Ameliorative Effects of Biochar for Cadmium Stress on Bean ( Phaseolus vulgaris L.) Growth

: In order to investigate the changes in the morphological and biochemical characteristics of bean plants in response to biochar treatment under cadmium (Cd) stress, a pot experiment was conducted in a greenhouse. Bean plants were subjected to different amounts of Cd (0, 100, 150 and 200 mg kg − 1 ) and biochar applied at different doses (0, 2.5 and 5%). Under Cd stress, the growth and development of bean seedlings were remarkably inhibited, whereas the biochar treatment could effectively improve the heavy metal tolerance of bean seedlings. Cd stress caused an increase in the hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), proline and sucrose content, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activity of leaves. However, biochar treatments reduced the CAT, POD and SOD activity of bean seedlings. Growing beans on Cd medium led to a signiﬁcant reduction in plant nutrient element content. However, biochar amendment to the soil elevated the plant nutrient element content compared to untreated soil. Cd content of the bean seedlings increased with increasing Cd doses. There was a sharp decrease in available concentration for Cd with the addition of biochar. In conclusion, biochar incorporation into the soil can alleviate the adverse impacts of Cd stress on the growth of bean seedlings.


Introduction
Heavy metals are often associated with anthropogenic activities. Heavy metal pollution in agricultural areas has become an important issue, especially in developed countries [1]. They accumulate in the environment due to sewage sludge, rock phosphate, mining applications and industrial activities [2]. Heavy metals such as Cd have a toxic effect on plants and adversely affect their morphological, physiological and biochemical properties [3]. Compared to other heavy metals, Cd is more mobile in soil. It can endanger plant, animal and human health by accumulating both in the soil and plants [4].
Cadmium is known to be a toxic element that poses serious human health risks through the ingestion of contaminated agricultural produce if found in high concentrations in soil [5]. Contamination of agricultural soil with Cd has become a global concern because of its adverse effects on health and food safety. Increased heavy metal levels are an important stress factor for the environment and humans. Heavy metals can mix with water, be taken up by plants and released into the atmosphere as a gas, or be adsorbed by soil components such as clay or organic matter. These heavy metals, which are included in the food chain with plant and animal production, adversely affect human health [6]. Today, heavy metal toxicity and accumulation in plants is a serious environmental problem. Heavy metals such as Cd have been reported to affect plant growth and productivity in many agricultural products negatively. Moreover, it has been reported that cadmium increases ROS (Reactive Oxygen Species) and decreases photosynthetic activity in plants [3].

Experimental Material
The study was conducted at Atatürk University in Erzurum (39 • 54 N; 41 • 15 E), Turkey, in 2022, under greenhouse conditions where the average photosynthetically available radiation measured at noon ranged from 1098 to 1490 µmol m −2 s −1 . Bean (Phaseolus vulgaris L. Hınıs cv.) plants were maintained under natural light conditions, the average day/night temperatures of 27/20 • C and 65% relative humidity during the span of the experiment.
Bean (Phaseolus vulgaris L. Hınıs cv.) seeds were used as plant material. Biochar produced by using the Thermal Conversion Process (TCP™) was obtained from SYNPET Technologies (Istanbul, Turkey). The chemical properties of the biochar are presented in Table 1. The soil used in this study was sampled to a depth of 15 cm from agricultural fields

Experimental Set-Up
Biochar was weighed at the rate of 0%, 2.5% and 5% by soil weight and mixed thoroughly with soil before filling the pot. Three biochar doses (sandy loam soil in 2.5 L pots, 2.5% (w/w) biochar mixed thoroughly with the sandy loam soil prior to filling into 2.5 L pots, 5% (w/w) biochar mixed thoroughly with the sandy loam soil prior to filling into 2.5 L pots) were applied under different cadmium levels. For the control treatment, only soil was filled in the pots. For heavy metal stress treatments, cadmium (CdSO 4 ·8H 2 O) Sustainability 2022, 14, 15563 4 of 13 was mixed with the medium at three different concentrations (0, 100, 150 and 200 mg kg −1 ) and incubated for 3 weeks. Irrigation was done at intervals of 2-3 days. Three bean seeds were sown for each pod 21 days later. After emergence, one vigorous seedling was left for each pod. The treatments consisted of three different doses of biochar (0%, 2.5% and 5% w/w) and four different doses of Cd (0, 100, 150, 200 mg kg −1 ) arranged in a completely randomized block design with three replications and four plants in each replicate.

