1 Abstract

2 Air pollution and soil contamination have caused major environmental damage in the

3 industrial complex of Gabes. This study aimed to evaluate the abilities of biochar to modify

4 soil properties and assess the adaptation of alfalfa (Medicago sativa L.) plants in contaminated

5 soils from the Gabes Region. The experiment was executed with soil samples from three sites

6 (S1, S2 and S3) located at different distances from the industrial zone of Gabes. Additionally,

7 a control soil was included for comparison. Pot experiments were performed under controlled

8 conditions, with or without biochar. After 60 days, the accumulation of heavy metals in plants

9 (roots, shoots and nodules) was determined. Moreover, oxidative stress biomarkers, such as

10 malondialdehyde (MDA) content, glutathione-S-transferase (GST) and catalase (CAT), were

11 evaluated. Soil microbiological properties, including bacterial functional diversity and

12 fluorescein diacetate hydrolytic (FDA) activity, were analysed, along with soil chemical

13 properties. Our results revealed that biochar supplementation can improve microbial functions

14 and cation-exchange capacity (CEC), thereby increasing the availability of nutrients to plants.

15 Interestingly, the application of biochar resulted in decreased concentrations of copper (Cu)

16 and zinc (Zn) in plants, which may be attributed to a reduction in their bioavailability in the

17 soil. The accumulation of heavy metals in alfalfa organs was positively correlated with the

18 levels of MDA and antioxidant enzymes in both leaves and roots. In this study, the addition of

19 biochar reduced the antioxidant mechanisms of alfalfa and mitigated the negative effects of

20 metals, resulting in a positive impact on growth and chlorophyll content. Our data highlights

21 the beneficial effects of biochar on enhancing crop productivity and remediating contaminated

22 soil.

23 Keywords: Gabes’ industrial area, alfalfa, biochar, heavy metals, bioavailability, microbial

24 functions, oxidative stress, enzyme activities

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29 1. Introduction

30 The Gulf of Gabes is a Mediterranean coastline located in the eastern part of Tunisia. The gulf

31 approximately covers the coast from Sfax to Djerba. At the head of the gulf is the city of

32 Gabes (Ghannouch). Over the past few decades, industrial activities and manufacturing

33 production located in this region have frequently been considered the major source of

34 environmental pollution (Gonzalez-Fernandez et al., 2011; Gao and Chen, 2012; Missawi et

35 al., 2020), notably after the establishment of the chemical industry between the commercial

36 and fishing harbours, near Gabes City (Armstrong-Altrin et al., 2015).

37 Mainly, soil contamination is derived from the local Chemistry Group, which produces

38 phosphoric acid, sulfuric acid and other phosphorus derivatives. One of the principal

39 discharged pollutants, phosphogypsum (PG), is a byproduct of the phosphate fertiliser

40 industry. It is generated during the production of phosphoric acid from phosphate rocks

41 (Rutherford et al., 1994). In fact, each tonne of phosphoric acid produces five tonnes of PG

42 (Ayadi et al., 2014). The production of PG is estimated at 112,500 tonnes (Bejaoui et al.,

43 2004).

44 Interestingly, PG is composed of lead (Pb, ~5 ppm), silica (5–10%), zinc (Zn), aluminium

45 (Al, ~0.1%), fluoride (F, ~1%), cadmium (Cd, ~0.3 ppm), phosphorus (P, ~0.5%), selenium

46 (Se, ~1 ppm), barium (Ba, 50 ppm), iron (Fe, ~0.1%) and chromium (Cr, ~3 ppm) (Gouidera

47 et al., 2009; Zaghden et al., 2014). Besides, 90% of radium (Ra) and polonium (Po) were

48 retained in PG (Carbonell-Barrachina et al., 2002). Indeed, its release into the environment

49 represents a serious threat to humans, plants, animals and natural ecosystems (Jin et al., 2019).

50 However, industries near agricultural soils and coastlines release wastewater directly into the

51 environment, posing a significant danger to soil quality, properties and plant viability

52 (Bermudez et al., 2010; Belon et al., 2012). At concentrations greater than 40 mg/kg of soil,
53 PG severely impairs soil fertility and limits plant growth (Sancho et al., 2009; Sengupta and

54 Dhal, 2021). This may be attributed to a decrease in soil nutrient status and the uptake of

55 essential nutrient elements by plants (Elloumi et al., 2015). Consequently, nutrient deficiency

56 significantly impairs the catabolic activity of indigenous microorganisms in a PG-

57 contaminated soil environment (Sengupta and Dhal, 2021).

58 As a model for evaluating soil fertility and the toxicity of chemical compounds, alfalfa plants

59 are widely used organisms (Li et al., 2013; Hattab et al., 2016; Kamran et al., 2016; Helaoui et

60 al., 2020a; Senovilla et al., 2018; Chen et al., 2022). In addition, its oxidative activities could

61 serve as potent biomarkers for ecotoxicological studies, such as catalase (CAT), superoxide

62 dismutase (SOD) and glutathione-S-transferase (GST). Also, several products of the

63 interaction between plants and pollutants could be generated. For example, malondialdehyde

64 (MDA) can result from peroxidation of lipidic membranes, and micronuclei frequency can be

65 a product of DNA damage (Hu et al., 2010; Gill and Tuteja, 2010; Sforzini et al., 2015;

66 Dourado et al., 2015).

67 In this context, bioremediation and phytoremediation are environmentally friendly techniques

68 used to remediate pollutants from contaminated soils, as proven in several studies (Pavel and

69 Gavrilescu, 2008; He et al., 2019). Bioremediation is a method aimed at removing

70 contaminants from soils using plants, invertebrates and/or microorganisms (Sherafatmand,

71 2015; Ojuederie and Babalola, 2017; Bose et al., 2020). Phytoremediation is a class of

72 bioremediation that involves the use of green plants for in-situ remediation, which aims to

73 clean up toxic substances from soils (Ullah et al., 2015; Rajendran and Gunasekaran, 2019).

74 The process of phytoremediation includes phytoextraction, phytodegradation,

75 phytovolatilisation and phytostabilisation (Chen et al., 2019). In addition to the traditional

76 concept of remediation, nowadays, several natural or synthetic organic compounds, such as

77 biochar, are being used for the immobilisation of other organic and inorganic pollutants (Hale
 78 et al., 2011; Beesley and Marmiroli, 2011; Arif et al., 2020; Helaoui et al., 2022). In fact,

79 biochar is known for its ability to improve soil physical structure in the soil restoration

80 process (Zhou et al., 2009; Ghosh et al., 2011; Ashfaq et al., 2021). Also, it improves the

81 physicochemical and biological characteristics of the soil, water-holding capacity and

82 aeration, ultimately decreasing the risk of contaminant absorption by plant roots (Puga et al.,

83 2016).

84 Furthermore, biochar has an excellent capacity to adsorb toxic pollutants from the soil

85 solution, making them unavailable for fauna (Uchimiya et al., 2010; Fahd et al., 2016).

