compared to the single application of biochar in the soil.

The decrease in the availability of the DTPA-extractable metals in the soil amended with both biochar and biochar–fertilizer can be linked to the increase in the soil pH, CEC, available nutrients, and high biochar surface area (Figure 1 and Figure 2). With the increase in the pH of the soil and continuous oxidation with time, the abundance of the polar functional groups (such as –OH, –COOH, –CO, and phenolic groups) reportedly increased [109]. Due to the increased polar functional groups on the biochar surface, the adsorption of cationic metals might have occurred through the surface complexation process [106]. Additionally, the high surface area and the presence of carbonates and minerals on the biochar surface might have also facilitated the adsorption of heavy metals through the formation of metal-carbonate or metal ions precipitate on the biochar surface [107]. Perhaps the pH_pzc of the biochar also has a key role in the sorption of heavy metals. It has been reported that the surface of the biochar remains positively charged if the pH of the aqueous solution lies below the pH_pzc of the biochar; otherwise, it is negatively charged [40,110]. In the present study, the initial pH of the soil was acidic, with pH = 5.86, which is less than the pH_pzc of the biochar (Table S1); however, with time, due to the liming effect, the pH of the soil was increased up to 8.2 (Figure 1). Hence, the surface of the biochar, which was initially positively charged, might have progressively increased the surface negative charge to promote the sorption of the metals through electrostatic attraction. The application of biochar, as well as the biochar–fertilizer mixture, effectively reduced the availability of the metals below the permissible limit in the soil and reduced their toxicity to the plants.

3.2. Effect of Biochar and Fertilizer Amendment on the Plant’s Physiological and Biochemical Properties

3.2.1. Effect of Biochar and Fertilizer Amendment on Plant’s Growth and Biomass

The plant biomass is an important indicator to mark changes in the physiological characteristics of any vegetation. The plant biomass (A. auriculiformis) of both shoot and root parts was significantly increased (p < 0.05) with the incorporation of biochar and biochar–fertilizer in the soil compared to the controls (C1 and C2) (Figure 5). Moreover, the shoot biomass was significantly increased by 37.02 and 38.11% (p < 0.05, Figure 5) by increasing the application rate of both 600 and 400 EB up to 5% (w/w), respectively. The co-application of biochar and fertilizer significantly increased the shoot and root biomass beyond a 0.5% (w/w) application rate compared to the single application of the biochar (Figure 5). The increase in the plant biomass (shoot and root) in the pots accompanied by the co-application of biochar–fertilizer might be related to the increase in the availability of the nutrients, neutralization of acidic pH, high CEC and WHC, and reduction in the availability of the toxic metals in the soil. A similar observation in the increase in the plant biomass with the application of biochar was reported in the earlier studies [92,111]. Likewise, the shoot and root length in the A. auriculiformis also significantly increased (p < 0.05, Figure 5) with the increase in the application rate of biochar compared to the controls (C1 and C2). Additionally, the shoot and root lengths were significantly increased by increasing the application rate of 400 and 600 EB from 0.5 to 5% (w/w). The increase in the shoot and root length with the increase in the biochar application rate might be linked to the fact that the presence of biochar in large volumes in the soil may have increased the availability of major and minor nutrients to be utilized by the plants. This fact is in line with the previously reported studies, in which biochar has shown a high affinity towards the sorption when major and minor nutrients applied in the soil [40,112,113].

3.2.2. Effect of Biochar and Fertilizer Amendment on the Heavy Metals Toxicity in the Plant

Concerning the heavy metals in the shoot and root parts of the A. auriculiformis, the application of 400 and 600 EB, as well as the biochar and fertilizer significantly (p < 0.05, Table S6) reduced the heavy metals content compared to the controls (C1 and C2). Increasing the application rate of 400 and 600 EB, as well as the biochar–fertilizer mixture up to 5% (w/w) significantly (p < 0.05, Table S8) reduced the heavy metals in both the shoot and root parts of the plant. A similar observation of a reduction in the heavy metals toxicity in the plant’s shoot and root biomass with the application of the biochar was reported in the earlier studies [113,114]. The reduction in the heavy metals toxicity in the plant’s shoot and root biomass might be associated with the increase in the sorption of the metals within the biochar–soil interface following the complex adsorption mechanism, as shown in Figure 4 [103,112]. The decrease in the heavy metals in shoot and root biomass can also be linked to the decrease in the content of DTPA-extractable heavy metals in the soil (Table S5).