Plant Analyses
Bean seedlings were harvested 40 days after sowing. In this study, morphological (plant height, plant stem diameter, leaf area, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight) and biochemical (mineral elements (N, P, K, Ca, Mg, S, Mn, Fe, Zn, B and Cd), H 2 O 2 , MDA, proline, sucrose, CAT, POD, SOD) parameters were investigated. During harvest, plants were cut from the soil level, and above-ground biomass (shoot) and roots were separated for measurements and analysis.
The leaf area was determined by a leaf area meter (CI-202 Portable Laser Leaf Area Meter by CID Bio-Science, Camas, WA, USA). Assays of MDA, H 2 O 2 and antioxidant enzyme activity were performed by UV/Vis spectrophotometer according to Sarafi et al. [16].
For the determination of mineral nutrition content, the leaves of the bean cultivars were dried at 68 • C for 48 h and grounded. Mineral contents were analyzed by a coupled plasma spectrophotometer (Optima 2100 DV; Perkin-Elmer, Shelton, CT, USA) [17].

Statistical Analyses
Data obtained from the measurements were evaluated statistically using analysis of variance (ANOVA). For statistical analysis of data, the SPSS program was used. The differences among the means were compared using the Duncan multiple range test (DMRT) (p < 0.05).

Results
The variation analysis results are given in Table 3. According to the results, it was determined that the effects of biochar, heavy metal stress, and their interaction were significant. Under Cd stress, the growth and development of bean seedlings were remarkably inhibited, whereas the biochar treatment could effectively improve the heavy metal tolerance of bean seedlings. In fact, biochar incorporation into soil improved the growth properties of the bean seedlings under non-stressed conditions. A total of 200 mg kg −1 Cd stress led to a decrease in seedling height, stem diameter, leaf area, shoot fresh weight, shoot dry weight, root fresh weight and root dry weight by 22.52%, 15.23%, 23.61%, 50.81%, 51.10%, 53.07% and 54.35%, respectively, compared to non-stressed control. However, 5% biochar treatment increased seedling height, stem diameter, leaf area, shoot fresh weight, shoot dry weight, root fresh weight and root dry weight by 20.76%, 16.07%, 25.32%, 46.68%, 38.67%, 37.41% and 36.36%, respectively, compared to the control (Table 4).  There were significant interactions between Cd treatments and biochar application rates on N, P, K, Ca, Mg, S, Mn, Fe, Zn, B and Cd content of the bean seedlings ( Figure 1) 42.33% and 87.76%, respectively, compared to untreated soil. The Cd content of the plant increased with the increase in Cd application dose. The highest Cd content was obtained from 200 mg kg −1 Cd application doses without BC application. Notably, 5% biochar is more effective in decreasing uptake than the other biochar concentration (Figure 1).
Each value is the mean of three replicates ± SD. Data followed by a different letter in column were significantly different according to the DMRT **: p < 0.01. Cd: Cadmium, BC: biochar.
There were significant interactions between Cd treatments and biochar application rates on N, P, K, Ca, Mg, S, Mn, Fe, Zn, B and Cd content of the bean seedlings ( Figure 1). Growing bean under 200 mg kg −1 Cd stress medium led to a significant reduction for N, P, K, Ca, Mg, S, Mn, Fe, Zn and B by 31 42.33% and 87.76%, respectively, compared to untreated soil. The Cd content of the plant increased with the increase in Cd application dose. The highest Cd content was obtained from 200 mg kg −1 Cd application doses without BC application. Notably, 5% biochar is more effective in decreasing uptake than the other biochar concentration (Figure 1).

Discussion
Heavy metals can cause serious dangers for living organisms, even at very low concentrations, since they do not have a biodegradable structure such as other pollutants. Heavy metals such as Cd have attracted the attention of many researchers not only because of their phytotoxicity but also because they can be absorbed into plant bodies and thus enter the food chain. The findings of the present study indicated that excess Cd caused a significant reduction in the growth of the bean. This lower growth may be due to Cd being toxic to plants [18]. However, biochar incorporation statistically significantly reduced bioinhibition of the growth of bean seedlings compared to non-treatment ( Table  3). The findings of the study pointed out an increase in the growth of bean seedlings with applied biochar. Yousaf et al. [19] reported that the addition of biochar to the soil had a positive effect on plant growth because it reduced the bioavailability of cadmium in the