86 Additionally, it is considered a favourable receptacle for microorganisms and fungal species

87 such as mycorrhizae and Trichoderma. This, in turn, provides a source of nutrients (Atkinson

88 et al., 2010; Sohi et al., 2010; Agegnehu et al., 2017; Dai et al., 2019). Hence, several studies

89 have shown that the application of biochar increases soil organic matter. This is due to the

90 sorption of organic compounds and the disruption of microbial enzymes that decompose

91 organic matter (Major et al., 2009; Beesley et al., 2010). These effects could be achieved by

92 influencing microbial metabolisms, which in turn induce changes in the structures of

93 microbial communities (Lehmann et al., 2011; O’Connor et al., 2018). However, the effects

94 of biochar on contaminated soils in the presence of alfalfa plants have not been evaluated.

95 Therefore, the objective of the present work is to assess the effects of biochar on heavy metal

96 mobility, soil properties and the growth of alfalfa plants in polymetallic-contaminated soils.

97 The specific aims are to (i) analyse how the addition of biochar (5%) to contaminated soils

98 affects agronomic, physiological and biochemical parameters of alfalfa plants; (ii) assess the

99 impact of biochar on the heavy metal content of alfalfa roots and shoots; (iii) determine the

100 effect of biochar on soil properties, including fluorescein diacetate hydrolytic (FDA) activity,

101 bacterial functional diversity and physicochemical properties; and (iv) evaluate the
102 relationship between variations in soil properties and plant parameters under biochar

103 amendment.

104 2. Materials and Methods

105 2.1. Field site and soil sampling

106 In the present investigation, four sites were chosen following a distance gradient from the

107 industrial zone of the Gabes region (Ghannouch) (Haydar et al., 1986). The sampling sites

108 were predominantly agricultural, with olive trees being the main crop for over 20 years (Fig.

109 S1). The first site, S1, is located approximately 2 km from the industry, S2 is 4.6 km away, S3

110 is 9.5 km away, and the control (C) organic soil was sampled from the Higher Institute of

111 Agronomy of Chott Meriem in Tunisia.

112 o Site 1 (S1): lbled – 33.895336, 10.100494, located 2 km from CGT.

113 o Site 2 (S2): Chentech – 33.868062, 10.108281, located 4.6 km from CGT.

114 o Site 3 (S3): Teboulbou – 33.828628, 10.128975, located 9.5 km from CGT.

115 o Control (C): Organic soil sampled from the Higher Institute of Agronomy of Chott

116 Meriem in Tunisia.

117 In accordance with the approach employed by Bayouli et al. (2021), the soils examined in the

118 study were predominantly composed of sand particles. The physicochemical characteristics of

119 the soils are presented in Table 1. The pH of the soil was determined to be alkaline. The

120 cation-exchange capacity (CEC) values of the contaminated soils were significantly lower

121 than those of the control. Also, Table 1 shows the concentrations of heavy metals in soils S1,

122 S2, S3 and C. The highest concentration of copper (Cu) was observed in soil S1, with a value

123 of 382.85±19.62 mg/kg, compared to the control value of 25.23±3.52 mg/kg. The most

124 polluted soil (S1) also had the highest Zn content at 1200.89±100 mg/kg, significantly higher

125 than the amounts observed at other sites. On the other hand, contaminated soils have higher

126 nickel (Ni) contents between 4.67±0.87 mg/kg and 8.09±0.77 mg/kg. In the case of Cd and
127 Pb, the highest concentrations were observed in S1, where concentrations reached 9.09±0.98

128 mg/kg and 0.89±0.03 mg/kg, respectively.

129 Soils were collected in March 2021 at a depth of 0–20 cm using a zigzag method (Mkhinini et

130 al., 2019). Each sample consists of a mix of four random sub-samples. Soils were

131 homogenised, sieved at 2 mm, air-dried and conserved at room temperature until

132 experimentation.

133 2.2. Plant material and growth conditions

134 In brief, each plastic pot measuring 16 cm in height and 16 cm in diameter was filled with 1

135 kg of dry soil (S1, S2, S3 and C). Biochar was added to the soil at a dose of 5% w/w (Abbas

136 et al., 2017a) and thoroughly mixed. The experiment was duplicated, with each treatment

137 being repeated five times, both in the presence and absence of biochar.

138 ● SB: soils cultured with alfalfa plants in the presence of 5% biochar (Li et al., 2018a).

139 ● S: soils cultured with alfalfa plants without biochar.

140 As reported by Zhang et al. (2010), biochar used for soil amendment was obtained from olive

141 sticks through thermal decomposition via pyrolysis between 350°C and 550°C using a

142 continuous production technology in a vertical kiln (Pan et al., 2011). Table S2 presents the

143 physicochemical properties of biochar.

144 Alfalfa seeds (cultivar Gabes) were soaked in sterile water for 48 hours, and then seedlings

145 were grown in pots. The plants were subsequently transported to the greenhouse, where the

146 average temperature was maintained at 20.5 ± 1.5°C, and the photoperiod was set at 14 hours.

147 They were placed on plastic plates in order to prevent the contaminant element from being

148 lost through leaching during irrigation, which was done using deionised water two or three

149 times a week.

150 2.3. Soil analysis

151 2.3.1. Chemical analysis

152 Soil samples were oven-dried overnight at 60°C and sieved (<0.02 µm). Next, 1 g of dry soil

153 was mixed with 37% HCl and 63% HNO3 (3/1:v/v) at 100°C (Zhao et al., 1994). The resulting

154 solution was diluted with 2% HNO3. Heavy metal analysis (Cu, Zn, Ni, Cd and Pb) was

155 conducted using inductively coupled plasma atomic emission spectrometry (ICP-AES; Fisons

156 ARL Accuris) (PerkinElmer 2380). The mean metal concentrations were calculated from five

157 replicates. The limit of detection (LD) for metals was 0.05 mg/kg.

158 2.3.2. Soil pH

159 Dried soil samples were mixed with water in a 1:2 ratio (sample: water), shaken for 30

160 minutes, and then left to stand for 60 minutes. Then, the pH of each sample was measured

161 using a pH metre (MetrOhm 744) (ISO 10390).

162 2.3.3. Cation exchange capacity (CEC)

163 The CEC of the soil sample was determined using the method described by Gillman (1979).

164 In brief, 2.5 g of soil was added to 30 ml of a 0.1 M BaCl2 solution. Then, the solution was

165 shaken for 60 minutes and centrifuged three times with 20 ml of 0.025 M BaCl2. The

166 supernatant was pipetted and used for barium analysis. After that, 20 ml of 0.025 M MgSO4

167 was added to the rest of the mixture and left overnight. The mixture was then centrifuged at

168 3000 rpm for 10 minutes. The CEC was calculated using coupled plasma optical emission

169 spectroscopy (ICP-OES, Spectroblue).

170 2.3.4. The fluorescein diacetate (FDA) assay

171 The FDA activity was measured in microplates following the method of Taylor et al. (2002).

172 The obtained activity (490 nm) was expressed as μg of fluorescein/g of dry matter (DM) of

173 soil/h, referring to a fluorescein calibration curve.