The translocation factor (TF) is an important index for the phytoavailability from the root to the shoot part of the plant, whereas the bioaccumulation factor (BAF) is an index for the translocation of metals from the soil to the plant [114,115]. A TF factor value higher than one indicates translocation of the heavy metals from the root to the shoot part of the plant, whereas a TF value less than one indicates the root part has a higher metal concentration than the shoot part [85]. It can be observed from Figure 6a that the TF values for the heavy metals in the plants that grew in the soil amended with biochar and fertilizer were less than the TF values of controlled studies indicating effective sorption of heavy metals within the soil root zone. The TF values for Zn, Ni, Cr, and Cu, which were less than unity, were further decreased with the increase in the application rate of 400 and 600 EB up to 5% (w/w) in the soil. The TF values for Zn, Ni, Cr, and Cu were further reduced with the joint application of biochar and fertilizer in the soil. This indicated that the application of the biochar and biochar–fertilizer effectively immobilized them within the soil-root zone through the various sorption processes [103].

Instead, the TF value of Co was increased with the increase in the application rate, indicating that the excess of Co²⁺ ions was translocated from the root to the shoot parts due to their poor sorption in the biochar-soil matrix. Moreover, the co-application of biochar and fertilizer further increased the TF values due to the poor sorption of Co²⁺ ions in the soil matrix. Such a reduction in the sorption of Co²⁺ ions in the soil matrix amended with both biochar and fertilizer can be related to the reduction in the adsorption sites in the competitive environment.

The bioaccumulation factor (BAF) for the heavy metals was lower (<1) in the plants that grew in the soil amended with the biochar and biochar–fertilizer mixture compared to the controls (C1 and C2, Figure S7). Moreover, the BAF_shoot for heavy metals was further reduced in the plants grown in the soil amended with the co-applied biochar and fertilizer except for Zn. The reduction in the BAF_shoot due to the co-application of biochar and fertilizer can be linked to the increase in the CEC and the presence of functional groups on the biochar surface that might have immobilized the metals within the soil [116]. However, the existence of cations might also have reduced the relative affinity for Zn²⁺ ions within the soil matrix due to increased competence on the adsorption sites onto the biochar’s surface. A similar observation of lower Zn sorption affinity for the biochar in a multimetal system was reported in an earlier study [117].

The BAF_shoot for Ni, Co, and Cu were increased with the increase in the application rate of both 400 and 600 EB from 0.5 to 5% (w/w) (Figure S7), whereas, for Zn and Cr, it was decreased by 31.92 and 14.28% and 13.95 and 16.67%, respectively. This indicated the higher translocation of Ni, Co, and Cu ions from the soil to the shoot part due to the presence of an excess of these ions within the soil matrix with the increase in the biochar’s application rate. Such an increase in BAF_shoot for these heavy metals can be associated with a decrease in the sorption capacity due to an increase in the biochar volume in the soil. As the biochar also intrinsically contains these metals in its structure to some extent (Table S3), the existence of the metals might have increased with the increase in the application rate, which, in turn, may have reduced their sorption in the soil matrix. Conversely, the Zn and Cr ions, in which Cr ions mostly occur in Cr (VI) form (HCrO₄⁻, CrO₄²⁻, and Cr₂O₇²⁻) under oxidizing conditions and at the acidic pH [118,119,120], might have adsorbed within the soil matrix through electrostatic attraction and abundance of cations and high organic matter content within the soil matrix.

The application of the 400 and 600 EB substantially reduced the BAF_root values for heavy metals in the root part of the plant (Table S8). This can be linked to the decrease in the concentration of the available form of the metals by the formation of metal complexes with the functional groups present on the biochar [103]. Additionally, the joint application of the biochar with fertilizer further reduced the BAF_root values for heavy metals in the root zone due to the presence of additional ions (K⁺, PO₄³⁻, and NO₃⁻), as well as a relative increase in the pH compared to the single application of the biochar, which might have facilitated the precipitation of the metal ions.