Discussion
Heavy metals can cause serious dangers for living organisms, even at very low concentrations, since they do not have a biodegradable structure such as other pollutants. Heavy metals such as Cd have attracted the attention of many researchers not only because of their phytotoxicity but also because they can be absorbed into plant bodies and thus enter the food chain. The findings of the present study indicated that excess Cd caused a significant reduction in the growth of the bean. This lower growth may be due to Cd being toxic to plants [18]. However, biochar incorporation statistically significantly reduced bioinhibition of the growth of bean seedlings compared to non-treatment ( Table 3). The findings of the study pointed out an increase in the growth of bean seedlings with applied biochar. Yousaf et al. [19] reported that the addition of biochar to the soil had a positive effect on plant growth because it reduced the bioavailability of cadmium in the soil. Biochar is rich in aromatic and humic substances, so it improves the physical, biochemical and biological properties of soils [9].
The negative effects on respiration, carbohydrate metabolism, chlorophyll formation, Calvin cycle, plant nutrient uptake and biosynthesis of DNA, RNA and other proteins are effective in the decrease of growth in plants grown under Cd toxicity. Since Cd is a phytotoxic element for soil organisms and plants [20,21], plant growth has been adversely affected, and a continuous decrease in the amount of dry matter has been observed. Cadmium toxicity decreased in the yield of different crops; at 30% for maize (Zea mays L.) [22], at 28.9% for chickpea (Cicer arietinum) [23] and at 37.0% for mungbean (Vigna radiata) [24]. Cadmium in the plant; reduces nitrate assimilation [25], causes closure of stomas [26], reduces the rate of photosynthesis and chlorophyll synthesis [27], changes their metabolism and inactivates enzymes [26]. It causes a decrease in chlorophyll synthesis [28], an increase in lipid peroxidation, and inhibition of N and S metabolism [29]. It was determined that the opening of stomata, respiration and photosynthesis activities decreased in plants exposed to Cd toxicity [30]. Stobort et al. [31] reported that chloroplast metabolism was impaired, and the activities of enzymes that played an important role in CO 2 fixation were inhibited because Cd reduced chlorophyll biosynthesis.
The negative effect of Cd applications on the growth of bean seedlings was partially eliminated with increasing amounts of biochar. In the conditions where Cd is not applied, the amount of the above-ground part and root dry matter of the bean seedlings has increased continuously with biochar ( Table 3). The increase in the amount of dry matter created by biochar, even in the presence of Cd, can be explained by the fact that biochar provides an important adsorbent feature due to its porous and void structure, negatively charged surfaces and functional groups such as carboxyl, hydroxyl, phenoxyl and carbonyl [32]. Thanks to this feature of biochar, the adsorption and immobilization of biochar, especially in the reclamation of soils contaminated with heavy metals, the usefulness and mobility of these heavy metals decreases, and the negative effects of heavy metals are prevented [33,34].
Cd stress led to ROS accumulation in various crops, enhancing the malondialdehyde (MDA) contents [35][36][37]. Trovato et al. [38] pointed out that proline is involved in response to environmental stress conditions. Cd stress increased H 2 O 2 , MDA, proline and sucrose content and antioxidant activity of bean (Phaseolus vulgaris L.) seedlings. A total of 3 µM Cd was applied to the bean plant for 48 h, and it was observed that the expansion of the leaf cells was inhibited, and the cell wall elasticity decreased [39]. It has been reported that Cd causes oxidative damage in plants by increasing free oxygen radicals or reducing the amount of enzymatic or non-enzymatic antioxidants under Cd toxicity [40,41]. The excess Cd has been reported to lead to genotoxicity in various crops [42].
Correa et al. [43] applied 0, 6.25, 12.5 and 50 mg kg −1 Cd to the soil in their greenhouse experiment to determine the effect of Cd on the development, fresh weight, germination rate and antioxidative enzyme activity of lettuce (Lactuca sativa), cabbage (Brassica oleracea) and oat (Avena sativa) plants. At the end of the experiment, it was determined that the effects of Cd applications on plant growth and biomass in lettuce, cabbage and oat plants had a negative effect, while the enzyme activities (catalase, peroxidase, superoxide dismutase and glutathione reductase) increased significantly compared to the control. Similarly, Yildirim et al. [44] showed that Cd contamination increased H 2 O 2 , MDA, proline and sucrose content and antioxidant activity of garden cress (Lepidium sativum). In the study, we observed biochar incorporation decreased H 2 O 2 , MDA, proline and sucrose content and antioxidant activity of bean seedlings. This may be due to the fact that biochar applications reduce the negative effect of Cd stress. In addition, biochar has strong interaction mechanisms with active surfaces, including ionic and covalent bonds [45]. With these properties, they bind heavy metals to their functional groups [34,[46][47][48] and prevent the toxic effects of heavy metals.