174 2.3.5. Functional diversity

175 BiologEcoplate™, a 96-well microplate (Biolog Inc., USA), filled in triplicate with 32 carbon

176 substrates, was employed to monitor the carbon source utilisation patterns of soil microbial
177 communities (Garland and Mills, 1991). Microtiter plates were inoculated following the

178 Harris-Hellal (2008) method. A 1 g soil sample was mixed with 9 ml of sterile saline (0.9%

179 NaCl), shaken for 30 minutes, and centrifuged at 1500 rpm for five minutes. The supernatant

180 was then diluted with sterile NaCl solution (0.85%) and added to each of the 96 wells of

181 BIOLOG1 Eco microtiter plates (150 ml). The plate was incubated at 25°C in the dark for

182 seven days. After incubation, the substrate utilisation (colour development) in the wells was

183 measured at 590 nm using a microplate E Max reader (Molecular Devices, Sunnyvale, CA).

184 Catabolic or functional diversity was measured by counting the number of positive wells. A

185 well was considered positive if it had an absorbance 0.125 higher than the blank (B), as

186 described by Garland (1997). The chromogenic average well-colour development (AWCD)

187 represents the overall metabolic activity of the bacterial community in the soil.

188 2.4. Plant sample analysis

189 2.4.1. Chemical analysis

190  Heavy metal accumulation in plants

191 The total heavy metal content of the plant samples was determined using the method

192 described by Zhao et al. (1994). In summary, 1 g of dried samples (leaves, roots and nodules)

193 was mixed with 37% HCL and 63% HNO3 (3/1: v/v) at 100°C. Then, the mixture was diluted

194 with 2% HNO3. Heavy metal content (Cu, Zn, Ni, Cd and Pb) was determined using

195 inductively coupled plasma atomic emission spectrometry (ICP-AES) with the Fisons ARL

196 Accuris instrument (PerkinElmer 2380). The means of metal concentrations were calculated

197 from five replicates. The limit of detection (LD) for metals was 0.05 mg/kg.

198  Heavy metal translocation index

199 The translocation factor (TF) depends on the transfer of heavy metals (such as Cu, Zn, Ni, Cd

200 and Pb) from the soil to the roots and from the roots to the shoots. TF may include the
201 phytoremediation potential of plants and the potential risks of heavy metals in plant biomass

202 (Phusantisampan et al., 2016).

203 TF = Croot/Csoil or Cshoot/Croot, where C represents the concentration of metal in soil,

204 shoot or root.

205 The bioconcentration factor (BCF) indicates the ability of plant roots to accumulate metals

206 from contaminated soil (Phusantisampan et al., 2016).

207 BCFR = Cplant/Csoil, where C represents the concentration of metals in their extractable

208 form in either the plant or the soil.

209 2.4.2. Plant harvesting and biometric parameters

210 After 60 days, alfalfa plants were transferred to the laboratory immediately after harvesting

211 and separated into roots and shoots. Plant parts were thoroughly washed with distilled water.

212 The length of shoots and roots was measured using a graduated ruler. Then, the analytical

213 balance Precision Scale ‘Sartorius Entris224-1S’ was used to weigh shoots and roots.

214 Subsequently, from each sample, fresh plants were dried in an oven set to low heat (60°C) for

215 72 hours. The percentage of DM was then measured as follows:

DM
216 DM (%) = ( ) * 100
FM

217 *DM: Dry matter of plants; FM: Fresh matter of plants.

218 2.4.3. Chlorophyll (Chl) content

219 Chlorophyll a (Chla), chlorophyll b (Chlb) and total chlorophyll (Chla+b) measurements were

220 taken from fresh leaves of plants. In fact, 0.5 g of leaves were weighed and digested in 20 ml

221 of 80% acetone. After filtration, the pigment content was determined using a VWR UV-3100

222 PC spectrophotometer at 663 nm for Chla and 645 nm for Chlb (Arnon, 1949; Sobrino-Plata

223 et al., 2013).

224 2.4.4. Analysis of antioxidant enzymatic activities

225  Protein extraction

226 The protein extraction was determined as described by Laemmli (1970) and detailed in

227 Supplementary Material S3. The protein content was quantified according to the method of

228 Bradford (1976) using a calibration curve with bovine serum albumin as a standard.

229  Catalase (CAT) activity

230 The CAT activity was determined using the Aebi (1984) method.

231  Glutathione-S-transferase (GST) activity

232 The activity of GST was determined according to the protocol of Habig et al. (1974).

233  Lipid peroxidation

234 The level of lipid peroxidation in alfalfa tissues was evaluated by measuring the MDA

235 content, which is a product of lipid peroxidation. The method used to determine MDA content

236 was based on the approach described by Ortega-Villasante et al. (2005).

237 2.5. Statistics

238 The results of this study were expressed as the mean ± standard deviation. Normality was

239 tested using the Shapiro-Wilk test. A student’s test was used to compare treatments with and

240 without biochar. A one-way analysis of variance (ANOVA) was performed on the data to

241 compare treatments without biochar against control treatments, as well as treatments in the

242 presence of biochar against control treatments. We also performed principal component

243 analysis (PCA) to assess the effects of different treatments on the detected biomarkers using R

244 software (R Development Core Team, 2011) and the FactoMineR and Factoshiny packages.

245 Heatmap correlation matrices were generated using the Corrplot and ggplot2 packages.

246 3. Results

247 3.1. Heavy metal analysis

248 3.1.1. Heavy metal accumulation in plants

249 The effects of biochar addition on heavy metal accumulation in different parts of Medicago

250 sativa L. (root, shoot and nodules) are presented in Table 2. Results demonstrated that Cu and
251 Zn are the most accumulated heavy metals in the plant, and this accumulation is greater

252 depending on the degree of soil contamination, with a maximum observed in nodules.

253 However, Ni, Cd and Pb were below the limit of detection (LOD), with heavy metal levels

254 measuring less than 0.05 mg/kg. Indeed, the levels of Cu in plants are showing a significant

255 increase, with a maximum of 19.95±0.9 mg/kg, 30.73±0.45 mg/kg and 321.26±22.49 mg/kg

256 in shoots, roots and nodules of alfalfa plants exposed to S1. The Cu content in shoots

257 decreased by 27%, 29%, 21% and 28% in the presence of biochar in S1B, S2B, S3B and C,

258 respectively, compared to the treatment without biochar. Also, in roots, we observed a

259 significant decrease in Cu content in S1B, S2B and C when biochar was present, with rates of

260 16%, 33% and 31%, respectively, compared to the treatment without biochar. For nodules, we

261 also observed a significant decrease in S1B, S3B and C, with rates of 28%, 49% and 25%,

262 respectively, compared to the treatment without biochar. In the case of Zn, higher

263 concentrations were detected in nodules, roots and shoots of plants exposed to S1. The values

264 reached 937.79±79.98 mg/kg, 121.76±6.51 mg/kg and 50.39±8.99 mg/kg, respectively. In the

265 presence of biochar, the Zn content decreased in shoots by 19% and 18% for S2B and S3B,

266 respectively, compared to the treatment without biochar. Also, the application of biochar

267 decreases the Zn content in the roots for S1B, S3B and CB compared to the treatment without

268 biochar, with percentages of 32%, 37% and 33%, respectively. In addition, Zn accumulation

269 decreased in nodules after the application of biochar in S1B, S3B and CB compared to the

270 treatment without biochar, with reductions of 23%, 24% and 26%, respectively.