3.2.3. Effect of Biochar and Fertilizer Amendment on a Plant’s Biochemical Hormones

As discussed earlier, the contaminated soil was deprived of nutrients and had elevated levels of heavy metals. Plants that grew in such soil were subjected to the drought condition along with having the stress of metal toxicity [121]. Under such poor environmental conditions, overproduction of reactive oxygen species (ROS) occurs, which indicates damage to the plant cells [122]. Under such circumstances, a defense mechanism comes into action to prevent the plant cells from being damaged, which leads to the production of antioxidants such as proline and total glutathione (GSH_t) [123,124]. The excessive production of proline and GSH_t are the indicators of the plant being subjected to abiotic stress conditions. It can be observed from Figure 6 that the application of 400 and 600 EB, as well as the joint application of biochar and fertilizer in the soil, significantly (p < 0.05) reduced the level of proline and GSH_t in the plants compared to the controls (C1 and C2) due to the inhibition of the production of ROS. Moreover, with the increasing application rate of the 400 and 600 EB from 0.5 to 5% (w/w), the proline level in the plant cells was significantly (p < 0.05) reduced by 36.57 and 33.48%, respectively. Likewise, the GSH_t level was reduced by 47.78 and 50.32%, respectively. The co-application of biochar–fertilizer further significantly (p < 0.05, Figure 6) reduced the proline and GSH_t levels in the plant tissues at the various application rates (0.5 to 5% (w/w). A similar observation of the reduction in the proline and GSH_t due to the application of the biochar in the mung bean reported in an earlier study [125]. The reduction in the proline and GSH_t levels in the plant tissues with the application of 400 and 600 EB, as well as the co-applied biochar–fertilizer might be related to the increase in the plant-available nutrients, improvement in the soil pH, and a reduction in the plant-available heavy metals. The relation among proline, GSH_t, and soil physicochemical properties is evident in the correlation matrix Figure S6, where the proline and GSH_t levels in the plant tissues have shown a strong positive correlation (r² > 0.85) with the TF and BAC for the heavy metals, and strong negative correlation (r² > 0.90) with the soil pH. Chlorophyll is the green pigment present in the plant leaves primarily responsible for the photosynthesis in the plants. It can be observed from Figure 6 that the incorporation of 400 and 600 EB, as well as the jointly applied biochar–fertilizer, significantly (p < 0.05) improved the chlorophyll content in the plants compared to the controls (C1 and C2). Moreover, with the increase in the application rate of biochar in the soil from 0.5 to 5% (w/w), the chlorophyll content in the plant leaves was also significantly (p < 0.05) improved. The increase in the chlorophyll content in the plants that grew in the biochar and biochar–fertilizer amended soil might be associated with the increase in the availability of the major and minor nutrients, and a decrease in the metal toxicity as indicated by TF and BAF values discussed earlier (Figure S6).

4. Conclusions

The following conclusions can be obtained from the results of the present study:

• Incorporation of the biochar in the soil improved its physicochemical properties, such as an increase in pH, available nutrients, organic matter, and soil enzymatic activities.
• An increase in the rate of application of biochar up to 5% (w/w) significantly improved the physicochemical properties of the soil. This can be evinced by the increase in the pH, exchangeable nutrients, organic matter, CEC, and the soil’s enzymatic activities. Simultaneously, it has reduced the plant-available content of the heavy metals in the soil.
• The soil fertility index was significantly increased with the application of both biochar and the biochar–fertilizer mixture compared to the controls. Furthermore, the co-application of the biochar with NPK fertilizer increased its efficacy to be used as a soil-amending material.
• High-temperature biochar (600 EB) showed better sorption of heavy metals in the soil compared to 400 EB indicating better efficacy of the 600 EB in reducing the metal toxicity in the soil.
• Plant analysis results showed that the co-application of biochar with fertilizer substantially reduced metal toxicity and water stress effect in the plants as evidenced by the BAF, TF, proline, and glutathione (GSH_t) data of the plant analysis.