Different applications have been used to prevent Cd toxicity, and it has been reported that biochar applications give good results in increasing plant tolerance against heavy metal stress. It has been noted that the toxicity effect on crops such as soybean (Glycine max), corn (Zea mays L.), rice (Oryza sativa) and spinach (Spinacia oleracea) is reduced, and the yield increased with the application of biochar in heavy metal polluted soils [48]. The biochar amendment into soil has been shown to affect the physical, chemical and biological processes of the soil. It has a positive effect on the microbiological activity in the soil and increases the efficiency of mycorrhizae by providing nutrients in particular [49,50].
Biochar application significantly reduced the Cd concentration of the spinach plant aerial parts and roots. Mckenna et al. [51], Naik et al. [52] and Alia et al. [53] determined that the Cd concentrations of the spinach plant increased depending on the Cd doses applied in their greenhouse experiments. Similar results with bean plant [39], five different pepper (Capsicum annuum) varieties [54], two different rice varieties [55], lettuce, cabbage and oat plants [43], and lettuce [56] and tomato (Solanum lycopersicum) plants [57] have also been reported in previous experiments.
The most effective factor in reducing the amount of Cd available to plants from biochar is due to the binding of Cd on high cation exchange surfaces [58]. It has been stated that N, P, K and other plant nutrients, which are found at high levels in biochar, compete with Cd in the pellet and prevent the decrease of Cd concentration and its transport to the plant [59]. An obvious positive feature of biochar is that it provides nutrients directly to the plants, prevents the nutrient elements in the soil from being washed away, and indirectly improves the soil quality, increasing the nutrient utilization of the plants and, accordingly, increasing the efficiency of fertilizer use. It has been determined that the presence of Cd in the plant growth medium has negative effects on the intake of plant nutrients necessary for the development of plants. Higher uptake and mobility of Cd by plants significantly reduce Fe intake, causing chlorosis formation and nutrient deficiency by limiting K and Ca intake [60][61][62]. Inal et al. [63] reported that biochar applications increased the development of corn and bean plants, and caused an increase in N, P, K, Ca, Fe, Zn, Cu and Mn concentrations in bean and corn plants. Biochar holds six times its own weight in water in the soil, which makes it a good nutrient for the soil by enabling the plants to absorb elements such as phosphorus and nitrogen from the soil more easily [10].
Activated black carbon and humic substances can immobilize heavy metals such as Cd in the soil and inhibit their availability to plants [64][65][66]. Biochar can be employed as fertilizer or soil amendment because of its highly porous carbon structure. It can improve the chemical, physical and biological structure of the soil for plant cultivation. The physical characteristics of agriculture soil are positively and directly affected by biochar as a soil conditioner [67]. The porosity and surface functional groups of biochar have shown that it will be a suitable adsorbent for the removal of heavy metals and phenolic compounds in soil and water [11].
The reducing/inhibiting effect of biochar on the uptake of Cd is due to its functional groups. In addition to its alkaline character, biochar retains Cd in the soil significantly and prevents its uptake by plants, thanks to its high ion exchange capacity and functional groups [6]. In addition, biochar has strong interaction mechanisms, including ionic and covalent bonds on the inner sphere surfaces [45] and functional groups that heavy metals can bind to biochar [34,68]. With these properties, biochar prevents the decrease of their usefulness in the soil solution and thus their uptake by plants by forming/fixing bonds with heavy metals, especially Cd. Furthermore, the precipitation of biochar with heavy metals can be an important factor in preventing the uptake of heavy metals [69].

Conclusions
The findings of the study indicated that biochar amendment of the Cd-contaminated soil mitigated the negative impacts of Cd stress on bean plants. In conclusion, biochar incorporation into the soil can alleviate the adverse impacts of Cd stress on the growth of bean seedlings. Biochar application increased the mineral element uptake, whereas it reduced Cd content under Cd stress conditions. It has the capacity to enable sustainable plant production with the stabilization technique of soils, especially in Cd metal pollution.