271 On the other hand, when alfalfa plants are associated with biochar, they can reduce the

272 amount of heavy metals in contaminated soils compared to their initial state. Indeed, the Cu

273 content in S1B and S2B was significantly reduced with the addition of biochar compared to

274 soil without biochar. The proportions of Cu were 29% and 22% in S1B and S2B, respectively.

275 The application of biochar significantly reduced the concentration of Zn in CB, with an
276 average value of 6.03±0.99 mg/kg, compared to C (9.98±0.8 mg/kg). Then, the Ni values

277 significantly decreased in CB by 38%. Significant decreases were also found in S1B for Cd

278 and Pb, with a percentage of 19% and 21%, respectively, compared to the treatment in the

279 absence of biochar.

280 3.1.2. Heavy metal translocation index

281 The TF and the BCF of heavy metals are presented in Table S4. Our results showed that

282 significant concentrations of Cu and Zn were accumulated in roots with a TF < 1. For Cu,

283 BCF values significantly increased in plants exposed to S1, S2 and S3, with values of

284 34.12±2.15, 20.54±1.02 and 18.23±1.04, respectively, compared to a control value of

285 12.82±0.82. Biochar treatment significantly decreased BCF in S1B, S3B and CB, where

286 values reached 27.34±2.98, 14.41±0.93 and 10.68±0.99, respectively.

287 In the case of Zn, the BCF of alfalfa plants increased when exposed to different contaminated

288 soils. Indeed, the values for S1, S2 and S3 were 34.22±0.93, 32.48±1.73 and 8.71±0.93,

289 respectively, compared to a control value of 3.87±0.82. However, in the presence of biochar,

290 the BCF significantly decreased by 27%, 19% and 38% in plants exposed to S1B, S2B and

291 S3B, respectively.

292 3.2. Soil analysis

293 3.2.1. Soil pH variation

294 The pH variation measured in contaminated soils after 60 days of cultivation is illustrated in

295 Fig. 1a. Our results revealed, firstly, that pH values decreased slightly in contaminated soils

296 compared to the control. The decrease was more pronounced in the soil with the highest

297 contamination (S1), with a value of 8.01±0.0023 compared to a control value of 8.07±0.005.

298 Also, the addition of biochar increased the pH levels in S1B, S2B, S3B and CB. The values

299 reached 8.06±0.007, 8.13±0.003, 8.22±0.002 and 8.03±0.03, respectively, compared to the

300 treatment without biochar.

301 3.2.2. Determination of CEC

302 The lowest CEC was recorded in soil S1, where the value reached 5.69±0.43 mg/kg,

303 compared to a control value of 14.09±0.08 mg/kg. But the CEC values after biochar addition

304 were significantly increased in S1B, S2B and CB compared to soils without biochar, with a

305 percentage increase of 19%, 15% and 12%, respectively (Fig. 1b).

306 3.2.3. The FDA assay

307 The FDA activity is presented in Fig. 2a. The activity decreased significantly in S1, S2 and

308 S3, reaching 0.03±0.001, 0.05±0.0009 and 0.06±0.002 μg of fluorescein/g/h, respectively.

309 This is in comparison to a control value of 0.086±0.005 μg of fluorescein/g/h. On the other

310 hand, in the presence of biochar, values increased significantly, with rates of 17%, 27% and

311 23%, respectively, in S1B, S2B and S3B compared to the treatment in the absence of biochar.

312 3.2.4. Average well-colour development (AWCD)

313 AWCD decreased significantly after 60 days in S1 and S2, with values of 0.002±0.0002 and

314 0.003±0.0002, respectively, compared to a control value of 0.005±0.0003. However, there

315 was a 29% increase recorded in S3 compared to the control soil (Fig. 2b). On the other hand,

316 the application of biochar significantly enhanced the AWCD in S1B, S3B and CB, with mean

317 values of 0.003±0.0001, 0.014±0.0006 and 0.011±0.0003, respectively.

318 3.2.5. Functional diversity

319 Regarding functional diversity in soils after 60 days of cultivation (Fig. 2c), the values in

320 contaminated soils were generally lower compared to the control. The values for S1 and for

321 S2 were 3.33±0.57 and 4.32±0.41, respectively, while the control value was 5.5±0.71.

322 However, a significant increase was clearly noted in S3, and the mean reached a maximum of

323 11.33±0.56. Additionally, functional diversity increased in contaminated soils in the presence

324 of biochar, with rates of 25%, 28% and 61%, respectively, in S1B, S2B and S3B, compared to

325 the treatment without biochar.

326 3.2.6. Substrate utilisation pattern

327 The substrate utilisation pattern is shown in Fig. 2d. We found that all soils contain bacteria

328 with the ability to degrade polymers, carboxylic acids, carbohydrates, amino acids and

329 amine/amide substrates. The degradation function of the polymer’s substrate decreased with

330 S1, S2 and S3, with values of 7.75±1.08, 15.5±1.99 and 19.37±1.02, respectively, compared

331 to the control value of 23.25±1.23. But with the addition of biochar, polymer degradation

332 increased by 60% in S1B, 37% in S3B and 14% in CB. The carboxylic acid then showed a

333 gradual increase and rose in S2 with a value of 9.68±1.02, compared to a control value of

334 7.75±0.99. However, the degradation of the carboxyl group in S1 reached a value of

335 3.87±0.87. The highest mean value was observed in plants exposed to S3B, with a value of

336 31±2.01. The maximum degradation of carbohydrates was observed in S3, with a value of

337 13.95± 1.87, compared to 8.26±0.89 for the control. Significant increases were observed in

338 S1B and S3B after the application of biochar, with an increase of about 60% and 55%,

339 respectively, compared to the treatment without biochar. In addition, S3 had the highest rate

340 of amino acid degradation, with a value of 13.28±1.01, compared to 4.42±0.97 for the control.

341 Our results showed a significant increase in carbohydrates after the application of biochar in

342 S2B and S3B, with mean values reaching 12.57±0.99 and 31±2.77, respectively. On the other

343 hand, bacteria in group S3 exhibited the highest degradation of amines/amides, with a value

344 of 18.65±0.94, compared to 15.5±0.97 for the control group. The addition of biochar had a

345 significant effect on amines/amides, especially in the case of S3B, which had an average

346 value of 31±2.22 compared to soil without biochar.

347 3.3. Plant sample analysis

348 3.3.1. Biometric parameters

349 Table 3 presents the biometric parameters of alfalfa plants after 60 days of exposure to four

350 contaminated soils from the Ghannouch site of Gabesin, both in the presence and absence of

351 biochar.

352  Length of plants

353 The plants that were exposed to a contamination gradient exhibited a reduction in growth

354 parameters after 60 days of exposure to Pb. Our results showed that the greatest impact was

355 observed in the shoots and roots that were exposed to S1, resulting in significant inhibition of

356 73% and 85%, respectively, when compared to the control plants. Biochar addition

357 significantly increased shoot length in S1B, S2B, S3B and CB, with mean lengths of

358 26.26±0.35 cm, 17.56±0.49 cm, 14.6±0.55 cm and 10.16±0.28 cm, respectively. In addition,

359 the treatment with biochar increases root length in plants of S1B, S2B and S3B compared to

360 the treatment without biochar. After 60 days of exposure, the percentage of length

361 augmentation was 22%, 23% and 11%, respectively.

362  Weight of plants

363 A significant decrease in shoot and root weight was observed along with the contamination

364 gradient. The greatest reduction was observed in plants exposed to S1, with levels of

365 4.16±0.66 g in shoots and 2.9 ± 0.26 g in roots, compared to 19.2±0.9 g in control shoots

366 and 21.76±1.36 g in control roots. On the other hand, the weight of shoots increased in plants

367 exposed to biochar of S1B, S2B, S3B and CB compared to the treatment without biochar.

368 After 60 days of exposure, the elevation rates were 38%, 19%, 14% and 18%, respectively.

369 Also, root weight increased by 46%, 24%, 28% and 27%, respectively, in S1B, S2B, S3B and

370 CB when exposed to contaminated soil in the presence of biochar, compared to the treatment

371 without biochar.

372  Dry matter

373 Interestingly, several decreases were noticed, with a rate of about 90% in shoots and 89% in

374 roots overall. In the presence of biochar, the DM in the roots increased progressively. The

375 mean DM percentages were 5.78±0.089% for S1B, 17.02±1.2% for S2B and 21.38±1.087%

376 for S3B.

377 3.3.2. Chl content

378 Chl content in alfalfa plants exposed to gradually contaminated soils for 60 days is reported in

379 Table 4. Our results indicate a crucial decrease in both Chla and Chlb content, especially

380 when exposed to S1. The mean Chla content reached 0.47±0.038 mg/g FM, while the Chlb

381 content reached 0.95±0.04 mg/g FM. Chla content increased in S1B and passed from

382 0.47±0.038 mg/g FM in S1 to 0.72±0.06 mg/g FM. Otherwise, in the presence of biochar, the

383 Chlb content increased by 40% in plants exposed to S2B compared to S2 contents.

384 3.3.3. Analysis of antioxidant enzymatic activities

385  CAT activity

386 CAT activity variation in alfalfa plants after 60 days is presented in Fig. 3. Regarding CAT

387 activity in shoots (Fig. 3a), our results showed a significant increase (p < 0.05) in plants

388 cultured in S1, S2 and S3. The mean values were 0.40±0.012, 0.17±0.008 and 0.02±0.0006

389 µmol min−1 mg−1 of protein, respectively, compared to a control value of 0.005±0.0007 µmol

390 min−1 mg−1 of protein. In contrast, the CAT activity of plants’ shoots cultured in biochar-

391 amended soil decreased gradually. The average values were 0.3±0.004, 0.08±0.003,

392 0.017±0.001 and 0.002 ±0.0002 µmol min−1 mg−1 of protein for S1B, S2B, S3B and CB,

393 respectively. Exposure of plants to a gradient of three contaminated soils for 60 days resulted

394 in an increase in CAT activity in roots (Fig. 3b), reaching a maximum in the plants exposed to

395 the highest contaminated soil (S1), with a value of 1414.55±67.31 µmol min−1 mg−1 of protein,

396 compared to a control value of 131.97±6.74 µmol min−1 mg−1 of protein. In the presence of
397 biochar, the CAT activity decreased by 41%, 14% and 35%, respectively, in B, Cd1B and

398 Cd2B compared to the treatment without biochar.

399  GST activity

400 GST activity in alfalfa plants’ shoots and roots is shown in Fig. 4. The highest activity was

401 observed in the case of exposure to S2, with a value of 0.39±0.05 µmol min−1 mg−1 of protein,

402 compared to a control value of 0.06±0.008 µmol min−1 mg−1 of protein. However, a significant

403 decrease was clearly observed in shoots exposed to soils with biochar, with mean values of

404 0.02±0.002, 0.1±0.002 and 0.12±0.008 µmol min−1 mg−1 of protein for S1B, S2B and S3B,

405 respectively (Fig. 4a). Otherwise, the most important activity was observed in roots exposed

406 to S2, with a value of 0.25±0.012 µmol min−1 mg−1 of protein, compared to a control value of

407 0.11±0.004 µmol min−1 mg−1 of protein. However, GST activity decreased by 87% in S1

408 compared to control plants. Then, a significant decrease in roots was observed after biochar

409 application, with a reduction of 50% in S1B, 28% in S2B, 28% in S3B and 36% in CB

410 compared to soils without biochar amendment (Fig. 4b).

411  Lipid peroxidation

412 Lipid peroxidation was determined by evaluating the accumulation of MDA in alfalfa plants

413 (Fig. 5). MDA content increased significantly in shoots exposed to S1, S2 and S3 after 60

414 days of exposure, reaching 4.87±0.11, 1.67±0.12 and 0.99±0.18 nmol g-1 FM, respectively,

415 compared to a control value of 0.24±0.07 nmol g-1 FM. Indeed, in the presence of biochar, the

416 means reached 3.98±0.11, 1.26±0.06 and 0.76±0.05 nmol g-1 FM, respectively, for S1B, S2B

417 and S4B, compared to a value of 0.23±0.05 nmol g-1 FM for CB (Fig. 5a). In the roots, the

418 highest level of MDA was observed in plants exposed to S1, with a rate of up to 7.54±0.42

419 nmol g-1 FM, compared to the control MDA content of 0.14±0.02 nmol g-1 FM. In the

420 presence of biochar, the MDA content decreased by 30% and 24%, respectively, in S1B and

421 S2B compared to non-amended soils (Fig. 5b).

422 3.4. Principal component analysis (PCA) and correlation matrix

423  Correlation analysis (Pearson’s coefficient)

424 Based on the statistical analysis of the correlation between different accumulated metals and

425 root activity (Fig. S5a), we discovered a strong negative correlation between root weight and

426 length, respectively, with Ni, Cu and Zn in soils and nodules. Also, we noticed a negative

427 correlation between MDA/CAT levels in roots and biometric data in bean plants (-0.87; -

428 0.84). In contrast, there is a strong positive correlation between heavy metals in soils and

429 nodules and CAT/MDA activities.

430 As illustrated in Fig. S5b, which represents the correlation matrix linking biochemical and

431 physiological data in bean plants with heavy metals in shoots. A slight negative correlation

432 was observed between GST levels in shoots and other biochemical activities, as well as the

433 accumulation of heavy metals in shoots. In contrast, an MDA correlated negatively with

434 Chla/b and the other biometric measurements. Interestingly, correlation analysis confirmed

435 the results obtained from this study, which revealed that heavy metals enhanced MDA and

436 CAT activity and contributed to the decrease in the biometric properties of bean plants.

437 Regarding the physicochemical properties of soils and the presence of heavy metals

438 originating from nearby industries at the sampling sites, the correlation matrix (Fig. S5c)

439 indicated a strong negative correlation between the heavy metals and both microbial and

440 physicochemical properties. A relatively positive correlation has been recorded between

441 AWCD (functional diversity), FDA (pH) and CEC.

442  PCA

443 In Fig. S6a, it is clearly shown that the accumulation of metals in the soil in bean roots had a

444 negative impact on the biochemical activities and morphological parameters of alfalfa roots.

445 Soils are separated from the control group, as demonstrated on the PCA plot in Fig. S6a’. The

446 most contaminated soils, S1 and S2, were positioned opposite Dim2 (10.63%). At the same
447 time, S3 was found to correlate negatively with Dim1 (78.87%) and was slightly separated

448 from the control group. The addition of biochar had a minor impact on soil S1, but a

449 pronounced effect was observed in S2B and S3B, as they are located near the origin of the

450 map.

451 Interestingly, Figs. S6b and b’ illustrate the PCA of heavy metals and biometric and

452 biochemical measurements in alfalfa shoots. It is clear that the accumulation of heavy metals

453 had a negative impact on the Chl content, weight and length of shoots after exposure while

454 also increasing biochemical activities. Biochar amendment had a significant effect, as

455 demonstrated in the PCA plot, where soils were clearly distinguished from one another.

456 Concerning microbial and physicochemical measurements in soils, Figs. S6c and c’

457 summarise the impact of metallic stress, plant cultivation and biochar amendment on soil

458 fertility. Metal accumulation has limited some bacterial functions as they are opposed to

459 Dim2 (13.93%) and have promoted soil pH and CEC. To confirm scientific findings and refer

460 to the PCA plot (Fig. S6c’), the application of biochar enhanced bacterial diversity and

461 resistance to metallic stress in soils. In addition, the cultivation of contaminated soils with

462 biochar amendment has intensified bacterial activities to alleviate heavy metal toxicity.

463  Multiple factor analysis (MFA)

464 Multiple factor analysis (MFA) (Fig. S7) is a multivariate data analysis technique used to

465 summarise and visualise complex data tables. It is particularly useful when describing

466 individuals using multiple sets of variables, both quantitative and qualitative, that are

467 structured into groups (Pagès, 2002).

468 Referring to individuals-MFA (Fig. S7a), soils with similar profiles were found in close

469 proximity to each other on the factor map. S1 and C soils oppose the second axis (17.68%),

470 while the first axis (57.12%) is primarily associated with S3. Based on the biochar

471 amendment, groups B and WB are situated near the origin of the MFA map, indicating that it
472 has no significant effect on the control soils. In contrast, other soil groups were discovered in

473 contrast to group B, demonstrating that biochar has influenced plant soil activities.

474 Briefly, the graph of variables-MFA (Fig. S7b) shows a relationship between variables,

475 representing the quality of representation and the correlation between variables within the

476 same group. However, the first axis (17.65%) is mainly correlated with certain biochemical

477 activities (such as GST in shoots and roots) and some bacterial properties. On the other hand,

478 the second axis (57.12%) represents the presence of heavy metals in soils and plants, as well

479 as biometric measurements and microbial properties of the different soils.

480 4. Discussion

481 The main objective of this work was to assess the impact of biochar amendment on soils that

482 have been gradually contaminated in an industrial region of Gabes Governorate in Tunisia. In

483 this work, we used alfalfa plants (Medicago sativa L.) as a bioindicator to evaluate their

484 oxidative balance under metallic stress, both with and without the presence of biochar. In

485 addition, the microbial properties of the different soils were examined to investigate the

486 ability of biochar to enhance phytoremediation in contaminated soils.

487 Extensive literature has investigated the effects of heavy metals on alfalfa; however, most of

488 the research has been conducted on artificial soils (Bian et al., 2016; Kamran et al., 2016;

489 Igalavithana et al., 2017; Morsy et al., 2022). Only a few studies have been conducted in the

490 field on natural agricultural soils contaminated with heavy metals near the Gulf of Gabes,

491 Tunisia (Li et al., 2015) that have focused on physicochemical analysis (Tayibi et al., 2009;

492 Kabata-Pendias and Szteke, 2015). The originality of this work lies in the use of biochar to

493 assess and improve the chemical and microbial quality of soil in the Gulf of Gabes, where

494 alfalfa plants are present.

495 Firstly, our data suggest that alfalfa plants can accumulate significant amounts of Cu and Zn

496 when exposed to highly contaminated soils from S1. However, alfalfa plants cannot be
497 considered hyperaccumulators because the [shoot-C]/[root-C] ratio of Zn and Cu is less than

498 one, and the highest concentrations are found in the roots, especially in nodules. These results

499 indicate that Cu and Zn are preferentially accumulated in nodules, suggesting limited

500 translocation into leaves (Hattab et al., 2013; Soussou et al., 2013). In addition, Reeves and

501 Baker (2000) found that Carpatica plants accumulate high concentrations of Zn in their roots

502 but not in their shoots. The same results were observed in alfalfa plants exposed to Ni

503 (Helaoui et al., 2020b). Likewise, Helaoui et al. (2022a) found that rhizospheric bacteria

504 interacted with roots, and these interactions increased the accumulation of heavy metals (such

505 as Cu, Zn, Ni, Cd, Pb and Hg) in plant biomass. In fact, nodules in alfalfa plants produce

506 organic acids and other secretions that make heavy metals more bioavailable to plants (Ma et

507 al., 2011; Massalha et al., 2017). According to Khan et al. (2009), symbiotic bacteria

508 associated with plants have the ability to remediate contaminated soil. On the other hand,

509 Podar and Maathuis (2021) demonstrated that nodules serve as a barrier against the transfer of

510 metals to other plant organs, possibly due to tolerance mechanisms. In addition, Sharma

511 (2021) showed that alfalfa plants have a high capacity to accumulate heavy metals through

512 nodular fixation, which subsequently restricts their migration to the aerial parts.

513 Interestingly, our results revealed that the presence of biochar reduced the accumulation of

514 heavy metals in plants. The adsorption of Cu and Zn on the surface of biochar is considered

515 an important mechanism for reducing metal mobility and bioavailability (Shen et al., 2020).

516 Moreover, Nie et al. (2018) showed that biochar has the ability to decrease the buildup of

517 heavy metals in plants and their availability in soil. Recently, several studies have shown that

518 biochar can play a role in removing metals from soil and water, thereby reducing the

519 bioavailability of toxic substances from soil to plants and other organisms, including humans

520 (Yao et al., 2012). Furthermore, biochar reduced the uptake of Cu, Ni, Zn, Pb and Cd through

521 surface chelation, precipitation and ion exchange (Akhtar et al., 2015; Wiqar et al., 2022). It
522 has been reported that biochar has a strong ability to adsorb heavy metals, which may be

523 another reason for the reduction in the concentration of heavy metals (Xie et al., 2018). It

524 appears that the most polluted soils exhibited the highest percentage change when biochar

525 was added. Under high metal content, the surface functional groups and adsorption sites of

526 biochar can increase the cation exchange capacity of the soil. This, in turn, increases the

527 soil’s metal exchange capacity by forming important complexes with metals (Paz-Ferreiro et

528 al., 2014).

529 Also, the present investigation reported that soil pH and CEC increased in the different soils

530 after biochar addition. These results are consistent with those of Yang et al. (2016), who

531 found that adding biochar at a 5% concentration increased soil pH and CEC. Further, the

532 decreased levels of extractable Cu and Zn in biochar-applied soils, compared to the control

533 group, may be attributed to several factors, including elevated soil pH and CEC (Fahmi et al.,

534 2018). Indeed, Zhang et al. (2017) revealed that an increase in the CEC and pH of

535 contaminated soil enhanced metal adsorption due to the increase in adhesive surface area. On

536 the other hand, Lucchini et al. (2014) demonstrated that the alkalinity of the biochar and the

537 release of basic cations into the soil solution could explain the increase in soil pH. Likewise,

538 increasing the soil pH enhances the immobilisation of heavy metals by biochar particles

539 (Bashir et al., 2018).

540 Concerning soil microbiological activities, the hydrolysis of the FDA is a good indicator of

541 total microbial activity because it is performed by various enzymes produced by

542 microorganisms such as bacteria and fungi (Li et al., 2017). FDA activity decreased

543 significantly along with the contamination gradient after 60 days of cultivation. The decrease

544 in enzymatic activity caused by heavy metals is a complex issue that depends on factors such

545 as metal concentration, speciation, enzyme type, soil characteristics and plant species

546 (Mkhinini et al., 2020). While the activity of FDA in soils with biochar was generally higher
547 compared to soils without biochar. According to Egamberdieva et al. (2018), biochar can

548 provide a favourable habitat for microbes because of its porous structure, significant internal

549 surface area and ability to adsorb organic matter and mineral nutrients. Consequently, it can

550 enhance the enzymatic activity of microorganisms (Lehmann et al., 2015; Khadim et al.,

551 2021a). In fact, biochar reduces the loss of soil organic matter by decreasing greenhouse gas

552 emissions from soils, enhancing nitrogen retention and increasing the efficiency of fertiliser

553 utilisation (Liu et al., 2016; Agyarko-Mintah et al., 2017).

554 The ability of bacteria to degrade the BiologEcoplate™ substrates is significantly reduced in

555 highly contaminated soil. These results are consistent with those observed by Azarbad et al.

556 (2013), who found that the degradation of carbon substrates by microbial biomass is affected

557 in soil contaminated with metals such as Cu, Zn, Pb and Ni. In the presence of biochar, high

558 catabolic potential activity was observed, especially in soil S2, indicating important microbial

559 activity in this contaminated soil. This result could be related to previous studies that have

560 highlighted the positive impact of biochar application on the metabolic growth of bacteria and

561 the creation of beneficial habitats for microorganisms. Biochar improves water retention

562 capacity, nutrient availability and carbon sequestration ability, thereby protecting

563 microorganisms from toxic pollutants (Zimmerman et al., 2011; Chen et al., 2017; Zafar et al.,

564 2020; Khadim et al., 2021b). Also, Gabhane et al. (2020) showed that the addition of biochar

565 increases nodule biomass and nitrogenase activities.

566 In general, soil biofertility is an important concept encompassing physical, chemical and

567 biological flow. Thus, evaluating the biological functioning of the soil after contamination

568 could indirectly indicate soil quality (Venturino et al., 2019). In the same vein, plants could be

569 used to analyse soil fertility and test the effects of metals on soil activities. This could be

570 assessed through studies of agronomic, photosynthetic and biochemical parameters (Hattab et

571 al., 2014; Helaoui et al., 2020). It has also been proven that certain plant species, such as
572 alfalfa plants, can be utilised in toxicological investigations due to several advantages. These

573 include their ability to maintain soil structure, their simple cultivation techniques and their

574 tolerance to heavy metals in polluted soils (Carrasco Gill et al., 2012; Helaoui et al., 2022a).

575 In our study, the growth of alfalfa plants was significantly affected by contaminated soil.

576 Indeed, Cd, Ni, Pb and Hg inhibited the biomass production of alfalfa and caused a decrease

577 in root and shoot length (Ghelich et al., 2014; Montero-Palmero et al., 2014; Helaoui et al.,

578 2020). Thus, soybean plants under 50 mg/kg Cu contamination have been shown to be

579 susceptible to reducing their biomass (Bernal et al., 2007). As demonstrated by numerous

580 authors, the decrease in plant growth can be explained by the fact that a high concentration of

581 Cu impacts essential processes such as cell viability, photosynthesis and mineral nutrition

582 transport (Kopittke and Menzies, 2006). Also, a reduction in root biomass and shoot biomass

583 was observed in Anthyllis vulneraria L. exposed to 1000 mg/kg of Zn (Soussou et al., 2013).

584 Therefore, it has been proven that the inhibition of plant growth is not caused by the direct

585 effect of Cu and Zn accumulation but rather by nutrient deficiencies that result from impaired

586 absorption of essential nutrients due to damaged roots (Dimkpa et al., 2019). On the other

587 hand, our results indicated that the supply of biochar significantly enhanced alfalfa’s growth

588 in various soil conditions. Previous research has reported that biochar can increase plant

589 growth by improving the bioavailability of mineral nutrients and enhancing soil’s physical

590 and biological properties (Saifullah et al., 2018; Irfan et al., 2021; Muhammad et al., 2022). In

591 fact, the physical and hydraulic properties of the soil influence macro- and micro-scale

592 processes such as root growth, pore volume, aeration and nutrient uptake (Zemanova et al.,

593 2017). Additionally, biochar seems to be a favourable substrate for remediating contaminated

594 soil (Tang et al., 2013; Fahad et al., 2016; Ibad et al., 2022). It has been reported that the

595 addition of biochar can improve the soil’s ability to retain and absorb plant nutrients while

596 also reducing nutrient loss caused by leaching. Therefore, biochar produced from various
597 organic residues is an effective method for improving soil fertility and crop productivity in the

598 long term (Kapoor et al., 2022). The application of biochar to agricultural soils has shown

599 potential agronomic and environmental benefits, including enhanced soil fertility, crop

600 productivity and carbon sequestration (Pandit et al., 2021).

601 Photosynthesis is a crucial mechanism in plants that plays an important role in their growth

602 and development (Xu, 2015). Our results revealed that Chl contents decreased gradually in

603 plants along with the contamination gradient. This decrease could also be attributed to the

604 presence of heavy metals such as Zn and Cu, which are known to reduce synthesising

605 enzymes, modify chloroplast numbers and induce nutrient deficiencies such as Fe and Mg

606 (Yruela, 2005; Parlak and Yilmaz, 2012). In addition, Faizan et al. (2021) demonstrated that

607 Cu contamination decreased the number of stomata in the leaves and reduced intercellular

608 CO2 levels, stomatal conductance and transpiration rate (Faizan et al., 2021). However, under

609 biochar addition, a remarkable increase was noted in Chl contents, which were more

610 pronounced in highly contaminated soils. Our findings are consistent with previous

611 investigations that have demonstrated an increase in the photosynthesis process in rice plants.

612 This increase could be attributed to the reduction of metal bioavailability in plants (Rizwan et

613 al., 2016; Hesham and Fahad, 2020). Also, Zoghi et al. (2019) demonstrated that the supply of

614 biochar significantly increased Chl content by 59%, transpiration by 60% and stomatal

615 conductance by 85%. Biochar enhances the stability of heavy metals in soil, thereby reducing

616 their negative effects on photosynthesis activities and Chl content (Kamran et al., 2019). As

617 expected, the application of biochar increased nitrogen absorption. In fact, nitrogen is an

618 important component of Chl and protein contents, which are well-known to enhance plant

619 biomass (Ullah et al., 2018).

620 The accumulation of heavy metals in alfalfa plants after their exposure to contaminated soils

621 near the industrial region of Gabes, Tunisia, has induced severe oxidative damage. In fact, Cu
622 is highly toxic to plant growth, potentially causing damage and resulting in the

623 overproduction of ROS and a reduction of antioxidant enzyme activities. This imbalance

624 between the generation and elimination of ROS may be induced by Cu (Schutzendubel and

625 Polle, 2002; Talebi et al., 2019). Furthermore, the combination of Cu and Zn stress increased

626 lipid peroxidation of the cell membrane and H2O2 production, thereby activating CAT and

627 GST activities. These results are similar to previous investigations, which demonstrated that

628 contamination with Cu, Cd, Pb, Ni and Hg caused severe oxidative stress in alfalfa plants

629 (Hattab et al., 2016). Additionally, MDA accumulation has also been observed in other

630 species such as Glycine max L. (Hao et al., 2006), Vicia faba L. (Mkhinini et al., 2018;

631 Helaoui et al., 2022b), Cicer arietinum L. (Ahmad et al., 2016) and Ocimum basilicum L.

632 (Georgiadou et al., 2018). However, the addition of biochar reduced the MDA content,

633 especially in plants from S1 and S2. In fact, there are various factors that could be involved in

634 the modification of the plant-soil environment by biochar, such as: (I) decrease of metal

635 accumulation in plants; (II) stimulation of the symbiotic association of bacteria with plants;

636 (II) increase in the production of regulating substances in plants; (IV) change in the structure

637 of soil; and (V) enhancement of the availability of essential mineral nutrients (Medyńska-

638 Juraszek and Ćwieląg-Piasecka, 2020).

639 Meanwhile, antioxidative enzymes play a key role in the tolerance of plants to heavy metal

640 stress (Abdal et al., 2021). First, CAT transforms hydrogen peroxide (H2O2) into oxygen (O2)

641 and water (H2O) (Sun and Zhou, 2008). Our results indicate that CAT activity significantly

642 increased in both the shoots and roots of plants exposed to contaminated soil. Also, several

643 studies have established that excessive accumulation of Zn in plants can lead to an increase in

644 CAT activities in Solanum lycopersicum L. (Faizan et al., 2020), Pisum sativum L. (Tripathy

645 et al., 2015) and Bacopa monnieri L. (Krishnaraj et al., 2012). In this study, the addition of

646 biochar decreased the activity of CAT in both leaves and roots. This finding is consistent with
647 the results reported by Younis et al. (2016). Several authors showed that the presence of

648 biochar caused a reduction in oxidative stress (Abbas et al., 2017b). This decline might be due

649 to the reduction in Cu and Zn accumulation in plants cultivated in soil with biochar.

650 Second, GST activity in the alfalfa plants of S2 and S3 was significantly higher compared to

651 the control plants. In fact, GST plays an important role in detoxification and metal chelation

652 because GSH peroxidase forms complexes with reactive metabolites such as heavy metals,

653 lipid peroxides and xenobiotic substrates (Hussain et al., 2013). In rice plants, higher levels of

654 GST activity could be associated with a greater ability to chelate Cu (Li et al., 2018b).

655 However, the exposure of alfalfa plants to highly contaminated soil resulted in a significant

656 decrease in GST activity in both shoots and roots. Similarly, Wang et al. (2009) reported that

657 the shoots and roots of rapeseed seedlings showed a significant increase when exposed to 0.28

658 mg/kg of Zn but a significant decline when exposed to 1.12 mg/kg. Nevertheless, our results

659 regarding alfalfa’s response to heavy metal accumulation in the presence of biochar indicated

660 a crucial decrease in GST activity in both shoots and roots. Interestingly, biochar enhances

661 plant tolerance against oxidative stressors and reduces the sorption of heavy metals (Schulz

662 and Glaser, 2012; Paz‐Ferreiro et al., 2017). Due to its porosity and adhesive surface, biochar

663 is characterised by its ability to sorb high amounts of metals, which provides notable

664 advantages for photosynthesis activities and the overall growth of plants (Jeffery et al., 2011).

665 Although there is considerable knowledge about the effects of metal toxicity on soil

666 functioning, less is known about the impact of biochar on the remediation processes of

667 contaminated soils. For instance, biochar has been reported to contribute to the improvement

668 of soil enzymatic activities, plants’ metabolic processes and the remediation of heavy metals

669 (Thies et al., 2011; Sun et al., 2017; Sayyadian et al., 2019; Mailakeba and Rajashekhar Rao,

670 2020). In the presence of biochar, the high cation exchange capacity has the potential to

671 provide sufficient nutrients to plants (Puga et al., 2015). It is possible that biochar plays a
672 crucial role in enriching the nutrient content of plant tissues and, as a result, reducing the

673 formation of ROS caused by soil metal toxicity. This, in turn, leads to increased synthesis of

674 photosynthetic pigments and improves the performance of alfalfa plants when exposed to

675 these harmful metals. These results provide the first insight into the role of biochar in

676 reducing metal uptake and promoting the growth of alfalfa. Therefore, our work proposes the

677 use of biochar and alfalfa crops to remediate contaminated agricultural soils.

678 5. Conclusion

679 Overall, the present investigation revealed that exposure to contaminated soil from the Gabes

680 region induces damage to alfalfa plants as well as soil microbiological parameters. These

681 adverse effects were alleviated by the application of biochar, which significantly reduced

682 heavy metal accumulation, increased plant growth and biomass, reduced Chl content and

683 reduced oxidative stress. Furthermore, biochar stimulates soil microbiological activities,

684 which in turn influence nutrient uptake and promote plant growth. Based on our results, this

685 study could provide a clear understanding for researchers/scientists regarding the role of

686 biochar in the remediation of polluted soil. It also offers perspectives on ecological solutions

687 that require further research and elucidation. In order to gain a better understanding of the

688 connections between alfalfa plants and bacterial communities in the presence of biochar,

689 metagenomics analyses are required to identify the rhizospheric microbes that are affected by

690 the addition of biochar.